Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcripts by excessive ethanol drinking.

The underlying genetic and epigenetic mechanisms driving functional adaptations in neuronal excitability and excessive alcohol intake are poorly understood. Small-conductance Ca2+-activated K+ (KCa2 or SK) channels encoded by the KCNN family of genes have emerged from preclinical studies as a key contributor to alcohol-induced functional neuroadaptations in alcohol-drinking monkeys and alcohol-dependent mice. Here, this cross-species analysis focused on KCNN3 DNA methylation, gene expression, and single nucleotide polymorphisms, including alternative promoters in KCNN3, that could influence surface trafficking and function of KCa2 channels. Bisulfite sequencing analysis of the nucleus accumbens tissue from alcohol-drinking monkeys and alcohol-dependent mice revealed a differentially methylated region in exon 1A of KCNN3 that overlaps with a predicted promoter sequence. The hypermethylation of KCNN3 in the accumbens paralleled an increase in the expression of alternative transcripts that encode apamin-insensitive and dominant-negative KCa2 channel isoforms. A polymorphic repeat in macaque KCNN3 encoded by exon 1 did not correlate with alcohol drinking. At the protein level, KCa2.3 channel expression in the accumbens was significantly reduced in very heavy-drinking monkeys. Together, our cross-species findings on epigenetic dysregulation of KCNN3 represent a complex mechanism that utilizes alternative promoters to potentially impact the firing of accumbens neurons. Thus, these results provide support for hypermethylation of KCNN3 as a possible key molecular mechanism underlying harmful alcohol intake and alcohol use disorder.

Interaction of chronic intermittent ethanol and repeated stress on structural and functional plasticity in the mouse medial prefrontal cortex.

Stress is a risk factor that plays a considerable role in the development and maintenance of alcohol (ethanol) abuse and relapse. Preclinical studies examining ethanol-stress interactions have demonstrated elevated ethanol drinking, cognitive deficits, and negative affective behaviors in mice. However, the neural adaptations in prefrontal cortical regions that drive these aberrant behaviors produced by ethanol-stress interactions are unknown. In this study, male C57BL/6J mice were exposed to chronic intermittent ethanol (CIE) and repeated forced swim stress (FSS). After two cycles of CIE x FSS, brain slices containing the prelimbic (PrL) and infralimbic (IfL) cortex were prepared for analysis of adaptations in dendritic spines and synaptic plasticity. In the PrL cortex, total spine density was increased in mice exposed to CIE. Immediately following induction of long-term potentiation (LTP), the fEPSP slope was increased in the PrL of CIE x FSS treated mice, indicative of a presynaptic adaptation on post-tetanic potentiation (PTP). In the IfL cortex, CIE exposure regardless of FSS experience resulted in an increase in spine density. FSS alone or when combined with CIE exposure increased PTP following LTP induction. Repeated FSS episodes increased IfL cortical paired-pulse facilitation, a second measure of presynaptic plasticity. In summary, CIE exposure resulted in structural adaptations while repeated stress exposure drove metaplastic changes in presynaptic function, demonstrating distinct morphological and functional changes in PrL and IfL cortical neurons. Thus, the structural and functional adaptations may be one mechanism underlying the development of excessive drinking and cognitive deficits associated with ethanol-stress interactions.

Bioinformatics identification and pharmacological validation of Kcnn3/KCa2 channels as a mediator of negative affective behaviors and excessive alcohol drinking in mice.

Mood disorders are often comorbid with alcohol use disorder (AUD) and play a considerable role in the development and maintenance of alcohol dependence and relapse. Because of this high comorbidity, it is necessary to determine shared and unique genetic factors driving heavy drinking and negative affective behaviors. In order to identify novel pharmacogenetic targets, a bioinformatics analysis was used to quantify the expression of amygdala K+ channel genes that covary with anxiety-related phenotypes in the well-phenotyped and fully sequenced family of BXD strains. We used a model of stress-induced escalation of drinking in alcohol-dependent mice to measure negative affective behaviors during abstinence. A pharmacological approach was used to validate the key bioinformatics findings in alcohol-dependent, stressed mice. Amygdalar expression of Kcnn3 correlated significantly with 40 anxiety-associated phenotypes. Further examination of Kcnn3 expression revealed a strong eigentrait for anxiety-like behaviors and negative correlations with binge-like and voluntary alcohol drinking. Mice treated with chronic intermittent alcohol exposure and repeated swim stress consumed more alcohol in their home cages and showed hypophagia on the novelty-suppressed feeding test during abstinence. Pharmacologically targeting Kcnn gene products with the KCa2 (SK) channel-positive modulator 1-EBIO decreased drinking and reduced feeding latency in alcohol-dependent, stressed mice. Collectively, these validation studies provide central nervous system links into the covariance of stress, negative affective behaviors, and AUD in the BXD strains. Further, the bioinformatics discovery tool is effective in identifying promising targets (i.e., KCa2 channels) for treating alcohol dependence exacerbated by comorbid mood disorders.

Kv7 channels in the nucleus accumbens are altered by chronic drinking and are targets for reducing alcohol consumption.

Alcohol use disorders (AUDs) are a major public health issue and produce enormous societal and economic burdens. Current Food and Drug Administration (FDA)-approved pharmacotherapies for treating AUDs suffer from deleterious side effects and are only effective in a subset of individuals. It is therefore essential to find improved medications for the management of AUDs. Emerging evidence suggests that anticonvulsants are a promising class of drugs for treating individuals with AUDs. In these studies, we used integrative functional genomics to demonstrate that genes that encode Kv7 channels (i.e. Kcnq2/3) are related to alcohol (ethanol) consumption, preference and acceptance in rodents. We then tested the ability of the FDA-approved anticonvulsant retigabine, a Kv7 channel opener, to reduce voluntary ethanol consumption of Wistar rats in a two-bottle choice intermittent alcohol access paradigm. Systemic administration and microinjections of retigabine into the nucleus accumbens significantly reduced alcohol drinking, and retigabine was more effective at reducing intake in high- versus low-drinking populations of Wistar rats. Prolonged voluntary drinking increased the sensitivity to the proconvulsant effects of pharmacological blockade of Kv7 channels and altered surface trafficking and SUMOylation patterns of Kv7.2 channels in the nucleus accumbens. These data implicate Kcnq2/3 in the regulation of ethanol drinking and demonstrate that long-term drinking produces neuroadaptations in Kv7 channels. In addition, these results have identified retigabine as a potential pharmacotherapy for treating AUDs and Kv7 channels as a novel therapeutic target for reducing heavy drinking.

Homer2 deletion alters dendritic spine morphology but not alcohol-associated adaptations in GluN2B-containing N-methyl-D-aspartate receptors in the nucleus accumbens.

Repeated exposure to ethanol followed by withdrawal leads to alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc) in both clinical and preclinical models of ethanol exposure. Homer2 is a member of a family of postsynaptic density (PSD) scaffolding proteins that functions in part to cluster N-methyl-D-aspartate (NMDA) signaling complexes in the PSD, and has been shown to be critically important for plasticity in multiple models of drug and alcohol abuse. Here we used Homer2 knockout (KO) mice and a chronic intermittent intraperitoneal (IP) ethanol injection model to investigate a potential role for the protein in ethanol-induced adaptations in dendritic spine morphology and PSD protein expression. While deletion of Homer2 was associated with increased density of long spines on medium spiny neurons of the NAc core of saline treated mice, ethanol exposure had no effect on dendritic spine morphology in either wild-type (WT) or Homer2 KO mice. Western blot analysis of tissue samples from the NAc enriched for PSD proteins revealed a main effect of ethanol treatment on the expression of GluN2B, but there was no effect of genotype or treatment on the expression other glutamate receptor subunits or PSD95. These data indicate that the global deletion of Homer2 leads to aberrant regulation of dendritic spine morphology in the NAc core that is associated with an increased density of long, thin spines. Unexpectedly, intermittent IP ethanol did not affect spine morphology in either WT or KO mice. Together these data implicate Homer2 in the formation of long, thin spines and further supports its role in neuronal structure.

KCNN Genes that Encode Small-Conductance Ca2+-Activated K+ Channels Influence Alcohol and Drug Addiction.

Small-conductance Ca(2+)-activated K(+) (KCa2) channels control neuronal excitability and synaptic plasticity, and have been implicated in substance abuse. However, it is unknown if genes that encode KCa2 channels (KCNN1-3) influence alcohol and drug addiction. In the present study, an integrative functional genomics approach shows that genetic datasets for alcohol, nicotine, and illicit drugs contain the family of KCNN genes. Alcohol preference and dependence QTLs contain KCNN2 and KCNN3, and Kcnn3 transcript levels in the nucleus accumbens (NAc) of genetically diverse BXD strains of mice predicted voluntary alcohol consumption. Transcript levels of Kcnn3 in the NAc negatively correlated with alcohol intake levels in BXD strains, and alcohol dependence enhanced the strength of this association. Microinjections of the KCa2 channel inhibitor apamin into the NAc increased alcohol intake in control C57BL/6J mice, while spontaneous seizures developed in alcohol-dependent mice following apamin injection. Consistent with this finding, alcohol dependence enhanced the intrinsic excitability of medium spiny neurons in the NAc core and reduced the function and protein expression of KCa2 channels in the NAc. Altogether, these data implicate the family of KCNN genes in alcohol, nicotine, and drug addiction, and identify KCNN3 as a mediator of voluntary and excessive alcohol consumption. KCa2.3 channels represent a promising novel target in the pharmacogenetic treatment of alcohol and drug addiction.

Withdrawal from chronic intermittent alcohol exposure increases dendritic spine density in the lateral orbitofrontal cortex of mice.

Alcohol use disorders (AUDs) are associated with functional and morphological changes in subfields of the prefrontal cortex. Clinical and preclinical evidence indicates that the orbitofrontal cortex (OFC) is critical for controlling impulsive behaviors, representing the value of a predicted outcome, and reversing learned associations. Individuals with AUDs often demonstrate deficits in OFC-dependent tasks, and rodent models of alcohol exposure show that OFC-dependent behaviors are impaired by chronic alcohol exposure. To explore the mechanisms that underlie these impairments, we examined dendritic spine density and morphology, and NMDA-type glutamate receptor expression in the lateral OFC of C57BL/6J mice following chronic intermittent ethanol (CIE) exposure. Western blot analysis demonstrated that NMDA receptors were not altered immediately following CIE exposure or after 7 days of withdrawal. Morphological analysis of basal dendrites of layer II/III pyramidal neurons revealed that dendritic spine density was also not affected immediately after CIE exposure. However, the total density of dendritic spines was significantly increased after a 7-day withdrawal from CIE exposure. The effect of withdrawal on spine density was mediated by an increase in the density of long, thin spines with no change in either stubby or mushroom spines. These data suggest that morphological neuroadaptations in lateral OFC neurons develop during alcohol withdrawal and occur in the absence of changes in the expression of NMDA-type glutamate receptors. The enhanced spine density that follows alcohol withdrawal may contribute to the impairments in OFC-dependent behaviors observed in CIE-treated mice.