Glycinergic Inhibitory Synaptic Currents and Related Receptor Channels in the Zebrafish Brain

Abstract

To extend our study of the inhibitory synaptic network we have developed an isolated whole-brain preparation of the 52-h-old zebrafish (Brachydanio rerio) in which the structural and functional integrity of the brain is preserved. We report the characterization of quantal inhibitory events and the correlation of their properties with those of the underlying activated channels. During whole-cell recordings of the Mauthner cells, applications of 10(-6) M tetrodotoxin greatly reduced the frequency and amplitude of the spontaneously occurring synaptic events, which were dominated by Cl--dependent inhibitory postsynaptic currents (IPSCs). Lowering Ca2+ and adding Mg2+ to tetrodotoxin-containing solutions resulted in a further decrease in amplitude of the recorded synaptic currents, the remaining ones being considered as miniature IPSCs (mIPSCs). Applications of 0.5-1 microM strychnine in the presence of tetrodotoxin eliminated > 90% of the inhibitory currents in the preparation. The amplitude histograms of these mIPSCs exhibited two initial equally spaced peaks, followed by a skewed distribution for higher values. The first two components were well fitted by the sum of two Gaussian curves, giving a mean quantal amplitude of 35.7 pA (at a holding potential of -50 mV) and a coefficient of variation of 0.25 for the first peak. Outside-out recordings showed at least two classes of glycine receptor channels, one having multiple conductance levels with a main state of 81-86 pS and another displaying only one opening level of 41-43 pS. These two mean conductance states had similar mean open times, of 0.6-1 and 4.5-6 ms respectively. In addition, three mean closed times were observed for the 41-43 pS level. The shortest group (0.6-1 ms) was considered as representing gaps within bursts. Burst analysis revealed three mean burst durations, of 0.6, 4 and 35 ms. Comparisons of the amplitude of the first class of mIPSCs and of the open channel conductances indicated that one quantum opens 14-22 channels. Moreover, the correspondence between the mean decay time of mIPSCs and the mean open time or medium burst duration (4-5 ms) suggests that glycine-activated channels open only once in response to a single exocytosis. The pre- and postsynaptic origins of mIPSCs amplitude fluctuations are discussed in the context of multivesicular release versus the hypothesis of postsynaptic receptor saturation.