Place-Specific Memory Engram Cells: Recovery from Place-Specific Retrograde Amnesia

Place-Specific Memory Engram Cells: Recovery from Place-Specific Retrograde Amnesia

Lehigh University
Bios 385 with Julie Haas

Nicholas Gans
12/20/17

Abstract:

Recent studies have made large steps in our understanding of memory formation,
retention, and recall; more work is necessary, however, to elucidate how the specific
information pertaining to a memory is synchronized. Memory engram cells coordinate
memory retrieval upon natural cue reactivation. These cells are susceptible to spine density degradation leaving them in a silent, amnesic state, wherein optogenetic stimulation, but not natural cues, can elicit the encoded memory. Recovery from silent state via PAK1-CA treatment restores the ability to recall a memory from natural cue. Place cells, which encode the organism’s specific location in space may form a subset of different engram cell types. Investigation of the disruption and recovery of location-specific memory may uncover new insights into how spatial memory operates, and potential treatment options for retrograde amnesia.

Introduction:

Understanding mechanics of memory is an extremely important task for helping us
better understand ourselves and the various diseases and disorders that cause amnesia or
dementia. While memory research has been of great interest to neuroscience and
psychology, only recently has there been dramatic progress in our understanding of the
biochemical and neurological processes which underlie the basis for memory formation and recollection.

The concept of memory engrams, the bio-physical representations of memories, can
be credited to Richard Semon dating back to 1911 with the publication of his book Die
Recovery from Place-Specific Retrograde Amnesia 2 Mneme. Recent research has identified evidence for the existence of memory specific cell ensembles (engram cells) in the hippocampus for recalling memories (Josselyn, S., et al., 2015; *Tonegawa, S. et al., 2015). These cells are learning activated and natural cue reactivated units which are necessary to recruit and coordinate the activity of the various brain regions which encode the specific information associated with a given experience. The cue induced re-activation (Tanaka, K., et al., 2014) or optogenetic stimulated activation (Liu, X., et al., 2012) of these cells induces memory associated behavior, a strong indication of cognitive memory recall.

These engram cell, and the memories that they grant access to, are susceptible to disruption. Exposure to the protein synthesis inhibitor anisomycin causes retrograde amnesia, and prevents natural cues from eliciting the memory behavior (** Tonegawa, S., et al., 2015). The engram cell, however, still connected with its downstream partners, exists in a silent state. Optogenetic stimulation of silent engram cells elicits the forgotten learning induced memory (Roy, D., et al., 2017) that natural cues cannot. Further, there exists a way to recover these memories from their amnesic (silent) state back to their normal (active) state. The silent state is accompanied by significantly reduced engram cell specific CA1 spine density. Overexpression of a constitutively active PAK1 (PAK1-CA) brings CA1 spine density back to normal levels and correlates with long term memory impairment recovery at least 8 days after training.

Also located in the hippocampus: place cells are neurons whose activity relates to the organism’s location within a context-specific space (Eichenbaum, H., et al., 1999). The particular location that a group of place cells respond to is a receptive field. The location each cell responds to within a given context remains the same each time the animal re-enters that context. These cells are reactivated bi-directionally during consolidation in sleep. A genetic technique involving fluorescent proteins coupled to a potassium channel was developed in 1977 by Siegel and Isacoff which allowed for imaging of membrane potential. This tool has since been improved and adapted to allow for more precise special and temporal imaging of action potentials (Jin, L., et al., 2012). Recovery from Place-Specific Retrograde Amnesia 3 could theoretically record the action potentials from place cells in real time without use of micro-electrode arrays. This has the advantage of being non-invasive and easily scalable. Simply drive the protein complex in as many or as few cell types that you want. Recent work has furthered our understanding of the dynamics of receptive fields and place cells (Mehta, M., et al., 2000), but more research is required to connect how they are related to memory. This paper addresses this disconnect and examines a potential experiment that could help uncover the role that place cells might play in place-specific memory formation, retention, lose, and recovery.

Experiment:

The proposed experiment aims to study if place cells are a specific type of engram cell or a downstream partner of one, which might tag the spatial valiance of a memory, coordinating the location in space at time of encoding with the other experience-relevant information. This experiment is building off the Roy, D. et al., 2017 paper, Silent Memory Engrams as the Basis for Retrograde Amnesia, by addressing a specific aspect of a memory: one’s location in space associated with such memory. This experiment will test if place cells are recruited and conserved in the processes of memory recall, induced amnesia, and recovery from amnesia.

To conduct this test, researchers would need to generate or obtain c-fos-tTA transgenic mice and cross them with mice containing an RFP tagged voltage-sensitive K+ channel system called ArcLight A242 (see Jin, L., et al., 2012) expressed only in CA1 pyramidal cells. This will provide a target for the AAV9-TRE-ChR2-EYFP virus and will cause fluorescent red light to be immitted during action potentials among the CA1 pyramidal cells using laser detection technology, respectively.

Next, target the DG with the AAV9-TRE-ChR2-EYFP virus and an optical fiber implant targeting this region. It is important that the fluorescent waves emitted by the CA1 cells do not induce stimulation of the DG cells tagged by the ChR2-EYFP complex. The virus will couple to the promoter of c-fos to the tetracycline trans-activator, which is activated in response to doxycycline (Dox). Dox inhibits c-fos-promoter-driven-tTA from binding to its Recovery from Place-Specific Retrograde Amnesia 4 target tetracycline-responsive element (TRE) site, preventing it driving ChR2-EYFP expression. In the absence of Dox, then, training stimulated neuronal activity will selectively label c-fos-expressing DG cells with the ChR2-EYFP which can be stimulated by optical fiber light onset.

Using this paradigm, allow habituation in context A. Then place the mice in a novel location, context B. Record the activity from RFP immitted at all locations within this context taking into account the direction of each mouse’s movement. This will generate a place field map from the corresponding active place cells. Identify each place cell and field associated with the location. Be sure to retest and ensure that the same place fields exist each time the mouse is exposed to the context.

Re-habituate the mice in context A. Next, train mice to fear context B with a foot shock. Observe freezing in response to the foot shock. Ensure that the fear conditioning manipulation worked by waiting 48 hours and placing a mouse back at the location of the foot shock and observe freezing behavior indicative of fear memory retrieval. Confirm the fear memory can be optogenetically stimulated via engram cells active during fear conditioning by optogenetically stimulating a mouse in context A and observing fear behavior.

Given genetic and behavioral manipulations are confirmed, rodents will have engram cells corresponding to their fear memory of the shock in context B tagged by ChR2-EYFP and specific place cells that light up corresponding to the specific place field of the location of the shock which light up in response to re-exposure to the shock location (indicating memory recall). Test to see if optogenetic stimulation of the engram cells elicits a RFP response from the mice. If so, this would suggest that the fear memory being stimulated optogenetically is recruiting the place cells associated with the location of the shock. Be sure that the RFP signal is originating from the place cells associated with the location of the shock and not the place cells associated with the mouse’s current location.

If these tests are successful, then they provide evidence that location in space is a specific type of information activated by engram cells such that a unique memory causes the Recovery from Place-Specific Retrograde Amnesia 5 firing of the place cell(s) associated with it. Next, researchers could confirm previous findings that exposure to a protein synthesis inhibitor after training blocks the memory from being recalled by natural cues by placing mice treated with anisomycin back in context B and not observing fear response (as the engram cells are in the silent state). However, if consistent with previous results, fear response would still be observed in anisomycin treated
animals after optogenetic stimulation.

Provided anisomycin causes retrograde amnesia, and optogenetic stimulation (but not cue) induces memory recall indicative behavior, we should also be able to convert the engram cells back into their active state by introducing constitutively active PAK1 (PAK1-CA). Overexpression of PAK1-CA should allow for the spine density to recover to normal levels and return the cells back to an active state. To test is PAK1-CA treatment allowed the memory to be recovered from retrograde amnesia, place mice back into context B and observe the return of freezing behavior. Given freezing behavior is returned when animals are exposed to natural cues, we can conclude that the memory itself is likely being recalled once again. Next researchers could test to see if the specific location assigned to the context was unaffected by these tests by looking at the RFP light patterns when placing the animal back in context B.

Discussion:

Memory engram cells act to initiate memory formation during an experience. A sufficient sensory or conceptual cue can then reactivate the memory through activation of the pertinent engram cells. Spatial context is a common natural cue used to elicit the recall of a memory, and behavior is a common measurement of this recall. The experiment discussed attempts to determine the relationship between a place cell which fire at a particular location and the recall of a memory.

Although it is directly building upon a previous paper, this experiment requires exhaustive resources and time, and has the potential to be stopped early at several steps. Some unexpected results that should be considered are: the distortion, silencing, or elimination of the place cell’s ability to fire following protein synthesis inhibition. While this recovery from Place-Specific Retrograde Amnesia 6 would change the subsequent experiments I explore, it might suggest that place cells are a type of engram cell. This finding might also help us better direct further investigations into how spatial cues for memory recall function. Following this trail of logic, a follow up test could look at if optogenetic stimulation of a place cell, or all of the place cells that make up a place field, can induce memory indicative behavior.

If, however place cells are not altered by protein synthesis inhibition in the same way that the engram cells have been shown to be, and the experimental design of combining fluorescent imaging with optogenetic stimulation works, then this design could be of great use to study many other brain regions and cognitive processes. Using these techniques in tandem, one could study the effects on specific down stream partners from any neuron by
optogenetic stimulation in culture, thus eliminating surrounding noise and isolating the individual extent of a neurons downstream influence.

Other alternatives to using an RFP action potential indicator system include using micro electrode arrays, calcium imaging, or receptor expression as measurements of activation. These techniques have a larger body of literature behind them, and are more common in many electro-physiology labs. Another alternative protocol that could be
explored is using a different form of natural cue. By using a paired tone to shock design instead of context specific recall there might be more simplistic conclusions to be made about the activation of a place cell during a memory.

Overall this experiment addresses only one aspect of a memory, its spatial valiance. Much work will need to be conducted to form a representative picture of how engram cells work to integrate different brain regions associated with a given memory. Understanding these components of memory integration may help us to develop treatments for memory loss and neurodegenerative diseases. Overexpression of PAK1-CA would not be a viable method for recovering a memory in humans, but perhaps a prevention from engram cells entering the silent state too often could be discovered.

References:

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**Tonegawa, Susumu, et al. "Memory engram storage and retrieval." Current opinion
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Disclaimer: This was used as an assignment at Lehigh University in a Synapses and Plasticity Course. Please let me know if there are any major typos, errors, formatting problems, or other issues. Thanks!