Development of spontaneous glycinergic currents in the Mauthner neuron of the zebrafish embryo

We used whole cell and outside-out patch-clamp techniques with reticulospin al Mauthner neurons of zebrafish embryos to investigate the developmental c hanges in the properties of glycinergic synaptic currents in vivo from the onset of synaptogenesis. Miniature inhibitory postsynaptic currents (mIP-SC s) were isolated and recorded in the presence of TTX (1 mu M), kynurenic ac id (1 mM), and bicuculline (10 mu M) and were found to be sensitive to stry chnine (1 mM). The mIPSCs were first observed in 26-29 h postfertilization (hpf) embryos at a very low frequency of similar to 0.04 Hz, which increase d to similar to 0.5 Hz by 30-40 hpf, and was similar to 10 Hz in newly hatc hed (>50 hpf) larvae, indicating an accelerated increase in synaptic activi ty. At all embryonic stages, the amplitudes of the mIPSCs were variable but their means were similar (similar to 100 pA), suggesting rapid formation o f the postsynaptic matrix. The 20-80% rise times of mIPSCs in embryos were longer (0.6-1.2 ms) than in larvae (similar to 0.3 ms), likely due to slowe r diffusion of glycine at the younger, immature synapses. The mIPSCs decaye d with biexponential (tau(off1) and tau(off2)) time courses with a half-wid th in 26-29 hpf embryos that was longer and more variable than in older emb ryos and larvae. In 26- to 29-hpf embryos, tau(off1) was similar to 15 ms a nd tau(off2) was similar to 60 ms, representing events of intermediate dura tion; but occasionally long mIPSCs were observed in some cells where tau(of f1) was similar to 40 ms and tau(off2) was similar to 160 ms. In 30-40 hpf embryos, the events were faster, with tau(off1) similar to 9 ms and tau(off 2) similar to 40 ms, and in larvae, events declined somewhat further to tau (off1) similar to 4 ms and tau(off2) similar to 30 ms. Point-per-point ampl itude histograms of the decay of synaptic events at all stages resulted in the detection of similar single channel conductances estimated as similar t o 45 pS, indicating the presence of heteromeric glycine receptors (GlyRs) f rom the onset of synaptogenesis. Fast-flow (1 ms) application of a saturati ng concentration of glycine (3-10 mM) to outside-out patches obtained at 26 -29 hpf revealed GlyR currents that decayed biexponentially with time const ants resembling the values found for intermediate and long mIPSCs; by 30-40 hpf, the GlyR currents resembled fast mIPSCs. These observations indicate that channel kinetics limited the mIPSC duration. Our data suggest that gly cinergic mIPSCs result from the activation of a mixture of fast and slow Gl yR subtypes, the properties and proportion of which determine the decay of the synaptic events in the embryos.