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

KEY POINTS We transfected preBotC SST+ neurons, which modulate respiratory pattern but are not rhythmogenic, with channelrhodopsin to investigate phase- and state-dependent modulation of breathing pattern in anesthetized and freely behaving mice in normoxia, hypoxia, and hypercapnia. In anesthetized mice, photostimulation during inspiration increased inspiratory duration and amplitude regardless of baseline breathing frequency, f, yet the effects were more robust at higher f. In anesthetized mice with low f (<2.5 Hz), photostimulation during expiration evoked either phase advance or phase delay, whereas in anesthetized 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 preBotC SST+ neurons was unexpected, requiring reconsideration of current models of preBotC function, which neither predict nor can readily account for such responses. ABSTRACT As neuronal subtypes are increasingly categorized, delineating their functional role is paramount. The preBotzinger Complex (preBotC) 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 anesthetized and freely moving mice. In anesthetized mice, short photostimulation (100 ms) of preBotC 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. Moreover, we observed a biphasic effect of photostimulation during expiration that is probabilistic, i.e., 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 preBotC that were not predicted and cannot be readily accounted for in current models of preBotC 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 anesthetized mice when f≥2.5 Hz. We conclude that preBotC SST+ neurons are a key mediator of the extraordinary and essential lability of breathing pattern. We optogenetically stimulated preBotC SST+ neurons to investigate phase- and state-dependent modulation of breathing pattern in anesthetized mice. In the same mouse, the same stimulus evoked either phase advance (shortening of the cycle), phase delay (prolongation of the cycle) or no change. Using cophase plots (phase ϕ vs. cophase θ) we determined that the transition between a bimodal effect (phase advance or delay) to a unimodal effect (phase advance) occurred at baseline breathing f <2.5Hz. This article is protected by copyright. All rights reserved.