Optogenetic manipulations of preBötC μ‐opioid receptor expressing neurons reveal dual mechanisms of opioid‐induced respiratory depression

The analgesic utility of opiates is limited by the risk for adverse and life‐threatening side effects, including respiratory depression. Opioid‐induced respiratory depression (OIRD) is characterized by a pronounced decrease in the frequency and regularity of inspiratory efforts. The inspiratory rhythm originates from the preBotzinger Compex (preBötC), a region in the ventral medulla that contains neurons that express the μ‐opioid receptor (μOR) and is intrinsically sensitive to opioids, including the synthetic μOR agonist DAMGO. However, the cellular‐level mechanisms of opioid‐induced suppression of rhythmogenesis within the preBötC are controversial. Some evidence suggests opiates regulate synaptic transmission while other evidence suggests membrane potential (V m ) is the primary effector. At the network level, the frequency and regularity of the inspiratory rhythm is primarily determined by the interburst interval IBI which is regulated, in part, by excitatory neurons with “pre‐inspiratory” (pre‐I) spiking activity, but the network‐level effects of opiates have not been well defined. To shed light on these cellular‐ and network‐level processes, we utilized Oprm1 Cre mice to optogenetically manipulate μOR neurons in vitro and in vivo . In brainstem slice preparations from Oprm1 Arch neonatal mice, unit recordings from pre‐I neurons revealed that 37% of these neurons express μOR. In the presence of increasing concentrations of DAMGO, pre‐I spiking was eliminated initially, followed by suppression of spiking activity during inspiratory bursts. Next, using intracellular recordings, laser power was tuned such that photoinhibition hyperpolarized μOR neurons by ~5–10mV, mimicking the effect of DAMGO on V m when bath applied at concentrations (100–300nm) that dramatically suppress inspiratory frequency (>90%). However, bilateral photoinhibition of μOR neurons reduced frequency by 36%, only partially mimicking the suppression of frequency by DAMGO. In slices from Oprm1 ChR2 mice, photostimuation of μOR neurons under control conditions increased frequency by 204%, indicating that these neurons are integrated within this rhythmogenic network. However, following suppression of the rhythm with DAMGO, ChR2‐mediated depolarization of μOR neurons could not rescue inspiratory frequency. These findings in vitro were largely corroborated by experiments in anesthetized mice in vivo , and suggest that hyperpolarization of μOR neurons at the cellular‐level only partially contributes to OIRD in the preBötC. Based on these initial results, we propose a model of preBötC OIRD in which the effects of opioids are twofold: 1) a modest hyperpolarization of μOR neurons at the cellular‐level reduces pre‐I spiking activity within the network and causes the duration between inspirations to become longer and more irregular, followed by 2) a crash in network‐driven rhythmogenesis, potentially through a μOR‐mediated reduction in the efficacy of excitatory synaptic transmission. Support or Funding Information K99 HL145004 (Baertsch)R01 HL144801 (Ramirez)