Endogenous Opioid Receptor Activation in the Caudal Medullary Raphe Depresses Respiratory Rate in Decerebrate Rabbits

Abstract

Background The caudal medullary raphe (CMR) is an essential structure in controlling pain, cardiovascular, and respiratory function (1). Partial reversal of systemic opioid-induced respiratory depression was achieved with naloxone injection into the CMR (2). This study investigated the presence of endogenous opioid activity in the CMR. Methods The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3-4kg) were anesthetized, tracheotomized, ventilated (FiO2 0.6, PCO2 45-55 mmHg), decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory (TI) and expiratory (TE) duration, breaths per minute (BPM), and peak phrenic activity (PPA). Using a multibarrel glass micropipette, we performed the following injection protocols into the CMR in the midline between 2 mm caudal and 1.5 mm rostral to the functionally identified preBötzinger Complex (preBC, (3)) and into the middle medullary raphe (middle MR (2), 2.5-3.5mm rostral to the preBC) in separate sets of animals: i) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 50 μM, 70 nl), stepsize 0.5mm rostro-caudal, 0.5mm dorso-ventral. ii) The non-selective opioid antagonist naloxone (1-5 mM, 700 nl), stepsize 0.5mm rostro-caudal, 1st injection at the ventral edge of neuronal activity, 2nd injection 1.5-2mm dorsal; and iii) the µ-opioid antagonist CTAP and δ-opioid antagonist naltrindole (each 1 mM, 350-700 nl), stepsize same as ii). Statistical comparison with Wilcoxon signed rank test. Median (25-75% range). Results i) In 6 animals, AMPA injection into the CMR caudal to and at the level of the preBC increased respiratory rate by up to 59% and PPA by up to 38% at multiple dorso-ventral levels. This was due to a decrease in TI and TE. In the middle MR, AMPA injection depressed respiratory rate, at times to apnea, through an increase in TI and TE, and also depressed PPA. ii) Naloxone injection into the CMR increased respiratory rate from 30 (23-34) to 37 (30-6) BPM (p=0.031, n=6) through a decrease in TI from 0.9 (0.8-1.0) to 0.7 (0.6-0.8) sec (p=0.031) and TE from 1.2 (1.0-1.6) to 0.9 (0.7-1.2) sec (p=0.031). We observed effects with injections caudal to, at the level of and rostral to the preBC. iii) CTAP injection increased respiratory rate from 26 (22-27) to 34 (29-34) BPM, and subsequent naltrindole injection increased respiratory rate to 43 (37-44) BPM (n=3). In the middle MR, CTAP + naltrindole increased respiratory rate between 2-5 BPM. Conclusion Excitation of the CMR increased respiratory rate over an area of several millimeters. During quiet breathing under moderate hyperoxic hypercapnia in our decerebrate rabbit preparation, respiratory raphe neurons were depressed by endogenous opioid activity. It will be important to further investigate the role of the CMR in opioid-induced respiratory depression. (1) Holtman et al., J Neurosci 1986, 6: 1185-93 (2) Zhang et al., Anesthesiology 2007, 107: 288-97 (3) Cook-Snyder et al., Resp Physiol Neurobiol 2019, 260: 37-52