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

1. Axonal [Ca2+] transients evoked by action potential (AP) propagation wer e studied by monitoring the fluorescence of the high-affinity calcium-sensi tive 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 an alysed in well-focused axonal stretches a few tens of micrometres long. AP- evoked Ca2+ signals were heterogeneously distributed along axone, with the largest and fastest responses appearing in hot spots on average similar to 5 mu m apart. 3. The spatial distribution of fluorescence responses was independent of th e 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 o f 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 omega-conotoxin MVIIC (5-6 mu M), and by 17 -65% after application of omega-agatoxin IVA (500 nM). omega-Conotoxin GVIA (1 mu M) had a variable, small effect (0-31% inhibition), and nimodipine ( 5 mu M) had none. Somatically recorded voltage-gated currents during depola rising pulses were unaffected in all cases. These data indicate that P/Q-ty pe Ca2+ channels, and to a lesser extent N-type channels, are responsible f or a large fraction of the [Ca2+] rise in axonal hot spots. 5. [Ca2+] responses never failed during low-frequency (less than or equal t o 0.5 Hz) stimulation, indicating reliable AP propagation to the imaged sit es. Axonal branching points coincided with a hot spot in similar to 50% of the cases. 6. The spacing of presynaptic varicosities, as determined by a morphologica l analysis of Neurobiotin-filled axons, was similar to 10 times larger than the one measured for hot spots. The latter is comparable to the spacing re ported 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 C a2+ channels, and possibly other [Ca2+] sources, marking the position of de veloping presynaptic terminals before the formation of en passant varicosit ies.