Candice Contet - US grants

SUMMARY Alcohol use disorders are in part characterized by the loss of control over alcohol drinking and the emergence of negative emotionality during abstinence from alcohol. While the brain regions and neurotransmitter systems affected by chronic alcohol exposure are well characterized, our understanding of the specific neural circuits driving drinking escalation and negative affect during withdrawal remains limited. We have generated data indicating that neurons located in the parasubthalamic nucleus (PSTN), a small region of the posterior lateral hypothalamus whose function in addiction is currently unknown, may be a critical component of this circuitry. Specifically, we show that chemogenetic stimulation of PSTN neurons expressing the neuropeptide corticotropin- releasing factor (CRF) replicates the behavioral symptomatology of withdrawal and that PSTN neurons become activated in ethanol-dependent mice experiencing withdrawal. A first aim of this proposal is to explore the mechanisms underlying the phenotypes resulting from the chemogenetic activation of PSTN CRF neurons. We will evaluate the contribution of PSTN neurons projecting to the central nucleus of the amygdala (as opposed to other targets of the PSTN) and the role of CRF signaling (as opposed to other neurotransmitters co-released by PSTN CRF neurons). Another goal is to test the hypothesis that the PSTN is a critical node of the neuronal network driving the behavioral symptomatology of ethanol withdrawal. We will determine whether the activation of PSTN neurons is necessary to the expression of withdrawal symptoms in dependent mice, whether it drives the activation of downstream central amygdala neurons, and which neurotransmitter is implicated. Finally, we aim to understand how PSTN neurons become activated during ethanol withdrawal. We will use retrograde tracing combined with c-Fos mapping as well as electrophysiological recordings to investigate the mechanisms controlling the activity of PSTN neurons in the context of ethanol withdrawal. Throughout the project, we will leverage state-of-the-art genetic tools for the functional manipulation of specific neural pathways, local silencing of gene expression and neuronal mapping, along with whole-brain imaging. In addition, we will use a well- validated mouse model of ethanol dependence that we have refined with novel measures of affective perturbation. Altogether, the proposed experiments are designed to enhance our understanding of the neural circuitry that contributes to motivational and emotional dysfunction in alcohol use disorders and may provide novel avenues for the development of more efficacious treatments.

SUMMARY ? Molecular Component The TSRI-ARC as an integrated whole will focus on the neurobiology of protracted abstinence and its role in relapse and recovery. The Molecular Component will test the hypothesis that alterations in the composition and/or dynamics of the microtubule (MT) cytoskeleton contribute to the motivational and emotional symptomatology of abstinence by driving the structural remodeling of neurons in key brain regions. The first Specific Aim will be to determine alterations in MT composition and dynamics, using state-of-the-art proteomics methodology to quantify the abundance of ?- and ?-tubulin isotypes, MT-associated proteins known to regulate MT dynamics, and tubulin posttranslational modifications indicative of MT stability. These analyses will be performed in the medial prefrontal cortex (mPFC), anterior insula, and amygdala. Mice will be exposed to chronic intermittent ethanol inhalation (CIE) and brains will be collected 1 and 4 weeks into withdrawal to explore the development and persistence of molecular adaptations in early and late abstinence, respectively. The second Specific Aim will be to test the functional implication of MT alterations in ethanol drinking escalation, negative affect and dendritic remodeling during abstinence. A first approach will involve local knockdown of tubulin isotypes and MT-associated proteins to mimic or reverse abundance changes identified in Specific Aim 1. Another approach will entail systemic treatment with the synthetic pregnenolone derivative MAP4343 to stimulate MT dynamics and accelerate recovery from withdrawal. The third Specific Aim will be to complement research conducted by other TSRI-ARC components. A first experiment will probe the involvement of glucocorticoid receptor signaling in the effect of CIE on MT dynamics. A second experiment will test the role of serotonergic projections to the central amygdala and mPFC in the anxiety-like behavior associated with abstinence from CIE. Altogether, this project is anticipated to identify a molecular mechanism for the homeostatic failure?or allostasis?that characterizes alcohol use disorders and underlies relapse vulnerability during protracted abstinence. The Molecular Component will have strong interactions with the Animal Models Core, will involve collaborations with the Neurophysiology and Neurocircuitry Components, and will provide a mechanistic basis for the testing of compounds by the Clinical Component.

SUMMARY Large conductance, voltage- and calcium-activated (BK) channels are a molecular target of ethanol. Our data indicate that ethanol-induced activation of BK channels facilitates the escalation of voluntary ethanol consumption in mice made dependent to ethanol. We therefore hypothesize that molecular adaptations resulting from chronic activation of BK channels by ethanol facilitate the progression to dependence, possibly by lowering ethanol sensitivity. Accordingly, our project aims to elucidate the molecular identity of BK-dependent adaptations (Aim 1, R21 phase) and to test their functional implication in the transition to dependence (Aim 2, R33 phase) and in the control of ethanol sensitivity (Aim 3, R33 phase). We will take advantage of a knockin mouse expressing BK channels that are insensitive to ethanol but function normally otherwise to identify molecular adaptations to chronic ethanol that selectively result from the action of ethanol on BK channels. Molecular adaptations that emerge in brain regions relevant to the motivational and affective effects of ethanol (ventral tegmental area, amygdala, prelimbic prefrontal cortex, and habenula) will be examined in a well-validated mouse model of ethanol dependence. We will leverage the unprecedented sensitivity and accuracy of data-independent acquisition mass spectrometry to quantify changes in protein abundance across the entire proteome. Furthermore, we will implement weighted correlation network analysis to identify proteins that are the most likely to drive concerted changes in abundance across modules of co-expressed proteins. Nine of these proteins will be selected at the end of the R21 phase for functional analysis during the R33 phase. We will use virally mediated RNA interference to knock down candidate proteins in targeted brain regions and evaluate the influence of these proteins on the time-course, amplitude and persistence of drinking escalation in ethanol-dependent mice. We predict that some of the proteins controlling drinking escalation will also control acute sensitivity to ethanol, such that their up- or down-regulation in ethanol-dependent mice would progressively decrease sensitivity to ethanol. Accordingly, we will also examine the impact of local protein knockdown on the reinforcing and anxiolytic effects of ethanol. Altogether, the proposed experiments are designed to identify novel molecular determinants of vulnerability to alcohol use disorders. Our proposal is relevant to the focus of FOA PAR-18-659 because the proteins identified in this project may pinpoint molecular mechanisms underlying differential sensitivity to alcohol in the human population.

SUMMARY Childhood adversity increases the vulnerability to develop an alcohol use disorder later in life, and variations in genes involved in corticotropin-releasing factor (CRF) signaling modulate this risk. However, the CRF-dependent mechanism(s) mediating the facilitation of alcohol abuse by early life stress is unknown. We recently discovered that CRF neurons located in the parasubthalamic nucleus (PSTN) may play a critical role in this mechanism because (1) they control voluntary ethanol drinking, and (2) they are enduringly altered by early life stress. Notably, these neurons send excitatory projections to the central nucleus of the amygdala (CeA), where CRF signaling is known to promote ethanol intake escalation in dependent animals. Accordingly, the present project will test the hypothesis that dysregulated CRF transmission from the PSTN to the CeA may facilitate the transition to alcohol dependence following early life stress. This project will leverage a novel mouse model of the interaction between early life stress and excessive ethanol intake by limiting bedding and nesting (LBN) materials during early postnatal development, a naturalistic model of simulated poverty, and exposing adult mice to voluntary ethanol drinking sessions alternated with chronic intermittent ethanol vapor inhalation (CIE). We established that LBN rearing accelerates ethanol intake escalation in male mice exposed to CIE and elicits negative affect during withdrawal. In Aim 1, we will determine the influence of sex on these phenotypes and test the hypothesis that, in vulnerable mice, LBN and CIE exert synergistic effects on the activity of PSTN?CeA CRF neurons. In Aim 2, we will attempt to reverse the enduring consequences of LBN on CIE-induced motivational and affective phenotypes by inhibiting PSTN?CeA CRF neurons using chemogenetics. Our approach capitalizes on the complementary expertise of two experienced collaborating researchers and leverages state- of-the-art methodology for the manipulation of neuronal activity in vivo. Our discoveries will pave the way for the identification of molecular mechanisms underlying the life-long pathological consequences of early life stress, which may ultimately enable the development of personalized therapeutic strategies for individuals who experienced childhood adversity and suffer from an alcohol use disorder.