Cocaine addiction is a health concern globally. Although both men and women get addicted to cocaine, women transition from casual drug use to addition faster than men, have greater difficulty remaining abstinent and demonstrate greater propensity to relapse. In our laboratory, we found that cocaine craving is potentiated in female rats specifically in the estrus phase of the hormonal cycle. The lateral habenula (LHb), an epithalamic nucleus, plays an important role in cocaine taking and seeking and also mediates several estrogen-dependent behaviors in female rats. In the brain, the effects of estrogen are primarily mediated by estrogen receptors alpha and beta. The goals of this study are - a) to determine if there are hormone-dependent changes in estrogen receptor plasticity in LHb neurons and, b) to define the neuroanatomical organization of estrogen receptors in LHb projection neurons. Firstly, brains from freely-cycling female rats were collected during all phases of the hormonal cycle and analyzed for LHb estrogen receptor expression. Preliminary results indicate that majority of estrogen receptor alpha expressing neurons are located in the ventromedial aspect of the medial to caudal LHb. Analyses are currently underway to determine if cyclical fluctuations in ovarian hormones across the estrus cycle influence estrogen receptor expression in LHb neurons. Secondly, rats were injected with canine adenovirus (CAV2) expressing ZsGreen into either the ventral tegmental area (VTA), dorsal raphe nucleus (DRN) or rostromedial tegmental nucleus (RMTg), three LHb targets. CAV2-ZsGreen vector was retrogradely transported to neuronal cell bodies in the LHb where ZsGreen transgene was expressed. RNAScope in situ hybridization studies are in progress to determine the co-localization of ZsGreen positive neurons in the LHb with estrogen receptors. Understanding the cyclicity of LHb estrogen receptor expression will guide our studies targeted at understanding sex differences in neuronal mechanisms of cocaine taking and seeking behaviors.

An important issue in addressing the cocaine addiction problem is the tendency and probability of recovering addicts to relapse to cocaine after a significant period of time not-using. External cues that have been conditioned to be associated with cocaine use in the past are powerful triggers that cause relapse to cocaine-seeking behaviors. Drug relapse is often caused by experiencing “craving,” a subjective affective state that motivates one to seek drugs. Cocaine craving increasing as a function of time has been shown in the laboratory setting in rats and subsequently dubbed as the “incubation of cocaine craving.” This phenomenon shows that craving increases as a function of time in response to these conditioned cues. Although both women and men have a path to addiction with cocaine, women experience a faster transition from casual use to drug addiction, have a harder time remaining drug-free, and display a greater probability of relapse compared to men. Pre-clinical studies showed that female rats demonstrate an enhanced incubation response when re-exposed to conditioned cocaine cues and display conditioned place preference at lower doses of cocaine than male rats. However, the brain regions that underlie the enhanced incubation response are unknown. We trained cycling female and male rats in the laboratory to self-administer cocaine on a continuous long-access (6h), fixed-ratio 1 reinforcement schedule (reinforcement administered after one correct response) for 10-14 days. After self-administration training, rats were exposed to the cocaine self-administration environment on abstinence days 1 and 35. Our aim for this experiment is to investigate the brain regions that underlie incubation. We are quantifying the immediate early gene c-Fos immunoreactivity in cells across various brain regions in order to determine the similarities and differences in the neural activation during incubation of cocaine craving in female and male rats. We expect to see increased c-Fos expression in animals that are incubuating craving.