1Center for Research in Neuroscience, McGill University; and Montreal General Hospital Research Institute, Montreal, Quebec H3G 1A4, Canada; and 2Institut des Neurosciences, Centre National de la Recherche Scientifique Unité Mixte de Recherche C7624, Universite Pierre et Marie Curie, 75252 Paris Cedex 05, France
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ABSTRACT |
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Ali, Declan W.,
Pierre Drapeau, and
Pascal Legendre.
Development of Spontaneous Glycinergic Currents in the Mauthner
Neuron of the Zebrafish Embryo.
J. Neurophysiol. 84: 1726-1736, 2000.
We used whole cell and
outside-out patch-clamp techniques with reticulospinal Mauthner neurons
of zebrafish embryos to investigate the developmental changes in the
properties of glycinergic synaptic currents in vivo from the onset of
synaptogenesis. Miniature inhibitory postsynaptic currents (mIPSCs)
were isolated and recorded in the presence of TTX (1 µM), kynurenic
acid (1 mM), and bicuculline (10 µM) and were found to be sensitive
to strychnine (1 µM). The mIPSCs were first observed in 26-29 h
postfertilization (hpf) embryos at a very low frequency of ~0.04 Hz,
which increased to ~0.5 Hz by 30-40 hpf, and was ~10 Hz in newly
hatched (>50 hpf) larvae, indicating an accelerated increase in
synaptic activity. At all embryonic stages, the amplitudes of the
mIPSCs were variable but their means were similar (~100 pA),
suggesting rapid formation of the postsynaptic matrix. The 20-80%
rise times of mIPSCs in embryos were longer (0.6-1.2 ms) than in
larvae (~0.3 ms), likely due to slower diffusion of glycine at the
younger, immature synapses. The mIPSCs decayed with biexponential
(off1 and
off2) time
courses with a half-width in 26-29 hpf embryos that was longer and
more variable than in older embryos and larvae. In 26- to 29-hpf
embryos,
off1 was ~15 ms and
off2 was ~60 ms, representing events of intermediate duration; but occasionally long mIPSCs were observed in
some cells where
off1 was ~40 ms and
off2 was ~160 ms. In 30-40 hpf embryos, the
events were faster, with
off1 ~ 9 ms and
off2 ~ 40 ms, and in larvae, events declined
somewhat further to
off1 ~ 4 ms and
off2 ~ 30 ms. Point-per-point amplitude
histograms of the decay of synaptic events at all stages resulted in
the detection of similar single channel conductances estimated as ~45
pS, indicating the presence of heteromeric glycine receptors (GlyRs)
from the onset of synaptogenesis. Fast-flow (1 ms) application of a
saturating concentration of glycine (3-10 mM) to outside-out patches
obtained at 26-29 hpf revealed GlyR currents that decayed biexponentially with time constants resembling the values found for
intermediate and long mIPSCs; by 30-40 hpf, the GlyR currents resembled fast mIPSCs. These observations indicate that channel kinetics limited the mIPSC duration. Our data suggest that glycinergic mIPSCs result from the activation of a mixture of fast and slow GlyR
subtypes, the properties and proportion of which determine the decay of
the synaptic events in the embryos.
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INTRODUCTION |
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Glycine is a major inhibitory
neurotransmitter in mammals (Aprison 1990), where
glycine receptors (GlyRs) are pentameric with various isoforms
(
1,
2,
3,
4, and
) whose properties depend on the
subunit composition (Bormann et al. 1993
). GlyR subtypes are expressed in the mammalian brain at different locations and with
different developmental patterns (for review, see Vannier and
Triller 1997
). The maturation of glycinergic synapses in
mammals is characterized by a progressive decrease in the duration of glycinergic inhibitory responses, and it has been proposed that this
reflects a progressive molecular switching of
2 for
1 subunits (Krupp et al. 1994
; Singer et al. 1998
;
Takahashi et al. 1992
), although
2 subunit can still
be present at adult age (Racca et al. 1998
) and synaptic
responses mediated solely by
2 GlyRs have not been detected.
It is still unclear whether this progressive decrease in the time
course of glycinergic responses during synaptic maturation is due to
the delayed appearance of synaptic endings with faster postsynaptic
GlyRs or reflects an increase in the proportion of mature over immature
GlyR subtypes at individual synapses. As growth cones can release
neurotransmitter (Allen and Brown 1996; Young and
Poo 1983
), it is possible that changes in the time course of
glycinergic responses in the developing brain also reflect maturation
of the presynaptic endings.
Previous developmental studies of embryonic GlyRs examined the channel
kinetics in stationary conditions (Takahashi et al. 1992), which do not allow the prediction of their behavior when activated in nonequilibrium conditions, as is the case during synaptic
vesicular release (Frerking and Wilson 1996
). In the zebrafish larva, glycinergic miniature inhibitory postsynaptic currents
(mIPSCs) have a complex time course with two decay components (Legendre 1998
, 1999
). This was also observed in
postnatal and juvenile rat brain stem motoneurons (Singer and
Berger 1999
; Singer et al. 1998
). This time
course is due to a complex behavior of transiently activated GlyRs, and
it cannot simply be related to the mean open duration of the channels
(Legendre 1998
). As nonstationary analyses in
mammals have been limited mainly to postnatal development (Singer and Berger 1999
; Singer et al.
1998
), it remains to be determined how the properties of
glycinergic synapses change from their initial appearance during the
earliest period of synaptogenesis in the embryo.
Zebrafish embryos provide a means to address these developmental issues
by an in vivo analysis of mIPSC amplitude fluctuations, time course
variability, and single GlyR channel kinetics. Zebrafish embryos
develop quickly, showing limited motor activity at <26 h
postfertilization (hpf) and first showing a touch-induced escape response from 26-29 hpf that matured by 30-40 hpf prior to hatching of the larvae at >50 hpf (Saint-Amant and Drapeau
1998). This suggests that neural networks involved in this
motor behavior develop progressively before birth. Glycine is also the
major inhibitory transmitter in the hindbrain of teleosts (Faber
and Korn 1982
; Legendre and Korn 1994
). It is
particularly involved in the control of the Mauthner-cell (M-cell)
activity mediating the escape response in zebrafish (Eaton et
al. 1977
) and other teleosts (Faber and Korn
1978
).
Morphological development of synapses was previously studied in the
hindbrain of the zebrafish embryo (Kimmel et al. 1990). In 2-day-old larvae the glycinergic synapses seen by immuno-electron microscopy on the M-cell body, and growing dendrites appear to be
morphologically mature although endings with an immature appearance are
also observed (Triller et al. 1997
). Immature endings
(with unidentified transmitter) are observed on the M cell at early embryonic stages (Kimmel et al. 1990
), but their
functional significance remains unknown.
To determine the developmental process of glycinergic synapse
development and maturation on embryonic zebrafish Mauthner cells, we
analyzed in vivo the changes in mIPSC-like properties in embryos at the
four stages described in the preceding text, i.e., at <26, 26-29,
30-40, and >50 hpf. The mIPSCs were compared with the kinetics of
GlyR currents evoked in outside-out patches with a fast-flow application system (Legendre 1998).
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METHODS |
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Preparation
Embryos and larvae were raised at 28.5°C and were obtained
from a zebrafish colony maintained according to established procedures (Westerfield 1995). Ages are given as hours
postfertilization (hpf). The animals hatch at ~50 hpf. All procedures
were carried out in compliance with the guidelines stipulated by the
Canadian Council for Animal Care and McGill University. Embryos and
newly hatched larvae were anesthetized in 0.02% tricaine (MS-222;
Sigma Chemical, St. Louis, MO) in recording solution prior to and
during dissections (see following text). The preparation was then moved to the recording setup, and the chamber was continuously perfused with
an oxygenated (95% O2-5%
CO2) recording solution that contained D-tubocurarine (15 µM) to paralyze the preparations but
that lacked tricaine. The recording solution contained (in mM) 145 NaCl, 1.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2 PO4, and 10 glucose, 330 mosM, pH 7.4. For high KCl solution, 10.5 NaCl was replaced with
KCl to maintain osmolarity.
Whole cell recordings
The ventral surface of the hindbrain was exposed after removing
rostral regions, as described by Drapeau et al. (1999),
and the embryos were pinned on their side through the notochord being careful to avoid damaging the neighboring spinal cord. Standard whole
cell recordings (Hamill et al. 1981
) were performed in
vivo within 5-15 min after dissection. Preparations were placed on the
stage of a Nikon Optiphot microscope and continuously perfused with
recording solution. Mauthner neurons were easily identified with a
×40 immersion lens using Hoffman modulation optics. Patch-clamp electrodes (3-5 M
) were pulled from thin-walled, Kimax-51
borosilicate glass and filled with a CsCl intracellular solution
composed of (in mM) 135 CsCl, 2 MgCl2, 10 HEPES,
10 EGTA, and 4 Na3 ATP, 290 mosM, pH 7.2. All
drugs were dissolved in the recording solution and applied by bath
perfusion while the preparation was continuously perfused. Tetrodotoxin
(1 µM), strychnine (1 µM), and D-tubocurarine (15 µM)
were obtained from Sigma Chemical while kynurenic acid (KYN; 1 mM) and
bicuculline (10 µM) were obtained from RBI.
Whole cell currents were recorded using an Axopatch 1A amplifier (Axon
Instruments) and were low-pass filtered at 5-10 kHz and digitized at
20-25 kHz. The series resistance was monitored during whole cell
recordings by applying 2-mV hyperpolarizing pulses. Neurons that had
input resistances <100 M were discarded. Synaptic activity was
monitored using pClamp 6 software (Axon Instruments), and the detection
of synaptic events was performed with Axograph software version 3.56 (Axon Instruments) on data re-filtered at 2 kHz using a second-order
Chebychev digital filter. Some traces were filtered at 1 kHz for
display purposes only. Histograms and scatter plots were constructed
using Kaleidagraph 3.1 software. Detailed analysis of their decay time
courses were performed by averaging 25 isolated single events (filter
cutoff frequency of 5 kHz). The decay phase was fitted with a sum of exponential curves, and the presence of one or more exponential components was tested by comparing the sum of squared errors of the
fits (Clements and Westbrook 1991
; Legendre
1998
). The equation with two exponential components always
resulted in a significantly better fit (Legendre 1998
).
Single-channel recordings
Standard outside-out recordings from the membrane of the
Mauthner cell in an isolated ex vivo hindbrain preparation
(Legendre and Korn 1994) were achieved under direct
visualization with differential interference contrast (Nikon Optiphot
microscope). The preparation was continuously perfused at room
temperature (20°C) with oxygenated solution. Patch-clamp electrodes
(10-15 M
) were pulled from thick-wall borosilicate glass and were
filled with CsCl intracellular solution. Outside-out single-channel
currents were evoked using a fast-flow operating system (Franke
et al. 1987
; Lester et al. 1990
) as previously described (Legendre 1998
). Briefly, drugs were dissolved
in a control solution containing (in mM) 145 NaCl; 1.5 KCl; 2 CaCl2; 1 MgCl2; 10 glucose,
and 10 HEPES; pH 7.2; 330 mosM. Control and drug solutions were gravity
fed into the two channels of a thin-wall glass theta tube (2 mm OD,
Hilgenberg, Germany) with a tip diameter of 200 µm. The solution
exchange was performed by rapidly moving the solution interface across
the tip of the patch pipette, using a piezo-electric translator (Physic
Instrument, model P245.30). Concentration steps of glycine were applied
every 5-10 s. The exchange time was determined after rupturing the
seal by monitoring the change in the liquid junction evoked by the
application of a 10% diluted control solution on the open tip of the
patch pipette. The exchange time on the patch (~0.1 ms) was estimated
using the method published by Maconochie and Knight
(1989)
(see Legendre 1998
for detailed analysis).
Patch currents were recorded using an Axopatch 1D amplifier (Axon
Instruments), filtered at 10 kHz using a eight-pole Bessel filter
(Frequency Devices), sampled at 50 kHz and stored on computer using
pClamp 6 software (Axon Instruments). Outside-out currents were
analyzed off-line with Axograph 3.56 software (Axon Instruments). The
time courses of outside-out responses were analyzed by averaging 10-15
single events, and the decay phase of the responses was fitted with a
sum of exponential curves using simplex algorithm. As for mIPSCs
analysis, the presence of more than one exponential component was
tested by comparing the sum of the squared errors of the fits
(Legendre 1998).
Glycine-gated channel behavior was simulated using a chemical kinetic
modeling program (Axograph 3.56, Axon Instruments). This program first
calculated the evolution of the number of channels in each given state
for given rate constants (Clements and Westbrook 1991).
The kinetic model used to generate the theoretical trace was previously
proposed to explain the behavior of GlyRs at mature glycinergic synapse
(Legendre 1998
). Results are presented as means ± SD throughout unless otherwise noted.
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RESULTS |
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Recordings were made from Mauthner neurons in embryos
corresponding to four developmental stages (see
INTRODUCTION): <26, 26-29, 30-40, and >50 hpf. The
results for the stages at which glycinergic mIPSCs-like currents were
observed are summarized in Table 1. With
CsCl electrodes the input resistance was consistently high in embryos
at all stages (1-2 G) and fell (to 0.3-0.5 G
) in larvae. In all
recordings, we applied 1 mM kynurenic acid to block glutamatergic
events.
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Properties of mIPSCs
Whole cell recordings from Mauthner cells of embryos <26 hpf
revealed a general lack of spontaneous synaptic activity (not shown).
In some cases, we observed very small strychnine-sensitive events that
were composed of two to three superimposed single-channel openings
(46 ± 6 pS; n = 9 events from 3 cells),
suggesting the presence of rare and very immature synapses but
excluding the presence of GlyRs containing a subunit variant like that
of the mammalian embryonic 2 subunit (
2*), which confers little
strychnine sensitivity to GlyRs (Kushe et al. 1990
).
Spontaneous synaptic currents appeared as early as 26-29 hpf, 2 h
after presynaptic nerve endings on embryonic Mauthner cells have been
detected (24 hpf) (Kimmel et al. 1990
). To examine in
isolation mIPSC-like evoked by the release of single quanta, we
perfused the preparation with 1 µM TTX to block action potential
discharge, 1 mM kynurenic acid to block ionotropic glutamate receptors,
and 10 µM bicuculline to block GABAA receptors. We
detected mIPSCs with rapid rise times and clear exponential decays in
embryos
26 hpf. Spontaneous, glycinergic mIPSC-like activity occurred
with a very low frequency (Fig.
1A; 0.04 ± 0.02 Hz;
n = 4) in 26-29 hpf embryos. To determine if
depolarization of presynaptic terminals could enhance the frequency of
glycinergic mIPSCs at this stage and improve our ability to detect
events, we applied an elevated (12 mM) KCl solution. Under these
conditions, the frequency of mIPSCs increased by an order of magnitude
to 1.4 ± 0.3 Hz (n = 4 cells; Fig.
1B). In older embryos (30-40 hpf), the average frequency of
mIPSCs was also an order of magnitude greater (0.45 ± 0.46 Hz;
n = 6 cells) than in 26-29 hpf embryos in normal (1.5 mM) KCl. All mIPSCs disappeared in the presence of 1 µM strychnine
(Fig. 1C) and returned during washout of strychnine (not
shown), confirming that the miniature events we detected were
effectively due to the activation of strychnine-sensitive GlyRs. The
frequency of mIPSCs increases by another order of magnitude to ~10 Hz
in newly hatched larvae (Legendre and Korn 1994
; Table 1), presumably reflecting an accelerated period of synaptogenesis prior
to hatching.
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The amplitude of most events in 26-29 hpf embryos was clustered
between 10 and 150 pA, with a distribution that was more or less
Gaussian (Fig. 2A) around a
mean of 75 ± 28 pA (n = 6 cells, Fig.
2D and Table 1). Occasionally (in 2 of 6 preparations), larger amplitude events (>200 pA) were recorded in 26- to 29-hpf embryos, but these were infrequent and represented <5% of the events.
Amplitude distributions of mIPSCs were skewed toward larger values
(means 100-150 pA) in animals >30 hpf (Fig. 2, B and
C, and Table 1) as larger events (>200 pA) became more
frequent. This is in the range of mIPSC amplitude fluctuations
previously described in larvae (Legendre and Korn 1994).
The coefficient of variation (CV = SD/mean) was large and
increased at later stages, ranging from 0.6-0.9 (Fig. 2E).
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To obtain a rough estimate of the decay duration, we measured the
half-width of each event and plotted the cumulative distributions for
eight recordings from 26-29 hpf embryos in Fig.
3A and for seven recordings
from 30- to 40-hpf embryos in Fig. 3B. The mean half-widths
were over twice as long at the younger stage and were briefest in the
larvae (Fig. 3C). Although the CVs were comparable (~0.5)
at all stages (Fig. 3D), it is clear from the distributions in Fig. 3, A and B, that the half-widths were far
more variable for the younger (26-29 hpf) embryos. A detailed analysis
of the individual mIPSCs in 26- 29 hpf embryos revealed two separate classes of events (Fig. 4). The vast
majority of events in six recordings decayed biexponentially (see Table
1) within 100-200 ms (Fig. 4A, 1 and 2) with
each time constant (mean values for all events:
off1 = 15 ± 6 ms;
off2 = 63 ± 24 ms) fitting about half of
the events. We term these events "intermediate" in duration. The
20-80% rise times of intermediate mIPSCs were slower (mean = 0.57 ± 0.16) than in larvae (mean ~ 0.3 ms; Fig.
2F and Table 1) (Legendre 1998
).
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We also detected events that were several-fold longer in duration (Fig.
4B, 1 and 2) and decayed over several hundred
milliseconds that we refer to as "slow" events. Slow events were
present in highly variable proportions. They were not detected in two
of the six recordings. In two other recordings with intermediate events, the slow events represented <1 and ~25% of synaptic-like activity and resulted in noticeably skewed distributions in some of the
plots in Fig. 3A. In two other experiments. they accounted for most of the events (i.e., these lacked intermediate events). Each
time constant described half of the events but with
off1 = 38 ± 11 ms (57%) and
off2 = 157 ± 75 ms. The rise times of the long events were also slow with a mean of 1.2 ± 0.6 ms. The slow rise times of intermediate and long events were not due to errors
in space clamping (embryonic Mauthner neurons are smaller and less
complex than larval cells) as they were not correlated to the
half-widths (not shown) and well-resolved single-channel openings could
occasionally be observed during the decay phase in cells with a low
noise level (see following text).
In older (30-40 hpf) embryos and in larvae, the mIPSCs remained
biexponential but decayed more rapidly with what are referred to as
"fast" events. In 30-40 hpf embryos,
off1 = 9.4 ± 2.9 ms (74 ± 20%;
n = 7 cells) and
off2 = 40 ± 14 ms (Fig. 5A, 1 and 2). A similar proportion of each component was observed
in newly hatched larvae but the decay times were briefer (Fig.
5B, 1 and 2), as previously observed
(Legendre 1998
):
off1 = 4.2 ± 0.9 ms (73%), and
off2 = 29 ± 7 ms (n = 10 cells). The rise times of fast events
were still slower than in larvae (~0.3 ms) (Legendre 1998
), with a mean = 0.74 ± 0.13 ms.
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Outside-out patch currents from young (<30 hpf) embryos
To determine whether or not the variability in the duration of mIPSCs reflects intrinsic kinetics of the postsynaptic receptors, we analyzed the time course of outside-out responses evoked by short (1 ms) applications of a saturating concentration (3-10 mM) of glycine (see METHODS). We did not record any channel activity in response to application of glycine in almost all (45 out of 46 cells) of the outside-out patches from embryos <26 hpf. In one patch from a 20-hpf embryo, we were able to obtain a short recording of long-lasting, glycine-gated channel openings (2-3 GlyRs) with a main conductance state of 48 pS (data not shown).
Short applications of glycine onto outside-out patches obtained from
somewhat older (26-29 hpf) embryos evoked single-channel activity in
the majority of the cases (14 of 16). In these patches, the outside-out
current greatly exceeded the duration of glycine application (Fig.
6). As observed for mIPSCs, three types
of outside-out responses (fast, intermediate, and slow) were recorded.
Slow responses were observed in 4 of the 14 patches (Fig.
6A) and were similar to what we observed for slow mIPSCs.
They were characterized by a long decay phase that was well fitted by a
sum of two exponential curves (Table 1) with decay time constants
off1 = 80 ± 16 ms (n = 4) and
off2 = 306 ± 57 ms
(n = 4). The first decay component represented
the majority (68 ± 3%, n = 4) of the total
current. The 20-80% rise time of these outside-out transient currents
was close to 0.2 ms (10 mM glycine; 0.19 ± 0.03 ms;
n = 4). It became maximal for glycine concentrations
3 mM, as observed on larvae where the mature form of GlyRs is
dominant (Legendre 1998
). In eight other patches, the
application (1 ms) of 3-10 mM glycine evoked a transient current of
intermediate duration (Fig. 6B) similar to that observed for
intermediate mIPSCs. These responses evoked by 1-ms application of 10 mM glycine had a similar 20-80% rise time (0.16 ± 0.02 ms,
n = 8) and a biexponential deactivation phase (Fig.
7B and Table 1). The decay
time constants were
off1 = 17 ± 3 ms
(n = 8) and
off2 = 146 ± 44 ms (n = 8), respectively, with the first
deactivation component representing the majority (74 ± 9%) of
the total current (n = 8). We also observed in two patches fast outside-out currents resembling those described in larvae
(Legendre 1998
) where mature inhibitory synapses were
described (Triller et al. 1997
). These outside-out
currents evoked by a 10 mM glycine application (1-ms pulse duration;
not shown) had a 20-80% rise time of 0.18 and 0.17 ms and a
biexponential decay with time constants
off1 = 6.4 and 4.3 ms and
off2 = 81 and 36 ms, with
the first component representing the majority (65 and 75%) of the peak
current.
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As single openings can be resolved during the decay phase of
outside-out currents evoked by 1-ms application of glycine and mIPSCs
in young embryos, we compared measured single-channel currents underlying outside-out responses and mIPSCs in 26-29 hpf embryos to
determine if extrasynaptic and synaptic immature GlyRs share the same
main conductance level. The main conductance state of GlyRs underlying
outside-out currents was measured from the decay phase of a single
transient current evoked by the application of 10 mM glycine, using
point-per-point amplitude histograms (Fig. 6, C and
D, and Table 1). Outside-out currents of long duration (Fig.
6C) could be seen to result from the activation of GlyRs with a main conductance state of 46 ± 4 pS (n = 3). Similarly, responses of intermediate duration (Fig. 6D)
reflected the activation of GlyRs with a main conductance state of
48 ± 3 pS (n = 7). We also selected examples of
long and intermediate mIPSCs in which single-channel openings could be
resolved. The long events were examined in one cell (29 hpf) in which
they represented ~25% of the events. In this cell many of the long
events decayed with resolvable step-like currents (Fig.
7A1). Point-per-point histograms of slow events (Fig.
7A2) revealed a principal peak current at 2.4 ± 0.3 pA (n = 10), which, at the holding potential of
50 mV
and calculated reversal potential for chloride of 0 mV, represents a
single-channel conductance of 48 ± 6 pS. Deactivation phase analyses of intermediate embryonic events (Fig. 7B, 1 and
2) revealed a first peak current at
2.2 ± 0.2 pA
(n = 11), which represents a conductance of 44 ± 4 pS. Two types of GlyRs were previously described on the zebrafish
M-cell larva according to their main conductance state and their
subconductance levels. One GlyR subtype was characterized by a single
conductance level of 44-48 pS while the other subtype had three
subconductance states with a main conductance level of 84-88 pS
(Legendre 1997
; Legendre and Korn 1994
).
Similarly, they can also be identified during concentration jump
experiments as their corresponding main conductance states occurred
during the deactivation phase of the outside-out responses (Legendre 1998
). Our present results therefore suggest
that GlyRs with the higher main state conductance (84-88 pS) described
previously in newly hatched larvae (Legendre 1997
, 1998
;
Legendre and Korn 1994
) were extremely rare or
presumably not present at the earlier stages examined here. In larvae
GlyRs with a conductance state of 84-88 pS were also rarely observed
as they were recorded in only 18% of the patches tested
(Legendre and Korn 1994
).
Outside-out patch currents from older (30-40 hpf) embryos
As observed for mIPSCs, outside-out currents evoked in patches
from M cells of 30-40 hpf embryos had faster decays than those observed in 26-29 hpf embryos. The deactivation phase of the
outside-out currents evoked by a 1-ms application of 10 mM glycine
(Fig. 8A) could be fit with
the sum of two exponential curves (Fig. 8B) with decay time
constants off1 = 8.6 ± 4.3 ms
(n = 19; 64 ± 14% of the total current) and
off2 = 66 ± 38 ms. These
outside-out currents had a fast 20-80% rise time of 0.16 ± 0.05 ms, which is not significantly different from that measured in 26-29
hpf embryos (unpaired t-test) and similar to that previously
described in larvae (Legendre 1998
) (see Table 1). The
main conductance state of GlyRs underlying fast outside-out currents
was measured from the decay phase of single transient currents (Fig.
8C) as described in the preceding text. Outside-out currents
resulted from the activation of GlyRs with a main conductance state of 47 ± 3 pS (n = 19), similar to that observed in
the 26-29 hpf embryos (Table 1).
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The mean rise time value of these outside-out currents was
several-fold faster than those measured for mIPSCs recorded in 30-40
hpf embryos (Table 1). The mIPSCs rise time in larvae were also more
than twice as fast as those in embryos and were proposed to correspond
to a peak concentration of glycine of 1 mM released at mature
synapses (Legendre 1998
). The slower mIPSC rise times observed in embryos can therefore be due to slow release of a lower
concentration of glycine, slower intrinsic kinetics of the postsynaptic
receptors (Singer and Berger 1999
), or both. To
discriminate between these possibilities, we measured the rise times of
outside-out responses evoked by 1 ms application of 0.3, 1, or 10 mM
glycine (Fig. 9A). Responses
evoked by 1 mM glycine application had a 20-80% rise time of
0.34 ± 0.05 ms (n = 9; Fig. 9, B and
C), similar to that described in larvae (Legendre
1998
) (see Table 1). As expected under nonstationary conditions
(Legendre 1998
), decreasing the concentration of glycine
to 0.3 mM had only a slight effect on the 20-80% rise time as it was
0.36 ± 0.03 ms (n = 9; Fig. 9, B and
C). It is therefore unlikely that the slow mIPSC rise time
was due to slow activation kinetics of the GlyRs at immature synapses.
A 1 ms application pulse is a relatively long pulse with respect to the
clearance speed of the transmitter from the synaptic clef at mature
synapses (Clements 1996
). It is therefore possible that
the slow mIPSC rise times we observe in embryos are due to slow release
of glycine at immature synapses.
|
Simulation of outside-out currents
Transient currents evoked by 3 mM glycine in patches from larval
Mauthner cells decay with fast time constants
(off1 ~ 4 ms and
off2 ~ 30 ms, Table 1) and effectively mimic
mIPSCs occurring from mature inhibitory synapses. This reflects the
activation of GlyRs with
1/
-like subunit composition
(Legendre 1997
, 1998
). In the zebrafish embryo,
mIPSCs and evoked outside-out currents have long deactivation phases
(as long as
off1 ~ 40 ms and
off2 ~ 150 ms). These are likely to be due
to the activation of a separate GlyR subtype. It is therefore possible
that the mIPSCs and the glycine-evoked outside-out currents of an
intermediate duration reflect postsynaptic receptor aggregates with a
variable proportion of slow, embryonic and fast, mature GlyR subtypes.
To test this hypothesis, we simulated outside-out currents with a decay
phase with time constant values corresponding to the fast and slow
glycinergic events. The kinetic model we used to simulate mIPSCs was
previously proposed to predict fast GlyR behavior in the zebrafish
larval M-cell (Legendre 1998
, 1999
).
The mean open time appeared longer for GlyRs underlying slow
glycinergic events (Fig. 6, C and D) than for
fast GlyRs (Fig. 8A). As a first approximation, slow
synaptic-like events were mimicked using the same GlyRs Markov model
but with the closing rate constant values reduced by 5- to 10-fold.
These slower closing rate constants gave theoretical outside-out
currents with decay time constants off1 = 38.5 ms (70.1%) and
off2 = 154.2 ms, which are
similar to our experimental data (Fig.
10A and Table 1). Simulation of outside-out currents with various relative proportions of the two
GlyRs (Fig. 10A) gave responses of intermediate duration
with a time course similar to outside-out experimental data when the relative proportion of fast GlyR was 50-80% (Fig. 10B).
The decay time constants increased significantly when the proportion of immature GlyRs was ~10%. The deactivation phase of these theoretical responses can be fit by the sum of two exponential curves, although the
beginning of the slower component was more poorly fit (Fig. 10A) as was also observed for some experimental outside-out
data (Fig. 8B). Our model predicts that the increase in the
relative proportion of fast GlyRs should progressively reduce the
duration of the decay time constant (Fig. 10C). It also
predicts a small increase in the relative amplitude of the fast
deactivation component (Fig. 10D) when the proportion of
fast GlyRs was increased above 30%. These general predictions are
consistent with our experimental findings (Table 1).
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DISCUSSION |
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Glycinergic inhibitory synapses developed quickly in the zebrafish
embryo, being first detected by 26 hpf and maturing by the time the
larvae hatch 1 day later (at ~50 hpf). The main characteristics of
mIPSCs recorded, for the first time, from early embryos in vivo were a
reduced amplitude fluctuation, a slower rise time, a long decay phase
for some events, and a large variability in the duration of the
miniature glycinergic synaptic-like events. Slow outside-out responses
and mIPSCs (off1 = 38 ms;
Table 1) were never described before. To the contrary, outside-out
responses and synaptic-like events of intermediate duration (see Table
1) recorded at 26-29 hpf resemble the outside-out response obtained from perinatal motoneurons in the rat brain stem (Singer and
Berger 1999
). They however differ from miniature synaptic-like
events recorded in perinatal motoneurons (Singer et al.
1998
). Responses observed at an intermediate stage (30-40 hpf)
were more homogenous in duration and resembled outside-out responses
recorded from brain stem juvenile motoneurons (Singer and Berger
1999
).
GlyR kinetics control the deactivation time course of mIPSC during development
At all developmental stages tested, the deactivation phase of
glycinergic outside-out responses and mIPSCs have a complex time course
with a doubly exponential decay. This was first described in zebrafish
larva (Legendre 1998). This apparently contrasts with
previously analyses of mIPSCs time course on M cell of adult goldfish
(Korn and Faber 1990
) using intracellular recordings. However, goldfish GlyR channel kinetics are still unknown, and the
frequency of occurrence of mIPSCs in the goldfish M-cell is high
(33-127 Hz) (Mintz and Korn 1991
), which precludes a
good resolution of a biphasic decay phase with a possible slow decay component. In the adult zebrafish (Hatta and Korn 1998
),
the decay phase of glycinergic mIPSCs was not analyzed and the presence of one or two decay components remains unknown, although evoked responses decayed monoexponentially.
Differences in mIPSC duration between early zebrafish embryos and
larvae are likely to reflect a transition from slow to fast zebrafish
GlyRs. Our data on deactivation time constants of outside-out currents
and mIPSCs suggested that two types of synaptically activated GlyRs are
present in the Mauthner cell of the zebrafish embryo. Changes in
glycinergic mIPSC duration during development was previously proposed
to be related to a switch in 2 and
1 GlyRs subunits mRNAs in
mammals (Singer et al. 1998
; Takahashi et al.
1992
). However,
2 mRNA can still be detected in adult
(Racca et al. 1998
), and due to the lack of specific
antibody, it is not possible to demonstrate whether or not
2,
1/
are present and in what proportion at glycinergic synapses
throughout their development. Nevertheless, there is indirect evidence
to suppose that long, immature synaptic-like events may be due to the
activation of
2 GlyRs (Takahashi et al.
1992
). In a previous study of larval GlyRs, channels
were observed to re-open in bursts before the dissociation of glycine
due to the complex kinetics, resembling mammalian
1/
heteromeric
GlyRs (Legendre 1998
). In mammals, the
2 subunits
confer GlyR activity with bursts of long openings (Takahashi et
al. 1992
; Virginio and Cherubini 1996
). We
speculate that slow mIPSCs-like may represent synaptically driven
2-like GlyRs while fast events are likely to reflect
1-like GlyR
activation (Legendre 1998
). A GlyR subunit has been
cloned recently from the adult zebrafish brain (
Z1)
(David-Watine et al. 1999
; Fucile et al.
1999
) and has structural and functional features resembling more the mammalian
1 subunit than other subunit types. However, the
molecular properties of embryonic zebrafish GlyRs, such as the presence
of
2 subunits, remain unknown.
Most embryonic mIPSCs fell between the extremes discussed in the
preceding text as their time courses were described by biexponential functions of intermediate duration. According to our model, a mixture
of fast and slow GlyRs could yield intermediate events similar in
duration to those observed experimentally. As a switch in subunit
expression has been described in other preparations (for review, see
Vannier and Triller 1997
), and so this mechanism would
seem feasible. An alternative explanation would be that individual
GlyRs with several types of
subunit exist and undergo a similar
transition in relative content at a molecular (rather than synaptic)
level since for example
1/
2 heteromers can be obtain in
transfected cells (Kushe et al. 1993
). Another
alternative is that a single type of GlyR (which maintains its subunit
composition) changes its kinetic properties progressively during
development by a posttranslational maturation such as protein
phosphorylation. However, GlyR current decay was stable in isolated
patches, and others have found that substrates, which promote
phosphorylation have no effect on GlyR current decay times
(Singer and Berger 1999
). We therefore conclude that a
variable switch from purely slow to mostly fast GlyRs may occur during
development of zebrafish glycinergic synapses.
Change in glycinergic synapse efficacy during development
The rise time of mIPSCs can give a rough estimation of the peak
concentration of agonist reaching the postsynaptic receptors and/or its
clearance speed after the release of one vesicle (Clements 1996; Jones and Westbrook 1995
; Legendre
1998
; but see Frerking and Wilson 1996
). The
slower 20-80% rise times of embryonic mIPSCs-like (1 ms), which were
not due to slower activation of embryonic GlyRs, cannot be accounted
for by a reduction in glycine concentration only, as at concentrations
<0.3 mM, it becomes relatively constant at <0.5 ms even for a 1 ms
pulse (Legendre 1998
). Thus this result suggests that at
embryonic synaptic-like contacts, the peak concentration of glycine
appears to be reached over a relatively prolonged period of
1 ms with
a slow clearance. This can be explained by a large cleft between the
M-cell membrane and axonal endings at immature synapses and/or the
presence of growth cone-like terminals.
Such mIPSCs-like with a slow 20-80% rise time were not observed in
>50-hpf M-cell larva where most of the detected synapses on M cells
appeared to be mature (Triller et al. 1997). These observations apparently contrast with previous morphological studies performed on zebrafish M-cell during development (Kimmel et al. 1981
). Effectively, Kimmel et al. (1981)
observed mature synaptic contact on 96-hpf zebrafish only. This
apparent discrepancy can be explained by a difference in zebrafish
development between the two studies. Effectively, the study of
Triller et al. (1997)
and ours were performed in
zebrafish larva raised at the standard temperature of 28.5°C
(Westerfield 1995
). To the contrary, the older
study of Kimmel et al. (1981)
was performed on embryos
raised at 25.5°C. Accordingly, the animals hatch at ~4 days in the
Kimmel et al. (1981)
study, whereas in the present study
and that of Triller et al. (1997)
, the analyses were
performed on animals hatching at 2 days. This creates difficulties in
comparing these two morphological studies; but using the hatching
period as a reference, it appears that synaptic contacts do not fully
mature until the animals hatch (Kimmel et al. 1981
). In
the early embryo, many of the boutons are not well differentiated, and
they give rise to immature chemical synaptic junctions some with a
defined synaptic cleft (Kimmel et al. 1981
). This could
give mIPSCs with slow rise times.
The amplitude of mIPSCs was remarkably constant in embryos, from
the onset of synaptogenesis at 26 hpf until 40 hpf, and became more
variable in larvae while their frequency increased dramatically with
the age of the animal. Changes in mIPSC amplitude and CV during
development were also observed in mammals (Singer and Berger 1999); but in the zebrafish larvae, it was associated with an increase in their frequency. This is likely to be partly due to the
increase in the number of mature synaptic boutons on the developing M-cell (Kimmel et al. 1981
). It can also partly reflect
the maturation of the presynaptic terminal as the release probability,
and the number of neurotransmitter molecules released by growth cones and immature synapses in vitro is very low and variable (Allen and Brown 1996
; Young and Poo 1983
). Amplitude
fluctuations of mIPSCs recorded in embryos might thus reflect a
variation in the number of glycine molecules released, but we cannot
exclude that it also reflects a variation in the number of postsynaptic
receptors. Accordingly, the largest events could reflect release of
more glycine or denser postsynaptic receptors.
Physiological significance
In general, inhibitory synaptic events with long decay times
produce sustained changes in the membrane potential resulting in an
increased efficacy of tonic synaptic activity. As in other vertebrate
embryos, zebrafish embryonic neurons have elevated chloride levels such
that glycinergic events are depolarizing (Saint-Amant and
Drapeau 2000). Thus GlyRs or GABAA
receptors induce cell membrane depolarization; this allows calcium
influx into immature neurons (Boehm et al. 1997
;
Singer et al. 1998
; Wang et al. 1994
) as
a prelude to synapse maturation (Kirsch and Betz 1998
)
and may thus play key roles during synaptogenesis (Lévi et
al. 1998
). By overcoming the effect of the long electrotonic time constant of the embryonic zebrafish neurons, long glycinergic synaptic events may also more efficiently evoke cell depolarization and
hence promote maturation of inhibitory synapses. Moreover inhibitory
synaptic events have other functions in addition to their control of
the resting membrane potential as they can prevent the electrotonic
propagation of excitatory events by decreasing the input resistance. In
the embryo, long glycinergic synaptic events may compensate for the
small number of glycinergic synapses on the embryonic Mauthner cell, as
reflected by the low frequency of events we detected, allowing
long-lasting, shunting inhibition. When inhibitory synaptic contacts
become more numerous or their release probability increases, a switch
between GlyR subtypes may then take place. In conclusion,
morpho-functional maturation of the pre- and postsynaptic elements
occurs during synaptogenesis, leading to a functional diversity of the
glycinergic inhibitory synapses aposed to the mature M-cell. This
diversity appears to be mainly related to the morpho-functional
heterogeneity of the postsynaptic GlyRs clusters.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Richard Miles for valuable help and discussions.
This work was supported by grants from the National Sciences and Engineering Research Council of Canada (P. Drapeau) and Institut National de la Santé et de la Recherche Médicale and Association Contre les Miopathies of France (P. Legendre), a Fonds de la Recherche en Santé du Québec Canada/INSERM France Collaborative Exchange (P. Legendre and P. Drapeau) and an NSERC Canada Fellowship (D. W. Ali).
Present address of D. W. Ali: Program in Brain and Behavior, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.
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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).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 February 2000; accepted in final form 14 June 2000.
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REFERENCES |
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