Faculty Bibliography

Simultaneous recording of complex spikes from multiple Purkinje cells (up to 44) in the rat cerebellum was used to examine the effects of 5-hydroxytryptamine (serotonin, 5-HT) on olivocerebellar function. Microinjection into the inferior olive was found to increase the average firing rate of inferior olivary neurons while slowing their oscillation frequency and increasing the coherence of their oscillations. Indeed, while the normal rostrocaudal band of synchronous activity remained unchanged, the degree of synchrony between Purkinje cell complex spikes within this band was enhanced following the 5-HT injections. Multiple-electrode recordings obtained from crus Ila and vermal lobule Vlb yielded qualitatively similar results; however, the effects on vermal activity were more pronounced. The effects of the 5-HT microinjection decayed with a time course of 75 min. The half-maximum effective concentration of 5-HT was between 10 and 100 microM. Injections of various 5-HT agonists and antagonists demonstrated that a 5-HT type-2A (5-HT2A) receptor is the main mediator for the 5-HT effect, which was very similar to the effect produced by injections of harmaline. However, 5-HT and harmaline appear to have independent mechanisms since the action of harmaline was not blocked by the 5-HT2A antagonist LY53857. A possible role for 5-HT, as a physiological enhancer of the timing of motor function of the olivocerebellar system, is discussed

The time course for the calcium entry that triggers release was studied at the squid giant synapse by imaging light emission from n-aequorin-J intracellularly injected into the presynaptic terminal. The imaging utilized a video system capable of acquiring 4000 frames per second. The results indicate that the calcium entry triggered by action potentials reaches a peak within 200 microseconds and has an overall duration of close to 800 microseconds, closely matching the duration of the presynaptic calcium current determined by voltage clamp results under similar conditions

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles

Ever since the initial measurements of presynaptic calcium currents it has been evident that calcium triggers transmitter release quite rapidly. Several models indicate, as did the initial voltage clamp measurements, that the calcium concentration triggering such release could be very high at the entry site and that this concentration should be very short lasting. In order to determine this time course, calcium entry was studied at the squid giant synapse by imaging light emission from n-aequorin-J, intracellularly injected into the presynaptic terminal. The imaging utilized a video system capable of acquiring 4000 frames per sec. The results indicate that the calcium entry, triggered by action potentials, reaches a peak within 200 musec and has an overall duration of close to 800 musec, closely matching the duration of the presynaptic calcium current determined by voltage clamp results under similar conditions

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release