Project 1: Role of parabrachial neurons in response to hypoxia, hypercapnia, and OIRD

Project Summary (Project 1) The parabrachial nucleus (PB) and adjacent Kölliker-Fuse nucleus (KF) play a key role in brain circuitry that controls both respiratory efforts and awakening during hypoxia and hypercarbia, such as occurs during obstructive sleep apnea (OSA) and which fail during opioid-induced respiratory depression (OIRD). In this P01, we found that during hypercarbia cells expressing the peptide CGRP in the external lateral PB (PBelCGRP neurons) drive waking responses, but a separate population of cells expressing the transcription factor FoxP2 in the KF and central lateral PB (PBcl/KFFosP2 neurons) drive respiratory responses. Our goal is to manipulate these neurons and their targets pharmacologically, to improve respiration and patient outcomes in both OSA and OIRD. Aim 1 uses single nucleus RNA-sequencing (snRNA-Seq) to identify the genes expressed by PB/KF neurons that show cFos responses during prolonged hypoxia and hypercarbia (as in OIRD) and which project to forebrain (arousal) or brainstem (respiratory) targets. This will allow us to determine the cell types, marker genes, and potential receptor types that can be used to manipulate them. Aim 2 will use 2-photon microscopy in vivo with calcium imaging to examine 100’s of neurons throughout the entire PBel/cl/KF region simultaneously during brief (30 sec) hypoxia and hypercarbia to observe the dynamics of their responses and how they are affected by morphine. The brains will then be subjected to EASI-FISH, to allow us to identify the mRNA markers for the cell types whose activities were observed in vivo. Aim 3 will use optogenetics to selectively activate or inhibit the different cell types identified in Aims 1 and 2 and which project to key forebrain arousal or medullary respiratory targets, to determine their effects on respiration at baseline, during hypoxia or hypercarbia, and how this is modified by morphine. Aim 4 will then perform slice physiology on those same pathways, using patch-clamp recording from target neurons in the forebrain or medulla, in combination with activating terminals from those inputs optogenetically, to determine the synaptic mechanisms used by the PB/KF neurons to cause EEG and respiratory activation in response to hypoxia and hypercarbia and how these are affected by mu-opioid agonists. These studies will identify the key cell types that respond to hypoxia and hypercarbia in OSA and OIRD and form the basis for rational pharmacological design for therapies for these conditions.