Author ORCID Identifier

Date Available


Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation


Arts and Sciences



First Advisor

Dr. Michael Bardo


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 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. The aims of this research were to determine if contingent fentanyl escalation alters basal ex vivo CeA activity following either acute withdrawal (Experiment 1) or protracted withdrawal (Experiment 2). The effect of ex vivo application of fentanyl on the neuronal activity of CeA slices obtained following acute or protracted withdrawal was also determined.

Male and female Sprague Dawley rats were divided into two groups: contingent fentanyl or saline. Rats in the fentanyl and saline groups underwent 7 days of fentanyl (2.5 ug/kg/inf) or saline acquisition and 21 days of 6 h SA. In preparation for calcium (Ca2+) imaging, rats were euthanized either 17 h into withdrawal or after 30 days of forced abstinence. Ca2+ imaging was 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.

Fentanyl rats quickly acquired and escalated their intake across acquisition and self-administration respectively, whereas saline rats did not. During acute withdrawal, fentanyl rats expressed lethargy and somatic withdrawal through a greater number of wet dog shakes and a reduced number of line crosses, rears, and digs compared to saline rats. During protracted withdrawal, fentanyl rats expressed similar behavior to saline rats. There were minimal differences in baseline CeA Ca2+ activity between fentanyl and saline rats in acute and protracted withdrawal, including only one difference where fentanyl rats expressed lower cell-weighted video mean (CWVM) frequency compared to saline rats. Although, fentanyl exposure at 0.1 and/or 1 uM reduced CWVM frequency, duration, and the video mean (VM) number of active cells for fentanyl and saline rats across both acute and protracted withdrawal. During acute withdrawal, these fentanyl exposure-induced reductions in CWVM frequency, duration, and the VM number of active cells was lower for fentanyl than saline rats at 0.1 and/or 1 uM fentanyl exposure conditions compared to 0 uM. However, during protracted withdrawal, these fentanyl exposure-induced reductions in CWVM frequency, duration, and the VM number of active cells were greater for fentanyl than saline rats at 0.1 and/or 1 uM fentanyl exposure conditions compared to 0 uM. There was no effect of self-administration condition on CWVA log(amplitude) during both acute and protracted withdrawal, and CWVA log(amplitude) only reduced in response to acute ex vivo fentanyl exposure at 0.1 and 1 uM during protracted withdrawal.

These results suggest that fentanyl rats demonstrate tolerance to the neuronal Ca2+ activity-reducing effects of acute ex vivo fentanyl exposure during acute withdrawal but demonstrate supersensitivity during protracted withdrawal. While there were minimal changes in baseline CeA neuronal Ca2+ activity resulting from fentanyl escalation, escalated fentanyl self-administration altered CeA neuronal Ca2+ reactivity to fentanyl exposure in a way that changed across withdrawal and may promote the time-dependent incubation of craving.

Digital Object Identifier (DOI)

Funding Information

National Institutes of Drug Abuse F31 DA057050

Substance Use Priority Research Area Graduate/Professional Student Grant