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

Declan W. Ali,1 Pierre Drapeau,1 and Pascal Legendre2

 1Center for Research in Neuroscience, McGill University; and Montreal General Hospital Research Institute, Montreal, Quebec H3G 1A4, Canada; and  2Institut des Neurosciences, Centre National de la Recherche Scientifique Unité Mixte de Recherche C7624, Universite Pierre et Marie Curie, 75252 Paris Cedex 05, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ali, Declan W., Pierre Drapeau, and Pascal Legendre. Development of Spontaneous Glycinergic Currents in the Mauthner Neuron of the Zebrafish Embryo. J. Neurophysiol. 84: 1726-1736, 2000. 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 (tau off1 and tau off2) 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, tau off1 was ~15 ms and tau off2 was ~60 ms, representing events of intermediate duration; but occasionally long mIPSCs were observed in some cells where tau off1 was ~40 ms and tau off2 was ~160 ms. In 30-40 hpf embryos, the events were faster, with tau off1 ~ 9 ms and tau off2 ~ 40 ms, and in larvae, events declined somewhat further to tau off1 ~ 4 ms and tau off2 ~ 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glycine is a major inhibitory neurotransmitter in mammals (Aprison 1990), where glycine receptors (GlyRs) are pentameric with various isoforms (alpha 1, alpha 2, alpha 3, alpha 4, and beta ) whose properties depend on the subunit composition (Bormann et al. 1993). GlyR subtypes are expressed in the mammalian brain at different locations and with different developmental patterns (for review, see Vannier and Triller 1997). The maturation of glycinergic synapses in mammals is characterized by a progressive decrease in the duration of glycinergic inhibitory responses, and it has been proposed that this reflects a progressive molecular switching of alpha 2 for alpha 1 subunits (Krupp et al. 1994; Singer et al. 1998; Takahashi et al. 1992), although alpha 2 subunit can still be present at adult age (Racca et al. 1998) and synaptic responses mediated solely by alpha 2 GlyRs have not been detected.

It is still unclear whether this progressive decrease in the time course of glycinergic responses during synaptic maturation is due to the delayed appearance of synaptic endings with faster postsynaptic GlyRs or reflects an increase in the proportion of mature over immature GlyR subtypes at individual synapses. As growth cones can release neurotransmitter (Allen and Brown 1996; Young and Poo 1983), it is possible that changes in the time course of glycinergic responses in the developing brain also reflect maturation of the presynaptic endings.

Previous developmental studies of embryonic GlyRs examined the channel kinetics in stationary conditions (Takahashi et al. 1992), which do not allow the prediction of their behavior when activated in nonequilibrium conditions, as is the case during synaptic vesicular release (Frerking and Wilson 1996). In the zebrafish larva, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) have a complex time course with two decay components (Legendre 1998, 1999). This was also observed in postnatal and juvenile rat brain stem motoneurons (Singer and Berger 1999; Singer et al. 1998). This time course is due to a complex behavior of transiently activated GlyRs, and it cannot simply be related to the mean open duration of the channels (Legendre 1998). As nonstationary analyses in mammals have been limited mainly to postnatal development (Singer and Berger 1999; Singer et al. 1998), it remains to be determined how the properties of glycinergic synapses change from their initial appearance during the earliest period of synaptogenesis in the embryo.

Zebrafish embryos provide a means to address these developmental issues by an in vivo analysis of mIPSC amplitude fluctuations, time course variability, and single GlyR channel kinetics. Zebrafish embryos develop quickly, showing limited motor activity at <26 h postfertilization (hpf) and first showing a touch-induced escape response from 26-29 hpf that matured by 30-40 hpf prior to hatching of the larvae at >50 hpf (Saint-Amant and Drapeau 1998). This suggests that neural networks involved in this motor behavior develop progressively before birth. Glycine is also the major inhibitory transmitter in the hindbrain of teleosts (Faber and Korn 1982; Legendre and Korn 1994). It is particularly involved in the control of the Mauthner-cell (M-cell) activity mediating the escape response in zebrafish (Eaton et al. 1977) and other teleosts (Faber and Korn 1978).

Morphological development of synapses was previously studied in the hindbrain of the zebrafish embryo (Kimmel et al. 1990). In 2-day-old larvae the glycinergic synapses seen by immuno-electron microscopy on the M-cell body, and growing dendrites appear to be morphologically mature although endings with an immature appearance are also observed (Triller et al. 1997). Immature endings (with unidentified transmitter) are observed on the M cell at early embryonic stages (Kimmel et al. 1990), but their functional significance remains unknown.

To determine the developmental process of glycinergic synapse development and maturation on embryonic zebrafish Mauthner cells, we analyzed in vivo the changes in mIPSC-like properties in embryos at the four stages described in the preceding text, i.e., at <26, 26-29, 30-40, and >50 hpf. The mIPSCs were compared with the kinetics of GlyR currents evoked in outside-out patches with a fast-flow application system (Legendre 1998).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation

Embryos and larvae were raised at 28.5°C and were obtained from a zebrafish colony maintained according to established procedures (Westerfield 1995). Ages are given as hours postfertilization (hpf). The animals hatch at ~50 hpf. All procedures were carried out in compliance with the guidelines stipulated by the Canadian Council for Animal Care and McGill University. Embryos and newly hatched larvae were anesthetized in 0.02% tricaine (MS-222; Sigma Chemical, St. Louis, MO) in recording solution prior to and during dissections (see following text). The preparation was then moved to the recording setup, and the chamber was continuously perfused with an oxygenated (95% O2-5% CO2) recording solution that contained D-tubocurarine (15 µM) to paralyze the preparations but that lacked tricaine. The recording solution contained (in mM) 145 NaCl, 1.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2 PO4, and 10 glucose, 330 mosM, pH 7.4. For high KCl solution, 10.5 NaCl was replaced with KCl to maintain osmolarity.

Whole cell recordings

The ventral surface of the hindbrain was exposed after removing rostral regions, as described by Drapeau et al. (1999), and the embryos were pinned on their side through the notochord being careful to avoid damaging the neighboring spinal cord. Standard whole cell recordings (Hamill et al. 1981) were performed in vivo within 5-15 min after dissection. Preparations were placed on the stage of a Nikon Optiphot microscope and continuously perfused with recording solution. Mauthner neurons were easily identified with a ×40 immersion lens using Hoffman modulation optics. Patch-clamp electrodes (3-5 MOmega ) were pulled from thin-walled, Kimax-51 borosilicate glass and filled with a CsCl intracellular solution composed of (in mM) 135 CsCl, 2 MgCl2, 10 HEPES, 10 EGTA, and 4 Na3 ATP, 290 mosM, pH 7.2. All drugs were dissolved in the recording solution and applied by bath perfusion while the preparation was continuously perfused. Tetrodotoxin (1 µM), strychnine (1 µM), and D-tubocurarine (15 µM) were obtained from Sigma Chemical while kynurenic acid (KYN; 1 mM) and bicuculline (10 µM) were obtained from RBI.

Whole cell currents were recorded using an Axopatch 1A amplifier (Axon Instruments) and were low-pass filtered at 5-10 kHz and digitized at 20-25 kHz. The series resistance was monitored during whole cell recordings by applying 2-mV hyperpolarizing pulses. Neurons that had input resistances <100 MOmega were discarded. Synaptic activity was monitored using pClamp 6 software (Axon Instruments), and the detection of synaptic events was performed with Axograph software version 3.56 (Axon Instruments) on data re-filtered at 2 kHz using a second-order Chebychev digital filter. Some traces were filtered at 1 kHz for display purposes only. Histograms and scatter plots were constructed using Kaleidagraph 3.1 software. Detailed analysis of their decay time courses were performed by averaging 25 isolated single events (filter cutoff frequency of 5 kHz). The decay phase was fitted with a sum of exponential curves, and the presence of one or more exponential components was tested by comparing the sum of squared errors of the fits (Clements and Westbrook 1991; Legendre 1998). The equation with two exponential components always resulted in a significantly better fit (Legendre 1998).

Single-channel recordings

Standard outside-out recordings from the membrane of the Mauthner cell in an isolated ex vivo hindbrain preparation (Legendre and Korn 1994) were achieved under direct visualization with differential interference contrast (Nikon Optiphot microscope). The preparation was continuously perfused at room temperature (20°C) with oxygenated solution. Patch-clamp electrodes (10-15 MOmega ) were pulled from thick-wall borosilicate glass and were filled with CsCl intracellular solution. Outside-out single-channel currents were evoked using a fast-flow operating system (Franke et al. 1987; Lester et al. 1990) as previously described (Legendre 1998). Briefly, drugs were dissolved in a control solution containing (in mM) 145 NaCl; 1.5 KCl; 2 CaCl2; 1 MgCl2; 10 glucose, and 10 HEPES; pH 7.2; 330 mosM. Control and drug solutions were gravity fed into the two channels of a thin-wall glass theta tube (2 mm OD, Hilgenberg, Germany) with a tip diameter of 200 µm. The solution exchange was performed by rapidly moving the solution interface across the tip of the patch pipette, using a piezo-electric translator (Physic Instrument, model P245.30). Concentration steps of glycine were applied every 5-10 s. The exchange time was determined after rupturing the seal by monitoring the change in the liquid junction evoked by the application of a 10% diluted control solution on the open tip of the patch pipette. The exchange time on the patch (~0.1 ms) was estimated using the method published by Maconochie and Knight (1989) (see Legendre 1998 for detailed analysis).

Patch currents were recorded using an Axopatch 1D amplifier (Axon Instruments), filtered at 10 kHz using a eight-pole Bessel filter (Frequency Devices), sampled at 50 kHz and stored on computer using pClamp 6 software (Axon Instruments). Outside-out currents were analyzed off-line with Axograph 3.56 software (Axon Instruments). The time courses of outside-out responses were analyzed by averaging 10-15 single events, and the decay phase of the responses was fitted with a sum of exponential curves using simplex algorithm. As for mIPSCs analysis, the presence of more than one exponential component was tested by comparing the sum of the squared errors of the fits (Legendre 1998).

Glycine-gated channel behavior was simulated using a chemical kinetic modeling program (Axograph 3.56, Axon Instruments). This program first calculated the evolution of the number of channels in each given state for given rate constants (Clements and Westbrook 1991). The kinetic model used to generate the theoretical trace was previously proposed to explain the behavior of GlyRs at mature glycinergic synapse (Legendre 1998). Results are presented as means ± SD throughout unless otherwise noted.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recordings were made from Mauthner neurons in embryos corresponding to four developmental stages (see INTRODUCTION): <26, 26-29, 30-40, and >50 hpf. The results for the stages at which glycinergic mIPSCs-like currents were observed are summarized in Table 1. With CsCl electrodes the input resistance was consistently high in embryos at all stages (1-2 GOmega ) and fell (to 0.3-0.5 GOmega ) in larvae. In all recordings, we applied 1 mM kynurenic acid to block glutamatergic events.


                              
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Table 1. Summary of mIPSC and outside-out patch data

Properties of mIPSCs

Whole cell recordings from Mauthner cells of embryos <26 hpf revealed a general lack of spontaneous synaptic activity (not shown). In some cases, we observed very small strychnine-sensitive events that were composed of two to three superimposed single-channel openings (46 ± 6 pS; n = 9 events from 3 cells), suggesting the presence of rare and very immature synapses but excluding the presence of GlyRs containing a subunit variant like that of the mammalian embryonic alpha  2 subunit (alpha 2*), which confers little strychnine sensitivity to GlyRs (Kushe et al. 1990). Spontaneous synaptic currents appeared as early as 26-29 hpf, 2 h after presynaptic nerve endings on embryonic Mauthner cells have been detected (24 hpf) (Kimmel et al. 1990). To examine in isolation mIPSC-like evoked by the release of single quanta, we perfused the preparation with 1 µM TTX to block action potential discharge, 1 mM kynurenic acid to block ionotropic glutamate receptors, and 10 µM bicuculline to block GABAA receptors. We detected mIPSCs with rapid rise times and clear exponential decays in embryos >= 26 hpf. Spontaneous, glycinergic mIPSC-like activity occurred with a very low frequency (Fig. 1A; 0.04 ± 0.02 Hz; n = 4) in 26-29 hpf embryos. To determine if depolarization of presynaptic terminals could enhance the frequency of glycinergic mIPSCs at this stage and improve our ability to detect events, we applied an elevated (12 mM) KCl solution. Under these conditions, the frequency of mIPSCs increased by an order of magnitude to 1.4 ± 0.3 Hz (n = 4 cells; Fig. 1B). In older embryos (30-40 hpf), the average frequency of mIPSCs was also an order of magnitude greater (0.45 ± 0.46 Hz; n = 6 cells) than in 26-29 hpf embryos in normal (1.5 mM) KCl. All mIPSCs disappeared in the presence of 1 µM strychnine (Fig. 1C) and returned during washout of strychnine (not shown), confirming that the miniature events we detected were effectively due to the activation of strychnine-sensitive GlyRs. The frequency of mIPSCs increases by another order of magnitude to ~10 Hz in newly hatched larvae (Legendre and Korn 1994; Table 1), presumably reflecting an accelerated period of synaptogenesis prior to hatching.



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Fig. 1. Isolation of miniature inhibitory currents in 26- to 29-hours postfertilization (hpf) embryos. A: a very low frequency of miniature synaptic currents was detected during bath application of 1 µM TTX, 1 mM kynurenic acid (KYN), and 10 µM bicuculline. B: effect of a high KCl solution (12 mM KCl) on mIPSCs. C: the bath application of 1 µM strychnine blocks miniature inhibitory postsynaptic currents (mIPSCs).

The amplitude of most events in 26-29 hpf embryos was clustered between 10 and 150 pA, with a distribution that was more or less Gaussian (Fig. 2A) around a mean of 75 ± 28 pA (n = 6 cells, Fig. 2D and Table 1). Occasionally (in 2 of 6 preparations), larger amplitude events (>200 pA) were recorded in 26- to 29-hpf embryos, but these were infrequent and represented <5% of the events. Amplitude distributions of mIPSCs were skewed toward larger values (means 100-150 pA) in animals >30 hpf (Fig. 2, B and C, and Table 1) as larger events (>200 pA) became more frequent. This is in the range of mIPSC amplitude fluctuations previously described in larvae (Legendre and Korn 1994). The coefficient of variation (CV = SD/mean) was large and increased at later stages, ranging from 0.6-0.9 (Fig. 2E).



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Fig. 2. Changes in the amplitude and rise time of mIPSCs with the age of the embryos. A: example of an amplitude distribution of mIPSCs recorded in 26- 29 hpf embryos (n = 747; binwidth = 10 pA). B: amplitude distribution of mIPSCs recorded from 30- 40 hpf embryo (n = 326; binwidth = 10 pA). C: amplitude distribution of mIPSCs recorded in a >50 hpf larva (n = 1013; binwidth = 10 pA). D: changes in the mean amplitude distribution of mIPSCs as a function of the age of the animal. E: changes in the coefficient of variation of the mIPSC amplitudes as a function of the age of the animal. F: changes in the 20-80% rise time of the mIPSCs with the age of the embryo. Note that both the mean amplitude and the coefficient of variation increase while the 20-80% rise time decreases with the age of the animals (26-29 hpf, n = 8; 30-40 hpf, n = 7; >50 hpf, n = 8). Values are means ± SE.

To obtain a rough estimate of the decay duration, we measured the half-width of each event and plotted the cumulative distributions for eight recordings from 26-29 hpf embryos in Fig. 3A and for seven recordings from 30- to 40-hpf embryos in Fig. 3B. The mean half-widths were over twice as long at the younger stage and were briefest in the larvae (Fig. 3C). Although the CVs were comparable (~0.5) at all stages (Fig. 3D), it is clear from the distributions in Fig. 3, A and B, that the half-widths were far more variable for the younger (26-29 hpf) embryos. A detailed analysis of the individual mIPSCs in 26- 29 hpf embryos revealed two separate classes of events (Fig. 4). The vast majority of events in six recordings decayed biexponentially (see Table 1) within 100-200 ms (Fig. 4A, 1 and 2) with each time constant (mean values for all events: tau off1 = 15 ± 6 ms; tau off2 = 63 ± 24 ms) fitting about half of the events. We term these events "intermediate" in duration. The 20-80% rise times of intermediate mIPSCs were slower (mean = 0.57 ± 0.16) than in larvae (mean ~ 0.3 ms; Fig. 2F and Table 1) (Legendre 1998).



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Fig. 3. Cumulative histograms of the half-width of mIPSCs recorded in zebrafish embryos. A: the histogram is highly variable from cell to cell in the 26-29 hpf embryos (n = 8). B: the half-width is smaller in 30- to 40-hpf embryo and is far less variable from cell to cell (n = 7). C: histogram showing the change of the half-width of mIPSCs with the age of the animal. Note that half-width decreases dramatically in 30-40 hpf embryos. D: this change in the half-width is not associated with a change in the coefficient of variation of the cumulative distributions (26-29 hpf embryos, n = 8; 30-40 hpf, n = 7; >50 hpf, n = 8). Values are means ± SE.



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Fig. 4. Individual mIPSCs recorded from 26-29 hpf embryos. A1: superimposed individual events with an intermediate decay phase duration. A2: averaged traces of the events shown in A1 (tau off1 = 15.9 ms and tau off2 = 64 ms). B1: superimposed long events that lasted for several hundred ms. B2: averaged trace of the long events showing the biexponential decay (tau off1 = 52.9 ms and tau off2 = 258.7 ms). Note the different time scales for the traces shown in A and B.

We also detected events that were several-fold longer in duration (Fig. 4B, 1 and 2) and decayed over several hundred milliseconds that we refer to as "slow" events. Slow events were present in highly variable proportions. They were not detected in two of the six recordings. In two other recordings with intermediate events, the slow events represented <1 and ~25% of synaptic-like activity and resulted in noticeably skewed distributions in some of the plots in Fig. 3A. In two other experiments. they accounted for most of the events (i.e., these lacked intermediate events). Each time constant described half of the events but with tau off1 = 38 ± 11 ms (57%) and tau off2 = 157 ± 75 ms. The rise times of the long events were also slow with a mean of 1.2 ± 0.6 ms. The slow rise times of intermediate and long events were not due to errors in space clamping (embryonic Mauthner neurons are smaller and less complex than larval cells) as they were not correlated to the half-widths (not shown) and well-resolved single-channel openings could occasionally be observed during the decay phase in cells with a low noise level (see following text).

In older (30-40 hpf) embryos and in larvae, the mIPSCs remained biexponential but decayed more rapidly with what are referred to as "fast" events. In 30-40 hpf embryos, tau off1 = 9.4 ± 2.9 ms (74 ± 20%; n = 7 cells) and tau off2 = 40 ± 14 ms (Fig. 5A, 1 and 2). A similar proportion of each component was observed in newly hatched larvae but the decay times were briefer (Fig. 5B, 1 and 2), as previously observed (Legendre 1998): tau off1 = 4.2 ± 0.9 ms (73%), and tau off2 = 29 ± 7 ms (n = 10 cells). The rise times of fast events were still slower than in larvae (~0.3 ms) (Legendre 1998), with a mean = 0.74 ± 0.13 ms.



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Fig. 5. Individual mIPSCs recorded from 30-40 hpf embryos. A1: superimposed fast mIPSCs recorded from a 30-40 hpf embryo. A2: the average trace of the events shown in A1 had a biexponential deactivation phase decay with tau off1 = 8.8 ms and tau off2 = 32.3 ms. B1: example of fast mIPSCs recorded from a newly hatched larva. D: the average trace of these fast events had 2 decay components with tau off1 = 5.2 ms and tau off2 = 23.8 ms.

Outside-out patch currents from young (<30 hpf) embryos

To determine whether or not the variability in the duration of mIPSCs reflects intrinsic kinetics of the postsynaptic receptors, we analyzed the time course of outside-out responses evoked by short (1 ms) applications of a saturating concentration (3-10 mM) of glycine (see METHODS). We did not record any channel activity in response to application of glycine in almost all (45 out of 46 cells) of the outside-out patches from embryos <26 hpf. In one patch from a 20-hpf embryo, we were able to obtain a short recording of long-lasting, glycine-gated channel openings (2-3 GlyRs) with a main conductance state of 48 pS (data not shown).

Short applications of glycine onto outside-out patches obtained from somewhat older (26-29 hpf) embryos evoked single-channel activity in the majority of the cases (14 of 16). In these patches, the outside-out current greatly exceeded the duration of glycine application (Fig. 6). As observed for mIPSCs, three types of outside-out responses (fast, intermediate, and slow) were recorded. Slow responses were observed in 4 of the 14 patches (Fig. 6A) and were similar to what we observed for slow mIPSCs. They were characterized by a long decay phase that was well fitted by a sum of two exponential curves (Table 1) with decay time constants tau off1 = 80 ± 16 ms (n = 4) and tau off2 = 306 ± 57 ms (n = 4). The first decay component represented the majority (68 ± 3%, n = 4) of the total current. The 20-80% rise time of these outside-out transient currents was close to 0.2 ms (10 mM glycine; 0.19 ± 0.03 ms; n = 4). It became maximal for glycine concentrations 3 mM, as observed on larvae where the mature form of GlyRs is dominant (Legendre 1998). In eight other patches, the application (1 ms) of 3-10 mM glycine evoked a transient current of intermediate duration (Fig. 6B) similar to that observed for intermediate mIPSCs. These responses evoked by 1-ms application of 10 mM glycine had a similar 20-80% rise time (0.16 ± 0.02 ms, n = 8) and a biexponential deactivation phase (Fig. 7B and Table 1). The decay time constants were tau off1 = 17 ± 3 ms (n = 8) and tau off2 = 146 ± 44 ms (n = 8), respectively, with the first deactivation component representing the majority (74 ± 9%) of the total current (n = 8). We also observed in two patches fast outside-out currents resembling those described in larvae (Legendre 1998) where mature inhibitory synapses were described (Triller et al. 1997). These outside-out currents evoked by a 10 mM glycine application (1-ms pulse duration; not shown) had a 20-80% rise time of 0.18 and 0.17 ms and a biexponential decay with time constants tau off1 = 6.4 and 4.3 ms and tau off2 = 81 and 36 ms, with the first component representing the majority (65 and 75%) of the peak current.



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Fig. 6. Glycine-evoked current in outside-out patches obtained from a 26- 29 hpf embryo. A: example of an averaged patch current (20 epochs) with a long deactivation phase evoked by a brief pulse (1 ms) of 10 mM glycine. B: another example of an averaged outside-out current (15 epochs) but with an intermediate decay evoked in an embryo at the same stage as in A. Note that the decay of the long and the intermediate glycine-evoked transient currents can be fit by the sum of two exponential curves with the indicated parameters. C and D: point-per-point amplitude histograms of the deactivation phase (-20 to 0 pA) of the transient currents (inset) with long (C) and intermediate (D) decay phases (binwidth = 0.05 pA). Note that the single-channel currents measured at the end of the deactivation phases of both long-lasting and intermediate lasting outside-out currents have similar amplitudes with a main conductance state of 43 pS (C) and 47 pS (D), respectively (Vh = -50 mV; reversal potential = 0 mV). The 1st 5 peaks of the histogram shown in C have a mean value of -2.2, -4.5, -6.1, -8.2, and -10.1 pA while the 4 distinguishable peaks of the distribution shown in D have a mean value of -2.4, -5.0, -7.4, and -10.0 pA.



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Fig. 7. Single-channel conductances underlying the deactivation phases of embryonic (26-29 hpf) mIPSCs. A1: a single glycinergic mIPSC with a long decay time. A2: point-per-point histogram of currents in the deactivation phase with a principal peak at -2.2 pA corresponding to a single-channel conductance of 44 pS (binwidth = 0.05 pA). B1: a single glycinergic mIPSC of intermediate duration. B2: point-per-point amplitude histogram of currents in the deactivation phase with a principal peak at -2.4 pA, which corresponds to a singe channel conductance of 48 pS (binwidth = 0.2 pA).

As single openings can be resolved during the decay phase of outside-out currents evoked by 1-ms application of glycine and mIPSCs in young embryos, we compared measured single-channel currents underlying outside-out responses and mIPSCs in 26-29 hpf embryos to determine if extrasynaptic and synaptic immature GlyRs share the same main conductance level. The main conductance state of GlyRs underlying outside-out currents was measured from the decay phase of a single transient current evoked by the application of 10 mM glycine, using point-per-point amplitude histograms (Fig. 6, C and D, and Table 1). Outside-out currents of long duration (Fig. 6C) could be seen to result from the activation of GlyRs with a main conductance state of 46 ± 4 pS (n = 3). Similarly, responses of intermediate duration (Fig. 6D) reflected the activation of GlyRs with a main conductance state of 48 ± 3 pS (n = 7). We also selected examples of long and intermediate mIPSCs in which single-channel openings could be resolved. The long events were examined in one cell (29 hpf) in which they represented ~25% of the events. In this cell many of the long events decayed with resolvable step-like currents (Fig. 7A1). Point-per-point histograms of slow events (Fig. 7A2) revealed a principal peak current at -2.4 ± 0.3 pA (n = 10), which, at the holding potential of -50 mV and calculated reversal potential for chloride of 0 mV, represents a single-channel conductance of 48 ± 6 pS. Deactivation phase analyses of intermediate embryonic events (Fig. 7B, 1 and 2) revealed a first peak current at -2.2 ± 0.2 pA (n = 11), which represents a conductance of 44 ± 4 pS. Two types of GlyRs were previously described on the zebrafish M-cell larva according to their main conductance state and their subconductance levels. One GlyR subtype was characterized by a single conductance level of 44-48 pS while the other subtype had three subconductance states with a main conductance level of 84-88 pS (Legendre 1997; Legendre and Korn 1994). Similarly, they can also be identified during concentration jump experiments as their corresponding main conductance states occurred during the deactivation phase of the outside-out responses (Legendre 1998). Our present results therefore suggest that GlyRs with the higher main state conductance (84-88 pS) described previously in newly hatched larvae (Legendre 1997, 1998; Legendre and Korn 1994) were extremely rare or presumably not present at the earlier stages examined here. In larvae GlyRs with a conductance state of 84-88 pS were also rarely observed as they were recorded in only 18% of the patches tested (Legendre and Korn 1994).

Outside-out patch currents from older (30-40 hpf) embryos

As observed for mIPSCs, outside-out currents evoked in patches from M cells of 30-40 hpf embryos had faster decays than those observed in 26-29 hpf embryos. The deactivation phase of the outside-out currents evoked by a 1-ms application of 10 mM glycine (Fig. 8A) could be fit with the sum of two exponential curves (Fig. 8B) with decay time constants tau off1 = 8.6 ± 4.3 ms (n = 19; 64 ± 14% of the total current) and tau off2 = 66 ± 38 ms. These outside-out currents had a fast 20-80% rise time of 0.16 ± 0.05 ms, which is not significantly different from that measured in 26-29 hpf embryos (unpaired t-test) and similar to that previously described in larvae (Legendre 1998) (see Table 1). The main conductance state of GlyRs underlying fast outside-out currents was measured from the decay phase of single transient currents (Fig. 8C) as described in the preceding text. Outside-out currents resulted from the activation of GlyRs with a main conductance state of 47 ± 3 pS (n = 19), similar to that observed in the 26-29 hpf embryos (Table 1).



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Fig. 8. Glycine-evoked current in an outside-out patch obtained from 30- 40 hpf embryos. A: example of 10 superimposed responses to 1-ms application of 10 mM glycine. Note the variability of the deactivation phase duration. B: an averaged patch current (15 epochs, same patch as in A). Note that the decay of the glycine-evoked transient current can be fit by the sum of 2 exponential curves with the indicated parameters. C: point-per-point amplitude histograms of the deactivation phase (-20 to 0 pA) of the transient current shown in the insert (binwidth = 0.05 pA). Note that the single-channel current measured at the end of the deactivation phases represents a main conductance state of 49 pS (Vh = -50 mV; reversal potential = 0 mV). The 1st 5 peaks of the histogram shown in C have a mean value of -2.5, -5.0, -7.5, -10.2, and -12.4 pA.

The mean rise time value of these outside-out currents was several-fold faster than those measured for mIPSCs recorded in 30-40 hpf embryos (Table 1). The mIPSCs rise time in larvae were also more than twice as fast as those in embryos and were proposed to correspond to a peak concentration of glycine of >= 1 mM released at mature synapses (Legendre 1998). The slower mIPSC rise times observed in embryos can therefore be due to slow release of a lower concentration of glycine, slower intrinsic kinetics of the postsynaptic receptors (Singer and Berger 1999), or both. To discriminate between these possibilities, we measured the rise times of outside-out responses evoked by 1 ms application of 0.3, 1, or 10 mM glycine (Fig. 9A). Responses evoked by 1 mM glycine application had a 20-80% rise time of 0.34 ± 0.05 ms (n = 9; Fig. 9, B and C), similar to that described in larvae (Legendre 1998) (see Table 1). As expected under nonstationary conditions (Legendre 1998), decreasing the concentration of glycine to 0.3 mM had only a slight effect on the 20-80% rise time as it was 0.36 ± 0.03 ms (n = 9; Fig. 9, B and C). It is therefore unlikely that the slow mIPSC rise time was due to slow activation kinetics of the GlyRs at immature synapses. A 1 ms application pulse is a relatively long pulse with respect to the clearance speed of the transmitter from the synaptic clef at mature synapses (Clements 1996). It is therefore possible that the slow mIPSC rise times we observe in embryos are due to slow release of glycine at immature synapses.



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Fig. 9. Changes in the 20-80% rise time of outside-out responses of a patch from a 30-40 hpf embryo with different glycine concentrations. A: averaged traces of currents evoked by 1-ms applications of 0.3, 1, and 10 mM glycine. Traces are the average of 15-30 epochs. B: onset of the responses shown in A on an expanded time scale. Note that the 20-80% rise time increased when the glycine concentration was decreased. C: averaged 20-80% rise times at different glycine concentrations (0.3 mM, 7 patches; 1 mM, 9 patches; 10 mM, 19 patches). Data are presented as means ± SD.

Simulation of outside-out currents

Transient currents evoked by 3 mM glycine in patches from larval Mauthner cells decay with fast time constants (tau off1 ~ 4 ms and tau off2 ~ 30 ms, Table 1) and effectively mimic mIPSCs occurring from mature inhibitory synapses. This reflects the activation of GlyRs with alpha 1/beta -like subunit composition (Legendre 1997, 1998). In the zebrafish embryo, mIPSCs and evoked outside-out currents have long deactivation phases (as long as tau off1 ~ 40 ms and tau off2 ~ 150 ms). These are likely to be due to the activation of a separate GlyR subtype. It is therefore possible that the mIPSCs and the glycine-evoked outside-out currents of an intermediate duration reflect postsynaptic receptor aggregates with a variable proportion of slow, embryonic and fast, mature GlyR subtypes. To test this hypothesis, we simulated outside-out currents with a decay phase with time constant values corresponding to the fast and slow glycinergic events. The kinetic model we used to simulate mIPSCs was previously proposed to predict fast GlyR behavior in the zebrafish larval M-cell (Legendre 1998, 1999).

The mean open time appeared longer for GlyRs underlying slow glycinergic events (Fig. 6, C and D) than for fast GlyRs (Fig. 8A). As a first approximation, slow synaptic-like events were mimicked using the same GlyRs Markov model but with the closing rate constant values reduced by 5- to 10-fold. These slower closing rate constants gave theoretical outside-out currents with decay time constants tau off1 = 38.5 ms (70.1%) and tau off2 = 154.2 ms, which are similar to our experimental data (Fig. 10A and Table 1). Simulation of outside-out currents with various relative proportions of the two GlyRs (Fig. 10A) gave responses of intermediate duration with a time course similar to outside-out experimental data when the relative proportion of fast GlyR was 50-80% (Fig. 10B). The decay time constants increased significantly when the proportion of immature GlyRs was ~10%. The deactivation phase of these theoretical responses can be fit by the sum of two exponential curves, although the beginning of the slower component was more poorly fit (Fig. 10A) as was also observed for some experimental outside-out data (Fig. 8B). Our model predicts that the increase in the relative proportion of fast GlyRs should progressively reduce the duration of the decay time constant (Fig. 10C). It also predicts a small increase in the relative amplitude of the fast deactivation component (Fig. 10D) when the proportion of fast GlyRs was increased above 30%. These general predictions are consistent with our experimental findings (Table 1).



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Fig. 10. Simulated mixtures of fast and slow glycinergic synaptic-like events, according to Legendre (1998). A: when a 1 ms application of 10 mM glycine was simulated it generated a theoretical transient response with 2 decay time constants tau off1 = 4.3 ms (78.3%) and tau off2 = 30.3 ms, as previously described (Legendre 1998). Slow glycine events were generate by decreasing the closing rate constants. The other traces are for simulations of glycine-evoked currents (1 ms step application of 3 mM glycine) showing variation in the deactivation phase duration with changes in the proportion of channels having short or long mean open times (see RESULTS). Each trace represents a 10% relative increase in the number of channels with long mean open times incremented from 0 to 100%. Note that the decay of the 2 simulated traces due the activation of 1 class of GlyRs can be well fitted by biexponential curves. B: example of a simulated glycine-evoked current with 60% of fast glycine-gated channel and 40% of slow glycine-gated channels. Note that the decay phase can be fitted by biexponential curves. C: plot of the fast (tau off1) and the slow (tau off2) deactivation time constants vs. the proportion of glycine-gated channels with long mean open times. D: plot of the relative amplitude of the 2 decay components vs. the proportion of glycine-gated channels with long mean open times.


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Glycinergic inhibitory synapses developed quickly in the zebrafish embryo, being first detected by 26 hpf and maturing by the time the larvae hatch 1 day later (at ~50 hpf). The main characteristics of mIPSCs recorded, for the first time, from early embryos in vivo were a reduced amplitude fluctuation, a slower rise time, a long decay phase for some events, and a large variability in the duration of the miniature glycinergic synaptic-like events. Slow outside-out responses and mIPSCs (tau off1 = 38 ms; Table 1) were never described before. To the contrary, outside-out responses and synaptic-like events of intermediate duration (see Table 1) recorded at 26-29 hpf resemble the outside-out response obtained from perinatal motoneurons in the rat brain stem (Singer and Berger 1999). They however differ from miniature synaptic-like events recorded in perinatal motoneurons (Singer et al. 1998). Responses observed at an intermediate stage (30-40 hpf) were more homogenous in duration and resembled outside-out responses recorded from brain stem juvenile motoneurons (Singer and Berger 1999).

GlyR kinetics control the deactivation time course of mIPSC during development

At all developmental stages tested, the deactivation phase of glycinergic outside-out responses and mIPSCs have a complex time course with a doubly exponential decay. This was first described in zebrafish larva (Legendre 1998). This apparently contrasts with previously analyses of mIPSCs time course on M cell of adult goldfish (Korn and Faber 1990) using intracellular recordings. However, goldfish GlyR channel kinetics are still unknown, and the frequency of occurrence of mIPSCs in the goldfish M-cell is high (33-127 Hz) (Mintz and Korn 1991), which precludes a good resolution of a biphasic decay phase with a possible slow decay component. In the adult zebrafish (Hatta and Korn 1998), the decay phase of glycinergic mIPSCs was not analyzed and the presence of one or two decay components remains unknown, although evoked responses decayed monoexponentially.

Differences in mIPSC duration between early zebrafish embryos and larvae are likely to reflect a transition from slow to fast zebrafish GlyRs. Our data on deactivation time constants of outside-out currents and mIPSCs suggested that two types of synaptically activated GlyRs are present in the Mauthner cell of the zebrafish embryo. Changes in glycinergic mIPSC duration during development was previously proposed to be related to a switch in alpha 2 and alpha 1 GlyRs subunits mRNAs in mammals (Singer et al. 1998; Takahashi et al. 1992). However, alpha 2 mRNA can still be detected in adult (Racca et al. 1998), and due to the lack of specific antibody, it is not possible to demonstrate whether or not alpha 2, alpha 1/beta are present and in what proportion at glycinergic synapses throughout their development. Nevertheless, there is indirect evidence to suppose that long, immature synaptic-like events may be due to the activation of alpha 2 GlyRs (Takahashi et al. 1992). In a previous study of larval GlyRs, channels were observed to re-open in bursts before the dissociation of glycine due to the complex kinetics, resembling mammalian alpha 1/beta heteromeric GlyRs (Legendre 1998). In mammals, the alpha 2 subunits confer GlyR activity with bursts of long openings (Takahashi et al. 1992; Virginio and Cherubini 1996). We speculate that slow mIPSCs-like may represent synaptically driven alpha 2-like GlyRs while fast events are likely to reflect alpha 1-like GlyR activation (Legendre 1998). A GlyR subunit has been cloned recently from the adult zebrafish brain (alpha Z1) (David-Watine et al. 1999; Fucile et al. 1999) and has structural and functional features resembling more the mammalian alpha 1 subunit than other subunit types. However, the molecular properties of embryonic zebrafish GlyRs, such as the presence of alpha 2 subunits, remain unknown.

Most embryonic mIPSCs fell between the extremes discussed in the preceding text as their time courses were described by biexponential functions of intermediate duration. According to our model, a mixture of fast and slow GlyRs could yield intermediate events similar in duration to those observed experimentally. As a switch in alpha  subunit expression has been described in other preparations (for review, see Vannier and Triller 1997), and so this mechanism would seem feasible. An alternative explanation would be that individual GlyRs with several types of alpha  subunit exist and undergo a similar transition in relative content at a molecular (rather than synaptic) level since for example alpha 1/alpha 2 heteromers can be obtain in transfected cells (Kushe et al. 1993). Another alternative is that a single type of GlyR (which maintains its subunit composition) changes its kinetic properties progressively during development by a posttranslational maturation such as protein phosphorylation. However, GlyR current decay was stable in isolated patches, and others have found that substrates, which promote phosphorylation have no effect on GlyR current decay times (Singer and Berger 1999). We therefore conclude that a variable switch from purely slow to mostly fast GlyRs may occur during development of zebrafish glycinergic synapses.

Change in glycinergic synapse efficacy during development

The rise time of mIPSCs can give a rough estimation of the peak concentration of agonist reaching the postsynaptic receptors and/or its clearance speed after the release of one vesicle (Clements 1996; Jones and Westbrook 1995; Legendre 1998; but see Frerking and Wilson 1996). The slower 20-80% rise times of embryonic mIPSCs-like (1 ms), which were not due to slower activation of embryonic GlyRs, cannot be accounted for by a reduction in glycine concentration only, as at concentrations <0.3 mM, it becomes relatively constant at <0.5 ms even for a 1 ms pulse (Legendre 1998). Thus this result suggests that at embryonic synaptic-like contacts, the peak concentration of glycine appears to be reached over a relatively prolonged period of >= 1 ms with a slow clearance. This can be explained by a large cleft between the M-cell membrane and axonal endings at immature synapses and/or the presence of growth cone-like terminals.

Such mIPSCs-like with a slow 20-80% rise time were not observed in >50-hpf M-cell larva where most of the detected synapses on M cells appeared to be mature (Triller et al. 1997). These observations apparently contrast with previous morphological studies performed on zebrafish M-cell during development (Kimmel et al. 1981). Effectively, Kimmel et al. (1981) observed mature synaptic contact on 96-hpf zebrafish only. This apparent discrepancy can be explained by a difference in zebrafish development between the two studies. Effectively, the study of Triller et al. (1997) and ours were performed in zebrafish larva raised at the standard temperature of 28.5°C (Westerfield 1995). To the contrary, the older study of Kimmel et al. (1981) was performed on embryos raised at 25.5°C. Accordingly, the animals hatch at ~4 days in the Kimmel et al. (1981) study, whereas in the present study and that of Triller et al. (1997), the analyses were performed on animals hatching at 2 days. This creates difficulties in comparing these two morphological studies; but using the hatching period as a reference, it appears that synaptic contacts do not fully mature until the animals hatch (Kimmel et al. 1981). In the early embryo, many of the boutons are not well differentiated, and they give rise to immature chemical synaptic junctions some with a defined synaptic cleft (Kimmel et al. 1981). This could give mIPSCs with slow rise times.

The amplitude of mIPSCs was remarkably constant in embryos, from the onset of synaptogenesis at 26 hpf until 40 hpf, and became more variable in larvae while their frequency increased dramatically with the age of the animal. Changes in mIPSC amplitude and CV during development were also observed in mammals (Singer and Berger 1999); but in the zebrafish larvae, it was associated with an increase in their frequency. This is likely to be partly due to the increase in the number of mature synaptic boutons on the developing M-cell (Kimmel et al. 1981). It can also partly reflect the maturation of the presynaptic terminal as the release probability, and the number of neurotransmitter molecules released by growth cones and immature synapses in vitro is very low and variable (Allen and Brown 1996; Young and Poo 1983). Amplitude fluctuations of mIPSCs recorded in embryos might thus reflect a variation in the number of glycine molecules released, but we cannot exclude that it also reflects a variation in the number of postsynaptic receptors. Accordingly, the largest events could reflect release of more glycine or denser postsynaptic receptors.

Physiological significance

In general, inhibitory synaptic events with long decay times produce sustained changes in the membrane potential resulting in an increased efficacy of tonic synaptic activity. As in other vertebrate embryos, zebrafish embryonic neurons have elevated chloride levels such that glycinergic events are depolarizing (Saint-Amant and Drapeau 2000). Thus GlyRs or GABAA receptors induce cell membrane depolarization; this allows calcium influx into immature neurons (Boehm et al. 1997; Singer et al. 1998; Wang et al. 1994) as a prelude to synapse maturation (Kirsch and Betz 1998) and may thus play key roles during synaptogenesis (Lévi et al. 1998). By overcoming the effect of the long electrotonic time constant of the embryonic zebrafish neurons, long glycinergic synaptic events may also more efficiently evoke cell depolarization and hence promote maturation of inhibitory synapses. Moreover inhibitory synaptic events have other functions in addition to their control of the resting membrane potential as they can prevent the electrotonic propagation of excitatory events by decreasing the input resistance. In the embryo, long glycinergic synaptic events may compensate for the small number of glycinergic synapses on the embryonic Mauthner cell, as reflected by the low frequency of events we detected, allowing long-lasting, shunting inhibition. When inhibitory synaptic contacts become more numerous or their release probability increases, a switch between GlyR subtypes may then take place. In conclusion, morpho-functional maturation of the pre- and postsynaptic elements occurs during synaptogenesis, leading to a functional diversity of the glycinergic inhibitory synapses aposed to the mature M-cell. This diversity appears to be mainly related to the morpho-functional heterogeneity of the postsynaptic GlyRs clusters.


    ACKNOWLEDGMENTS

We thank Dr. Richard Miles for valuable help and discussions.

This work was supported by grants from the National Sciences and Engineering Research Council of Canada (P. Drapeau) and Institut National de la Santé et de la Recherche Médicale and Association Contre les Miopathies of France (P. Legendre), a Fonds de la Recherche en Santé du Québec Canada/INSERM France Collaborative Exchange (P. Legendre and P. Drapeau) and an NSERC Canada Fellowship (D. W. Ali).

Present address of D. W. Ali: Program in Brain and Behavior, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.


    FOOTNOTES

Address for reprint requests: P. Legendre, Institut des Neurosciences, Bat B. 6eme étage, boite 8, Université Pierre et Marie Curie, 7 Quai Saint Bernard, 75252 Paris Cedex 05, France (E-mail: pascal.legendre{at}snv.jussieu.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4 February 2000; accepted in final form 14 June 2000.


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