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Action potential‐evoked Ca 2+ signals and calcium channels in axons of developing rat cerebellar interneurones
Author(s) -
Forti Lia,
Pouzat Christophe,
Llano Isabel
Publication year - 2000
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2000.00033.x
Subject(s) - bapta , biophysics , chemistry , fluorescence , electrophysiology , depolarization , voltage dependent calcium channel , calcium , cerebellum , neuroscience , biology , physics , organic chemistry , quantum mechanics
1 Axonal [Ca 2+ ] 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 Ca 2+ ‐dependent fluorescence changes was analysed in well‐focused axonal stretches a few tens of micrometres long. AP‐evoked Ca 2+ 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 Ca 2+ channels, and to a lesser extent N‐type channels, are responsible for a large fraction of the [Ca 2+ ] rise in axonalhot spots. 5 [Ca 2+ ] 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 Ca 2+ channels, and possibly other [Ca 2+ ] sources, marking the position of developing presynaptic terminals before the formation of en passant varicosities.