A key issue in the alcohol field is identification of the neuronal circuits responsible for the loss of control over alcohol drinking in dependent individuals. Loss of control has long been hypothesized to result from dysfunction of the frontal lobes and subsequent disinhibition (or activation) of subcortical systems that underlie stress, anxiety, reward, pain, and habits. Our recent work has also shown that activation of a neuronal ensemble in the central nucleus of the amygdala (CeA) is required for excessive drinking and negative emotional behavior in dependent rats.
Based on data we obtained in the previous funding period and by others, the goal of the Neurocircuitry Component is to integrate these views by identifying the cortical pathways upstream (corticoamygdalar; Specific Aim 1) and downstream (amygdalocortical; Specific Aim 2) of the CeA that contribute to the compulsivity of alcohol-drinking and negative emotional behavior. The infralimbic cortex (IL), which normally flexibly inhibits emotional and behavioral responses when actionoutcome contingencies change, and the anterior insula (AI), which represents interoceptive states and has been causally implicated in drug withdrawal and relapse as well as compulsive self-administration behavior, are hypothesized, based on data obtained in the previous funding period, to form key interconnected components of these cortico-amygdalo-cortical loops.
SPECIFIC AIM 1
To identify the role of infralimbic- and insular-amygdala projections on compulsive ethanol drinking and negative emotional behavior in dependent rats
Specific Aim 1 tests the hypothesis that reduced infralimbic (IL; Aim 1A) and greater anterior insula (AI; Aim 1B) glutamatergic outflow to the amygdala promotes more compulsive drinking and negative emotional behavior in abstinence. Specific Aim 1 will use “retro-DREADD” viral recombination to express inhibitory (KORD) and excitatory (M3[Gq]) DREADDs selectively in IL-amygdala and AI-amygdala projections to bidirectionally test the hypotheses that greater activity in IL-amygdala projections reduces the compulsivity of ethanol self-administration and anxiety- and irritability-like behavior, whereas greater AI-amygdala activity increases them. Fos and ex vivo patch clamp analysis will be used to identify differences in pathway activity of dependent vs. nondependent rats and to confirm chemogenetic action.
SPECIFIC AIM 2
To identify the brain regions and neuronal pathways under the control of the CeA neuronal ensemble in dependent rats
Loss of control and dysfunction of the PFC occurs during conditions that are known to activate the CeA, such as intense stress and anxiety, but the neuronal circuitry underlying this effect is unknown. In the previous funding period, we identified a neuronal ensemble recruited in the CeA during withdrawal that is required for excessive drinking. We have preliminary data suggesting that this neuronal ensemble in the CeA may control the PFC through activation of the basolateral amygdala (BLA) and ventral tegmental area (VTA) possibly through activation of the bed nucleus of the stria terminals (BNST). The main hypothesis under test in Specific Aim 2 is that activation of a neuronal ensemble in the CeA during abstinence recruits CeA-VTA-IL and CeA-BLA-AI circuits that promote compulsive ethanol drinking, anxietylike behavior, and irritability. We will use chemogenetic (cfos-lacZ), targeted recombination in transiently active neurons (targeted recombination in active populations [TRAP]), whole-brain imaging (iDisco+), and assessment of compulsive alcohol drinking, anxiety-like behavior, and irritability to test this hypothesis.
SPECIFIC AIM 3
Integration with Center
Specific Aim 3 involves close interactions and integration with other TSRI-ARC components to understand better the translational significance and local circuitry of the identified cortico-amygdalo-cortical loops. One goal is to test the translationally-oriented hypothesis that chronic antagonism of stress-related glucocorticoid receptor signaling or activation of microtubule-associate proteintype 2 (MAP-2) to normalize microtubule homeostasis will normalize dysfunctional activation in identified cortico-amygdalo-cortical projections. The same compounds we are studying are under parallel preclinical (Contet, Molecular Component) and clinical (Mason, Clinical Component) investigation for their therapeutic potential to reduce drinking and negative emotional symptoms during protracted abstinence. A second goal is, with close collaboration with the Roberto Neurophysiology Component, to dissect the indirect connectivity of the IL to the CeA, via the basomedial amygdala (BMA) and capsular infralimbic-targeted zone (CITZ) of the amygdala using channelrhodopsin+fluorescent retrobead-assisted circuit-mapping.