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Cellular/Molecular
Two Loci of Expression for Long-Term Depression at
Hippocampal Mossy Fiber–Interneuron Synapses
Saobo Lei and Chris J. McBain
Laboratory of Cellular and Synaptic Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda,
Maryland 20892-4495
Two distinct forms of long-term depression (LTD) exist at mossy fiber synapses between dentate gyrus granule cells and hippocampal
CA3 stratum lucidum interneurons. Although induction of each form of LTD requires an elevation of postsynaptic intracellular Ca
2⫹
,at
Ca
2⫹
-impermeable AMPA receptor (CI-AMPAR) synapses, induction is NMDA receptor (NMDAR) dependent, whereas LTD at Ca
2⫹
-
permeable AMPA receptor (CP-AMPAR) synapses is NMDAR independent. However, the expression locus of either form of LTD is not
known. Using a number of criteria, including the coefficient of variation, paired-pulse ratio, AMPA–NMDA receptor activity, and the
low-affinity AMPAR antagonist
␥
-D-glutamyl-glycine, we demonstrate that LTD expression at CP-AMPAR synapses is presynaptic and
results from reduced transmitter release, whereas LTD expression at CI-AMPAR synapses is postsynaptic. The N-ethylmaleimide-
sensitive fusion protein–AP2-clathrin adaptor protein 2 inhibitory peptide pep2m occluded LTD expression at CI-AMPAR synapses but
not at CP-AMPAR synapses, confirming that CI-AMPAR LTD involves postsynaptic AMPAR trafficking. Thus, mossy fiber innervation of
CA3 stratum lucidum interneurons occurs via two parallel systems targeted to either Ca
2⫹
-permeable or Ca
2⫹
-impermeable AMPA
receptors, each with a distinct expression locus for long-term synaptic plasticity.
Key words: AMPA; depression; hippocampus; interneuron; synapse; glutamate
Introduction
The mossy fiber axons of dentate gyrus granule cells innervate
CA3 pyramidal neurons via large, complex boutons with multi-
ple release sites, whereas inhibitory interneurons of the CA3 stra-
tum lucidum receive innervation via small en passant or filopo-
dial extensions that often comprise a single release site (Acsady et
al., 1998). AMPA receptors (AMPARs) at mossy fiber–interneu-
ron synapses comprise a continuum that ranges from glutamate
receptor 2 (GluR2)-lacking, Ca
2⫹
-permeable (CP)-AMPAR syn-
apses to GluR2-containing, Ca
2⫹
-impermeable (CI)-AMPAR
synapses (Toth et al., 2000; Bischofberger and Jonas, 2002; Law-
rence and McBain, 2003). NMDA receptor (NMDAR) expres-
sion also differs between the two extremes of this continuum,
which in part provide distinct mechanisms for mossy fiber exci-
tation of postsynaptic inhibitory interneurons (Lei and McBain,
2002).
High-frequency mossy fiber stimulation induces long-term
potentiation at CA3 pyramidal neuron synapses (Harris and Cot-
man, 1986; Zalutsky and Nicoll, 1990; Nicoll and Malenka, 1995;
Yeckel et al., 1999), whereas, the same stimulation paradigm in-
duces two forms of long-term depression (LTD) at mossy fiber–
CA3 interneuron synapses. Each form of LTD is associated with a
distinct AMPAR synapse type (Toth et al., 2000; Lei and McBain,
2002). Induction of LTD at CI-AMPAR synapses requires activa-
tion of NMDARs and an elevation of postsynaptic Ca
2⫹
. In con-
trast, although LTD at CP-AMPAR synapses also requires a
postsynaptic Ca
2⫹
elevation, induction is NMDAR independent
(Laezza et al., 1999). This differential dependence of LTD induc-
tion on NMDAR activation likely arises as a consequence of the
dissimilar NMDAR:AMPAR ratios present at either synapse and
the resulting sources of Ca
2⫹
(Bischofberger and Jonas, 2002; Lei
and McBain, 2002).
In the hippocampus, LTD expression at principal neuron syn-
apses is typically associated with a reduction in presynaptic trans-
mitter release, a reduction in postsynaptic AMPAR density, or
sensitivity to a fixed concentration of glutamate (Sheng and Kim,
2002; Song and Huganir, 2002). At hippocampal interneuron
synapses, the expression locus for either form of LTD is unex-
plored, but evidence suggests that LTD at mossy fiber– dentate
gyrus basket cell synapses has a presynaptic expression locus (Alle
et al., 2001). In the present study, we investigated the expression
locus of both forms of mossy fiber–interneuron LTD using three
approaches. First, we used conventional methods, including
measuring the coefficient of variation (CV), the paired-pulse ra-
tio (PPR), and NMDAR activity, to assess potential changes in
presynaptic transmitter release probability. Then we probed the
LTD expression locus further using a low-affinity, competitive
AMPAR antagonist,
␥
-D-glutamyl-glycine (
␥
-DGG), to detect
potential changes in transmitter profile in the cleft (Liu et al.,
1999; Wadiche and Jahr, 2001; Shen et al., 2002). Finally, inde-
Received Oct. 14, 2003; revised Dec. 17, 2003; accepted Jan. 6, 2004.
C.J.M. was supported by the National Institute of Child Health and Human Development Intramural Research
Program and the Human Frontiers Science Program (C.J.M.). We thank Drs. John Isaacs and Jeremy Henley for
discussions concerning the NSF inhibitory peptide and Drs. Josh Lawrence and Jeff Diamond for their constructive
criticism of this manuscript.
Correspondence should be addressed to Chris J. McBain, Laboratory of Cellular and Synaptic Neurophysiology,
National Institutes of Health, Room 5A72, Building 49, Convent Drive, Bethesda, MD 20892. E-mail:
mcbainc@mail.nih.gov.
DOI:10.1523/JNEUROSCI.4645-03.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/242112-10$15.00/0
2112 •The Journal of Neuroscience, March 3, 2004 •24(9):2112–2121
pendent confirmation of these results came from experiments
demonstrating that NMDAR-dependent LTD was occluded by
postsynaptic infusion of an N-ethylmaleimide-sensitive fusion
protein (NSF)–AP2-clathrin adaptor protein inhibitory peptide,
pep2m (KRMKVAKNAQ) (Lu¨scher et al., 1999; Lu¨thi et al.,
1999; Lee et al., 2002), suggesting a mechanism involving
postsynaptic AMPAR trafficking at CI-AMPAR synapses. In con-
trast, pep2m neither changed the amplitude of control synaptic
transmission nor occluded LTD expression at CP-AMPAR syn-
apses, further supporting a presynaptic expression locus.
Materials and Methods
Hippocampal slice preparation. Transverse hippocampal slices (300
m)
were obtained from 16- to 20-d-old Sprague Dawley rats, as described
previously (Lei and McBain, 2002, 2003). Briefly, rats were deeply anes-
thetized with isoflurane and rapidly decapitated. The brain was dissected
out in ice-cold saline solution that contained the following (in mM): 130
NaCl, 24 NaHCO
3
, 3.5 KCl, 1.25 NaH
2
PO
4
, 1.0 CaCl
2
, 5.0 MgCl
2
, and 10
glucose, saturated with 95% O
2
and 5% CO
2
, pH 7.4. All animal proce-
dures conformed to National Institutes of Health animal welfare guide-
lines. Slices were incubated in the same solution for periods of at least 1 hr
at room temperature before their use.
Electrophysiology. Whole-cell patch-clamp recordings were made at
room temperature (⬃22–24°C) from visually identified interneurons
located within the stratum lucidum of CA3 using an Axopatch 200A
amplifier (Axon Instruments, Foster City, CA) in voltage-clamp mode
(Lei and McBain, 2002). Recording electrodes were filled with the follow-
ing solution (in mM): 100 Cs-gluconate, 0.6 EGTA, 5 MgCl
2
, 8 NaCl, 2
ATP
2
Na, 0.3 GTPNa, 40 HEPES, 0.4 spermine, and 1 QX-314, (N-(2,6-
dimethylphenylcarbomoylmethyltriethylammonium bromide), pH 7.2–
7.3. Biocytin (0.2%) was routinely added to the recording electrode so-
lution to allow post hoc morphological processing of recorded cells.
Unless otherwise stated, the extracellular solution comprised the follow-
ing composition (in mM): 130 NaCl, 24 NaHCO
3
, 3.5 KCl, 1.25 NaHPO
4
,
1.5 MgCl
2
, 2.5 CaCl
2
, 10 glucose, and 0.1 bicuculline methobromide,
saturated with 95% O
2
and 5% CO
2
, pH 7.4. At the end of all recordings,
the group II metabotropic GluR (mGluR) agonist (2S,2⬘R,3⬘R)-2-(2⬘,3⬘-
dicarbocyclopropyl) glycine (DCG-IV) (1
M) was added to the perfus-
ate to confirm that synaptic events were mossy fiber in origin (Kamiya et
al., 1996; Toth and McBain, 1998; Lawrence et al., 2004). Series resistance
was rigorously monitored by the delivery of 5 mV voltage steps after each
evoked EPSC. Experiments were discontinued if the series resistance
changed by ⬎15%. LTD of evoked synaptic transmission was induced by
a high-frequency stimulation (HFS) paradigm (100 Hz for 1 sec, repeated
three times at an interval of 10 sec).
Synaptic responses were evoked by low-intensity stimulation (dura-
tion, 80
sec; intensity, 40 –80
A) of dentate gyrus granule cells or by
placement directly in the stratum lucidum of the CA3 hippocampus via a
constant-current isolation unit (A360; World Precision Instruments,
Sarasota, FL) connected to a patch electrode filled with oxygenated ex-
tracellular solution. For each experiment, the stimulus intensity was set
to the lowest value that reliably evoked a multifiber EPSC waveform with
minimal failures; no attempt was made to stimulate single fiber re-
sponses. Synaptic responses were included in the analysis if the rise times
and decay time constants were monotonic and possessed no apparent
polysynaptic waveforms. In most experiments, a solution containing (in
M) 100 dl-APV, 100 bicuculline, and 10 DNQX was added at the end of
the experiment to record the isolated stimulus artifact, which was then
averaged and subtracted from synaptic records to obtain stimulus
artifact-free records of EPSCs for accurate amplitude measurement. In
those experiments in which this was not possible, the stimulus artifact
was blanked from the trace for clarity. Rise times were measured as
20 –80% duration. Current traces shown in the figures are the averages of
10 –15 EPSCs recorded at the corresponding time points indicated in the
figures.
In experiments designed to monitor changes in NMDAR activity be-
fore and after LTD expression (see Fig. 2 A1,B1), we initially recorded the
composite-evoked EPSC mediated by both NMDARs and AMPARs at a
holding potential of ⫹40 mV for 1 min (20 EPSCs) and then switched the
holding potential to ⫺60 mV to record the basal AMPA EPSC. LTD was
then induced by HFS, and after a period to allow stabilization of plastic-
ity, the composite EPSCs mediated by both NMDARs and AMPARs were
again recorded at ⫹40 mV. The Ca
2⫹
-permeable nature of AMPARs was
then identified by bath application of philanthotoxin (Toth et al., 2000;
Lei and McBain, 2002). A time point 50 msec after the end of the stimu-
lation artifact was chosen for measurement of the NMDAR component,
because at this time point, NMDARs were still open, but AMPARs were
almost completely deactivated.
Data analysis. The rectification index (RI) of the evoked EPSC was
generated from averaged (20–30 traces) AMPA EPSC amplitudes at a
series of holding potentials between ⫺60 and ⫹40 mV (Lei and McBain,
2002). EPSC amplitudes recorded at negative holding potentials (from
⫺60 to ⫺20 mV) were fit by a linear regression. The RI of the I–V
relationship was then defined as the ratio of the actual current amplitude
at ⫹40 mV to the predicted linear value at ⫹40 mV (Liu and Cull-Candy,
2000). The PPR was calculated as the mean P2/mean P1 (Kim and Alger,
2001), where P1 was the amplitude of the first evoked current and P2 was
the amplitude of the second synaptic current, measured after subtraction
of the remaining P1 “tail”current. The CV of synaptic currents was
calculated as the SD of current amplitude divided by mean (x)ofthe
current amplitude (CV ⫽SD/x).
Data are presented as means ⫾SEM and, unless stated otherwise, were
analyzed using a paired Student’sttest; pvalues are reported throughout
Results.
Chemicals.
␥
-DGG was purchased from Tocris (Ellisville, MO).
Pep2m and pep-⌬A849-Q853 (Lee et al., 2002) were synthesized by
Sigma (St. Louis, MO) (purity, ⬎95%). All other chemicals were prod-
ucts of Sigma.
Results
LTD induction at mossy fiber–CP-AMPAR and –CI-AMPAR in-
terneuron synapses requires postsynaptic calcium elevation (Lei
and McBain, 2002). However, the locus and mechanism(s) of
LTD expression at either synapse type are completely unex-
plored. In the first series of experiments, we used conventional
methods of analysis to explore the presynaptic or postsynaptic
locus of each type of mossy fiber–interneuron LTD.
Mossy fiber EPSCs were evoked (V
hold
⫽⫺60 mV, 0.33 Hz)
by placement of a stimulating electrode in the dentate gyrus gran-
ule cell layer or in the stratum lucidum (Toth and McBain, 1998;
Lei and McBain, 2002). The RI of AMPARs was used to identify
CI-AMPAR and CP-AMPAR synapses using criteria established
previously (Toth and McBain, 1998; Lei and McBain, 2002). We
defined AMPA EPSCs possessing an RI of ⬎0.7 as CI-AMPAR
synapses and those with an RI of ⬍0.3 as CP-AMPAR synapses.
At the end of all recordings, the group II mGluR agonist DCG-IV
(1
M) was added to the perfusate to reduce evoked synaptic
events confirming their mossy fiber origin (Kamiya et al., 1996;
Lei and McBain, 2002; Lawrence et al., 2004). Although biocytin
filling confirmed that all cells recovered were stratum lucidum
interneurons, attempts to correlate the presence of CI-AMPAR
LTD or CP-AMPAR LTD with cell morphology proved inconclu-
sive when based solely on the somatodendritic–axonal arbors.
Consistent with our previous observations (Toth and McBain,
1998), CP-AMPARs and CI-AMPARs were found on almost all
morphologically defined subgroups of the stratum lucidum
interneuron.
LTD expression does not change the rectification index of
AMPA receptors
In cerebellar interneurons, the subunit composition of postsyn-
aptic AMPARs is activity dependent, such that repetitive synaptic
activation of Ca
2⫹
-permeable AMPARs rapidly reduces Ca
2⫹
Lei and McBain •Two-Expression Loci for Interneuron LTD J. Neurosci., March 3, 2004 •24(9):2112–2121 • 2113
permeability and changes EPSC amplitude by the selective incor-
poration of GluR2-containing AMPARs (Liu and Cull-Candy,
2000, 2002). For the experiments that follow, it was important to
determine whether a similar mechanism exists at mossy fiber–
interneuron synapses (i.e., does LTD expression alter the relative
Ca
2⫹
permeability of evoked synaptic events?). HFS of mossy
fibers (100 Hz for 1 sec, repeated three times at an interval of 10
sec) induced an LTD (Fig. 1A,B) similar to that described previ-
ously (Maccaferri et al., 1998; Toth et al., 2000; Lei and McBain,
2002). After LTD expression, the RI was not significantly altered
at either CI-AMPAR (control, 0.94 ⫾0.05; LTD, 0.97 ⫾0.05; p⫽
0.67; n⫽9) (Fig. 1 A) or CP-AMPAR (control, 0.17 ⫾0.03; LTD,
0.21 ⫾0.02; p⫽0.40; n⫽6) (Fig. 1B) synapses, suggesting that
the molecular composition and Ca
2⫹
permeability of AMPARs
are unchanged after LTD expression.
LTD expression locus differentiated by CV and PPR analysis
To determine whether LTD expression at CI-AMPAR and CP-
AMPAR synapses shares a similar locus, we compared the CV and
the PPR of AMPA EPSCs before and after LTD expression. At
CI-AMPAR synapses, the CV was unaltered after LTD expression
(control, 0.37 ⫾0.04; LTD, 0.39 ⫾0.05; n⫽9; p⫽0.29) (data
not shown), whereas LTD expression at CP-AMPAR synapses
was associated with an increased CV (control, 0.34 ⫾0.06; LTD,
0.50 ⫾0.08; n⫽6; p⫽0.01), suggesting that LTD at CP-
AMPARs but not CI-AMPARs may be related to changes in pre-
synaptic transmitter release.
We next examined the PPR (mean P2/mean P1) (Kim and
Alger, 2001) before and after LTD expression. Consistent with
previous results (Toth et al., 2000), either paired-pulse depres-
sion (PPD) or paired-pulse facilitation (PPF) was detected at
both CI-AMPAR and CP-AMPAR synapses (Fig. 1C,D). In all
CI-AMPAR synapses tested, the mean PPR was not altered after
LTD expression (control, 1.04 ⫾0.11; LTD, 1.04 ⫾0.12; n⫽10;
p⫽0.79) (Fig. 1C). However, the mean PPR was significantly
increased after LTD expression at CP-AMPAR synapses (control,
1.03 ⫾0.12; LTD, 1.20 ⫾0.13; n⫽10; p⫽0.0001) (Fig. 1D).
LTD expression locus evaluated by NMDAR activity
If LTD expression at CP-AMPAR synapses arises from a reduc-
tion in presynaptic glutamate release, then a reduction in the
NMDAR-mediated EPSCs concomitant with changes in both CV
and PPR would also be expected. Representative experiments
from CI-AMPAR and CP-AMPAR synapses are shown in Figure
2, A1 and B1, respectively. Cells were held at ⫹40 mV to measure
the initial NMDAR-mediated component (measured at 50 msec
after the stimulus artifact). Cells were then voltage clamped at
⫺60 mV, and LTD was induced by the HFS paradigm. After LTD
expression (15–20 min), cells were again voltage clamped at ⫹40
mV to monitor changes in NMDAR EPSC. At both synapse types,
the NMDAR component was significantly reduced after LTD ex-
pression (Fig. 2 A1,A2,B1
,
B2). Similar to AMPA EPSCs, the CV of
NMDA EPSCs at CI-AMPAR synapses was not significantly al-
tered after LTD expression (Fig. 2A3), whereas the CV of NMDA
EPSCs at CP-AMPAR synapses was significantly increased (Fig.
2B3). Together, these data suggest that LTD expression at CP-
AMPAR synapses is consistent with a presynaptic locus, whereas
LTD expression at CI-AMPAR synapses may be postsynaptic.
Changes in synaptic glutamate concentration probed with the
low-affinity AMPAR antagonist
␥
-DGG
Next, we explored the nature of transmitter release at mossy fi-
ber–interneuron synapses. Taking advantage of the low-affinity,
competitive AMPAR antagonist
␥
-DGG, we probed for varia-
tions in transmitter glutamate concentration during paired-pulse
paradigms and after LTD expression at both synapse types (Liu et
al., 1999; Wadiche and Jahr, 2001; Shen et al., 2002). These ex-
periments are based on the observation that a certain fraction of
synaptic receptors continually exposed to
␥
-DGG and then sub-
jected to the rapid synaptic glutamate transient will replace
bound
␥
-DGG for glutamate because of the low affinity of
␥
-DGG. Consequently, the degree of
␥
-DGG inhibition can be
used to assess changes in the glutamate transient concentration in
the synaptic cleft during paired-pulse protocols (Wadiche and
Jahr, 2001) and after induction of LTD (Shen et al., 2002).
First, to confirm that changes in
␥
-DGG inhibition reliably
reflect alterations in cleft glutamate concentration, we changed
transmitter release probability by altering the extracellular Ca
2⫹
concentration ([Ca
2⫹
]
o
) and examined the corresponding
changes in
␥
-DGG (1 mM) inhibition of evoked EPSCs. When
[Ca
2⫹
]
o
was elevated from 2.5 to 4 mM, the magnitude of
␥
-DGG
inhibition was significantly reduced at both CI-AMPAR (39.9 ⫾
Figure 1. LTD expression does not change the RI of either CI-AMPAR or CP-AMPAR synapses
but increases PPR at CP-AMPAR synapses. A, B, The rectification index is unaltered after LTD
expression at both CI-AMPAR ( A) and CP-AMPAR ( B) synapses. Top, Control AMPAR-mediated
EPSCs evoked at different holding potentials (left) and their corresponding I–V relationship
(right). Middle, AMPAR EPSCs (left) and corresponding I–V curve (right) after LTD expression.
Bottom, Time course of LTD at each synapse. C, D, LTD expression does not change the PPR at
CI-AMPAR synapses ( C) but increases the PPR at CP-AMPAR synapses ( D). Top left, Paired EPSC
evoked by two stimuli at an interval of 50 msec before (thick) and after (thin) LTD expression.
Top right, EPSCs recorded before and after LTD expression normalized to the first EPSC peak.
Note the change in PPR at the CP-AMPAR synapse. Bottom, Pooled data for PPRs from different
synapses (open circles) and pooled mean data (filled circles). Note that LTD expression signifi-
cantly increases PPR only at CP-AMPAR synapses.
2114 •J. Neurosci., March 3, 2004 •24(9):2112–2121 Lei and McBain •Two-Expression Loci for Interneuron LTD
5.7 vs 62.0 ⫾3.1% of controls; n⫽5; p⫽0.003) (Fig. 3A,B) and
CP-AMPAR (47.5 ⫾2.4 vs 64.1 ⫾2.5% of controls; n⫽5; p⫽
0.002) (Fig. 3C,D) synapses. However, a similar increase in
[Ca
2⫹
]
o
failed to alter the magnitude of EPSC inhibition by the
high-affinity AMPAR antagonist DNQX (0.2
M) at either CI-
AMPAR (48.9 ⫾4.2 vs 45.9 ⫾3.8%; n⫽4; p⫽0.67) or CP-
AMPAR (51.2 ⫾5.3 vs 47.4 ⫾3.3%; n⫽3; p⫽0.53) synapses
(data not shown). Conversely, reducing [Ca
2⫹
]
o
from 2.5 to 0.5
mMsignificantly increased
␥
-DGG inhibition at both CI-AMPAR
(47.4 ⫾2.8 vs 22.6 ⫾2.7% of controls; n⫽5; p⫽0.004) and
CP-AMPAR (50.5 ⫾4.4% vs 21.6 ⫾3.8% of controls; n⫽4; p⫽
0.003) synapses (data not shown). Again, this manipulation
failed to change the percentage of inhibition by the high-affinity
antagonist DNQX at either CI-AMPAR (49.3 ⫾4.1 vs 50.2 ⫾
3.9%; n⫽3; p⫽0.89) or CP-AMPAR (53.1 ⫾4.4 vs 48.8 ⫾3.7%;
n⫽3; p⫽0.76) synapses. The above results are consistent with
previous reports (Liu et al., 1999; Wadiche and Jahr, 2001; Shen et
al., 2002) and suggest that the magnitude of inhibition by the
low-affinity antagonist
␥
-DGG (but not the high-affinity antag-
onist) is sensitive to variation in synaptic glutamate concentra-
tion at both CP-AMPAR and CI-AMPAR synapses.
We next tested whether the magnitude of
␥
-DGG block de-
tected changes in synaptic cleft glutamate concentration during
paired-pulse stimulation. Because interneuron synapses possess
either facilitation or depression in response to paired-pulse stim-
ulation (Fig. 1C,D) (Toth et al., 2000), we treated the data sepa-
rately. For CI-AMPAR synapses displaying PPD, application of
␥
-DGG (1 mM) inhibited EPSC
1
by 47.2 ⫾1.8% and EPSC
2
by
57.8 ⫾0.7% (n⫽5; p⫽0.001) (Fig. 4A
1
). The larger degree of
␥
-DGG block suggests that a lower glutamate transient is evoked
by the second stimulus. Consistent with previous results (Liu et
al., 1999; Wadiche and Jahr, 2001),
␥
-DGG increased the EPSC
1
and EPSC
2
rise times. In addition,
␥
-DGG decreased the
weighted decay kinetics of EPSC
1
(Table 1, Fig. 4A
1,
middle)
without significantly changing the decay kinetics of EPSC
2
. This
faster rate of EPSC
1
decay in the presence of
␥
-DGG likely arises
from the more effective block by
␥
-DGG of the synaptic tail cur-
rent, resulting from lower concentrations of glutamate, com-
pared with those occurring at the EPSC peak (Wadiche and Jahr,
2001). For CI-AMPAR synapses exhibiting PPF,
␥
-DGG inhib-
ited EPSC
1
by 44.5 ⫾1.4% and EPSC
2
by 37.6 ⫾1.5% (n⫽5; p⫽
0.007) (Fig. 4A
2
). Similar to synapses showing PPD,
␥
-DGG
slowed the rise times of EPSC
1
and EPSC
2
without changing their
decay kinetics (Table 1).
For CP-AMPAR synapses showing PPD,
␥
-DGG inhibition of
EPSC
2
(44.5 ⫾1.3%) was significantly larger than EPSC
1
(29.7 ⫾
1.6%; n⫽5; p⫽0.002) (Fig. 4B
1
). Similarly,
␥
-DGG increased
Figure 2. LTD expression is correlated with a reduction in NMDAR activity at both CI-AMPAR
and CP-AMPAR synapses, but an increase in CV was observed only at CP-AMPAR synapses. A1,
Time course of EPSCs recorded at ⫹40 mV (1 and 4) or ⫺60 mV (2, 3, and 5) from a CI-AMPAR
synapse. Traces in the top panels are EPSCs recorded at the time points indicated in the dot plot
(bottom).Inthis series of experiments,philanthotoxin(PhTx;5
M)wasadded at the endofthe
experimenttoconfirmthe Ca
2⫹
-permeablenatureofthe synapse being studied. A2,Themean
NMDAR-mediated EPSC (measured at 50 msec after the stimulus artifact indicated by the ver-
tical dotted lines in both A1 and B1) is significantly reduced after LTD expression at CI-AMPAR
synapses. A3, The mean NMDAR–EPSC CV is unaltered after LTD expression at CI-AMPAR syn-
apses. B1–B3, Corresponding data from CP-AMPAR synapses arranged as described above for
CI-AMPAR synapses. B3, Note that in addition to a reduction in the NMDAR–EPSC amplitude,
the NMDAR–EPSC CV was significantly increased after LTD expression. **p⬍0.01.
Figure 3. Increasing transmitter release probability reduces the magnitude of
␥
-DGG inhi-
bition at both CI-AMPAR and CP-AMPAR synapses. A, Dot plot shows the time course of EPSC
amplitude from a CI-AMPAR synapse recorded in the presence of low (2.5 mM) and high (4 mM)
extracellular Ca
2⫹
. Top, AMPA EPSCs recorded at different holding potentials (left) and the
corresponding I–V curve (right) from a CI-AMPAR synapse. Middle, AMPA EPSCs recorded at the
time points indicated in the dot plot (bottom). B, Normalized time course of
␥
-DGG (1 mM)
inhibition in low and high [Ca
2⫹
]
o
from five representative CI-AMPAR synapses. Note that the
magnitude of
␥
-DGG block is significantly less in elevated extracellular Ca
2⫹.
C, D, The corre-
sponding data from CP-AMPAR synapses arranged as described above for CI-AMPAR synapses.
Lei and McBain •Two-Expression Loci for Interneuron LTD J. Neurosci., March 3, 2004 •24(9):2112–2121 • 2115
the rise times of EPSC
1
and EPSC
2
and reduced the decay kinetics
of EPSC
1
(Fig. 4B
1
, middle trace) without altering that of EPSC
2
(Table 1). In contrast, at CP-AMPAR synapses showing PPF,
␥
-DGG inhibition of EPSC
1
(47.3 ⫾2.1%) was significantly
larger than EPSC
2
(36.4 ⫾2.3%; n⫽5; p⫽0.003) (Fig. 4B
2
). In
addition,
␥
-DGG increased the rise times of EPSC
1
and EPSC
2
without changing their decay kinetics (Table 1). It is unlikely that
␥
-DGG directly altered glutamate release probability, because the
CV of AMPA EPSCs was not altered by
␥
-DGG at either CI-
AMPAR (control, 0.31 ⫾0.04;
␥
-DGG, 0.32 ⫾0.03; n⫽5; p⫽
0.81) or CP-AMPAR (control, 0.34 ⫾0.05;
␥
-DGG, 0.37 ⫾0.06;
n⫽5; p⫽0.93) synapses. Together, the differential magnitude of
␥
-DGG block of EPSC
1
versus EPSC
2
is consistent with the hy-
pothesis that differing glutamate transients are released by the
first and second stimuli, possibly as a result of multivesicular
release during transmission (Wadiche and Jahr, 2001), changes in
fusion pore dynamics (Choi et al., 2000; Aravanis et al., 2003;
Gandhi and Stevens, 2003), or glutamate pooling from multiple
adjacent sites (Barbour and Hausser, 1997; Rusakov and Kull-
mann, 1998). It is important to point out that although these data
are consistent with
␥
-DGG detecting changes in the synaptic glu-
tamate transient, low-affinity antagonists can also reduce both
postsynaptic receptor saturation and desensitization during
paired-pulse protocols (Neher and Sakaba, 2001; Taschenberger
et al., 2002). Thus, caution must be exercised when interpreting
such data.
␥
-DGG reveals a presynaptic locus of expression for
CP-AMPAR LTD
Having determined that the low-affinity antagonist
␥
-DGG reli-
ably detects changes in glutamate transient concentrations at
mossy fiber–interneuron synapses, we next determined whether
LTD expression at CP-AMPAR and CI-AMPAR synapses re-
sulted from a change in transmitter release. Specifically, if the
released glutamate concentration in the synaptic cleft decreases
after LTD expression, then the degree of
␥
-DGG inhibition
would be expected to increase relative to controls. Therefore,
comparison of the percentage change of
␥
-DGG inhibition of
EPSCs before and after LTD induction may further elucidate the
LTD expression loci at both CP-AMPAR and CI-AMPAR
synapses.
To compare the magnitude of
␥
-DGG inhibition of mossy
fiber-evoked AMPAR EPSCs before and after LTD expression,
the experiment requires that we apply
␥
-DGG twice. As a control,
we first confirmed that the sensitivity of
␥
-DGG-mediated inhi-
bition was unchanged by repeated application. Application of
␥
-DGG (1 mM) reduced mossy fiber-evoked AMPA EPSCs by
52.9 ⫾1.7% (n⫽5). The effect of
␥
-DGG was fully reversible
after 10–15 min of washout, and a second application of
␥
-DGG
inhibited EPSCs to a similar extent (53.9 ⫾1.6% reduction; n⫽
5; p⫽0.87), indicating that
␥
-DGG sensitivity was unaltered
after repeated applications.
Next, we measured the magnitude of
␥
-DGG inhibition be-
fore and after induction of LTD. At CI-AMPAR synapses (RI,
0.92 ⫾0.02; n⫽8), application of
␥
-DGG (1 mM) reduced
EPSCs by 54.6 ⫾3.7% (n⫽8) in controls (Fig. 5A). After wash-
ing out
␥
-DGG, HFS induced LTD; EPSC amplitude was reduced
to 49.5 ⫾4.7% of controls (n⫽8; p⫽0.001) measured 10 min
after the end of the induction protocol (Fig. 5A, bottom). After
LTD expression, a second application of
␥
-DGG inhibited EPSCs
to 46.9 ⫾3.5% of controls (n⫽8), a value similar to controls
(p⫽0.4) (Fig. 5B,C). This result suggests that the glutamate
concentration in the synaptic cleft was not changed after LTD
expression at CI-AMPAR synapses.
At CP-AMPAR synapses (RI, 0.19 ⫾0.02; n⫽6),
␥
-DGG
inhibited EPSCs by 43.7 ⫾4.3% (n⫽6) in controls (Fig. 6A).
High-frequency stimulation induced LTD and reduced EPSC
amplitude to 58.7 ⫾2.9% of controls (n⫽6; p⫽0.002). After
LTD expression,
␥
-DGG now inhibited the EPSC amplitude by
72.1 ⫾2.1% (n⫽6), a value significantly higher than that ob-
served in controls ( p⫽0.0008) (Fig. 6B,C), suggesting that the
glutamate concentration in the synaptic cleft was reduced after
LTD expression.
Thus far, for ease of discussion, we focused on those synapses
at the ends of the continuum that ranges from GluR2-lacking,
CP-AMPAR synapses to GluR2-containing, CI-AMPAR syn-
apses (Lei and McBain, 2002). For completeness, it is important
to determine the LTD expression locus at that population of syn-
apses comprising intermediate RIs [i.e., 0.7–0.3 (⬃25% of the
total synapses studied)]. It is important to point out that we
cannot determine whether these intermediate synapses reflect a
homogeneous population of AMPA receptors with intermediate
Ca
2⫹
permeability or whether they represent synapses compris-
ing both CI-AMPARs and CP-AMPARs in varying ratios, or mul-
tiple fibers activating different ratios of synapses composed of
CP-AMPA or CI-AMPA receptors. In nine synapses (RIs between
0.3 and 0.7), the degree of change of
␥
-DGG block after LTD
Figure 4.
␥
-DGGdifferentiallyblocksEPSCs evoked during paired-pulse stimulation.A1, A2,
Effects of
␥
-DGG on PPR at CI-AMPAR synapses displaying PPD (A1) or PPF (A2). Left traces,
EPSCs evoked by two stimuli at an interval of 50 msec in the absence (thick trace) and presence
(thintrace)of
␥
-DGG(1mM).Middle traces, EPSCs normalized to thepeakofthe first EPSC show
significant differences in the degree of block of the second EPSC. Right, Summarized data, from
synapses showing PPD (n⫽5) and PPF (n⫽5) reveal a differential block by
␥
-DGG of the first
and second EPSC. B1, B2, Corresponding data from CP-AMPAR synapses showing PPD (n⫽5)
(B1) or PPF (n⫽5) (B2). Data are arranged as described for CI-AMPAR synapses in A1 and A2.
Double asterisks indicate that the degree of block by
␥
-DGG of the first EPSC is significantly
different from the second EPSC ( p⬍0.01).
2116 •J. Neurosci., March 3, 2004 •24(9):2112–2121 Lei and McBain •Two-Expression Loci for Interneuron LTD
expression was intermediate (49.7 ⫾1.7% for control
␥
-DGG
block vs 63.1 ⫾3.1% after LTD; p⫽0.007) to the values obtained
for synapses at the two extremes. These data suggest that a pre-
synaptic reduction in transmitter release also contributes to ex-
pression of LTD at those synapses with weakly rectifying I–V
relationships; however, a contribution of postsynaptic mecha-
nisms cannot be fully excluded. These data raise an important
issue that suggests that mixed (presynaptic and postsynaptic)
forms of LTD cannot be excluded at individual synapses, and
although the locus of expression is mainly presynaptic at CP-
AMPAR synapses and mainly postsynaptic at CI-AMPAR syn-
apses, overlapping contributions from both forms of LTD cannot
be ruled out.
Table 1. EPSC parameters in 2.5 mm Ca
2ⴙ
EPSC
1
EPSC
2
Control
␥
-DGG (1 mm) p Control
␥
-DGG (1 mm) p
CI-AMPAR
PPD
Rise time (msec) 0.56 ⫾0.07 0.63 ⫾0.08 0.019 0.76 ⫾0.06 0.79 ⫾0.07 0.048
Decay time (msec)
a
17.5 ⫾1.6 14.4 ⫾1.8 0.002 21.8 ⫾3.5 13.6 ⫾1.2 0.06
PPF
Rise time (msec) 0.61 ⫾0.05 0.67 ⫾0.05 0.019 0.72 ⫾0.05 0.77 ⫾0.06 0.037
Decay time (msec) 12.3 ⫾1.5 12.0 ⫾1.6 0.6 9.8 ⫾2.1 13.0 ⫾1.2 0.22
CP-AMPAR
PPD
Rise time (msec) 0.54 ⫾0.05 0.60 ⫾0.05 0.02 0.66 ⫾0.04 0.72 ⫾0.05 0.02
Decay time (msec) 7.1 ⫾0.6 5.8 ⫾0.3 0.02 9.8 ⫾1.1 8.4 ⫾1.3 0.32
PPF
Rise time (msec) 0.53 ⫾0.04 0.73 ⫾0.03 0.001 0.62 ⫾0.04 0.79 ⫾0.04 0.003
Decay time (msec) 8.2 ⫾1.5 8.3 ⫾1.3 0.75 16.2 ⫾0.40 15.1 ⫾1.9 0.61
a
The decay time constant represents the weighted mean. n⫽5 for all conditions. pvalues were determined by paired ttest.
Figure5. LTDat CI-AMPAR synapsesdoesnot involve a changein transmitter release. A,Top,
EPSCsrecordedat holding potentials between ⫺60and⫹40 mV (left) andtheircorresponding
I–V relationship (right). A linear I–V curve (RI, 1.19) suggests that this synapse comprises
CI-AMPARs. In all experiments, dl-APV was initially included to block NMDARs for the construc-
tion of the AMPAR–EPSC I–V relationship. After determination of the RI, APV was then washed
out. Middle, Averages of 10 consecutive EPSCs taken at time points indicated in the bottom dot
plot.Therightpanel shows the normalization ofcontrolaveraged EPSCs (1) and averagedEPSCs
recordedduringthefirst (2) and second(4)application of
␥
-DGG.Bottom,Meantime courses of
EPSCsaveragedfrom eight neurons.Inthe control epoch,
␥
-DGG(1mM) was appliedtomeasure
thebasallevel of inhibition.Aftercomplete washout, thehigh-frequencyLTD induction protocol (HFS,
100Hzfor 1 sec, repeated3times at an intervalof10 sec) was applied.AfterLTD had stabilized (3),the
same concentration of
␥
-DGG was applied for a second time. B, EPSCs obtained during the two appli-
cations of
␥
-DGG were normalized to the controls and overlain to illustrate the degree of
␥
-DGG
inhibition before and after induction of LTD. A comparable degree of
␥
-DGG inhibition was observed
before and after induction of LTD. C,
␥
-DGG-mediated inhibition from eight individual CI-AMPAR
synapses before and after the induction of LTD. The filled circles are averages of
␥
-DGG inhibition
before and after the induction of LTD. Open circles are data obtained from individual experiments.
Note that the percentage of inhibition by
␥
-DGG was not significantly different before and after the
induction of LTD.
Figure 6. LTD at CP-AMPAR synapses involves a reduction in transmitter release. A, Top,
EPSCs recorded at holding potentials between ⫺60 and ⫹40 mV (left) and the corresponding
I–V relationship (right). An inwardly rectifying I–V relationship suggests that this synapse
comprised CP-AMPARs. dl-APV was included for the entire period of the experiment to isolate
CP-AMPAR-dependent LTD. Middle, Averaged current traces from 10 EPSCs taken at the time
points indicated in the dot plot (bottom). The right traces show the normalization of control
EPSCs (1) and the EPSCs recorded in the presence of
␥
-DGG (2 and 4). Bottom, Time course of
inhibition by
␥
-DGG (1 mM) (2 and 4) before (1) and (3) after induction of LTD from six neurons.
B, EPSCs recorded during the two applications of
␥
-DGG were normalized to their respective
controls for ease of comparison of the degree of
␥
-DGG block before and after the induction of
LTD.C,
␥
-DGG-mediatedinhibitionfromsix individual CP-AMPAR synapses beforeandafter the
induction of LTD. The filled circles are averages of
␥
-DGG inhibition before and after the induc-
tion of LTD. Open circles are data obtained from individual experiments. Note that the percent-
age of inhibition by
␥
-DGG was significantly increased after the induction of LTD at CP-AMPAR
synapses.
Lei and McBain •Two-Expression Loci for Interneuron LTD J. Neurosci., March 3, 2004 •24(9):2112–2121 • 2117
NSF–AP2 inhibitory peptide confirms a postsynaptic locus of
expression for NMDAR-dependent LTD at
CI-AMPAR synapses
Postsynaptic AMPAR translocation is an important mechanism
in NMDAR-dependent LTD expression at synapses onto pyrami-
dal neurons (Carroll et al., 1999; Lu¨scher et al., 1999; Lu¨thi et al.,
1999; Man et al., 2000; Matsuda et al., 2000; Wang and Linden,
2000; Sheng and Kim, 2002; Song and Huganir, 2002). At Schaf-
fer collateral–CA1 pyramidal neuron synapses, NMDAR-
dependent LTD expression involves a pool of AMPARs regulated
by both NSF–GluR2 (Lu¨scher et al., 1999; Lu¨thi et al., 1999; Noel
et al., 1999) and clathrin adaptor AP2–GluR2 (Lee et al., 2002)
interactions. NMDAR-dependent LTD-induced endocytosis of
AMPARs is dependent on both Ca
2⫹
and the activity of protein
phosphatase 1 or 2A (Beattie et al., 2000; Ehlers, 2000). We next
tested whether expression of LTD at either CI-AMPAR or CP-
AMPAR synapses similarly involved a translocation of AMPARs
using the broad-spectrum NSF–AP2 inhibitory peptide, com-
monly referred to as pep2m (Lu¨scher et al., 1999; Lu¨thi et al.,
1999; Shi et al., 2001; Lee et al., 2002).
Infusion of pep2m into the cells via the recording pipette did
not significantly influence evoked AMPA EPSC amplitudes (note
that it only affected CI-AMPAR EPSCs; see below for details)
until ⬎8 min after formation of the whole-cell recording. Taking
advantage of this initial control period, we first constructed I–V
curves and calculated the RI of evoked EPSCs to determine the
Ca
2⫹
-permeable nature of AMPARs. At CI-AMPAR synapses
(RI, 0.96 ⫾0.05; n⫽7) (Fig. 7A, top), infusion of pep2m (0.5
mM) not only decreased EPSC amplitude to 67.7 ⫾4.5% of con-
trols (measured 38 min after the formation of whole-cell record-
ing; n⫽7; p⫽0.0004) (Fig. 7B) but occluded LTD expression
(Fig. 7A,B). EPSC amplitude was 67.7 ⫾4.5% of controls before
LTD induction versus 64.5 ⫾6.8% of controls at 20 min after
LTD expression (n⫽7; p⫽0.35) (Fig. 7B). Together with the
results from the
␥
-DGG inhibition experiment, these data
strongly suggest that the expression of NMDAR-dependent LTD
at CI-AMPAR synapses is postsynaptic and involves a pool of
AMPARs regulated by the NSF–AP2–GluR2 interaction.
In contrast, pep2m did not appreciably inhibit EPSC ampli-
tude at CP-AMPAR synapses even ⬃40 min after formation of
the whole-cell recording (102.2 ⫾9.9% of the initial EPSC am-
plitude; n⫽5; p⫽0.84) (Fig. 7C,D), suggesting that there was no
NSF–AP2–GluR2 interaction at CP-AMPAR synapses. These
data are consistent with results obtained from CA1 hippocampal
pyramidal neurons containing a deletion of the GluR2 subunit
(Shi et al., 2001) and confirm that the effect of the NSF–AP2
inhibitory peptide is specific for interactions mediated by GluR2
at GluR2-containing receptors (Sheng and Kim, 2002; Song and
Huganir, 2002). Furthermore, application of the LTD induction
protocol still depressed transmission at CP-AMPAR synapses
(RI, 0.19 ⫾0.03; n⫽5) (Fig. 7C,D). EPSC amplitude was re-
duced by 49.5 ⫾7.7% (n⫽5; p⫽0.003) 20 min after LTD
expression. Together with the result from the
␥
-DGG inhibition
experiment, these results indicate that the LTD expression at CP-
AMPAR synapses is consistent with a reduction in transmitter
release with no involvement of postsynaptic AMPA receptor
translocation.
Finally, in an attempt to separate the NSF–GluR2 interaction
(i.e., the EPSC amplitude “rundown”) from the clathrin adaptor
AP2-dependent translocation of AMPARs (i.e., one potential
mechanism for LTD expression), we used an AP2-specific block-
ing peptide (pep-⌬A849-Q853) (Lee et al., 2002). At Schaffer
collateral–CA1 pyramidal neuron synapses, this peptide does not
affect basal synaptic transmission but completely abolishes low-
frequency stimulation-induced LTD. In contrast, at mossy fiber–
interneuron synapses, although pep-⌬A849-Q853 did not im-
pact basal synaptic transmission (CI-AMPAR synapse, 97.6 ⫾
2.1%, n⫽5, p⫽0.32; CP-AMPAR synapse, 92.0 ⫾6.0%, p⫽
0.26, n⫽5), it also failed to block LTD expression at both synapse
types (CI-AMPAR synapse, 52.3 ⫾6.8%, n⫽5, p⫽0.002; CP-
AMPAR synapse, 49.5 ⫾6.1%, p⫽0.001, n⫽5), suggesting that
the AP2-dependent pathway may not be necessary for LTD ex-
pression at interneuron synapses, consistent with the lack of de-
tectable protein phosphatase 2B at these synapses (Sik et al.,
1998).
Discussion
We used three approaches to explore the expression locus of two
forms of mossy fiber–inhibitory interneuron LTD: (1) conven-
Figure 7. Postsynaptic AMPAR trafficking contributes to expression of LTD at CI-AMPAR
synapses. The NSF–AP2-inhibitory peptide pep2m (0.5 mM) was included in the recording
pipettes for all experiments. A, CI-AMPAR synapse. Top, EPSCs evoked at holding potentials
between ⫺60 and ⫹40 mV, recorded during the first 8 min after formation of whole-cell
configuration (left) and the corresponding I–V relationship (right) from a CI-AMPAR synapse.
dl-APVwasincluded for the periodofRI determination. Middle, Averagedcurrenttraces from 10
EPSCs taken at the time points indicated in the dot plot below. Bottom, Dot plot of EPSC ampli-
tude indicating the time course of the experiment. The first 8 min after the formation of whole-
cell recording were used to construct the I–V relationship and identify the Ca
2⫹
-permeable
nature of the AMPAR-mediated EPSC. Inclusion of the NSF inhibitory peptide inhibited EPSCs at
thisCI-AMPARsynapse and occluded expressionofLTD.DCG-IV was included intheextracellular
solution at the end of the recording to confirm that EPSCs were mossy fiber in nature. B, Aver-
aged data from seven CI-AMPAR synapses illustrate the reduction of EPSC amplitude and the
concomitant occlusion of LTD. C, CP-AMPAR synapse. The I–V relationship and the time course
of evoked EPSCs at a CP-AMPAR synapse are shown. Organization of data is identical to that
shown in Aand B. Inclusion of the NSF inhibitory peptide was without effect on both the EPSC
amplitude and the ability to express LTD at CP-AMPAR synapses. D, Averaged data from five
CP-AMPAR synapses.
2118 •J. Neurosci., March 3, 2004 •24(9):2112–2121 Lei and McBain •Two-Expression Loci for Interneuron LTD
tional analysis of synaptic parameters, including CV analysis,
PPR, and analysis of both AMPA and NMDA receptor-mediated
EPSCs; (2) application of a low-affinity glutamate receptor an-
tagonist to probe changes in the synaptic glutamate transient
concentration; and (3) infusion of NSF–GluR2- or AP2–GluR2-
interfering peptides into cells to explore potential postsynaptic
mechanisms. All three approaches point to the same conclusion:
that the expression locus of each form of mossy fiber–interneu-
ron LTD occurs on opposite sides of the synapse.
LTD expression at CP-AMPAR synapses was associated with
increases in both the CV and PPR. Furthermore, the concentra-
tion of cleft glutamate, assessed by measuring changes in
␥
-DGG
inhibition, was reduced after LTD expression. Finally, postsyn-
aptic introduction of pep2m, an NSF–AP2 inhibitory peptide,
failed to influence the expression of LTD at CP-AMPAR syn-
apses, suggesting that postsynaptic AMPAR translocation is un-
likely to be involved. Together, these results indicate that LTD
expression at CP-AMPAR synapses is presynaptic in origin and is
reminiscent of mossy fiber–basket cell LTD (Alle et al., 2001). In
contrast, LTD expression at CI-AMPAR synapses did not alter
the CV or paired-pulse ratio, and the magnitude of
␥
-DGG inhi-
bition was also unaltered after LTD expression, arguing against
presynaptic expression. Postsynaptic expression was further sup-
ported by experiments involving postsynaptic infusion of pep2m,
which occluded LTD expression. Together, these results suggest
that LTD expression at CI-AMPAR synapses is postsynaptic and
involves a pool of AMPARs regulated by NSF–AP2–GluR2
interaction.
What is the mechanism whereby LTD results in decreased
glutamate release at mossy fiber–CP-AMPAR synapses? If trans-
mission at CP-AMPAR synapses occurred via univesicular re-
lease [i.e., a “one release site–one vesicle”mechanism (Redman
1990; Korn et al., 1994)] and LTD expression involved a reduc-
tion in transmitter release probability, then a change in the mag-
nitude of
␥
-DGG block would not be expected to occur after LTD
expression. Clearly, such a mechanism is unlikely to account for
the increased block by
␥
-DGG after CP-AMPAR LTD expression.
We suggest three possible scenarios to explain the present obser-
vations: (1) Under control conditions, transmission at CP-
AMPAR synapses may result from multivesicular release [i.e.,
more than one vesicle can be released from a single synapse after
a stimulus (Tong and Jahr, 1994; Auger et al., 1998; Prange and
Murphy, 1999)]. LTD expression may therefore arise from a re-
duced likelihood of multiple exocytotic events at a single release
site as the probability of release is reduced after LTD induction
(Wadiche and Jahr, 2001), resulting in a lowered glutamate tran-
sient. Evidence for multivesicular release (Jahr, 2003) has been
provided at an increasing number of disparate synapses, includ-
ing the neuromuscular junction (Heuser et al., 1979), cerebellar
stellate, and basket cell inhibitory synapses (Auger et al., 1998),
excitatory synapses onto hippocampal neurons (Tong and Jahr,
1994; Oertner et al., 2002; Hallermann et al., 2003), and climbing
fiber–Purkinje cell synapses (Wadiche and Jahr, 2001). Recent
evidence suggests that the release rate at the large mossy fiber–
CA3 pyramidal neuron terminal is also consistent with multive-
sicular release (Hallermann et al., 2003). The differential degree
of block by
␥
-DGG during paired-pulse protocols also suggests
that multivesicular release may occur at the smaller mossy fiber–
interneuron synapses under basal conditions. (2) Alternatively,
CP-AMPAR LTD may arise from changes in fusion pore dynam-
ics or reversal of the “kiss and run”hypothesis (Choi et al., 2000;
Aravanis et al., 2003; Gandhi and Stevens, 2003), where LTD
results from an incomplete emptying of vesicular glutamate. In-
creasing evidence suggests that multiple exocytotic states exist
within single synapses and that mechanisms that shift the prob-
ability of each state will shape transmitter release profile at indi-
vidual synapses. (3) Under normal conditions, mossy fiber trans-
mission at CP-AMPAR synapses may result from glutamate
pooling from multiple adjacent sites. A reduction in release prob-
ability will reduce the likelihood that adjacent synapses release
transmitter simultaneously, consequently reducing the transmit-
ter pool (Barbour and Hausser, 1997; Rusakov and Kullmann,
1998). Support for this hypothesis comes from consideration of
the mossy fiber–filopodial architecture (Acsady et al., 1998) and
their known quantal properties (Lawrence et al., 2004). Filopodia
primarily synapse onto smooth, spine-free dendritic segments or
arrange in clusters onto the characteristic long and thin dendritic
spines of inhibitory interneurons. On occasion, multiple filopo-
dia emanating from a single large mossy bouton make contact
with a single postsynaptic interneuron. Qualitatively, the low-
affinity antagonist technique by itself cannot distinguish between
these three interpretations, and future experiments will attempt
to elucidate the precise mechanism of LTD expression at CP-
AMPAR synapses.
Postsynaptic Ca
2⫹
elevation, presumably through NMDA
and Ca
2⫹
-permeable AMPA receptors, is required for LTD in-
duction at CI-AMPAR and CP-AMPAR synapses, respectively
(Lei and McBain, 2002). Our results suggest that Ca
2⫹
influx
through NMDARs leads to the translocation of AMPARs and
LTD expression only at CI-AMPAR synapses. This mechanism is
reminiscent of the NMDAR-dependent LTD observed at Schaffer
collateral–CA1 pyramidal neuron synapses, which involves
postsynaptic AMPAR trafficking (Lu¨scher et al., 1999; Lu¨thi et al.,
1999; Beattie et al., 2000; Lee et al., 2002; Sheng and Kim, 2002;
Song and Huganir, 2002). However, it is unclear how Ca
2⫹
entry
via NMDARs leads to the translocation of AMPARs at
interneuron–CI-AMPAR synapses. In cultured hippocampal
neurons, activation of NMDARs triggers AMPAR endocytosis
through Ca
2⫹
influx and activation of the Ca
2⫹
-dependent pro-
tein phosphatase calcineurin (Beattie et al., 2000). However, cal-
cineurin is absent from hippocampal interneurons (Sik et al.,
1998), making it an unlikely target for Ca
2⫹
influx at CI-AMPAR
synapses. Consistent with this hypothesis was the failure of the
AP2–GluR2-interfering peptide (Lee et al., 2002) to block LTD
induction. This suggests that although NMDAR-dependent LTD
shares many of the features of LTD observed at principal cell
synapses, an identical mechanism would appear unlikely. Given
that diverse intracellular signals are involved in AMPAR endocy-
tosis (Beattie et al., 2000; Chung et al., 2000; Ehlers, 2000; Lin et
al., 2000; Matsuda et al., 2000), it would not be surprising to see
alternative intracellular signals involved in synaptic trafficking of
AMPARs at CI-AMPAR synapses.
Postsynaptic NMDAR-dependent LTD of excitatory trans-
mission onto CA1 pyramidal neurons is associated with an in-
crease in failure rate (Stevens and Wang, 1994) and a change in
CV (Selig et al., 1995), consistent with the silencing of “active”
synapses by receptor internalization (Malenka and Siegelbaum,
2001; Sheng and Kim, 2002). In contrast, the NMDAR-
dependent LTD observed at interneuron synapses was not asso-
ciated with changes in the CV. How can we explain these two
disparate observations? The presence of “silent”synapses on
principal cells has been supported by considerable anatomical
and physiological evidence, whereas little evidence exists for an-
atomically silent synapses onto interneurons. Furthermore, exci-
tatory synapses onto interneurons have an AMPAR distribution
with a relatively small variability and contain on average four
Lei and McBain •Two-Expression Loci for Interneuron LTD J. Neurosci., March 3, 2004 •24(9):2112–2121 • 2119
times as many receptors as excitatory synapses onto CA3 pyrami-
dal cell spines (Nusser et al., 1998). Such an anatomical arrange-
ment suggests that postsynaptic LTD at interneuron synapses
may reduce AMPAR content but fail to “silence”active synapses.
In this scenario, LTD would result as a reduction in EPSC ampli-
tude without a change in CV.
Interestingly, Ca
2⫹
influx via CP-AMPARs does not engage
translocation of AMPA receptors, and LTD expression appears to
be entirely presynaptic. What then is the link between postsynap-
tic induction and presynaptic expression? Laezza et al. (1999)
speculated that entry of Ca
2⫹
through CP-AMPARs at collateral
synapses onto stratum radiatum interneurons may trigger a syn-
aptic shape change that enables access of released glutamate to
presynaptic mGluRs, or cause release of a retrograde messenger
that cooperates with presynaptic mGluR activation to suppress
transmitter release, perhaps via the recently described trans-
synaptic EphB–Ephrin receptor signaling system (Contractor et
al., 2002). Future experiments will elucidate these mechanisms.
Finally, these experiments raise an intriguing hypothesis that
mossy fiber innervation of CA3 stratum lucidum interneurons
occurs via two parallel systems: one linked to CP-AMPAR syn-
apses that contain low levels of NR2B-containing NMDARs and a
second mossy fiber system that engages CI-AMPAR synapses,
which contain a significant NMDAR contribution. High-
frequency stimulation of CP-AMPAR synapses results in a pre-
synaptic form of LTD expression associated with a reduction in
both synaptic glutamate transient and release probability, which
alters short-term plasticity at these synapses. However, LTD in-
duction and expression at CI-AMPAR synapses appears to be
entirely postsynaptic and to involve translocation of AMPAR
subunits. Of particular interest at synapses with an intermediate
RI profile (i.e., RIs of 0.3–07), the presynaptic form of LTD ex-
pression predominates, suggesting that these synapses may “de-
fault”to a plasticity involving an alteration in transmitter release
probability. How these properties of distinct short-term and
long-term plasticity map onto the mossy fiber–CA3 network di-
alogue is unclear, but mechanisms that alter the mean EPSC am-
plitude without changing the variance (postsynaptic LTD) will
have a profoundly different impact from depression associated
with a concomitant change in variance (presynaptic LTD) (Aradi
et al., 2002).
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