The current opioid crisis occurring in the US emphasizes the need for a more comprehensive understanding of pain control mechanisms and improved therapies for chronic pain. While opioids are effective for pain control, they come with adverse side effects, including abuse liability and tolerance to the analgesic effects with repeated use. Thus, novel targets for pain treatment should be identified. 

In the CNS, Acetylcholine (ACh) and its receptors (AChRs) are implicated in the descending pain modulation pathway in the ventrolateral periaqueductal gray (vlPAG), a key site of opioid action. The vlPAG neurons have been shown to express cholinergic receptors, specifically 𝛼7-nicotinic acetylcholine receptors (𝛼7-nAChRs), that produce strong analgesic effects with an efficacy similar to opioids. While mRNA assays and electrophysiological studies have demonstrated presence of other AChRs in the vlPAG, a comprehensive study of AChR expression, the cell-types that express these AChRs, and the extent to which their agonists/antagonists modulate pain has not been investigated.

Using fluorescence in-situ hybridization, these experiments explored which AChRs are expressed in the vlPAG. We identified strong expression of M2 mAChRs, along with 𝛼7 nAChRs and weak expression of M1, M3, M4, and M5 mAChRs. Measuring expression of the immediate early gene, c-fos, as a marker of neuronal activity, We found that M2 mAChRs are expressed on neurons that increase activity in response to pain.  We also explored the neurotransmitter phenotype of M2 mAChR expressing neurons and found that these receptors are expressed on GABAergic vlPAG neurons that co-express opioid receptors.

Our experiments have comprehensively identified AChR subtypes in the vlPAG. These explorations and the discovery or synthesis of selective M2 agonists might result in novel non-opioid pain treatment strategies.