Activation of Gs Signaling in Cortical Astrocytes Does Not Influence Formation of a Persistent Contextual Memory Engram.

Formation and retrieval of remote contextual memory depends on cortical engram neurons that are defined during learning. Manipulation of astrocytic Gq and Gi associated G-protein coupled receptor (GPCR) signaling has been shown to affect memory processing, but little is known about the role of cortical astrocytic Gs-GPCR signaling in remote memory acquisition and the functioning of cortical engram neurons. We assessed this by chemogenetic manipulation of astrocytes in the medial prefrontal cortex (mPFC) of male mice, during either encoding or consolidation of a contextual fear memory, while simultaneously labeling cortical engram neurons. We found that stimulation of astrocytic Gs signaling during memory encoding and consolidation did not alter remote memory expression. In line with this, the size of the mPFC engram population and the recall-induced reactivation of these neurons was unaffected. Hence, our data indicate that activation of Gs-GPCR signaling in cortical astrocytes is not sufficient to alter memory performance and functioning of cortical engram neurons.

Shaping Memories via Stress: A Synaptic Engram Perspective.

Stress modulates the activity of various memory systems and can thereby guide behavioral interaction with the environment in an adaptive or maladaptive manner. At the cellular level, a large body of evidence indicates that (nor)adrenaline and glucocorticoid release induced by acute stress exposure affects synapse function and synaptic plasticity, which are critical substrates for learning and memory. Recent evidence suggests that memories are supported in the brain by sparsely distributed neurons within networks, termed engram cell ensembles. While the physiological and molecular effects of stress on the synapse are increasingly well characterized, how these synaptic modifications shape the multiscale dynamics of engram cell ensembles is still poorly understood. In this review, we discuss and integrate recent information on how acute stress affects synapse function and how this may alter engram cell ensembles and their synaptic connectivity to shape memory strength and memory precision. We provide a mechanistic framework of a synaptic engram under stress and put forward outstanding questions that address knowledge gaps in our understanding of the mechanisms that underlie stress-induced memory modulation.

Retraction of Astrocyte Leaflets From the Synapse Enhances Fear Memory.

BACKGROUND: The formation and retrieval of fear memories depends on orchestrated synaptic activity of neuronal ensembles within the hippocampus, and it is becoming increasingly evident that astrocytes residing in the environment of these synapses play a central role in shaping cellular memory representations. Astrocyte distal processes, known as leaflets, fine-tune synaptic activity by clearing neurotransmitters and limiting glutamate diffusion. However, how astroglial synaptic coverage contributes to mnemonic processing of fearful experiences remains largely unknown. METHODS: We used electron microscopy to observe changes in astroglial coverage of hippocampal synapses during consolidation of fear memory in mice. To manipulate astroglial synaptic coverage, we depleted ezrin, an integral leaflet-structural protein, from hippocampal astrocytes using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing. Next, a combination of Föster resonance energy transfer analysis, genetically encoded glutamate sensors, and whole-cell patch-clamp recordings was used to determine whether the proximity of astrocyte leaflets to the synapse is critical for synaptic integrity and function. RESULTS: We found that consolidation of a recent fear memory is accompanied by a transient retraction of astrocyte leaflets from hippocampal synapses and increased activation of NMDA receptors. Accordingly, astrocyte-specific depletion of ezrin resulted in shorter astrocyte leaflets and reduced astrocyte contact with the synaptic cleft, which consequently boosted extrasynaptic glutamate diffusion and NMDA receptor activation. Importantly, after fear conditioning, these cellular phenotypes translated to increased retrieval-evoked activation of CA1 pyramidal neurons and enhanced fear memory expression. CONCLUSIONS: Together, our data show that withdrawal of astrocyte leaflets from the synaptic cleft is an experience-induced, temporally regulated process that gates the strength of fear memories.

The CADM2 Gene and Behavior: A Phenome-Wide Scan in UK-Biobank.

The cell adhesion molecule 2 (CADM2) gene has appeared among the top associations in a wide range of genome-wide association studies (GWASs). This study aims to: (1) examine how widespread the role of CADM2 is in behavioural traits, and (2) investigate trait-specific effects on CADM2 expression levels across tissues. We conducted a phenome-wide association study in UK Biobank (N = 12,211-453,349) on 242 psycho-behavioral traits, both at the SNP and the gene-level. For comparison, we repeated the analyses for other large (and high LD) genes. We found significant associations between CADM2 and 50 traits (including cognitive, risk taking, and dietary traits), many more than for the comparison genes. We show that many trait associations are reduced when taking geographical stratification into account. S-Predixcan revealed that CADM2 expression in brain tissues was significantly associated with many traits; highly significant effects were also observed for lung, mammary, and adipose tissues. In conclusion, this study shows that the role of CADM2 extends to a wide range of psycho-behavioral traits, suggesting these traits may share a common biological denominator.

Extinction of Cocaine Memory Depends on a Feed-Forward Inhibition Circuit Within the Medial Prefrontal Cortex.

BACKGROUND: Cocaine-associated environments (i.e., contexts) evoke persistent memories of cocaine reward and thereby contribute to the maintenance of addictive behavior in cocaine users. From a therapeutic perspective, enhancing inhibitory control over cocaine-conditioned responses is of pivotal importance but requires a more detailed understanding of the neural circuitry that can suppress context-evoked cocaine memories, e.g., through extinction learning. The ventral medial prefrontal cortex (vmPFC) and dorsal medial prefrontal cortex (dmPFC) are thought to bidirectionally regulate responding to cocaine cues through their projections to other brain regions. However, whether these mPFC subregions interact to enable adaptive responding to cocaine-associated contextual stimuli has remained elusive. METHODS: We used antero- and retrograde tracing combined with chemogenetic intervention to examine the role of vmPFC-to-dmPFC projections in extinction of cocaine-induced place preference in mice. In addition, electrophysiological recordings and optogenetics were used to determine whether parvalbumin-expressing inhibitory interneurons and pyramidal neurons in the dmPFC are innervated by vmPFC projections. RESULTS: We found that vmPFC-to-dmPFC projecting neurons are activated during unreinforced re-exposure to a cocaine-associated context, and selective suppression of these cells impairs extinction learning. Parvalbumin-expressing inhibitory interneurons in the dmPFC receive stronger monosynaptic excitatory input from vmPFC projections than local dmPFC pyramidal neurons, consequently resulting in disynaptic inhibition of pyramidal neurons. In line with this, we show that chemogenetic suppression of dmPFC parvalbumin-expressing inhibitory interneurons impairs extinction learning. CONCLUSIONS: Our data reveal that vmPFC projections mediate extinction of a cocaine-associated contextual memory through recruitment of feed-forward inhibition in the dmPFC, thereby providing a novel neuronal substrate that promotes extinction-induced inhibitory control.

Glucocorticoids Promote Fear Generalization by Increasing the Size of a Dentate Gyrus Engram Cell Population.

BACKGROUND: Traumatic experiences, such as conditioned threat, are coded as enduring memories that are frequently subject to generalization, which is characterized by (re-) expression of fear in safe environments. However, the neurobiological mechanisms underlying threat generalization after a traumatic experience and the role of stress hormones in this process remain poorly understood. METHODS: We examined the influence of glucocorticoid hormones on the strength and specificity of conditioned fear memory at the level of sparsely distributed dentate gyrus (DG) engram cells in male mice. RESULTS: We found that elevating glucocorticoid hormones after fear conditioning induces a generalized contextual fear response. This was accompanied by a selective and persistent increase in the excitability and number of activated DG granule cells. Selective chemogenetic suppression of these sparse cells in the DG prevented glucocorticoid-induced fear generalization and restored contextual memory specificity, while leaving expression of auditory fear memory unaffected. CONCLUSIONS: These results implicate the sparse ensemble of DG engram cells as a critical cellular substrate underlying fear generalization induced by glucocorticoid stress hormones.

A neural substrate of compulsive alcohol use.

Alcohol intake remains controlled in a majority of users but becomes "compulsive," i.e., continues despite adverse consequences, in a minority who develop alcohol addiction. Here, using a footshock-punished alcohol self-administration procedure, we screened a large population of outbred rats to identify those showing compulsivity operationalized as punishment-resistant self-administration. Using unsupervised clustering, we found that this behavior emerged as a stable trait in a subpopulation of rats and was associated with activity of a brain network that included central nucleus of the amygdala (CeA). Activity of PKCδ+ inhibitory neurons in the lateral subdivision of CeA (CeL) accounted for ~75% of variance in punishment-resistant alcohol taking. Activity-dependent tagging, followed by chemogenetic inhibition of neurons activated during punishment-resistant self-administration, suppressed alcohol taking, as did a virally mediated shRNA knockdown of PKCδ in CeA. These findings identify a previously unknown mechanism for a core element of alcohol addiction and point to a novel candidate therapeutic target.

A Synaptic Framework for the Persistence of Memory Engrams.

The ability to store and retrieve learned information over prolonged periods of time is an essential and intriguing property of the brain. Insight into the neurobiological mechanisms that underlie memory consolidation is of utmost importance for our understanding of memory persistence and how this is affected in memory disorders. Recent evidence indicates that a given memory is encoded by sparsely distributed neurons that become highly activated during learning, so-called engram cells. Research by us and others confirms the persistent nature of cortical engram cells by showing that these neurons are required for memory expression up to at least 1 month after they were activated during learning. Strengthened synaptic connectivity between engram cells is thought to ensure reactivation of the engram cell network during retrieval. However, given the continuous integration of new information into existing neuronal circuits and the relatively rapid turnover rate of synaptic proteins, it is unclear whether a lasting learning-induced increase in synaptic connectivity is mediated by stable synapses or by continuous dynamic turnover of synapses of the engram cell network. Here, we first discuss evidence for the persistence of engram cells and memory-relevant adaptations in synaptic plasticity, and then propose models of synaptic adaptations and molecular mechanisms that may support memory persistence through the maintenance of enhanced synaptic connectivity within an engram cell network.

A persistent alcohol cue memory trace drives relapse to alcohol seeking after prolonged abstinence.

Alcohol use disorder is characterized by a high risk of relapse during periods of abstinence. Relapse is often triggered by retrieval of persistent alcohol memories upon exposure to alcohol-associated environmental cues, but little is known about the neuronal circuitry that supports the long-term storage of alcohol cue associations. We found that a small ensemble of neurons in the medial prefrontal cortex (mPFC) of mice was activated during cue-paired alcohol self-administration (SA) and that selective suppression of these neurons 1 month later attenuated cue-induced relapse to alcohol seeking. Inhibition of alcohol seeking was specific to these neurons as suppression of a non-alcohol-related or sucrose SA-activated mPFC ensemble did not affect relapse behavior. Hence, the mPFC neuronal ensemble activated during cue-paired alcohol consumption functions as a lasting memory trace that mediates cue-evoked relapse long after cessation of alcohol intake, thereby providing a potential target for treatment of alcohol relapse vulnerability.

Engram-specific transcriptome profiling of contextual memory consolidation.

Sparse populations of neurons in the dentate gyrus (DG) of the hippocampus are causally implicated in the encoding of contextual fear memories. However, engram-specific molecular mechanisms underlying memory consolidation remain largely unknown. Here we perform unbiased RNA sequencing of DG engram neurons 24 h after contextual fear conditioning to identify transcriptome changes specific to memory consolidation. DG engram neurons exhibit a highly distinct pattern of gene expression, in which CREB-dependent transcription features prominently (P = 6.2 × 10-13), including Atf3 (P = 2.4 × 10-41), Penk (P = 1.3 × 10-15), and Kcnq3 (P = 3.1 × 10-12). Moreover, we validate the functional relevance of the RNAseq findings by establishing the causal requirement of intact CREB function specifically within the DG engram during memory consolidation, and identify a novel group of CREB target genes involved in the encoding of long-term memory.

Memory strength gates the involvement of a CREB-dependent cortical fear engram in remote memory.

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.

The Extracellular Matrix Protein Brevican Limits Time-Dependent Enhancement of Cocaine Conditioned Place Preference.

Cocaine-associated environmental cues sustain relapse vulnerability by reactivating long-lasting memories of cocaine reward. During periods of abstinence, responding to cocaine cues can time-dependently intensify a phenomenon referred to as 'incubation of cocaine craving'. Here, we investigated the role of the extracellular matrix protein brevican in recent (1 day after training) and remote (3 weeks after training) expression of cocaine conditioned place preference (CPP). Wild-type and Brevican heterozygous knock-out mice, which express brevican at ~50% of wild-type levels, received three cocaine-context pairings using a relatively low dose of cocaine (5 mg/kg). In a drug-free CPP test, heterozygous mice showed enhanced preference for the cocaine-associated context at the remote time point compared with the recent time point. This progressive increase was not observed in wild-type mice and it did not generalize to contextual-fear memory. Virally mediated overexpression of brevican levels in the hippocampus, but not medial prefrontal cortex, of heterozygous mice prevented the progressive increase in cocaine CPP, but only when overexpression was induced before conditioning. Post-conditioning overexpression of brevican did not affect remote cocaine CPP, suggesting that brevican limited the increase in remote CPP by altering neuro-adaptive mechanisms during cocaine conditioning. We provide causal evidence that hippocampal brevican levels control time-dependent enhancement of cocaine CPP during abstinence, pointing to a novel substrate that regulates incubation of responding to cocaine-associated cues.

Optogenetic dissection of medial prefrontal cortex circuitry.

The medial prefrontal cortex (mPFC) is critically involved in numerous cognitive functions, including attention, inhibitory control, habit formation, working memory and long-term memory. Moreover, through its dense interconnectivity with subcortical regions (e.g., thalamus, striatum, amygdala and hippocampus), the mPFC is thought to exert top-down executive control over the processing of aversive and appetitive stimuli. Because the mPFC has been implicated in the processing of a wide range of cognitive and emotional stimuli, it is thought to function as a central hub in the brain circuitry mediating symptoms of psychiatric disorders. New optogenetics technology enables anatomical and functional dissection of mPFC circuitry with unprecedented spatial and temporal resolution. This provides important novel insights in the contribution of specific neuronal subpopulations and their connectivity to mPFC function in health and disease states. In this review, we present the current knowledge obtained with optogenetic methods concerning mPFC function and dysfunction and integrate this with findings from traditional intervention approaches used to investigate the mPFC circuitry in animal models of cognitive processing and psychiatric disorders.

Neural ECM in addiction, schizophrenia, and mood disorder.

The extracellular matrix (ECM) has a prominent role in brain development, maturation of neural circuits, and adult neuroplasticity. This multifactorial role of the ECM suggests that processes that affect composition or turnover of ECM in the brain could lead to altered brain function, possibly underlying conditions of impaired mental health, such as neuropsychiatric or neurodegenerative disease. In support of this, in the last two decades, clinical and preclinical research provided evidence of correlations and to some degree causal links, between aberrant ECM function and neuropsychiatric disorders, the most prominent being addiction and schizophrenia. Based on these initial observations of involvement of different classes of ECM molecules (laminin, reelin, and their integrin receptors, as well as tenascins and chondroitin sulfate proteoglycans), ECM targets have been suggested as a novel entry point in the treatment of neuropsychiatric disorders. Hence, understanding how ECM molecules contribute to proper neuronal functioning and how this is dysregulated in conditions of mental illness is of pivotal importance. In this chapter, we will review available literature that implicates the different classes of brain ECM molecules in psychiatric disorders, with a primary focus on addiction (opiates, psychostimulants, and alcohol), and we will compare these ECM adaptations with those implicated in schizophrenia and mood disorders.

Manipulating a "cocaine engram" in mice.

Experience with drugs of abuse (such as cocaine) produces powerful, long-lasting memories that may be important in the development and persistence of drug addiction. The neural mechanisms that mediate how and where these cocaine memories are encoded, consolidated and stored are unknown. Here we used conditioned place preference in mice to examine the precise neural circuits that support the memory of a cocaine-cue association (the "cocaine memory trace" or "cocaine engram"). We found that a small population of neurons (∼10%) in the lateral nucleus of amygdala (LA) were recruited at the time of cocaine-conditioning to become part of this cocaine engram. Neurons with increased levels of the transcription factor CREB were preferentially recruited or allocated to the cocaine engram. Ablating or silencing neurons overexpressing CREB (but not a similar number of random LA neurons) before testing disrupted the expression of a previously acquired cocaine memory, suggesting that neurons overexpressing CREB become a critical hub in what is likely a larger cocaine memory engram. Consistent with theories that coordinated postencoding reactivation of neurons within an engram or cell assembly is crucial for memory consolidation (Marr, 1971; Buzsáki, 1989; Wilson and McNaughton, 1994; McClelland et al., 1995; Girardeau et al., 2009; Dupret et al., 2010; Carr et al., 2011), we also found that post-training suppression, or nondiscriminate activation, of CREB overexpressing neurons impaired consolidation of the cocaine memory. These findings reveal mechanisms underlying how and where drug memories are encoded and stored in the brain and may also inform the development of treatments for drug addiction.

Ventromedial prefrontal cortex pyramidal cells have a temporal dynamic role in recall and extinction of cocaine-associated memory.

In addicts, associative memories related to the rewarding effects of drugs of abuse can evoke powerful craving and drug seeking urges, but effective treatment to suppress these memories is not available. Detailed insight into the neural circuitry that mediates expression of drug-associated memory is therefore of crucial importance. Substantial evidence from rodent models of addictive behavior points to the involvement of the ventromedial prefrontal cortex (vmPFC) in conditioned drug seeking, but specific knowledge of the temporal role of vmPFC pyramidal cells is lacking. To this end, we used an optogenetics approach to probe the involvement of vmPFC pyramidal cells in expression of a recent and remote conditioned cocaine memory. In mice, we expressed Channelrhodopsin-2 (ChR2) or Halorhodopsin (eNpHR3.0) in pyramidal cells of the vmPFC and studied the effect of activation or inhibition of these cells during expression of a cocaine-contextual memory on days 1-2 (recent) and ∼3 weeks (remote) after conditioning. Whereas optical activation of pyramidal cells facilitated extinction of remote memory, without affecting recent memory, inhibition of pyramidal cells acutely impaired recall of recent cocaine memory, without affecting recall of remote memory. In addition, we found that silencing pyramidal cells blocked extinction learning at the remote memory time-point. We provide causal evidence of a critical time-dependent switch in the contribution of vmPFC pyramidal cells to recall and extinction of cocaine-associated memory, indicating that the circuitry that controls expression of cocaine memories reorganizes over time.

The synaptic pathology of drug addiction.

A hallmark of drug addiction is the uncontrollable desire to consume drugs at the expense of severe negative consequences. Moreover, addicts that successfully refrain from drug use have a high vulnerability to relapse even after months or years of abstinence. In this chapter, we will discuss the current understanding of drug-induced neuroplasticity within the mesocorticolimbic brain system that contributes to the development of addiction and the persistence of relapse to drug seeking. I particular, we will focus at animal models that can be translated to human addiction. Although dopaminergic transmission is important for the acute effects of drug intake, the long-lived behavioral abnormalities associated with addiction are thought to arise from pathological plasticity in glutamatergic neurotransmission. The nature of changes in excitatory synaptic plasticity depends on several factors, including the type of drug, the brain area, and the time-point studied in the transition of drug exposure to withdrawal and relapse to drug seeking. Identification of drug-induced neuroplasticity is crucial to understand how molecular and cellular adaptations contribute to the end stage of addiction, which from a clinical perspective, is a time-point where pharmacotherapy may be most effectively employed.

Extracellular matrix plasticity and GABAergic inhibition of prefrontal cortex pyramidal cells facilitates relapse to heroin seeking.

Successful treatment of drug addiction is hampered by high relapse rates during periods of abstinence. Neuroadaptation in the medial prefrontal cortex (mPFC) is thought to have a crucial role in vulnerability to relapse to drug seeking, but the molecular and cellular mechanisms remain largely unknown. To identify protein changes that contribute to relapse susceptibility, we investigated synaptic membrane fractions from the mPFC of rats that underwent 21 days of forced abstinence following heroin self-administration. Quantitative proteomics revealed that long-term abstinence from heroin self-administration was associated with reduced levels of extracellular matrix (ECM) proteins. After extinction of heroin self-administration, downregulation of ECM proteins was also present in the mPFC, as well as nucleus accumbens (NAc), and these adaptations were partially restored following cue-induced reinstatement of heroin seeking. In the mPFC, these ECM proteins are condensed in the perineuronal nets that exclusively surround GABAergic interneurons, indicating that ECM adaptation might alter the activity of GABAergic interneurons. In support of this, we observed an increase in the inhibitory GABAergic synaptic inputs received by the mPFC pyramidal cells after the re-exposure to heroin-conditioned cues. Recovering levels of ECM constituents by metalloproteinase inhibitor treatment (FN-439; i.c.v.) prior to a reinstatement test attenuated subsequent heroin seeking, suggesting that the reduced synaptic ECM levels during heroin abstinence enhanced sensitivity to respond to heroin-conditioned cues. We provide evidence for a novel neuroadaptive mechanism, in which heroin self-administration-induced adaptation of the ECM increased relapse vulnerability, potentially by augmenting the responsivity of mPFC GABAergic interneurons to heroin-associated stimuli.

Prefrontal cortex plasticity mechanisms in drug seeking and relapse.

Development of pharmacotherapy to reduce relapse rates is one of the biggest challenges in drug addiction research. The enduring nature of relapse suggests that it is maintained by long-lasting molecular and cellular adaptations in the neuronal circuitry that mediates learning and processing of motivationally relevant stimuli. Studies employing the reinstatement model of drug relapse in rodents point to an important role of the medial prefrontal cortex (mPFC), with distinct contributions of the dorsal and ventral regions of the mPFC to drug-, stress- and cue-induced drug seeking. Whereas drug-induced neuroadaptations in the dorsal mPFC function to enhance excitatory output and drive expression of drug seeking, recent evidence suggests that plasticity in the ventral mPFC leads to reduced glutamatergic transmission in this region, thereby impairing response inhibition upon exposure to drug-conditioned stimuli. Treatments aimed at restoring drug-induced neuroadaptations in the mPFC may help to reduce cue-reactivity and relapse susceptibility.