PROJECT SUMMARY Opioid induced respiratory depression (OIRD) is the major cause of death associated with opioid use and drugs of abuse. Although the mortality risk increases in a dose-dependent manner, individual vulnerability makes opioids particularly dangerous, and no dose of opioids is without risk. Although multiple brainstem sites are involved in OIRD, an opioid sensitive subregion, known as the preBötzinger Complex (preBötC), constitutes the minimal circuitry necessary for respiratory rhythmogenesis since this network continues to generate a respiratory rhythm when isolated in-vitro, and lesions of this region in-vivo result in respiratory failure. Within this network rhythm is generated via two primary mechanisms, 1) a synaptic-based mechanism in which spontaneous spiking in some neurons leads to a chain reaction of excitatory synaptic interactions that culminates in a synchronized population burst, and 2) an intrinsic persistent sodium (INaP) based mechanism in which persistent sodium currents in a subgroup of neurons builds up excitability to initiate a burst. Within a functional network, these two mechanisms do not operate independently. However, we propose that the balance between these mechanisms is dynamic and shifts in this balance underlies variability in the sensitivity of the network to OIRD. The overarching goal of this project is to determine whether shifts in the balance between synaptic- and INaP-based mechanisms within the preBötC underlie variability in the susceptibility to OIRD. Based on or previous work showing that opioids presynaptically suppress synaptic transmission among preBötC neurons, we hypothesize that rhythmogenic states skewed towards synaptic-based mechanisms are more susceptible to OIRD than rhythmogenic states in which INaP is the dominant mechanism. We will test this hypothesis using powerful electrophysiological, optogenetic, pharmacological and imaging techniques in-vitro to specifically isolate the preBötC and ventral respiratory column (Aim 1,2), and in-vivo in anesthetized and freely behaving mice (Aim 3). We expect that integration of these preparations will provide a unique perspective to examine issues that remain unresolved in the fields of both OIRD and respiratory rhythm generation. The training plan within this proposal is specifically tailored to gain expertise in each of the techniques required to test our overall hypothesis. However, training would not be comprehensive if only exposed to the techniques immediately relevant to this project. Thus, immersion into the multidisciplinary, collaborative academic setting within the Center for Integrative Brain Research at Seattle Children's will expand upon this training to cover all aspects of modern neurobiology – from cellular biology, optogenetics, network dynamics, and translational neuroscience that covers all the levels from bench to bedside.

PROJECT NARRATIVE Opioid induced respiratory depression is particularly dangerous due to its unpredictability. This makes the condition difficult to manage clinically and creates a significant barrier for scientists trying to unravel the underlying mechanisms. This project utilizes novel experimental preparations to unravel the mechanisms underlying the unpredictability of opioid overdose.