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

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 preBotzinger complex (preBotC) in generating inspiratory rhythms in vitro and in vivo. However, the influence of timed activation of the preBotC network in vivo is as yet unknown given the experimental approaches previously used. By unilaterally infecting preBotC 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 preBotC 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 preBotC 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 (approximate to 100ms). A 200ms refractory period following inspiration was also identified. These data provide strong evidence for a fine control of inspiratory activity in the preBotC and provide further evidence that the preBotC network constitutes the fundamental oscillator of inspiratory rhythms.