Project Summary Fentanyl and other ultrapotent synthetic opioids cause respiratory depression, a potentially fatal acute toxic effect that is mediated by activation of mu opioid receptors. Naloxone, an antagonist of mu opioid receptors, can reverse fentanyl-induced respiratory depression, but must compete with ultrapotent opioids for receptor binding. A respiratory stimulant that acts independently of opioid receptors would be an appealing approach to combat ultrapotent synthetic opioid poisoning. Serotonin generally provides a net stimulatory effect on breathing due, in part, to 5HT2A/2C receptors in respiratory controlling areas in the brainstem. In addition, the emerging interest in serotonergic psychedelics for the treatment of various neuropsychiatric disorders, including opioid use disorder, has led to renewed interest in development of serotonin agents, particularly targeting the 5HT2A receptor. Activation of 5HT2A receptors in the dorsolateral pons can counter opioid- induced respiratory depression and microinjection studies implicate medullary raphe neurons, which produce serotonin, in opioid-induced respiratory depression, but mechanisms for these effects are unknown. The overall goal of this proposal is to fill a major void in our understanding of serotonergic mechanisms in fentanyl- induced respiratory depression. We hypothesize that fentanyl interferes with serotonin mediated mechanisms that support breathing, which can be reversed by activation of 5HT2A receptors. We will test this hypothesis at the cellular, circuit and whole animal level using approaches that selectively identify and modulate serotonin neurons. Aim 1 will determine the effect of fentanyl on medullary raphe serotonin neuron activity mediated by pre- and/or post-synaptic opioid receptors by recording from genetically labeled serotonin neurons contained in acute brain slices. The role of post-synaptic opioid receptors on serotonin neurons in fentanyl-induced respiratory depression will be determined in awake behaving mice. Aim 2 will determine the cellular and synaptic mechanisms by which 5HT2A receptors and endogenous serotonin neurons counter opioid inhibition of dorsolateral pontine neurons using ex vivo electrophysiology and a unique arterially perfused pontomedullary circuit preparation. Aim 3 will determine the effect of systemic 5HT2A/2C agonists on fentanyl- induced respiratory depression alone and in combination with a sub-therapeutic dose of naloxone in awake behaving mice. Together, this R01 project will improve our understanding of serotonin mechanisms in fentanyl- induced respiratory depression that will inform how to best leverage the serotonin system to counter fentanyl toxicity in future studies.

Project Narrative Fentanyl and other ultrapotent opioids interfere with the normal functioning of brainstem circuitry that controls breathing leading to potentially fatal respiratory depression. The proposed studies will provide mechanistic details on serotonergic mechanisms involved in respiratory depression to inform how the serotonin system could be leveraged to combat acute fentanyl toxicity. The outcomes of this research will advance our understanding of the pontomedullary circuitry that controls breathing and provide timely assessment of the safety of co-use of drugs activating 5HT2A receptors, such as serotonergic psychedelics, with fentanyl.