GLYCINERGIC INHIBITORY SYNAPTIC CURRENTS AND RELATED RECEPTOR CHANNELS IN THE ZEBRAFISH BRAIN

To extend our study of the inhibitory synaptic network we have develop ed an isolated whole-brain preparation of the 52-h-old zebrafish (Brac hydanio rerio) in which the structural and functional integrity of the brain is preserved. We report the characterization of quantal inhibit ory events and the correlation of their properties with those of the u nderlying activated channels. During whole-cell recordings of the Maut hner 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 curren ts (IPSCs). Lowering Ca2+ and adding Mg2+ to tetrodotoxin-containing s olutions resulted in a further decrease in amplitude of the recorded s ynaptic currents, the remaining ones being considered as miniature IPS Cs (mlPSCs). Applications of 0.5 - 1 mu M strychnine in the presence o f tetrodotoxin eliminated > 90% of the inhibitory currents in the prep aration. The amplitude histograms of these mlPSCs exhibited two initia l equally spaced peaks, followed by a skewed distribution for higher v alues. The first two components were well fitted by the sum of two Gau ssian 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 glyc ine receptor channels, one having multiple conductance levels with a m ain 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 mea n closed times were observed for the 41 - 43 pS level. The shortest gr oup (0.6 - 1 ms) was considered as representing gaps within bursts. Bu rst analysis revealed three mean burst durations, of 0.6, 4 and 35 ms. Comparisons of the amplitude of the first class of mlPSCs and of the open channel conductances indicated that one quantum opens 14-22 chann els. Moreover, the correspondence between the mean decay time of mlPSC s 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 mlPSCs amplitude flu ctuations are discussed in the context of multivesicular release versu s the hypothesis of postsynaptic receptor saturation.