ENDOGENOUS CALCIUM BUFFERING IN MOTONEURONS OF THE NUCLEUS HYPOGLOSSUS FROM MOUSE

1. Simultaneous patch clamp and rapid microfluorometric calcium measur ements were performed on sixty-five motoneurones in slices of the nucl eus hypoglossus in the brainstem of 2- to 6-day-old mice. 2. Hypogloss al motoneurones were particularly vulnerable to mechanical or metaboli c stress during isolation of in vitro slice preparations. Therefore, e xperimental conditions were optimized for functional integrity, as jud ged by spontaneous rhythmic activity of hypoglossal nerves (XII). 3. C alcium 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 m onochromator (switching tau < 10 ms), a photomultiplier tube and the c alcium sensitive dyes fura-2 and mag-fura-5. 4. Dynamics of somatic ca lcium transients were investigated as a function of the concentration of calcium indicator dye in the cell. Decays of calcium transients wer e approximated to a single exponential component and decay time consta nts showed a linear dependence on dye concentration. The extrapolated decay time in the absence of indicator dye was 0.7 +/- 0.2 s, suggesti ng 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 co mparable size or geometry. Accordingly, our measurements suggest that the selective vulnerability of hypoglossal motoneurones to calcium-rel ated excitotoxicity might partially result from low concentrations of calcium buffers in these cells.