Phase- and state-dependent modulation of breathing pattern by preBötzinger complex somatostatin expressing neurons

Abstract

As neuronal subtypes are increasingly categorized, delineating their functional role is paramount. The preBötzinger complex (preBötC) subpopulation expressing the neuropeptide somatostatin (SST) is classified as mostly excitatory, inspiratory-modulated and not rhythmogenic. We further characterized their phenotypic identity: 87% were glutamatergic and the balance were glycinergic and/or GABAergic. We then used optogenetics to investigate their modulatory role in both anaesthetized and freely moving mice. In anaesthetized mice, short photostimulation (100 ms) of preBötC SST⁺ neurons modulated breathing-related variables in a combinatory phase- and state-dependent manner; changes in inspiratory duration, inspiratory peak amplitude (Amp), and phase were different at higher (≥2.5 Hz) vs. lower (<2.5 Hz) breathing frequency (f). Moreover, we observed a biphasic effect of photostimulation during expiration that is probabilistic, that is photostimulation given at the same phase in consecutive cycles can evoke opposite responses (lengthening vs. shortening of the phase). These unexpected probabilistic state- and phase-dependent responses to photostimulation exposed properties of the preBötC that were not predicted and cannot be readily accounted for in current models of preBötC pattern generation. In freely moving mice, prolonged photostimulation decreased f in normoxia, hypoxia or hypercapnia, and increased Amp and produced a phase advance, which was similar to the results in anaesthetized mice when f ≥ 2.5 Hz. We conclude that preBötC SST⁺ neurons are a key mediator of the extraordinary and essential lability of breathing pattern. KEY POINTS: PreBötzinger complex (preBötC) SST⁺ neurons, which modulate respiratory pattern but are not rhythmogenic, were transfected with channelrhodopsin to investigate phase- and state-dependent modulation of breathing pattern in anaesthetized and freely behaving mice in normoxia, hypoxia and hypercapnia. In anaesthetized mice, photostimulation during inspiration increased inspiratory duration and amplitude regardless of baseline f, yet the effects were more robust at higher f. In anaesthetized mice with low f (<2.5 Hz), photostimulation during expiration evoked either phase advance or phase delay, whereas in anaesthetized mice with high f (≥2.5 Hz) and in freely behaving mice in normoxia, hypoxia or hypercapnia, photostimulation always evoked phase advance. Phase- and state-dependency is a function of overall breathing network excitability. The f-dependent probabilistic modulation of breathing pattern by preBötC SST⁺ neurons was unexpected, requiring reconsideration of current models of preBötC function, which neither predict nor can readily account for such responses.

Keywords: breathing pattern; optogenetics; preBötC; somatostatin.