Time to learn: The role of the molecular circadian clock in learning and memory.

The circadian system plays an important role in aligning biological processes with the external time of day. A range of physiological functions are governed by the circadian cycle, including memory processes, yet little is understood about how the clock interfaces with memory at a molecular level. The molecular circadian clock consists of four key genes/gene families, Period, Clock, Cryptochrome, and Bmal1, that rhythmically cycle in an ongoing transcription-translation negative feedback loop that maintains an approximately 24-hour cycle within cells of the brain and body. In addition to their roles in generating the circadian rhythm within the brain's master pacemaker (the suprachiasmatic nucleus), recent research has suggested that these clock genes may function locally within memory-relevant brain regions to modulate memory across the day/night cycle. This review will discuss how these clock genes function both within the brain's central clock and within memory-relevant brain regions to exert circadian control over memory processes. For each core clock gene, we describe the current research that demonstrates a potential role in memory and outline how these clock genes might interface with cascades known to support long-term memory formation. Together, the research suggests that clock genes function locally within satellite clocks across the brain to exert circadian control over long-term memory formation and possibly other biological processes. Understanding how clock genes might interface with local molecular cascades in the hippocampus and other brain regions is a critical step toward developing treatments for the myriad disorders marked by dysfunction of both the circadian system and cognitive processes.

The circadian clock gene Per1 modulates context fear memory formation within the retrosplenial cortex in a sex-specific manner.

Context memory formation is a complex process that requires transcription in many subregions of the brain including the dorsal hippocampus and retrosplenial cortex. One critical gene necessary for memory formation is the circadian gene Period1 (Per1), which has been shown to function in the dorsal hippocampus to modulate spatial memory in addition to its well-documented role in regulating the diurnal clock within the suprachiasmatic nucleus (SCN). We recently found that alterations in Per1 expression in the dorsal hippocampus can modulate spatial memory formation, with reduced hippocampal Per1 impairing memory and overexpression of Per1 ameliorating age-related impairments in spatial memory. Whether Per1 similarly functions within other memory-relevant brain regions is currently unknown. Here, to test whether Per1 is a general mechanism that modulates memory across the brain, we tested the role of Per1 in the retrosplenial cortex (RSC), a brain region necessary for context memory formation. First, we demonstrate that context fear conditioning drives a transient increase in Per1 mRNA expression within the anterior RSC that peaks 60 m after training. Next, using HSV-CRISPRi-mediated knockdown of Per1, we show that reducing Per1 within the anterior RSC before context fear acquisition impairs memory in both male and female mice. In contrast, overexpressing Per1 with either HSV-CRISPRa or HSV-Per1 before context fear acquisition drives a sex-specific memory impairment; males show impaired context fear memory whereas females are not affected by Per1 overexpression. Finally, as Per1 levels are known to rhythmically oscillate across the day/night cycle, we tested the possibility that Per1 overexpression might have different effects on memory depending on the time of day. In contrast to the impairment in memory we observed during the daytime, Per1 overexpression has no effect on context fear memory during the night in either male or female mice. Together, our results indicate that Per1 modulates memory in the anterior retrosplenial cortex in addition to its documented role in regulating memory within the dorsal hippocampus, although this role may differ between males and females.

Special Issue "Molecular Mechanisms of Memory Formation and Modification".

Memory is vital to human functioning and controls future behavioral responses [...].

HDAC3-Mediated Repression of the Nr4a Family Contributes to Age-Related Impairments in Long-Term Memory.

Aging is accompanied by cognitive deficits, including impairments in long-term memory formation. Understanding the molecular mechanisms that support preserved cognitive function in aged animals is a critical step toward identifying novel therapeutic targets that could improve memory in aging individuals. One potential mechanism is the Nr4a family of genes, a group of CREB-dependent nuclear orphan receptors that have previously been shown to be important for hippocampal memory formation. Here, using a cross-species approach, we tested the role of Nr4a1 and Nr4a2 in age-related memory impairments. Using a rat model designed to identify individual differences in age-related memory impairments, we first identified Nr4a2 as a key gene that fails to be induced by learning in cognitively impaired male aged rats. Next, using a mouse model that allows for genetic manipulations, we determined that histone deacetylase 3 (HDAC3) negatively regulates Nr4a2 in the aged male and female hippocampus. Finally, we show that overexpression of Nr4a1, Nr4a2, or both transcripts in the male mouse dorsal hippocampus can ameliorate age-related impairments in object location memory. Together, our results suggest that Nr4a2 may be a key mechanism that promotes preserved cognitive function in old age, with HDAC3-mediated repression of Nr4a2 contributing to age-related cognitive decline. More broadly, these results indicate that therapeutic strategies to promote Nr4a gene expression or function may be an effective strategy to improve cognitive function in old age.SIGNIFICANCE STATEMENT Aging is accompanied by memory impairments, although there is a great deal of variability in the severity of these impairments. Identifying molecular mechanisms that promote preserved memory or participate in cognitive reserve in old age is important to develop strategies that promote healthy cognitive aging. Here, we show that learning-induced expression of the CREB-regulated nuclear receptor gene Nr4a2 is selectively impaired in aged rats with memory impairments. Further, we show that Nr4a2 is regulated by histone deacetylase HDAC3 in the aged mouse hippocampus. Finally, we demonstrate that hippocampal overexpression of either Nr4a2 or its family member, Nr4a1, can ameliorate age-related memory impairments. This suggests that promoting Nr4a expression may be a novel strategy to improve memory in aging individuals.

Molecular Mechanisms of Reconsolidation-Dependent Memory Updating.

Memory is not a stable record of experience, but instead is an ongoing process that allows existing memories to be modified with new information through a reconsolidation-dependent updating process. For a previously stable memory to be updated, the memory must first become labile through a process called destabilization. Destabilization is a protein degradation-dependent process that occurs when new information is presented. Following destabilization, a memory becomes stable again through a protein synthesis-dependent process called restabilization. Much work remains to fully characterize the mechanisms that underlie both destabilization and subsequent restabilization, however. In this article, we briefly review the discovery of reconsolidation as a potential mechanism for memory updating. We then discuss the behavioral paradigms that have been used to identify the molecular mechanisms of reconsolidation-dependent memory updating. Finally, we outline what is known about the molecular mechanisms that support the memory updating process. Understanding the molecular mechanisms underlying reconsolidation-dependent memory updating is an important step toward leveraging this process in a therapeutic setting to modify maladaptive memories and to improve memory when it fails.

Exercise opens a temporal window for enhanced cognitive improvement from subsequent physical activity.

The beneficial effects of exercise on cognition are well established; however specific exercise parameters regarding the frequency and duration of physical activity that provide optimal cognitive health have not been well defined. Here, we explore the effects of the duration of exercise and sedentary periods on long-term object location memory (OLM) in mice. We use a weak object location training paradigm that is subthreshold for long-term memory formation in sedentary controls, and demonstrate that exercise enables long-term memories to form. We show that 14- and 21-d of running wheel access enables mice to discriminate between familiar and novel object locations after a 24 h delay, while 2- or 7-d running wheel access provides insufficient exercise for such memory enhancement using the subthreshold learning paradigm. After 14- and 21-d of wheel running, exercise-induced cognitive enhancement then decays back to baseline performance following 3-d of sedentary activity. However, exercise-induced cognitive enhancement can be reactivated by an additional period of just 2 d exercise, previously shown to be insufficient to induce cognitive enhancement on its own. The reactivating period of exercise is capable of enhancing memory after three- or seven-sedentary days, but not 14-d. These data suggest a type of "molecular memory" for the exercise stimulus, in that once exercise duration reaches a certain threshold, it establishes a temporal window during which subsequent low-level exercise can capitalize on the neurobiological adaptations induced by the initial period of exercise, enabling it to maintain the benefits on cognitive function. These findings provide new information that may help to guide future clinical studies in exercise.

CREST in the Nucleus Accumbens Core Regulates Cocaine Conditioned Place Preference, Cocaine-Seeking Behavior, and Synaptic Plasticity.

Epigenetic mechanisms result in persistent changes at the cellular level that can lead to long-lasting behavioral adaptations. Nucleosome remodeling is a major epigenetic mechanism that has not been well explored with regards to drug-seeking behaviors. Nucleosome remodeling is performed by multi-subunit complexes that interact with DNA or chromatin structure and possess an ATP-dependent enzyme to disrupt nucleosome-DNA contacts and ultimately regulate gene expression. Calcium responsive transactivator (CREST) is a transcriptional activator that interacts with enzymes involved in both histone acetylation and nucleosome remodeling. Here, we examined the effects of knocking down CREST in the nucleus accumbens (NAc) core on drug-seeking behavior and synaptic plasticity in male mice as well as drug-seeking in male rats. Knocking down CREST in the NAc core results in impaired cocaine-induced conditioned place preference (CPP) as well as theta-induced long-term potentiation in the NAc core. Further, similar to the CPP findings, using a self-administration procedure, we found that CREST knockdown in the NAc core of male rats had no effect on instrumental responding for cocaine itself on a first-order schedule, but did significantly attenuate responding on a second-order chain schedule, in which responding has a weaker association with cocaine. Together, these results suggest that CREST in the NAc core is required for cocaine-induced CPP, synaptic plasticity, as well as cocaine-seeking behavior.SIGNIFICANCE STATEMENT This study demonstrates a key role for the role of Calcium responsive transactivator (CREST), a transcriptional activator, in the nucleus accumbens (NAc) core with regard to cocaine-induced conditioned place preference (CPP), self-administration (SA), and synaptic plasticity. CREST is a unique transcriptional regulator that can recruit enzymes from two different major epigenetic mechanisms: histone acetylation and nucleosome remodeling. In this study we also found that the level of potentiation in the NAc core correlated with whether or not animals formed a CPP. Together the results indicate that CREST is a key downstream regulator of cocaine action in the NAc.

Medial habenula cholinergic signaling regulates cocaine-associated relapse-like behavior.

Propensity to relapse, even following long periods of abstinence, is a key feature in substance use disorders. Relapse and relapse-like behaviors are known to be induced, in part, by re-exposure to drug-associated cues. Yet, while many critical nodes in the neural circuitry contributing to relapse have been identified and studied, a full description of the networks driving reinstatement of drug-seeking behaviors is lacking. One area that may provide further insight to the mechanisms of relapse is the habenula complex, an epithalamic region composed of lateral and medial (MHb) substructures, each with unique cell and target populations. Although well conserved across vertebrate species, the functions of the MHb are not well understood. Recent research has demonstrated that the MHb regulates nicotine aversion and withdrawal. However, it remains undetermined whether MHb function is limited to nicotine and aversive stimuli or if MHb circuit regulates responses to other drugs of abuse. Advances in circuit-level manipulations now allow for cell-type and temporally specific manipulations during behavior, specifically in spatially restrictive brain regions, such as the MHb. In this study, we focus on the response of the MHb to reinstatement of cocaine-associated behavior, demonstrating that cocaine-primed reinstatement of conditioned place preference engages habenula circuitry. Using chemogenetics, we demonstrate that MHb activity is sufficient to induce reinstatement behavior. Together, these data identify the MHb as a key hub in the circuitry underlying reinstatement and may serve as a target for regulating relapse-like behaviors.

Deleting HDAC3 rescues long-term memory impairments induced by disruption of the neuron-specific chromatin remodeling subunit BAF53b.

Multiple epigenetic mechanisms, including histone acetylation and nucleosome remodeling, are known to be involved in long-term memory formation. Enhancing histone acetylation by deleting histone deacetylases, like HDAC3, typically enhances long-term memory formation. In contrast, disrupting nucleosome remodeling by blocking the neuron-specific chromatin remodeling subunit BAF53b impairs long-term memory. Here, we show that deleting HDAC3 can ameliorate the impairments in both long-term memory and synaptic plasticity caused by BAF53b mutation. This suggests a dynamic interplay exists between histone acetylation/deacetylation and nucleosome remodeling mechanisms in the regulation of memory formation.

Distinct roles for the deacetylase domain of HDAC3 in the hippocampus and medial prefrontal cortex in the formation and extinction of memory.

Histone deacetylases (HDACs) are chromatin modifying enzymes that have been implicated as powerful negative regulators of memory processes. HDAC3has been shown to play a pivotal role in long-term memory for object location as well as the extinction of cocaine-associated memory, but it is unclear whether this function depends on the deacetylase domain of HDAC3. Here, we tested whether the deacetylase domain of HDAC3has a role in object location memory formation as well as the formation and extinction of cocaine-associated memories. Using a deacetylase-dead point mutant of HDAC3, we found that selectively blocking HDAC3 deacetylase activity in the dorsal hippocampus enhanced long-term memory for object location, but had no effect on the formation of cocaine-associated memory. When this same point mutant virus of HDAC3 was infused into the prelimbic cortex, it failed to affect cocaine-associated memory formation. With regards to extinction, impairing the HDAC3 deacetylase domain in the infralimbic cortex had no effect on extinction, but a facilitated extinction effect was observed when the point mutant virus was delivered to the dorsal hippocampus. These results suggest that the deacetylase domain of HDAC3 plays a selective role in specific brain regions underlying long-term memory formation of object location as well as cocaine-associated memory formation and extinction.

CaMKII regulates proteasome phosphorylation and activity and promotes memory destabilization following retrieval.

Numerous studies have suggested that memories "destabilize" and require de novo protein synthesis in order to reconsolidate following retrieval, but very little is known about how this destabilization process is regulated. Recently, ubiquitin-proteasome mediated protein degradation has been identified as a critical regulator of memory trace destabilization following retrieval, though the specific mechanisms controlling retrieval-induced changes in ubiquitin-proteasome activity remain equivocal. Here, we found that proteasome activity is increased in the amygdala in a CaMKII-dependent manner following the retrieval of a contextual fear memory. We show that in vitro inhibition of CaMKII reversed retrieval-induced increases in proteasome activity. Additionally, in vivo pharmacological blockade of CaMKII abolished increases in proteolytic activity and activity related regulatory phosphorylation in the amygdala following retrieval, suggesting that CaMKII was "upstream" of protein degradation during the memory reconsolidation process. Consistent with this, while inhibiting CaMKII in the amygdala did not impair memory following retrieval, it completely attenuated the memory impairments that resulted from post-retrieval protein synthesis blockade. Collectively, these results suggest that CaMKII controls the initiation of the memory reconsolidation process through regulation of the proteasome.

The retrosplenial cortex is involved in the formation of memory for context and trace fear conditioning.

The retrosplenial cortex (RSC) is known to play a role in the retrieval of context memory, but its involvement in memory formation and consolidation is unclear. To better characterize the role of the RSC, we tested its involvement in the formation and retrieval of memory for trace fear conditioning, a task that requires the association of two cues separated by an empty period of time. We have previously shown that trace fear extinction requires the RSC (Kwapis, Jarome, Lee, Gilmartin, & Helmstetter, 2014) and have hypothesized that trace memory may be stored in a distributed cortical network that includes prelimbic and retrosplenial cortices (Kwapis, Jarome, & Helmstetter, 2015). Whether the RSC participates in acquiring and storing cued trace fear, however, is currently unknown. Here, we demonstrate that blocking protein synthesis in the RSC before, but not after acquisition impairs rats' memory for trace CS and context fear without affecting memory for the CS in standard delay fear conditioning. We also show that NMDA receptor blockade in the RSC transiently impairs memory retrieval for trace, but not delay memory. The RSC therefore appears to critically contribute to formation of trace and context fear memory in addition to its previously recognized role in context memory retrieval.

The role of the medial prefrontal cortex in trace fear extinction.

The extinction of delay fear conditioning relies on a neural circuit that has received much attention and is relatively well defined. Whether this established circuit also supports the extinction of more complex associations, however, is unclear. Trace fear conditioning is a better model of complex relational learning, yet the circuit that supports extinction of this memory has received very little attention. Recent research has indicated that trace fear extinction requires a different neural circuit than delay extinction; trace extinction requires the participation of the retrosplenial cortex, but not the amygdala, as noted in a previous study. Here, we tested the roles of the prelimbic and infralimbic regions of the medial prefrontal cortex in trace and delay fear extinction by blocking NMDA receptors during extinction learning. We found that the prelimbic cortex is necessary for trace, but not for delay fear extinction, whereas the infralimbic cortex is involved in both types of extinction. These results are consistent with the idea that trace fear associations require plasticity in multiple cortical areas for successful extinction. Further, the infralimbic cortex appears to play a role in extinction regardless of whether the animal was initially trained in trace or delay conditioning. Together, our results provide new information about how the neural circuits supporting trace and delay fear extinction differ.

Extinguishing trace fear engages the retrosplenial cortex rather than the amygdala.

Extinction learning underlies the treatment for a variety of anxiety disorders. Most of what is known about the neurobiology of extinction is based on standard "delay" fear conditioning, in which awareness is not required for learning. Little is known about how complex, explicit associations extinguish, however. "Trace" conditioning is considered to be a rodent model of explicit fear because it relies on both the cortex and hippocampus and requires explicit contingency awareness in humans. Here, we explore the neural circuit supporting trace fear extinction in order to better understand how complex memories extinguish. We first show that the amygdala is selectively involved in delay fear extinction; blocking intra-amygdala glutamate receptors disrupted delay, but not trace extinction. Further, ERK phosphorylation was increased in the amygdala after delay, but not trace extinction. We then identify the retrosplenial cortex (RSC) as a key structure supporting trace extinction. ERK phosphorylation was selectively increased in the RSC following trace extinction and blocking intra-RSC NMDA receptors impaired trace, but not delay extinction. These findings indicate that delay and trace extinction require different neural circuits; delay extinction requires plasticity in the amygdala whereas trace extinction requires the RSC. Anxiety disorders linked to explicit memory may therefore depend on cortical processes that have not been traditionally targeted by extinction studies based on delay fear.

NR2A- and NR2B-containing NMDA receptors in the prelimbic medial prefrontal cortex differentially mediate trace, delay, and contextual fear conditioning.

Activation of N-methyl-D-aspartate receptors (NMDAR) in the prelimbic medial prefrontal cortex (PL mPFC) is necessary for the acquisition of both trace and contextual fear memories, but it is not known how specific NR2 subunits support each association. The NR2B subunit confers unique properties to the NMDAR and may differentially regulate these two fear memories. Here we show that NR2A-containing NMDARs mediate trace, delay, and contextual fear memories, but NR2B-containing NMDARs are required only for trace conditioning, consistent with a role for PL mPFC in working memory.

The ubiquitin-specific protease 14 (USP14) is a critical regulator of long-term memory formation.

Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme that is thought to regulate protein degradation in neurons; however, it is unknown if USP14 is involved in learning-dependent synaptic plasticity. We found that infusion of a USP14 inhibitor into the amygdala impaired long-term memory for a fear conditioning task, suggesting that USP14 is a critical regulator of long-term memory formation in the amygdala.

Long-lasting effects of disturbing the circadian rhythm or sleep in adolescence.

Circadian rhythms are endogenous, near 24-hour rhythms that regulate a multitude of biological and behavioral processes across the diurnal cycle in most organisms. Over the lifespan, a bell curve pattern emerges in circadian phase preference (i.e. chronotype), with children and adults generally preferring to wake earlier and fall asleep earlier, and adolescents and young adults preferring to wake later and fall asleep later than their adult counterparts. This well-defined shift speaks to the variability of circadian rhythmicity over the lifespan and the changing needs and demands of the brain as an organism develops, particularly in the adolescent period. Indeed, adolescence is known to be a critical period of development during which dramatic neuroanatomical changes are occurring to allow for improved decision-making. Due to the large amount of re-structuring occurring in the adolescent brain, circadian disruptions during this period could have adverse consequences that persist across the lifespan. While the detrimental effects of circadian disruptions in adults have been characterized in depth, few studies have longitudinally assessed the potential long-term impacts of circadian disruptions during adolescence. Here, we will review the evidence that disruptions in circadian rhythmicity during adolescence have effects that persist into adulthood. As biological and social time often conflict in modern society, with school start times misaligned with adolescents' endogenous rhythms, it is critical to understand the long-term impacts of disrupted circadian rhythmicity in adolescence.

Pharmacological HDAC3 inhibition alters memory updating in young and old mice.

Long-term memories are not stored in a stable state but must be flexible and dynamic to maintain relevance in response to new information. Existing memories are thought to be updated through the process of reconsolidation, in which memory retrieval initiates destabilization and updating to incorporate new information. Memory updating is impaired in old age, yet little is known about the mechanisms that go awry. One potential mechanism is the repressive histone deacetylase 3 (HDAC3), which is a powerful negative regulator of memory formation that contributes to age-related impairments in memory formation. Here, we tested whether HDAC3 also contributes to age-related impairments in memory updating using the Objects in Updated Locations (OUL) paradigm. We show that blocking HDAC3 immediately after updating with the pharmacological inhibitor RGFP966 ameliorated age-related impairments in memory updating in 18-m.o. mice. Surprisingly, we found that post-update HDAC3 inhibition in young (3-m.o.) mice had no effect on memory updating but instead impaired memory for the original information, suggesting that the original and updated information may compete for expression at test and HDAC3 helps regulate which information is expressed. To test this idea, we next assessed whether HDAC3 inhibition would improve memory updating in young mice given a weak, subthreshold update. Consistent with our hypothesis, we found that HDAC3 blockade strengthened the subthreshold update without impairing memory for the original information, enabling balanced expression of the original and updated information. Together, this research suggests that HDAC3 may contribute to age-related impairments in memory updating and may regulate the strength of a memory update in young mice, shifting the balance between the original and updated information at test.

The timing of multiple retrieval events can alter GluR1 phosphorylation and the requirement for protein synthesis in fear memory reconsolidation.

Numerous studies have indicated that maintaining a fear memory after retrieval requires de novo protein synthesis. However, no study to date has examined how the temporal dynamics of repeated retrieval events affect this protein synthesis requirement. The present study varied the timing of a second retrieval of an established auditory fear memory and followed this second retrieval with infusions of the protein synthesis inhibitor anisomycin (ANI) into the basolateral amygdala. Results indicated that the memory-impairing effects of ANI were not observed when the second retrieval occurred soon after the first (within 1 h), and that the inhibitor gradually regained effectiveness as the retrieval episodes were spaced further apart. Additionally, if the second of the closely timed retrievals was omitted prior to ANI infusions, long-term memory deficits were observed, suggesting that the altered effectiveness of ANI was due specifically to the second retrieval event. Further experiments revealed that the second retrieval was not associated with a change in Zif268 protein expression but did produce a rapid and persistent dephosphorylation of GluR1 receptors at Ser845, an AMPAR trafficking site known to regulate the stability of GluR2 lacking AMPARs, which have been shown to be important in memory updating. This suggests that the precise timing of multiple CS presentations during the reconsolidation window may affect the destabilization state of the memory trace.

Connecting the Dots: Potential Interactions Between Sex Hormones and the Circadian System During Memory Consolidation.

Both the circadian clock and sex hormone signaling can strongly influence brain function, yet little is known about how these 2 powerful modulatory systems might interact during complex neural processes like memory consolidation. Individually, the molecular components and action of each of these systems have been fairly well-characterized, but there is a fundamental lack of information about how these systems cooperate. In the circadian system, clock genes function as timekeeping molecules that convey time-of-day information on a well-stereotyped cycle that is governed by the suprachiasmatic nucleus. Keeping time is particularly important to synchronize various physiological processes across the brain and body, including those that regulate memory consolidation. Similarly, sex hormones are powerful modulators of memory, with androgens, estrogens, and progestins, all influencing memory consolidation within memory-relevant brain regions like the hippocampus. Despite clear evidence that each system can influence memory individually, exactly how the circadian and hormonal systems might interact to impact memory consolidation remains unclear. Research investigating either sex hormone action or circadian gene function within memory-relevant brain regions has unveiled several notable places in which the two systems could interact to control memory. Here, we bring attention to known interactions between the circadian clock and sex hormone signaling. We then review sex hormone-mediated control of memory consolidation, highlighting potential nodes through which the circadian system might interact during memory formation. We suggest that the bidirectional relationship between these two systems is essential for proper control of memory formation based on an animal's hormonal and circadian state.