Inspiratory and Phase‐Spanning Dorsolateral Pontine Neurons Are Silent During Fentanyl‐Induced Apnea

Abstract

Overdoses caused by the opioid agonist fentanyl have increased exponentially in recent years. These overdose deaths are the result of inadequate ventilation. While much research has focused on opioid modulation of medullary respiratory nuclei, pontine mediated effects of opioids have received less attention. Given the interconnectivity of the pontine Kölliker-Fuse/Parabrachial complex (KF/PB) with rhythmogenic nuclei in the medulla, their involvement in controlling respiratory rate and pattern, and their sensitivity to opioids, we investigate the role they play in opioid mediated respiratory depression and hypothesize that dorsolateral pontine inspiratory, phase-spanning, and post-inspiratory neurons are silenced by opioids. To address this, we have done single unit recordings in the working heart brainstem in situ rat preparation. Dorsolateral pontine neural units (n=48) were recorded extracellularly and systemically exposed to fentanyl and then the opioid antagonist naloxone. A phase-fraction 2-D histogram (heatmap) was generated to compare all unit's action potential spiking over respiratory phases and during periods following fentanyl administration. Each neural unit was categorized at baseline into 4 main groups of discharge identity: Inspiratory, Expiratory, Phase-spanning, and Tonic. Systemic application of fentanyl (300nM) resulted in a consistent decline in fictive respiratory output, transitioned to fast shallowing breathing, and eventually resulted in cessation of respiratory output (apnea). Out of 48 extracellular single unit recordings in the dorsolateral pons, 25 (52%) were completely silent (firing frequency of 0 Hz) suggesting post-synaptic opioid receptor activation during fentanyl-induced apnea; 17/20 inspiratory, 6/12 phase spanning (4/9 IE & 2/3 EI), 1/13 expiratory (1/5 Post I Dec) and 1/3 tonic neurons. To note, many neurons that were not completely silenced by opioids did fire at a much lower firing frequency, suggesting pre-synaptic opioid receptors also contribute to opioid-induced loss of pontine neural activity. Our hypothesis was partially supported, as the majority of inspiratory neurons (17/20) and a subpopulation of phase-spanning neurons (6/12) were completely silenced by fentanyl. Surprisingly post-inspiratory neurons were mainly insensitive to opioids (4/5). These results suggest that loss of post-inspiratory activity with DAMGO injection into the KF may depend critically on post-synaptic inhibition of inspiratory and a subset of IE phase-spanning neurons, which in turn, could decrease local pre-synaptic excitation of post-inspiratory neurons. The majority of pontine expiratory neurons were not silenced by fentanyl (12/13). Due to the KF/PB role in expiratory duration, sustained activity of pontine expiratory neurons may facilitate sustained apnea during opioid overdose. These results warrant further investigation into the contribution of the dorsolateral pons in respiratory pattern formation and opioid-induced respiratory depression.