Endogenous calcium buffering in motoneurones of the nucleus hypoglossus from mouse

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Abstract
1 Simultaneous patch clamp and rapid microfluorometric calcium measurements were performed on sixty-five motoneurones in slices of the nucleus hypoglossus in the brainstem of 2- to 6-day-old mice. 2 Hypoglossal motoneurones were particularly vulnerable to mechanical or metabolic stress during isolation of in vitro slice preparations. Therefore, experimental conditions were optimized for functional integrity, as judged by spontaneous rhythmic activity of hypoglossal nerves (XII). 3 Calcium concentrations in the cell soma were monitored with a temporal resolution in the millisecond time domain during depolarizing voltage steps. Ratiometric fluorescence measurements were made using a rapid monochromator (switching τ < 10 ms), a photomultiplier tube and the calcium sensitive dyes fura-2 and mag-fura-5. 4 Dynamics of somatic calcium transients were investigated as a function of the concentration of calcium indicator dye in the cell. Decays of calcium transients were approximated to a single exponential component and decay time constants showed a linear dependence on dye concentration. The extrapolated decay time in the absence of indicator dye was 0·7 ± 0·2 s, suggesting rapid somatic calcium dynamics under physiological conditions. 5 By a process of back-extrapolation, the ‘added buffer’ method, a calcium binding ratio of 41 ± 12 (9 cells) was obtained indicating that 98 % of the calcium ions entering a hypoglossal motoneurone were bound by endogenous buffers. 6 Endogenous calcium binding ratios in hypoglossal motoneurones were small compared with those of other neurones with comparable size or geometry. Accordingly, our measurements suggest that the selective vulnerability of hypoglossal motoneurones to calcium-related excitotoxicity might partially result from low concentrations of calcium buffers in these cells.