Neuronal Colocalization of μ-Opioid Receptor, κ-Opioid Receptor, and Oxytocin Receptor mRNA in the Central Nucleus of the Amygdala in Male and Female Mice.

Given the observed interaction and reports of oxytocin, μ-opioid receptor, or κ-opioid receptor expression in brain regions important to emotion regulation (i.e., the central amygdala), we hypothesized that oxytocin (), μ-opioid (), and κ-opioid () receptor mRNA were colocalized to the same cells in the central amygdala. RNAScope in situ hybridization using fresh frozen coronal brain sections was used to label cells containing , , and/or The coronal sections were imaged using a 40X objective (widefield fluorescence) on a Leica Thunder Fluorescent Microscope and the images were processed using open-source ImageJ/Fiji software and analyzed using Imaris software. The central amygdala was identified using Paxinos and Watson's The Mouse Brain in Stereotaxic Coordinates (Paxinos and Franklin 2019). Eight distinct cell populations were enumerated; i.e., only, only, only, + only, + only, + only, + + , and non-transcript cells. Our findings demonstrated that 47% of cells in the central amygdala express with and/or Of the -expressing cells, 38% colocalized only and 56% of -expressing cells colocalized both and Whereas 53% of -expressing cells colocalize and 61% of -expressiong cells colocalize These findings suggest that opioid and oxytocin receptors can function at the cellular level through morphological interactions. Future work will examine the physiological basis for the interaction between opioid and oxytocin receptors using transgenic behavior and electrophysiological assays. We report novel findings that a large proportion of central amygdala cells co-express and receptor mRNA, and a substantial proportion of central amygdala cells co-express , , and These data are significant and support our hypothesis of an interaction between opioid and oxytocin receptors, an interaction that has not yet been explored in the central nervous system. These findings underscore the unique and intricate role of opioids in regulating oxytocinergic systems, providing valuable insights into their therapeutic implications for anxiety disorders. This interaction is particularly interesting considering that both systems may have efficacy in mitigating some neuroadaptations that are associated with substance use disorders, particularly alcohol misuse and alcohol use disorder, which are often comorbid with anxiety.