Chronic ethanol exposure modifies the dendritic arbor and spine density of various neuronal populations in the brain. Such morphological remodeling is hypothesized to form the structural basis for functional changes in synaptic strength and plasticity that underlie behavioral abnormalities associated with ethanol dependence. Microtubules (MTs) are highly dynamic cytoskeletal elements that play a critical role in shaping dendritic arborization. Microtubules also invade dendritic protrusions in an activity-dependent manner and support the intracellular transport of cargo proteins to synaptic sites. In vivo, chronic stress increases markers of MT stability, while pharmacological enhancement of MT dynamics attenuates depressive- and anxiety-like behaviors.
We propose to investigate whether altered MT dynamics also underlies negative affect in ethanol dependence. We speculate that MT hyperstability may represent a molecular mechanism for the homeostatic failure—or allostasis—that characterizes alcohol use disorders (AUDs) and causes relapse vulnerability during protracted abstinence. Accordingly, restoring MT dynamics could be a viable strategy to enable the physiological readjustment of neuronal circuits to the absence of ethanol, thereby accelerating recovery.
In support of this premise, ethanol can interfere with MT polymerization in vitro, and tubulin abundance and stability are drastically altered in the prefrontal cortex (PFC) in alcoholics. We also found that β5-tubulin is significantly downregulated in the medial prefrontal cortex (mPFC) in mice exhibiting excessive ethanol drinking. Furthermore, the TSRI-ARC Animal Models Core generated compelling evidence of the ability of a compound promoting MT dynamics to reduce excessive ethanol self-administration in dependent rats. Building on these findings, the present proposal aims to test the hypothesis that alterations of MT composition and/or dynamics directly contribute to the behavioral symptoms and cellular hallmarks of ethanol dependence. We propose to test this hypothesis in C57Bl/6J mice using chronic intermittent ethanol vapor inhalation (CIE) to induce dependence, along with measures of voluntary ethanol intake, anxiety-like behavior, and irritability to evaluate drinking escalation and negative affect during early and late abstinence.
SPECIFIC AIM 1
Identify alterations in MT composition and dynamics during withdrawal from CIE
We will use mass spectrometry, immunoblotting, and immunohistochemistry to quantify the abundance of tubulin isotypes, MT-associated proteins known to regulate MT dynamics, and posttranslational modifications indicative of MT stability. Brains will be collected 1 and 4 weeks into withdrawal from CIE. Analyses will be conducted in brain regions that are neural substrates for motivational and emotional dysfunction in AUDs and represent the common anatomical focus of TSRI-ARC preclinical research components (i.e., mPFC, anterior insula, and amygdala). We predict that CIE will alter the isotype composition of MTs and will increase their stability, with the mPFC possibly being more vulnerable to these changes.
SPECIFIC AIM 2
Test the functional implication of MT alterations in the behavioral and cellular symptomatology of ethanol dependence
We hypothesize that treatments altering MT composition or dynamics can mimic or reverse the effects of CIE on voluntary ethanol intake, anxiety-like behavior, and irritability. One approach will be to use virally mediated RNA interference to locally manipulate the expression of tubulin isotypes or MT-associated proteins identified in Specific Aim 1. Another approach will entail systemic treatment with the synthetic pregnenolone derivative MAP4343 to stimulate MT dynamics. We predict that chronic treatment with MAP4343 will reduce excessive drinking and alleviate negative affect in CIE-exposed mice. Furthermore, we will evaluate whether treatments that have a positive impact on behavior also reverse CIE-induced dendritic remodeling and synaptic transmission changes.
SPECIFIC AIM 3
Integration within TSRI Alcohol Research Center—Interactions with other components
Our project will complement research conducted in the Neurophysiology, Neurochemistry, and Neurocircuitry Components by providing a mechanistic basis for the dysregulation of neuronal activity in the mPFC, anterior insula, and amygdala during withdrawal from CIE. Furthermore, our component will provide preclinical validation for the testing of MAP4343 in humans by the Clinical Component. Within our component, we will directly collaborate with the Animal Models Core (CIE exposure in mice, rat brain samples), Neurocircuitry Component (whole-brain immunohistochemistry), and Neurophysiology Component (electrophysiological recordings). In Specific Aim 3, we will further bridge with the Neurocircuitry and Clinical Components by probing the involvement of glucocorticoid receptor signaling in the effect of CIE on MT dynamics. In addition, we will complement work from the Neurophysiology Component with a chemogenetic experiment testing the role of serotonergic projections to the central amygdala and mPFC in the anxiety-like behavior associated with CIE withdrawal.