Opioid Suppression of Serotonin Signaling in Ponto-medullary Breathing Circuitry

Opioid-induced respiratory depression (OIRD) is due to mu-opioid receptor (MOR) activation and can cause fatality in overdose. Stimulation of brainstem serotonergic raphe neurons and 5HT2A/C receptors has been shown to stimulate breathing and may serve as a MOR-independent target for treating OIRD. Despite this, the actions of opioids on raphe serotonin neurons and their inputs to OIRD-implicated lateral parabrachial neurons are poorly understood. Herein, we hypothesized that medullary serotonin neurons innervate lateral parabrachial neurons forming a circuit that is sensitive to opioids and important to OIRD. To test this hypothesis, we first crossed PetCre mice with MOR fl/fl mice to delete MORs on serotonin neurons. Whole-body plethysmography revealed no difference in breathing rate between wild-type and PetCre-MOR fl/fl mice following three different doses of fentanyl (0.3, 1.0, and 3.0 mg/kg), indicating post-synaptic MORs on serotonin neurons do not contribute to OIRD. To determine if serotonin neurons are regulated by opioids at the cellular level, we made whole-cell voltage clamp recordings from TdTomato-labeled medullary raphe serotonin neurons contained in mouse brain slices. The opioid agonist met-enkephalin hyperpolarized fewer than 5% of serotonin neurons, but reduced spontaneous excitatory post-synaptic current frequency with no change in amplitude. These data indicate that serotonin neurons are likely regulated by pre-synaptic opioid receptors. To characterize serotonin inputs to lateral parabrachial neurons, we used acute brain slice electrophysiology with slices containing the lateral parabrachial area from mice selectively expressing channelrhodopsin-2 on serotonin neurons. We observed optically evoked slow outward currents that were blocked by the 5HT1A receptor antagonist WAY100635. Surprisingly, we also observed optically evoked fast inhibitory post-synaptic currents that were attenuated by met-enkephalin and blocked by the GABA-A receptor antagonist gabazine. These data indicate that serotonin neurons may modulate breathing through complex inhibitory actions via GABA and serotonin release onto lateral parabrachial neurons. The distinct contributions of serotonin neurons to breathing within this ponto-medullary circuit will be evaluated in vivo utilizing optogenetics and whole-body plethysmography. Supported by NIH R01DA061320 and T32DA007281-28. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.