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.

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.

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.

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.

The clock gene Per1 may exert diurnal control over hippocampal memory consolidation.

The circadian system influences many different biological processes, including memory performance. While the suprachiasmatic nucleus (SCN) functions as the brain's central pacemaker, downstream "satellite clocks" may also regulate local functions based on the time of day. Within the dorsal hippocampus (DH), for example, local molecular oscillations may contribute to time-of-day effects on memory. Here, we used the hippocampus-dependent Object Location Memory task to determine how memory is regulated across the day/night cycle in mice. First, we systematically determined which phase of memory (acquisition, consolidation, or retrieval) is modulated across the 24 h day. We found that mice show better long-term memory performance during the day than at night, an effect that was specifically attributed to diurnal changes in memory consolidation, as neither memory acquisition nor memory retrieval fluctuated across the day/night cycle. Using RNA-sequencing we identified the circadian clock gene Period1 (Per1) as a key mechanism capable of supporting this diurnal fluctuation in memory consolidation, as learning-induced Per1 oscillates in tandem with memory performance in the hippocampus. We then show that local knockdown of Per1 within the DH impairs spatial memory without affecting either the circadian rhythm or sleep behavior. Thus, Per1 may independently function within the DH to regulate memory in addition to its known role in regulating the circadian system within the SCN. Per1 may therefore exert local diurnal control over memory consolidation within the DH.

The clock gene Per1 is necessary in the retrosplenial cortex-but not in the suprachiasmatic nucleus-for incidental learning in young and aging male mice.

Aging impairs both circadian rhythms and memory, though the relationship between these impairments is not fully understood. Circadian rhythms are largely dictated by clock genes within the body's central pacemaker, the suprachiasmatic nucleus (SCN), though these genes are also expressed in local clocks throughout the body. As circadian rhythms can directly affect memory performance, one possibility is that memory deficits observed with age are downstream of global circadian rhythm disruptions stemming from the SCN. Here, we demonstrate that expression of clock gene Period1 within a memory-relevant cortical structure, the retrosplenial cortex (RSC), is necessary for incidental learning, and that age-related disruption of Period1 within the RSC-but not necessarily the SCN-contributes to cognitive decline. These data expand the known functions of clock genes beyond maintaining circadian rhythms and suggests that age-associated changes in clock gene expression modulates circadian rhythms and memory performance in a brain region-dependent manner.

Investigating Memory Updating in Mice Using the Objects in Updated Locations Task.

In the laboratory, memory is typically studied as a de novo experience, in which a naïve animal is exposed to a discrete learning event that is markedly different from its past experiences. Most real-world memories, however, are updates-modifications or additions-to existing memories. This is particularly true in the aging, experienced brain. To better understand memory updating, we have developed a new behavioral paradigm called the objects in updated locations (OUL) task. OUL relies on hippocampus-dependent spatial learning and has the advantage of being able to test both the original memory and the updated information in a single test session. Further, OUL relies on incidental learning that avoids unnecessary stress that might hinder the performance of aging animals. In OUL, animals first learn the location of two identical objects in a familiar context. This memory is then updated by moving one object to a new location. Finally, to assess the animals' memory for the original and the updated information, all animals are given a test session in which they are exposed to four copies of the object: two in the original training locations, one in the updated location, and one in a novel location. By comparing exploration of the novel location to the familiar locations, we can infer whether the animal remembers the original and updated object locations. OUL is a simple but powerful task that could provide new insights into the cellular, circuit-level, and molecular mechanisms that support memory updating. © 2020 by John Wiley & Sons, Inc.