the importance of forgetting

Posted comment on ´The importance of forgetting` written by L. Gravitz and published in Nature 2019 vol 571 25th July 2019 S12-S14 doi 10.1038/d41586-019-02211-5

SUMMARY

   Gravitz introduces her article by describing the development of the idea that forgetting is not a passive process of memory decay and recall failure, but an active functional process. Therefore, it was said that the standard state of the brain is not to remember, but to forget. The forgetting process is one largely overlooked by researchers and it is possible that a better understanding of it may lead to breakthroughs in the treatments of conditions such as anxiety, post-traumatic stress disorder and Alzheimer`s disease where dysfunctional forgetting play a role.

   The article begins with a general summary of the memory processes and its requirement for neuronal plasticity which strengthens the connectivity and firing of the active neurons representing the event characteristics. This event representation (referred to in the article as an engram) may consist of synaptic connections across several areas of brain and each neuron and synapse of the network can be involved in multiple engrams.  Recall strengthens the neural network of firing cells and Gravitz describes the consistent recall as encoding the memory in both hippocampus and cortex areas until eventually the long-term storage reflects only cortex activity. The article continues by looking at the various aspects of the memory mechanisms from the viewpoint of forgetting and not memory formation.

   The discussion begins by looking at the association of neurotransmitters with forgetting. Gravitz describes the first reports of active forgetting as work reported in 2012 from Davis. Davis used fruit flies in his studies and looked at the action of dopamine released from other neurons on the mushroom bodies, which are dense networks of neurons in insect brains that store olfactory and other sensory information. Conditioning experiments were performed with flies avoiding odours associated with electric shocks. It was found that dopamine blocked the conditioned avoidance response, but inhibition of the same neurons preserved the memory. Therefore, Davis concluded that dopamine regulated how memories could be expressed. Hence, dopamine was said to provide the ´forget` signal. It was also found that dopamine neurons were active for long periods and this observation was interpreted as the brain always trying to forget the information it has learnt. An association between forgetting and neurotransmitters was also described for rats. In this case, the active process of forgetting was shown by Hardt to involve glutamatergic AMPA receptors. AMPA receptors are known to be part of the memory storage process by being trafficked to the post-synapse membrane and forgetting was described as the destruction of the connections at the synapse. Again, the premise that forgetting is not a failure of memory, but a function of it was stated.

   Gravitz`s article then went on to describe the association between forgetting and neurogenesis, which is a process normally associated with memory formation. In this case, the research by Frankland and neurogenesis of hippocampal cells in mice was cited. Using the knowledge that memory formation requires newly formed neurons, Frankland looked at whether increasing neurogenesis would aid memory recall in adult mice. The team found instead that under these conditions increased neurogenesis which would normally provide a greater neuronal capacity actually led to increased memory loss. This was explained by the new neurons in the hippocampus integrating into already established neuronal network actually causing ´disruption` of the network and making information harder to access. Although not always beneficial, it is used to advantage because of the constantly changing environment that the individual lives in and the need for continual learning of new information and adaptation of learnt material by over-writing.

   The article then continues by looking at studies on human memory. Gravitz cites Richards who states that the ability to generalise new experiences is due to the brain`s ability to carry out controlled forgetting. This Richards says prevents ´overfitting` which relates to artificial intelligence where the mathematical model is so good at matching programmed information that the AI is unable to predict what comes next. Richards explains that memory of gist rather than detail allows generalisation of experiences and use of that information in novel situations. This view is supported by Levine with reference to real-life situations where people can have highly superior autobiographical memory (HSAM) or severely deficient autobiographical memory (SDAM). In HSAM, an extraordinary level of detail is remembered, but there is limited informational processing capability and an increased tendency to obsessiveness. These characteristics are attributed to a person´s lack of ability to extract themselves from the pure data. In SDAM the individual is unable to vividly recall specific events and has a problem with projection into future events, but they are good at problem solving and abstract thinking.

   Work by Anderson and team on how active forgetting occurs is cited in the article. They found that in situations where thoughts are actively suppressed then high GABA levels are linked with greater suppression of the prefrontal cortex on the hippocampus. Gravitz says in her article that work on GABA and this type of suppression may explain the action of anti-anxiety benzodiazepines (eg. diazepam) which are known to enhance GABA receptors. The action of benzodiazepines is explained by the prefrontal cortex commanding the hippocampus to inhibit the thought. However, if the hippocampus does not have enough GABA then it cannot carry out the command and therefore, the increase in GABA receptors by anti-anxiety drugs will increase the hippocampus response. Gravitz goes on to say that GABA´s role in suppressing unwanted thoughts hence may be part of the mechanisms linked to phobias, schizophrenia and depression. These conditions present with various symptoms (eg. flashbacks, obsessive and/or depressive thoughts and difficulty in controlling thoughts) which may be linked to an overactive hippocampus. Anderson`s observations and the switch off mechanism orchestrated by GABA may play a role in removing these cognitive problems. Anderson also suggests that this might have implications for treatment of post-traumatic distress syndrome which may be failure to forget. It has been found that people who report more traumatic experiences are particularly good at repressing specific memories. The hypothesis may also apply to Alzheimer´s disease where memories are lost. Hardt thinks that the malfunction of forgetting lies with an overactive forgetting mechanism erasing more than it should may be at play rather than with a dysfunctional recall mechanism.  

  Gravitz concluded her article with a description of the shift of focus of research from the brain´s ability to form memories to the brain`s ability to forget, hence indicating this aspect`s rise in importance. The importance is said to be understandable since in a changing environment adaptive memory with the capability of updating knowledge enables individuals to move forwards and for adaptive memory to exist, forgetting is required.

COMMENT

   What makes this article so interesting is that it discusses ´forgetting` which is one of the many mechanisms involved in memory. Unlike learning, forgetting cannot be thought of as a single type of process because it applies to at least two different situations. The first is where forgetting is ´failure to remember`, a situation such as that occurring with Alzheimer disease and is essentially regarded as negative. This situation refers to when information is no longer available for recall even under prompting due to destruction of the neuronal network required for its representation. And the second situation is where ´forgetting` means that stored information cannot be remembered because it has been ´overwritten`. In this case, the original information has been updated because perhaps it is no longer relevant or needed. This type of situation occurs in for example, extinction in conditioning experiments or more simply knowing how to operate your first mobile of over 20 years ago. In this case, ´forgetting` may mean that the new information supercedes the old information and that it is truly gone or the new information has higher priority in the recall process. Here, the old information is still there, but only comes to mind when prompted to a high degree or when deliberately and specifically searched for. So, here is the first point about using the word ´forgetting`. Forgetting refers to how we, the individual, refers to the recall of the information and not how the brain as a physiological structure views the recall of the information. This indicates how we should look at forgetting in terms of neurochemical mechanisms.

If we look at the first situation, where forgetting is ´failure to remember` this occurs because the information is no longer there or accessible to us due to physiological destruction. This is a different situation to not being able to remember something because it was never there (ie. long-term memories were never formed from the real-time neural representations independent of the reason). Therefore, for this forgotten information we can assume that the physiological mechanisms required to form long-term memories were at some point carried out. This also means that the long-term memories are located in the relevant cortical areas or cerebellum and are in the form of a network of cells (neuronal assembly) that represent the event. The cells of the network fire together in response to a particular stimulus leading to the recall of the characteristics they represent.  Firing of the network is ameliorated (´fire together, wire together`) by the physiological changes that occur to its constituent cells on event storage, eg. increased AMPA receptors (long-term potentiation), increased connectivity through dendrite formation as well as gene modulation that aids neurotransmitter production, vesicle recycling, energy supply and glial cell functioning for example. Two things are assumed here: the first is that the neuronal assembly stays relatively stable for a period of time from its conception to its first use; and secondly, that the hippocampus is purely a relay station in charge of the recall stimulus and timing of event recall, real-time input and real-time informational processing. (Its role in binding of event characteristics and synchronising firing of different areas in the formation of the neuronal assembly is no longer applicable when the information is recalled.) Therefore, in the case of forgetting where forgetting is ´failure to remember` the overlying physiological cause is the decay of the ´silent` neuronal assembly so that a stimulus no longer initiates the firing of the neurons that made it up and that represented the event experienced in the past. The event can be forgotten in its entirety or only part as in the forgetting of certain characteristics.

   If we assume that memory retrieval involves the reinstatement of the neuronal firing patterns, then memory decay involves the physiological destruction or dysfunction of the relevant neurons and the connectivity between them. There are a number of different causes for such failure. The obvious one is the large scale physical destruction of the neuronal cells and this is seen in situations such as injuries, lesions and Alzheimer`s disease. It is clear therefore, that if the cells are no longer present then the network cannot be reactivated and the event cannot be remembered.

  Another important cause of memory failure is the destruction of neuronal connectivity which leads to synchronisation and timing issues. The cells representing the event act together and to a large extent retrieval of information relies on the reactivation of the same end cells that fired during encoding. The connectivity of the network relies on the activity of a number of brain areas, eg. greater similarity between patterns of firing of the dorsal lateral prefrontal cortex during encoding and retrieval relate to better memory recall performance (Javadi) and the increased connectivity between the perirhinal cortex and other areas relate to object recognition performance (Staresinaln). The specificity of this brain area connectivity is sometimes associated with particular information content of the memory, eg. autobiographical memory preferentially activates the areas of the default mode network (Chen) whereas visual memories activate parietal and frontoparietal areas (Chen and Ye) with both memory types having functional connectivity to the hippocampus (Westphal). Therefore, destruction of the connectivity or dysfunctional connectivity can lead to decay of neuronal networks specific for the event representation with the result that recall of the specific information fails. Dysfunctional connectivity also links to the failure of the instigation of appropriate brain waves consistent with memory reenactment, eg. beta waves between visual regions and parahippocampal cortex being required for the reinstatement of neural patterns matching the retrieval of visual information (Staudigl).

   Failure to remember can also be due to micro-scale dysfunction at the neuron level since firing of each relevant cell is a necessary process for event characteristic retrieval. Since there are many components and processes required for satisfactory neuronal function in both encoding and recall of event characteristics there are many points at which dysfunction or deficit can prohibit the firing process necessary for re-enactment. For example, loss of memory reported in traumatic brain injury is related to loss of dendritic spines (Sen) which would diminish the capacity to receive the transmitted signal whereas forgetting of spatial memories is said to be caused by the removal of the AMPA receptors at the neuronal cell membrane (Migues). These are normally required for long-term plasticity of the neurons required for the event representation.

  It is clear therefore, that forgetting can be due to failure to re-instigate the event representation due to dysfunction and deficits of the neurons and networks that contribute to it. However, forgetting can also be due to failure to adapt existing memory when new information is presented. In this situation, the old information is remembered, but the failure to update with the new information means that this will be ´forgotten` at the next demand for retrieval. Updating event representations is an important part of the memory process and requires a number of correctly operating processes in the appropriate order. For example, it requires retrieval of past information (event representation re-enactment), simultaneous input of the ´new` information, ´comparison to or addition to` type decision mechanisms, and physiological processes for long-term storage as part of the re-consolidation of the ´old` information and binding and storage of the ´new` information.

   The correct functioning of two brain areas in particular, the hippocampus and the prefrontal cortex, appears to be required if ´overwriting` is successful. In the case of the hippocampus, this area plays a vital role in the relay of information and its binding during the event`s encoding. With regards to ´overwriting`, it is likely that it plays equivalent roles not only in the relay of the new information to the upper cortical areas, but also maintains that firing in order for the ´comparison` mechanisms to be carried out. This view is supported by research that shows the hippocampus`s requirement in matching of ´old` information to ´new` information as seen in face-diagnosis memory with connectivity with the left middle temporal gyrus (Brod) and contextual fear conditioning where learning is required if the recalled information does not match the new input (Bernier). Theta gamma rhythms involving the hippocampus at the time of retrieval appear to be important as to whether ´new` information overwrites the ´old` in fear memory consolidation (Radiske). Therefore, failure to overwrite manifesting as ´forgetting` may be caused by a dysfunctional hippocampus activity during the encoding and recall processes.

  The other important brain area relating to failure to overwrite the old information with new information is the functioning of the prefrontal cortex. This is a key area in decision-making processes which relates to the question whether the ´new` information is assessed as being ´valuable` and hence, an overwrite is necessary or irrelevant and classed as ´interference` and ignored. Research shows that the prefrontal cortex performs this role in the case of reward prediction errors with connectivity to the ventral tegmental area with the decision whether to maintain or deviate from previously learned cue-reward interactions (Ellwood). It is likely that both the dorsolateral prefrontal cortex (known for its role in strategic control and working memory – value assessment of incoming stimuli) and the ventromedial prefrontal cortex (known for its role in value assessment and comparison, decision-making awareness of choice  and switching of attention) are involved. Therefore, failure to overwrite can be caused by the failure of the prefrontal cortex to bring about the decision to update.

   Apart from dysfunctioning at one or both of these areas, another reason for failure is that the neuronal firing attributed to the new information is not strong enough to overcome the recalled information if it is contrasting, or not strong enough to form part of the event representation if the characteristic is to be added. Similarity to the ´old` information may be an advantage with greater similarity to the stored information being shown to be more easily encoded so leading to an improvement in memory performance with time (seen with face-diagnosis pairs – Brod). This may be due to the sharing of cells already exhibiting strong firing from the similar characteristic. However, the lack of strength of the firing for the new event characteristics may be due to input deficiencies, eg. poor visual input due to changing gaze, interference of visual details by flashing lights. Two factors may play roles here and these are emotional status and age which are both attributed as causing changes in memory performance. For example, negative emotional status such as from fear or stress is known not only to negatively impact informational input by changing attentional performance in general, but can also affect specific content. In the case of stress, there is interference with long-term memory for associated material (Trammel) and impaired memory selective for content, eg. memory for items is deficient, but not for background information (Steinmetz). The highest state of anxiety is shown to cause the inhibition of retrieval for both threatening and non-threatening informational categories (Nunez) and therefore, this will have an impact on whether event representations are updated or not. This appears to be the case also with avoidance strategy where an ability to suppress unwanted, upsetting memories. This is shown to have effects on the recall not only of the distressing event, but also of other details (Quang) and low arousal emotion can facilitate the recall for peripheral information if directly relating to it (Davidson). Therefore, if information is not being recalled, the capability to ´overwrite` it is disrupted. Positive emotions can also affect memory retrieval as shown in the case of episodic memory where performance is lower following a highly positive event even though the executive control performance is unaffected (Lagner). Therefore, as described, memory enhancement or impairment by emotion depends on the nature of the information to be retrieved and the circumstance (McKenzie).

   Another factor that can affect the performance of updating memories appears to be the age of the individual. Ageing has been shown to have an effect on memory performance attributed to physiological changes of the neuronal network, eg. aged rats exhibit defective recognition memory and alterations in hippocampal synaptic plasticity through defective LTP and enhanced LTD (Arias-Cavieres) and episodic memory declines with age, an observation that correlates to regional connectivity of the default mode network plus the medial prefrontal cortex (Huo). However, the situation is not clear cut, since age is not the reason for changes in specificity of detail retrieved for autobiographical memories since both young and older age groups show deficits (Aizpurua) and the frequency of involuntary autobiographical remembering appears not to decline with age (Berntsen). Reduced capability levels may be attributed to an inability to inhibit the incoming and storage of irrelevant material which is shown to increase with age rather than the more severe long-term physiological deficiencies. This view is supported by the reported positive effects of cognitive training which may increase older individuals` abilities to inhibit irrelevant material whereas for younger individuals it leads to an improvement in their cue-integration capability (Cappelleti). Therefore, forgetting may be due to selective failure to store new information to overwrite the old.

  Therefore, this comment shows that the topic of forgetting cannot be regarded as a simple failure to remember. It may involve different mechanisms depending on the circumstances in which it occurs or is demanded. Gravitz in her article supports the view of Davis that the brain is always trying to forget, but this is not strictly correct. The brain tries to remember but only remembers what it can when given the right conditions to do so, or what it is demanded to remember through conscious control. However, the transient physiological nature of neuronal cells means that the memory system is always in flux and cells are destroyed and formed to try to maintain what is important to the individual and recalled information normally means ´important`. This demand is not always fulfilled and success at memory retrieval is not always possible with information being lost or not being updated so effectively ´lost`. Therefore, as Hardt says, this latter type of forgetting is a function of memory and not a failure of it. It is possible that a through a greater understanding of the mechanisms that contribute to desired forgetting, solutions to the unwanted forgetting as seen in dementia for example may come to light.

Since we`re talking about the topic………………………

                ….a single electroconvulsive therapy (ECT) application is known to disrupt reactivated, but not non-reactivated memory recall for an emotional event in patients suffering from depression (Kroes). Would controlled staged recall of a highly emotional event interrupted with ECT applications lead to forgetting of the event in its entirety over time or would the repetition of the recall at each state consolidate the memory even more?

                ….–recent memories are generally recalled from a first person perspective and older memories from a third person perspective (Butler). The repeated retrieval of visual details in the first person is shown to lead to better retention of material and slowing of the shift from first person to third (Butler). Can we assume that by directing retrieval with imagery or cues (Harris) and instructing recounting in the first person that recall performance may show an improvement?

                ….the capability to update information into beliefs relies to some extent on whether the information is desirable or undesirable, with the former greater than the latter (Garrett). This biasness does not exist when there is perceived threat to the environment (Garrett). Therefore, would ensuring that the way information is ´phrased` is positive lead to an improvement of event characteristic recall?

synaptic plasticity of proximal and distal dendrites of CA1 pyramidal neurons

Posted comment on ´Synaptic plasticity depends on the fine-scale input pattern in thin dendrites of CA1 pyramidal neurons` written by A. Magó, J.P: Weber, B.B. Ujfalussy and J.K. Makara and published in Journal of Neuroscience 40 (13) p. 2593 doi 10.1523/JNEUROSCI.2071-19.2020

SUMMARY

In this article, Magó and colleagues describe the findings of their investigation into the plasticity of glutamate excitatory input patterns located in the peri-somatic dendrites of CA1 pyramidal neurons of the rat hippocampus. Their aim was to explore the spatially structured forms of synaptic potentiation in those dendrites since this activity and long-term plasticity are considered as important for shaping experienced-related information processing.

    In their experiments, Magó and team used suitably prepared transverse slices from the CA1 area of the hippocampus. Current clamp whole cell recordings were taken from the somata of hippocampal pyramidal cells (CA1PCs) and only cells whose resting potentials were more negative than minus 55mV were used. The neurons were imaged with a dual galvanometer based two-photon scanning system which was also used to uncage glutamate (2PGU) at the individual dendritic spines. Two ultrafast pulsed laser beams were used for imaging the fluorophore and to photo-lyse the MNI caged glutamate and the emitted light was collected by multi-alkali or GaAsP photomultipliers.  The neurons used in the experiment had complete apical oblique dendritic arbors (35%, proximal stratum radiatum) and basal dendritic arbors (65%, stratum oriens). Stimulation via uncaging glutamate occurred laterally to the heads of the visually identified spines which were separated by at least 1.1micrometers for 0.5msecs. The time interval between stimulation of spines was 200ms or 0.1 for the LTP (long-term potentiation) protocol. Repeated excitatory post-synaptic potentials (EPSPs) were measured. The LTP experiments involved recording the EPSPs evoked by 2PGU in the whole cell voltage clamp experiments so that photo-damage could be taken into account (damaged or unusually acting cells were excluded) and EPSPs were evoked regardless of the depth of the spines used. To avoid the problem of washout of the intracellular components by whole cell dialysis, the method used with the LTP protocol started within 10 minutes from the time of the measurement of the control whole cell configuration. Four test spines were used as the sample and the laser power was kept the same for all homo-synaptic experiments, but it was increased 15% for some hetero-synaptic neurons so that d-spikes were evoked. The presence of d-spikes by at least one stimulus was confirmed also by visual inspection and these were most clearly detected at the first stimulus of the LTP induction protocol whereas later the results were more ambiguous and therefore, not taken into account.

   Magó and colleagues performed data analyses on all their results using various methods. Several conditions were used. For example, the mean normalised change in EPSP amplitude of all test spines averaged between 30 and 40 minutes after the LTP protocol indicated the magnitude of plasticity; the potentiation of an individual spine was said to occur via a normalised EPSP greater than 1.3 after LTP; and spines that were retracted, that failed to produce the normalised EPSP, that exhibited other unusual firing behaviour, or moved closer to other spines were not included in the analysis. Other analytical methods used included Image J which was used to measure the cell morphology and distance using dye-loaded neurons. A detailed biophysical CA1PC model was also used to reproduce the dendritic processing. Default passive parameters were used plus adjustments for ion channel activation (eg. voltage gated sodium, potassium) and AMPA and NMDA excitation. Therefore, the somatic and dendritic properties of the sample cells were determined including: the generation and propogation of Na+ action potentials at the soma and along the dendritic trunk; the generation of local Na+ spikes in thin dendritic branches; the amplitude distribution of synaptic responses; nonlinear integration of inputs via NMDA receptors; the similar voltage threshold for Na+ and NMDA nonlinearities; and the major role of A type K+ channels in limiting dendritic excitability. In vivo, stimulation produced the same parameters, but with place-selective activity.

   Magó and colleagues found with their experimental set-up that regenerative d-spikes are required for efficient cooperative LTP at proximal dendritic locations. The EPSP amplitudes of four test spines at either proximal or distal locations along individual branches were measured and then short PGU pulses (the LTP protocol) were applied. The d-spikes if present were measured in comparison to the amplitude and kinetics of the expected EPSPs. Previous experiments had shown that no subthreshold LTP was evoked with the co-activation of only 4 test synapses located at proximal sites along the peri-somatic dendrites and therefore, the synapse cluster was increased to 12-16 by including neighbouring spines. In this case, synchronous stimulation evoked substantial somatic EPSPs (3.9) with small peak EPSP non-linearity (0.7), but no regenerative d-spikes occurred. This was explained as being likely due to low impedance of proximal dendritic segments. However, no LTP was observed at the test synapses and the mean EPSP amplitude of test spines decreased.

  Magó and colleagues repeated the experiment, but changed the locations of the spines and in this case, d-spikes were evoked. For this to occur, 4 proximal test spines were co-activated during the LTP protocol with another 11 spines located more distally, but on the same dendrite. The d-spikes evoked were probably due to the extension of higher impedance of the dendritic segments. They were observed via transient increases in the rate of compound EPSP rise (indicative of dendritic Na+ spikes) and/or peak somatic non-linearity greater than 2mV (indicative of NMDA spikes). Somatic APs were excluded since they were prevented by slight hyperpolarisation during the LTP protocol. The result of the evoked d-spikes was the induced robust long-lasting increase in the mean EPSP amplitude of the 4 proximal test spines (1.98). When APs were also evoked by at least 1 of the 50 LTP stimulus pulses then EPSP amplitude increased to 215, similar LTP was recorded and potentiated synapses were found in all experiments. This suggested to the authors that large depolarisation involving regenerative d-spikes is required for the cooperative LTP induction of synapses located in their sample of proximal segments of peri-somatic dendrites.

   Previous investigations by the authors showed that repeated co-activation of 4 clustered test spines located instead distally increased their EPSP amplitude (1.32) and potentiation occurred in at least 1 spine. However, d-spikes were not detected during the LTP protocol. Experiments described here showed subthreshold LTP observed occurred with at least 3 synapses (increased EPSP amplitude – 1.35) dependent on location. No subthreshold LTP was produced when 4 co-activated test spines were distributed evenly on longer dendritic stretches (EPSP amplitude 0.91), but tight clusters of more than 3 coactive distal inputs could be strengthened by subthreshold LTP even in the absence of regenerative d-spikes. This demonstrated spatial selectivity. This led to further investigation as to whether subthreshold LTP changed in the presence of local d-spikes in distal dendritic segments. Therefore, during the LTP protocol then additional neighbouring spines were co-activated together with the 4 clustered test inputs to trigger d-spikes. Eight synchronous synapses evoked in general at least 1 d-spike during the LTP protocol without somatic AP and induced LTP in at least 1 test spine. There was no significant difference between these results in terms of magnitude of LTP or ratio of potentiated synapses to those obtained with only 3-4 clustered inputs. Magó and team also found that LTP with d-spikes depends on the spatial arrangement of the inputs with additional proximal synapses. In this case, they distributed the 4 test spines and co-activated them with 4 more additional proximal synapses to trigger the d-spikes. In 4-5 experiments at least one synapse was potentiated with an average LTP of 1.52 and therefore, the effect was significantly stronger than with only 4 distributed sub-threshold synapses. Therefore, it was concluded that the more extended propagation of d-spikes especially towards the sealed tip allows more distributed input patterns to potentiate.

   Magó and colleagues then continued their investigation by exploring the results of previous research that reports that using electrical stimulation d-spikes can trigger synaptic potentiation with fewer stimulus repetitions than other LTP-inducing activity patterns. The authors therefore, tested with a short LTP protocol consisting of only 5 co-activations of 4 subthreshold for d-spikes, or 8 suprathreshold clustered spines. The team found that suprathreshold clustered inputs developed robust LTP (1.5) whereas 5 asynchronous activations of subthreshold clustered inputs (0.79) did not. Therefore, d-spikes although not necessarily required for LTP at distal dendritic segments they can alleviate the tight spatial clustering requirement and reduce the number of synchronous events needed to induce LTP.

  The authors continued their investigation by looking at the crosstalk involved in local plasticity. Co-activation of a group of 8 spines during LTP induction which triggered d-spikes produced variable effects on a set of 4 test spines nearby. The long-term change in test spine EPSP amplitude was smaller than that for homo-synaptic LTP with d-spikes (1.43), but still signs of potentiation in the test spines were detected. In the majority of experiments, EPSP increased more than 30% in at least 1 test spine as well as in 12 out of 30 experiments the mean EPSP amplitude changes in the test spines were larger than the mean control with no LTP protocol. These changes were independent of test spine location (ie. proximal or distal) from the LTP induction spines. The crosstalk potentiation was not observed when the test spine and LTP induction spine groups were located at short distances on different dendrites of the cell and the EPSP amplitude was decreased when compared to the spines all being on the same dendrite. These observations were interpreted as the crosstalk potentiation affecting only the activated dendritic segment and involving intracellular rather than extracellular signals.  

   The next set of experiments described by Magó and colleagues was carried out to investigate whether the observations recorded could be attributed to a general change in electrical properties of the test cells due to the repeated stimulations in the LTP protocol. The strength of the dendritic sodium ion spikes showed no change in value and therefore, it was concluded that there was no change to dendritic excitability during the course of the experiments. Using the biophysical CA1PC model, strong stimulations elicited local dendritic sodium ion and NMDA spikes in the model which were visualised as small fast spikelets and slow plateaus in the soma. Increasing the local excitability of the branch (eg. by changing the passive parameters such as increasing local membrane resistivity and decreasing axial resistivity) led to increased amplitude of the individual EPSPs and increased the dV/dt of the somatic spikelets. When the local excitability was changed by locally eliminating potassium ion channels the EPSPs did not increase, but the dV/dt of the somatic spikelets did. Increasing the AMPA conductance of the synapses by 40% led to increases in the amplitude of the EPSPs, but the dV/dt did not. This showed the authors that crosstalk was likely to have occurred and could not be explained by changes in local dendritic excitability evoked by the LTP induction protocol.

  Crosstalk was further investigated since previous reports showed that LTP at a single spine could lower the LTP induction threshold at nearby spines. Therefore, Magó and colleagues tested whether crosstalk plasticity may be related to the weak test stimuli applied to monitor the EPSPs. It was found that the number of pre-LTP stimulation events did not affect the magnitude of the LTP by individual spines, but alternative analysis of groups of cells showed that the spine group receiving less than 15 pre-LTP stimuli produced a smaller EPSP amplitude change (1.02) than those receiving more (1.43). This indicated to the authors that the activation of synapses before LTP induction by other synapses may facilitate crosstalk potentiation.

   The last set of experiments carried out related to the neurochemical mechanisms of the crosstalk observed. The absence of potentiation in the presence of d-AP5 indicated that the mechanism appears to be NMDA receptor dependent. Using VGCC inhibitors, it was found that there was an initial increase in EPSP amplitude, but this was followed by a gradual decline. Therefore, although NMDA receptors are required the calcium ion channels VGSCCs are not. They were however, suggested as being beneficial for the stabilisation of the neurochemical process. Another requirement appeared to be the MEK/ERK pathway since crosstalk was eliminated by the inhibitor U0126.

   Magó and colleagues concluded that several mechanisms can lead to local, spatially structured LTP and found that there are rules about cooperation were based on dendritic location. Even low numbers of clustered inputs on distal segments of thin dendrites, providing they were close, cooperated and co-potentiated without generating d-spikes. However, local d-spikes play a role to induce LTP at proximal dendritic segments and it appears that the potentiation is most likely when input patterns are distributed throughout the dendrite.  Therefore, it was suggested that subthreshold and suprathreshold LTP may be hierarchically organised so that initial gradual potentiation of repeated activated small distal input clusters would help to reach d-spike threshold leading to a second spatially less constrained and faster mechanism evoking somatic action potential firing leading to neuronal cell assembly plasticity. Hetero-synaptic potentiation of inputs in the vicinity of synapses evoking d-spikes, although less prominent, was observed with both firing potentiation and depression. This crosstalk plasticity was suggested as being NMDA receptor dependent and mediated via the MEK/ERK pathway. The function of the crosstalk was suggested as binding together temporally separate informational characteristics of a common event onto the dendritic segment so that subsequent synchronous recurrence of those characteristics would be achieved more efficiently. In this way, the d-spikes and dependent LTP cooperation would form part of the neuronal tuning required in the formation of the event representation and its memory. 

COMMENT

  What makes Magó and team´s article interesting is that it describes dendritic firing which is an aspect of neuronal post-synaptic firing that is normally overlooked in the rush to investigate the more exciting topics of changes to glutamate receptors and ion channels. However, measurements of dendritic firing is an indication of neuronal firing and cell status and can as reported in Magó and team`s article be influenced by the cell`s environment and functional demands.

  Magó and colleagues looked at dendritic firing of one type of cell, the pyramidal cell in the area of CA1 of the hippocampus. This type of cell is ideal for studying dendritic firing because the cells can be described as a ´tree-like` with ´branches` (the apical arbor, proximal since away from the soma, but widely linked to the ´branches` of its neighbouring cells), the ´trunk` (long, with the possibility of discerning segments experimentally) and ´roots` as distal dendrites (closer to the soma, but also widely connected to neighbouring cells). Although pyramidal cells are found in the cerebral cortex, cerebellum and hippocampus, it is the CA1 area which the authors used as their tissue source. The advantage of using this particular area is the uniqueness of neuronal firing mechanisms, the connectivity of the area (within the hippocampus itself and externally) and the consequences of these two aspects on cognitive function (ie. information input, processing and memory). Therefore, this comment explores the influences of dendritic firing in relation to CA1 cognitive functionality.

   The comment begins with a general look at dendritic firing and activity at the synapse and it centres on firing initiated by the action potential signal travelling in the direction of the post-synaptic membrane to the soma. It should be noted that back propogation firing can also occur in the dendrite with firing in the opposite direction, but this is not discussed here (see Role of action back propogation in pyramid cell apical tuft dendrites – April 2020).

  In general, firing of the dendrite as part of the signal transmission process begins at the post-synaptic membrane with the binding of the released neurotransmitter (normally glutamate in the case of the CA1) to the glutamate receptors at the membrane surface. A simplified explanation of the process is that binding to NMDA type receptors or AMPA type receptors leads to the opening of the associated sodium ion channels so that there is an influx of sodium ions into the post-synaptic area. This depolarisation results in calcium ion channel opening and the instigation of the post-synaptic cascade of mechanisms that make up signal transmission propogation and regulation of the synaptic receptor number (eg. IP3 production, CaMKII activation and protein kinase C activation). Binding of glutamate can also occur to NMDA receptors that are linked to SK channels which on depolarisation lead to potassium ion channel opening, release of bound magnesium ions on the outer membrane surface which then bind to the attached NMDA receptor and blocks its activity. Therefore, there is a forward motion of the action potential signal from the post-synaptic membrane to the soma. The strength of the signal depends amongst other factors on the amount of neurotransmitter released and the number of glutamate receptors present on the surface. One method of regulating dendritic firing with relation to synaptic physiology is the presence of SK channel linked NMDA receptors at the synapse (Ngo-Anh).  This is shown to be the case in Schaffer collateral-CA1 synapses where a difference in neuronal firing between dorsal and ventral placed pyramidal cells is linked to the number of SK channels present (Babiec).

  The dendritic firing is observed as burst firing, ie. d-spikes and characteristics of this type of firing is linked to long-term potentiation (LTP) which is indicative of plasticity, are described in the article by Magó and colleagues. If it is assumed that dendritic firing mirror that observed by Magó and colleagues, then location and number of dendritic spines are important for the appearance of the d-spikes. In the case of proximal apical dendrites then according to Magó and team more than 4 test spines are required to evoke the d-spikes. The induction of d-spikes in their experiments requires 4 proximal test spines and a further 11 spines which are more distally located on the same dendrite.  This strong depolarisation achieved by spatial summation of firing from the proximal and distal located spines of the same neuron causes a transient increase in the rate of compound EPSPs rise indicative of a sodium ion spike. (The calcium ion effect appears not to be involved since PKA blockers significantly inhibit NMDAR-mediated calcium ion rises in activated dendritic spines but have no significant effect on synaptic current.) Therefore, the d-spikes evoked, indicative of the transmission of the signal in a forward direction, require cooperative behaviour from spines in proximal locations which are likely to receive input from the directly connected neurons from the CA3 hippocampal area supplemented by firing of those spines located distally. These may also receive input from the CA3 area or perhaps from other brain areas. In this case, the firing pattern representative of the current event may have characteristics from many sources.  

  Magó and team also found in their study that between there has to be synchronous stimulation of between 12 and 16 test spines if these are located on neighbouring spines rather than the same one before there is substantial EPSPs of the cells. However, no d-spikes are observed. This indicates a difference in functionality since the d-spikes are associated with the forward transmission of the action potential signal and when relevant, the induction of LTP. It is likely that in this case the contribution of the cells to the firing pattern lies in the strengthening of the local firing for a purpose other than direct signal transmission, eg. induced ion level changes for vesicle transport or enzyme activity regulation.

   When only distally located spines are taken then according to Magó and team the spines have to be clustered tightly in order that the increase in EPSPs translates into d-spikes, forward sweep and induced LTP. This may indicate that the firing response is specific rather than random and that input comes from one specific area and has to be strong.

   Linked to the forward transmission of the signal is the association of the dendritic d-spikes with the adaptive firing response, that of LTP (Magó). Repeated stimulation strengthens firing patterns and allows encoding of them via long-term physiological changes so that they can be reactivated at a later date. The increased firing sensitivity allows lower levels of firing to induce event representation of previously experience events and allows past events to influence the present. The dendritic d-spikes are associated with LTP (Magó) and hence, provide a means of experimentally investigating neuronal plasticity in response to repetitive stimulation. As described before, the induction of LTP requires a long-lasting rise in EPSP amplitude in the case of proximal cells as long as the spines are on the same dendrite plus preferably the supplementation of the signal with firing from more distal spines. The distally located spines need to be located in tight clusters in order to induce both d-spikes and LTP (Magó). This spatial summation and spine location specificity may be linked to the brain waves observed since dendritic firing is associated with gamma wave burst firing and theta brain wave oscillations (Tominga). These two brain waves appear to be associated with two different LTP systems in the CA1 pyramidal cells (Zhu). The gamma waves are reported to require a system involving adenosine A2 receptors, PKA activity and actin polymerisation whereas the theta brain waves with calpain-1-mediated suprachiasmatic nucleus circadian oscillatory protein degradation, ERK activation and actin polymerisation (Zhu). Therefore, the differences in LTP systems induced may reflect the contributions of dendritic firing from the distal and proximal pyramidal areas to the overall event representation.  

   The LTP observed in pyramidal cells of CA1 is likely to follow the mechanisms employed to induce plasticity in the mossy fibres of the CA3 (Nicoll), ie. released glutamate activates the post-synaptic NMDARs and AMPARs. Calcium permeable AMPARs are associated with calcium ion influx through L-type channels. This type of LTP is associated with the induction of PKA (Park) and to one form of NMDAR dependent LTP. This PKA dependent form requires multiple firing events linked to gamma burst firing or burst firing in the theta range. Both lead to the insertion of calcium-permeable AMPARs into the post-synaptic membrane (Ehringer, Koener, Plant) and the induced plasticity via this method results in strengthened firing.

   The LTP observed in addition to the different levels of SK channels and the distal or proximal contributions of different signal sources described above can also be influenced by the number of NMDARs and AMPARs present since these would have effects on firing, ion influx and plasticity. In an extreme example it is possible that there are no AMPARs and only NMDARs on the spines, the so-called ´silent synapses`. Repeated stimulation of this type of neuron leads to LTP by inserting AMPARs into the membranes just like with normal LTP (Arendt). Such an insertion has the effect of ´switching on` the synapse. Therefore, d-spikes may occur via NMDAR binding in a cell with no AMPARs which then promotes LTP and the insertion of AMPARs into the membrane. This results in the action potential threshold being in subsequent events achieved and the cell firing. The event representation in this case will include these characteristics and hence, be expanded.  This therefore, changes the contribution that a particular cell can make to the overall firing pattern. Hence, a dendrite that is previously subthreshold will become a dendrite that is part of the transmitted signal due to the insertion of AMPARs and spatial summation. The resulting firing pattern will be expanded to include this information in the event representation.

   The CA1 also experiences plasticity in the form of long-term depression (LTD) which is the natural progression from LTP in memory formation (consolidation of fear memories, but not acquisition – Liu). However, there are no specific reports of d-spikes linked to LTD in the CA1 area. This is unexpected since LTD is thought to be associated with extracellular calcium ion levels and NMDAR calcium ion channels (Babiec). The NMDAR receptor mediated activation of p38-MAPK and dephosphorylation of the GluA1 subunit of the AMPAR and subsequent internalization of the receptor implicated in LTD is also shown to be involved. Therefore, the dendritic firing and action potential induced because of spatial summation would be expected to cause the shift from LTP to LTD as expected. The observation that LTD prevents subsequent LTP of AMPAR transmission (Bhouri) may explain why. The aim of dendritic firing is to promote signal transfer and event representation and therefore, the calcium input is high, LTP is welcomed, but the switch off mechanism of LTD is not. If LTD does not occur then the forward signal is continued. The alternative view that LTD is said to be caused by the selective activation of a group of presynaptic metabotropic glutamate receptors (mGlu) instead at the Schaffer collateral-CA1 synapses (Rosenberg) may also provide an explanation as to why LTD is not associated with dendritic firing in this area.

  Therefore, d-spikes and LTP are linked to CA1 cognitive function capability and this can be used to advantage by specific input and output connectivity of the area. Stimulation of the CA1 leads to excitation of the entorhinal cortex, to the dentate gyrus (DG) of the hippocampus plus the prefrontal cortex and other areas and these all play roles in the event representation, memory and information processing. Different connectivity patterns support the signal transmission of one type of information over another. For example, there is greater connectivity between the amygdala and hippocampus in emotionally arousing material (Fastenrath); input from the lateral entorhinal cortex into the distal CA1 is important for information about objects, whereas the input into proximal pyramidal cells from the medial entorhinal cortex provides information about space (Hartzell); the inputs from the medial entorhinal cortex to the CA1 are also required for the temporal organization of the hippocampal firing patterns and therefore, are important for sequences of information (Schlesiger); and synchronisation of the theta wave oscillations of the CA1 and beta wave oscillations of the ventral striatum indicate reward expectancy (Lansink).

   The combined effect of firing mechanisms and connectivity allow the hippocampus to be a major player in the neuronal representation of event information, processing of that information and memory. Therefore, as expected any physiological changes to the particular areas can have wide-ranging effects on a cognitive scale, eg. epilepsy induces unusual resting connectivity between the anterior and posterior hippocampal areas which is associated with memory decline (Voets). In the examples given above it can be seen that specific areas of the hippocampus have different functions relating to event features, eg. distal CA1 receives input from the lateral entorhinal cortex and is important for information about objects, whereas the proximal pyramidal cells receive input from the medial entorhinal cortex and provide information about space (Hartzell); and there is differential encoding of spatial location and numeric object characteristics in the CA1 and CA3 areas (Opris).  The temporal interval between neuronal firing is important because this indicates the boundary of the event representation. This allows binding of appropriate characteristics together which are represented by simultaneously firing neurons and allows sequential events to be recorded. Dendritic firing with proportions of proximal and distal firing allows spatial summation to occur for features that under normal circumstances would be too weak to be part of the event representation or makes certain features stronger by increasing their firing contribution to the overall neuronal firing pattern in preference to others. Two examples come to mind: events where both object characteristics and subtleties of location are required, eg. in spatial encoding and memory dependent on three dimensional interpretation of viewpoint, placement and direction (Kim) where it has already been shown that there is a scale of spatial information between the dorsal and ventral poles of the CA1 with the threshold for LTP induction higher in the dorsal CA1 neurons (Malik); and in event representation where emotional information plays an important role, eg. in fear situations. Working memory may also benefit from the distal and proximal specificity of the dendritic firing since this requires real-time processing, maintenance and possible matching of multiple simultaneous event representations whose origins of material can be both real-time and reactivated. This hippocampal function (Vila-Ballo) requires both glutamate and NMDAR activation (Takadi, Gage). 

   The concepts of sequences and temporal order may also be elicited via CA1 pyramidal firing patterns (Allen) which may or may not involve dendritic firing contributions of the distal and proximal spines. It is known that there is a general link between hippocampus cell firing and timing (Jacobs). Sequence coding of non-spatial events has been associated with CA1 and gamma wave oscillations observed there are modulated more strongly by temporal context than theta waves (Allen, Salz) with theta wave activity attributed to clock-like activity of small groups of CA1 neurons (Zhang).

   The contributions of the distal and proximal areas in order to elicit forward firing of the signal or to widen the informational value of the event representation may have relevance when the mechanisms relating to memory formation on the basis of real-time neuronal firing patterns is considered. The hippocampus is known to be important for encoding of the short term event representations (mice create what-when-where memories – Fellini) into longer term memories and this process relies on the LTP changes at the synapse described above (Winocur, Hawkins). The CA1 plays a role in this by aiding the encoding of new information in the face of retrieved information stored from previous experiences (Douchanps, Kuhl). Two mechanisms involving the CA1 have been suggested for this priority. The first is that acetycholine released in firing relating to the input of new information selectively suppresses the excitatory projections from the CA3 which is involved with the reactivation of the past experience to the CA1. It has no effect on the entorhinal inputs in to the CA1 which is involved with the new material (Douchanps). The second mechanism involves the preferential encoding of the new material at the pyramidal layer associated with the theta brain wave oscillation peak relating to the input from the entorhinal cortex whereas retrieval occurs at the trough of the theta wave coincident with the input from the CA3. Therefore, there is a shift towards encoding of new information. This is not the case with reactivated memories where familiar events lead to bigger responses (Fried). In this case, the antagonism of acetylcholine firing shifts the theta firing phase to the theta wave oscillation trough associated to retrieval and involves both CA3 and DG areas (Douchanps, Reigh). Reactivation in the hippocampus also leads to patterns in the cortex and stabilizes the connections between the hippocampus and the cortex (Frankland).

   The shift in CA3, CA1 firing dominance according to whether the input relates to reactivated or novel material may correspond to the contributions of the dendritic firing action potential from the distal and proximal located sources as well as spatial summation. For example, a higher proportion of distal firing may reflect a shift to the higher proportion of reactivated features whereas a high proportion of proximally sourced action potential may represent the real-time novel input.

   This balance of novel information and reactivated information elicited through the different contributions of distal and proximal located spine activity may be part of the explanation of how the hippocampus undertakes its varying cognitive functions relying on memory. For example in object recognition there is a requirement for real-time input of the object presented and memory reactivation in order to identify it. The hippocampus, as part of the firing pattern of entorhinal cortex to hippocampus following on to fornix, mammalian bodies and anterior thalamus, plays the role of comparator capable of individualizing representations of overlapping units of familiar versus novel events (Zeamer, Jeneson, Smith).  In the case of emotionally charged events, the incoming information is judged in accordance to past experiences and therefore, forward sweep gamma firing may be supplemented by distal firing representing the emotional component. In anxiety, there is neural oscillation connectivity between the hippocampus and medial prefrontal cortex (Khmeka) with theta brain wave modulating the level of threat probability, but not its magnitude indicating a fear memory effect where incoming information is processed along with reactivated memory information (Khmeka). In the case of spatial memory, LTP of the hippocampus plays an important role with impaired AMPAR subunits leading to defective spatial working memory task performance (Schmidt) and LTP deficits (Rowland).

   One possible explanation as to the function of specific localization of dendritic firing contributions relating to memory can be ruled out however and that is that dendritic firing of the CA1 is unlikely to be related to neurogenesis that is required for memory storage. Memory formation is known to require newly formed cells ((Bischofburger, Kee, Toni), but this is likely to involve the DG area and the CA3. Newly formed synapses between the two can compete with and displace the established synapses demonstrating plasticity. Therefore, the neurogenesis destabilizes the past memory reactivation and shifts the dominance to the new input (Martinez). Therefore, the dendritic firing of the CA1 would be indirectly involved, but neurogenesis of the CA1 is not required.

   However, the dendritic firing of the CA1 could play a role in memory consolidation undertaken during sleep. The reactivations of hippocampal place cell assemblies during sleep (De Lavilion) lead to a shift from the hippocampus to cortex memories according to Oswald`s sleep theory (Mehta, Ji) as well as memory consolidation (Maingret) and better memory performance (Mednick). During the NREM stages there is functional global spike-based connectivity between the hippocampus and cortex whereas in REM there are increases at the neuronal level with increases in cAMP, MAPK and CREB phosphorylation (Luo). The hippocampus demonstrates sharp-wave/ripple complexes which are short periods of increased activity with high frequency oscillations (Maingret, Jahnke). These occur simultaneously with spike based firing representing the replay of previous experiences (Jahnke). Both are the product of dendritic firing (Jahnke) and sodium spikes in the CA1 and CA3. The hippocampus also demonstrates theta oscillations and spindle firing (Sullivan) with hippocampal sub-regions demonstrating more synchronized firing in the latter than the former. The spindle firing of the CA1 is phase locked to the medial entorhinal cortex in sleep stages 2 and 3 (Sullivan) and this modulates the strength of gamma oscillations. It is the sleep spindles that are linked to the transfer of local information to the cortex and memory consolidation (Staresina).

   Therefore, dendritic firing of the hippocampus is a requirement for neurotransmitter based signal transmission. The flexibility in sources of firing whether proximal via forward signal transmission or distal means that spatial summation allows only strong signals to be propogated further. Strong depolarisation can come from firing of multiple spines located either proximally and distally or distally alone, but only if clustered. This flexibility from an information point of view means that only dominant features continue or a wider range of strong features are bound together so that the event representation may be made up of a number of different features, eg. object characteristics, location, timing even emotional value. The adaptive response of the CA1 to action potential and post-synaptic firing of a repetitive or continued stimulatory nature involves the strengthening of the post-synaptic physiological area to future firing. The result is a priority for forward signaling and dendritic firing. Although the post-synaptic physiological changes often dominate research, dendritic firing should also be considered, not as a secondary feature, but as being a factor responsible for the continuation and modulation of forward signal transmission.  

Since we`re talking about the topic……………………

                ….since ageing is known to be associated with deficits in spatial memory in mice, can we assume that if Magó and team`s experiments were repeated, but with the CA1 pyramidal cells of 24 month or 29 month old mice (aged) we may see a difference in dendrite firing and even a change in the contributions of the distal and proximal located spines?

                ….adult rats undergoing locomotive stress (ie. elevated platform) exhibit an increase in gamma burst activity in the hippocampus of whereas the administration of diazepam decreases the gamma burst activity due to its inhibiting interaction with GABA receptors Takillah). If Magó and team`s experiments were repeated with tissues from 8month old rats, can we assume that the level of dendritic firing will be affected in general by the administration of diazepam?

                ….sodium ion channel activity is associated with dendritic firing (Magó). Would the administration of tetradoxin, a known sodium channel inhibitor, confirm the association between sodium ion entry and the evoking of d-spikes? Would the administration of specific inhibitors of the R-type calcium ion channels and T-type calcium ion channels both linked to lower voltage evoked calcium influx confirm that calcium ion transport is not required for d-spike activity in the same way as the higher voltage VGCCs?

near-death experience memories compared to flashbulb memories

Posted comment on ´Near-death experience memories include more episodic components than flashbulb memories` written by H. Cassol, E.A.C. Bonin, C. Bastin, N. Puttaert, V. Charland-Verville, S. Laureys and C. Martial  and published in Frontiers in Psychology 13^th May 2020 doi 10.3389/fpsyg.2020.0088

SUMMARY

   Cassol and colleagues investigated whether memories of near-death experiences (NDEs) are comparable to aspects of flashbulb memories (FBs). In their investigation they identified and compared episodic and non-episodic information, phenomenological characteristics and the centrality of memories of participant`s verbal recollections of their NDE, flashbulb and control autobiographical memories.

  The experiment was set up so that the 25 participants who had lived through a life-threatening situation (eg. anoxia, trauma) and who had met the accepted criteria of having experienced a NDE, first took part in a screening task.  This was to detect and exclude memories biases relating to negative mood (according to Positive and Negative Affect Schedule) and mild cognitive impairments (Montreal Cognitive Assessment). The participants then performed the Autobiographical Memory Interview (AMI).  Each participant was asked to describe in detail three target memories that occurred in the same time period of their life. These were: the memory of their NDE; a flashbulb memory (these followed particular criteria such as that they were either very surprising and consequential, or emotionally arousing, eg. Man`s first steps on the moon, September 11<sup>th</sup> terrorist attacks); and an autobiographical memory. The interviewers were allowed to probe when required to encourage a full description of the events. The narratives were recorded, transcribed and analysed using an established manual scoring procedure so that the episodic details could be separated from the non-episodic details. Episodic details (termed ´internal details`) related to descriptions of the events such as the event details describing the unfolding of the story (eg. listed as the happenings, persons involved, reactions/emotions of oneself and other people, one`s clothing and the weather), the time (eg. life epoch as well as defined physical time) and place plus the sensory or mental state details (eg. thoughts and emotions) specific for the event. The non-episodic details (termed the ´external details` related to semantic or factual information not specific for the event). Based on the final classification of reliability between scores from independent scorers, the details generated were summed across the memory types.  The data of the AMI was analysed for each type of memory using appropriate software and statistical methods and significance was defined.

  After each memory recall the participants were given a short version of the Memory Characteristics Questionnaire (sMCQ – 16 items) and the Centrality of Event Scale (CES – 20 items) to fill in. The sMCQ assessed memory clarity, sensory details, self-referential and emotional information, reactivation frequency and confidence in memory on a Likert scale of 1 to 7. Data analyses were carried out using appropriate statistical software. The CES assessed how central the event was to the person`s identity and life story using agreement (scale 1-5) to the following statements of the event as: considered a reference point for generation of expectations to other events in life story; considered as a central component of a person`s identity; or considered as a turning point in the person`s life story. Both sets of results were analysed using appropriate statistical methods.

   The results of Cassol and team`s experiments showed that the level of negative mood was no higher for the NDE group than the control and was not related to the amount of internal or external details of the NDE, flashbulb and autobiographical memories. The age of the interviewee and the time of the interview in relation to the NDE event also did not correlate to the amount of internal or external details of the three memory types. However, the overall amount of details given for the NDE memory was higher than for both the flashbulb and autographical memories which were the same.

   The results also showed a significant interaction between the type of detail and the type of memory. The number of internal details reported for NDE memories was higher than the internal details reported for flashbulb and autographical memories. The number of internal details was also higher than the amount of external details reported for the NDE and again, this level was higher than for both the flashbulb and autobiographical memories.

   With regards to the results of the sMCQs, Cassol and colleagues found that NDE memories exhibited higher, but not statistically significant, ratings for internal perceptual details relating to emotional status compared to the autobiographical and flashbulb memories. They found that there was a memory difference in the feeling of mentally reliving the event and the sensation of feeling the emotions felt during the event while remembering. This feeling was higher in both NDE and autobiographical memories than with the flashbulb memories. The authors also reported a memory difference in the visual perspective taken while remembering. The NDE and autobiographical memories did not demonstrate statistical differences to each other, but they did show a difference to flashbulb memories. The results indicated that NDE and autobiographical memories were more likely to be remembered from the first person perspective. The emotions felt at the time of the event, ie. the valence, also produced a memory difference. Again, flashbulb memories gave lower scores than NDE and autobiographical memories and because NDE and autobiographical memories did not differ, this indicated to the authors that the latter were more positive in average. Two further observations were made that indicated a trend (but were not statistically significant) towards higher scores for NDE memories and autobiographical memories than flashbulb memories and these were: personal importance attached to the event; and frequency of sharing and reactivation. Scores also not reaching significance for the memory types were those relating to the amount of sensory details, clarity (time location and coherence), confidence in memory and the ability of the individuals to remember their own actions/words/thoughts.

   Cassol and colleagues found more significant differences with the results of their Centrality of Event Scale studies, ie. how central an event was to a person`s identity and life story. They found that the mean score for the NDE memory was significantly higher than for the autobiographical memory which was higher than that for the flashbulb memory. They also found a significant positive correlation between CES scores and a number of the reported internal details given above (eg. the feeling of re-experiencing the event, the feeling of re-experiencing the emotions felt at the time of the event , the importance of the event and the level of sharing of the event and reactivation frequency). However, there was no correlation between the CES and the external details. This indicated that the NDE memories are the most central memories to identity, more than the control autobiographical memories and with flashbulb memories the least.

   Cassol and colleagues concluded their article with a discussion of their findings. They found that NDE memories were not the same as flashbulb memories and presented explanations as to why this might be. The authors suggested several reasons why there is a higher overall amount of details and higher amount of internal/episodic details in the NDE memories compared to the autobiographical memories and flashbulb memories. The first reason related to the association between NDE and the unusual perceptions of the individuals at the time such as leaving the physical body, being in an unknown spatial-temporal dimension and the intense sensation of ´reality` reported with them. This, the authors suggested could lead to the vivid memories of the NDE in comparison to flashbulb memories.

   The second reason given suggested that the difference lies with the ´weakness` of flashbulb memories. The authors explained that flashbulb memories were more subject to forgetting in comparison to other emotionally arousing events. The severity of the emotions felt and the degree of involvement would maintain consistency and amplify the memories rather than for the flashbulb memories where the participants were not normally personally involved. This was supported by the strength of the NDE memories in terms of details. Cassol and colleagues found that the NDE narratives became richer in time compared to flashbulb memories which they said declined. They found that certain subtypes of recalled details were increased, eg. the event details describing the unfolding of the story and the perceptual details (eg. sensory and spatial-temporal information). This was attributed to the self-referential nature of the information which would have a positive effect on encoding, processing and storage of the memory.

   The authors also linked the self-referential nature with the differences relating to emotions and importance. They explained that the strength of the NDEs because of the event being highly emotional and the emotional relevance of autobiographical memories meant that the phenomenological characteristics for the NDEs and the autobiographical memories were higher than those for the flashbulb memories. The levels for autobiographical memories which were on a par with the NDEs were explained as due to the autobiographical memories being temporally linked in time in some way to the NDE which would strengthen them both. Again the self-referential aspect of both the NDEs and autobiographical memories would mean that they would be shared more often and communicated more often than the flashbulb memories and so would be strengthened because of it.

   With regards to the perspective taken during recollection, the authors also used the self-referential nature of the NDEs as being reflected by the individual using the first person compared to the third person when recalling the flashbulb memory. The autobiographical memories were shown as being recalled either in the first or third person perspective. Cassol and colleagues suggested that the use of the third person perspective for the NDE or autobiographical memories could be an avoidance strategy to set up distancing between the individual and the memory and hence, emotional intensity would be reduced. Therefore, the difference in the adopted visual perspective could be due to the emotions felt at the time of the event (eg. valence) that are overall very positive in NDE and autobiographical, but more negative in flashbulb. Cassol and colleagues also gave the results of the CES and centrality tests as support for the NDEs having a higher impact on the individual`s life story than the flashbulb memory.

   Therefore, Cassol and team`s experiments show that NDE memories have different characteristics to flashbulb memories with richer internal details and a propensity to self-reference and importance. They concluded their article with a few suggestions as to how to extend their experimentation such as increasing the number of subjects who had experienced NDEs, controlling for false memories and using autobiographical and flashbulb memories from different time periods.

COMMENT

   What makes this article interesting is that it describes two of the more unusual types of long-term memory: that of near-death experience memory (NDE) and flashbulb memory (FB). Both of these relate to memories formed of events experienced at particular points in an individual`s life and whereas the former is rare (hence, difficult to find experimental subjects) and extremely personal, the other is more common and relates to the individual and his/her social environment.  The NDE is an experienced situation of intense physical or emotional danger where some individuals report a dissociative consciousness state, vivid extraordinary perceptions (eg. out-of-body – OBE), or an intense feeling of peacefulness and calm. (This is similar to, but not the same as, near-death-like experiences where there are similar phenomenological experiences, but without the physical and/or emotional danger. NDE-like experiences are sometimes associated with meditation, or depression.) In comparison, flashbulb memories are experiences where the individual is given a ´piece of news`. This may be consequential to the individual, but it is unlikely that the individual is in physical danger at that time. Therefore, although the memories are autobiographical and relate to the individual at specific time points in their life story they are formed in different circumstances. Therefore, this comment looks at the similarities and differences between the two with reference to Cassol and team`s findings and looks at which neurochemical mechanisms may promote NDEs being formed.  

   The first difference that springs to mind between the two types of experience and memory from a neurochemical point of view is the level of acute stress and stress response experienced at the time of the event. The intense physical or emotional danger experienced by the NDEers (individuals who report a NDE) is likely to promote a physiological  ´defence` response in order to secure survival. If we look at Schauer and Elbert`s sequence of stages in response to traumatic stress (ie. Stages 1-6 -freeze, flight, fight, fright, flag, faint) the NDE is likely to begin to occur at the end of Stage 3 (the fight phase) when sympathetic activation has mobilised resources, but since the ´fight` is not successful the individual slips into the next stage, Stage 4, fright. Sympathetic activation brings about a range of physiological signs such as dizziness, light-headedness, palpitations, numbing and important for this topic, feelings of non-reality. Stage 4, although associated with unresponsive immobility but tachycardia and hypertension, consolidates the change in mental approach to the danger situation with signs of hyper-alertness, high emotional arousal and fear. This continues into the ´flag` phase where there is a physiological ´shut-down` instigated via the activation of the parasympathetic system and symptoms such as bradycardia, vasodilation and hypotension and relating to the NDE, a drop in arousal, feeling of surrender, cognitive failure and ´numbing` of all emotions (perhaps, the feeling of ´inner calm` experienced). Therefore, it appears that the reported experiences of the NDE event (eg. dissociative consciousness and reported increases in awareness leading to inner calm and peace) can be explained by the staged physiological responses to stress according to Schauer and Elbert . In comparison, flashbulb memories (FB) are associated with a minimal or temporarily raised stress level since the individual is not in physical danger.   

  The different stages of the stress response induce changes in the brain neurochemistry and these we assume bring about the cognitive changes observed with the NDE and highly emotional situations. These neurochemical changes are linked to cortisol (or glucocorticoid) production, which is known to cause effects in some cognitive mechanisms. Cortisol is eventually produced in response to the immediate threat to health. This manifests stimulation of a number of different routes, eg. vagus nerve to medulla to periventricular nucleus of the hypothalamus; somatosensory stressors via the tegmentum and the reticular formation to the paraventricular nucleus of the hypothalamus; painful stressors via the periacqueductal gray PAC to the paraventricular nucleus; locus coeruleus and changes to heart rate and blood pressure leading to increased release of noradrenaline leading to paraventricular nucleus; emotional stressors mediated via the raphe nucleus leading to the paraventricular nucleus; and finally and more importantly linked to cognitive function stimulation of the hippocampus, septum and amygdala. This stress pathway is involved in changes to emotional and cognitive input. The amygdala releases corticotrophin-releasing hormone (CRH) which activates the autonomic and endocrine systems which mobilise the energy for the fight or flight response.

   The next stage of the neurochemical response to stress is the activation of the sympathetic adrenomedullary axis (SAM) and more importantly for cognitive functions, the hypothalamic-pituitary adrenocortical axis (HPA). The SAM route involves the release of adrenaline, noradrenaline and stress hormones and increases arousal and vigilance. The HPA route involves actions of the hypothalamus via the paraventricular nucleus. This when activated leads to the release of corticotrophin releasing factor which enters the anterior pituitary gland where it binds to CRh R1 receptors and stimulates the production of adrenocorticotropic hormone (ACTH). ACTH stimulates the cortex of the adrenal gland leading to the synthesis and release of glucocorticoids, which spread through the body causing a number of different effects (eg. increased availability of blood glucose, decreased immune system and in the brain, inhibition of eating, sexual behaviour and growth). The main glucocorticoid in humans is cortisol and it binds to receptors found in the cytoplasm of many neurons. The activated receptor travels to the nucleus where it can stimulate gene transcription and appropriate protein synthesis. One role of cortisol is the increased influx of calcium ions via increased activation of voltage gated channels (shown by administration of GC R antagonist RU38486 – Karst). It is the influence on calcium ion concentrations on many neurochemical mechanisms within the neuron which cause the changes to neuronal functioning and ultimately can lead to cognitive effects associated with NDE. Therefore, whereas individuals that are experiencing NDE may have a physical stress response and release cortisol because of the threat to life which then has an effect on many cognitive functions, individuals that have FB memories are not under threat and therefore, the effects on their cognitive functions occur by different means.

   The first cognitive effect associated with NDE which may be linked to cortisol production and neuronal calcium ion effects is the informational content of the memories formed at the time of the event. Cassol and team found in their experiments that the amounts of information (total, external and internal) in NDE were greater than FB memories with both demonstrating a high level of context information not greatly relevant to the situation at the time. It is likely that this increase in informational content comes about by a change in attention, which is known to be a controlling factor of the quality and quantity of sensory information and this may be induced by the cortisol released as part of the response to the threat situation. Cortisol has been reported to increase attention (Banks), but what does this mean to the information being inputted and stored? We know that the fear attention state (and the corresponding fear emotional state since they are interrelated) can increase the quantity of informational input, but decrease its quality (ie. more gist rather than high level detail). This is possibly due to a change in the balance of task-relevant and task-irrelevant material where the latter under normal circumstances would not be learnt, but in NDE are included in a significant amount. This is also a possible explanation for FB memories too, since these memories are also known to include not only task-relevant information, but also a host of irrelevant information such as trivial sensory details (Brown and Kulik).

   Therefore, in both cases central and task relevant information is given the same weighting as irrelevant, non-central, peripheral information and this change in balance is likely to be achieved through alterations to the attentional system. For example, Cukor showed that attention dynamically alters visual representation to optimize the processing of behaviorally relevant objects during natural vision. The likely brain areas involved in such as shift would be: the intraparietal sulcus, precuneus and dorsolateral prefrontal cortex shown to be involved in bilateral attentional control in selecting the extent of relevancy and updating the representation (Niv); the anterior thalamus  which appears to direct attention to task-relevant stimuli that will bring reward (Wright); the amygdala involved in the fear state; and the hippocampus which is required for sustained activation of the relevant neurons without repetition and is important for memory formation. There is also likely to be an involvement of the NMDA receptors since the NMDA R antagonist, ketamine is reported to strongly impair the ability to ignore irrelevant task information (Stoets).

   The shift in informational content of the NDE and FB memories is also likely to include as well a shift in the balance of attended to unattended information (information of which the individual is aware to information that the individual has no awareness of). This is suggested with NDE memories since they are described by NDErs as being ´realer than real`.Such a shift could be explained by a widening of the limits of ´awareness` since the experience not only includes task relevant and attended information, but also, task irrelevant and what would normally be, unattended information. Hence, the experience would be described as ´realer than real`.

   The original models of Cherry, Broadbent etc. relating to levels of attended and unattended information describe them in terms of perception and processing. This leads onto factors affecting the balance and characteristics with for example, decreased processing of unattended information (Wojceulik) and greater processing of unattended information when the perceptual load is low (Lavie). Therefore, even if there is no awareness of unattended information there can still be processing (Wright, Nee and Norman). The situation with NDE is that this level of information would not normally be consciously experienced or be the first information recalled.  Therefore, the NDE condition changes the balance so that more information is attended. The question is how can this be achieved?

   One possible suggestion is that the stress response causes changes not only in the amount of information inputted as described above, but how the information is grouped or bound together. This has an effect on the capacity of working memory, which we know is limited to a certain number of event characteristics. Perception and Gestalt theories group visual elements such as lines, curves together into unitary objects such as forms and shapes which are then processed by working memory. By increasing the ´group size`, processing capability can therefore, be ´stretched` to more elements. We know of this through memory improvement techniques where individuals instead of trying to remember 6 different numbers group them together to represent years or months for example to increase recall efficiency. In the case of NDE experiences, the different sensory input may be grouped together as a single ´unit`. This has been shown in a similar manner by Jang who described event representation as containing not only the prime features of the event, but also more abstract, more internal features of the event such as attentional control states and category information. Such grouping was said to require the activation of the hippocampus and putamen as well as other regions representing visual feature-selective event information (primarily visual cortex), category-selective information (posterior frontal cortex) and control demand-selective event information (insula, caudate, anterior cingulate, and parietal cortex) event information.Therefore, brain areas affected by the stress response such as the hippocampus would, like the other attentional system brain areas, lead not only to changes in informational quality and quantity, but also awareness and processing capability. This would explain both the NDE event content and the content of FBs where both task relevant and task irrelevant information form part of the event representation.

   Although the stress response may then explain the change in conscious awareness relating to information and informational processing, can it also explain the dissociative consciousness state reported with NDE experiences? The mechanics of conscious awareness observed for visual input 170msec after presentation (Thierry) involves synchronized activation of many areas including the DMN, insular cortex, anterior cingulate cortex, medial prefrontal cortex and dorsal thalamic nuclei. However, conscious awareness under normal circumstances does not involve some of the transcendental, out-of-body type awareness reported by NDErs. In this case there is a dissociation from ´reality`, ie. the real-time sensory input information achieved via eyes, ears etc. One explanation for this could be the near-complete separation of top-down and bottom-up processing leading so that the top-down ´I, SELF` thinking part is distanced from the ´sensing, input` bottom-up part. This idea may be acceptable since imagining and dreaming are both examples where top-down processing and bottom-up input are ´distinct`. In the case of NDE, however the separation is likely to be achieved by the attentional system, which we have already seen is under the influence of cortisol released in the stress response. Top-down attentional modulation appears to suppress the incoming visual information via inhibition of firing at the V1 level (Jacob) and dissociation of attention and awareness is observed in the case of failure of applied chromatic flicker to reach awareness, but not alerting and orienting effects – also associated with V1 visual area effects (Lu). This separation is also apparent through the conscious awareness of an isolated object (or gist of a scene) in the near-absence of top-down attention (van Boxtel). (However, in this particular research example it appears that the explanation is against the proposed one for NDE since attention is described as top-down and ´thought` and consciousness is the information coming in and bottom-up. Possibly the way to approach the results of this study is to view conscious awareness as top-down and subject to top-down attention whereas sensory information is bottom up and subject to bottom-up attention with attentional modulation being the ´tool` and not the ´result`.)

   Therefore, one possible explanation for the more transcendental aspects of the dissociative consciousness reported by NDErs is the separation of top-down processing from bottom-up processing. The advantage of such a separation is protection of the higher brain areas from excessive information and high processing load. This may also be attributed to the emotional state at the time of the experience which is interrelated to attentional system functioning (Gregory). Cassol and team reported that the emotional stateduring the NDE experience was greater than for the autobiographical memory and both were greater than that recorded for the FB experience and it is clear that NDE are profound psychological events with highly emotional and self-related content. The increase in information is associated with this highly emotional state since it has been found that the link between episodic memory,  the feeling of ´being there` and the higher emotional experience is linked to better factual memory via an improved attentional focus on the stimuli, hence increasing memory encoding (Makowski). The same is reported with FB memories which also carry factual memory with strong emotions that can be recalled years later after the initial event (Brown, Finkenauer).

   The increased fear emotional state as the individual experiences the near-death event in Schauer and Elbert`s Stages 3 (fight) and 4 (fright) of the stress response is responsible for the increased levels of relevant and irrelevant material for the task (Newenhause, Stoets) and requires the activation of brain areas such as prefrontal cortex, amygdala, cingulate cortex and thalamus. Anxiety is shown to produce elevated cortisol levels linked to the hyperactivity of the HPA axis (Lenze) and there is a direct ventral connection between the amygdala and prefrontal cortex (Eden), both affected by anxiety.

   However, as the stress response progresses this fear emotional state shifts to a period of ´calm`. This relates to Schauer and Elbert`s Stage 5, ´flag` phase where there is a physiological ´shut-down` instigated via the activation of the parasympathetic system with symptoms such as drop in blood pressure and heart rate, but more importantly to the emotional experience of the NDE, a drop in arousal, a feeling of surrender, cognitive failure and a ´numbing` of all emotions, ie. the reported ´calm`. This can be brought about by the reduction in cortisol production and hence, cortisol effects on the brain areas involved in instigating the emotional state (eg. the Papez-Mclean limbic model involving activity of the amygdala, hippocampus and cingulate cortex and the two track Le-Doux theory with rapid emotional response based on information from the thalamus to the amygdala leading to autonomic and endocrine changes interpreted by the cortex and the slower, direct transmission of information from the thalamus to the cortex).  Reports show that the strength of the amygdala-hippocampus connectivity indicative of fear is enhanced with cortisol release (Hakamata) and cortisol production will re-instate fear after its extinction by activating this pathway (Kinner) and hence, reduced cortisol production will instigate a reduction in the emotional experience of fear. Also there is a possible switch off via the ventromedial prefrontal cortex in Schauer and Elbert`s Stages 5-6 since when stress is deemed controllable (ie. through the numbing of the emotions) then the stress induced activation of the dorsal raphe nucleus is inhibited by the ventromedial prefrontal cortex and the behavioural effects of stress are blocked because of the reduction in cortisol production (Amat). Therefore, the Stage 5, ´flag` stage is achieved and a reassessment of ´value` of the event occurs, eg. ´fear, threat` to ´calm, acceptance`.  

   What all of the Cassol`s observations and the above explanations show is that the functioning status of the brain during the NDE forming experience is not damaged to the extent that normal sensory input, processing and memory formation mechanisms do not occur. Therefore, there is support for the explanation of NDE from a psychological perspective of the ´biological/psychological` framework (Braithwaite). This framework supposes as supported here from looking at the memory characteristics and mechanism that the NDE memories are a consequence of the brain`s functional changes that can occur as a response to a perceived threat of death. Whether they form when death occurs for obvious reasons cannot be determined. Therefore, the sensory experience, memory characteristics and mechanism can be explained by normal cognitive physiology and functioning and even the OBEs and other more transcendental experiences can also be given explanations from normal neurochemical principles, eg. tunnels and lights caused by the disinhibition of the visual cortex and positive emotions and lack of pain attributed to the action of endorphins.

   Up to this point, we have only discussed the NDE from the perspective of the experience at the time of the event, but for both the NDE and FM, recall is important. Recall reflects the memory formed at the time and the level of forgetting between the event and the retrieval stage and since NDE and FM can be recalled years later this could have a significant effect if these memories follow the normal trajectories of other memory types. We have to assume that the actual physiological mechanisms for memory formation and storage are the same whether for NDE, FM or autobiographical memories, even if levels vary. This has been shown for the case of acute stress and cortisol which is relevant to the topic discussed here for the NDE and FM memories. Cortisol has been shown to be linked to memory changes due to its action on the hippocampus, an important area in the formation and recall of memories (Osborne). Acute stress (and even much later after the stress event) has been shown to cause rapid release of norepinephrine and glucocorticoids (cortisol) which bind to the hippocampal receptors. The resulting change in function of the hippocampus leads to the promotion of memory formation and consolidation, but appears to inhibit memory retrieval(Wais). This has been expanded in the case of cortisol increase by showing that the decreased memory level being observed is restricted to specific task relevant information whereas background memory is not affected (Steinmetz). This is contrary to what Cassol and team found where both internal and external information are remembered to a greater extent in NDE memories, but may support the ´change in grouping` hypothesis given above. 

   The demands for an ´undamaged` brain with NDE is reinforced by the need for connectivity of brain areas during the recall process. It is found that recall requires connectivity between the frontoparietal network, the default mode network and the hippocampus (Xiao, Westphal). Connectivity is demonstrated by brain wave activity and an increase in alpha brain wave activity is associated with NDE memory recall which is indicative of attentional control (van Schouwenburg). However, gamma waves normally associated with hippocampus activity during retrieval is not seen in NDE retrieval, but instead theta wave activity is observed which is linked to recall of emotional episodic events and the required hippocampal activity (Palmieri) and an increase in delta wave activity also associated with recall and personal feelings of reward (Knyazev). Therefore, the retrieval mechanism appears to be the same for the different memory types independent of quality or quantity of information recalled.

   Cassol and team however, expanded their exploration of the NDE memory retrieval by looking at the effect of emotional state during the recall session. We have already given an explanation as to how the actual quality and quantity of the memory formed was increased by the stress experienced by the individual during the NDE event, but Cassol and team looked into whether or not inducing the same contextual and emotional states during the recall as experienced during the event itselfwould strengthen the recall and reactivation of the NDE memory.  This exploration was indicated because it has been found that memory retrieval is increased when there is a match between the consciousness state at encoding and the consciousness state at retrieval (state-dependent memory – Overton). Researchers have also found that NDE memories are better retrieved when delta wave connectivity is greater because this appears to be the dominate wave pattern during the encoding NDE experience phase (Hartman). (Delta wave activity is also associated with transcendental type experiences (Hartman) which would correlate with the OBE and other dissociative consciousness events linked to the NDE). This appears to be supported by Cassol and team`s results since they found that the NDEr experienced the sensation of feeling the emotions felt during the event while they recalled the NDE and these feelings were greater for the NDE and autobiographical memories than for the FB memories. The retrieval of NDE induces the same emotional states of fear, ´realer than real` emotionality, but then calm, positive emotions.  Therefore, whereas fear can lead to memory impairment normally this is counteracted by the positive, calming emotional response which would lead to normalised recall (McKenzie). The expression of emotion during recall was found to aid both central and peripheral information retrieval (Davidson). In the case of the FB memories, which are more than just emotional memories,  emotions can help or impair recall just like with the NDE, eg.  people experiencing high levels of stress and distress show significantly higher levels of recall of context information than individuals with lower levels of stress after a period of 3 years (Bohannon). The emotional reactions however, to the event may differ in that the reaction could remain over the long-term, be short lived, or only appear after the event’s impact is fully appreciated by the individual in reference to his own consequentiality.

   This question of consequentiality and relevance to SELF and self-identity brings to light another factor that may or may not strengthen NDE memories and FB memories through recall. NDE experiences and memories are considered very important to the individual`s life story, greater than for autobiographical memories, which are more important than FB memories. These findings are understandable since NDEs are from living through a near-death, extremely personal and significant life threatening event and so should be more important than the other two memory types, but FB memories are also important to the individual otherwise why would the individual record it.

  The relationship between NDE memory and the SELF is that the NDE memory is characterised by the rich phenomenology recorded at the time which we have already described as being due to the stress response, increase in cortisol production and increased attentional and emotional systems affecting central and peripheral information input and storage. This richness of the NDE memory is associated with the SELF and self-identity as observed by the centrality scores (CES scores) in Cassol and team`s experiments and the effect of the NDE event may affect the SELF and self-identity for a much longer time and to a much greater degree than a FB memory. It has been reported that the prevalence of meaning attached to the NDE event may bring about changes in behavioural patterns such as for example a reduced fear of death, a greater level of compassion for others or a lower value placed on materialistic positions. Therefore, recall of the NDE memory strengthens the event details and if not changed, strengthens the relevance to the SELF and self-identity. The FB memories must also serve some role in SELF or self-identity since they are recorded for a particular reason. However, this is disputed by some who say that the FB memory does not serve personal consequentiality, but serves consequentiality for the community, hence shaping social identity rather than self-identity. However, the same event characteristics may be inputted and stored for a vast number of individuals, but the exact memory details may be different due to the incorporation of the internal, phenomenological information. Therefore, this renders only part of the memory having consequentiality for the community and all to the SELF and self-identity.

   This factor of consequentiality and association with the SELF and self-identity reinforces the personal nature of the NDE and FB memories and brings into discussion the topic of retrieval accuracy. Recall of memories can reinforce event characteristics through their reactivation and strengthening of firing of the neuronal assemblies that make up the event representation (internal and external features). Therefore, the thinking or re-telling of the experiences may change or strengthen the characteristics recorded. Cassol and team reported that in the case of the NDE that reactivation sharing and its frequency were found to be important and this is as expected. It was also found to influence the memory accuracy. The problem with judging whether the recall of NDE events is accurate is that the individual doing the recall is the only person who has experienced it and who has recorded the internal and external event characteristics. Therefore, the accuracy of the recall for at least the first reactivation cannot be verified totally by a third party. Subsequent retrievals can be judged to some extent via consistency of reporting and to this extent it has been reported that NDErs remember the event clearly and accounts are not modified over a period of two decades. In this way it appears that memory traces do not fade like other autobiographical memories and narratives remain consistent if no post-event processing, construction and embellishment takes place.

   In the case of FBs, judgement of accuracy would be deemed easier to establish since part of the event characteristics recorded are shared by a large number of people and therefore, there should be many accounts of these particular facts. However, the details may be affected by who and how events are recalled by others and reports have shown how media attention can affect the recall of these shared features. Again like NDE however, FB memory has a quantity of external peripheral facts that may not be common to all and a quantity of internal event characteristics which are entirely individual. Therefore, it would be difficult for a third person to check the accuracy of these features against the original encoding event and consistency accuracy is the only measurement possible. Researchers have found once an inconsistency emerges which usually occurs within the first year then it remains (Hirst) and like NDE events, FB memory recall may be years later after the event. Again, FB recall makes the memory subject to change via post-event processing, construction, embellishment and the influence of others.

   Therefore, this exploration of NDE and FB memories has shown that they are more examples of how emotional state can affect informational input and memory. The uniqueness of the memories with their increased level of peripheral, internal information can be attributed to the change in attentional state induced particularly in the case of NDE to a rise in cortisol production due to the perceived threat to life. Their value to memory research is via their strong association to the SELF and self-identity and this in the case of FB memories can be advantageous.

Since we`re talking about the topic……….

            …..dexamethasone is a synthetic glucocorticoid known to lead to the instigation of the stress response. Is it possible that the NDE-like responses can be induced by its use and these responses can be observed with neuroimaging and compared to those experienced by practiced meditators?

            …. If NDE-like memory can be induced by hypnosis, can neurochemical studies on cortisol production, calcium influx and neuroimaging of brain area activity confirm whether or not, cortisol is the cause of the memory changes observed?

            …. ketamine-induced anaesthesia is said to be similar to NDEs in the level of wakefulness and connectedness, could this be used to study the influence of external environmental changes such as loud noise and smells on memories if formed by experienced meditators?

near-death experience memories compared to flashbulb memories

Posted comment on ´Near-death experience memories include more episodic components than flashbulb memories` written by H. Cassol, E.A.C. Bonin, C. Bastin, N. Puttaert, V. Charland-Verville, S. Laureys and C. Martial  and published in Frontiers in Psychology 13^th May 2020 doi 10.3389/fpsyg.2020.0088

SUMMARY

   Cassol and colleagues investigated whether memories of near-death experiences (NDEs) are comparable to aspects of flashbulb memories (FBs). In their investigation they identified and compared episodic and non-episodic information, phenomenological characteristics and the centrality of memories of participant`s verbal recollections of their NDE, flashbulb and control autobiographical memories.

  The experiment was set up so that the 25 participants who had lived through a life-threatening situation (eg. anoxia, trauma) and who had met the accepted criteria of having experienced a NDE, first took part in a screening task.  This was to detect and exclude memories biases relating to negative mood (according to Positive and Negative Affect Schedule) and mild cognitive impairments (Montreal Cognitive Assessment). The participants then performed the Autobiographical Memory Interview (AMI).  Each participant was asked to describe in detail three target memories that occurred in the same time period of their life. These were: the memory of their NDE; a flashbulb memory (these followed particular criteria such as that they were either very surprising and consequential, or emotionally arousing, eg. Man`s first steps on the moon, September 11<sup>th</sup> terrorist attacks); and an autobiographical memory. The interviewers were allowed to probe when required to encourage a full description of the events. The narratives were recorded, transcribed and analysed using an established manual scoring procedure so that the episodic details could be separated from the non-episodic details. Episodic details (termed ´internal details`) related to descriptions of the events such as the event details describing the unfolding of the story (eg. listed as the happenings, persons involved, reactions/emotions of oneself and other people, one`s clothing and the weather), the time (eg. life epoch as well as defined physical time) and place plus the sensory or mental state details (eg. thoughts and emotions) specific for the event. The non-episodic details (termed the ´external details` related to semantic or factual information not specific for the event). Based on the final classification of reliability between scores from independent scorers, the details generated were summed across the memory types.  The data of the AMI was analysed for each type of memory using appropriate software and statistical methods and significance was defined.

  After each memory recall the participants were given a short version of the Memory Characteristics Questionnaire (sMCQ – 16 items) and the Centrality of Event Scale (CES – 20 items) to fill in. The sMCQ assessed memory clarity, sensory details, self-referential and emotional information, reactivation frequency and confidence in memory on a Likert scale of 1 to 7. Data analyses were carried out using appropriate statistical software. The CES assessed how central the event was to the person`s identity and life story using agreement (scale 1-5) to the following statements of the event as: considered a reference point for generation of expectations to other events in life story; considered as a central component of a person`s identity; or considered as a turning point in the person`s life story. Both sets of results were analysed using appropriate statistical methods.

   The results of Cassol and team`s experiments showed that the level of negative mood was no higher for the NDE group than the control and was not related to the amount of internal or external details of the NDE, flashbulb and autobiographical memories. The age of the interviewee and the time of the interview in relation to the NDE event also did not correlate to the amount of internal or external details of the three memory types. However, the overall amount of details given for the NDE memory was higher than for both the flashbulb and autographical memories which were the same.

   The results also showed a significant interaction between the type of detail and the type of memory. The number of internal details reported for NDE memories was higher than the internal details reported for flashbulb and autographical memories. The number of internal details was also higher than the amount of external details reported for the NDE and again, this level was higher than for both the flashbulb and autobiographical memories.

   With regards to the results of the sMCQs, Cassol and colleagues found that NDE memories exhibited higher, but not statistically significant, ratings for internal perceptual details relating to emotional status compared to the autobiographical and flashbulb memories. They found that there was a memory difference in the feeling of mentally reliving the event and the sensation of feeling the emotions felt during the event while remembering. This feeling was higher in both NDE and autobiographical memories than with the flashbulb memories. The authors also reported a memory difference in the visual perspective taken while remembering. The NDE and autobiographical memories did not demonstrate statistical differences to each other, but they did show a difference to flashbulb memories. The results indicated that NDE and autobiographical memories were more likely to be remembered from the first person perspective. The emotions felt at the time of the event, ie. the valence, also produced a memory difference. Again, flashbulb memories gave lower scores than NDE and autobiographical memories and because NDE and autobiographical memories did not differ, this indicated to the authors that the latter were more positive in average. Two further observations were made that indicated a trend (but were not statistically significant) towards higher scores for NDE memories and autobiographical memories than flashbulb memories and these were: personal importance attached to the event; and frequency of sharing and reactivation. Scores also not reaching significance for the memory types were those relating to the amount of sensory details, clarity (time location and coherence), confidence in memory and the ability of the individuals to remember their own actions/words/thoughts.

   Cassol and colleagues found more significant differences with the results of their Centrality of Event Scale studies, ie. how central an event was to a person`s identity and life story. They found that the mean score for the NDE memory was significantly higher than for the autobiographical memory which was higher than that for the flashbulb memory. They also found a significant positive correlation between CES scores and a number of the reported internal details given above (eg. the feeling of re-experiencing the event, the feeling of re-experiencing the emotions felt at the time of the event , the importance of the event and the level of sharing of the event and reactivation frequency). However, there was no correlation between the CES and the external details. This indicated that the NDE memories are the most central memories to identity, more than the control autobiographical memories and with flashbulb memories the least.

   Cassol and colleagues concluded their article with a discussion of their findings. They found that NDE memories were not the same as flashbulb memories and presented explanations as to why this might be. The authors suggested several reasons why there is a higher overall amount of details and higher amount of internal/episodic details in the NDE memories compared to the autobiographical memories and flashbulb memories. The first reason related to the association between NDE and the unusual perceptions of the individuals at the time such as leaving the physical body, being in an unknown spatial-temporal dimension and the intense sensation of ´reality` reported with them. This, the authors suggested could lead to the vivid memories of the NDE in comparison to flashbulb memories.

   The second reason given suggested that the difference lies with the ´weakness` of flashbulb memories. The authors explained that flashbulb memories were more subject to forgetting in comparison to other emotionally arousing events. The severity of the emotions felt and the degree of involvement would maintain consistency and amplify the memories rather than for the flashbulb memories where the participants were not normally personally involved. This was supported by the strength of the NDE memories in terms of details. Cassol and colleagues found that the NDE narratives became richer in time compared to flashbulb memories which they said declined. They found that certain subtypes of recalled details were increased, eg. the event details describing the unfolding of the story and the perceptual details (eg. sensory and spatial-temporal information). This was attributed to the self-referential nature of the information which would have a positive effect on encoding, processing and storage of the memory.

   The authors also linked the self-referential nature with the differences relating to emotions and importance. They explained that the strength of the NDEs because of the event being highly emotional and the emotional relevance of autobiographical memories meant that the phenomenological characteristics for the NDEs and the autobiographical memories were higher than those for the flashbulb memories. The levels for autobiographical memories which were on a par with the NDEs were explained as due to the autobiographical memories being temporally linked in time in some way to the NDE which would strengthen them both. Again the self-referential aspect of both the NDEs and autobiographical memories would mean that they would be shared more often and communicated more often than the flashbulb memories and so would be strengthened because of it.

   With regards to the perspective taken during recollection, the authors also used the self-referential nature of the NDEs as being reflected by the individual using the first person compared to the third person when recalling the flashbulb memory. The autobiographical memories were shown as being recalled either in the first or third person perspective. Cassol and colleagues suggested that the use of the third person perspective for the NDE or autobiographical memories could be an avoidance strategy to set up distancing between the individual and the memory and hence, emotional intensity would be reduced. Therefore, the difference in the adopted visual perspective could be due to the emotions felt at the time of the event (eg. valence) that are overall very positive in NDE and autobiographical, but more negative in flashbulb. Cassol and colleagues also gave the results of the CES and centrality tests as support for the NDEs having a higher impact on the individual`s life story than the flashbulb memory.

   Therefore, Cassol and team`s experiments show that NDE memories have different characteristics to flashbulb memories with richer internal details and a propensity to self-reference and importance. They concluded their article with a few suggestions as to how to extend their experimentation such as increasing the number of subjects who had experienced NDEs, controlling for false memories and using autobiographical and flashbulb memories from different time periods.

COMMENT

   What makes this article interesting is that it describes two of the more unusual types of long-term memory: that of near-death experience memory (NDE) and flashbulb memory (FB). Both of these relate to memories formed of events experienced at particular points in an individual`s life and whereas the former is rare (hence, difficult to find experimental subjects) and extremely personal, the other is more common and relates to the individual and his/her social environment.  The NDE is an experienced situation of intense physical or emotional danger where some individuals report a dissociative consciousness state, vivid extraordinary perceptions (eg. out-of-body – OBE), or an intense feeling of peacefulness and calm. (This is similar to, but not the same as, near-death-like experiences where there are similar phenomenological experiences, but without the physical and/or emotional danger. NDE-like experiences are sometimes associated with meditation, or depression.) In comparison, flashbulb memories are experiences where the individual is given a ´piece of news`. This may be consequential to the individual, but it is unlikely that the individual is in physical danger at that time. Therefore, although the memories are autobiographical and relate to the individual at specific time points in their life story they are formed in different circumstances. Therefore, this comment looks at the similarities and differences between the two with reference to Cassol and team`s findings and looks at which neurochemical mechanisms may promote NDEs being formed.  

   The first difference that springs to mind between the two types of experience and memory from a neurochemical point of view is the level of acute stress and stress response experienced at the time of the event. The intense physical or emotional danger experienced by the NDEers (individuals who report a NDE) is likely to promote a physiological  ´defence` response in order to secure survival. If we look at Schauer and Elbert`s sequence of stages in response to traumatic stress (ie. Stages 1-6 -freeze, flight, fight, fright, flag, faint) the NDE is likely to begin to occur at the end of Stage 3 (the fight phase) when sympathetic activation has mobilised resources, but since the ´fight` is not successful the individual slips into the next stage, Stage 4, fright. Sympathetic activation brings about a range of physiological signs such as dizziness, light-headedness, palpitations, numbing and important for this topic, feelings of non-reality. Stage 4, although associated with unresponsive immobility but tachycardia and hypertension, consolidates the change in mental approach to the danger situation with signs of hyper-alertness, high emotional arousal and fear. This continues into the ´flag` phase where there is a physiological ´shut-down` instigated via the activation of the parasympathetic system and symptoms such as bradycardia, vasodilation and hypotension and relating to the NDE, a drop in arousal, feeling of surrender, cognitive failure and ´numbing` of all emotions (perhaps, the feeling of ´inner calm` experienced). Therefore, it appears that the reported experiences of the NDE event (eg. dissociative consciousness and reported increases in awareness leading to inner calm and peace) can be explained by the staged physiological responses to stress according to Schauer and Elbert . In comparison, flashbulb memories (FB) are associated with a minimal or temporarily raised stress level since the individual is not in physical danger.   

  The different stages of the stress response induce changes in the brain neurochemistry and these we assume bring about the cognitive changes observed with the NDE and highly emotional situations. These neurochemical changes are linked to cortisol (or glucocorticoid) production, which is known to cause effects in some cognitive mechanisms. Cortisol is eventually produced in response to the immediate threat to health. This manifests stimulation of a number of different routes, eg. vagus nerve to medulla to periventricular nucleus of the hypothalamus; somatosensory stressors via the tegmentum and the reticular formation to the paraventricular nucleus of the hypothalamus; painful stressors via the periacqueductal gray PAC to the paraventricular nucleus; locus coeruleus and changes to heart rate and blood pressure leading to increased release of noradrenaline leading to paraventricular nucleus; emotional stressors mediated via the raphe nucleus leading to the paraventricular nucleus; and finally and more importantly linked to cognitive function stimulation of the hippocampus, septum and amygdala. This stress pathway is involved in changes to emotional and cognitive input. The amygdala releases corticotrophin-releasing hormone (CRH) which activates the autonomic and endocrine systems which mobilise the energy for the fight or flight response.

   The next stage of the neurochemical response to stress is the activation of the sympathetic adrenomedullary axis (SAM) and more importantly for cognitive functions, the hypothalamic-pituitary adrenocortical axis (HPA). The SAM route involves the release of adrenaline, noradrenaline and stress hormones and increases arousal and vigilance. The HPA route involves actions of the hypothalamus via the paraventricular nucleus. This when activated leads to the release of corticotrophin releasing factor which enters the anterior pituitary gland where it binds to CRh R1 receptors and stimulates the production of adrenocorticotropic hormone (ACTH). ACTH stimulates the cortex of the adrenal gland leading to the synthesis and release of glucocorticoids, which spread through the body causing a number of different effects (eg. increased availability of blood glucose, decreased immune system and in the brain, inhibition of eating, sexual behaviour and growth). The main glucocorticoid in humans is cortisol and it binds to receptors found in the cytoplasm of many neurons. The activated receptor travels to the nucleus where it can stimulate gene transcription and appropriate protein synthesis. One role of cortisol is the increased influx of calcium ions via increased activation of voltage gated channels (shown by administration of GC R antagonist RU38486 – Karst). It is the influence on calcium ion concentrations on many neurochemical mechanisms within the neuron which cause the changes to neuronal functioning and ultimately can lead to cognitive effects associated with NDE. Therefore, whereas individuals that are experiencing NDE may have a physical stress response and release cortisol because of the threat to life which then has an effect on many cognitive functions, individuals that have FB memories are not under threat and therefore, the effects on their cognitive functions occur by different means.

   The first cognitive effect associated with NDE which may be linked to cortisol production and neuronal calcium ion effects is the informational content of the memories formed at the time of the event. Cassol and team found in their experiments that the amounts of information (total, external and internal) in NDE were greater than FB memories with both demonstrating a high level of context information not greatly relevant to the situation at the time. It is likely that this increase in informational content comes about by a change in attention, which is known to be a controlling factor of the quality and quantity of sensory information and this may be induced by the cortisol released as part of the response to the threat situation. Cortisol has been reported to increase attention (Banks), but what does this mean to the information being inputted and stored? We know that the fear attention state (and the corresponding fear emotional state since they are interrelated) can increase the quantity of informational input, but decrease its quality (ie. more gist rather than high level detail). This is possibly due to a change in the balance of task-relevant and task-irrelevant material where the latter under normal circumstances would not be learnt, but in NDE are included in a significant amount. This is also a possible explanation for FB memories too, since these memories are also known to include not only task-relevant information, but also a host of irrelevant information such as trivial sensory details (Brown and Kulik).

   Therefore, in both cases central and task relevant information is given the same weighting as irrelevant, non-central, peripheral information and this change in balance is likely to be achieved through alterations to the attentional system. For example, Cukor showed that attention dynamically alters visual representation to optimize the processing of behaviorally relevant objects during natural vision. The likely brain areas involved in such as shift would be: the intraparietal sulcus, precuneus and dorsolateral prefrontal cortex shown to be involved in bilateral attentional control in selecting the extent of relevancy and updating the representation (Niv); the anterior thalamus  which appears to direct attention to task-relevant stimuli that will bring reward (Wright); the amygdala involved in the fear state; and the hippocampus which is required for sustained activation of the relevant neurons without repetition and is important for memory formation. There is also likely to be an involvement of the NMDA receptors since the NMDA R antagonist, ketamine is reported to strongly impair the ability to ignore irrelevant task information (Stoets).

   The shift in informational content of the NDE and FB memories is also likely to include as well a shift in the balance of attended to unattended information (information of which the individual is aware to information that the individual has no awareness of). This is suggested with NDE memories since they are described by NDErs as being ´realer than real`.Such a shift could be explained by a widening of the limits of ´awareness` since the experience not only includes task relevant and attended information, but also, task irrelevant and what would normally be, unattended information. Hence, the experience would be described as ´realer than real`.

   The original models of Cherry, Broadbent etc. relating to levels of attended and unattended information describe them in terms of perception and processing. This leads onto factors affecting the balance and characteristics with for example, decreased processing of unattended information (Wojceulik) and greater processing of unattended information when the perceptual load is low (Lavie). Therefore, even if there is no awareness of unattended information there can still be processing (Wright, Nee and Norman). The situation with NDE is that this level of information would not normally be consciously experienced or be the first information recalled.  Therefore, the NDE condition changes the balance so that more information is attended. The question is how can this be achieved?

   One possible suggestion is that the stress response causes changes not only in the amount of information inputted as described above, but how the information is grouped or bound together. This has an effect on the capacity of working memory, which we know is limited to a certain number of event characteristics. Perception and Gestalt theories group visual elements such as lines, curves together into unitary objects such as forms and shapes which are then processed by working memory. By increasing the ´group size`, processing capability can therefore, be ´stretched` to more elements. We know of this through memory improvement techniques where individuals instead of trying to remember 6 different numbers group them together to represent years or months for example to increase recall efficiency. In the case of NDE experiences, the different sensory input may be grouped together as a single ´unit`. This has been shown in a similar manner by Jang who described event representation as containing not only the prime features of the event, but also more abstract, more internal features of the event such as attentional control states and category information. Such grouping was said to require the activation of the hippocampus and putamen as well as other regions representing visual feature-selective event information (primarily visual cortex), category-selective information (posterior frontal cortex) and control demand-selective event information (insula, caudate, anterior cingulate, and parietal cortex) event information.Therefore, brain areas affected by the stress response such as the hippocampus would, like the other attentional system brain areas, lead not only to changes in informational quality and quantity, but also awareness and processing capability. This would explain both the NDE event content and the content of FBs where both task relevant and task irrelevant information form part of the event representation.

   Although the stress response may then explain the change in conscious awareness relating to information and informational processing, can it also explain the dissociative consciousness state reported with NDE experiences? The mechanics of conscious awareness observed for visual input 170msec after presentation (Thierry) involves synchronized activation of many areas including the DMN, insular cortex, anterior cingulate cortex, medial prefrontal cortex and dorsal thalamic nuclei. However, conscious awareness under normal circumstances does not involve some of the transcendental, out-of-body type awareness reported by NDErs. In this case there is a dissociation from ´reality`, ie. the real-time sensory input information achieved via eyes, ears etc. One explanation for this could be the near-complete separation of top-down and bottom-up processing leading so that the top-down ´I, SELF` thinking part is distanced from the ´sensing, input` bottom-up part. This idea may be acceptable since imagining and dreaming are both examples where top-down processing and bottom-up input are ´distinct`. In the case of NDE, however the separation is likely to be achieved by the attentional system, which we have already seen is under the influence of cortisol released in the stress response. Top-down attentional modulation appears to suppress the incoming visual information via inhibition of firing at the V1 level (Jacob) and dissociation of attention and awareness is observed in the case of failure of applied chromatic flicker to reach awareness, but not alerting and orienting effects – also associated with V1 visual area effects (Lu). This separation is also apparent through the conscious awareness of an isolated object (or gist of a scene) in the near-absence of top-down attention (van Boxtel). (However, in this particular research example it appears that the explanation is against the proposed one for NDE since attention is described as top-down and ´thought` and consciousness is the information coming in and bottom-up. Possibly the way to approach the results of this study is to view conscious awareness as top-down and subject to top-down attention whereas sensory information is bottom up and subject to bottom-up attention with attentional modulation being the ´tool` and not the ´result`.)

   Therefore, one possible explanation for the more transcendental aspects of the dissociative consciousness reported by NDErs is the separation of top-down processing from bottom-up processing. The advantage of such a separation is protection of the higher brain areas from excessive information and high processing load. This may also be attributed to the emotional state at the time of the experience which is interrelated to attentional system functioning (Gregory). Cassol and team reported that the emotional stateduring the NDE experience was greater than for the autobiographical memory and both were greater than that recorded for the FB experience and it is clear that NDE are profound psychological events with highly emotional and self-related content. The increase in information is associated with this highly emotional state since it has been found that the link between episodic memory,  the feeling of ´being there` and the higher emotional experience is linked to better factual memory via an improved attentional focus on the stimuli, hence increasing memory encoding (Makowski). The same is reported with FB memories which also carry factual memory with strong emotions that can be recalled years later after the initial event (Brown, Finkenauer).

   The increased fear emotional state as the individual experiences the near-death event in Schauer and Elbert`s Stages 3 (fight) and 4 (fright) of the stress response is responsible for the increased levels of relevant and irrelevant material for the task (Newenhause, Stoets) and requires the activation of brain areas such as prefrontal cortex, amygdala, cingulate cortex and thalamus. Anxiety is shown to produce elevated cortisol levels linked to the hyperactivity of the HPA axis (Lenze) and there is a direct ventral connection between the amygdala and prefrontal cortex (Eden), both affected by anxiety.

   However, as the stress response progresses this fear emotional state shifts to a period of ´calm`. This relates to Schauer and Elbert`s Stage 5, ´flag` phase where there is a physiological ´shut-down` instigated via the activation of the parasympathetic system with symptoms such as drop in blood pressure and heart rate, but more importantly to the emotional experience of the NDE, a drop in arousal, a feeling of surrender, cognitive failure and a ´numbing` of all emotions, ie. the reported ´calm`. This can be brought about by the reduction in cortisol production and hence, cortisol effects on the brain areas involved in instigating the emotional state (eg. the Papez-Mclean limbic model involving activity of the amygdala, hippocampus and cingulate cortex and the two track Le-Doux theory with rapid emotional response based on information from the thalamus to the amygdala leading to autonomic and endocrine changes interpreted by the cortex and the slower, direct transmission of information from the thalamus to the cortex).  Reports show that the strength of the amygdala-hippocampus connectivity indicative of fear is enhanced with cortisol release (Hakamata) and cortisol production will re-instate fear after its extinction by activating this pathway (Kinner) and hence, reduced cortisol production will instigate a reduction in the emotional experience of fear. Also there is a possible switch off via the ventromedial prefrontal cortex in Schauer and Elbert`s Stages 5-6 since when stress is deemed controllable (ie. through the numbing of the emotions) then the stress induced activation of the dorsal raphe nucleus is inhibited by the ventromedial prefrontal cortex and the behavioural effects of stress are blocked because of the reduction in cortisol production (Amat). Therefore, the Stage 5, ´flag` stage is achieved and a reassessment of ´value` of the event occurs, eg. ´fear, threat` to ´calm, acceptance`.  

   What all of the Cassol`s observations and the above explanations show is that the functioning status of the brain during the NDE forming experience is not damaged to the extent that normal sensory input, processing and memory formation mechanisms do not occur. Therefore, there is support for the explanation of NDE from a psychological perspective of the ´biological/psychological` framework (Braithwaite). This framework supposes as supported here from looking at the memory characteristics and mechanism that the NDE memories are a consequence of the brain`s functional changes that can occur as a response to a perceived threat of death. Whether they form when death occurs for obvious reasons cannot be determined. Therefore, the sensory experience, memory characteristics and mechanism can be explained by normal cognitive physiology and functioning and even the OBEs and other more transcendental experiences can also be given explanations from normal neurochemical principles, eg. tunnels and lights caused by the disinhibition of the visual cortex and positive emotions and lack of pain attributed to the action of endorphins.

   Up to this point, we have only discussed the NDE from the perspective of the experience at the time of the event, but for both the NDE and FM, recall is important. Recall reflects the memory formed at the time and the level of forgetting between the event and the retrieval stage and since NDE and FM can be recalled years later this could have a significant effect if these memories follow the normal trajectories of other memory types. We have to assume that the actual physiological mechanisms for memory formation and storage are the same whether for NDE, FM or autobiographical memories, even if levels vary. This has been shown for the case of acute stress and cortisol which is relevant to the topic discussed here for the NDE and FM memories. Cortisol has been shown to be linked to memory changes due to its action on the hippocampus, an important area in the formation and recall of memories (Osborne). Acute stress (and even much later after the stress event) has been shown to cause rapid release of norepinephrine and glucocorticoids (cortisol) which bind to the hippocampal receptors. The resulting change in function of the hippocampus leads to the promotion of memory formation and consolidation, but appears to inhibit memory retrieval(Wais). This has been expanded in the case of cortisol increase by showing that the decreased memory level being observed is restricted to specific task relevant information whereas background memory is not affected (Steinmetz). This is contrary to what Cassol and team found where both internal and external information are remembered to a greater extent in NDE memories, but may support the ´change in grouping` hypothesis given above. 

   The demands for an ´undamaged` brain with NDE is reinforced by the need for connectivity of brain areas during the recall process. It is found that recall requires connectivity between the frontoparietal network, the default mode network and the hippocampus (Xiao, Westphal). Connectivity is demonstrated by brain wave activity and an increase in alpha brain wave activity is associated with NDE memory recall which is indicative of attentional control (van Schouwenburg). However, gamma waves normally associated with hippocampus activity during retrieval is not seen in NDE retrieval, but instead theta wave activity is observed which is linked to recall of emotional episodic events and the required hippocampal activity (Palmieri) and an increase in delta wave activity also associated with recall and personal feelings of reward (Knyazev). Therefore, the retrieval mechanism appears to be the same for the different memory types independent of quality or quantity of information recalled.

   Cassol and team however, expanded their exploration of the NDE memory retrieval by looking at the effect of emotional state during the recall session. We have already given an explanation as to how the actual quality and quantity of the memory formed was increased by the stress experienced by the individual during the NDE event, but Cassol and team looked into whether or not inducing the same contextual and emotional states during the recall as experienced during the event itselfwould strengthen the recall and reactivation of the NDE memory.  This exploration was indicated because it has been found that memory retrieval is increased when there is a match between the consciousness state at encoding and the consciousness state at retrieval (state-dependent memory – Overton). Researchers have also found that NDE memories are better retrieved when delta wave connectivity is greater because this appears to be the dominate wave pattern during the encoding NDE experience phase (Hartman). (Delta wave activity is also associated with transcendental type experiences (Hartman) which would correlate with the OBE and other dissociative consciousness events linked to the NDE). This appears to be supported by Cassol and team`s results since they found that the NDEr experienced the sensation of feeling the emotions felt during the event while they recalled the NDE and these feelings were greater for the NDE and autobiographical memories than for the FB memories. The retrieval of NDE induces the same emotional states of fear, ´realer than real` emotionality, but then calm, positive emotions.  Therefore, whereas fear can lead to memory impairment normally this is counteracted by the positive, calming emotional response which would lead to normalised recall (McKenzie). The expression of emotion during recall was found to aid both central and peripheral information retrieval (Davidson). In the case of the FB memories, which are more than just emotional memories,  emotions can help or impair recall just like with the NDE, eg.  people experiencing high levels of stress and distress show significantly higher levels of recall of context information than individuals with lower levels of stress after a period of 3 years (Bohannon). The emotional reactions however, to the event may differ in that the reaction could remain over the long-term, be short lived, or only appear after the event’s impact is fully appreciated by the individual in reference to his own consequentiality.

   This question of consequentiality and relevance to SELF and self-identity brings to light another factor that may or may not strengthen NDE memories and FB memories through recall. NDE experiences and memories are considered very important to the individual`s life story, greater than for autobiographical memories, which are more important than FB memories. These findings are understandable since NDEs are from living through a near-death, extremely personal and significant life threatening event and so should be more important than the other two memory types, but FB memories are also important to the individual otherwise why would the individual record it.

  The relationship between NDE memory and the SELF is that the NDE memory is characterised by the rich phenomenology recorded at the time which we have already described as being due to the stress response, increase in cortisol production and increased attentional and emotional systems affecting central and peripheral information input and storage. This richness of the NDE memory is associated with the SELF and self-identity as observed by the centrality scores (CES scores) in Cassol and team`s experiments and the effect of the NDE event may affect the SELF and self-identity for a much longer time and to a much greater degree than a FB memory. It has been reported that the prevalence of meaning attached to the NDE event may bring about changes in behavioural patterns such as for example a reduced fear of death, a greater level of compassion for others or a lower value placed on materialistic positions. Therefore, recall of the NDE memory strengthens the event details and if not changed, strengthens the relevance to the SELF and self-identity. The FB memories must also serve some role in SELF or self-identity since they are recorded for a particular reason. However, this is disputed by some who say that the FB memory does not serve personal consequentiality, but serves consequentiality for the community, hence shaping social identity rather than self-identity. However, the same event characteristics may be inputted and stored for a vast number of individuals, but the exact memory details may be different due to the incorporation of the internal, phenomenological information. Therefore, this renders only part of the memory having consequentiality for the community and all to the SELF and self-identity.

   This factor of consequentiality and association with the SELF and self-identity reinforces the personal nature of the NDE and FB memories and brings into discussion the topic of retrieval accuracy. Recall of memories can reinforce event characteristics through their reactivation and strengthening of firing of the neuronal assemblies that make up the event representation (internal and external features). Therefore, the thinking or re-telling of the experiences may change or strengthen the characteristics recorded. Cassol and team reported that in the case of the NDE that reactivation sharing and its frequency were found to be important and this is as expected. It was also found to influence the memory accuracy. The problem with judging whether the recall of NDE events is accurate is that the individual doing the recall is the only person who has experienced it and who has recorded the internal and external event characteristics. Therefore, the accuracy of the recall for at least the first reactivation cannot be verified totally by a third party. Subsequent retrievals can be judged to some extent via consistency of reporting and to this extent it has been reported that NDErs remember the event clearly and accounts are not modified over a period of two decades. In this way it appears that memory traces do not fade like other autobiographical memories and narratives remain consistent if no post-event processing, construction and embellishment takes place.

   In the case of FBs, judgement of accuracy would be deemed easier to establish since part of the event characteristics recorded are shared by a large number of people and therefore, there should be many accounts of these particular facts. However, the details may be affected by who and how events are recalled by others and reports have shown how media attention can affect the recall of these shared features. Again like NDE however, FB memory has a quantity of external peripheral facts that may not be common to all and a quantity of internal event characteristics which are entirely individual. Therefore, it would be difficult for a third person to check the accuracy of these features against the original encoding event and consistency accuracy is the only measurement possible. Researchers have found once an inconsistency emerges which usually occurs within the first year then it remains (Hirst) and like NDE events, FB memory recall may be years later after the event. Again, FB recall makes the memory subject to change via post-event processing, construction, embellishment and the influence of others.

   Therefore, this exploration of NDE and FB memories has shown that they are more examples of how emotional state can affect informational input and memory. The uniqueness of the memories with their increased level of peripheral, internal information can be attributed to the change in attentional state induced particularly in the case of NDE to a rise in cortisol production due to the perceived threat to life. Their value to memory research is via their strong association to the SELF and self-identity and this in the case of FB memories can be advantageous.

Since we`re talking about the topic……….

            …..dexamethasone is a synthetic glucocorticoid known to lead to the instigation of the stress response. Is it possible that the NDE-like responses can be induced by its use and these responses can be observed with neuroimaging and compared to those experienced by practiced meditators?

            …. If NDE-like memory can be induced by hypnosis, can neurochemical studies on cortisol production, calcium influx and neuroimaging of brain area activity confirm whether or not, cortisol is the cause of the memory changes observed?

            …. ketamine-induced anaesthesia is said to be similar to NDEs in the level of wakefulness and connectedness, could this be used to study the influence of external environmental changes such as loud noise and smells on memories if formed by experienced meditators?

action video game experience linked to improved attention driven perceptual exploration in categorisation learning

Posted comment on ´Play to win: action video game experience and attention driven perceptual exploration in categorisation learning` written by S. Schenk, C. Bellebaum, R.K. Lech, R. Heinen and B. Suchan and published in Frontiers in Psychology 13^th May 2020 doi 10.3389/fpsyg.2020.00933

SUMMARY

   Schenk and colleagues investigated the difference between action video gamers and non-gamers in visual exploration and attention driven perception during a categorisation learning task. Their subjects, 17 right-handed non-gamers (mean age nearly 23, nearly all female) and 16 action video gamers (mean age nearly 24, nearly all male) performed 490 random trials (5 blocks of 98) of a visual categorisation task. This consisted of distinguishing 14 ring stimuli which had the same structure, but different colour combinations. These had to be divided into 2 categories, seven in each and made up of a prototype, 5 typical stimuli and one exception. The typical stimuli shared 5 colour features with the prototype of the category, whereas the exception shared five colour features with the prototype of the other category.  The subjects did not know of the existence of the exceptions and had to first work out that they were present and then had to explicitly remember both exceptions in order to categorise all the stimuli presented. The stimuli were presented and the response made by pressing the left and right control keys designated for the two categories. The key press was followed by an immediate feedback of right or wrong. The subjects were required to react within 1.8 secs of stimulus presentation and failure to do so resulted in a verbal warning to quicken response time. Following the feedback the subjects were presented with a fixation cross for 1-2 secs before being allowed a short break before the next trial started.  EEG recordings were taken during the experiment and eye-tracking recordings of the subject`s right eye were also made during the stimulus presentation.  

   Data analyses were performed on the recordings. In the case of the EEG recordings, the datasets were analysed with software packages. Blink artefacts and vertical eye movements were removed from the EEG data and components reflecting eye movements were removed from the EEG signal. The recordings for the prototypes and typical stimuli were combined because of their similarity (prototypical stimuli) and the recordings were adjusted so that the segments for both stimulus types (prototypical stimuli and exceptions) were followed by a baseline correction relative to the 200ms preceding the stimulus presentation. In the case of the P150 ERP component, data from the parietal-occipital electrodes was recorded and the amplitude was taken as that within 120-180ms after stimulus presentation. The maximum negative peak amplitude within the time frame 150-190ms after stimulus presentation was taken and defined as the Peaks of the N170 amplitude. The analysis of the N170 component was carried out using different electrodes to those of the P150.

    The eye-tracking data was analysed using appropriate software. Fixations were visualised on the basis of heat maps with the ring stimuli divided into areas. Percentages of the fixation rates and number of fixations were calculated for all areas of the ring and for the centre and the differences between prototypical and exceptions calculated. Eye movements (taken as changes in velocity of the eye position exceeding 40 degrees per second and displacement of at least 1.5 degree visual angle) were also analysed with saccades detected within 800ms of the presentation period. Statistical analyses of the various sets of behaviour data were carried out using ANOVA with repeated measures (including Greenhouse-Geisser procedure and Bonferroni), paired T test and G Power.

   The results of Schenk and colleagues experiments showed that the learning process relied on the immediate feedback of right or wrong response. The ANOVA analysis carried out for the percentage of correct responses yielded significant main effects for both the factors ´stimulus` and ´block` as well as showing significant interaction. The pairwise comparisons revealed for both groups more correct categorisations in the last blocks, especially for the prototypical stimulus. Using the paired t-test it was shown that interaction was based on a high number of correctly categorised prototypical stimuli compared to a low number of correctly categorised exceptions at the early stage of the experiment. A high increase in correctly categorised exceptions in block 3 showed that the successful categorisation of the exceptions occurred later. The differences between the blocks between percentage correct responses for exceptions and prototypical stimuli were shown by paired t tests to yield significance between block 2 and 3. All other results showed no significant differences.

    The results from the ANOVA analysis of behavioural data of the two groups showed a significant interaction between ´stimulus` and ´group`. This showed that action video gamers had superior categorisation performance for the exceptions than the non-gamers. The interaction effect was particularly enhanced in block 2 which indicated that action video gamers perform correct categorisations of exceptions earlier than non-gamers. They also categorised the exceptions better than the non-gamers at the beginning of each of the five blocks.

   The studies of Schenk and colleagues also showed gender and reaction time differences. The female gamers categorised better than other female non-gamers and male gamers categorised better than other non-gamers. However, female gamers categorised better than male gamers and female non-gamers performed better than male non-gamers. In the case of reaction times, reaction times decreased during the course of the experiment for both stimulus types and the largest difference was observed in block 3 onwards.  The subjects categorised the prototypical stimuli in total faster than exceptions.

   The results of the eye-tracking studies showed that in general there were higher fixation rates at the stimulus centre. At the beginning of the learning process, that is at the beginning of the experiment, data showed scattered fixations over the whole stimulus with more focused eye movements for the exceptions than for the prototype stimuli. At the end of the learning process, eye movements were more concentrated for both stimulus types and focussed on specific colour segments. Action video gamers exhibited less scattered and more centre-focused eye movements compared to non-gamers and this was particularly apparent in the last block of the experiment. Significant interaction between stimulus and block began at block 3.

   The results of the ANOVA investigation into the saccadic eye movements supported the findings of the fixation rates. The mean numbers of saccades per trial were negatively affected by increasing block number with some participants having no saccades during the first 800ms of stimulus presentation toward the end of the experiment. Schenk and colleagues also found increases in saccade latencies during the course of experiment supporting the reduced number of saccades performed.

    The analysis of the EEG results of the P150 amplitude showed that action video gamers (both male and female) generally exhibited higher P150 amplitudes than non-gamers (both male and females) for both stimulus types. The analysis of N170 amplitude gave a significant between-subject effect with the amplitude more negative for non-gamers than for action video gamers. The analysis also yielded a main effect for the factors electrodes with the PO7 and PO8 amplitudes more negative than the others. Different profiles were shown for both groups. Only for the non-gamers was there a significant effect for the factors electrodes. For the video gamers there were no significant main effects or interactions. The analysis of the N170 latency showed that the action video gamers had higher latencies on the right side and non-gamers almost significant higher latencies on the left. For the exceptions, non-gamers showed significant higher PO7 latency whereas action video gamers had higher latencies at the PO8 position for prototypical stimuli. Both the PO7 and PO8 latencies were shorter for the prototypical stimuli and the P8 and PO8 latencies were shorter for exceptions.

    Schenk and colleagues concluded their article with a discussion on their findings. They concluded that a learning process took place for the stimulus material by both groups of subjects. Both groups categorised the prototypical stimuli in earlier blocks and with faster reaction times than the exceptions. A change appeared to occur in the third block. For example; in the third block the number of correctly categorised exceptions increased; the difference in accuracy only became significant between the second and third block; and the highest difference between reaction times for the two stimulus types was detected in the third block. These results suggested that the observations were due to different learning strategies for the stimulus types (not demonstrated) or a need to explicitly remember exceptions.

   Further analyses showed that there were differences between the two groups for exceptions. The action video gamers showed better categorisation performance at the beginning of each block throughout the experiment and also demonstrated superior categorisation performance especially in the second and third block, although it became balanced towards the end. The non-video gamers needed more trials to correctly categorise the exceptions. Analysis of the fixation rates showed that they were not dependent on stimulus construction, but were associated with central or peripheral location of the stimulus segments. In the first block there were more fixations on the stimulus segments and in the last block more on the stimulus centre independent of subject group. The fixation rates decreased with increasing number of blocks for both groups, as did the number of saccades supported by increases in saccade latencies from the beginning to the end of the experiment. In the case of the exceptions, one or two segments that were decisive for the correct categorisation showed higher fixation rates at the end of the experiment.

  Schenk and colleagues concluded that both groups instigated a learning process regarding the stimulus material. It was possible that in the early learning phase, both groups tried to learn the stimuli based on their different colour features (showed more fixations on stimulus segments) and in later blocks, showed more fixations on the stimulus centres. This meant that they had learnt the stimulus structure and did not need to explore anymore, going directly for the ´change` site. In the case of the exceptions, only action video gamers showed higher fixation rates on the stimulus centre possibly indicating covert peripheral processing. (And the authors noted that this was the advantage of their experimental design and that this advantage might disappear if the critical features were indeed placed in the centre.) However, it was also proposed that the action video gamers could be faster in capturing visual information per se, or more adept at processing complex stimuli. This was supported by the studies on the ERP components P150 and N170 which reflected differential processing for the stimulus material of the two subject groups.

   Therefore, it was concluded that action video gamers have enhanced attentional and visual processing capabilities and non-gamers normal. The action video gamers show different stimulus exploration (more centre vs peripheral) and use an enhanced early perceptual analysis of the stimulus material and hence, may detect changes in objects faster and learn the belonging of the stimuli to their categories earlier. Schenk and colleagues findings support the work by others (West) that action video gamers show enhanced counting and remembering of specific sequences, features and locations which could be an advantage when learning the exceptions in Schenk`s visual categorisation task. The authors concluded that there were two areas worthy of further exploration: the first, testing with other types of video games; and secondly, more in-depth study of differences in gender with this type of learning.

COMMENT

   What makes this article interesting is that supports the view that cognitive skills gained from performing one task can be applied advantageously to other tasks that are not direct imitations of the performed one. In Schenk and team`s experiments, expertise in action video gaming gained from long-term practice proved a benefit to individuals in their performance of a categorisation task which involved visual input, working memory, short-term memory and hand-eye coordination. Therefore, it appears that action video gaming which can be a solo activity and can be performed anywhere (provided the computer hardware and software are available) may give individuals who have limited access to training opportunities in social settings the chance to maintain or restore various cognitive skills.

   The experimental set-up of Schenk and colleagues involved general skills in attention, working memory, short-term memory and decision-making and more task-specific skills related to visual input (colour and location) and visual search.  Some cognitive skills were not required such as those relating to emotions (value, empathy), other senses and information-type factors such as temporal order and movement. In general, it was found that both subject groups demonstrated similar performance regarding short-term memory and decision-making. For example, both groups showed earlier responses to discount prototypical stimuli than exceptions which implies that the routine was learnt, the decision-making method decided upon and the categorisation grouping recalled independent of whether the individuals had prior gaming experience or not. The third block of tests appeared to be the turning point for both groups and therefore, it is likely that the advantages of prior gaming experience did not lie with these particular cognitive skills. The areas where the action video gamers gained an advantage from their training appeared to be directly and mainly related to the visual processing and perception areas (eg. capable of identifying the key colour segment characteristic as being more centrally located, higher fixations on the stimuli to support short-term memory demands), which lead to the indirect advantages of faster speeds at categorising and category learning. This comment focusses on these types of systems and mechanisms where the gaming experience appears to give an advantage.

  We begin by hypothesising whether the hierarchical physiological structure of the visual input pathway itself is a key point to the training improvement. Since the experiment was associated with colour segment recognition, it can be assumed that the forward sweep of the incoming visual information occurred in all subjects within 100msecs of the stimulus onset and retinal cone firing. This would be followed by the relevant firing of the retinal bipolar cells with their on/off centres and surrounds leading to greater discrimination of the colour at the next stage involving firing of cells in the retinal ganglion layer. Here, 95% of the cells are P-type ganglion cells, responsible for shape determination although some are sensitive to wavelength. The other 5% of cells, the nonM-nonP ganglion cells are sensitive to wavelength and have the on/off centre and surround type structure. The response to one wavelength in the centre can be cancelled by the response to another wavelength in the surround and this is where the colour signal becomes established in a firing pattern. This colour signal is passed further up the visual pathway to the complex structure of the lateral geniculate nucleus (LGN) located in the dorsal thalamus. Information about colour is transferred through the activation of appropriate nonM-nonP ganglion cells of the six layer LGN structure to the tiny neurons of the koniocellular layers, which lie just ventral to each parvocellular layer. The receptive fields of the cells of the koniocellular layers are also of a centre-surround type structure and have either light/dark or colour opponency and it is these cells that define colour (the blob pathway). It should be noted that input from both eyes is kept separate in this area through the layering structure, but as far as colour is concerned there appears to be no binocularity disparity. It may be that shadow/contrast and the other qualities of colour, brightness and saturation, may be eye-dependent though.

    Projections from the LGN lead to the V1 visual cortex and then colour determination seems to follow the psychologists WHAT pathway (P pathway or temporal pathway), which takes the ventral route into the infero-temporal lobe via the cortical regions V2, V4, (V8) and area IT. A quarter of the complex V1 structure deals with information from the retinal fovea, ie. 25% of incoming information is about colour and firing follows a characteristic retinotopic colour map which is maintained as the colour pathway continues upwards via the ventral stream to areas V2 and V4. Further discrimination of the colour signal occurs at the area IT (in the interior temporal lobe). This area is known to be stimulated by a wide variety of colours and abstract shapes and is said to be important for both visual perception and visual memory. A small patch of the area IT is particularly responsive to faces with some faces being more effective stimuli than others. Therefore, since the categorisation test was performed by all individuals successfully then it is likely that the hierarchical physiological structure of the visual pathways of the individuals were the same independent of whether they had gaming experience or not and the visual information and colours were inputted and perceived by the system described above. Deficits in eye sight, or particular brain area deficiencies (eg. lesions of the visual cortex) would have had an effect on categorisation performance and these reasons can be excluded since the individuals that took part in Schenk and colleagues experiments were all described as healthy and had perfect eyesight.

   Therefore, at which points of the visual input and processing could the gamers gain an advantage? One such area could be the determination and appropriation of colour to the segments. This relies on an increased capability to recognise colour and depends on the concept of colour constancy. The feature of colour appears to have debatable value when action is the individual`s response to the visual input. A degree of memory involvement occurs in the assessment of colour as colour is considered constantand the perception of it appears to be reliant on visual pathway detection and on previous experience and expectation. Colour constancy, attributed to certain cells in the V4 and hence, dependent on an individual`s V4 performance, is where there is a tendency for a surface or object to appear to have the same colour even when there is a change in the wavelengths contained in the lighting source, eg. an object does not appear ´redder` when seen in artificial light. This is because colour constancy is found to be influenced by top-down processes (Bloj and the retinex theory of Land) where individuals use their own knowledge to interpret incoming visual stimuli and so colour is defined according to an individual`s own interpretation according to their own perceptions and experience and not the true colour determined by the colour`s physical wavelength. It was found that the colour constancy factor was responsible for global (compared to the whole visual scene) and more relevant here to local contrast, which involves comparing the retinal cone responses from the target surface with those of the immediate background (Kraft). This is likely to apply to the experiments of Schenk and team where the target colours would be compared to the neighbouring colours. Therefore, colour constancy and the personal definition of colour may be an area where the gamers have an advantage. Action video games are fast and rely on the gamers identifying shapes quickly. Therefore, the gamers may have developed cognitive skills in fast assessment of colour according to their own definitions (their own individual measurement of colour constancy). This capability of fast perception could be applied to the categorisation task and lead on to more efficient and accurate formation of short-term memories of the target colours of the visual stimuli presented in the experiments. 

   Another skill that relates to the visual target that is likely to be increased in gamers theoretically and was actually shown by Schenk and team`s experiments is the increase in control of eye movement. This is shown by the change in fixation rates observed in Schenk and team`s experiments for those subjects with prior gaming experience and the observation supports work by others who show that eye movements can enhance sensitivity to the target (Ennis). The association comes from the coupling between eye movement and deliberate goal-directed focus (Walcher). In Schenk`s experiments this means that eye movement control is associated with the establishment of target colour and manifests as fewer saccades and longer fixations. The experiments of Schenk and team`s were made slightly easier for subjects since the target colours were in the centre of the shape and hence, focus of the visual field and not in the peripheral regions. Hence, their determination was maximised since most retinal cones of the first stage of the visual pathway are situated in the central point of the lens at the fovea where the light rays pass in a straight line through from the lens to the retinal cell layer. Therefore, gamers with their prior experience of keeping targets centralised in the focus of the visual field will optimise the firing patterns of the presented stimuli (Carrillo-Reid) whereas non-gamers are likely to be less focussed (searching central and peripheral) until they have learnt where to concentrate their focus. The advantage of keeping the target in the focus of the visual field may be even more specific in that targeting might be dependent on only a small proportion of the total segment available. This is supported by visual search hypotheses, which say in a large stimulus, parts of the stimulus are processed quicker than the whole (Kinchla). Therefore, the gamers could specifically target not only the central segment where they know that the target colour changes are located, but narrow it down to even a smaller proportion of that target area to reduce processing load.

 A topic linked to eye movement and one that may play a role in the categorisation task given by Schenk and team is that of visuomotor responses. It is possible that through their prior gaming experience, the gamers have developed faster eye-hand coordination. Once the visual process is stopped, recognition is made and the decision taken, the end result is a motor movement consisting of a key press action. Therefore, efficient and quick eye-hand coordination may extend the gamer`s visual advantage, ie. will process faster and carry out the required response faster. This improved eye-hand coordination may occur via strengthened connectivity between multiple cortico-cortical and cortico-subcortical frontal cortex networks (Brovelli) and the temporal locking of firing oscillations of the early visual processing with the early motor planning controlling the execution of the hand response (Tomassini). Therefore, training may give the gamers an advantage by speedier movements once the decision has been made.

   Gamers may also have an advantage in Schenk and team`s experiments by being able to maintain concentration and process relevant information faster during the learning phase and then shifting this to non-conscious processing at a later stage, albeit faster than the non-gamers. This type of capability would be gained by playing many hours of action video games where fast changing situations are constantly being presented and decisions have to be made. Gamers then when presented with a simpler categorisation task are likely to shift to non-conscious processing at an earlier stage than non-gamers and since non-conscious processing is faster than conscious, then response times of the gamers would be quicker. Conscious information processing which occurs early on in the trial blocks involves a number of skills relating to awareness of what is required and this conscious awareness is demonstrated by the N170 component (Thierry). Two cognitive processes come into play, working memory and attention, and these capabilities would have been honed to cope with the task demands by the prior gaming experience which could be considered as priming. From a physical perspective, priming would help the individual to target specifically (ie. only a proportion of the colour segment in the visual search part of the task) leading to improved cue utilisation for example. A greater level of cue utilisation leads to consistently greater response latencies consistent with strategies that maintain accuracy, but reduce the demands on cognitive resources (Brouwers). This means that priming would positively affect demands on attention and information processing relating to working memory performance.

   With regards to attention, the high level of conscious awareness particularly at the beginning of the trial blocks leads to higher levels of top-down attention which is likely to result in strong alpha brain wave activity in the fronto-parietal network (Van Schouwenburg). Various areas are responsible in the selection and maintenance of attention on stimuli eg. there is co-activation of neurons within 50-200ms across the anterior cingulate cortex and prefrontal cortex during stimulus selection in a spatial attention task (Oemisch); and firing in the ventral intraparietal sulcus maintains attention to a specific location (Capotosto). However, experience (ie. by prior gaming experience) would affect how targets are consciously selected and attentional selection would respond. This is supported by Corradi-Dell´Acqua who showed that the ability to select, within the complexity of sensory input, the information most relevant for the purpose of the task was influenced by both internal settings (ie. top-down control and conscious awareness) and the relevant features of the external stimuli (ie. bottom-up control and visual input). This capability to switch to relevant information comes from firing of the areas involved in top-down control (frontal, parietal and sensory cortices) and the lateral intraparietal neurons (Kumano). Lateral intraparietal neurons appear to accumulate relevant information depending on context to decide which eye movements to carry out to maximise it. The selection of the target would imply attended stimuli and this would enhance the evoked firing potentials for these stimuli in comparison to non-attended (Andersen). The effect of increased firing with reference to Schenk and team`s experiments is that colour (the target characteristic) would exhibit general sharpened selectivity by initial gain at the V1 level and within 100msec a sharper tuned profile in the posterior ventral cortex (Bartsch).

   This increased attention and selectivity of target afforded by conscious awareness and the ability of gamers to know faster what is required leads to working memory performance gain. Gaming experience, if considered as priming, would bring about benefits to working memory as seen through pre-stimulus alpha brain wave activity (Myers) and theta-band oscillations representing multiple predictions that are dynamically coordinated in time (Huang). The training of the individuals although unlikely to produce long-term changes in the physiological structure of the visual pathway are likely to induce changes to the physiological structures involved in working memory if they follow the pattern of other cases where training has been carried out prior to testing. For example, Caeyenberghs has reported that adaptive working memory training led to improvement on non-training related working memory tasks and tasks of reasoning and inhibition and these improvements were related to increased global integration within the fronto-parietal attention network observed by increased structural white matter network connectivity. Astle also reported that in children, after training, connectivity between the fronto-parietal networks and both lateral occipital complex and inferior temporal cortex areas exhibited increased strength of connectivity at rest. These were mirrored by an increased working memory performance.

    The problem with working memory is that it is of a limited capacity with regards to maintaining and manipulating the objects held in it (Myers) with resolution decreasing as the number of items increases above 3 (Anderson). This is in spite of the work load being spread over different areas (eg. specific brain areas have particular working memory functions such as post parietal cortex – manipulation of information and item maintenance; lateral occipital cortex – item maintenance and even different areas of the prefrontal cortex, with ventrolateral prefrontal cortex involved in face and vocal information; IT, V4 with visual information – Plakke). Therefore, conscious awareness and attention focused on relevant and targeted material aids the overall performance of working memory. The characteristics featured in object based attention are present in the working memory (Peters) and are strengthened (Soto) and sharpened by this overlapping (Lim). Therefore, attention selectively updates and maintains the relevant material (Blauracke, Heuer) required for the task at hand. This would mean that although both gamers and non-gamers would employ the same working memory processes the degree of efficiency of the former would be higher at least in the earlier trial blocks.

    Therefore, we can see that several areas of visual input and processing may be advantageously changed by training involving playing action video games. The value of playing action video games by increasing the selection of relevant material, maintaining it in working memory and then carrying out a response of a planned motor movement is significant for circumstances where these cognitive skills need to be maintained or improved and where the individual`s environment presents it as a valid learning option. The proviso is that the game used has to be challenging, capable of repetition without loss of motivation, set-up to give feedback preferably immediately and relevant to the required cognitive skill.

Since we`re talking about the topic…………………..

            …… achromatopsia sufferers have damage in the V2, V3 and V4 areas and therefore, can process information about colour implicitly but are unable to use the information explicitly when the judgement concerns colour. Therefore, if Schenk and team`s experiments were repeated would the advantages of the gamers be negated?

            …… serotonin administration causes a mainly multiplicative decrease of visual responses and a slight increase in stimulus-selective response latency (Seillier). Can we assume that if serotonin was administered prior to the test, both groups would suffer a loss in performance, but the gamers would still show the training advantage?

            …..……….mood is reported to have an effect on working memory performance with positive mood enhancing verbal and impairing spatial working memory, whereas negative mood enhances spatial and impairs verbal working memory (Storbeck). If Schenk and team`s experiments were repeated with anxiety levels increased for both groups, would we see any change in performance of the two groups in general particularly at block 3, the turning point between learning and automatic response?

action video game experience linked to improved attention driven perceptual exploration in categorisation learning

Posted comment on ´Play to win: action video game experience and attention driven perceptual exploration in categorisation learning` written by S. Schenk, C. Bellebaum, R.K. Lech, R. Heinen and B. Suchan and published in Frontiers in Psychology 13^th May 2020 doi 10.3389/fpsyg.2020.00933

SUMMARY

   Schenk and colleagues investigated the difference between action video gamers and non-gamers in visual exploration and attention driven perception during a categorisation learning task. Their subjects, 17 right-handed non-gamers (mean age nearly 23, nearly all female) and 16 action video gamers (mean age nearly 24, nearly all male) performed 490 random trials (5 blocks of 98) of a visual categorisation task. This consisted of distinguishing 14 ring stimuli which had the same structure, but different colour combinations. These had to be divided into 2 categories, seven in each and made up of a prototype, 5 typical stimuli and one exception. The typical stimuli shared 5 colour features with the prototype of the category, whereas the exception shared five colour features with the prototype of the other category.  The subjects did not know of the existence of the exceptions and had to first work out that they were present and then had to explicitly remember both exceptions in order to categorise all the stimuli presented. The stimuli were presented and the response made by pressing the left and right control keys designated for the two categories. The key press was followed by an immediate feedback of right or wrong. The subjects were required to react within 1.8 secs of stimulus presentation and failure to do so resulted in a verbal warning to quicken response time. Following the feedback the subjects were presented with a fixation cross for 1-2 secs before being allowed a short break before the next trial started.  EEG recordings were taken during the experiment and eye-tracking recordings of the subject`s right eye were also made during the stimulus presentation.  

   Data analyses were performed on the recordings. In the case of the EEG recordings, the datasets were analysed with software packages. Blink artefacts and vertical eye movements were removed from the EEG data and components reflecting eye movements were removed from the EEG signal. The recordings for the prototypes and typical stimuli were combined because of their similarity (prototypical stimuli) and the recordings were adjusted so that the segments for both stimulus types (prototypical stimuli and exceptions) were followed by a baseline correction relative to the 200ms preceding the stimulus presentation. In the case of the P150 ERP component, data from the parietal-occipital electrodes was recorded and the amplitude was taken as that within 120-180ms after stimulus presentation. The maximum negative peak amplitude within the time frame 150-190ms after stimulus presentation was taken and defined as the Peaks of the N170 amplitude. The analysis of the N170 component was carried out using different electrodes to those of the P150.

    The eye-tracking data was analysed using appropriate software. Fixations were visualised on the basis of heat maps with the ring stimuli divided into areas. Percentages of the fixation rates and number of fixations were calculated for all areas of the ring and for the centre and the differences between prototypical and exceptions calculated. Eye movements (taken as changes in velocity of the eye position exceeding 40 degrees per second and displacement of at least 1.5 degree visual angle) were also analysed with saccades detected within 800ms of the presentation period. Statistical analyses of the various sets of behaviour data were carried out using ANOVA with repeated measures (including Greenhouse-Geisser procedure and Bonferroni), paired T test and G Power.

   The results of Schenk and colleagues experiments showed that the learning process relied on the immediate feedback of right or wrong response. The ANOVA analysis carried out for the percentage of correct responses yielded significant main effects for both the factors ´stimulus` and ´block` as well as showing significant interaction. The pairwise comparisons revealed for both groups more correct categorisations in the last blocks, especially for the prototypical stimulus. Using the paired t-test it was shown that interaction was based on a high number of correctly categorised prototypical stimuli compared to a low number of correctly categorised exceptions at the early stage of the experiment. A high increase in correctly categorised exceptions in block 3 showed that the successful categorisation of the exceptions occurred later. The differences between the blocks between percentage correct responses for exceptions and prototypical stimuli were shown by paired t tests to yield significance between block 2 and 3. All other results showed no significant differences.

    The results from the ANOVA analysis of behavioural data of the two groups showed a significant interaction between ´stimulus` and ´group`. This showed that action video gamers had superior categorisation performance for the exceptions than the non-gamers. The interaction effect was particularly enhanced in block 2 which indicated that action video gamers perform correct categorisations of exceptions earlier than non-gamers. They also categorised the exceptions better than the non-gamers at the beginning of each of the five blocks.

   The studies of Schenk and colleagues also showed gender and reaction time differences. The female gamers categorised better than other female non-gamers and male gamers categorised better than other non-gamers. However, female gamers categorised better than male gamers and female non-gamers performed better than male non-gamers. In the case of reaction times, reaction times decreased during the course of the experiment for both stimulus types and the largest difference was observed in block 3 onwards.  The subjects categorised the prototypical stimuli in total faster than exceptions.

   The results of the eye-tracking studies showed that in general there were higher fixation rates at the stimulus centre. At the beginning of the learning process, that is at the beginning of the experiment, data showed scattered fixations over the whole stimulus with more focused eye movements for the exceptions than for the prototype stimuli. At the end of the learning process, eye movements were more concentrated for both stimulus types and focussed on specific colour segments. Action video gamers exhibited less scattered and more centre-focused eye movements compared to non-gamers and this was particularly apparent in the last block of the experiment. Significant interaction between stimulus and block began at block 3.

   The results of the ANOVA investigation into the saccadic eye movements supported the findings of the fixation rates. The mean numbers of saccades per trial were negatively affected by increasing block number with some participants having no saccades during the first 800ms of stimulus presentation toward the end of the experiment. Schenk and colleagues also found increases in saccade latencies during the course of experiment supporting the reduced number of saccades performed.

    The analysis of the EEG results of the P150 amplitude showed that action video gamers (both male and female) generally exhibited higher P150 amplitudes than non-gamers (both male and females) for both stimulus types. The analysis of N170 amplitude gave a significant between-subject effect with the amplitude more negative for non-gamers than for action video gamers. The analysis also yielded a main effect for the factors electrodes with the PO7 and PO8 amplitudes more negative than the others. Different profiles were shown for both groups. Only for the non-gamers was there a significant effect for the factors electrodes. For the video gamers there were no significant main effects or interactions. The analysis of the N170 latency showed that the action video gamers had higher latencies on the right side and non-gamers almost significant higher latencies on the left. For the exceptions, non-gamers showed significant higher PO7 latency whereas action video gamers had higher latencies at the PO8 position for prototypical stimuli. Both the PO7 and PO8 latencies were shorter for the prototypical stimuli and the P8 and PO8 latencies were shorter for exceptions.

    Schenk and colleagues concluded their article with a discussion on their findings. They concluded that a learning process took place for the stimulus material by both groups of subjects. Both groups categorised the prototypical stimuli in earlier blocks and with faster reaction times than the exceptions. A change appeared to occur in the third block. For example; in the third block the number of correctly categorised exceptions increased; the difference in accuracy only became significant between the second and third block; and the highest difference between reaction times for the two stimulus types was detected in the third block. These results suggested that the observations were due to different learning strategies for the stimulus types (not demonstrated) or a need to explicitly remember exceptions.

   Further analyses showed that there were differences between the two groups for exceptions. The action video gamers showed better categorisation performance at the beginning of each block throughout the experiment and also demonstrated superior categorisation performance especially in the second and third block, although it became balanced towards the end. The non-video gamers needed more trials to correctly categorise the exceptions. Analysis of the fixation rates showed that they were not dependent on stimulus construction, but were associated with central or peripheral location of the stimulus segments. In the first block there were more fixations on the stimulus segments and in the last block more on the stimulus centre independent of subject group. The fixation rates decreased with increasing number of blocks for both groups, as did the number of saccades supported by increases in saccade latencies from the beginning to the end of the experiment. In the case of the exceptions, one or two segments that were decisive for the correct categorisation showed higher fixation rates at the end of the experiment.

  Schenk and colleagues concluded that both groups instigated a learning process regarding the stimulus material. It was possible that in the early learning phase, both groups tried to learn the stimuli based on their different colour features (showed more fixations on stimulus segments) and in later blocks, showed more fixations on the stimulus centres. This meant that they had learnt the stimulus structure and did not need to explore anymore, going directly for the ´change` site. In the case of the exceptions, only action video gamers showed higher fixation rates on the stimulus centre possibly indicating covert peripheral processing. (And the authors noted that this was the advantage of their experimental design and that this advantage might disappear if the critical features were indeed placed in the centre.) However, it was also proposed that the action video gamers could be faster in capturing visual information per se, or more adept at processing complex stimuli. This was supported by the studies on the ERP components P150 and N170 which reflected differential processing for the stimulus material of the two subject groups.

   Therefore, it was concluded that action video gamers have enhanced attentional and visual processing capabilities and non-gamers normal. The action video gamers show different stimulus exploration (more centre vs peripheral) and use an enhanced early perceptual analysis of the stimulus material and hence, may detect changes in objects faster and learn the belonging of the stimuli to their categories earlier. Schenk and colleagues findings support the work by others (West) that action video gamers show enhanced counting and remembering of specific sequences, features and locations which could be an advantage when learning the exceptions in Schenk`s visual categorisation task. The authors concluded that there were two areas worthy of further exploration: the first, testing with other types of video games; and secondly, more in-depth study of differences in gender with this type of learning.

COMMENT

   What makes this article interesting is that supports the view that cognitive skills gained from performing one task can be applied advantageously to other tasks that are not direct imitations of the performed one. In Schenk and team`s experiments, expertise in action video gaming gained from long-term practice proved a benefit to individuals in their performance of a categorisation task which involved visual input, working memory, short-term memory and hand-eye coordination. Therefore, it appears that action video gaming which can be a solo activity and can be performed anywhere (provided the computer hardware and software are available) may give individuals who have limited access to training opportunities in social settings the chance to maintain or restore various cognitive skills.

   The experimental set-up of Schenk and colleagues involved general skills in attention, working memory, short-term memory and decision-making and more task-specific skills related to visual input (colour and location) and visual search.  Some cognitive skills were not required such as those relating to emotions (value, empathy), other senses and information-type factors such as temporal order and movement. In general, it was found that both subject groups demonstrated similar performance regarding short-term memory and decision-making. For example, both groups showed earlier responses to discount prototypical stimuli than exceptions which implies that the routine was learnt, the decision-making method decided upon and the categorisation grouping recalled independent of whether the individuals had prior gaming experience or not. The third block of tests appeared to be the turning point for both groups and therefore, it is likely that the advantages of prior gaming experience did not lie with these particular cognitive skills. The areas where the action video gamers gained an advantage from their training appeared to be directly and mainly related to the visual processing and perception areas (eg. capable of identifying the key colour segment characteristic as being more centrally located, higher fixations on the stimuli to support short-term memory demands), which lead to the indirect advantages of faster speeds at categorising and category learning. This comment focusses on these types of systems and mechanisms where the gaming experience appears to give an advantage.

  We begin by hypothesising whether the hierarchical physiological structure of the visual input pathway itself is a key point to the training improvement. Since the experiment was associated with colour segment recognition, it can be assumed that the forward sweep of the incoming visual information occurred in all subjects within 100msecs of the stimulus onset and retinal cone firing. This would be followed by the relevant firing of the retinal bipolar cells with their on/off centres and surrounds leading to greater discrimination of the colour at the next stage involving firing of cells in the retinal ganglion layer. Here, 95% of the cells are P-type ganglion cells, responsible for shape determination although some are sensitive to wavelength. The other 5% of cells, the nonM-nonP ganglion cells are sensitive to wavelength and have the on/off centre and surround type structure. The response to one wavelength in the centre can be cancelled by the response to another wavelength in the surround and this is where the colour signal becomes established in a firing pattern. This colour signal is passed further up the visual pathway to the complex structure of the lateral geniculate nucleus (LGN) located in the dorsal thalamus. Information about colour is transferred through the activation of appropriate nonM-nonP ganglion cells of the six layer LGN structure to the tiny neurons of the koniocellular layers, which lie just ventral to each parvocellular layer. The receptive fields of the cells of the koniocellular layers are also of a centre-surround type structure and have either light/dark or colour opponency and it is these cells that define colour (the blob pathway). It should be noted that input from both eyes is kept separate in this area through the layering structure, but as far as colour is concerned there appears to be no binocularity disparity. It may be that shadow/contrast and the other qualities of colour, brightness and saturation, may be eye-dependent though.

    Projections from the LGN lead to the V1 visual cortex and then colour determination seems to follow the psychologists WHAT pathway (P pathway or temporal pathway), which takes the ventral route into the infero-temporal lobe via the cortical regions V2, V4, (V8) and area IT. A quarter of the complex V1 structure deals with information from the retinal fovea, ie. 25% of incoming information is about colour and firing follows a characteristic retinotopic colour map which is maintained as the colour pathway continues upwards via the ventral stream to areas V2 and V4. Further discrimination of the colour signal occurs at the area IT (in the interior temporal lobe). This area is known to be stimulated by a wide variety of colours and abstract shapes and is said to be important for both visual perception and visual memory. A small patch of the area IT is particularly responsive to faces with some faces being more effective stimuli than others. Therefore, since the categorisation test was performed by all individuals successfully then it is likely that the hierarchical physiological structure of the visual pathways of the individuals were the same independent of whether they had gaming experience or not and the visual information and colours were inputted and perceived by the system described above. Deficits in eye sight, or particular brain area deficiencies (eg. lesions of the visual cortex) would have had an effect on categorisation performance and these reasons can be excluded since the individuals that took part in Schenk and colleagues experiments were all described as healthy and had perfect eyesight.

   Therefore, at which points of the visual input and processing could the gamers gain an advantage? One such area could be the determination and appropriation of colour to the segments. This relies on an increased capability to recognise colour and depends on the concept of colour constancy. The feature of colour appears to have debatable value when action is the individual`s response to the visual input. A degree of memory involvement occurs in the assessment of colour as colour is considered constantand the perception of it appears to be reliant on visual pathway detection and on previous experience and expectation. Colour constancy, attributed to certain cells in the V4 and hence, dependent on an individual`s V4 performance, is where there is a tendency for a surface or object to appear to have the same colour even when there is a change in the wavelengths contained in the lighting source, eg. an object does not appear ´redder` when seen in artificial light. This is because colour constancy is found to be influenced by top-down processes (Bloj and the retinex theory of Land) where individuals use their own knowledge to interpret incoming visual stimuli and so colour is defined according to an individual`s own interpretation according to their own perceptions and experience and not the true colour determined by the colour`s physical wavelength. It was found that the colour constancy factor was responsible for global (compared to the whole visual scene) and more relevant here to local contrast, which involves comparing the retinal cone responses from the target surface with those of the immediate background (Kraft). This is likely to apply to the experiments of Schenk and team where the target colours would be compared to the neighbouring colours. Therefore, colour constancy and the personal definition of colour may be an area where the gamers have an advantage. Action video games are fast and rely on the gamers identifying shapes quickly. Therefore, the gamers may have developed cognitive skills in fast assessment of colour according to their own definitions (their own individual measurement of colour constancy). This capability of fast perception could be applied to the categorisation task and lead on to more efficient and accurate formation of short-term memories of the target colours of the visual stimuli presented in the experiments. 

   Another skill that relates to the visual target that is likely to be increased in gamers theoretically and was actually shown by Schenk and team`s experiments is the increase in control of eye movement. This is shown by the change in fixation rates observed in Schenk and team`s experiments for those subjects with prior gaming experience and the observation supports work by others who show that eye movements can enhance sensitivity to the target (Ennis). The association comes from the coupling between eye movement and deliberate goal-directed focus (Walcher). In Schenk`s experiments this means that eye movement control is associated with the establishment of target colour and manifests as fewer saccades and longer fixations. The experiments of Schenk and team`s were made slightly easier for subjects since the target colours were in the centre of the shape and hence, focus of the visual field and not in the peripheral regions. Hence, their determination was maximised since most retinal cones of the first stage of the visual pathway are situated in the central point of the lens at the fovea where the light rays pass in a straight line through from the lens to the retinal cell layer. Therefore, gamers with their prior experience of keeping targets centralised in the focus of the visual field will optimise the firing patterns of the presented stimuli (Carrillo-Reid) whereas non-gamers are likely to be less focussed (searching central and peripheral) until they have learnt where to concentrate their focus. The advantage of keeping the target in the focus of the visual field may be even more specific in that targeting might be dependent on only a small proportion of the total segment available. This is supported by visual search hypotheses, which say in a large stimulus, parts of the stimulus are processed quicker than the whole (Kinchla). Therefore, the gamers could specifically target not only the central segment where they know that the target colour changes are located, but narrow it down to even a smaller proportion of that target area to reduce processing load.

 A topic linked to eye movement and one that may play a role in the categorisation task given by Schenk and team is that of visuomotor responses. It is possible that through their prior gaming experience, the gamers have developed faster eye-hand coordination. Once the visual process is stopped, recognition is made and the decision taken, the end result is a motor movement consisting of a key press action. Therefore, efficient and quick eye-hand coordination may extend the gamer`s visual advantage, ie. will process faster and carry out the required response faster. This improved eye-hand coordination may occur via strengthened connectivity between multiple cortico-cortical and cortico-subcortical frontal cortex networks (Brovelli) and the temporal locking of firing oscillations of the early visual processing with the early motor planning controlling the execution of the hand response (Tomassini). Therefore, training may give the gamers an advantage by speedier movements once the decision has been made.

   Gamers may also have an advantage in Schenk and team`s experiments by being able to maintain concentration and process relevant information faster during the learning phase and then shifting this to non-conscious processing at a later stage, albeit faster than the non-gamers. This type of capability would be gained by playing many hours of action video games where fast changing situations are constantly being presented and decisions have to be made. Gamers then when presented with a simpler categorisation task are likely to shift to non-conscious processing at an earlier stage than non-gamers and since non-conscious processing is faster than conscious, then response times of the gamers would be quicker. Conscious information processing which occurs early on in the trial blocks involves a number of skills relating to awareness of what is required and this conscious awareness is demonstrated by the N170 component (Thierry). Two cognitive processes come into play, working memory and attention, and these capabilities would have been honed to cope with the task demands by the prior gaming experience which could be considered as priming. From a physical perspective, priming would help the individual to target specifically (ie. only a proportion of the colour segment in the visual search part of the task) leading to improved cue utilisation for example. A greater level of cue utilisation leads to consistently greater response latencies consistent with strategies that maintain accuracy, but reduce the demands on cognitive resources (Brouwers). This means that priming would positively affect demands on attention and information processing relating to working memory performance.

   With regards to attention, the high level of conscious awareness particularly at the beginning of the trial blocks leads to higher levels of top-down attention which is likely to result in strong alpha brain wave activity in the fronto-parietal network (Van Schouwenburg). Various areas are responsible in the selection and maintenance of attention on stimuli eg. there is co-activation of neurons within 50-200ms across the anterior cingulate cortex and prefrontal cortex during stimulus selection in a spatial attention task (Oemisch); and firing in the ventral intraparietal sulcus maintains attention to a specific location (Capotosto). However, experience (ie. by prior gaming experience) would affect how targets are consciously selected and attentional selection would respond. This is supported by Corradi-Dell´Acqua who showed that the ability to select, within the complexity of sensory input, the information most relevant for the purpose of the task was influenced by both internal settings (ie. top-down control and conscious awareness) and the relevant features of the external stimuli (ie. bottom-up control and visual input). This capability to switch to relevant information comes from firing of the areas involved in top-down control (frontal, parietal and sensory cortices) and the lateral intraparietal neurons (Kumano). Lateral intraparietal neurons appear to accumulate relevant information depending on context to decide which eye movements to carry out to maximise it. The selection of the target would imply attended stimuli and this would enhance the evoked firing potentials for these stimuli in comparison to non-attended (Andersen). The effect of increased firing with reference to Schenk and team`s experiments is that colour (the target characteristic) would exhibit general sharpened selectivity by initial gain at the V1 level and within 100msec a sharper tuned profile in the posterior ventral cortex (Bartsch).

   This increased attention and selectivity of target afforded by conscious awareness and the ability of gamers to know faster what is required leads to working memory performance gain. Gaming experience, if considered as priming, would bring about benefits to working memory as seen through pre-stimulus alpha brain wave activity (Myers) and theta-band oscillations representing multiple predictions that are dynamically coordinated in time (Huang). The training of the individuals although unlikely to produce long-term changes in the physiological structure of the visual pathway are likely to induce changes to the physiological structures involved in working memory if they follow the pattern of other cases where training has been carried out prior to testing. For example, Caeyenberghs has reported that adaptive working memory training led to improvement on non-training related working memory tasks and tasks of reasoning and inhibition and these improvements were related to increased global integration within the fronto-parietal attention network observed by increased structural white matter network connectivity. Astle also reported that in children, after training, connectivity between the fronto-parietal networks and both lateral occipital complex and inferior temporal cortex areas exhibited increased strength of connectivity at rest. These were mirrored by an increased working memory performance.

    The problem with working memory is that it is of a limited capacity with regards to maintaining and manipulating the objects held in it (Myers) with resolution decreasing as the number of items increases above 3 (Anderson). This is in spite of the work load being spread over different areas (eg. specific brain areas have particular working memory functions such as post parietal cortex – manipulation of information and item maintenance; lateral occipital cortex – item maintenance and even different areas of the prefrontal cortex, with ventrolateral prefrontal cortex involved in face and vocal information; IT, V4 with visual information – Plakke). Therefore, conscious awareness and attention focused on relevant and targeted material aids the overall performance of working memory. The characteristics featured in object based attention are present in the working memory (Peters) and are strengthened (Soto) and sharpened by this overlapping (Lim). Therefore, attention selectively updates and maintains the relevant material (Blauracke, Heuer) required for the task at hand. This would mean that although both gamers and non-gamers would employ the same working memory processes the degree of efficiency of the former would be higher at least in the earlier trial blocks.

    Therefore, we can see that several areas of visual input and processing may be advantageously changed by training involving playing action video games. The value of playing action video games by increasing the selection of relevant material, maintaining it in working memory and then carrying out a response of a planned motor movement is significant for circumstances where these cognitive skills need to be maintained or improved and where the individual`s environment presents it as a valid learning option. The proviso is that the game used has to be challenging, capable of repetition without loss of motivation, set-up to give feedback preferably immediately and relevant to the required cognitive skill.

Since we`re talking about the topic…………………..

            …… achromatopsia sufferers have damage in the V2, V3 and V4 areas and therefore, can process information about colour implicitly but are unable to use the information explicitly when the judgement concerns colour. Therefore, if Schenk and team`s experiments were repeated would the advantages of the gamers be negated?

            …… serotonin administration causes a mainly multiplicative decrease of visual responses and a slight increase in stimulus-selective response latency (Seillier). Can we assume that if serotonin was administered prior to the test, both groups would suffer a loss in performance, but the gamers would still show the training advantage?

            …..……….mood is reported to have an effect on working memory performance with positive mood enhancing verbal and impairing spatial working memory, whereas negative mood enhances spatial and impairs verbal working memory (Storbeck). If Schenk and team`s experiments were repeated with anxiety levels increased for both groups, would we see any change in performance of the two groups in general particularly at block 3, the turning point between learning and automatic response?

complexity of area-specific synapse structure in branched posterior nucleus axons

Posted comment on ´Area-specific synapse structure in branched posterior nucleus axons reveals a new level of complexity in thalamocortical networks` written by J. Rodriguez-Moreno, C. Porrero, A. Rollenhagen, M. Rubio-Teves, D. Casas-Torremocha, L. Alonso-Nanclares, R. Yakoubi, A. Santuy, A. Merchan-Perez, J. DeFelipe, J.H.R. Luebke and F. Clasca and published in Journal of Neuroscience 2020 40(13) p. 2663 doi 10.1523/JNEUROSCI.2886-19.2020

SUMMARY

Rodriguez-Moreno and colleagues described in their article their results from their investigation into the synapse structures of two specific synaptic areas, that of the rat thalamocortical posterior nucleus (Po) branched axons innervating both the somatosensory cortex (S1) and motor cortex (MC) linked with snout whisker (vibrissae) movement associated with environment exploration.

Using male C57BL/6 mice, BDA was iontophoretically injected to selectively label the Po axons. Five days later, tissues were prepared according to the imaging technique to be used and samples of the regions of interest were sliced and identified by appropriate staining. Light microscopy and fine-scale 3D-electron microscopy (serial sectioning transmission electron microscopy, ssTEM and focused ion beam milling scanning electron microscopy, FIB-SEM) were carried out and software programmes used to reconstruct the images and provide measurements. In order to directly visualise the complete axonal tree of an individual Po neuron, the isolated Po neurons were transfected by in vivo electroporation or a RNA construct designed to drive expression of the enhanced green fluorescent protein eGFP fused with a distinctive motif from the growth-associated protein 43(GAP43) under the promoter Sind-Pal-eGFP. Appropriate microscopy, imagings and statistical analyses were carried out on the samples taken.

The first set of results using light microscopy showed the Po axon terminals in the S1 and MC. Two distinct bands of axonal arborisations were visualised in the S1, one in the L5a and other in the upper half of the L1 whereas in the MC only a single band was visualised from the upper L5a to the lower L3. Gogli-like staining showed frequent varicosities of variable size.

The second set of results involved ssTEM and FIB-SEM of the heaviest anterograde labelled thalamocortical Po axons of both the S1 and MC.  S1 samples were taken from the L1 and L5a since they are readily delineated. The MC samples included the L4 and adjacent deep parts of the L3 since these MC regions are not so readily delineated and therefore, the samples were termed MC-L4/3. The analysis of ssTEM images gave a high resolution 3D volume reconstruction of the overall geometry of the synaptic complexes. Subsequent quantification of post-synaptic density (PSD) surface area and vesicle size could be carried out, but was limited in that it only sampled axon varicosities. Individual synaptic vesicles and other fine details could be clearly observed and counted. FIB-SEM analysis instead, because of it slightly lower resolution, allowed complete visualisation of post-synaptic elements and inter-bouton axonal segments.

The third set of results indicated the ultrastructural features of the Po axons in the S1 and MC regions. The majority of boutons/varicosities (termed together to describe the axonal swellings of greater than 0.5micrometres or exceeding 50% of the typical variation of the adjacent axonal segments) were synaptic sites (93-98%) as defined by being associated to at least one PSD. Most of them (88-92%) contained at least one mitochondrion. Few boutons were non-synaptic and these were swollen axon regions each containing one mitochondrion and some presynaptic vesicles.  A total of 220 Po synapses were established by labelled Po axons and their respective target structures reconstructed and analysed by ssTEM and FIB-SEM techniques. Synaptic contacts were defined as asymmetric, excitatory and glutamatergic and were indicated by the presence of distinct, parallel presynaptic and post-synaptic membranes separated by the synaptic cleft and an electron-dense band adherent to the cytoplasmic surface of the PSD. A total of 192 axon boutons were constructed and analysed with ssTEM or FIB-SEM. Most (70-90%) were found to be monosynaptic. There were also synapses (25% – identified by FIB-SEM) in the non-varicose inter-bouton segments which were always monosynaptic. Approx. 30% of boutons in the MC-L4/3 and approx. 10% in the S1 were in contact with two different post-synaptic structures, but the expected Po boutons with 3 or more synapses common in the VPM S1-L4 were not observed. There were few non-varicose synaptic sites (approx. 5%) in the VPM. Most Po boutons contained one or several mitochondria (88-92%) of different shapes and sizes and these contributed to the bouton volume substantially (25% in S1-L5a; 16% in S1-L1; and 20.5% in MC-L4/3). It was also possible to distinguish the synaptic vesicles. In both S1-L1 and S1-L5a, about 7% of Po axonal boutons contained a mitochondrion and some synaptic vesicles, but had no synaptic contact whereas only 2% existed in the MC-L4/3 Po axons. These are not observed in the VPM S1-L4 axons. FIB-SEM analysis of a few samples was possible (26 Po axonal segments – approx. 283micrometres axonal length from a sampled area of 2435cubic micrometres). The analysis to determine synaptic structure features produced consistent results between the FIB-SEM and ssTEM. Some boutons contained a mitochondrion, but were not associated with a PSD. About 25% of synapses occurred in long inter-bouton axonal segments whereas few synapses are said to be observed in the non-varicose segments of the VPM S1-L4 region.

The fourth set of results recorded by Rodriguez-Moreno and team related to the ultrastructural features of elements post-synaptic to the Po axons in both the MC and S1 regions. A majority of synapses (83-96%) were found on the dendritic spines in all three. Synapses visualised on the shafts were attributed by the authors to be non-spiny cortical interneurons and these were less frequent on the S1-L5a (17%) and even less on the S1-L1 and MC-L4/3 (4-6%). No synapses were visualised on the soma. The PSDs were also observed as being of different sizes and shapes. The mean PSD surface area of the S1-L5a was similar to the VPM-L4 synapses at approx. 0.11square micrometres whereas the PSD surface area of the MC-L4/3 and S1-L1 regions were 60% larger. Morphology also differed between the areas with 65% of PSDs on the S1-L5a having a disc-like morphology whereas most MC-L4/3 (59%) were complex horseshoe shaped, perforated or fragmented. About 9% of the Po bouton post-synapses on spines in MC-L4/3 region, about 3% in S1-L1 and 18% in S1-L5 were unlabelled and symmetric and were possibly inhibitory synapses of unknown origins. The majority of post-synaptic spines in the MC-L4/3 (70%) had spine apparatus (an endoplasmic reticulum derivative responsible for spine motility and stabilisation of the presynaptic and post-synaptic apposition zone during synaptic transmission) compared to 30% in Po bouton post-synapses of the S1 (L5a and L1). Mitochondria were also observed in 2 out of 68 spines of the S1-L1 region, although these are not reported in rat somatosensory cortex.

Further investigation of the ultrastructural features of the post-synaptic regions showed that dendritic spine heads post-synaptic to the Po boutons appeared as having thick finger like protrusions embedded into the presynaptic thalamocortical Po boutons. (These appear as a common feature of mammalian thalamocortical synapses on spiny stellate and pyramidal cells.) The protrusions were more frequent in the MC-L4 (13-19%) than in the S1 with S1-L4 VPM synapses appearing to be at same level as that observed for the MC. These protrusions were seen to be located in spines with widely different PSD sizes, but were not found near those of less than 0.05square micrometres in size. The spine PSDs were always external to the protrusions and their edges, but could be adjacent to them. The invaginated inter-membrane surface was found to be large at almost 20 times the size of the active zone. They were found to be smooth, closely apposed, lacked membrane specialisations and probably allowed non-linear diffusion of secreted molecules free of glial interaction and electric field conditions associated with tightly apposed membrane surfaces.

The fifth set of results recorded by Rodriguez-Moreno and colleagues related to the quantitative analyses carried out and their comparison to recorded results for the VPM. The Po boutons in the MC-L4/3 were found to be significantly larger (60%) than those in the S1-L5a. The mitochondrial volume per bouton was recorded as being significantly larger (33%) in the MC-L4/3 than the S1-L5a. Vesicle pools in both the MC-L4/3 and the S1-L1 boutons were significantly larger (approx. twice as large) as those in the S1-L5a. Head volume and PSD surface area of the spines post-synaptic to the Po boutons in the MC-L4/3 and the S1-L1 were significantly larger than those of the S1-L5a.

Quantitative comparisons of the results of the Po boutons of the S1 to the VPM thalamic nucleus axons were then carried out. It was found that the VPM-L4 boutons were 90% larger in volume, approx. 50% larger in mitochondrial volume and had more than twice the number of synaptic vesicles than the Po-L5a, but there was no significant difference between these results and those for the MC-L4/3. Therefore, the authors concluded that axons from two different thalamic nuclei can form structurally different presynaptic specialisations in adjacent layers of the same cortical column as well as to separate areas or layers. Quantitative comparisons of the results of the Po S1 axons and the VPM S1 were then carried out. It was found that there were almost identical head volumes and PSD sizes. However, spines post-synaptic to the Po axons were much larger in the S1-L1 (83%, 44%) or MC-L4/3 (50%, 45%). Therefore, it was concluded that post-synaptic element differences reflect specific cell types and/or dendritic domains present in the cortical layers of areas. Comparison of frequencies of extra-synaptic mitochondria gave significant differences between the VPM-L4 and Po axons as a whole and in Po S1-L1 or L5a. The authors also compared the distribution of synapses situated in the inter-bouton segments greater than 2mm away from any mitochondrion and found there were significant differences between the VPM-L4 and Po axons in both the MC and S1 as a whole and Po axons of S1-L5a in particular. Rodriguez-Moreno and team therefore, concluded that axon mitochondria of Po axons in S1 and MC are far less bound to synaptic sites than in VPM S1-L4 axons.

Quantitative analyses of the results continued with correlation analyses and cluster cross-comparisons. It was found that in all three areas particularly in the MC-L4/3 the volume of Po synaptic boutons was positively correlated with that of the mitochondria, but less so with the size of the vesicle pool or the total surface of the PSD. Therefore, it was concluded that this might be a feature of thalamocortical boutons and their concentration of mitochondria at the synapses and/or to the prevalence of the multi-synaptic boutons.

Rodriguez-Moreno and colleagues performed transfection experiments to see if structural differences in MC to S1 reflected the existence of 2 Po cell populations each projecting to 1 area or were of an area-specific synapse structure in divergent axon branches of the same individual Po neurons. The boutons in the MC branches of Po axons were found to be consistently larger than those found in the branches of the same axons in the S1. It was also visualised that in three cells that an axonal branch specifically arborized in the MC and also a collateral branch arborized in the S1. Boutons of the MC-L4/3 were found to be up to twice as large as those in the S1-L5a or S1-L1 areas.

Rodriguez-Moreno and team concluded from their investigations that S1 and MC synapses have different structural features both pre-synaptically (bouton and active zone size, neurotransmitter vesicle pool size, distribution of mitochondria around synapses) and post-synaptically (proportion of synapses established on dendritic spines and shafts). They found that there were differences between those of the Po S1 region and VPM S1 region, but similarities between the Po MC and VPM S1. They described the differences as actually occurring on the MC versus the S1 branches of individual Po cell axons that innervate both areas. Large MC boutons and small S1-5a boutons could occur on separate branches of individual axons. The structural differences observed could be associated with different efficacies to transmit neuronal signals with Po MC synapses higher than S1. This they attributed to the wider functions of the area and went on to report that thalamocortical shaft synapses in rat demonstrate connectivity to cortical inhibitory neurons leading to strong feed-forward inhibition in parallel to excitation on cortical neurons. It was found that in general thalamocortical levels were comparable to Po S1 axons whereas fewer were demonstrated with the MC. This indicated that there is facilitation of firing by the MC area whereas there is depression of the S1 in response to rapid repetitive activation of the Po axons.

When the authors compared the VPM synapses in the Po S1 to the VPM S1 they found that there were relatively the same proportion of post-synaptic shafts to spines and they had similar PSD sizes. There were however, presynaptic structural differences relating to size, mitochondria number and placement, number of active zones and number of non-synaptic boutons. The pattern observed for S1 was given as being similar to that described for hippocampal Schaffer collaterals. Rodriguez-Moreno and team also indicated that the structure may be linked to the capacity of the Po S1-L5a for delayed, but stable potentiation as a result of conditional learning that is not observed in VPM S1-L4 synapses. This was linked to the ability of the area to dock mobile mitochondria to the synapse in times of high firing activity. This was said not to be possible with VPM which has a high basal mitochondria concentration. Differences were also attributed to the involvement of selective receptor activation at different times. The VPM synapses fire only ionotrophic receptors and the EPSCs depress markedly by repetitive stimulation whereas the Po fire both ionotrophic receptors and metabotrophic receptors and display EPSC facilitation. The boosted local ATP production and calcium ion buffering lead to docking and fusion of synaptic vesicles, exocytosis and endocytosis. Therefore, the VPM with its mitochondria rich synapses is capable of maintaining high release probability over a wide range of firing rates and repetitive high frequency firing. The Po synapses instead produce smaller currents with slower rise and decay times. However, in comparison to the MC, the VPM and MC was observed to share structural similarities even if located in different areas and derived from embryological and functionally different thalamic nuclei. Both elicit large EPSCS with paired pulse depression and only fire ionotropic receptors. The larger sizes of the PSDs of the Po MC versus the VPM were attributed to higher synaptic strength when activated or less tight regulation by feed-forward inhibition.

Rodriguez-Moreno and colleagues concluded their discussion by saying that the differences between the Po synapses (MC and S1) and the VPM plus other research into efficacy and plasticity of firing demonstrates a new subcellular level of complexity in the thalamocortical circuits. Investigation into these differences could give in general an indication of the functional complexity of other branched projection axonal systems.

COMMENT

What makes this article interesting is that it is about a topic that is not so well discussed when we are talking about synaptic firing and that is the role of neuronal mitochondria. Rodriguez-Moreno and colleagues investigated the synaptic structures of the rat thalamocortical posterior nucleus (Po) branched axons that innervate both the somatosensory cortex (S1, L5a and upper L1) and motor cortex (MC – upper L5a to lower L3) and compared these to the axons of the ventral posterior medial thalamic nucleus VPM (L4 of S1). Their study included such things as number of synapses, post-synaptic shape and size, but also included information about the number and location of mitochondria present. Hence, information about the energy supply at the synapse can be inferred.

This comment begins with a look at why mitochondria are so important to cell functioning and naturally, the first function we should discuss is energy supply. Living cells require a continual input of free energy for three major purposes: the performance of mechanical work such as in muscle contraction and other cellular movements, the synthesis of molecules from simple precursors and the active transport of molecules and ions. Neuronal cells are no different: the performance of mechanical work strictly speaking not in the form of muscle contraction, but mechanical work internally through the movement of vesicles along the cytoskeleton; the synthesis of biomolecules referring to the synthesis of neurotransmitters for example; and the active transport of ions across membranes for example such as those maintaining the ionic gradients and neuronal excitability, eg. Na+-K+-ATPases and Ca2+-ATPases. In the CNS 60-80% of the ATP required is for the latter with 10-20% of the energy demand for neurotransmission such as the synthesis, packaging and transport of neurotransmitters in vesicles.

The biochemical mechanism of energy supply involving the mitochondria of the neuronal cell follows the same path, conditions and requirements as for other living cells in that they too are responsible for the Tricarboxylic Acid Cycle (TCA, or citric acid cycle) and oxidative phosphorylation processes that transform pyruvate into usable energy. Investigation of the NAD(P)H production (Kann) showed that stimulus evoked neuronal activity resulted in characteristic biphasic transients with an initial ´dip` component and a subsequent prolonged ´overshoot` component. These were attributed to different contributors: the dip to the neuronal response (demand for energy) and the overshoot to the astrocytic response linked to the possible astrocyte-neuron lactate shuttle hypothesis.

Regulation of the TCA in neuronal mitochondria is carried out by two additional mechanisms and these are both so that extra energy demands of the cell are fulfilled, ie. those above that required for normal ´housekeeping` functions. The first mechanism is the change in ADP/ATP ratio and this is understandable in that as ATP levels fall then ATP production would be stimulated. This would apply for example in the activation of the cell during firing and signal transmission. The second regulatory method is the change in mitochondrial calcium concentration and this brings into a play a relationship between cytosolic calcium concentration and cell firing. The ultimate action of calcium on energy demand is that the activities of the TCA dehydrogenases (particularly the acetyl co A to pyruvate controlling enzyme, the pyruvate dehydrogenase PDH) are functionally modulated by calcium ions and hence, enzyme activities are linked to calcium concentration in the internal mitochondrial environment.

The firing of neuronal cells leads to depolarisation of the cell and axon-induced ion changes. With respect to calcium, the low calcium ion concentration of the resting cell is boosted by transport of calcium into the cells via voltage gated channels at the plasma membrane and increased release from intracellular stores. These result in increased calcium concentration in the cytosol and translates into calcium ions being taken into the mitochondrion via the mitochondrial calcium ion uniporter (Uni). The effect of the calcium within the mitochondria, as said above, is to modulate the TCA dehydrogenases functioning as well as possibly changing the potential difference across the inner mitochondrial membrane responsible for the oxidative phosphorylation reactions. However, the level of internal calcium in the mitochondria is below the value for the opening of the mitochondrial permeability transition pore (mptp) which is responsible for calcium efflux from the mitochondria so that concentration of calcium in the mitochondria will rise. The overall effect will be an effect on the energy production of that mitochondrion. Therefore, a change in cytosolic calcium concentration will lead to many mitochondria being affected in the area where the calcium concentration rise occurs. This translates into a wider effect on energy supplied as shown by the correlation between amplitude of mitochondrial depolarisation correlated to intensity of synaptic activity (Kann). Therefore, there is high ATP formation in places where there is high energy demand.

The association with mitochondrial population and energy demand appears not to be clear cut. On the one hand, microscope studies suggest that the distribution of the mitochondrial population in neurons and glial cells is largely heterogeneous and is a dynamically changing network and neuronal mitochondria can be positioned and retained in neuronal segments where there is high metabolic demand such as active growth cones and pre- and postsynaptic structures. This is understandable since then the mitochondria can provide energy and metabolites essential to the formation, maintenance and function of the neural connections. This is supported by the findings that mitochondrial trafficking is more sensitive to ADP concentration increases than ATP concentration depletion and that local increases in ADP will promote trafficking (Mirinov). It is also supported by the investigations into the transport mechanisms of the mitochondria. There are findings that individual mitochondria are highly mobile in anterograde and retrograde directions in the neurons with the velocity of the directed movements dependent on temperature. The anterograde transport mechanism appears to be microtubule based (Mandel and Drerup) with kinesin motor protein and membrane anchor protein RhoT and motor adaptors Trak1 and Trak2 involvement. The retrograde transport mechanism is actin based with dynein motor protein involvement (Schnapp) and an apparent disruption by Trak2.

However, it appears that most mitochondria have a stationary positioning in the cell (Mandel and Drerup) and this requires specific docking proteins. This goes against the view that mitochondria move according to energy demand relating to the cell`s need for ion transport and vesicle exocytosis and endocytosis because of the time frames involved. However, it has been found that stationary mitochondria are often found in areas of ATP demand (Mironov) and areas of high calcium. High levels of calcium affect the conformation of the RhoT membrane anchoring protein which modulates the affinity of the kinesin motor protein and anterograde movement. It also appears to affect the retrograde movement of mitochondria too (Saotome) so that the overall effect of elevation of calcium, observed with depolarisation and receptor binding is the prohibition of all mitochondrial movement. Therefore, it is possible that in developing neurons and those where there is synaptic plasticity developing because of repeated firing there is formation and transport of mitochondria to the areas of high energy demand which in the case of the neuron is likely to be at the plasma membranes. Once located the mitochondria are docked so that energy demands are met at this time and also in the future. This would be part of the increased sensitivity to firing a neuronal cell would achieve on repeated firing and be part of the whole range of physiological changes taking place with plasticity.

Another function of the mitochondria with relation to firing and calcium elevation has been suggested and that is that the mitochondria also aids in the buffering of calcium within the local synaptic environment by acting as a buffering chamber which takes up the excess cytosolic calcium evoked by increased calcium influx through the cell`s plasma membrane in response to neurotransmitter receptor binding and release from the intracellular stores. It was suggested that mitochondrial mobility was related to the need for calcium buffering (Mandel and Drerup), but we have already established that this might not be the case in every situation as mitochondria appear to be stationary and local elevations in calcium stops all mitochondrial movement. The buffering capability appears to come about from uptake of the calcium from the immediate environment and slower efflux. The uptake of calcium into the mitochondria, carried out by the calcium uniporter, was found to lag slightly behind that of the cytosolic rise in calcium concentration and repetitive electrical stimulation evoked mitochondrial calcium concentration transients that had an elevated plateau phase not seen in the cytoplasm. However, it appears that it is the efflux process that determines the buffering capability.

The main extrusion pathway for calcium ions from the mitochondria is the Na+-Ca+ exchanger which is responsible for the low resting concentrations of the ion and is believed to exchange one calcium ion with two (or some think) three incoming sodium ions. The exchanger has a low transport rate and is exceeded by the calcium influx uniporter so that calcium accumulates in the mitochondrion. Two other efflux mechanisms exist: that of the mitochondrial H+-Ca2+ exchanger which saturates at an even lower rate than the Na+-Ca2+ exchanger and is slow and therefore, has limited contribution to fast mitochondrial calcium ion cycling; and the transient low-conductance opening of the mitochondrial permeability transition pore (MPTP) which is linked to efflux during toxic high internal mitochondrial calcium levels. Therefore, on neuronal activation there is mitochondrial calcium uptake which is in the range for activation of the mitochondrial dehydrogenases, but is not at the level where MPTP is activated so calcium ions concentrate in the mitochondrion. Therefore, the ions are allowed in, but not to the same level out, leading to buffering in the cytoplasm. The calcium exuded is thought to be taken up by the intracellular stores.

Therefore, we ask what did Rodriguez-Moreno and colleagues find out about the mitochondrial populations in their samples and what can be inferred from them with relation to energy demand and calcium buffering capability. The complexity of firing from one set of axons is achieved through different means such as connectivity and synaptic area selectivity which could involve neurotransmitter type and receptor number as well as vesicular capability for example. Rodriguez-Moreno and colleagues looked at the synaptic structure of the thalamocortical posterior nucleus (Po) branched axons innervating the somatosensory cortex (S1) and motor cortex (MC) and compared some of the features to those found in the ventral posterior medial thalamic nucleus (VPM). They saw differences in structure which they attributed to firing demands and connectivity differences and which we will see later matches the mitochondrial populations and their functional role in fulfilling energy demand and calcium buffering in response to  neuronal activation.

With regards to the thalamocortical posterior nucleus (Po) branched axons innervating the somatosensory cortex (S1, L5a and upper L1), there were few non-synaptic boutons although this number was higher than for the other branched axons connecting with the MC. Those present also had a single mitochondrion. A quarter of the boutons were non-varicose (ie.  not associated with a synapse) and were found to be in inter-bouton segments of the axon. However, most of the boutons were synaptic with at least 1 associated post-synaptic area (PSD) and 1 mitochondrion. Few boutons had more than 1 PSD so that in total 88-92% of the boutons were synaptic. The size of the Po S1 boutons was on average 60% smaller than those found in the MC and they also had smaller mitochondrial volumes than those of the MC (33%). In comparison to the VPM-L4 boutons, the boutons found in the S1 were 90% smaller in volume and had approx. 50% smaller mitochondrial volume.

Further investigation by the Rodriguez-Moreno team showed that the pre-synaptic areas of the Po S1 axons were of different shapes and sizes and comparison of the frequencies of extra-synaptic mitochondria gave significant differences between the VPM-L4 and Po axons. A comparison of distribution of synapses situated in the inter-bouton segments greater than 2mm away from any mitochondrion showed that there were significant differences between the VPM-L4 and Po axons in both the MC and S1 as a whole and Po axons of S1-L5a in particular. It was therefore concluded that axon mitochondria of Po axons in the S1 and MC are far less bound to synaptic sites than in the VPM S1-L4 axons. Investigation of the post-synaptic areas of the Po S1 showed that the majority of synapses (83-96%) in this area were found on the post synaptic dendritic spines. Only 30% of these dendritic spines had spine apparatus compared to 70% in MC which implied that there was less spine motility and lower stabilisation of the presynaptic and post-synaptic apposition zone during synaptic transmission in the S1 post-synaptic areas compared to those of the MC.  The PSDs located on the dendritic shafts were attributed by Rodriguez-Moreno and colleagues to be non-spiny cortical interneurons and these were at a higher level in S1-L5a layer (17%) than on the S1-L1. Other structural differences between the two S1 layers were seen with the mean PSD surface area of the S1-L5a being similar to the VPM-L4 synapses (approx. 0.11square micrometres) whereas for S1-L1 and MC-L4/3 the regions were 60% larger. Also 65% of the PSDs on the S1-L5a were reported as having a disc-like morphology whereas most PSD`s on the MC-L4/3 (59%) were complex horseshoe shaped, perforated or fragmented. Further differences were observed with the number of unlabelled post-synaptic areas which were found to be about 3% in the S1-L1 dendrites, but higher in the S1-L5 at 18%. These were suggested as being possibly inhibitory, but their origin was unknown.

The structural morphology of the Po S1 axons and post-synaptic areas has to be in accordance to the firing demands of the area and the area`s function. Rodriguez-Moreno and colleagues described the features of this particular group of neurons as having the capability of eliciting smaller currents instigated by ionotropic (mainly by glutamate – excitatory) and metabotropic glutamate receptors and firing has slower rise and decay times. The firing tends to be excitatory if the post-synaptic area is on the dendrite spine and therefore, post-synaptic areas reflect this type of firing response. When situated on the dendritic shaft, then it is likely that firing is inhibitory and relies on the spiny interneurons. The firing capability is linked to the Po S1 axons function and a rapid increase in firing capability is observed with learning. This is consistent with plasticity associated with sustained or repetitive firing.

The results of the investigation into the thalamocortical posterior nucleus (Po) branched axons that innervate the motor cortex (MC – upper L5a to lower L3) showed differences to the structural morphology of the boutons of the S1 although firing was perceived to be relatively the same. In the MC area, there were fewer non-synaptic boutons (2%) also with 1 mitochondrion than the S1, but again 25% were found to be non-varicose located in the inter-bouton segments of the axon. Like S1 most boutons were synaptic with at least 1 post-synaptic area (PSD) and 1 mitochondrion, but 30% had more than 1 PSD which was twice as high as that found in S1. The Po boutons in the MC-L4/3 were also found to be significantly larger (60%) than those in the S1-L5a, but had similar bouton volumes and mitochondrial volumes to those found in the VPM-L4. With regards to the pre-synaptic areas of the MC, Rodriguez-Moreno and colleagues found that just like with the S1, the axon mitochondria are far less bound to the synapse zones than those in the VPM S1-L4 axons.

Differences in the features of the post-synaptic areas of the MC to the S1 were far more apparent with 30% of the MC boutons having more than 1 PSD, twice as many as the S1. The PSDs were also larger by 60% and of a different shape (horseshoe, perforated or fragmented versus disc-like) and the majority contained the spine apparatus necessary for spine motility and stabilisation of the pre-synaptic and post-synaptic active zones during nerve transmission. They were also more susceptible to inhibition since about 9% of the Po bouton post-synapses on the spines in the MC-L4/3 were unlabelled and considered inhibitory compared to about 3% in the S1-L1 and 18% in the S1-L5. However, the synapses visualised on the dendritic shafts MC-L4/3 as being attributed by the authors to be non-spiny cortical interneurons were only 4-6%, much less frequent than the S1-L5a at 17%.  As far as bouton volume and mitochondria volume were concerned, correlation analyses found that in all three areas particularly in the MC-L4/3 the volumes of Po synaptic boutons was positively correlated with that of the mitochondria, but less so with the total surface of the PSD.

Again, the structural morphology of the Po MC axons and post-synaptic areas has to be in accordance to the firing demands of the area and the area`s function and Rodriguez-Moreno and colleagues described the firing of the MC as the same as for the Po S1. However, the structural morphology differences show that this in general may be correct, but another factor must be in play.

Although not specifically investigated, Rodriguez-Moreno and colleagues compared their Po S1 and MC results to those of the ventral posterior medial thalamic nucleus (VPM S1-L4) axons. In this case, a greater number of VPM boutons are in contact with more than 3 synapses and the morphology was considerably different. The VPM-L4 boutons were found to be 90% larger in volume, approx. 50% larger in mitochondrial volume and had more than twice the number of synaptic vesicles than the Po-L5a, but there was no significant difference between them and those for the MC-L4/3. Although Rodriguez-Moreno and team reported 25% of non-varicose boutons, other groups report the same level as the S1 and MC (approx. 5%). With regards to extra-synaptic mitochondria then the authors found significant differences between the VPM-L4 and Po axons and concluded that the mitochondria of the VPM S1-L4 axons were far more bound to the synaptic sites than those of the other areas. The majority of synapses (83-96%) were found to be post-synaptic on the VPM dendritic spines and that PSD volume positively correlated to that of mitochondria just like with the S1. The structural differences are reflected by the firing differences with firing associated with the VPM as being excitatory with large currents due to ionotropic receptor binding only that can depress rapidly under repetitive stimulus and exhibits considerable resistance to sensory experience dependent changes. This implies an inhibitory input which has on repetitive firing the effect that in learning plasticity is unlikely to be achieved.

Rodriguez-Moreno and colleagues said that the VPM S1 and Po S1 bouton and post-synaptic differences probably reflected the specific cell types and this view is supported here in that the S1, MC and VPM may all have slightly different structures, but they share a common function, ie. the forward transmission of sensory information relating to the animal`s immediate environment. There are however, different sources of the information with for example: the thalamocortical posterior nucleus – snout whisker movement versus ventral posterior medial thalamic nucleus – more general sensory information from head/face;  S1 input – somatosensory cortex responsible for processing the somatic sensations, in this case touch from the rat`s whiskers; and MC input – motor cortex responsible for processing sensations in order to plan and execute movement. Therefore, it would be expected that the VPM would instigate stronger firing matching its more general function, ie. face/head vs whiskers. This is supported by the larger currents observed and that the ionotropic induced currents are stimulus dependent. As with other sensory input, it is likely that repetition or sustaining of tactile stimuli lead to a shift in priority of the neuronal firing to the unattended information to overcome the refractory periods of the neural representations of the real-time attended tactile information. The VPM is no different and is known to exhibit rapid depression of firing under repetitive stimuli. Therefore, structural morphology of the VPM boutons supports the general but high chance of firing with a large number of the boutons having 3 or more contacts, large bouton sizes and large PSD areas with few non-synaptic boutons. With regards to mitochondria, the number (and volume) should reflect the high demand for energy and calcium buffering requirement relating to this general but high rate of firing and this appears to be supported with the VPM having equal mitochondrial volume to the MC area.

So, what about the Po S1 and Po MC? These differ to the VPM in that the source of the information differs (as given above – the thalamocortical posterior nucleus Po – snout whisker movement versus ventral posterior medial thalamic nucleus – more general sensory information from head/face) as well as the functionality of the receivers (the somatosensory cortex S1 responsible for somatic sensory processing and the MC motor planning and execution in response). In general, both demonstrate firing differences, eg. smaller currents to the VPM relating to the more specialised nature of the information being passed forward, tactile information from snout whiskers to processing of that information and planned movements in response. This is reflected in general in the morphology of the boutons with most having only 1 PSD and the capability of storing information of experiences (learning) through neuronal plasticity mechanisms.

There are however, structural differences between the S1 and MC, eg. larger number of boutons with more than 1 PSD in the MC than the S1, larger bouton size than S1, larger mitochondrial volume, higher stabilisation of active zones during transmission. This is possibly due to the specificity of information received, ie. S1 relates to sensory information processed about the snout whiskers, whereas  MC motor movements that could be shared from a variety of inputs. This shared capability would mean that the active zones of the MC need to be more stabile and are likely to demonstrate plasticity with repeated stimulation to some degree. The inhibition of feed forward transmission also shows this difference and even differences between S1 layers with 9% of the boutons of the post-synaptic dendrite shafts of the MC attributed by Rodriguez-Moreno and colleagues to inhibitory non-spiny cortical interneuron input compared to 17% for the S1-L5a layer compared to only 3% in the S1-L1. Therefore, with relation to energy demands and calcium buffering, the mitochondrial volume is likely to reflect the needs of the cells they serve. MC with its larger size and shared function is likely to have higher demands than its S1 counterpart and this is demonstrated by the higher mitochondrial volume in the MC tissues.

Another factor that could indicate the energy demands of the cell is the link to vesicle recycling. It has already been said that 10-20% of energy required by neurotransmission includes that required for the synthesis, packaging and release of neurotransmitters from vesicles, which is part of the firing mechanism in some neurons. Vesicles are released on stimulus in an energy dependent mechanism and then are recycled via endocytosis which is also an energy dependent mechanism. Therefore, firing demand should be reflected by energy supply (ie. mitochondrial volume) as well as vesicle populations. Rodriguez-Moreno and colleagues` investigation showed that the VPM had probably the highest mitochondrial demand, probably followed by MC neurons which were likely to be higher than the S1 due to the selectivity of functioning. This should also be reflected by the vesicle populations in the tissues. Rodriguez-Moreno and team`s experiments showed that in both S1-L1 and S1-L5a, about 7% of the Po axonal boutons contained a mitochondrion and some synaptic vesicles. The vesicle pools appeared to be similar in both the MC-L4/3 and the S1-L1 boutons whereas the pools in the VPM-L4 were found to be significantly larger (approx. twice as large) as those in the S1-L5a. This supports the view that the VPM is a mitochondrial and vesicular rich synaptic area and is capable of maintaining high release over a wide range of firing rates as well as repetitive high frequency firing.  The MC neurons were also good and better than the S1 and that they too, support the view that the vesicle populations reflect the mitochondrial functional status of the cells in question. Rodriguez-Moreno and team also saw protrusions in their tissue samples that could be part of the exocytosis and recycling process of the vesicles, although no membrane specialisations were observed. They were perceived however, as being linked to the secretion of molecules.

Therefore, it can be concluded that energy supply matches energy demand of the neurons independent of their location or function. The energy supply needs to be either at a ´housekeeping level`, eg. normal levels required for the cell`s protein synthesis or raised when the neurons are fired and require higher levels of energy for ion transport or vesicular recycling for example. Therefore, in this case the mitochondria need to be where the high energy demand is and likely to be and that is around the active zones. This is achieved by ´building` the mitochondria and transporting them to these sites and this is carried out during the development of the neuron. Fully developed neurons have stationary mitochondria docked where they are primarily needed and this need comes not just from energy supply demands, but also as described above for the buffering of calcium ions whose concentrations rise during depolarisation. Repeated or sustained firing is known to cause physiological changes to the neuron so that there is increased firing and therefore, it is likely that changes in mitochondrial number are part of the effects forming plasticity. It is also likely that mitochondrial number and functioning level respond to neuronal deficiencies and this is seen for example in the increased number of damaged mitochondrial observed with ROS formation. Therefore, the roles of mitochondria in energy supply and calcium buffering is just as important to the correct functioning of the neuronal cell as vesicle recycling for example and this ´housekeeping` tool should not be underestimated. Rodriguez-Moreno and team`s investigation also shows that measurements of mitochondria and energy components could also be used as indicators of neuron functionality and levels of damage in addition to measurements of firing and neurotransmitter levels.

Since we`re talking about the topic………

…..using high resolution confocal laser scanning microscopy would it be possible to establish whether mitochondria were located or not in the regions of high calcium concentration when the Po axons were electrically stimulated?

……can we assume that changes in NADPH fluorescence would show whether mitochondria present in the tissue samples were active energy suppliers or not when the cells were electrically stimulated and whether or not changes in mitochondrial function are observed when the cells undergo repetitive stimulation?

……if mitochondria were visualised using tetramethlyrhodamineethylester and their movements analysed by applying single-particle tracking (Mironov) and intracellular ATP concentrations were imaged using luciferase luminescence would it be possible to show that stimulation of the Po axons would lead to a change in ATP/ADP ratio as expected for an active area?

….if the fission protein Drp1 is inhibited in the Po axons and the cells stimulated would a lack of transport show whether the energy supply was elicited through stationary mitochondria only?

contribution of AMPA and NMDA receptors of the dorsal lateral prefrontal cortex in working memory

Posted comment on ´The contribution of AMPA and NMDA receptors to persistent firing in the dorsolateral prefrontal cortex in working memory` written by B. Van Vugt, T. Van Kerkoerle, D. Vartak and P.R. Roeslfsema and published in Journal of Neuroscience 2020 40(12) p. 2458 doi 10.1523/JNEUROSCI.2121-19.2020

SUMMARY

Van Vugt and team report in their article the results of their investigation into the contributions played by the different glutamate receptors, AMPARs and NMDARs, of the dorsal lateral prefrontal cortex (DLPFC) in the persistent firing observed during a working memory task. The current theory says that NMDARs because of their long time constants play a greater role than AMPARs which have shorter time constants. Van Vugt and colleagues tested the hypothesis that in the oculomotor delayed response task (ODR) the NMDARs maintained the information associated with the cue as a pattern of persistent neuronal firing during the memory delay whereas the AMPARs activated the neurons during the sensory stimulus and persistent firing phase. Hence, they found that both NMDARs and AMPARs contributed to the persistent activity observed.

For their experiments, three male macaque monkeys performed an oculomotor delayed response task (ODR). The monkeys were first trained with a fixation point of a red circle on a grey background and had to gaze at a fixation window centred on the fixation point. After 300ms they were then presented with a visual cue of a white circle at either the appropriate neuron`s receptive field (RF) or at what was termed the anti-preferred location which was the fixation point`s mirrored location. The visual cue was turned off after 150ms, but the monkey had to maintain the fixation window for another 100ms before the fixation it was extinguished. This was the signal for the monkey to make a memory guided eye movement back to the target window which was centred on the location of the previous visual cue. Correct responses were rewarded with apple juice. Trials where the monkeys failed to maintained fixation before the fixation point was extinguished were stopped. All stimuli were generated by computer software and eye movements were recorded with a video eye-tracker. Iontophoresis was used to eject small amounts of glutamate receptor antagonists (APV for NMDARs and CNQX for AMPARs), which were enough to disrupt, but not abolish neuronal activity.

Neural activity was recorded from the dorsolateral prefrontal cortex (DLPFC) including frontal eye fields and surrounding cortex. Only selected neurons that showed sustained firing were used. Three blocks of approx. eighty trials were recorded: first without drug delivery by maintaining holding current (pre-drug recordings); a recording block when drugs were administered by applying the ejection current (during drug recordings); and finally a recording block without drug delivery, but again with the holding current maintained (post-drug recordings). These phases began at different times after the effect of the antagonist drug produced noticeable spiking activity.

Data analyses were performed on the results obtained. The ODR task was divided into two time periods: spontaneous activity (lasted from 300ms before the stimulus onset up to stimulus onset); and task-related activity (from the stimulus onset up to saccade onset, with ´saccade` intended here as equivalent to the neural firing pattern formation). Values calculated included cue-driven activity in the time window from 50-250ms, persistent activity from 300-1150ms after cue onset (starting 150ms after cue onset) and saccade related activity from 200ms before the onset of saccade. Spatial selectivity for each individual cell was quantified using the measurement d´ which showed how well a single neuron could distinguish between the memories formed for the two locations. Statistical analyses were performed including two-sided t tests and three-way repeated measures ANOVA. The authors also performed a stratification analysis to see if APV and CNQX differences were due to relatively low firing rates in cells when dealing with anti-preferred locations and the results led to only certain cells being used in the analyses.

The results of Van Vugt and team`s experiments showed that overall performance on the task was high. Small doses of the NMDAR antagonist, APV, applied so as to perturb activity, but not to abolish it produced no consistent effects on performance accuracy. Small doses of the AMPAR antagonist, CNQX, when applied again like APV so as to perturb activity, but not to abolish it, produced instead small increases and decreases in performance accuracy. These were interpreted by the authors as possible changes in the motivation of the animals to perform the tests under these conditions.

The sets of experiments carried out involved the recording of neural activities of single DLPFC neurons during the ODR task. It was found that most neurons exhibited persistent firing during the memory periods. Neurons showed increased firing during the full duration of the trial when the visual cue was presented at the preferred location of their receptive fields (RFs) and a suppression of firing when the visual cue was presented at the anti-preferred location. The iontophoretic application of APV (the NMDAR antagonist) suppressed baseline activity before the visual cue onset as well as spiking activity in both monkeys at the preferred location during visual cue responses, during the persistent activity phase (time window 300-1150 ms) and the saccade window (200ms before the saccade). The suppression for the preferred location responses was greater than for the anti-preferred locations. Therefore, the antagonism of the NMDARs was shown to weaken spatial selectivity. Computed d´ measurements of how well a single neuron could distinguish between the memories for the two locations showed that the values decreased in both monkey subjects. The cessation of APV administration led to a return of spiking values, although not to the original values and neither was it observed for both monkeys.

Van Vugt and colleagues looked at the time course of the APV suppression effect by plotting the difference between the spiking activity before and during its administration. For the preferred location, the main effect of APV was found to be on the firing rate, but there were no significant interactions between the drug effect and the different test periods (eg. spontaneous, visual, delay and saccade activity periods). The decrease of the cue-driven response supported the hypothesis of a general multiplicative effect of NMDARs on spiking activity, but appeared not to support the hypothesis that the NMDARs have a specific role in the generation of persistent activity. Investigation of small subsets of neurons with a visual response without the delay activity found that APV suppressed the visually driven activity of these neurons supporting the view that NMDARs play a more general role in both cue-driven and persistent activity.

In the case of the iontophoretic administration of CNQX (the antagonist of AMPARs) Van Vugt and team found that there was a decreased level of persistent firing associated with the memories of neurons for both the preferred and anti-preferred spatial locations. The baseline spiking activity before the visual cue onset was also suppressed as well as the spiking activity in the cue window, memory window when the visual cue was presented in the preferred location and saccade window. When the visual cue was at the anti-preferred location then significant decreases of activity were observed in the cue window, memory window and saccade window in one monkey, but not in the other. The decrease in d´ (performance of a single neuron for each location) was observed with both monkeys to different degrees and there was no effect on the Fano factor (defined here as the variance of firing divided by the mean spiking activity averaged within a specified time window). Cessation of the iontophoretic administration of CNQX did not restore the spiking activity to pre-drug levels and with some neurons no trend of recovery was even observed. Therefore, the authors concluded that CNQX administration induces long-lasting effects. The influence on firing rates was found to be significant for the preferred location, but there were no interaction effects between the drug and time period indicating that the drug effects were similar across the different time periods.

Comparison of the effect of APV and CNQX on delay activity showed that the decrease in d´ was greater for NMDAR antagonist administration than for AMPAR antagonistic administration. No definitive conclusions were made by the authors because of the differences in ejection currents, poor controls of factors such as efficiencies of drugs, distance between neuron and pipette, diffusion and clearance of drugs for example. Therefore, the authors determined how well the influence on delay activity for the preferred cue location predicted the influence on delay activity for the non-preferred location. The authors found that in the case of APV administration the value was not significant and therefore, it was concluded that the decreased activity in the preferred direction was not a good predictor for the activity decrease in the anti-preferred direction. This interaction was not seen with CNQX where the value was significant and therefore, the prediction statement was valid. The difference between the two antagonists was also found to be significant. This was interpreted as the two likely demonstrating the distinct actions of the NMDARs and AMPARs. The authors explained that when glutamate binds to the AMPAR then the associated ion channel opens and the cell is activated. However, when glutamate binds to the NMDARs, the associated ion channels and receptors are normally non-functional due to the binding of magnesium, which has to be removed before the ion channel functioning occurs. This could explain the observation that a decrease in delay activity in the preferred direction was not always accompanied by a comparable decrease in the anti-preferred location since in this case the receptors may still be blocked because of the lower neuronal firing rate associated with the non-preferred location. Therefore, the administered APV could not exert its full effect.

Van Vugt and team concluded that both AMPARs and NMDARs contribute to persistent activity during working memory tasks. The administration of APV, the NMDAR antagonist, led to a decrease in persistent firing associated with those active neurons representing the preferred spatial location in short-term memory, but had little effect on the neural representation of the anti-preferred location. Therefore, APV decreased the information conveyed by persistent firing about the memorised location. The administration of CNQX, AMPAR antagonist, gave a different effect with decreased persistent firing associated with the actively firing neurons in the formation of short-term memories for both the preferred and anti-preferred spatial locations with the baseline spiking activity suppressed even before visual cue onset. Van Vugt and colleagues stated that their study could provide a base for further studies on the effects of excitation of the dorsolateral prefrontal cortex area and demonstrate its connectivity to other brain areas as well as explaining how task-relevant information is kept online during memory recall delays.

COMMENT

What makes this paper interesting is that it looks at the roles of NMDARs and AMPARs in the dorsolateral prefrontal cortex (DLPFC) during the transient world of working memory involving information processing and short-term memory. The general experimental set-up that of the oculomotor delayed response task (the ODR) determines like any other experimentation what systems are required for the successful performance of the task set.  The ODR used by Van Vugt and team in their experiments was basically a conditioning task. The monkeys were trained to look at visual cues and perform eye movements in response to changing locations. Failure was determined by the monkey not performing the learnt eye movement at the correct time.

Each stage of the experimental set-up of this ODR task required a number of sensory and cognitive processes. The first stage, that of training to gaze at a visual cue (the red circle on a grey background) at a particular location (fixation window centred on the fixation point) on a screen, required at least the application of the visual input system and attentional system. Short-term memory formation of the visual cue`s location was also required in order that the task could be performed successfully later. The type of memory employed is probably spatial memory and not particularly visual memory since the red/white colours of the cues were essentially immaterial to test success since the monkey only needed to know about the location of the lighted point. Then, after 300ms the monkey was presented with another visual cue (this time white) for a time period of 150ms at a location either at the same point as the first (the preferred location) or at its mirrored location (termed the anti-preferred location). For this stage, a number of sensory and cognitive capabilities were in play, eg. the visual system (looking at the visual cue), attention system (shifting attention to the cue, maintaining attention on the visual cue and avoiding distraction), motor system (shifting the gaze) and short-term memory of this position (spatial memory) whilst also remembering the location of initial target (working memory and short-term memory). The monkey had to keep its gaze on the location first for the time period of 150ms when the visual cue was lit and for a further 100ms once the visual cue was extinguished guided by the placement of the fixation window. This 100ms period provided the ´delay` where both neural representations in the form of short-term memories had to be maintained in the absence of the visual test stimuli. (Although, it could be said that the fixation window also produced a visual signature and real-time sensory systems relating to visual information were still being stimulated.) The extinguishing of the fixation window was the conditioned signal for the monkey to move its gaze back to the initial location (termed ´saccade` by the Van Vugt team) and this stage employed a number of different systems including motor movements and the employment of spatial memory and comparisons of temporarily stored information. The success of the experiment was the shift of gaze back to the preferred location and failure was effectively any other eye movements (ignored completely as test results) or the movement to the anti-preferred location. Therefore, the experimental set-up required overall a number of different sensory and cognitive systems (eg. visual system and visual memory, motor systems and motor memory – head and eye movements, attentional system, working memory – information processing and short-term memory as well as spatial memory) and since the DLPFC area was used as the test area, results of Van Vugt`s experiments should indicate the role that this particular area plays in this type of task and more specifically, what roles the two glutamate receptor types have in this particular task.

What Van Vugt and colleagues found from their ODR task based experiments was that both AMPARs and NMDARs of the DLPFC area contribute to the persistent neuronal activity required for successful working memory employment and task completion. Contribution of the two sets of receptors appeared not to be the same. The administration of APV, the NMDAR antagonist, produced decreased persistent firing associated with the firing of neurons required to produce the neuronal representation of the preferred spatial location in short-term memory, but had little effect on the neural representation of the anti-preferred location. Therefore, it was said that the APV decreased the information conveyed by persistent firing about the memorised location required. This indicates that NMDARs are essential for the short-term memory formation of the relevant information. CNQX, the AMPAR antagonist, however, showed a different effect since decreased persistent firing was observed associated with the short-term memories for both the preferred and anti-preferred spatial locations with the baseline spiking activity suppressed even before visual cue onset. So, how does this fit in with what we know about the DLPFC area and NMDAR and AMPAR contributions associated with the task of working memory (information processing) and short-term memory formation?

As far as working memory goes, there are different types eg. visual, tactile and spatial which all share the main aims of information processing and the holding of multiple simultaneous informational units. Working memory should perhaps be considered more of a ´state` allowing these functions to occur and therefore, the requirements of the system relate to information in and out, maintenance of information and assessment of information (whether internally within the active state or sent to somewhere else for assessment). The location of the active ´state` plus connectivity of this location to other areas is likely to follow the function.  Therefore, separate senses have been reported as having differing working memory locations (eg. visual – infero-temporal area, V4 and medial temporal area; tactile – S1,S2; and auditory where even time, frequency and intensity of sound are all located to separate modules – work by Pasternak). The location of working memory and its connectivity appears to be traditionally located to the prefrontal cortex following the connected pathway to the anterior cingulate cortex, then hippocampus followed by the anterior cingulate cortex, but other areas also appear to be involved. For example, the lateral intraparietal cortex (Takeichi – working memory training led to increased myelination in white matter neurons of the intra parietal sulcus and anterior corpus callosum), plus areas associated with attention (guided eye movements, saccades), manipulation of information (post-parietal cortex) and item maintenance (post-parietal cortex and lateral occipital cortex).

   So, what roles does the DLPFC play in working memory? It is likely that the DLPFC is associated with executive control, the maintenance of items and value assessment that are in play in the particular working memory state being considered. The executive control function which is required in the conditioning task appears to be a common function of lateral prefrontal cortical areas (D`Espisito, Macdonald) and can even demonstrate differences in function according to hemisphere (eg. D`Espisito – right hemisphere, plan generation; left hemisphere, plan execution). Within the DLPFC itself, flexible neuronal tuning supports top-down modulation of task-relevant processes and neuroimaging has shown that the DLPFC can carry out cognitive control adjustments based on the detection of conflict. This could be because of its connectivity to the anterior cingulate cortex, an area known to monitor for conflict.

With regards to spatial working memory, most researchers support the view that the DLPFC is essential for this type of working memory whereas its ventral lateral counterpart is required for the processing of language, communication signals such as facial expressions and audiovisual working memory (Plakke). However, studies to transfer this function to humans appear to show that spatial working memory (as required in Van Vugt`s ODR task) in the human requires not only the DLPFC, but also the precentral sulcus. Part of the working memory function is the requirement to maintain the neural representation of the item or items within the working memory state so that the required processing of the information can be carried out. This appears to be a function associated with the general prefrontal cortex area (Baier). In the ODR task, two neural representations have to be maintained – two visual cues (one red, one white) which have two locations (one preferred, one anti-preferred). It has been found that working memory can hold simultaneously three items decreasing as the number of items stored is increased (Anderson). Therefore, the two pieces of information relating to the ODR task undertaken by Van Vugt`s subjects, bearing in mind that one piece of neural information with regards to spatial memory has feature plus location characteristics, is well within the capability of the working memory state. The quantity is attenuated by the content overlapping (visual cues may only differ by colour) and both being goal-relevant (Soto).

The third function of the DLPFC and the working memory state is the assignment and comparison of ´value`. In the ODR task, the ´value` is deemed according to location, ie. preferred location versus anti-preferred location. The first brings reward, whereas the second (as well as no movement) does not. (As said above, the colour of the visual cue is probably immaterial and it is only the location and relevant eye movement that is counted as success.) Recognition for this type of function relating to this area is well known, eg. increased activity in the DLPFC when actions are selected and initiated (Spencer); neuronal representation of information relevant for credit assignment reported in the DLPFC with specifically, the neuronal activity reflecting both the relevant cues and outcomes at the time of feedback and were stable over time (Asaad); and DLPFC activity denotes comparison of two strategy values (Wan). Value is to some extent recorded since the monkey knows that one particular movement instigates on retrieval, reward. This value is assigned and recorded during the training period and those physiological changes associated with long-term memory are reflected in the control values of the NMDARs and AMPARs at the beginning of the test periods. This value assignment is likely to involve the connectivity of the DLPFC with the anterior cingulate cortex (ACC). It was found that a larger proportion of neurons were activated in low motivational conditions in the DLPFC than in the ACC and the onset of this activity was significantly earlier in this region (Amemori). Therefore, this indicated that motivation and value judgement required firing of both the DLPFC and ACC, but to different degrees and at differing timings.

The ability of the DLPFC to perform these functions relating to working memory is dependent on the area`s physiology and connectivity. From a connectivity perspective, it is known that the DLPFC is part of a general network of frontal, parietal and insular brain regions activated in response to a wide range of demanding task conditions (Brosman). In particular, there is competition between the DLPFC, PFC (there are reciprocal connections to the orbitofrontal cortical area OFC) and striatal regions (Daw). With reference to spatial attention, cortico-striatal pathways are particularly important with the identification two bilateral convergence zones (in the caudate and putamen) receiving input from the OFC, DLPFC and parietal regions.

The functionality of the area also relies on its neuronal firing capability and relating to this, Van Vugt and team explored the capabilities of the two glutamate receptors, NMDAR and AMPAR located within the DLPFC. It has been shown that the PFC contains neurons that can perform multiple tasks simultaneously in the form of both working memory and attention functions (Messenger). This would be a valid explanation of how neuronal activity is controlled for informational processing demands. From a neurotransmitter perspective, working memory is normally associated with the neurotransmitters, dopamine and GABA. It has been reported that there is dopamine connectivity between the left and right fronto-parietal brain networks (Cassidy) that may adapt flexibly to cognitive demand. Poor working memory performance was related to deficient cortical dopamine release. However, with regards to amphetamine, a U-shaped functional modulation of working memory performance and the medial PFC effect was observed (Lapish) where moderate increases in monoamine efflux would increase attractor stability, but high frontal levels would diminish it. The role of GABA in working memory is clearer with a report showing that GABAergic dysfunction in the PFC observed with increasing age is linked to decreased working memory capability (Banuals) and in particular with the DLPFC that lower GABA elicits a greater loss of performance particularly with high task demands (Yoon).

However, Van Vugt and team`s article concentrated on the roles of the two glutamate receptors, NMDAR and AMPAR on working memory function. In general, there are known stages to glutamate functioning at the neuronal level.  The first is the binding of the neurotransmitter to the receptor protein eliciting the activation of its associated G protein. This is followed by the activation of the effector systems through the G protein signal cascade and finally, the post-synaptic neuronal functioning. In the case of the NMDAR, bound magnesium ions are released on arrival of the action potential on cell firing and neurotransmitter binding and this release causes the associated calcium channel to open. This calcium ion permeability of the neuronal NMDAR is under the control of the cAMP protein kinase A signaling cascade of the post-synaptic area (Skerbidis, Nicolls). Some NMDARs however, are associated with sodium channels like the AMPAR and these are then opened when glutamate binds. Others are associated with SK channels and potassium ion channels causing an influx of potassium ions. Binding of glutamate released by the action potential reaching the presynaptic area can also occur to the post-synaptic AMPARs and these are associated mainly with sodium channels, although some are linked to calcium channels. Both lead to the appropriate ion influx. If firing is sustained then long term potentiation (LTP) may occur. LTP occurs primarily because of long-term AMPAR changes causing increased sensitivity of the neuron to the firing stimulus. There are an increased number of AMPARs at the membrane surface resulting in higher sensitivity and strength of firing to stimulus. The alterations in AMPAR functional properties are coupled to trafficking, cytoskeletal dynamics and local protein synthesis (Derkach – PIP3 turnover required at synapse to maintain the clustering of AMPAR; Bacaj – AMPAR nanodomains are often, but not systematically, colocalized with clusters of the scaffold protein PSD95).

Therefore, the main difference between the NMDAR and AMPAR is their response to sustained firing – a condition required in the formation of memories. With regards to Van Vugt`s experiments, long-term physiological changes are likely to have occurred in the training periods before the ODR test phase. Those training periods would have led to LTP of the relevant neurons meaning that insertion of AMPARs in the post-synaptic membrane would have occurred (Derkath, Plant, Ehninger, Rauer). The LTP would result in increased synaptic strength and increased susceptibility to depolarization for neurons associated with the visual cues, but from an experimental perspective the changes would be reflected by the higher control values compared to non-trained animals. LTP is not always observed in sustained firing conditions and sometimes long-term depression (LTD) results. This means that there is increased sensitivity of the neuron to inhibit firing on input. However, LTD is not likely to have occurred here since the DLPFC neurons were associated with excitation and it should be remembered that any changes would have been observed and accounted for by the control.

The role of glutamate receptors in working memory is well known. For example, NMDARs containing the NR2A subunit in the PFC (particularly the layer 2/3 pyramidal neurons) were found to be required (McQuail) and the level of task irrelevant information is affected by NMDAR antagonists (Cage). Van Vugt and team`s experiments investigated the roles of both NMDARs and AMPARs in the DLPFC during a task that required working memory. They found that the AMPARs and NMDARs contributed to the persistent neuronal activity in the DLPFC during their working memory tasks. The antagonist of NMDARs, APV, decreased persistent firing associated with the memory of neurons firing to represent the preferred spatial location, but had little effect on the neural representation of the anti-preferred location. Therefore, the APV decreased the information conveyed by persistent firing about the memorised preferred location.

The antagonist of the AMPARs, CNQX, showed a different effect with decreased persistent firing associated with the memories of the firing neurons for both the preferred and anti-preferred spatial locations with the baseline spiking activity suppressed even before visual cue onset. Therefore, Van Vugt and team concluded that the receptors provided different contributions with NMDARs showing a general multiplicative effect on spiking activity with no specific role in the generation of persistent activity, but a specific role in visually driven activity of those neurons supporting the desired cue location, whereas the AMPARs were associated with the memories of neurons for both the preferred and anti-preferred spatial locations. This is supported to some extent by work on the NMDARs of the lateral PFC again of macaque monkeys during a working memory task (Ma). Here it was found that that acute injections of the NMDAR antagonist, ketamine, both weakened the rule signal across all delay periods and amplified the trial-to-trial variance in neural activities (i.e., noise), both within individual neurons and at the neuronal assembly level. This resulted in impaired working memory performance. In the minority of post-injection trials when the animals responded correctly, the preservation of the signal strength during the delay periods was predictive of their subsequent success. These findings suggested that the NMDA receptor function would be critical for establishing the optimal signal-to-noise ratio in information representation by assemblies of PFC neurons. This supported Van Vugt`s study where application of APV reduced neuronal spiking and this was also reflected by the success of the memory being maintained so that the eye gaze moved back to the preferred location at the correct time.

The results of Van Vugt`s experiments can also lead us to make further observations about the actions of both NMDARs and AMPARs of the DLPFC during this type of task. Van Vugt termed the firing of DLPFC cells responsive to spatial information as ´delay cells`. These neuronal cells are activated by the visual cue and remain active during the period between it being extinguished and the time when the gaze shifts back to the preferred location. This is termed as the ´memory delay`.  In neurochemical terms, this would accounted for by the formation of sensory stores relating to the visual cue and then maintenance of these firing neurons in a short-term memory store representing their neuronal firing pattern. This would be held in the working memory state whilst the distracting task of the alternate visual cue is presented and extinguished. Van Vugt and team concluded that there was a subset of the firing population which was activated by the stimulus, but switched off when the stimulus was no longer visible and a set of ´delay cells` that exhibited persistent activity. They suggested that this persistent firing could result in reverberatory excitation within the area and between cortical areas eg. between the cortex and subcortical structures including the thalamus and cerebellum.

So, the first question is whether subsets of neurotransmitter receptor populations within an area exist or not? The answer is affirmative. We know that glutamate is a single neurotransmitter which can bind to and activate different receptors and multiple forms of receptors. Even in the case of the NMDARs, some are linked to sodium channels, others potassium channels for example. Receptors can also have different characteristics and functions dependent on their locations within an area, eg. TANS. In the experiments described by Van Vugt, the NMDAR populations respond to transient visual cue appearance as well as maintaining the neural representation in its absence. Therefore, it is likely that it does not require the whole population to maintain the stimulus`s characteristics and selectivity could depend on the type of channel associated with it or even on the receptors location. Van Vugt and team hypothesised that with NMDAR involvement, membrane depolarisation leads to neuronal firing with strong effects on neurons driven by the stimulus and smaller effects for weakly activated cells. In persistent firing, ie. that maintaining the neural representation of the visual cue at the preferred location, then Van Vugt says that glutamate needs to bind and the neuron has to be depolarised to release the magnesium ions from the NMDAR channel to unlock the block which exists at rest. In this case, it is likely that the NMDARs referred to here are SK channel linked and respond with an influx of potassium ions. If this is the case, then firing is abolished and recovery of the cell instigated. Therefore, these neurons could represent those responding to the presence of the actual visual cue whereas the other firing subset would maintain the neural representation in the working memory state (essentially the short-term memory) whilst the distracting cue is present. This is because at one time period of the experiment, two sets of active neurons exist: one maintaining the preferred location in the working memory and the other the real-time visual stimulus. Therefore, the functions of the NMDAR populations could be temporarily split. This has been observed in the premotor cortex where two targets representing two movements of the sequence are represented in the working memory by two subpopulations of the neurons (Sanechi). Also, with more relevance to the experiments of Van Vugt and team, by findings from Andersen, who found that spatial attention can be divided effectively between separate locations whereas non-spatial features have a so-called ´global effect` whereby items having the attended features may be preferentially processed throughout the entire visual field. Therefore, when cues are of the same colour then even if spatially independent then the firing for both is improved since one helps the other rather than when different and competition occurs. Task similarity, task difficulty, practice, training, age and anxiety are all observed to influence the quality and quantity of information in divided attention conditions.

In the experiments of Van Vugt, the working memory content refers in general to one task-relevant group (the visual cue at the preferred location) and one task irrelevant (a visual cue at the anti-preferred location). The division goes further by the task relevant being associated with reward and therefore, firing is likely to be more robust. This was demonstrated by Donahue who showed that neurons in the DLPFC only encoded task-relevant memory signals with their congruent choice signals and these were more robustly encoded following rewarded outcomes. This is supported by work by Suzuki who says that the DLPFC performance closely correlates to the suppression of distractor stimuli and therefore, mediates selection of information.

However, Van Vugt`s experiments also looked at the contribution of AMPARs of the DLPFC in their working memory task. These were also shown to play a role in persistent activity independent of visual cue location. This demonstrates a likely general firing response to calcium ion influx mediated by the action potential. Van Vugt and team state that the AMPARs always depolarise the post-synaptic neurons in an additive manner. Therefore, NMDAR activity is induced by sensory input and NMDAR channels and ion flow occurs resulting in the activation of the AMPARs leading to persistent activity from sufficient depolarisation even in the absence of stimulus. This predominance of the NMDAR effect is supported by others. It has been shown that regarding persistent firing in the DLPFC, NMDAR antagonists almost abolished persistent activity whereas AMPAR antagonists were weak during the start of the delay period and stronger towards the end of the delay period indicating that NMDARs had a role in working memory (Wang). However, it was also said that it could demonstrate a difference in the efficacy of the receptor antagonists since the NMDAR antagonist produced stronger effects than the AMPAR antagonist in all experimental periods.

The second question raised as a result of the differences in glutamate receptor contributions is whether the persistent activity observed is due to differing contributions of and to connectivity between the DLPFC and other areas. We have already seen that working memory requires the connectivity between this area and others. In particular, the DLPFC and OFC demonstrate strong reciprocal connectivity. Connectivity is important since working memory requires an active attentional system and this is dependent on activity of multiple brain areas. The attentional system already described above as playing a role in the priority of task relevant and task irrelevant information in working memory, also provides informational selection and updating processes on the basis of strength of neuronal firing. In the case of information relevancy, for example working memory allows individuals to pay only three quarters of the maximum level of attention to relevant stimuli and to ignore unwanted stimuli and for this, connectivity of areas such as the PFC and globus pallidus are said to be important (Klingsberg). It is known that the selection process retrieving the relevant item requires the brain areas of the superior frontal gyrus, post-cingulate cortex and precuneus (Blauracke, Bledowski). Also the parieto-medial-temporal connectivity was found to be important when working memory benefits attention by strategic control when the contents overlap in goal relevant stimulus features (Soto). Therefore, connectivity to other areas is important to working memory performance and since some NMDAR populations are connected to ion channels that initiate post-synaptic signal transmission and others not, this may provide another reason for subset firing differences.

Therefore, we have established an association between working memory performance and NMDAR and AMPAR populations of the DLPFC. This is further supported by observed changes to working memory performance with reported DLPFC effects. For example, specialized cognitive training has been shown to increase working memory performance and this has been linked to the volume of the rostral part of the left DLPFC being able to predict an individual`s reponse to training (Verghese). Also transcranial stimulation (tDCS) of the DLPFC has been shown to prevent the deficits observed in working memory with stress (visuospatial worse than verbal – Bogdanov). In this case, stress was shown to impair working memory performance by decreasing the activity of the area which could be alleviated by anodal tDCS. The administration of oestrogen also restored DLPFC functional capability in surgically induced menopausal rats (Hara). This increase was linked to positive changes in the structure of the presynaptic area (frequency of boutons and mitochondrial number).

We also ask whether factors such as emotions and age which are known influences on working memory performance can be associated with changes in DLPFC functioning. In the case of emotions, it is known that emotions, working memory task demands and individual cognitive differences predict behavior and cognitive effort. Negative emotional effects promote cognitive tendencies that are goal incompatible with task demands so that greater cognitive effort is required to perform well (Stobeck). This is supported by the observations that there is a negative impact of anxiety on working memory functioning (Chuderski) whereas positive feelings facilitate working memory and complex decision making among older adults (Carpenter). In this case, the effects of emotions could be explained by the close relationship between attention and working memory. It is known that changes to attentional awareness will elicit changes in informational quantity and quality of neural representations. From a neurochemical perspective, this translates to changes in firing and connectivity of appropriate brain areas and it has already been demonstrated above that the brain area important for personal values, the OFC, and the area discussed here, the DLPFC, demonstrate strong reciprocal connectivity. Therefore, any effects on OFC behaviour will influence DLPFC activity and it is possible that these changes can be affected through the NMDARs and AMPARs populations.

The other factor known to affect working memory performance is ageing. It is has been shown that the Gabergic dysfunction of the PFC in general with age leads to decreased working memory performance (Banuals). More specifically, we have already described that age-related cognitive decline is linked to the accessibility of NMDARs of the DLPFC of the particular sub-type that have the NR2A subunit. Therefore, if the activity of the DLPFC area is dependent on particular sub-populations of NMDARs then ageing may play a role in any performance change shown.

Therefore, we can conclude that NMDARs and AMPARs of the DLPFC play slightly differing roles during the transient world of working memory. The type of task reported on here required a number of different cognitive and sensory systems, eg. visual system, motor systems – head and eye movements, attentional system, working memory – information processing, short-term memory – visual memory, spatial memory and motor memory based and therefore, there are ample areas where NMDARs and AMPARs in the DLPFC could play  a role. Van Vugt showed that AMPARs and NMDARs of the DLPFC area contributed to the persistent neuronal activity required for successful working memory employment during their experimental tasks. From their results they were able to conclude that NMDARs were essential for the short-term memory formation of the relevant information whereas AMPARs were involved in both relevant and irrelevant information whether transient or sustained. This was accounted for in neurochemical terms by the formation of sensory stores relating to the visual cue and then maintenance of these firing neurons in a short-term memory store representing their neuronal firing patterns. This is held in the working memory state whilst the distracting task of alternate visual cue is presented and extinguished. It was therefore, suggested that there was a subset of the firing population which was activated by the stimulus, but switched off when the stimulus was no longer visible and a set of ´delay cells` that exhibited persistent activity. And it is possible that these, as suggested by Van Vugt, are the sub-population of NMDARs that are linked to potassium channels, that of the SK channel which respond on binding of glutamate with an influx of potassium ions. It is assumed that if this is the case, then firing would be abolished and recovery of the cell instigated. Therefore, these neurons would represent those neurons responding to the presence of the actual visual cue whereas the other firing subset (ion channel type not determined) would maintain the neural representation in the working memory state (essentially the short-term memory). The contribution of AMPARs of the DLPFC in their working memory task were to the neural representation of the visual cues independent of location since they were shown to play a role in persistent activity.  The functioning of the NMDARs and AMPARs of the DLPFC cells in response to visual stimuli in addition to the connectivity of the area would lead to the overall performance of working memory and other cognitive capabilities, such as executive control, the maintenance of items and value assessment. Therefore, studies on it are important and may provide subtle ways in which cognitive capability can be influenced.

Since we`re talking about the topic ………

….training has been shown to increase the strength of neuronal connectivity and working memory performance (Astle). Therefore, if subjects undergo a period of cognitive training before carrying out the ODR task can we assume that increases in firing potential and possibly differences to NMDAR and AMPAR contributions may be observed if Van Vugt`s experiments are repeated?

…Van Vugt`s experiments were performed without the monkeys being subjected to interference or distracting elements. If these were introduced and Van Vugt`s experiments were repeated, would we see as expected a decrease in performance accuracy due to working memory impairment, but no change in the relative NMDAR and AMPAR populations contributions in those subjects that performed the given tasks successfully?

…lesions of the basal ganglia lead to working memory being susceptible to irrelevant information (Baier). If these lesions were carried out and Van Vugt`s experiments repeated would we see the number of experimental failures increase as the monkeys were unable to carry out the demanded eye movements back to the target location due to the new visual stimulus location being given higher priority?

 

microtubule and actin differentially regulate synaptic vesicle cycling in calyx of Held tissue

Posted comment on ´Microtubule and actin differentially regulate synaptic vesicle cycling to maintain high frequency neurotransmission` written by L. Piriya, A. Babu, H.-Y. Wang, K. Eguchi, L. Guillaud and T. Takahashi and published in Journal of Neuroscience January 2020 40(1) 131 doi 10.1523/JNEUROSCI.1571-19.2019

SUMMARY

Piriya and colleagues reported in their article the results of their investigation into the presence and activity of microtubules (MT) and filamentous actin (F-actin – FA) with regards to vesicle cycling occurring in the presynaptic terminals of rat calyx of Held. Their work was in response to controversy surrounding the function of these cytoskeleton elements in the presynaptic terminals with some researchers reporting that they are involved in the transport of synaptic vesicles (SVs) that are present in the active zones (such as in the Drosophila) and others finding that MTs do not co-localise with the synaptic vesicles (such as in the lamprey).

The experiments were carried out using the fast synapse of the calyx of Held of male and female juvenile rats. This area is a large brainstem terminal important for the localisation of sound and brain stem slices of between 175 and 250 micrometres were prepared. Before electrophysiological recordings of the excitatory post-synaptic currents (EPSCs) of the brain-stem slices were recorded, the tissue was pre-incubated with strychnine to block GABA A receptor and glycine receptor activities. In order to measure presynaptic membrane capacitance (Cm) a pre-incubating solution containing tetrodoxin, bicuculline and strychnine was used whereas for recording the postsynaptic Cm the solution contained EGTA, magnesium chloride and QX314-chloride. For recording of the simultaneous pre- and post-synaptic potentials (AP) the pre-synaptic pipette solution contained potassium gluconate, potassium chloride, EGTA, glutamate and magnesium chloride whereas the post-synaptic pipette solution also contained arginine. Depolymerisation of the MTs was carried out by incubating the brainstem slices with vinblastine for up to 60 minutes at 35-37 degrees whereas depolymerisation of F-actin was induced by incubation with latrunculin A for 60 mins.

The effect of depolymerisation of the cytoskeleton filaments was assessed using confocal and stimulated emission depletion (STED) microscopy. Appropriate antibody administrations (eg. against beta3-tubulin, alpha-tubulin, VGluT1 and Nucblue) and tissue preparations were carried out for the fixed tissue imaging, live imaging and confocal imaging methods. The Region of Interest for quantifying the fluorescence intensity levels was limited to the calyx of Held terminals. Three-dimensional reconstruction of STED confocal stacks was carried out to estimate the distance between the identified synaptic vesicles (SVs – coloured red) and microtubules (MTs – coloured green). The EPSCs were evoked in the principal neurons of the medial nucleus of the trapezoid body (MNTB) cells by afferent fibre stimulation and whole cell recordings of the excitatory post-synaptic currents (EPSCs) were recorded using the voltage patch clamp technique. Continual stimulation of the EPSCs (STD) was induced by a train of 30 stimuli at 100 Hz and recovery was measured by the EPSCs evoked by the stimulations at different intervals. The calyceal terminals were voltage clamped with evoked pre-synaptic calcium ion currents. The rate of endocytosis was taken as 50% decay time of membrane capacitance change. All of the electrophysiological data acquired was at a sampling rate of 50kHZ and was analysed off-line using standard data software. Data fitting was performed using the least-squares method. The imaging data was analysed using appropriate data management software. Statistical significance was determined by the two-tailed unpaired t-test or one-way ANOVA with a post hoc Sheffe´s test.

Using specific antibodies against alpha- and beta3-tubulin and confocal and STED microscopy methods, Piriya and colleagues found in the brain slices tubulin polymer bundles (green colour) running along axons and going into the calyceal terminals surrounding the post-synaptic MNTB neurons. The tubulin bundles were found using STED to be in proximity with the SVs labelled with vesicular glutamate transporter 1 (VGluT1 – magenta colour) or synaptophysin. Three dimensional reconstruction of STED confocal stacks found that 59% of the SVs were localised on or alongside the MT lattice within 100nm of the whole terminal. In presynaptic swellings, 64% of the SVs were distributed at a distance of greater than 100nm away from the MTs with an average distance of 408nm and 36% of SVs were localised within 100nm of the MTs with an average distance of 44 nm. These results indicated that the MTs extend into the calyceal terminal swellings and that they are partially co-localised with a subset of glutamatergic SVs.

Depolymerisation of the MTs using a vinblastine pre-incubation was carried out and the extent of the MT depolymerisation was monitored by the intensity of  SiR-tubulin fluorescence. The authors found that incubation with vinblastine led to a gradual decrease in SiR-tubulin fluorescence (red colour) in calyceal terminals with time (20min pre-treatment – approx. 30% intensity decrease, 120mins – 90%) and dose. Control experiments using HeLa cells confirmed the result. Immunostaining of vinblastine pre-incubated calyceal cells with antibodies specific for alpha- or beta3-tubulin produced reduced fluorescence intensity staining (30-40%), but VGluT1 fluorescence was not changed. Incubation of cells with the F-actin depolymerising agent, latrunculin A, also reduced SiR-actin fluorescence, but the results were not reported in the given article.

Piriya and team then investigated the effects of depolymerisation of MTs or F-actin on basal synaptic transmission. Pre-treatment with either vinblastine or latrunculin A caused no change in the amplitude, in rise-time kinetics (10-90% rise), or in decay time constant of the EPSCs measured. Neither did they produce a change in the frequency or amplitude of miniature EPSCs. Then, the authors investigated whether depolymerisation of MTs and F-actin affected SV exocytosis or endocytosis. Presynaptic calcium ion currents produced changes in the exocytic-endocytic membrane capacitance (-80 to 10mV), but pre-treatment with either vinblastine or latrunculin A did not produce any changes in calcium ion current and the magnitude of exocytosis or endocytosis kinetics. Therefore, Piriya and team concluded that the depolymerisation of MTs and F-actin had no effect on exocytosis or endocytosis of SVs or basal synaptic transmission in their sample of calyx of Held brain slices.

The authors went on to explore the effect of prolonged stimulation on SVs. STD induced by a brief train of stimulations was not affected by pre-incubation with vinblastine, but the magnitude of the STD did show a general increase with the treatment. This was not shown with F-actin depolymerisation. The size of the readily releasable vesicles (RRPs – around 20nA) estimated from cumulative EPSC amplitudes also showed no difference with the pre-treatments of either vinblastine or latrunculin A. However, an investigation into the recovery kinetics of the EPSCs after STD did show different effects between the MT and F-actin depolymerised samples. Piriya and colleagues found that after STD, the EPSCs underwent a bi-exponential recovery with a component having a sub-second time constant (ifast) followed by a component with a second time constant (islow – approx. 2 secs). Pre-treatment with vinblastine produced no effect on the ifast component or on STD magnitude, but produced a marked and prolonged recovery of the islow component. The effect was strongly correlated to the degree of MT depolymerisation. On the other hand, pre-treatment with latrunculin A prolonged the ifast component, but had no effect on the islow component. The ratio of ifast component to islow component remained the same for both vinblastine and latrunculin A pre-treatments. Co-treatment with both vinblastine and latrunculin A caused prolonged recovery from STD for both fast and slow components indicating that MTs and F-actin differentially contribute to the slow and fast recoveries of the EPSCs after STD.

Piriya and team then investigated whether or not MTs and F-actin play roles in the maintenance of long-lasting high frequency transmission even if there is no significant effect on basal synaptic transmission. EPSCs were evoked at 100HZ for 45s and Piriya and colleagues found that EPSCs continuously underwent a depression during stimulation which was greater in slices treated with either vinblastine or latrunculin A. Both were found to reduce the regularity of the neurotransmission by increasing the number of action potential (AP) failures in the post-synaptic membranes with vinblastine pre-treatment causing the greater effect. Therefore, the results indicated that MTs and F-actin play roles in the high-frequency synaptic transmission of the calyx of Held.

Piriya and colleagues concluded from their studies that their results confirmed previous findings and the findings of others that axonal MTs extend into the terminals of the fast synapses of the calyx of Held and are partially co-localised with SVs.  MTs and F-actin independently and differentially contribute to the slow and fast SV replenishment (essential roles) occurring with STD. Hence, MTs and F-actin microfilaments play roles in the maintenance of the high frequency neurotransmission in this area. The authors claim that the time constant of the slow recovery component found was much faster than that of the SV refilling with glutamate (approx. 7 secs) which suggested that the slow recovery component observed reflected the replenishment of SVs already filled with neurotransmitter from the reserve pool to the RRP. Vinblastine slowed SV replenishment from the reserve pool to RRP, but did not affect RRP size during the brief train stimulations. However, during a long train stimulation vinblastine significantly depressed the EPSC amplitude because of likely RRP depletion and hence, neurotransmission was blocked. Little is known about the slow recovery component except that it requires GTP.

The authors then went on to discuss the role of MTs in this SV transport. Several possibilities were given. For example it was suggested that MTs may directly transport SVs to release sites although MTs have been observed in cultured calyceal terminals to be selectively involved in long-distance SV movements. It was also suggested that mitochondria which are transported along MTs may provide ATP close to SVs to promote their movement. This was considered unlikely because during simultaneous pre-synaptic and post-synaptic AP recordings even with available ATP meant that pre-synaptically administered vinblastine impaired transmission. Also, in cultured hippocampal synapses ATP is uniformly available whether mitochondria are present or not. It was also suggested that SVs tethered to MTs in the terminal may serve as a reserve pool for SV re-use and that these may be scattered after MT depolymerisation, hence reducing the number of SVs available for recycling at the plasma membrane.

The discussion continued with a look at the role of F-actin in the calyx of Held. The authors found in their studies that the fast component of neuronal recovery from STD was prolonged by the depolymerisation of F-actin. The fast recovery component is observed with a large calcium ion influx induced by high frequency stimulation or by pre-synaptic depolarisation and is found to be dependent on calcium ions, calmodulin, F-actin and intersectin. It was found to be absent when EPSCs are at low frequency under normal conditions, at a high frequency without potassium ion channel blockers or when endocytosis is blocked. The fast recovery component was proposed as reflecting the super-priming of SVs through release site clearance and/or fast SV replenishment.

It has been reported by others that F-actin plays a negative regulatory role on exocytotic SV fusion in cultured hippocampal synapses (Morales) and when genetic ablation blocks both clathrin-dependent and -independent endocytosis. Piriya and team found that in the calyx of Held F-actin depolymerisation had no effect on the magnitude of SV exocytosis or EPSC amplitude, prolonged the fast recovery component after STD and impaired the regularity of high-frequency transmission. This indicated to the authors that the depolymerising drug was accessible to the presynaptic target, but elicited no effect on SV endocytosis. Hence, it was thought that more extensive F-actin disassembly might be needed before SV endocytosis would be seen to be disrupted. Ultrafast endocytosis (taking tens of seconds to re-form SVs from endosomes to be re-used) is known to be blocked by depolymerisation of F-actin and this may also contribute to release site clearance as in the case of clathrin-mediated endocytosis. However, these views were disputed by Piriya and team who found that capacitance measurements in the calyx of Held after pre-treatment with latrunculin A had no appreciable effect on SV exocytosis-endocytosis. This indicated that ultrafast endocytosis appears not to operate significantly in the calyx of Held area.

The authors concluded by saying that high-precision, high-frequency neurotransmission plays important roles in brain cognitive functions such as sensory processing, cognition, memory formation and motor control functions. The depolymerisation of MTs in the brain area, calyx of Held, indicates that pre-synaptic MTs normally accelerate SV recycling which is an important factor in the maintenance of integrative synaptic functions forming the basis of cognitive processes.

COMMENT

What makes this article interesting is that it explores the topic of the vesicle transport and recycling systems that play an important role in the normal functioning of neurons. These systems were investigated in Piriya and team`s article in one particular brain area, that of the rodent calyx of Held and under two firing conditions, normal and sustained excitation and gives an indication of the tubulin and actin dependent processes particularly relating to synaptic vesicle (SV) recycling / recovery phase of endocytosis of the neurons studied. The authors chose this brain area because of the ease of experimentation on SV recycling and also it makes a good choice because of its function and overall relevance to the auditory capability of the animal. The calyx of Held receives input from globular bushy cells of the anteroventral cochlear nucleus and sends out fast excitatory signals to the principal neurons of the medial nucleus of the trapezoid body (MNTB) which then sends out fast excitatory signals to the superior olivary complex (SOC) leading to its hyperpolarisation. This fast excitation and eventual hyperpolarisation of the end site (the SOC) are linked to the overall function of the area, that of the location of sound. This capability is important in spatial processing and memory (eg. the learning of the location of food stuffs).

Synaptic vesicle (SV) recycling is important for neuronal functioning and memory and hence, it is likely that the prime function of the vesicles in the calyx of Held neurons is the transport and release of neurotransmitters. It is also likely that there could be a link to ATP since ATP has been found to be concentrated in SVs with neurotransmitters in many synapses. ATP is released into the synapse by the presynaptic action potential spikes in a calcium ion dependent manner. (ATP packaging could explain the observations that it was considered unlikely that mitochondria which are transported along MTs may provide ATP close to SVs to promote their movement because during simultaneous presynaptic and post-synaptic AP recording even with ATP available presynaptically administered vinblastine impaired transmission. Also, in cultured hippocampal synapses it was observed that ATP is uniformly available whether mitochondria were present or not.) It is likely that the SVs and molecular motors and cytoskeleton found in these neurons follow physiological and neurochemical expectations and therefore, before we can understand what effect depolymerising agents have on the actin and tubulin cytoskeleton structure and associated vesicle cycling system we have to understand the processes in general.

The synaptic vesicle recycling process consists of 2 mechanisms: exocytosis and endocytosis. Exocytosis begins with the arrival of the action potential or any other stimulation that leads to the neuron firing. An action potential arriving at the neuron leads to an influx of calcium ions in the presynaptic area to a concentration greater than 0.1mM in the active zone microdomain and this is related to the transmission of the signal further when neurotransmitter release and post-synaptic binding is involved. The neurotransmitters are manufactured in the presynapse and need to be exported from the production site (eg. Golgi Apparatus or linked to local translation sites) to the plasma membrane at the active zone via SVs using the cytoskeleton machinery. The calcium ion increase causes activation of enzymes that phosphorylate particular proteins that cause this movement and subsequent docking and fusion of the SVs at the active zone of the plasma membrane. This fusion occurs by changes in conformation of the SV complexes and docking proteins resulting in the lipid bilayers of the SV and the presynaptic membranes fusing together. The vesicle membranes continue to expand until they are fully incorporated into the synaptic plasma membrane and a pore is formed so that the neurotransmitters contained in the SVs are released into the synapse (the exocytosis process).

The SVs that undergo exocytosis appear to be different sizes and have different functions (Guarnieri). Although imaging indicates that the small vesicles are morphologically similar, it was found that some were more prone to being released than others. Hence, three major functional pools were identified (Guarnieri): the readily releasable pool (RRPs) – a few vesicles docked at the plasma membrane and primed for immediate fusion on neuron AP stimulation so that the neurotransmitters are immediately released into the synaptic cleft; the recycling pool – about 10-20% of all vesicles located away from the membrane, but available for recruitment when the RRP is depleted during moderate neuronal stimulation; and the resting pool – the large majority of SVs with the pool only being recruited when the neuron experiences intense high frequency stimulation or prolonged low-frequency stimulation (such as that seen in the STD experimental set-up of Piriya and team).

The process of exocytosis begins with the action potential causing a flow of calcium ions into the presynaptic area. This can trigger the exocytosis process that is linked to soluble N-ethylmaleimide-sensitive factor attachment protein receptors group (SNARE) consisting of molecules such as synatobrevin, SNAP25 and syntaxin. The exocytosis process also requires other factors such as synapsin, synaptophysin and synaptotagmin proteins (syt1, syt4, syt7, syt11). Each has its own role to play in the process. For example, syntaxin possesses a SNARE domain known as H3 which binds to both synaptobrevin (also known as vesicle-associated membrane proteins, VAMPs) and SNAP25 to form the core SNARE complex which is thought to generate the free energy required to initiate fusion between the vesicle membrane and the plasma membrane. The H3 domain can also bind synaptogamin in a calcium ion dependent manner and interacts with the calcium ion and potassium ion channels on the pre-synaptic membranes and is therefore, linked to fluxes of these two ions. Synapsin plays a role by organising the resting pool (Guarnieri). The increased level of calcium ions in the presynapse is associated with the binding of them to calmodulin to form a calcium ion/calmodulin complex which activates a kinase which phosphorylates synapsin 1 (Syn1). Synapsin 1 normally prevents the SV from moving towards the presynaptic membrane by reversibly tethering the SVs to each other and to the actin filaments of the cytoskeleton. The phosphorylated synapsin 1 releases its associated synaptic vesicle so that fusion of the SV to the membrane can occur. This important phosphorylation can occur with a number of kinases such as the one described above, the calcium ion/calmodulin dependent kinase (CaMK) as well as protein kinase A (PKA), mitogen-activated protein kinase (MAPK) and cyclin-dependent protein kinase 1 (Cdk1). Phosphorylation of Src or Cdk5 increases the ability of Syn1 to bind SVs and actin filaments and hence, hinders the release of SVs.

However, the exocytotic process is not solely dependent on the phosphorylation of Syn 1 as shown by Syn 1 knock-out mice where Syn deletion does not cause the full disappearance of SVs (Siksou). Other factors also play a role eg. synaptophysin and synaptogamins. Synaptophysin is normally associated with the endocytosis mechanism, but it, like the synaptogamin group, is described as being part of the exo-endocytotic coupling determining timing and quantity. The synaptogamin proteins (Syt) are described as calcium ion sensors that are linked to SNARE dependent vesicle fusion, but are more commonly associated in the literature with clathrin-mediated endocytosis (CME). The different Syt isoforms have different emphasis on the two vesicular systems. For example, Syt 1 – more endocytosis, but also rapid calium ion dependent exocytosis (Lu); Syt7 –described as high affinity calcium ion sensor for SV replenishment, but also mediates a slow component of exocytosis (asynchronous release). Therefore, the exocytotic process is in general complex and relies on factors working together.

But the action at the plasma membrane of active neurons relates to two strands of the vesicle recycling process: the pre-synaptic part of vesicle fusion (exocytosis) as described above and signal transmission involving the post-synapse; and the pre-synaptic part of vesicle recovery (endocytosis), which is carried out in order that the firing process can be repeated and the homeostasis of the synaptic area (ie. the vesicle pool) is maintained (Liang, Xie). The relationship between exocytosis and endocytosis is well known (exo-endocytosis coupling – Liang, Xie) and is believed to be timed according to evoked action potentials and fusion of the SV and plasma membranes during the exocytosis phase (Xie). Endocytosis occurs immediately after exocytosis in order to retrieve the SV membrane and it appears that this exo-endocytosis coupling involves some of the same factors, eg. the calcium ion-calmodulin-calcineurin pathway, synaptophysin and SNARE proteins, Syt molecules as well as generic factors such as the membrane lipid structures and proteins involved in phosphoinositide metabolism.  Therefore, the pre-synaptic neuron is not only responsible for exocytosis, but is also the site for the recovery of vesicles (endocytosis).

Endocytosis is a complex set of mechanisms and there area at least three main endocytic pathways, eg. clathrin-mediated endocytosis (CME), activity-dependent bulk endocytosis and the kiss-and-run mode of fast endocytosis. It has already been said that the timing of endocytosis is pre-determined to the time the vesicle fuses to the plasma membrane (Liang) and the release of the neurotransmitters. Again, the basis of the endocytosis mechanism relies on the plasma membrane`s lipid structure and associated proteins and cytoskeleton (Xie). The endocytosis process begins with the expansion of the plasma membrane and the reduction in surface tension following exocytosis which is then believed to initiate the curvature of the membrane in the local area in order for internalisation to occur (Chassefayre). Multivesicular body proteins (Chmp1-7) are believed to play a role by transiently assembling into helical polymers (Chassefayre). The membrane folds to make small pockets and then deepens to form vesicles which separate from the membrane to move into the cell.

Again, a number of factors required, many of which are also linked to the exocytosis process. A general one is the requirement of particular myosins II and IV which are part of the SV replenishment scheme (Xie). Myosin light-chain kinase accelerates both the slow and fast forms of exo-endocytosis through the activity dependent phosphorylation of myosin. Sometimes, however, the factors required depend on the type of endocytosis taking place. For example, bulk endocytosis where a large area of plasma membrane is internalized in response to activity. The membrane forms an endosome-like endocytotic form which eventually turns into releasable SVs by an unknown mechanism; versus kiss-and-run where SVs are rapidly retrieved without the collapse into the plasma membrane.

Clathrin- mediated endocytosis (CME) is probably the most characterized endocytic pathway since it is the predominant route of vesicle recycling although the speed of this type of recycling is slow (10-30 seconds). Clathrin coated pits and other structures associated with bulk endocytosis increase soon after depolarisation and a decrease of the docked SVs occurs. Whereas the Cm readings of a cell rise with depolarisation it then decays with endocytosis. The extent of the values indicates that the amounts of endocytosis relate similarly to the amounts of exocytosis with reference to membranes. Contrary to exocytosis, the role of calcium ions in endocytosis is less clear. It has been reported that a rise in calcium ion concentration is associated with both clathrin-dependent and clathrin-independent endocytosis in neurons (Xie). There are several endocytic calcium ion sensors and factors such as calmodulin which as described above is also involved in the exocytosis process. Calmodulin has been reported to be involved in most forms of endocytosis and calcineurin functions as a key modulatory of the calcium ion/calmodulin complex by dephosphorylating the endocytic proteins, dephosphins. Many proteins in the CME process are dephosphorylated to go from the inactive form to active form eg. dynamin, synaptojanin, amphiphysin and epsin, Eps15.  However, endocytosis can also occur independently of calcium ions and increasing its concentration can also slow the process in some cases.

The sharing of factors between exocytotic and endocytotic processes allows the exo-endocytosis coupling to be controlled to some extent. For example, the SNARE proteins, synatobrevins, syntaxin and SNAP 25 are important for endocytic membrane fusion as well as for that in the exocytosis phase (Xie). The synaptobrevin-2 (VAMP2) is known to be required for the fast component of endocytosis and fast replenishment of SVs in hippocampal neurons (Deak) whereas VAMP2 and VAMP3 is linked to both the fast and slow modes of endocytosis in the calyx of Held. VAMP4 is linked to activity dependent bulk endocytosis in hippocampal neurons. SNAP 25 appears to be associated with the slow endocytic process in the same area whereas again in calyx of Held cells it is linked to both fast and slow modes (Zhang). Synaptophysins interact with dynamin in a calcium ion dependent process linked to the fast endocytic method, CME (Xie) and synaptotagmins (Syts) also are reported to play a role. The C2 domains of their protein structure are well known calcium ion sensors that initiate the SNARE dependent vesicle fusion process. They too are linked to the fast CME endocytosis mode since all Syt isoforms bind the clathrin-adaptor protein, AP2 with high affinity. Hence, Syt 1 is reported as a major calcium ion sensor that promotes CME. Syt11 has been shown to inhibit CME and bulk endocytosis by likely acting as a clamp protein to ensure the coupling of exo-endocytosis is not achieved. Knock-down of Syt11 removes this inhibition and induces excessive membrane retrieval, accelerated vesicle pool replenishment and sustained neuronal transmission (Wang). Synaptojanin (Synj) is also believed to play a role (Geng). This molecule is a phosphatase of the membrane phosphoinositides and promotes the uncoating of clathrin following synaptic vesicle uptake. This action demands that the synaptojanin to be phosphorylated and it appears to regulate positively the number of vesicles in the reserve pool in preference to the active recycling pool.

Other pathways of endocytosis have other demands. For example, the kiss-and-run mode is influenced by Syt7 and Syt11. The kiss-and-run mode is thought to be the fast component of SV endocytosis during which SVs release their neurotransmitters through a transient nano-meter sized fusion pore and then the SVs are rapidly retrieved without the collapse into the plasma membrane observed with the CME mode. Dynamin and the calcineurin dependent dynamin-syndapin interaction appear to be required (Xie) as well as Syt7 and Syt4. Syt7 binds calcium ions with high affinity and slow kinetics, but functions as a slow calcium ion sensor in the slow phase of exocytosis (asynchronous release). It is likely to be involved in the expansion process of the fusion-pore formation and opening via the binding of calcium ions to the C2A domain of the Syt7 molecule (Segovia). However, the resulting pore is unstable and leads to a rapid increase in complementary kiss-and-run mode endocytosis. Binding to the C2B domain however, facilitates the continuous expansion of fusion pores and hence, is likely to be involved in other endocytoc modes of fusion and vesicle recycling (Xie).  On the other hand, Syt4 does not bind calcium ions and have differing effects on the endocytic process. The overexpression of Syt4 leads to kiss-and-run type endocytosis and increases the duration of pore opening in PC12 cells (Wang).

Electron microscope and membrane capacitance recordings have also indicated the existence of an ultra fast endocytosis (Xie). However, the nature of this endocytic pathway is unknown although we can probably assume that it also uses the same factors as those indicated above for the already documented exocytotic and endocytotic mechanisms.

But, with relation to Piriya and team`s work, synaptic vesicle transport and recycling is not randomly organised. It is guided along a network of microtubules and microfilaments that make up the cytoskeleton of the cell and the vesicles are driven along the cytoskeleton by molecular motor proteins. It is the physiology of this network that the experiments of Piriya and team exploit by using agents that specifically disrupt it. In their experiments the depolmerising properties of two agents were used: vinblastine that depolymerises tubulin polymers that make up the microtubules; and latrunculin A that depolymerises the actin polymers that make up the filamentous actin (F-actin or microfilaments).

Microtubules functions include providing the controlled structure of cells (cytoskeleton), as motor protein tracks and is well recognized as important in determining the shapes of cells, separating daughter chromosomes in mitosis and in neurons and relevant to this work, the transport of neurotransmitter from production site to synaptic cleft. The basic building block of the microtubule, which is between 20-30nm in diameter, is the molecule tubulin which exists in two 50Kd forms, the subunits alpha and beta-tubulin. The alpha and beta forms have similar tertiary conformation and are members of the N-Loop ATPases so they contain nucleoside triphosphate binding sites in the P loop. In this case, the nucleoside triphosphate bound is GTP in preference to ATP. The tubulin molecules are assembled in helical arrays of alternating types to form a cylinder and the process of joining them is polymerization. Hence, depolymerisation is the disassembly of the microtubulin structure by chemical action on the tubulin molecules which in the case of Piriya and team`s experiments is induced by administration of vinblastine. Other proteins are also involved in maintaining the structural integrity such as microtubule-associated proteins (MAPs) which can anchor the microtubules to each other or other parts of the neurons and any disruption of these can also cause dysfunction of the cytoskeleton network (eg. pathological changes in axonal MAPs form tau linked to Alzheimer disease). Another example is the mitochondrial GTPase Miro protein which binds Milton/TRAK adaptor proteins linking motor proteins to mitochondria (Babic).

Regarding neurons, the microtubules run longitudinally down the neurites and it appears that there is an organization centre since many appear in a particular region in the cell. This provides the basis of a cytoskeletal network so that vesicles can be transported from one part of the cell to another. In the case of microtubules, vesicles are rapidly ´walked down` the axons from the soma to the terminal using the molecular motor protein kinesin which is powered by ATP (anterograde transport) or ´walked up` from the terminal to the soma using another motor protein, dynein (reterograde transport). The key to this transport is the polarity of the microtubule with the minus end anchored near the centre of the cell and the plus end towards the cell surface. This is important because of the way the two motor proteins function biochemically. There appears however, to be some flexibility in the system with most axons having the terminal as plus whereas dendrites have mixed polarity orientations in the hippocampus and cortex (Yau).

The other component of the cytoskeleton is the microfilament (or as Piriya termed F-actin or filamentous actin) which is found throughout the neuron, but particularly numerous in neurites. This filament is only 5nm in diameter and consists of thin strands of the actin monomer braided together. Each monomer consists of 4 domains coming together to surround a bound nucleotide and the actin monomer is positioned relative to the preceeding one in the helical strand by a rotation of 166 degrees. Other proteins can also be associated, eg. myosin in muscle cells and myosins II an VI which are described as actin-based cytoskeletal motors (Hayshadi). Just like with tubulin and microtubules, microfilaments consisting of actin also exhibit polarity with plus or minus ends and this is what drives biochemically the motor proteins that allow the vesicles to be transported along this cytoskeleton network. The filaments are closely associated with the membrane and attached to the membrane with specific proteins that line the inside of the membrane.

Depolymerisation of the actin filament is exploited in the experiment`s of Piriya and team in the same way as microtubules, but in this case, the depolymerizing agent, latrunculin A is used. This too relies on the capability of the actin filament to assemble and disassemble constantly into and out of strands which is regulated by signals from the neuron eg. actin tyrosine 53 phosphorylation in dendritic spines of rat hippocampus appears increased by induction of LTP (Bertling). The F-actin forms normally by self-assembly and will polymerise until the monomer concentration is reduced to the value of Kd. If the values however, are below Kd then the filament will depolymerize. The critical concentration for the actin-ATP complex is 20 times lower than for actin-ADP complex and hence, actin-ATP polymerises more readily than actin-ADP. The concentration of free actin is controlled by for example, actin sequestering proteins such a beta-thymosin which binds actin monomers and inhibits polymerization. In Piriya`s experiments, the depolymerizing agent latrunculin A is used to promote depolymerisation of the F-actin.

Now that we have given a brief summary of the knowledge regarding vesicle recycling and the role of the cytoskeleton in the neuron, we can go on to discuss what Piriya and team`s experiments added to the overall scheme. Piriya and colleagues performed experiments relating to cytoskeleton functioning and vesicle recycling using brainslices of the rodent calyx of Held. Two activation scenarios were investigated: single excitatory stimulation and high frequency sustained stimulation (STD). The authors found as expected a fast excitatory effect on the post-synaptic membrane and EPSCs were recorded. Administration of the depolymerising agents of microtubules and microfilaments (F-actin) of the cytoskeleton in that area produced no change in the amplitude, in rise-time kinetics (10-90% rise), or in decay time constant of the EPSCs measured. Neither was there any change in the frequency or amplitude of miniature EPSCs, nor in the calcium ion influx currents reported. This showed that the presynaptic neurons acted as expected in response to excitatory firing.  It was therefore, assumed that the vesicles investigated also acted according to expectation, ie. vesicles performed exocytosis and endocytosis with all the elements described above and according to exo-endocytosis coupling rules. It was also likely that the presynaptic vesicles released neurotransmitter for post-synaptic firing and further transmission of the signal. There was no spontaneous vesicle release as the neurons were stimulated and these observations and the firing ones were supported by others.

Imaging studies performed by Piriya and team showed that there was a distribution of vesicles within the presynaptic axons and therefore, we can hypothesise to some extent what type of vesicles these are and what functions they perform. Piriya and team found using their imaging method of 3D reconstruction of STED confocal stacks that 59% of the synaptic vesicles (SVs) observed were localised on or alongside the microtubule (MT) network within 100nm of the whole terminal. Therefore, it can be assumed that the majority of vesicles were likely to be in the ready releasable pool (RRP) attached to the plasma membrane and contained neurotransmitter to be released immediately on stimulation. These would be seen attached to the membrane and others observed further away, could be suggested to be in the other two vesicle pools, ie. in the reserve pool and ready to be released with moderate stimulation (those at distance of greater than 100nm and released under single activation conditions) and possibly also a lower number within the resting pool (ready for release after high frequency or prolonged stimulation). However, this is unlikely to be the case since neither vinblastine nor latrunculin A depolymerising agents of the microtubules and microfilaments affected firing. Therefore, disruption of the vesicle movement scaffolding system which would transport these vesicles to the plasma membrane in response to firing did not effect the function of the neuron ie. the release of neurotransmitter and transmission of the signal. Hence, the response to the firing observed can be assumed to be limited to only the RRPs located at the presynaptic plasma membrane surface.

This observation is supported by others. It has been found that high stimulation of the calyx of Held leads to a decrease in number of the neurotransmitter containing vesicles and in the appearance of labelled endosome-like structures (De Lange). Recovery led to the number of labelled vesicles increasing with a simultaneous disappearance of these endosome-like structures. This was interpreted as the endosome-like structures not participating in the vesicle-neurotransmitter release cycle supporting Piriya and team`s experiments. It was proposed that only a pool of approximately 5% of the vesicles present in total provided sufficient vesicles for release providing exo-endocytotic coupling rules are followed. If this is correct, we have to ask what the functions of the other vesicles are. There are several possibilities. Distribution could mean that the depolymerising agents destroyed the centralised location of the vesicles in pools so that they were distributed throughout the cytoplasm. They could have also disrupted the organisation centre so that strands of actin filaments and microtubules were floating in the cytoplasm with vesicles attached. The vesicles themselves may still contain neurotransmitter in a functional or pre-functional state, but the cytoskeleton structure would not be capable of moving both of these to the plasma membrane. We should also consider that not all vesicles function as neurotransmitter transporters eg. local translation is also linked to endosomes and these may contain the RNA machinery required for protein synthesis. This might be supported by Piriya and team`s observation that 41% of the vesicles were not attached to the microtubules and that a large majority of the SVs (64%) were distributed at a distance of greater than 100nm away from the microtubules with an average distance of 408nm.

Piriya and team also performed sustained activation of the calyx of Held neurons (STD). In this case, they found that sustained activation led to post-synaptic depression and pre-administration of the depolymerising agents increased this depression due to a decrease in the regularity of the neurotransmission. This occurred as a result of an increase in the number of action potential failures. From a neurochemical perspective, this would be understandable if the number of vesicles actually performing the signal function ie. release of neurotransmitters is restricted to only 5% of the total number and exist in the RRP pool. The presence of the depolymerising agents disrupting the cytoskeleton would mean that the transfer of vesicles from the resting pool to the RRP as expected in times of sustained acvitation would not be possible. Neither would the transfer from reserve pool to RRP occur. Therefore, the function of the presynaptic area would be limited to only those vesicles involved directly in the exocytosis-endocytosis coupling cycle. Again, the lack of change in firing in the STD condition confirms that vesicle use and recycling is the same as that seen with normal firing. This is also supported by Piriya and team`s observation that there was no size difference in the RRP vesicles (around 20nA) after either vinblastine or latrunculin A pre-treatment confirming that the RRPs involved were not from the resting pool as expected.

A point to consider is that Piriya`s experiments focused on those vesicles attached to the microtubules and whose function appeared to be linked to neurotransmitter release. Vesicles in the pre-synaptic area away from the plasma membrane could have also been present attached to the actin microfilaments. Experiments with specific actin binding or tagging experiments to identify the specific pools would clarify the matter. For example, the SV-associated protein synapsin 1 organises the resting pool by reversibly tethering SVs to each other and to the actin filaments in a phosphorylation-dependent manner and therefore, imaging using agents specifically identifying synapsin 1 would elucidate whether actin filaments were involved and the location of the vesicles, themselves. Piriya and team did perform experiments with latrunculin A the depolymerising agent against actin, but the results were not reported in detail athough it was said that actin-dependent fluorescence was also observed to decrease.

So, where did the depolymerising agents have an effect in Piriya and team`s experiments? The effects of disruption of the cytoskeleton were principally observed in the calyx of Held relating to vesicle recovery after sustained activation (STD). We know that STD demands vesicle release from the RRP pool and resting pool in exocytosis and that exocytosis and endocytosis are coupled. Piriya`s experiments showed that the process of recovery ie. endocytosis and vesicle recycling involves two processes. They found that there was an initial fast recovery (sub-second time constant – ifast) dependent on actin microfilaments followed by a slow component (second time constant – islow approx. 2 secs) dependent on microtubules. There appeared to be no cross talk between the two which implies that the two separate systems are in play simultaneously. If we look at the first component, the fast endocytosis based on actin involvement, we can hypothesise that this relates to recycling of vesicles likely via the kiss-and-run mode of endocytosis. This is where the SVs release their neurotransmitters through a transient nano-meter sized fusion pore in the plasma membrane (exocytosis) and then the SVs are rapidly retrieved without the collapse into the plasma membrane. This type of endocytosis as seen above requires probably a rise in calcium ions, dynamin and is calcineurin dependent (Xie) as well as factors such as Syt7 and Syt4. The expansion process of the fusion-pore formation and the opening via the binding of calcium ions to the C2A domain of the Syt7 (Segovia) leads to a unstable pore being formed and hence, to a rapid increase in the kiss-and-run mode endocytosis. It could also indicate that the endocytosis mode of ultra fast endocytosis (Xie) could be occurring, but since the nature of this endocytic pathway is unknownit would be difficult to prove.

The observation that in the calyx of Held fast based endocytosis is actin dependent supports the findings of Piriya and team and others eg. Sakaba and Neher who proposed that the endocytosis observed is dependent on calcium ions and calmodulin. The function of the fast endocytosis measured was suggested to reflect the superpriming of the SVs through release site clearance (yes – fusion with membrane to release neurotransmitters as part of the exocytosis process) and fast SV replenishment via kiss-and-run endocytosis mode to promote the recovery of the vesicle system for future neuronal activation.

The second stage of the recovery process was seen by Piriya and team to be slow and dependent on microtubules. A likely endocytotic mode for this slow component is clathrin-mediated endocytosis (CME) which is generally associated with slow recycling speeds (10-30 seconds) and bulk endocytosis. In slow endocytosis, the number of clathrin coated pits and other structures increase soon after depolarisation and there is a decrease in the number of docked SVs. Membrane conductance measurements reflect the exo-endocytotic coupling with a rise with depolarisation and the exocytotic phase and a decay with endocytosis. This type of endocytosis might also be associated with an increase in calcium ions, an involvement of calmodulin and the function of calcineurin acting as a key modulatory of the calcium ion/calmodulin complex by dephosphorylating endocytic proteins, eg. dephosphins. The sharing of factors between the exocytotic and endocytotic processes allows the exo-endocytosis coupling to be controlled to some extent, eg. SNARE proteins, synatobrevins, syntaxin and SNAP 25. The involvement of such a mode in endocytic vesicle recycling could also be assessed by looking for changes in specific factors, eg. synaptobrevin-2 (VAMP2). This factor is known to be required for the fast component of endocytosis and fast replenishment of SVs in hippocampal neurons (Deak) whereas VAMP2 and VAMP3 is linked to both the fast and slow modes of endocytosis in calyx of Held and VAMP4 for activity dependent bulk endocytosis in hippocampal neurons. SNAP 25 appears to be associated with the slow endocytic process in hippocampal neurons whereas again in calyx of Held cells it is linked to both fast and slow modes (Zhang).

The authors interpreted their results relating to the slow component of vesicle recovery after STD as the replenishment of SVs already filled with transmitter and transported from reserve pool to RRP. This fitted in with the CME endocytotic mode where a large proportion of the membrane is excised to become new vesicles as part of the exo-endocytotic coupling vesicle recycling programme. The cytoskeleton component required for such a task appears in the case of calyx of Held tissue as microtubules and this supports other reports, eg. microtubules were found to be involved in long-distance movements of SVs in cultured calyceal terminals (Guillaud) and that during intense stimulation, the presynaptic areas of the calyx of Held elicits bulk endocytosis (De Lange). Therefore, in the case of endocytosis in the calyx of Held 2 endocytotic modes are used: the fast kiss-and-run mode to replenish vesicle number because it does not excise the whole membrane and the slower CME mode associated with more intensive disruption of the membrane. Both show individual demands on the cytoskeleton structure which could reflect individuality of function (eg. actin dependent SVs finally refilled with neurotransmitters and microtubule dependent SVs transferred to the soma for local translation purposes) or of location (eg.  actin dependent SVs recycled to the plasma membrane and the RRP and microtubule dependent SVs recycled further afield and relocated to the resting pool or reserve pool). This hypothesis may explain the observation in the cat calyx of Held where microtubules are observed in the presynaptic terminals and depolymerisation impairs long distance SV movements whereas depolymerisation of actin microfilaments had no effect (Guillard).

From Piriya and team`s results we have extended our knowledge of vesicle recycling relating to neuronal firing and we have found that vesicle recycling requirements can be different due to a number of different factors: single stimulation versus sustained stimulation and tissue specificity. Therefore, like other neurochemical mechanisms there is no single mode or mechanism and the systems have to be flexible according to the demands placed upon it. This makes the study of vesicle recycling an ideal target for further exploration of the effects on neuronal firing.

Since we`re talking about the topic……….

…..can we assume that imaging of brain slices stained with selective markers of synapsin 1 (Guarnieri) should show where the majority of vesicles seen distributed away from the plasma membrane are located?

….chronic incubation with picrotoxin (a GABA A inhibitor) appears to increase tomosyn1 phosphorylation (Guarnieri) and prevents the prolonged inhibition of neuronal activity (synaptic scaling) as a measure of neurons adjusting the strength of excitatory synapses to restore normal activity in the face of chronic disruption. Would a repeat of this experiment with pre-incubation with depolymerising agents show a difference in vesicle recycling in the calyx of Held brain slices?

…..administration of botulinum neurotoxin abolishes endocytosis by cleaving SNARE proteins (Xie). Would the co-administration of botulinum and either vinblastine or latrunculin A demonstrate the roles the different cytoskeleton components have on the exocytosis side of the vesicle recycling process? If vesicular proteins were tagged with PHluorin (a pH sensitive green fluorescent protein) would direct visualisation of the resulting exocytosis be possible?

….can we assume that administration of brain-derived neurotrophic factor (BDNF) which is said to regulate synaptic function and plasticity by slowing down calcium channel activation (Xie) also show that the exocytosis and endocytosis mechanisms observed with calyx of Held tissue are also dependent on calcium ion influx?

implicit and explicit processing of time impacts numerical processing

Posted comment on ´Can implicit or explicit time processing impact numerical representation? Evidence from a dual task paradigm` written by M. G. Di Bono, C. Dapor, S. Cutini and K. Priftis and published in Frontiers in Psychology 8^th January 2020 doi.org 10.3389/fpsyg.2019.02882

SUMMARY

In their article, Di Bono and colleagues described the results of their investigation into the effects of implicit and explicit processing of time on a numbers task with the aim of elucidating whether or not time and numbers share neuronal representations.

Twenty-nine participants were divided into two groups. One group of 15 performed the Single task condition involving comparisons of numbers. Implicit time processing trials were divided into 4 blocks each consisting of 144 trials which were separated from each other by a blank display given for over 1 second. The participants started the trial by being shown a fixation cross for 500ms and then were given an auditory cue (a single tone – the prime) for 300ms followed by the presentation of the target number (termed Forward Stimulus Onset Asynchrony – SOA). For half of the trials, the onset of the target visually presented number corresponded to the offset of the prime auditory cue and this was termed the no-overlap condition. In these cases the SOA could be 100ms or 150ms for the short duration trials or 300ms or 350 ms for the long duration trials. The other half of the participants were presented with the primary auditory cue and the visual target number overlapping for the last 100ms of the prime presentation time (termed the overlap condition). The participants were then asked to perform a number comparison task. On presentation of the target number the participant had to decide whether the target number (2, 3, 4, 6, 7, or 8) was bigger or smaller than 5. Target numbers of greater than 5 required a verbal response of the non-word ´To` and those less than 5 the non-word ´Ti`. For half of the participants these non-words were reversed to disregard unfamiliarity with the response words as a reason for any time differences observed.

The other test group of 14 participants carried out a Dual task condition involving for the first part the same number task as in the Single task condition (the primary implicit processing task) followed a second, explicit processing task where judgement of time was required. The experimental set-up was identical to the Single task condition with the exception that when the response of the number comparison had been given the participants then had to judge whether or not the duration of the presented auditory tone was long or short. The reaction times for all trials were measured and computer analyses of the results including mixed-measures ANOVA were carried out. Some trials were omitted from the overall analysis because of errors, omissions or time delays longer than the required standard.

Di Bono and colleagues found that in general the type of the task was significant with the Single task condition on average faster (541ms) compared to the reaction times of the Dual task (864ms). They also found that reaction time responses were faster when there was no overlap between the two stimuli (no-overlap – Single task condition approx. 528ms, Dual task condition 839 ms whereas with overlap – Single task condition approx.. 554ms, Dual task condition 891ms). The durations of the presented auditory tone were also found to affect the reaction times. Responses were faster with long durations than short durations (long – Single task condition approx. 520ms whereas Dual task condition, 837ms whereas short durations – Single task condition approx. 562ms whereas Dual task condition, 892ms).

Statistical analyses carried out on the results of the reaction time responses indicated differences in interaction between the various conditions. It was found that an interaction between the SOA and task according to ANOVA analysis was significant (F 1,27 = 34.45) and this interaction was more pronounced for the overlap condition than the no-overlap condition. The interaction between SOA and duration of the auditory tone was also found to be significant (F 1,27 = 8.50) with the interaction more pronounced with short duration tasks and in the overlap condition rather than no-overlap. ANOVA analysis also showed the expected significant interaction between number and duration (F 1,27 = 4.82). Paired-samples t-test analysis showed a congruency effect for large numbers and authors found that participants were faster at processing large numbers if target number presentation was preceded by long durations (672ms) of the prime auditory tone compared to short durations (728ms). There was however, no congruency effect for small numbers. Analysis of a three-way interaction of SOA, duration and task also gave a significant result (F 1,27 = 5.55). Analysis of two-way interactions produced a significant result for SOA and duration for the Single task condition (F 1,14 =22.9), but not for the Dual task condition. Other statistical analyses carried out found no significant interactions of three-way or two-way interactions. Statistical analyses of accuracy of the various studies supported the results of the studies on reaction times.

Di Bono and colleagues discussed their findings on the basis of whether or not the results fitted their hypotheses. They first discussed the a priori effect of SOA and duration and their interaction. The authors found in their study that participants were faster and more accurate with long rather than short durations as given by other research in the form of the Foreperiod effect (FP). The SOA effect seen was a result of greater overlapping between time and number processing so that slower reaction times were observed when stimuli overlapped rather than not. The results obtained were only significant for the Single task condition. In this case, the authors decided that the time stimulus would be seen as a distractor. However, when time was specifically processed as in the Dual task condition then the effect disappeared. The authors interpreted this as the effect of duration being related to the ordinal dimension of time (ie. before/after). The accuracy experiments supported the hypotheses and brought according to the authors a new element to the discussion.

The authors then continued their discussion with the effects of implicit and explicit processing of time on the numbers task. They found with their experiments that the only congruency between numbers and time durations were with the Dual task condition (ie. when time was explicitly processed) and with large numbers (ie. those greater than the 5). Faster and more accurate responses were observed for large numbers with long durations compared to the short duration of auditory tone. Their findings led the authors to extend the hypothesis of number-space interaction by showing that time duration modulates the explicit processing of numbers. The classification of duration in terms of short and long was said to enhance the representation of left/right spatial locations along the Mental Number Line (MNL) even if the verbal response did not indicate it specifically. The interaction of time and number was only observed for large numbers and two possible explanations were given: the FP effect would cancel out the congruent effect; or that no congruent effect would be seen with large numbers. Since the FP effect was seen with both large and small numbers the authors concluded that the likely explanation was the former. There are opposite patterns of response for small numbers with FP effect against congruency effect (ie. faster responses for smaller numbers paired with short durations rather than longer).

Di Bono and colleagues continued the discussion with an examination of the asymmetric relationship between time and numbers demonstrated with large numbers. This was said not to indicate a common system for quantity representation, but instead that the representations of time and numbers are partially independent. This view was supported by spatial attention orienting where number processing leads to an automatic shift of spatial attention resulting in increased attention on a specific side of physical space (left – small numbers, right – large numbers) and increased time perception (left – short amount of time, right – long amount of time). However, since Di Bono and team`s experiments showed that only long durations led to a shift of orienting representing large numbers/long durations, it was speculated that there is hierarchical representation for numbers and time where both are explicitly expressed. This is not supported completely by others. For example Dehaene states that spatial attention modulates numbers whether explicitly or implicitly processed and Casarotti shows that numbers modulate the allocation of spatial attention only when numbers are explicitly expressed. From their experimental findings Di Bono and colleagues supported the view that time duration modulates number processing only when explicitly expressed whereas the influence of numbers on time seems to be more automatic. This suggested to the authors that number-time or number-space interaction could be hierarchical on the basis of the level of distraction. They concluded their article by indicating that further investigation of the hierarchical representation of space, numbers and time is required and whether if it is the case, how space and number-space representations depending on specific tasks are used for coding time.

COMMENT

What makes this article interesting is that it looks at the perception of time and how a judgement as to how much time has passed can have an effect on the response time for a simple assessment task. From a biochemical point of view it is essentially a question of attention and deployment of attentional resources and how time is from a neurochemical perspective  perceived and recorded.

Before we discuss what Di Bono and colleagues found out with their study we should look at what their experiments showed from the perspective of cognitive demands. For both groups of participants the first stimulus given was the auditory tone which activated the auditory system and then came the presentation of the visual stimulus in the form of a written number between 2 and 9. Attention would be shifted from the sound stimulus to the visual event because task instructions given to the participants before the experiment began required them to concentrate on the visual event because the following question would relate to their assessment of it. (It is likely that those that were not able to do this and therefore, employed cross modal attention would be removed from the experimental group since their response times would likely fall outside the designated ´standards` and hence, be disregarded.) In the case of where the primary task would be the identification of the visual stimulus as a number greater than 5 (the Single task condition) then activation of the visual system on presentation of the number would lead to object recognition and short term memory formation. Decision-making systems would be stimulated either prior to the question being given since it was predicted following task instruction or at the time of enquiry. This would involve working memory activation in order that the task of assessing whether or not the presented digit was greater than 5. The decision-making process would require the recall of basic maths rules learnt from past experience, the holding of the two (5 and the target number) in working memory (Sanechi) and a decision based on comparison (5 versus the target number – bigger or smaller). The response was given according to instruction and the next trial began. Attentional resources would be likely to remain on the task at hand throughout the trial period.

The attentional demands however, in the Dual task condition where the participants were asked two consecutive questions would be different. In this case, the primary task was just like in the Single task condition ie. to assess whether the presented number was greater than 5, but then the participants had to perform a secondary task of judging whether the time of the overlap between auditory tone and visual stimulus was short or long. The first part of the Dual task condition followed the pattern of the Single task including the type of response given. The secondary task however, involved another decision-making task but this time it was an objective judgement decision of whether the participant thought something was long or short. For this particular task the perception of passed time was required. The other difference between the Single task condition and the Dual task condition related to attentional demands during them. As described above, in the Single task condition task the individual`s attention remained on the task for the entire time. Attention was first on the sound stimulus and then a simple shift (orienting) and reengagement on the visual stimulus was carried out. From a neurochemical point of view, this process requires cortical medial temporal connectivity (Connor).  Disengagement of attention from one event requires activity in the parietal areas and engaging the new stimulus requires activity in the pulvinar nucleus of thalamus. Ventral attention and oculomotor stimulus was shown by Liu to require reciprocal connections with most visual cortex areas of the occipital, temporal, and parietal lobes and involved GABA firing.

Attentional resources for the Dual task condition, however, are different. The task begins with attention on the sound stimulus then disengagement and a shift (orienting) and re-engagement to the presented visual stimulus. However, since time perception is required and the participant knows this from the outset then attentional resources are divided between the recognition of the number during its presentation, sound perception during the duration of its presentation and an overall perception of time passed. The model of time duration judgements by Zakay says that temporal relevance determines the amount of attentional resources allocated. If this is correct then with Di Bono`s experiments full attentional resources would be employed here on the three factors. From a neurochemical perspective, Salz suggested that temporal processing within the hippocampus may be exclusive to CA1 and CA2, but not CA3, and may occur only for a memory demanding task and therefore, the hippocampus in Di Bono`s experiments would be involved not only in the binding of the information of the event together, but also in the perception of time. The situation would also require activity in multiple sensory systems so cross-modal attention is employed. Attention would shift then back to the single visual task when the auditory tone ends and the response is given and would remain on the processing in the working memory whilst the decision-making tasks are carried out even though the content of the working memory would change (ie. presented number and 5 for the first task and length of time for the second). The working memory mechanisms are believed to be the same for the visual, auditory and temporal stimuli (Manohar) and therefore, the strength of firing would be important for determination of the decision.

Bearing this in mind, Di Bono and colleagues observed differences in the times for the tasks as would be expected if attentional demands play a role. If there was an overlap of sound stimulus and visual stimulus then there would be an increased overall time for the task completion consistent with divided attention theories. Although cross modal attention would be utilised with reference to the sensory stimuli since they require different pathways and therefore are not competing for same resources, the holding of the two forms of information in working memory and the switching between the two for the decision-making tasks would mean that the attention switches from one to the other. What is new from Di Bono`s studies is that in the Dual task condition the shorter the overlap, the greater the time required for task completion and vice versa the longer the overlap duration, the shorter the reaction time.  This feels like it is against logical thinking since attention would be divided for longer when the overlap is longer and therefore, longer duration should give a longer reaction time. However, it can be explained if we see the longer overlap as being an advantage to attentional demand switching in the face of competing stimuli. With a longer overlap there would be essentially a processing readiness for, or anticipation of, the required visual stimulus in the presence of the irrelevant, but still attended auditory tone. It could also indicate a faster processing speed once the visual stimulus is observed. Ogden found in relation to pain that there was increased arousal and attention when anticipating and experiencing pain which led to a longer perceived duration of time. This could be because of faster processing in the fear state. With regards to Di Bono`s experiments, the faster processing could be accounted for by a switch to relevant information processing (the numbers instead of the sound) and faster, unconscious processing since the numbers presented were relatively familiar and simple numbers.

Therefore, the shorter overlap duration could be seen as a disadvantage to the working memory processing required in order to fulfil the numbers task. The disengagement from irrelevant auditory tone, switch (orienting) and re-engagement to relevant visual number takes time and the response is required quickly with the short overlap condition. Therefore, the short overlap condition could be disadvantageous for processing as shown by Miller for example. Miller and team investigated temporal summation of event characteristics and found that information close together in time led to an increased change of response since near simultaneous cross modal cues from the same event generated neural inputs more strongly than those temporarily displaced. This could as Tuennermann and team found make it more difficult to ascertain that the auditory cue was not in fact the target cue. Tuennermann and colleagues found that peripheral cues produced target confusion when cueing intervals used were shorter, but the cue increased the processing speed of the target. Also in the case of the Dual task condition, the short overlap time could mean that the numerical task could be seen as a distractor for the required time task and hence, have a bearing on processing speed. Falquez and team found that the presentation of negative or positive distractor words had an effect on reaction times of prime trials because of interference.

Now, that we have established reasons for why a longer duration is advantageous to reactions times for sequential tasks where the target task is the latter one, Di Bono and team added to their findings by showing that the larger the number in the number task then the quickest response in the Dual task condition came with the larger overlap durations. Di Bono and team gave explanations for this relating to the Mental Numbers Line and Mental Time Line. The largest numbers correlated to the longest times and the left visual field along the line and vice versa. The authors believed that there is a hierarchical representation of numbers and time when both are explicitly expressed, a view as stated above in the summary as not agreed by all researchers.  Our view of the Di Bono`s results is that their findings may actually indicate that numbers and time are interrelated concepts with relation to neurochemical firing and brain activity. It is possible that numbers are neuronally coded in the same way as timing with respect to location in the perception and storage of spatial information. If this is correct then the Mental Time Line and Mental Numbers Line may not be left to right on a straight line where left is the shortest time/lowest number and right the longer time/larger number, but forward to back with forward being the shortest time/lowest number and further back the longer time/larger number. This concept could come about from the way the eyes process visual information relating to spatial position and is cognitively important in the processing and planning of movement and actions. It relates to rules about distance and retinal expansion in response to movement of visual stimuli (Lee). If this is the case, up close is likely to represent the smaller number because the eye accommodates lower numbers of objects, but of a greater size. This would be equivalent to shorter times if we equate this to the time to contact. Larger numbers would be represented by distant or far locations. It is easier to see a crowd of people from a distance rather than close up where only an individual or two would be visible in the visual field. From a distance the time to contact is longer. Therefore, numbers and time could be related to location and this provides for numbers a neurochemical means to its cognitive representation and for time, a second means of expression. (Time can also be from a neurochemical perspective represented by event order such as in sequences and hippocampal firing). Support for spatial attention and spatial working memory relating to time comes from some neuronal firing studies. For example, Xu found using transcranial magnetic stimulation that if it was administered to the right posterior parietal cortex which is known to be involved in visual spatial attention then slower mean reaction times in target presentation following a spatial cue were recorded. Administration of transcranial magnetic stimulation to the left side led instead to faster response times due to increased alerting and spatial orienting functions. Hallock found that both medial prefrontal cortex and ventral hippocampal activity was required for spatial working memory functioning and activity in these areas produced theta oscillations (Lim) when connectivity was recorded. And Santangelo reported increased parieto-temporal functional connectivity where integration of sensory cues and the memory for storing objects locations was required to encode object related spatial information in working memory.

This view that the representation of numbers and time are interrelated and therefore, larger numbers with the overlap period of the longest duration giving the quickest response can indicate not only the positive changes in attentional demands as described above for the Dual task condition, but also the positive effects on response time due to task similarity. Both targets of numbers and time use the same neurochemical mechanisms and therefore, activation of the same mechanisms would result in quicker responses. It could also be explained by our learnt ways of dealing with numbers and the attentional resources we associate with numbers presented relating to size. For example, the larger numbers would be subjected to ´gist processing` associated with simply ascertaining whether the number is greater than 5 whereas the smaller numbers would be processed accurately and detailed as normally carried out for lower numbers of the 1-9 range. Attentional resources would then benefit from the longer duration. The fact that short overlap durations produced no significant results for small or large numbers points to the attentional demand differences described above and the difficulty in processing the two competing event characteristics in view of their interrelated neuronal systems. Morrone found that successive stimuli led to compression of time and an underestimation of the time interval between two stimuli and therefore, it is possible that for the low numbers/short overlap group the results were omitted from the mathematical analysis as inaccurate times outlying the standards.

Therefore, to conclude we know that time perception can be altered by a number of different factors such as language, fear, ADHD and even boredom and we showed in the blog post of March 2020 that our perception of the passage of time can influence how we approach future orientated events. Di Bono and team`s article gives another possible aspect to time perception in that it could also be related to number processing. If this is true then it may provide other possible approaches for research into both topics.

Since we`re talking about the topic………

…sufferers of hemispatial neglect are reported as not being capable of drawing on a time line corresponding to the side of their defect. If Di Bono and team`s experiments were repeated can we assume we would see a difference in the larger number/longer duration / quickest response results if the Mental Time / Number Line hypotheses are correct, but would see no difference if the forward/back hierarchical representation view is correct?

…mindful meditation leads to an increased sensitivity to time and a lengthening of perceived time (Colzato). Can we assume that if subjects who practised mindful meditation on a regular basis performed the experiments of Di Bono and team differences in results for for both Single and Dual task conditions would be observed? Pain is also reported to increase the perception of time (Ogden) and therefore, would these results also be different if moderately but acceptable painful stimuli (ie. one hand in iced water) were administered before and during the experiments?

…..it has been shown that the precision of memory recall is worse for the presentation of a single item when more items were expected than if more items are expected and received (Monohar). If Di Bono`s experiments were repeated and two digits were presented instead (even if in the same numerical range of above or below 5) would reaction times be longer because of interference?