Alcohol excitation of the ventral tegmental area (VTA) is important in neurobiological processes related to the development of alcoholism. The ionotropic receptors on VTA neurons that mediate ethanol-induced excitation have not been identified. Quinidine blocks ethanol excitation of VTA neurons, and blockade of two-pore potassium channels is among the actions of quinidine. Therefore two-pore potassium channels in the VTA may be potential targets for the action of ethanol. Here, we explored whether ethanol activation of VTA neurons is mediated by the two-pore potassium channel KCNK13. Extracellular recordings of the response of VTA neurons to ethanol were performed in combination with knockdown of Kcnk13 using a short hairpin RNA (shRNA) in C57BL/6 J mice. Real-time PCR and immunohistochemistry were used to examine expression of this channel in the VTA. Finally, the role of KCNK13 in binge-like drinking was examined in the drinking in the dark test after knockdown of the channel. Kcnk13 expression in the VTA was increased by acute ethanol exposure. Ethanol-induced excitation of VTA neurons was selectively reduced by shRNA targeting Kcnk13. Importantly, knockdown of Kcnk13 in the VTA resulted in increased alcohol drinking. These results are consistent with the idea that ethanol stimulates VTA neurons at least in part by inhibiting KCNK13, a specific two-pore potassium channel, and that KCNK13 can control both VTA neuronal activity and binge drinking. KCNK13 is a novel alcohol-sensitive molecular target and may be amenable to the development of pharmacotherapies for alcoholism treatment.

Alcohol excitation of the ventral tegmental area (VTA) is important in neurobiological processes related to the development of alcoholism. The ionotropic receptors on VTA neurons that mediate ethanol-induced excitation have not been identified. Quinidine blocks ethanol excitation of VTA neurons, and blockade of two-pore potassium channels is among the actions of quinidine. Therefore two-pore potassium channels in the VTA may be potential targets for the action of ethanol. Here, we explored whether ethanol activation of VTA neurons is mediated by the two-pore potassium channel KCNK13. Extracellular recordings of the response of VTA neurons to ethanol were performed in combination with knockdown of Kcnk13 using a short hairpin RNA (shRNA) in C57BL/6 J mice. Real-time PCR and immunohistochemistry were used to examine expression of this channel in the VTA. Finally, the role of KCNK13 in binge-like drinking was examined in the drinking in the dark test after knockdown of the channel. Kcnk13 expression in the VTA was increased by acute ethanol exposure. Ethanol-induced excitation of VTA neurons was selectively reduced by shRNA targeting Kcnk13. Importantly, knockdown of Kcnk13 in the VTA resulted in increased alcohol drinking. These results are consistent with the idea that ethanol stimulates VTA neurons at least in part by inhibiting KCNK13, a specific two-pore potassium channel, and that KCNK13 can control both VTA neuronal activity and binge drinking. KCNK13 is a novel alcohol-sensitive molecular target and may be amenable to the development of pharmacotherapies for alcoholism treatment.

Alcohol use disorder (AUD) and major depressive disorder (MDD) are prevalent, debilitating, and highly comorbid disorders. The molecular changes that underlie their comorbidity are beginning to emerge. For example, recent evidence showed that acute ethanol exposure produces rapid antidepressant-like biochemical and behavioral responses. Both ethanol and fast-acting antidepressants block N-methyl-D-aspartate receptor (NMDAR) activity, leading to synaptic changes and long-lasting antidepressant-like behavioral effects. We used RNA sequencing to analyze changes in the synaptic transcriptome after acute treatment with ethanol or the NMDAR antagonist, Ro 25-6981. Ethanol and Ro 25-6981 induced differential, independent changes in gene expression. In contrast with gene-level expression, ethanol and Ro 25-6981 produced overlapping changes in exons, as measured by analysis of differentially expressed exons (DEEs). A prominent overlap in genes with DEEs indicated that changes in exon usage were important for both ethanol and Ro 25-6981 action. Structural modeling provided evidence that ethanol-induced exon expression in the NMDAR1 amino-terminal domain could induce conformational changes and thus alter NMDAR function. These findings suggest that the rapid antidepressant effects of ethanol and NMDAR antagonists reported previously may depend on synaptic exon usage rather than gene expression.

Two independent lines of High Drinking in the Dark (HDID-1, HDID-2) mice have been bred to reach high blood alcohol levels after a short period of binge-like ethanol drinking. Male mice of both lines were shown to have reduced sensitivity to develop a taste aversion to a novel flavor conditioned by ethanol injections as compared with their unselected HS/NPT founder stock. We have subsequently developed inbred variants of each line. The current experiments established that reduced ethanol-conditioned taste aversion is also seen in the inbred variants, in both males and females. In other experiments, we asked whether HDID mice would ingest sufficient doses of ethanol to lead to a conditioned taste aversion upon retest. Different manipulations were used to elevate consumption of ethanol on initial exposure. Access to increased ethanol concentrations, to multiple tubes of ethanol, and fluid restriction to increase thirst motivation all enhanced initial drinking of ethanol. Each condition led to reduced intake the next day, consistent with a mild conditioned taste aversion. These experiments support the conclusion that one reason contributing to the willingness of HDID mice to drink to the point of intoxication is a genetic insensitivity to the aversive effects of ethanol.

Two independent lines of High Drinking in the Dark (HDID-1, HDID-2) mice have been bred to reach high blood alcohol levels after a short period of binge-like ethanol drinking. Male mice of both lines were shown to have reduced sensitivity to develop a taste aversion to a novel flavor conditioned by ethanol injections as compared with their unselected HS/NPT founder stock. We have subsequently developed inbred variants of each line. The current experiments established that reduced ethanol-conditioned taste aversion is also seen in the inbred variants, in both males and females. In other experiments, we asked whether HDID mice would ingest sufficient doses of ethanol to lead to a conditioned taste aversion upon retest. Different manipulations were used to elevate consumption of ethanol on initial exposure. Access to increased ethanol concentrations, to multiple tubes of ethanol, and fluid restriction to increase thirst motivation all enhanced initial drinking of ethanol. Each condition led to reduced intake the next day, consistent with a mild conditioned taste aversion. These experiments support the conclusion that one reason contributing to the willingness of HDID mice to drink to the point of intoxication is a genetic insensitivity to the aversive effects of ethanol. 

Nucleus accumbens dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) have been implicated in the formation of dependence to many drugs of abuse including alcohol. Previous studies have revealed that acute alcohol exposure suppresses glutamatergic signaling within the accumbens and repeated alcohol exposure enhances glutamatergic signaling. D1-MSNs receive glutamatergic input from several brain regions and it is not currently known how individual inputs onto D1-MSNs are altered by alcohol experience. To Address this, we used virally mediated expression of Channelrhodopsin (ChR2) in ventral hippocampal (vHipp) glutamate neurons to selectively activate vHipp to D1-MSN synapses and compared synaptic adaptations in response to low and high alcohol experience in vitro and in vivo. Alcohol experience enhanced glutamatergic activity and abolished long-term depression (LTD) at ventral hippocampal (vHipp) to D1-MSN synapses. Following chronic alcohol experience GluA2-lacking AMPA receptors, which are Ca-permeable, were inserted into vHipp to D1-MSN synapses. These alcohol-induced adaptations of glutamatergic signaling occurred at lower levels of exposure than previously reported. The loss of LTD expression and enhancement in glutamatergic signaling from the vHipp to D1-MSNs in the nucleus accumbens may play a critical role in the formation of alcohol dependence and enhancements in ethanol consumption. Reversal of alcohol-induced insertion of Ca-permeable AMPA receptors and enhancement of glutamatergic activity at vHipp to D1-MSNs presents potential targets for intervention during early exposure to alcohol.

SIGNIFICANCE STATEMENT The work presented here is the first to elucidate how an individual glutamatergic input onto D1-MSNs of the accumbens shell (shNAc) are altered by repeated ethanol exposure. Our findings suggest that glutamatergic input from the ventral hippocampus (vHipp) onto D1-MSNs is enhanced following drinking in a two-bottle choice (2BC) paradigm and is further enhanced by chronic intermittent ethanol (CIE) vapor exposure which escalated volitional ethanol intake. A critical finding was the insertion of Ca-permeable AMPA receptors into vHipp-shNAc D1-MSN synapses following CIE exposure, and more importantly following ethanol consumption in the absence of vapor exposure. These findings suggest that enhancements of glutamatergic input from the vHipp and insertion of Ca-permeable AMPARs play a role in the formation of ethanol dependence.

A growing number of studies implicate alterations in glutamatergic signaling within the reward circuitry of the brain during alcohol abuse and dependence. A key integrator of glutamatergic signaling in the reward circuit is the nucleus accumbens, more specifically, the dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) within this region, which have been implicated in the formation of dependence to many drugs of abuse including alcohol. D1-MSNs receive glutamatergicinput from several brain regions; however, it is not currently known how individual inputs onto D1-MSNs are altered by alcohol experience. Here, we investigate input-specific adaptations in glutamatergic transmission in response to varying levels of alcohol experience. Virally mediated expression of Channelrhodopsin in ventral hippocampal (vHipp) glutamate neurons of male mice allowed for selective activation of vHipp to D1-MSN synapses. Therefore, we were able to compare synaptic adaptations in response to low and high alcohol experience in vitro and in vivo Alcohol experience enhanced glutamatergic activity and abolished LTD at vHipp to D1-MSN synapses. Following chronic alcohol experience, GluA2-lacking AMPARs, which are Ca permeable, were inserted into vHipp to D1-MSN synapses. These findings support the reversal of alcohol-induced insertion of Ca-permeable AMPARs and the enhancement of glutamatergic activity at vHipp to D1-MSNs as potential targets for intervention during early exposure to alcohol. SIGNIFICANCE STATEMENT Given the roles of the nucleus accumbens (NAc) in integrating cortical and allocortical information and in reward learning, it is vital to understand how inputs to this region are altered by drugs of abuse such as alcohol. The strength of excitatory inputs from the ventral hippocampus (vHipp) to the NAc has been positively associated with reward-related behaviors, but it is unclear whether or how ethanol affects these inputs. Here we show that vHipp-NAc synapses indeed are altered by ethanolexposure, with vHipp glutamatergic input to the NAc being enhanced following chronic ethanol experience. This work provides insight into ethanol-induced alterations of vHipp-NAc synapses and suggests that, similarly to drugs such as cocaine, the strengthening of these synapses promotes reward behavior.

A growing number of studies implicate alterations in glutamatergic signaling within the reward circuitry of the brain during alcohol abuse and dependence. A key integrator of glutamatergic signaling in the reward circuit is the nucleus accumbens, more specifically, the dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) within this region, which have been implicated in the formation of dependence to many drugs of abuse including alcohol. D1-MSNs receive glutamatergic input from several brain regions; however, it is not currently known how individual inputs onto D1-MSNs are altered by alcohol experience. Here, we investigate input-specific adaptations in glutamatergic transmission in response to varying levels of alcohol experience. Virally mediated expression of Channelrhodopsin in ventral hippocampal (vHipp) glutamate neurons of male mice allowed for selective activation of vHipp to D1-MSN synapses. Therefore, we were able to compare synaptic adaptations in response to low and high alcohol experience in vitro and in vivo. Alcohol experience enhanced glutamatergic activity and abolished LTD at vHipp to D1-MSN synapses. Following chronic alcohol experience, GluA2-lacking AMPARs, which are Ca permeable, were inserted into vHipp to D1-MSN synapses. These findings support the reversal of alcohol-induced insertion of Ca-permeable AMPARs and the enhancement of glutamatergic activity at vHipp to D1-MSNs as potential targets for intervention during early exposure to alcohol.

Genetically engineered animals are powerful tools that have provided invaluable insights into mechanisms of alcohol action and alcohol-use disorder. Traditionally, production of gene-targeted animals was a tremendously expensive, time consuming, and technically demanding undertaking. However, the recent advent of facile methods for editing the genome at very high efficiency is revolutionizing how these animals are made. While pioneering approaches to create gene-edited animals first used zinc finger nucleases and subsequently used transcription activator-like effector nucleases, these approaches have been largely supplanted in an extremely short period of time with the recent discovery and precocious maturation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. CRISPR uses a short RNA sequence to guide a non-specific CRISPR-associated nuclease (Cas) to a precise, single location in the genome. Because the CRISPR/Cas system can be cheaply, rapidly, and easily reprogrammed to target nearly any genomic locus of interest simply by recoding the sequence of the guide RNA, this gene-editing system has been rapidly adopted by numerous labs around the world. With CRISPR/Cas, it is now possible to perform gene editing directly in early embryos from every species of animals that is of interest to the alcohol field. Techniques have been developed that enable the rapid production of animals in which a gene has been inactivated (knockout) or modified to harbor specific nucleotide changes (knockins). This system has also been used to insert specific DNA sequences such as reporter or recombinase genes into specific loci of interest. Genetically engineered animals created with the CRISPR/Cas system (CRISPy Critters) are being produced at an astounding pace. Animal production is no longer a significant bottleneck to new discoveries. CRISPy animal studies are just beginning to appear in the alcohol literature, but their use is expected to explode in the near future. CRISPy mice, rats, and other model organisms are sure to facilitate advances in our understanding of alcohol-use disorder.

Genetically engineered animals are powerful tools that have provided invaluable insights into mechanisms of alcohol action and alcohol-use disorder. Traditionally, production of gene-targeted animals was a tremendously expensive, time consuming, and technically demanding undertaking. However, the recent advent of facile methods for editing the genome at very high efficiency is revolutionizing how these animals are made. While pioneering approaches to create gene-edited animals first used zinc finger nucleases and subsequently used transcription activator-like effector nucleases, these approaches have been largely supplanted in an extremely short period of time with the recent discovery and precocious maturation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. CRISPR uses a short RNA sequence to guide a non-specific CRISPR-associated nuclease (Cas) to a precise, single location in the genome. Because the CRISPR/Cas system can be cheaply, rapidly, and easily reprogrammed to target nearly any genomic locus of interest simply by recoding the sequence of the guide RNA, this gene-editing system has been rapidly adopted by numerous labs around the world. With CRISPR/Cas, it is now possible to perform gene editing directly in early embryos from every species of animals that is of interest to the alcohol field. Techniques have been developed that enable the rapid production of animals in which a gene has been inactivated (knockout) or modified to harbor specific nucleotide changes (knockins). This system has also been used to insert specific DNA sequences such as reporter or recombinase genes into specific loci of interest. Genetically engineered animals created with the CRISPR/Cas system (CRISPy Critters) are being produced at an astounding pace. Animal production is no longer a significant bottleneck to new discoveries. CRISPy animal studies are just beginning to appear in the alcohol literature, but their use is expected to explode in the near future. CRISPy mice, rats, and other model organisms are sure to facilitate advances in our understanding of alcohol-use disorder.

Chronic alcohol abuse alters the molecular structure and function of brain cells. Recent work suggests adaptations made by glial cells, such as astrocytes and microglia, regulate physiological and behavioral changes associated with addiction. Defining how alcohol dependence alters the transcriptome of different cell types is critical for developing the mechanistic hypotheses necessary for a nuanced understanding of cellular signaling in the alcohol-dependent brain. We performed RNA-sequencing on total homogenate and glial cell populations isolated from mouse prefrontal cortex (PFC) following chronic intermittent ethanol vapor exposure (CIE). Compared with total homogenate, we observed unique and robust gene expression changes in astrocytes and microglia in response to CIE. Gene co-expression network analysis revealed biological pathways and hub genes associated with CIE in astrocytes and microglia that may regulate alcohol-dependent phenotypes. Astrocyte identity and synaptic calcium signaling genes were enriched in alcohol-associated astrocyte networks, while TGF-β signaling and inflammatory response genes were disrupted by CIE treatment in microglia gene networks. Genes related to innate immune signaling, specifically interferon pathways, were consistently up-regulated across CIE-exposed astrocytes, microglia, and total homogenate PFC tissue. This study illuminates the cell-specific effects of chronic alcohol exposure and provides novel molecular targets for studying alcohol dependence.

Chronic alcohol abuse alters the molecular structure and function of brain cells. Recent work suggests adaptations made by glial cells, such as astrocytes and microglia, regulate physiological and behavioral changes associated with addiction. Defining how alcohol dependence alters the transcriptome of different cell types is critical for developing the mechanistic hypotheses necessary for a nuanced understanding of cellular signaling in the alcohol-dependent brain. We performed RNA-sequencing on total homogenate and glial cell populations isolated from mouse prefrontal cortex (PFC) following chronic intermittent ethanol vapor exposure (CIE). Compared with total homogenate, we observed unique and robust gene expression changes in astrocytes and microglia in response to CIE. Gene co-expression network analysis revealed biological pathways and hub genes associated with CIE in astrocytes and microglia that may regulate alcohol-dependent phenotypes. Astrocyte identity and synaptic calcium signaling genes were enriched in alcohol-associated astrocyte networks, while TGF-β signaling and inflammatory response genes were disrupted by CIE treatment in microglia gene networks. Genes related to innate immune signaling, specifically interferon pathways, were consistently up-regulated across CIE-exposed astrocytes, microglia, and total homogenate PFC tissue. This study illuminates the cell-specific effects of chronic alcohol exposure and provides novel molecular targets for studying alcohol dependence.

The orphan receptor GPR88 is highly expressed in D1R- and D2R-medium spiny neurons (MSNs) and has been associated to striatum-dependent functions in rodents. The total deletion of Gpr88 in mice was shown to decrease anxiety-like behaviors, increase stereotypies and locomotion, and impair motor coordination and motor learning. Knowing the opposing role of D1R- and D2R-MSNs, we here investigated the respective roles of GPR88 in the two MSN subtypes for these behaviors. To do so, we compared effects of a conditional Gpr88 gene knockout (KO) in D1R-MSNs (D1R-Gpr88 mice) or D2R-MSNs (A2AR-Gpr88 mice) with effects of the total Gpr88 KO (CMV-Gpr88 mice). Overall, most phenotypes of CMV-Gpr88 mice were recapitulated in A2AR-Gpr88 mice, including reduced marble burying, increased social interactions, increased locomotor activity and stereotypies in the open field, and reduced motor coordination in the rotarod. Exceptions were the reduced habituation to the open field and reduced motor skill learning, which were observed in CMV-Gpr88 and D1R-Gpr88 mice, but not in A2AR-Gpr88 mice. D1R-Gpr88 mice otherwise showed no other phenotype in this study. Our data together show that GPR88 modulates the function of both D1R- and D2R-MSNs, and that GPR88 activity in these two neuron populations has very different and dissociable impacts on behavior. We suggest that GPR88 in D2R-MSNs shapes defensive and social behavior and contributes in maintaining the inhibition of basal ganglia outputs to control locomotion, stereotypies and motor coordination, while GPR88 in D1R-MSNs promotes novelty habituation and motor learning.

Significance Statement GPR88, an orphan G-protein-coupled receptor, has been implicated in the regulation of striatum-dependent behaviors. In the striatum, GPR88 is most abundant in both medium spiny neurons expressing dopamine D1 and D2 receptors. We compared effects of a conditional Gpr88 gene knockout in D1R-MSNs or D2R-MSNs with effects of the total Gpr88 deletion. Our data suggest that GPR88 in D2R-MSNs shapes defensive and social behavior and contributes in maintaining the inhibition of basal ganglia outputs to control locomotion, stereotypies and motor coordination, while GPR88 in D1R-MSNs promotes novelty habituation and motor learning. Gpr88 therefore plays very distinct roles in modulating D1R- and D2R-type neurons function and the related behaviors.

The orphan receptor GPR88 is highly expressed in D1 receptor (D1R)- and D2R-medium spiny neurons (MSNs) and has been associated to striatum-dependent functions in rodents. The total deletion of Gpr88 in mice was shown to decrease anxiety-like behaviors, increase stereotypies and locomotion, and impair motor coordination and motor learning. Knowing the opposing role of D1R- and D2R-MSNs, we here investigated the respective roles of GPR88 in the two MSN subtypes for these behaviors. To do so, we compared effects of a conditional Gpr88 gene knock-out (KO) in D1R-MSNs (D1R-Gpr88 mice) or D2R-MSNs (A_2AR-Gpr88 mice) with effects of the total Gpr88 KO (CMV-Gpr88 mice). Overall, most phenotypes of CMV-Gpr88 mice were recapitulated in A_2AR-Gpr88 mice, including reduced marble burying, increased social interactions, increased locomotor activity and stereotypies in the open field, and reduced motor coordination in the rotarod. Exceptions were the reduced habituation to the open field and reduced motor skill learning, which were observed in CMV-Gpr88 and D1R-Gpr88 mice, but not in A_2AR-Gpr88 mice. D1R-Gpr88 mice otherwise showed no other phenotype in this study. Our data together show that GPR88 modulates the function of both D1R- and D2R-MSNs, and that GPR88 activity in these two neuron populations has very different and dissociable impacts on behavior. We suggest that GPR88 in D2R-MSNs shapes defensive and social behavior and contributes in maintaining the inhibition of basal ganglia outputs to control locomotion, stereotypies and motor coordination, while GPR88 in D1R-MSNs promotes novelty habituation and motor learning.

The profile of brain structural dysmorphology of individuals with Alcohol Use Disorders (AUD) involves disruption of the limbic system. In vivo imaging studies report hippocampal volume loss in AUD relative to controls, but only recently has it been possible to articulate different regions of this complex structure. Volumetric analysis of hippocampal regions rather than total hippocampal volume may augment differentiation of disease processes. For example, damage to hippocampal subfield cornu ammonis 1 (CA1) is often reported in Alzheimer's disease (AD), whereas deficits in CA4/dentate gyrus are described in response to stress and trauma. Two previous studies explored the effects of chronic alcohol use on hippocampal subfields: one reported smaller volume of the CA2+3 in alcohol-dependent subjects relative to controls, associated with years of alcohol consumption; the other, smaller volumes of presubiculum, subiculum, and fimbria in alcohol-dependent relative to control men. The current study, conducted in 24 adults with DSM5-diagnosed AUD (7 women, 53.7 ± 8.8) and 20 controls (7 women, 54.1 ± 9.3), is the first to use FreeSurfer 6.0, which provides state-of-the art hippocampal parcellation, to explore the sensitivity of hippocampal sufields to alcoholism. T1- and T2- images were collected on a GE MR750 system with a 32-channel Nova head coil. FreeSurfer 6.0 hippocampal subfield analysis produced 12 subfields: parasubiculum; presubiculum; subiculum; CA1; CA2+3; CA4; GC-ML-DG (Granule Cell (GC) and Molecular Layer (ML) of the Dentate Gyrus (DG)); molecular layer; hippocampus-amygdala-transition-area (HATA); fimbria; hippocampal tail; hippocampal fissure; and whole volume for left and right hippocampi. A comprehensive battery of neuropsychological tests comprising attention, memory and learning, visuospatial abilities, and executive functions was administered. Multiple regression analyses of raw volumetric data for each subfields by group, age, sex, hemisphere, and supratentorial volume (svol) showed significant effects of svol (p < .04) on nearly all structures (excluding tail and fissure). Volumes corrected for svol showed effects of age (fimbria, fissure) and group (subiculum, CA1, CA4, GC-ML-DG, HATA, fimbria); CA2+3showed a diagnosis-by-age interaction indicating older AUD individuals had a smaller volume than would be expected for their age. There were no selective relations between hippocampalsubfields and performance on neuropsychological tests, likely due to lack of statistical power. The current results concur with the previous study identifying CA2+3 as sensitive to alcoholism, extend them by identifying an alcoholism-age interaction, and suggest an imaging phenotype distinguishing AUD from AD and stress/trauma.

The profile of brain structural dysmorphology of individuals with Alcohol Use Disorders (AUD) involves disruption of the limbic system. In vivo imaging studies report hippocampal volume loss in AUD relative to controls, but only recently has it been possible to articulate different regions of this complex structure. Volumetric analysis of hippocampal regions rather than total hippocampal volume may augment differentiation of disease processes. For example, damage to hippocampal subfield cornu ammonis 1 (CA1) is often reported in Alzheimer's disease (AD), whereas deficits in CA4/dentate gyrus are described in response to stress and trauma. Two previous studies explored the effects of chronic alcohol use on hippocampal subfields: one reported smaller volume of the CA2+3 in alcohol-dependent subjects relative to controls, associated with years of alcohol consumption; the other, smaller volumes of presubiculum, subiculum, and fimbria in alcohol-dependent relative to control men.

The current study, conducted in 24 adults with DSM5-diagnosed AUD (7 women, 53.7 ± 8.8) and 20 controls (7 women, 54.1 ± 9.3), is the first to use FreeSurfer 6.0, which provides state-of-the art hippocampal parcellation, to explore the sensitivity of hippocampal sufields to alcoholism. T1- and T2- images were collected on a GE MR750 system with a 32-channel Nova head coil. FreeSurfer 6.0 hippocampal subfield analysis produced 12 subfields: parasubiculum; presubiculum; subiculum; CA1; CA2+3; CA4; GC-ML-DG (Granule Cell (GC) and Molecular Layer (ML) of the Dentate Gyrus (DG)); molecular layer; hippocampus-amygdala-transition-area (HATA); fimbria; hippocampal tail; hippocampal fissure; and whole volume for left and right hippocampi. A comprehensive battery of neuropsychological tests comprising attention, memory and learning, visuospatial abilities, and executive functions was administered.

Multiple regression analyses of raw volumetric data for each subfields by group, age, sex, hemisphere, and supratentorial volume (svol) showed significant effects of svol (p < .04) on nearly all structures (excluding tail and fissure). Volumes corrected for svol showed effects of age (fimbria, fissure) and group (subiculum, CA1, CA4, GC-ML-DG, HATA, fimbria); CA2+3 showed a diagnosis-by-age interaction indicating older AUD individuals had a smaller volume than would be expected for their age. There were no selective relations between hippocampal subfields and performance on neuropsychological tests, likely due to lack of statistical power.

The current results concur with the previous study identifying CA2+3 as sensitive to alcoholism, extend them by identifying an alcoholism-age interaction, and suggest an imaging phenotype distinguishing AUD from AD and stress/trauma.

Withdrawal from chronic alcohol drinking can cause depression, leading to an inability to function in daily life and an increased risk for relapse to harmful drinking. Understanding the causes of alcohol withdrawal-related depression may lead to new therapeutic targets for treatment. Epigenetic factors have recently emerged as important contributors to both depression and alcohol use disorder (AUD). Specifically, acetylation of the N-terminal tails of histone proteins that package DNA into nucleosomes is altered in stress-induced models of depression and during alcohol withdrawal. The goal of this study was to examine depression-like behavior during alcohol withdrawal and associated changes in histone acetylation and expression of histone deacetylase 2 (HDAC2) in the hippocampus, a brain region critical for mood regulation and depression. Male Sprague–Dawley rats were treated with the Lieber-DeCarli ethanol liquid diet for 15 days and then underwent withdrawal. Rats were treated with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), during withdrawal and were tested for depression-like behavior. In a separate group of rats, the hippocampus was analyzed for mRNA and protein expression of HDAC2 and levels of histone H3 lysine 9 acetylation (H3K9ac) during chronic ethanol exposure and withdrawal. Rats undergoing ethanol withdrawal exhibited depression-like behavior and had increased HDAC2 and decreased H3K9ac levels in specific structures of the hippocampus. Treatment with SAHA during withdrawal ameliorated depression-like behavior and normalized changes in hippocampal HDAC2 and H3K9ac levels. These results demonstrate that ethanol withdrawal causes an altered epigenetic state in the hippocampus. Treatment with an HDAC inhibitor can correct this state and alleviate depression-like symptoms developed during withdrawal. Targeting histone acetylation may be a novel strategy to reduce ethanol withdrawal-induced depression.

Withdrawal from chronic alcohol drinking can cause depression, leading to an inability to function in daily life and an increased risk for relapse to harmful drinking. Understanding the causes of alcohol withdrawal-related depression may lead to new therapeutic targets for treatment. Epigenetic factors have recently emerged as important contributors to both depression and alcohol use disorder (AUD). Specifically, acetylation of the N-terminal tails of histone proteins that package DNA into nucleosomes is altered in stress-induced models of depression and during alcohol withdrawal. The goal of this study was to examine depression-like behavior during alcohol withdrawal and associated changes in histone acetylation and expression of histone deacetylase 2 (HDAC2) in the hippocampus, a brain region critical for mood regulation and depression. Male Sprague-Dawley rats were treated with the Lieber-DeCarli ethanol liquid diet for 15 days and then underwent withdrawal. Rats were treated with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), during withdrawal and were tested for depression-like behavior. In a separate group of rats, the hippocampus was analyzed for mRNA and protein expression of HDAC2 and levels of histone H3 lysine 9 acetylation (H3K9ac) during chronic ethanol exposure and withdrawal. Rats undergoing ethanol withdrawal exhibited depression-like behavior and had increased HDAC2 and decreased H3K9ac levels in specific structures of the hippocampus. Treatment with SAHA during withdrawal ameliorated depression-like behavior and normalized changes in hippocampal HDAC2 and H3K9ac levels. These results demonstrate that ethanol withdrawal causes an altered epigenetic state in the hippocampus. Treatment with an HDAC inhibitor can correct this state and alleviate depression-like symptoms developed during withdrawal. Targeting histone acetylation may be a novel strategy to reduce ethanol withdrawal-induced depression.

GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR-Cas9-mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.

GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR-Cas9-mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.