blocking hippocampal AMPAR removal prevents forgetting

Posted comment on ´Blocking Synaptic Removal of GluA2-Containing AMPA Receptors Prevents the Natural Forgetting of Long-Term Memories` by P.V. Migues, L. Liu, G.E.B. Archbold, E.O. Einarsson, J. Wong, K. Bonasia, S.H. Ko, Y.T. Wang and O. Hardt and published in Journal of Neuroscience 36(12) p. 3481 doi.org/10.1523/JNEUROSCI.3333-15.2016

SUMMARY

Migues and colleagues investigated whether blocking the removal of GluA2-containing AMPA receptors (GluA2/AMPAR) of neuronal cell membranes prevented the loss of long term memories. They looked at two areas in particular: the rat dorsal hippocampus and infralimbic cortex and used two peptides, GluA23Y (control GluA23A ) and G2CT fused to specific peptide sequences for cell permeability, to introduce interference with the receptor endocytotic process.

Long Evans rats were used in most of Migues and colleagues memory experiments and these underwent bilateral cannula placement using the standard reference atlas. The rats were infused with either control substance or the interference peptides, GluA23Y or G2CT. Habituation to the experimental conditions was followed by specific sets of experiments. For the object location recognition memory test, the rats were subjected to 7 times 2 copies of the same object. After a probe with 1 object removed, Migues and colleagues carried out their experiments. The rats were tested for object location memory and were scored according to the time they spent carrying out exploratory behaviour of the novel and familiar objects. An interval of 14 days was introduced between the learning phase and the recall/testing phase. In the case of the appetitive conditioned place preference (CPP) test, 2 compartments were used with Fruit Loops as reward. Performance was assessed by measuring the time the rats spent in each compartment, hence preference for one or the other was recorded. Contextual fear conditioning was carried out using vanilla and peppermint oils and electric foot shocks and performance was measured according to the amount of freezing (blocks of 30 seconds) exhibited.  The auditory fear conditioning experiments were carried out in a similar manner to the contextual fear experiments except tones (3 tones at 5HZ) were used. For the electrophysiological studies, Sprague Dowley rats were used instead of the Long Evans rats. Long term potentiation (LTP) was induced in whole hippocampal CA1 cells by applying 200 pulses of repetition stimulus and voltage clamping cells at -5mV whereas cells were exposed to 300 pulses at – 45mV for depotentiation. Results were assessed using one way ANOVA for EPSC amplitude at 5 mins for basal transmission, 20 mins for LTP and 60 mins for depotentiation.

Migues and colleagues found in their object location recognition memory tests that control rats were more likely to explore the novel location if they still had the memory for the old object. They found that this preference for the old object decreased with time until 7 days after learning and concluded that no long term memories existed after 8 days. Therefore, control rats and rats that had received the infusion of the interference peptide, GluA23Y , were tested 14 days after training. Migues and colleagues found that rats where the dorsal hippocampal GluA2/AMPAR removal had been blocked with the interference peptide GluA23Y  preferentially explored old objects at new locations. Hence, the normal time-associated forgetting of established, long-term spatial memories relating to object location had been prevented by the administration of the AMPAR removal blocker. Repetition of the experiments at 7 days after learning produced the same results. When the peptide G2CT which binds to a different site in the removal process was used instead, the authors again detected a significant preference for exploration of the old object moved to the new location. Therefore, blocking the removal of dorsal hippocampal GluA2/AMPARs with interference peptides GluA23Y or G2CT during an interval after learning did not affect acquisition of new object locations in a spatial memory test, but did prevent loss of established memories.

Blocking the removal of dorsal hippocampal GluA2/AMPARs with interference peptides GluA23Y or G2CT also prevented loss of associative memories of food-reward in the authors` conditioned place preference (CPP) test. The use of the AMPAR antagonist, CNQX, showed that AMPAR activation was linked to conditioning in the hippocampus. After 10 days no conditioned response was visible under normal conditions, but with infused GluA23Y the authors found that the CPP forgetting had been blocked. This was demonstrated by the rats showing preference for the side that had previously been supplied with food.

The authors also investigated if dorsal hippocampal GluA2/AMPAR removal was involved in behavioural changes observed with time. In auditory fear conditioning experiments, Migues and colleagues found after 2 weeks fear expression was the same. When they infused GluA23Y into the dorsal hippocampus during the 2 week retention interval, they found the rats exhibited generalization of contextual fear expression ie.  the loss of context discrimination had been blocked. Rats infused with the control substance showed the same contextual fear levels. Infusion of GluA23Y into the infralimbic cortex area instead of the hippocampus after extinction of auditory fear prevented the spontaneous recovery of the fear memory conditioned response after the extinction period.

Migues and colleagues also investigated possible physiological mechanisms of memory decay using hippocampal slices and the electrophysiological patch clamp technique. EPSCs were evoked in CA1 cells and LTP and then depotentiation were induced. The authors found that bath application of GluA23Y prevented depotentiation, but not the induction of LTP or basal transmission. Application of the control substance, GluA23A, did not prevent depotentiation.

The authors concluded following their experiments that removal of GluA2/AMPARs erased consolidated long-term memories in the hippocampus and other brain structures over time and this contributes to the displayed behaviour. Blocking the activity dependent removal process using infusion of the peptides GluA23Y and G2CT that interfere with the internalization process of AMPARs prevented the time-associated loss of memory. This suggested to them that dysregulation of the AMPAR removal process could hinder the time-associated decline of spatial memory and improve cognition in this area.

COMMENT

This article is interesting because it links memory retrieval with hippocampal AMPAR population. It is known that hippocampal AMPARs are required for memory formation of spatial memories and conditioning memories and according to Migues and colleagues, forgetting of this type of information is linked to the removal of these AMPARs in a time-dependent manner. By maintaining AMPAR number in this region then loss of memories of these memory types is prevented, ie. information learnt at a previous time can still be recalled. Information learnt in these cases relates to the memories of object and place for spatial memory and for conditioning an extension of this by adding a temporal element followed by additional information relating to the reward (or fear). Forgetting spatial memory and the extinction of conditioning occurred naturally in Migues and colleagues experiments with no information retained 14 days after learning had stopped. This was demonstrated by the exploration of the old object in the new location for the spatial memory task being greater than the old object in its old location. Administration of an AMPAR removal blocker led to equal exploration of the old object in both new and old locations. Therefore, it was concluded that blocking the removal of the hippocampal receptors meant that long-term spatial and conditioning memories had been maintained.

The question is how does this occur. We know that AMPARs are linked to spatial or sequence memory (object and ´reward` as in conditioning) and are required for information input, processing and memory formation. Their capability is linked to the physiology of the hippocampal region itself. In the case of spatial memory two pieces of information, ie. place and object stimulate activity in specialized hippocampal neurons called place cells (work by McNaughton, O´Keefe), and these cells exist alongside neurons representing the object information, location and timing. Firing of the neurons leads to AMPAR activity as expected in normal neuronal firing mechanisms. Information coming in fires neurons in the entorhinal cortex area (EC2) leading to firing of the dentate gyrus (DG) followed by the CA3 region, or straight from the EC2 to the CA3 region. DG to CA3 firing relies on the activation of large mossy fibres and glutamate release, but more important in terms of AMPAR is the firing of the small terminals and filopodial cells. This firing results in activation of GABA interneurons with GABA neurons providing the most information in this region. These neurons produce short term responses to repetitive stimulation leading to glutamate release which causes the post-synaptic AMPAR activation reported in the literature. Ca2+ permeable or impermeable AMPAR leads to NMDAR independent or dependent LTP and AMPAR downregulation (work by Nicoll and colleagues). This will have an influence on information input and processing of future events. My own view is that stimulation of the CA3 leads to neurogenesis and glutamate receptors formation leading to LTP in newly formed cells which then switch to LTD (or the area switches to cells favouring LTD) and finally endocytosis of the receptors and cell apoptosis. This is supported by reports that as far as the CA3 to CA1 network is concerned there are multiple synapses;  in the CA1 both LTP and LTD are demonstrated; and the areas show differences in processing new and old information. New information is given priority and causes the effect with repetition leading to memories formed higher up the brain area hierarchy based on the priority given at this stage. The area and neuronal cells then switch into LTD mode once these long-term memories are established higher up in the appropriate cortical areas.

The LTP observed in the CA1 region does not reflect memory storage in the region itself as some proponents think, but instead is likely to act more like a signal and the area then as a relay station. A glutamate receptor link to memory function is well known here demonstrated by gene targeting mice leading to defective glutamate receptor formation demonstrating impaired maze learning. More specifically, the associated LTP is linked to the insertion of AMPAR (work by Derkath, Plant, Ehninger, Rauer for example) resulting in increased synaptic strength and increased susceptibility of cells to depolarization. Plant reported that calcium permeable AMPAR inserted first are without the Glu2R subunit which appears later which could explain the time difference using the Glu2R subunit specific peptide. Increased synthesis of AMPAR occurs via increased synthesis of both GluA1 and GluA2 subunits by 4E-PB2 deletion and AMPAR trafficking to the cell membrane requires, for example, the kinesin-kinesin superfamily 16B (ILIF 16R) for the microtubules; palmitoylation of AMPAR linked scaffold protein kinase A anchoring protein (AKAP) 79/150, RAb 11 and other effectors as well as  A2R, PKA and actin polymerization (work by Zhu – stimulation and trafficking). PIP3 turnover is required at the synapse to maintain the clustering of AMPAR  into what is thought to be nano-domains and this is assisted by MAGUK proteins. Calcium ion entry is linked to the membrane situated AMPAR and the calcium ion channel is reported as a L-type channel which leads to slow after hyperpolarization (Kim). These

calcium-permeable AMPA receptors (CP-AMPARs) contribute to the synaptic plasticity of the area (McGee). The capability of the CP-AMPAR lies in its structure with the prototypical transmembrane AMPAR regulatory protein stargazin, which acts as an auxiliary subunit, enhancing the receptor function by increasing single-channel conductance, slowing channel gating, increasing calcium permeability, and relieving the voltage-dependent block by endogenous intracellular polyamines. The overall effect of the glutamate release on cell activation is that the AMPAR trafficking and membrane insertion results in the cell being more sensitive to the firing stimulus (LTP).

However, the initial LTP observed in the CA1 and CA3 is replaced by LTD which is where the active synapses decrease in firing effectiveness. LTD is normally linked to sequence memory in cerebellum, but it is also linked to changes in memory and learning associated with hippocampus functioning in a mechanism similar to that followed in the cerebellum system. In the cerebellum, LTD arises when 3 intracellular signals occur at same time: a rise in internal calcium ion concentration due to climbing fibre activation, a rise in internal sodium ion concentration due to AMPAR activation and activation of protein kinase c due to metabotropic glutamate R activation. These signals result in a decrease in AMPAR, and hence a decrease in the opening of postsynaptic AMPAR channels. Similar results are obtained with the hippocampus. Riazo and colleagues showed that inflammation in other parts of the body leads to cognitive impairment and this was attributed to induced changes in the hippocampus synaptic transmission. Altered postsynaptic effects were observed. Increased AMPAR and NMDAR currents were demonstrated with increased mGluR2 receptors and decreased LTD and LTP. In other experiments LTP/LTD changes were observed in AD mouse models which may reflect defects in the neuronal plasticity processes. Experimenters found that in young 1 month old Tg mice, LTP is enhanced at the expense of LTD, but in 6 month old adults the phenotype is reversed to promote LTD and reduce LTP. These observations were attributed to altered AMPAR phosphorylation and the appearance of calcium ion permeable AMPAR.

In the CA1, LTD and decreased cellular PSD95 levels are observed and in my view is part of the mechanism to ´switch off` the CA1 neurons after stimulus firing and memory formation. Such cells are then culled (or more susceptible to culling) by the microglia in order to maintain the excitability of the area. The process is linked to the level of internal calcium. Increases are due to modest NMDAR activation and prolonged increases in calcium ion concentration ie. due to prolonged stimulation lead to activation of protein phosphatases resulting in AMPAR dephosphorylation and the induction of LTD. The post synaptic AMPARs are internalized at the synapse leading to decreased PSD95. This view is not totally supported with Babiec suggesting that LTD induction is instead attributed to an ion channel-independent, metabotropic form of NMDAR signaling. It was found that the induction of LTD in the adult hippocampus is highly sensitive to extracellular calcium ion levels and that MK-801 blocks NMDAR-dependent LTD in the hippocampus of both adult and immature mice. In addition,  MK-801 inhibits NMDAR-mediated activation of p38-MAPK and dephosphorylation of AMPAR GluA1 subunits at sites implicated in LTD. Therefore, these results indicate that the induction of LTD in the hippocampal CA1 region is instead dependent on ionotropic, rather than metabotropic, NMDAR signalling. These differences may be attributed to the amount of stimulation occurring at the time since differences in receptor internalisation appear to exist. Glebov showed that rapid forms of internalisation of AMPAR  during LTD require clathrin and dynamin, whereas they were not required in slow homeostatic forms of synaptic plasticity. In this case AMPAR trafficking is blocked by a Rac1 inhibitor and is regulated by a dynamic nonstructural pool of F-actin. In the experiments conducted by Migues and colleagues, receptor removal is prevented by the administration of either GluA23Y or G2CT interference peptides, hence the cell remains extra sensitive to the stimulus. When forgetting occurs, memories are still ´there`, but cannot be accessed and this has been proven with experiments carried out with genetically modified genes responsive to blue light. The memory is forgotten, but as soon as the cell is shone with blue light then the conditioned fear returns.

Therefore, the hippocampus AMPARs are responsible for information input and processing in initial memory formation and this is attributed to the areas unique structure, functioning and connectivity.  Information is ferried from the EC to the hippocampus to the fornix to the mammillary bodies to the anterior thalamus. Beta brain wave synchronisation of the lateral EC and hippocampus is required for initial information encoding and beta wave synchronization of the LEC and mPFC areas for remote memory recall (work by Vetere). It was found that dendritic plateau potentials were produced by an interaction between properly timed input from the EC and hippocampal CA3 region. These conjunctive signals positively modulate the firing of previously established place fields and rapidly induce new place field formation to produce feature selectivity in the CA1 which is a function of both entorhinal cortex and CA3 input. Bittner says that such selectivity could allow mixed network level representations that support context-dependent spatial maps. This functioning is susceptible to age differences. The appearance of different field maps in the CA1 of hippocampus means that older rats ´remap` their environment each time they are exposed to it and there is a reliance on self-motion whereas younger rats use previously formed models and rely on visual stimuli.

This experiment gives an indication of how AMPARs influence memory recall. Memory recall is linked to hippocampal functioning, but not with memory storage since hippocampal lesions leads to the loss of new information, but retention of familiar environments (Winucor). This supports the views of Pinel who stated that the hippocampus is involved in the consolidation of long-term memory for spatial location, but not in its storage. Although storage of the memories occurs at the higher cortical areas, the hippocampus is still required in memory retrieval. It has been found that the both the medial prefrontal cortex (mPFC) and the hippocampus are required for the retrieval of spatial memory since, for example the blockade of 5HT2Rs in the mPFC affects the retrieval of the object in context memory, but not in a single object recognition task. Connectivity between the mPFC and the hippocampus is required during the retrieval task (Beckenstein). In Migues and colleagues experiments object recognition is part of the spatial memory and conditioning tasks and this requires appropriate functioning of the hippocampus and the involvement of AMPARs. This was proven by the observation that inactivation of the dorsal hippocampus after training impairs object-place recognition and memory, but enhances novel object recognition. Repeated exposure is not affected by the inactivation of the dorsal hippocampus (work by Olioveirol and colleagues) because long-term memories have been formed elsewhere and retrieval requires activation of these areas. Haetting refined the observation by showing that inactivation of the dorsal hippocampus with muscimol prior to retrieval had no effect on long-term memory in object recognition experiments, but completely blocked the long-term memory for object location demonstrating that place cells and cells stimulated by object features were not involved, but the cells involved in location were. Memory consolidation and reconsolidation is a constantly evolving process with remapping occurring on each reencounter as seen in Migues and colleagues experiments where the same object is constantly being moved to new locations. For such consolidation and reconsolidation processes protein synthesis is required and NMDAR activity mediates trafficking of AMPARs taking place during the recall process (Lopez).

The continuing matching of new information to reactivated old information requires working memory which requires hippocampal activity. Maintenance of multiple items are associated with hippocampus activation (hippocampus-dependent working memory) while maintenance of individual items induces deactivation. Processing leads to long term memory if the hippocampal activity patterns match those previously observed (Fixmacher) and the link to AMPARs comes through the observation that NMDAR antagonists decrease working memory (Takadi) and the level of task irrelevant information is affected by NMDA antagonists (Gage). In the experiments carried out by Migues and colleagues there is a certain level of informational overlap in the form of new location/old location, but the object information is the same. It is believed that the DG plays a role here with pattern separation linked to neurogenesis (Deng). The DG role is at a maximum when there is maximum similarity between object and place pairs and minimal when there is little overlap (Lee). This supports earlier experiments where the hippocampus is known to play a role in familiarity experiments as well as recollection (remember/know) (work by Song, Jeneson, and Smith for example). Here the hippocampus plays a comparator role capable of individualizing representations of overlapping inputs (Zeamer).

So far we have discussed only the hippocampal AMPAR role in spatial memory tasks, but it is clear from Migues and colleagues experiments that they are also involved in fear conditioning. Fear memories are normally linked to amygdala functioning, but the hippocampus also plays a role since there is for each conditioning event information input and processing. There is continual analysis of sensory input to learnt material and in the case of reward, if none is presented there is no reinforcement of the memory and extinction occurs. Migues and colleagues experiments show that extinction of the conditioned response involves AMPAR removal since once it was blocked with the AMPAR removal interference peptide then the fear memory was retained. This means that post-synaptic AMPAR population was maintained and played a role in retrieval of the fear memory.This is against the established view since normally fear conditioning (consolidation, but not acquisition – Liu) is linked to CA1 activity and regulation of NMDAR  receptor number. It is reported that in this case, NMDARs are induced and LTD requires activation of caspase 3 by cytochrome c released from mitochondria in a process promoted by BAX. (BAX induces cell death by apoptosis normally, but does not play this role in LTD.)  Liu showed that in fear extinction a new memory is actually formed rather than erasing the original fear memory. Exposure to novelty (environment) facilitates the transfer of short-term extinction memory to long-lasting memory, but the mechanisms are to date not understood. Therefore, the established view that extinction of fear memory occurs through NMDAR down regulation is extended by Migues and colleagues experiments which show that down regulation of AMPARs also plays a significant role and if the endocytosis of the AMPAR is prevented then extinction cannot occur.

Therefore, Migues and colleagues experiments confirm what we already know about role of glutamate receptors in synaptic plasticity and their role in spatial memory and conditioning. However, it also demonstrates the critical role of the AMPAR aside from its function in LTP and memory formation. Migues and colleagues experiments show that time-related forgetting associated with down-regulation of this type of receptor can be prevented by blocking their removal from the post-synaptic membrane. However, it is unlikely that control of memory loss is focused completely on the removal of this one type of receptor. It is possible that they play a role in the initial stages and this may be important if we consider the case of Alzheimer illness. In Alzheimer illness there is reported hyperexcitability of the EC and hippocampal areas which should theoretically be linked to AMPAR up-regulation and PSD95 increase representing increased synaptic sensitivity. However, Alzheimer illness is linked at later stages with loss of memory which suggests a lower level of AMPARs supporting Migues experiments and therefore, the situation may not be clear cut. AMPAR down regulation in the early stages of the disease (ie. before the symptoms show) may provide an answer. Administration of AMPAR endocytosis blocker may if practically possible provide a beneficial short term effect only. The other conclusion from Migues and colleagues experiments is that the mechanisms for object vs spatial memory are probably slightly different. Old object/ old location vs old object/new location place cells in rat may have different control mechanisms to the human where visual information, key and location are not olfactory linked. Promotion of one type may aid the memory of the other. This may provide another mechanism by which forgetting may be prevented.

Since we`re talking about the topic…..

…..brain waves such as delta, gamma and beta are reported as showing temporal coordination, therefore can we assume that the use of the infused interference peptides would produce a measurable change in brain wave firing if observed during Migues and colleagues experiments?

 

…. LTD is found to be sensitive to calcium extracellular MK801 which blocks NMDAR  mediated p38 MAPK and dephosphorylation of the AMPAR subunit Glu A1. Therefore, if MK801 is administered would the same results be seen as with the interference peptides?

……can we assume that the action of tetrahydrocannabinoids which increase spatial memory by altering dopaminergic pathway activity in the PFC (Makele) would increase spatial learning, but have no effect on spatial memory forgetting?

…..since the stimulation of the perirhinal cortex at 10-15Hz causes animals to treat a novel image as familiar (observed by decreasing the time spent looking at an image – Ho), would using this type of stimulation have any effect on the experiments carried out here?

 

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