astrocytic calcium ion surges and tDCS

Posted comment on ´Calcium imaging reveals glial involvement in transcranial direct current stimulation-induced plasticity in mouse brain` by H. Monai, M. Ohkura, M. Tanaka, Y. Oe, A. Konno, H. Hirai, K. Mikoshiba, S. Itohara, J. Nakai, Y. Iwai and H. Hirase and published on 22nd March 2016 in Nature Communications 7:11100 doi: 10.1038/ncomms11100 (2016).

SUMMARY

In their article, Monai and colleagues discuss a possible neurochemical mechanism involved in transcranial direct current stimulation (tDCS), which has been described for example as helpful in alleviating depression and enhancing learning. Work by others using in vitro brain slices has shown that NMDA receptors are likely to play a role in the mechanism, which in general is unclear. One possibility put forward by Monai`s group and others is that astrocytes are involved in the NMDA receptor plasticity and the mechanism includes activation of the astrocytes resulting in rising intracellular calcium levels which may lead to the secretion of signaling molecules into the synapse. This release eventually leads to glutamate receptor plasticity at the post-synaptic membrane. Hence, Monai and colleagues investigated whether tDCS produces its therapeutic effects by causing astrocytic activation.

In their study, Monai and team used transgenic mice (mouse line G7NG817) which expressed G-CaMP7 (a green fluorescent protein Ca2+ indicator protein) based in the astrocytes and a subpopulation of excitatory neurons. They found high level of expression of the fluorescent protein in the astrocytes of the cortex, hippocampus (particularly the CA3 region), thalamus and striatum. Expression of G-CaMP7 was also observed in the neurons, but not in the cortical GABA cells. A level of tDCS of 0.1 mA direct current for 10 min induced large amplitude calcium ion surges in the astrocytes of the cortex. These surges exhibited higher amplitudes than spontaneous calcium ion events, but led to no changes in local field potential. There was also no change in local travelling wave propagation. Similar results were obtained for the anodal, contra-anodal and distal regions of the cortex. Long calcium ion surges were found to be more frequent during tDCS. The amplitude changes observed were the same for awake and anaesthetized mice, but in the latter onset of the surge was found to be more variable. In awake mice, the surges began several seconds after the tDCS onset whereas the calcium ion surges were found to be of a lower frequency in anaesthetized mice.

Monai and colleagues found that the t-DCS induced calcium surges in awake mice could be blocked by the administration of an alpha 1 adrenergic receptor inhibitor, prazosin, or by destroying the noradrenergic innervation with DSP-4. The surges were also blocked in vivo by local application of prazosin. Increases in astrocytic calcium ions were also not observed in IP3R2 (inositol triphosphate receptor type 2) knock-out mice and therefore, it was concluded that astrocytic GPCR activation is the prevalent mechanism of tDCS-induced Ca2+ surges. This view was further supported by behavioural investigations. Transcranial DCS was sufficient to alleviate a mouse model of depression by chronic restraint stress, but could not produce the same therapeutic effect after prazosin administration, DSP-4 treatment or IP3R2 deficiency.

To prove that the calcium surges were linked to astrocytes and not neurons, Monai and colleagues used two-photon imaging in layer 2/3 of the primary visual cortex. These experiments showed that the calcium surges were linked to SR101-positive astrocytes. Transcranial DCS evoked astrocytic calcium ion responses which had significantly higher amplitudes than spontaneous events, whereas the neuronal calcium ion events during tDCS had similar amplitudes to spontaneous events. The astrocytic calcium surges also occurred nearly seven times more frequently during the course of tDCS (10 min) than during the baseline, while neuronal activity did not show any obvious changes. The authors also used transgenic mice to confirm that the astrocytes were responsible for the calcium surge. They used transgenic mice with expressed G-CAMP7 in neurons or astrocytes using cell-type-specific recombinant adeno-associated viruses (AAV2.1-hSyn1-G-CaMP7 and AAV9-hGFAP-G-CaMP7, respectively) in C57BL/6 mice. Astrocytic soma, but not neuronal soma, were found to give rise to the long-lasting calcium ion surges associated with tDCS.

Monai and colleagues also investigated the role of NMDA receptors in calcium surges. They looked at visual evoked potential (VEP) of primary visual cortex of anaesthetized C57BL/6 mice after a flash stimulation of 60 secs before and after tDCS. It was found that the VEP slope increased by 50% after tDCS and remained at this increased level for at least 2hours after application. The effect was blocked by AP-5, an NMDA receptor antagonist and topical application of the alpha 1 adrenergic receptor antagonist, prazosin, but was not affected by the application of the muscarinic receptor, atropine. This indicated that NMDA receptors and alpha 1 adrenergic receptors play roles in the calcium surges induced with tDCS. There was also no VEP slope enhancement in IP3R2 knock out mice. Therefore, it was concluded that astrocytic calcium ion rises were involved in the tDCS enhancement of the VEP effect.

Therefore, Monai and colleagues concluded that tDCS induced astrocytic activity brings about plasticity changes in the cortex through calcium ion and Ip3 signalling. They showed that the tDCS-induced enhancement of a sensory evoked response is NMDAR dependent and as astrocyte calcium ion levels are positively related to the extracellular level of the NMDAR co-agonist d-serine, tDCS-induced astrocytic Ca2+ elevations possibly lead to NMDAR-dependent synaptic plasticity. The team also concluded that tDCS induced plasticity could be blocked by prazosin or DSP-4 treatment, thus indicating an involvement of alpha 1 adrenergic receptors which induce the G signaling cascade for IP3 production. The authors quote in their conclusion supporting work by Panktratov and Lalo who showed that application of noradrenaline raised extracellular d-serine and ATP levels and lowers the threshold for LTP induced plasticity in mouse cortical slices. They also conclude that the activation of A1AR is the prevalent mechanism for astrocytic calcium ion elevation in awake mice and suggested that the tDCS induced noradrenergic drive includes activation of the locus coeruleus and/or direct induction of transmitter release from noradrenegeric axon terminals in the cortex.

COMMENT

What makes this article interesting is that glial cells are again found to be more than just support cells for the all-important neuron and neuronal synapse. We know that different types of glial cells are important for different functions, eg. microglia are important in the degeneration of neuronal cells and oligodendrocytes are important in myelin production. This article focuses on another type of glial cell, the astrocyte, and expands its function in the action potential recovery phase and later on in the development of neuronal plasticity. The authors in their study showed that not only do astrocytes ´mop up` excess released neurotransmitters, binding of these neurotransmitters can cause intracellular astrocytic calcium surges that have an influence ultimately on neuronal NMDA dependent plasticity. This action can occur even spontaneously and hence, the excitable nature of the astrocytic glial cell provides yet another aspect to neuronal function that could go dysfunction and evoke deleterious changes in the neuronal and synaptic area. This additional role of the astrocyte has provoked lots of discussion simply because of the discrepancies in reported results, which have primarily arisen from experimenters using different brain areas, in vivo and in vitro samples and various experimental conditions.

Monai and colleagues have shown in their experiments that astrocytes produce internal calcium ion surges on binding of neurotransmitters to G protein linked receptors on the glial cell membrane. Using this finding, their aim was to show that these same calcium surges and resulting events could be produced by applying transcranial direct current stimulation (tDCS) and hence, one mechanism (or even, the mechanism) that is involved in the tDCS effect would be elucidated. Their finding supports observations about tDCS that it is effective in alleviating neuropsychiatric and neurological conditions such as depression in humans, and enhances learning and memory formation. Previous work on cortical slices shows that tDCS increases the excitability of the motor cortex in a NMDA receptor dependent manner, but the mechanisms involved in vivo are largely unknown with the exception that astrocytic calcium ion/IP3 signalling appears to play a significant role in synaptic plasticity in the cortex and hippocampus. Monai and colleagues` investigation confirmed this and showed that the tDCS induced plasticity was NMDA receptor dependent. They also found that the mechanism involved alpha 1 noradrenergic receptors which they believed transduced the G protein signaling cascade for IP3 production and was linked to the resulting calcium surges. This noradrenergic drive involved activation of the locus coeruleus area and/or direct induction of transmitter release from noradrenergic axon terminals in the cortex.

However, since there are discrepancies between the findings relating to astrocytic action it is not possible to say definitively what is going on in the case of tDCS. So, what do we know about astrocytic function? We know that astrocytes are one type of glial cell with the others being oligodendrites for myelination, microglia (shape shifters) as scavengers removing dead and damaged tissue from the nervous system, nerve/glial antigen 2 (NG2)-positive glia, which include oligodendrocyte and astrocyte progenitor cells as well as NG2+ cells that persist in the mature brain and astrocytes. It is likely that astrocytic cells share many of the characteristics and mechanisms of neurons. In fact, Liu showed that a single transcriptions factor, ACII, can convert astrocytes into functional neurons. Astrocytes are part of the action potential/firing stage of an active cell networking system and they can influence whether a neurite grows or retracts and regulate the content of extracellular space eg. they surround the synapse, remove excess neurotransmitter and control potassium ion concentration after the action potential has occurred, or in times of neuronal stress.

Astrocytes are capable of such actions by their physiology. The most common type are protoplasmic astrocytes and these cells have a very complex morphology and contact most, if not all, other cell types in the brain. The cells form from their soma (diameter 7-9 μm) elaborate and dense, fine non-overlapping processes that interact closely with the synapses present and supporting blood vessels (greater than 99% of the cerebrovascular surface is sheathed by astrocyte processes). It has been said that processes from a single astrocyte can envelop approximately 140,000 synapses which means that one astrocytic cell can occupy a ´working` volume of approximately 66,000 cubic μm. The cells are linked with each other by gap junctions and patch clamping experiments with a gap-junction permeable dye show that a single astrocyte rapidly leads to the filling of hundreds, even thousands of other astrocytes. Therefore, astrocytes likely function as a ´syncytium` contacting essentially all other cellular elements in the brain, including neurons, oligodendrocytes, NG2+ cells, microglia, and blood vessels.

In addition to this abundabt communication, there is also diversity within individual astrocytes with respect to interactions with the local environment. For example, it is possible that within a single astrocyte, a subset of processes (microdomains) can interact autonomously with neuronal synapses within its immediate environment while other regions of the same astrocyte interact with different groups of synapses or with other synaptic and neuronal elements such as blood vessels. These microdomains may not communicate with each other which implies that one astrocytic cell can carry out multiple functions simultaneously. However, this is unclear at this time and requires further investigation.

Known astrocytic functions appear to be linked to two mechanisms. The first is that described by Monai and colleagues that of glial cell surface receptor binding and calcium surges. Astrocytes are assumed to be like other glial cell types in that they have many signaling proteins similar to those found in neurons, eg. ion channels and receptors such as those for glutamate, GABA and noradrenaline. These specific neurotransmitter receptors are on the cell membranes and can trigger events within the glial cells and Monai and colleagues reported this in the case of noradrenergic receptors. This finding confirmed work from the mid 1990s which showed that activation of G protein-coupled receptors on the astrocytic cell membrane surface by synaptically released neurotransmitters produces rises in intracellular calcium concentration. An IP3 signalling cascade is involved and this demonstrates not only that astrocytes display a form of excitability like the neuron, but also that astrocytes may be active participants in brain information processing. The ultimate consequence of such a binding is the increase in NMDA receptor plasticity on the neuronal post-synaptic membrane which will affect overall neuronal area functioning.

Although the authors and others report a link to NMDA receptor plasticity, some researchers (eg. Goldman) report instead an increase in AMPA receptor trafficking and increased plasticity of the neuronal area. Goldman found that grafting human cells onto mice cells led to a 4 times increase in synaptic activity. Han and colleagues, as reported in this blog`s post of January 2015, supports this observation since they found that grafting of human glial progenitor cells in the mouse forebrain led to increased synaptic plasticity and learning in the adult linked to glial cell increases.  An increase in human astrocytes was observed at 4-5 months in the hippocampus and deep neocortex layers, but by 12-20 months it was also observed in other areas such as the amygdala, thalamus, neostriatum and cortex. An investigation into synaptic activity in neuronal cells from the hippocampal dentate granule layer, an area used because of its large number of engrafted cells and the region´s known role in spatial memory, found a significant increase in the engrafted human glia cell`s  basal level of excitatory synaptic transmission. This long term potentiation (LTP) enhancement was not linked to increased NMDA receptor activity (or increased glutamate release), altered adenosine concentrations or, changed D-serine release but instead to increased TNF alpha which induces the addition of AMPA receptors to neuronal membranes and AMPA GluR1. The insertion is regulated through protein kinase C (PKC)-mediated phosphorylation of appropriate sites. Such increased neuronal activity was mirrored by enhanced learning in the chimeric mice with increased spatial memory and quicker contextual fear and tone conditioning. This observation was supported by Hennessey and colleagues who showed that astrocytes in degenerating brains caused by acute sterile inflammatory insult are primed to produce exaggerated responses via strong nucleur localization of NK-kB subunit p65 and increased synthesis of the chemokines, CXCL1 and CCL2. Administration of IL-1beta and TNF-alpha produced a more robust response in degenerating rat brain than the control.

Another function of astrocytes is potassium spatial buffering which is where extracellular potassium ions occuring during stimulation are taken up. The potassium ions enter through potassium channels causing the astrocyte to depolarize. The specialized inwardly rectifying potassium channels involved in this are also known to be linked to GPC receptors and are ATP sensitive. Potassium ion entry increases the internal concentration which is dissipated over a large area by the extensive network of astrocyte processes. There is no evidence that these GPC receptors are linked to the calcium surges that induce higher sensitivity to neuronal firing, but if they are this could mean that the internal calcium ion surge may not be caused just by neurotransmitter binding on the astrocytic surface, but also by the inward current of potassium ions released by presynaptic cells on neuronal activation.

Therefore, astrocytes appear to be stimulated by the activation and release of neurotransmitters from the activated presynaptic neuronal cell, or possibly by the internalization of potassium ions excluded during firing. These actions affect G protein receptors activities and cause internal calcium surges which presumably activate protein kinases, phosphorylate relevant proteins and cause specific gene transcription changes just like in neuronal cells. It is possible that the result of this is the release of gliotransmitters (signal molecules that could be noradrenaline, glutamate or GABA) from the glial cells which will then bind to the relevant receptors on the post-synaptic membrane. The overall result of this is that there is possibly a ´second wave` stimulation of the post-synaptic membrane, the first being the direct binding of neurotransmitters released pre-synaptically directly on firing. This second wave is linked to AMPA receptor addition to the post-synaptic membrane which is associated with LTP. The implication of this ´second wave` response is the slight delay observed in post-synaptic effects. Therefore, the post-synaptic neuronal response is augmented and temporally extended by the astrocytic response to both neurotransmitter and potassium ion presence in the synaptic cleft. Using this hypothesis, we can suggest that if tDCS can cause astrocytic calcium ion surges, then it could work by ultimately causing effects that are normally associated with neurotransmitter firing triggered by other means. The excess of electrons administered by the direct current stimulation can cause changes in presynaptic membrane electron fields which can result in the release of neurotransmitters from the presynaptic neuronal cells and/or firing of the cell. The neurotransmitters released or the excluded potassium ions can then bind to the astrocytic G protein linked receptors and cause the effects described above. In this way, the ultimate result is AMPA receptor addition to the neurons and induced LTP. This changed plasticity will present as the changes in learning and depression suggested as being associated with tDCS.

Therefore, the role of astrocytes in the neuronal firing scenario and neuronal plasticity appears to be important and can possibly be manipulated by applying direct current. This may provide a mechanism by which neuronal areas understimulated or defective in normal stimulation can be induced to fire, but it may be detrimental in areas where hyperexcitability is being reported eg. in the hippocampus and entorhinal areas as recorded in dementia. Therefore, more research is needed to investigate this astrocytic effect.

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

………would using radioactively marked AMPA receptors in the hippocampal area of transgenic mice show that tDCS is linked to AMPA receptor insertion into the cell membrane?

……….can we assume that removal of astrocytic cellular calcium ions with EDTA will have an effect on neurotransmitter release?

 

Posted in astrocytes, calcium ions, glutamate receptors, tDCS, Uncategorized | Tagged , , ,

link between Self and neural responses to heartbeats

Posted comment on ´ Neural Responses to Heartbeats in the Default Network Encode the Self in Spontaneous Thoughts` by M. Babo-Rebelo, C.G. Richter and C. Taillon-Baudry and published in Journal of Neuroscience 27 July 2016 36 830) 7829 doi 10.1523/JNEUROSCI,0262-16.2016

SUMMARY

Babo-Rebelo, Richter and Taillon-Baudry`s article describes the Default Network (DN) as being associated with self-related cognition and physiological processes such as bodily state monitoring and autonomic regulation. These are described as functionally coupled because Selfhood is grounded in the neural monitoring of internal organs such as the heart.  In their study, the authors measured neural responses evoked by heartbeats (HERs) when 16 right-handed subjects who had been pre-trained in the test method, allowed their minds to freely wander after a period of visual fixation (a black circle with a black dot in the centre on a grey background). This period was interrupted by a visual stimulus (8 white dots presented for 200ms) at random intervals (13.5 to 30 secs) and the subjects were asked to score the self-relatedness of their thoughts occurring at the time of the interruption.

Self-relatedness was assessed from the first-person perspective, the subject (I) in the thought and this was termed ´Actor/Author` and the extent to which the ´author` was thinking about himself, termed ´Me` and ´Content`. The subjects were asked to use high ratings in the case of consideration from their own viewpoint and low when considering from someone else`s viewpoint or in the case of the ´Me` thoughts something external to them.  The authors found that both ´I` and ´Me` results were significantly positive suggesting that the scales used may have represented the same notion of Self. The interrupted thoughts were also rated according to emotional intensity (pleasant or unpleasant and termed ´Valence`), and relevance to past, present or future events on a scale from a few hours to several weeks (termed ´Time`). In these cases the responses were recorded by moving a cursor along a scale or by ignoring it if the subject had no response or were unable to quantify the response. Babo-Rebelo, Richter and Taillon-Baudry found that there was slight biasness towards high self-relatedness in the ´I` test to both the present when rating Time and pleasantness in rating Valence.

The test results produced showed that HERs scores were significantly different for high and low trials for ´I` thoughts and also for ´Me` thoughts over medial frontal sensors. No difference was found for the Time and Valence studies. There were spatial and temporal variations between the ´I` and ´Me` results and so further investigation was required since the study could have been capturing an unified self-relatedness of thoughts with any differences the result of personal rating differences of the subjects. A re-classification of the ratings on the Self scale gave no significant differences, but a study of heartbeat-by-heartbeat cluster amplitude and the raw Self-related rating at each investigated thought gave significant differences. The authors found no covariation however between self-relatedness and peripheral autonomic measures such as heart rate, heart rate variability, pupil diameter and blink response, electrodermal activity, respiration rate and phase, or alpha brain waves.

Babo-Rebelo, Richter and Taillon-Baudry continued their study by relating the interrupted thoughts to neural measurements of two brain areas of the DN that of the ventral precuneus and the ventromedial prefrontal cortex. They found HERs differed significantly along the ´I` scale in the left ventral precuneus with activity centering on the left precuneus and extending dorsally and posteriorly to the cuneus and calcarine sulcus. However, the HERs could only be detected in the left ventromedial prefrontal cortex when the Self was the subject of the ongoing thought. In the case of ´Me` thoughts then the study produced only differential HERs when the subject was the object of the ongoing thought and in this case activity was located in the left ventromedial prefrontal cortex centering on the left frontal medial orbital gyrus and extending posteriorly and dorsally to the left anterior cingulate and rectus gyri. The results were verified with surrogate heartbeats, ECG, personality (aspects such as self-consciousness scale, daydreaming frequency scale, trait anxiety inventory) and interoceptive ability.

Therefore, Babo-Rebelo, Richter and Taillon-Baudry concluded that the two areas, ventral precuneus and ventral medial prefrontal cortex, are differentially activated by heartbeats and part of the DN and that there is a link between Selfhood and neural responses. Therefore, they suggested that physiological and cognitive functions have to be considered jointly in the DN. They found that the ventral precuneus is associated with ´I` thoughts and this correlates to activity in episodic memory retrieval, perspective, body ownership, self-location, spatial navigation, imagination and future planning.  In comparison, the ventral medial prefrontal cortex is related to ´Me` thoughts and it is known that this area is important for monitoring of the visceral organs (including heartbeats). It is not a direct target of visceral inputs, but is functionally connected to the visceral centres of the brain and is involved in autonomous functions. Therefore, the ventral medial prefrontal cortex may be receiving visceral information through one or more cortical relays, which could explain the longer latency of the effect observed in the ventral precuneus. It was not possible for the authors to determine how the latency difference in transient neural responses to heartbeats in both areas directly relate to a differential time course of the ´I` and ´Me` dimensions in spontaneous thought that probably developed over seconds and this remains a topic for debate.

The authors concluded that the functional coupling between HERs and self-relatedness could stem from different mechanisms. HERs could directly contribute to the specification of the Self. It would contribute to the constant update of a neural reference frame centered on the subject’s body and hence, would serve as a basis for the development of self-relatedness. However, although the authors` results support this view other hypotheses were also considered. For example, self-related thoughts could induce an internally directed shift of attention leading to an amplification of processing of internal signals including heartbeats. This hypothesis was discounted by the authors since explicit orienting of attention towards heartbeats would alter the activity in other brain areas such as the insula, somatomotor sensory system and dorsal anterior cingulate cortices. No changes in activity were observed by Babo-Rebelo, Richter and Taillon-Baudry. Another explanation was given in that the HER covariation with self-relatedness was a byproduct of self-related processing. The neurons responding to heartbeats were being modulated by the neurons encoding self-relatedness. If this hypothesis is correct then the authors surmised that the HERs are modulated by the self-relatedness of spontaneous thoughts but have no direct consequence on the contents of those thoughts. This was determined by the authors as being very difficult to achieve. For now, they concluded that certain areas of the DN are engaged in physiological regulation, thus providing an explanation for its high basal metabolic rate, persistent activity in early sleep stages and its conservation across species.

COMMENT

After so many years of research and discussion, we are still not able to give definitive biological scientific explanations for consciousness, thinking and memories for example. Therefore, what makes this article interesting is that it links consciousness, something that is to date a biological mechanistic mystery, to a physiological process that is explainable, observable and adaptable, that of heartbeats. The authors of the article, Babo-Rebelo, Richter and Taillon-Baudry, found in their investigation that the activities of certain brain areas are associated with a change in consciousness according to thoughts occurring at the time and that, neural responses of these specific areas to heartbeats were affected. Therefore, something that is physical is ´rooted` to something that is mental and because the latter affects the former then concerns about the way some experimentation is carried out must arise. The interpretation of brain area activity and connectivity results maybe needs to be adjusted or experimental controls put in place that are robust enough to account for discrepancies of the thought processes of the various test participants at the time of experimentation.

For now we assume that study results are correct and therefore that the neural responses to heartbeats in two particular brain areas are different depending on the types of thinking being carried out by the subject in the test period. This article involves the Default Network (DN) also known as the Default Mode Network which is a set of brain regions which have a high metabolic rate even at ´idle` and which is switched off in task-orientated processing, but important where awareness of the Self is required. The authors looked at two areas in particular: that of the ventral precuneus (PC) and the ventromedial prefrontal cortex (vMPFC). The PC is located in the posteromedial of parietal lobe and is a highly connected area and a hub between the prefrontal and parietal areas. It consists of many distinctive areas which are linked to specific functions.  General functions include responsibility for visual sensory attentional information, episodic memory (linked to PFC), visuo-spatial processing, the Self and certain aspects of consciousness (eg. reflective Self-consciousness). The ventral PC is linked with the Self, the past and future, spatial navigation (motor imagery and shifting attention to motor targets whereas the dorsal part is associated with involuntary awareness and arousal. In comparison, the vMPFC is a part of the larger prefrontal cortex which is associated with lots of cognitive functions including memory and attention. The vMPFC itself (otherwise known as the orbitofrontal cortex) is linked with Self processing, emotional information, reward and value eg. the ability to assess according to one`s own objectives, formulating criteria etc. for decision-making, and control of stress through inputs from the PFC.

Babo-Rebelo, Richter and Taillon-Baudry found that elements of DN heartbeat connectivity were different according to the thoughts in ´idle` ie.  neuronal awareness of the bodily process was different according to what angle of thought was occurring at the time. They found that ´I` thoughts were linked to neural responses to the heartbeats in the area of the precuneus whereas ´Me` thoughts produced a response in the vMPFC. For their experiment, the authors defined Self-relatedness as the ´I` when the participant was the agent or subject in the thought and therefore this group included thoughts from the first-person perspective. A high ´I` rating was given for the thought ´I am thirsty` for example and low for thoughts such as ´It´s cloudy`. ´I` thoughts defined what the participant (the ´I`) wanted. On the other hand, ´Me` thoughts were defined as when the participant thought about himself/herself. For example, ´I am thirsty` or ´I should be more concerned` were rated high whereas thoughts directed towards something or someone else such as ´It´s raining` or ´She`s coming here on Monday` were given a low rating. ´Me ` thoughts defined the Self as what I am in the community. I must admit that I am not clear about distinguishing between ´I` and ´Me` thoughts in this way since I believe that both relate to the person in question. They may relate to real things either past or present, relate to unreal things such as things that will happen in the future or intentions, but they both relate to me, the Self. Therefore, defining ´I` and ´Me` thoughts appears to me more semantics. I accept that ´I am thirsty` requires bodily awareness and relates to the present and ´I will go to the bank tomorrow` involves more higher order brain areas with no physical awareness, but both relate to ´I/Me`, the person, the individual and both require my memories, my processing capability, my physiological and emotional awareness and personality for example. However, if I accept the results presented in this paper I have to accept the difference in definition of the ´I` and ´Me` thoughts according to the definition given by the psychologists. It is possible that the difference lies in that in some thoughts the particular brain areas are defined as being part of the DN whereas in other types of thought the same brain areas are regarded as essential for cognitive thought. For example, DN is known not to be involved in task orientated processing and therefore the thought ´I will go to the bank tomorrow` counts as forward planning and decision-making and value requiring known vPFC involvement according to cognitive functioning definition and ´Me` thought as defined here. Maybe here therefore, activity cannot be defined as part of the DN.

The authors did see however, a difference in neural response to heartbeats according to the content of thoughts at the time. The question must be asked therefore, what value would heart beat perception have to consciousness? To answer this question, we must look at the function of consciousness with reference to bodily awareness. In 1944, consciousness started out as a rare phenomenon associated with sudden super-alertness required in reacting to sudden emergencies (Claxten). Later on in 1977, Baars defined consciousness as a supremely functional adaptation and that, somewhere in our evolutionary past, consciousness would have saved us from danger. Therefore, an awareness of heartbeat in this case would be important. Consciousness also extended the ability of the brain to create transient states and McGovern and Baars linked cognitive functions to more higher order processing with definition and context setting, adaptation and learning. However, still important was consciousness`s role in error detection and awareness to things in memory for example. Therefore, heartbeat awareness and changes to it associated with biological learning and processing would be important. It would allow a quick response with less conscious thought then the other physiological body awareness, that of emotions.

The idea that awareness of heartbeat might be part of the conscious experience is clear if we consider that in a frightening situation people may feel their heart beating and explains the observations of the neural responses to heartbeats by the authors. This is supported by theories about the Self, Self-relatedness and Self-hood. In Baars Global Workplace Theory, the Default Network extends the realms of it. In this case, ´I` and ´Me` are used to distinguish between the Self-concept (includes values and beliefs about oneself) from the more fundamental Self-system (includes Self as the observer or agent). There is no conscious awareness of heartbeat. This theory led on to Damasio in 1999 defining the Self by distinguishing between the Proto-Self, the Core-Self, and the Autobiographical Self. The Proto-Self is described as a set of neural patterns which map the state of an organism moment by moment. In this case there is only unconscious awareness of the heartbeat and this is supported by other non-conscious bodily functions such as pupil dilation which have been in the past used to deduce attention. The basic kind of consciousness is the Core Self which is not dependent on memory, language or reasoning and provides the organism with a sense of Self in the present time. This is a transient entity ceaselessly re-created for each and every object with which the brain interacts. Memories form the third Damasio Self, the autographical self.

In 2003 and 2009 Metzinger established the representational view of Self by describing an inner tool called the Phenomenal Self-model (PSM). This is a distinct and coherent pattern of neural activity that allows the individual to integrate parts of the world into an inner image of him- or herself as a whole. Therefore, because of this Self model the individual can experience his own arms and legs as his own arms and legs, certain cognitive processes as his own thoughts and certain events in the motor parts of your brain as his own intentions and acts of will. In this case, the heartbeat and acknowledgment of the heartbeat would become part of the Phenomenal Self-model. A similar model, the concept of the Neural Subjective Frame has been proposed by Taillon-Baudry (one of the author`s of the article described here). This describes ´what I am` and is based on constantly updated neural maps of the internal state of the body. It constitutes a neural referential form from which the first person experience (the ´I` experience) can be created. Taillon-Baudry used this concept to form the basis of the investigation described here since the neural subjective form is rooted in the neural representation of visceral information which is transmitted through multiple anatomical pathways to a number of target sites, including posterior insula, ventral anterior cingulate cortex, amygdala and somatosensory cortex. In the experiments described here the conscious experience was linked to bodily function (that of heartbeats) and the the cortical processing associated with the signals from the cardiovascular system was studied by measuring the heart-evoked response (HER) using EEG. Co-variance of brain activity and heart rate was measured.

The problem with the theory is that the neural response to heartbeat is unconscious and thought is a conscious experience. Conscious perception to the heartbeat is not required. There is no conscious awareness of the heartbeat whilst the mind wanders and perhaps this is an example of the Self ´pampering` itself. It can think about things outside the normal routine functioning of the body which is carried on unconsciously. This is also observed with other signals such as pupil diameter.  Attention at this time is directed on higher thought processes and it is likely that according to Koch (2007) at the beginning there is a link between attention and consciousness, but later on they are highly or even entirely dissociated capabilities.  There is good evidence that attentional processes can operate without the attended stimuli ever reaching consciousness (Bressan, 2008) and this correlates to Franklin and Baars (2012) hypotheses about two types of unconscious process, one being preconscious and the other never-conscious. Distraction can impair the efficiency of unconscious processing. In mind wandering then there is no conscious awareness of heartbeats. Therefore, sensory and bodily information is processed in a wide variety of ways with different consequences depending on circumstances and cognitive involvement. Perception of the heartbeat can be trained and hence, the balance of unconscious and conscious processes can be adapted according to the wishes of the individual.

The other problem with the results of the experiments is the implications for the definition of a unified conscious experience. The binding problem of conscious experience appears to be intimately related to memory and attention. Crick and Koch investigated binding and consciousness and found 35-75 HZ oscillations (called gamma oscillations) or 40 Hz oscillations for groups of firing neurons. Crick suggested that these might be the neural correlates of visual awareness. It was argued that consciousness depends crucially on some form of rather short-term memory and also on some form of serial attentional mechanism and it was suggested that the thalamus controls attention by selecting the features to be bound together by synchronisation of firing. Crick suggested that consciousness only exists if certain cortical areas have reverberatory circuits (involving cortical layers 4 and 6) that project strongly enough to produce significant reverberations. Later in 2003, Crick and Koch changed their view saying that 40HZ oscillations were not a sufficient condition for the neural correlates of consciousness and instead argued that the primary role of synchronous firing was to assist one nascent firing coalition in its competition with others. The features of one single object or event are bound together when they form one part of one coalition and that coalition may involve neurons widely distributed over the brain. This view was supported by Taillon-Baudry. Zeki in 2007 extended this view with his Microconsciousness theory by saying that there is no single consciousness, but instead multiple consciousnesses (microconsciousnesses) distributed in time and space.

The problem with reference to these theories and the observations found in Babo-Rebelo, Richter and Taillon-Baudry`s study lies with the time difference described by Libet between neural firing (including those of the Global Workspace participating areas and neural responses to heartbeats) and conscious experience (Global Workspace thoughts occurring after the interruption). Libet produced his half-second delay theory where events become conscious only when the neurons involved in somatosensory cortex have been firing for a sufficient length of time (half a second). The majority of neural activity remains unconscious because it is too fleeting (needs at least 0.5 sec) or too unstable for neuronal adequacy. Paulignan also showed ´consciousness catch-up` when he  asked subjects to look back at where they thought a change had occurred and found that they reported the change much later than its actual occurrence, hence conscious awareness was too late for causal action. Therefore, in this case of neural responses and ´I` or ´Me` thinking, the heartbeat monitoring would occur in real-time, but the conscious thoughts according to Global Workspace Theory would be half a second slower. Therefore, the conscious thoughts are not related to the heartbeat responses recorded at that time, instead ones recorded half a second before. This supports the view of Marcel (1993) who looked at different reporting modalities such as blink and finger tap and found that they produced conflicting reports about the conscious experience. He argued ´slippage` in the unity of consciousness and therefore said opposing global interaction theories that there is no unified Self.

Therefore, although the experiments of Babo-Rebelo, Richter and Taillon-Baudry appear to suggest that neural responses to heartbeats are different in terms of brain area depending on what type of conscious thought is being processed at the time, the problem with time delay means that this is unlikely to be so clear-cut.

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

………transient changes have also been reported in heart rate with perceptual decision-making. It was found that in the case of forewarned perceptual reaction-time tasks in response to warning stimuli, the heart followed a typical pattern of deceleration followed by an acceleration. Would this same pattern be followed if participants were asked instead to concentrate on  ´I`  or ´Me` thoughts?

…….it has been reported that looking into someone`s eyes for 10 minutes causes a feeling of being ´spaced out` as well as decreased colour intensity and sounds being louder than expected. What would happen to neural responses to heartbeats if the subject had to concentrate on only ´I` or ´Me` thoughts whilst looking into someone`s eyes for 10 minutes before being asked to report?

…..can we assume that if deep TMS is used and the experiment repeated that the activity would remain with the precuneus independent of experiment condition because deep TMS decreases self-awareness, and therefore only unconscious processing would occur?

Posted in consciousness, default network, heart-evoked response, prefrontal cortex, Self, Uncategorized | Tagged , , , ,

PDE4A5 signalling impairs hippocampal synaptic plasticity and long-term memory

Posted comment on ´Compartmentalized PDE4A5 Signaling Impairs Hippocampal Synaptic Plasticity and Long-Term Memory` by B.Y. R Havekes, A.J. Park, R.E. Tolentino, V.M Bruinenberg, J.C. Tudor, Y. Lee, R.T. Hansen, L.A. Guercio, E. Linton, S.R. Neves-Zaph, P. Meerlo, G.S. Baillie, M.D. Houslay and T. Abel and published in Journal of Neuroscience 24 Aug 2016 36(34) 8936 – doi 10.1523/JNEUROSCI.0248-16.2016

SUMMARY

Havekes and colleagues investigated the link between the binding of specific compartmentalized cAMP-specific phosphodiesterase 4 (PDE4) isoforms in mouse excitatory hippocampal neurons and cognitive changes associated with some neurological disorders. Expression levels of PDE4 isoforms are known to be altered in traumatic brain injury, autism, schizophrenia, bipolar disorder for example as well as being affected by ECT and antidepressant treatment. It is also known that the PDE4 isoforms exert their influence on cognitive capability by binding via their N terminals to specific protein complexes and affecting degradation of cAMP in specific intracellular compartments. In order to investigate the effect of the PDE4 isoforms in the hippocampal cells, Havekes and colleagues altered PDE4A5 and PDE4A1 expression in mice and performed various in vivo cognitive tests eg. object–place recognition task, fear-conditioning task, open field task, and zero maze task and several in vitro tests on cultured cells such as electrophysiology and fluorescence resonance energy transfer sensory imaging.

Havekes and colleagues found that virally induced PDE4A5 expression was observed in the excitatory neurons in hippocampus, but not in the astrocytes. This expression led to increased PDE4 activity in the hippocampus inducing reduced cAMP levels in this area, but not in the prefrontal cortex or cerebellum. The cAMP effect was not overall, but specific for certain intracellular compartments. The increased PDE4A5 protein levels were found not to alter basal synaptic transmission in the Schaffer collateral-CA1 pathway, but decreased synaptic potentiation.

They also investigated the link between PDE4A5 level and long-term context-shock associations and found that selective overexpression of PDE4A5 attentuated long-term memory. Increased protein levels did not affect freezing levels during training, but decreased freezing levels were observed when the mice were re-exposed after the conditioning training period. This result was explained by short-term memories not needing cAMP signaling whereas long-term memories did. Hence, PDE4A was said to lead to impairment of hippocampal plasticity resulting in long-term memory problems. Repeating the context shock associations with tone-cued fear conditioning instead (a process that uses the amygdala region rather than the hippocampus due to the fear element of the electric shock) found similar freezing levels under all conditions. Therefore, it was concluded that tone-cued fear conditioning is not affected by PDE4A5 levels in the hippocampus.

The authors also looked at the effect of PDE4A5 levels on performance of the object-location memory task in mice. They found that mice expressing eGFP or PDE4A5 reduced exploratory behaviour during training as they learnt the locations of objects. After learning, eGFP mice could remember the locations, but mice overexpressing the PDE4A5 protein demonstrated reduced memory and explored all of the objects to the same extent. In the case of the novel object recognition task, mice with both eGFP and PDE4A5 over-expression demonstrated the same exploration of novel objects showing that they could determine novel objects from familiar ones. An investigation of cAMP responses using the ICUE3 biosensor in hippocampal neurons expressing a control vector and full-length PDE4A5 found that baseline FRET responses were not affected by the overexpression. The attenuated forskolin-mediated FRET response could be normalized by application with the PDE inhibitor IBMX which suggested that the decrease in FRET response was due to the overexpression of PDE4A5 and not a result of nonspecific alterations in PDE/cAMP signaling.

It is known that the N terminal of the PDE4 isoforms is important for PDE4 binding to complex groups and Havekes and colleagues investigated if PDE4A5 also requires the N terminal for the context-shock results. The PDE4 isoform was truncated at the N terminal at 303bp and no impairment of long-term memory was found in this test. A repetition of the object-place memory test also found no difference between the eGFP and PDE4A5 over-expression animals. The investigation of cAMP responses using ICUE3 biosensor in hippocampal neurons expressing a control vector and full-length PDE4A5 which led to the attenuated forskolin-mediated FRET response which could be normalized by the application of the PDE inhibitor IBMX was also not observed with the truncated version. These investigations supported the view that the N terminal was important for the placement of the PDE4 isoform in the cell. Using fluorescent imaging, Havekes and colleagues found there was different intracellular distribution between the full and truncated versions. The full length form was found in discrete perinucleur areas and the dendritic compartments, whereas the truncated version was found predominately only in the former.

Havekes and colleagues also investigated another PDE4 isoform that of the PDE4A1. They found differences between PDE4A5 and PDE4A1 with the PDE4A5 isoform being membrane associated, whereas 4A1 was located in the Golgi. The investigators also found that overexpression of PDE4A1 produced no change in memory when tested using the object-location memory test. Hence, it was suggested that PDE4A1 does not target protein complexes critical for the formation of object location memories and that the two 4A5 and 4A1 isoforms affect different cellular compartments.

With the link between PDE4A5, its overexpression, cAMP increase and cognitive disorders being established, the authors concluded their article by suggesting that instigating N terminal changes would produce an alternative method of regulating the PDE4A5 cellular level. This method would be welcomed as an alternative to using the broad PDE4 inhibitors which cause such undesirable side effects such as diarrheoa and emesis.

COMMENTS

What makes Havekes and colleagues article interesting is that it investigates indirectly the role of cyclic adenosine monophosphate (cAMP) in hippocampal cells and memory and perhaps gives an indication of one of the elements required in the process of ´switching off` an active cell once the synaptic stimulation is over. The article looks at the binding of cAMP-specific phosphodiesterase 4 (PDE4) isoforms to specific proteins in identified compartments of the post-synaptic regions of excitatory neurons in the mouse hippocampus. The authors found that expression of the PDE 4 gene leads to production of the protein and its subsequent specific binding to intracellular proteins results in a reduction in cellular cAMP level. Further investigation by the authors showed that this was a specific effect to one isoform of the PDE4 protein (the A5) and binding required a functional N terminal. Negative effects on cognition were attributed to this N terminal binding such as interaction with beta-arrestins, a molecular element critical for learning and memory and association with certain proteins containing the SH3 domain such as src tyrosyl kinase family, the inhibition of which also leads to memory defects.

From a biochemical point of view, PDE4A5 provides a tool by which cAMP functioning within the synaptic area can be investigated. Cyclic AMP is a multifunctional second messenger and its production from adenylate cyclase within the neuron is linked with the opening of chloride ion channels, protein kinase (PK) activation and gene transcription (eg. CREB phosphorylation). Therefore, if cAMP levels are reduced then either the PDE4A5 protein reduces cAMP production by binding directly to the adenylate cyclase enzyme (AC) and eliciting conformational changes that prevent the enzyme from working, or it increases the level at which the cAMP formed by a normal acting AC is degraded. Since the PDE4A5 protein is described as a phosphodiesterase (breakdown of cAMP to AMP) then the latter seems to be how this protein functions in the normal cell. Therefore, it can be said that if the level of this common second messenger is reduced on PDE4A5 binding then the protein is likely to play a role in the ´switching off` mechanisms of the neuronal cell after stimulation (e.g. in hyperpolarization for example). The question is which natural cAMP dependent neuronal functions is PDE4A5 likely to have an effect on?

Havekes and colleagues found in their study that PDE4A5 binding was perinucleur, dendritic and inter-compartmentalised. Therefore, the known role of cAMP in chloride ion channel functioning can be ruled out as a location for the PDE4A5 effect since chloride ion channels are situated on the cell membrane surface. Under normal activation, cAMP would increase the opening of the chloride channels to aid hyperpolarization and this is linked with GABA binding. The hippocampal CA3 area contains GABA interneurons and increased GABA receptor binding in this area is linked with fear memory which correlates with the observation that increased PDE4A5 expression results in anxiety and emotional memory changes. Hence, an increase in GABA binding leading to increased long-term depression of the relevant interneurons may mean that hyperexcitability of the CA1 area may occur. This would be consistent with the cognitive effects observed. However, since a membrane effect is not attributed to the PDE4A5 action then an influence on chloride channel opening by affecting cAMP level can probably be ruled out and it can be assumed that the cognitive effects observed with increased PDE4 expression come from other factors.

It is more likely that the PDE4A5 protein instigates its effect on cognition by influencing the performance of the various protein kinases involved in neuronal functioning. Cyclic AMP activates protein kinases by altering the enzyme`s quartenary structure and therefore, reduced cAMP would reduce the level of functioning protein kinases within the cell. For example, in the presence of PDE4A5 binding there would possibly be reduced activation of calcium calmodulin protein kinase which leads to decreased phosphorylation of the synapsin proteins and synaptogamin synaptic vesicular proteins. This would result in for example less vesicular transport in the synapse leading to less release and degradation of neurotransmitters and lower receptor trafficking. Therefore, it may be suggested that this could be a pathway by which the ´switching off ` of the synapse post-stimulation might occur.

A similar rationale could also be applied to the actin binding protein, girdin, which is one of the proteins responsible for the neuron`s actin-based cytoskeleton. This protein interacts with Src tyrosyl kinase which acts on the NR2B subunit of the NMDA receptor in the hippocampus. This type of glutamate receptor is linked to normal neuronal functioning after stimulation and long-term potentiation of the area. Therefore, a reduction in cAMP level induced by the PDE4A5 binding could lead to an effect on the actin cytoskeleton of the pre- and post-synaptic areas resulting in less vesicular transport and trafficking of proteins, receptors etc. as well as an effect on the very receptors that are linked with long-term potentiation and memory. A more direct influence of cAMP on the NMDA receptor also comes from its effect on post-synaptic protein kinase A (PKA) activation. This enzyme would normally phosphorylate a particular residue of the GluN2 subunit of the NMDA receptor and this subunit has been found to be critical for correct synaptic targeting of the receptor. Therefore, a reduced level of cellular cAMP would mean less protein kinase A phosphorylation of the subunit and lower NMDA receptor numbers at the cell membrane. It is likely that in this case long-term potentiation would not occur and this would result in lower or non-existent memory formation. Therefore, PDE4A5 binding would reduce neuronal functioning after stimulation and this effect would mean binding is located in the neuronal dendrites.

Havekes and colleagues also found that PDE4A5 binding was located in the perinuclear region of the cell and this could be explained by decreased PKA functioning, too. In this case, the PKA phosphorylates the cAMP response binding protein (CREB) which binds to the DNA. Activation of this protein results in changes in gene transcription, eg. nuclear factors such as Bdnf. There is evidence of CREB involvement in PDE4A5 binding and hence, reduced cAMP levels could ultimately affect the amount of gene transcription occurring at the nuclear level.

Therefore, it appears that PDE4A5 could be involved in the ´switching off` of the active neuronal cell and it is likely that this effect is brought about by the reduced cAMP level influencing protein kinase activity at both the perinuclear and dendritic locations. Since there is less known about the mechanisms involved in ´rebalancing` the cells after firing in readiness for the next firing stimulus, identification of elements such as PDE4A5 helps to elucidate the process. This is important because it may be possible in the case of cognitive disorders which involve the hyperexcitability of areas that manipulation of such an element can induce the cell to ´switch` off  thus returning the area to its correct firing level and restoring appropriate cognitive function.

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

……if PDE4A5 function is linked with protein kinase activity then can we assume that use of a PK inhibitor such as staurosporine would have no additional effect on cell functioning and cAMP level if PDE4A5 gene expression was increased?

…….can we assume that the administration of entomidate which effects GABA receptor binding and hyperpolarization through chloride ion channel opening confirms the non-involvement of cAMP at chloride ion channels in the presence of increased PDE4A5 expression?

….is it possible that investigation of neuronal activity of schizophrenic sufferers who are reported to have disrupted N terminal binding of PDE4A5 would demonstrate unusual protein kinase functioning and that further investigation of the areas and particular protein kinases would elucidate exactly where the PDE4A5 works?

 

Posted in hippocampus, long-term memory, PDE4A5, Uncategorized | Tagged , ,

visual imagery deficiency

Posted comment on ´Blind in the Mind` by D Grinnell and published in New Scientist 23rd April 2016 3070 p34

SUMMARY

The author of the article, D. Grinnell, has never been able to carry out mental imagery, but claims he has no problems with tasks that are usually aided by it, eg. navigation and people recognition. He appears not to be unique with 2-3% of people also lacking the capability according to a study using the test, Vividness of Visual Imagery Questionnaire, where various scenes have to be imagined and the clarity of the mental picture rated. The idea that some people are not capable of forming mental images is not new with Sir Francis Galton reporting it as early as 1880. He asked his study participants to imagine things on a breakfast table and found that some were unable to carry out the required task.

Grinnell in his article quotes a study by Zeman and colleagues whose subject, MX, was a 65 year old building surveyor who reported losing the capability to form mental images after heart surgery. MRI scans showed that when pictures of recognizable things were shown to MX, firing patterns were produced in visual areas towards the back of brain and these patterns were both expected and distinctive. Attempts by MX to imagine the same pictures however, produced no such firing patterns. Although the mental images could not be formed, it was found that MX could still give relevant information about the objects such as the number of windows in a particular house. The condition of lack of mental imagery was named as aphantasia by Zeman and his colleagues who also found in their study a further 21 people suffering from it, all of whom appeared to have had it from birth. Zeman concluded that a person does not have to see something to ´live it`, they just needed to be aware of it and Grinnell, himself a sufferer, in his article agrees with this view.

In his article, Grinnell went on to describe the psychological hypotheses relating to visual imagery. The cognitive neuroscientist, Kosslyn, described visual imagery as depictive/ quasipictorial representations and that spatial organization of brain activity resembles the object imagined. Kosslyn explained visual imagery from a physiological perspective by saying that visual imagery is not constructed in a single way in the brain because the separate visual circuits for shape, colour and spatial relationships are not all switched off in aphantasia. Grinnell found on questioning aphantasics that visual imagery was replaced by imaginary drawing and therefore, there was control of physical movements such as finger movements. This hypothesis was supported by Zeman and team. In their tests on MX, Zeman´s group also found that MX`s spatial rotation skills were faster than average. Spatial rotation requires the subject to say which images are the same as the guide image, only rotated and hence, the greater the rotation, the longer the time required to work out if there is a match because of the need for mental image manipulation. To explain their observations, Zeman believes that everyone has visual capabilities and people with mental imagery rely on this visual information whereas aphantasics are given other information or representations. This is supported by evidence that aphantasics dream in pictures and some see flashes of imagery under certain conditions, eg. before they fall asleep. Therefore, aphantasics may not be able to consciously control their mental pictures, but the capability to carry them out may not itself have vanished.

Grinnell continues in his article by citing Zeman`s hypothesis of the parallels between aphantasia and blindsight. In blindsight, there is visual information, but no conscious awareness of it. De Vito and Bartolomeo extended this by saying that aphantasics still have the capability to imagine, but just believe they cannot thus supporting Zeman`s hypothesis. It was proposed that extreme stress could induce a change to aphantasia and evidence from a study of Monsieur X in 1883, who after a period of intense anxiety developed aphantasia, was given. However, this could not be said to apply to other well-known cases including MX whose aphantasia was caused by brain injury and by Grinnell himself who was born with the condition.

Grinnell in his article also discussed whether aphantasia was reversible. Pearson in Australia looked at whether mental imagery could be reset. In 2008, a test was developed that objectively measured peoples` mental imagery capability. Subjects` fields of vision were divided so that they saw a set of horizontal red stripes through one eye and a set of vertical green stripes through the other. Normally, one set is perceived first, but if flash cards are displayed quickly several times then for most people the probability of perceiving that particular colour the first time increased. This was explained by the formation of the picture in the subject`s mind`s eye which led to priming of the participant to see it again. However, studies on aphantasics gave inconsistent results. Pearson then coached those participants that demonstrated the unconscious mind`s eye by saying that they had to try visualizing either the green or red striped pattern for a few seconds every day for 5 days. The process was then repeated in the laboratory and the participants were asked to rate the strength of the image. Immediately afterwards, Pearson flashed the red pattern in one eye and the green in the other and measured whether people had perception bias. In some cases, the objective rating was found to remain constant, but the subjective rating had improved suggesting that the training had helped people to begin to access the previously subconscious mind`s eye. Grinnell himself found shapeless lights flashed into his mind, but decided not to continue with the training.

Grinnell`s article concluded with him saying that aphantasia had given him an unique way of seeing the world which he did not want to relinguish. Others also stressed the importance of aphantasia and the unique skills required for people lacking mental imagery to carry out cognitive processes. This capability could be used to determine alternative ways of information processing and thinking which could aid those suffering from neurological disorders.

COMMENT

What makes this article interesting is that aphantasia appears to go against what we think is happening with the neurochemical mechanisms in the cases of certain cognitive capabilities such as complex decision-making or navigation. In such examples we believe that imagined visual information built in the mind, albeit based on ´real` information whether in real-time or from memories, helps the brain to carry out the required tasks. However, it is clear that there are certain people, the aphantasics, who have no visual imagery, possess neurochemical mechanisms that are obviously different to others, but are still able to perform normal cognitive tasks such as decision-making. Therefore, there is a need to investigate the neurochemical mechanisms of this minority of people. For  97-98% of people capable of seeing, visual information plays an important role in memories, thinking and informational processing and to carry out these functions there are various visual systems and mechanisms employed including: the physiological visual neuronal pathway from input in the eye to the higher cortical areas; visual short term memory where visual information is held as an electrical firing pattern for a very short period of time (less than 10 seconds) and the person may be conscious of the experience or unconscious; visual long term memory where neuronal cell assemblies are formed from the short term visual firing patterns and the information is stored as memories to be consciously or unconsciously recalled at a later date; the visual buffer which is part of the Baddeley and Hitch working memory model and is the processing ´work space` of the cognitive brain; and finally, and obviously not for everyone, visual imagery which is defined as where there is a visual memory representation when the stimulus is not actually being viewed, ie. ´seeing with the mind`s eye`.

The various visual systems and mechanisms are well-researched and new knowledge is continually being added and from this collection of knowledge we know that visual representations are part of decision-making for example, thought (Aristotle`s view that they are the ´medium of thought`), problem-solving, prospective memory planning and memory techniques such as method of loci. The visual images are formed in the V1 with involvement of the V2, slightly elongated fields and have close similarities to perception even though they are lower in detail than those spawned from ´real` stimuli. In the majority of people the visual representations formed in the V1 are likely to follow Kosslyn`s perceptual anticipation theory with the images being quasipictorial representations.

However, aphantasics show that visual imagery is not necessary in their case for the same cognitive capabilities to be demonstrated as those having this capability and therefore, their neurochemical mechanisms are likely to be different to those of the majority. In fact, they are examples of support for the Pylyshyn`s propositional theory for visual imagery where the visual image is not dependent on depictive/quasi pictorial representations, but a tacit knowledge of how the subject would ´look` in the situation. This could possibly be explained by considering the information at V1 not as solely visual, but as instead electrical representations that are capable of being interpreted into a visual image if required or more likely a multi-sensory representation with input included from the other sensory systems as well. Such a representation would override the need for visual dominance in cognitive functioning and allow aphantasics to process information in the absence of conscious visual imagery, but using the unconscious information from visual pathways and other senses. Therefore, aphantasics have a lack of awareness that visual information is being used, only that an electrical representation is formed. This view is supported by observations that: the visual imagery capability is still present in aphantasics since studies have shown that people can be trained to some degree to use it; conscious visual information is not always required since in others there are plenty of examples of unconscious visual processing such as moving before knowing why you have to move; and the cases of blind sight and visual working memory where visual information is being processed without conscious awareness.

What makes this topic interesting is that aphantasics provide a relatively large subject group in experimental terms that are likely not to suffer from mental health issues, or brain injuries and who could allow the conditions and mechanisms of mental tasks to be explored to the full, eg. decision-making or prospective memory. Not only could objective research methods be employed, but introspection could be considered more repeatable and reliable. Studies using techniques such as imaging, temporary incapacitation of certain brain areas with tDCS for example or local anaesthetics could explore the mechanisms involved in the cognitive processing of aphantasics and perhaps shed light on new approaches to, for example, the treatment of mental disorders affected by deficient information processing. In his article, Grinnell refuses to continue with the training to overcome his lack of visual imagery preferring his uniqueness and he may be helping the rest of us by doing so!

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

……since visual imagery is presumed to be required for matching objects that have been rotated to some degree, would accurate imaging studies show the mechanisms that aphantasics employ in the carrying out this skill?

……if training with flash cards changes the performance of aphantasics to ´seeing images`, could training using images of clocks aid prospective memory performance in those suffering from disorders where information binding is problematic?

Posted in Uncategorized, visual imagery | Tagged

frequency selective control of cortical networks by thalamus using optogenetics

Posted comment on ´Frequency-selective control of cortical and subcortical networks by central thalamus` by  J. Liu, H.J. Lee, A.J. Weitz, Z. Fang, P. Lin, M. Choy, R. Fisher, V. Pinskiy, A. Tolpygo, P. Mitra, N. Schiff and J.H. Lee published in eLife 2015;4:e09215 (doi.org/10.7554/eLife.09215)

SUMMARY

The authors of this paper explored the network connections of the central thalamus which is known to play a role in arousal and organized behaviour. They used optogenetics (20 s periods of light stimulation every minute for 6 min at 10, 40, or 100 Hz) with fMRI to form the ofMRI technique which provided whole brain spatial and temporal information.  A stereotactic injection was given to the subject in the right CL and PC intralaminar nuclei of central thalamus with the adeno-associated virus carrying channelrhodopsin-2 (ChR2) and the fluorescent reporter protein EYFP under the control of the CaMKIIa promoter. This promoter was used since it is expressed primarily in excitatory neurons which in the thalamus are mostly relay cells. Liu and colleagues found that nearly 34% of cells were EYFP-positive, co-expressing CaMKIIa which showed that the technique was highly selective for excitatory neurons and hence ideal for neuronal stimulation experiments. Targeted stimulation of the intralaminar nuclei area was achieved by MR-validated stereotactic fiber placement and using a small volume of excited tissue. Electrophysiology and video EEG monitoring was also used to investigate the network connections. Ex vivo fluorescence microscopy images of ChR2-EYFP expression were also carried out.

Liu and colleagues found in their experiments that EYFP-expressing axons could be seen throughout the forebrain, including areas such as the frontal cortex and striatum with the medial prefrontal, lateral prefrontal, cingulate, motor, and sensory cortices all receiving strong projections from the thalamus. Input was found to be highly convergent at the superficial layers, with moderate but weaker projections also present in the middle layers. Furthermore, projections were significantly restricted to the hemisphere ipsilateral to the virus injection for both the cortex and striatum.

The authors also found using the ofMRI technique at all 3 frequencies strong positive blood-oxygen-level-dependent (BOLD) signals at the site of stimulation that was highly synchronized to light delivery, increased upon optical activation, and gradually returned to baseline following the end of stimulation. Local neuronal firing was also observed. A much larger volume of brain tissue was activated by stimulation at 40Hz and 100 Hz compared to 10 Hz as was the frontocortical areas and striatum in particular. The difference in activation volume between the low 10 Hz stimulation and the higher 40 or 100 Hz stimulation frequencies was significant for the thalamus, striatum, and medial prefrontal, lateral prefrontal, cingulate, motor, and sensory cortical areas. Striatal activity was found to be primarily localized to the dorsal sector, with negligible activity occurring in the ventral region and BOLD activation was generally restricted to the ipsilateral hemisphere, although activation volumes in the contralateral striatum, lateral prefrontal cortex, motor cortex, and sensory cortex were all significantly greater during 100 Hz stimulation compared to the low 10 Hz stimulation. The rapid 40 and 100HZ stimulations of the central thalamus causing the widespread activation of the forebrain caused a state of arousal in the sleeping rats and the increase in neuronal firing rate observed during the 100 Hz stimulation was generally maintained throughout the 20 s stimulation period.

With the slower 10Hz stimulation, Liu and colleagues found that even though the excitatory neurons had been targeted for activation the somatosensory cortex exhibited a strong negative BOLD signal during 10 Hz stimulation which suggested that baseline activity had been suppressed. This was supported by the results of the ofMRI technique which showed that 10 Hz stimulation had decreased the neuronal firing rate between pre-stimulation and stimulation period and this decrease occurred mainly between 5 to 15 s after initiation of the stimulation. Spiking events which occurred during this inhibition had a non-uniform distribution over time suggesting that only sometimes did the glutaminergic thalamocortical input generate action potentials. The resulting lower activation of the forebrain and inhibition of the sensory cortex led to seizure-like unconsciousness of the test subject.

Using the ofMRI technique, Liu and colleagues could identify a group of inhibitory neurons in the central thalamus in the zona incerta (ZI) region which sends direct GABAergic projections to the somatosensory thalamic nuclei and sensory cortex and whose activity is linked to whisker stimulation. The authors found that the majority of the ZI cells exhibited increases in firing rate during the central thalamus stimulation at 10Hz and 40Hz. Spindle like oscillations (SLOs) were evoked at the lower 10Hz stimulation, but not at 40Hz and these oscillations exhibited an inter-event interval centered around 6.6 s similar to those observed in the thalamus during the onset of sleep. The suppressed ZI firing during the 10Hz stimulation was found to lead to a reduction of evoked cortical inhibition. Simultaneous EEG recordings in the frontal cortex revealed strong spike-wave modulation during the 10 Hz stimulation associated with the loss of consciousness and lower amplitude, fast oscillations during 40 Hz stimulation associated with aroused brain states.

Liu and colleagues investigated if the evoked activity in ZI plays a causal role in driving the frequency-dependent inhibition of the somatosensory cortex. They injected the inhibitory opsin halorhodopsin (eNpHR) fused to the mCherry fluorescent marker and controlled by the pan-neuronal hSyn promoter into the ZI of four animals expressing ChR2-EYFP in the central thalamus. The light stimulation at 10Hz of halorhodopsin was found to be successful in suppressing ZI activity and this had a net inhibitory effect on somatosensory cortex activity. The authors suggested that this was brought about by hyperpolarization of the neuronal cells in this area.

The results found with ofMRI were supported by the simultaneous video and EEG recordings. During the 10 Hz stimulation, the majority of animals exhibited behavior indicative of an absence seizure, including freezing and behavioral arrest throughout stimulation leading to sleep onset. The most common EEG response was a shift to slow spike-wave discharges indicative of a loss of consciousness. The higher 40 and 100 Hz stimulations led to the awake state and an EEG pattern associated with cortical activation and desynchronization.

Therefore, the authors concluded that the awake or unconscious (or sleep) state is promoted by the ZI area of the central thalamus and how fast these neurons are stimulated. Differences in time could reflect the short-term plasticity of the thalamocortical pathway which has frequency-dependent properties. Their experiments show that neuronal cells in a single population can have different firing patterns and promote different effects on connecting areas depending on the temporal code of their stimulation. Since there are GABAergic projections from the ZI to central thalamus, activity in ZI may also limit forebrain activation through incertal-thalamic feedback. Therefore, the hypothesized feedforward and feedback inhibition via ZI both suggest a direct projection from central thalamus to ZI, which the fluorescence imaging data supported. However, there is no thalamic input specifically from the intralaminar nuclei to ZI and therefore arousal regulation is driven by the central thalamus which has a causal and frequency-dependent influence on ZI. Suppression of the ZI activity modulates the activity of the overall brain which is susceptible to thalamus stimulation eg. inhibitory signals from the ZI lead to frequency-dependent depression of cortical activity. This type of information can be important in the treatment of traumatic brain injury and the minimization of cognitive defects.

COMMENT

What makes this paper interesting is the use of the newly popular technique of optogenetics to further investigate a brain area with relation to a well-known function. It has been known for a long time that the central thalamus is an important area relating to arousal/alertness and sleep/wakefulness and that damage to this area can be lead to not only excessive sleeping and coma, but also cognitive problems such as loss of memory. The study described here in this Blog post uses optogenetics to investigate the arousal and sleep function of the thalamus further. It can be seen that the central thalamus and intraluminar nuclei when stimulated at low frequencies leads to the subject losing consciousness, limited forebrain functioning, strong inhibition of the somatosensory cortex and  EEG spindle bursts. Alternatively, high frequency stimulation leads to arousal of the subject, attention and goal directed behaviour and is supported by desynchronized EEG cortical signals.

Using optogenetics with its high sensitivity to spatial and temporal changes, these different effects can be attributed to activity in a specific thalamus region, that of the zona incerta (ZI). This is a grey matter area located in the subthalamus under the thalamus and gates sensory input and synchronized cortical and subcortical brain rhythms. It is known that this area has a wide variety of cells all merging areas into one another and is divided into sectors eg. rostral, dorsal, ventral (known to be GABergic cells) and caudal known as the ´motor sector` and an area bringing research attention because of targeting by tDCS in sufferers of Parkinson`s disease.

ZI is also known to have numerous connections some outgoing (eg. to cerebral cortex, hypothalamus), others incoming (eg. cingulate cortex, frontal lobe, parietal lobe, cerebellum, raphe nuclei, thalamic reticular nucleus, super colliculus, the last three being cholinergic) and some bidirectional such as the thalamus (eg. intraluminar and central lateral nucleus) substantia nigra (linked to DOPA and Parkinson`s disease) and globus pallidus (linked to reward). The capability of the area appears to be linked to the frequency at which it and the thalamus are stimulated. The stimulation either removes the inhibition placed upon the area (high frequency) or activates it (low frequency).  Sensory suppression means hyperpolarization of thalamus leading to GABAergic IPSP and depression in the ZI area. Sensory activation means likely glutaminergic depolarization of the thalamus leading to EPSP of the ZI. Hence, depression of ZI is inhibited by the depolarization of the thalamus. Therefore, the optogenetics study of Liu and colleagues shows that the frequency of stimulation has a wide-ranging neuronal firing affect. Similar to work on the medial leminiscus tract and the thalamus, frequency of stimulation changes subsequent firing such as short EPSP leads to longer IPSP (Castro-Alamancos). Further investigation of the firing within smaller frequency ranges is likely to reiterate the results of Bartho et al. who used anaesthetized rats. They showed that slow cortical 1-3HZ waves become synchronized to depth-negative phasing of cortical waves to a degree comparable to thalamocortical neurons; paroxysomal high voltage spindles display highly rhythmic activity in tight synchrony with cortical oscillations; and 5-9HZ oscillations respond with a change in interspike interval distribution. Hence, the optogenetics technique can be used to further investigate the neural networks existing in the brain and the effect on firing of specific frequency stimulation.

However, herein lies some problems with optogenetics. Is this technique only repeating, albeit more accurately, studies that were carried out many years in the past?  We may be able to pinpoint areas more accurately and say where and with what these areas are networking, but does that add to previous knowledge to sufficiently answer the questions about how memory and consciousness are formed for example? Or how neurodegenerative diseases start? Optogenetics is expensive, there are small sample numbers and the technique has an element of risk with human subjects. Plus it requires cell alterations (the neurons have to express the gene encoding the light sensitive ion channel) so can we guarantee that what we are seeing is actually real and not the result of this insertion? The benefit of this technique could be in cases where it is linked with other techniques such as cell targeting of chemotherapy drugs or in cases like Parkinson`s disease where we can override the effects of limited DOPA in one area and consequential reduced firing by stimulating with light the next area in the motor system. Another benefit of the technique could be in cases where we can compare the molecular complexity of mechanisms investigated by other means for areas lit up due to firing from the targeted area. It is clear that the technique is here to stay and can offer new experimental avenues to explore, but the talked about panacea for human mental disorders is in my opinion not yet proven.

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

………………..if Alzheimer`s disease is linked to hyperexcitability of the hippocampus, could optogenetics with illumination at intervals be used to suppress activation in this area and hence, reduce the build-up of beta amyloid?

………………could the use of gold nanoparticles attached to specific antibodies as suggested by Bezanilla instead of gene therapy be used to study other membrane molecules where the transport of electrons is a part of their function and not just neurons?

Posted in neuronal firing, optogenetics, thalamus, Uncategorized | Tagged , ,

iron levels and memory performance

Posted comment on ´Iron Level and Myelin Content in the Ventral Striatum Predict Memory Performance in the Aging Brain` by T.K. Steiger, N. Weiskopf, and N. Bunzeck published in The Journal of Neuroscience, 23rd March 2016, 36(12): p.3552

SUMMARY

Steiger, Weiskopf and Bunzeck`s paper looks at the relationship between iron accumulation, the degeneration of neuronal myelin sheaths and brain memory performance in the elderly. They examined the performance at the verbal learning memory test (VLMT) against the degree of myelination and iron accumulation for a group of 17 participants aged 18-32 against a group of 31 participants aged 55-79. Grey matter volume was also measured using voxel-based morphometry (VBM) and MT maps were segmented into grey matter (GM), white matter (WM) and CSF. To test for differences in R2* and MT parameters, a voxel-based quantification (VBQ) analysis was used and statistical analysis was applied to all results. Brain memory performance was measured using VLMT where a list of 15 non-related items (List A) was learnt and immediately recalled five times to give the parameter ´VLMT total learning`. This process was repeated using an alternative word list (List B) which was immediately recalled once. The participants were then asked to recall List A and then again after a delay of 30 mins. The difference between the first and second recall session was designated ´VLMT consolidation`. Cued recall was then performed using words taken from both List A and List B plus additional similar words. Participants were required to identify components of List A and these items were termed ´VLMT recognition`. Regression analyses were performed on the relationship between GM, MT, R2* maps and VLMT performance.

Steiger, Weiskopf and Bunzeck found in their investigation that there was a decrease in grey matter volume in the elderly brain relative to the young. Bilateral decreases were observed in the putamen, orbitofrontal cortex (OFC), precentral and postcentral gyri, supplementary motor area, left supramarginal gyrus, right occipital cortex, right superior temporal gyrus, the right medial frontal gyrus and right inferior parietal gyrus. A decrease in myelin was also observed in the elderly relative to the young as shown by VBQ on MT maps and this was registered for areas within the left hippocampus, right thalamus, caudate, cerebellum, post central and precentral gyri, right colliculi superior, left occipital cortex, and widespread WM tracts.

The authors also found using VBQ on R2* maps an increase in iron levels in the elderly brains and this was observed within widespread brain regions including the basal ganglia, bilaterally in the putamen, pallidum, caudate, ventral striatum, and partly within the occipital cortex. A further investigation of the basal ganglia of elderly participants using the mean R2* and MT found negative correlation between myelin and iron in the area of the ventral striatum in the elderly. However, direct comparison between both correlations for elderly and young participants did not give a significant result and this was explained by the authors as possibly due to the small sample size.

Both markers of iron and myelin (and the ratio) were found by the authors to predict VLMT performance for the elderly participants. The VLMT scores of the elderly were used as covariates in whole brain linear regression models on GM, R2* and MT maps. The authors found within the ventral striatum a positive correlation between VLMT and MT, but negative VLMT and R2*. The VLMT performance predicted by the ratio MT/R2* in the ventral striatum showed increased iron levels in the elderly participants. Steiger, Weiskopf and Bunzeck also reported significant effects within the vicinity of the corpus callosum with learning correlating to MT white and grey matter. However, whole brain regression analysis on grey matter volume and VLMT scores was found not to be significant.

From their results the authors of this paper concluded that there are age related decreases in grey matter volume and this finding was supported by reports from other researchers who stated that the grey matter volume decrease is due to loss of neurons, changes in synaptic density and /or axonal or dendritic arborization. Steiger, Weiskopf and Bunzeck concluded that decreases in myelin in the elderly as seen in lower levels of white matter tracts and in subcortical regions indicates less macromolecular content (mainly myelin) and probably demonstrates demyelination and dysfunctional re-myelination in the aging brain. This provides an understandable link to VLMT performance since myelin is a factor in the speed of neuronal signal conduction and interconnectivity between brain areas important for learning. Also, learning induces myelination linked to oligodendrocytic function, which has been found to decrease with age. The decreased myelin level could also be due to damaged oligodendrocytes releasing iron into the surroundings. The authors found increased iron in the elderly brain mainly in the basal ganglia, a reason for which, although unclear, was suggested by some that it is triggered by an attempt by the cell to maintain a declining system through increasing metabolic processes. The rise in iron accumulation was found to be region specific and ventral striatum iron accumulation was found to be linked to demyelination and impairments in declarative memory in the elderly. The authors explained this by citing the role of the ventral striatum in encoding novel information into long-term memory. Therefore, any change in myelin brings about a change in the hippocampal learning mechanism. On investigation of the whole brain, iron and myelin within the basal ganglia was found to account for individual VLMT performance and not the grey matter volume.

In order to provide an explanation for the link between iron accumulation, Steiger, Weiskopf and Bunzeck discussed an association between iron levels and the neurotransmitter, dopamine. In the healthy brain there is a homeostatic balance between dopamine and iron, but this does not exist when the iron levels are high. Therefore, the negative correlation observed in the ventral striatum in these experiments was suggested as indicating that decreased dopamine levels led to decreased memory performance. This hypothesis was supported by experiments involving iron chelation which was shown to reverse any memory impairments.

Therefore, Steiger, Weiskopf and Bunzeck demonstrated in their paper that iron accumulation and myelin reduction seen in elderly brains can lead to observed cognitive impairments measured by the verbal memory learning test.

COMMENT

This article is interesting because it tenuously links a dietary mineral, iron, which should be part of our normal nutritional intake to neurodegenerative disease. It appears that iron accumulates in the brain naturally with age, but can also occur in some neurodegenerative diseases, which are linked with impaired memory and other cognitive skills. Hence, if this tenuous link is correct then there must be a link between iron and the physiology and mechanisms associated with brain memory. Therefore, the question has to be asked where does the mineral iron fit in with the brain memory hypotheses for neurotransmission and cognition?  Immediately, obvious connections with brain neuronal efficiency come to mind, for example: the role of iron in myelin production and oligodendrocytes functioning with myelin giving the neurons signal transmission protection; the role of iron in the synthesis of cholesterol, hence affording the neuronal cells with membrane fluidity essential in efficient and correct neuronal functioning; the role of iron in the synthesis of the neurotransmitters, hence providing the instigators of the firing from cell to cell. And it does not stop there because there are less obvious roles of iron in the normal ´housekeeping` carried out in living cells such as energy metabolism (eg. respiratory chain, citric acid cycle associations) and biosynthesis of amino acids and nucleotides for example.

The wide range of roles played by the mineral iron is indicative of its chemical ´flexibility` which gives it biochemical advantages. It allows energy state changes of the molecules in which it is a part due to its inherent capability of being in either an oxidized, or reduced state. Electron transfer whether donation or acceptance can lead to structural conformational changes of the molecules that include the iron ion in their structure and these changes in conformation can be part of the functioning mechanisms of that particular molecule. Heme groups and iron-sulphur clusters are good examples of this. Owing to this electron transfer capability, it is not good for a cell to have iron ions free in the cytoplasm and hence, iron is ´wrapped up` in the form of ferritin (storage), transferrin (serum) and transferrin receptor (entry to cells). The balance of these forms is important and this has been shown with reports of reactive oxygen species (ROS) production and modification of lipids, proteins, carbohydrates, DNA etc. when the balance is disturbed.

Therefore, it is clear that a system relying on signaling transfer such as that found in neurotransmission can be influenced by iron concentrations and this is supported by evidence that memory and cognitive skills can be affected by iron availability. Free iron accumulation has been reported in neurodegenerative diseases such as Alzheimer`s disease. Iron chelation has been found to lead to decreased symptoms, increased memory and inhibited beta-amyloid accumulation, a major contributor to Alzheimer pathology and symptoms. Therefore, is iron accumulation a cause or consequence of Alzheimer`s disease? This is important because if it is a cause then therapy based on modulating iron availability could lead to a reduction in occurrences of the disease.

A look at where iron fits in with the neurotransmission mechanism shows that iron probably plays a role (in addition to those described above) with the beta-amyloid led endocytosis of neurotransmitters into the lysosomal vesicles which forms part of the neuronal cell regeneration after the action potential phase. Disruption of this endocytotic phase by the effects of the dysfunctional amyloid-precursor-protein/beta-amyloid in sufferers of Alzheimer`s disease could explain the observed iron effects. In the hypothetical version of neurotransmission advocated by the author of this blog, ferroportin (the iron transporter for efflux) is attached to the amyloid precursor protein (APP) found in the lipid raft of the presynaptic membrane. PICALM, AP2 and clathrin are all in close proximity. (Other APPs also exist in the neuronal membrane, but are outside the lipid raft and linked to the potassium channel). In normal functioning APP is cleaved by beta-secretase and y-secretase to produce beta-amyloid that is capable of normal conformational changes, and neurotransmitter and metal ion binding. This, hypothetically, leads to the endocytosis of excess neurotransmitter not bound to the post-synaptic membrane by the beta-amyloid aggregate forming vesicular structures from the lipid raft area and transferred within the presynaptic body from cell membrane to endoplasmic reticulum via microtubules and dynein action. The neurotransmitters and membrane components undergo appropriate lysosomal degradation during the transport process and the vesicles are recycled back to the membrane for the next signaling phase. Under normal conditions the conversion of the membrane bound APP to beta-amyloid causes the ferroportin channel to open and reduced iron floods from the cell to be picked up by the oligodendrocytes in the synaptic cleft. This is then used for myelin production, an important mechanism especially in the case of the hippocampus with its high levels of neurogenesis that occurs there during memory formation.

In Alzheimer`s disease it is possible that the unusual cleavage of the membrane bound APP and the formation of the excess beta-amyloid does not produce the membrane conformational changes that leads to the opening of the ferroportin iron transporter resulting in the accumulation of free iron in the cell. Such an accumulation can cause ROS production which is reported in Alzheimer`s disease. Therefore, iron ions are part of the dysfunctions observed at the neuronal level that eventually end in cell death and the peculiar pattern of pathology observed in Alzheimer`s disease. It is also possible that reduced iron ions themselves bind to the abnormal beta amyloid sat on the presynaptic membrane and is part of the dysfunctional endocytotic vesicle formed at the membrane surface. This is supported by the observation that presynaptic iron induces aggregates of inert alpha synuclein and beta-amyloid to form toxic aggregates. Therefore, it is clear that in this case iron is not the cause of Alzheimer`s disease, but a consequence if this hypothesis of neuronal functioning is correct. The limiting factor of the disease appears to be associated more with the formation of excess beta amyloid and dysfunctional APP cleavage.

Iron, however, is not the only metal ion with a role in neurotransmission. Zinc is also an essential mineral that has important biochemical links to efficient neuronal function particularly in the hippocampus where deficiency is associated with causing lethargy and cognitive difficulty for example. A rise in zinc level has been found in Alzheimer sufferers and it is a known blocker of ferroportin, the iron transporting protein whose role in neurotransmission is described above. It is also known that vesicular release and the zinc transporter (Zn-T3) are required for beta amyloid targeting. Therefore, like iron, could zinc be a cause or consequence of the Alzheimer disease?

Zinc is a constituent of many enzymes and in particular the metalloproteases and plays an important role in vesicles and in autophagy (the neuronal endocytosis described before with the breakdown of the neurotransmitters and membranes to be recycled for future synaptic activity). With zinc, the link to neurotransmission is with calcium ions and calcium ion influx which is observed with neuronal excitation. In Alzheimer`s disease, hyperexcitation in the hippocampus leads to massive calcium influx and excess glutamate release. The increase in zinc leads to increased zinc in the lysosomes resulting in membrane disintegration, release of cathepsins and other lysosomal enzymes and increased caspase induced apoptosis. This brings about the neuronal pathology observed in the disease. Again like iron, although zinc deficiency leads to cognitive effects it appears it is not the limiting factor in the causation of Alzheimer`s disease, but a consequence. In this case, hyperexcitation of the neuronal system in this neurodegenerative disease appears to precede the zinc effects.

Therefore, what can we conclude about the role of metal ions in neuronal transmission? We can see that both iron and zinc play a number of specific roles in neuronal signaling and neuronal cell functioning and deficiency can cause abnormal physiological effects that influence the overall functioning of the cell. It is also clear that the causes and physiology of Alzheimer`s disease are complicated with effects observed in the multiple systems, enzymes etc that make up neurotransmission and cognitive functioning, eg. relating to action potential, neurotransmitter synthesis and release, exocytosis and endocytosis, receptor trafficking just to name just a few. Therefore, the likelihood is low that positive changes in the neurotransmission of elderly people with something as simple as iron or zinc administration can cancel out the negative changes seen with Alzheimer pathology leading to retention and improvement of cognitive skills. However, this does not mean that there is not a link between iron and zinc deficiency in the very early stages of Alzheimer`s disease, ie. before the distinctive beta-amyloid accumulation and oligomer pathology is observed. Since the pre-Alzheimer stage develops over many years, who knows what the real instigators are and wouldn`t it be nice if the solution was as easy as administration of zinc or iron! More research is obviously required, but until then maybe everyone should make sure that their daily mineral intake is sufficient.

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

…..are mouse models of Alzheimer`s disease the best models when dietary considerations are being investigated and should it not be that ´elderly` mice are preferentially used for testing for this particular neurodegenerative disease?

……is it that in iron deficient mice, myelin production in the hippocampus is reduced and this can be linked to synchronization problems between this area and others relating to spatial memory and conditioning. In this case, would neuroimaging experiments and brain wave monitoring show the defective connectivity between the hippocampus and other areas linked to memory for example?

Posted in iron, memory recall, neuronal firing, Uncategorized | Tagged , ,

neuroimaging of connectome changes after working memory training

Posted comment on ´Dynamics of the Human Structural Connectome Underlying Working Memory Training` by K. Caeyenberghs, C. Metzler-Baddeley, S. Foley and D.K. Jones published in The Journal of Neuroscience, 6th April 2016, 36(14) p. 4056

SUMMARY

Neuroimaging studies of the brain normally involve showing functional areas of the brain and that functioning responding to some change in condition. The work of Caeyenberghs and colleagues is no different except they have found that the results of neuroimaging studies relating to memory capability pre- and post-cognitive training can be dependent on the metrics used. Most of the previous research looking at this topic uses diffusion tensor MRI studies and the metric of fractional anisotropy (FA) looking at white matter and results obtained are inconsistent. In Caeyenberghs and colleagues study 40 healthy participants underwent either an adaptive training program for working memory (Cogmed – 45 sessions; 40 sessions in total with training for verbal and spatial memory) or non-adaptive training. The participants were assessed using MRI neuroimaging and computerized working memory and executive function tests.

The neuroimaging techniques used in the reported study combined well established (although nonspecific) diffusion tensor MRI metrics with both MRI relaxometry-based metrics (an indirect measure of myelin, but corrected for motion and distortion artifacts for example) and metrics derived from advanced models of diffusion tensor studies (gives estimates of axonal density and corrected for distortions induced by the diffusion-weighted gradients and motion of the head for example). Quantitative maps of axonal morphology were constructed using the CHARMED protocol and maps of myelin level using the mcDESPOT protocol. A total of eleven different kinds of networks were generated with a network being defined as a set of nodes denoting anatomical regions and interconnecting edges denoting undirected tractography-reconstructed fiber trajectories interconnecting the nodes. These were weighted using the mcDESPOT protocol. Network areas included the inferior and superior parietal cortex, supramarginal gyrus, caudal and rostral middle dorsolateral prefrontal cortex, superior frontal cortex, inferior ventrolateral prefrontal cortex, insula and anterior cingulate cortices and the subcortical regions of the basal ganglia i.e. caudate, putamen, globus pallidum and thalamus. The volumes of grey matter of 30 regions of interest were used to construct structural correlation networks. FreeSurfer was used for cortical reconstruction and volumetric segmentation reconstruction of the brain’s surface to compute cortical thickness. The authors then performed graph and theoretical analyses to obtain their imaging results.

In order to ascertain the possible relationship between various cognitive skills in their behavioural testing methods, Caeyenberghs and colleagues ran prior to training test exploratory principal component analysis and this showed three significant behavioral components that together amounted to 59% of the total variance. The first component, complex span working memory, accounted for 34% and related to all of the tasks in which information had to be actively maintained in short-term memory, eg. the automated symmetry span task, the spatial span task, and the odd one out task. The second component accounting for 13% of the variance was associated with tasks involving a verbal component and included the double trouble task, digit span tasks, and the grammatical reasoning task. Tasks requiring general reasoning including the Hampshire tree task and the self-ordered spatial span, related to the third component and this accounted for 12% of the variance. Statistical analyses of the results were carried out by combining the scores.

Caeyenberghs and colleagues found that the Cogmed training program carried out produced positive cognitive changes. The main effect of time (pre and post-training) and group (adaptive and non-adaptive training) produced significant results, but there was no significance between the three cognitive skills (ie. complex working memory, verbal or general reasoning). Both sets of results for time against group and time against cognitive skill were significant and a three-way interaction between factor, group, and time was also found to be significant. Varying levels of performance improvement were obtained with the highest levels for the complex working memory and the verbal component in the post-training session for the adaptive group compared to the non-adaptive group.

Using graph theoretical network analysis of the working memory training effects, the authors found that there were significant changes for the interaction of group against time using the mcDESPOT protocol. This demonstrated that there had been an increase in global efficiency between the nodes of the network in the adaptive group from pre- to post-training. Marginal significant interaction effects were observed for the global efficiency of the graphs weighted by different diffusion-derived parameters, including FA, 1/mean diffusivity (MD), axial diffusivity, tissue volume fraction, 1/radial diffusivity, and a number of streamlines. The parameter that best captured the effect was the relaxation rate. No significant interaction effects were observed for the graph weighted by the total restricted fraction derived from the CHARMED protocol, or in the graph derived from the covariance of gray matter volumes.

Analysis of the neuroimaging results obtained for different regions led to the identification of the nodes responsible for the effects of the working memory training. The authors found that right anterior rostral cingulate gyrus, an area associated with attentional control and mental effort, showed a significant group against time interaction. Using a different method, significant results for a group against time interaction effect was also achieved for the right inferior ventrolateral prefrontal cortex which is associated with attentional orienting processes. These observations were supported by post hoc two-sided t test results.

Caeyenberghs and authors also found that correlation analyses between the changes in global efficiency from prior training periods to training and afterwards and the composite scores of the behavioral parameters showed little direct association between changes in structural network metrics and the improved performance on the cognitive tests. However,  using an exploratory uncorrected threshold of p < 0.05, correlations were observed between the changes in Cogmed tasks performance and changes in global efficiency of the R1-weighted networks. This was associated with better working memory performance on the Cogmed pairing with higher efficiency of information transfer (i.e. more global integration). None of the correlations were significant when the necessary correction for multiple comparisons was carried out.

The scores of global efficiency of the networks constructed with different metrics as ´connection strengths` were found to be highly intercorrelated at the baseline and therefore the authors interpreted this as representing non-independent observations. For example, the global efficiency of the network whose connections strengths were defined by the quantitative relaxation rate R1 (1/T1) derived from the mcDESPOT protocol correlated strongly with global efficiency of the network weighted by the TRF derived from the CHARMED protocol. However, the difference scores in global efficiency of the networks weighted by R1 were found not to correlate significantly with difference scores in the efficiency of the network weighted by the TRF metric or MWF-weighted network. Therefore, Caeyenberghs and authors concluded that although all metrics correlated at the baseline and that the reduction in correlation post-training suggested that the white matter network underwent changes during training, these changes are better detected with R1 than the other metrics tested. Since the different metrics relate to different aspects of the white matter microstructure and because relaxation times T1 and T2 are affected by changes in water, lipid, and protein content, T2 by iron within the oligodendrocytes and MWF by lipid myelin content, the changes observed with training in Caeyenberghs and colleagues study are suggested as being linked to alterations in these cell components.

Therefore, Caeyenberghs and authors showed in their neuroimaging study that changes occur in the structural connectome as a result of adaptive cognitive training. These changes relate to improved performance of working memory and verbal tasks and less so the far-transfer tasks involving general reasoning. The positive performance changes relate to increased global efficiency and there are likely to be white matter changes as shown by relaxation-rated network increased neuroimaging sensitivity. Since the authors discovered that some MRI metrics are not ideal for this type of neuroimaging of these particular cognitive skills (eg. techniques normally used for observing global efficiency changes rely on FA or MD), Caeyenberghs and authors concluded their paper by emphasizing the need for using specific microstructural markers for this type of experimentation.

COMMENT

What makes this paper and others like it interesting is to see the shift over the years of emphasis in neuroscience from laboratory test-tube experiments of long-term physiological changes in single brain area samples to the current fields of neuroimaging of real-time neuronal firing and functional networks. This gives another dimension to cognitive research and this paper takes advantage of these modern real-time techniques to demonstrate how training can affect neuronal cell firing and neural networks. However, every technique has its problems and drawbacks and neural imaging is no exception with the authors here demonstrating that not all metrics are ideal for every cognitive situation and that some can mask effects that would normally be seen or give results different to those obtained by other means. Coupled with these experimental problems are others relating to the fact that living subjects are being used and so with all such experiments of this nature stress, anxiety, timing, previous medication etc. can all affect the imaging results obtained. Even providing enough participants to produce significant results can be a problem.

However, the results obtained from neuroimaging can expand the neuroscientific knowledge front and what was observed here and in other studies of this nature are the functioning changes in neuronal firing and neural networking relating to cognitive training. The authors here found a positive training effect suggested as a result of a positive effect on axonal connectivity within certain areas and between certain areas. Caeyenberghs and authors identified 11 different kinds of networks and found a significant result for the group containing the right anterior rostral cingulate gyrus, an area normally associated with attentional control and mental effort. Using a different method significant results were also obtained for a group with the right inferior ventrolateral prefrontal cortex, an area normally associated with attentional orienting processes and decision-making. These results supported observations by Dreher and Grafman who investigated task switching and dual-task performance using fMRI. They showed that performing two tasks successively or simultaneously activated a common prefrontal-parietal neural network relative to performing each task separately. Performing two tasks simultaneously brought about activation in the rostral anterior cingulate cortex whereas switching between two tasks activated the left lateral prefrontal cortex and the bilateral intra-parietal sulcus region. The results were interpreted as indicating that the rostral anterior cingulate cortex serves to resolve conflicts between stimulus–response associations when performing two tasks simultaneously (attentional control and mental effort), whilst the lateral prefrontal cortex dynamically selects the neural pathways needed to perform a given task during task switching (attentional orienting processes).

The involvement of more brain areas during specific tasks and the effect of training has also been observed by other researchers, too. In August 2015, a post was published on this blog about the Cogmed program and a meta-analysis of the results which had been performed by Spencer-Smith and Klingberg, the authors of the paper. In their paper, they reported an average improvement of 16% after participation in the Cogmed training program independent of participant health status. The repetitive nature of the training program with participants learning through adaptation during the program time span, applying routine, use of memory chunking, improving attention and concentration, and taking note of feedback all possibly providing reasons why the training program led to cognitive improvement. The authors also noted that there was a greater effect on visuospatial memory than verbal suggesting that the improvement could be associated to increased working memory. The improvements obtained were also sustained for 2-8 months after training had finished.

From a neuroscientific point of view, studies have shown that training increases connectivity in frontoparietal and parietal occipital networks (Kunden). We also know that working memory requires acetylcholine and glutamate and the involvement of many brain areas. For example, neurons in the prefrontal cortex are associated with multi-tasking, working memory and attention (Messinger) and visual working memory requires activity in the inferotemporal cortex, V4, medial temporal cortex, prefrontal cortex and globus pallidus, lateral infero parietal cortex (guided eye movements in attention), post-parietal cortex (Koenigs – manipulation of information in working memory), as well as fronto-hippo connectivity (Cordesa-Cruiz). Working memory activity sees changes in prefrontal oscillations with  theta oscillations increasing during temporal order maintenance and alpha oscillations increasing over the posterior parietal and lateral occipital regions for item maintenance (Hsieh) with these alpha oscillations being used to maintain the relevant memory contents rather than suppressing unwanted or no longer relevant memory traces (Manza). In spatial working memory, theta oscillations occur in the medial prefrontal cortex with the ventral hippocampus playing a role in synchronization (O`Neill).

Cogmed training has also been linked to increased attention as well as working memory. In fact, it is likely that Cogmed decreases inattention and increases how much verbal and visuospatial information a subject can temporarily work with (Slezak). What part is effected by training must be ascertained and it could be an increase in efficiency of Posner`s control networks of alerting, orienting or central executive or to the components of orienting (Posner and Petersen) with disengagement (responsibility of the parietal area), shifting and reengagement of focus (responsibility of the What-Where pathway, cortical medial temporal cortex or pulvinar nuclei of thalamus). Another area that exhibits higher efficiency after training is the selection process that retrieves the relevant items from memory (activation in the rostral superior frontal sulcus and posterior cingulate cortex) or the updating process that changes the focus of attention on it (caudal superior frontal sulcus and post parietal cortex). Training could also shift the top-down, bottom-up balance of the control systems (control – stimulus driven and goal directed – Asphland, or top-down and bottom up system – Corbetta and Shulman) with training leading to better control of the top-down attentional systems or by improving working memory biases of attention by initiating the novel parieo-medial-temporal pathway proposed by Soto. The interconnectivity of all these areas can be observed by neuroimaging.

It is also possible that training programs change the balance of task relevant to task irrelevant (or attended to unattended) information, hence improving overall performance this way. It is known that working memory performance is dependent on effectively filtering out irrelevant information through neural suppression (Zonto). The dorsal parietal cortex exhibits influence on top-down attention and ventral parietal cortex on bottom up (Curicella) with prefrontal cortex playing a role (Nieuwenhaus – 5HT and dopamine amplify task relevant information rather than inhibiting distraction), and cingulate cortex (Egner – increase in task relevant information rather than inhibiting task irrelevant). This balance in task relevant information and task irrelevant can be affected by various factors. For example, there appears to be a decreased inhibition of task irrelevant information with age (Blair; Heshier; Redrick); anxiety appears to decrease information with relevance (Weltman), but on a positive note effects can be mitigated by training (Matzell) and computer games (Gofper; Green).

In order to see how the training altered the activation levels at the physiological level, the authors of this paper used different neuroimaging metrics. They found that working memory training brings about effects in water, lipid, and protein content, T2 by iron within the oligodendrocytes and MWF by lipid myelin content and associated the changes observed with training to alterations in these cell components. Other researchers have found that working memory training is associated with variability in white matter (Golestani) and more specifically, increased myelination in white matter neurons in the intraparietal sulcus and anterior body of corpus callosum (Takeichi). Chapman and colleagues also reported changes in blood flow. They found in their MRI studies (arterial spin labeling MRI, functional connectivity, and diffusion tensor imaging) healthy seniors tested pre, mid, and post training (12 weeks) had significant training-related changes in the brain in the resting state. Specifically there were increases in global and regional cerebral blood flow and connectivity particularly in the Default Mode Network and the central executive network and that there was increased white matter integrity in the left brain areas connecting parts of the limbic system in the temporal lobe (eg. hippocampus, amygdala) with parts of the frontal lobes such as the orbitofrontal cortex. They suggested that cognitive training enhanced resting-state neural activity and connectivity and increased the blood flow to certain brain areas, an idea supported by the results given in Caeyenberghs and colleagues paper.

Therefore, it can be summarized that training can induce positive effects on certain types of memory and processing and that these effects are likely to be associated with improved task-relevant information levels, increased attention and increased connectivity between brain areas responsible for attentional systems, visual systems and working memory. However, there is a word of caution in that training of this type does not give an unlimited positive effect, ie. the more you train the more you improve. Even after 8 weeks of training which is a long time and requires a high level of commitment to the training program, the improvement in cognitive performance lies only at less than 25% for healthy individuals. The advantage appears to come when cognitive problems are evident pre-training. It would be interesting to see how the neuroimaging results reported here correlate to training programs undertaken under these conditions.

Since we`re talking about the topic…….

….can we assume that the neuroimaging techniques applied here can be used to demonstrate neural functioning and neural connectivity in other types of memory which have a time element eg. conditioning?  Should connectivity between the areas relating to emotions and decision-making also appear strengthened?

…using this neuroimaging technique would provide interesting comparisons of functioning and neural connectivity if knock-out mice, or transgenic mice, are compared to controls.

….would the consumption of caffeine which increases alertness and consolidation of memory, or other stimulants shortly before each participation in the training program have a noticeable effect on the neuroimaging results as well as performance post-training?

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