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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ABSTRACT |
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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 (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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INTRODUCTION |
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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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METHODS |
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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 M
(whole cell) or 10-15 M
(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 m
) 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 G
. 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 M
, 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 105 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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RESULTS |
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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 /
GlyRs
(Legendre 1997
) from the Mauthner cell (M-cell). GlyR
resembling homomeric
1 and heteromeric
1/
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
offfast = 5.6 ± 0.4 ms and
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
offfast and
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
offfast and
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, offfast and
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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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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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 offfast and
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
offfast from 4.2 ± 1.04 ms to 9.7 ± 2.4 ms (Fig. 2B) and
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
offslow in a concentration-dependent
manner (paired t-test, P = 0.01). For
example, the relative amplitude of
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
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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The zinc EC50 was obtained by fitting the data
using a single binding isotherm of the form
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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
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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 () or a decrease of the closing rate constant (
)
(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 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
is the main determinant of the rising time of the
responses (Legendre 1998
). A decrease in the GlyR
closing rate constant
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
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
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|
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
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|
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
s1 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
offfast but slightly higher (
45 nM)
for
offslow and, a Hill coefficient of
0.8, a koffmax of 2,600 s
1 and
koffmax/koffmin
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
45 nM for
offslow, a Hill coefficient of 0.8, a
koffmax of 2,600 s
1, and a
koffmax/koffmin
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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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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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).
|
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 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 (
10 µm large and
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).
|
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.
|
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.
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DISCUSSION |
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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
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
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 (
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
0.24 ms in the absence of zinc and
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
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
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.
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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.
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FOOTNOTES |
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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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REFERENCES |
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