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 (<i>oxtr</i>), μ-opioid (<i>oprm1</i>), and κ-opioid (<i>oprk1</i>) receptor mRNA were colocalized to the same cells in the central amygdala. RNAscope in situ hybridization performed on fresh-frozen coronal brain sections was used to label cells containing <i>oxtr</i>, <i>oprm1</i>, and/or <i>oprk1</i> The coronal sections were imaged using a 40× objective (widefield fluorescence) on a Leica Thunder fluorescent microscope, and the images were processed using open-source ImageJ/Fiji software and analyzed using the Imaris software. The central amygdala was identified using Paxinos and Watson's <i>The Mouse Brain in Stereotaxic Coordinates</i> ( Paxinos and Franklin, 2019). Eight distinct cell populations were enumerated (i.e., <i>oxtr</i>-only, <i>oprm1</i>-only, <i>oprk1</i>-only, <i>oxtr</i> + <i>oprm1</i>-only, <i>oxtr</i> + <i>oprk1</i>-only, <i>oprm1</i> + <i>oprk1</i>-only, <i>oxtr</i> + <i>oprm1</i> + <i>oprk1</i>, and nontranscript cells). Our findings demonstrated that 47% of cells in the central amygdala express <i>oxtr</i> with <i>oprm1</i> and/or <i>oprk1</i> Of the <i>oxtr</i>-expressing cells, 38% colocalized only <i>oprm1</i>, and 56% of <i>oxtr</i>-expressing cells colocalized both <i>oprm1</i> and <i>oprk1</i> However, 53% of <i>oprm1</i>-expressing cells colocalized <i>oxtr</i>, and 61% of <i>oprk1</i>-expressiong cells colocalized <i>oxtr</i> 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.