Drowning a frog respiratory rhythm generator in a wash of excitation: State-dependent architecture of a ventilatory oscillator

Abstract Breathing begins at birth and continues to death, providing a unique system to understand the architecture of rhythm-generating circuits in vertebrates. In mammals and frogs, three discrete rhythmogenic brainstem nuclei appear essential for generating different phases of breathing, with individual neurons intrinsically tuned to each phase. This discrete multi-oscillator architecture differs fundamentally from the distributed spinal locomotor networks, where rhythmogenesis spans multiple segments and the active phase of individual neurons depends on network state. However, whether these discrete respiratory oscillators are strictly essential and whether the phase of their neurons remains constant across states is coming under increasing scrutiny. Here, we focus on the frog buccal oscillator, which shares anatomical and functional similarities with the mammalian preBötzinger Complex. We developed a technique to silence motor neurons in frogs and mapped premotor unit activity within and surrounding the putative buccal oscillator under standard conditions and during heightened network excitability. We found that increasing network excitation caused the rhythmically active area to expand, in part by converting premotor lung units into buccal units. Next, we located the buccal area using excitatory and inhibitory microinjections and established the minimum number of inhibitory injections required to suppress buccal bursts. We then increased network excitability and again tested whether inhibitory microinjections could suppress the rhythm. While inhibitory microinjections could reverse local excitatory effects, they were unable to abolish the buccal rhythm under elevated excitation. Our data shows that not only does the region containing rhythmic premotor buccal units enlarge as network excitation increases, but the essential requirement for a discrete, rudimentary buccal oscillator is also lost. Thus, although rostral-caudal segments of the frog brainstem generally contribute to discrete phases of ventilatory motor patterns, our findings suggest that oscillator identity should be refined: oscillators should be viewed as promiscuous flexible functional entities that expand or contract to occupy different extents of the brainstem, rather than as fixed anatomical nuclei. The respiratory rhythm is hypothesized to be generated by specialized brainstem circuits comprised of individual non-redundant, discrete, and intrinsically rhythmic oscillators. These oscillators are essential in the impoverished network, often generating rhythmic output in isolation. The inspiratory preBötzinger Complex (preBötC) is the most important of these oscillators in mammals. Isolated brainstem preparations in frogs have revealed a ventilatory rhythm-generating circuit closely resembling that of mammals. Particularly, the Buccal Area shares anatomical and functional similarities to the preBötC. We demonstrate that the discrete Buccal Area is rearranged with mild increases in network excitation to a diffuse network that appears to generate the buccal rhythm instead. These findings support the broader hypothesis that respiratory rhythm-generating circuits can switch to being diffuse and redundant, with discrete oscillators quickly drowning in a sea of excitation. Competing Interest Statement The authors have declared no competing interest. Abbreviations - aCSF - artificial Cerebral Spinal Fluid - CN - cranial nerve - IBI - inter buccal interval - IF - instantaneous frequency - preBötC - preBötzinger Complex - SD - standard deviation - SN - spinal nerve