Quantification of presynaptic Ca²⁺ concentration is essential to understand the mechanisms of neurotransmitter release and short-term plasticity. Therefore, we aimed at characterizing the Ca²⁺ transients at the cerebellar mossy fibre bouton (cMFBs) to granule cell synapse, which can transmit signals at high frequencies and with high fidelity. We established whole-cell patch-clamp recordings and quantitative two-photon calcium imaging at cMFBs. Boutons were filled with a Ca²⁺ insensitive dye (Atto 594, 10 µM) and the Ca²⁺ sensitive dyes OGB-1 (50 and 100 µM), Fluo-5F (50 and 200 µM) or OGB-5N (200 µM). Ca²⁺ signals were quantified using experiments in which the intra-bouton Ca²⁺ concentration was clamped to 0 or 10 mM [1]. Single action potentials had a peak amplitude of 217 ± 18 nM and a decay time constant of 175 ± 28 ms (n = 12) at 36°C using 200 µM OGB-5N. Extrapolating the data from the different dyes and dye concentrations [2] gave estimates of the calcium transient elicited by a single action potential to be at 300 nM, decaying with a time constant of 157 ms. From these data the endogenous fixed buffer capacity can be estimated to be in the range of 10-45. A train of 20 action potentials at 300 Hz led to an increase in intracellular calcium concentration of about 4.3 µM. These data suggest linear Ca²⁺ summation within high-frequency transmission and will provide important constraints to understand the Ca²⁺ dynamics at the release site.

1 Yasuda, R. et al. Sci. STKE 2004 219: pl5

2 Helmchen, F. et al. Biophys. J. 1997 72:1458-1471