High-Affinity Zinc Potentiation of Inhibitory Postsynaptic Glycinergic Currents in the Zebrafish Hindbrain

Hiroshi Suwa,1 Louis Saint-Amant,3 Antoine Triller,2 Pierre Drapeau,3 and Pascal Legendre1

 1Institut des Neurosciences, Université Pierre et Marie Curie, 75252 Paris Cedex 05;  2Institut National de la Santé et de la Recherche Médicale U 497, Ecole Normale Superieure, 75005 Paris, France; and  3Center for Research in Neuroscience, McGill University, and Montreal General Hospital Research Institute, Montreal, Quebec H3G 1A4, Canada


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Suwa, Hiroshi, Louis Saint-Amant, Antoine Triller, Pierre Drapeau, and Pascal Legendre. High-Affinity Zinc Potentiation of Inhibitory Postsynaptic Glycinergic Currents in the Zebrafish Hindbrain. J. Neurophysiol. 85: 912-925, 2001. Zinc has been reported to potentiate glycine receptors (GlyR), but the physiological significance of this observation has been put in doubt by the relatively high values of the EC50, 0.5-1 µM, since such concentrations may not be attained in the synaptic cleft of glycinergic synapses. We have re-evaluated this observation in the frame of the hypothesis that contaminant heavy metals present in usual solutions may have lead to underestimate the affinity of the zinc binding site, and therefore to underestimate the potential physiological role of zinc. Using chelators either to complex heavy metals or to apply zinc at controlled concentrations, we have examined the action of zinc on GlyR kinetics in outside-out patches from 50-h-old zebrafish Mauthner cells. Chelating contaminating heavy metals with tricine or N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreased the duration of the currents evoked by glycine, confirming that traces of heavy metals alter the GlyR response in control conditions. Using tricine- (10 mM) buffered zinc solution, we then showed that zinc increases the amplitude of outside-out responses evoked by 0.1-0.5 mM glycine with an EC50 of 15 nM. In contrast zinc had no effect on the amplitude of currents evoked by a saturating concentration (3-10 mM) of glycine. This suggests that zinc enhances GlyR apparent affinity for glycine. The study of the effects of zinc on the kinetics of the response indicates that this increase of apparent affinity is due to a decrease of the glycine dissociation rate constant. We then analyzed the effects of zinc on postsynaptic GlyRs in whole cell recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Chelation of contaminant heavy metals decreased the amplitude and the duration of the mIPSCs; inverse effects were observed by adding zinc in buffered solutions containing nanomolar free zinc concentrations. Zinc plus tricine or tricine alone did not change the coefficient of variation (approx 0.85) of the mIPSC amplitude distributions. These results suggest that postsynaptic GlyRs are not saturated after the release of one vesicle.


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Zinc ions modulate several voltage-gated and ligand-gated postsynaptic receptors, and these effects may have functional significance, in particular because zinc can be released with glutamate into the synaptic cleft of excitatory synapses (see for review Smart et al. 1994). Zinc has opposite effects on glycine-receptor (GlyR) activity depending on its concentration (see for review Betz et al. 1999). At low concentrations (<= 10 µM) zinc enhances chloride currents evoked by glycine while at concentrations higher than 10 µM, zinc depresses GlyR activation. While the binding sites for zinc on GlyRs are not yet localized, the site involved in potentiation seems to involve histidine residues on the NH2 terminals of the alpha subunits (Harvey et al. 1999). Zinc has recently been shown to enhance GlyR currents via allosteric interactions with the agonist gating process that depend in part on an apparent increase in glycine binding affinity (Laube et al. 2000; Lynch et al. 1998), but the details of these effects are still not fully understood.

At many excitatory synapses, zinc is co-released with glutamate, and it has been estimated that the extracellular zinc concentration may reach several micromolar (Smart et al. 1994). However, it is not clear that glycinergic terminals release zinc, and therefore it is generally assumed that at glycinergic synapses zinc comes from adjacent nonglycinergic synaptic terminals and that its concentration must be much lower. For this reason the apparent affinity of the GlyR zinc facilitation described previously (0.5-1 µM) seems not high enough for zinc to actually have a modulatory effect (Bloomenthal et al. 1994; Doi et al. 1999; Han et al. 1999; Harvey et al. 1999; Laube et al. 1995, 2000; Lynch et al. 1998; Tapia and Aguayo 1998; Zhang and Berg 1995). GlyRs would need to possess a high-affinity zinc binding site, as described for the N-methyl-D-aspartate (NMDA) NR1-NR2A receptor subtype (EC50 = 15 nM) using heavy metal chelators (Paoletti et al. 1997).

In previous studies we observed a large variability in amplitude of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded from the Mauthner cell (M-cell) of zebrafish larva (Legendre 1998; Legendre and Korn 1994). The amplitude of mIPSCs whether GABAergic, glycinergic, or mixed is generally highly variable with a coefficient of variation of 0.7-0.8 (Lim et al. 1999; Oleskevich et al. 1999). This can be due to a large variation in the number of neurotransmitter molecules released per vesicle at inhibitory synapses (Frerking et al. 1995), which implies that saturation of postsynaptic receptors does not occur. If saturation of postsynaptic receptors does not occur at glycinergic synapses, the application of an allosteric potentiator known to enhance the apparent affinity for glycine, such as zinc, will increase the amplitude of glycinergic mIPSCs. It was also proposed that variable postsynaptic receptor cluster size might also explain mIPSC amplitude variations (Alvarez et al. 1997; Lim et al. 1999; Nusser et al. 1997; Oleskevich et al. 1999). In mammalian spinal cord and brain stem neurons (Lim et al. 1999; Oleskevich et al. 1999) the amplitude of mIPSCs and the surface areas of GlyRs vary in parallel, but this does not demonstrate that postsynaptic receptors are fully saturated by the release of a single vesicle as shown for GABA synapses (Nusser et al. 1997).

In the present study we asked whether traces of heavy metals in extracellular solutions might enhance GlyR activity in outside-out patches obtained from the M-cell (Legendre 1998). The effects of zinc on GlyRs kinetics were then characterized, using fastflow application techniques. To estimate the occupancy of postsynaptic GlyRs after the release of a vesicle, we analyzed the effect of zinc on mIPSCs amplitude. We then compared mIPSCs amplitude fluctuations with morphologically determined values for the surface area of GlyR clusters.

Portions of these data have appeared in a meeting abstract (Suwa and Legendre 1999).


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Isolated intact brain preparation

The isolated hindbrain of the zebrafish was prepared as previously described (Legendre and Korn 1994). Briefly, the hindbrains of 50- to 60-h-old larvae were dissected out and glued to a coverslip using a plasma-thrombin embedding procedure. Before starting experiments, brain preparations were stored for 15 min in an oxygenated (100% O2) bathing solution containing (in mM) 145 NaCl, 1.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.3) with the osmolarity adjusted to 300 mOsm.

Whole cell and outside-out patch-clamp recordings

Standard whole cell and outside-out recordings (Hamill et al. 1981) were achieved under direct visualization (Nikon Optiphot microscope) on the Mauthner cell (M-cell) as described previously (Legendre 1998). The isolated hindbrain was continuously perfused at room temperature (20°C) with an oxygenated bathing solution (2 ml/min) in the recording chamber (0.5 ml). Patch-clamp electrodes were pulled from thick-wall borosilicate glass with a resistance of 1-2 MOmega (whole cell) or 10-15 MOmega (outside-out). They were fire-polished and filled with (in mM) 135 CsCl, 2 MgCl2, 4 Na3ATP, 10 EGTA, and 10 HEPES, pH 7.2. The osmolarity was adjusted to 290 mOsm to enhance seal formation. During whole cell recordings the series resistance (4-10 mOmega ) was monitored using 2 mV pulses, and it was 50-80% compensated. Outside-out patches were obtained by slowly retracting the pipettes. The resistance of outside-out patches ranged from 2 to 10 GOmega . Currents were recorded using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA), filtered at 10 kHz, and stored using a digital tape recorder (DAT DTR 1201, SONY).

Drug delivery

During whole cell recordings, drugs were applied to the bath via a single pipette (400 µm diameter) positioned 500 µm away from the preparation. To allow the application of different solutions, the pipette was connected to six different reservoirs using a manifold. Applications were controlled using solenoid valves. Drugs were dissolved in the bathing solution.

Outside-out single-channel currents were evoked using a fastflow application system (Franke et al. 1987; Legendre 1998; Lester et al. 1990). Drugs were dissolved in the same solution used during whole cell recordings. Control and drug solutions were gravity fed into the two channels of a glass theta tube (2 mm OD; Hilgenberg, Germany). The tip diameter was 200 µm. One lumen of the tube was connected to reservoirs filled with control solutions. The solution exchange was performed by rapidly moving the solution interface across the tip of the patch pipette, using a piezo-electric translator (Physics Instrument, model P245.30). Concentration steps of glycine lasting 1-200 ms were applied every 5-10 s. The exchange time (0.08-0.1 ms) was determined after rupturing the seal by monitoring the change in the liquid junction potential evoked by the application of a control solution diluted by 10% to the open tip of the patch pipette (Legendre 1998). The theoretical limit to the speed of solution change was estimated using the method published by Maconochie and Knight (1989; see Legendre 1998 for detailed analysis). With a patch electrode resistance >10 MOmega , the estimated absolute exchange time was found to be close to the <= 0.1 ms.

Buffered zinc solutions

Two heavy metals chelators were used: N-tris(hydroxymethyl) methylglycine (tricine, Sigma) and N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN, Sigma) (Paoletti et al. 1997). Ten micromolar TPEN and 1-10 mM tricine applications were tested on glycine-evoked outside-out current to determine whether traces of heavy metals in the bath solution can influence GlyRs activity. Since TPEN is membrane permeant (Arslan et al. 1985), experiments to examine the potential effects of extracellular zinc on mIPSCs fluctuations were performed with tricine only.

The concentration-dependent effects of zinc were analyzed using buffered zinc solutions with concentrations of zinc added to the tricine solution assuming a Kd for zinc of 10-5 M (Paoletti et al. 1997). Adding 0.026, 0.26, 2.6, 26, and 254 µM zinc to 10 mM tricine was estimated to result in free zinc concentrations of 0.1, 1, 10, 100, and 1,000 nM, respectively. TPEN was not used to buffer zinc solutions since its affinity is too high (<10-10 M) (Paoletti et al. 1997).

Analysis of miniature postsynaptic current

Spontaneous synaptic activity was digitized off-line with a Macintosh G3 computer at 25 kHz using Axograph 4.1 software (Axon Instruments). The detection of synaptic events was automatically performed using a typical event as a template function (Clements and Bekkers 1997). Event detection was optimized using a signal-to-noise ratio with a threshold value equal to 3 times the SD noise. The 20-80% rise time, the half-amplitude duration, and the amplitude of each mIPSC was analyzed on isolated detected events using Axograph 4.1. The deactivation time course of mIPSCs was analyzed by averaging 25 isolated single events using Axograph 4.1 (filter cutoff frequency: 10 kHz). The decay phase of mIPSCs was always best fitted with the sum of two exponential curves (Legendre 1998).

Outside-out patch current analysis

Single-channel currents were filtered at 10 kHz using an eight-pole Bessel filter (Frequency Devices), sampled at 50 kHz (Digidata 1200 interface, Axon Instruments), stored on an IBM AT-compatible computer using Pclamp software 6.03 (Axon Instruments), and analyzed off-line with Axograph 4.2 software (Axon Instruments).

The time course of outside-out responses was analyzed by averaging 10-20 single events using Axograph 4.2 (Axon Instruments; filter cutoff frequency: 10 kHz). The activation time constants of currents evoked by a low concentration of glycine (0.1 mM) applied for 100-200 ms were estimated by fitting the onset of the responses with a sum of two sigmoidal curves (Legendre 1998) (filter cutoff frequency of 10 kHz). The first 250 ms of the decay phase of the outside-out currents, evoked by a brief (1 ms) application of 0.5-10 mM glycine, was fitted with a sum of exponential curves. 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, 1999).

Kinetic modeling programs

A previously developed kinetic model of M-cell glycine receptors was used (Legendre 1998). Glycine evoked currents were analyzed off-line using chemical kinetic modeling programs (Axograph 4.2, Axon Instruments) on a G4 Macintosh to adjust the rate constants to obtain theoretical responses of similar time course to the experimental data. This program first calculated the evolution of the number of channels in each given state for given rate constants. Simulated traces were obtained using Axogaph 4.2 software by varying the dissociation rate constant with zinc concentrations according to the experimental measurements. Patch currents represent the average of 10 or more traces as specified in the figure legends or the text.

Confocal microscopy

GlyR immunohistological staining and confocal image analysis were performed as previously described (Triller et al. 1990). Isolated brains were fixed by immersion for 15 min in a 4% paraformaldehyde in 0.1 M phosphate-buffered solution (pH 7.4), passed through NH4Cl, dehydrated and rehydrated in graded (0, 1, 25, and 50%) ethanol. They were incubated overnight with mouse monoclonal antibody GlyR 4a (Pfeiffer et al. 1984) (1:50 in 0.01 M phosphate-buffered solution). Antibody binding was visualized using CY3-coupled rabbit anti mouse (dilution 1:200).

Morphological data were visualized with confocal scanning microscopy (Molecular Dynamics confocal microscope). The pixel size was 0.1 µm (×60 oil objective N.A. 1.4) and a z step of 0.29 m to allow an oversampling. Data were acquired through the whole M-cell. A Gaussian filter (3 × 3 × 3) was used to eliminate random noise and to increase contrast. Clusters of GlyR immunoreactivity were analyzed using National Institutes of Health Image (Bethesda, MD). The surface areas were measured for immunofluorescent clusters on every five digitized optical sections to avoid multiple measurements of the same cluster. Labeling each cluster with a marker checked this point. GlyR clusters, in 50-h-old zebrafish larvae, predominate on the dorsal surface of the M-cell body. For the illustration (Fig. 9A), the top surface of the M-cell was reconstructed using a projection of the stack of optical sections (Adobe Photoshop software 3.0).

Results are presented as means ± SD unless otherwise noted.


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Traces of heavy metals and zinc modify GlyR kinetics

Using fastflow application techniques, we examined the effects of TPEN, tricine, and zinc on the activation and deactivation kinetics of membrane patch currents of native heteromeric alpha /beta GlyRs (Legendre 1997) from the Mauthner cell (M-cell). GlyR resembling homomeric alpha 1 and heteromeric alpha 1/beta mammalian receptors have been functionally characterized on the zebrafish M-cell (Legendre 1997). These two receptors are segregated from each other and can be discriminated by their mean conductance states. In the present study, we focused our analysis on heteromeric-like GlyRs. These GlyRs possess a single conductance state of 40-46 pS and represents more than 80% of the postsynaptic receptors encountered on the M-cell (Legendre 1997, 1998).

In the absence of tricine in the recording solution, zinc modified the time course and amplitude of outside-out responses to glycine only at concentrations of >= 0.1 µM only (data not shown). But traces of heavy metals may mask a high-affinity zinc binding site on GlyRs, as previously observed for some NMDA receptor subtypes (Paoletti et al. 1997). We tested this hypothesis by comparing the effects of the heavy metal chelators tricine and TPEN on transient outside-out currents evoked by 1 ms applications of a saturating concentration of glycine (3-10 mM). In the absence of TPEN or tricine, 1 ms application of 3-10 mM glycine evoked a transient current with fast and slow decay components with time constants tau offfast = 5.6 ± 0.4 ms and tau offslow = 39.1 ± 4.6 ms (mean ± SD, n = 16). The slow decay component represented 39 ± 4.4% of the total current (n = 16). Both TPEN and tricine shortened ouside-out the currents decay time constants but had little effect on the peak amplitude of the responses (5.6 ± 0.4% decrease; n = 16; Fig. 3, A and B). Ten micromolar TPEN significantly decreased tau offfast and tau offslow to 4.5 ± 0.5 ms and 26 ± 6.7 ms, respectively (paired t-test, P = 0.01). It also decreased the relative amplitude of the slow decay component to 31.2 ± 5.2% of the total current (n = 6). In the presence of 10 mM tricine (paired t-test, P = 0.01) the deactivation time constants tau offfast and tau offslow were 3.7 ± 0.9 ms and 28.4 ± 6.1 ms, respectively, while the relative amplitude of the slow decay component decreased to 31.6 ± 7.1% (n = 10).

Nanomolar concentrations of zinc increased GlyRs reponses

The effects of tricine and TPEN may result from their chelation of heavy metals. If so, adding a known concentration of zinc in the presence of tricine (see METHODS) should induce opposite effects to those evoked by tricine or TPEN. Figure 1C shows that this was the case. The addition of zinc (free concentration: 100 nM) to the tricine solution significantly slowed the decay of transient outside-out currents evoked by 1 ms applications of 3-10 mM glycine. In the presence of 100 nM free zinc, tau offfast and tau offslow were 8.9 ± 1.5 ms and 75.6 ± 22.8 ms (paired t-test, P = 0.01), respectively, while the slow decay component represented 46 ± 9% of the total current (n = 10), for applications of a saturating concentration of glycine (3-10 mM).



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Fig. 1. Traces of heavy metals enhance the duration of glycine-evoked responses in outside-out patches. To ensure stationary conditions, tricine and N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) were present before, during, and after glycine applications. The decay phase of the outside-out currents, evoked by a brief (1 ms) application of glycine, was fitted with a sum of two exponential curves. The values of the two decay time constants, tau offfast and tau offslow, are given in the figure. The relative amplitude of the slow decay component is given in % of the total current, below the corresponding tau off. A and B: heavy metal chelators TPEN (10 µM) and tricine (10 mM) decrease the decay time constants of outside-out responses evoked by 1-ms application of a saturating concentration of glycine (3 mM). The decay phase of the glycine-evoked response was fitted with the sum of two exponential curves with fast and slow decay time constants. C: in contrast, addition of zinc to the tricine solution (100 nM free zinc; see METHODS) only increased the duration of 10 mM glycine-evoked outside-out current.

The same zinc applications on the same patch had little effect on either the peak response amplitude (5.1 ± 0.4% increase; n = 6 patches; Fig. 1C) or the 20-80% rise time (0.14 ± 0.03 ms in control, 0.13 ± 0.04 ms in the presence of 100 nM free zinc; n = 6; paired t-test, P = 0.1).

Effects of zinc on the amplitude of outside-out currents evoked by glycine became significant (paired t-test, P = 0.01) when nonsaturating concentrations were examined (1 ms applications of 0.5 mM Gly). In these conditions, the addition of 100 nM free zinc to the tricine solution evoked a 32 ± 10% increase (n = 4) in amplitude (Fig. 2A, arrow). The 20-80% rise time of these glycine-evoked responses was not significantly changed. It was 0.49 ± 0.11 ms in the absence of zinc and 0.48 ± 0.09 ms in the presence of 100 nM free zinc (n = 4; paired t-test, P = 0.1).



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Fig. 2. Zinc increases the deactivation time of glycine-evoked chloride current in a concentration-dependent manner. A: responses of patch to 1-ms step application of 0.5 mM glycine in the presence of 10 mM tricine and in the presence of 10 nM and 100 nM free zinc. Note that zinc application increased the amplitude (arrows) and the duration of the evoked-outside currents. B: the fast decay time constant (tau offfast) increased with the zinc concentration. C: the slow decay time constant (tau offslow) also increased with the zinc concentration, and its relative amplitude was increased (D). Each point is the average of 23 (tricine), 9 (1 nM zinc), 8 (10 nM zinc), 10 (100 nM zinc), and 9 (1 µM zinc) measurements.

Zinc increased the decay time of the responses evoked by 1 ms application of 0.5-10 mM glycine in a concentration-dependent way. In the presence of tricine alone (n = 23), the decay time constants tau offfast and tau offslow were 3.7 ± 0.6 ms and 27.3 ± 7.7 ms, respectively (Fig. 2, B and C). Increasing free zinc concentrations from 1 nM (n = 9) to 1 µM (n = 9) increased tau offfast from 4.2 ± 1.04 ms to 9.7 ± 2.4 ms (Fig. 2B) and tau offslow from 31.8 ± 7.5 ms (1 nM free zinc) to 87.6 ± 34.4 ms (1 µM free zinc; Fig. 2C; paired t-test, P = 0.01). Zinc also significantly increased the relative amplitude of tau offslow in a concentration-dependent manner (paired t-test, P = 0.01). For example, the relative amplitude of tau offslow was 28 ± 8% in the presence of tricine alone (n = 23) and was increased to 42 ± 6% in the presence of 10 nM free zinc (n = 9) and to 62 ± 9% in the presence of 1 µM free zinc (n = 9; Fig. 2D).

The apparent affinity of GlyRs for zinc was determined by analyzing the effect of increasing free zinc concentrations on the amplitude of outside-out responses evoked by 0.1 mM glycine applications. The duration of the glycine application was adjusted to obtain steady-state responses (Fig. 3A). Zinc was present before, during, and after glycine application. Two concentrations of free zinc were usually tested with each patch, and response amplitudes were normalized to those obtained in the absence of added zinc. As shown in Fig. 3B, increasing free zinc concentration increased the amplitude of the glycine-evoked outside-out currents. Threshold effects of free zinc were observed at concentrations as low as 1 nM (8 ± 3.5% increased; n = 9). A maximum effect was obtained for concentrations of free zinc >= 1 µM (Fig. 3B). One micromolar free zinc evoked a 65 ± 20% increase in outside-out currents (n = 13) while 10 µM free zinc evoked an 66 ± 15% increase (n = 5). The maximum increase of approx 65% seen at free zinc concentration >= 1 µM is similar to values obtained in mammalian cell cultures (Tapia and Aguayo 1998), and in transfected cells (Harvey et al. 1999; Lynch et al. 1998).



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Fig. 3. Zinc has a high-affinity effect on 0.1 mM glycine-evoked responses. A: an outside-out response evoked by 100 ms application of 0.1 mM glycine in the presence of tricine and 10 and 100 nM zinc. Each trace is the average of 10 outside-out responses. B: concentration-response curve of zinc effects on glycine-evoked current amplitude. Responses evoked by tricine are used as the control responses. Each point is the average of 5-12 measurements. Experimental data were fitted with a single isotherm function with a EC50 of 15 nM and a Hill coefficient of 0.8. C: concentration-response plot of the amplitude of responses to glycine in the presence of tricine () and in the presence of 100 nM zinc (open circle ). Each point is the average of 4-9 measurements. Data obtained in the presence of tricine and zinc were fitted with a single isotherm (see RESULTS).

The zinc EC50 was obtained by fitting the data using a single binding isotherm of the form
<IT>I</IT><IT>=</IT><IT>I</IT><SUB><IT>max</IT></SUB><IT>/</IT>[<IT>1+</IT>(<IT>EC<SUB>50</SUB>/</IT>[<IT>zinc</IT>])<SUP><IT>h</IT></SUP>]
where I is the % increase of the outside-out current, Imax is the maximum %increase, EC50 is the free zinc concentration [zinc] producing 50% of the maximum increase, and h is the Hill coefficient. This fit produced a Hill coefficient of 0.8 and an EC50 of 15 nM (Fig. 3B). A Hill coefficient close to 1 suggests that the potentiation involves the binding of one zinc molecule as previously proposed (Lynch et al. 1998). These results also suggest that GlyRs possess a high-affinity allosteric binding site for zinc, similar to that observed in studies on NMDA receptors (Paoletti et al. 1997).

Since zinc increases the amplitude of responses to low concentrations of glycine, we examined its effect on the glycine EC50. We compared the concentration-response curves for glycine obtained in the presence of 100 nM free zinc and in the absence of added zinc (10 mM tricine). A concentration of 100 nM free zinc was used to avoid distortions due to inhibitory effects mediated at low-affinity zinc binding sites (Lynch et al. 1998). Three to four glycine concentrations were usually tested with each patch. The duration of glycine concentration steps was adjusted to ensure that steady-state amplitudes were measured. The concentration-response curves in the absence or in the presence of zinc were obtained for seven and nine experiments, respectively. The normalized data were fitted using a single isotherm function of the form
<IT>I</IT><IT>/</IT><IT>I</IT><SUB><IT>max</IT></SUB><IT>=1/</IT>[<IT>1+</IT>(<IT>EC<SUB>50</SUB>/</IT>[<IT>glycine</IT>])<SUP><IT>h</IT></SUP>]
where I/Imax is the normalized current amplitude, EC50 is the glycine concentration [glycine] producing 50% of the maximum current amplitude, and h is the Hill coefficient. This fit produced a glycine EC50 of 146 µM with a Hill coefficient of 1.19 in the absence of zinc and an EC50 of 41 µM with a Hill coefficient of 1.21 in the presence of zinc.

Effect of nanomolar concentrations of zinc on GlyR kinetics

An increase in the amplitude and in the deactivation time constants of the glycine-evoked outside-out currents evoked by zinc might in principle reflect either an increase of the fast opening rate constants (beta ) or a decrease of the closing rate constant (alpha ) (Legendre 1998, 1999), but it could also reflect a decrease in the dissociation constant for glycine (Kd = koff/kon), which can increase the number of openings per bursts (decrease in koff) (Legendre 1998, 1999).

However, it is unlikely that zinc causes an increase in beta  since we have shown there was no significant change in the rising phase of the currents evoked by the applicatons of a saturating concentration of glycine (3-10 mM). At these concentrations of glycine the opening rate constant beta  is the main determinant of the rising time of the responses (Legendre 1998). A decrease in the GlyR closing rate constant alpha  was previously proposed to account for the voltage-dependent change in the deactivation phase of glycine-evoked transient current (Legendre 1999). But, in this case, decreasing alpha  resulted in an increase of the two deactivation time constants of the patch currents without a significant change of the relative amplitude of these two components (Legendre 1999). This is not consistent with the observations (Fig. 2) showing that the relative amplitude of the two decay time constants depended on free zinc concentration. The most probable explanation for the zinc effects on GlyR activity is therefore an increase in the affinity of glycine for its binding site.

To check whether modifications in the Kd for glycine can be evoked by zinc, we compared the time course of the activation phase of outside-out responses evoked by the application of 0.1 mM glycine in the absence of zinc and in the presence of 100 nM free zinc. The two rate constants kon and koff effectively determine the time course of the activation phase of responses evoked by the application of a nonsaturating concentrations of glycine (Legendre 1998, 1999). To ensure stationary conditions, tricine or zinc and tricine were present before, during, and after glycine applications. The activation phase of the responses evoked by 0.1 mM glycine is complex, but it was shown that it could be fitted with the sum of two sigmoidal functions (Legendre 1998) of the form
{<IT>a</IT><IT> ∗ </IT>[<IT>1−exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>on1</SUB></IT>)]<SUP><IT>2</IT></SUP>}<IT>+</IT>{<IT>b</IT><IT> ∗ </IT>[<IT>1−exp</IT>(−<IT>t</IT><IT>/&tgr;<SUB>on2</SUB></IT>)]<SUP><IT>2</IT></SUP>}
where a and b are the relative amplitudes of the two components of the activation phase and tau on1 and tau on2, the corresponding activation time constants (Legendre 1998, 1999). Figure 4A shows that zinc accelerated the activation of the responses. The fast component was unaffected by zinc (Fig. 4B), whereas the slow activation phase was selectively decreased (Fig. 4C). In the presence of tricine alone, tau on1 and tau on2 had values of 2.24 ± 0.25 and 9.7 ± 2.8 ms, respectively (n = 8). Addition of 100 nM free zinc did not change tau on1 (tau on1 = 2 ± 0.4 ms; n = 8) but significantly decreased tau on2 values (paired t-test, P = 0.01) to 6.0 ± 1.7 ms (n = 8; Fig. 4, B and C). The relative amplitude of the two components was not changed in the presence of zinc (Fig. 4D). tau on1 had a relative amplitude of 54 ± 10% in the presence of tricine and 57 ± 11% in the presence of 100 nM free zinc (n = 8). These results suggest that zinc does not modify kon since an increase in kon will both increase the two activation time constants and decrease the relative amplitude of the slow component (Legendre 1998). A decrease in the dissociation rate constant koff is therefore more likely to be the major effect of zinc on GlyRs kinetics. It accounts for the increase in the amplitude of the responses evoked by the application of a nonsaturating concentration of glycine, for the slowing of the deactivation phase (Legendre 1998, 1999) and for the selective decrease of the slow activation phase component of responses evoked by 0.1 mM glycine application (Legendre 1999), as also observed.



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Fig. 4. Zinc dependence of the activation time course of outside-out responses evoked by the application of 0.1 mM glycine. A: example of normalized averaged trace currents (n = 10 per trace) evoked by the application of 0.1 mM glycine in the presence of tricine () or in the presence of 100 nM zinc (open circle ). Each 30th data point only is plotted for clarity. The onset of the responses was fitted by the sum of 2 sigmoïdal curves with 2 activation time constants (see RESULTS). B: zinc application does not significantly affect the fast activation time constant (tau on1) of glycine-evoked currents (n = 8). C: in contrast zinc decreases the slow activation time constant (n = 8). D: comparison of the relative amplitude of the fast sigmoidal components in the presence of tricine or in the presence of zinc. Note that the relative amplitude of the fast components is insensitive to 100 nM zinc application (n = 8).

The degree in koff change with zinc was estimated using the Markov model (Fig. 5A), proposed for M-cell heteromeric GlyRs (Legendre 1998, 1999). This estimation was performed comparing our experimental data to simulated patch responses. The GlyR model had two equivalent binding sites and two open states linked to two interconnected doubly liganded closed states (Fig. 5A). This model can predict the biphasic deactivation of the patch current and mIPSCs in the absence of an accumulation of the receptor-channel in a fast desensitized state by assuming that this is a reluctant close state leading to a new open state (Fig. 5A) (Legendre 1998). It also predicts a biphasic rising phase for currents evoked by applications of nonsaturating glycine concentrations (Legendre 1998). The rate constants were first adjusted to obtain simulated outside-out currents with time courses similar to experimental measurements. Simulated traces were then compared with responses evoked by the 1-ms application of 10 mM and 0.1 mM glycine in the presence of tricine and in the presence of zinc. Changes in koff with zinc were estimated using the following equation
<IT>k</IT><SUB>off</SUB> = <IT>k</IT><SUB>offmax</SUB>/(1 + {[(<IT>k</IT><SUB>offmax</SUB> − <IT>k</IT><SUB>offmin</SUB>)/<IT>k</IT><SUB>offmin</SUB>]/[1 + (EC<SUB>50</SUB>/[zinc])<SUP>h</SUP>]})
where koffmax is the maximum dissociation rate constant in the absence of zinc, koffmin is the minimum dissociation rate constant obtained for a saturating concentration of free zinc ([zinc]), EC50 is the free zinc concentration producing 50% of the maximum effect on koff and h is the Hill coefficient.



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Fig. 5. Examples of theoretical responses generated using a GlyR Markov model. A: Markov model reproducing the properties of the glycine-gated channel of the zebrafish hindbrain reticular neurons (Legendre 1998). This model has 2 equivalent agonist binding steps, yielding A + AC and 2AC. The doubly liganded closed state (2AC) provides access to a reluctant closed state 2AC* and each doubly liganded closed state provides access to independent open states (O1 and O2). B: simulated responses to 1 ms applications of 10 mM glycine, showing changes in the deactivation phase when the dissociation rate constant koff was decreased by zinc (1-1,000 nM) applications (see RESULTS). The rate parameters used to generates glycine-evoked responses in the presence of tricine were kon = 5 µM-1 s-1 koff = 2,605 s-1, alpha 1 = 630 s-1, beta 1 = 8,940 s-1, alpha 2 = 1,500 s-1, beta 2 = 3,200 s-1, d = 1,000 s-1 and r = 140 s-1. The model predicts that decreasing koff causes an increase in the 2 decay time constants tau offfast (C) and tau offslow (D) and in the relative amplitude of the slow decay component (E). Theoretical data points in C-E were fitted by a single isotherm of the form: {(max - min)/[1 + (EC50/[zinc])h]}, where [zinc] is the zinc concentration, min is the minimum value, max is the maximum value obtained for a saturating [zinc] and h is the Hill coefficient. F: simulated rising phase of responses to 0.1 mM glycine in the presence of tricine () and in the presence of 100 nM zinc (open circle ).

This model gave a good approximation to our experimental data. Simulated data with time courses similar to experimentally observations were obtained by setting koffmax and koffmin values to 2,300-3,100 s-1 and 220-280 s-1, respectively (see Fig. 5). As shown in Fig. 5 decreasing koff reduced the two decay time constants and increased the relative amplitude of the slow decay phase component. Experimental results of the effects of zinc on the decay phase were best simulated using a zinc EC50 of 15 nM for tau offfast but slightly higher (approx 45 nM) for tau offslow and, a Hill coefficient of 0.8, a koffmax of 2,600 s-1 and koffmax/koffmin approx  11 (Fig. 5, C-E). Our model can also predict that decreasing the Koff value for 100 nM free zinc application should increase the slow activation time constant only of responses to concentration steps of glycine to 0.1 mM (Fig. 5F). As experimentally observed, the relative amplitude of the two rising phase components did not change, but our model predicts a slight increase in the fast rise time constant for higher free zinc concentrations. There was good agreement between the experimental measurements of onset and simulated data with a zinc EC50 of approx 45 nM for tau offslow, a Hill coefficient of 0.8, a koffmax of 2,600 s-1, and a koffmax/koffmin approx  11. This is consistent with values used to analyze the effect of zinc on decay time constants.

Tricine decreases glycinergic mIPSC amplitude and duration

Since nanomolar concentrations of free zinc modify the amplitude and the time course of glycine currents, it is likely that traces of zinc in extracellular space also control the mIPSC time course and amplitude. Data obtained from outside-out patches show that zinc increases the affinity of the glycine binding site but has little effect on GlyR maximum open probability. If all postsynaptic GlyRs are saturated following the release of transmitter from a single vesicle, then changes in free zinc concentration should alter mIPSC duration without affecting their amplitude.

This hypothesis was first tested by analyzing the effects of the heavy metal chelator tricine on glycinergic synaptic activity in records of mIPSCs made in the presence of TTX (1 µM), 2-amino-5-phosphonovaleric acid (APV; 10 µM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM), and bicuculline (20 µM). Ten millimolar tricine was applied for 10 min. The isolated hindbrain preparation was then washed for 10 min to ensure recovery from tricine effects and stationary conditions. mIPSCs were collected for 3 min, 5-6 min after the beginning of the application.

As shown in Fig. 6, 10 mM tricine shifted to the left both mIPSCs amplitude and half-width cumulative distributions. In the 11 cells tested at Vh = -50 mV, the addition of tricine to the bath solution caused a significant 19.7 ± 13.2% decrease in the mean amplitude of mIPSCs and a 20.5 ± 18% reduction in their mean half-width (paired t-test, P = 0.01; Fig. 5, A and C). Depolarizing the cell to +50 mV (Fig. 5, B and D) did not prevent the effects of tricine showing that it does not act to block open GlyRs. Adding tricine to the bath solution decreased averaged mean amplitude by 19 ± 7.5% and the mean duration by 25.3 ± 12.4% (n = 4). These results confirm that traces of heavy metal present in the extracellular medium influence postsynaptic GlyRs occupancy.



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Fig. 6. The heavy metal chelator tricine decreases the amplitude and the duration of glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Glycinergic mIPSCs were recorded in the presence of 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 µM 2-amino-5-phosphonovaleric acid (APV) and 20 µM bicuculline. A and B: cumulative histogram amplitude of mIPSCs recorded in the presence and in the absence of 10 mM tricine at negative (A) and positive (B) holding potentials (Vh). At Vh = -50 mV, 444 events were measured in the absence and 430 in the presence of tricine. At Vh = +50 mV cumulative distributions were calculated for 399 events in the presence and 355 events in the absence of tricine. C and D: effect of tricine on mIPSC half duration recorded at negative and positive Vh. Note that tricine decreases the amplitude and the duration of mIPSCs in a voltage-independent manner. The inset shows averaged traces obtained from 45 consecutive isolated mIPSCs. Data shown in A-D are from the same M-cell.

Nanomolar free zinc increases mIPSC amplitude and duration

We then examined how the application of 10-1,000 nM free zinc concentrations affected the mIPSC time course and amplitude distributions. Data obtained in the presence of zinc were compared with those obtained in the absence of zinc.

mIPSCs were recorded in the "zinc free" solution for 10 min, and then a given free zinc concentration was applied for 10 min. The preparation was then washed for 10 min in the presence of tricine before switching to another free zinc concentration. mIPSCs were collected over a period of 3 min starting 5-6 min after the beginning of a new application. Cumulative amplitude and half-width distributions for mIPSCs recorded before and after zinc application were compared with those collected during control recording conditions to ensure stationarity and full recovery from zinc effects (Fig. 7, C and D).



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Fig. 7. Zinc reversibly increases the amplitude and the duration of mIPSC. A and B: superimposed mIPSCs recorded in the presence of 10 mM tricine (n = 25) and during 100 nM zinc application (n = 25). Note that both amplitude and duration were increased by zinc application. C: cumulative amplitude histograms before (mean, 145.3 ± 128 pA; n = 709), during (mean, 229 ± 192 pA; n = 767) and after (mean, 137.3 ± 120 pA; n = 728) 100 nM zinc application (3-min recordings). Zinc reversibly increased mIPSC amplitude. D: cumulative histogram of mIPSC half-width before (mean, 1.85 ± 0.9 ms), during (mean, 3.97 ± 2.2 ms), and after (mean, 1.97 ± 0.9 ms) zinc application. Zinc also reversibly increased mIPSC half-width. Data shown in A-D are from the same M-cell (Vh = -50 mV).

Zinc reversibly increased mIPSC amplitude and duration (Figs. 7 and 8). Greater than or equal to 10 nM free zinc applications caused a leftward shift of both amplitude (Fig. 7C) and half-width (Fig. 7D) cumulative distributions for mIPSCs. The averaged mean mIPSCs amplitude was 169 ± 47 pA in all cells tested (n = 13) in the absence of tricine or zinc, while it was reduced to 142 ± 38 pA (n = 14) in the presence of 10 mM tricine and reached 182 ± 56 pA in the presence of 10 nM free zinc (n = 7).



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Fig. 8. Summary of tricine and zinc effects on mIPSCs mean amplitude and time course. A: effect of free zinc concentrations on the mean mIPSC amplitude. Zinc significantly increased the mean mIPSC amplitude (paired t-test, P = 0.01). B: effect of free zinc concentration on the coefficient of variation of the mIPSC amplitude histogram. The coefficient of variation (CV) is not significantly modified by zinc application. C: mean mIPSC half-width is significantly increased by zinc application (paired t-test, P = 0.01). D: mean 20-80% rise time of mIPSCs in the presence or in the absence of tricine and in the presence of 10-1,000 nM zinc concentrations. Free zinc concentrations >10 nM significantly increased the 20-80% rise time of mIPSCs (paired t-test, P = 0.01). In the absence of tricine, traces of heavy metals increased the amplitude and the half duration of mIPSC to a extent similar to that observed for <= 10 nM zinc. Each point is the average of 14 (tricine), 7 (10 nM zinc), 12 (100 nM zinc), 8 (1 µM zinc), and 11 (control) measurements. Data in A-D are means ± SE.

There were no further significant changes in the mIPSCs averaged mean amplitude for free zinc concentrations >= 10 nM (paired t-test, P = 0.1). Application of 1 µM free zinc (Fig. 8A) increased the averaged mean mIPSC amplitude to a maximum of 207 ± 54 pA (n = 8; tricine plus zinc). Zinc did not significantly change the coefficient of variation (CV = SD/mean) of mIPSC amplitude distributions (Fig. 8B) in the presence of zinc free solution. The CV was 0.85 ± 0.05 (n = 14) while in the presence of 1 µM free zinc, it was 0.81 ± 0.04 (n = 8; paired t-test, P = 0.1).

The effects of zinc were associated with a small increase in mIPSC frequency. In the presence of tricine alone, mIPSCs averaged frequency was 5.2 ± 1.6 Hz (n = 14). Wash out of tricine did not significantly increase mIPSCs frequency (2.5 ± 4%, n = 11). The frequency was increased by 5.2 ± 3.9% in the presence of 10 nM free zinc (n = 7), 13.3 ± 8.5% with 100 nM free zinc (n = 12) and by 21.8 ± 8.8% increased in the presence of 1 µM free zinc (n = 8).

The half-width of mIPSCs increased with free zinc concentrations greater than or equal to 10 nM (Fig. 8C), as predicted from outside-out patch responses to glycine. The half-width was 2.1 ± 0.6 ms in the absence of zinc (10 mM tricine; n = 14 cells) and 3.9 ± 0.5 ms (n = 8) in the presence of 1 µM free zinc concentration (Fig. 8C).

Increasing the free zinc concentration also increased the 20-80% rise times (20-80% RT) of mIPSCs. In the presence of tricine, 20-80% RT was 0.24 ± 0.03 ms (n = 14), while it was 0.27 ± 0.04 ms (n = 11) in the absence of the heavy metal chelator. In the presence of 10 nM free zinc concentration, it was 0.28 ± 0.02 ms (n = 6). Increasing the free zinc concentration from 100 nM to 1 µM siginificantly increased the 20-80% RT to 0.32 ± 0.05 ms (n = 12) and 0.33 ± 0.05 ms (n = 8), respectively (paired t-test, P = 0.01). This effect, which would not be predicted from our experiments with glycine responses in outside-out patches, might depend on a rapid clearance of glycine at the synaptic cleft (see DISCUSSION).

Comparison of data obtained in the presence of zinc or tricine with those obtained in control conditions permitted an estimate for basal heavy metals concentrations in the extracellular space. As shown in Fig. 8, the effects of zinc on mIPSP amplitude fluctuations and half duration suggest that contamination by heavy metals was equivalent to <= 10 nM free zinc. This is consistent with estimates from outside-out experiments for the concentration of heavy metals in the recording medium.

Nanomolar free zinc concentrations change the occupancy level of postsynaptic GlyRs

In the absence or in the presence of tricine, mIPSC amplitude histograms were skewed with a CV approx 0.84. This variability might result from differences in the postsynaptic cluster size from one synapse to another. Strong correlations have previously been described between the size of gephyrin clusters and fluctuations in mIPSC amplitude (Lim et al. 1999; Oleskevich et al. 1999).

In our experiments zinc enhanced the mIPSC amplitudes without changing the CV of amplitude distributions (Fig. 8B). By increasing the occupancy of postsynaptic GlyRs, zinc might therefore increase the correlation between the distributions for GlyR cluster areas and mIPSC amplitudes. This has been observed for GABA synapses of cerebellar stellate cells in the presence of flurazepam (Nusser et al. 1997). This hypothesis was tested by comparing distributions of mIPSC amplitudes obtained in the presence and in the absence of zinc with the size of GlyR clusters.

GlyR immunoreactivity was examined over the surface of the Mauthner cell (M-cell). The M-cell is easily recognized in 50-h-old zebrafish larva hindbrain (Metcalfe et al. 1986). It is the only large reticular interneuron (approx 10 µm large and approx 100 µm long) located in the fourth rhombomere. In the 13 cells analyzed, punctuate GlyR-IR aggregates of different sizes were observed (Fig. 1A). The number of clusters per M-cell varied from 83 to 254 (178 ± 57; n = 13), but the mean cluster surface did not strongly vary from one M-cell to another. It ranged from 0.14 to 0.18 µm2. The averaged mean surface area was 0.158 ± 0.015 µm2 (n = 13). For our computation, we considered clusters with surfaces in the 0.03-0.8 µm2 range. Some larger clusters (up to 1.7 µm2) were observed but represented only 1.5 ± 1.2% of the total GlyR clusters per cell (0 to 6 clusters in different cells), and it was difficult to determine whether they represented single clusters or were formed by an artifactual fusion of smaller clusters since their shape was usually very complex (see Fig. 9A, inset). In all cells examined, the distribution of GlyR cluster areas was skewed (Fig. 9B). The extent of this variability was relatively constant from cell to cell with a coefficient of variation of 0.86 ± 0.06 (n = 13). Therefore the mean cluster surface area was poorly correlated with the CV (R = 0.11; Fig. 9C).



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Fig. 9. GlyR cluster size varies to a similar extent as mIPSC on the 50-h-old M-cell. A: confocal image showing the variable size of GlyR-ir clusters on the soma (S) and the lateral dendrite (LD). Inset: higher magnification of the boxed region (scale bar: 2 µm). B: histogram of GlyR-IR cluster surface area (µm2) for the neuron shown in A. GlyRs-IR clusters larger than 0.8 mm2 (n = 3) were not included (see RESULTS). C: mean GlyR-ir coefficient of variation (CV = SD/mean) plotted against the cluster size (n = 13 M-cell). Note the poor correlation between these 2 parameters (R = 0.11). D: example of the amplitude distribution of mIPSCs recorded in zebrafish larva M-cell. Note that the skewness of the mIPSC amplitude histogram is similar to that of the distribution of areas of GlyRs-ir clusters. E: mean CV of the amplitude distribution plotted against the amplitude of mIPSCs (n = 13). As observed for GlyR-IR clusters, these 2 parameters were poorly correlated (R = 0.12).

A large variability has been observed in the amplitude of mIPSCs impinging on the M-cell (Legendre and Korn 1994). In the absence of zinc and tricine, mIPSC amplitude distributions were skewed as were surface area distributions for GlyR clusters (Fig. 9D). In the 13 M-cell recordings made in the absence of zinc or tricine, the averaged mean amplitude of mIPSCs was 169 ± 47 pA, and their CV (0.83 ± 0.04) was close to that for the surface area of GlyR clusters. As observed for GlyR cluster surface area distribution, there was poor correlation between the mean amplitude values and CV (R = 0.12; Fig. 9E).

To determine whether the zinc-dependent enhancement of mIPSC amplitude depends on cluster surface area, we first compared the averaged cumulative amplitude distributions of mIPSCs from all cells tested in the absence or in the presence of tricine and in the presence of 1,000 nM free zinc. Figure 10A shows as expected that the addition of tricine to the bath solution clearly shifted the averaged cumulative amplitude distribution to the left.



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Fig. 10. Nanomolar zinc concentrations can influence the occupancy level of the postsynaptic GlyRs, depending on their cluster size. A: averaged cumulative mIPSC amplitude histograms obtained from 13 (no tricine), 14 (tricine), and 8 (1 µM free zinc) M-cells. In the absence of tricine the cumulative amplitude distribution reflects a basal heavy metal presence. B: standardized cumulative averaged histogram of mIPSCs in the absence of zinc (10 mM tricine) compared with a standardized cumulative histogram of all GlyR-IR cluster sizes (n = 2,315) measured on M-cells. C: standardized cumulative histogram of mIPSCs in the control condition (no tricine added) compared with the standardized cumulative histogram of GlyR-IR cluster sizes. D: standardized cumulative histogram of mIPSCs in the presence of 1 µM free zinc compared with standardized cumulative histogram of GlyR-IR cluster sizes. Note that mIPSCs distributions obtained in the absence or in the presence of zinc parallel the GlyR-IR cluster sizes distribution. The distributions in B-D were normalized by dividing each data point by the mean value of their respective distribution.

To analyze correlations between zinc-dependent mIPSC amplitude fluctuations and GlyR cluster surface areas, we then compared normalized averaged mIPSC amplitude distributions obtained in the absence of zinc (10 mM tricine; n = 14 records), in control condition (n = 13 records), and in the presence of 1 µM free zinc (n = 8 records) with a normalized distribution of GlyR cluster surface areas (n = 2,315 clusters from all measurements). The distributions were normalized by dividing each data point by the mean value of their respective distribution. This method does not change the CV value of each cumulative distribution in contrast to the approach used by Frerking et al. (1995). In our experiments the two methods demonstrated that the CV of the mIPSC amplitude distribution and that of the cluster surface area distributions were very similar. As shown in Fig. 10, B-D, the shapes of the distributions for mIPSC amplitudes in the presence or in the absence zinc and for GlyR-IR cluster surface areas are not statistically different (Kolmogorov-Smirnov test, P = 0.1). This suggests that the size of GlyR clusters and the mIPSC amplitudes strongly co-vary when zinc is absent and when it is present. These results also suggest that no postsynaptic GlyR cluster can be fully occupied after the release of one vesicle.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results showed that zinc causes an increase in glycine-evoked currents with an EC50 of 15 nM. This potentiating effect results from a decrease in the glycine dissociation rate constant. Our results also suggest that postsynaptic GlyRs are not fully occupied, at least in the absence of zinc, after the release of one vesicle at M-cell inhibitory synapses.

Nanomolar free zinc concentrations increase GlyRs affinity for glycine

The effects of the heavy metal chelators suggest that a nanomolar basal contamination by zinc or other metals suffices to modify GlyR activation and inhibitory synaptic efficacy. Similar effects have been described for some recombinant NMDA receptors (Paoletti et al. 1997). The contaminant remains unknown since at estimated basal concentrations near 10 nM, classical detection procedures are ineffective. The zinc binding site on GlyRs is recognized by Pb2+ and La3+ with the potency sequence Zn2+ > Pb2+ > La3+ (Kumamoto and Murata 1996). These three metals are equally probable as contaminants of water or salts. Such a contamination could explain why the potentiation effect of zinc has been previously observed in only a narrow range of concentrations (0.1-10 µM) (Bloomenthal et al. 1994; Doi et al. 1999; Han and Wu 1999; Laube et al. 1995, 2000; Lynch et al. 1998; Tapia and Aguayo 1998; Zhang and Berg 1995).

Our results show that zinc potentiates glycine-gated currents in zebrafish by decreasing the glycine dissociation rate constant koff. These findings are consistent with the proposed increase in glycine binding affinity by zinc described previously in [3H] strychnine binding assays (Lynch et al. 1998). In one study of recombinant human homomeric alpha 1 glycine receptors, Laube et al. (2000) have recently postulated that a threefold decrease of glycine koff underlies the maximum potentiation effect of free zinc application (10 µM). This calculation was based on data obtained in the absence of heavy metal chelators by analysis of single channel openings recorded in stationary conditions. In our experiments, an 11-fold decrease of koff was necessary to simulate experimental data. This apparent discrepancy and the fact that the EC50 for zinc potentiating effects that Laube et al. calculated was close to 1 µM can be explained by a basal contamination of the external solution by zinc or other heavy metals in their experiments.

Lynch et al. (1998; but see Laube et al. 2000) suggested, from mutagenesis and expression studies on human GlyR alpha 1-subunit cDNA, that zinc-induced changes in the apparent binding affinity were not causally related to a potentiation of glycinergic currents. Their hypothesis was based on the observation that mutations located on the N-terminal (D80A) or on the M2-M3 loop (L274A) impair zinc-evoked potentiation of glycine-evoked current amplitude but do not change zinc-evoked enhancement of glycine binding affinity or the potentiation of taurine-evoked respones. Effects of zinc on the deactivation time course of glycine-evoked responses of D80A or L274A mutated GlyRs must be known for a rigorous test of the hypothesis (Lynch et al. 1998). Effectively, zinc might still modify koff for this GlyR mutation and, if so, might be expected to increase the duration of glycine-evoked responses (see RESULTS and Fig. 5). Zinc effects on response amplitude might be counteracted by alterations in the allosteric mechanisms that link the zinc binding site to the agonist transduction pathway.

Zinc modifies postsynaptic GlyR occupancy

Our results suggest that, in the absence of zinc, postsynaptic GlyRs are not saturated by synaptically released glycine. This might seem to contradict previous work in which we proposed that peak concentrations of extracellular glycine within the synaptic cleft can reach 1-3 mM (Legendre 1998). However, the transmitter concentration needed to saturate postsynaptic GlyRs depends on multiple other parameters including neurotransmitter clearance, synaptic geometry, and the size of postsynaptic receptor clusters (see for review Clements 1996).

Although synaptic glycine transporters have slow kinetics (Titmus et al. 1996), Monte Carlo modeling studies suggest that clearance is fast and generally biphasic, due to diffusion barriers (Clements 1996; Faber et al. 1992). Such a fast clearance may account for the low occupancy level of postsynaptic GlyRs suggested by the effect of zinc on mIPSCs amplitude. We tested this hypothesis using a GlyR Markov model (Fig. 5A) to simulate synaptic currents in the absence or in the presence of zinc. The rate constant values used to simulate synaptic currents were those given in Fig. 5, and koff was decreased 10 times to mimic the effect of 1 µM free zinc. The time course of glycine concentration at the synapse was modeled assuming a 3-mM peak concentration of glycine released with two decay components (Clements 1996). The optimal parameters were 2.7 and 0.3 mM for the initial exponential amplitude and 0.1 and 1 ms for the decay time constants (Clements 1996). Our simulations predict an open probability of 0.39 in the absence of zinc and 0.68 in the presence of 1 µM free zinc. This is far from the maximum open probability observed for outside-out responses evoked by 1 ms applications of 3 mM of glycine (approx 0.9) (Legendre 1998). Simulating a decrease of glycine koff by zinc also results in an increase of the 20-80% rise time, from 0.22 ms for simulations without zinc to 0.38 ms for those corresponding to 1 µM free zinc. This is similar to our observations on mIPSCs (20-80% rise time approx 0.24 ms in the absence of zinc and approx 0.33 ms in the presence of 1 µM free zinc). This analysis suggests that the lack of a saturation of postsynaptic GlyRs after the release of a single vesicle partly depends on a fast glycine clearance from the synaptic cleft.

It is difficult to completely exclude that mIPSC amplitude fluctuations result from variations in the number of released neurotransmitter molecules per vesicle, as postulated at GABAergic synapses terminating on retinal amacrine cells (Frerking et al. 1995). However, this seems unlikely to be the only factor underlying the large variation in mIPSC amplitudes. If so, the 20-80% RT values of mIPSCs would be correlated with mIPSC amplitude fluctuations, which was not experimentally observed (Legendre 1998). Variations in the number of molecules released per vesicle can be simulated assuming that all synaptic vesicles are filled with the same transmitter concentration but that their diameters vary according to a Gaussian distribution with CV approx 0.15 (Bekkers et al. 1990; Frerking et al. 1995). The vesicle volume distribution should then follow a cubed distribution (Bekkers et al. 1990). Frerking et al. (1995) used a Gaussian distribution raised to the sixth power since two molecules of GABA are needed to open GABAA channels as for GlyRs. To determine how potential variations in the number of released glycine molecules affect mIPSC amplitude, we used the same vesicle volume distribution as proposed by Frerking et al. (1995), assuming an average peak concentration of glycine released of 0.5 mM. mIPSCs were simulated using the same GlyR Markov model and the same time course of glycine clearance as described above. Simulations produced a skewed amplitude distribution for mIPSCs with a CV of approx 0.86, similar to that observed in the presence of tricine. However, simulating a decrease of glycine koff by 1 µM free zinc reduced the CV to 0.68, a change that was not experimentally observed (Fig. 8B).

GlyR cluster size and mIPSC amplitude variability

The mean surface area of GlyR clusters reported here is 3.3 and 10 times smaller than those present on the soma and the lateral dendrite of the goldfish M-cell, respectively (Triller et al. 1990). This difference may explain why mIPSCs recorded in M-cell goldfish (Korn et al. 1982) have a much larger amplitude than those observed in M-cell zebrafish larva (Legendre and Korn 1994). It may also reflect a difference in the level of synapse maturation between the zebrafish larva and adult goldfish. The surface areas of the latter are also smaller than those determined on rat spinal cord motoneuron somata (Alvares et al. 1997; Oleskevich et al. 1999) but are similar to those measured on brain stem neurons (Lim et al. 1999). We should note, however, that comparisons between the size of GlyR clusters measured using an antibody against GlyRs (our study) and using antibodies against gephyrin (Alvares et al. 1997; Lim et al. 1999; Oleskevich et al. 1999) are not straightforward. Gephyrin has recently been shown to be co-localized with GABAA receptors (Baer et al. 1999; Giustetto et al. 1998; Kneussel et al. 1999; Todd et al. 1996). For this reason, gephyrin cluster sizes may not be tightly related to those of GlyRs.

Our experiments show that mIPSC amplitudes co-vary with the surface area of GlyR clusters both in the absence of zinc and in the presence of high free zinc concentration. A correlation between mIPSCs fluctuation and GlyR cluster sizes was observed at other glycinergic inhibitory synapses (Lim et al. 1999; Oleskevich et al. 1999). Although this co-variation does not imply that postsynaptic GlyRs are saturated after the release of one vesicle, it nevertheless suggests that the postsynaptic GlyR cluster size can be one of the factors involved in the large mIPSC amplitude fluctuation.

Physiological role of high-affinity binding site for zinc

The basal concentrations of free and bound zinc in the extracellular space remain unknown. According to our experiments, the inhibitory synaptic efficacy is already sensitive to applied zinc, at concentrations above 1 nM. Intracerebral zinc does increase after drinking contaminated water (Rabchevsky et al. 1998) to levels that may influence postsynaptic GlyRs. Interestingly, chelation of zinc by injection of dipilonic acid in the extracellular fluid of the spinal cord produces hyperalgesia, whereas zinc injection produced antinociception, which suggests that zinc is involved in sensory processing (Larson and Kitto 1997). As glycinergic synapses in the spinal cord are involved in pain control (Dickenson 1996), it is tempting to speculate that such an effect of zinc on nociception is due in part to the modulation of glycinergic synaptic efficacy.

The co-release of zinc with glycine has not yet been described and may not even exist. On the other hand zinc has been shown to be co-released with glutamate (Smart et al. 1994), and glycinergic synapses are intermingled with GABA synapses and excitatory glutamatergic synapses on the M-cell soma (Sur et al. 1994). Accordingly, the presence of a high-affinity zinc binding site on GlyRs raises the possibility that an increase in activity at glutamatergic synapses might be counteracted by an increase in glycine synapse efficacy due to lateral zinc diffusion, in addition to direct effects of zinc on glutamate receptors.


    ACKNOWLEDGMENTS

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

This work was supported by INSERM and INSERM-Fonds de la Recherche en Santé du Québec (P. Legendre and P. Drapeau). H. Suwa was supported by a Human Frontier Science Program Organization fellowship. L. Saint-Amant was supported by a Medical Research Council of Canada Studentship.


    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).

Received 6 June 2000; accepted in final form 16 October 2000.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society