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School of Biochemistry and Immunology and Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin, IrelandFlorey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, University of Melbourne, Parkville, Victoria, AustraliaChild & Brain Development Program, Canadian Institute for Advanced Research (CIFAR), Toronto, Ontario, Canada
Memory, defined as the storage and use of learned information in the brain, is necessary to modulate behavior and critical for animals to adapt to their environments and survive. Despite being a cornerstone of brain function, questions surrounding the molecular and cellular mechanisms of how information is encoded, stored, and recalled remain largely unanswered. One widely held theory is that an engram is formed by a group of neurons that are active during learning, which undergoes biochemical and physical changes to store information in a stable state, and that are later reactivated during recall of the memory. In the past decade, the development of engram labeling methodologies has proven useful to investigate the biology of memory at the molecular and cellular levels. Engram technology allows the study of individual memories associated with particular experiences and their evolution over time, with enough experimental resolution to discriminate between different memory processes: learning (encoding), consolidation (the passage from short-term to long-term memories), and storage (the maintenance of memory in the brain). Here, we review the current understanding of memory formation at a molecular and cellular level by focusing on insights provided using engram technology.
). In short, memory involves four different phenomena: encoding, consolidation, storage, and recall. Encoding is the process by which information reaching the brain through perception is written in the brain. Consolidation allows information to be selected and made stable for long-term periods. The stable storage of memory involves permanent modifications to retain the information, and recall is the process that enables the reactivation of the pertinent information upon specific and precise cues to allow the modification of behavior (
) proposed the idea of the “engram” and defined it as “the enduring though primary latent modification in the irritable substance produced by a stimulus (from an experience).” An engram, sometimes understood as a synonym for memory trace, is formed by a group of neurons that (1) become activated by a specific learning experience, (2) are modified by this experience, and (3) are reactivated by re-exposure to the same experience, inducing a change in the behavior of the animal (
). Engram cells, therefore, are at least a part of the physical place or substrate where learning leaves imprints in the brain. Sets of engram cells can be found sparse in many areas of the brain, forming an engrome, or engram complex (
To understand memory, it is necessary, but not sufficient, to describe the biological mechanisms that enable memory formation, maintenance, and expression. As well as explaining the processes required for memory, we must also explain how specific memories are formed as discrete engrams. In other words, we need to understand how specific pieces of information translate into the brain in a way that allows the animal to manage separate memories associated with concrete events. This question of how mental representations are organized in the brain is a long-standing problem. Descartes (1641) (
), the mechanism of inheritance was one of the most fascinating mysteries of biology. How is a single cell, or an embryo, able to carry the astonishingly complex information package to drive the formation of a full organism? Where in that cell is all that information stored and what is the mechanism that translates it into cells, tissues, and organs?
We understand now how biological information is precisely organized in living organisms. After the progress of Gregor Mendel (
). The golden era of molecular genetics had finally described how biological information was written and read in living organisms. Arguably, the neurobiology of the memory field is still waiting for its golden era.
Neuroscience and experimental psychology have focused on the biology of learning and memory acquisition and retention (
). Whilst we know a great deal about plasticity mechanisms required for learned behavior in general (which will be reviewed later), we are still far from identifying the “double helix” of memory—if one even exists. We do not have a clear idea of how long-term, specific information may be stored in the brain, into separate engrams that can be reactivated when relevant. Understanding engram organization would be the equivalent to understanding how genes are organized in the genome.
General considerations for the study of the neurobiology of memory
Taking a step back and looking at the whole picture of the neurobiology of memory can help to identify the next scientific questions. Some general considerations must first be clarified.
Information specificity: A goal in mind
Within a biological system, memory is a form of information. As discussed previously, the molecular and cellular mechanisms that underly memory function must ultimately explain how the information is specific to one experience and not others. Engram technology allows the study of how specific memories translate into neuronal changes.
Time: For now, forever
When considering how memory functions from a psychological point of view, it is crucial to consider the timescale of these processes. Any change or group of changes, whether functional or structural, that are responsible for memory should be fast enough so that learning is immediate (e.g., an unpleasant experience will immediately create aversion) and potentially permanent (this negative association will persist over time) (
). Moreover, learning is a constant process that does not cease. Therefore, the mechanisms that allow it must be constantly active and ready to function.
What memory mechanism exactly?: Call it by its name
An additional consideration in interpreting molecular studies of memory is the necessity to distinguish between the different mechanisms involved in memory function. We must clearly differentiate between the mechanisms of encoding, (re)consolidation, storage, recall, and forgetting when considering a particular molecular pathway. The lack of behavioral manifestation of a particular memory in an individual, such as an animal model, does not allow differentiation between encoding, consolidation, storage, or recall deficits (
). This inherent challenge needs to be considered when investigating the precise role of molecular pathways and mechanisms involved in each of these processes. This caveat has been partly overcome by the development of engram technology.
In the past decade, the development of memory engram technology opened the door to identify the engram cells for a given experience, manipulate them, and study the biochemical changes that underlie engram formation and therefore, memory function.
Development of the technology
Memory engram technology involves the combination of several techniques: transgenic manipulation, optogenetics or chemogenetics, electrophysiology, and behavioral techniques (Fig. 1). It is based on the use of endogenous markers of neuronal activity, immediate early genes (IEGs) such as c-fos or mammalian activity-regulated cytoskeleton-associated protein (Arc), as drivers to target and manipulate neurons that respond to an experience—putative engram neurons. The first use of engram technology engineered viral vectors that hijacked the promoter of the IEG c-fos to drive the expression of both a fluorescent reporter (GFP) and a light-sensitive channel, channelrhodopsin-2 (ChR2) in the mouse hippocampus (
) allows the labeling window to be reduced to target only cells that are active during a particular event, this is, the engram cells of a specific episodic memory. Finally, to evaluate memory function, mice were trained in a contextual fear conditioning paradigm, where animals learn to associate a particular contextual environment with an electric footshock. Memory reactivation was later measured by assessing the animal fear response of freezing in the same training context. Since engram cells become tagged with ChR2, they can be later artificially reactivated by light delivery resulting in memory recall. The mice, therefore, froze in a neutral context upon reactivation of the engram associated with a fearful experience (
). These studies described that optogenetic inhibition via inhibitory opsins prevented the elicitation of the fearful response—when silencing the activation of the engram, animals did not freeze to the context associated with a footshock. Engrams were identified and labeled in several brain areas, such as the amygdala (
) created a false association between a neutral context and a fearful experience. Engram technology was used to label and later artificially activate an engram for a neutral context while the animal was given a footshock. The simultaneous activation of these two engrams, one by optogenetics and one by natural contextual cues, created a false association, an artificial memory. Despite never being shocked in context A, animals were now freezing in that context—and crucially, not in an unmanipulated third context (
). In short, they are neurons that fire when an animal occupies a particular given location within a context. Its discovery initiated a revolution in the field and changed how the hippocampus was conceived (
). This is, engram cells encode experience instead of place. An engram is formed by a subset of the place cells firing in the context (if the memory carries a contextual component), but it is also formed in the absence of place cells firing if the memory task does not include a spatial component (
). This theory posits that the hippocampus function as a spatial framework that links and associates the items that constitute the experience and its location on time. Following this view, engrams would act as indices that would hold the information of what parts of the memory were involved in an event (
Numerous advances have been made on the technology in the last decade (Fig. 2), and a versatile and sophisticated set of tools is now available to target and study neurons activated by a given experience. Chemogenetic tools such as designer receptors exclusively activated by designer drugs have been extensively used as an alternative to optogenetics to induce both neuronal activation or neuronal inhibition in engram cells (
). In such system, a fusion protein containing a mutated estrogen receptor and Cre recombinase (Cre-ERT2) is sequestered in the cytoplasm until tamoxifen presence induces its translocation to the nuclei leading to recombination of the desired transgene. Viral vectors can be entirely or partially substituted by the use of transgenic mice (
), the IEG c-fos promoter controls the expression of a tamoxifen-inducible Cre-ERT2 recombinase. The technology has been applied to other species via adeno-associated virus to achieve the gene delivery—that is, Fos-CreERT2 constructs (
) uses an artificial activity-induced sequence that combines the DNA sequence of the activator protein 1 and the binding motif of the neuronal-specific activity-dependent gene Npas4. The enhanced synaptic activity–regulated element system (
), on the other hand, uses a synthetic promoter that gets activated by activity-dependent transcription factors, such as cAMP response element–binding protein (CREB), myocyte enhancer factor 2, and serum response factor.
Apart from changes in gene expression, other activity-triggered processes have also been used to engineer activity-dependant reporters to investigate engram cells (
), respectively. These are molecular probes that rapidly and reversibly emit fluorescent signal in response to action potentials. In particular, genetically encoded calcium indicators have been used extensively to image large populations of neuronal activity in behaving animals when combined with florescence-detecting tools such as fiber optic recording, widefield microscopy, confocal microscopy, two-photon microscopy, or head-mounted miniature microscopes (
) and monitor the response of neuronal populations. Other reporters such as calcium-modulated photoactivatable ratiometric integrator (CaMPARI) allow permanent labeling of active cells at a given time point (
). CaMPARI is a fluorescent indicator that switches from green to a red fluorescent state in the presence of both Ca2+ and a pulse of light, allowing the tagging of specific populations related to specific experiences. Although CaMPARI allows for the rapid capture of tight time windows of activity, it can only be used for identifying and studying the cells in question and does not enable the manipulation of those cells.
A similar strategy based on combining two requirements to target engram cells with higher precision has also been used to label engram cells with exogenous constructs that allow for subsequent manipulation. (1) The capturing activated neuronal ensembles (CANE) technology combines molecular tagging with engineered viruses that selectively infect activated neurons (
). (2) In the Cal-Light technology, intracellular Ca2+ binds to calcium sensors calmodulin (CaM) and M13, which then bind to each other and rescue the proteolytic activity of the tobacco etch virus protease. At the same time, light delivered by the experimenter induces the unmasking of the tobacco etch virus protease target sequence, the subsequent cleavage of tetracycline transactivator (tTA), and the expression of the reporter gene (
), rely on the modification of a transcription factor to respond to both increase of intracellular Ca2+ and a pulse of light and drive the expression of a reporter gene. (4) Fast Light and Calcium-Regulated Expression (FLiCRE) technology (
) is similarly based on the induction of a reporter transcript in cells activated by intracellular Ca2+ at the time of blue light application, with higher sensitivity and temporal precision. As opposed to simply identifying or imaging neurons activated at a certain time point (such as CaMPARI), these tools allow to drive the expression of genes of interest to these cells.
Recently, a new resource has become available to investigate the history of activation of neurons over time. The expression recording island (
) is a reporter that encodes biological information of neuronal activity in the form of self-assembling protein chains. In this system, tag-labeled monomers are incorporated in a sequential order that mimics the sequence of activation states a neuron undergoes in response to experiences. The posterior analysis of such sequence reflects the involvement of individual cells in different engrams.
Overall, this powerful set of tools and its constant upgrades are approaching the field to the goal of manipulating the population of neurons associated with a particular memory with an even higher level of precision and accuracy. Because we can now artificially activate and inhibit memories, manipulate them, tag them, and study them, it is possible to address questions about the molecular and cellular mechanisms associated with memory to help us understand memory function and neurobiological correlates of memory (
We review here the current understanding of the neurobiology of memory, specifically at a cellular and molecular level, and with an emphasis on what we have learnt from engram studies. We discuss how these mechanisms can be interpreted to form a coherent neurobiological theory of memory that could offer verifiable explanations for each phenomenon of memory function. We classify the mechanisms as those involved in the (1) formation of an engram during learning, the (2) consolidation of an engram from short-term memory to long-term memory, and the (3) storage of the information in the brain. Finally, we also consider the role of non-neuronal cells in these processes and reflect on some open questions in the memory field. The molecular and cellular mechanisms underlying other memory phenomena such as reconsolidation (a process by which memories are susceptible to modification or updating after recall) (
Molecular and cellular mechanisms that allow the formation of the engram
Learning is the process of acquiring new information that culminates in the creation of a memory. The encoding of information for a particular event within a specific neuronal ensemble takes seconds to minutes (
) postulated in his Neuron Theory that neurons form a contiguous, instead of a continuous structure. They are physically separated but functionally connected by synapses, a term coined by the physiologist Charles Scott Sherrington (
). The electric impulse coming from a presynaptic neuron is transmitted through a synapse to the postsynaptic neuron, where it travels toward the axon. Going even further, Cajal predicted that synaptic strength underlies the storage of information (
) described that memory lies in the strengthening of the connections between neurons that were simultaneously activated. Since then, the field has substantially advanced on our understanding of the molecular and cellular mechanisms involving memory function (
Neuronal plasticity can be defined as the mechanism that allows connections involved in a particular response to specifically strengthen, facilitating neuronal transmission between them, whereas others may weaken (
) described a long-lasting modification in synaptic strength induced by electrical stimulation, also known as long-term potentiation (LTP). The opposite effect, long-term depression, was also described (
)). During early-LTP, glutamate is released from the presynaptic neuron and activates the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) in the postsynaptic membrane, allowing Na+ and K+ influx into the cell. N-methyl-d-aspartate receptors (NMDARs) are another type of ionotropic glutamate receptors that are sensitive to the depolarization state of the postsynaptic element. They detect coincident presynaptic and postsynaptic activation and open to permeate Ca2+ inside the postsynaptic neuron.
This increase of Ca2+ leads to a plethora of intracellular signaling events. First, it activates the Ca2+/CaM-dependent protein kinase II (CaMKII) (
)). This family of signaling proteins includes members such as Rac1 (Ras-related C3 botulinum toxin substrate 1), Cdc42, and RhoA (Ras homologous member A), which then modulate effector molecules such as cofilin or the complex Arp2/3. Cofilin is an actin-binding protein that depolymerizes actin filaments and that is inhibited by RhoA (
Through these processes, synaptic plasticity ultimately ties salient experiences to changes in the efficiency of the synaptic transmission between neurons, modifying the synapse accordingly for future reactivation. However, there are still significant questions opened to explain how synaptic plasticity could be understood to mediate learning, and they relate precisely to the three general considerations discussed in the introduction.
First, the goal in mind is to decipher the molecular and cellular mechanism/s that allow for one particular memory to be encoded and then ultimately escalate from that to understand how the system works for the collection of memories that a brain holds. Engram technology offers the resolution needed to understand the mechanism for a specific component of memory functionality—for a particular salient experience and not other/s.
Second, plastic changes are necessary for memory function, but they are not the only players in the game. Information in a brain is long-lasting, and yet it is constantly susceptible to modifications that allow memory updating to occur (
). While an engram is necessarily formed by a process of plasticity, it must be also preserved and endure across the life of an animal, by a constant process of homeostasis. It must be maintained in a state that can be reactivated during recall when it became relevant. Are plastic changes of synapses able to perform both roles, encode now and store forever?
Third, the role of plasticity in learning can now be better assessed with engram technology since it confers enough technical resolution to discriminate from its role in consolidation, recall, or storage. To illustrate this point with an example, studies based on engram technology have demonstrated that memory survives under certain types of amnesia. Though apparently lost, the memory is still retrievable by optogenetic reactivation, showing that the memory deficit is neither due to disruption of learning or storage mechanisms, but impaired recall (
). It is now possible, as we discuss later, to precisely characterize the physiological process affected and call it by its name.
Creating the engrams
Engram technology has contributed to better understanding the role of synaptic and structural plasticity in the encoding of a memory.
The use of engram technology has demonstrated that engram-to-engram synapses exhibit plasticity of synaptic strength. In the hippocampus, patch-clamp recording of excitatory postsynaptic currents in engram cells upon depolarization of presynaptic input cells showed that engram to engram synapses are specifically strengthened relative to nonengram synapses, showing higher excitatory postsynaptic current amplitude, a higher spontaneous excitatory postsynaptic current amplitude, and higher ratio AMPA to NMDA (
). This increase in plasticity between engram cells has been validated and refined by Choi et al. using a more advanced methodology. By combining engram technology with GFP Reconstitution Across Synaptic Partners (dual-eGRASP) technique (
), synapses between engram cells have been characterized. This work described that engram cells in CA1, a subarea of the hippocampus, establish more synapses with engram cells in CA3 than nonengram cells, and that the strength of memory expression correlates with the strength of the connections between these cells. Engram-to-engram cell synapses were potentiated since they showed an increase in the presynaptic transmitter release and AMPAR levels (
If learning induces changes in a population of neurons that respond to an experience, then a question that must be answered is which neurons and why? In other words, what are the mechanisms that select neurons to form the engram?
One possibility is that a cue-specific afferent randomly activates a high excitable given ensemble that later becomes the engram (
). Artificial overexpression of the CREB in a subpopulation of neurons in the lateral amygdala increased preferential recruitment of these cells to the engram and enhanced auditory fear memory after training (
). Concordantly with this hypothesis, it has been described how two experiences that occur close in time are encoded in overlapping populations, in such a way that recall of the first one will provoke the recall of the second one (
) recorded neuronal activity while the animals explored different contexts using in vivo calcium imaging, describing how the more separated in time the memories for two contexts were, the less overlapping their engrams are in the hippocampal region CA1. In aged mice, where neuronal excitability, as well as other forms of plasticity, is impaired, this overlapping phenomenon did not occur. Furthermore, the effect on overlapping and shared recall in aged mice was rescued with artificial activation of the neurons with designer receptors exclusively activated by designer drugs.
However, another study has shown that recall (and likely learning) increases engram excitability but does not increase the likelihood of memories being linked or coallocated (
). Rather, it seems that increased excitability itself enhances short-term memory precision by increasing engram cell accessibility and the ability to discriminate between similar but distinguishable contexts (pattern separation), thus keeping similar experiences separated. Taken together, it seems that the overexpression of CREB influences the allocation of an engram to targeted cells and also alter the excitability state of those cells in parallel. What is the mechanism whereby natural learning decides to which neurons an engram is allocated?
While this question is largely unanswered, it is reasonable to hypothesize that the excitation state of cells at the time of learning may influence their probability of allocation to an engram. In other words, if a cell lies in an anatomical region that is innervated by a receptive field activated by a relevant perceptual experience, then its mode of excitation during that experience may influence how the engram is assembled. In addition, stable cellular properties, such as molecular subtype, location within the area (
), also likely determine which ones are selected to the engram. Recent studies using two-photon microscopy in engram cells have described preferential selection to neurons that receive more stable connections (
). Finally, the epigenetic stage of the cell also influences eligibility for recruitment to the engram (Box 2). Future studies that monitor neuronal activity of ensembles longitudinally, before and after engram formation, and combine this analysis with physiological and structural analysis of the allocated cells will likely generate insights into the mechanisms of engram allocation.
). Therefore, it is not surprising they play a role in memory function. Learning induces a battery of epigenetic changes, including reversible modulation of both histones and DNA. These include histone methylations (
)). The resolution needed to understand the mechanisms at a singular memory level has been achieved by incorporating engram labeling technology into epigenetic tools.
A study of the epigenome architecture of engram cells based on the engram technology has indeed demonstrated that memory encoding modifies chromatin structure to increase the accessibility of chromatin on enhancers that will interact with promoters during memory recall (
). This way, engram cells become epigenetically primed after encoding. Later, during memory recall, when the engram is reactivated, the primed engram cells undergo transcriptional changes. This study demonstrates that the epigenetic modifications on the engram during learning are stable and persist. These results also confirm those reported by a parallel transcriptomic and epigenetic study of activated neurons. One hour after exploration of a novel context or an artificial activation triggered by kainic acid, activated hippocampal neurons reorganized their chromatin to allow accessibility of activity-regulated genes, enhancers, and transcription factors (
). Overexpression of the DNA methyltransferase 3a2 (Dnmt3a2) specifically in engram cells increased the precision of the engram reactivation during recall and strengthen memory in a context-specific manner. On the other hand, isoform Dnmt3a has been described to play a role in engram allocation: overexpression of Dnmt3a in a random sparse population of the DG increased intrinsic excitability and biased engram allocation toward them (
).The epigenetic code theory explains how, by means of epigenetic reversible modification of chromosomal regions and histones, it is possible to label activity states in a permanent way in the neuron (
). Crucially, these changes can be maintained by homeostatic mechanisms. It has been proposed that, after learning and plasticity take place and the gene expression changes and structural modifications have occurred, the neurons switch from a permissive epigenetic state to a maintenance transcriptome that facilitates the long-term storage of information (
These studies evidence that engram cells suffer important epigenetic regulations that control cellular processes behind learning, consolidation, and storage. Engram technology, and related methodologies, will continue to help us clarify these molecular mechanisms and answer the open questions in the field.
Another open question is how some synapses consolidate, whereas others stay available for learning-induced plasticity? This is the problem that has been commonly referred to as synaptic allocation or synaptic specificity (reviewed in Ref. (
It has been suggested that synaptic specificity could be achieved thanks to the information that neurons share extrasynaptically. This noncanonical way of communication may confine plasticity mechanisms to particular synapses (
), as it is trafficked interneuronally. Arc mRNA and protein appear to be self-assembled into virus-like capsids, released from neurons, and transferred into other cells. In the target cell, it is required for synaptic plasticity in the postsynaptic neuron and activity-dependent synapse formation in Drosophila (
), it can be speculated that Arc plays a role in defining which synapses will be modulated by plasticity.
Either by extrasynaptic or synaptic communication, only specific synapses are formed between a given subset of engram cells after experiences. The specificity of the connections formed during learning has precisely been hypothesized to be the mechanism at the center of memory storage, as it will be discussed later (
Molecular and cellular mechanisms that allow the consolidation of the engram
Memory consolidation is the mechanism that transforms temporary or short-term memories into stable long-term memories. It can be subdivided into two very different processes: synaptic consolidation (at the cellular level) and system consolidation (at the circuit level) (
). On the other hand, system consolidation, which is out of the scope of this review as it does not necessitate a molecular explanation, involves the gradual reorganization of the engram from hippocampal to neocortical structures and takes from days to weeks (
). Overall, the final product of memory consolidation is the persistence of a modified synaptic structure and function.
During synaptic consolidation, the synapses that encoded the information after learning go through a group of plasticity mechanisms referred as the L-LTP (Fig. 5). This phase involves (1) activation of the protein synthesis machinery in both soma and dendrites (local translation) to translate pre-existing mRNA and (2) de novo mRNA transcription (
). The increase in intracellular Ca2+ provoked by early-LTP activates signaling cascades mediated by PKA, PKC, and mitogen-activated protein kinases (MAPKs), which then activate transcription factors. The most studied one, CREB (discussed in previous sections), is a transcription factor that initiates transcription of a group of genes containing CREB-responsive elements (
), in a tightly regulated way, a repertoire of genes gets transcribed. These include IEGs such as Arc, Homer, c-fos or Zif268, kinases such as CaMKII and protein kinase Mzeta, and cytoskeletal proteins (see Ref. (
) has been proposed to explain this phenomenon. According to this theory, the activation of synapses during early-LTP tags the synapses and will induce the capture of plasticity-related proteins (PRPs), activated by the CaMKII–CREB pathway. Synapses in the same cell share PRPs. Therefore, a weak stimuli can induce L-LTP by tagging a synapse and then recruiting available PRPs derived from a strongly stimulated second synapse (
Engram technology has helped us understand how this de novo mRNA and protein synthesis modify the molecular profile of the engram synapses. Studies based on single-cell sequencing methods have characterized the transcriptomic response of engram cells (
) to identify changes associated with L-LTP. Attempts have studied engram cell transcriptomics using single-nuclei RNA-Seq, demonstrating that engram cell activation results in the expression of genes from the MAPK family, and IEG transcriptional regulators, such as Atf3, Egr1, Fosb, Homer1, and Junb (
). Further studies found a heterogeneous response within the population of engram cells, depending on the area and cell type. Even within the hippocampus, DG and CA1 neurons, as well as a subtype of inhibitory interneurons (vasoactive intestinal polypeptide positive neurons) show different profiles of gene expression after activation by learning (
). To investigate if the transcriptomic response supports reactivation during retrieval, mice were re-exposed to the same context 4 h after the first exposition. In the DG, subsets of engram cells express either (1) a characteristic early activation transcriptomic signature, (2) a late-activation signature still present 5 h after the experience, and, interestingly, (3) a particular signature that predicts that the particular engram cell will be reactivated upon exposure to the same context (
). The role of this functional response is still open to speculation.
During later consolidation into long-term memory, the transcriptomic profile of engram cells also undergoes changes. Thanks to the fact that engram cells can be permanently tagged, the mechanisms taking part in each stage of the memory process can be evaluated. Rao-Ruiz et al. (
) used RNA-Seq to demonstrate that, 1 day after training, engram cells activate a gene signature mediated mainly by CREB-induced genes. Weeks after learning, consolidated memory is known to be more dependent on cortical areas to be reactivated than on hippocampal areas (
). As memory undergoes consolidation into cortical areas, engram cells, labeled in the medial prefrontal cortex 16 days after an experience is encoded, also activate specific transcriptional programs. As happens with postlearning transcriptomic profiles, consolidation transcriptomic profiles are also specific to cell type. They contain genes involved in transcriptional and translational regulation, vesicle exocytosis, transmembrane transport, dendritic spine organization, and long-range intracellular transport. Interestingly, the genes in this consolidation signature do not seem to be directly controlled by canonical transcriptional regulators, evidenced by the lack of regulatory motifs of Creb, Nfkb, Cbp, and C/ebp. Overall, this study demonstrates that activity-specific changes in engram cells occur during weeks after encoding (
), which is accompanied by an extensive chromatin reorganization, indicating epigenetic modulations are involved in the modification of the response (discussed in Box 2).
Gene expression can also be modulated during consolidation through noncoding RNAs. Located at the synapses, noncoding RNAs modify gene expression locally and adjust it to neuronal activity requirements. They modify mRNA stability and translation, regulate transcription and trigger epigenetic modifications (reviewed in Refs. (
)) by targeting transcription factors. For example, neuronal activity in Aplysia increases expression of miRNA miR-124 that modulates Creb expression inducing plasticity changes, such as dendrite morphogenesis, synaptogenesis, and glutamate receptor modulation (
). In Aplysia neurons, the piRNA piR-F was shown to respond to the neurotransmitter serotonin, important for learning and plasticity, inducing the epigenetic silencing of transcription factor CREB2, which in turn provoked plasticity changes (
Finally, lncRNAs interact with RNA-binding proteins to remodel chromatin and modulate alternative splicing at the nuclear level. Locally at the synapses, lncRNAs control plasticity mechanisms by regulating mRNA stability and protein synthesis (
), and its spatiotemporal regulation supports their role in synaptic specification. In an interesting example, the expression of the lncRNA BC1 has been proved inducible by neuronal activity in hippocampal primary cultures (
). Recently, an elegant study characterized a novel lncRNA, termed ADEPTR, involved in structural plasticity. ADEPTR was isolated from the synaptic fraction of hippocampal neuronal cultures treated with the secondary messenger cAMP. Using a loss-of-function analysis, knocking down of ADEPTR prevented the increase in the number of dendritic spines and in spontaneous excitatory postsynaptic currents measured by whole-cell patch clamp induced by neuronal activation in vitro. ADEPTR interacts with the actin-scaffolding regulators spectrin/ankyrin complex to drive them to the synapse and promote structural changes induced by activity (
), and it shapes the dendritic spine, a structure that concentrates the functional components of the synapse (see the later section). Inside the spine, actin monomers (or G-actin) organize in filamentous polymers (F-actin) to form a branched network (
). F-actin polymerizes on the barbed end of the filament, whereas G-actin monomers disassemble at the other end, in a dynamic process. The velocity of polymerization and depolymerization of actin monomers, as well as their orientation and stability, determine the structure and stability of the dendritic spine, therefore modifying structural plasticity (
). Actin is enriched at the PSD to anchor receptors via interaction with scaffolding proteins. On the other hand, actin depolymerization, in close interaction with lipids on the exterior side of the membrane, increases the motility of nonstabilized molecules at the PSD (
The cytoskeleton dynamics are critical to consolidation mechanism of memories in engram cells. Engram cells express Rac1 during memory consolidation. One day after contextual fear conditioning, Rac1 expression was found in 80% of the engram population in CA1, labeled by engram technology, and was absent in nonengram cells. This expression, sustained for 10 days, induces the natural forgetting of the memory; and pharmacological inhibition of Rac1, as well as overexpression of its negative regulator α2-chimaerin, restored the manifestation of the memory, suggesting a recall deficit instead of consolidation or storage (
During memory consolidation, changes initiated by learning induce a more permanent and long-term adaptation, characterized by a regulation of gene expression and a structural reorganization and that culminate with an increase in synaptic plasticity and structural plasticity. Short-term memory becomes stabilized into long-term memory. But more importantly, besides which particular neurons or synapses (where) and the changes they undergo (how), an open question is what is really the substrate of the information in the brain. What is the change that grasps specific information for a certain experience in our brains?
Molecular and cellular mechanisms that allow the persistence of the engram
Learned information needs a solid system to be secured. As molecular and cellular mechanisms provide the basis for physiological mechanisms, which is the mechanism behind memory storage that maintain the information?
An early engram study provided a starting point to help clarify this question by demonstrating that L-LTP of synaptic strength is not strictly required for memory storage. Abolishing the mechanism of L-LTP by means of a protein synthesis inhibitor right after the encoding of an episodic memory did not alter its storage in the hippocampus. Injection of the protein synthesis inhibitor anysomicin induced amnesia to the mice, evidenced by the absence of freezing behavior in a context previously associated with a shock. Using optogenetics and engram-specific technology, the engram, labeled with ChR2, was reactivated and the behavioral response elicited. In the absence of L-LTP, the presence of the memory was observed (
). In the amnesic mice, the increase in engram-to-engram connectivity strength was prevented, as well as the increase in spine density between engram cells, but memory was not lost. Therefore, memory must rely on storage in other changes that are independent of L-LTP (
). This study also evidences the necessity of a fine characterization of the elements of the memory process when interpreting molecular studies of memory. A subset of studies have replicated and advanced on these results (
If not synaptic plasticity, what then is the mechanism that retains the memory in the traces in the long term? At this point, it is important to make a distinction between plasticity mechanisms that modify synaptic weight (the strength of a particular synapse) and plasticity mechanisms that modify synaptic wiring (the flowchart diagram of connections in between neurons or structural plasticity) (
). Molecular mechanisms involved in specifically creating and maintaining this connectivity are still to be fully understood. Two processes are particularly relevant: the formation of new connections and the activity of adhesion molecules specifying which connections must be formed.
Formation of new connections
New connections can be formed by (a) adding synapses to the system or synaptogenesis in between new or existing dendrites and/or axons or by (b) unsilencing synapses (Fig. 3).
Dendritic spines are bulbous shapes that connect to the dendrite by a neck and act as the postsynaptic component of excitatory synapses (
)). Engram-specific manipulation demonstrated that the spine density of engram cells correlated with the temporal progression of the memories. A recent study based on eGRASP technology characterized spine morphology between engram neurons in the cortex to amygdala areas (
). Memory encoding was associated with higher spine size and that parameter correlated with the maintenance of memory (Fig. 3). After memory extinction (the targeted suppression of a learned behavioral response), spines returned to their original state (