Opioids produce profound and diverse effects on a range of behaviors, many driven by brainstem activity; however, the presence of opioid and opioid-like receptors at this level has been poorly studied outside of nociceptive structures and components of respiratory circuitry. While previous studies identified expression of these receptors in the brainstem, patterns have not been fully delineated and neither has coexpression of receptors nor the behavioral implications of their expression in most structures. We aimed to elucidate expression of all four receptors across somatosensory-motor, auditory, and respiratory brainstem circuits; identify recurring themes to provide insight into the mechanisms by which exogenous opioids affect broader brainstem circuits; and characterize the function of endogenous opioids in subcortical processing and behavior modulation. Using a fluorescent reporter mouse line for each receptor, we created a three-dimensional, comprehensive atlas of brainstem receptor distribution and identified novel expression patterns in modality-specific circuits. Each receptor showed unique expression patterns across the brainstem with minimal correlation between receptors. Orofacial somatosensory-motor circuits expressed all four receptors, though generally in distinct nuclei, suggesting differential opiate modulation of afferent and efferent trigeminal signaling. Within the auditory circuit, receptors segregated along the vertical and horizontal processing pathways with minimal colocalization. Finally, the respiratory circuit strongly expressed the μ opioid receptor in multiple crucial structures with minimal presence of the other three receptors. We further assessed the functional significance of this expression pattern in the respiratory circuitry by characterization respiratory responses to selective opioid agonists, finding that each agonist caused unique alterations in breathing pattern and/or breath shape. Together, the results establish a comprehensive atlas of opioid and opioid-like receptor expression throughout the brainstem, laying the essential groundwork for further evaluation of opioid neuromodulation across the spectrum of behaviors.

Microglia take on an altered morphology during chronic opioid treatment. This morphological change is broadly used to identify the activated microglial state associated with opioid side effects, including tolerance and opioid-induced hyperalgesia (OIH). Following chronic opioid treatment and peripheral nerve injury (PNI) microglia in the spinal cord display similar morphological responses. Consistent with this observation, functional studies have suggested that microglia activated by PNI or opioids engage common molecular mechanisms to induce hypersensitivity. Here we conducted deep RNA sequencing of acutely isolated spinal cord microglia from male mice to comprehensively interrogate transcriptional states and mechanistic commonality between multiple OIH and PNI models. Following PNI, we identify a common early proliferative transcriptional event across models that precedes the upregulation of histological markers of activation, followed by a delayed and injury-specific transcriptional response. Strikingly, we found no such transcriptional responses associated with opioid-induced microglial activation, consistent with histological data indicating that microglia number remain stable during morphine treatment. Collectively, these results reveal the diversity of pain-associated microglial transcriptomes and point towards the targeting of distinct insult-specific microglial responses to treat OIH, PNI, or other CNS pathologies.