HDAC3-selective inhibitor enhances extinction of cocaine-seeking behavior in a persistent manner.

Nonspecific histone deacetylase (HDAC) inhibition has been shown to facilitate the extinction of drug-seeking behavior in a manner resistant to reinstatement. A key open question is which specific HDAC is involved in the extinction of drug-seeking behavior. Using the selective HDAC3 inhibitor RGFP966, we investigated the role of HDAC3 in extinction and found that systemic treatment with RGFP966 facilitates extinction in mice in a manner resistant to reinstatement. We also investigated whether the facilitated extinction is related to the enhancement of extinction consolidation during extinction learning or to negative effects on performance or reconsolidation. These are key distinctions with regard to any compound being used to modulate extinction, because a more rapid decrease in a defined behavior is interpreted as facilitated extinction. Using an innovative combination of behavioral paradigms, we found that a single treatment of RGFP966 enhances extinction of a previously established cocaine-conditioned place preference, while simultaneously enhancing long-term object-location memory within subjects. During extinction consolidation, HDAC3 inhibition promotes a distinct pattern of histone acetylation linked to gene expression within the infralimbic cortex, hippocampus, and nucleus accumbens. Thus, the facilitated extinction of drug-seeking cannot be explained by adverse effects on performance. These results demonstrate that HDAC3 inhibition enhances the memory processes involved in extinction of drug-seeking behavior.

HDAC3 is a negative regulator of cocaine-context-associated memory formation.

Cocaine-induced neuroplasticity mediated by histone acetylating and deacetylating enzymes may contribute to addiction-like behaviors. For example, overexpression of histone deacetylases (HDACs) 4 or 5 in the nucleus accumbens suppresses cocaine-induced conditioned place preference (CPP) acquisition in mice. HDAC4 and HDAC5 are known to interact with HDAC3, but the role of HDAC3 in cocaine-induced behaviors has never been examined. In this study, we address the hypothesis that HDAC3 is a negative regulator of cocaine-context-associated memory formation in mice. We examined the role of HDAC3 during the conditioning phase of CPP, when the mouse has the opportunity to form an associative memory between the cocaine-paired context and the subjective effects of cocaine. To address this hypothesis, Hdac3(flox/flox) and Hdac3(+/+) mice (generated from a C57BL/6 background) were infused into the nucleus accumbens with adeno-associated virus expressing Cre recombinase to create focal, homozygous Hdac3 deletions. Hdac3(flox/flox) mice exhibit significantly enhanced CPP acquisition, which is correlated with increased gene expression during the consolidation phase of acquisition. Increased gene expression of c-Fos and Nr4a2 is correlated with decreased HDAC3 occupancy and increased histone H4 lysine 8 acetylation at their promoters. The results from this study demonstrate that HDAC3 negatively regulates cocaine-induced CPP acquisition.

Differential contribution of CBP:CREB binding to corticotropin-releasing hormone expression in the infant and adult hypothalamus.

Corticotropin-releasing hormone (CRH) contributes crucially to the regulation of central and peripheral responses to stress. Because of the importance of a finely tuned stress system, CRH expression is tightly regulated in an organ- and brain region-specific manner. Thus, in the hypothalamus, CRH is constitutively expressed and this expression is further enhanced by stress; however, the underlying regulatory mechanisms are not fully understood. The regulatory region of the crh gene contains several elements, including the cyclic-AMP response element (CRE), and the role of the CRE interaction with the cyclic-AMP response element binding protein (CREB) in CRH expression has been a focus of intensive research. Notably, whereas thousands of genes contain a CRE, the functional regulation of gene expression by the CRE:CREB system is limited to ∼100 genes, and likely requires additional proteins. Here, we investigated the role of a member of the CREB complex, CREB binding protein (CBP), in basal and stress-induced CRH expression during development and in the adult. Using mice with a deficient CREB-binding site on CBP, we found that CBP:CREB interaction is necessary for normal basal CRH expression at the mRNA and protein level in the nine-day-old mouse, prior to onset of functional regulation of hypothalamic CRH expression by glucocorticoids. This interaction, which functions directly on crh or indirectly via regulation of other genes, was no longer required for maintenance of basal CRH expression levels in the adult. However, CBP:CREB binding contributed to stress-induced CRH expression in the adult, enabling rapid CRH synthesis in hypothalamus. CBP:CREB binding deficiency did not disrupt basal corticosterone plasma levels or acute stress-evoked corticosterone release. Because dysregulation of CRH expression occurs in stress-related disorders including depression, a full understanding of the complex regulation of this gene is important in both health and disease.

HDAC3 is a critical negative regulator of long-term memory formation.

Gene expression is dynamically regulated by chromatin modifications on histone tails, such as acetylation. In general, histone acetylation promotes transcription, whereas histone deacetylation negatively regulates transcription. The interplay between histone acetyltranserases and histone deacetylases (HDACs) is pivotal for the regulation of gene expression required for long-term memory processes. Currently, very little is known about the role of individual HDACs in learning and memory. We examined the role of HDAC3 in long-term memory using a combined genetic and pharmacologic approach. We used HDAC3-FLOX genetically modified mice in combination with adeno-associated virus-expressing Cre recombinase to generate focal homozygous deletions of Hdac3 in area CA1 of the dorsal hippocampus. To complement this approach, we also used a selective inhibitor of HDAC3, RGFP136 [N-(6-(2-amino-4-fluorophenylamino)-6-oxohexyl)-4-methylbenzamide]. Immunohistochemistry showed that focal deletion or intrahippocampal delivery of RGFP136 resulted in increased histone acetylation. Both the focal deletion of HDAC3 as well as HDAC3 inhibition via RGFP136 significantly enhanced long-term memory in a persistent manner. Next we examined expression of genes implicated in long-term memory from dorsal hippocampal punches using quantitative reverse transcription-PCR. Expression of nuclear receptor subfamily 4 group A, member 2 (Nr4a2) and c-fos was significantly increased in the hippocampus of HDAC3-FLOX mice compared with wild-type controls. Memory enhancements observed in HDAC3-FLOX mice were abolished by intrahippocampal delivery of Nr4a2 small interfering RNA, suggesting a mechanism by which HDAC3 negatively regulates memory formation. Together, these findings demonstrate a critical role for HDAC3 in the molecular mechanisms underlying long-term memory formation.

The role of histone acetylation in cocaine-induced neural plasticity and behavior.

How do drugs of abuse, such as cocaine, cause stable changes in neural plasticity that in turn drive long-term changes in behavior? What kind of mechanism can underlie such stable changes in neural plasticity? One prime candidate mechanism is epigenetic mechanisms of chromatin regulation. Chromatin regulation has been shown to generate short-term and long-term molecular memory within an individual cell. They have also been shown to underlie cell fate decisions (or cellular memory). Now, there is accumulating evidence that in the CNS, these same mechanisms may be pivotal for drug-induced changes in gene expression and ultimately long-term behavioral changes. As these mechanisms are also being found to be fundamental for learning and memory, an exciting new possibility is the extinction of drug-seeking behavior by manipulation of epigenetic mechanisms. In this review, we critically discuss the evidence demonstrating a key role for chromatin regulation via histone acetylation in cocaine action.

Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter.

Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action.

Regulation of CART peptide expression by CREB in the rat nucleus accumbens in vivo.

Production of mRNA from the cocaine- and amphetamine-regulated transcript (CART) gene is regulated by cocaine and other drugs of abuse in the nucleus accumbens (NAc), a brain reward region. Current hypotheses postulate that CART peptides there oppose the rewarding actions of cocaine by opposing the effects of dopaminergic transmission. Since over expression of CREB was shown to decrease cocaine-mediated reward, we hypothesized that CART could be a target gene for CREB in the NAc and that over expression of CREB would increase CART peptide levels. Transcription factor (TF) binding to DNA is influenced by sequences adjacent to consensus TF binding sites and other factors. We thus examined CREB binding to a 27mer oligonucleotide containing the CRE sequence from the CART gene proximal promoter. Using electrophoretic mobility shift assays and TF-antibody super shift assays, CREB was found to bind to the CRE sequence from the CART promoter. To test if over expression of CREB in the NAc affected CART peptide levels, Herpes simplex virus-1 vectors over expressing CREB (HSV-CREB), or a vector that expressed LacZ (HSV-LacZ) as a control, were injected into the NAc of rats. Western blotting and in situ hybridization showed that HSV-CREB injections increased CART mRNA and peptide levels. Injections of a dominant negative CREB mutant (HSV-mCREB) did not alter either CART mRNA or peptide levels. The finding that CREB can regulate the levels of CART mRNA and peptides in vivo in the NAc supports a role for CART peptides in psychostimulant-induced reward and reinforcement.