Advertisement

Understanding the physical basis of memory: Molecular mechanisms of the engram

  • Clara Ortega-de San Luis
    Correspondence
    For correspondence: Clara Ortega-de San Luis; Tomás J. Ryan
    Affiliations
    School of Biochemistry and Immunology and Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland
    Search for articles by this author
  • Tomás J. Ryan
    Correspondence
    For correspondence: Clara Ortega-de San Luis; Tomás J. Ryan
    Affiliations
    School of Biochemistry and Immunology and Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin, Ireland

    Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, University of Melbourne, Parkville, Victoria, Australia

    Child & Brain Development Program, Canadian Institute for Advanced Research (CIFAR), Toronto, Ontario, Canada
    Search for articles by this author
Open AccessPublished:March 25, 2022DOI:https://doi.org/10.1016/j.jbc.2022.101866
      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.

      Keywords

      Abbreviations:

      AAV (adeno-associated virus), AMPAR (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor), CaM (calmodulin), CAM (cell adhesion molecule), CaMKII (CaM-dependent protein kinase II), CaMPARI (calcium-modulated photoactivatable ratiometric integrator), ChR2 (channelrhodopsin-2), CREB (cAMP response element–binding protein), CS (conditioned stimulus), DG (dentate gyrus), Dnmt3a2 (DNA methyltransferase 3a2), eGRASP (engram technology with GFP Reconstitution Across Synaptic Partners), FLARE (Fast Light– and Activity-Regulated Expression), IEG (immediate early gene), IgCAM (immunoglobulin superfamily cell adhesion protein), L-LTP (late phase of LTP), lncRNA (long noncoding RNA), LTP (long-term potentiation), MAPK (mitogen-activated protein kinase), NCAM (neural cell adhesion molecule), NMDAR (N-methyl-d-aspartate receptor), piRNA (Piwi-interacting RNA), PNN (perineuronal net), PRP (plasticity-related protein), PSD (postsynaptic density), Rac1 (Ras-related C3 botulinum toxin substrate 1), RhoA (Ras homologous member A), tTA (tetracycline transactivator)
      Animals extract information from the environment through learning and modify their behavior accordingly. Memory is the ability to store and recall that knowledge (
      • Kandel E.R.
      • Mack S.
      • Jessell T.M.
      • Schwartz J.H.
      • Siegelbaum S.A.
      • Hudspeth A.J.
      Principles of Neural Science, Fifth Edition.
      ,
      • Tulving E.
      Episodic memory: From mind to brain.
      ). 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 (
      • Dudai Y.
      • Roediger H.L.
      • Tulving E.
      Memory concepts.
      ,
      • McGaugh J.L.
      Memory - a century of consolidation.
      ,
      • Schacter D.L.
      • Tulving E.
      Memory Systems 1994.
      ,
      • Squire L.R.
      • Genzel L.
      • Wixted J.T.
      • Morris R.G.
      Memory consolidation.
      ).
      In 1904, Richard Semon (
      • Semon R.
      Die mneme [The mneme].
      ) 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 (
      • Josselyn S.A.
      • Tonegawa S.
      Memory engrams: Recalling the past and imagining the future.
      ). 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 (
      • Tonegawa S.
      • Liu X.
      • Ramirez S.
      • Redondo R.
      Memory engram cells have come of age.
      ).

      The coding problem

      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) (
      • Descartes R.
      Meditations on First Philosophy.
      ) proposed that the mind was organized in ideas, which are material representations of both what is presented in front of the mind and resulting from the operation of the mind itself, a thought (
      • Descartes R.
      Meditations on First Philosophy.
      ,
      • Smith K.
      Descartes’ Theory of Ideas.
      ). The problem of mental representations, and how to operationalize it into experimental design, remains a core challenge in modern neuroscience (
      • Tulving E.
      Episodic memory: From mind to brain.
      ,
      • Semon R.
      Die mneme [The mneme].
      ,
      • Josselyn S.A.
      • Tonegawa S.
      Memory engrams: Recalling the past and imagining the future.
      ,
      • Krakauer J.W.
      • Ghazanfar A.A.
      • Gomez-Marin A.
      • MacIver M.A.
      • Poeppel D.
      Neuroscience needs behavior: Correcting a reductionist bias.
      ).
      Early in the 1910s, before the discovery of the DNA double helix (
      • Watson J.D.
      • Crick F.H.C.
      Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid.
      ), 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 (
      • Mendel G.
      Versuche uber pflanzen-hybriden.
      ) on understanding inheritance, the physical substrate of the information was discovered: the gene-bearing chromosomes (
      • Morgan T.H.
      Sex limited inheritance in drosophila.
      ). DNA was found to carry genetic traits that were trafficked between bacteria during transformation (
      • Avery O.T.
      • Macleod C.M.
      • McCarty M.
      Studies on the chemical nature of the substance inducing transformation of pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III.
      ), and genes were discovered to be made of DNA (
      • Hershey A.D.
      • Chase M.
      Independent functions of viral protein and nucleic acid in growth of bacteriophage.
      ). The discovery of the DNA structure (
      • Watson J.D.
      • Crick F.H.C.
      Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid.
      ) was the Rosetta stone that made everything else comprehensible. The structure of genes was understood (
      • Benzer S.
      On the topology of the genetic fine structure.
      ,
      • Benzer S.
      On the topography of the genetic fine structure.
      ), and the genetic code was finally solved (
      • Crick F.H.C.
      • Barnett L.
      • Brenner S.
      • Watts-Tobin R.J.
      General nature of the genetic code for proteins.
      ). 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 (
      • Abraham W.C.
      • Jones O.D.
      • Glanzman D.L.
      Is plasticity of synapses the mechanism of long-term memory storage?.
      ,
      • Asok A.
      • Leroy F.
      • Rayman J.B.
      • Kandel E.R.
      Molecular mechanisms of the memory trace.
      ,
      • Chklovskii D.B.
      • Mel B.W.
      • Svoboda K.
      Cortical rewiring and information storage.
      ,
      • Johansen J.P.
      • Cain C.K.
      • Ostroff L.E.
      • LeDoux J.E.
      Molecular mechanisms of fear learning and memory.
      ,
      • Kandel E.R.
      The molecular biology of memory storage: A dialogue between genes and synapses.
      ,
      • Kandel E.R.
      • Dudai Y.
      • Mayford M.R.
      The molecular and systems biology of memory.
      ) but have largely sidestepped the question of the coding problem: how the specific information is written in the brain (
      • Gallistel C.R.
      The coding question.
      ,
      • Gallistel C.R.
      The physical basis of memory.
      ,
      • Queenan B.N.
      • Ryan T.J.
      • Gazzaniga M.S.
      • Gallistel C.R.
      On the research of time past: The hunt for the substrate of memory.
      ). 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) (
      • Gallistel C.R.
      • Matzel L.D.
      The neuroscience of learning: Beyond the hebbian synapse.
      ). 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 (
      • Josselyn S.A.
      • Köhler S.
      • Frankland P.W.
      Finding the engram.
      ,
      • Miller R.R.
      Failures of memory and the fate of forgotten memories.
      ). 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.

      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 (
      • Liu X.
      • Ramirez S.
      • Pang P.T.
      • Puryear C.B.
      • Govindarajan A.
      • Deisseroth K.
      • Tonegawa S.
      Optogenetic stimulation of a hippocampal engram activates fear memory recall.
      ). Adding the doxycycline-inducible elements tet-ON and tet-OFF to the system (
      • Shockett P.E.
      • Schatz D.G.
      Diverse strategies for tetracycline-regulated inducible gene expression.
      ) 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 (
      • Liu X.
      • Ramirez S.
      • Pang P.T.
      • Puryear C.B.
      • Govindarajan A.
      • Deisseroth K.
      • Tonegawa S.
      Optogenetic stimulation of a hippocampal engram activates fear memory recall.
      ), despite never having a negative experience in this context.
      Figure thumbnail gr1
      Figure 1Engram labeling technology. (1) The immediate early gene c-fos promoter drives the expression of tTA. Doxycycline (DOX), delivered through diet, prevents tTA-TRE element binding. (2) An electric shock is delivered when the animal is in a particular context (context A). An engram for that episodic experience is formed (purple cells). In the absence of DOX, engram neurons that are active during the encoding of that fear memory express channelrhodopsin-2 (ChR2) transgene and GFP reporter. (3) Return to context A will reactivate the engram and will induce a freezing response in the animal—behavior associated with fear. (4) The engram cells tagged with ChR2 can be optogenetically reactivated by delivering light into the brain of the animal. This artificial recall of the memory induces freezing behavior in the animal in a neutral context B. TRE, tetracycline response element; tTA, tetracycline transactivator.

      What has research with engram technology taught us about how memory works?

      Engram technology triggered a revolution in the memory field (
      • Josselyn S.A.
      • Tonegawa S.
      Memory engrams: Recalling the past and imagining the future.
      ,
      • Tonegawa S.
      • Liu X.
      • Ramirez S.
      • Redondo R.
      Memory engram cells have come of age.
      ). Rapidly, numerous engram studies capitalized on the ability to manipulate memories and study engram physiology, and our understanding of engram biology grew rapidly.
      Activity of memory engram cells was demonstrated to be sufficient for the recall of a contextual memory associated with fear conditioning (
      • Liu X.
      • Ramirez S.
      • Pang P.T.
      • Puryear C.B.
      • Govindarajan A.
      • Deisseroth K.
      • Tonegawa S.
      Optogenetic stimulation of a hippocampal engram activates fear memory recall.
      ,
      • Ramirez S.
      • Liu X.
      • Lin P.-A.
      • Suh J.
      • Pignatelli M.
      • Redondo R.L.
      • Ryan T.J.
      • Tonegawa S.
      Creating a false memory in the hippocampus.
      ). Later, engram cells were demonstrated to be necessary for the reactivation of the memory (
      • Denny C.A.
      • Kheirbek M.A.
      • Alba E.L.
      • Tanaka K.F.
      • Brachman R.A.
      • Laughman K.B.
      • Tomm N.K.
      • Turi G.F.
      • Losonczy A.
      • Hen R.
      Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis.
      • Tanaka K.Z.
      • Pevzner A.
      • Hamidi A.B.
      • Nakazawa Y.
      • Graham J.
      • Wiltgen B.J.
      Cortical representations are reinstated by the hippocampus during memory retrieval.
      ). 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 (
      • Han J.H.
      • Kushner S.A.
      • Yiu A.P.
      • Hsiang H.L.
      • Buch T.
      • Waisman A.
      • Bontempi B.
      • Neve R.L.
      • Frankland P.W.
      • Josselyn S.A.
      Selective erasure of a fear memory.
      ,
      • Kim J.
      • Kwon J.T.
      • Kim H.S.
      • Josselyn S.A.
      • Han J.H.
      Memory recall and modifications by activating neurons with elevated CREB.
      ,
      • Redondo R.L.
      • Kim J.
      • Arons A.L.
      • Ramirez S.
      • Liu X.
      • Tonegawa S.
      Bidirectional switch of the valence associated with a hippocampal contextual memory engram.
      ,
      • Reijmers L.G.
      • Perkins B.L.
      • Matsuo N.
      • Mayford M.
      Localization of a stable neural correlate of associative memory.
      ,
      • Trouche S.
      • Sasaki J.M.
      • Tu T.
      • Reijmers L.G.
      Fear extinction causes target-specific remodeling of perisomatic inhibitory synapses.
      ,
      • Zelikowsky M.
      • Hersman S.
      • Chawla M.K.
      • Barnes C.A.
      • Fanselow M.S.
      Neuronal ensembles in amygdala, hippocampus, and prefrontal cortex track differential components of contextual fear.
      ), dentate gyrus (DG) (
      • Liu X.
      • Ramirez S.
      • Pang P.T.
      • Puryear C.B.
      • Govindarajan A.
      • Deisseroth K.
      • Tonegawa S.
      Optogenetic stimulation of a hippocampal engram activates fear memory recall.
      ,
      • Denny C.A.
      • Kheirbek M.A.
      • Alba E.L.
      • Tanaka K.F.
      • Brachman R.A.
      • Laughman K.B.
      • Tomm N.K.
      • Turi G.F.
      • Losonczy A.
      • Hen R.
      Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis.
      ,
      • Deng W.
      • Mayford M.
      • Gage F.H.
      Selection of distinct populations of dentate granule cells in response to inputs as a mechanism for pattern separation in mice.
      ,
      • Ramirez S.
      • Liu X.
      • MacDonald C.J.
      • Moffa A.
      • Zhou J.
      • Redondo R.L.
      • Tonegawa S.
      Activating positive memory engrams suppresses depression-like behaviour.
      ,
      • Ryan T.J.
      • Roy D.S.
      • Pignatelli M.
      • Arons A.
      • Tonegawa S.
      Engram cells retain memory under retrograde amnesia.
      ), CA1 (
      • Tanaka K.Z.
      • Pevzner A.
      • Hamidi A.B.
      • Nakazawa Y.
      • Graham J.
      • Wiltgen B.J.
      Cortical representations are reinstated by the hippocampus during memory retrieval.
      ), CA3 (
      • Denny C.A.
      • Kheirbek M.A.
      • Alba E.L.
      • Tanaka K.F.
      • Brachman R.A.
      • Laughman K.B.
      • Tomm N.K.
      • Turi G.F.
      • Losonczy A.
      • Hen R.
      Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis.
      ), cortex (
      • Trouche S.
      • Sasaki J.M.
      • Tu T.
      • Reijmers L.G.
      Fear extinction causes target-specific remodeling of perisomatic inhibitory synapses.
      ,
      • Cowansage K.K.
      • Shuman T.
      • Dillingham B.C.
      • Chang A.
      • Golshani P.
      • Mayford M.
      Direct reactivation of a coherent neocortical memory of context.
      ,
      • DeNardo L.A.
      • Liu C.D.
      • Allen W.E.
      • Adams E.L.
      • Friedmann D.
      • Fu L.
      • Guenthner C.J.
      • Tessier-Lavigne M.
      • Luo L.
      Temporal evolution of cortical ensembles promoting remote memory retrieval.
      ,
      • Kitamura T.
      • Ogawa S.K.
      • Roy D.S.
      • Okuyama T.
      • Morrissey M.D.
      • Smith L.M.
      • Redondo R.L.
      • Tonegawa S.
      Engrams and circuits crucial for systems consolidation of a memory.
      ), or nucleus accumbens (
      • Koya E.
      • Golden S.A.
      • Harvey B.K.
      • Guez-Barber D.H.
      • Berkow A.
      • Simmons D.E.
      • Bossert J.M.
      • Nair S.G.
      • Uejima J.L.
      • Marin M.T.
      • Mitchell T.B.
      • Farquhar D.
      • Ghosh S.C.
      • Mattson B.J.
      • Hope B.T.
      Targeted disruption of cocaine-activated nucleus accumbens neurons prevents context-specific sensitization.
      ). The connection between engram and place cells was also investigated (Box 1). Engram cell dynamics were successfully manipulated, and false memories were created in animals (
      • Ramirez S.
      • Liu X.
      • MacDonald C.J.
      • Moffa A.
      • Zhou J.
      • Redondo R.L.
      • Tonegawa S.
      Activating positive memory engrams suppresses depression-like behaviour.
      ,
      • Garner A.R.
      • Rowland D.C.
      • Hwang S.Y.
      • Baumgaertel K.
      • Roth B.L.
      • Kentros C.
      • Mayford M.
      Generation of a synthetic memory trace.
      ,
      • Ohkawa N.
      • Saitoh Y.
      • Suzuki A.
      • Tsujimura S.
      • Murayama E.
      • Kosugi S.
      • Nishizono H.
      • Matsuo M.
      • Takahashi Y.
      • Nagase M.
      • Sugimura Y.K.
      • Watabe A.M.
      • Kato F.
      • Inokuchi K.
      Artificial association of pre-stored information to generate a qualitatively new memory.
      ,
      • Vetere G.
      • Tran L.M.
      • Moberg S.
      • Steadman P.E.
      • Restivo L.
      • Morrison F.G.
      • Ressler K.J.
      • Josselyn S.A.
      • Frankland P.W.
      Memory formation in the absence of experience.
      ). In an early study, Ramirez et al. (
      • Ramirez S.
      • Liu X.
      • Lin P.-A.
      • Suh J.
      • Pignatelli M.
      • Redondo R.L.
      • Ryan T.J.
      • Tonegawa S.
      Creating a false memory in the hippocampus.
      ) 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 (
      • Ramirez S.
      • Liu X.
      • Lin P.-A.
      • Suh J.
      • Pignatelli M.
      • Redondo R.L.
      • Ryan T.J.
      • Tonegawa S.
      Creating a false memory in the hippocampus.
      ).
      Engram cells and place cells
      Place cells are neurons in the hippocampus that encode spatial information by tuning its firing to particular locations (
      • O'Keefe J.
      • Dostrovsky J.
      The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat.
      ). 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 (
      • Moser E.I.
      • Moser M.B.
      • McNaughton B.L.
      Spatial representation in the hippocampal formation: A history.
      ).
      The relation between engram cells and place cells has been the focus of several discussions that ultimately question how the hippocampus work and how it processes memories (
      • Goode T.D.
      • Tanaka K.Z.
      • Sahay A.
      • McHugh T.J.
      An integrated index: Engrams, place cells, and hippocampal memory.
      ,
      • Tanaka K.Z.
      Heterogeneous representations in the hippocampus.
      ). One view is that the activity of engram cells reflects the contextual identity of the memory, instead of specific locations (
      • Hainmueller T.
      • Bartos M.
      Dentate gyrus circuits for encoding, retrieval and discrimination of episodic memories.
      ,
      • Tanaka K.Z.
      • He H.
      • Tomar A.
      • Niisato K.
      • Huang A.J.Y.
      • McHugh T.J.
      The hippocampal engram maps experience but not place.
      ). 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 (
      • Tanaka K.Z.
      • He H.
      • Tomar A.
      • Niisato K.
      • Huang A.J.Y.
      • McHugh T.J.
      The hippocampal engram maps experience but not place.
      ). Another conceptual framework, not mutually exclusive, is based on the cognitive map theory (
      • O’keefe J.
      • Nadel L.
      The Hippocampus as a Cognitive Map.
      ). 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 (
      • Goode T.D.
      • Tanaka K.Z.
      • Sahay A.
      • McHugh T.J.
      An integrated index: Engrams, place cells, and hippocampal memory.
      ).

      The expanded engram toolbox

      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 (
      • Kim J.
      • Kwon J.T.
      • Kim H.S.
      • Josselyn S.A.
      • Han J.H.
      Memory recall and modifications by activating neurons with elevated CREB.
      ,
      • Yiu A.P.
      • Mercaldo V.
      • Yan C.
      • Richards B.
      • Rashid A.J.
      • Hsiang H.-L.L.
      • Pressey J.
      • Mahadevan V.
      • Tran M.M.
      • Kushner S.A.
      • Woodin M.A.
      • Frankland P.W.
      • Josselyn S.A.
      Neurons are recruited to a memory trace based on relative neuronal excitability immediately before training.
      ). Compared with optogenetics, chemogenetics manipulations offer an easier alternative for sustained manipulations (
      • Vlasov K.
      • Van Dort C.J.
      • Solt K.
      Optogenetics and chemogenetics.
      ). The tamoxifen-driven inducible Cre-recombinase system (
      • Metzger D.
      • Clifford J.
      • Chiba H.
      • Chambon P.
      Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase.
      ) has been applied instead of the doxycycline-regulated system to achieve more precise temporal control (
      • Denny C.A.
      • Kheirbek M.A.
      • Alba E.L.
      • Tanaka K.F.
      • Brachman R.A.
      • Laughman K.B.
      • Tomm N.K.
      • Turi G.F.
      • Losonczy A.
      • Hen R.
      Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis.
      ,
      • DeNardo L.A.
      • Liu C.D.
      • Allen W.E.
      • Adams E.L.
      • Friedmann D.
      • Fu L.
      • Guenthner C.J.
      • Tessier-Lavigne M.
      • Luo L.
      Temporal evolution of cortical ensembles promoting remote memory retrieval.
      ,
      • Guenthner C.J.
      • Miyamichi K.
      • Yang H.H.
      • Heller H.C.
      • Luo L.
      Permanent genetic access to transiently active neurons via TRAP: Targeted recombination in active populations.
      ). 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 (
      • Denny C.A.
      • Kheirbek M.A.
      • Alba E.L.
      • Tanaka K.F.
      • Brachman R.A.
      • Laughman K.B.
      • Tomm N.K.
      • Turi G.F.
      • Losonczy A.
      • Hen R.
      Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis.
      ), in order to target bigger areas, which are logistically difficult to be reached via adeno-associated virus injections. For example, in the targeted recombination in active populations mice (
      • DeNardo L.A.
      • Liu C.D.
      • Allen W.E.
      • Adams E.L.
      • Friedmann D.
      • Fu L.
      • Guenthner C.J.
      • Tessier-Lavigne M.
      • Luo L.
      Temporal evolution of cortical ensembles promoting remote memory retrieval.
      ,
      • Guenthner C.J.
      • Miyamichi K.
      • Yang H.H.
      • Heller H.C.
      • Luo L.
      Permanent genetic access to transiently active neurons via TRAP: Targeted recombination in active populations.
      ,
      • Allen W.E.
      • Denardo L.A.
      • Chen M.Z.
      • Liu C.D.
      • Loh K.M.
      • Fenno L.E.
      • Ramakrishnan C.
      • Deisseroth K.
      • Luo L.
      Thirst-associated preoptic neurons encode an aversive motivational drive.
      ), 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 (
      • Koya E.
      • Golden S.A.
      • Harvey B.K.
      • Guez-Barber D.H.
      • Berkow A.
      • Simmons D.E.
      • Bossert J.M.
      • Nair S.G.
      • Uejima J.L.
      • Marin M.T.
      • Mitchell T.B.
      • Farquhar D.
      • Ghosh S.C.
      • Mattson B.J.
      • Hope B.T.
      Targeted disruption of cocaine-activated nucleus accumbens neurons prevents context-specific sensitization.
      ). Engineered artificial promoters have been designed to improve the labeling specificity and efficiency. The Robust Activity Marking system (
      • Sørensen A.T.
      • Cooper Y.A.
      • Baratta M.V.
      • Weng F.J.
      • Zhang Y.
      • Ramamoorthi K.
      • Fropf R.
      • Laverriere E.
      • Xue J.
      • Young A.
      • Schneider C.
      • Gøtzsche C.R.
      • Hemberg M.
      • Yin J.C.P.
      • Maier S.F.
      • et al.
      A robust activity marking system for exploring active neuronal ensembles.
      ) 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 (
      • Kawashima T.
      • Kitamura K.
      • Suzuki K.
      • Nonaka M.
      • Kamijo S.
      • Takemoto-Kimura S.
      • Kano M.
      • Okuno H.
      • Ohki K.
      • Bito H.
      Functional labeling of neurons and their projections using the synthetic activity–dependent promoter E-SARE.
      ), 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.
      Figure thumbnail gr2
      Figure 2The expanded engram toolbox. Engram cell tagging has been achieved with several strategies or tools that can be used individually or combined. A, temporal control allows labeling engrams responsible for encoding a particular experience and can be achieved with different strategies. The tTA/TRE or Cre-ERT2 (targeted recombination in active populations [TRAP]) genetic strategies are temporally controlled by the delivery of doxycycline or tamoxifen, respectively. The capturing activated neuronal ensembles (CANE) technology allows high temporal precision by engineering viruses to specifically infect activated neurons. Other tools (Cal-Light, Fast light–regulated and activity-regulated expression [FLARE] and its improved version single-chain FLARE [scFLARE], and fast light–regulated and calcium-regulated expression [FLiCRE]) rely on the combination of two requirements to achieve temporal control: increase in intracellular Ca2+ and delivery of light. A similar strategy based on the coincidence of activity and light is used by CaMPARI to label only activated cells. B, transgenes can be delivered by generating transgenic mice models or by the use of vectors such as adeno-associated viruses (AAVs). C, only activated cells (engram cells, purple) are tagged; thanks to several spatial control strategies. The expression of immediate early genes (IEGs) such as c-fos can be used to manipulate only activated cells. Engineered artificial promoters have been also used in the robust activity marking (RAM) or the enhanced synaptic activity–regulated element (E-SARE) systems. Intracellular Ca2+ levels are detected by genetically encoded calcium or voltage indicators (GECI and GEVI, respectively) as well as reporters that detect specific neurotransmitter (Nt) release. CaMPARI is a fluorescent indicator that responds to intracellular Ca2+ levels. D, engram cells can be tagged with reporters (e.g., GFP or mCherry fluorescent reporters). They can also be tagged with tools that will allow future manipulation by light (optogenetics) or by drugs (chemogenetics). They can be imaged with several techniques such as two-photon or head-mounted miniature microscopes (miniscopes). Their activity can be monitored by techniques that allow activity readings such as fiber photometry. Activation history over time can be investigated by the use of the expression recording island (XRI) technology. CaMPARI, calcium-modulated photoactivatable ratiometric integrator; GECI, genetically encoded calcium indicator; GEVI, genetically encoded voltage indicator; tTA, tetracycline transactivator.
      Apart from changes in gene expression, other activity-triggered processes have also been used to engineer activity-dependant reporters to investigate engram cells (
      • Wang W.
      • Kim C.K.
      • Ting A.Y.
      Molecular tools for imaging and recording neuronal activity.
      ). Membrane depolarization and elevation of intracellular Ca2+ can be monitored with genetically encoded voltage indicators (
      • Xu Y.
      • Zou P.
      • Cohen A.E.
      Voltage imaging with genetically encoded indicators.
      ) and genetically encoded calcium indicators (
      • Mank M.
      • Griesbeck O.
      Genetically encoded calcium indicators.
      ), 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 (
      • Wang W.
      • Kim C.K.
      • Ting A.Y.
      Molecular tools for imaging and recording neuronal activity.
      ,
      • Kim T.H.
      • Schnitzer M.J.
      Fluorescence imaging of large-scale neural ensemble dynamics.
      ). Similarly, fluorescent neurotransmitter indicators (
      • Wang H.
      • Jing M.
      • Li Y.
      Lighting up the brain: Genetically encoded fluorescent sensors for imaging neurotransmitters and neuromodulators.
      ) are also available to detect specific neurotransmitter release, such as glutamate (
      • Marvin J.S.
      • Scholl B.
      • Wilson D.E.
      • Podgorski K.
      • Kazemipour A.
      • Müller J.A.
      • Schoch S.
      • Quiroz F.J.U.
      • Rebola N.
      • Bao H.
      • Little J.P.
      • Tkachuk A.N.
      • Cai E.
      • Hantman A.W.
      • Wang S.S.H.
      • et al.
      Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.
      ), acetylcholine (
      • Jing M.
      • Zhang P.
      • Wang G.
      • Feng J.
      • Mesik L.
      • Zeng J.
      • Jiang H.
      • Wang S.
      • Looby J.C.
      • Guagliardo N.A.
      • Langma L.W.
      • Lu J.
      • Zuo Y.
      • Talmage D.A.
      • Role L.W.
      • et al.
      A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies.
      ), or γ-aminobutyric acid (
      • Marvin J.S.
      • Shimoda Y.
      • Magloire V.
      • Leite M.
      • Kawashima T.
      • Jensen T.P.
      • Kolb I.
      • Knott E.L.
      • Novak O.
      • Podgorski K.
      • Leindenheimer N.J.
      • Rusakov D.A.
      • Ahrens M.B.
      • Kullmann D.M.
      • Looger L.L.
      A genetically encoded fluorescent sensor for in vivo imaging of GABA.
      ) 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 (
      • Fosque B.F.
      • Sun Y.
      • Dana H.
      • Yang C.T.
      • Ohyama T.
      • Tadross M.R.
      • Patel R.
      • Zlatic M.
      • Kim D.S.
      • Ahrens M.B.
      • Jayaraman V.
      • Looger L.L.
      • Schreiter E.R.
      Labeling of active neural circuits in vivo with designed calcium integrators.
      ). 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 (
      • Sakurai K.
      • Zhao S.
      • Takatoh J.
      • Rodriguez E.
      • Lu J.
      • Leavitt A.D.
      • Fu M.
      • Han B.X.
      • Wang F.
      Capturing and manipulating activated neuronal ensembles with CANE delineates a hypothalamic social-fear circuit.
      ). (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 (
      • Lee D.
      • Hyun J.
      • Jung K.
      • Hannan P.
      • Kwon H.
      A calcium- and light-gated switch to induce gene expression in activated neurons.
      ). (3) Fast Light– and Activity-Regulated Expression (FLARE) (
      • Wang W.
      • Wildes C.P.
      • Pattarabanjird T.
      • Sanchez M.I.
      • Glober G.F.
      • Matthews G.A.
      • Tye K.M.
      • Ting A.Y.
      A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.
      ), and its improved version, single-chain FLARE (
      • Sanchez M.I.
      • Nguyen Q.A.
      • Wang W.
      • Soltesz I.
      • Ting A.Y.
      Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease.
      ), 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 (
      • Kim C.K.
      • Sanchez M.I.
      • Hoerbelt P.
      • Fenno L.E.
      • Malenka R.C.
      • Deisseroth K.
      • Ting A.Y.
      A molecular calcium integrator reveals a striatal cell type driving aversion.
      ) 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 (
      • Linghu C.
      • An B.
      • Shpokayte M.
      • Celiker O.T.
      • Shmoel N.
      • Zhang C.
      • Park W.M.
      • Ramirez S.
      • Boyden E.S.
      Recording of cellular physiological histories along optically readable self-assembling protein chains.
      ) 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 (
      • Josselyn S.A.
      • Tonegawa S.
      Memory engrams: Recalling the past and imagining the future.
      ).
      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) (
      • Eisenberg M.
      • Kobilo T.
      • Berman D.E.
      • Dudai Y.
      Stability of retrieved memory: Inverse correlation with trace dominance.
      ,
      • Przybyslawski J.
      • Sara S.J.
      Reconsolidation of memory after its reactivation.
      ) or forgetting (the inability to recall a memory that was successfully learned) (
      • Ryan T.J.
      • Frankland P.W.
      Forgetting as a form of adaptive engram cell plasticity.
      ,
      • Tulving E.
      Cue-dependent forgetting.
      ) are out of the scope of this review.

      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 (
      • Dudai Y.
      Consolidation: Fragility on the road to the engram.
      ). What are the changes that the neurons undergo to form the engram?

      Learning and plasticity

      Shortly before Semon described the concept of the engram, Santiago Ramón y Cajal (
      • Ramon y Cajal S.
      Estructura de los centros nerviosos de las aves.
      ) 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 (
      • Sherrington C.
      The Integrative Action of the Nervous System.
      ). 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 (
      • Ramon y Cajal S.
      The Croonian lecture.—La fine structure des centres nerveux.
      ). His student, Rafael Lorente de Nó, described how neurons form “multiple chains of transmission through which impulses circulate” (
      • Lorente De Nó R.
      Analysis of the activity of the chains of internuncial neurons.
      ), and Donald Hebb (
      • Hebb D.O.
      The Organisation of Behaviour.
      ) 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 (
      • Abraham W.C.
      • Jones O.D.
      • Glanzman D.L.
      Is plasticity of synapses the mechanism of long-term memory storage?.
      ,
      • Asok A.
      • Leroy F.
      • Rayman J.B.
      • Kandel E.R.
      Molecular mechanisms of the memory trace.
      ,
      • Chklovskii D.B.
      • Mel B.W.
      • Svoboda K.
      Cortical rewiring and information storage.
      ,
      • Johansen J.P.
      • Cain C.K.
      • Ostroff L.E.
      • LeDoux J.E.
      Molecular mechanisms of fear learning and memory.
      ,
      • Kandel E.R.
      • Dudai Y.
      • Mayford M.R.
      The molecular and systems biology of memory.
      ,
      • Markram H.
      • Lübke J.
      • Frotscher M.
      • Sakmann B.
      Regulation of synaptic efficacy by coincidence of postsynaptic APs and EPSPs.
      ,
      • Markram H.
      • Gerstner W.
      • Sjöström P.J.
      Spike-timing-dependent plasticity: A comprehensive overview.
      ).
      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 (
      • Bliss T.V.P.
      • Collingridge G.L.
      A synaptic model of memory: Long-term potentiation in the hippocampus.
      ). After Hebb (
      • Hebb D.O.
      The Organisation of Behaviour.
      ) postulated that neurons that are functionally active at the same time adapt their synapses to become linked forming an ensemble, Kandel and Tauc (
      • Kandel E.R.
      • Tauc L.
      Heterosynaptic facilitation in neurones of the abdominal ganglion of Aplysia depilans.
      ) demonstrated that learning influenced the synapses. Later, Bliss and Lomo (
      • Bliss T.V.
      • Lomo T.
      Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path.
      ) 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 (
      • Ito M.
      Cerebellar control of the vestibulo-ocular reflex--around the flocculus hypothesis.
      ).
      The first phase of the LTP, early-LTP (Fig. 3), lasts from seconds to a few hours and relies mainly on covalent modifications of already existing proteins (reviewed in Refs. (
      • Asok A.
      • Leroy F.
      • Rayman J.B.
      • Kandel E.R.
      Molecular mechanisms of the memory trace.
      ,
      • Malenka R.C.
      • Bear M.F.
      LTP and LTD: An embarrassment of riches.
      )). 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.
      Figure thumbnail gr3
      Figure 3Molecular mechanisms of learning. When an animal encodes a new memory (such as the encounter of food in a particular arm of a Y-maze, upper panel), a subset of neurons gets activated and an engram is formed (purple neurons, middle panel). Certain synapses in between engram cells (dotted lines) undergo synaptic plasticity changes (early long-term potentiation [LTP]). The (1) neurotransmitter glutamate (Glu) is released from the presynaptic neuron, Glu binds to (2) AMPARs in the postsynaptic membrane, at the level of the dendritic spine, allowing K+ and Na+ to enter the postsynaptic neuron and depolarizing it (3). The positive charges inside the postsynaptic neurons allow the release of the Mg2+ ion from the NMDARs and if the Glu release is sustained enough, it will open the channel (4). Ca2+ enters the postsynaptic neuron, activating the (5) Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates AMPARs (6) to increase their sensitivity to Glu and drives more active channels to the membrane, AMPA trafficking (7). The number and shape of dendritic spines also get modified during learning. Spine remodeling to strengthen the synapse involves cytoskeletal modification such as (A) actin polymerization and (B) actin branching. Engram cells become unsilenced by the trafficking of AMPAR to the synapses by (C) secretion from the intracellular pool and (D) diffusion from other membrane areas. AMPAR, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor; NMDAR, N-methyl-d-aspartate receptor.
      This increase of Ca2+ leads to a plethora of intracellular signaling events. First, it activates the Ca2+/CaM-dependent protein kinase II (CaMKII) (
      • Malenka R.C.
      • Bear M.F.
      LTP and LTD: An embarrassment of riches.
      ,
      • Bayer K.U.
      • Schulman H.
      CaM kinase: Still inspiring at 40.
      ,
      • Lisman J.
      • Schulman H.
      • Cline H.
      The molecular basis of CaMKII function in synaptic and behavioural memory.
      ,
      • Lisman J.
      • Yasuda R.
      • Raghavachari S.
      Mechanisms of CaMKII action in long-term potentiation.
      ). CaMKII is a central regulator of plasticity that increases postsynaptic responsiveness. It phosphorylates the GluA1 subunit of AMPAR, which increases single-channel conductance (
      • Benke T.A.
      • Luthl A.
      • Isaac J.T.R.
      • Collingridge G.L.
      Modulation of AMPA receptor unitary conductance by synaptic activity.
      ,
      • Derkach V.
      • Barria A.
      • Soderling T.R.
      Ca2+/calmodulin-kinase II enhances channel conductance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors.
      ,
      • Kristensen A.S.
      • Jenkins M.A.
      • Banke T.G.
      • Schousboe A.
      • Makino Y.
      • Johnson R.C.
      • Huganir R.
      • Traynelis S.F.
      Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating.
      ), and it allows more active channels in the membrane in a process known as AMPAR trafficking. AMPARs diffuse to the synapses from other membrane areas (
      • Penn A.C.
      • Zhang C.L.
      • Georges F.
      • Royer L.
      • Breillat C.
      • Hosy E.
      • Petersen J.D.
      • Humeau Y.
      • Choquet D.
      Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors.
      ), as well as getting secreted from the existing intracellular pool (
      • Shirke A.M.
      • Malinow R.
      Mechanisms of potentiation by calcium-calmodulin kinase II of postsynaptic sensitivity in rat hippocampal CA1 neurons.
      ), facilitating further impulse transmission.
      Immediate cytoskeletal modifications (Fig. 4) independent of protein synthesis are also triggered by an increase in intracellular Ca2+ (
      • Matus A.
      Actin-based plasticity in dendritic spines.
      ). They contribute to enlarging the spine in the case of LTP and retracting the spine in the case of long-term depression (
      • Matsuzaki M.
      • Honkura N.
      • Ellis-Davies G.C.R.
      • Kasai H.
      Structural basis of long-term potentiation in single dendritic spines.
      ,
      • Nägerl U.V.
      • Eberhorn N.
      • Cambridge S.B.
      • Bonhoeffer T.
      Bidirectional activity-dependent morphological plasticity in hippocampal neurons.
      ,
      • Okamoto K.I.
      • Nagai T.
      • Miyawaki A.
      • Hayashi Y.
      Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity.
      ,
      • Zhou Q.
      • Homma K.J.
      • Poo M.M.
      Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses.
      ). Intracellular Ca2+ activates a transduction signal cascade mediated by tyrosine kinases and Src family kinases that in turn activate Rho GTPases and Ras GTPases (reviewed in Refs. (
      • Carlisle H.J.
      • Kennedy M.B.
      Spine architecture and synaptic plasticity.
      ,
      • Hotulainen P.
      • Hoogenraad C.C.
      Actin in dendritic spines: Connecting dynamics to function.
      ,
      • Spence E.F.
      • Soderling S.H.
      Actin out: Regulation of the synaptic cytoskeleton.
      ,
      • Zhang H.
      • Ben Zablah Y.
      • Zhang H.
      • Jia Z.
      Rho signaling in synaptic plasticity, memory, and brain disorders.
      )). 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 (
      • Arber S.
      • Barbayannis F.A.
      • Hanser H.
      • Schnelder C.
      • Stanyon C.A.
      • Bernards O.
      • Caroni P.
      Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase.
      ,
      • Ben Zablah Y.
      • Merovitch N.
      • Jia Z.
      The role of ADF/cofilin in synaptic physiology and Alzheimer’s disease.
      ). Arp2/3 is an actin nucleation factor that regulates actin branching, polymerization, and recycling (
      • Goley E.D.
      • Welch M.D.
      The ARP2/3 complex: An actin nucleator comes of age.
      ,
      • Hotulainen P.
      • Llano O.
      • Smirnov S.
      • Tanhuanpää K.
      • Faix J.
      • Rivera C.
      • Lappalainen P.
      Defning mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis.
      ). On top of their structural role, Rho GTPases also regulate AMPAR trafficking. They modulate both AMPAR insertion in the postsynaptic membrane and AMPAR stability in the membrane (
      • Zhang H.
      • Ben Zablah Y.
      • Zhang H.
      • Jia Z.
      Rho signaling in synaptic plasticity, memory, and brain disorders.
      ). Cdc42, for example, triggers the phosphorylation of the AMPAR subunit GluA1, which induces the insertion of AMPAR in the membrane (
      • Hussain N.K.
      • Thomas G.M.
      • Luo J.
      • Huganir R.L.
      Regulation of AMPA receptor subunit GluA1 surface expression by PAK3 phosphorylation.
      ). Via these signaling pathways that regulate actin polymerization and AMPAR trafficking, the synapse structure gets quickly modified in a process known as structural plasticity (
      • Nägerl U.V.
      • Eberhorn N.
      • Cambridge S.B.
      • Bonhoeffer T.
      Bidirectional activity-dependent morphological plasticity in hippocampal neurons.
      ,
      • Okamoto K.I.
      • Nagai T.
      • Miyawaki A.
      • Hayashi Y.
      Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity.
      ,
      • Zhou Q.
      • Homma K.J.
      • Poo M.M.
      Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses.
      ,
      • Lamprecht R.
      • LeDoux J.
      Structural plasticity and memory.
      ). With the emergence of more dendritic spines, the synapse is strengthened (
      • Johansen J.P.
      • Cain C.K.
      • Ostroff L.E.
      • LeDoux J.E.
      Molecular mechanisms of fear learning and memory.
      ,
      • Engert F.
      • Bonhoeffer T.
      Dendritic spine changes associated with hippocampal long-term synaptic plasticity.
      ).
      Figure thumbnail gr4
      Figure 4Cytoskeletal modifications after learning. The increase in intracellular Ca2+ provoked by neuronal activation activates tyrosin kinases (TKs) and Src kinases. These activate members of the Rho family small GTPases: RhoA, Cdc42, and Rac1. RhoA activates ROCK and subsequently LIMK kinases, which in turn inhibits effector cofilin and eventually inhibits actin depolymerization. Rac1 and Cdc42 activate Arp2/3, an actin nucleation factor that induces actin polymerization, the elongation of actin filaments, and actin branching, and the formation of new ramifications in the actin filaments. Rho GTPases also anchor, stabilize, phosphorylate, and insert AMPARs to the postsynaptic density area of the membrane. AMPAR, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor; LIMK, LIM-domain kinases; NMDAR, N-methyl-d-aspartate receptor; Rac1, Ras-related C3 botulinum toxin substrate 1; RhoA, Ras homologous member A; ROCK, Rho-associated coiled-coil kinase.

      Still much to learn

      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 (
      • Dudai Y.
      • Eisenberg M.
      Rites of passage of the engram: Reconsolidation and the lingering consolidation hypothesis.
      ). Therefore, there must be an equilibrium between malleability and fidelity (
      • Raman D.V.
      • O'Leary T.
      Optimal plasticity for memory maintenance during ongoing synaptic change.
      ,
      • Turrigiano G.G.
      • Nelson S.B.
      Homeostatic plasticity in the developing nervous system.
      ). The problem of how plasticity, a mechanism based on the presence and amount of turnover-sensitive proteins, is sustained, is an old discussion in the field (
      • Crick F.
      Neurobiology: Memory and molecular turnover.
      ). 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 (
      • Ryan T.J.
      • Roy D.S.
      • Pignatelli M.
      • Arons A.
      • Tonegawa S.
      Engram cells retain memory under retrograde amnesia.
      ,
      • Guskjolen A.
      • Kenney J.W.
      • de la Parra J.
      • Yeung B.R.A.
      • Josselyn S.A.
      • Frankland P.W.
      Recovery of “lost” infant memories in mice.
      ,
      • Perusini J.N.
      • Cajigas S.A.
      • Cohensedgh O.
      • Lim S.C.
      • Pavlova I.P.
      • Donaldson Z.R.
      • Denny C.A.
      Optogenetic stimulation of dentate gyrus engrams restores memory in Alzheimer’s disease mice.
      ,
      • Poll S.
      • Mittag M.
      • Musacchio F.
      • Justus L.C.
      • Giovannetti E.A.
      • Steffen J.
      • Wagner J.
      • Zohren L.
      • Schoch S.
      • Schmidt B.
      • Jackson W.S.
      • Ehninger D.
      • Fuhrmann M.
      Memory trace interference impairs recall in a mouse model of Alzheimer’s disease.
      ,
      • Roy D.S.
      • Arons A.
      • Mitchell T.I.
      • Pignatelli M.
      • Ryan T.J.
      • Tonegawa S.
      Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease.
      ,
      • Zhao B.
      • Sun J.
      • Zhang X.
      • Mo H.
      • Niu Y.
      • Li Q.
      • Wang L.
      • Zhong Y.
      Long-term memory is formed immediately without the need for protein synthesis-dependent consolidation in Drosophila.
      ). 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 (
      • Ryan T.J.
      • Roy D.S.
      • Pignatelli M.
      • Arons A.
      • Tonegawa S.
      Engram cells retain memory under retrograde amnesia.
      ). Structurally, engram neurons also showed an increase in dendritic spine density (
      • Ryan T.J.
      • Roy D.S.
      • Pignatelli M.
      • Arons A.
      • Tonegawa S.
      Engram cells retain memory under retrograde amnesia.
      ,
      • Kim W.B.
      • Cho J.H.
      Encoding of discriminative fear memory by input-specific LTP in the amygdala.
      ,
      • Kim W.B.
      • Cho J.H.
      Encoding of contextual fear memory in hippocampal–amygdala circuit.
      ). 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 (
      • Feinberg E.H.
      • VanHoven M.K.
      • Bendesky A.
      • Wang G.
      • Fetter R.D.
      • Shen K.
      • Bargmann C.I.
      GFP reconstitution across synaptic partners (GRASP) defines cell contacts and synapses in living nervous systems.
      ), 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 (
      • Choi J.-H.
      • Sim S.-E.
      • Kim J.
      • Choi D.I.
      • Oh J.
      • Ye S.
      • Lee J.
      • Kim T.
      • Ko H.-G.
      • Lim C.-S.
      • Kaang B.-K.
      Interregional synaptic maps among engram cells underlie memory formation.
      ). Very recent studies also using dual-eGRASP from the same laboratory corroborated the increase in structural connectivity between engram cells in the auditory cortex to lateral amygdala circuit (
      • Choi D.I.
      • Kim J.
      • Lee H.
      • Kim J.
      • Sung Y.
      • Choi J.E.
      • Venkat S.J.
      • Park P.
      • Jung H.
      • Kaang B.-K.
      Synaptic correlates of associative fear memory in the lateral amygdala.
      ). Increases in structural plasticity and functional plasticity have been found specifically in engram-to-engram cells in many other circuits and areas: hippocampus to amygdala (
      • Kim W.B.
      • Cho J.H.
      Encoding of contextual fear memory in hippocampal–amygdala circuit.
      ), cortex to amygdala (
      • Kim W.B.
      • Cho J.H.
      Encoding of discriminative fear memory by input-specific LTP in the amygdala.
      • Nonaka A.
      • Toyoda T.
      • Miura Y.
      • Hitora-Imamura N.
      • Naka M.
      • Eguchi M.
      • Yamaguchi S.
      • Ikegaya Y.
      • Matsuki N.
      • Nomura H.
      Synaptic plasticity associated with a memory engram in the basolateral amygdala.
      ), or hippocampus to nucleus accumbens (
      • Zhou Y.
      • Zhu H.
      • Liu Z.
      • Chen X.
      • Su X.J.
      • Ma C.
      • Tian Z.
      • Huang B.
      • Yan E.
      • Liu X.
      • Ma L.
      A ventral CA1 to nucleus accumbens core engram circuit mediates conditioned place preference for cocaine.
      ).

      Neuronal allocation

      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 (
      • Josselyn S.A.
      • Frankland P.W.
      Memory allocation: Mechanisms and function.
      ). 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 (
      • Han J.H.
      • Kushner S.A.
      • Yiu A.P.
      • Hsiang H.L.
      • Buch T.
      • Waisman A.
      • Bontempi B.
      • Neve R.L.
      • Frankland P.W.
      • Josselyn S.A.
      Selective erasure of a fear memory.
      ,
      • Han J.
      • Kushner S.A.
      • Yiu A.P.
      • Cole C.J.
      • Matynia A.
      • Brown R.A.
      • Neve R.L.
      • Guzowski J.F.
      • Silva A.J.
      • Josselyn S.A.
      Neuronal competition and selection during memory formation.
      ). The transcription factor CREB, one of the best-studied genes in the memory field with a critical role in the late phase of LTP (L-LTP; see later), modulates intrinsic excitability (
      • Barco A.
      • Pittenger C.
      • Kandel E.R.
      CREB, memory enhancement and the treatment of memory disorders: Promises, pitfalls and prospects.
      ,
      • Benito E.
      • Barco A.
      CREB’s control of intrinsic and synaptic plasticity: Implications for CREB-dependent memory models.
      ). Without increasing the size of the engram, CREB increases the intrinsic excitability of the neuron—the propensity to generate an action potential upon reception of the input (
      • Yiu A.P.
      • Mercaldo V.
      • Yan C.
      • Richards B.
      • Rashid A.J.
      • Hsiang H.-L.L.
      • Pressey J.
      • Mahadevan V.
      • Tran M.M.
      • Kushner S.A.
      • Woodin M.A.
      • Frankland P.W.
      • Josselyn S.A.
      Neurons are recruited to a memory trace based on relative neuronal excitability immediately before 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 (
      • Cai D.J.
      • Aharoni D.
      • Shuman T.
      • Shobe J.
      • Biane J.
      • Song W.
      • Wei B.
      • Veshkini M.
      • La-Vu M.
      • Lou J.
      • Flores S.E.
      • Kim I.
      • Sano Y.
      • Zhou M.
      • Baumgaertel K.
      • et al.
      A shared neural ensemble links distinct contextual memories encoded close in time.
      ,
      • Rashid A.J.
      • Yan C.
      • Mercaldo V.
      • Hsiang H.-L.L.
      • Park S.
      • Cole C.J.
      • De Cristofaro A.
      • Yu J.
      • Ramakrishnan C.
      • Lee S.Y.
      • Deisseroth K.
      • Frankland P.
      • Josselyin S.A.
      Competition between engrams influences fear memory formation and recall.
      ). Cai et al. (
      • Cai D.J.
      • Aharoni D.
      • Shuman T.
      • Shobe J.
      • Biane J.
      • Song W.
      • Wei B.
      • Veshkini M.
      • La-Vu M.
      • Lou J.
      • Flores S.E.
      • Kim I.
      • Sano Y.
      • Zhou M.
      • Baumgaertel K.
      • et al.
      A shared neural ensemble links distinct contextual memories encoded close in time.
      ) 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 (
      • Ryan T.J.
      • Roy D.S.
      • Pignatelli M.
      • Arons A.
      • Tonegawa S.
      Engram cells retain memory under retrograde amnesia.
      • Pignatelli M.
      • Ryan T.J.
      • Roy D.S.
      • Lovett C.
      • Smith L.M.
      • Muralidhar S.
      • Tonegawa S.
      Engram cell excitability state determines the efficacy of memory retrieval.
      ). 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 (
      • Chawla M.K.
      • Penner M.R.
      • Olson K.M.
      • Sutherland V.L.
      • Mittelman-Smith M.A.
      • Barnes C.A.
      Spatial behavior and seizure-induced changes in c-fos mRNA expression in young and old rats.
      ,
      • Erwin S.R.
      • Sun W.
      • Copeland M.
      • Lindo S.
      • Spruston N.
      • Cembrowski M.S.
      A sparse, spatially biased subtype of mature granule cell dominates recruitment in hippocampal-associated behaviors.
      ), or the age of the neuron (
      • Kee N.
      • Teixeira C.C.M.
      • Wang A.H.A.
      • Frankland P.W.
      Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus.
      ), 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 (
      • Castello-Waldow T.P.
      • Weston G.
      • Ulivi A.F.
      • Chenani A.
      • Loewenstein Y.
      • Chen A.
      • Attardo A.
      Hippocampal neurons with stable excitatory connectivity become part of neuronal representations.
      ). 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.
      Epigenetic mechanisms in memory
      The environment can influence gene expression by modulating epigenetic mechanisms (
      • Sweatt J.
      • Meaney M.
      • Nestler E.
      • Akbarian S.
      Epigenetic Regulation in the Nervous System: Basic Mechanisms and Clinical Impact.
      ). 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 (
      • Gupta-Agarwal S.
      • Franklin A.V.
      • DeRamus T.
      • Wheelock M.
      • Davis R.L.
      • McMahon L.L.
      • Lubin F.D.
      G9a/GLP histone lysine dimethyltransferase complex activity in the hippocampus and the entorhinal cortex is required for gene activation and silencing during memory consolidation.
      ,
      • Oliveira A.M.M.
      DNA methylation: A permissive mark in memory formation and maintenance.
      ), histone acetylations (
      • Levenson J.M.
      • O'Riordan K.J.
      • Brown K.D.
      • Trinh M.A.
      • Molfese D.L.
      • Sweatt J.D.
      Regulation of histone acetylation during memory formation in the hippocampus.
      ), or DNA methylations (
      • Day J.J.
      • Childs D.
      • Guzman-Karlsson M.C.
      • Kibe M.
      • Moulden J.
      • Song E.
      • Tahir A.
      • Sweatt J.D.
      DNA methylation regulates associative reward learning.
      ,
      • Guo N.
      • Soden M.E.
      • Herber C.
      • Kim M.T.W.
      • Besnard A.
      • Lin P.
      • Ma X.
      • Cepko C.L.
      • Zweifel L.S.
      • Sahay A.
      Dentate granule cell recruitment of feedforward inhibition governs engram maintenance and remote memory generalization.
      ,
      • Miller C.A.
      • Sweatt J.D.
      Covalent modification of DNA regulates memory formation.
      ,
      • Miller C.A.
      • Gavin C.F.
      • White J.A.
      • Parrish R.R.
      • Honasoge A.
      • Yancey C.R.
      • Rivera I.M.
      • Rubio M.D.
      • Rumbaugh G.
      • Sweatt J.D.
      Cortical DNA methylation maintains remote memory.
      ) among others (reviewed in Refs. (
      • Campbell R.R.
      • Wood M.A.
      How the epigenome integrates information and reshapes the synapse.
      ,
      • Gräff J.
      • Tsai L.-H.
      Histone acetylation: Molecular mnemonics on the chromatin.
      ,
      • Sweatt J.D.
      The emerging field of neuroepigenetics.
      ,
      • Zovkic I.B.
      • Guzman-Karlsson M.C.
      • Sweatt J.D.
      Epigenetic regulation of memory formation and maintenance.
      )). 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 (
      • Marco A.
      • Meharena H.S.
      • Dileep V.
      • Raju R.M.
      • Davila-Velderrain J.
      • Zhang A.L.
      • Adaikkan C.
      • Young J.Z.
      • Gao F.
      • Kellis M.
      • Tsai L.H.
      Mapping the epigenomic and transcriptomic interplay during memory formation and recall in the hippocampal engram ensemble.
      ). 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 (
      • Fernandez-Albert J.
      • Lipinski M.
      • Lopez-Cascales M.T.
      • Rowley M.J.
      • Martin-Gonzalez A.M.
      • del Blanco B.
      • Corces V.G.
      • Barco A.
      Immediate and deferred epigenomic signatures of in vivo neuronal activation in mouse hippocampus.
      ).
      Another interesting example of engram technology to understand epigenetic mechanisms in memory consolidation has been described by Gulmez Karaca et al. (
      • Gulmez Karaca K.
      • Kupke J.
      • Brito D.V.C.
      • Zeuch B.
      • Thome C.
      • Weichenhan D.
      • Lutsik P.
      • Plass C.
      • Oliveira A.M.M.
      Neuronal ensemble-specific DNA methylation strengthens engram stability.
      ). 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 (
      • Odell S.C.
      • Taki F.
      • Klein S.L.
      • Chen R.J.
      • Levine O.B.
      • Skelly M.J.
      • Nabila A.
      • Brindley E.
      • Gal Toth J.
      • Dündar F.
      • Sheridan C.K.
      • Fetcho R.N.
      • Alonso A.
      • Liston C.
      • Landau D.A.
      • et al.
      Epigenomically bistable regions across neuron-specific genes govern neuron eligibility to a coding ensemble in the Hippocampus.
      ).
      Finally, the storage of the information at a cellular level by maintaining certain epigenetic signature has been hypothesized (
      • Aoued H.S.
      • Sannigrahi S.
      • Doshi N.
      • Morrison F.G.
      • Linsenbaum H.
      • Hunter S.C.
      • Walum H.
      • Baman J.
      • Yao B.
      • Jin P.
      • Ressler K.J.
      • Dias B.G.
      Reversing behavioral, neuroanatomical, and germline influences of intergenerational stress.
      ,
      • Dias B.G.
      • Ressler K.J.
      Parental olfactory experience influences behavior and neural structure in subsequent generations.
      ,
      • Gräff J.
      • Mansuy I.M.
      Epigenetic codes in cognition and behaviour.
      ).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 (
      • Day J.J.
      • Sweatt J.D.
      Epigenetic mechanisms in cognition.
      ). 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 (
      • Kyrke-Smith M.
      • Williams J.M.
      Bridging synaptic and epigenetic maintenance mechanisms of the engram.
      ).
      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.

      Synaptic allocation

      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. (
      • Rogerson T.
      • Cai D.J.
      • Frank A.
      • Sano Y.
      • Shobe J.
      • Lopez-Aranda M.F.
      • Silva A.J.
      Synaptic tagging during memory allocation.
      )).
      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 (
      • Abraham W.C.
      • Jones O.D.
      • Glanzman D.L.
      Is plasticity of synapses the mechanism of long-term memory storage?.
      ). Neurons communicate nonsynaptically via soluble factors (such as growth factors or cytokines), gap junctions (or electrical synapses) (see Ref. (
      • Söhl G.
      • Maxeiner S.
      • Willecke K.
      Expression and functions of neuronal gap junctions.
      ) for a review), tunneling nanotubes (see Ref. (
      • Ariazi J.
      • Benowitz A.
      • De Biasi V.
      • Den Boer M.L.
      • Cherqui S.
      • Cui H.
      • Douillet N.
      • Eugenin E.A.
      • Favre D.
      • Goodman S.
      • Gousset K.
      • Hanein D.
      • Israel D.I.
      • Kimura S.
      • Kirkpatrick R.B.
      • et al.
      Tunneling nanotubes and gap junctions–their role in long-range intercellular communication during development, health, and disease conditions.
      ) for a review), or RNA-containing exosomes (see Ref. (
      • Smalheiser N.R.
      Exosomal transfer of proteins and RNAs at synapses in the nervous system.
      ) for a review). Transmitted RNAs include coding RNAs (such as Arc) (
      • Ashley J.
      • Cordy B.
      • Lucia D.
      • Fradkin L.G.
      • Budnik V.
      • Thomson T.
      Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons.
      ) or noncoding RNAs, particularly miRNAs and Piwi-interacting RNAs (piRNAs) (
      • Landry C.D.
      • Kandel E.R.
      • Rajasethupathy P.
      New mechanisms in memory storage: piRNAs and epigenetics.
      ) (see the later section). A particularly fascinating example is Arc (
      • Shepherd J.D.
      • Bear M.F.
      New views of Arc, a master regulator of synaptic plasticity.
      ), 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 (
      • Ashley J.
      • Cordy B.
      • Lucia D.
      • Fradkin L.G.
      • Budnik V.
      • Thomson T.
      Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons.
      ,
      • Pastuzyn E.D.
      • Day C.E.
      • Kearns R.B.
      • Kyrke-Smith M.
      • Taibi A.V.
      • McCormick J.
      • Yoder N.
      • Belnap D.M.
      • Erlendsson S.
      • Morado D.R.
      • Briggs J.A.G.
      • Feschotte C.
      • Shepherd J.D.
      The neuronal gene Arc encodes a repurposed retrotransposon gag protein that mediates intercellular RNA transfer.
      ), which potentially explains how Arc could mediate synapse specificity. Since Arc expression is induced by synaptic activity, is located in the dendritic spine, and modulates local translation (
      • Farris S.
      • Lewandowski G.
      • Cox C.D.
      • Steward O.
      Selective localization of Arc mRNA in dendrites involves activity- and translation-dependent mRNA degradation.
      ), 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 (
      • Ryan T.J.
      • Ortega-de San Luis C.
      • Pezzoli M.
      • Sen S.
      Engram cell connectivity: An evolving substrate for information storage.
      ,
      • Tonegawa S.
      • Pignatelli M.
      • Roy D.S.
      • Ryan T.J.
      Memory engram storage and retrieval.
      ).

      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) (
      • Dudai Y.
      • Eisenberg M.
      Rites of passage of the engram: Reconsolidation and the lingering consolidation hypothesis.
      ). The first one involves biochemical and morphological changes at the synapses and in the cell, lasting from minutes to hours, with consequences even several weeks after (
      • Squire L.R.
      • Genzel L.
      • Wixted J.T.
      • Morris R.G.
      Memory consolidation.
      ,
      • Staubli U.
      • Lynch G.
      Stable hippocampal long-term potentiation elicited by “theta” pattern stimulation.
      ). 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 (
      • Squire L.R.
      • Genzel L.
      • Wixted J.T.
      • Morris R.G.
      Memory consolidation.
      ,
      • Dudai Y.
      Consolidation: Fragility on the road to the engram.
      ,
      • Tonegawa S.
      • Morrissey M.D.
      • Kitamura T.
      The role of engram cells in the systems consolidation of memory.
      ). 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 (
      • Kandel E.R.
      The molecular biology of memory storage: A dialogue between genes and synapses.
      ,
      • Raymond C.R.
      • Thompson V.L.
      • Tate W.P.
      • Abraham W.C.
      Metabotropic glutamate receptors trigger homosynaptic protein synthesis to prolong long-term potentiation.
      ,
      • Steward O.
      mRNA localization in neurons: A multipurpose mechanism?.
      ,
      • Sutton M.A.
      • Schuman E.M.
      Dendritic protein synthesis, synaptic plasticity, and memory.
      ). 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 (
      • Dash P.K.
      • Hochner B.
      • Kandel E.R.
      Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation.
      ). Rapidly, induced by CREB and other transcription factors such as C/EBP (
      • Alberini C.M.
      • Ghirardl M.
      • Metz R.
      • Kandel E.R.
      C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia.
      ), 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. (
      • Abraham W.C.
      • Jones O.D.
      • Glanzman D.L.
      Is plasticity of synapses the mechanism of long-term memory storage?.
      ) for a review).
      Figure thumbnail gr5
      Figure 5Molecular mechanisms of consolidation. After the encoding of a memory (the encounter of food in a Y-maze, upper panel), memory undergoes a series of processes that allow its consolidation into long-term memories resistant to the passage of time. Engram synapses (purple, middle panel) undergo synaptic plasticity changes (late phase of long-term potentiation [or late-LTP]). Triggered by the intracellular increase in Ca2+, adenylyl cyclase is activated and the intracellular concentration of cAMP increases. The cAMP increase triggers the activation of protein kinase A (PKA) and mitogen-activated protein kinases (MAPKs). PKA translocates to the nucleus, phosphorylates the transcription factor CREB, and ultimately triggers the transcription of genes containing CREB-responsive element (CRE), such as immediate early genes (c-fos, Arc, or Zif268), kinases such as Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase Mzeta (PKMζ). In the dendrites, protein synthesis occurs locally by ribosomes translating mRNAs in a localized manner in the dendritic spines.
      How are these changes, occurring at the whole-cell level, able to specifically be targeted only at some synapses in the cell? A theory known as Synaptic Tagging and Capture (
      • Frey U.
      • Morris R.G.M.
      Synaptic tagging and long-term potentiation.
      ,
      • Frey U.
      • Morris R.G.
      Weak before strong: Dissociating synaptic tagging and plasticity-factor accounts of late-LTP.
      ) 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 (
      • Rogerson T.
      • Cai D.J.
      • Frank A.
      • Sano Y.
      • Shobe J.
      • Lopez-Aranda M.F.
      • Silva A.J.
      Synaptic tagging during memory allocation.
      ,
      • Martin K.C.
      • Kosik K.S.
      Synaptic tagging — who’s it?.
      ).

      The repertoire of genes

      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 (
      • Lacar B.
      • Linker S.B.
      • Jaeger B.N.
      • Krishnaswami S.R.
      • Barron J.J.
      • Kelder M.J.E.
      • Parylak S.L.
      • Paquola A.C.M.
      • Venepally P.
      • Novotny M.
      • O’Connor C.
      • Fitzpatrick C.
      • Erwin J.A.
      • Hsu J.Y.
      • Husband D.
      • et al.
      Nuclear RNA-seq of single neurons reveals molecular signatures of activation.
      ) 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 (
      • Lacar B.
      • Linker S.B.
      • Jaeger B.N.
      • Krishnaswami S.R.
      • Barron J.J.
      • Kelder M.J.E.
      • Parylak S.L.
      • Paquola A.C.M.
      • Venepally P.
      • Novotny M.
      • O’Connor C.
      • Fitzpatrick C.
      • Erwin J.A.
      • Hsu J.Y.
      • Husband D.
      • et al.
      Nuclear RNA-seq of single neurons reveals molecular signatures of activation.
      ). 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 (
      • Jaeger B.N.
      • Linker S.B.
      • Parylak S.L.
      • Barron J.J.
      • Gallina I.S.
      • Saavedra C.D.
      • Fitzpatrick C.
      • Lim C.K.
      • Schafer S.T.
      • Lacar B.
      • Jessberger S.
      • Gage F.H.
      A novel environment-evoked transcriptional signature predicts reactivity in single dentate granule neurons.
      ). 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 (
      • Jaeger B.N.
      • Linker S.B.
      • Parylak S.L.
      • Barron J.J.
      • Gallina I.S.
      • Saavedra C.D.
      • Fitzpatrick C.
      • Lim C.K.
      • Schafer S.T.
      • Lacar B.
      • Jessberger S.
      • Gage F.H.
      A novel environment-evoked transcriptional signature predicts reactivity in single dentate granule neurons.
      ). 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. (
      • Rao-Ruiz P.
      • Couey J.J.
      • Marcelo I.M.
      • Bouwkamp C.G.
      • Slump D.E.
      • Matos M.R.
      • van der Loo R.J.
      • Martins G.J.
      • van den Hout M.
      • van IJcken W.F.
      • Costa R.M.
      • van den Oever M.C.
      • Kushner S.A.
      Engram-specific transcriptome profiling of contextual memory consolidation.
      ) 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 (
      • Kitamura T.
      • Ogawa S.K.
      • Roy D.S.
      • Okuyama T.
      • Morrissey M.D.
      • Smith L.M.
      • Redondo R.L.
      • Tonegawa S.
      Engrams and circuits crucial for systems consolidation of a memory.
      ,
      • Tonegawa S.
      • Morrissey M.D.
      • Kitamura T.
      The role of engram cells in the systems consolidation of memory.
      ). 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 (
      • Chen M.B.
      • Jiang X.
      • Quake S.R.
      • Südhof T.C.
      Persistent transcriptional programmes are associated with remote memory.
      ). Finally, during recall, engram cells seem to exhibit a particular transcriptional program distinctive from the one associated with encoding (
      • Marco A.
      • Meharena H.S.
      • Dileep V.
      • Raju R.M.
      • Davila-Velderrain J.
      • Zhang A.L.
      • Adaikkan C.
      • Young J.Z.
      • Gao F.
      • Kellis M.
      • Tsai L.H.
      Mapping the epigenomic and transcriptomic interplay during memory formation and recall in the hippocampal engram ensemble.
      ), which is accompanied by an extensive chromatin reorganization, indicating epigenetic modulations are involved in the modification of the response (discussed in Box 2).

      Noncoding RNAs

      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. (
      • Mercer T.R.
      • Dinger M.E.
      • Mariani J.
      • Kosik K.S.
      • Mehler M.F.
      • Mattick J.S.
      Noncoding RNAs in long-term memory formation.
      ,
      • Smalheiser N.R.
      The RNA-centred view of the synapse: Non-coding RNAs and synaptic plasticity.
      )). They are classified into noncoding miRNAs, piRNAs, and long noncoding RNAs (lncRNAs) (
      • Landry C.D.
      • Kandel E.R.
      • Rajasethupathy P.
      New mechanisms in memory storage: piRNAs and epigenetics.
      ).
      miRNAs bind to complementary mRNAs preventing their translation into proteins (
      • Bartel D.P.
      MicroRNAs: Genomics, biogenesis, mechanism, and function.
      ). They shape the cellular response after plasticity (reviewed in Ref. (
      • Bredy T.W.
      • Lin Q.
      • Wei W.
      • Baker-Andresen D.
      • Mattick J.S.
      MicroRNA regulation of neural plasticity and memory.
      )) 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 (
      • Rajasethupathy P.
      • Fiumara F.
      • Sheridan R.
      • Betel D.
      • Puthanveettil S.V.
      • Russo J.J.
      • Sander C.
      • Tuschl T.
      • Kandel E.
      Characterization of small RNAs in Aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB.
      ). miRNAs can also modulate synaptic plasticity through the protein complex translin/trax—two dendritic proteins with RNAse activity (
      • Finkenstadt P.M.
      • Kang W.-S.
      • Jeon M.
      • Taira E.
      • Tang W.
      • Baraban J.M.
      Somatodendritic localization of translin, a component of the translin/trax RNA binding complex.
      ). Neuronal activity triggers the RNAse activity of translin/trax, which then degrade miRNAs, releasing the silencing of PRPs (
      • Park A.J.
      • Havekes R.
      • Fu X.
      • Hansen R.
      • Tudor J.C.
      • Peixoto L.
      • Li Z.
      • Wu Y.C.
      • Poplawski S.G.
      • Baraban J.M.
      • Abel T.
      Learning induces the translin/trax RNase complex to express activin receptors for persistent memory.
      ).
      A very particular subtype of noncoding RNAs, piRNAs, are small RNA molecules involved in repressing transposons (
      • Landry C.D.
      • Kandel E.R.
      • Rajasethupathy P.
      New mechanisms in memory storage: piRNAs and epigenetics.
      ,
      • Ishizu H.
      • Siomi H.
      • Siomi M.C.
      Biology of PIWI-interacting RNAs: New insights into biogenesis and function inside and outside of germlines.
      ). 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 (
      • Rajasethupathy P.
      • Antonov I.
      • Sheridan R.
      • Frey S.
      • Sander C.
      • Tuschl T.
      • Kandel E.R.
      A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity.
      ).
      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 (
      • Grinman E.
      • Espadas I.
      • Puthanveettil S.V.
      Emerging roles for long noncoding RNAs in learning, memory and associated disorders.
      ,
      • Liau W.-S.
      • Samaddar S.
      • Banerjee S.
      • Bredy T.W.
      On the functional relevance of spatiotemporally-specific patterns of experience-dependent long noncoding RNA expression in the brain.
      ), 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 (
      • Muslimov I.A.
      • Banker G.
      • Brosius J.
      • Tiedge H.
      Activity-dependent regulation of dendritic BC1 RNA in hippocampal neurons in culture.
      ). Analysis of BC1 sequence allowed identification of a motif that interacts with the heterogeneous nuclear ribonucleoprotein A2 to drive BC1 exportation to the dendrite (
      • Muslimov I.A.
      • Iacoangeli A.
      • Brosius J.
      • Tiedge H.
      Spatial codes in dendritic BC1 RNA.
      ). 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 (
      • Grinman E.
      • Nakahata Y.
      • Avchalumov Y.
      • Espadas I.
      • Swarnkar S.
      • Yasuda R.
      • Puthanveettil S.V.
      Activity-regulated synaptic targeting of lncRNA ADEPTR mediates structural plasticity by localizing Sptn1 and AnkB in dendrites.
      ).

      The cytoskeleton

      The activation of a neuron modifies the actin cytoskeleton architecture to modulate the structure, composition, and physiology of the synapses (reviewed in Refs. (
      • Lamprecht R.
      • LeDoux J.
      Structural plasticity and memory.
      ,
      • Basu S.
      • Lamprecht R.
      The role of actin cytoskeleton in dendritic spines in the maintenance of long-term memory.
      )).
      The cytoskeleton at the postsynaptic site is mainly formed by actin (
      • Landis D.M.D.
      • Reese T.S.
      Cytoplasmic organization in cerebellar dendritic spines.
      ), 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 (
      • Korobova F.
      • Svitkina T.M.
      Molecular architecture of synaptic actin cytoskeleton in hippocampal neurons reveals a mechanism of dendritic spine morphogenesis.
      ). 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 (
      • Star E.N.
      • Kwiatkowski D.J.
      • Murthy V.N.
      Rapid turnover of actin in dendritic spines and its regulation by activity.
      ). Several neuropathies and disorders that lead to memory impairments have been associated with deficits in cytoskeletal function (
      • Kommaddi R.P.
      • Das D.
      • Karunakaran S.
      • Nanguneri S.
      • Bapat D.
      • Ray A.
      • Shaw E.
      • Bennett D.A.
      • Nair D.
      • Ravindranath V.
      Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer’s disease.
      ,
      • Pelucchi S.
      • Stringhi R.
      • Marcello E.
      Dendritic spines in Alzheimer’s disease: How the actin cytoskeleton contributes to synaptic failure.
      ).
      Apart from being a structural component, actin modulates postsynaptic proteins in the postsynaptic density (PSD) in response to activation (
      • Kuriu T.
      • Inoue A.
      • Bito H.
      • Sobue K.
      • Okabe S.
      Differential control of postsynaptic density scaffolds via actin-dependent and -independent mechanisms.
      ). The PSD is the area in the postsynaptic neuron located immediately opposite the presynaptic contact that organizes receptors, adhesion molecules, and signaling molecules (
      • Sheng M.
      • Kim M.J.
      Postsynaptic signaling and plasticity mechanisms.
      ). 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 (
      • Renner M.
      • Choquet D.
      • Triller A.
      Control of the postsynaptic membrane viscosity.
      ) and contributes to disperse AMPARs and NMDARs (
      • Allison D.W.
      • Gelfand V.I.
      • Spector I.
      • Craig A.M.
      Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: Differential attachment of NMDA versus AMPA receptors.
      ). On the presynaptic side, actin contributes to modulating the synaptic vesicle cycle and neurotransmitter release, organizing the fusion of synaptic vesicles with the active zone membrane (
      • Cingolani L.A.
      • Goda Y.
      Actin in action: The interplay between the actin cytoskeleton and synaptic efficacy.
      ).
      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 (
      • Lv L.
      • Liu Y.
      • Xie J.
      • Wu Y.
      • Zhao J.
      • Li Q.
      • Zhong Y.
      Interplay between α2-chimaerin and Rac1 activity determines dynamic maintenance of long-term memory.
      ).
      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 (
      • Ryan T.J.
      • Roy D.S.
      • Pignatelli M.
      • Arons A.
      • Tonegawa S.
      Engram cells retain memory under retrograde amnesia.
      ). 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 (
      • Tonegawa S.
      • Pignatelli M.
      • Roy D.S.
      • Ryan T.J.
      Memory engram storage and retrieval.
      ). 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 (
      • Abdou K.
      • Shehata M.
      • Choko K.
      • Nishizono H.
      • Matsuo M.
      • Muramatsu S.
      • Inokuchi K.
      Synapse-specific representation of the identity of overlapping memory engrams.
      ,
      • Chen S.
      • Cai D.
      • Pearce K.
      • Sun P.Y.W.
      • Roberts A.C.
      • Glanzman D.L.
      Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in Aplysia.
      ,
      • Shrestha P.
      • Ayata P.
      • Herrero-Vidal P.
      • Longo F.
      • Gastone A.
      • LeDoux J.E.
      • Heintz N.
      • Klann E.
      Cell-type-specific drug-inducible protein synthesis inhibition demonstrates that memory consolidation requires rapid neuronal translation.
      ). A recent study based on genetic blockage of protein translation substantially expanded on these findings in the amygdala (
      • Shrestha P.
      • Ayata P.
      • Herrero-Vidal P.
      • Longo F.
      • Gastone A.
      • LeDoux J.E.
      • Heintz N.
      • Klann E.
      Cell-type-specific drug-inducible protein synthesis inhibition demonstrates that memory consolidation requires rapid neuronal translation.
      ). In agreement with these findings, studies based on the use of amnesia-inducing drugs in Aplysia also demonstrated that memory does not depend on L-LTP to be stored (
      • Chen S.
      • Cai D.
      • Pearce K.
      • Sun P.Y.W.
      • Roberts A.C.
      • Glanzman D.L.
      Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in Aplysia.
      ). Other studies covered other types of amnesia, where memory was also hidden but not destroyed: Alzheimer’s disease (
      • Perusini J.N.
      • Cajigas S.A.
      • Cohensedgh O.
      • Lim S.C.
      • Pavlova I.P.
      • Donaldson Z.R.
      • Denny C.A.
      Optogenetic stimulation of dentate gyrus engrams restores memory in Alzheimer’s disease mice.
      ,
      • Poll S.
      • Mittag M.
      • Musacchio F.
      • Justus L.C.
      • Giovannetti E.A.
      • Steffen J.
      • Wagner J.
      • Zohren L.
      • Schoch S.
      • Schmidt B.
      • Jackson W.S.
      • Ehninger D.
      • Fuhrmann M.
      Memory trace interference impairs recall in a mouse model of Alzheimer’s disease.
      ,
      • Roy D.S.
      • Arons A.
      • Mitchell T.I.
      • Pignatelli M.
      • Ryan T.J.
      • Tonegawa S.
      Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease.
      ) or infantile amnesia (
      • Guskjolen A.
      • Kenney J.W.
      • de la Parra J.
      • Yeung B.R.A.
      • Josselyn S.A.
      • Frankland P.W.
      Recovery of “lost” infant memories in mice.
      ).

      Storing as connections

      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) (
      • Chklovskii D.B.
      • Mel B.W.
      • Svoboda K.
      Cortical rewiring and information storage.
      ). One hypothesis is that memory is stored in the stable connectivity pattern between engram cells (
      • Ryan T.J.
      • Ortega-de San Luis C.
      • Pezzoli M.
      • Sen S.
      Engram cell connectivity: An evolving substrate for information storage.
      ,
      • Tonegawa S.
      • Pignatelli M.
      • Roy D.S.
      • Ryan T.J.
      Memory engram storage and retrieval.
      ), which remains unaltered in memories that can be reactivated by artificial (induced by light) but not natural recall (induced by contextual cues) (
      • Ryan T.J.
      • Roy D.S.
      • Pignatelli M.
      • Arons A.
      • Tonegawa S.
      Engram cells retain memory under retrograde amnesia.
      ). 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 (
      • Harris K.M.
      Structure, development, and plasticity of dendritic spines.
      ). They have been proposed to be relevant for memory storage since they undergo structural remodeling after plastic changes. Their remodeling, determined by the actin cytoskeleton (
      • Cingolani L.A.
      • Goda Y.
      Actin in action: The interplay between the actin cytoskeleton and synaptic efficacy.
      ), contributes to both modifying synaptic weight and synaptic wiring or connectivity. Dendritic turnover, although higher during development, also happens in adult brains (
      • Bourne J.
      • Harris K.M.
      Do thin spines learn to be mushroom spines that remember?.
      ,
      • Holtmaat A.J.G.D.
      • Trachtenberg J.T.
      • Wilbrecht L.
      • Shepherd G.M.
      • Zhang X.
      • Knott G.W.
      • Svoboda K.
      Transient and persistent dendritic spines in the neocortex in vivo.
      ,
      • Yu X.
      • Zuo Y.
      Spine plasticity in the motor cortex.
      ). Rapidly formed after an experience, they have been suggested to act as a lasting structural ground for memory storage (
      • Attardo A.
      • Fitzgerald J.E.
      • Schnitzer M.J.
      Impermanence of dendritic spines in live adult CA1 hippocampus.
      ,
      • Yang G.
      • Pan F.
      • Gan W.-B.
      Stably maintained dendritic spines are associated with lifelong memories.
      ,
      • Yuste R.
      • Bonhoeffer T.
      Morphological changes in dendritic spines associated with long-term synaptic plasticity.
      ). Shape-wise thin-shaped spines have been associated with learning mechanisms (
      • Bourne J.
      • Harris K.M.
      Do thin spines learn to be mushroom spines that remember?.
      ,
      • Xu T.
      • Yu X.
      • Perlik A.J.
      • Tobin W.F.
      • Zweig J.A.
      • Tennant K.
      • Jones T.
      • Zuo Y.
      Rapid formation and selective stabilization of synapses for enduring motor memories.
      ), whereas mushroom-shaped dendrites have been associated with memory storage since they anchor more postsynaptic molecules to their membrane (reviewed in Ref. (
      • Bourne J.
      • Harris K.M.
      Do thin spines learn to be mushroom spines that remember?.
      )). 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 (
      • Choi D.I.
      • Kim J.
      • Lee H.
      • Kim J.
      • Sung Y.
      • Choi J.E.
      • Venkat S.J.
      • Park P.
      • Jung H.
      • Kaang B.-K.
      Synaptic correlates of associative fear memory in the lateral amygdala.
      ). 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 (
      • Choi D.I.
      • Kim J.
      • Lee H.
      • Kim J.
      • Sung Y.
      • Choi J.E.
      • Venkat S.J.
      • Park P.
      • Jung H.
      • Kaang B.-K.
      Synaptic correlates of associative fear memory in the lateral amygdala.
      ). In the DG, engram cells showed a higher proportion of mushroom spines 5 days after encoding than engrams analyzed 24 h after encoding (
      • Marco A.
      • Meharena H.S.
      • Dileep V.
      • Raju R.M.
      • Davila-Velderrain J.
      • Zhang A.L.
      • Adaikkan C.
      • Young J.Z.
      • Gao F.
      • Kellis M.
      • Tsai L.H.
      Mapping the epigenomic and transcriptomic interplay during memory formation and recall in the hippocampal engram ensemble.
      ). Then hippocampal engram cells decrease their spine density as memory consolidates into a different area of the brain, the cortex, where engram neurons increase their spine density (
      • Kitamura T.
      • Ogawa S.K.
      • Roy D.S.
      • Okuyama T.
      • Morrissey M.D.
      • Smith L.M.
      • Redondo R.L.
      • Tonegawa S.
      Engrams and circuits crucial for systems consolidation of a memory.
      ), which suggests that they associate with memory storage in a long term and ability to recall a memory.
      New connections between engram cells might also be due to unsilencing synapses (Fig. 3). Silent synapses are synapses that are functionally silent—having NMDARs, they lack AMPA ones (
      • Isaac J.T.
      • Nicoll R.A.
      • Malenka R.C.
      Evidence for silent synapses: Implications for the expression of LTP.
      ,
      • Liao D.
      • Hessler N.A.
      • Malinow R.
      Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice.
      ). Supporting this view, engram cells have shown a higher presence of silent synapses than nonengram cells in the nucleus accumbens (
      • Koya E.
      • Cruz F.C.
      • Ator R.
      • Golden S.A.
      • Hoffman A.F.
      • Lupica C.R.
      • Hope B.T.
      Silent synapses in selectively activated nucleus accumbens neurons following cocaine sensitization.
      ,