DESCRIPTION (provided by applicant): This proposal explores consequences of chronic alcohol (ethanol; EtOH) exposure and withdrawal on sex- specific astrocyte activation, expression and function. Chronic EtOH abuse is associated with neurotoxicity and reactive astrogliosis, but the mechanisms and cell types involved remain poorly characterized. Furthermore, brain damage associated with alcoholism is sexually dimorphic, yet the sex-specific effects of chronic EtOH exposure have not been fully explored. This proposal extends our previous analysis that compared males vs. females using gene expression transcriptional profiling, to characterize neuroadaptive changes that are associated with withdrawal from chronic EtOH exposure. Notably, these studies demonstrated that sex is a more powerful determinant of neuroadaptation and the transcriptional response after chronic exposure than genotype and/or withdrawal severity phenotype. Biological confirmation of gene expression differences revealed that females were more vulnerable to the resulting alcohol-induced brain damage, consistent with some (but not all) clinical studies. Among the genes significantly regulated by chronic EtOH in the prefrontal cortex are some that are predominantly or exclusively expressed by astrocytes. Furthermore, EtOH-induced brain damage is associated with excitotoxicity resulting from increased glutamate release, likely from astrocyte sources. GABA receptors are also a target of neuroadaptation to chronic EtOH abuse, and have been implicated in astrocyte function. Because astrocytes are able to regulate neuronal activity and synaptic neurotransmission (both excitatory and inhibitory signals) to influence neurotoxicity, and astrocyte activation is observed with alcohol abuse, investigations into the effects of chronic EtOH on astrocyte function are timely and important. Given the evidence that EtOH withdrawal differentially damages female vs. male brain, the present application examines the hypothesis that chronic alcohol exposure and withdrawal targets astrocyte function in a sex- specific fashion, resulting in changes in gene expression and function, resulting in elevated excitotoxic signaling. Aim 1 characterizes the cellular effects of chronic ethanol ex vivo on cortical astrocyte cultures from males vs. females. This aim will develop primary single sex cortical astrocyte culture methodologies to directly test the response to chronic EtOH exposure and withdrawal in male vs. female astrocyte populations in a controlled environment. This aim also characterizes astrocyte proliferation, apoptosis, glutamate uptake and release, GABAergic signaling and the contribution of calcium stores after chronic ethanol exposure and withdrawal. Aim 2 will define sex-specific gene expression differences in astrocytes after chronic EtOH insult. Methods include qRT-PCR analysis of transcripts important in glutamatergic and GABAergic signaling with Western or immunocytochemical analysis for confirmation of expression differences. Utilizing the primary cortical astrocyte male vs. female ex vivo culture methodologies and chronic EtOH treatment paradigms developed in this developmental R21 application, these studies will lead to a better understanding of the specific effects of alcohol on astrocyte function to potentially identify therapeutic targets for the sex-specific treatment of brain damage associated with chronic EtOH abuse in both males and females. PUBLIC HEALTH RELEVANCE: Chronic alcohol abuse is a major public health problem. Although many abused drugs, including alcohol, cause neurotoxicity and brain damage, the mechanisms and the contribution of specific cell types involved are poorly characterized. In addition, there are many differences between males and females with regard to EtOH behaviors and sex-specific brain damage has been reported, but there is little analysis of the pathways involved. Astrocytes are able to regulate neuronal activity and synaptic neurotransmission (both excitatory and inhibitory signals) to influence neurotoxicity, and astrocyte activation is observed with alcohol abuse. However, the influence of chronic alcohol exposure and withdrawal on astrocyte expression and function has not been determined. The present application utilizes sex-specific primary cortical astrocyte cell culture as an ex vivo model to elucidate how alcohol influences astrocyte viability, gene expression and function in males vs. females, with a focus on glutamatergic and GABAergic signaling. These studies will provide for a better understanding of the consequences of chronic alcohol exposure and withdrawal on astrocyte function in both males and females, and may lead to novel targets for the sex-specific treatment or amelioration of brain damage associated with chronic ethanol abuse.

Narrative: Chronic alcohol abuse is a major public health problem. Although many abused drugs, including alcohol, cause neurotoxicity and brain damage, the mechanisms and the contribution of specific cell types involved are poorly characterized. In addition, there are many differences between males and females with regard to EtOH behaviors and sex-specific brain damage has been reported, but there is little analysis of the pathways involved. Astrocytes are able to regulate neuronal activity and synaptic neurotransmission (both excitatory and inhibitory signals) to influence neurotoxicity, and astrocyte activation is observed with alcohol abuse. However, the influence of chronic alcohol exposure and withdrawal on astrocyte expression and function has not been determined. The present application utilizes sex-specific primary cortical astrocyte cell culture as an ex vivo model to elucidate how alcohol influences astrocyte viability, gene expression and function in males vs. females, with a focus on glutamatergic and GABAergic signaling. These studies will provide for a better understanding of the consequences of chronic alcohol exposure and withdrawal on astrocyte function in both males and females, and may lead to novel targets for the sex-specific treatment or amelioration of brain damage associated with chronic ethanol abuse.