Glutamate co-release from catecholaminergic neurons shapes breathing and is inhibited during opioid-induced respiratory depression

Breathing is controlled by a distributed brainstem network that includes multiple catecholaminergic nuclei. The locus coeruleus (LC), the brains primary source of noradrenaline (NA), projects to several respiratory centers, including the Kolliker-Fuse (KF) nucleus in the pons. While LC neurons are predominantly noradrenergic (NAergic), many co-release glutamate, which may contribute to the state-dependent modulation of breathing, particularly during opioid exposure. Here, we examined how opioids affect NAergic and glutamatergic signaling in the LC-KF circuit using optogenetics and whole-cell patch clamp recordings in mouse brain slices. Optogenetic activation of LC terminals evoked glutamatergic excitatory postsynaptic currents (EPSCs) in KF neurons that were presynaptically inhibited by the opioid receptor agonist Met-enkephalin. Additionally, [~]36% of glutamate-responsive KF neurons exhibited postsynaptic opioid inhibition via GIRK currents, while KF neurons receiving excitatory NAergic input showed minimal opioid sensitivity. To assess the behavioral role of glutamate release from all catecholaminergic neurons, we compared breathing in awake VGluT2fl/fl::TH-Cre mice (lacking VGluT2 in tyrosine hydroxylase-positive neurons) to control littermates and TH-Cre hemizygous mice using whole-body plethysmography. The conditional VGluT2 knockout mice exhibited prolonged inspiratory duration, increased tidal volume, and reduced respiratory rate during baseline breathing, with state-dependent differences emerging during hypercapnia. Systemic morphine administration diminished these genotype differences, and machine learning analysis using dynamic time warping confirmed that genotype-specific breathing patterns were distinguishable at baseline, but not after morphine. These findings demonstrate that glutamate co-release from catecholaminergic neurons modulates respiratory patterning in a state-dependent manner and is selectively vulnerable to opioid inhibition.