Solving the paradox of neural inhibition in pacing breathing - SUMMARY Breathing is one of our most important behaviors. Each breath is composed of two alternating phases; inspiration when air is drawn into the lungs and expiration when the air is expelled. Here we will dissect if the subconscious pacing mechanism is a reciprocity of these two phases or an autonomous oscillator for just one whereby the other phase passively follows. Right now, the breathing rhythm is thought to arise from the latter model. A cluster of just several thousand neurons in the brainstem, called the preBötzinger Complex (PBC), trigger each inspiration and expiration simply follows. The PBC produces an autonomous rhythm by local excitatory neurotransmission this is thought to be sufficient to pace breathing. Yet nearby to the PBC is a node of inhibitory neurons active during expiration, the Bötzinger Complex (BötC), and the PBC and BötC are connected via reciprocal inhibition. This forms the scaffold for the classic ‘reciprocal inhibitory oscillator’ which has previously been proposed to be the mechanism that paces breathing. These two hypotheses conflict, and here we will use intersectional mouse genetics to selectively target and then probe the neural types in the PBC and BötC which we anticipate to pace breathing. We hope this study will determine how subconscious breathing arises in vivo. Our preliminary data is consistent with the ‘reciprocal inhibition’ model and have led to our central hypothesis. Up to now, we have found that briefly silencing all PBC inhibitory neurons locks breathing in tonic inspiration, while silencing just a subset of molecularly defined PBC inhibitory neurons, ~150 in total, locks breathing in expiration. In fact, ablation of just these few neurons in adult animals is lethal and demonstrates their vital role which cannot be compensated for. In this proposal, we have designed three aims to independently test the ‘reciprocal inhibition’ model and thereby define the cellular basis of breathing. We will determine if the specialized inhibitory neurons are bonified PBC neurons, if they are necessary to “turn-off” BötC activity and expiration, and then we will characterize the complementary BötC neurons that complete the reciprocal inhibitory coupling. This proposal is significant as it will establish the mechanism for pacing breathing in vivo. Furthermore, the identification of the cellular basis will enable future studies to characterize the biophysical properties that underlie it. This study will serve as a model when exploring other rhythmic mammalian behaviors like licking or chewing, as well as other oscillatory microcircuits in the brain. Ultimately, such knowledge could enable the discovery of novel pharmacological approaches to control breathing, as well as key cell types perhaps impacted in common and devastating breathing conditions like sleep apnea and sudden infant death.
