Development of Spontaneous Glycinergic Currents in the Mauthner Neuron of the Zebrafish Embryo

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Abstract
We used whole cell and outside-out patch-clamp techniques with reticulospinal Mauthner neurons of zebrafish embryos to investigate the developmental changes in the properties of glycinergic synaptic currents in vivo from the onset of synaptogenesis. Miniature inhibitory postsynaptic currents (mIPSCs) were isolated and recorded in the presence of TTX (1 μM), kynurenic acid (1 mM), and bicuculline (10 μM) and were found to be sensitive to strychnine (1 μM). The mIPSCs were first observed in 26–29 h postfertilization (hpf) embryos at a very low frequency of ∼0.04 Hz, which increased to ∼0.5 Hz by 30–40 hpf, and was ∼10 Hz in newly hatched (>50 hpf) larvae, indicating an accelerated increase in synaptic activity. At all embryonic stages, the amplitudes of the mIPSCs were variable but their means were similar (∼100 pA), suggesting rapid formation of the postsynaptic matrix. The 20–80% rise times of mIPSCs in embryos were longer (0.6–1.2 ms) than in larvae (∼0.3 ms), likely due to slower diffusion of glycine at the younger, immature synapses. The mIPSCs decayed with biexponential (τoff1and τoff2) time courses with a half-width in 26–29 hpf embryos that was longer and more variable than in older embryos and larvae. In 26- to 29-hpf embryos, τoff1was ∼15 ms and τoff2was ∼60 ms, representing events of intermediate duration; but occasionally long mIPSCs were observed in some cells where τoff1was ∼40 ms and τoff2was ∼160 ms. In 30–40 hpf embryos, the events were faster, with τoff1∼ 9 ms and τoff2∼ 40 ms, and in larvae, events declined somewhat further to τoff1∼ 4 ms and τoff2∼ 30 ms. Point-per-point amplitude histograms of the decay of synaptic events at all stages resulted in the detection of similar single channel conductances estimated as ∼45 pS, indicating the presence of heteromeric glycine receptors (GlyRs) from the onset of synaptogenesis. Fast-flow (1 ms) application of a saturating concentration of glycine (3–10 mM) to outside-out patches obtained at 26–29 hpf revealed GlyR currents that decayed biexponentially with time constants 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 glycinergic mIPSCs result from the activation of a mixture of fast and slow GlyR subtypes, the properties and proportion of which determine the decay of the synaptic events in the embryos.