The Effects of Cholinergic Mu-Opioid Receptor Modulation on Preference and Reward

Research Team

Introduction

Research Question

Methods

Results/Findings

Conclusions and Future Study

Acknowledgements

NACE Career Readiness Competencies

References

Research Team

Matthew Wittlich

Biology, Economics, Neuroscience, Pre-Medical Studies

Dr. Anna Radke

Department of Psychology

Molly Jacobson

Psychology Graduate Student

Introduction

● Opioid Use Disorder (OUD) is a critical and escalating public health crisis in the United States, affecting approximately 6.1 million Americans aged 12 or older.

● Acetylcholine (ACh) from cholinergic interneurons (CIN) in the nucleus accumbens (NAc) plays an important role in neural responses and adaptation, underlying cocaine reinforcement and addiction.

● Acetylcholine (ACh) released from CINs triggers dopaminergic axon activity, reinforcing addictive behaviors.

● Previous studies have shown that activation of mu-opioid receptors (MORs) in the striatum inhibits cholinergic interneurons.

●This study aims to discover the role that mu-opioid receptors (MORs) play within cholinergic interneurons within the context of opioid use disorder, specifically for reward and reinforcement.

Research Question

How do mu-opioid receptors (MORs) on cholinergic interneurons (CINs) impact reward, preference, and locomotion?

Methods

Experiment Subjects: A group of control mice, or wild type (WT) (N=9 male and 9 female), and experimental mice with a genetic deletion of MOR in cholinergic cells (ChAT-Cre/Oprm1fl/fl) (HET) (N=9 male and 9 female) were used in the CPP paradigm. Three cohorts, each consisting of 12 mice, were used, with an equal distribution of males and females divided between the experimental knockout and control groups.

Conditioned Place Preference (CPP): The place preference apparatus is a two-chamber, plexiglass box with visually and spatially discriminable stimuli (Panlab Spatial Place Preference Box, Harvard Apparatus). In each box, there was a striped, dotted, and a neutral zone. The context of the rooms was paired with the administered drug (morphine) to develop a preference. On Day 1, mice had access to free roam all three chambers of a spatial preference box. In each chamber, the time spent, locomotion, and the number of entries was tracked by AnyMaze software. Days 2-5 were conditioning days where the mice received morphine (30 mg/kg) conditioned to one environment and saline conditioned to the opposing environment. On Day 6, mice were allowed to roam freely across all chambers, and the resulting data was compared to Day 1 to calculate the CPP score. Each day, the mice were in the CPP box for 25 minutes.

Figure 2. Timeline for Conditioned Place Preference Paradigm

Figure 3. Deletion of MOR in cholinergic interneurons

Results/Findings

Figure 4: Time Spent

Time spent in drug compartment on Day 1 (Baseline) and Day 6 (Test). In both males and females, there was an increase in the time spent in the drug-paired compartment compared to the saline-paired compartment (p < 0.001). There was no significant difference between genotype or sex (three-way ANOVA).

Figure 5: Distance Traveled

Distance traveled within each paired compartment. In females, there was an increase in the distance traveled in the drug-paired compartment compared to the saline-paired compartment in both genotypes (p < 0.01). The females also showed a significant genotype effect, with HETs traveling a shorter distance on test day (p < 0.001). Males did not elicit a change in the distance traveled between compartments or genotype (three-way ANOVA).

Figure 6: Number of Entries

Number of compartment entries during test day and the change in number of compartment entries from baseline to test day. Female mice had an increased number of entries in the drug compartment compared to males. There were significant differences between sexes and treatment. * p < 0.05 (three-way ANOVA).

Figure 7: Average Speed

Average speed during test day and change in average speed from baseline to test day. In females, the WT mice exhibited an increase in the average speed compared to the HET mice in the drug-paired compartment and saline-paired compartment (p < 0.0001). Males did not elicit a change in average speed between compartments or genotype (three-way ANOVA).

Conclusions and Future Study

Conclusions:

● CPP for morphine (30 mg/kg) was observed in all groups.

● MOR deletion exhibited no significant effect on CPP; specifically time spent.

● There was a significant sex difference for locomotor variables distance traveled and compartment entries. However, no significant difference was observed between sexes for average speed.

● There was a significant genotype difference for distance traveled and average speed.

● In particular, HET females seemed to demonstrate decreased locomotion compared to their female WT counterparts, for distance traveled on test day, compartment entries, and average speed.

● The male mice exhibited resistance to changes in locomotion.

● MOR deletion on CIN does not affect opioid reward, but may prevent increases in locomotion following repeated morphine conditioning, particularly in females.

Future Directions

● Investigate the sex effects that may be modulating differences in locomotor behavior following deletion of MORs on CINs.

● Further studies into conditioned place aversion (CPA) could further explain how the deletion of MORs on CINs modulates aversion compared to preference.

● Future studies could use naloxone to demonstrate the behavioral effects of CIN MOR deletion during acute withdrawal.

The following is an image of the poster presented at the 2026 Undergraduate Research Forum.

Acknowledgements

The Department of Psychology and the Office of Research for Undergraduates at Miami University, for allowing me to conduct this research with the funds from the Undergraduate Research Award.

Dr. Anna Radke, for mentoring my research in her lab and for facilitating my academic growth throughout the past two years.

Molly Jacobson, for her help in methodology training, data analysis, and general support throughout the completion of this project.

NACE Career Readiness Competencies

Career + Self-Development: Conducting this research has given me deep exposure to the field of translational research. Also, it has expanded my interests in the field of addiction, as I continue my education in medical school next fall.

Critical Thinking: This project has challenged me to look at data from multiple different perspectives. Additionally, I had to navigate a complex methodology and address problems as they occurred.

Teamwork: Leading this project increased my effectiveness as a communicator and improved my ability to tackle problems as a team member.

Technology: I gained significant knowledge in navigating data analysis software and data collection software.

References

Cachope R, Mateo Y, Mathur BN, et al. Selective activation of cholinergic interneurons enhances Accumbal phasic dopamine release: Setting the tone for reward processing. Cell Reports. 2012;2(1):33-41. doi:10.1016/j.celrep.2012.05.011
Hikida T, Kitabatake Y, Pastan I, Nakanishi S. Acetylcholine enhancement in the nucleus accumbens prevents addictive behaviors of cocaine and morphine. Proceedings of the National Academy of Sciences. 2003;100(10):6169-6173. doi:10.1073/pnas.0631749100
Jabourian M, Venance L, Bourgoin S, et al. Functional mu opioid receptors are expressed in cholinergic interneurons of the rat dorsal striatum: Territorial specificity and diurnal variation. European Journal of Neuroscience. 2005;21(12):3301-3309. doi:10.1111/j.1460-9568.2005.04154.x
Liu C, Cai X, Ritzau-Jost A, et al. An action potential initiation mechanism in distal axons for the control of dopamine release. Science. 2022;375(6587):1378-1385. doi:10.1126/ science.abn0532
Opioid use disorder. Johns Hopkins Medicine. November 21, 2023. Accessed February 9, 2025. https://www.hopkinsmedicine.org/health/conditions-and-diseases/opioid-use-disorder.

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