Raphe Neurons Stimulate Respiratory Circuit Activity by Multiple Mechanisms via Endogenously Released Serotonin and Substance P

Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphe 5-HT neurons on respiratory network activity including at the level of the pre-Botzinger complex (pre-BotC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem-spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT2A/2C, 5-HT4, and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems. The midline raphe obscurus contained spontaneously active 5-HT neurons, some of which projected to the pre-BotC and hypoglossal motoneurons, colocalized 5-HT and SP, and received reciprocal excitatory connections from the pre-BotC. Experimentally augmenting raphe obscurus activity increased motor output by simultaneously exciting pre-BotC and motor neurons. Biophysical analyses in vitro demonstrated that 5-HT and SP modulated background cation conductances in pre-BotC and motor neurons, including a nonselective cation leak current that contributed to the resting potential, which explains the neuronal depolarization that augmented motor output. Furthermore, we found that 5-HT, but not SP, can transform the electrophysiological phenotype of some pre-BotC neurons to intrinsic bursters, providing 5-HT with an additional role in promoting rhythm generation. We conclude that raphe 5-HT neurons excite key circuit components required for generation of respiratory motor output.