Initial release of [3H]dopamine from rat striatal synaptosomes: correlation with calcium entry

Calcium entry and dopamine (DA) release were compared in rat striatal synaptosomes, to evaluate the role of Ca in mammalian CNS presynaptic neurotransmission. After equilibrating the synaptosomes with 0.1 PM [“HIDA, release was measured at O.l-min intervals by collecting superfusion effluents; release was measured over lto lo-set intervals by a novel rapid filtration technique in which release was stopped by adding a solution containing Ni, a Ca channel blocker. During the initial second, the rate coefficient of high Kand Ca-evoked [3H]DA release (0.067 + 0.003 set-‘) was 40-fold greater than basal release (0.0017 + 0.003 set-‘) and much greater than estimated in previous superfusion studies. Evoked release declined at longer intervals (0.009 + 0.001 set-’ between 1 and 10 set), but was still evident after 1 min of depolarization. The fast phase of [3H]DA release, but not subsequent release, showed voltage-sensitive inactivation. A similar time course of Ca uptake was observed, i.e., there was both a fast, inactivating phase and a slow, non-inactivating phase, as described previously in forebrain synaptosomes (Nachshen, D. A., and M. P. Blaustein (1980) J. Gen. Physiol. 76: 709-728). Ca-evoked [3H]DA release during land lo-set incubations increased as the K concentration was raised above 10 mM and levelled off at 75 mM K. The K0.5 for Ca of evoked [3H]DA release was 0.2 I? IM during the fast phase and 0.7 mM for release between 1 and 10 sec. The effects of varying the K concentration (at 1 and 10 set) and Ca concentration (at 1 set) on DA release resembled their effects on Ca uptake into forebrain synaptosomes reported previously. Sr and Ba increased the basal rate of [3H]DA release, but Mg, Co, Ni, La, and Cd were ineffective. Sr, Ba, and La substituted for Ca during K-evoked [“HIDA release, whereas the other divalent cations reduced Ca-dependent evoked release. Micromolar concentrations of La and Cd selectively blocked the fast phase of Ca entry and inhibited evoked [3H]DA release in the presence of low (0.2 mM) external Ca concentrations. We conclude that Ca entry is a limiting step for DA release in synaptosomes during the first few seconds of depolarization. In 1967, Katz and Miledi proposed a “calcium hypothesis” to explain their observations on neurotransmitter release at the frog neuromuscular junction (NMJ). According to this hypothesis, the Ca that enters during nerve terminal depolarization triggers neurotransmitter release. The calcium hypothesis has been confirmed by electrophysiological studies on a number of preparations, ’ We are very grateful to Dr. D. A. Nachshen for reading the manuscript and for his helpful suggestions and criticisms throughout the course of this study. We thank Ms. Arvette Wilder for preparing the typescript. P. D. is a Postdoctoral Fellow of the Medical Research Council of Canada. This work was supported by National Institutes of Health Grant NS-16106. ’ Present address: Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305. ,’ To whom correspondence should be addressed. including the squid giant synapse and the vertebrate NMJ (see Llinas, 1977; Martin, 1977). The small size of the terminals in the mammalian central nervous system precludes intraterminal microelectrode recording. Nevertheless, studies on brain slices and isolated nerve ending (synaptosome) preparations from mammalian brain (DeRobertis et al., 1962; Gray and Whittaker, 1962) indicate that transmitter release at central synapses is Ca-dependent. Synaptosomes retain many of the functional properties of in situ nerve terminals: for example, they can maintain relatively large resting membrane potentials and can synthesize neurotransmitters and release them upon depolarization in the presence of Ca (Blaustein, 1975; Bradford, 1975; Levi and Raiteri, 1976; Blaustein et al., 1977). Synaptosomes may be ideally suited for the study of the molecular events that couple the entry of Ca to neurotransmitter release.