ORCID

https://orcid.org/0000-0002-0914-8592

Date of Award

8-18-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Endogenous opioid peptides and their receptors perform critical roles in reward, aversion, and drug exposure processes. Both the peptides and the receptors perform distinct roles in modulating behavior depending on the brain region and stimulus in question. It has long been a challenge to study opioid peptides specifically because they are structurally similar to each other and occur at very low concentrations. Therefore, understanding their roles in stress, drug exposure, and drug withdrawal has presented difficulty for researchers over the years. To address this difficulty, I helped develop a method for the detection of enkephalins, a class of opioid peptides, in vivo with high specificity and sensitivity in mice. Enkephalins bind to both the mu and delta opioid receptors. In our study, we applied the method to measure these peptides in stress. However, this method can be broadly applicable to other behavioral questions. I found that both Met- and Leu-Enkephalin are released in response to stress in the Nucleus Accumbens (NAc) shell, a region in the ventral striatum. Using fiber photometry, I also showed that the release of these peptides is also coupled with calcium activity in neurons expressing the enkephalin precursor. This method will prove useful for translational efforts to human samples and for the study of other opioid peptides specifically dynorphin, the endogenous ligand to the Kappa Opioid Receptor. In addition to understanding endogenous opioid peptides, we are also interested in correlating these mechanisms with behavioral outputs such as drug withdrawal. Modeling opioid withdrawal in rodents has presented several challenges due to the wide variability of drugs, routes of exposure, and behavioral methods. We sought to make use of the advances in machine-learning based algorithms for behavioral analysis to better score the somatic signs of withdrawal in mice. We used the pose-estimation toolkit Deeplabcut (DLC) and the supervised machine learning behavioral classification tool, Simple Behavioral Analysis (SimBA) to label body-parts and behaviors after withdrawal from fentanyl. We observed differences between acute and chronic fentanyl exposure. We also found that the opioid receptor antagonist, naloxone, causes changes in the duration and frequency of the behaviors regardless of prior fentanyl exposure. In addition to our assessment of the behavioral differences during withdrawal, we also interrogated the neural underpinnings of fentanyl exposure and withdrawal. It has previously been shown that the kappa opioid receptor and its endogenous ligand dynorphin mediate negative affective states, and that withdrawal increases endogenous dynorphin transcript levels. We hypothesized that withdrawal leads to the activation of dynorphin expressing neurons in the ventral striatum, the nucleus accumbens shell. Using fiber photometry, we tested how fentanyl exposure affects calcium activity of dynorphin neurons as well as how withdrawal affects the activity of the same neurons. We found that fentanyl exposure briefly suppresses dynorphin neuron activity likely through direct action on mu opioid receptors on dynorphin neuron cell bodies. We also found that withdrawal from chronic fentanyl exposure leads to long-lasting suppression of calcium transient frequency while the amplitude of calcium transients increases during withdrawal. In the future, we would like to couple our method for peptide detection with monitoring calcium activity in dynorphin neurons. In addition to their role in withdrawal behaviors, the dynorphin and Kappa Opioid Receptor systems are potential modulators of neuroimmune interactions. Opioids directly impact the immune system likely through opioid receptors on immune cells. Recent literature has demonstrated that immune cells regulate anxiety-like and depressive behaviors, which are also features of opioid withdrawal in humans. Interestingly, studies have shown that the expression of somatic withdrawal symptoms relies on an intact immune system in rodents yet whether this effect is mediated by endogenous opioid signaling remains unknown. Here, we investigate the immunomodulatory consequences of fentanyl treatment and withdrawal in mice. We demonstrate decreased cortical CD4+ and CD8+ T cells following chronic fentanyl treatment. We also show decreased expression of MHCII in myeloid cells in the blood both after chronic fentanyl treatment and during abstinence, suggesting potentially impaired antigen presentation capabilities. We hypothesized that the lasting immunomodulatory effects of fentanyl withdrawal may be mediated by the Kappa Opioid Receptor (KOR). Therefore, in KOR knockout animals, we observed changes in both lymphoid and myeloid cells after fentanyl exposure and withdrawal suggesting that KOR is necessary for some of the alterations in the immune system. We also investigated the role of T cells during withdrawal behaviors and found that T cells may be necessary for the manifestation of the somatic signs of withdrawal in mice. In the future, we will investigate whether the KOR knockout animals show decreased somatic signs of withdrawal and whether withdrawal behaviors are mediated by KOR activity on immune cells. Our studies demonstrate complex interactions between the opioid and immune systems, offering insight into the utility of these interactions in designing better treatments. Finally, we were also interested in understanding neonatal opioid withdrawal syndrome (NOWS), so we developed a mouse model to determine the long-term consequences on adult behaviors using Oxycodone (Oxy). We compared the effects of Oxy exposure until birth (Short Oxy) to the impact of continued postnatal opioid exposure (Long Oxy) spanning gestation through birth and lactation. Short Oxy exposure was associated with a sex-specific increase in weight gain trajectory in adult male mice. Long Oxy exposure caused an increased weight gain trajectory in adult males and alterations in nociceptive processing in females. Importantly, there was no evidence of long-term social behavioral deficits, anxiety, hyperactivity, or memory deficits following Short or Long Oxy exposure. Our findings suggest that offspring with prolonged opioid exposure experienced some long-term sequelae compared to pups with opioid cessation at birth. These results highlight the potential long-term consequences of opioid administration as a mitigation strategy for clinical NOWS symptomology and suggest alternatives should be explored. Together, the studies described in this dissertation provide a general framework for a better understanding of endogenous opioids and the mechanisms of opioid withdrawal through peptide detection and enhanced behavioral quantification. Additionally, this work suggests a novel role for the Kappa Opioid Receptor as a key mediator of neuroimmune consequences. Finally, it also suggests that NOWS leads to long-term behavioral consequences that warrant mechanistic exploration in the context of endogenous opioid peptides and receptors.

Language

English (en)

Chair and Committee

Ream Al-Hasani

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