Presynaptic and Somatic Mechanisms of Opioid Action in Respiratory Circuits
Authors: Wei Aguan Daniel , Agosto Ibis , Ramirez Jan‐Marino 
Published: November 2023

Opioid‐mediated respiratory suppression (ORS) is a significant cause of death from the use of opioid analgesics and drugs of abuse. We are interested in identifying molecular and cellular mechanisms underlying ORS, in the context of circuits underlying respiratory central pattern generation.We demonstrated that inspiratory rhythms generated by in vitro brain slices containing the preBötzinger Complex (preBötC) are suppressed by DAMGO, a mu‐opioid receptor (MOR1) specific agonist. DAMGO‐suppressed rhythms were rescued by blockers of KCNQ K+ channels (XE991, Chromanol 293B), but not by a GIRK‐specific blocker (TertiapinQ). Genetic removal of presynaptic P/Q‐type calcium channels (CACNA1A), sensitized preBötC inspiratory rhythms to DAMGO suppression; heterozygous CACNA1A KO/+ slices were ~10‐fold more sensitive to DAMGO suppression compared to WT, suggesting a presynaptic site of action for ORS. Direct evidence for a presynaptic component of ORS was provided by recording mEPSCs from genetically labelled Dbx‐1 inspiratory interneurons in the presence of TTX. Spontaneous mEPSCs frequency was suppressed by DAMGO (70% of cells) and restored by subsequent block of KCNQ channels by XE991 (~83% of cells). In vivo plethysmography combined with systemic injections with morphine, KCNQ blocker (XE991) and activator (Retigabine) and GIRK‐specific activator (ML297), in un‐anesthetized neonatal and adult mice largely corroborated these in vitro findings. These data suggests that a large component of ORS in the preBötC inspiratory circuit results from presynaptic inhibition of excitatory transmission via MOR1‐mediated suppression of presynaptic calcium channels. This suppressive effect can be modulated by presynaptic KCNQ K+ channels, with a surprisingly modest contribution by GIRK K+ channels.Although our data show a significant presynaptic component underlying ORS, an unknown outwardly rectifying somatic K+ current activated by DAMGO was previously reported in preBötC neurons (Gray, et al., 1999). TWK‐type (2‐pore) K+ channels are an attractive class of K+ channels to mediate these somatic currents, since they conduct outwardly rectifying currents and many are directly modulated by volatile anesthetics and diverse GPCRs. Co‐expression of MOR1 with TREK‐2 in Xenopus oocytes produced currents which were augmented ~2‐fold by DAMGO and blocked by fluoxetine (500 uM), indicating that MOR1 activation can couple to gate TREK‐2 channels. To provide evidence for an in vitro role of TREK‐2 in ORS, recordings were made from horizontal slices containing preBötC and PiCo, a post‐inspiratory rhythm generator which is hypersensitive to DAMGO suppression. Rescue of DAMGO‐suppressed respiratory rhythms was observed by fluoxetine in preBötC but not PiCo, indicating differential involvement of TREK‐2 in ORS in these two distinct oscillators. TREK‐2 is thus a third type of ion channel coupled to MOR1 signaling, in addition to presynaptic calcium channels and GIRK K+ channels. These ion channel effector systems act differentially to produce ORS in two distinct respiratory oscillators, along with modulation by presynaptic KCNQ K+ channels.Support or Funding InformationNIH and Seattle Children's Research Institute Seed Funds.