Contextual Memory Formation Research Articles

Contextual Fear Conditioning: Connecting Brain Glucose Sensing and Hippocampal-Dependent Memories

Ensembles of spatial-temporal cues determine the context associated with a given episodic memory, and efficient encoding of such cues is critical for producing appropriate behavioral reactions to emotionally charged contexts. Growing evidence suggests that deficiencies in the neural mechanisms regulating contextual memory formation constitute a central component of posttraumatic stress disorder. Because the hippocampus plays a critical role in consolidating contextual spatialtemporal cues, it is plausible to assume that impaired hippocampal function may be causally linked to poor trauma contextual memories and to the etiology of posttraumatic stress disorder. Rodent studies have convincingly demonstrated that glucose administration greatly enhances memory performance in numerous challenging tasks (1). In this issue of Biological Psychiatry, an elegant study by Glenn et al. (2) extended this principle to fear learning in humans, where evidence is provided to support the concept that glucose consumption leads to superior retention of hippocampal-dependent contextual learning—as shown by acoustic startle responses and expectancy ratings for an aversive (mild shock) unconditioned stimulus. Contextual fear conditioning thus appears to link neuronal glucose sensing to hippocampaldependent memory formation in humans. Although glucose may improve hippocampus-dependent context encoding, much remains to be learned about the mechanisms by which hippocampal neurons sense local variations in the levels of glucose. But how may neurons sense local changes in glucose availability in the first place? The so-called glucosensing neurons are cells whose membrane potential appears to be under direct control of glucose availability. Such neurons are named “glucose-excited” or “glucose-inhibited” according to whether their firing rate counts increase or decrease, respectively, in response to local changes in glucose provision (3). The mechanisms controlling glucose-excited neuronal firing are believed to be similar to the mechanisms operating in insulin-secreting beta cells of the pancreas (4). In these cells, intracellular glucose metabolism is controlled by glucokinase (GK), the rate-limiting factor in neuronal glycolysis (4). The action of GK on glucose activates an intracellular cascade of signaling events leading to increases in cytosolic adenosine triphosphate (ATP)/adenosine diphosphate ratios, with consequent closure of ATP-sensitive potassium (KATP) channels, the net effect of which is membrane depolarization (3). These KATP channels appear to be critical for neurons to respond to local changes in glucose availability. Evidence exists favoring a central role for KATP channels in controlling hippocampal function and specifically in contextual

Regulation of the neuronal transcription factor NPAS4 by REST and microRNAs

NPAS4 is a brain restricted, activity-induced transcription factor which regulates the expression of inhibitory synapse genes to control homeostatic excitatory/inhibitory balance in neurons. NPAS4 is required for normal social interaction and contextual memory formation in mice. Protein and mRNA expression of NPAS4 is tightly coupled to neuronal depolarization and most prevalent in the cortical and hippocampal regions in the brain, however the precise mechanisms by which the NPAS4 gene is controlled remain unexplored. Here we show that expression of NPAS4 mRNA is actively repressed by RE-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) in embryonic stem cells and non-neuronal cells by binding multiple sites within the promoter and Intron I of NPAS4. Repression by REST also appears to correlate with the binding of the zinc finger DNA binding protein CTCF within Intron I of NPAS4. In addition, we show that the 3′ untranslated region (3′UTR) of NPAS4 can be targeted by two microRNAs, miR-203 and miR-224 to further regulate its expression. miR-224 is a midbrain/hypothalamus enriched microRNA which is expressed from an intron within the GABAA receptor epsilon (GABRE) gene and may further regionalize NPAS4 expression. Our results reveal REST and microRNA dependent mechanisms that restrict NPAS4 expression to the brain.

Neuronal Per Arnt Sim (PAS) Domain Protein 4 (NPAS4) Regulates Neurite Outgrowth and Phosphorylation of Synapsin I

Neuronal Per Arnt Sim domain protein 4 (NPAS4), a brain-specific basic helix-loop-helix transcription factor, has recently been shown to regulate the development of the GABAergic inhibitory synapses and transcription program for contextual memory formation in the hippocampus. We previously reported that chronic social isolation or restriction stress in mice resulted in an impairment in memory and emotional behavior, which was associated with a decrease in Npas4 mRNA levels. In this study, we investigated the role of NPAS4 in neuronal function in vitro and in vivo. Differentiation medium-induced neurite outgrowth was inhibited in Npas4 knockdown Neuro2a cells, whereas overexpression of NPAS4 accelerated the neurite outgrowth in Neuro2a cells. Furthermore, depolarization-induced neurite outgrowth was abolished in Npas4 KO hippocampal neurons. NPAS4 overexpression increased cyclin-dependent kinase 5 (CDK5)-dependent synapsin I phosphorylation in Neuro2a cells and primary cultured hippocampal neurons. A CDK5 inhibitor, roscovitine, inhibited the neurite outgrowth and the increase in phosphorylated synapsin I (p-SYN I) levels in Npas4-overexpressed Neuro2a cells. Interaction of NPAS4 with promoters of Cdk5 and NeuN genes was demonstrated by a chromatin immunoprecipitation assay. In an in vivo study, pentylenetetrazole-induced convulsions in mice resulted in an increase in NPAS4 and p-SYN I levels in the prefrontal cortex of wild-type mice, although no changes in p-SYN I levels were observed in Npas4 knock-out mice. These results suggest that NPAS4 plays an important role in the structural and functional plasticity of neurons.

Long‐lasting regulation of hippocampal Bdnf gene transcription after contextual fear conditioning

Long-term memory formation requires de novo protein synthesis and gene transcription. During contextual long-term memory formation brain-derived neurotrophic factor (BDNF) gene expression changes in conjunction with alterations of DNA methylation in the Bdnf gene. However, little is known about the molecular mechanisms underlying the maintenance and persistence of contextual long-term memory. Here, we examined the transcription of specific Bdnf exons in the hippocampus for long periods after contextual fear conditioning. We found changes in transcription lasting for at least 24 h after contextual fear conditioning, with some sex-specific effects. In addition, hypomethylation at a CpG site in CpG island 2 located at the end of Bdnf exon III sequence was detected at 0.5 h and maintained for up to 24 h after contextual fear conditioning. The identification of these long-lasting changes in transcription and DNA methylation at the Bdnf gene suggests that BDNF might have a role for storage of contextual long-term memory in the hippocampus.

Npas4 Regulates a Transcriptional Program in CA3 Required for Contextual Memory Formation

The rapid encoding of contextual memory requires the CA3 region of the hippocampus, but the necessary genetic pathways remain unclear. We found that the activity-dependent transcription factor Npas4 regulates a transcriptional program in CA3 that is required for contextual memory formation. Npas4 was specifically expressed in CA3 after contextual learning. Global knockout or selective deletion of Npas4 in CA3 both resulted in impaired contextual memory, and restoration of Npas4 in CA3 was sufficient to reverse the deficit in global knockout mice. By recruiting RNA polymerase II to promoters and enhancers of target genes, Npas4 regulates a learning-specific transcriptional program in CA3 that includes many well-known activity-regulated genes, which suggests that Npas4 is a master regulator of activity-regulated gene programs and is central to memory formation.

Up-regulation of dopamine D 1 receptor in the hippocampus after establishment of conditioned place preference by cocaine

The hippocampus plays an important role in the formation of contextual memory between the environment and the rewarding effect of abused drugs. The dopaminergic neural transmission in the hippocampus seems to be critical for such memory. Using conditioned place preference in rats, we found that the protein level of the dopamine D 1 receptor and its prerequisite mRNA in the hippocampus increased in animals that showed a clear preference for the environment paired with cocaine. The increase was not a simple reflection of the repeated administration of cocaine. Instead, it is attributable to conditioning, because systematic contingency between drug administration and exposure to a particular environment was necessary for the increase. Furthermore, we found that the mRNA of the dopamine D 1 receptors increased in the granule cell layer of the dentate gyrus. These results suggest that the alteration of dopamine D 1 receptor in the hippocampus, especially in the dentate gyrus, is related to the induction of drug-induced contextual memory. The finding implicates the relevance of the dopaminergic signal transduction in the hippocampus to drug dependence.

Properties of Contextual Memory Formed in the Absence of αCaMKII Autophosphorylation

The alpha-isoform of calcium/calmodulin-dependent kinase II (αCaMKII) is a major synaptic kinase that undergoes autophosphorylation after NMDA receptor activation, switching the kinase into a calcium-independent activity state. This αCaMKII autophosphorylation is essential for NMDA receptor-dependent long-term potentiation (LTP), induced by a single tetanus, in hippocampal area CA1 and in neocortex. Furthermore, the αCaMKII autophosphorylation is essential for contextual long-term memory (LTM) formation after a single training trial but not after a massed training session. Here, we show that in the absence of αCaMKII autophosphorylation contextual fear conditioning is hippocampus dependent and that multi-tetanus-dependent late-LTP cannot be induced in hippocampal area CA1. Furthermore, we show that in the absence of αCaMKII autophosphorylation contextual LTM persists for 30 days, the latest time point tested. Additionally, contextual, but not cued, LTM formation in the absence of αCaMKII autophosphorylation appears to be impaired in 18 month-old mice. Taken together, our findings suggest that αCaMKII autophosphorylation-independent plasticity in the hippocampus is sufficient for contextual LTM formation and that αCaMKII autophosphorylation may be important for delaying age-related impairments in hippocampal memory formation. Furthermore, they propose that NMDA receptor-dependent LTP in hippocampal area CA1 is essential for contextual LTM formation after a single trial but not after massed training. Finally, our results challenge the proposal that NMDA receptor-dependent LTP in neocortex is required for remote contextual LTM.

An Odyssey of Fear: Homer Stresses New Mechanisms

An Odyssey of Fear: Homer Stresses New Mechanisms

Impaired Terminal Differentiation of Hippocampal Granule Neurons and Defective Contextual Memory in PC3/Tis21 Knockout Mice

Neurogenesis in the dentate gyrus of the adult hippocampus has been implicated in neural plasticity and memory, but the molecular mechanisms controlling the proliferation and differentiation of newborn neurons and their integration into the synaptic circuitry are still largely unknown. To investigate this issue, we have analyzed the adult hippocampal neurogenesis in a PC3/Tis21-null mouse model. PC3/Tis21 is a transcriptional co-factor endowed with antiproliferative and prodifferentiative properties; indeed, its upregulation in neural progenitors has been shown to induce exit from cell cycle and differentiation. We demonstrate here that the deletion of PC3/Tis21 causes an increased proliferation of progenitor cells in the adult dentate gyrus and an arrest of their terminal differentiation. In fact, in the PC3/Tis21-null hippocampus postmitotic undifferentiated neurons accumulated, while the number of terminally differentiated neurons decreased of 40%. As a result, PC3/Tis21-null mice displayed a deficit of contextual memory. Notably, we observed that PC3/Tis21 can associate to the promoter of Id3, an inhibitor of proneural gene activity, and negatively regulates its expression, indicating that PC3/Tis21 acts upstream of Id3. Our results identify PC3/Tis21 as a gene required in the control of proliferation and terminal differentiation of newborn neurons during adult hippocampal neurogenesis and suggest its involvement in the formation of contextual memories.

Inhibitors of Class 1 Histone Deacetylases Reverse Contextual Memory Deficits in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by cognitive impairments that progress to dementia and death. The earliest symptoms of AD present as a relatively pure deficit in memory retrieval. Therefore, drug treatments that intervene in the early stages of AD by rescuing memory deficits could be promising therapies to slow, or even reverse progression of the disease. In this study, we tested the potential of systemic histone deacetylase inhibitor (HDACi) treatment to rescue cognitive deficits in a mouse model of AD. APPswe/PS1dE9 mice showed pronounced contextual memory impairments beginning at 6 months of age. Chronic HDACi injections (2-3 weeks) did not alter contextual memory formation in normal mice, but had profound effects in transgenic animals. Injections of sodium valproate, sodium butyrate, or vorinostat (suberoylanilide hydroxamic acid; Zolinza) completely restored contextual memory in these mutant mice. Further behavioral testing of the HDACi-treated transgenic mice showed that the newly consolidated memories were stably maintained over a 2-week period. Measurement of the HDAC isoform selectivity profile of sodium valproate, sodium butyrate, and vorinostat revealed the common inhibition of class I HDACs (HDAC1, 2, 3, 8) with little effect on the class IIa HDAC family members (HDAC4, 5, 7, 9) and inhibition of HDAC6 only by vorinostat. These preclinical results indicate that targeted inhibition of class I HDAC isoforms is a promising avenue for treating the cognitive deficits associated with early stage AD.

Failures to reconsolidate memory in a mouse model of Alzheimer’s disease

Previous studies have demonstrated that the formation of spatial, contextual and trace conditioning memories are impaired in animal models of Alzheimer’s disease (AD), consistent with the observations that the first sign of cognitive decline in AD includes difficulties in the acquisition of new information or memory formation. Evidence is accumulating that memory retrieval is a dynamic process in which stored information becomes labile again and needs to be restabilized. However, it is poorly understood how this process referred to as memory reconsolidation is affected in animal models of AD. The present study was designed to use contextual fear conditioning to compare the changes in memory formation and subsequent reconsolidation processes in transgenic mice that overexpress human APP and PS1 harboring five familial AD mutations (5XFAD model). The results clearly demonstrate that cognitive dysfunction starts to occur primarily as reduced levels of contextual learning or memory formation in 5XFAD mice, but it is exacerbated by additional retrieval-dependent retrograde amnesia due to deficient reconsolidation as disease further develops.

RNA interference in hippocampus demonstrates opposing roles for CREB and PP1α in contextual and temporal long‐term memory

We injected small interfering RNAs (siRNAs) directly into the hippocampus of wild-type mice, knocking down expression of cyclic AMP responsive element-binding protein (CREB) and disrupting long-term, but not short-term, memory after both contextual and trace fear conditioning. In contrast, similar knockdown of siRNA for protein phosphatase 1 (PP1) was sufficient to enhance contextual and temporal memory formation, thereby demonstrating with such a gain-of-function effect a lack of any general deleterious effect for this method of RNAi-mediated gene knockdown. Our findings clearly confirm that contextual memory formation involves CREB and PP1 as positive and negative regulators, respectively, and show for the first time that temporal memory formation shares this mechanism. More generally, we establish that direct injection of siRNA into identified adult brain regions yields specific gene knockdowns, which can be used to validate in vivo candidate genes involved in behavioral plasticity.

Activation of nociceptin opioid peptide (NOP) receptor impairs contextual fear learning in mice through glutamatergic mechanisms

The present study investigated whether the selective nociceptin opioid peptide (NOP) receptor agonist, Ro64-6198, impairs acquisition of fear conditioning through glutamatergic mechanisms. Systemic administration of Ro64-6198 (0.3 and 1 mg/kg) or the non-competitive NMDA receptor antagonist, MK-801 (0.03 and 0.1 mg/kg) prior to conditioning severely impaired contextual but not cued fear learning in C57BL/6N mice. When administered together at sub-effective doses, Ro64-6198 (0.5 mg/kg) and MK-801 (0.05 mg/kg), synergistically impaired contextual fear learning, but left cued fear learning intact. We next used the immediate shock deficit paradigm (ISD) to examine the effects of Ro64-6198 and MK-801 on contextual memory formation in the absence of the foot-shock. As expected, naive mice that were shocked briefly after being placed in the training chamber displayed no contextual fear conditioning. This learning deficit was elevated by prior exposure of mice to the training context. Furthermore, administration of Ro64-6198 and MK-801, either separately at amnesic doses (1 mg/kg and 0.1 mg/kg, respectively) or concomitantly at sub-effective doses (0.5 mg/kg and 0.05 mg/kg, respectively) significantly reduced the facilitating effects of context preexposure. These findings demonstrate the existence of functional antagonism between NOP and NMDA receptors that predominantly contributes to modulation of conditioned fear learning which involves spatial-processing demands.

Hypothalamic sites responding to predator threats – the role of the dorsal premammillary nucleus in unconditioned and conditioned antipredatory defensive behavior

In this study we provide a comprehensive analysis of the hypothalamic activation pattern during exposure to a live predator or an environment previously associated with a predator. Our results support the view that hypothalamic processing of the actual and the contextual predatory threats share the same circuit, in which the dorsal premammillary nucleus (PMd) plays a pivotal role in amplifying this processing. To further understand the role of the PMd in the circuit organizing antipredatory defensive behaviors, we studied rats with cytotoxic PMd lesions during cat exposure and examined the pattern of behavioral responses as well as how PMd lesions affect the neuronal activation of the systems engaged in predator detection, in contextual memory formation and in defensive behavioral responses. Next, we investigated how pharmacological blockade of the PMd interferes with the conditioned behavioral responses to a context previously associated with a predator, and how this blockade affects the activation pattern of periaqueductal gray (PAG) sites likely to organize the conditioned behavioral responses to the predatory context. Behavioral observations indicate that the PMd interferes with both unconditioned and conditioned antipredatory defensive behavior. Moreover, we have shown that the PMd influences the activation of its major projecting targets, i.e. the ventral part of the anteromedial thalamic nucleus which is likely to influence mnemonic processing, and PAG sites involved in the expression of antipredatory unconditioned and conditioned behavioral responses. Of particular relevance, this work provides evidence to elucidate the basic organization of the neural circuits integrating unconditioned and contextual conditioned responses to predatory threats.

Dorsal hippocampus involvement in delay fear conditioning depends upon the strength of the tone‐footshock association

The hippocampus is important for the formation of spatial, contextual, and episodic memories. For instance, lesions of the dorsal hippocampus (DH) produce demonstrable deficits in contextual fear conditioning. By contrast, it is generally agreed that the DH is not important for conditioning to a discrete cue (such as a tone or light) that is paired with footshock in a temporally contiguous fashion (delay conditioning). There are, however, some reports of hippocampus involvement in delay conditioning. The present series of experiments was designed to assess the conditions under which the hippocampus-dependent component of delay fear conditioning performance may be revealed. Here, we manipulated the number of conditioning trials and the intensity of the footshock in order to vary the strength of conditioning. The results indicate that the DH contributes to freezing performance to a delay conditioned tone when the conditioning parameters are relatively weak (few trials or low footshock intensity), but not when strong parameters are used. The results are discussed in terms of two parallel memory systems: a direct tone-footshock association that is independent of the hippocampus and a hippocampus-dependent memory for the conditioning session.

Emotion Enhances Learning via Norepinephrine Regulation of AMPA-Receptor Trafficking

Emotion enhances our ability to form vivid memories of even trivial events. Norepinephrine (NE), a neuromodulator released during emotional arousal, plays a central role in the emotional regulation of memory. However, the underlying molecular mechanism remains elusive. Toward this aim, we have examined the role of NE in contextual memory formation and in the synaptic delivery of GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors during long-term potentiation (LTP), a candidate synaptic mechanism for learning. We found that NE, as well as emotional stress, induces phosphorylation of GluR1 at sites critical for its synaptic delivery. Phosphorylation at these sites is necessary and sufficient to lower the threshold for GluR1 synaptic incorporation during LTP. In behavioral experiments, NE can lower the threshold for memory formation in wild-type mice but not in mice carrying mutations in the GluR1 phosphorylation sites. Our results indicate that NE-driven phosphorylation of GluR1 facilitates the synaptic delivery of GluR1-containing AMPARs, lowering the threshold for LTP, thereby providing a molecular mechanism for how emotion enhances learning and memory.

Cholinergic activity in the insular cortex is necessary for acquisition and consolidation of contextual memory

Experiences with a high emotional content (aversive) tend to be stored as long-term memories; however, there are also contextual recollections, which form a significant part of our memories. Different research has shown that the insular cortex (IC) plays an important role during aversive memory formation, yet its role during incidental/non-aversive learning like pre-exposure contextual memory formation has received little attention. The objective of this research was to establish the role of cholinergic activity in the IC through its muscarinic receptors during the formation of inhibitory avoidance (IA) memory, as well as during pre-exposure contextual memory, using a paradigm such as latent inhibition (LI). Rats with bilateral cannulae directed into the IC were trained in the LI paradigm of IA or IA task alone. The muscarinic antagonist receptor scopolamine was infused bilaterally into the IC 5 min before the pre-exposure into the dark chamber of the IA cage, one day before the conventional IA training or during the IA training day. During the IA test, the entrance latency into the dark chamber of the IA cage was measured as an index of contextual memory. The results showed that scopolamine infused before and after IA training disrupts inhibitory avoidance memory. Also, it showed that the pre-exposed saline-infused animals (LI) had a lower entrance latency compared to the group not pre-exposed (IA). However, the group that received scopolamine into the IC before, but not after, the pre-exposure to the dark chamber, presented a similar latency to the IA group, showing a blockade of the latent inhibition of the IA. These results suggest that cholinergic activity in the insular cortex is necessary during the acquisition and consolidation of avoidance memory, but appears necessary only during the acquisition of pre-exposure non-aversive contextual memory.

Polysialylated Neural Cell Adhesion Molecule Is Involved in Induction of Long-Term Potentiation and Memory Acquisition and Consolidation in a Fear-Conditioning Paradigm

Polysialic acid (PSA) regulates functions of the neural cell adhesion molecule (NCAM) during development and in neuroplasticity in the adult; the underlying mechanisms at different phases of learning and memory consolidation are, however, unknown. To investigate the contributions of PSA versus the extracellular domain of the NCAM glycoprotein backbone to synaptic plasticity, we applied NCAM, PSA-NCAM, and PSA to acute slices of the hippocampal CA1 region of NCAM-deficient mice and measured their effects on long-term potentiation (LTP). Remarkably, only PSA and PSA-NCAM, but not NCAM restored normal LTP. Application of these molecules to the dorsal hippocampus of wild-type mice showed that PSA-NCAM and PSA, but not NCAM, injected before fear conditioning, impaired formation of hippocampus-dependent contextual memory. Consolidation of contextual memory was affected by PSA-NCAM only when injected during its late, but not early phases. None of the tested compounds disturbed extrahippocampal-cued memory. Mice lacking the polysialyltransferase (ST8SialV/PST) responsible for attachment of PSA to NCAM in adulthood showed a mild deficit only in hippocampal contextual learning, when compared with NCAM-deficient mice that were disturbed in both contextual and cued memories. Contextual and tone memory in NCAM-deficient mice could be partially restored by injection of PSA-NCAM, but not of NCAM, into the hippocampus, suggesting that the impact of PSA-NCAM in synaptic plasticity and learning is not mediated by modulation of NCAM-NCAM homophilic interactions. In conclusion, our data support the view that polysialylated NCAM is involved in both formation and late consolidation of contextual memory.

A bioinformatics analysis of memory consolidation reveals involvement of the transcription factor c-rel.

Consolidation of long-term memory (LTM) is a complex process requiring synthesis of new mRNAs and proteins. Many studies have characterized the requirement for de novo mRNA and protein synthesis; however, few studies have comprehensively identified genes regulated during LTM consolidation. We show that consolidation of long-term contextual memory in the hippocampus triggers altered expression of numerous genes encompassing many aspects of neuronal function. Like contextual memory formation, this altered gene expression required NMDA receptor activation and was specific for situations in which the animal formed an association between a physical context and a sensory stimulus. Using a bioinformatics approach, we found that regulatory elements for several transcription factors are over-represented in the upstream region of genes regulated during consolidation of LTM. Using a knock-out mouse, we found that c-rel, one of the transcription factors identified in our bioinformatics study, is necessary for hippocampus-dependent long-term memory formation.

Hippocampal CRE-mediated gene expression is required for contextual memory formation.

One of the molecular events associated with contextual long-term memory (LTM) formation is the induction of cyclic AMP response element (CRE)-mediated transcription1. Here we report that activation of NMDA receptors and of extracellular signal–regulated kinase (ERK) were necessary for stimulation of CRE-mediated transcription during contextual fear conditioning. In addition, we found that inhibition of CRE-regulated transcription during learning blocked LTM, which indicates that this transcriptional activity is critical for memory formation.