Research Component 4: Sex specificity of corticolimbic circuit activity and anxiety-like behavior after alcohol exposure
Research Component 4: Sex specificity of corticolimbic circuit activity and anxiety-like behavior after alcohol exposure
Primary Investigator: Dr. Melissa Herman
Co-Investigator: Dr. Leslie Morrow
The prelimbic prefrontal cortex (PrL) and central amygdala (CeA) have been implicated in both anxiety and alcohol dependence. Our previous work revealed sex-specific differences in PrL and CeA activity and anxiety-like behavior following chronic ethanol exposure. Alcohol is acutely anxiolytic, but prolonged exposure produces an anxiogenic phenotype, particularly in withdrawal1, 2. Notably, young women are more likely to initiate drinking to alleviate anxiety3 and women may be more susceptible to stress-induced relapse4, suggesting that sex differences in neurobiological mechanisms may contribute to alcohol dependence pathology5. The impact of basal anxiety levels in sex-specific alcohol sensitivity in PrL and CeA and the role of PrLàCeA in sex differences in anxiety-like behavior following chronic alcohol exposure remains unknown and offers a potential target for improved understanding of alcohol use disorders (AUDs). Preclinical and emerging clinical evidence indicates that the neurosteroid allopregnanolone may be a potential therapeutic strategy for the treatment of AUDs via actions on GABAA receptor-mediated inhibitory control6. As our previous work demonstrated sex-specific adaptations in inhibitory control in PrL and CeA, we will also investigate the impact of allopregnanolone on ethanol-induced dysregulation.
Specific Aim 1. Test the hypothesis that sex differences in neuronal activity in PrL and CeA neurons and differences in neuronal reactivity in the context of anxiety-like behavior are related to acute sensitivity to ethanol and allopregnanolone. Neuronal activity and synaptic transmission in male and female rats will be measured in parallel under basal conditions and following ethanol (5 g/kg; IG in vivo / 44 mM in slices) or allopregnanolone (15 mg/kg in vivo / 500 nM in slices). Neuronal activity in layer V/VI PrL and CeA will be assessed using: 1) immunohistochemical measures of the immediate early gene cFos in conjunction with cell type-specific markers, 2) electrophysiological measures of firing activity and excitatory/inhibitory (E/I) balance in PrL and CeA neurons, 3) optogenetic recordings of PrLàCeA synaptic strength, and 4) fiber photometry recordings of changes in calcium activity in PrLàCeA during an aversive stimulus and time-locked anxiety-like behavior. We predict that there will be sex differences in PrL and CeA activity in response to acute ethanol and allopregnanolone and that these changes will be reflected in sex-specific differences in anxiety-like behavior.
Specific Aim 2. Test the hypothesis that chronic ethanol exposure and withdrawal produce sex differences in inhibitory control, activity, and sensitivity to acute ethanol and allopregnanolone in PrLàCeA that mediate sex differences in ethanol-induced anxiety-like behavior. Neuronal activity and synaptic transmission in male and female rats will be measured following chronic ethanol exposure by oral gavage (5g/kg, 14 days, 24 hr withdrawal). Neuronal assessments will be performed as outlined in Specific Aim 1 comparing chronic ethanol to water gavage in male and female rats. Immunohistochemical assessments will determine the role of sex in global activity changes in PrL and CeA following chronic ethanol exposure. Immunohistochemical and electrophysiological assessments will examine the changes in excitability and E/I balance in PrL and CeA neurons. Optogenetic assessments will determine the changes in PrLàCeA synaptic strength following chronic ethanol exposure. Fiber photometry assessments will determine the changes in calcium activity in PrLàCeA during time-locked anxiety-like behavior following chronic ethanol exposure. All studies will include basal assessments, responses to acute ethanol (5 g/kg, IG in vivo / 44 mM in slices) and allopregnanolone (15 mg/kg in vivo / 500 nM in slices). We predict that there will be sex differences in PFC and CeA activity, sensitivity to acute ethanol, and time-locked anxiety-like behavior following chronic ethanol exposure, and that these changes will be reversed or ameliorated by allopregnanolone.
Specific Aim 3. Test the hypothesis that chronic ethanol exposure produces sex-specific alterations in brain-wide functional connectivity, specifically corticolimbic circuit connectivity and E/I balance. Studies for this Aim will assess sex differences in PrLàCeA circuit activity following chronic ethanol exposure (5g/kg, IG, 14 days, 24 hr withdrawal). As our previous data showed loss of inhibitory control in the PrLà CeA projection neurons, studies will be performed at the whole brain level using resting state functional connectivity magnetic resonance imaging (rs-fcMRI) and magnetic resonance spectroscopy (MRS) in male and female rats. The rs-fcMRI studies will be performed in conjunction with Center Research Components 2 and 3 to provide an integrative multi-Component assessment of brain-wide changes in functional connectivity and transmitter reserve levels. We predict that PFCàCeA circuitry will be dysregulated following ethanol exposure, resulting in impairments in functional connectivity and disrupted E/I balance as evidenced by altered transmitter levels in discrete brain regions.
The results of these studies will provide important information on sex differences in the activity of PFC and CeA circuits and anxiety-like behavior and uncover the potential effects of a pharmacological approach targeting inhibitory circuitry to reverse specific circuit dysfunction following chronic ethanol exposure.