Dissertations, Theses, and Capstone Projects

Date of Degree

6-2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biology

Advisor

Robert Ranaldi

Committee Members

Carolyn Pytte

Richard Bodnar

Ewa Galaj

Kenneth Carr

Subject Categories

Neuroscience and Neurobiology

Abstract

Background: Relapse remains the greatest barrier to long-term recovery and the consequential rising overdose-related mortality in heroin use disorder (HUD). Currently available behavioral and pharmacological interventions fail to extend their benefits beyond the duration of the treatment. Additionally, opioid agonist-based medications accompany harmful neurobiological risks and propensity to promote further drug-seeking post-treatment. We have shown that environmental enrichment (EE), as a therapeutic strategy applied after drug intravenous-self administration (IVSA), can effectively facilitate abstinence and reduce reinstatement of drug-seeking in animal models of drug abuse. Moreover, dopamine receptors like D1 and D3, and mu-opioid (MO) receptors are well known for their implication in HUD, and there are recent studies also implicating the ghrelin system and receptor GHS-R1a in drug reward. However, the robustness of EE has not been demonstrated in a compulsive heroin use model yet, and the associated neuronal adaptations mediating effects of EE on reinstatement are also understudied.

Objective: To determine if EE can have anti-relapse effects after prolonged heroin use, it is crucial to investigate if EE can extend its positive effects to the long-access (LA) drug IVSA model that captures characteristic compulsive drug taking in HUD, and to identify potential translational neurobiological targets for relapse-focused pharmacotherapies. The aims of this dissertation thesis were to determine if EE can also reduce cue-induced reinstatement of heroin seeking when applied after LA heroin IVSA, and to begin to identify the neurobiological mechanisms underlying the effectiveness of EE.

Methods: 1) To determine effects of EE in the LA model, rats were moved to non-EE or EE housing following LA IVSA, followed by extinction and cue-induced reinstatement. Lever presses were measured for all phases to compare cue-induced reinstatement between groups. 2) To quantify heroin- and EE- induced changes in D1R, D3R and MOR mRNA expression, we conducted two experiments: in Exp. 1, rat brains were analyzed following saline or heroin IVSA; in Exp. 2, brains were analyzed from rats that underwent heroin IVSA, followed by 15 days of either non-EE or EE housing assignment. 3) To determine heroin- and EE- induced region-specific changes in GHS-R1a expression in non-EE and EE rats, brains were collected and analyzed after the cue-induced reinstatement test from the LA IVSA experiment (1).

Results: EE, applied after LA heroin IVSA, facilitated extinction and reduced cue-induced reinstatement of heroin seeking. Using RNAscope, we found that heroin-use leads to D1R mRNA upregulation in the nucleus accumbens (NAc) but downregulation in the insular cortex (IC). Interestingly, EE significantly reversed the accumbal D1R changes but not in the IC. The Western Blotting assays showed that in the ventral tegmental area (VTA) and NAc, heroin upregulated GHS-R1a expression and, interestingly, EE reversed these changes. We did not find any significant differences in D3R and MOR mRNA with heroin or EE in the NAc and IC. There were also no significant differences observed in GHS-R1a expression in the substantia nigra (SN), amygdala (AMY), hypothalamus (HYPOTH) and hippocampus (HIPPO) regions.

Conclusion: Altogether, these results confirm the efficacy of EE in reducing reinstatement after prolonged heroin use and offers a deeper understanding of the associated underlying neurobiological substrates. Our findings highlight the translational scope of EE implementation in addiction treatments and provide promising avenue for advancement in potential addiction behavioral and neurobiological therapies aimed at reducing relapse.

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