Anna Berezovskaia ^1,2 Morgan Thomsen ^1,2,3 Anders Fink-Jensen ^1,2,4 Gitta Wörtwein ^1,2,5,6*

• ¹ Psychiatric Center Copenhagen, Region Hovedstad Psychiatry, Copenhagen, Denmark
• ² Copenhagen University Hospital, Copenhagen, Denmark
• ³ Department of Neuroscience, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
• ⁴ Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
• ⁵ University of Copenhagen, Copenhagen, Denmark
• ⁶ Department of Public Health, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Objective: Acetylcholine modulates the activity of the direct and indirect pathways of the striatum through muscarinic M4 and M1 receptors. M4 receptors are uniquely positioned to regulate plasticity within the direct pathway and play a substantial role in addiction-related behaviors. However, the role of M4 autoreceptors on cholinergic neurons is less explored. This study aims to fill this gap. Methods: To investigate the significance of M4-dependent signaling in cholinergic neurons we created mutant mice that lack M4 receptors on cholinergic neurons. Cholinergic neuron-specific depletion was confirmed using in situ hybridization. We aimed to untangle the possible contribution of M4 autoreceptors to the effects of the global M4 knockout by examining aspects of basal locomotion and dose-dependent reactivity to the psychostimulant and rewarding properties of cocaine, haloperidol-induced catalepsy, and examined both the anti-cataleptic and locomotion-inducing effects of the nonselective anticholinergic drug scopolamine. Results: Basal phenotype assessment revealed no developmental deficits. Cocaine stimulated locomotion in both genotypes, with no differences observed at lower doses. However, at the highest cocaine dose tested, male knockout mice displayed significantly less activity compared to wild type littermates (p=0.0084). Behavioral sensitization to cocaine was similar between knockout and wild type mice. Conditioned place preference tests indicated no differences in the rewarding effects of cocaine between genotypes. In food-reinforced operant tasks knockout and wild type mice successfully acquired the tasks with comparable performance results. M4 receptor depletion did not affect haloperidol-induced catalepsy and scopolamine reversal of catalepsy but attenuated scopolamine induced locomotion in females (p=0.04). Our results show that M4 receptor depletion attenuated the locomotor response to high doses of cocaine in males and scopolamine in females, suggesting sex-specific regulation of cholinergic activity. Conclusion: Depletion of M4 receptors on cholinergic neurons does not significantly impact basal behavior or cocaine-induced hyperactivity but may modulate the response to high doses of cocaine in male mice and the response to scopolamine in female mice. Overall, our findings suggest that M4dependent autoregulation plays a minor but delicate role in modulating specific behavioral responses to pharmacological challenges, possibly in a sex-dependent manner.