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 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 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 (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 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?

 

Posted in glutamate receptors, prefrontal cortex, Uncategorized, working memory | Tagged , ,

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?

Posted in endocytosis, exocytosis, neuronal firing, Uncategorized, vesicle recycling | Tagged , , ,

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 8th 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?

Posted in attention, time, Uncategorized, working memory | Tagged , ,

age-related differences in short-term memory capability and confidence in decision-making

Posted comment on ´False recognition in short-term memory – age differences in confidence`  written by B. Sikora-Wachowicz, K. Lewandowska, A. Keresztes, M. Werkle-Bergner, T. Marek and M. Fafrowicz and published in Frontiers in Psychology 13th December 2019 doi 10.3389/fpsyg.2019.02785

SUMMARY

Sikora-Wachowicz and colleagues described in their article their investigation into age-related differences in short term memory (STM) from the perspective of the level of incorrect recognitions (false alarms, FA) in a visual object matching task and the levels of confidence in their judgements.

Sixty one participants (31 with an average age of 21 – the younger group and 30 with an average age of 60 – the older group) performed a short term memory task based on visual recognition of an object and matching. The target item was a single relatively complex abstract object and a visual mask was used instead of a distractor. The objects chosen were to prevent long-term memory being a factor. Each participant performed 120 trials each of which consisted of the following procedure. A single abstract object was presented for 2000ms followed by inter-stimulus interval (ISI) of 800-1200ms. The visual mask was then presented for 2000ms followed by a second interval for 800-1200ms. Then, the participants performed a memory test for 2000ms where they were asked to judge whether the second presented object was the same or different to the memorised object. This was followed by a blank interval of 300-700ms and then the participants were asked to express their confidence in the correctness of their judgement by rating their response on a scale of 1 to 3 where 1 was unsure of correctness and 3 certain. A period of 2000ms was allowed for this followed by another interval of 2500-4500ms and then a flashing fixation point was presented for 550ms. This was followed by the next trial. In all 40 of the visual masks matched the target, 40 were similar to the target memory (Lure) and 40 were clearly distinct (Foil). Correct identification of the mask matching the target was termed a Hit and an incorrect answer a Fail (false alarm, FA). The overall STM performance was analysed according to the sensitivity index (d´) which is a measurement of accuracy derived from Signal Detection Theory and calculated as the subtraction of Hit minus FA rates. In order to assess confidence in the response the metacognitive sensitivity index, type two d´ was used instead. This involved calculating high confidence correct responses as a proportion for all correct responses for both targets and Lures (type two Hit) and high confidence incorrect responses as a proportion of all incorrect responses for the same group (type 2 FA) and subtracting the two values.  In order to investigate whether confidence was attributed at the same level for true recognitions (Hit) and false recognitions (same responses to Lures, FA) a mixed measures ANOVA analysis was then carried out. In order to investigate reaction times (RT), a further two mixed measures ANOVA analyses were performed. The first involved within person factor response type (Hits versus FAs) and the between person factor age (Younger, Y vs Older, O) and the second regarding confidence RTs with the same parameters.

Sikora-Wachowicz and colleagues found when they compared with their sensitivity index d` the FA to Lures versus the FA to Foils and the between-person factor age (Y versus O) that there was a significant effect of trial type, a significant effect of age and an interaction. Further mathematical analysis using HSD Tukey showed age differences resulted from changes in d´ related values for FA to Lures, but not FA to Fails. HSD Tukey analysis also found no significant age difference in the rate of FAs or in the rates of Hits. The interaction result came from the Hit rate being higher than that for FAs for each age group.  These results were confirmed with further mathematical analysis using mixed measures ANOVA accuracy with response type (Hit versus FA to Lures) forming within-person factor and age as a between-person factor (Y versus O). Significant effects were seen with response type (ie. false to correct recognitions, Fail to Hit) and an interaction effect, but not an effect with age. Therefore, the authors concluded that their experimental procedure was effective in eliciting false recognitions based on perceptual similarity. The observed differences in STM accuracy, measured by the sensitivity index d´ involving Hits and FAs to Lures but not to Foils, was contrary to expectation and not brought about by a higher FA rate for older subjects, but to a non-significant age difference in performance of both positive and Lure trials.

The second part of the experiment involved the participants judging personal confidence in the correctness of their answers. Sikora-Wachowicz and team ensured by their experimental set-up that any changes observed were not attributed to personal monitoring differences since no differences were seen between the younger and older groups in the use of the confidence scale, confidence distribution and ISIs after reported errors.  The type two d´ analyses carried out involved calculating high confidence correct responses as a proportion for all correct responses for both targets and lures (type two Hit) and high confidence incorrect responses as a proportion of all incorrect responses for the same groups (type 2 FA). Then, the two values were subtracted. The result showed that the type two d´ values for the Y and O groups were significantly different and therefore, indicated significant age differences with the older adults presenting lower sensitivity to inaccuracy than the younger ones. The mixed measure ANOVA tests with within-person factor response type (Hit versus FA) and the between-person factor age (Y versus O) also showed significant effects of age, response type and an interaction effect. In this case, the HSD Tukey analysis showed that average confidence for FA responses by the younger adults was significantly lower than confidence by the older participants. Confidence was also lower in FA compared to Hit responses in the younger adults as well as in the older ones. The confidence in Hit responses was comparable between age groups. Therefore, it was concluded that older adults demonstrate generally poorer metacognitive abilities, but higher confidence in their inaccurate acceptance of Lure items as target memories.

A possible cause for the observed differences in confidence between the age groups was an age related difference in response reaction times and so this was investigated by the team. An ANOVA mixed measure analysis showed significant effects of response type and of age, but not an interaction effect. It was therefore, concluded that older adults presented slower responses, but both groups were slower for FA than Hits. This indicated that age differences in confidence were not explained by age differences in trade-offs between speed and accuracy. The second ANOVA mixed measure analysis also showed a significant main effect of response type (Hits versus FA) and an interaction effect, but no effect of age. Further analysis using HSD Tukey showed that response times of confidence judgements differed significantly between Hits and FA for younger adults, but not for older ones. Therefore, Sikora-Wachowicz and colleagues concluded that there was no age difference in recognition response times, but age did affect the response times of confidence judgements. Since recognition RTs for Hits were faster than FAs for both age groups then the levels of monitoring and associative processes used in both age groups were similar. However, the authors also concluded that since the younger participants showed the same RT pattern (slower RTs for FA compared to Hits) in confidence judgements then they demonstrated greater engagement and/or effectiveness of monitoring processes than the older participants did.

In conclusion, Sikora-Wachowicz and team found with their experiments that the lower STM performance observed with older people was not due to a greater susceptibility for incorrect recognitions, since they found, in contrast to other researchers working on episodic memory, that the errors with visual STM did not differ between the older adults and the younger ones. The observed age difference was due to the older participants` lack of capability to discriminate target and Lure, but not the unrelated Foil. This was attributed age differences in executive functioning and reduced or distorted retrieval of details. This is supported by other reports which have linked decreased memory capability with age with working memory reduction and information binding difficulty. With regards to confidence, Sikora-Wachowicz did find, just like with other researchers of episodic memory, that older adults were more confident with false responses than their younger counterparts. It was suggested that with regards to STM older adults do not lower their confidence after errors to the same extent as younger adults and this was attributed to both associative and monitoring impairments. These were linked to top down control and strategic monitoring being necessary for effective working memory and both of these have been reported as being impaired with age (eg. reduced retrieval of perceptual details accompanied by prefrontal monitoring during correct recognitions, but not FAs -Dennis; and the overreliance on familiarity based monitoring being strengthened in older adults and therefore, demonstrating an  overconfidence in FAs -Yonelinas). The observations were supported by the results of the response times measured in Sikora-Wachowicz and team`s experiments. It was found that there is no age modulation with RT in recognition tasks, but in confidence and this difference could not be attributed to different information processing strategies or associative impairments. Younger adults appeared to make faster confidence decisions after Hits than after errors, an effect that was not observed for the older adults. The difference appeared to be because the older adults have diminished and/or ineffective monitoring and cognitive control which could not compensate for the reduced retrieval of event details. The authors ended their article suggesting that further research is required in a number of areas to clarify their age modulation observations.

COMMENT

What makes this article interesting is that it continues the investigation into ageing and effects on cognitive capability in what would be a simple everyday type task. An individual is asked whether something matches something else and then has to express confidence in their position. The cognitive processes that have to go on to achieve this are wide-ranging. An individual has to employ visual processes, attention, short term memory, retrieval and recognition and then decision-making processes for example. The one cognitive capability not put under test is long-term memory which we know from many reports can suffer from age modulation. Long-term memory is excluded because the experimental set-up is such that the individual is presented with a single abstract visual event and given time to see it and store it in short term memory as gist and details to an extent that relates to personal capability. From the outset, the individual knows that this neural representation needs to be retained for only a short amount of time ie. until he/she has been asked to decide whether it matches the current presented object. This whole procedure occurs within a minimum of only 3600ms (the confidence question is not essentially part of the short-term memory since it can be regarded as a feeling which is less time sensitive) and therefore, long-term memory mechanisms are not needed.

Therefore, using their experimental set-up, Sikora-Wachowicz and team were able to make some observations with relation to memory performance and age. The first conclusion made related to the recognition success for what they termed Hits. This meant that the subjects recognised the second image as being the same as the memorised one and responded as such. The authors found that this capability was relatively the same for older and younger participants and there was greater success at recognising complete matches than anything else. Many processes and mechanisms are required for this such as short term memory processes (storage and retrieval ) plus holding of information,  divided attention, shifting attention, working memory capability as well as the decision-making process of matching ie. a simple yes/no type question and answer decision. Therefore, the authors determined that with their experimental set-up that age was not likely to be a factor in determining performance.

   However, certain neurochemical deficiencies are known to occur with ageing and therefore these were not particularly seen in Sikora-Wachowicz and team`s experiments. For example, an age-related decrease in working memory performance has been observed related to GABA dysfunction in the prefrontal cortex (Banuelos) and associated with elevated hypothalamic-pituitary-adrenal (HPA) axis activity and impaired hippocampal function (Anderson). There is also a reported age effect with regards to multitasking (Motluk) which may be said to be occurring here since there has to be retention of a neural representation of one image whilst another image is being processed.

There are a few possible reasons why age related modulations were not observed with Sikora-Wachowicz and colleagues` experimentation.  The first indicates the stability of neuronal population firing. This means that minor changes in one region`s firing capability can compensated for by firing in another. For example, Spaak found that working memory depends on the persistence of stable neural representations, but the evidence suggests that the neural states are highly dynamic. Spaak looked at the lateral prefrontal cortex in a memory task and found that there was dynamic population coding associated with working memory and therefore, ageing may affect local neuronal firing capability. However, the dynamic coding could mean that other areas take over this function. This is also supported by work by Tsventanov who looked at how cognitive capability is maintained from decay with ageing by extrinsic and intrinsic brain network connectivity. Tsventanov and colleagues found that successful cognition is determined by the interaction both within and between large scale functional networks such as the salience network, dorsal attention network and default mode network. Age was said to influence connectivity both within and between these networks and that cognitive performance relied on neural dynamics more strongly in older adults than younger. The effects are driven in part by the reduced stability of the neural activity within the networks. Lighthall was even more specific giving the ventromedial prefrontal cortex area as the area that experiences age-related increases in activity to compensate for the decline in memory dependent decision-making performance relating to choice.

Another possible reason why age related modulations were not observed with Sikora-Wachowicz and colleagues` experimentation is that the cognitive demands of their task did not involve areas of working memory affected by age. For example, Gayet found that visual working memory is used to maintain visual information available for subsequent goal-directed behaviour. The content of this visual working memory is known to affect the behavioural response to simultaneously occurring visual input. This suggested that the visual working memory representation and the sensory input representation had shared neural populations. According to Gayet, the visual working memory therefore, enhanced the neural response to the simultaneous visual input in a content-specific way. Therefore, if ageing had in the individual caused localised deficits with certain visual characteristics for example then presentation of the new second image would be compensated for by the working memory representation of the first and therefore, age related modulation would not be observed.

This is further supported by work by two other research groups. For example, Cogan found that verbal working memory involves storing and manipulating information in phonological sensory input. The manipulation is carried out by a number of central executive systems and storage is performed by two interacting systems: a phonological input buffer for sound based information and an articulatory rehearsal system that controls speech motor output. Therefore, in this case the experimental set-up of Sikora-Wachowicz and team may not involve areas where age modulation has occurred and if it has as stated above, compensatory mechanisms may be in place.

In the same vein, the simplicity of the images presented may not be enough to indicate the extent of age-related modulations. For example work by Ji and team on implicit and explicit working memory showed that these two capabilities have separate mechanisms. Ji`s experiments involved a visual search task where the subject individual was presented with four consecutive search displays with locations of a pre-determined pattern and then a fifth which could follow the same pattern or be different. The response time for the fifth search was quicker when the implicit working memory had followed the pattern compared to when it had not. These results shows that in the case of Sikora-Wachowicz`s experiments, implicit working memory may have had a controlling influence on the matching performance and this may or may not be subject to age modulations. The images presented may be of a simplicity that the difference in reaction times could not be discerned between the faster implicit working memory performance and the more deliberate consciously aware explicit working memory. In a similar vein, work by Arias-Cavieres supports the lack of effect on short term memory performance with age because of different processes being engaged. In their experiments older rats demonstrated deficient recognition memory between hippocampus associated object location tasks and perirhinal cortex associated novel-object recognition tasks when long-term memory was tested, but there was no defect observed with short-term memory. In this case, the two different task demands of retrieval and familiarity judgements involve different brain areas which may or may not be subject to age modulations in individuals.

Another possible reason why age related modulations were not observed with Sikora-Wachowicz and colleagues` experimentation is that the average age of the older group at 60 was in general not old enough to see advanced neurochemical degeneration of a type that affects overall performance. Therefore, expected changes were not particularly observed. As a control a group of much more senior participants would have to be included in the experiment.

A more obvious result of age modulation was achieved by Sikora-Wachowicz and team in their experiments where the second image was similar to, but not the same as the first (the Lures). In this case, over 50% more older people gave the same response as the younger for the Lure even though this was still only 10% of the Hit rate in general. Also over 50% more older people had missing responses for the Lure than their younger counterparts. The high rate of responses matching the younger adults´ responses indicated that in general the success rate of identification of similar object appearance by the older participants was still at an acceptable level. This showed that, just like with the Hit success, that the functional interactions of the distributed brain areas required for successful recollection of specific context as indicated by King were to the most extent functioning adequately. However, the higher rate of Lure responses with the older group means that the more senior participants were slightly less capable of identifying the Lure as definitely not the same as the original first image that they memorised. The difference probably lies in the level of detail of the characteristics that the original memory had rather than the more general outline or colour/contrasts. This view is supported by work on multitasking where performance is lower where there is task similarity. It is also more relevantly supported by the work of Aizpurua and colleagues on autobiographical memory who found that there were modest age-related changes in the specificity of autobiographical recall observed. In this way, the older participants of the Sikora-Wachowicz`s experiments could not distinguish accurately whether the images presented matched 100% (the Hit) or not (anything less than 99% the same) and therefore, the level of Lure responses for the older group would be higher. But also from Aizpurua`s work, the success of both age groups was affected relating to detail. Aizpurua`s work on autobiographical memory showed as said above modest age-related changes in the specificity of autobiographical recall, but a more robust effect was for both age groups on the proportion of specific details retrieved.

This deficit in memory detail leading to poorer discrimination between same and similar images is also supported by the results of the omissions scored in Sikora-Wachowicz and team`s experiments. The higher omission result of the older participants means that they were unable to make a decision within the time given whether from conscious uncertainty of the answer or unwillingness to commit to the answer they believe unconsciously is not correct. This observation is supported by work by Guidi and colleagues on the functioning of the medial prefrontal cortex where an age related increase in omissions was related to decreased glutaminergic transmission in this particular brain area. The younger adults in Sikora-Wachowicz`s experiments also demonstrated a 60% increase in omissions for the Lure trials than for the Hit ones which also indicates that they too prefer to omit to answer than to commit to an answer that they suspect is incorrect. It could be suggested that that the time limit given for responses did not allow the processing mechanisms to complete satisfactorily with the older participants. Ford and colleagues found that there is an age-related decrease in the recruitment of posterior sensory regions with an increase in recruitment of prefrontal cortex regions and a temporal shift in which younger and older adults recruit the same neural areas, but at different points when faced with multiple cognitive tasks. The later elaboration stage relating to detail of the memory retrieval task undertaken led to increases in posterior region activation in the older participants as with the activation of the prefrontal cortex regions. Therefore, time limits on responses may prohibit the engagement of these posterior regions in the case of the older participants. However, this can be discounted in Sikora-Wachowicz and team`s experiments since response times showed that although the older participants were slower there was not a significant difference with age for the Lure responses.

The results with the Foils ie. those second images that were distinctly different from the first memorised objects reaffirm the view that the age modulation observed in Sikora-Wachowicz`s experiments was not at a level that would cause significant life-style changes. A very small percentage of older participants failed to make the same judgements as the younger ones with regards to Foils, but the levels of omissions and different responses were the same. This means that the Foils were in general clearly recognised. The difference between older participants and younger capabilities was shown to be instead with the level of confidence that the two groups associated with the decisions they made. With regards to Hits, then both older and younger participants performed roughly the same. This supports the results and views given above with image recognition that both groups were certain and had high levels of confidence in their matching capability with objects that were the same. However, the difference lies with the confidence that the participants showed with images that were not similar rather than distinctly different. In this case, the older participants demonstrated a confidence level roughly the same whether the image was the same or incorrectly assessed as being different whereas the younger adults showed the same level of confidence in their answers to the same images, but were less confident for those that were incorrect. This was interpreted as the older participants having a lower sensitivity to inaccuracy than their younger counterparts.

So, what can we deduce from this from a neurochemical perspective. The question about confidence also requires processes and mechanisms relating to a variety of different cognitive capabilities. Short term memory processes plus holding, divided attention, shifting attention, working memory capability are all required, but also those relating to emotional awareness eg. How do I feel about my memory about the object and my success at my matching performance? The older participants appear more confident in their memory capability and decision-making approach whether correct or not and therefore, it was concluded that the monitoring capabilities of the older participants is impaired. Either were they incapable of noticing differences in the image or they were more prepared to take a riskier decision based on uncertain knowledge than admit they did not know. In this way we can associate confidence with risk.

When we look at the effect of age on risk in decision-making, previous research has shown us that the approach of older participants to risk is different to that of younger adults and this may provide an explanation as to why confidence differences were observed in Sikora-Wachowicz and team`s experiments. Goh found that ageing could compromise in some individuals the frontal, striatal and medial temporal areas of the reward system and impede accurate value representation and feedback processing critical for decision-making. Older participants in their lottery choice type task made less optimal decisions and accepted stakes when losses were likely and declined stakes when gains were likely. This inability to assess risk optimally was associated with frontal activity differences. Fernades also showed that there were age-related differences in some individuals in decision-making where there is uncertainty and outcomes were learnt through feedback from previous choices. Older participants were more likely to make riskier choices when learning should have shown them to take the opposite approach. However, they were also more prone to not taking risks when they should have been more able to take risk. Therefore, decision-making in cases where there is not a clear option was different for older than younger adults. Decisions were there were clear cases of risk showed that both younger and older adults had similar decision-making behaviour. We can associate these observations with the results of Sikora-Wachowicz and team`s experiments in confidence. The older participants took riskier decisions in the form of stating similarity of the Lure to the target image than their younger counterparts. However, in the case of the Foils, the images that were distinctly different and clear cases of risk, then both older and younger adults had the same decision-making behaviour.

Another aspect of this higher risk taking is possibly the need to succeed being higher for the older adults than the younger ones. Talmi found that emotional stimuli exert parallel effects on attention and memory -induced arousal by varying the participants` goals. In their experiments, hungry and sated participants encoded food and clothing images under divided attention conditions. The sated participants were found to attend to and recall food and clothing images equivalently whereas the hungry participants performed worse on the simultaneous discrimination task when they viewed food images relative to clothing images. This suggested that attention was enhanced to food images and that recall was higher for the food images than for the clothing ones and attention to the simultaneous task was distracted. Therefore, memory capability can be enhanced when the need for something is increased by more distinctive processing of the relevant images. In Sikora-Wachowicz`s experiments, the need to be successful at the task performance especially with the known link between age and reduced memory performance would lead to more distinctive processing of imaging and a higher level of confidence in capability being admitted by the older participants even if misplaced.

Therefore, we can conclude that as in most things relating to the brain, individual capability need not reflect group`s capability and that the plasticity and differences in physiology and functioning level will allow some people to function at levels independent of age and others to suffer from impairments, whether minor or severe. From a short-term memory point of view, the age of 60 does not appear to be a milestone in short-term memory performance, but it does indicate that an individual`s attitude and therefore, emotional state may play a role. A more confident emotional state may be misplaced when actual capability does not support it. With emotional status being an influencing factor of cognitive processes, maybe we should approach this with reference to increased age as being positive and a means of boosting neuronal mechanisms or compensating for deficiencies that may be beginning to appear.

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

….. can we assume that if Sikora-Wachowicz and team`s experiments were repeated with altered conditions such as increased number of stimuli, more limited response times and the introduction of stress or fear that the age-related effects on confidence and differences in cognitive capabilities with older participants would be more apparent? Would measurements of neural activity using fMRI of the prefrontal cortex show reductions or increases in activity relating to function as expected during the progress of the task under such conditions?

…. It has been reported that the systemic administration of a GABA B receptor antagonist, CGP55845, increases in a dose-dependent manner working memory capability in aged rats (Banuelos). If GABA antagonists could be administered and the experiments of Sikora-Wachowicz and team repeated would the performance of the older participants match that of the younger in every aspect?

…the cumulative exposure to glucocorticoids has been cited as a central underlying process in age-related prefrontal impairment (Anderson). Would the administration of glucocorticoids to the younger participant group lead to cognitive performance levels decreasing comparable to their older counterparts?

… it has been said that deficits in working memory can be alleviated by training (Matzell). Toril showed that video game training had positive effects on visuospatial working memory and episodic memory performances with older adults. If the older participants of Sikora-Wachowicz´s study were subjected to training sessions of this nature before the experiments were carried out can we assume that the recognition performance of this older group would be enhanced?

…it is known that anxiety has a negative effect on multitasking performance (Wicken). Can we assume that anxiety would have different effects on the performances of the older and younger participants if the experiments of Sikora-Wachowicz and team were repeated due to the susceptibility of particular brain area functioning?

Posted in ageing, emotions, memory recall, short-term memory, Uncategorized, working memory | Tagged , , , ,

possible role of action potential backpropogation in pyramid cell apical dendrite tufts

Posted comment on ´The stochastic nature of action potential backpropogation in apical tuft dendrites` written by S.M. Short, K.D. Oikonomou, W.-L. Zhou and C.D. Acker and published in Journal of Neurophysiology 2017 doi.org/10.1152/jn.00800.2016

SUMMARY

Short and colleagues investigated the variability of calcium ion signals relating to action potential backpropogation (bAP) found in some apical dendrite tufts of pyramidal neurons found in the prefrontal cortical layer 5. They began their article with a description of apical bAP in the L1 layer of the cortex which is known to be involved in processing of information from other cortical areas as well as emotional states and this exists via glutaminergic and hormone modulatory input. Modulation of the bAP in distal apical dendrite areas was found to be brought about by small changes in single voltage-gated dendritic conductance and therefore, the authors continued with a description of factors that could affect the bAP eg. distance of the AP initiation site from the soma. Short and colleagues` aim was to investigate the bAPs found in another cortical prefrontal cortex (PFC) layer, that of layer 5 (L5) as well as another brain area, the somatosensory cortex.

In their experiments, backpropogation was induced in rat brain slices and regions of interest from whole cell recordings were identified. Optical recordings of the AP-associated calcium ion conductance in the apical tuft dendrites were taken for periods of 2-5 minutes for an approximate total time of 100 minutes. Three APs were induced by brief current injections and the calcium ion influx was recorded in the distal apical tuft branches. In experiments where potassium ion antagonists were administered, these were applied locally. Then, dendritic calcium and voltage imaging were carried out and the optical signals computer analysed. In the case of the calcium ion concentration changes, not all the background values were substracted because levels of the change were thought too low, eg. in the distal regions. In the case of the voltage imaging experiments, 3 to 9 trials were averaged to improve signal to noise ratio. Modelling of the tuft PFC layer 5 pyramidal cell dendrites was carried out using the computer modelling program NEURON. The distribution of sodium ion and potassium ion (including A-type potassium ion  channels)  channels was carried out according to previously published studies.

Short and colleagues` results showed that somatic current injections of the L5 PFC apical dendrites induced triplets of APs with interspike intervals of 120ms and a low AP frequency of 8.3HZ. The action potential produced appeared to be similar to that observed elsewhere since it involved sodium ion channel activation in the apical tuft dendrites. Application of glutamate triggered local depolarisation in the apical tuft dendrites (termed dendritic plateau potentials) which was preceded by fast sodium ion spikes. These fast sodium ion spikelets peaked before the somatic AP and therefore, could be distinguished from the bAP. When tetradoxin was used to block the sodium ion channels it was found that a local distal application reduced the amplitudes of the large voltage transients in the tuft dendrites and also caused the loss of the second and third spikes. No effect was observed for those cells that did not display APs.  Therefore, the authors concluded that voltage gated sodium ion channels are present in apical tuft dendrites of PFC layer 5 and these had activity related to the generated AP. Regarding the AP-inducing calcium ion signal, nearly 70 out of 196 pyramidal cells failed to produce any calcium ion signal in the apical dendrite tuft and 127 produced a signal only in the middle sections of thin branches. The peak calcium ion signal was achieved with the first AP with signals from the 2nd and 3rd APs either smaller or not detectable. The calcium ion signals did not summate.  Further investigation of the triplets of APs induced, but with an interspike interval slightly longer at 125HZ and a higher frequency (200HZ), showed that the calcium ion signal waveform exhibited 3 characteristic features. These were:  the calcium ion signal peak could be identified as being due to bAP or to the summation of two successive calcium ion transients; the inflection on the rise phase of the calcium ion transient was due to the non-linear temporal summation of dendritic AP voltage waveforms; and the peaks were due to the successful initiation of dendritic calcium ion spikes by the activation of the local dendritic calcium ion channels. Therefore, the authors found that the calcium ion signal was not always all-or-none in the distal apical tufts, but there was an intermediary range of dendritic signal amplitudes with low frequency APs on one side and calcium ion spikes on the other. These were due to non-linear temporal summation of dendritic AP voltage waveforms unrelated to the calcium ion related firing.

Short and colleagues continued with their investigation of the calcium ion conductance by looking at the timing of the signals. When the used an interspike frequency of 120 to 5 msecs at 125-200HZ they found that there was a time delay between the peak of somatic AP voltage to peak dendritic calcium ion invasion which was a function of interspike interval (eg. interspike interval  ISI range 5-20msecs – average peak latency 41msecs; range 30-60msecs – latency 32msecs; range 90-120msecs – latency 21msecs). Fast dendritic voltage imaging at 2,700HZ gave a peak voltage signal in distal areas with a delay of less than 2msecs and this increased with the temporal order of spikes in the train (first – time delay of 1.09; second 1.134, third 1.72).

An investigation into the reliability of the calcium ion signals along the same neurons found that the signals of different dendritic segments did not show synchronous peaks. In 20 neurons, at least 1 tuft branch was more delayed (8.1 msecs) than the trunk indicating that the calcium ion signal peaked first in the trunk then in the tuft. It was also found that somatic slow frequency AP trains produced calcium ion peaks in the apical tuft branches sooner than high frequency ones.

Short and colleagues also found different results when they looked at the level of the calcium ion signal in relation to the critical AP frequency. In 3 cells out of the tested 11, it was not possible to determine the critical frequency at all and in 8 cells out of the tested 11, the level of the signal was greatly increased (550%) to that achieved when the AP firing frequency was below the critical threshold. In this case, the dendritic calcium ion influx was variable (270% to 1,486%). The cells where critical frequency could be determined displayed in different trials weak propogation into the apical tuft, strong boosting of signal amplitude at the critical frequency and a large increase in dendritic length affected by strong calcium ion influx. The cells where no critical frequency could be determined exhibited strong propogation at low AP frequency and therefore, these cells had small amplitude boosting with increasing frequency but no discernible critical frequency. The gradual increase in AP firing frequency from 8 to 200HZ caused very small effective changes in dendritic membrane area (dendritic length) receiving the strong calcium ion influx. Independent of backpropogation strength, increasing the AP firing rate invariably caused the calcium ion front to advance along the apical axis of the cortical pyramidal neuron, immersing more and more L1 synapses. Therefore, it was concluded that somatic AP frequency regulates the depth of the cerebral cortex in which the synaptic afferents are subjected to large internal calcium ion concentrations in the post-synaptic pyramidal neurons.

Although the calcium ion influx was demonstrated with somatic AP frequency in most apical tuft cells, it was necessary to check the stability of the influx and this was carried out by setting up 10-33 repeating trials. In 63% of neurons the dendritic calcium signals were stable across the trials and in 37% the results deviated by more than 20%. Some deviations were between the amplitudes of the calcium signal in the apical tuft branches of the same cell, others had large amplitude deviations in only 1 branch. Experiments indicated that AP flickering (defined as successful invasion alternating from trial to trial with complete failure to invade) had occurred in some cells, but this was random and suggested to the authors that calcium ion invasion relating to dendritic AP was more likely in some phases than in others. It was found that the AP flickering of the second and third spikes did not follow the typical response of the first. 52 out of 127 cells had multiple peaks suggesting more than one AP waveform. One apical tuft branch (designated ROI2) experienced 2 and another (ROI1) experienced 3. This suggested to the authors that AP flickering in one branch can occur independently of other branches. The difference between branches of the apical tuft and responses was further supported by imaging using the voltage sensitive dye, JPW-3028, which showed frequency dependent amplitude adaptation of the bAP. The average AP signal was greater at the apical tuft proximal to the principal bifurcation than those distal and a distance dependent decline in AP amplitude was observed from trunk to tuft. Varying levels were seen in different tufts, but in general it was possible to conclude that the imaging experiments showed that bAP successfully caused calcium ion invasion in some apical tuft branches whereas in other cases the APs failed to cause the invasion.

The authors continued to investigate the AP-calcium ion signal with regards to distribution of it along the dendritic apical length. At a firing rate of 8HZ, 65% of cells showed unambiguous AP-calcium ion conductance in proximal segments of the apical tuft branches whereas 35% of neurons demonstrated no AP-calcium ion signal. Out of the 65%, 34% also showed AP-calcium ion transients in the distal segments of at least 1 apical tuft brunch and 40% had multiple calcium ion peaks indicating more than one invasion into the tuft region. AP flickering was obser ved in 37%. Therefore, the authors tested to see if the AP flickering seen was an experimental artefact. They performed 10 to 21 trials per neuron and found large differences in calcium ion spikes between consecutive trials. This was concluded as being due to the cells being positioned at different depths within the brain slices and hence, the differences in results were caused by positioning of cells and the level of damage induced by the experimental preparation process.

Short and colleagues continued their investigation of the bAP-calcium ion signal by determining whether or not somatic AP afterdepolarisation (ADP) was linked to the AP calcium ion invasion into the L5 PFC apical tuft branches. This was carried out since it had been previously reported that activation of dendritic voltage-gated calcium ion channels (VGCCs) are manifested in the cell body in the form of ADP. In 2 samples it was found that the AP-calcium ion signal amplitudes were significantly larger with successful invasion linked to broader somatic ADP. However, in others there was no such correlation and hence, it was concluded that somatic ADP does not reliably indicate successful AP invasion. Neither was it found that the AP-calcium ion invasion was linked to spontaneous synaptic inputs into the synaptic branches. If occurring this could have an affect on the level of depolarisation. However, the authors found that: the average peak amplitude per minute of recording was 0.68 and was relatively stable for all 24 cells; the average number of synaptic events per minute was 203 with a variation of plus or minus 20; and the average instantaneous frequency of synaptic inputs per minute was 36 and it varied minute to minute. Therefore, they concluded that minute to minute synaptic input could influence AP flickering. This was investigated further by repeating the experiments in the presence of glutamine and dopamine antagonists. In these cases, no AP flickering behaviour was observed confirming suspicion that spontaneous synaptic inputs may have an affect on the stability of AP amplitude in apical tuft dendrites. However, a repeat of the experiments with faster AP firing (ISI 12ms instead of 120ms) produced AP flickering showing that the synaptic blockers did not prevent the apparently random nature of the AP-induced calcium ion spike firing.

The authors then went on to investigate the effect of potassium ion channel functioning on the AP-calcium ion conductance. The channel investigated was that of the A-type potassium ion channel which had been linked to calcium ion current in hippocampal CA1 pyramidal neurons. Short and colleagues used the potassium ion channel antagonist, 4-aminopyridine (4-AP) which has been reported to selectively block the A-type potassium ion current in apical tuft dendrites of the somatosensory cortex. They found with their test sample of the apical tuft branches of the L5 PFC that thepresence of the antagonist caused a significant change in the amplitude of the AP-calcium ion current close to the drug application site, but not in dendritic branches away from it. This indicated a local reaction. Multiple applications of 4-AP on the same dendritic branch saw the amplitudes of the dendritic AP-calcium ion signals increase significantly following each drug application. Therefore, it was concluded that A-type potassium ion channels populate thin dendritic branches of the PFC layer 5 and that these channels have a strong impact on the efficacy of AP-calcium ion invasion into the PFC apical tuft.

Since A-type potassium ion channels can exist in different phosphorylation states that affect its activity or inactivity, Short and colleagues investigated the ion channels potassium ion conductance (gka) as a way of explaining the amplitude fluctuations of AP observed in model neurons. Short and colleagues used a computer simulation of a PFC L5 neuron and entire apical tuft and with the exception of 1 apical tuft branch assigned global voltage gated sodium ion and A-type potassium ion conductances. The exception had only potassium ion conductance. They found that bAP failed to invade the selected apical tuft branch because the peak AP was below the threshold for active propogation from trunk to tuft. The attenuated AP amplitude in the tuft caused a small activation of local VGCCs and a small increase in calcium ion concentration. This appeared to mimic real neurons where lower states of dendritic membrane excitability produced small AP induced calcium ion transients in the tuft branches. Downregulation of the input current produced a reduced density of A-type potassium ion conductance in one dendritic branch only with the bAP invading the branch more strongly. The amplitude and duration of the dendritic voltage transient increased in tandem and therefore, more efficient activation of local VGCCs was indicated and hence, a large increase in internal dendritic calcium. A plot of peak calcium ion signal against decreasing values of local gka produced a sharp transition around a gka value of 400pS/um2 from failure to invasion. This non-linear relationship was found for each apical tuft branch, but at different gka values. Therefore, it was concluded that the transition between AP failure and AP invasion could be induced by very small alterations in one dendritic conductance, in this case the A-type potassium ion conductance. It was also concluded that this transition was possible even ifthe conductance change was spatially restricted to one segment of the apical tuft and that individual branches had unique gka thresholds. This was said to possibly explain AP flickering of individual tuft branches on the same neuron. Most neurons (38 out of 47) produced moderate amplitude differences (20-50%) with only 9 producing large amplitude differences (100% changes).

The computer model of the neuron was also used to investigate how the AP-calcium ion signal could be modulated. In one experiment using the computer model, the dendritic AP-calcium ion signal was altered to reflect a greater proportion of low-threshold calcium ion channels instead of the normal high and also the fast sodium ion conductance was reduced by 12.5% and the A–type potassium ion conductance by 28.5%. Short and colleagues found that the amplitude of the dendritic calcium ion signal increased moderately in response to a relatively large decrease in gka. There was also a clear discrepancy between the trends of voltage and calcium ion peaks in apical truft dendrites in response to gradually changing local gka. For a range of gka values between 550 and 300pS/um2 the peak of the dendritic calcium ion signal decreased whereas the peak of dendritic voltage increased in the same dendritic compartment. This was attributed to slow activation dynamics of dendritic VGCCs which cannot activate when the duration of the sharp spike (large amplitude, but short duration) are not long enough. Smaller spikes have longer durations and therefore, can activate VGCCs more strongly. Therefore, smaller and not longer waveforms produce stronger calcium ion transients in comparison to large amplitude sharp spikes. At the three different levels of global gka gradual changes in gka produced sigmoidal curves with moderate amplitudes and distinct thresholds for each apical tuft branch as with the large amplitude changes and the magnitude of change in dendritic signal (20 – 100%) matched the real neurons.

Computer modelling was also used to investigate manipulation of the AP signal by boosting the amplitude and the effect of temperature. In the case of boosting the amplitude of AP in real neurons, it was found that repeated voltage sensitive dye imaging of AP waveforms in the apical tuft showed at first failed invasion, but later APs in the train demonstrated successful invasion consistent with the presence of rapidly inactivating A-type potassium ion current in the apical tufts. The membrane depolarisation induced by the first AP was thought to inactivate the local A-type conductance so that later APs were not opposed by the potassium ion current. Using the model, the authors found the same results in that in a train of 3APs at ISI 12ms, the first AP failed, but there were successful invasions of the second and third spikes. These observations opposed previous research carried out by others who had shown that AP activity had instead induced depression of later spikes in the train. Both behaviours were said to take place in the same pyramidal neuron albeit at different frequencies of somatic AP firing, ie. at lower frequencies, AP amplitudes decrease whereas at higher frequencies, AP amplitudes increase with spike order.

The computer modelling system was also used to investigate the effect of raising the temperature slightly in model neurons since it is known that the activation and inactivation of voltage gated currents in the model depend on temperature. Short and colleagues found that in the two degree temperature range investigated (27 to 28.3 degrees), the apical tuft branches showed different AP outcomes – sometimes failing and sometimes successful. Second and third spikes successfully invaded the apical tuft branch at some but not all temperatures for all but one trial. In this case, the second spike failed, but the third was successful. This variation in AP-calcium ion signalling activity was attributed to the temperature changes causing small alterations in the kinetics of the biophysical system, eg. via spontaneous synaptic inputs and down regulation or upregulation of voltage-gated channels activity relating to temperature-dependent factors.

Although the authors` experiments centred on the PFC brain area, Short and colleagues also investigated the bAP in the somatosensory cortex. In these brain slices, voltage imaging of bAP in distal tuft dendrites of the somatosensory cortex showed backpropogation success and failure and the variability of the results across the trials were similar to the findings using the PFC tissue. The somatosensory cortex slices gave high frequency AP trains with boosting of the second and third spikes followed by calcium ion spikes in the apical trunk. Boosting of the late spikes had occurred already in the apical trunk and was more prominent in the trunk than in the tuft. The voltage waveform of the regenerative calcium ion spike was present from the proximal apical trunk near the cell body all the way up to the principal bifurcation. Passive propogation of the calcium ion spike from the apical trunk through the bifurcation manifested as significant depolarisation in distal apical tuft branches. In the somatosensory cortex, the first AP often failed to invade the apical tuft and amplitude of the first AP varied considerably. Voltage transients of small amplitudes had wide waveforms and vice versa. Therefore, Short and colleagues concluded that L5 pyramidal neurons of the somatosensory cortex are a highly heterogeneous group of neurons giving many AP outcomes. Early spikes in the AP train are prone to failure and inconsistent over trials, but the second APs produce variable outcomes even within the same cell on a moment to moment basis. The apical tuft branches of L5 pyramidal neurons show variation in voltage waveforms during the AP firing train. They have similar waveforms near the AP initiation site at the cell body, but have unique and highly unpredictable voltage waveforms when entering the apical tuft.

Short and colleagues concluded their article with a discussion of their results. They concluded that the bAP-calcium ion signal was an additional mechanism by which pyramidal neurons could combine input arriving at different cortical layers. By bringing calcium ions to synaptic contacts on distal dendrites the necessary conditions for changes in local dendritic membrane excitability could be achieved. It was hypothesised that the bAP is on the point of failing when it enters the apical nexus, but then sodium ion and potassium ion conductances can support local regenerative spikelets in the tuft and these small changes in dendritic voltage waveform would lead to AP success or failure of invasion in the terminal tuft branch. Short and colleagues` investigation gave insight into the characteristics of the bAP-calcium ion signal such as frequency of AP and A-type potassium ion conductance involvement and highlighted the variability in the signal eg. dependent on position in the AP train and temperature of the apical dendrite from successful invasion to failure (AP-calcium ion flickering). This all led to support for the  authors` argument that although there is enormous amount of variability in properties, the bAP-calcium ion signal is deemed one of the ´random elements` of cortical firing. They went on to conclude that any neuronal ´decision` is more certain when these random elements are taken into account and are active participants in the mechanisms involved.

COMMENT

What makes Short and colleagues` article interesting is that it explores another one of the subtelties of the neuronal firing mechanisms, that of the calcium ion signal induced by backpropogation. This particular mechanism is related to a specific type of cell, the pyramid cell, and may not be considered as a mainstream neuronal firing process, but one that must have importance in some conditions. Short and colleagues investigated the backpropogation calcium ion signal found in apical dendrites of pyramid cells found in two brain areas, the prefrontal cortex (PFC) and the somatosensory cortex. They proposed that this particular mechanism is a ´random element` of neuronal firing physiology that is part of the ´decision-making` machinery of the neuron as to whether it fires or not. We do not particularly agree with such an expression as it gives a feeling of ´self-will`, ie. the cell can ´decide` whether it ignores the signal or considers it and acts on it and this type of decision-making is not especially shown. Backpropogation occurs and although variable, there is no information about whether this variability is deliberately steered or not. Therefore, we have to assume that it is there and occurs when physiological conditions in the neuron deem it should occur. When it does, it is not random, but occurs to the cell`s advantage. No cell carries out processes that are not advantageous, because processes are too energy consuming and too distracting from primary function to be catered for.

Short and colleagues experiments determined the characteristics of the bAP calcium ion signal and therefore, it is unnecessary to repeat these. This comment concentrates on what we can infer about it from the consideration of the type of cell where it is reported to occur (the pyramidal cell); and secondly the mechanism itself and the advantage it brings. As we have already said bAP and related calcium ion influx occurs in pyramidal cells which are found in the cerebral cortex (in Short`s article – PFC and somatosensory cortex), the hippocampus, entorhinal cortex and olfactory cortex. All of these brain areas are involved in information and memory. In the case of the hippocampus and entorhinal cortex, one of the roles these areas play is the timing and binding of information so that characteristics of the event are bound together in an event representation. For this to be to be committed to memory, the whole neuronal firing of the cells representing the information needs to be synchronised and sustained whilst local cellular adaptations can be made. What makes the pyramidal cell type special is its single axon meaning that the ´message`  from that cell is sent in one direction from the cell body (soma or where the nucleus is) and normally its single dendrite which has multiple branches meaning that it receives messages from multiple sources. Dendrites appear in 2 types: basilar and apical and it is the apical dendrite where bAPs occur. The apical single dendrite branches into tufts and so the cell becomes accessible to receive messages from a wide number of other neurons via the synapses. These neurons can be at different levels of the brain area (eg. PFC layer 5 or 1) and therefore, it gives a good interrelated network which is ideal for synchronised information electrical representation and binding.

The particular mechanism related to bAP is the induced calcium ion influx (termed here invasion). And we agree with Short as to why such a mechanism is present. Short and colleagues stated that bAP-calcium ion signal is a physiological process that is there to serve the neuron by providing another means or a supplementary means by which excitability of the neuron is achieved when this is not possible through direct firing mechanisms (ie. in the post-synaptic case via receptor binding of neurotransmitter). Therefore, its presence biases the cell towards firing and its consequential function of transmitting a weaker signal further from the dendritic postsynapse to the soma/cell body. Another point in agreement with Short is that a consequence of this is that it provides another means by which the functioning of the neuron can be modulated to its advantage. Because of this, we contest as given above, Short´s argument that bAP is random and believe more that it is deliberate and there to provide conditions to maximise neuronal signal transmission especially if weak.

So, how and why is backpropogation different from normal signal transfer? The two main differences are the direction of travel and the fact that it is carried out when conditions for AP are not optimal under axon transmission rules. In normal apical dendritic firing, the signal is transmitted from the synapse to the soma where the nucleus is. The binding of neurotransmitter to receptors, conformational changes, ion channels opening particularly those relating to receptor binding like sodium ions, potassium and calcium are all part of this post-synaptic/ dendritic neuronal firing machinery. Backpropogation is different since it comes theoretically from the soma and travels to the dendritic end ie. the post-synaptic area. This means that the message goes from the soma and nucleus and theoretically can alter the conditions at the ´business end` of the dendrite.  There are number of possible reasons why this is advantageous or necessary.  One reason given is that it is signal to the dendrite from the soma that the AP spike has been received and therefore, the message initiation process at the synapse can be stopped. It is is unlikely that this is the most plausible reason since such a message would have to be fast and not slow as in the case of bAP and it would be easier to send a hyperpolarising message down rather than a calcium signal. The most likely reasons for the bAP and calcium ion signal as given above is that it induces positive feedback towards firing and hence, to the excitatory or inhibitory cell`s advantage. It could as said before, weight the post-synapse to fire and carry out actions relating to its depolarisation. Therefore, it could possibly lead to a re-set of or compensation for previously downregulated processes. It could also possible promote synchrony between the pre-synaptic axon and the post-synaptic dendrite when the conditions in the post-synapse are not ideal for firing. And as others have suggested, bAP induced calcium ion signal could help in the provision of conditions for long-term potentiation (LTP) to occur (eg. leading to calcium ion dependent activation of enzymes linked to AMPA receptor regulation) which is of course the link between the pyramidal cell containing brain areas and memory.

For backpropogation to have an effect, it must have a mechanism which fits in with its surroundings and it has been shown that the heterogeneity of dendritic cell physiology determines the effectiveness of bAP on promoting calcium ion influx and hence, its effect. The bAP achieves its ´assistant-firing status` because it`s greatest effect comes from slow AP or weak propogation. This fits in with its function of providing the extra factor to an already active neuron so that excitability or inhibition results.  A further indication is that the AP signal itself is sodium ion influx, just like with the action potential of axons. However, the sodium ion channels found on the dendrites have a higher threshold, which means that they are only triggered to open when instructed to be from the AP signal. They cannot open spontaneously. In Short`s experiments the AP of the dendrites of the brain slices was induced by injections of glutamate. The signal travels down the dendrite from its injection site at the soma end and is stronger at the proximal areas, weakening with distance. The advantage of having a secondary effect is that each time the signal begins to fade, the opening of locally distributed calcium ion channels and the calcium ion influx reinforces the signal in each segment. The caveat is to this that the factor determining the signal`s strength or even effectiveness is the number of calcium ion channels present along the length of the dendrite. The number of calcium ion channels is also believed to be linked to the burst properties of firing as reported in the hippocampal CA3 regions. Burst properties are due to to high number of calcium ion channels and this was also observed in the PFC slices in Short`s experiments. The first AP produced normally good spikes, but the later ones in the train not. This indicates that the signal does not rely on summation for effect and that the first signal achieves its aims. If the second or third APs in the train produce successful invasion then this could relate to the time delay of the secondary system (ie. these too need time themselves to reach activation threshold concentration such as cAMP for example). Therefore, the bAP-calcium ion signal can demonstrate lots of variability including AP flickering all to the advantage of the cell so that it reaches firing threshold even if conditions at the synapse do not directly cause it.

This is also supported by the presence and level of the hyperpolarising mechanism that stops it. Hyperpolarisation just like with axonal transmission involves potassium ion influx and in the case of the bAP-calcium ion signal, specific A-type potassium ion channel opening is involved.  It has been shown that the density of these A-type channels increases as distance from the soma to the synapse decreases and therefore, this implies that the function of the calcium ion signal is likely to be local and that the signal can be controlled not only by the number of calcium ion channels, but also by the number of potassium ion channels ready to switch it off.

So, why is calcium used as the second messenger for the bAP? The first reason is simplicity in regards to biological systems, mechanisms and protein synthesis. Calcium ion channels and calcium ion signalling is a known part of the axonic action potential mechanism and so it is easier to use a system in play rather than formulate a new unique one. The only thing required is to tweak it to work towards the aim required. In this way, the calcium ion pool in the dendrite is normally maintained at a low concentration and therefore, the bAP signal results in calcium ion influx and a rise in local calcium ion concentration. Calcium stored internally is not used along the length of the dendrite and for the purpose of the bAP, not in the synapse either. The influx comes via L-type calcium ion channels and it appears that STIM2 can regulate the calcium ion entry. The calcium ion invasion and rise in calcium ions has effects presumably at points along the dendritic length and in or close to the post-synaptic area.  The mechanism likely to be affected away from the synapse could possibly relate to the activity of the pyruvate dehydrogenase enzyme (PDH) which is involved in converting pyruvate to acetyl coA as part of the energy providing mechanism in the cell. Calcium ions stimulate PDH activity and hence, local increases in calcium ion concentration could lead to increased energy production along the dendritic length. This energy production may be important in restoring the dendritic housekeeping mechanisms after the normal dendrite to soma signal transmission.Towards the synapse the calcium ion invasion and local rise in calcium ions is likely to be linked more with processes associated with firing and signal transmission. Calcium ions are a common secondary messenger and therefore, a rise in level would lead to increased activity of a number of systems. For example, calcium ions are linked to calmodulin activity which is related to the transport of vesicles to the post-synaptic membrane and hence, may be associated with AMPA receptor trafficking. Since AMPA trafficking is required for LTP this may be how LTP is induced in areas where the external firing influence is below the normal threshold. Another way in which a calcium ion influx can aid the firing mechanism is that calcium ions are also linked to CaMKII activity, which results in increased PKA and PKC activity and which may increase the amount of active phosphorylated proteins in the synapse for example. A third possibility is that proteins required for the transmission of the signal may be synthesised under conditions where the post-synaptic membrane effect does not indicate it. Calcium ions are believed to control the elongation phase of mRNA transcription via elongation factor 2 kinase activity and therefore, a rise in calcium ion concentration independent of receptor binding would lead to a possible positive influence on local transcription at the synapse and protein synthesis. This is supported by the observation that apical dendrites exhibit local protein synthesis.

Therefore, we can conclude that the bAP calcium ion signal is one of those smaller, regulatory systems that aids neuronal firing in circumstances where the threshold is not reached by normal, post-synaptic neurotransmitter activation. It is a simple switch on/switch off mechanism which uses local rises in calcium ions which can have a number of different effects. Since it is observed in brain regions where memory and the demand for sustained firing is important it is likely that it may help in maintaining firing briefly until the local adaptations to the synaptic area occur. For this reason, bAP calcium ion signalling may not be mainstream, but it should not be ignored.

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

….if a calcium ion chelator is administered at the same time as the AP-inducer in a dendrite with known successful invasion, would the AP waveform be observed, but the calcium ion influx not since this particular ion channel would be blocked? Would the use of specific antagonists for different Cav subunits of the L-type receptor determine the exact nature of the channel involved in bAP and the related calcium ion influx in the different brain areas containing pyramidal cells?

…can we assume that if aptiganel (a NMDA receptor antagonist binding to the magnesium ion site) is administered and the AP experiments repeated that any calcium ion influx occurring would be independent of NMDA receptor activation at the post-synaptic areas?

….if the function of the bAP calcium ion influx is to stimulate CaMKII function as thought in the case of tetanus stimulation then would an increase in CaMKII concentration be observed if Short and colleagues experiments were repeated?

 

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investigation of the division between present time end and future time start

Posted comment on ´When does the present end and the future begin` written by H.E. Hershfield, S.J. Maglio and published in Journal of Experimental Psychology: General

SUMMARY

In their article, Hershfield and Maglio investigated the division separating the conscious awareness of present time from future time. They began their article with an introduction into the concept of these two time frames and described different studies of how motivation, language and personal connection to future time could influence time passage. The article continued with a description of the six studies performed by the researchers and their results.

Hershfield and Maglio began their investigation by looking at when people believe that present time ends and the future begins (Study 1). The study involved using an online sample of 203 participants to report when they felt that the present ended and to choose a code that best described the answer they wrote. The responses reported could be categorised into groups: those reporting that the present ended immediately (20% of the participants); others reporting between 1 second and a minute from time zero (18%); and then groups describing between one minute and one hour, between one hour and one day, between one day and one week, between one week and one month, between one month and one year and finally longer than a year and at some future event.  The results showed that there was a variation in the perception of when present time ended, but in general there was a biasness towards the view that present time ended immediately (20%) or up to a minute (18%) and almost 50% of the subjects perceived that present time would within one hour. From the subject group taken, 15% of participants said that the present ended at some future event and that death was the common reason given for this separation. From this, Hershfield and Maglio concluded that their results were noisy owing to not measuring exclusively the intended goal of the experiment which was ´feeling` the end of present time rather than attributing time to specific life or experienced events. They did establish, however, that there were no gender differences in the perception of time, but age produced a significant negative relationship.

Study 2 was carried out to test the reliability over time of when people believe that present time ends and future begins. 508 participants were asked the same question 3 times over a period of 4 months. The participants were shown a horizontal line of 30 clickable dots and told to think of them as representing the passage of time. They were instructed to click the one dot which they thought represented when the present time ends and then a second to represent when the future starts. Like in Study 1, the participants were asked to choose a code that best described their perception and they were also asked by open-ended prompt to indicate how they made their decision. Hershfield and Maglio found with this study as in Study 1 a biasness towards the perception that present time ended immediately and more than half (51.6%) believed before one hour. The average position of the dots (7.01) clicked was towards the left (ie. sooner) for present time ending, but the mean score of future time beginning was at 12.31 dots suggesting that the participants in general perceived a buffer (termed ´grey area`) between present  time ending and future time starting. 54.3% of participants indicated the presence of a grey area of a median value of 2 dots.  The results for all 3 time periods were similar suggesting that there was stability in how people conceptualise the passage of time. When asked to describe their thoughts as they made their decision, the authors identified 4 categories of responses; continuous time (ie. present ends, then future starts) – 85% of participants for present, 82% for future; intuition (ie. thought when present moment would end) – 4% of participants for present, 2% for future; event-based (ie. thought about specific event and life after that event)- 3% of participants for present, 4% for future; and lifetime-based (ie. period of life and after that period – such as old age so cannot look after oneself) – 7% of participants for present, 12% for future. Therefore, it was concluded that the findings supported the hypothesis that the majority of participants interpreted and performed the tasks of Study 1 and 2 in the author`s intended manner ie. the majority of participants ´felt` the passage of time and did not attribute time to lifetime or specific events. Those that did were treated as ´noise` in the subsequent studies.

Study 3 was set up to investigate what constructs relate to the perception of present time ending and future time beginning and what downstream behaviours such perceptions might predict. 524 participants who believed that present time ends sooner rather than later were asked to report when they felt present time ends whilst the authors examined a number of other constructs. These were:  personality traits such as openness, extraversion, emotional stability and agreeableness (Ten-Item Personality Inventory); general tendency to think in more concrete and abstract terms (Behaviour Identification Form); examination of conceptualisation of the Self over time (Future Self-Continuity Scale); measurement of how long participants time horizons were (Future Time Perspective Scale); measurement of how long people consider the duration between one future period and another future period (Temporal Duration Estimate);  assessment of how emotionally reactive the participants thought their future Selves would be to rewards (Future Anhedonia scale); and whether the participants viewed time periods positively or negatively (Short Zimbardo Time Perspective Inventory). The participants performed the same dots task of Study 2 and were then asked to complete a monetary allocation task. This consisted of the participants having to imagine that after paying bills and expenses they had 1000 dollars left. They were then asked whether they would use the money to buy immediately something fun or special or whether they would put it in a savings account. Then, the tests given above were carried out in order with finally, the participants being asked to indicate when they felt present time ends. Hershfield and Maglio found that the longer the present seemed to last the less emotional the participants felt towards the future, the longer they felt a year lasted and they also exhibited higher scores on extraversion, Future Time Perspective scale and on Short Zimbardo Time Perspective Inventory (SZTPI) past positive, present fatalism and present hedonistic. The perception of when present time ended was not linked to the tendency to think abstractly, conscientiousness, agreeableness, emotional stability, openness, future self-continuity, or with the SZTPI past negative and future subscales. In the second part of the Study 3, the authors found that the perception of when present ends is not a significant predictor of monetary allocation independent of age, gender, education and abstract thinking. When present was viewed as short then participants tended to put money into long-term savings. This was interpreted by Hershfield and Maglio as demonstrating future-orientated self-regulatory behaviour.

In Study 4, the ending of present time perceived by the participants was manipulated and its effect on real choice was investigated. This was carried out to provide support for the findings of Study 3. Hershfield and Maglio put 905 participants into 2 groups: one presented with a short present, the other a long present. The participants were shown a bar that faded from blue to red labelled ´present ends` at the point of fading out of the blue colour and ´future starts` where red faded. The ´short-present` group gave the bar with the fading at a third along from the left and the ´long-present` group, two-thirds. The participants were also shown a screen with lists of creative ways to save money for the future and asked if they wanted to read it. Agreement to read it indicated successful self-regulation by the participant giving personal time to consider long-term financial prospects. Then, the participants were asked to complete the SZTPI test and the clickable dot task of Study 2. The results of this part of the study showed no difference to the findings of the original Study 2. The results of the rest of Study 4 showed that participants viewing the short present were more likely to read the saving tips (65.9%) and these results were independent of the SZTPI findings.

Study 5 was an adaptation of Study 4 to test whether experimentally manipulated variation in the duration of the present would generalise to a more consequential outcome in an applied setting.  The experiment was set-up in same way as in Study 4 with the coloured bar (in this case, green fading to red) presented to 126 participants of a financial wellness group.  The participants were asked to examine whether a message that framed the present as short would encourage more people to enrol in a financial education seminar hence demonstrating self-regulation by considering long-term financial prospects. The results obtained supported the findings of Study 4 in that about 31% in the short-present group and about 10% in the long-present group, independent of whether the participant thought the advertising for the seminar was persuasive or not, decided to enrol.

Hershfield and Maglio`s final study, Study 6, was set up to investigate the mechanism between time perception and meaningful outcome. Other researchers had conflicting views. It was hypothesised that sharpness or blurriness rather than location of the division between present and future would prove relevant to future orientated behaviour. And one set of views proposed that blurry division would heighten motivation and self-regulation because the present would continue seamlessly into the future. On the other hand, however, sharp division would enhance the desire to act in the service of the future Self since people would be aware of the contrast between present state and future state and the individual would be spurred on to future-orientated action. Therefore, in order to clarify the situation, Hershfield and Maglio performed 3 sets of tests. Study 6a consisted of the present being framed as either short or long and the participants being asked to see the division as either sharp or blurry. Studies 6b and 6c were set up to manipulate both location and sharpness/blurriness to see how they impacted on future-orientated behaviour in both behavioural intentions (Study 6b) and incentive-compatible choices (Study 6c).

Study 6a involved 304 participants being shown 2 images of the time bar with varying levels of sharpness or blurriness at the division and being asked which they thought best suited the separation between present end and future start. The results showed that the participants in general (72%) believed a blurrier division between present and future time. Short-present preferred a sharp division (34%) compared to long-present (22.9%) which supported the findings of Study 3 and indicated future-orientated self-regulatory behaviour (Studies 4, 5). Study 6b was set up to investigate whether this relationship (short-/long-present and sharpness/blurriness) could play a role in decision-making. Over a thousand participants who perceived short-present or long-present and sharp/blurry divisions were asked how motivated they were to take future-orientated financial action (ie. contribute to a savings account, order automatic deposits into a savings account, speak to a financial advisor on long-term savings).  The results showed that participants who perceived short-present/sharp division were more motivated to take future financial action than long-present with both sharp and blurry divisions. In the case of long-present perception there was no difference between sharp or blurry division views. This finding was tested in Study 6c where a real financial incentive in the form of a gift voucher for a ´buy now` action or for an investment service (demonstrates future-orientated behaviour) was used instead. Over a thousand participants took part and the results showed that 54.8% of participants with short present/sharp division views chose the investment choice compared to 47.9% with short present/blurry division, 44.7% with long present/sharp division and 45.5% with long present/blurry division. Again, the results supported the hypothesis that participants demonstrate successful future-orientated self-regulation.

Hershfield and Maglio concluded their article with a discussion on the ramifications of their findings. Their view consists of people perceiving time passage as self-jumping through a never-ending succession of temporal bubbles each of which consists of a new present moment and their study investigated the size of the bubbles. They attempted to explain the rationale behind their observations by saying that the perception of a short-present leads to more future-orientated decisions in that short-present may bring about a longer future which would demand more financial resources so that people would need to act to secure this. A shorter-present could also indicate a greater sense of urgency to act before the ´window of opportunity` closes. They also asked the question as to how might variation in the current present moment affect how people conceptualise the duration of the present moments in general. It has been shown that filling a given time interval with a variation of activities for example makes that time period seem longer to the individual and the authors` experiments did appear to change the perception of time for their participants. Those participants exposed to manipulations of present end and future start by creating variation in one moment saw it as relatively long or short. The question was therefore asked would this apply to all future periods of time and this was ear-marked for further study. Other ideas for future research included division not on demand as in Hershfield and Maglio`s reported experiments and also investigation into the effect of the past on the perception of present end and future start and the future-orientated decisions made.

COMMENT

What makes this article interesting is that it describes how people view time boundaries and this supports to some extent the neurochemical findings. Neurochemical findings relating to physiological present or ´real time` involve the formation, maintenance and degradation of the event ´percept` represented by neuronal firing assemblies. This is the culmination of the firing of relevant neurons and binding of this information into a percept which can be conscious or unconscious. The event representation can then be used to dictate feelings, decisions for example (present time), and/or can be stored (ie. can represent the past in future time), and/or dictate actions (future time). It can also degrade if the neuronal firing is not sustained and hence, a new percept of the ´present` moment is formed. In a previous post, we have discussed this shift in percept and time passage by looking at event boundaries with relation to sequences of events (Yakov and Henson`s ´The hippocampal film editor: sensitivity and specificity to event boundaries in continuous experience` posted comment  July 2019) and this shift from one event to another was found to be associated with firing of the hippocampus, an important area for informational timing, signal relay and binding.

So, physiological timing is relatively simple to explain, but people consciously view time in a different way. It can be roughly seen as ´physical` time and ´mental` time. We have already described ´physical` time from the physiological perspective of neuronal firing, but it can also include ageing, which we view as physiological changes associated with the passage of time during our life span. It can also be linked to our association with regulated time as dictated by clocks or the environment, eg. the sun rising and setting and we share this regulated time with others. The game ´say when you think a minute has passed` anchors our thoughts to this physical constraint, but also links it to the other form of time, that of ´mental` time. Mental time is different in that it is personal, determined by the individual`s own thought processes. It still has present (´what we are experiencing now`), past (´what we have experienced`) and future (´what we will experience`) phases and to a large extent it is dependent on the physiological processes being carried out. But, mental time can be disassociated from physical time simply by for example consciously thinking about something irrelevant to the environment we are presently in. In this example, physiological time is dictated unconsciously by the event being portrayed in the brain`s own electrical representation and conscious awareness is on a mental construct formed from reactivating memories and feelings. The neurochemical capability of divided attention allows us to be active in two or more ´scenarios` and sometimes these are two physical-based events, or it can be one physical and one thought-related.

In Hershfield and Maglio`s study, participants were asked to perform this type of task. Physical time carried on regardless of conscious awareness and the participants were asked to say when they felt that the present ended and the future started, ie. when they consciously felt their personal present ended and their future began. They reported this using time lines portrayed visually by dots or coloured bars. It was interesting that only 20% of participants felt that the present ended immediately and a further 18% between 1 second and 1 minute, the latter long periods in neurochemical terms.  In our view, it is unlikely that considering the environment the participants were in at the time of the experiment and the task itself, that the present would be considered from a physiological point of view because the visual neurochemical mechanisms have automatic cessation points built into them, such as blinking and switching of the visual field as shown by looking at the Neckar cube for example. These would be activated long before 1 minute of constant viewing or thought. Therefore, it is likely that the decision of when present time ended and the future started would reflect mental time and this would be associated with the personal view of present and future. For the vast majority of participants it is clear that this was linked to concrete personal events ie. the end of the task or experiment itself, the next activity such as lunch or starting work rather than feelings.

Hershfield and Maglio went on to describe the nature of the division itself. Their experiments showed that faced with a timeline portrayed by clickable dots that the average end of present time was roughly a quarter of the way along the timeline (about 7 dots of a total 30) and future time started roughly a third along (12 dots). This could be interpreted as indicating that there was a gap between the two time periods which was contrary to the results of further questioning which showed that most participants believed time to be continuous, seamlessly flowing from one phase to another. This was visually portrayed as the division between present end and future start was perceived to be ´blurry` (72% of participants) and not ´sharp`. Two explanations can be given for what appears as opposing views. The first relates to cognitive processing. Present time could be considered as the period when the participant thinks about his feelings and future time could be considered as when he acts, which obviously comes later. The difference would represent the physical translation of thought to action. Since the thought and action would be regarded as ´one event` then the division could also be described as ´blurry`. This would also be the case if the task was regarded from a neurochemical point of view. The periods would relate to the neuronal firing relating to thinking and the action and would be subjective. The ´blurriness` of the division would relate to the similarity of the ´percepts` of now and later as described by Yakov and Henson`s study on event boundaries in continual experiences (comment July 2019).  Event boundaries are the shifts/breaks/intervals between 1 event and another and subjects are aware of the changes of events and report it. In sequences of events, the similarity of event characteristics between one period and the next is reflected by the firing of approximately the same neuronal groupings ie. firing is sustained. Therefore, in the case of feelings about time, most of the characteristics of these percepts would be similar and hence, the participant`s percepts would change little giving the impression of ´blurred` division. It should be noted that the subjective event boundaries of Yakov and Henson (comment July 2019) occur later than the actual neuronal firing responses ie. conscious awareness comes as a result of global workplace activity.

Once Hershfield and Maglio had established conscious awareness of present end and future start they investigated the psychological consequences of such attributes. Their studies (Studies 3-6) looked at how time perception was linked with future concerns about financial matters. Such an area of thought is a product of learning, environment and personal attitude and Hershfield and Maglio were able to show that regard for the present (whether long or short) brought about preparation for future financial stability, but to different extents. Both showed self-regulation, but participants that viewed the present as long, were less concerned about whether time was meaningful or not, believing that new information was less likely to affect or have a high impact on their personal future events. Participants who viewed the present as short treated the access to financial information or the financial incentive with a greater sense of urgency. They believed that the future would come quicker and probably last longer and therefore, there would be a greater need for appropriate financial resources.

It is probably this link between perception of passage of time and future orientated self-regulation that gives this study value. Although Hershfield and Maglio`s results support to some extent neurochemical findings relating to time periods and event representations, it is more useful in providing a control in experiments looking at self-regulation of future-orientated events and provides a means of affecting how monitoring and manipulation of time lines can bring about long-range behavioural changes.

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

…..it has been reported that certain conditions bring about changes in awareness of the personal duration of time. For example caffeine, mindful meditation, pain, fear and ADHD all lead to individuals perceiving a longer duration of personal time. Therefore, if the studies of Hershfield and Maglio were repeated under conditions where participants were exposed to those conditions can we assume that the assessment of the timelines (whether by clickable dots or coloured bars) would show a shift to the right (ie. a longer time to the present end)? Is it possible that this would also affect the perception of the start of the future and affect how individuals approach future orientated situations and events?

…..Hershfield and Maglio`s experiments involved only visual portrayals of the passage of time. Would the use of sound instead affect the portrayal of time? And, can we assume that the use of sound with visual information would lead to attentional splitting which would prolong the duration of present time and hence, be shown in the visual time bars?

…..if the portrayal of the time line is manipulated by either increasing the distance between the dots or by lengthening the fading of the colour bar, can we assume that awareness of time would show conflict and that it would be difficult for the individual`s to determine when present ends and future starts?

 

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regulation of long-term memory and behaviour by CREB1 in the worm

Posted comment on ´Differential regulation of innate and learned behaviour by Creb1/crh-1 in Caernohbditis elegans` written by Y. Dahiya, S. Rose, S. Thapliyal, S. Bhardwaj, M.Prasad and K. Babu and published in Journal of Neuroscience 2019 39(40) p. 7934 doi 10.1523/JNEUROscI.006-19.2019

SUMMARY

Dahiya and colleagues investigated the effects of different creb1/ crh-1 mutations on the ability of the worm, Caenorhabditis elegans to form long-term memory (ltm) and perform and adapt innate behaviours. (Crh-1 is the C. elegans homolog of the mammalian cAMP binding protein 1, CREB1). The authors used the premise that mutations that affect innate behaviour could also alter the animal`s ability to modify behaviour in response to experience.

In their experiments, Dahiya and team used wild type N2 C. elegans and crh-2(tz2) mutant lines. The animals were exposed to a dry heating block at 37C on a glass plate covered with either isoamyl alcohol (IAA) or diacetyl alcohol for a period of 2 minutes. The training periods were repeated 5 times with an interval of 10 minutes between each. For the chemotaxis behaviour assay, the worms were placed at the centre of agar plates without food and then IAA (or diacetyl, benzaldehyde) was placed at one end of the plate. The chemotaxis behaviour was recorded and quantified according to the displacement of the animal along the IAA gradient (positive for travelling up the gradient and negative for down) over the distance travelled in 10minutes in the absence of the gradient. The procedure was repeated with the various crh-1 isoform mutations which had been created by CRISPR and identified by quantitative PCR. Various imaging techniques were used such as; fluorescence microscopy;  optogenetics  with animals expressing the channelrhodopsin-2, ChR2 which was excited by blue light; and calcium imaging using the genetically encoded calcium ion indicator GCaMP-expressing strain prig.3::GCaMP5 to show the calcium ion transients in the AVA command interneuron. All imaging results were statistically analysed. In order to study the temporal requirement of crh-1e expression, silencing of the expression was carried out using histamine. WT worms were taken and HisCl1 was expressed under the control of the crh-1e promoter, pcrh-1e. The HisCl1-expressing neurons were silenced by growing the worms on histamine-containing plates while training took place or during the chemotaxis experiment. The training/learning and chemotaxis behavioural experiments were carried out as before.

Dahiya and colleagues first looked at established ltm-based conditioning behaviours. It has been previously shown that C. elegans exhibits associative ltm formation by pairing the presence or absence of food (unconditioned stimulus) with a variety of cues eg. temperature. The memories formed were retained for up to 40 hours. Starvation alone can induce expression of CREB/Crh-1 and therefore, the authors used a training set-up that was independent of the feeding state of the worm ie. the conditioning experiments were based on IAA and high temperature. Exposure to IAA and 37C temperature produced no change in the worms` crawling speeds, but significantly reduced the chemotaxis index (CI) values whereas singly CI values were comparable to naïve animals. The specificity of learning to the cues provided in training was guaranteed by control experiments where chemotaxis behaviour in response to diacetyl and chemotaxis to IAA was unaffected, but chemotaxis behaviour to diacetyl when paired with heat led to a reduction to diacetyl. Worms trained with IAA and heat showed normal chemotaxis to exposure to benzaldehyde demonstrating that there was no down-regulation of AWC neurons from the training programme. The authors found that when they repeated their experiments with null creb1/crh-1 mutants, these animals gave higher CI values than the WT worms. Therefore, it was said that the null creb1/crh-1 mutants demonstrated defective ltm formation and exhibited defects in innate chemotaxis behaviour.

The authors then went on to investigate the different isoforms of the crh-1 gene. Cloned cDNA for the six isoforms (crh-1 a-f) was used and quantitative PCR gave variable results. This indicated functional diversity even when the proteins were structurally similar eg. CRH – 1a to isoform ´-e` were full length proteins having both the kinase inducible domain (KID) and bZIP binding domain with differences limited to 30 amino acids at the N terminal, whereas CRH-1f was a truncated protein with the N terminal KID absent. When cloned under the pan-neuronal rab-3 promoter, only CRH-1c and CRH-1e expressing animals were able to rescue the ltm defect in the creb1/crh-1 null mutants. CRH-1f expressing animals demonstrated inhibited memory formation.  The results were confirmed using CRISPR mutants.

Further investigation on the restoration of learning of the creb1/crh-1 mutants was carried out with regards to interneuron subtype. Normal movement behaviour in response to attractants or repellants (ie. random movement normally, but the presence of an attractant/repellent can lead to a suppression or enhancement of reversal and turn frequency in order to move towards or away from the attractant/repellent) is controlled by the command AVA, AVD and AVE interneurons and the RIM interneurons. In the presence of the IAA gradient, the creb1/crh-1 null mutants did not modify their reversal behaviour in response to the changing IAA attractant concentration. The transgenic line expressing CRH-1e in the AVE and the RIM interneurons brought back the reversal behaviour and the memory deficits in the creb1/crh-1 null mutants.

The low mRNA expression levels and the presence of a unique 5`UTR on the CRH-1e mRNA suggested to the authors that the promoter sequence was different in this particular mutant from the other CRH-1 expressing mutants. Therefore, Dahiya investigated the expression of GFP positioned upstream from the CRH-1e translation start site (termed pcrh-1e) and found it was only observed in a few head neurons with an overlap in the expression pattern of pnmr-1. This localisation was found in the AVA, AVE and RIM interneurons when tested for co-expression of the pcrh-1e::GFP with prig3::mCherry (neuron-specific promoter marker line for AVA  interneurons), popt-3::mCherry (AVE interneurons) and pgcy-13::mCherry (RIM interneurons). Using the co-expression promoters, it was found that the crh-1e, the gcy-13 and opt-3 promoters (albeit the latter at a lower level) restored the associative learning for IAA and heat in the creb1/crh-1 null mutants. No such affect was seen using the rig-3 promoter to express CRH-1e in the AVA subtype of interneuron. A similar experiment using the CRH-1a isoform was performed, but no overlaps of the pcrh-1a:GFP and the various interneuron promoter sites were found supporting the authors view of CRH-1 diversity.

Once the requirement of CRH-1e expression for memory formation was established, Dahiya and colleagues investigated whether or not the expression was needed at the time of training, or later.  It was found that the worms with silenced HisCl1-expressing neurons during training could not form memories, whereas those silenced during the chemotaxis part displayed defective chemotaxis. This was also found to be the case for the naïve animals, too.  The requirement for CRH-1e expression for learning was also supported by the results of experiments using creb1/crh-1 mutants expressing the CRH-1e under the control of the heat shock promoter hsp-16.41.  In this case, induction of the CRH-1e before training (3hours) rescued the learning defects whereas induction before chemotaxis did not. This confirmed that CRH-1e expression is required before acquisition and/or consolidation of memory formation.

Dahiya and colleagues went on to explore the N terminus regions of the CRH-1e and CRH-1c using the CRISPR technique. The two isoforms were identified as being important for memory formation and having smaller N regions than the other isoforms. An inert 3xHA tag was added to the N terminus and it was found that this modification caused defective memory formation. The authors also investigated whether key amino acids associated with CREB1 in mice were also required for memory formation in the C. elegans. The authors created K247R/K266R mutant worms where K247 and K266 are equivalent to the K285 and K304 lysine residues of mice. It was found that the mutant worms were unable to form ltm whereas the naïve animals performed to the same level as the WT. It was therefore concluded that the mutations of K247R and K266R affect the learning process. This was further supported by the optogenetic experiments involving the activated AWC neuron and recorded reversal probability of naive WT worms and K247R/K266R mutants compared to trained. It was found that there was no effect on reversal frequencies of the mutant worms compared to the naive WT ones, but trained worms demonstrated increased reversal frequencies with the mutant forms lower than the WT. Simultaneous imaging of AVA interneurons with the optogenetic activation of the AWC showed that the WT worms had an increased probability of AVA firing, but there was no such increase with the mutant worms. Therefore, the authors` results demonstrated an involvement of the K247 and K266 lysine residues in regulating AVA firing and reversal probability in their training experiments, but concluded that these residues were not necessary for innate chemotaxis behaviour.

Dahiya and colleagues concluded their article with a discussion about the implications of their study on the creb1/crh-1 gene of the C. elegans worm. The creb1/crh-1 gene is known to be involved in a variety of signalling pathways such as cellular energy metabolism, ageing and circadian rhythm and the null mutants of the creb1/ crh-1 demonstrated that it also is plays a role in long-term memory formation, too. In the case of naive creb1/ crh-1 mutants there was reduced innate chemotaxis behaviour compared to the WT because of the inability of the mutant worms to modulate reversal frequency in response to attractant gradients. The observed learning defects in training compounded the deficiency of the innate behaviour. Dahiya and team found functional specialisation of the CRH-1 with the absence of CRH-1c or CRH-1e leading to defective ltm formation, but normal innate chemotaxis behaviour. The null creb1/crh-1 mutants not only demonstrated abolished ltm formation, but also showed reduced innate behaviour.

The authors then carried out further investigation of the CRH-1 homologs since although these homologs have similar structural sequencing they demonstrate functional diversity eg. the crh-1a and crh-1e. Spatial differences due to variations in expression were suggested as a reason for this functional diversity. However, this was proven not to be the sole reason since the pan-neuronal expression of crh-1e could restore memory defects in the null creb1/crh-1 mutants, but expression of the crh-1a could not even though it did help to restore ageing effects. Dahiya and team also showed that the addition of small amino acid sequences to the N terminus of the CRH-1c and CRH-1e proteins both disrupted memory formation. This led to the conclusion that tertiary structural changes resulting from the amino acid additions at the N terminus could cause functional changes not shared by the other homologs.

Experiments with the CRISPR mutants with K247R/K266R also demonstrated that in the case of C.elegans lysine247 and lysine266 are important for learning behaviour, but not for innate behaviour. However, the Dahiya team questioned the validity of the results due to the optogenetic  experimental set-up. Channelrhodopsin-mediated AWC activation was used, but it is known that AWC neurons are OFF type neurons that are activated in response to decreased attractant concentrations. Therefore, the results obtained should not have been observed. This suggested that the promoter used, pstr-2, only expresses in AWC- ON type neurons and therefore, only activate one AWC neuron. This was considered not to be a natural occurrence for worms in an IAA environment. Also, it was thought not possible to localise ChR2 to the subcellular position of the IAA receptor whose exact location is not known. Therefore, it was assumed that the CrH2 had a similar position to the other chemo-sensors, eg. diacetyl GPCR is considered as being primarily localised in the ciliated endings of AWA neurons. Since the worm senses information from its environment using a number of different neurons then localisation of the optogenetically active neuron may not represent the whole output of the neurons.

Dahiya and colleagues concluded their article by discussing the requirement of specific interneuron types in associative memory. AVE and RIM interneurons were considered as functioning together in memory formation and this was supported by the presence of functional CRH-1e found in both.  AVA activity is also known to be correlated to reversal behaviour in C. elegans and therefore, the authors were surprised that the expression of CRH-1e in RIM and AVE interneurons was sufficient to restore ltm in the null creb1/crh-1 mutants. This was explained as being due to neuronal connectivity since the AVA interneurons are widely connected with AVE and RIM interneurons and therefore any change in the latter two would produce similar effects on the AVA interneurons. This suggested that the memory restoration was an artefact of the experimental methods used ie. one factor being observed whereas the cause lies with another or that learning is a function of CRH-1, but not its specificity and therefore, using these methods, only the memory process before the execution of the relevant behaviour can examined.

COMMENT

What makes this article interesting is that it explores the topic of the neuronal cAMP response element binding protein, CREB (albeit in the Caernohbditis elegans) and its role in long-term memory (ltm). The mechanisms behind the long-term storage of neural representations of events as well as how these memories are recalled and reconsolidated have not been elucidated fully and are really not much further forward than when Kandel received his Nobel Prize in 2000. It is still recognised that CREB is involved and we know more about some of pathways linked to it (especially those relating to genetic transcription), but the exact nature of memory formation and what memories are from a physiological perspective is still unclear. The comment here considers further CREB and its role in memory from the neurochemical perspective.

Long-term memory storage has been linked to structural changes in the neurons and synapses relevant to the information it concerns. Zatorre reported changes not only in the brain`s white matter, but also in the grey matter and therefore, extensive neural representations in real-time equivalent to external or internal events are translated from temporary neuronal changes into a permanent representation that is wide-ranging and widespread. It is known that CREB leads to increased synaptic functioning on learning and this may require event repetition in order for storage to occur. Yger reported that in fact new sensory stimuli may be learned with a single or few presentations and that cortical responses indicative of long-term storage and physiological changes may be increased after just a few repetitions. Such conditions would normally produce only weak synaptic plasticity and Yger showed that there are actually only very small changes in synaptic strength of cortical neurons after a pair of presynaptic and post-synaptic spikes. The strong synaptic changes thought necessary for permanent changes are provided instead by the large numbers of synapses onto the neuron which counteracts the weak synaptic plasticity induced by the frequency of spikes. Therefore, long-term changes of the neuronal structure can be induced by not only repetition, but also by extensive connectivity.

CREB is believed to be involved in these physiological changes and classic work has been done on the long-term memory formation pathways of the Aplysia and Drosophila species as well as humans. For example, Kim found that amygdala neurons express increased levels of CREB in the learning and expression of fear memories and even artificial activation of CREB in these cells leads to recall of the established fear memory plus strengthened activation in the reconsolidation reorganisation process. CREB is capable of doing this because it exists in a number of different forms: CREB2, CREB1 non-activated and a phosphorylated activated form. This phosphorylation is carried out by protein kinase A (PKA) which is stimulated by a rise in intracellular calcium ion concentration, and hence, the link to neuronal firing and learning is established. CREB2 protein sits on the CRE (cyclic AMP response elements) site of the DNA strand blocking it so that the transcription of appropriate genes located downstream from it cannot occur. The stimulation of expression of CREB1 by PKA leads to the CREB1 protein replacing the CREB2 on the DNA strand and phosphorylation of the CREB1 also again by PKA at specific serine residues activates. This means that the CREB binding protein (CBP) can now bind and transcription of downstream genes can occur. This dominance of CREB2 over CREB1 for binding to the CRE site probably relates to the higher affinity for the binding site of the CREB1 due to better conformational fit and stronger bond formation. The resulting CBP binding can be considered as the cue for promotion of DNA transcription and hence, probably causes a conformational change of the DNA structure that opens it up to allow DNA polymerase access to occur and gene transcription to begin. Therefore, long-term physiological change in the cell stems from its initial firing which causes intracellular ionic changes which activate particular enzymes.

The reliance on PKA activation and phosphorylation is linked in particular to the calcium ion rise due to NMDA R stimulation for example. There are many studies on this topic including work by Zhang who showed that PKA and extracellular signal-regulated kinase (ERK) cascades were necessary for long-term synaptic facilitation in Aplysia with training enhancing the increased PKA and ERK leading to increased ltm. McCampbell`s work demonstrated the process`s complexity since the team also showed that in the Aplysia, either spaced or continuous exposure to serotonin induced a form of intermediate-term facilitation (ITF) that required new protein synthesis, but not gene transcription. This spaced and continuous ITF was thought to use distinct molecular mechanisms to maintain increased synaptic strength with spaced activation dependent on persistent PKA activity and continuous on persistent protein kinase C (PKC). Continuous activation leads to a PKC dependent increase in phosphorylation of the eukaryotic elongation factor 2 (eEF2) which is responsible for the catalysation of GTP-dependent translocation of the ribosome during protein synthesis. Spaced IFT resulted from a PKA-dependent decrease in the phosphorylation of eEF2. Therefore, continuous activation produces a 5-HT-dependent increase in synaptic strength requiring translation elongation, but not translation initiation, whereas the spaced 5HT-dependent increase in synaptic strength is partially dependent on translation initiation. This links in with the CREB1 influence on gene transcription.

So, although we know that CREB is linked to gene transcription it is not fully known what permanent changes to the functioning or structure of the cell this gene transcription controls. It is in fact easier to determine what it probably does not control and this is for example: increased glutamate receptor number particularly the AMPA R which is known to be associated with memory learning and recall; sleep mechanisms since sleep is not required for long-term memory storage in the Aplysia and Drosophila species and neither is attention; and the extent of memory content since CREB controlled gene transcription does not appear to be related to information exposure or updating. On the other hand, CREB is linked possibly to gene transcription changes relating to structural change as thought necessary for long-term memory storage such as dendritic spine growth and remodelling and/or increased glucose uptake as well as for production of a number of proteins thought linked to memory formation.

The link between CREB and dendritic spine growth and remodelling comes from its involvement with brain-derived neurotrophic factor (BDNF) and Ras-related C3 botulinum toxin substrate 1 (Rac1) functioning.  CREB expression and binding to the CRE site as described above is initiated and regulated by firing of the neuron and NMDAR binding instigating a rise in intracellular calcium ion concentration. The binding of phosphorylated active CREB1 to the CRE site causes the transcription of the BDNF gene which lies downstream of it. The BDNF formed is parcelled in vesicles and transferred along the tubulin cytoskeleton to the cell surface where it is released. This released BDNF then binds to its unique receptor on the cell surface, the TrkB (Guo). This binding and the TrkB`s de-ubiquination at the K460 site in the juxtamembrane domain (Guo) then has a positive effect on the internal Rac1 protein and Cdc42 protein. Both of these proteins are activated by the CaMKIIa enzyme which is also controlled by neuronal firing. Serine phosphorylation of the BDNF regulates its interaction with the Rac1-specific guanine nucleotide exchange factor TIAM1 (Lai) which leads to phosphorylation of Rac1-GDP to active Rac1-GTP. This results in phosphorylation of the S6 ribosomal protein and phosphorylation of the PAK which is involved in actin remodelling and the outgrowth of neurites. Hence, structural remodelling of the neuron which leads to increased firing has occurred and this is consistent with the neuronal advantage of learning eg. hippocampal LTP and memory theories eg. spatial memory. Sen confirmed the link between CREB and BDNF by investigating the case of the endoplasmic reticulum version of ER kinase, Perk. Perk is a transmembrane protein and activation of it is associated with ER stress such as that seen in traumatic brain injury and associated memory deficits. When the activation of Perk is inhibited by for example, GSK2656157, dendritic spine loss is prevented and the memory deficits are rescued. It was found that Perk phosphorylates CREB at the serine 129 residue preventing it from binding to the CREB-binding protein and hence, a reduction in BDNF level was observed.

The link between CREB and dendritic spine density is also confirmed by its activation by glucocorticoids. Conor showed that excessive glucocorticoid exposure during chronic stress causes synapse loss and learning impairment. Under normal circumstances, the hippocampus glucocorticoid receptor is engaged during the formation of long-term avoidance memory and is coupled to the downstream activation of CAMK11alpha, TrkB, Akt, PLCgamma, CREB genes and induction of Arc and synaptic GluA1 (Dillon). The glucocorticoid activity oscillates with circadian rhythms where peaks promote post-synaptic dendritic spine growth in the cortex after learning (via non-transcriptional LIM-kinase-cofilin pathway) whereas troughs are required for stabilising the newly formed spines that are important for long-term memory retention. Chronic and excessive exposure to glucocorticoids eliminates dendrite spine formation with learning (via transcriptional mechanisms involving mineral-corticoid receptor activation) and disrupts previously acquired memories. Therefore, it is assumed that chronic and excessive exposure to glucocorticoids has a negative effect on CREB as well as the other factors influenced by it so that the dendritic remodelling required and indicative of long-term memory formation is not able to occur.

Synaptic remodelling involving CREB may also occur via its activation of its target genes, the c-fos or Arc intermediate early genes which have been shown to be required for the encoding and storing of information in memories (Minatohara). This view is supported by He who showed that cyclin-dependent kinase Cdk7 inhibition leads to suppressed levels of these intermediate early genes in the hippocampus resulting in impaired long-term synaptic plasticity and deficits in the formation of long-term memories. Also, it was shown that social recognition memory requires CREB transcription and significant inductions of those particular CREB target immediate early genes occurred not only in the hippocampus, but also in the medial prefrontal cortex, amygdala and anterior cingulate cortex (Tanimizu).

CREB may also indirectly affect synaptic remodelling and other processes required for successful long-term memory formation by affecting gene transcription of other proteins that play roles in the transition of temporary neuronal effects to permanent. For example, CREB could also influence storage mechanisms indirectly by playing a role in the provision of energy for the cell. Kong stated that the learning in fear conditioning and the formation of long-term memories require the glucose transporter, GLUT3 for the uptake of glucose into the dorsal hippocampal neurons and Rac1 regulates the translocation of GLUT4 containing vesicles to the membrane. Therefore, CREB via its influence on Rac1 can indirectly affect glucose metabolism in the firing cells. This general influence maybe wide-ranging affecting many different processes. For example, it is known that learning and consolidation of memories require protein synthesis (Lopez) and there is an accumulation of polyribosomes in the dendritic spines of amygdala neurons during aversive conditioning (Ostroff). This induction means that conditioning memory involves in this case local translation during learning and this requires cap-dependent initiation. Therefore, it is possible that CREB activation leads to the transcription of genes that are involved in this local translational control.

Another suggestion for CREB`s indirect effect on the long-term memory storage mechanism is that it could be linked to LSD1n functioning which is known to be required for spatial learning and memory (Wang). LSD1n binds to neuronal gene enhancers, promoters and transcribed coding regions and is required for the initiation of transcription and elongation. It is possible that CREB promotes the transcription of genes leading to a protein similar to its own CBP that activates the LSD1n so that it can carry out its promoting function more efficiently. In a similar vein, CREB may also play this role of producing proteins that promote the transcription of other genes for example for: controlling Brd4 which is part of the bromo-domain and binds acetylated histones and is critical for the regulation of transcription linked to learning and memory (Korb); or aids the decrease of histone deacetylase HDAC7 after contextual fear conditioning so that histone acetylation (of possibly gene Nur77) can occur leading to long-term memory formation. Jing showed that histone acetylation plays a role in long-term memory formation. HDAC7 decreases via ubiquition-dependent degradation of CBX4, one of the HDAC7 ligases involved in process; or CREB could aid the regulation of BAF53b expression which is a post-mitotic neuron specific subunit of the BAF nucleosome-remodelling complex. Yoo found that the functional loss of BAF53b leads to a deficit of consolidation of hippocampus dependent memory and in the amygdala; or CREB could aid the activation of NK-kB and translocation to the cell nucleus during consolidation. Salles found that NF-kB transcription factor activity was increased in the hippocampus, amygdala and nucleus accumbens during fear learning. Therefore, CREB as a promoter of gene transcription could play a number of roles in the production of proteins required for the necessary physiological changes linked to changing short-term, temporary neuronal representations into permanent memories.

So, it is clear that the structural physiology and mechanisms behind long-term storage of memories needs much more research. It is clear that firing and connectivity changes experienced by the neurons under the conditions of transferring from short-term responses to something more permanent have to be recorded with the possibility of accurate recall, alterations and re-storage and it is clear that CREB is involved in this process in some way. This may be by influencing the required structural remodelling of the synapse and neuronal cell capability directly via BDNF or it may be by influencing indirectly other required processes such as histone methylation of other gene promoters. It is fair to say we are only at the beginning of understanding this complicated process.

Since we`re talking about the topic…….

…..it has been reported that training increases learning (Cappelletti). Is this connected to CREB performance changes and does it involve BDNF?

….caffeine is said to produce an enhancing effect on long-term memory consolidation for a behavioural discrimination task (Borota). It is known that caffeine can cause calcium ion release and since calcium ions affect CREB production and activation, can we assume caffeine would have a positive effect on CREB functioning in the long-term memory consolidation?

….the accumulation of iron leads to the degeneration of myelin sheaths in the elderly and is linked to alleged reduced verbal learning memory test performance (Steiger). Can we confirm that iron does not affect CREB functioning and therefore, structural changes caused by it are not related to BDNF directed synaptic remodeling?

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effects of sleep deprivation on neuronal connectivity

Posted comment on ´Intrinsic brain connectivity after partial sleep deprivation in young and older adults: results from the Stockholm Sleepy Brain study` written by G. Nilsonne, S. Tamm, J. Schwarz, R. Almeida, H. Fischer, G. Kecklund, M. Lekander, P. Fransson and T. Akerstedt and published in Scientific Reports 2017 7 article no. 9422

SUMMARY

Nilsonne and colleagues investigated the effect of partial sleep deprivation on brain area connectivity and whether or not it was affected by age. Their experiments were cross-over comparisons between individuals who were subjected to partial sleep deprivation (which meant that they were asked to go to bed 3 hours before they would normally get up) and those who had full sleep. All participants slept in their own homes and the sleep state was monitored by polysomnography. Fifty three participants took part and these fell into two age groups: 20-30 years old and 65-75. Two runs of functional magnetic resonance imaging (fMRI) were performed in the evening in order to accommodate for circadian rhythms. One was performed at the beginning of the session and one at end and during the latter the participants were asked to rate on a scale of 1 to 9 their level of ´sleepiness`. During all imaging sessions, eye tracking was monitored in order to determine eye closures and subjects were woken if eye movement had not occurred for more than 5 seconds. Various analyses were performed on the results. Data was pre-processed in order to remove task-related interference and two sets of Independent Component Analysis (ICA) were performed. Seed-region analyses of functional connectivity for the default mode network (DMN) and anti-correlated task positive (TPN) network were carried out. Regions of interest taken were the thalamus, amygdala and DMN. Regional homogeneity was measured using the ReHo toolbox. Global signal variability was assessed using the pre-processed data and was taken as the standard deviation of grey matter signal during each run. The amplitude of low-frequency fluctuations (ALFF) and fractional amplitude of low frequency fluctuations (fALFF) were also measured using the pre-processed data and the DPARSFA toolbox.

Nilsonne and colleagues experiments showed that partial sleep deprivation led to an increased level of subjective sleepiness particularly in the younger participants and those that were longer in the imaging scanner. Head motion was not a factor. The ICA analyses for resting state networks and connectivity gave similar results for data assessed according to pre-processed/grey matter masking and temporal filtering absence/no grey matter masking. Out of 30 components, the investigators found only 10 components of interest. Partial sleep deprivation did not cause changes in connectivity within the networks of interest, but the younger participants produced a pattern of greater connectivity than their older counterparts. The more senior participants showed higher connectivity in small scattered foci. Therefore, the authors concluded that partial sleep deprivation produced no significant differences in network connectivity with age.

Nilsonne and team also carried out seed-based analyses of connectivity with the thalamus and amygdala and within the DMN. They found functional connectivity with the thalamus in the cingular cortices (posterior and anterior), occipital cortex and, cerebellum. There was also functional connectivity with the amygdala and large parts of brain including contralateral amygdala, basal ganglia and cortical areas such as the parietal, temporal and frontal. In the case of the DMN, the authors found an overall pattern of correlation between DMN pairs and anti-correlation between DMN nodes and task-positive (TPN) nodes. Partial sleep deprivation caused no changes although there was lower connectivity within the DMN and reduced anti-correlation levels between the DMN and TPN. Older participants produced a pattern of generally lower connectivity for the 3 DMN node pairs and partial sleep deprivation meant that the pattern observed was further reduced even if there were no node pair differences. Therefore, again partial sleep deprivation produced no changes in participant`s connectivity of the thalamus, amygdala and DMN regardless of age.

Regional homogeneity (ReHo) was also investigated by Nilsonne and colleagues. The hypothesised decreases were not observed with the partial sleep deprivation groups of either age. However, the younger participants did demonstrate higher ReHo in the areas of the cerebral cortex and basal ganglia particularly the medial prefrontal cortex, superior temporal cortex and bilateral insula. The older participants showed higher ReHO values for areas prone to imaging and motion artefacts and these included the orbitofrontal cortex and anterior and posterior outer edges of the brain.

Owing to the unreliable measurements for the global signal itself, global signal variability was used instead. Nilsonne and team found that partial sleep deprivation caused higher global signal variability in the older participants which was even higher for the younger ones. There were no notable associations between predictors and variability except that longer total sleep time (TST) in the sleep deprivation condition was associated with less global variability. Investigations of the amplitude of low-frequency fluctuations, ALFF and fALFF also produced no significant correlations between the control groups and sleep deprivation and age.

Therefore, Nilsonne and team concluded that partial sleep deprivation only increases global signal variability. It had been hypothesised that the older participants would have lower functional connectivity and be less sensitive to sleep deprivation, but major interactions between the age groups and partial sleep deprivation were not observed. The authors did not find any major effects in intrinsic connectivity (including ICA derived networks, seed-based connectivity in DMN and task-positive networks) and from thalamus and amygdala to the rest of brain, regional homogeneity and amplitude of fluctuations.  They did find however, that younger participants had higher connectivity in most examined networks.

The authors went on to discuss possible reasons for their observations. In the case of the increase seen with global signal variability, the authors stated that this was probably due to wake-sleep state instability. It was suggested that partial sleep deprivation increases sleepiness with an increase in propensity to drift in and out of sleep. This view is supported by observations by others that show that global availability is: reduced by caffeine; correlates to EEG measurements of vigilance; increases with sleep; and is increased by the sedative midazolam. It is also supported by results that were achieved without monitoring for eye closures. For example, that associated resting state networks fluctuations relate to cortical electrophysiological indices of arousal; networks are more active in low and wake wakefulness states and wake-state instability causes effects of respiration, heart rate and movement which are associated with global signal variability.

With regards to connectivity within the DMN, Nilsonne and colleagues found reduced connectivity and also reduced anti-correlation values between the DMN and TPN with sleep deprivation. This supported work by others although it appeared not to be at the same level. The difference was attributed to the experimental set-up. The authors also found lower connectivity in ICA derived networks of interest in older participants which supported the results of others. Nilsonne and team found that older participants had lower regional homogeneity in the medial PFC, superior temporal lobes and bilateral insula and this was discussed as a possible reflection of using the partial sleep deprivation condition and not full. The level of sleepiness reported was only equivalent to that seen during night work or night driving and therefore, was considered as possibly not strong enough to induce the hypothesised intrinsic network connectivity changes. Length of sleep deprivation was suggested as one way in which the experimental set-up could be changed as well as including assessment for monitoring head motion and EEG measurements to identify micro-sleeps. However, the authors concluded that even with partial sleep deprivation an increase in global signal variability was observed as reported by others and they attributed this change to sleep-wake state instability affecting neural activity as well as respiration, heart rate and head movements. Major effects of partial sleep deprivation on network connectivity were not observed regardless of age.

COMMENT

What makes Nilsonne and colleagues` article interesting is that it confirms what we already suspect about lack of sleep and that is although when we are tired we aware of lower personal cognitive performance and difficulty in keeping our concentration on certain tasks especially if they are boring or repetitive, we do not suffer the same effects as when we go without sleep altogether for a day or two. There are many reports about the effect of full sleep deprivation with relation to emotional and memory disturbances, but Nilsonne and team`s work investigated the different condition that of partial sleep deprivation, which was set as the participants going to bed three hours before they would normally get up. Under these conditions and experimental set-up the authors found that nothing much occurred to neuronal firing and connectivity from the 3 hour sleep pattern regardless of age. The global network signal was slightly weaker and more variable especially with the older subjects, but the network organisation remained even in the task-related situation.  Nilsonne and colleagues` experiments confirmed that the cognitive mechanisms going on achieved their function even within the available short period of sleep time. A possible explanation for this is that usually larger scale cognitive effects occur when sleep has been deprived for a period of longer than 48 hours and not once as under the experimental set-up of Nilsonne and team and that since normal sleep consists of between 1 and 5 so-called BRAC cycles (each about 90 minutes long with different amounts of time spent in each stage) the omission of 3-4 cycles appears to allow sufficient normal or nearly normal neuronal functioning levels. The observation that partial sleep deprivation does not have major effects long-term or otherwise on cognitive capability is comforting news to those who are unable once in a while not to get their full sleep quota.

In this comment we look at why sleep is important, what happens to certain cognitive capabilities with full sleep deprivation and why partial sleep deprivation is unlikely to have any major effect. Sleep is important because it amongst other things allows certain brain cells and neuronal pathways to carry out ´housekeeping` functions. These are functions that are not related to task response directly, but are necessary to keep the system working properly. For example, it includes neuronal cell proliferation (eg. oligodendrocyte precursor cell proliferation – Bellesi); the pruning of unnecessary neuronal connections in order to uptake and store further events (Tononi); and the repair of damage from free radicals (Lawton). The specific cognitive effects associated with sleep relate to neuronal firing and network connectivity changes. In particular and well documented is the formation and consolidation of information in memories and the mechanisms relating to the emotional system, emotional state elicitation and storage of that status. Stopenich showed that events are better remembered with sleep and this increased memory requires stronger correlations between the ventromedial prefrontal cortex, precuneus, amygdala and occipital cortex. The effect of sleep on the emotional state affects both the present and future since emotional state determines real-time emotional expression, but also what is stored. The emotional memories (´emotional tags`) can give personal ´values` which can influence behaviour eg. seeking out something that pleases and avoiding danger and decision-making.

If we look at memory in more detail we see that full sleep deprivation can bring about long term disturbances in performance. The reason for this is that during the sleep process there is consolidation of learning and memories which is achieved through replay, as well as the addition of new input to previously stored material. The various stages in the sleep process play different roles in the memory mechanism. In general, the stages of sleep are mainly NREM (non-rapid eye movement, 4 stages or under the new nomenclature only 3 stages) and REM (rapid eye movement, 1 stage). NREM is thought to be important for replenishing the synapse, inserting the receptors for example and other housekeeping functions. In the NREM, the thalamo-cortical network generates low frequency oscillations and modulation by GABAergic inhibitory inputs from the thalamic reticular nucleus can occur (Herrera). A removal of this modulation leads to an increase in the duration of sleep and its activation to a rapid arousal. These are not the only areas where functional connectivity during this stage plays a role. Olcese found that loss of global connectivity between the cortex and hippocampus in NREM stages was coupled to the preservation of local connectivity.

More specifically, effects relating to the memory mechanism can be seen in all stages of the sleep cycle to a greater or lesser degree. In Stage 1 of sleep, which is seen as the transitional sleep stage, brain waves of the waking state become less regular with a period of transition from the relatively non-synchronised beta and gamma waves that are normal for the wake state to the more synchronised but slower alpha wave followed by theta. This stage lasts less than 10mins, is less than 5% of the total time of sleep, is easily disrupted and from a memory mechanism perspective is not particularly significant.

Stage 2, however is. This stage, as conscious awareness fades, is about 45-50% of the total sleep time. Brain waves are mainly theta and include occasional sleep spindles (8-14Hz, lasting about half a second) which are generated by the thalamic pacemaker. High amplitude K complex waves (theta) also occur which are short negative high voltage peaks followed by a slower positive complex and then a final negative peak with each complex lasting 1-2 mins. Stage 2 serves to protect the sleep state and suppresses responses to outside stimuli as well as to aid in sleep-based memory consolidation and information processing. It does this by network connectivity shown by particular brain wave activity. The sleep spindles, seen in both Stages 2 and 3 are known to coordinate interregional cortical connectivity and particularly that of the hippocampus (CA1 to CA3 and DG- Sullivan) and this has functional relevance for memory performance. Such a function is associated with not only excitatory firing, but also inhibitory firing, ie. via GABA. The administration of a short-acting GABA agonist can increase sleep spindle density, decrease REM sleep causing increased verbal memory, decrease perceptual memory and have no effect on motor learning (Mednick). The K complex waves that also occur in Stage 2 aid the memory process, too.

The next sleep stage, Stage 3, is known as deep, delta or slow wave sleep (SWS) and is characterized by delta brain waves. It occurs in longer periods during the first half of the night and is 15-20% of the total sleep time. Stage 3 has some sleep spindles, fewer than Stage 2, but the majority of the wave forms are slow with large amplitudes. In this stage the sleeper is unresponsive to the outside environment and is unaware of any sounds or stimuli. The slow wave sleep oscillations or ripples (SWS) can be found in the first three stages and oscillate in relatively high synchrony. They are important to the memory process because they are associated with memory formation and consolidation (Ngo, Leonard). This function is achieved by coordinating interregional cortical communication (Cox) between the thalamo-cortical network, which receives input from the hippocampus (Wei). Wei found that external input mimicking the hippocampal input led to input-specific changes in synaptic firing and promoted replay of specific firing sequences in the cortex. Such firing is thought to involve nicotinic acetylcholine receptors (Sigalas), but just like in Stage 2, GABA plays an important role. In SWS inhibitory GABAergic firing of two areas of thalamus (nucleus reticularis thalami and thalamocortical neurons) is due to T type calcium channel activation. This causes long term depression of input areas when coupled with excitatory firing (Pigeat). However, memory processing, formation and consolidation appear to require hippocampus-cortical connectivity (Maingret, Jahnke) with SWS replay modulating both the strength and precision of the memories formed (Barnes). It has also been found that new information can also be integrated into the memory traces (Tamminen, Arzi) and this can occur during SWS and sleep spindles.

The next stage if using the old nomenclature, Stage 4, appears to have no great significance to the memory process. It consists of delta waves and is greater than 50% of the total sleep time. However, the last stage does. Stage 5, the REM dream phase, linked to beta brain waves is associated with physiological synaptic changes required for memory consolidation (Luo), eg. increases in cAMP, MAPK Activity and CREB phosphorylation. It, like the other stages is associated with excitatory and inhibitory firing as shown by a requirement for GABA. Connectivity between certain brain areas is also observed, eg. possible increase in connectivity between nodes of the DMN in REM even if the reason is unknown.

   Therefore, in summary, sleep is important to the memory process and the individual stages have particular functions. For example, there is a requirement for hippocampal SWS for memory formation with consolidation through replay.  Connectivity of particular areas is also important, eg. the hippocampus, thalamus and cortex for the quality and quantity of information and this requires theta brain wave activity. Firing and connectivity in real-time produces neural representations of the information, but this is only transient and therefore, more permanent storage of the representation has to occur to form the long-term memories. To do this, fundamental long-term physiological changes in the synapses have to be brought about such as increases in cAMP, MAPK activity and CREB phosphorylation.

So, what happens to the memory process with full sleep deprivation? We know that full sleep deprivation elicits memory deficits. This is probably in general due to long-term changes in the firing capabilities of particular cells and groups leading to alterations in the network connectivity. Specific effects are also observed. For example, full sleep deprivation leads to a region specific homeostatic increase in theta activity (associated with Stage 1) suggesting that theta waves represent the transient neuronal ´off` (Bernardi). Since Stage 1 is affected, it is unlikely that the individual stays in the sleep cycle so that the vital memory formation and consolidation processes cannot occur later on in the sleep cycle. Another example relates to Stage 3, the delta SWS. If the SWS are disturbed by for example sleep deprivation occurring at an earlier time in the overall sleep time (SWS occurs in longer periods during the first half of the night and is 15-20% of the total sleep time) then the memory process such as event replay and memory formation and consolidation cannot be successfully carried out. A lack of Stage 5, the REM stage can also lead to the disruption of the memory processes. Deficient REM is known to have a negative effect on DMN connectivity and task-positive networking. The role that the DMN plays in the memory formation and consolidation is not known for certain, but it is possibly related to neuronal ´housekeeping` functions and maintaining the firing of the appropriate areas independent of tasks.

Sleep deprivation not only has effects on neuronal firing and network connectivity, it can also have effects on the physiological synaptic changes, eg. increases in cAMP and CREB phosphorylation that are required for memory consolidation (Luo). In nocturnal mice woken during the day, increased levels of gamma waves appeared, but more fundamentally cFOs gene changes resulted plus increased GABA receptor number and decreased AMPA receptor number on CAMKII responsive neurons (Cid-Pellitero). Therefore, any change of the neurochemical characteristics of the cell would have an effect on the performance of the memory mechanism. In this capacity, GABA and GABA firing have also been reported as being affected by sleep deprivation conditions. Cid-Pellitero in line with the changes reported above showed that GABA Rs were also increased with sleep deprivation in correlation to the decreased AMPA Rs. Therefore, the excitatory neurons were scaled down during sleep deprivation and conversely, GABA inhibitory firing was increased.  This could be important in that in the NREM stages, the thalamo-cortical network generates low frequency oscillations with the modulation by GABA inhibitory inputs from the thalamic reticular nucleus (Herrera). Silencing of the modulation leads to an increase in the duration of sleep and activation towards rapid arousal and a change in the level of the GABAergic connectivity involved going from the thalamus to the hippocampus and ultimately to the cortex.

Other cell changes that are more general are also observed in sleep deprivation. For example, there are many gene expression changes. Narwade and colleagues found that the expression of genes involved in for example DNA methylation, synaptic regulation and neuronal plasticity were all altered in selective REM sleep deprivation. Neuronal cell proliferation was also found to be decreased in the hippocampal dentate gyrus region with full sleep deprivation although the apoptosis rate remained constant. This leads to a decrease in cell number in that area. An increase in astrocytic phagocytosis after both acute and chronic sleep deprivation that was not linked to neuroinflammation has also been reported (Bellesi).

Therefore, the establishment of physiological conditions required for memory formation and consolidation at the levels of networks, neurons and synapses are not met for a number of reasons in full sleep deprivation. This was found not to be the case in partial sleep deprivation which produced only few, transient effects that appear not to be significant to the memory process.  Global signal variability is likely to reflect decreased firing levels and a transient increase in inhibitory firing via GABA as seen with full sleep deprivation. The reduced firing of the various elements leads to reduced levels of intraregional and interregional connectivity and hence, the required theta brain wave formation is not achieved within the network.

In Nilsonne and teams experiment the early phases of the normal total sleep time is absent and therefore, the memory consolidation process relies on one or two BRAC cycles rather than the normal 4-5. Also Stage 3, the SWS, occurs in longer periods during the first half of the night and therefore, if sleep is not possible during this time as it was in Nilsonne`s experimental set-up, then this phase may be shorter than normal. However, the lack of effect from partial sleep deprivation indicates that memory consolidation occurs relatively normally and therefore, there is with sleep, just like with other cognitive processes,  a cognitive ´reserve` or ´excess` . It is unlikely that there is no REM stage in partial sleep deprivation and therefore, any physiological synaptic changes eg. increases in cAMP, MAPK activity, and CREB phosphorylation required for memory consolidation (Luo) are still possible even if the total sleep time is reduced.

Before, we leave the topic of memory, we should talk about the hippocampus and what happens in sleep deprivation and this is because the hippocampus plays central roles in sleep itself and in memory. In fact, the Sleep theory of Oswald states that the hippocampus is linked to shifting memories from it to the cortex in the sleep cycle (Mehta). In the NREM stages 2 and 3 this occurs through sleep spindles and interregional connectivity (Sullivan) and it has been found that the better the sleep spindle density then the better the memory (Hennies) through the disengagement of the hippocampus. In Stage 3, the SWS, the hippocampus promotes global connectivity from it to the thalamus-cortical areas (Wei) plus promoting connectivity within its own local regions (Olcese). The SWS and the hippocampus are linked in particular to spatial memory (Leonard) for example and on a more general scale, this particular functioning and connectivity are responsible for memory consolidation through replay of the neural representations (Maingret, Jahnke). Long-term synaptic physiological changes in the hippocampus are also observed during the REM stage (Luo) and this is consistent with memory formation and consolidation views. Therefore, any change in hippocampal functioning can have an extensive effect on long-term memory and this is found to be the case in sleep deprivation where: the lack of sleep can lead to stress hormones being released that leads to an effect on the hippocampus in the form of decreased neurogenesis (Gould); and long-term detrimental effects on spatial memory can occur (Varga, Soto-Rodriguez) because of a decrease in cell proliferation in the DG area for example although this is disputed.

So far we have discussed the effect of sleep deprivation on one cognitive capability that of memory. However, we know that tiredness and lack of sleep also has an effect on our emotional state and this can influence our mental performance and our behaviour in real-time and also in the future. We know that tiredness can make us for example more ´fragile`, less patient, more aggressive or less ambitious and therefore, sleep is important because it allows us to form and maintain appropriate emotional and motivational states and aids cognitive performance in the learning and application of suitable emotional tags. This has importance in recall because of the influence on behaviour and decision-making for example by our application of personal values.

   With regards to sleep deprivation, any effects are probably due to long-term changes in the firing of particular neuronal cells and groups of cells that are involved in the emotional pathways. These could lead to alterations in quality of firing and extent of network connectivity which would result in changing real-time emotional status and records of that status in the form of emotional tags and values. This view is supported by work by Liu who showed that sleep deprivation has an effect on emotional state by increasing the motivation for reward. The effect is due to a decrease in the ratio of the overall excitatory over inhibitory synaptic inputs onto the nucleus accumbens. This shift is partly mediated by reduced glutaminergic transmission via a selective reduction of glutamate release at the synapses of the medial prefrontal cortex that link to the nucleus accumbens. Such an effect is specific since no change of the input to the hippocampus, thalamus or amygdala is recorded. Lack of sleep was also found to increase pre-emptive responding in the amygdala and anterior insula during anticipation especially for cues associated with high certainty (Goldstein) and especially in individuals who demonstrated high levels of anxiety.

Sleep deprivation could also affect the emotional status by changing the values of the events and this can change recall for example. Carr found that the recall rates for REM daytime naps (96%) and NREM daytime naps (89%) were elevated compared to typical recall rates for night-time dreams (80% and 43% respectively) which suggested an increased circadian influence. NREM dreams were shown to have lower ratings for emotional intensity and sensory experience while REM dreams had higher ratings for bizarreness and sensory experience. Also it was found that it was harder to suppress unpleasant memories if sleep came after learning. This led to the idea that sleep deprivation might be one way in which unpleasant memories could be suppressed and therefore, research has been carried out to investigate whether it this method could be used in disorders such as post-traumatic stress disorder for example.

Sleep deprivation can also affect cognitive performance by enhancing the inability to ignore neutral distracting information, but not emotional information so that both are processed similarly (Simon). This renders emotional values to be neutral (Simon) and ineffective in distinguishing information value. This change was found to be associated with the REM sleep stage and decreased PFC connectivity. Therefore, the balance of emotional status, correct assessment of personal values and use of both and association with information requires appropriate sleep cycles and sleep time. Sleep deprivation can lead to dysfunction of the processes relating to the emotions and is likely to occur just like with memory in the failure to instigate appropriate firing, network connectivity and long-term physiological changes. Partial sleep deprivation is likely to have the same transient effects on the emotional system as for the memory mechanism and therefore, real-time emotional status and worth may be affected by the change in global signal variability, but nothing will be long-term.

Nilsonne and colleagues also looked at the difference between the effects of partial sleep deprivation on the cognitive capabilities of older individuals compared to their younger counterparts and found only minor differences. The authors determined that the subjective assessment of sleepiness was found to be reduced in the older individuals whether sleeping normally or after sleep deprivation. This correlated with the older individuals also having reduced sleep duration and ability to produce sleep under normal conditions. Taken together the study implies that older individuals attain less sleep and require less sleep to achieve their ´normal` cognitive performance and this is supported by the observation that younger individuals are more susceptible to sleep loss both physiologically and with self-reported levels of tiredness. However, others have reported losses of spatial and episodic memory and learning capabilities in older people (Wang) which have been associated with physiological and functional changes in the hippocampus and PFC. For example, decreased glutaminergic transmission in the medial PFC is linked to a slow age-related decline in various capabilities such as attention, decision-making, perception of time (Guidi). However, this view is not supported by all since for example Parks has shown that visual, spatial and verbal memory performances actually decline with all ages.  Therefore, we have to assume that individuals can show a range of levels relating to cognitive capabilities depending on the extent and quality of their own individual brain physiology and the normal ageing process may reduce this functioning performance. Sleep deprivation is just another exacerbating effect and this is supported by Nilsonne and team`s study where the older participants exhibited functional connectivity that was in general lower than their younger counterparts. In this case, the reduced capability was said to be due to structural changes including white matter degeneration. Partial sleep deprivation also caused global signal variability with older participants having greater values and again this points to transient effects from the lack of sleep independent of age, eg. reduced network connectivity and temporary neuronal firing depression occurs.

Therefore, to conclude we can see that sleep is important for, as described above, the formation and consolidation of memories since it provides the conditions that promote connectivity and binding of information together and allows long-term physiological changes of these representations to occur. If this is the case it is understandable that full sleep deprivation will have major effects on certain cognitive and emotional capabilities that depend on information storage and recall. It is also comforting to see that in healthy individuals partial sleep deprivation may cause only transient effects and age is only a minor factor. Therefore, providing there is sufficient physiological capability for appropriate and relevant cognitive functioning, restoration of sleep patterns even if at levels lower than ideal should produce some positive cognitive effect.

Since we`re talking about the topic……

…..it is said that one way the circadian rhythm of sleep-wake cycle is modulated is via the level of adenosine. If Nilsonne and team`s experiments were repeated with administration of adenosine thus inducing towards the sleep state, but with deliberate waking using for example loud music or physical shaking would the same imaging results of network connectivity be produced? Would global signal variability be increased independent of age due to the conflicting conditions?

…it has been found that pairing pleasant and unpleasant odours with different tones during sleep caused learning of the novel associations (Arzi). Can we assume that this occurs even with partial sleep deprivation and there is no change in the quality of this learning?

…can we assume that all methods of partial sleep deprivation, eg. losing the first part of total sleep-time or being woken in the middle of the total sleep time produce the same effects on global signal variability as that shown in Nilsonne and colleagues experiments or does it have different effects? How does this relate to memory performance such as spatial or verbal memory? Would the administration of GABA agonists overcome any effect?

 

Posted in ageing, neuronal connectivity, neuronal firing, sleep, sleep deprivation, Uncategorized | Tagged , , , ,

striatal dopamine DA2 receptors modulating role in hippocampal functionality

Posted comment on ´Dopamine D2 receptor availability is linked to hippocampal-caudate functional connectivity and episodic memory`  written by L. Nyberg, N. Karalija, A. Salami, M. Andersson, A. Wahlin, N. Kaboovand, Y. Koehncke, J. Axelsson, A. Rieckmann, G. Papenberg, D.D. Garrett, K. Riklund, M. Loevden, U. Lindenberger and L. Baeckman and published in PNAS 2016 113(28) p. 7918 doi 10.1073/pnas.1606309113

SUMMARY

In their study, Nyberg and colleagues investigated the relationship between dopamine type 2 receptors (DA2R) in the striatum and hippocampus and certain cognitive functions such as episodic memory, working memory and speed of processing. The authors found that DA2R populations contribute only to episodic memory performance by influencing the activities of the striatal and hippocampal areas and their interrelated connectivity.

Nyberg and team`s experimental set-up had an effective sample of 174 subjects between the ages of 64 and 68. Assessment of the subjects` cognitive functions was carried out by using the COBRA (Cognition, Brain and Ageing) study design. Verbal, numerical and figurative capabilities were measured in order to ascertain episodic memory, working memory and speed of processing performances. The tests for episodic memory performance included word recall, number-word recall and word-location recall.  Working memory was tested by looking at letter-string updating, numerical performance by numerical 3-back and spatial updating and processing speed by performances at letter comparison, number comparison and figure comparison. The three scores for each capability were averaged to create one summary score for each cognitive domain. Neuronal functioning was assessed using images acquired from structural MRI and functional resting state MRI, perfusion experiments to measure blood flow and DA2R populations by DA2R binding potential  to non-displaceable tissue uptake using radioactive raclopride and PET (DA2R BP). The regions of interest (ROIs) studied included the caudate, putamen, hippocampus and cerebellum. For the main analyses the caudate area was divided into sub-regions, but for the whole brain analyses extrastriate areas especially the hippocampus were used. Functional connectivity studies relating to DA2R BP looked at the interactions between the ventral caudate and medial temporal areas.  Statistical analyses were carried out on all results.

Nyberg and colleagues found that there was a significant positive association between the binding potential of caudate DA2Rs and episodic memory, but not for working memory or processing speed. Controlling for caudate volume produced correlations of similar size between DA2R BP and performance and therefore, it was said that there were negligible partial volume effects.  Analysis of the DA2RBP of the inferior ventral caudate and episodic memory performance produced a significant association, but no such correlation was found between DA2R BP of the putamen and episodic memory even though caudate and putamen DA2R BPs are interrelated.

The authors then went on to investigate DA2R BP activity in the ROIs chosen. They found DA2R BP cognitive associations across the whole brain. In the left hippocampal complex a significant cluster of cells was discovered that showed a positive linear association to episodic memory performance. Whole brain analyses for working memory and processing speed showed no significant effects. Because of hippocampus result, whole brain analyses on ROI areas were then carried out. Hippocampal DA2R BP values were found to be normally distributed and produced a higher signal than for the other ROI, the cerebellum. The whole hippocampus (left and right) produced significant correlation to episodic memory when controlling for hippocampus volume and a direct test of caudate-hippocampus DA2R BP gave also a significant positive correlation. Therefore, the authors concluded that individuals with higher caudate DA2R BP had higher hippocampal DA2R PB and showed greater episodic memory performance.

The authors also found that caudate DA2R BP did not correlate to the grey matter volumes of the hippocampal, caudate or putamen areas, but hippocampal volume was correlated to episodic memory performance (even though there was no association between hippocampal perfusion and DA2R BP) and certain grey matter volumes were interrelated, eg, caudate-putamen, caudate-hippocampus and hippocampus-putamen.  Hippocampal and caudate DA2R availability was also found to be interrelated.  The functional MRI-based resting state functional connectivity maps for the inferior ventral caudate seed (VCi) included both left and right hippocampus and showed a significant relation between it and caudate DA2R BP. This demonstrated a positive association in the left hippocampal /parahippocampal gyrus and the anterior medial temporal lobe.  A study of episodic memory performance showed a link between the connectivity map of the VCi and a significant cell cluster in the adjacent medial temporal lobe region. This connectivity appeared to be the mediator between caudate DA2R BP and episodic memory performance.

Nyberg and colleagues therefore, concluded that although the DA1R is linked to prefrontal cortex activity and working memory and executive functioning, the DA2R is a contributing factor to hippocampus-based cognition by influencing striatal and hippocampal functioning and their connectivity.  The authors` study used more senior subjects in their experiments and found a positive association between caudate and hippocampal DA2R BPs and episodic memory performance, but not working memory or processing speed. Their MRI analyses of functional connectivity showed that participants with high caudate DA2R BP and high episodic memory performance had stronger functional connectivity between the ventral caudate seed (Vci) and medial temporal areas (MTL) eg. hippocampus. This observation supported work by others who showed positive associations between high caudate and hippocampal DA2R BP and larger hippocampal volumes. In general, the observations reported here support the view that DA2Rs contribute to hippocampal cognitive functions which is indicated by hippocampal DA2R modulation of long-term potentiation, long-term depression and learning and memory. Episodic memory appears to involve a considerable overlap of the functional connectivity between striatal sub-regions (especially the caudate) and the hippocampus and therefore, these regions are viewed as a shared functional network. This view is supported by others who for example have found that interconnecting hippocampus and ventral striatal areas produce increased striatal DA release upon hippocampal hyperactivity. Nyberg and team went on in their discussion to explore the roles for the association and suggest that in the case of episodic memory the ventral striatum is involved in the integration of inputs from several areas including the MTL. This input from the MTL to the striatum input can affect dopaminergic activity in the ventral tegmentum area. Therefore, the observed dopamine-episodic memory association may be related to the level of functional MTL input into the caudate. The MTL-VCi connectivity relating to DA2R functionality was therefore, suggested as a possible predictor to the observed differences in episodic memory performances between individuals. The DA2R BP level s were found in both the caudate and hippocampus to be associated with episodic memory performance and were also found to be interrelated. This, the authors suggested relates to crosstalk between different dopaminergic pathways since both the caudate and hippocampus are target areas for dopaminergic projections originating from different nuclei (eg. substantia nigra and VTA).

The authors concluded their discussion by looking at ageing and dopamine integrity. They suggested that this and hippocampal volume may be indicators of brain functioning level in ageing and that DA2Rs may exert protective effects against age- related processes such as neuroinflammation and excitotoxicity of hippocampal neurons.

COMMENT

What makes this article interesting is the association between striatal functioning which is normally linked directly to essentially emotional status (and values) and motor functioning and the cognitive capability of episodic memory and this link appears to be through the neurotransmitter, dopamine and its specific receptor population, the DA2R and the brain area, the hippocampus. This dopaminergic function appears to be modulating since Nyberg and colleagues show that the activity of striatal (particularly in the caudate) DA2Rs positively correlate to hippocampal activity and episodic memory performance.

So, we ask why should the striatum modulate the activity of the hippocampus  and firstly, why should anything affect the hippocampus at all? If we restrict the argument to episodic memory since Nyberg and team`s article refers to this particular type of cognitive function, the hippocampus plays a role in sensory input and timing and binding of that information within the immediate neural representation. This neural representation forms the basis of the episodic memory which can be said to be pure multisensory information bound together in time, but with a personal emotional value attached (the emotional tag). Therefore, any modulation of hippocampal activity affects the neural representation formed and the working memory informational processing state whether its roots are real-time in the form of sensory input or emotional status or from the past in the form of recalled stored information and attached emotional feelings. And this is where the striatum fits in as a modulator of hippocampal activity.

The striatum, so called because of its striped appearance, is located in the temporal lobe of the brain and consists of 2 main regions: the caudate nucleus (described in Nyberg and team`s article) and the lentiform nucleus which consists of the putamen and globus pallidus areas. References to the dorsal striatum relate to the caudate and putamen and the designated ventral striatum, the nucleus accumbens (NAc) and olfactory tubule. The main neuronal cell types are dopaminergic (the subject of Nyberg and team`s article) and glutaminergic. The principle cell type (95% of the population) is the medium spiny neuron (msn) which is either GABAergic and hence, an inhibitory neuron or dopaminergic of which 40% are DA1R related and 40% DA2R. However, other cell types do exist in the striatum which also contribute to the overall functioning of the area. These are: cholinergic interneurons which are large aspiny interneurons that are affected by dopamine via a DA5 R, release acetylcholine and respond to the salient environment with stereotypical responses temporally aligned with the dopaminergic responses of the substantia nigral neurons; the GABAergic interneurons of which there are many types with the best known being the parvalbumin expressing interneurons (also known as fast-spiking interneurons) which are responsible for the fast feedforward inhibition of the principle neurons; and the tonically activated interneurons (tans) which are cholinergic also leading to DA release and therefore are excitatory. Input to the area is varied with the largest input coming from cortical axons. Many parts of the neocortex innervate the dorsal striatum with cortical pyramidal neurons projecting to the striatum being located in layers II-VI, with the densest of projections coming from layer V. These axons end mainly on the dendritic spines of the medium spiny neurons which are glutamatergic and hence, excitatory. The ventral striatum also receives direct input from multiple regions in the cerebral cortex, but also has input from the limbic areas such as the amygdala, thalamus and hippocampus. The nigrostriatal connection comes from the neurons of the substantia nigra and the mesolimbic pathway projects from the ventral tegmentum (VTA) to the NAc. These nigral axons synapse mainly on spine shafts. Output from the striatum is also varied with important connections to the rest of the basal ganglia. Primary outputs of the ventral striatum project eventually to the medial dorsal nucleus of thalamus and there are also projections to the globus pallidus, substantia nigra pars reticulate, amygdala and lateral hypothalamus.

An indication of the cognitive functioning of the striatum can be obtained by looking at what deficiency of the region brings. A role in motor functioning is indicated by Parkinson`s disease which is caused by a loss of dopaminergic innervation to the dorsal striatum (and other basal ganglia areas) as well as an involvement in Huntingdon`s disease  and movement disorders such as chorea, and dyskinesias. A role in emotional and value systems is indicated by the involvement of the striatum in a number of neuropsychriatric disorders such as: addiction where there is a disorder of the brain’s reward system arising from the overexpression of deltaFosB  transcription factor  in the DA1R expressing msns of the ventral striatum; bipolar disorder where there is an association between the striatal expression of the PDE10A gene variants with bipolar I disorder and genes DISC1 and GNAS with the bipolar II disorder; the autism spectrum disorder which is characterized by cognitive inflexibility and poor understanding of social systems and originates with defects in the striatal and prefrontal cortex (PFC) circuits; and emotional disorders such as depression and obsessive compulsive disorder where striatal deficiency indicates the areas involvement in reward pathways. Therefore, the striatum plays roles in the health and well-being of an individual by being associated with physical effects using the motor systems eg. head-turning and Parkinson`s disease and mental effects using the cognitive and emotional systems.

The role of the striatum in emotions and emotional status lies with its functioning level and connectivity. Connectivity involves areas such as the PFC, amygdala and insular cortex and there are at least 3 integrated cortico-amygdala-striatal circuits (Cho). This type of connectivity provides a mechanism where emotional states are instigated and can provide input into the neural representations of the sensory information. One use of such an association involving striatal participation directly is in decision-making where striatal representations of value could bias responses towards performing actions of higher value for example (Tai). This is carried out via dopamine regulation (Tai) who found that DA1R and DA2R are involved in value-biased actions. Support for the role of the striatum and dopamine in emotions, value and reward also comes from Chowdhury for example who showed that expected reward value signals occur through structural connectivity between the substantia nigra, VTA and striatum and is dopaminergic since L-Dopa replacement increases task performance in elderly adults who naturally show a decline in reward processing capability. The ventral area in particular has been found to be linked to learning the value of stimuli (work by Rothenhoefer) and reward prediction error (work by Chien, Boehme and Wimmer).

But the striatum has also been found to be linked to different types of memory including the subject of Nyberg and team`s article, episodic memory. This role comes from the connectivity of the striatum to the hippocampus, which is fundamental not only for reward prediction error (Wimmer), but also for episodic memory formation and consolidation (Jahnke). This function is associated with dopamine, eg. repeated stimulus presentation causes a decrease in dopaminergic activity and cortico-striatal pathways are required for event characteristics integration (Jarbo) with the striatum having two zones, one in the caudate and the other in the putamen . Memory formation and consolidation can also be influenced by attention and this too can be linked to striatal and dopamine functioning. For example, changes in dopamine availability in the PFC, striatum and anterior cingulate lead to changes in attention (Settles) and Jang found that attentional state is encoded with factual and emotional information and requires the involvement of firing and connectivity of different temporal areas, eg. hippocampus and putamen integrate event characteristics such as concrete-feature-selective information (in their experiments primarily the visual cortex), category-selective information (posterior frontal cortex) and control demand-selective information (requires insula, caudate, anterior cingulate, and parietal cortex connectivity). The striatum has also been linked to conscious awareness (Slagter) which can also influence memory formation and consolidation. Slagter found that the ventral striatum modulates the flow of information to the cortex and hence, contributes to conscious perception.

The value of the striatum and dopamine is therefore clear for certain physical and mental systems, but, Nyberg and team in their article were discussing the striatum and dopamine in terms of neuromodulation and this is different to direct action. The effect of the neuromodulator is only, as the name suggests, used to alter the primary system which exists and functions elsewhere. This can be seen in certain disorders such as schizophrenia and ADHD. Therefore, we conclude this discussion of the striatum and its dopaminergic activity by looking at the various mechanisms by which neuromodulation of this nature can occur and we use the striatum and/or dopamine as examples.

    One the ´macroscale`, neuromodulation can occur by altering the number of dopaminergic responsive neurons or by altering the dopaminergic dominant area`s connectivity to other brain areas. In the case of the former, this would have the effect of changing the extent of the modulation possible ie. if the number of DA responsive neurons increases then modulation could be greater. An example of this is the effect of dopaminergic neurons on ADHD behaviour. Aside for changes in the regulation of dopamine production, it has been found that the circadian clock also regulates the genes that are involved in the development and/or maintenance of dopaminergic neurons in the ventral diencephalic posterior tuberculum (Huang) leading to regulation of the number and organisation of these particular neurons. Disruption of the circadian clock was found to produce ADHD-like behaviour (Huang) confirming the link between dopamine and this type of cognitive disorder. Effects on the number of specific cell populations would also have an effect on the extent of neuromodulation possible. For example, work by Owesson-Wright showed that distinct populations of NAc neurons affect reward responses. A subset of DA2Rs cells are affected after the cue whereas responses proximal to the response involve both DA1R and DA2R cells. Work by Edelmann showed that hippocampal Schaffer collateral-CA1 synapses also are subject to neuromodulation in such a manner. In this case, the dendrites might provide domain specific locations for multiple types of synaptic plasticity (LTD or LTP) in the same neuron brought about by the receptor populations present.

The other way in which neuromodulation can occur on a ´macroscale` is by the neuromodulator affecting the connectivity of that brain area to others. This can affect strength and extent of firing and hence, level of functional activity of the modulated area. This particular type of modulation has been described in the article by Nyberg and colleagues relating to the connectivity between the striatum and the hippocampus with regards to episodic memory performance with the striatum and dopamine acting as neuromodulators. Work by Piray gives another example where the striatum is instead being modulated by its connectivity with the ventromedial PFC. In this example, the extent and strength of the connectivity lies with the structural integrity of the white-matter tracts between the two areas whereas Nyberg`s example relies on the strength of the dopaminergic activity of the neuromodulating striatal area. This is supported also by work by Kahnt who investigated the modulating role of dopamine on the connectivity of the midbrain nuclei with the orbitofrontal cortex. It was found here that inhibition of the DA2R population caused a shift from the associative areas of the temporal and parietal areas to connectivity with the frontal cortex.

Physical modulation of the connectivity of brain areas may cause another effect and that is the effect on brain wave activity of the collective areas. It is known that certain brain wave frequencies are linked with particular cognitive functions or states, eg. sleep is associated with alpha, beta, theta and gamma brain waves in a pattern indicative of brain functioning at that time of the sleep cycle. It has been shown that theta-alpha brain wave oscillations bind the hippocampus, PFC and striatum during memory recall for example (Herwig) and therefore, any change in connectivity can lead to the disruption of the brain wave activity which may be an important condition for successful functioning.

Neuromodulation can also occur on the ´microscale` or cellular scale as we have seen in Nyberg and team`s article with a small cluster of hippocampal cells being the recipient of the striatal dopaminergic modulating influence. Modulation at the neuronal cellular level can be brought about by a number of different factors. For example, via control of the level of neurotransmitter which determines the excitatory or inhibitory firing of that particular cell or cell group. Referring to dopamine, Huang found that the circadian clock directly regulates the genes responsible for dopamine synthesis and hence, reduced levels of dopamine can be induced when the circadian rhythm is disrupted. This resulted in the appearance of ADHD-like symptoms. Work by Boehme supports this idea of neurotransmitter level since the dopamine synthesis capacity of pre-synapses in the striatum was found to be linked to the encoding of reward prediction error.

Neurotransmitter synthesis is not the only factor affecting the level of active transmitter in the cell or immediate area. Release and transport can also be areas where changes can be brought about that affect the activity of the cell and hence, its ability to modulate other areas. For example the release of dopamine can be inhibited by the binding of its own molecules and other agonists to appropriate receptors which can lead to second messenger Ginhibitory (Gi) functioning and lack of cAMP production. Yorgason found that the release of dopamine in the NAc is regulated by several factors such as voltage-gated ion channels, DA2R population and nicotinic acetylcholine receptor population. Cassidy found that in the case of schizophrenia, then working memory task performance deficits were linked to deficient cortical dopamine release whereas Kodoma found that in the case of ADHD, administration of methylphenidate led to an increase in DA release in the striatum. Even if the neurotransmitter is released it has to be transported and this too gives a point where overall cellular activity can be influenced in order to induce modulatory behaviour in connected areas. In the example given above about methylphenidate administration to treat ADHD (Kodoma) it was found that methylphenidate actually causes an increase in DA release by blocking the striatal DA transporter. This can be compared to a change in dopamine transporter number in the case of individuals without the dopamine transporter genotype DAT1 where lack of dopamine transporters leads to an ability to suppress task-irrelevant information (Newman).

Affecting the level of transmitter can alter the level of firing of the cell and hence, cause changes to the level of the cell`s activity and its scope for modulation on its connecting areas. Another way of producing such an effect is a change in neurotransmitter receptor number or functioning capability and this was described in Nyberg`s article with the DA2R population of the striatum affecting hippocampus activity and ultimately episodic memory and working memory performance. A decrease in receptor population results in lower firing and vice versa, but it can also mean that second messenger systems and subsequent cell plasticity are also affected and these too can result in overall changes in cellular activity and consequential effects. For example, the DA2R is primarily coupled to an inhibitory G protein (Gi) which directly inhibits the formation of cAMP by inhibiting adenylate cyclase action. This can have an effect, eg . Clarkson reported that the action potential excitability in infero-temporal regions such as the NAc and amygdala were through effects on the D3 dopamine receptor population (D3 is a member of the Gi-coupled DA family of receptors) and this was found to be linked to neuropsychiatric disorders. The reverse can also be found since the DA2R can also be linked to a stimulating G protein linked to adenylate cyclase action and cAMP formation. This can also result in the neuromodulating effects of the cells being instigated. For example, Robinson found that the dopaminergic influence on the PFC (the so-called afterdepolarisations) may be elicited initially through glutamate NMDAR activation causing calcium ion influx and DA2R activation via cAMP/PKA signalling.

Therefore, Nyberg and team`s article confirmed what we knew all along – that looking at single cell activity in the brain brings us only part of the way forward in our understanding of how the brain works and gives us the cognitive capabilities we know we have. Neuromodulation is just one way in which cells not only communicate with one another, but can influence one another and it needs to be investigated with the same vigour as that for changes in activity relating to single cells. The answers to memory and consciousness for example, and dementia and Parkinson`s disease no doubt lie not only with single cell functioning and performance, but also on interrelations with other cells locally and globally and therefore, any research into this complicated topic is welcome.

Since we`re talking about the topic…….

…a decrease in DA2R population has been reported with ageing and this is linked to certain cognitive effects. Steiger reported that age-related memory impairments were associated with structural changes in the dopaminergic system and this may be linked to the accumulation of iron causing a degeneration of neuronal cell myelin sheaths and a decrease in grey matter volume. Since the subjects of Nyberg and team`s experiments were of a more senior age, could the experiment be repeated using magnetic resonance imaging to quantify the degree of myelination and iron accumulation in this sample and compare it to a group of younger subjects and relate this to episodic memory performance effects?

… can we assume that a repetition of Nyberg`s experimental conditions, but with prior administration of the DA2R antagonist amisulpride or amphetamine (which promotes dopamine release) would cause changes in episodic memory performance plus changes in the hippocampal-striatal activity as observed using functional magnetic resonance imaging?

….Izumo investigated the link between dopamine and oestrogen level and found that oestrogen deficiency (OVX – overiectomy model) caused a decrease in voluntary activity in rats associated with reduced levels of dopamine and 5HT in the amygdala. Since there is a reported link between amygdala and striatal effects, would a repetition of Nyberg`s experiments using OVX rats also show decreased dopamine in the hippocampal-striatal pathway and decreased spatial and episodic memory as expected?

Posted in dopamine receptors, episodic memory, hippocampus, neuronal connectivity, striatum, Uncategorized | Tagged , , , ,

effect of attention on event characteristic selectivity and neuronal adaptation

Posted comment on ´Interactions between conscious and subconscious signals: selective attention under feature-based competition increases neural selectivity during brain adaptation` written by Y. Kikuchi, J. Ip, G. Lagier, J.C. Mossom, S. Kumar, C.I. Petkov, N.E. Barraclough and Q.C. Vuong and published in Journal of Neuroscience July 2019 39(28) p. 5506 doi.org/10.1523/JNEUROSCI.3052-18.2019

SUMMARY

The article by Kikuchi and team begins by describing efficient perception as a neural interaction between voluntary cognitive processes such as attention and automatic and subconscious processes such as repetition suppression due to adaptation from predictable input. The authors examined in their study how attention interacts with visual and auditory neural adaptation from the perspective of whether it counteracts adaptation reduction in neural gain as previously observed, or not and if it did whether attended stimulus features were in competition with each other.

Kikuchi and team`s experiments were based on the manipulation of attentional focus on visual stimuli (ie. faces) or auditory stimuli (ie. voices) and assessment of neuronal responses by comparing fMRI images. It was expected that the BOLD responses would be strongest for the repetition of identical stimuli and therefore, the face and voice sensitive regions of interest (ROIs) would demonstrate increases in activities for the second manipulated stimuli in comparison to the first un-manipulated ones. This observation was believed to demonstrate stimulus-related adaptation function.  Increases of equal amounts equivalent to the changing level of stimulus manipulation would, according to the authors, indicate that attention affected only neural gain and not selectivity whereas differential increases would indicate that attention affected neural selectivity. In their experiments, auditory stimuli were taken to be emotionally neutral Ah-like utterances and 12 samples were paired into six lots of one male-one female voice combinations. Each voice pair was manipulated using recording equipment until over 5,000 stimuli were possible. The visual stimuli used in the experiments were 6 male and 6 female faces with neutral expressions. In all 6 pairs were created with one male face and one female and these were ´morphed` using software into over 120 different facial images. Spatial location was manipulated by presenting the facial images in different parts of the screen.

In Experiment 1, spatial selective attention was compared to non-spatial selective attention. Seven spatial locations on the display monitor for the faces (or in the virtual acoustic space for voices) were selected for the behavioural experiment and 3 perceptual spatial distances were selected for the fMRI experiments. The test subjects had to discriminate the identity or spatial position of the presented face or voice pairs and had to attend to one of the required stimuli judging whether it had changed or not. The other presented feature had to be ignored. The identity and spatial location were manipulated during the course of the experiment and the experiment was calibrated in order to control for the participant`s expectancy of change. Therefore, only 50% of the stimulus repetitions were changed. The participant`s indicated their decision by pressing a computer key during the fixation period between the images.  In Experiment 2, attention was manipulated to voice identity compared to sound loudness. Different participants took part in this experiment to those in Experiment 1, but the participants were presented with the same voice stimuli as that of Experiment 1. Noise bursts were generated by pink acoustic sounds with 21 variations in intensity to simulate changes in loudness. In Experiment 2, subjects were asked to judge whether the identities of the voices were the same and were also asked to judge the loudness of two noise stimuli. The other stimuli were ignored. In both experiments fMRI images of the BOLD responses in the face and voice ROIs located in the temporal lobe were taken, processed and analysed.

Kikuchi and team found in Experiment 1 that attention to face or voice identity whilst ignoring the stimulus location increased only the neural gain of the face or voice stimuli ie. cortical firing was increased. The response increased as the identity level increased for both faces and voices (ie.  as the morphing difference between the two stimuli increased there was a release from the adaptation response).  There appeared to be no significant interaction between the types of stimulus. In the case of attention to spatial position, responses increased as the spatial position level increased for both stimulus types (ie. as the spatial displacement between the two stimuli increased and attention was focused on spatial information). However, no spatial adaptation effect was observed in the face or voice ROIs in the cortex. The main effect of stimulus type occurred as expected (face and voice ROIs are more involved in processing face or voice features than spatial location), but although attention to spatial position increased the gain of neural responses in the ROIs, it did not change the selectivity of these responses.

The results of Experiment 2 showed that attention to voice identity whilst ignoring stimulus loudness increased neural selectivity. The proportion of varying responses increased as the morphing differences increased only when the participant attended to and discriminated voice identity, but not loudness. This showed the authors that attention stopped the decrease caused by adaptation. However, there was significant interaction between task and identity levels which indicated a change in selectivity during attention to voice identity. Further analysis showed that attention to voice identity whilst ignoring loudness increased both gain and selectivity of the neural responses in the voice ROIs. Therefore, the subjects were able to discriminate identity and loudness. In this case, the proportion of different responses increased as the loudness level rose when subjects attended to loudness. Again, therefore, there appeared to be no adaptation effect for loudness. The significant interaction between task and loudness was driven by a quadratic trend and the authors interpreted this as although voice ROIs are insensitive to changes in noise intensity, attention to stimulus features including loudness moderated the selectivity of neural responses within the ROI. Further analyses showed a three-way interaction (attend identity, attend other – loudness or position, plus stimulus level). Therefore, the authors proposed that their experiments showed that the effect of focus of attention on adaptation responses is significantly different between their two experiments on auditory stimuli ie. gain effects from Experiment 1 and selectivity exhibited in Experiment 2.

In conclusion, Kikuchi and colleagues proposed that their experiments showed that neural populations responsible for visual and auditory features are separate and that voice and face ROIs will adapt to stimulus features and reduce their responses more to repetition of stimuli with similar features as a form of neural selection. Attention was found to amplify and counteract the adapted neural signal by a constant factor, ie. top-down attention affects adaptation depending on the level of feature-based competition. However, the authors also showed that attention does more than just affect neural gain in that it can also influence selectivity. In the case of auditory vocal feature and loudness, the voice sensitive cortex demonstrated no adaptation to loudness, but attention to voice or loudness alone led to a greater increase than expected by releasing the adaptation. It was therefore concluded that attention selectively boosts the reduced adaptation of stimuli that is crucial for discriminating differences in identity. The authors then went on to explain their findings in terms of mathematical models. They also considered in their discussion expectancy even though this was a feature controlled against in their experiments. (Expectation suppression is said to occur when a repetition is expected and when this is violated, an expectation prediction error results). Attention and expectation are believed to work together and can change feature selectivity, ie. can dampen or sharpen neural responses. Kikuchi and colleagues stated that further work is required on this topic even though in their experiments perceptual expectations were minimised by the test set-up. They reiterated to conclude their article the proposal that their experiments showed that the properties of the event that are attended determine whether attention in interaction with adaptation modulates the signal gain alone or also effects selectivity.

COMMENT

What makes this paper interesting is that confirms two observations associated with attention. The first is that deliberate focused attention on specific features can alter positively the extent of neural firing against the neurochemically expected reduction in firing associated with repeated events (Experiment 1). And the second is that it can also affect the selectivity of certain features where there is competition (Experiment 2). The experimental set-up of Kikuchi and colleagues to demonstrate these factors was quite simple: visual versus auditory stimuli in the form of faces and voices and hence, features associated with common person identity mechanisms; manipulation in the form of computer generated morphing of facial images and mechanical sound adjustment for changes to vocal characteristics and volume intensity; and fMRI imaging, processing and analysis of BOLD responses for quantitative assessment of the extent of neural firing. With such a set-up, the neurochemical mechanisms required included sensory input and processing and cognitive decision-making (eg. is this face different to the previous?), but did not require mechanisms involved with long-term memory formation and recall and those associated with emotions (eg. assessment of stimulus value). Both of which are also affected by attentional capability.

The first conclusion from Kikuchi and colleagues was that deliberate attention counteracts the neural firing decrease observed with adaptation, eg. their fMRI ´fatigue` model. This conclusion leads to a consideration of the effects of top-down attention (ie. conscious awareness and deliberate focusing of attention) versus subconscious/unconscious attention. Such a consideration already demonstrates the fact that attention and conscious awareness are not from a neurochemical perspective the same thing. Attention brings to and maintains objects in the working memory state whereas awareness brings objects into the personal SELF/cognitive thinking and processing arena. There can be consciousness without some form of attention eg. objects can be attended to that are perceptually invisible and vice versa there can be top-down attention without consciousness eg. one can become conscious of isolated objects or a gist of a scene in the virtual absence of top-down attention (van Boxtel). This is because attention itself can come in different forms aside from those relating to emotional status. The ´thinking` form, top-down attention means personal, cognitive involvement and requires frontoparietal connectivity and alpha brain wave firing synchrony (van Schouwenburg). Greater levels of attention mean greater awareness of selected objects, but it does not mean greater awareness of distractors (Oriet). Therefore, it results in memory benefits not just by reducing the cognitive load, but also by enhancing the level and quality of the task-relevant information.  On the other hand, unconscious attention means that characteristics of an event may be attended ie. inputted and processed, but they do not reach conscious awareness. Top-down attentional modulation may bring those features to conscious awareness and in doing so, the information normally becomes attended.

This is where the connection to adaptation of neural firing due to repeated input comes in. Attended information relating to a single event involves only features that occur within a temporal window and there may be a shift from unattended information to attended in order to expand the neural representation to optimise processing. Repeated firing of the same cells to maintain this neural representation leads to the cells going into their refractory periods in order for them to neurochemically recover and therefore, firing adaptation (the fMRI ´fatigue` model) is said to have occured. This results in other cell firing to dominate in this particular temporal window and hence, the neurochemical ´priority to the unattended` and ´inhibition of return` rules apply. Other features that may have been unattended during the initial firing phase may then reach conscious awareness through being attended. This is a natural process and is different to deliberate selection and suppression of feature input as carried out in Kikuchi and team`s experiments where the participants are told specifically to select or ignore certain sensory features. In this case, the instructions lead to top-down modulation of attention brought to particular features and therefore, the neurochemical firing returns to the cells representing these original features once their neurochemical recovery is completed in order that the event representation is restored. In this case, as described by Kikuchi, neural adaptation (ie. ´priority to unattended` and ´inhibition of return`) is overruled.

The second observation about attention confirmed by Kikuchi`s experiments is that attended stimuli and features compete and that focused attention can bring about selectivity of features (Kauramaeki). In Kikuchi`s study the team found that attention to voice/face with the instruction to ignore location leads to increased face/vocal responses; attention to loudness with an instruction to ignore voice leads to increased loudness; and attention to voice with an instruction to ignore loudness leads to increased selectivity, ie. voice. From a neurochemical point of view, in a natural situation it is clear that the strongest firing would come from the most dominant event feature and/or if appropriate the feature that is task-relevant. Neuronal populations responding to sensory input are localised separately and therefore, the event representation can consist of many independent characteristics eg. two primary features of auditory perception, pitch and timbre, are processed in overlapping auditory cortex regions, but are separable (Allen).  The event representation may also contain other information eg. auditory processing has been shown to encorporate information not only relevant to sounds such as frequency, level and amplitude modulation over time, but also unrelated aspects of the experience such as arousal level, past experience and motor planning (King). Top-down modulation of sensory input and processing by deliberate direction of attention would shift the normal selection of features, ie. a strong visual input may be suppressed by being told to ignore visual features and concentrate on the loudness of the voice. It would result in the natural order of competition relating to strength of firing to be overruled and is likely to involve suppression at the primary cortical level responsible for the sensory input (Jacob). Such a direction has been shown to be advantageous in general in some cases. For example, it has been found that: spatial attention brings about an increase in visual processing (Connor); sustained attention on one auditory object in a complex scene leads to improved attentional selectivity over time (Best);  attention will expand the attended features at the cost of the unattended to optimise processing (Cukor, Kuo); and the precision of the feature itself can be increased (Lim – deliberate attention to a auditory feature leads to decreased working memory load and increased task-relevant feature precision; Andersen – selection of one visual feature will optimize the firing of another; and Bartsch – sharpened selectivity for a particular colour arises from feedback processing at the V1 level). This correlates to the fMRI ´sharpening` model where changes in selectivity or timing increases some neuronal responses whilst decreasing others.

Therefore, we conclude that deliberate attention is just as important as attention elicited through bottom-up mechanisms and influenced by the emotional state. It too provides focus, timing, and conflict assessment to sensory input and working memory content and information processing. Although deliberate attention can have a negative effect on task performance, eg. by leading to focussing on task-irrelevant information, its value is that it can have a positive influence on feature strength and feature selectivity. Therefore, the amount of task-relevant information can be increased, distraction can be prevented and task performance aided. The use of deliberate attention can counteract not only the natural neurochemical mechanisms associated with firing adaptation, but also the defective personal cognitive capabilities associated with inappropriate object value, memory impairment as well as the cognitively detrimental physiological factors such as tiredness, glucocorticoid level and hormonal status.

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

….. Blair found that the ability to decrease task irrelevance is suppressed with age and Gazzeley found that not being able to decrease task irrelevant information leads to a detrimental effect on working memory performance. Can we assume that if Kikuchi and team`s experiments were repeated using participants of a senior age that although firing of the ROIs would be located the same as their younger counterparts the intensity of the firing and the specificity would be lower? In a similar manner, Weltman and Wegbreit for example showed that anxiety like ageing decreases information relevance. Therefore, would the same type of experiments as indicated above produce the same effects as ageing?

….. Caeyenberghs showed that adaptive working memory training led to improvement on working memory tasks and generalization to tasks of reasoning and inhibition. Would a period of attentional training before taking part in the experiment promote an increase in the fMRI BOLD responses, increase the speed of responses and/or improve task performance as expected if working memory capability is positively influenced by the training?

…. Carlson found that fearful facial expressions conveyed threat-related information and automatically captured spatial attention through increased attentional orienting to threat. Therefore, if Kikuchi and colleagues´ experiments were repeated but the faces had fearful expressions and the voices projected fear, would there be a change in the firing patterns observed and in the ability to follow the instructions to ignore certain stimuli?

…. Verleger and Garcia-Perez found that normally people are equally aware of events in both hemispheric fields, but when two streams of stimuli are rapidly presented left and right containing two targets, the second target is better identified in the left than in the right visual field. If Kikuchi and colleagues` experiments are repeated with the first stimuli placed in both hemispheres, but the second in either the right or left, would the fMRI images observed be affected?

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