Optogenetic excitation of preBötzinger complex neurons potently drives inspiratory activity in vivo

This study investigates the effects on ventilation of an excitatory stimulus delivered in a spatially and temporally precise manner to the inspiratory oscillator, the preBötzinger complex (preBötC). We used an adeno-associated virus expressing channelrhodopsin driven by the synapsin promoter to target the region of the preBötC. Unilateral optogenetic stimulation of preBötC increased respiratory rate, minute ventilation and increased inspiratory modulated genioglossus muscle activity. Unilateral optogenetic stimulation of preBötC consistently entrained respiratory rate up to 180 breaths min(-1) both in presence of ongoing respiratory activity and in absence of inspiratory activity. Unilateral optogenetic stimulation of preBötC induced a strong phase-independent Type 0 respiratory reset, with a short delay in the response of 100 ms. We identified a refractory period of ∼200 ms where unilateral preBötC optogenetic stimulation is not able to initiate the next respiratory event.

Understanding the sites and mechanisms underlying respiratory rhythmogenesis is of fundamental interest in the field of respiratory neurophysiology. Previous studies demonstrated the necessary and sufficient role of preBötzinger complex (preBötC) in generating inspiratory rhythms in vitro and in vivo. However, the influence of timed activation of the preBötC network in vivo is as yet unknown given the experimental approaches previously used. By unilaterally infecting preBötC neurons using an adeno-associated virus expressing channelrhodopsin we photo-activated the network in order to assess how excitation delivered in a spatially and temporally precise manner to the inspiratory oscillator influences ongoing breathing rhythms and related muscular activity in urethane-anaesthetized rats. We hypothesized that if an excitatory drive is necessary for rhythmogenesis and burst initiation, photo-activation of preBötC not only will increase respiratory rate, but also entrain it over a wide range of frequencies with fast onset, and have little effect on ongoing respiratory rhythm if a stimulus is delivered during inspiration. Stimulation of preBötC neurons consistently increased respiratory rate and entrained respiration up to fourfold baseline conditions. Furthermore, brief pulses of photostimulation delivered at random phases between inspiratory events robustly and consistently induced phase-independent (Type 0) respiratory reset and recruited inspiratory muscle activity at very short delays (∼100 ms). A 200 ms refractory period following inspiration was also identified. These data provide strong evidence for a fine control of inspiratory activity in the preBötC and provide further evidence that the preBötC network constitutes the fundamental oscillator of inspiratory rhythms.