Oscillations and hypoxic changes of mitochondrial variables in neurons of the brainstem respiratory centre of mice

1. We studied the functions of mitochondria and their hypoxic modulation in the brainstem slices of neonatal mice (postnatal day (P)G-ll). The measure ments were made in the preBotzinger complex (pBC), a part of the respirator y centre, and in the hypoglossal (XII) nucleus. Using a CCD camera, changes in the redox state were assessed from cell autofluorescence produced by NA DH and FAD, while alterations in mitochondrial membrane potential (Delta ps i) and free Ca2+ concentration ([Ca2+](m)) were obtained from fluorescence signals after loading the cells with Rh123 and Rhod-2, respectively. 2. In the pBC, the cells were functionally identified by correlating the os cillations in [NADH), [FAD, Delta psi anti [Ca2+](m) with the respiratory m otor output recorded, simultaneously from XII rootlets. In the inspiratory cells, NADH fluorescence showed a brief decrease followed by a slow and lon g-lasting increase during one oscillation period. The initial decrease in N ADH fluorescence was accompanied by an increase in FAD fluorescence and coi ncided with Delta psi depolarization. The slow secondary increase in NADH f luorescence had a time course similar to that of the Rhod-8 signal, indicat ing the role of Ca2+ uptake by mitochondria in NAD and FADH reduction. 3. Brief (2-4 min) hypoxia reversibly abolished rhythmic changes in mitocho ndrial variables and brought them to new steady levels. In parallel, ATP-se nsitive K+ (K-ATP) channels were activated and the respiratory output was d epressed. The hypoglossal neurons showed much bigger increases in Delta psi and [NADH] during hypoxia than the pBC neurons, which may explain their ex treme vulnerability to hypoxia. 4. We show here that mitochondrial function can be monitored in vitro in ne urons constituting the respiratory neural network in slice preparations. Si nce mitochondrial variables demostrate specific, stereotypic fluctuations d uring a respiratory cycle, we suggest that mitochondrial function is modula ted by spontaneous activity in the respiratory network. Therefore mitochond rial depolarization and Ca2+ uptake can contribute to the biphasic reaction of the respiratory network during hypoxia.