theta clock-spiking cells in the hippocampus

Posted comment on ´Theta rhythmic clock-like activity of single units in the mouse hippocampus` by L. Zhang, X. Ma, G. Chen, E. Barkai and L. Lin and published in Journal of Neuroscience 36(16) 2016 p.4415


In their article, Zhang and colleagues describe their finding of a small group of neurons in the mouse hippocampus that exhibit theta oscillations only during waking exploration and REM sleep. The cells were named theta clock-spiking cells and the theta oscillations exhibited by these cells differed to those oscillations normally found in the hippocampus as part of the information processing and memory functions for example.

Zhang and colleagues took 15 C57/6J freely behaving mice and used drivable microelectrode arrays to record the brain wave oscillations. Three 36-pin connector arrays were positioned in parallel and for the recording microdrive two independently movable electrode bundles of 12 tetrodes and 24 stereotrodes were implanted into both sides of the dorsal hippocampi. The connector pin arrays were connected to amplifiers and the extracellular signals were filtered through these amplifiers to separate neuronal activity and local field potentials (LFP).  Spike signals and the LFP signals were filtered on-line at different frequencies and spike waveforms, time stamps, and LFP signals were saved to Plexon data files. Individual neurons were identified and sorted by clustering methods using Offline Sorter version 2.8 software. Interneurons were identified by their greater than 5Hz firing rates and 100-300 μs spikes and interspike intervals (ISIs) were used to further isolate theta clock-spiking cells from the other cells. Oscillations were identified using bandpass filtering with theta oscillations LFP at 4–12 Hz, gamma 30–80 Hz and ripple oscillations 100–250 Hz. Stationary data was selected for further analysis using an augmented Dickey–Fuller test and power spectral density analyses were performed on both spike and LFP signals. Theta phase locking firing was analysed using a Hilbert transform to split into instantaneous amplitude and phase components, followed by spike phase and phase locking calculations. Animal behaviours during the testing process were digitally recorded and positions and firing rate maps constructed. Locomotion velocity and firing rates were also calculated.

Their investigation led Zhang and colleagues to identify a small subset of neurons (5 cells) in the stratum oriens region of the hippocampal CA1. The cells were named theta clock-spiking cells and they were found to only persistently fire during REM sleep and in the theta states of waking exploration. They were silent during slow-wave sleep. The theta oscillations exhibited by these theta clock-spiking cells had a simple clock-like spike firing pattern with one spike per theta cycle. There was a significant difference between the average firing rates of these cells and the peak frequencies of power spectra of corresponding LFP theta neurons and therefore, it was concluded that from the five cells, four bore no relationship to theta LFP. The authors calculated firing rates of the theta clock-spiking cells under different theta states and found that the average firing rate during waking exploration was about 9Hz which was slightly higher than the peak frequency of the power spectrum of the corresponding LFP theta rhythm in the hippocampus at 8Hz. The average firing rate during REM sleep was also found to be different for the 4 cells with the theta clock-spiking cells exhibiting a theta frequency of about 5Hz with other cells with the LFP theta rhythm having a slightly higher frequency of about 7Hz.

Zhang and colleagues also looked at other cells in the hippocampus CA1 area. Out of 508 cells, they found 44 theta locked interneurons, 30 theta unlocked interneurons in the stratum oriens and stratum pyramidale and 425 complex spiking cells in the stratum pyramidale. Therefore, their subset of theta clock-spiking cells was very small. The method of cell separation meant that these cells were together where neurons were sparsely distributed.  The authors also found ripple oscillations with these cells, but these were smaller than for cells of the stratum pyrimadale area and the theta oscillations had a delayed 3–24 ms phase, which indicated that the soma of the theta clock-spiking cells were probably located in the stratum oriens. The cells also showed a different peak interval of ISI distributions to the other theta oscillating cells of the area. A difference was found between the firing rates of the theta clock-spiking cells and the complex firing cells observed during wakeful exploration and also by differences in spike durations between the theta clock-spiking cells and interneurons.

An investigation into gamma oscillations led the authors to the conclusion that the theta clock-spiking cells were not locked to gamma oscillations in REM sleep nor wakeful exploration, or to ripple oscillations in SWS. The cells, although they showed non spatial preference, appeared however to be linked to locomotion velocity.

Therefore, Zhang and colleagues found in their study a very small subset of theta oscillating cells in the hippocampus CA1 which exhibited firing under two conditions; REM sleep and waking exploration. They hypothesized that these theta clock-spiking cells may provide a temporal reference in theta-related temporal coding or decoding of information in the hippocampal area, but unlike the place cells of the area, they did not encode spatial information. Owing to the correlation between the theta clock-spiking cells firing and locomotion velocity the authors hypothesized that there may be link between this small subset of cells and speed.


What makes this article interesting is firstly, the way in which a small population of cells can be detected and secondly, the complexity, both temporally and frequency-related, of brain waves. Accurate detection of small cell populations could lead to more precise attributes of function to brain area and could also lead to experimental and therapeutic methods where manipulation of small populations of cells only could lead to widespread functional and structural effects. Understanding brain waves and their functions in smaller cell populations could provide a means of manipulation, eg. by specific transcranial magnetism, that could result in widespread neuronal effects. Although, the main emphasis in Zhang and colleagues` study was the theta oscillation, their hypothesis could apply to all of the brain wave types.

Unfortunately, one of the problems with Zhang and colleagues experiments was the low sample number of theta clock-spiking cells found – only 5 cells out of 15 mice out of over 500 cells looked at and even one of the 5 was not the theta clock-spiking cell in question. Several reasons can be brought forward to explain such a low number of cells in an area known for its cognitive function. For example, the theta clock-spiking cells could represent anomalous readings. However, this is probably unlikely since theta oscillations were definitely different in the theta clock-spiking cell to the theta oscillations observed with other cells and those differences were observed over different situations, eg REM sleep and waking exploration. Support for such cells also comes from other species since clock spiking cells have also been reported in the optic lobes of insects as early as 1965. The small subset of cells identified by Zhang and colleagues could also represent immature cells or cells not at the same point of their life cycle relative to other theta oscillating cells in the area. This possibility is also unlikely since all theta clock-spiking cells were found in one area only, the stratum oriens, and the cells were not morphologically different to the other hippocampal cells. Another explanation is that the results represent cells active in a common behaviour which is not displayed by this specific mouse strain. This explanation could be considered possible since the hippocampus exhibits neurogenesis and cell function adapts to cope with the animal`s behavioural requirements. If this mouse strain is not very exploratory for example, maybe the number of cells responsible for this function is decreased relative to other mouse strains and therefore, a low number of cells would be observed. Similarly, the results could represent cells active with a very specific function. This is another possible explanation although in this case this specific function must be in low demand in this mouse strain or in the day-to-day life of these mice.

Therefore, since we assume that the small population of theta oscillating cells identified by Zhang and colleagues is functionally and detectably different to the other theta oscillating cells of the hippocampus it is necessary to determine why they are there. Zhang and colleagues investigated whether the firing pattern of these theta  clock-spiking cells could contribute to the hippocampal self-generated theta oscillations in general since several intrinsic, atropine-resistant (ie. not cholinergic cells) theta generators have been found in the CA1 using isolated rat hippocampal preparations. However, since the  author`s experiments showed that the theta clock-spiking cells demonstrated a different frequency of firing rate from the peak frequency of the theta oscillating cells contributing to the local field potential, their involvement in cognitive functioning such as information processing and memory from a content point of view was deemed unlikely. However, Zhang and colleagues did speculate that the activity of the theta clock-spiking cells may provide an overall time reference for the theta phase precession of intracellular membrane potential oscillations in place cells. Therefore, the theta clock spiking cells could play a temporal role. The authors also did find a specific function linked to the small subset of cells since there were correlations between the firing of the cells and the locomotive velocity of the animals during waking exploration. It was concluded then that there could be a link between theta oscillations and speed and this has been reported in other studies as well.

So, how can we explain theta oscillations, locomotive speed and the hippocampus? In this case of being awake and exhibiting exploratory behaviour, there are two sources of inputted information: visual speed (ie. the change in visual information inputted of the environment while the animal is exploring) and ´run` speed (ie. the speed of mouse movement). Previous research has shown that input of both occurs via the V1 visual cortex, with the input and interpretation of the information involving the hippocampus. The hippocampus is known to be strongly correlated to cognitive functions such as memory and spatial navigation, both required in exploratory behaviour and there is a link between sensory input (visual information in visual cortex) and object and location of spatial information in the hippocampal place cells during waking exploration. This link is not only demonstrated at the cellular mechanical level, but also through brain waves. Brain waves represent synchronous firing of cells and the frequency of the brain wave demonstrates the speed of neuronal firing at that time. By measuring the brain waves of one area or between areas, functioning and connectivity can be observed. For example, firing between the thalamus and cortex is activated in a specific temporal sequence and this connectivity can be modulated by inhibiting the inputs from the thalamus reticular nucleus which is GABA dependent. Another example involves the prefrontal cortex which is also important in memory recall and is linked to increased theta oscillations in temporal order maintenance whereas alpha oscillations are required for item maintenance. The hippocampal theta bursts drive the generation of prefrontal cortical theta-gamma dependent hippocampus coupling and firing of the enterorhinal cortex. Theta oscillations are also linked to memory and in the case of waking exploration, the mouse uses its spatial memory for interpretation as well as storage of information for future use. Formation of new memories involving the CA1 neurons occurs with encoding at pyramidal cells preferentially timed later than the theta oscillation peak coincident with input from the enterorhinal cortex and retrieval of memories occurring at the theta oscillatory trough coincident with firing input from the CA3 region. Lesions of the enterorhinal cortex lead to disruption of these theta oscillations and silencing of the CA3 neurons resulting in loss of temporal coding in the CA1. However, the authors demonstrated in their experiments that the theta oscillations observed from their theta clock-spiking cells were different to those of the pyramidal cells and therefore, it is unlikely that these cells are directly responsible for information processing and memory formation of the event. However, it is possible that the theta- clock spiking cells although not directly responsible for the content of the event during the active times of the waking exploration as is the normal function of the theta oscillations, provide instead a ´background pulse` for times of intervals in the exploratory behaviour, ie. akin to a drum beating time. Spontaneous firings of the 4 cells would keep the area in a state of ´readiness` whilst active place cells undergo the biochemical refractory periods necessary during continual firing periods. This is seen with saccades in retinal cells and incoming visual information. Refractory periods of the active visual cells means that priority of event characteristics is shifted to the unattended features and there is temporary inhibition of return so that the cells representing the important event characteristics can biochemically recover ready for the next wave of firing.

This explanation could also apply to the other scenario where theta clock-spiking cells are observed, that is in REM sleep. In this case, the mouse undergoes no exploratory behaviour, but is motionless with no visual input and therefore, functioning of this particular sub-group of cells cannot be contributed to visual speed and run speed, or place cell activity recording object and location. However, just like in the waking exploration scenario, in REM sleep there is predominately another brain wave frequency representing informational content and manipulation. In this case, the frequency of the brain wave activity is beta with interspersed low frequency theta oscillations. The function of the brain waves here is just like in the waking case, to represent synchronous firing of groups of cells, but in the case of REM sleep, the firing is linked to memory formation and consolidation. This function is supported by the observation that REM sleep is disrupted by inducing sleep apnoea and this leads to significant negative effects on spatial navigational memory. Therefore, what function could Zhang and colleagues population of theta clock-spiking cells have in REM sleep? Just like in waking exploration, this subset of cells could be the ´default` cell, providing the ´background pulse`, essentially active when the firing cells representing the event features during this memory formation period reach their refractory periods. In REM sleep, the frequency of the normal oscillatory rhythm for memory formation and consolidation is also beta with spiking theta rhythms. This combination of primary frequency and secondary frequency can also be observed under other circumstances. In NREM stage 2 sleep there are sleep spindles observed with theta oscillating cells as spikes, or in slow wave sleep there are bursts of sleep spindles where new information is being integrated, replay is seen and there is reconsolidation of memories. Therefore, like a temporal marker, the theta clock-spiking cells in REM sleep could be ´innate` markers spontaneously firing to maintain area ´readiness` whilst other cells biochemically recover from firing. This hypothesis is supported by the observation by Bernardi that sleep deprivation, known to be linked to poor memory recall, leads to region specific increases in theta oscillations suggesting that theta oscillations represent transient neuronal states unrelated to event content.

Therefore, this article is interesting because it demonstrates just how complicated neuronal firing is and how monitoring of brain wave functioning has to be carried out on much smaller scales if we are to determine how cell firing is linked to information processing and memory. It could be that the theta clock-spiking cells identified by Zhang and colleagues are just ´artifacts` or a spurious observation of a few hippocampal cells, but they could be the ´default` firing cells of this important area keeping it in ´readiness` whilst other cells biochemically recover. Input and binding of information and interplay between the hippocampus, enterorhinal cortex and cortical areas may focus research attention on the predominant brain waves and cell firing during event characteristic registration, but if the theta clock-spiking cells are linked to the ´default` state of the hippocampus then disruption of their functioning may prevent correct informational input and interpretation overall. For example, Alzheimer disease is linked to hyperexcitability of the hippocampal region and there are currently no hypotheses on how this comes about. It could be that the fault lies not with the neuronal cells involved with informational input, but with cells like the theta clock-spiking cells who are involved in the correcting functioning of the area, but who are not event related. Therefore, investigation of small groups of cells is important.

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

…..can we assume that if the theta clock-spiking cells are linked to a specific activity which is not in much demand during waking exploration, if the mice were trained to  perform a task dependent on mouse speed, then we should see a major increase in number of this subset of cells if their function is truly linked to locomotive speed?

…… since cannabinoids disrupt theta oscillations in the hippocampus and ketamine increases theta oscillations in the medial frontal cortex, if Zhang and colleagues` experiments were performed again with these drugs pre-administered would we see how the theta clock-spiking cells are linked to normal brain wave functioning in these areas?

……can we assume that if the mouse is exposed to anaesthetics and brain waves are monitored we would see more and more of the brain going into slow wave oscillations as normal, but we would see an effect on the theta clock-spiking group of cells?

…….sleep deprivation leads to region specific homeostatic increases in theta oscillatory activity and therefore, would there be an increased number of theta clock-spiking cells if these cells are linked to the ´default` firing state of the hippocampus?

…….is it possible that the GABA agonist, zolpidem, which leads to increased sleep spindles and increased REM would produce noticeable effects on the number of the theta clock-spiking cells?


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neuronal firing patterns with true and false memories

Posted comment on ´Neural Global Pattern Similarity Underlies True and False Memories` by Z. Ye, B. Zhu, L. Zhuang, Z. Lu, C. Chen and G. Xue and published in Journal of Neuroscience 22nd June 2016, 36 (25) 6792-6802; DOI:


Ye and colleagues used as a basis in their investigation of neuronal firing of memories the  computational global matching model that hypothesizes that memory strength of a given item is derived from the match (measured as similarity) between its representation and all other studied items (i.e memories) at that time. They looked at the strengths of true and false memories that arose from global similarity of each item`s neural activation pattern during retrieval to that of the groups during encoding and called it ER-nGPS.

In their experiments, Ye and colleagues used fMRI and the participants were scanned during both the encoding and retrieval phases of the memory task, which was an adapted version of the Deese-Roediger-McDermott (DRM) paradigm. The participants, who were 35 healthy college students, were presented visually with 9 word lists each containing 12 words that related to one particular theme. Eight of the 12 words were part of the group study and the other 4 were used as ´critical lures` (words encoded, but not presented in the recall part of the test). Thirty-six semantically unrelated words were also used as ´foils` (non-studied words) in the recognition test.

For the encoding phase, each word was presented for 3 seconds. Then, the participant was asked to perform a perceptual orientation task for 8 secs to prevent further processing of the recently presented word. They were asked to judge each word as pleasant or not and to give each a value from 1-4 by pressing a button. In the retrieval phase, the participants were first given a 2-back working memory task for 10 minutes as a distraction. Then, they were asked on the presentation of each word (36 studied, 36 critical lures and 36 foils) if they judged the word being presented as being definitely new (given a rating of 1) to definitely old (4). These confidence responses rated memory strength. The similarity of the global neuronal firing patterns between encoding and retrieval of the test items was assessed for all 72 studied items.

The participants were also asked to rate semantic similarity. For each task given, they had to assess pairwise semantic similarity to the tested items. In this case only 8 words and 4 critical lures from each word list were used. Judgement for semantic similarity between the two words was tested by rating using a value of 1 for a very weak semantic association to 6 for a very strong semantic association.

The information obtained from the experiments underwent behavioural and univariate activation analyses. In the behavioural analysis, the differences in the endorsement rates of target, lure, and foil items and associated reaction times in the recognition test were examined.

In the univariate activation analysis, single-item response estimation, neural global pattern similarity between encoding and retrieval (ER-nGPS), ROI analysis, mixed-effects model and mediation analysis were carried out.

Ye and colleagues found that in their behavioural tests there was a mean endorsement rate of 90% for targets, 46% for lures, and 11% for foils showing that the participants exhibited high accuracy for true memories, but also showed a high level of false memories. In the rating of items, the authors found that for the targets and lures that the memory strengths of the studied items related to semantic similarity. Target items exhibited high memory strength and semantic similarity was higher for lures than the targets.

For their fMRI-based results, Ye and colleagues used the calculated ER-nGPSs for the individual items which were the neural activation pattern similarities between each item during its retrieval with all other items during their encoding. They examined whether the ER-nGPS was associated with memory strength and found agreement. There was high memory strength in high ER-nGPS areas such as left inferior frontal gyrus (LIFG), left inferior parietal lobule (LIPL), left superior parietal lobule (LSPL), and left ventral lateral occipital complex (LvLOC). This was confirmed by linear mixed-model analysis where increased true memory strength increased the ER-nGPS observed. However, not all categories produced the same results. Therefore, the authors tested the lures, but not the foils because they showed very low memory strength. In the case of the lures, the ER-nGPS increased with memory strength in the LIFG, LIPL, and the LSPL, but not in the LvLOC or right ventral lateral occipital complex (RvLOC).  Within the word lists similarities produced similar results. Therefore, the authors concluded that the ER-nGPS of the frontoparietal regions was associated with the strength of both true and false memories, whereas the ER-nGPS in the visual cortex was only associated with the strength of a true memory.

Ye and colleagues also investigated the activity in the medial temporal lobe which is associated with memory. In these experiments they used whole brain searchlight analysis and found that the ER-nGPS was not associated with memory strength. Four regions of interest (ROI) were identified and they found that there was only a slight significant difference between a high memory strength item and one of low strength in the left hippocampus only.

In their experiments on sematic similarity, the ER-nGPS reflected the similarity and hence mediated the effect of semantic global ratings (sGS) on memory strength. They found the similarity in the LSPL, partially in the LIPL, but not in the LIFG and therefore, concluded that ER-nGPS is more sensitive to the content of episodic representation rather than univariate activation level.

In their experiments to investigate if ER-nGPS could differentiate between true and false memories, Ye and colleagues looked at the area which exhibited high strength for the lures, ie. the RvLOC.  In this area they found greater activation for targets judged as old than lures judged as old.  Two other areas showed the same results:  the right intracalcarine cortex extending to the right lingual gyrus and a small cluster in the right superior parietal lobe. When the authors looked at activations for correctly rejected lures and foils judged as new, they found strong activation in a large cluster of the left lateral prefrontal cortex (responsible for cognitive control and conflict resolution) and in a small cluster in the medial frontal cortex (responsible for conflict processing). They also found stronger activation for the group of  targets judged as old than that of  foils judged as new in the left lateral prefrontal cortex (LPFC), but found no difference between lures judged as old and lures judged as new,  or between targets judged as old and lures judged as old. Therefore, their experiments recorded strong positive association between the activation of LPFC and ER-nGPS in the LIPL for both true and false (ie. lure) items. This led to the authors concluding that the activation of the LPFC was associated with the discrepancy of the ER-nGPS in the LIPL and the visual cortex. When a mixed-effect model analysis was carried out, a strong positive association between the ER-nGPS difference (LIPL − RvLOC) and the left LPFC activation was found. Hence, it was concluded that the cognitive control process might result from a discrepancy between the ER-nGPS in the parietal and visual cortices.

The univariate activation level experiments in different brain areas also reflected the activation levels determined from the fMRI analyses. The authors used a mixed-effect regression model and found that after controlling for univariate activation levels, ER-nGPSs were still a significant predictor of true memory strength in all ROIs such as the LIFG, LIPL, LSPL, and LvROC and were a significant predictor for the strength of false memory in the LIPL and a marginally significant predictor in the LIFG. They also found using univariate analysis that there was greater activation for true memory than false memory in the left MFG, bilateral IPL, precuneus, and anterior and posterior cingulate cortices. However, these regions did not overlap with those showing differences in ER-nGPS between true and false memories.

In summary, Ye and colleagues showed in their experiments based on global matching computational model that memory strength of a given item depended on how it was encoded during learning and on its similarity of its neural activity pattern with other studied items. They showed multiple ER-nGPSs carried distinct information and contributed differentially to true and false memories. The location of the ER-nGPS was also found to be important. Parietal regions reflected semantic similarity and ER-nGPSs were scaled to the recognition strengths of both true and false memories, ie. to studied and unstudied items whereas activity in the visual cortex areas contributed solely to true memory. The differences between parietal and visual cortices correlated to frontal monitoring processes. Therefore, it was concluded that multiple neural mechanisms underlie memory strengths of events registered in the brain and this area requires further research and discussion.


Ye and colleagues looked at the neuronal activation patterns measured using fMRI that are associated with real, experienced objects (here, words) and correlated these to whether the participant has seen the word before (termed here a ´true` memory), or not (a ´false` memory) and to the degree of similarity the presented word had to others. They interpreted their results using mathematical modelling and statistical analysis. What makes this article interesting is first, that neuronal activation relating to encoding and retrieval is put on a mathematical modelling basis, and second how this type of measurement gives information about the neurochemical mechanisms involved in how objects are learnt and recalled. The experiments show that instead of researchers looking at neuronal activation patterns for single items, they can actually look at activation patterns for whole groups and see the difference when one member of that group is removed. Essentially, this is what is being used to determine where the default mode network of consciousness lies. Consciousness researchers take a neuronal activation picture of a conscious experience and keep removing the activity from the area with the lowest activation until only one area is left. This was said to be the root of the conscious experience. Ye`s experiments also confirm that activation patterns are strengthened by grouping items and that overall activity reflects the source of the stimulation as well as its actual content. This can be said since if only the content is reflected in the neuronal trace then there would be no discrepancy between the patterns achieved between the imagined or false words and the real images observed in past (ie. true).

So, how can the experiments be explained from the perspective of neurochemical mechanisms? The experiments begin with the encoding of the word lists. When the first word is shown to the participant, the visual pathways are stimulated leading to the activation of the sensory stores and then short term memory stores. The brain`s linguistic centres are activated since the word is recognized because all words used were known. Learning is achieved by repetition as the participant is given 3 seconds to commit the word to memory, essentially a reasonable time in learning terms. This results in the neurochemical mechanisms being activated for long term storage. (Long-term storage is assumed because recall takes over 10 minutes later.)

The first word presented can be said to be learnt ´pure`, ie. without processing because there is nothing to relate it to. Even if processing occurs it is likely to be at a low level and of a general nature inspired because of how the participant knows he will be tested in the future, ie. with the association of meaning. Learning is reinforced by the given task of rating the word according to ´pleasantness`. It is known that assigning an emotional value (the ´emotional tag`) to a memory can affect learning and its later retrieval. In Ye and colleagues experiment, learning is also reinforced by the multi-modality of the task, that is the participant sees the word (visual sense) and then must press a button to give the word the emotional value. Hence, different areas representing the visuomotor areas are activated and all added to the overall pattern of firing. The activity of these areas, however, remain the same for all the words, since only the visual information, emotional value and meaning of the word are different.

The presentation of the second word of the word list instigates essentially the same systems as for the first, ie. the same visual pathway, attentional, memory and motor mechanisms. Apart from the individual characteristics of the word, the presentation of the second word differs because of the level of processing carried out by the participant.  It is known that the second word has an apparent link to the first and this link is not visual, but in word meaning. The participant knows that the word belongs to a group and it is likely the word is processed unconsciously since the words used are familiar. This accounts for the speed of whole process. The type of processing carried out is categorization and therefore, the psychologist models of relatedness and schema likely come into play. Accretion probably also applies which is where the addition of a new example to relevant information already in memory (in this case, the previously learnt words) leads to tuning and restructuring if necessary so the schema is more accurate. Inferences could also be used.

It is known that processing and linking to other words results in stronger neuronal cell patterns. The presentation of visual information leads to known patterns of connectivity in neuronal cell firing. Guidotti found that spontaneous brain activity could be evoked by previously presented stimuli. Task evoked patterns to trained stimuli versus novel found patterns in several cortical regions such as the visual cortex, V3, V3A, V7, DFN, precuneus, inferior parietal lobe, dorsal attentional network (intraparietal sulcus which discriminated between trained and novel stimulus).  This agrees with areas demonstrating brain activity in categorization such as the V1, basal ganglia and bilateral intraparietal sulcus as shown by Seger. In Ye and colleagues experiments they found activity in areas such as the LIFG (an area associated with speech comprehension), LIPL (language, sensory motor control of writing – here probably the button pushing), LSPL (spatial orientation, sensory information from the hand – normally writing,  but here again the button pushing) and LvLOC (associated with the visual process). Parietal lobe activity is also associated with attention, the visual perception-action WHERE model which fits in with word recognition and the required motor processes and working memory with the inferior area associated with multi-modality and the lateral inferior being highly sensitive to memory learning recency, but not repetition. According to theories on the neurochemical mechanisms linked with object recognition, activity in the medial temporal lobe is associated with encoding success and so this V5 area is linked to form, sleep, movement, visual perception, and visual working memory. Therefore, Ye and colleagues results of no activity in this particular area was a surprise.

Again repetition aids the learning process of the second presented word and the whole procedure is repeated until all the words in the list are learnt. In the recall part of the test, on presentation of a word the participant asks himself if the word has been seen before and hence, was one learnt in the previous stage. This is an easier test of recall than one of asking the participant to remember each word presented. It is unlikely that a participant could recall all 12 words from the beginning as a list because he would have needed to have employed mnemonic methods in the learning phase and that is not likely considering the time frame of presentation and learning and the distraction task. Instead, the category is sought out and the features that make up that category and the participant has to rate the level of certainty about whether he has seen this word previously, or not. Jang provides a neurochemical explanation for this as it was shown that the  brain encodes experience in an integrative fashion by binding together various features of an event into what was termed an ´event file`. A subsequent reoccurrence of an event feature could then cue the retrieval of the memory file to ´prime` cognition and action. The ´event file` could also include attentional control states, emotional values etc. It was found that areas such as the hippocampus and putamen integrate event features across all these levels in conjunction with other regions representing concrete-feature-selective (primarily visual cortex) and category selective (posterior frontal cortex) and control demand selective (insula, caudate, anterior cingulate, parietal cortex) event information. Hence, according to Jahnke words are learnt as a group and the retrieval of one would mutually generate and support the rest of group. This was seen with sharp-wave ripple complexes (short episodes of increased activity with superimposed high frequency oscillations) occurring during rest and sleep which showed that replay and the SW were tightly interconnected. Such activity was attributed to dendritic sodium spikes found in the hippocampal CA3 and CA1 areas. Recognition of objects is also associated with activity in the perirhinal cortex (Malkora) and Ho showed that this area had a well-established role in familiarity based recognition of individual items. The area responds to novelty and familiarity by increasing or decreasing firing rates. Oscillatory activity occurs in the low beta and low gamma frequency bands in sensory detection, perception and recognition. Stimulation of this area at 30-40HZ causes old items to be treated as novel.

In Ye and colleagues experiments, presentation of each word in the recall part of the experiment requires the participant to make a decision of whether he has seen this word before, or not and perform a motor action. Therefore, neuronal traces also show activity in those areas involved in decision making, ie. the strength of the cortico-striatal pathway and prefrontal cortex (Daw, Chung-Chuan), parietal cortex (guidance of eye movements), basal ganglia, motor structures. This activation, just like those areas representing the visuomotor mechanisms of the button pushing, is the same whether the item has been encoded or not. Therefore, Ye and colleagues could conclude that the only difference between the neuronal traces observed, apart from visual attributes and meaning, was whether the word had been visually seen during encoding or not. Activity in the occipital cortex was observed for words presented during the encoding part of the test (´true` memories), whereas its absence denoted ´false` memories. This expands Fuentemilia`s observations that ´true` memories cause activity in the inferior longitudinal fascile (a major connective pathway of the medial temporal lobe), whereas ´false` memory relates to activity in the superior longitudinal fascile. In this case, the so-called ´false` items relate to visual imagery with the firing of multiple common features including general meaning, but not all are correct. Therefore, the task given is more difficult to get 100% correct and this was proven by the high number of false calls. Brascamp explains this by saying that when an individual knows he is faced with inconclusive or conflicting perception then there is a dominance of whatever perceptual interpretation was commonly reported on a previous encounter. In this case, by asking if a word had been seen before, the brain processed it as relating to the ´meaning` and the likelihood that it had been. Therefore, the word was deemed as being familiar. In order to achieve higher scores, the participant would need to divorce this feeling of familiarity with recognition of the word in a visual capacity only.

In Ye and colleagues experiment, there was no official feedback as to the level of correct or incorrect answers given either instantaneously or at a later date and therefore, with the former, no real-time feedback processing. The participants may have intuitively felt that an error had been made and firing activity would then be visible in the anterior cingulate cortices and amygdala areas. However, again likelihood of activity in these areas would be the same for all words and would enhance the overall neuronal activation pattern rather than be present and specific for either real or imagined word groups.

Therefore, it can be concluded that Ye and colleagues experiments show that neuronal activation relating to encoding and retrieval can be put on a mathematical modelling basis and activation is better if whole groups are considered with the required single object being removed from this group rather than just looking at the activation pattern of the object on its own. This type of measurement shows that retrieval appears to be improved when an object is learnt with its meaning and with others of the same category and not by word structure, or by order. This observation could lead to new learning techniques in the case of reminders for example which are important in prospective memory or for those suffering from forgetfulness.

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

……does emotional attachment to words change global activation patterns? Does the level of anxiety shown by a participant during the test change the activity patterns during encoding and retrieval and does it change the categorization of the words?

…….would instigation of instant feedback during retrieval, eg. giving a positive or negative visual sign change the speed or accuracy of the following replies and change the global activation patterns achieved?

……would a concurrent testing of brain waves within the parietal cortex, perirhinal cortex and hippocampus show the theta, gamma brain wave synchronicity and would these change during the course of test or by presentation of encoded or novel words?

…. would tests with patients with ventral medial prefrontal cortical lesions show that the number of false results is decreased compared to the control group since this brain area is linked to increasing the influence of schematically congruent memories (Warren)?

Posted in recall, Uncategorized | Tagged ,

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).


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.


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


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.


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


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.


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


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.


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 (


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.


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?

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