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 Previous Article | Next Article 
The Journal of Neuroscience, January 15, 2003, 23(2):377-383
Receptor Occupancy Limits Synaptic Depression at Climbing Fiber
Synapses
John
Harrison and
Craig E.
Jahr
Vollum Institute, Oregon Health and Science University, Portland,
Oregon 97201-3098
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ABSTRACT |
Postsynaptic responses to presynaptic stimulation are used
regularly to assess the amount of transmitter released from presynaptic release zones. At climbing fiber-to-Purkinje cell synapses, the number
of vesicles released per active zone follows changes in release
probability such that, normally, more than one vesicle is released per
presynaptic action potential. This leads to high occupation of
postsynaptic AMPA receptors by glutamate and thus may render the
postsynaptic response relatively insensitive to changes in release. We
find that paired-pulse depression of presynaptic release is not
accurately represented by postsynaptic responses because of receptor
saturation. By lowering vesicular glutamate concentrations or by using
nonsaturated Bergmann glial AMPA receptors to monitor presynaptic
release, we find that presynaptic depression of release is much greater
than suggested previously. In addition, densely expressed glutamate
transporters can shield Bergmann glial AMPA receptors and presynaptic
metabotropic glutamate receptors from activation.
Key words:
climbing fiber; Purkinje cell; receptor saturation; multivesicular release; glutamate; glutamate transporters
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Introduction |
At excitatory synapses in the
CNS, vesicular release of glutamate into the small volume of the
synaptic cleft results in the rapid activation of postsynaptic
ionotropic glutamate receptors. Diffusion of glutamate into the
extrasynaptic space, along with binding to glial and neuronal
transporters, rapidly reduces the cleft glutamate concentration
(Barbour et al., 1994 ; Bergles and Jahr, 1997; Bergles et al., 1997;
Clark and Barbour, 1997). The glutamate concentration transient in the
synaptic cleft after exocytosis of single vesicles is thought to occupy
approximately one-half or fewer of the postsynaptic AMPA receptors, on
average (Clements et al., 1992; Diamond and Jahr, 1995; Choi et al.,
2000; McAllister and Stevens, 2000). Thus, AMPA receptor
(AMPAR)-mediated EPSCs should follow variations in the size of the
glutamate transient. However, if postsynaptic AMPARs are close to fully
occupied by glutamate, as they are thought to be at climbing
fiber-Purkinje cell (CF-PC) synapses because of high release
probability (Pr) (Dittman and Regehr, 1998; Silver et al., 1998) and
multivesicular release (Wadiche and Jahr, 2001), the EPSC will not be
an accurate monitor of the glutamate transient. In the extreme case of
receptor saturation, the size of the EPSC will reflect the number of
receptors and not the amount of transmitter released into the cleft. In this condition, using the EPSC to estimate changes in presynaptic release will underestimate changes in release Pr during short trains or
paired stimulation of the presynaptic element.
Very large synaptic glutamate transients resulting from multivesicular
release may also temporarily overwhelm clearance mechanisms and allow
activation of extrasynaptic receptors. Because release probability and
therefore multivesicular release are diminished by repetitive
stimulation, clearance may become faster and limit extrasynaptic
receptor activation. Thus, actions of receptors located outside the
cleft may occur only during low-frequency presynaptic activity at
climbing fiber synapses.
In this study, we decreased synaptic vesicle glutamate concentrations
using inhibitors of the vacuolar type ATPase, bafilomycin A1 (Baf) and concanamycin A (Conca), to study
paired-pulse depression at the CF-PC synapse with a reduced synaptic
glutamate transient to determine the effect of receptor occupancy on
synaptic depression. We also used extrasynaptic AMPARs expressed by
Bergmann glial cells (BGs) as nonsaturable sensors of release as a
comparison. In addition, we examined the ability of glutamate
transporters to shield Bergmann glial AMPARs and presynaptic
metabotropic glutamate receptors (mGluRs) from transmitter at low and
high release probability.
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Materials and Methods |
Slices and solutions. Parasagittal slices of 8- to
16-d-old rat cerebella were cut at a thickness of 250-300 µm with a
vibrating microtome (VT 1000S; Leica, Wetzlar, Germany) in ice-cold
solution containing the following (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose (saturated with 95%
O2-5% CO2). The slices
were incubated in the same solution at 34°C for 15-30 min and then
stored at room temperature. During recording, slices were superfused
with the above solution with the addition of 100 µM picrotoxin. Outside-out patch experiments were performed with an external solution containing the following (in
mM): 140 NaCl, 1.8 CaCl2,
1.3 MgCl2, and 5 HEPES, pH of 7.4. For synaptic
experiments in Purkinje cells, the pipette solution contained the
following (in mM): 130 CsMeSO4, 4 NaCl, 1 MgCl2, 10 HEPES, and 10 EGTA, pH of 7.2. The sources of the chemicals are as
follows: concanamycin A, bafilomycin A1,
picrotoxin, monosodium glutamate, MgCl2, HEPES,
EGTA, CoCl2, and CaCl2 were
from Sigma (St. Louis, MO); (±)-amino-4-carboxy-methyl-phenylacetic
acid (MCPG), (RS)- -cyclopropyl-4-phosphonophenyl-glycine
(CPPG), 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline (NBQX), threo- -benzyloxyaspartate (TBOA), cyclothiazide (CTZ), and
D-600 were from Tocris Cookson (Ellisville, MO); NaCl, KCl, NaH2PO4,
NaHCO3, and glucose were from Mallinckrodt
(Paris, KY); and pardaxin and tetrodotoxin were from Alomone Labs
(Jerusalem, Israel).
Recording and perfusion techniques. Bergmann glia were
identified by their size and location using a 40× water-immersion
objective on an upright microscope (Axioskop FS; Zeiss, Oberkochen,
Germany) equipped with infrared-differential interference contrast
optics. Bergmann glial currents were recorded at their resting
potential. Purkinje cell bodies were identified by their large size,
layered arrangement, and large dendritic tree. Evoked CF-PC EPSCs were recorded at  10 to 20 mV; miniature EPSCs (mEPSCs) were
recorded at 70 mV in the presence of tetrodotoxin (1 µM). To isolate mEPSCs resulting from CF
release, slices from postnatal day 8 (P8) to P10 rats were used because
functional parallel fiber synapses are not usually present at this age
(Yamada et al., 2000); we were unable to evoke EPSCs in Purkinje cells
by stimulation in the molecular layer in these slices. Climbing fibers
were stimulated with a theta glass pipette pulled to a 5-10 µm tip
and filled with external solution. The stimulating electrode was placed
in the granule cell layer ~10-50 µm from the Purkinje cell layer. A constant-voltage isolated stimulator (Digitimer, Hertfordshire, UK)
was used to supply a 20-100 µsec pulse of 10-99 mV. Pipette position and stimulus intensity were adjusted until the current necessary to produce an all-or-none response was minimized. Climbing fiber failures were complete, indicating that there was no
contamination by parallel fiber synapses. Synaptic currents were
filtered at 2 kHz and digitized at 10 kHz, and patch currents were
filtered at 5 kHz and digitized at 30-50 kHz. Synaptic and patch
currents were recorded with an Axopatch 1C (Axon Instruments, Foster
City, CA). A four-barreled glass pipette mounted on a piezoelectric bimorph (Morgan Matroc, Bedford, OH) was used for agonist application to outside-out patches (Tong and Jahr, 1994). Data were acquired using
Axobasic software (Axon Instruments) and Igor Pro software (Wavemetrics, Lake Oswego, OR). Data analysis was performed using Axograph 4.6, and results were compiled and statistical analysis was
performed using Microsoft Excel (Microsoft, Redmond, WA) and GraphPad
(GraphPad Software, San Diego, CA). All experiments were performed at
34-36°C using an in-line heating device (Warner Instruments, Hamden,
CT). Values are given as mean ± SEM, and confidence limits were determined using Student's t tests.
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Results |
Bafilomycin A1 and concanamycin A reduce the frequency
and amplitude of PC mEPSCs
If high Pr (Dittman and Regehr, 1998; Silver et al., 1998)
results in high occupancy of AMPARs at CF-PC synapses, then
AMPAR-mediated EPSCs should be relatively insensitive to small changes
in the glutamate concentration transient in the synaptic cleft. We used two blockers of the vacuolar-type ATPase, Baf and Conca (Drose and
Altendorf, 1997), to reduce glutamate concentrations in synaptic vesicles (Zhou et al., 2000) by abolishing the proton and
voltage gradient required by vesicular glutamate transporters (Carlson and Ueda, 1990; Maycox et al., 1990). First, we tested the efficacy of
Baf or Conca to reduce the glutamate concentration in CF vesicles by
comparing the amplitude and frequency of mEPSCs recorded in PCs in
control P8-P10 cerebellar slices with those that had been incubated in
Baf (2 µM) or Conca (5 µM) for  1 hr.
Because the frequency of mEPSCs in PCs is very low, 1 µM
pardaxin (Bergles et al., 2000) was used to induce
asynchronous release. Both Baf and Conca resulted in a
significant decrease in pardaxin-induced mEPSC amplitude as
demonstrated by a leftward shift in the cumulative amplitude
distribution (Fig.
1A,B).
Incubation with Baf or Conca also dramatically decreased
pardaxin-stimulated mEPSC frequency (Fig.
1A,C) (control, 4.86 ± 0.74 Hz vs Baf, 0.426 ± 0.084 Hz; n = 3;
p < 0.0001; control, 3.03 ± 0.59 Hz vs Conca,
0.38 ± 0.055 Hz; n = 4; p < 0.003), consistent with the majority of mEPSC amplitudes decreasing to
undetectable levels (Zhou et al., 2000).
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Figure 1.
Bafilomycin A1 reduces the amplitude
and frequency of induced mEPSCs from Purkinje cells. A,
Representative consecutive traces from a cerebellar Purkinje cell,
showing pardaxin- (1 µM) induced mEPSCs, in control
(A1) and in slices pretreated with
bafilomycin A1 (2 µM) for 1 hr
(A2). Recorded in the presence of TTX
(1 µM) and picrotoxin (100 µM).
Holding potential, 70 mV. B, Cumulative probability
distribution of mEPSC amplitude before and after bafilomycin
A1 treatment. C, Frequency of mEPSCs before
and after bafilomycin A1 treatment.
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Bafilomycin A1 and concanamycin A increase paired-pulse
depression at CF-PC synapses
Paired-pulse depression at CF-PC synapses has been shown to
result from a lower Pr after the second stimulus than that after the
first stimulus (Dittman and Regehr, 1998; Hashimoto and Kano, 1998;
Silver et al., 1998). If AMPAR occupancy follows Pr (Silver et al.,
1998; Wadiche and Jahr, 2001), then we would expect that occupancy
during the first EPSC (EPSC1) would be higher than that during the
second EPSC (EPSC2) and thus that EPSC1 may be less sensitive to
changes in the glutamate concentration transient than EPSC2. Addition
of Baf (2 µM) or Conca (5 µM) to the
perfusate resulted in a reduction in the amplitudes of both EPSCs over
time (Fig. 2) (interstimulus interval, 50 msec). However, EPSC2 decreased sooner and to a greater extent than
EPSC1 (Fig. 2B,C) (time to 10% reduction in
amplitude in Baf, 320 ± 20 sec EPSC1 vs 183 ± 14 sec EPSC2;
p = 0.005; n = 3; in Conca, 535 ± 44 sec vs 451 ± 34 sec; p = 0.006;
n = 5), and, thus, Baf and Conca application caused a
decrease in the paired-pulse ratio (EPSC2/EPSC1; control, 0.72 ± 0.03 vs Baf, 0.36 ± 0.005; n = 3; control,
0.74 ± 0.01 vs Conca, 0.32 ± 0.04; n = 5).
This differential inhibition suggests that AMPARs are highly occupied
after the first stimulus. The much larger paired-pulse depression seen
after 30 min in Baf or Conca indicates that presynaptic
depression of release is far greater than predicted
from the paired-pulse ratio of EPSCs in control conditions. In addition
to the effects on paired-pulse depression, Baf treatment tended to
accelerate the decay of EPSC1 (control, 3.17 ± 0.51 msec vs Baf,
2.27 ± 0.20 msec, weighted decay time constants;
p = 0.07; n = 3), suggesting that the
large bolus of glutamate released by the first stimulus temporarily overwhelms clearance mechanisms.
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Figure 2.
Bafilomycin A1 reduces climbing
fiber-evoked EPSC amplitude and increases paired-pulse depression in
Purkinje cells. A, Sample traces of CF-evoked EPSCs at
0, 20, 25, and 30 min after addition of bafilomycin A1 (2 µM) to perfusate. Each trace is an average
of at least five sweeps. Holding potential, 20 mV. B,
Recordings in A scaled to the peak of the first EPSC at
time 0. C, Time course of the effect of bafilomycin
A1 on the normalized amplitude of EPSC1
(filled circles) and EPSC2 (open
circles).
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AMPARs expressed by Bergmann glial cells are more sensitive to
changes in Pr than those of PCs
Climbing fiber synapses are surrounded by sheets of BG membrane
(Palay and Chan-Palay, 1974), which express both AMPARs (Baude et
al., 1994) and glutamate transporters (Rothstein et al., 1994; Chaudhry
et al., 1995). Stimulation of CFs activates currents in BGs mediated by
both of these glutamate receptors (Bergles et al., 1997). Because the
peak concentration of glutamate reaching the BG AMPARs is ~200
µM (Dzubay and Jahr, 1999), it seems unlikely that
receptor saturation occurs. Thus, BG AMPARs may be more sensitive to
changes in the amount of glutamate released from CF synapses than those
in the PC postsynaptic membrane. To test this, responses of both cell
types to CF stimulation were compared in control and in the presence of
700 µM Co2+ to inhibit
presynaptic voltage-dependent Ca2+
currents and thereby decrease multivesicular release and the resulting
glutamate concentration transients. Co2+
decreased the CF-PC EPSC by only 7.4 ± 2.5% (n = 5), whereas the CF-BG AMPAR component (in CTZ, 200 µM and TBOA, 50 µM; see below) was inhibited by 49.7 ± 7.2% (n = 7)
(Fig. 3A). Because BG AMPARs
but not those of PCs are calcium permeable (Geiger et al., 1995), the
differential sensitivity could result from channel block of BG AMPARs
by Co2+. However, AMPAR currents evoked in
outside-out patches from BG by 10 mM glutamate
were not significantly altered by 700 µM
Co2+ (Fig. 3B). These results,
like those with Baf or Conca, are consistent with the proposal that
multivesicular release creates a large transient concentration of
glutamate in the cleft that results in the occupation of practically
all postsynaptic PC AMPARs. Because glutamate diffuses to BG AMPARs,
dilution in the extrasynaptic space is apparently sufficient to
decrease the glutamate concentration transient to nonsaturating
levels.
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Figure 3.
Reduction of climbing fiber release
probability has different effects on response amplitude of Purkinje
cells and Bergmann glia cells. A, Average reduction in
response amplitude by CoCl2 (700 µM) in
Purkinje cells and Bergmann glia cells. Inset,
Recordings from a Purkinje cell and a Bergmann glial cell before and
during CoCl2 (700 µM) application. Recordings
are averages of at least five sweeps. Bergmann glial responses were
recorded in the presence of CTZ (200 µM) and TBOA (50 µM). Holding potential, 80 mV for the Bergmann glial
cell and 20 mV for the Purkinje cell. *p < 0.05;
**p < 0.005. B, Addition of
CoCl2 (700 µM) had no significant effect on
Bergmann glial outside-out patch responses to 10 mM
glutamate in 200 µM CTZ.
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Paired-pulse depression is larger in BGs than in PCs
If BG glutamate receptors are more sensitive monitors of changes
in presynaptic release from CFs than PCs, we would expect them to
report greater paired-pulse depression than PCs in normal conditions. In fact, paired-pulse depression (150 msec
interstimulus interval) was dramatically greater in BGs than in PCs
(Fig. 4A,D). In
these recordings, the two components of the CF-BG response were
separated pharmacologically. The AMPAR-mediated component was isolated
by subtracting the synaptic transporter current (STC) (10 µM NBQX) (Fig. 4B) from the
control currents (Bergles et al., 1997; Dzubay and Jahr, 1999). The
paired-pulse ratio of the CF-BG AMPAR-mediated current was 0.14 ± 0.01 (Fig. 4A) (n = 12). This is a
sixfold larger depression than that of the CF-PC EPSC (Fig. 4D) (0.83 ± 0.025; n = 5).
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Figure 4.
Climbing fiber stimulation elicits both AMPA
receptor and glutamate transporter currents in Bergmann glial cells.
A, Comparison of the dual-component Bergmann glial
response (Control) and the isolated AMPA
receptor-mediated response (Subtracted) by subtraction
of the recording in B (interstimulus interval, 150 msec). B, CF-evoked transporter current from the same
cell as in A in the presence of NBQX (10 µM). C, Current remaining after the
addition of TBOA (50 µM). D, CF-PC EPSCs
evoked by stimuli at a 150 msec interval. All traces are
averages of at least five sweeps. Holding potential, 80 mV for the
Bergmann glial cell and 20 mV for the Purkinje cell.
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The CF-BG STC (Fig. 4B) accounted for less of the
first CF-BG current than the second (16 ± 2 and 39.5 ± 3.5%, respectively), indicating that the conductances mediated by BG
AMPA receptors and transporters do not have the same sensitivity to the
same presynaptic release events. In part, this may be the result of differential access of glutamate to AMPA receptors and transporters and
to nonlinear dose-response relationships (see below). The paired-pulse
ratio of the CF-BG STCs, as measured by their charge transfer, should
not be affected by a nonlinear dose-response relationship, because the
stoichiometry of uptake is fixed in the absence of permeable anions;
each glutamate molecule taken up results in two charges crossing the
membrane (Zerangue and Kavanaugh, 1996). Thus, the integral of the
current in time should represent the number of glutamate molecules
taken up by the BG. When expressed as charge transfer, the paired-pulse
ratio for the CF-BG STC was 0.59 ± 0.06 (range, 0.34-0.91;
n = 9), falling between the paired-pulse ratio of the
CF-PC EPSC (0.83) and the CF-BG AMPAR response (0.14). The
paired-pulse depression of the CF-BG STC amplitudes (0.51 ± 0.03; n = 9) was greater than that of charge transfer
because the decay time of the first STC (8.5 ± 0.7 msec) was
longer than that of the second STC (6.6 ± 0.7; n = 7; p < 0.01). The difference in these decay times
suggests that the larger glutamate transient after the first stimulus
takes longer to clear than that after the second stimulus and also
suggests that clearance mechanisms are taxed more by large glutamate
transients (see below).
Cyclothiazide decreases paired-pulse depression
The surprisingly large paired-pulse depression of the CF-BG AMPAR
response may result not only from depression of presynaptic release but
also from the nonlinear dose-response relationship of these AMPARs
(Dzubay and Jahr, 1999). CTZ (200 µM), a blocker of BG
AMPAR desensitization that also increases the apparent affinity of
AMPARs (Patneau et al., 1993; Yamada and Tang, 1993; Partin et al.,
1994, 1996; Koh et al., 1995; Dzubay and Jahr, 1999), significantly
increased the first and second CF-BG AMPAR responses to paired
stimulation by 6.43 ± 0.25 and 8.89 ± 0.42-fold,
respectively, as well as the paired-pulse ratio (0.18 ± 0.01;
n = 12). This effect is consistent with the action of
CTZ to potentiate AMPAR responses evoked by lower concentrations of
glutamate more than AMPAR responses evoked by higher concentrations
(Dzubay and Jahr, 1999). In addition, if AMPAR desensitization
contributed to paired-pulse depression at an interstimulus interval of
150 msec, then CTZ would inhibit this effect.
Glutamate transporters shield BG AMPARs
The paired-pulse depression of the CF-BG AMPAR response is also
influenced by surrounding glutamate transporters. In the presence of
CTZ, inhibition of glutamate transporters (TBOA, 50 µM)
increased the amplitude and slowed the decay phase of both the first
and second CF-BG AMPAR responses (Fig.
5A). However, the amplitude of
the second response was increased more (57%) than the first (23%),
resulting in an additional decrease in paired-pulse depression (control, 0.20 ± 0.017 vs TBOA, 0.26 ± 0.023;
p = 0.003; n = 9) (Fig. 5). This result
suggests that transport is overwhelmed by the large glutamate transient
resulting from the first CF stimulus and that this allows activation of
more BG AMPA receptors. The depressed release after the second CF
stimulus results in a smaller glutamate transient that is more
effectively curtailed by transport and causes less BG AMPA receptor
activation.
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Figure 5.
Inhibition of glutamate transporters decreases
paired-pulse depression of CF-Bergmann glial responses.
A, Paired-pulse depression of CF-evoked Bergmann glial
responses was decreased by TBOA (50 µM) in the presence
of CTZ (200 µM). Holding potential, 78 mV.
Traces are an average of at least five sweeps.
B, Average effects of TBOA on peak amplitudes and
paired-pulse ratio (PPR; response 1/response 2) of six
Bergmann glial cells.
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Glutamate transporters shield presynaptic metabotropic
glutamate receptors
The fact that transporters apparently shield BG AMPARs suggests
that they might shield other extrasynaptic glutamate receptors (Brasnjo
and Otis, 2001) that could alter paired-pulse depression. Activation of
mGluRs by exogenous agonists suppresses transmission at CF synapses by
a presynaptic mechanism (Hashimoto and Kano, 1998). However, these
presynaptic mGluRs are not activated by release of endogenous glutamate
(Hashimoto and Kano, 1998). In keeping with these results, CF-BG
responses recorded in the presence of CTZ (200 µM) were
not altered by inhibitors of mGluRs (Fig. 6A). However, if
glutamate transporters were first inhibited by TBOA, then paired-pulse
depression was decreased by mGluR antagonists at an interstimulus
interval of 150 msec but not 40 msec (Fig. 6B)
(control, 0.36 ± 0.04 vs mGluR antagonist, 0.43 ± 0.04;
p < 0.0001; n = 5). The response to
the first stimulus was not altered by mGluR antagonists, indicating
that basal levels of glutamate are too low to cause significant mGluR
activation. However, with glutamate transport inhibited, the transient
elevation of glutamate after release is sufficient to activate
presynaptic mGluRs, causing a decrease in subsequent release. The
linkage between mGluRs and their effects on the release process
apparently requires >40 msec.
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Figure 6.
Glutamate transporters shield presynaptic
metabotropic glutamate receptors. A, mGluR antagonists
(500 µM CPPG and 1 mM MCPG) had no effect on
the paired-pulse ratio (PPR) at an interstimulus
interval (ISI) of 40 msec in the presence
(filled circles; n = 5;
n = 2 for washout) or absence (open
circles; n = 5) of TBOA (50 µM). CTZ at 200 µM was present in both
conditions. Insets, Superimposed average currents, with
or without TBOA (50 µM), in the absence and presence of
mGluR antagonists. B, mGluR antagonists (500 µM CPPG and 1 mM MCPG) had no effect on the
paired-pulse ratio at an interstimulus interval of 150 msec in the
absence (open circles; n = 5) of
TBOA (50 µM), but, in the presence of TBOA, the
paired-pulse ratio was significantly increased (filled
circles; n = 5; n = 2 for washout). *p < 0.05. Insets,
Superimposed average currents, with or without TBOA (50 µM), in the absence and presence of mGluR antagonists.
Recordings from the same cells as in A. Each
trace is an average of at least five sweeps.
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Discussion |
AMPA receptor saturation at the climbing fiber-Purkinje
cell synapse
The frequency-dependent depression of CF-PC EPSCs has been
attributed to presynaptic reduction of transmitter release (Dittman and
Regehr, 1998; Hashimoto and Kano, 1998; Silver et al., 1998). Although
this is clearly true, our results show that paired-pulse depression of
release is greater than that reported by the EPSCs. This underestimate
of depression results from the high occupancy of postsynaptic AMPARs
caused by multivesicular release (Wadiche and Jahr, 2001). In these
conditions, the number of postsynaptic receptors, rather than the size
of the glutamate concentration transient in the cleft, limits the size
of the EPSC. Subsequent stimuli result in less multivesicular release,
lower receptor occupancy, and therefore a more accurate measure of the
glutamate concentration transient by the AMPAR EPSCs.
Multivesicular release overwhelms clearance mechanisms
The results of several experiments suggest that multivesicular
release results in such a large glutamate transient that clearance from
climbing fiber synapses is retarded. This is particularly apparent in
comparisons of the characteristics of EPSCs evoked by paired stimuli
40-150 msec apart. First, in control conditions, the decay time course
of the first EPSC is slower than that of the second, and this cannot be
attributed to voltage-clamp errors (Wadiche and Jahr, 2001). Second, as
Baf washed into the slice, the decay phase of EPSC1 tended to become
faster, because the quantity of glutamate in each vesicle presumably
decreases. Similar effects are found by lowering release probability by
decreasing external calcium (Wadiche and Jahr, 2001). Third,
low-affinity competitive antagonists of AMPARs, which are more
effective at blocking low rather than high concentrations of glutamate,
speed the decay of the first EPSC (Wadiche and Jahr, 2001). Fourth, Bergmann glial AMPAR responses to paired climbing fiber stimuli are
differentially altered by blocking transporters. The amplitude of the
first response is increased only slightly, whereas the second is
increased by >50%. This suggests that transporters are overwhelmed by
the first glutamate transient, whereas the transient resulting from the
second stimulus is readily curtailed by transporters. This finding is
corroborated by the comparison of paired-pulse depression of the
Bergmann glial AMPARs and the STC. Whereas the former displays very
large depression, the depression of the STC is less. Because all
glutamate released must eventually be taken up, the Bergmann glial
transporter current may offer a less biased measure of presynaptic
function. Indeed, the decay time constant of the first STC of a pair is
longer than that of the second, indicating that clearance can be
overwhelmed if enough glutamate is released.
In addition to shielding AMPARs expressed by Bergmann glia, glutamate
transporters apparently protect presynaptic mGluRs from released
transmitter. Similar results indicate that postsynaptic mGluRs are also
protected by glutamate transporters at parallel fiber synapses (Brasnjo
and Otis, 2001). Exogenous agonists of mGluRs have been shown to
decrease release from climbing fibers (Hashimoto and Kano, 1998), but
physiological activation of these receptors has not been shown. In the
presence of glutamate transporter blockers, we show that paired-pulse
depression of the BG AMPAR response is decreased by metabotropic
receptor antagonists without affecting the amplitude of the first
response. This indicates that the glutamate released by the first
stimulus is responsible for this effect rather than an elevation of
ambient glutamate. Because this phenomenon was observed with
interstimulus intervals of 150 msec but not 40 msec, it is consistent
with activation of a G-protein-coupled receptor, because the downstream
effectors of such receptors require a variable period after receptor
binding to assert their actions. Although the decrease of paired-pulse depression within 150 msec is rapid, it is in line with 
subunit-linked effects (Hille, 2001). Whether these mGluRs are ever
activated in vivo remains to be determined.
Physiology of receptor saturation
We suggest that multivesicular release results in the
near-saturation of AMPA receptors expressed by Purkinje cells at
climbing fiber synapses. What is the consequence of this seemingly
inappropriate waste of transmitter? By increasing receptor occupancy,
multivesicular release will augment charge transfer in the Purkinje
cell after climbing fiber stimulation and ensure that the Purkinje cell
will fire a complex spike. However, complex spikes generally are evoked by climbing fiber action potentials within a few tens of milliseconds of previous climbing fiber spikes, when release probability is far
lower. Because the frequency of climbing fiber action potentials reaches only 8 Hz (125 msec period), the safety factor at this synapse
is rather high. Indeed, it may be that the usefulness of multivesicular
release and the resulting underreporting of presynaptic depression is
to keep this safety factor high. Although presynaptic release is
dramatically lower at 150 msec after a previous stimulus, as judged by
paired-pulse depression in Bergmann glia, the Purkinje cell EPSC in
control conditions is decreased by only ~20%. This nonlinear
relationship between release and postsynaptic response ensures the
fidelity of transmission at this synapse, which is thought to provide
essential error correction capabilities to the motor system.
Whether the postsynaptic responses at other synapses similarly
underestimate changes in presynaptic release is uncertain. However,
recent reports that the incidence of multivesicular release follows Pr
at hippocampal excitatory synapses (Oertner et al., 2002) suggest that,
even at synapses that have much lower Pr than those of the climbing
fibers, postsynaptic responses may not faithfully report presynaptic
release. In addition, at some inhibitory synapses, both multivesicular
release (Auger et al., 1998) and high postsynaptic receptor occupancy
(Auger and Marty, 1997; Auger et al., 1998; Hajos et al., 2000)
can occur, the same conditions that lead to underreporting changes in
release at the climbing fiber-Purkinje cell synapse.
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FOOTNOTES |
Received July 29, 2002; revised Sept. 19, 2002; accepted Oct. 15, 2002.
This work was supported by National Institutes of Health Grant NS40056
and Human Frontier Science Program Grant 119/2000. We thank the
members of the Jahr laboratory for discussions and helpful comments.
Correspondence should be addressed to Craig E. Jahr, Vollum Institute,
Oregon Health and Science University, L474, 3181 S.W. Sam Jackson Park
Road, Portland, OR 97201-3098. E-mail: jahr{at}ohsu.edu.
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TOP
ABSTRACT
Introduction
