The goals of this study are to test hypotheses regarding the development of cellular and synaptic mechanisms underlying respiratory rhythm generation and the tolerance to anoxia in the amphibian brain. An in vitro brainstem preparation, brainstem slices and isolated neurons from North American bullfrogs (Rana catesbeiana) at various stages of development will be used in this study. The bullfrog brainstem preparation was chosen because it produces a quantifiable, spontaneous respiratory motor ouput in vitro at ajl stages of development. The metamorphic transition from an aquatic to a terrestrial habitat in amphibians is accompanied by major maturationai changes of the respiratory system, but very little is known about the neural mechanisms that underlie this ontogenetic maturation. The specific aims of this project are to: 1) Identify specific neuroanatomical locations of the amphibian respiratory network; 2) Examine the role of pacemaker mechanisms for respiratory rhythm generation; 3) Determine the role of oxygen-sensing mechanisms in the respiratory response to anoxia; 4) Determine the role of intracellular calcium as a neuroprotective signaling mechanism in anoxia. Isolated brainstem preparations, spontaneously active medullary slices and isolated neurons will be used to carry out the specific aims. A multifaceted approach that combines fluorescent dyes for neuroanatomical tracing, electrophysiological measurements from respiratory neurons, intracellular calcium measurements, bath application and microinjection of neurochemicals into discrete brain locations, will be used to test hypotheses related to the specific aims. The proposed research should provide greater insight into the role of development and evolutionarily conserved mechanisms in the neural regulation of ventilation and cellular mechanisms of neuroprotection in vertebrates. The results of this project may provide a greater understanding of developmental mechanisms that contribute to abnormal breathing states in developing mammals.