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The Journal of Neuroscience, May 1, 2003, 23(9):3633
Developmental Increase in Vesicular Glutamate Content
Does Not Cause Saturation of AMPA Receptors at the Calyx of Held
Synapse
Takayuki
Yamashita,
Taro
Ishikawa, and
Tomoyuki
Takahashi
Department of Neurophysiology, University of Tokyo Graduate School
of Medicine, Tokyo 113-0033, Japan
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ABSTRACT |
Whether a quantal packet of transmitter saturates postsynaptic
receptors is a fundamental question in central synaptic transmission. However, this question remains open with regard to saturation at mature
synapses. The calyx of Held, a giant glutamatergic synapse in
the auditory brainstem, becomes functionally mature during the fourth
postnatal week in rats. During postnatal development, the mean
amplitude of miniature (i.e., quantal) EPSCs (mEPSCs) becomes
significantly larger. Experiments using the rapidly dissociating glutamate receptor antagonist kynurenate suggested that vesicular glutamate content increases with development. To test whether AMPA
receptors are saturated by a packet of transmitter, we infused a high
concentration of L-glutamate into mature calyceal
terminals. This caused a marked increase in the mean amplitude of
mEPSCs. We conclude that a single packet of transmitter glutamate does not saturate postsynaptic AMPA receptors even at the mature calyx of
Held synapse with increased vesicular transmitter content.
Key words:
quantal EPSCs; calyx of Held; AMPA receptor; postnatal development; receptor saturation; vesicular transmitter
content
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Introduction |
Whereas saturation of postsynaptic
glutamate receptors by quantal transmitter has been a long-standing
hypothesis (Jack et al., 1981 ; Larkman et al., 1991; Clements et al.,
1992; Jonas et al., 1993; Tang et al., 1994; Tong and Jahr, 1994; for
review, see Frerking and Wilson, 1996), recent studies at putative
single-site synapses suggest that a single packet of transmitter does
not saturate postsynaptic AMPA receptors (Silver et al., 1996; Liu et
al., 1999) or NMDA receptors (Mainen et al., 1999; Umemiya et al.,
1999; McAllister and Stevens, 2000). At the calyx of Held synapse, it
has been demonstrated that a high concentration of L-glutamate directly loaded into the giant nerve terminal
significantly increases the mean amplitudes of quantal AMPA-EPSCs and
quantal NMDA-EPSCs, indicating that a single vesicular transmitter does not saturate postsynaptic glutamate receptors (Ishikawa et al., 2002).
However, synapses used for these studies are either in culture or in
slices of immature animals, where transmitter packaging might still be
under development. At the Xenopus neuromuscular junction in
culture, synaptic vesicles are incompletely filled with the transmitter
acetylcholine at the immature stage (Evers et al., 1989; Song et al.,
1997). At the cerebellar mossy fiber-granule cell synapse, the
variation in quantal size is large at immature synapses but
significantly decreases as animals mature (Wall and Usowicz, 1998).
These observations raise the possibility that quantal transmitter may
saturate postsynaptic glutamate receptors as animals mature. We
examined this possibility using simultaneous presynaptic and
postsynaptic recordings at the mature calyx of Held synapse.
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Materials and Methods |
Preparation and solutions. All experiments were
performed in accordance with the guidelines of the Physiological
Society of Japan. Transverse brainstem slices (125-300 µm thick)
containing the medial nucleus of the trapezoid body (MNTB) were
prepared from 6- to 29-d-old [postnatal day 6 (P6)-P29]
Wistar rats killed by decapitation under halothane anesthesia (Forsythe
and Barnes-Davies, 1993). Slices were incubated for 1 hr at 36-37°C
and maintained thereafter at room temperature (22-28°C). The
extracellular artificial CSF (aCSF) for perfusion contained (in
mM): 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 10 glucose,
3 myoinositol, 2 sodium pyruvate, and 0.5 ascorbic acid, pH 7.4 when
bubbled with 95% O2 and 5%
CO2, 310-320 mOsm. The aCSF routinely contained bicuculline methiodide (10 µM;
Sigma, St. Louis, MO) and strychnine hydrochloride (0.5 µM; Sigma) to block inhibitory
synaptic responses. Principal neurons in the MNTB and the presynaptic
terminal, the calyx of Held, were visually identified with a 60× water
immersion objective (Olympus Optical, Tokyo, Japan)
attached to an upright microscope (BX50WI; Olympus
Optical; or Axioskop 2; Zeiss, Oberkochen, Germany). Patch pipettes for postsynaptic recordings were filled with (in mM): 110 CsF, 30 CsCl, 10 HEPES, 5 EGTA,
and 1 MgCl2, pH adjusted to 7.3-7.4 with CsOH,
290-295 mOsm.
N-(2,6-Diethylphenylcarbamoylmethyl)-triethyl-ammonium chloride (5 mM; Alomone Labs,
Jerusalem, Israel) was routinely included in the postsynaptic pipette
solution to suppress action potential generation. Tetrodotoxin (TTX;
0.5-1 µM; Wako, Osaka, Japan) was added to the
aCSF for recording miniature EPSCs (mEPSCs). Presynaptic pipette
solutions contained (in mM): 105 potassium gluconate, 30 KCl, 10 HEPES, 0.5 EGTA, 12 phosphocreatine (Na salt), 3 ATP (Mg salt), 0.5 GTP (Na salt), and 1 MgCl2, pH
7.3-7.4, adjusted with KOH, 290-300 mOsm. The
L-glutamate solution for presynaptic infusion was
prepared by replacing potassium gluconate in the pipette solution by
L-glutamate to maintain constant osmolarity. Infusion of the L-glutamate solution into calyces
was made using a plastic tube installed in a presynaptic patch pipette
as reported previously (Hori et al., 1999; Ishikawa et al., 2002).
Briefly, a tube with an outer tip diameter of 50-70 µm was
fabricated from an Eppendorf yellow tip and back-filled with the
L-glutamate solution. The tube was inserted into
a patch pipette to 500-700 µm behind the tip of the pipette. The
L-glutamate solution in the tube was ejected with
positive pressure applied manually through a syringe.
Recording. Whole-cell recordings were made from
the MNTB principal neurons using a patch-clamp amplifier (Axopatch 200B
or Multiclamp 700A; Axon Instruments, Foster City, CA).
The postsynaptic pipette was pulled for the resistance of 2-3
M and had access resistance of 4-8 M. The presynaptic pipette
was pulled for the resistance of 6-10 M and had access resistance
of 18-30 M. No compensation was made for the access resistance. The
postsynaptic cells were voltage-clamped at a holding potential of  70
mV. Routinely, before recording mEPSCs, EPSCs were evoked at 0.04-0.1
Hz by extracellular stimulation with a bipolar tungsten electrode
positioned halfway between the midline and the MNTB (Forsythe and
Barnes-Davies, 1993) to ensure that the cells receive calyceal inputs.
EPSCs derived from the calyx of Held synapse were identified as those evoked in an all-or-none manner for graded stimulus intensity and
having amplitudes >1 nA at 70 mV (Forsythe and Barnes-Davies, 1993).
In simultaneous presynaptic and postsynaptic recordings, EPSCs were
evoked by presynaptic action potentials elicited by a 1 msec
depolarizing pulse (Takahashi et al., 1996), and spontaneous mEPSCs
were recorded in the presence of TTX with presynaptic terminals voltage-clamped at 70 mV.
Data analysis. Records were low-pass filtered at 5 kHz and
stored on digital audio tapes (sampling rate, 48 kHz). Data were digitized at 50 kHz by a Digidata 1320A analog-digital converter with
pClamp8 software (Axon Instruments, Foster City, CA) and analyzed off-line using Axograph (Axon Instruments).
Spontaneous and mEPSCs having an amplitude more than three times the
baseline noise level (SD, 3-4 pA) were detected using a sliding
template method implemented in Axograph. The templates were made by
averaging 40-50 mEPSCs. Overlapped mEPSCs were excluded from analyses
by visual inspection. The decay time of averaged mEPSCs was fitted to
double exponential functions, and its weighted mean
( m) was calculated from individual time
constants (1,
2) and their relative amplitude
(a1,
a2) as follows:
m = a11 + a22.
For the nonstationary fluctuation analysis, the difference between
individual mEPSCs and peak-scaled averaged mEPSCs at their decay phases
was calculated, and the ensemble variance was plotted against the
current amplitude (Traynelis et al., 1993). Assuming binomial
statistics of the channel opening, the
 2-I relationship is
expected to be a parabolic function [i.e., it can be fitted by the
following equation: 2 = iI (I2/N) + 2B, where I
represents the mean current, 2
represents the peak-scaled variance, N represents the
average number of channels open at the peak, i represents
the single-channel current amplitude, and
2B represents the
background noise variance]. Although plots of experimental data tended
to deviate from symmetrical parabola, the initial slope of the
2-I relationship provides a
reliable estimate for the weighted single-channel current (Silver et
al., 1996). All values are given as mean ± SEM. Statistical
analysis for two samples was made using Student's t test
unless otherwise noted. For multiple comparisons, the Sheffe test was
used unless otherwise noted. A value of p < 0.05 was
taken as the level of significance.
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Results |
Developmental changes in the quantal EPSCs
Spontaneous mEPSCs mediated by AMPA receptors (Futai et al.,
2001) were recorded from the MNTB principal neurons in the presence of
TTX. These mEPSCs likely derived from the calyx of Held terminal, because their amplitude distribution is indistinguishable from those
evoked by direct steady depolarizations of the nerve terminal in the
presence of TTX (Sahara and Takahashi, 2001). The mean amplitude of
mEPSCs remained similar from P6-P7 (40.3 ± 3.0 pA; n = 10 cells) to P13-P14
(47.1 ± 1.9 pA; n = 10) (Figs. 1, 2A), as
reported previously (Chuhma and Ohmori, 1998; Taschenberger and von
Gersdorff, 2000; Iwasaki and Takahashi, 2001), but significantly increased thereafter to P20-P21 (61.7 ± 3.7 pA;
n = 10) on average by 31% (between P13-P14 and
P20-P21; p < 0.02; Sheffe test). It then reached a
plateau with no additional increase to P28-P29 (59.1 ± 2.7 pA;
n = 10). The mean quantal frequency also increased by
4.3-fold from P6-P7 (0.26 ± 0.02 Hz) to P20-P21 (1.1 ± 0.2 Hz;
p < 0.04) and reached a steady level (1.3 ± 0.3 Hz) at P28-P29 (Fig. 2B).
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Figure 1.
Miniature EPSCs recorded from MNTB principal
neurons in developing rats. Representative amplitude distributions of
mEPSCs in P6, P14, P21, and P29 rats (number of events, 242, 370, 408, and 392) are shown with sample records (insets) of averaged mEPSCs
(from all events of each distribution) (top inset) and superimposed
records (4 traces) (bottom inset). Background noise
distributions (filled bars) were obtained from the baselines of records
with no clear events. Arrows indicate mean amplitude of mEPSCs. CV,
Coefficient of variation (SD/mean) for the mEPSC amplitude.
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Figure 2.
Developmental changes in the amplitude, frequency,
and kinetics of mEPSCs. Miniature EPSCs were recorded from the MNTB
principal neurons in rats at P6-P7, P13-P14, P20-P21, and P28-P29
(n = 10 for each). A, The mean
amplitude of mEPSCs increased from P13-P14 to P20-P21.
B, The mean frequency of mEPSCs increased from P6-P7 to
P20-P21. C, The 10-90% rise time of mEPSCs decreased
from P6-P7 to P13-P14. Sample records (inset) are the rising phases
of average mEPSCs normalized at the peak. D, The
weighted mean of decay time constants decreased significantly between
P6-P7 and P13-P14 and between P13-P14 and P20-P21. Sample records
(inset) are the decay phases of average mEPSCs normalized at the
peak.
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In addition to the mean quantal amplitude, the kinetics of mEPSCs
changed with postnatal development. The rise time (10-90%) of mEPSCs
became significantly shorter from P6-P7 (0.30 ± 0.02 msec) to
P13-P14 (0.15 ± 0.01 msec; p < 0.01; Sheffe
test) (Fig. 2C) as reported previously (Taschenberger and
von Gersdorff, 2000; Futai et al., 2001) and reached a minimal level
thereafter (0.13 ± 0.01 msec in P20-P21 and 0.13 ± 0.004 msec in P28-P29) (Futai et al., 2001). Such a phenomenon is not
observed for GABAergic (P4-P40) (Okada et al., 2000) or glycinergic
(P0-P18) (Singer et al., 1998) miniature synaptic currents where the
rise time remains constant throughout development. The weighted mean
time constant of mEPSCs (see Materials and Methods) decreased from P6-P7 (1.84 ± 0.12 msec) to P13-P14 (0.64 ± 0.05 msec),
as reported previously (Taschenberger and von Gersdorff, 2000; Futai et
al., 2001; Joshi and Wang, 2002). It then continued to decrease from P13-P14 to P20-P21 (0.33 ± 0.01 msec; p < 0.03) (Fig. 2D) [but see Futai et al. (2001) for
mice] and reached a minimum level at that time (0.36 ± 0.02 msec
at P28-P29). The developmental shortening of the decay time may arise
from changes in the subunit composition of AMPA receptors (Caicedo and
Eybalin, 1999), resultant speeding in the desensitization kinetics
(Wall et al., 2002), and/or a decrease in burst duration of
underlying channels (Takahashi et al., 1992).
Single-channel conductance of postsynaptic receptor channels
underlying mEPSCs
A developmental increase in quantal size might be a result of an
increase in the underlying single-channel conductance of AMPA
receptors. We examined this possibility by applying the nonstationary fluctuation analysis (Sigworth, 1980; Robinson et al., 1991; Traynelis et al., 1993) to mEPSCs. The variance during the decay phase of individual mEPSCs was plotted against their mean amplitude, and the
weighted mean single-channel current was estimated from the initial
slope of the peak-scaled mean-variance relationship (Fig. 3). Single-channel conductance was
21.5 ± 1.5 pS at P13-P14 (n = 8) as reported
previously (20.4 pS) (Sahara and Takahashi, 2001), which was
essentially the same as those in older (P28-P29; 19.4 ± 1.9 pS;
n = 7) and younger (P6-P7; 20.6 ± 1.7 pS;
n = 7) rats (p > 0.6;
ANOVA).
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Figure 3.
Single-channel conductance of AMPA receptors
underlying mEPSCs. Right column, Individual mEPSCs (top) (10 records superimposed after peak alignment) and averaged mEPSCs (bottom)
at P6, P14, and P28. Left column, Peak-scaled variance-mean current
plots obtained by the nonstationary fluctuation analysis for mEPSCs in
P6, P14, and P28 rats. These 2-I plots
were calculated from 162, 341, and 371 events, respectively. Mean
single-channel current was estimated from the initial slope of the
2-I relationships. To estimate
single-channel conductance ( ), reversal potentials of mEPSCs were
measured in each recording.
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Developmental increase in the synaptic cleft concentration of
transmitter liberated from each synaptic vesicle
Given that AMPA receptor channel conductance remains constant
throughout development, a developmental increase in the mean quantal
amplitude must arise from an increased number of receptors and/or
increased concentration of transmitter in the synaptic cleft. Whereas
the former possibility is technically difficult to assess, we examined
the latter possibility by testing the effect of the rapidly
dissociating AMPA receptor antagonist kynurenate on mEPSCs (Diamond and
Jahr, 1997; Wadiche and Jahr, 2001). The inhibitory effect of
kynurenate would be weaker if the glutamate concentration in the
synaptic cleft is higher. As illustrated in Figure
4, kynurenate (50 µM)
attenuated mEPSCs in both P13-P14 and P28-P29 rats, with the
magnitude of inhibition at P28-P29 (20.8 ± 0.8%;
n = 7) being significantly weaker than that at P13-P14 (26.9 ± 1.3%; n = 7; p < 0.01;
Sheffe test) (Fig. 4C). To examine whether kynurenate can
indeed detect changes in the glutamate concentration in the synaptic
cleft, we loaded 50 mM
L-glutamate into calyceal
terminals at P13-P14 via presynaptic patch pipettes. The mean
amplitude of mEPSCs recorded in this condition was larger than that of
controls at P13-P14 but comparable with that of controls at
P28-P29 (Fig. 4B) (Ishikawa et al., 2002).
The inhibitory effect of kynurenate after presynaptic
L-glutamate loading (17.8 ± 1.2%; n = 5) was significantly weaker than controls at
P13-P14 (p < 0.01) (Fig. 4C),
confirming that kynurenate can reliably detect increased vesicular
glutamate content. In contrast to kynurenate, the slowly dissociating
AMPA receptor antagonist
2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline (30 nM) attenuated mEPSCs in P13-P14 and
P28-P29 rats to a similar extent, with the magnitude of inhibition
being 31.1 ± 1.1% (n = 5) at P13-P14 and
31.6 ± 2.4% (n = 4) at P28-P29
(p > 0.8), indicating that the dissociation
rate of antagonist must be fast to detect different glutamate
concentrations in the synaptic cleft. These results also suggest that
the affinity of AMPA receptor antagonists may not change
much during development. Because the rise time of mEPSCs was
unchanged from P13-P14 to P28-P29 (Fig. 2C), the diffusion
distance from release sites to receptors seems unchanged. In fact, the
morphological architecture of this synapse is established at P14
(Kandler and Friauf, 1993). Higher glutamate concentration in the
synaptic cleft at mature synapses suggests that the amount of
transmitter in each synaptic vesicle increases with development.
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Figure 4.
Developmental increase in the quantal transmitter
concentration in the synaptic cleft detected by the low-affinity AMPA
receptor antagonist kynurenate (KYN) (50 µM).
A, Sample records (averaged from 243 events for each)
and amplitude histograms of mEPSCs recorded from an MNTB neuron, before
(thin line) and after (thick line) application of kynurenate
(superimposed), each at P14, P29, and P14 with 50 mM
L-glutamate (50Glu) loaded into a
calyceal terminal. Filled bars are background noise histograms.
B, C, Mean amplitude of mEPSCs
(B) and their percentage inhibition by kynurenate
(C) at P13-P14, P28-P29, and P13-P14 with 50 mM L-glutamate loaded into calyceal
terminals. Asterisks indicate a significant difference with
p < 0.01 (Sheffe test).
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Nonsaturation of postsynaptic AMPA receptors
A quantal packet of transmitter does not saturate postsynaptic
glutamate receptors at the calyx of Held synapse in P13-P15 rats
(Ishikawa et al., 2002). Our results suggest that vesicular transmitter
content increases with development after P13-P14. Therefore, the
increased transmitter might then saturate postsynaptic AMPA receptors.
We examined this possibility by loading a high concentration of
L-glutamate directly into the calyceal nerve terminal via
pipette perfusion in P28-P29 rats, while recording mEPSCs in the
presence of TTX (Ishikawa et al., 2002). With 1 mM
glutamate in the presynaptic pipette, amplitudes of mEPSCs were stable.
After confirming a stable baseline, we infused 100 mM
L-glutamate into the calyceal terminal via a
perfusion pipette installed inside the patch pipette (Hori et al.,
1999; Ishikawa et al., 2002). This caused a marked potentiation of
mEPSCs (p < 0.01) (Fig.
5A), with their amplitude
distribution clearly shifted toward larger events (Fig. 5B).
No significant change was observed for the mean frequency of mEPSCs
(99 ± 13%; n = 4). At four synapses, the mean
magnitude of potentiation was 50 ± 12% (measured 13-15 min
after glutamate infusion) (Fig. 5C). Despite the marked
increase in the amplitude of mEPSCs, neither the rise time (10-90%;
0.13 ± 0.006 msec before and 0.13 ± 0.005 msec after
switch; n = 4; p > 0.9) nor the decay
time constant (0.32 ± 0.03 msec before and 0.32 ± 0.02 msec
after switch; n = 4; p > 0.8) changed
significantly (Fig. 5A). Unchanged rise time argues against
the possibility that increased vesicular transmitter spilled over to
remote receptors and increased the mEPSC amplitude (DiGregorio et al.,
2002; Ishikawa et al., 2002). Whereas a positive correlation between
rise time and amplitude of mEPSCs is taken as evidence for multiquantal release at other synapses (Paulsen and Heggelund, 1996; Wall and Usowicz, 1998), no such correlation was observed at this synapse, both
before and after L-glutamate infusion
( r < 0.2; n = 4 synapses). Also, the
coefficient of variation of the mEPSC amplitude remained similar
(91 ± 3%; n = 4; p > 0.08)
after L-glutamate loading. These results argue
against an involvement of multivesicular release in the potentiation of
mEPSCs caused by presynaptic L-glutamate loadings. As expected from the results of mEPSCs, presynaptic loading of L-glutamate also potentiated
EPSCs evoked by presynaptic action potentials at P29 (Fig.
5D), on average by 71 ± 16% (n = 4)
(Fig. 5E). Spontaneous EPSCs recorded simultaneously with evoked EPSCs are equivalent to mEPSCs at this synapse, because TTX had
no effect on the amplitude (102 ± 4%; n = 4) or
frequency (104 ± 4%; n = 4) of spontaneous EPSCs
in P29 rats compared with P13-P15 rats (Ishikawa et al., 2002). In
addition to mEPSCs (Fig. 5A-C), spontaneous
EPSCs underwent a clear potentiation (60 ± 6%; n = 4) (Fig. 5E). These results strongly suggest that quantal packets of transmitter do not saturate postsynaptic AMPA receptors even
at the mature calyx of Held synapse.
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Figure 5.
Nonsaturation of postsynaptic AMPA receptors at
mature synapses. A, Miniature EPSCs recorded from a P28
MNTB neuron before (i) and after (ii) loading 100 mM
L-glutamate into a calyceal terminal. Each data
point represents the mean amplitude of mEPSCs sampled every 20 sec.
Sample records of mEPSCs before (i) and after (ii) the
L-glutamate loading at a slow sweep (top) and a fast sweep
(bottom left, superimposed), and those normalized in the amplitude
(bottom right, superimposed). B, Amplitude histograms of
mEPSCs recorded from the same neuron before (i) and after (ii) the
L-glutamate loading (number of events is 427 and 420, respectively). C, Summary data of mEPSCs from four
synapses at P28-P29. The mean amplitude of mEPSCs before the
L-glutamate loading was 54.3 ± 5.4 pA.
D, Potentiation of evoked EPSCs by presynaptic loading
of L-glutamate in a P29 rat. Sample records are averaged
presynaptic action potentials (top, superimposed) and EPSCs before (i)
and after (ii) the L-glutamate loading (bottom,
superimposed). E, Summary data for evoked EPSCs (open
circles) and spontaneous EPSCs (filled circles) from four synapses at
P29. Before L-glutamate loading, the mean amplitude of
evoked EPSCs was 4.37 ± 0.71 nA, and that of spontaneous EPSCs
was 64.0 ± 10.6 pA. Glu, Glutamate. Dotted lines in C
and E denote mean values of data points before
L-glutamate loadings.
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Discussion |
By making whole-cell recordings from presynaptic terminals and
postsynaptic cells at the mature calyx of Held synapse, we have
demonstrated that a quantal packet of transmitter does not saturate
postsynaptic AMPA receptors. This implies that vesicular transmitter
content can affect synaptic efficacy even in mature animals. Because
vesicular glutamate is controlled by vesicular glutamate transporters
(Burger et al., 1989) and indirectly via cytoplasmic glutamate
concentration by glutamine and glial glutamate transporters (Danbolt,
2001), modulation of these transporters may influence synaptic efficacy
at mature synapses as well as at immature synapses (Ishikawa et al.,
2002).
The calyx of Held synapse is formed at approximately P5 and undergoes
morphological transformations until P14, at which the adult-like
calyceal structure is established (Kandler and Friauf, 1993). Rodents
start to hear sounds during the second postnatal week (P10-P13) (Futai
et al., 2001). During this critical week, the calyx of Held synapse
undergoes various molecular and functional changes, such as
downregulation of NMDA receptors (Futai et al., 2001; Joshi and Wang,
2002), switch of voltage-dependent Ca2+
channel subtypes (Iwasaki and Takahashi, 1998), a decrease in the
transmitter release probability, and an increase in size of the readily
releasable pool of synaptic vesicles (Taschenberger and von Gersdorff,
2000; Iwasaki and Takahashi, 2001). After P14, postsynaptic NMDA
receptors continue to decrease toward P27, at which time the
high-fidelity synaptic transmission is established (Futai et al.,
2001). Our present results also demonstrate that the mean
amplitude, mean frequency, and kinetics of mEPSCs reach steady levels
at P20-P21, suggesting that the calyx of Held synapse becomes
functionally mature at this period.
A developmental increase in mean quantal amplitude can be caused by an
increase in vesicular transmitter content or in postsynaptic receptor
density. Our results with kynurenate support the presence of the former
mechanism but do not at all rule out the latter mechanism. Direct
loading of a high concentration of L-glutamate into the
calyces increased the quantal amplitude by 50% on average in P28-P29
rats, suggesting that the occupancy of postsynaptic AMPA receptors by a
single quantum is still <67% as estimated at P13-P15 (<60%)
(Ishikawa et al., 2002). It is possible that the postsynaptic density
of AMPA receptors increases with development, thereby maintaining low
receptor occupancy by quantal transmitter. Although quantal
transmitter does not saturate NMDA receptors in P13-P15 rats (Ishikawa
et al., 2002), it remains unknown whether it does so in mature animals
with decreased NMDA receptor expression (Futai et al., 2001).
Developmental reduction in the basal release probability during the
second postnatal week reduces synaptic depression, thereby increasing
synaptic efficacy for high-frequency transmission (Taschenberger and
von Gersdorff, 2000; Iwasaki and Takahashi, 2001). The developmental increase in the quantal amplitude observed in this study should increase the synaptic efficacy regardless of the frequency of transmission, thereby contributing to improving stability of
high-fidelity transmission for sound localization at the calyx of Held synapse.
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FOOTNOTES |
Received Jan. 2, 2003; revised Jan. 31, 2003; accepted Feb. 4, 2003.
This study was supported by a grant-in-aid for Specially Promoted
Research from the Ministry of Education, Culture, Sports, Science and
Technology to T.T. We thank Yoshinori Sahara and Tetsuhiro Tsujimoto
for helpful comments on this manuscript.
Correspondence should be addressed to Dr. Tomoyuki Takahashi,
Department of Neurophysiology, University of Tokyo Graduate School of
Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail:
ttakahas-tky{at}umin.ac.jp.
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TOP
ABSTRACT
Introduction
