The transition to alcohol dependence is associated with the concomitant dysregulation of executive function by the medial prefrontal cortex (mPFC) and stress systems within the central nucleus of the amygdala (CeA). The infralimbic (IL) subdivision of the mPFC exerts “top-down” control over the amygdala to regulate emotional aspects of goal-directed behaviors. Chronic ethanol exposure impairs IL function, leading to the overactivation of CeA stress-related systems and loss of control over drinking.
In particular, CeA recruitment of corticotrophin releasing factor (CRF) signaling drives anxiety-like behavior and excessive drinking via the CRF1 receptor-mediated enhancement of γ-aminobutyric acid (GABA) transmission in dependent animal. Excessive serotonin (5-hydroxytryptamine [5-HT]) transmission has also been implicated in the etiology of alcoholism and can elicit craving in abstinent alcoholics. In rodent models, chronic ethanol exposure increases the activity of dorsal raphe 5-HT neurons, and 5-HT2C receptor signaling in the bed nucleus of the stria terminalis (BNST) and amygdala contribute to anxiety-like behavior during ethanol withdrawal. Our preliminary data show that 5-HT, similarly to CRF11, dramatically increases GABA release in the CeA. These results are consistent with previously reported interactions between 5-HT and CRF in the BNST and mPFC.
Our general hypothesis is that 5-HT transmission contributes to the recruitment of CRF-CRF1 signaling in the CeA and mPFC to disrupt mPFC-amygdala connectivity and produce negative affect. Given the key role of the CeA in the transition to alcohol dependence particularly in the emergence of a negative emotional state, it is essential to characterize the neuroadaptations that occur within IL-CeA projections with chronic ethanol and withdrawal. To test whether ethanol dependence induces CRF and 5-HT alterations in the descending excitatory drive from pyramidal neurons in the mPFC to limbic areas, such as the CeA, we propose to pursue the following Specific Aims in both male and female rodents that are subjected to chronic intermittent ethanol (CIE) inhalation for 6-8 weeks followed by early (2-8 h) and late (2 weeks) withdrawal.
SPECIFIC AIM 1
To determine the effects of ethanol dependence and withdrawal on 5-HT modulation of inhibitory signaling and interaction with CRF in the CeA of male and female rats
To test the hypothesis that increased 5-HT transmission in the CeA dysregulates GABAergic transmission in ethanol-withdrawn rats, we will perform electrophysiological recordings in rat CeA slices to determine the effects of 5-HT and 5-HT receptor subtype-selective agonists and antagonists on membrane properties and GABA transmission in CeA neurons in the absence or presence of CRF1 antagonists and/or CRF.
SPECIFIC AIM 2
To assess the impact of ethanol dependence and withdrawal on 5-HT and CRF modulation of cortical descending drive to the CeA
We will test the hypothesis that ethanol dependence and withdrawal decrease the output of IL-CeA projection neurons by characterizing the downstream connectivity of the IL using tracing via retrograde labeling from the CeA. We will perform electrophysiological recordings from labeled IL-CeA projection neurons and focus on the effects of CRF and 5-HT signaling that contribute to the downstream dysregulation of amygdala function. Based on the electrophysiological results and the identified 5- HT receptor subtype(s) underlying the effects of 5-HT in the CeA and mPFC, we will systemically administer selective antagonists of the identified receptors to assess their contribution to the behavioral symptomatology of ethanol withdrawal (Animal Models Core). We will further study whether the transient chemogenetic inhibition of 5-HT projections to the CeA or mPFC (via activation of a Gi/o-coupled designer receptor, hM4Di or KORD, respectively) reverses anxiety-like behavior in CIE mice (Contet).
SPECIFIC AIM 3
To complement other ARC projects by providing in vitro electrophysiological assessments of synaptic function and projections between the CeA and mPFC
(1) We will assess CeA and mPFC function in animals that will be treated (Contet and Animal Models Core) with candidate drugs identified by Dr. Mason as translational targets. (2) We will perform studies on the interaction between CRF and hypocretin/orexin signaling in the IL to complement the behavioral studies proposed in the Martin-Fardon project. (3) We will perform electrophysiological analyses of the neuroadaptations induced by ethanol dependence and abstinence in the identified cortical-amygdalar circuits (George project). We anticipate that our electrophysiological analyses will provide mechanistic insight and cellular validation for the top-down investigation of common drugs and/or targets that are identified by other ARC investigators.