Shunichi Yamagishi

Differential effects of phospholipase inhibitors in long-term potentiation in the rat hippocampal mossy fiber synapses and Schaffer/commissural synapses

Bath application of the inhibitors of phospholipases, nordihydroguaiaretic acid (NDGA) and p-bromo-phenacyl bromide (BPB), to the rat hippocampal slices suppressed long-term potentiation (LTP) in Schaffer/commissural-CA1 pyramidal synapses. On the other hand, neither of the two inhibitors suppressed LTP in mossy fiber-CA3 pyramidal cell synapses. BPB did not suppress phosphatidylinositol-specific phospholipase C (PI-PLC) activity of the slices. These results suggested that the mechanisms of LTP were quite different in the CA1 and CA3 subfields of rat hippocampus: in CA1, the involvement of an arachidonate metabolism was strongly suggested, whereas in CA3, an arachidonic acid cascade may not be necessary for LTP.

Characterization of mRNA responsible for induction of functional sodium channels in Xenopus oocytes

When Xenopus laevis oocytes were microinjected with poly(A)+ mRNA isolated from adult rat brains or electric organs of Electrophorus electricus, the oocytes developed functional sodium channels. Upon application of veratrine, the microinjected oocytes exhibited transient depolarization, resulting in spontaneous repetitive spikes in some occasions, and action potentials. These responses were mediated mainly by external Na ions, prolonged by scorpion toxin, completely blocked by tetrodotoxin, and suppressed by local anesthetics. Thus the mRNA-induced sodium channels exhibited essentially all the functional properties expected for native sodium channels in nerve and muscle membranes. Rat brain mRNA was fractionated into 4 fractions by sucrose gradient centrifugation. Each fraction and various combinations of them were examined for the efficiency in inducing functional sodium channels in Xenopus oocytes. A fraction corresponding to mRNA of approximately 30S to 46S was found to contain all mRNA necessary for the expression of the channels, indicating that mRNA of smaller sizes expected to code for smaller polypeptides may not be required.

Characterization of glutamate receptors induced in Xenopus oocytes after injection of rat brain mRNA.

Xenopus oocytes in which poly(A)+ mRNA isolated from rat brains were previously injected, exhibited at least 3 categories of current responses to excitatory amino acids. They were oscillatory responses to glutamate (Glu) or quisqualate (QA), smooth large responses to kainate (KA), and smooth small responses to Glu and QA. Oscillatory responses were mediated by a metabotropic type of Glu receptor which is coupled to a G-protein but not directly to an ionic channel. Amplitudes of smooth Glu responses and smooth QA responses were similar in size, and were not additive to each other, suggesting a common receptor mediating both responses. L-Glutamylglycine inhibited KA responses in a competitive manner without affecting smooth Glu/QA responses, indicating that KA and smooth Glu/QA responses were mediated by separate receptors. From these results, it was concluded that the injection of rat brain mRNA induced at least 3 different glutamatergic receptors: receptors mediating (a) KA responses and (b) smooth Glu/QA responses, and (c) the metabotropic Glu receptor. The former two may most likely correspond to Glu receptor subtypes preferring KA and QA, respectively, as seen in the brain.

Evidence for l-glutamate as the neurotransmitter of the squid giant synapse

The effect of a spider toxin (JSTX), a specific blocker of glutamate receptors, was studied in the squid stellate ganglion. JSTX irreversibly blocked the excitatory postsynaptic potential in a dose-dependent manner. The toxin neither affected the spike in the postsynaptic nor the presynaptic fibers. Spontaneous miniature potentials recorded from thin stellate nerves were suppressed by the toxin. Iontophoretically applied L-glutamate depolarized the postsynaptic membrane of the giant axon and this potential was also blocked by JSTX. Kainic acid also depolarized the postsynaptic membrane but this was partially blocked by JSTX, indicating that JSTX differentiated kainate receptor from glutamate one. The results strongly suggest that L-glutamate is the neurotransmitter in the giant synapse of squid.