Calcium dynamics and lowering in motoneurones of the mouse spinal cord

Abstract

1. A quantitative analysis of endogenous calcium homeostasis was performed on 65 motoneurones in slices of the lumbar spinal cord from 2- to 8-day-old mice by simultaneous patch-clamp and microfluorometric calcium measurements. 2. Somatic calcium concentrations were monitored with a temporal resolution in the millisecond time domain. Measurements were performed by using a monochromator for excitation and a photomultiplier detection system. 3. Somatic calcium signalling was investigated during defined voltage-clamp protocols. Calcium responses were observed for membrane depolarizations positive to -50 mV. A linear relation between depolarization time and free calcium concentrations ([Ca2+](i)) indicated that voltage-dependent calcium influx dominated the response. 4. Endogenous calcium homeostasis was quantified by using the 'added buffer' approach. In the presence of fura-2 and mag-fura-5, calcium transients decayed according to a mono-exponential function. Decay-time constants showed a linear dependence on dye concentration and the extrapolated constant in the absence of indicator dye was 371 ± 120 ms (n = 13 cells, 21°C). 5. For moderate elevations (< 1 μM), recovery kinetics of depolarization-induced calcium transients were characterized by a calcium-independent, 'effective' extrusion rate γ = 140 ± 47 s-1 (n = 13 cells, 21°C). 6. The endogenous calcium binding ration for fixed buffers in spinal motoneurones was κ(B)' = 50 ± 17 (n = 13 cells), indicating that less than 2% of cytosolic calcium ions contributed to [Ca2+](i). 7. Endogenous binding ratios in spinal motoneurones were small compared to those found in hippocampal or cerebellar Purkinje neurones. From a functional perspective, they provided motoneurones with rapid dynamics of cytosolic [Ca2+](i) for a given set of influx, extrusion and uptake mechanisms. 8. With respect to pathophysiological conditions, our measurements are in agreement with a model where the selective vulnerability of spinal motoneurones during excitotoxic conditions and motoneurone disease partially results from low endogenous calcium buffering.