µ-Opioid Receptor Activation Reduces Glutamate Release in the PreBötzinger Complex in Organotypic Slice Cultures

The inspiratory rhythm generator, located in the brainstem preBötzinger complex (preBötC), is dependent on glutamatergic signaling and is affected profoundly by opioids. Here, we used organotypic slice cultures of the newborn mouse brainstem of either sex in combination with genetically encoded sensors for Ca²⁺, glutamate, and GABA to visualize Ca²⁺, glutamatergic and GABAergic signaling during spontaneous rhythm and in the presence of DAMGO. During spontaneous rhythm, the glutamate sensor SF-iGluSnFR.A184S revealed punctate synapse-like fluorescent signals along dendrites and somas in the preBötC with decay times that were prolonged by the glutamate uptake blocker (TFB-TBOA). The GABA sensor iGABASnFR showed a more diffuse fluorescent signal during spontaneous rhythm. Rhythmic Ca²⁺- and glutamate transients had an inverse relationship between the spontaneous burst frequency and the burst amplitude of the Ca²⁺ and glutamate signals. A similar inverse relationship was observed when bath applied DAMGO reduced spontaneous burst frequency and increased the burst amplitude of Ca²⁺, glutamate, and GABA transient signals. However, a hypoxic challenge reduced both burst frequency and Ca²⁺ transient amplitude. Using a cocktail that blocked glutamatergic, GABAergic, and glycinergic transmission to indirectly measure the release of glutamate/GABA in response to an electrical stimulus, we found that DAMGO reduces the release of glutamate in the preBötC but has no effect on GABA release. This suggest that the opioid mediated slowing of respiratory rhythm involves presynaptic reduction of glutamate release, which would impact the ability of the network to engage in recurrent excitation, and may result in the opioid-induced slowing of inspiratory rhythm.SIGNIFICANCE STATEMENT Opioids slow down breathing rhythm by affecting neurons in the preBötzinger complex (preBötC) and other brainstem regions. Here, we used cultured slices of the preBötC to better understand this effect by optically recording Ca²⁺, glutamate, and GABA transients during preBötC activity. Spontaneous rhythm showed an inverse relationship between burst frequency and burst amplitude in the Ca²⁺ and glutamate signals. Application of the opioid DAMGO slowed the rhythm, with a concomitant increase in Ca²⁺, glutamate, and GABA signals. When rhythm was blocked pharmacologically, DAMGO reduced the presynaptic release of glutamate, but not GABA. These data suggest the mechanism of action of opioids involves presynaptic reduction of glutamate release, which may play an important role in the opioid-induced slowing of inspiratory rhythm.