Action potential‐evoked Ca2+ signals and calcium channels in axons of developing rat cerebellar interneurones

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Abstract
1 Axonal [Ca2+] transients evoked by action potential (AP) propagation were studied by monitoring the fluorescence of the high-affinity calcium-sensitive dye Oregon Green 488 BAPTA-1, introduced through whole-cell recording pipettes in the molecular layer of interneurones from cerebellar slices of young rats. 2 The spatiotemporal profile of Ca2+-dependent fluorescence changes was analysed in well-focused axonal stretches a few tens of micrometres long. AP-evoked Ca2+ signals were heterogeneously distributed along axons, with the largest and fastest responses appearing in hot spots on average ∼5 μm apart. 3 The spatial distribution of fluorescence responses was independent of the position of the focal plane, uncorrelated to basal dye fluorescence, and independent of dye concentration. Recordings using the low-affinity dye mag-fura-2 and a Cs+-based intracellular solution revealed a similar pattern of hot spots in response to depolarisation, ruling out measurement artefacts or possible effects of inhomogeneous dye distribution in the generation of hot spots. 4 Fluorescence responses to a short train of APs in hot spots decreased by 41–76 % after bath perfusion of ω-conotoxin MVIIC (5–6 μM), and by 17–65 % after application of ω-agatoxin IVA (500 nM). ω-Conotoxin GVIA (1 μM) had a variable, small effect (0–31 % inhibition), and nimodipine (5 μM) had none. Somatically recorded voltage-gated currents during depolarising pulses were unaffected in all cases. These data indicate that P/Q-type Ca2+ channels, and to a lesser extent N-type channels, are responsible for a large fraction of the [Ca2+] rise in axonalhot spots. 5 [Ca2+] responses never failed during low-frequency (≤ 0.5 Hz) stimulation, indicating reliable AP propagation to the imaged sites. Axonal branching points coincided with a hot spot in ∼50 % of the cases. 6 The spacing of presynaptic varicosities, as determined by a morphological analysis of Neurobiotin-filled axons, was ∼10 times larger than the one measured for hot spots. The latter is comparable to the spacing reported for varicosities in mature animals. 7 We discuss the nature of hot spots, considering as the most parsimonious explanation that they represent functional clusters of voltage-dependent Ca2+ channels, and possibly other [Ca2+] sources, marking the position of developing presynaptic terminals before the formation of en passant varicosities.