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

摘要

We studied the functions of mitochondria and their hypoxic modulation in the brainstem slices of neonatal mice (postnatal day (P)6-11). The measurements were made in the preBotzinger complex (pBC), a part of the respiratory centre, and in the hypoglossal (XII) nucleus. Using a CCD camera, changes in the redox state were assessed from cell autofluorescence produced by NADH and FAD, while alterations in mitochondrial membrane potential ([Delta][psi]) and free Ca2+ concentration ([Ca2+]m) were obtained from fluorescence signals after loading the cells with Rh123 and Rhod-2, respectively. In the pBC, the cells were functionally identified by correlating the oscillations in [NADH], [FAD], [Delta][psi] and [Ca2+]m with the respiratory motor output recorded simultaneously from XII rootlets. In the inspiratory cells, NADH fluorescence showed a brief decrease followed by a slow and long-lasting increase during one oscillation period. The initial decrease in NADH fluorescence was accompanied by an increase in FAD fluorescence and coincided with [Delta][psi] depolarization. The slow secondary increase in NADH fluorescence had a time course similar to that of the Rhod-2 signal, indicating the role of Ca2+ uptake by mitochondria in NAD and FADH reduction. Brief (2-4 min) hypoxia reversibly abolished rhythmic changes in mitochondrial variables and brought them to new steady levels. In parallel, ATP-sensitive K+ (KATP) channels were activated and the respiratory output was depressed. The hypoglossal neurons showed much bigger increases in [Delta][psi] and [NADH] during hypoxia than the pBC neurons, which may explain their extreme vulnerability to hypoxia. We show here that mitochondrial function can be monitored in vitro in neurons constituting the respiratory neural network in slice preparations. Since mitochondrial variables demostrate specific, stereotypic fluctuations during a respiratory cycle, we suggest that mitochondrial function is modulated by spontaneous activity in the respiratory network. Therefore mitochondrial depolarization and Ca2+ uptake can contribute to the biphasic reaction of the respiratory network during hypoxia.