The long-term goal is to analyze the neural mechanisms of vertebrate respiration, using as an experimental model the isolated brainstem/spinal cord of newborn rats. This novel preparation presents significant experimental advantages such as motionless conditions for intracellular recording, unrestricted access to the brainstem, elimination of feedback mechanisms from the periphery, prompt responses to bath-applied drugs, and an automatic on-going respiratory rhythm produced by a central pattern generator. The aims of this proposal are first to validate the experimental model by analyzing the in vitro respiratory rhythm and comparing it to the in vivo activity of respiratory muscles in newborn rats. Next, the locations of key respiratory neurons in the medulla will be mapped using ejection of neurotransmitters to reset the rhythm timing, with reference to the well-known respiratory nuclei of other mammals. The most intensive part of the proposed research will be to record intracellularly from respiratory neurons defined as cells that produce impulse bursts or show synaptic activity coordinated with the phenic discharge. At present there are no published intracellular recordings from respiratory neurons in this rat preparation. Cells will be routinely marked by intracellular injection of a fluorescent dye. Research will focus on motorneurons, bulbospinal neurons, and novel groups of respiratory neurons. Their locations, activity patterns, morphology, synaptic inputs, projections, and certain cellular properties will be characterized. A more detailed study will be made of the serotonergic innervation of phrenic motorneurons from neurons in the medullary raphe nuclei. The in vitro rat preparation, in conjunction with the bath-application of specific pharmacological agents, will allow significant advances in understanding the loci, cellular mechanisms, and receptor types that mediate the powerful serotonergic modulation of respiration.