Drugs of abuse, such as cocaine, are known to cause long-term changes in reward circuitry and, ultimately, persistent changes in behavior. Although many critical nodes in the neural circuitry contributing to relapse of drug-seeking behavior have been identified and studied, a full characterization of the networks driving reinstatement of cocaine-seeking remains lacking. One area that may provide further insight to the mechanisms of relapse is the habenula complex, an epithalamic region composed of lateral (LHb) and medial (MHb) substructures. Recent research has demonstrated a role for the MHb in regulating drug-associated behavior; specifically, the MHb has been shown to be a regulator of nicotine-seeking behavior and nicotine withdrawal. Though there is mounting evidence suggesting the MHb is responsible for various behavioral responses to various drugs of abuse, the position of the MHb in regulating relapse-like behaviors remains tenuous. The present dissertation examined the role of the MHb in regulating reinstatement of cocaine-associated behavior. We demonstrate MHb activity is engaged during relapse of cocaine-associated behavior and driving activity in a subset of the MHb population mimics the reinstatement response. Moreover, we identify a unique epigenetic signature at transcription factor Nr4a2 that is critical for the reinstatement of cocaine-associated behaviors. My studies provide new insight into the role of molecular adaptations within the MHb in regulating substance use disorders.

Recent evidence have implicated epigenetic mechanisms in drug-associated memory formation. A possible role for one major epigenetic mechanism, nucleosome remodeling, in drug-associated memory formation has been largely unexplored. The present dissertation examined mice with genetic manipulations targeting a neuron-specific nucleosome remodeling complex subunit, BAF53b. These mice displayed deficits in cocaine-associated memory that were more severe in BAF53b dominant-negative transgenic mice compared to BAF53b heterozygous knockout mice. Similar to the memory deficits, theta-induced long-term potentiation (theta-LTP) in the nucleus accumbens (NAc) was significantly impaired in slices taken from BAF53b transgenic mutant mice but not heterozygous knockout mice. Further studies indicated that theta-LTP in the NAc is dependent on TrkB receptor activation, and that BDNF rescues theta-LTP and cocaine-associated memory in BAF53b transgenic mice. My studies suggest a role for BAF53b in NAc neuronal function required for cocaine-associated memories. Furthermore, targeting BDNF/TrkB activation in the nucleus accumbens may overcome memory and plasticity deficits linked to BAF53b mutations.

Epigenetic mechanisms regulate critical processes of neuroplasticity that drive cocaine- seeking. Previous work from our lab identified the class I histone deacetylase (HDAC) HDAC3 acts as a negative regulator of cocaine-associated memory formation in the nucleus accumbens (NAc). However, it remains unknown whether this occurs via HDAC3’s deacetylase function. It also unclear whether HDAC3 regulates cocaine-induced in a cell-type specific manner within the NAc. Cocaine induces distinct changes in the two major cell populations of the NAc (D1R- and D2R-medium spiny neurons) and activation of these cell types drive opposing behavioral responses to cocaine. In the present dissertation, we investigated whether HDAC3’s deacetylase activity acts in a cell-type specific manner within the NAc to regulate behavioral responses to cocaine. In these experiments, we demonstrate that HDAC3’s deacetylase activity drives molecular and cellular mechanisms of plasticity within the NAc. In addition, we show how HDAC3’s activity within D1R-MSNs, not D2R-MSNs, affects cocaine-associated memory formation and cocaine seeking behaviors. We also demonstrate how HDAC3 regulates histone acetylation to drive changes in gene expression and synaptic plasticity within D1R-MSNs. Overall, this work illustrates how cocaine disrupts HDAC3-dependent mechanisms in a cell-type specific manner to drive cocaine-seeking behaviors.

Exposure to drugs of abuse, such as cocaine, engages transcriptional machinery to alter gene expression profiles, ultimately giving rise to long-term changes in cellular function, synaptic plasticity, and drug-related behavior. Previous work from our lab has identified glucocorticoid-induced leucine zipper (Gilz) as an epigenetically regulated gene that may be responsible for orchestrating long-term transcriptional adaptations underlying such behaviors. However, it remains unknown whether the distinct mRNA splice variants encoded by the Gilz gene play unique roles in this context. It is also unclear whether Gilz splice variants in the brain differentially respond to stress-related events, despite the body of literature demonstrating Gilz as a downstream transcriptional regulator of glucocorticoid-signaling. In the present dissertation, we investigated whether Gilz splice variants exhibit differential responses to cocaine- or stress-exposure, and we investigated whether Gilz is necessary for (1) long-term potentiation (LTP) in the nucleus accumbens (NAc), (2) cocaine-conditioned place preference, and (3) cocaine self-administration behaviors. We demonstrate that Gilz acts in the NAc to regulate LTP, in the VTA to regulate CPP, and that Gilz is necessary for reinstatement of cocaine-seeking behavior in male mice. We also demonstrate that Gilz splice variants differentially respond to a variety of stress-related stimuli. Overall, this work illustrates how cocaine or stress may engage Gilz in distinct brain regions to regulate cellular and behavioral responses to maladaptive experiences.

The work in this presentation creates a completely novel dataset for exploring DNA methylation as a means for predicting epigenetic (biological) age and memory performance. In the first study, dorsal hippocampal (DHPC) tissue from young and aging mice, both male and female, was processed for reduced representation bisulfite sequencing (RRBS) to establish methylome signatures. We found aging male and female mice exhibited global hypomethylation, but diverging sex-dependent patterns in methylation enrichment specific to genomic context. Aging males displayed significant hypomethylation in 3’UTR regions while females displayed significant hypermethylation in the same region. In both sexes, hypomethylated regions were significantly associated with binding motifs integral to activity-dependent transcription. In a second study, male mice at five different ages underwent a memory updating behavior task followed by RRBS analysis of dorsal hippocampal tissue to investigate how methylation may correlate to age-related memory impairment. With this behavioral cohort, we created a highly accurate epigenetic age predictor, labeled an epigenetic clock. We also generated a second and third memory updating and testing behavior predictor, each with a unique set of CpG sites. DHPC DNA methylation correlation with behavior was strongest when tested for an original memory, but not an updated memory. Thus, this suggests that generation of behavior predictors may be time-dependent and strongest for retrieval of original information, influenced by methylation patterns in the dorsal hippocampus. In sum, we generated the first epigenetic clock, to our knowledge, specific to bulk tissue in the male mouse dorsal hippocampus. Our data suggests that separate but accurate behavior predictors of memory performance are a plausible and compelling link to age-related changes in DNA methylation.

Recent exome sequencing studies have implicated polymorphic BAF complexes (mammalian SWI/SNF chromatin remodeling complexes) in several human intellectual disabilities and cognitive disorders. However, it is currently unknown how mutations in BAF complexes result in impaired cognitive function. Post mitotic neurons express a neuron specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Mice harboring selective genetic manipulations of BAF53b have severe defects in long-term memory and long-lasting forms of hippocampal synaptic plasticity. We rescued memory impairments in BAF53b mutant mice by reintroducing BAF53b in the adult hippocampus, indicating a role for BAF53b beyond neuronal development. The defects in BAF53b mutant mice appear to derive from alterations in gene expression that produce abnormal postsynaptic components, such as spine structure and function, and ultimately lead to deficits in synaptic plasticity. My studies provide new insight into the role of dominant mutations in subunits of BAF complexes in human intellectual and cognitive disorders.