A central problem in the treatment of ethanol (EtOH) addiction is the prevalence of relapse to EtOH use even after protracted intervals of forced or self-imposed abstinence. Advances have been made in elucidating the neurocircuitry that mediates craving and EtOH seeking, which provides insights into the neurobiological basis of relapse. Functional brain imaging in humans and studies that use c-fos expression as a marker of neural activation in rodents implicate interconnected cortical and limbic brain regions in response to drug cue-, drug priming-, and stress-induced reinstatement.
Major components of this circuitry include the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST), ventral tegmental area (VTA), nucleus accumbens (NAC), hippocampus, thalamus (THAL), and dorsal striatum. The hypocretin (Hcrt) system regulates a wide range of physiological processes, including feeding, energy metabolism, arousal, and stress, and is recruited by drugs of abuse. Of interest for the present proposal, recent studies have demonstrated a critical role of Hcrt in the modulation of stress and a possible anxiolytic effect of Hcrt receptor (Hcrt-r) antagonism. Chronic drug use is well known to dysregulate stress responses that are mediated by corticotropin-releasing factor (CRF) in both the hypothalamic-pituitary-adrenal (HPA) axis and extrahypothalamic brain stress areas outside the HPA axis (e.g., CeA and BNST). With repeated cycles of drug use, the HPA axis becomes hyporesponsive, accompanied by an increase in the extrahypothalamic CRF stress system response (i.e., CRF-CRF1 receptors). Importantly, a Hcrt/CRF interaction exists. The modulation of CRF neurons by Hcrt participates in chronic relapsing, negative affective states that characterize drug addiction. Human and animal studies suggest that the impairment of mPFC function that is caused by exposure to drugs of abuse is a key factor in the transition from goal-directed to compulsive drug seeking. The mPFC contains CRF interneurons, and Hcrt neurons project to the mPFC. This proposal will test the hypothesis that a history of EtOH dependence dysregulates Hcrt and its interaction with CRF in the mPFC, particularly the infralimbic area (IL), and this dysfunction will predict compulsive EtOH seeking (relapse) that is precipitated by stress.
SPECIFIC AIM 1
To determine whether a functional interaction exists between Hcrt and CRF in the IL and whether this interaction is dysregulated by EtOH dependence, which could explain compulsive EtOH seeking that is induced by stress
Specific Aim 1 will determine whether CRF and Hcrt act concomitantly in the IL and mediate EtOH-seeking behavior. We will determine the extent to which intra-IL administration of a dual Hcrt-r1/r2 antagonist ± a CRF1 receptor antagonist can reverse stress-induced EtOH seeking in animals with or without a history of EtOH dependence at 8 h of abstinence (acute/early abstinence, [A-Abst]), 2 weeks of abstinence (late abstinence, [L-Abst]), and 6 weeks of abstinence (protracted abstinence, [P-Abst]). Using intracerebral microdialysis, Specific Aim 1 will then determine whether EtOH dependence changes Hcrt/CRF levels in the IL during A-Abst, L-Abst, and P-Abst and following stress-induced EtOH-seeking behavior.
SPECIFIC AIM 2
To establish whether the gene expression and posttranslational products of Hcrt/Hcrt-r and CRF/CRF1 change in EtOH-dependent rats during abstinence vs. nondependent
Specific Aim 2 will determine whether molecular changes that occur as a consequence of EtOH dependence are detectable in both the IL and LH/DMH/PFA (sources of Hcrt production), which could explain compulsive EtOH seeking through an enhanced CRF response that results from an enhanced Hcrt input. This will be achieved by measuring crh mRNA, crhr1 mRNA, Hcrtr1/r2 mRNA (and CRF, CRF1 receptor and Hcrt-r1/r2 peptides) in the IL and Hcrt mRNA (and Hcrt-1/2 peptides) in the LH/DMH/PFA at the same abstinence time points as in Specific Aim 1.
SPECIFIC AIM 3
To determine whether knocking down Hcrt in the hypothalamus during the development of EtOH dependence prevents compulsive EtOH-seeking behavior
Specific Aim 3 will explore the functional effect of Hcrt transmission on stress-induced EtOH-seeking behavior by testing whether knocking down the production of Hcrt in the LH/DMH/PFA using a short-hairpin RNA-encoding AAV5 vector is sufficient to prevent/decrease enhanced EtOH-seeking behavior during abstinence at A-Abst, L-Abst, and P-Abst.