UCLA

Inhibitory neurons are a substantial component of the rhythmic preB�tzinger Complex (preB�tC) microcircuit, a node in the ventrolateral medulla critical for generating eupneic breathing in mammals. The role of glycinergic preB�tC neurons in generating and modulating respiratory rhythm in spontaneously breathing anesthetized or awake mice was investigated by optogenetically-targeted excitation or inhibition. Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) were expressed in glycinergic neurons by injecting AAV2/1-Ef1α-DIO-ChR2-eYFP or AAV2/1-flex-CBA-Arch-GFP, respectively, into the preB�tC of GlyT2-Cre mice. In spontaneously breathing mice, inspiratory-phase bilateral laser photostimulation of ChR2-expressing preB�tC glycinergic neurons decreased tidal volume and shortened inspiratory duration, whereas expiratory-phase stimulation delayed the onset of the next inspiration. Prolonged stimulation produced apneas lasting as long as the light pulse (> 20s). Equivalent protocols producing photoinhibition of Arch-expressing glycinergic neurons during inspiration increased tidal volume without altering inspiratory duration, whereas expiratory-phase stimulation shortened the latency until the onset of the next inspiration. During Breuer-Hering lung inflation reflex-induced apneas, prolonged photoinhibition restored rhythmic breathing. We conclude that glycinergic preB�tC neurons modulate the pattern of inspiratory motor output and are important for production of reflex apneas. However, while modulatory, these neurons are not necessary for rhythmogenesis in intact (awake or anesthetized) mice. We also identified a novel marker, reelin, of excitatory preB�tC neuronal subpopulations and assessed its anatomical and functional implications for preB�tC neuroanatomy.