Sighs Originating in the PreBötC are Driven by Neuroglial Interactions

Sighs are large amplitude inspirations that occur periodically during normal breathing and are physiologically important for preventing alveolar collapse. Normal or “eupneic” inspirations and sighs originate from the same neural network, the preBötzinger Complex (preBötC), in the ventral medulla. Given that both sighs and eupnea arise from the same network, it has been challenging to differentiate the underlying mechanisms that concurrently generate both rhythmic behaviors. Using the in vitro brainstem slice preparation, we have recently demonstrated that this is possible through neuroglial coupling. We found that modulating purinergic signaling through P2Y1 receptors strongly alters sigh frequency without altering eupnea. Then, we used optogenetics in combination with extracellular population recordings to drive ChR2 expression under control of the astrocytic promoter, Aldh1l1. Light activation of Aldh1l1 expressing preBötC astrocytes evoked both eupnea and sigh bursts. Evoked sighs and eupnea had the same characteristics of spontaneous bursts, including comparable amplitudes, burst durations, and post‐burst intervals. Furthermore, optogenetic stimulations showed that sighs are tightly coupled to the eupneic frequency, as they occurred with a significantly higher probability when stimulated during the inspiratory cycle. Next, we demonstrated that light‐evoked sighs could be inhibited by applying P2Y1 blockade and low concentrations of the Ca2+ channel blocker cadmium (4μM, to selectively target P/Q‐type receptors). Sighs under control conditions and with application of the muscarinic receptor agonist oxotremorine were elicited within the limits defined by the refractory period. Utilizing calcium imaging with Fura‐2, we next identified a population of brainstem astrocytes that displayed an increase in intracellular calcium corresponding to both sigh and eupnea activity. We hypothesize that this increase in calcium facilitates neuroglial coupling through release of ATP, as preliminary data indicates that release of ATP from astrocytes and conversion to ADP contributes to this calcium change. Taken together, our experiments suggest that the timing characteristics underlying sigh generation are a coordinated action between glial calcium oscillations and the recruitment of preBötC neurons along the inspiratory column through purinergic signaling. Support or Funding Information NIH Ruth L. Kirschstein Predoctoral Individual National Research Service Award NIH R01 HL126523