Differential Contributions of Glutamatergic Hippocampal→Retrosplenial Cortical Projections to the Formation and Persistence of Context Memories.

Learning to associate stressful events with specific environmental contexts depends on excitatory transmission in the hippocampus, but how this information is transmitted to the neocortex for lasting memory storage is unclear. We identified dorsal hippocampal (DH) projections to the retrosplenial cortex (RSC), which arise mainly from the subiculum and contain either the vesicular glutamate transporter 1 (vGlut1) or vGlut2. Both vGlut1+ and vGlut2+ axons strongly excite and disynaptically inhibit RSC pyramidal neurons in superficial layers, but vGlut2+ axons trigger greater inhibition that spreads to deep layers, indicating that these pathways engage RSC circuits via partially redundant, partially differentiated cellular mechanisms. Using contextual fear conditioning in mice to model contextual associative memories, together with chemogenetic axonal silencing, we found that vGlut1+ projections are principally involved in processing recent context memories whereas vGlut2+ projections contribute to their long-lasting storage. Thus, within the DH→RSC pathway, engagement of vGlut1+ and vGlut2+ circuits differentially contribute to the formation and persistence of fear-inducing context memories.

Role of retrosplenial cortex in processing stress-related context memories.

This work summarizes evidence for the role of RSC in processing fear-inducing context memories. Specifically, we discuss molecular, cellular, and network mechanisms by which RSC might contribute the processing of contextual fear memories. We focus on glutamatergic and cholinergic mechanisms underlying encoding, retrieval, and extinction of context-dependent fear. RSC mechanisms underlying retrieval of recently and remotely acquired memories are compared to memory mechanisms of anterior cortices. Due to the strong connectivity between hippocampus and RSC, we also compare the extent to which their mechanisms of encoding, retrieval, and extinction show overlap. At a theoretical level, we discuss the role of RSC in the framework of systems consolidation as well as retrieval-induced memory modulation. Lastly, we emphasize the implication of these findings for psychopathologies associated with neurological and psychiatric disorders. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Network oscillatory activity driven by context memory processing is differently regulated by glutamatergic and cholinergic neurotransmission.

Memory retrieval requires coordinated intra- and inter-regional activity in networks of brain structures. Dysfunction of these networks and memory impairment are seen in many psychiatric disorders, but relatively little is known about how memory retrieval and memory failure are represented at the level of local and regional oscillatory activity. To address this question, we measured local field potentials (LFPs) from mice as they explored a novel context, retrieved memories for contextual fear conditioning, and after administration of two amnestic agents: the NMDA receptor antagonist MK-801 and muscarinic acetylcholine receptor antagonist scopolamine (SCOP). LFPs were simultaneously recorded from retrosplenial cortex (RSC), dorsal hippocampus (DH), and anterior cingulate cortex (ACC), which are involved in processing contextual memories, and analyzed for changes in intra-regional power and inter-regional peak coherence of oscillations across multiple frequency bands. Context encoding and memory retrieval sessions yielded similar patterns of changes across all three structures, including decreased delta power and increased theta peak coherence. Baseline effects of MK-801 and SCOP were primarily targeted to gamma oscillations, but in opposite directions. Both drugs also blocked memory retrieval, as indicated by reduced freezing when mice were returned to the conditioning context, but this common behavioral impairment was only associated with power and peak coherence disruptions after MK-801 treatment. These findings point to neural signatures for memory impairment, whose underlying mechanisms may serve as therapeutic targets for related psychiatric disorders.

Neurobiological correlates of state-dependent context fear.

Retrieval of fear memories can be state-dependent, meaning that they are best retrieved if the brain states at encoding and retrieval are similar. Such states can be induced by activating extrasynaptic γ-aminobutyric acid type A receptors (GABAAR) with the broad α-subunit activator gaboxadol. However, the circuit mechanisms and specific subunits underlying gaboxadol's effects are not well understood. Here we show that gaboxadol induces profound changes of local and network oscillatory activity, indicative of discoordinated hippocampal-cortical activity, that were accompanied by robust and long-lasting state-dependent conditioned fear. Episodic memories typically are hippocampus-dependent for a limited period after learning, but become cortex-dependent with the passage of time. In contrast, state-dependent memories continued to rely on hippocampal GABAergic mechanisms for memory retrieval. Pharmacological approaches with α-subunit-specific agonists targeting the hippocampus implicated the prototypic extrasynaptic subunits (α4) as the mediator of state-dependent conditioned fear. Together, our findings suggest that continued dependence on hippocampal rather than cortical mechanisms could be an important feature of state-dependent memories that contributes to their conditional retrieval.

Muscarinic acetylcholine receptors act in synergy to facilitate learning and memory.

Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on which mAChR subtypes are critical for memory processing. Using pharmacological and genetic approaches, we found that (1) encoding and retrieval of contextual memory requires mAChR in the dorsal hippocampus (DH) and retrosplenial cortex (RSC), (2) memory formation requires hippocampal M3 and cooperative activity of RSC M1 and M3, and (3) memory retrieval is more impaired by inactivation of multiple M1-M4 mAChR in DH or RSC than inactivation of individual receptor subtypes. Contrary to the view that acetylcholine supports learning but is detrimental to memory retrieval, we found that coactivation of multiple mAChR is required for retrieval of both recently and remotely acquired context memories. Manipulations with higher receptor specificity were generally less potent than manipulations targeting multiple receptor subtypes, suggesting that mAChR act in synergy to regulate memory processes. These findings provide unique insight into the development of therapies for amnestic symptoms, suggesting that broadly acting, rather than receptor-specific, mAchR agonists and positive allosteric modulators may be the most effective therapeutic approach.

Analysis of coherent activity between retrosplenial cortex, hippocampus, thalamus, and anterior cingulate cortex during retrieval of recent and remote context fear memory.

Memory for contextual fear conditioning relies upon the retrosplenial cortex (RSC) regardless of how long ago conditioning occurred, whereas areas connected to the RSC, such as the dorsal hippocampus (DH) and anterior cingulate cortex (ACC) appear to play time-limited roles. To better understand whether these brain regions functionally interact during memory processing and how the passage of time affects these interactions, we simultaneously recorded local field potentials (LFPs) from these three regions as well as anterior dorsal thalamus (ADT), which provides one of the strongest inputs to RSC, and measured coherence of oscillatory activity within the theta (4-12Hz) and gamma (30-80Hz) frequency bands. We identified changes of theta coherence related to encoding, retrieval, and extinction of context fear, whereas changes in gamma coherence were restricted to fear extinction. Specifically, exposure to a novel context and retrieval of recently acquired fear conditioning memory were associated with increased theta coherence between RSC and all three other structures. In contrast, RSC-DH and RSC-ADT theta coherence were decreased in mice that successfully retrieved, relative to mice that failed to retrieve, remote memory. Greater RSC-ADT theta and gamma coherence were observed during recent, compared to remote, extinction of freezing responses. Thus, the degree of coherence between RSC and connected brain areas may predict and contribute to context memory retrieval and retrieval-related phenomena such as fear extinction. Importantly, although theta coherence in this circuit increases during memory encoding and retrieval of recent memory, failure to decrease RSC-DH theta coherence might be linked to retrieval deficit in the long term, and possibly contribute to aberrant memory processing characteristic of neuropsychiatric disorders.

GABAergic mechanisms regulated by miR-33 encode state-dependent fear.

Fear-inducing memories can be state dependent, meaning that they can best be retrieved if the brain states at encoding and retrieval are similar. Restricted access to such memories can present a risk for psychiatric disorders and hamper their treatment. To better understand the mechanisms underlying state-dependent fear, we used a mouse model of contextual fear conditioning. We found that heightened activity of hippocampal extrasynaptic GABAA receptors, believed to impair fear and memory, actually enabled their state-dependent encoding and retrieval. This effect required protein kinase C-ßII and was influenced by miR-33, a microRNA that regulates several GABA-related proteins. In the extended hippocampal circuit, extrasynaptic GABAA receptors promoted subcortical, but impaired cortical, activation during memory encoding of context fear. Moreover, suppression of retrosplenial cortical activity, which normally impairs retrieval, had an enhancing effect on the retrieval of state-dependent fear. These mechanisms can serve as treatment targets for managing access to state-dependent memories of stressful experiences.

Double Dissociation of the Roles of Metabotropic Glutamate Receptor 5 and Oxytocin Receptor in Discrete Social Behaviors.

Social interactions in vertebrates are complex phenomena based on affective and cognitive processes. Multiple brain regions and neurotransmitter systems are involved in the expression of social behaviors, but their individual roles in specific aspects of social interactions are not well understood. Here we investigated how Gq-protein-coupled metabotropic glutamate receptor 5 (mGluR5) and oxytocin receptor (Oxtr) affect social affiliation and social memory. We used conditional genetic approaches in which the genes coding for these receptors were knocked out in the lateral septum by infusion of recombinant adeno-associated viral vectors containing Cre recombinase (AAV-Cre). Social behavior was assessed 2 weeks later using a three-chamber paradigm for sociability and preference for social novelty. Septal deletion of mGluR5 abolished sociability while leaving preference for social novelty intact. In contrast, deletion of Oxtr did not affect sociability but significantly impaired preference for social novelty. Nonsocial behaviors or memories, including novel object recognition or fear conditioning, were not affected by these genetic manipulations. Immunohistochemical analyses of the distribution of mGluR5 and Oxtr revealed non-overlapping localization of these receptors within the lateral septum, suggesting that not only different neurotransmitters but also different neuronal types contribute to sociability versus preference for social novelty. Our findings identify highly specialized roles of lateral septal mGluR5 and Oxtr in the the regulation of discrete social behaviors, and suggest that deficits in social interactions, which accompany many mental illnesses, would benefit from comprehensive treatments targeting different components of social functioning.

Co-activation of NR2A and NR2B subunits induces resistance to fear extinction.

Unpredictable stress is known to profoundly enhance susceptibility to fear and anxiety while reducing the ability to extinguish fear when threat is no longer present. Accordingly, partial aversive reinforcement, via random exposure to footshocks, induces fear that is resistant to extinction. Here we sought to determine the hippocampal mechanisms underlying susceptibility versus resistance to context fear extinction as a result of continuous (CR) and partial (PR) reinforcement, respectively. We focused on N-methyl-D-aspartate receptor (NMDAR) subunits 2A and B (NR2A and NR2B) as well as their downstream signaling effector, extracellular signal-regulated kinase (ERK), based on their critical role in the acquisition and extinction of fear. Pharmacological inactivation of NR2A, but not NR2B, blocked extinction after CR, whereas inactivation of NR2A, NR2B, or both subunits facilitated extinction after PR. The latter finding suggests that co-activation of NR2A and NR2B contributes to persistent fear following PR. In contrast to CR, PR increased membrane levels of ERK and NR2 subunits after the conditioning and extinction sessions, respectively. In parallel, nuclear activation of ERK was significantly reduced after the extinction session. Thus, co-activation and increased surface expression of NR2A and NR2B, possibly mediated by ERK, may cause persistent fear. These findings suggest that patients with post-traumatic stress disorder (PTSD) may benefit from antagonism of specific NR2 subunits.

Extinction of remotely acquired fear depends on an inhibitory NR2B/PKA pathway in the retrosplenial cortex.

As memories age, their processing increasingly relies upon cortical rather than hippocampal circuits, but the adaptive significance and mechanisms of this shift are not fully understood. Here we investigated the behavioral features and cortical mechanisms underlying extinction of remotely versus recently acquired context fear in mice. Behaviorally, extinction and reinstatement were similar, but re-extinction of remote fear was significantly faster, suggesting time-dependent engagement of mechanisms specific for processing remote memory. Using pharmacological manipulations of NMDA receptors and associated signaling pathways in the in the retrosplenial cortex, we demonstrated that extinction of remote fear uniquely required NR2B-mediated downregulation of the cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein pathway. Interestingly, NR2B/PKA interactions weakened independently of the age of the memory, but the functional significance of this molecular change was evident only as memory retrieval became PKA-dependent over time. Thus, cortical PKA signaling may provide a molecular signature of when a memory has become "remote," and inhibition of this pathway may open the door for modulation of remote memories.

Fear conditioning and extinction: emotional states encoded by distinct signaling pathways.

Conditioning and extinction of fear have traditionally been viewed as two independent learning processes for encoding representations of contexts or cues (conditioned stimuli, CS), aversive events (unconditioned stimuli, US), and their relationship. Based on the analysis of protein kinase signaling patterns in neurons of the fear circuit, we propose that fear and extinction are best conceptualized as emotional states triggered by a single CS representation with two opposing values: aversive and non-aversive. These values are conferred by the presence or absence of the US and encoded by distinct sets of kinase signaling pathways and their downstream targets. Modulating specific protein kinases thus has the potential to modify emotional states, and hence, may emerge as a promising treatment for anxiety disorders.

NMDA receptors in retrosplenial cortex are necessary for retrieval of recent and remote context fear memory.

Over time, memory retrieval is thought to transfer from the hippocampus to a distributed network of neocortical sites. Of these sites, the retrosplenial cortex (RSC) is robustly activated during retrieval of remotely acquired, emotionally valenced memories. It is unclear, however, whether RSC is specifically involved in memory storage or retrieval, and which neurotransmitter receptor mechanisms serve its function. We addressed these questions by inhibiting NMDARs in RSC via infusions of APV before tests for context fear in mice. Anterior cingulate cortex (ACC) and dorsal hippocampus (DH), which have been implicated in the retrieval of remote and recent memory, respectively, served as neuroanatomical controls. Surprisingly, infusion of APV only into RSC, but not ACC or DH, abolished retrieval of remote memory, as revealed by lack of freezing to the conditioning context. APV infused into RSC also impaired retrieval of recent memory, but had no effect on conditioning or memory storage. Within-subject experiments confirmed that the role of RSC in memory retrieval is not time limited. RSC-dependent context fear memory retrieval was mediated by NR2A, but not NR2B, subunit-containing NMDARs. Collectively, these data are the first demonstration that NMDARs in RSC are necessary for the retrieval of remote and recent memories of fear-evoking contexts. Dysfunction of RSC may thereby contribute significantly to the reexperiencing of traumatic memories in patients with posttraumatic stress disorder.

IQGAP1 regulates NR2A signaling, spine density, and cognitive processes.

General or brain-region-specific decreases in spine number or morphology accompany major neuropsychiatric disorders. It is unclear, however, whether changes in spine density are specific for an individual mental process or disorder and, if so, which molecules confer such specificity. Here we identify the scaffolding protein IQGAP1 as a key regulator of dendritic spine number with a specific role in cognitive but not emotional or motivational processes. We show that IQGAP1 is an important component of NMDAR multiprotein complexes and functionally interacts with the NR2A subunits and the extracellular signal-regulated kinase 1 (ERK1) and ERK2 signaling pathway. Mice lacking the IQGAP1 gene exhibited significantly lower levels of surface NR2A and impaired ERK activity compared to their wild-type littermates. Accordingly, primary hippocampal cultures of IQGAP1(-/-) neurons exhibited reduced surface expression of NR2A and disrupted ERK signaling in response to NR2A-dependent NMDAR stimulation. These molecular changes were accompanied by region-specific reductions of dendritic spine density in key brain areas involved in cognition, emotion, and motivation. IQGAP1 knock-outs exhibited marked long-term memory deficits accompanied by impaired hippocampal long-term potentiation (LTP) in a weak cellular learning model; in contrast, LTP was unaffected when induced with stronger stimulation paradigms. Anxiety- and depression-like behavior remained intact. On the basis of these findings, we propose that a dysfunctional IQGAP1 gene contributes to the cognitive deficits in brain disorders characterized by fewer dendritic spines.

ERK-associated changes of AP-1 proteins during fear extinction.

Extensive research has unraveled the molecular basis of learning processes underlying contextual fear conditioning, but the mechanisms of fear extinction remain less known. Contextual fear extinction occurs when an aversive stimulus that initially caused fear is no longer present and depends on the activation of the extracellular signal-regulated kinase (ERK), among other molecules. Here we investigated how ERK signaling triggered by extinction affects its downstream targets belonging to the activator protein-1 (AP-1) transcription factor family. We found that extinction, when compared to conditioning of fear, markedly enhanced the interactions of active, phospho-ERK (pERK ) with c-Jun causing alterations of its phosphorylation state. The AP-1 binding of c-Jun was decreased whereas AP-1 binding of JunD, Jun dimerization protein 2 (JDP2) and ERK were significantly enhanced. The increased AP-1 binding of the inhibitory JunD and JDP2 transcription factors was paralleled by decreased levels of the AP-1 regulated proteins c-Fos and GluR2. These changes were specific for extinction and were MEK-dependent. Overall, fear extinction involves ERK/Jun interactions and a decrease of a subset of AP-1-regulated proteins that are typically required for fear conditioning. Facilitating the formation of inhibitory AP-1 complexes may thus facilitate the reduction of fear.

Hippocampal NMDA receptor subunits differentially regulate fear memory formation and neuronal signal propagation.

Activation of NMDA receptors (NMDAR) in the hippocampus is essential for the formation of contextual and trace memory. However, the role of individual NMDAR subunits in the molecular mechanisms contributing to these memory processes is not known. Here we demonstrate, using intrahippocampal injection of subunit-selective compounds, that the NR2A-preferring antagonist impaired contextual and trace fear conditioning as well as learning-induced increase of the nuclear protein c-Fos. The NR2B-specific antagonist, on the other hand, selectively blocked trace fear conditioning without affecting c-Fos levels. Studies with cultured primary hippocampal neurons, further showed that synaptic and extrasynaptic NR2A and NR2B differentially regulate the extracellular signal-regulated kinase 1 and 2/mitogen- and stress-activated protein kinase 1 (ERK1/2/MSK1)/c-Fos pathway. Activation of the synaptic population of NMDAR induced cytosolic, cytoskeletal, and perinuclear phosphorylation of ERK1/2 (pERK1/2). The nuclear propagation of pERK1/2 signals, revealed by upregulation of the downstream nuclear targets pMSK1 and c-Fos, was blocked by a preferential NR2A but not by a specific NR2B antagonist. Conversely, activation of total (synaptic and extrasynaptic) NMDAR engaged receptors with NR2B subunits, and resulted in membrane retention of pERK1/2 without inducing pMSK1 and c-Fos. Stimulation of extrasynaptic NMDAR alone was consistently ineffective at activating ERK signaling. The discrete contribution of synaptic and total NR2A- and NR2B-containing NMDAR to nuclear transmission vs. membrane retention of ERK signaling may underlie their specific roles in the formation of contextual and trace fear memory.

Recalling safety: cooperative functions of the ventromedial prefrontal cortex and the hippocampus in extinction.

Anxiety disorders are commonly treated with exposure-based therapies that rely on extinction of conditioned fear. Persistent fear and anxiety following exposure therapy could reflect a deficit in the recall of extinction learning. Animal models of fear learning have elucidated a neural circuit for extinction learning and recall that includes the amygdala, ventromedial prefrontal cortex (vmPFC), and hippocampus. Whereas the amygdala is important for extinction learning, the vmPFC is a site of neural plasticity that allows for the inhibition of fear during extinction recall. We suggest that the vmPFC receives convergent information from other brain regions, such as contextual information from the hippocampus, to determine the circumstances under which extinction or fear will be recalled. Imaging studies of human fear conditioning and extinction lend credence to this extinction network. Understanding the neural circuitry underlying extinction recall will lead to more effective therapies for disorders of fear and anxiety.

Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears.

The amygdala has long been considered to be both necessary and sufficient for classical fear conditioning, but recent evidence suggests that the medial prefrontal cortex (mPFC) may also be involved. The prelimbic (PL) subregion of mPFC projects to the amygdala, and neurons in PL show fear-related increases in activity. It is unknown, however, whether PL activity is necessary for expression of learned fears, expression of innate fears, or the learning of fear associations. To address this, we used the sodium channel blocker tetrodotoxin to inactivate PL during fear learning or expression. Inactivation of PL reduced freezing to both a tone and a context that had been previously paired with footshock (learned fear) but had no effect on freezing to a cat (innate fear). Inactivation of PL before conditioning, however, did not prevent the formation of auditory or contextual fear memories. Thus, activity in PL is critical for the expression, but not the acquisition, of learned fears. We suggest that PL integrates information from auditory and contextual inputs and regulates expression of fear memories via projections to the basal nucleus of the amygdala.

Inactivation of the ventromedial prefrontal cortex reduces expression of conditioned fear and impairs subsequent recall of extinction.

Anxiety disorders are thought to reflect deficits in the regulation of fear expression. Evidence from rodent studies implicates the ventromedial prefrontal cortex (vmPFC) in the regulation of conditioned fear. Lesions of the vmPFC have had differing effects on the acquisition and expression of conditioned fear, as well as on recall of extinction. The use of permanent lesions, however, makes it difficult to assess the phase of training in which the vmPFC is acting and can trigger recruitment of other structures, thereby masking lesion deficits. To overcome these problems, we temporarily inactivated the vmPFC of rats with tetrodotoxin (10 ng in a 0.5-microl midline infusion) at one of four time points: prior to conditioning, prior to extinction, immediately after extinction or prior to recall of extinction. Consistent with lesion findings, inactivation of the vmPFC prior to acquisition had no effect but inactivation prior to extinction led to impaired recall of extinction the following day. In contrast to lesion findings, inactivation of the vmPFC decreased freezing at all time points, suggesting that some component of the vmPFC facilitates the expression of conditioned fear. These findings suggest that inactivation of the vmPFC can have opposite effects depending on the phase of training. The vmPFC appears to be involved both in stimulating the expression of conditioned fear and in serving as a site of extinction-related plasticity that inhibits fear during recall of extinction.

Contextual and temporal modulation of extinction: behavioral and biological mechanisms.

Extinction depends, at least partly, on new learning that is specific to the context in which it is learned. Several behavioral phenomena (renewal, reinstatement, spontaneous recovery, and rapid reacquisition) suggest the importance of context in extinction. The present article reviews research on the behavioral and neurobiological mechanisms of contextual influences on extinction learning and retrieval. Contexts appear to select or retrieve the current relationship of the conditional stimulus (CS) with the unconditional stimulus (US), and they are provided by physical background cues, interoceptive drug cues, emotions, recent trials, and the passage of time. The current article pays particular attention to the effects of recent trials and trial spacing. Control of fear extinction by physical context involves interactions between the dorsal hippocampus and the lateral nucleus of the amygdala. This interaction may be mediated by gamma-aminobutyric acid (GABA)-ergic and adrenergic mechanisms.