Utilizing opioid receptor expression to identify the neurons and molecules responsible for opioid respiratory depression and basal breathing.

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Between 1999 and 2018 more than 446,000 people died from profoundly slow and shallow breathing after opioid overdose. This number grows at a faster rate each year and in 2019 there were nearly 50,000 overdose deaths. Opioids are perhaps the most effective analgesic, therefore, despite concern, they will persist as a staple therapy in medicine and within our society into the future. Thus, novel solutions to safely use opioids must be developed. But to discover these, we must first understand the key cellular and molecular mechanisms for opioid depression of breathing, known as opioid induced respiratory depression (OIRD). Recently we demonstrated that opioids depress breathing mostly through their action upon µ-opioid receptor (MOR) expressing neurons in the same site where the breathing rhythm is created, the preBötzinger Complex (preBötC). In this proposal, a central goal is to define the key preBötC neural type and MOR signaling pathway(s) that cause OIRD. Results from both aims will have direct clinical and therapeutic impact. Here, we hypothesize that opioids cause OIRD by blocking synaptic vesicle release from just 140 MOR+ excitatory preBötC neurons. Additionally, we expect that these same neurons, despite being less than 10% of preBötC, are specialized for creating the pace of breathing in general, analogous to the cardiac pacemaker cells. If so, these will be the first neurons identified with such an important purpose. This is the second central goal of the proposal. To test these three hypotheses, we have designed a sophisticated intersectional genetic approach to selectively delete MOR in adult mice from either excitatory or inhibitory preBötC neurons or nearby upper airway motor neurons to then compare if and how much OIRD changes. Next, we will use a sensitive in vitro system to uniformly test the necessity of each primary MOR signaling pathway in suppressing preBötC rhythmicity. And last, we will use an intersectional genetic approach to measure changes in breathing after acutely, ectopically silencing the preBötC MOR+ excitatory neurons. The identification of the cellular and molecular mechanisms of OIRD will establish a framework for future studies to develop novel pharmacological approaches to block or rescue it. Moreover, this proposal aims to determine if MOR+ excitatory neurons are pacemakers for breathing. If so, we will have identified, perhaps, some of the most vital neurons in the brain.