Effects of Chronic Ethanol Exposure and Withdrawal on Glutamatergic Signaling at Two Afferent Projections into the Rat Lateral/Basolateral Amygdala
Authors: McGinnis Molly M , Morales Melissa , Alexander Nancy J , McCool Brian A 
Published: November 2023

IntroductionIncreased anxiety during alcohol withdrawal (WD) is a central characteristic of alcohol dependence and has been reported as a common, underlying cause of relapse. The lateral/basolateral amygdala (BLA) has been identified as a critical component of the neural circuitry that regulates a variety of emotional behaviors, including anxiety. Our laboratory has recently shown that chronic ethanol exposure and WD produce distinct synaptic alterations in the BLA that are input‐ and timing‐specific. However, little is known about how specific inputs from distinct upstream brain regions are affected by chronic ethanol exposure and how they regulate withdrawal‐induced anxiety. Therefore, this present study sought to examine glutamatergic inputs to the BLA from the medial prefrontal cortex (mPFC) and agranular insular cortex (AI), which project to the BLA via two anatomically distinct pathways.MethodsMale Sprague‐Dawley rats were co‐injected with AAV viral constructs containing channelrhodopsin and Gi‐coupled DREADDs into either the mPFC or AI. Following viral expression, rats were exposed to chronic intermittent ethanol (CIE) or air in vapor chambers for 7 consecutive days. Anxiety‐like behaviors were assessed using the elevated zero maze (EZM) 24 hours into WD and then rats were sacrificed for electrophysiology studies. A single i.p. injection of the DREADD agonist, Clozapine‐N‐Oxide (CNO), or saline, was given 30 minutes prior to behavioral testing. Optogenetically stimulated paired‐pulse ratios were recorded as a measure of presynaptic function.ResultsOur data indicates that rats exposed to CIE spend less time in the open areas of the EZM than air exposed control animals, suggesting an increase in anxiety‐like behavior during withdrawal. Interestingly, CIE exposed animals that received CNO tend to spend similar amounts of time in the open areas as air exposed controls, suggesting that activating the inhibitory DREADDs can attenuate the increased anxiety‐like behavior we see during withdrawal. Additionally, we found a significant increase in the paired‐pulse ratio in CIE animals as compared to air exposed controls for mPFC‐BLA projections suggesting an increase in glutamatergic release probability during withdrawal at these synapses. Importantly, bath applied CNO seems to normalize the paired‐pulse ratio of CIE animals to control levels. Furthermore, our preliminary data shows no difference in paired‐pulse ratios for AI‐BLA projections.ConclusionsGlutamatergic afferents arriving within the stria terminalis along the medial BLA boundary, which contain mPFC terminals, express a presynaptic plasticity in the form of increased glutamate release probability. In contrast, inputs arriving via the external capsule along the lateral boarder of the BLA, which contain AI synapses, do not. Optogenetic dissection of these inputs suggests that the effects of CIE on mPFC and AI inputs are consistent with their anatomical localization. Furthermore, mPFC inputs appear to be involved in the expression of withdrawal‐induced anxiety‐like behavior.Support or Funding InformationNIH grants R01 AA014445, R01 AA023999, and T32 AA007565