Abstract
Class A G-protein-coupled receptors (GPCRs) normally function as monomers, although evidence from heterologous expression systems suggests that they may sometimes form homodimers and/or heterodimers. This study aims to evaluate possible functional interplay of endogenous µ- and δ-opioid receptors (MORs and DORs) in mouse neurons. Detecting GPCR dimers in native tissues, however, has been challenging. Previously, MORs and DORs co-expressed in transfected cells have been reported to form heterodimers, and their possible co-localization in neurons has been studied in knock-in mice expressing genetically engineered receptors fused to fluorescent proteins. Here, we find that single cholinergic neurons in the mouse striatum endogenously express both MORs and DORs. The receptors on neurons from live brain slices were fluorescently labeled with a ligand-directed labeling reagent, NAI-A594. The selective activation of MORs and DORs, with DAMGO (µ-agonist) and deltorphin (δ-agonist) inhibited spontaneous firing in all cells examined. In the continued presence of agonist, the firing rate returned to baseline as the result of receptor desensitization with the application of deltorphin but was less observed with the application of DAMGO. In addition, agonist-induced internalization of DORs but not MORs was detected. When MORs and DORs were activated simultaneously with [Met5]-enkephalin, desensitization of MORs was facilitated but internalization was not increased. Together, these results indicate that while MORs and DORs are expressed in single striatal cholinergic interneurons, the two receptors function independently.
Highlights
Opioid receptors are a family of G protein-coupled receptors (GPCRs) targeted by endogenous peptides and exogenous opiate drugs
Naltrexamine-acylimidazole-alexa594 (NAI-A594; Figure 1A-a) is a labeling reagent that has been shown to bind opioid receptors and covalently tag them with the fluorophore Alexa-594 to MORs in brain slices (Arttamangkul et al, 2019). The labeling of this molecule is based on traceless labeling affinity approach (Hayashi and Hamachi, 2012; Shiraiwa et al, 2020), in which the naltrexamine moiety acts as a ligand that guides specific binding to opioid receptors
The results demonstrate that while endogenous MORs and
Summary
Opioid receptors are a family of GPCRs targeted by endogenous peptides and exogenous opiate drugs. The co-expression of opioid receptor-subtypes in individual neurons has steadily gained attention because possible cross-interactions between the receptors could provide new pharmacological targets for analgesia while avoiding the side effects associated with the activation of a single opioid receptor subtype (Fujita et al, 2015). There is evidence from biochemical and fluorescent imaging studies to support the existence of MOR-DOR heterodimers (Cahill and Ong, 2018), those studies required the use of heterologous expression and genetically modified receptors to enable experimental detection. The major challenge for testing these possible interactions in native tissue is the fact that opioid receptors are generally expressed in low densities in neuronal tissues, detection of subcellular co-localization is often ambiguous. Functional readout using electrophysiological measurement is a powerful tool to detect MOR-DOR co-expression in single neurons but there are few such studies (Egan and North, 1981; Chieng et al., 2006)
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