MULTIPLE TYPES OF CALCIUM CHANNELS MEDIATE TRANSMITTER RELEASE DURING FUNCTIONAL RECOVERY OF BOTULINUM TOXIN TYPE A-POISONED MOUSE MOTOR NERVE TERMINALS M. M. SANTAFE ´ ,* F. J. URBANO,† M. A. LANUZA* and O. D. UCHITEL†‡ *Unitat d’Histologia i Neurobiologia (UHN), Facultat de Medicina i Cie `ncies de la Salut, Universitat Rovira i Virgili, carrer St Llorenc ¸, num 21, 43201 Reus, Spain †Laboratorio de Fisiologı ´a y Biologı ´a Molecular (LFBM), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabello ´n II-2 do piso, 1428 Buenos Aires, Argentina Abstract —The involvement of different types of voltage-dependent calcium channels in nerve-evoked release of neurotransmitter was studied during recovery from neuromuscular paralysis produced by botulinum toxin type A intoxication. For this purpose, a single subcutaneous injection of botulinum toxin (1 IU; dl 50 ) on to the surface of the mouse levator auris longus muscle was performed. The muscles were removed at several time-points after injection (i.e. at one, two, three, four, five, six and 12 weeks). Using electrophysiological techniques, we studied the effect of different types of calcium channel blockers (nitrendipine, v-conotoxin-GVIA and v-agatoxin-IVA) on the quantal content of synaptic transmission elicited by nerve stimulation. Morpho- logical analysis using the conventional silver impregnation technique was also made. During the first four weeks after intoxication, sprouts were found at 80% of motor nerve terminals, while at 12 weeks their number was decreased and the nerve terminals were enlarged. The L-type channel blocker nitrendipine (1 mM) inhibited neurotransmitter release by 80% and 30% at two and five weeks, respectively, while no effects were found at later times. The N-type channel blocker v-conotoxin-GVIA (1 mM) inhibited neurotransmitter release by 50–70% in muscles studied at two to six weeks, respectively, and had no effect 12 weeks after intoxication. The P-type channel blocker v-agatoxin-IVA (100 nM) strongly reduced nerve-evoked transmitter release (90%) at all the time-points studied. Identified motor nerve terminals were also sensitive to both nitrendipine and v-conotoxin-GVIA. This study shows that multiple voltage-dependent calcium channels were coupled to transmitter release during the period of sprouting and consolidation, suggesting that they may be involved in the nerve ending functional recovery process.  1999 IBRO. Published by Elsevier Science Ltd. Key words: botulinum toxin type A, calcium channels, neuromuscular junction, v-conotoxin-GVIA, v-agatoxin-IVA, nitrendipine. The local injection of Clostridum botulinum toxin type A (BoTx-A) on skeletal muscles has long been known to block evoked quantal acetylcholine (ACh) release from motor nerve endings, producing profound but transient muscle paralysis. 1,11–13,34,51 BoTx-A is a zinc-containing protein that recognizes and cleaves the synaptosome-asso- ciated membrane protein of 25,000 mol. wt, 35 preventing exo- cytosis. Within a few weeks, motor nerve terminals (MNTs) develop “sprouts” (i.e. nerve terminal outgrowths) during muscle paralysis, 1,13,22,34 without degeneration of the axon. 12,34 Moreover, sprouts play a role in the functional recovery of neuromuscular transmission, 11,12 and it was suggested that they might contribute to ACh release from MNTs. 1,51 Hence, the injection of BoTx-A provides a model of synaptic plasticity. Furthermore, intramuscular injection of BoTx-A is currently used as treatment for several neuro- muscular disorders (i.e. dystonias, blepharospasm, strabis- mus, disphonia). 26 Neuromuscular transmission is highly dependent upon influx of calcium from the external media via presynaptic voltage-dependent calcium channels (VDCCs). 24 Neuronal VDCCs have been subdivided, on the basis of their electro- physiological and pharmacological properties, into low- voltage-activated or T-type channels 20 and high-voltage- activated (HVA) channels, a class that includes the L, N, P/ Q and R types. The HVA channel types have mainly been characterized by their different sensitivities to pharmacologi- cal modulators and inhibitory toxins. HVA channels are complexes composed of a pore-forming a 1 subunit together with modulatory b and a 2 /d subunits, and, at least in skeletal muscle, g subunits. 5,17 To date, six HVA a 1 subunits have been identified: a 1A , a 1B , a 1C , a 1D , a 1E and a 1S . It is clear that a 1B forms the N-type channel, a 1C and a 1D the L-type channel, a 1S the skeletal muscle channel/voltage sensor, a 1A the P/Q type and a 1E the R type. 17,43 In motoneurons, it is well known that the evoked ACh release from mature mammalian MNTs is mediated by the calcium influx through VDCCs of the P/Q type. 4,18,42,52 In contrast, neither the L- nor N-type VDCCs have been found to participate in the evoked release of ACh in these mature MNTs. 3,4,27,39,41 Some of these pharmacological results have been rein- forced using immunocytochemical techniques. 7,58 However, changes of the VDCC types coupled to neurotransmitter release have been described during development in avian, 16 amphibian 14 and mammalian MNTs, 44,47 and during reinner- vation in mammalian MNTs. 25 The aim of the present work was to study the involvement of different VDCCs in the excitation–secretion coupling Calcium channels at botulinum-poisoned motor nerve terminals 227 227 Neuroscience Vol. 95, No. 1, pp. 227–234, 2000 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00 PII: S0306-4522(99)00382-6 Pergamon ‡To whom correspondence should be addressed. Tel.: + 54-11-4576-3368; fax: + 54-11-4576-3321. E-mail address: odu@bg.fcen.uba.ar (O. D. Uchitel) Abbreviations: ACh, acetylcholine; v-Aga-IVA, v-agatoxin-IVA; a- BgTx–Rd, rhodamine-conjugated a-bungarotoxin; BoTx-A, botulinum toxin type A; v-CgTx-GVIA, v-conotoxin-GVIA; DMSO, dimethyl- sulphoxide; dTC, d-tubocurarine; EPP, endplate potential; HVA, high- voltage-activated; LAL, levator auris longus; MNT, motor nerve term- inal; VDCC, voltage-dependent calcium channel. www.elsevier.com/locate/neuroscience