Effect of cholinergic mu-opioid receptor deletion on locomotor sensitization in CMOR mice

The mu-opioid receptor (MOR) is a receptor in the endogenous opioid system, which is involved in the sensation of pain as well as reward and addiction. Activation of these receptors by opioids, including drugs such as fentanyl and morphine, causes pain relief, sedation and a feeling of euphoria. Repeated activation of these receptors can lead to tolerance and addiction. These receptors are found populated in the central nervous system, but are mainly found in the amygdala, nucleus accumbens, periaqueductal gray (PAG), ventral tegmental area  (VTA), as well as the hypothalamus. In this study, we used mice with  a genetic deletion of the MOR in cholinergic neurons to understand the role of this subpopulation of receptors. ChAT-Oprm Cre- (control) and Cre- (experimental) male and female mice were tested for locomotor sensitization to morphine.Mice were first habituated in open field chambers for 60 min. The next day, they were injected with morphine (20 mg/kg dose) and placed in the chamber for 120 min. Mice were then injected with morphine daily for 6 additional sessions. On Day 7, the locomotor test was repeated. Data from all three locomotor tests were analyzed using a three-way repeated measures ANOVA. There were differences present between sessions: morphine injection increased locomotion in both genotypes and sexes on Day 1 and Day 7 and sensitization was observed when comparing Day 1 vs. Day 7. The results suggest that MOR’s on cholinergic neurons do not have a significant effect on locomotor sensitization in mice. 

Subjects:

22 male and female ChAT-Cre/Oprm1fl/fl (CMOR) mice were used in this experiment (Beane et al., 2024). They were housed in standard shoebox cages with each cage having 1-4 mice. These mice were either wild type Cre- CMOR or het CMOR. The mice were fed regular chow (Lab Diet 5001) and had constant access to reverse osmosis-filtered (RO) water.  Mice were kept on a 12 hour light-dark cycle (7 am to 7 pm).

Drug exposure: 

Mice received one injection of 20 mg/kg morphine daily. The mice were weighed daily, and the amount of drug was changed accordingly..

Locomotor chambers: 

The locomotor chambers were open field 45x45 cm plexiglass boxes and were connected to ANYMAZE software. The body weight was measured every day before injections. Mice were first habituated during a 1 hour session. During testing days 1 and 7, the mice were placed in the chambers for 2 hours. They were injected and observed 30 minutes prior to being placed in the chambers. The mice were not placed in the chambers between day 2 to 6. 

Experimental outline: 

Habituation: Habituation of the mice: the mice were placed in the locomotor chambers for 1 hour 

Day 1: Morphine-induced locomotion: The mice were injected with morphine according to their body weight and placed in the chambers for two hours. 

Day 2 to day 6: The mice were injected according to their body weight, every day at the same time (in between 9 am to 11am) and returned to the home cage.

Day 7: The same procedure for day 1 was followed for day 7. 

 This timeline was repeated in a total of 3 cohorts of mice.  

Conclusions

●Locomotor sensitization was observed in male WT, but not HET, mice.

● Sensitization was not observed in female mice of either genotype.

● There were no differences in center distance between genotypes.

● The sharp decrease in the center zone distance after habituation shows that the mice stick to the walls of the chambers.

● Cholinergic mu-opioid receptors have an effect on locomotor sensitization in mice. For humans, this means that this subpopulation of MOR may have a role in opioid addiction and craving. 

Future Directions

• Investigate the role of estrogen and testosterone in these genotypes: the effect of these hormones on mu-opioid receptors. 

• Look at anxiety spikes via different tests: elevated maze test looking at behavioral anxiety. 

• Investigate the extent of effect of MOR deletion from cholinergic neurons.

Valjent, Emmanuel, et al. “Mechanisms of Locomotor Sensitization to Drugs of Abuse in a Two-Injection Protocol.” Neuropsychopharmacology, vol. 35, no. 2, 1 Jan. 2010, pp. 401–415, www.nature.com/articles/npp2009143#citeas, https://doi.org/10.1038/npp.2009.143. Accessed 12 Apr. 2021.

Riday, Thorfinn T, et al. “The Rewarding and Locomotor-Sensitizing Effects of Repeated Cocaine Administration Are Distinct and Separable in Mice.” Neuropharmacology, vol. 62, no. 4, 16 Dec. 2011, pp. 1858–1866, pmc.ncbi.nlm.nih.gov/articles/PMC3269519/, https://doi.org/10.1016/j.neuropharm.2011.12.011.

“μ-Opioid Receptor - an Overview | ScienceDirect Topics.” Www.sciencedirect.com, www.sciencedirect.com/topics/neuroscience/mu-opioid-receptor.

Beane, C. R., Lewis, D. G., Bruns Vi, N., Pikus, K. L., Durfee, M. H., Zegarelli, R. A., … Radke, A. K. (2024). Cholinergic mu-opioid receptor deletion alters reward preference and aversion-resistance. Neuropharmacology, 255, 110019. https://doi.org/10.1016/j.neuropharm.2024.110019