Opioid sensitivity of projection‐specific Kölliker‐Fuse neurons

Abstract

Opioid-induced respiratory depression is the primary cause of death in individuals who overdose on opioids. Respiration is controlled by a highly interconnected neural network located in the pons and medulla. The Kölliker-Fuse nucleus (KF), located in the dorsolateral pons, is an essential part of this network. It projects to core respiratory nuclei in the ventrolateral medulla, and is a key mediator of opioid-induced respiratory depression. We have previously shown that mu opioid receptor (MOR) agonists hyperpolarize a population of neurons in the KF due to activation of G protein-mediated inwardly rectifying conductance. However, we do not know the projection target of the opioid-sensitive KF neurons, or if they project at all. Given the extensive projections from the KF to medullary respiratory rhythm generators, we hypothesize that opioid-sensitive KF neurons project to respiratory rhythm generators. To test this hypothesis, we used two different approaches. First, retrograde AAV-DIO-eGFP (retrograde-AAV-hSyn-DIO-eGFP, 100 nL, Addgene) was unilaterally injected into the Bötzinger complex (BötC), pre-Bötzinger complex (preBötC), or rostral ventral respiratory group (rVRG) of Oprm1-cre mice to retrogradely-label MOR-expressing KF neurons. KF-containing brain slices were imaged for the presence or absence of retrogradely-labeled, GFP-expressing neurons. These results demonstrate that MOR-expressing KF neurons project to the BötC, preBötC and rVRG. Second, fluorescent beads (FluoSpheres 580/605, ThermoFisher) were injected into the BötC, preBötC or rVRG of wild-type mice. Whole-cell voltage-clamp recordings were made from retrogradely-labeled KF neurons contained in brain slices. MOR-mediated outward currents were identified in KF neurons that project to the BötC, preBötC, and rVRG, confirming that KF projection neurons are functionally inhibited by MORs. Together, these approaches demonstrate that opioid-sensitive KF neurons project to the BötC, preBötC, and rVRG. Opioid inhibition of these projections could affect respiratory output in different ways (i.e. respiratory rate, amplitude, gain, and pattern) depending on the projection target. The results of this study identify projection-related heterogeneity of dorsolateral pontine neurons vulnerable to opioid inhibition, and further unravel mechanisms of opioid-induced respiratory depression.