NRSA Fellowship for Samantha Malone: Calcium Imaging of Central Amygdala Activity after Fentanyl Escalation Year 2

Evidence suggests that rats given long access (LgA) sessions to self-administer (SA) opioids escalate their intake, while also showing greater withdrawal severity and drug-induced reinstatement compared to rats maintained on short access (ShA) daily SA sessions. Little is known about the neural changes that occur during contingent opioid escalation that may impact withdrawal and relapse. Past work examining opioid SA using ShA sessions in rodents has identified the central amygdala (CeA) as an area of interest that becomes hyperactive in acute withdrawal and may be involved in the incubation of craving that occurs after protracted withdrawal. However, these studies have not examined the impact of escalated opioid intake using LgA sessions on CeA activity during acute withdrawal, when individuals experience heightened craving and negative emotionality, or following protracted withdrawal during extended abstinence, when individuals experience an increased susceptibility to relapse. Furthermore, no study has determined if changes in CeA activity depend on the escalation of fentanyl intake that is self-administered contingently or merely from exposure to fentanyl per se, even if it were administered non-contingently. The specific aims of this research are to determine if contingent fentanyl escalation alters basal ex vivo CeA activity following either acute withdrawal (Aim 1) or protracted withdrawal (Aim 2). The effect of ex vivo application of fentanyl on the activity of CeA slices obtained following acute or protracted withdrawal will also be determined. Male and female Sprague Dawley rats will be divided into three groups: (1) contingent fentanyl, (2) yoked fentanyl, or (3) yoked saline. Rats in the contingent fentanyl group will undergo 7 days of fentanyl (2.5 ug/kg/inf) acquisition and 21 days of 6 h SA. Yoked rats will receive non-contingent infusions of fentanyl or saline dependent upon responding emitted by the contingent fentanyl rat. In preparation for calcium (Ca2+ ) imaging, rats will be euthanized either 17 h into withdrawal or after 30 days of forced abstinence. Ca2+ imaging will be conducted at baseline and in the presence of fentanyl (0.1 uM or 1 uM) to determine differences in CeA Ca2+ fluorescence, a measure of neural activity. We hypothesize that, at both stages of abstinence, fentanyl rats will demonstrate greater Ca2+ transients than yoked saline rats; contingent fentanyl rats may also display a greater effect than yoked fentanyl rats. We also hypothesize that, at both stages of abstinence, all rats will demonstrate a fentanyl, dose-dependent decrease in Ca2+ transients, an effect that will be greater in saline rats than fentanyl rats. Finally, we predict that greater basal CeA activity will be observed after protracted withdrawal compared to acute withdrawal, particularly for contingent fentanyl rats. Studying the mechanisms underlying differences in withdrawal and relapse after opioid escalation using Ca2+ imaging is highly novel and will elucidate the role of the CeA in driving withdrawal and relapse in OUD. The long-term goal of this work is to inform the development of novel treatments for OUD, particularly those that may target the CeA to reduce the negative emotionality and craving that occurs during acute withdrawal and throughout abstinence.