The Role of Dbx1‐Derived Cells in the Generation of Inspiration In Vivo

The preBötzinger Complex (preBötC) in the ventral medulla generates the inspiratory phase of breathing. Cells expressing the transcription factor Dbx1 during prenatal development have been proposed to give rise to the excitatory neurons that form the rhythmogenic core of the preBötC. When isolated in brainstem slices from neonatal DBx1/Ai27 mice, optogenetic stimulation of Dbx1‐derived cells reliably elicits inspiratory population bursts from the preBötC. However, it has recently been reported that presynaptic depression occurring during endogenous preBötC population bursts results in a refractory period lasting ~2 seconds, during which Dbx1 cell stimulation cannot elicit a burst (Kottick and Del Negro, 2015). In vivo, anesthetized mice breathe at a frequency that is typically 5 times faster (~1Hz) than that observed in vitro (~0.2Hz). We hypothesized that higher respiratory frequencies in vivo would minimize the effect of Dbx1 cell stimulation in anesthetized Dbx1/Ai27 mice. Using a ventral approach, the occipital bone was removed to expose the medulla, and the hypoglossal (XII) nerve was isolated to record inspiratory activity while mice were allowed to spontaneously breathe 100% O 2 . An optical fiber was placed unilaterally on the ventral surface of the medulla over the preBötC. During a 10 second continuous light pulse, a modest laser power‐dependent increase in respiratory frequency was observed (15–20% change from baseline at ~9mW). Dbx1 cell stimulation entrained the inspiratory rhythm when 200ms light pulses were delivered at or near the endogenous respiratory frequency (~1Hz); however entrainment was lost when the stimulation frequency was altered by ~ ±0.3Hz. Dbx1 cell stimulation had varied effects when light pulses were delivered at random time points in the inspiratory cycle. If a light pulse coincided with inspiration, the cycle duration was lengthened. When stimulation occurred during the first ~3/4 of the inspiratory cycle there was no effect on cycle duration. A burst could only be elicited and the cycle duration was shortened if stimulation occurred during the final ~1/4 of the cycle. To test if respiratory frequency influences the effect of Dbx1 stimulation, animals were placed on a ventilator and hyperventilated to slow and eventually stop breathing during random light stimulation. As breathing slowed, the probability of eliciting inspiration with Dbx1 stimulation increased; during apnea, inspiratory activity was reliably evoked with each stimulation. We conclude that Dbx1 stimulation in vivo can alter the respiratory pattern, but the effects are subtle compared to stimulation of Dbx1 in vitro. We hypothesize that the decreased Dbx1 influence is due to the elevated respiratory drive and frequency present in the intact animal. Support or Funding Information NIH RO1 HL 126523