PROJECT SUMMARY (See instructions): Overview: The preBotzinger Complex (preBotC) is the neuronal network that drives inspiratory rhythmogenesis whose activity is orchestrated in response to changes in homeostasis and is critical for maintaining and adapting breathing to the demands of the organism. Respiratory networks are constantly modulated by numerous neuromodulators through altering properties of neurons, synapses and networks. Understanding action of neuromodulation on respiratory networks is critical to understanding control of breathing. While there is extensive experimental effort on studying network effects of neuromodulation, the mechanisms underlying neuromodulatory action on respiratory network properties cannot be easily understood just by experimental work alone. Despite the well-established utility of computational approaches to studying the neural control of breathing, a knowledge gap exists for a computational understanding of the detailed mechanisms underlying how different neuromodulators interact to impact respiratory rhythmogenesis. The overall objective of this proposal is to uncover the mechanism by which the temporal order of neuromodulation with opposing actions (i.e., inhibitory and excitatory) differentially impact respiratory network dynamics. Our preliminary data suggest that such temporal sequencing can induce bifurcations and bistability in network states. We hypothesize that temporal order of opposing neuromodulators yields different changes in intrinsic and synaptic properties of a network which together produce qualitatively different network behaviors when the order is reversed. We will combine electrophysiological experiments and computational modeling across the levels of molecules (glutamates), neurons and networks to test this hypothesis via a focus on respiration, with three Specific Aims: (1) Identify how changes in inhibitory neuromodulation (e.g., opioids) impacts synaptic dynamics and properties in glutamatergic synapse. (2) Dissect the mechanisms by which excitatory neuromodulation (e.g., norepinephrine) regulates respiratory network dynamics through changes in both intrinsic and synaptic properties. (3) Examine the synaptic and intrinsic mechanisms by which temporal order of opposing neuromodulators produces different network states. We will explore and identify relevant bifurcations induced by temporal order of neuromodulators using dynamical systems theory.