Modulation of Presynaptic Calcium Transients by Metabotropic
Glutamate Receptor Activation: A Differential Role in Acute
Depression of Synaptic Transmission and Long-Term Depression
Guido C. Faas, Hita Adwanikar, Robert W. Gereau IV, and Peter Saggau
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030
Activation of group I metabotropic glutamate receptors (mGluRs)
can induce acute depression of excitatory synaptic transmission
and long-term depression (LTD) in area CA1 of the rat hippocam-
pus. The underlying mechanisms for both forms of depression are
unknown. By measuring presynaptic calcium transients, we show
that a reduction in the stimulation-induced presynaptic calcium
rise that triggers vesicular release causes the acute depression of
transmission by group I mGluRs. In contrast, the mechanism
underlying mGluR-induced LTD does not involve a persistent
change in stimulation-induced calcium influx. However, analysis of
paired-pulse facilitation experiments suggests a presynaptic loca-
tion for expression of this form of LTD. Furthermore, we show that
mGluR-induced LTD can be completely blocked by a specific
mGluR5 antagonist, whereas mGluR1 antagonists strongly atten-
uate the acute depression of transmission. These results support
the hypothesis that the acute depression of transmission caused
by activation of group I mGluRs involves regulation of stimulation-
induced presynaptic calcium transients, whereas mGluR-induced
LTD involves a distinct presynaptic modulation downstream of
calcium influx.
Key words: mGluR; DHPG; LTD; presynaptic; calcium;
hippocampus
Metabotropic glutamate receptors (mGluRs) play many impor-
tant roles in regulating neuronal excitability and synaptic trans-
mission (Conn and Pin, 1997). In hippocampal area CA1, activa-
tion of mGluRs can reduce both excitatory and inhibitory
transmission (Baskys and Malenka, 1991; Desai et al., 1992, 1994;
Gereau and Conn, 1995; Manzoni and Bockaert, 1995). In addi-
tion, activation of mGluRs is inducing a certain form of long-term
depression (LTD) of excitatory synaptic transmission at the
Schaffer collateral–CA1 (SCC) synapse (Stanton et al., 1991;
Bashir et al., 1993; Bolshakov and Siegelbaum, 1994; Oliet et al.,
1997; Kemp and Bashir, 1999; Huber et al., 2000; Fitzjohn et al.,
2001). This form of LTD (mGluR-LTD) is NMDA receptor
independent and can be induced simultaneously with NMDA
receptor-dependent LTD (Oliet et al., 1997).
mGluR-LTD can be selectively induced by (RS)-3,5-
dihydroxyphenylglycine (DHPG), an agonist that specifically ac-
tivates group I mGluRs, consisting of mGluR1 and mGluR5
(Palmer et al., 1997; Fitzjohn et al., 1999; Schnabel et al., 1999;
Huber et al., 2000, 2001; Fitzjohn et al., 2001; Schnabel et al.,
2001; Snyder et al., 2001) (for review, see Kemp and Bashir,
2001). The mechanisms underlying the expression of mGluR-
LTD are still unclear. Although some studies show that
stimulation-induced LTD is expressed presynaptically (Oliet et
al., 1997), others suggest a postsynaptic expression of mGluR-
LTD (Snyder et al., 2001) or that the expression is at least
dependent on postsynaptic protein synthesis (Huber et al., 2000,
2001). Based on paired-pulse facilitation (PPF) data, it has been
suggested recently that chemically induced mGluR-LTD is ex-
pressed presynaptically (Fitzjohn et al., 2001).
Several neurotransmitters, including adenosine, acetylcholine,
GABA, and neuropeptide Y, reduce stimulation-induced presyn-
aptic calcium influx at the CA3–CA1 synapse and, in this way,
strongly attenuate synaptic transmission (Wu and Saggau, 1994a,
1995, 1997; Qian and Saggau, 1997; Qian et al., 1997). Preliminary
findings in adult animals (Faas et al., 2000) and previous studies
in neonatal rats showed that activation of mGluRs can similarly
reduce synaptic transmission at this synapse by modulating pre-
synaptic calcium influx, although the mGluR subtypes involved in
this modulation of calcium influx were not known (Yoshino and
Kamiya, 1995). These investigations were limited to acute depres-
sion by mGluR activation and did not address mGluR-LTD. In
the present study, we sought to test the hypothesis that both acute
and long-term depression of transmission by DHPG at the CA3–
CA1 synapse involves modulation of stimulation-induced presyn-
aptic calcium influx.
MATERIALS AND METHODS
Sprague Dawley rats (4–6 weeks old) were anesthetized with halothane
and quickly decapitated in accordance with the guidelines of the National
Institutes of Health, as approved by the animal care and use committee
of Baylor College of Medicine. Transverse hippocampal slices of 400
m
were prepared on a tissue cutter (Vibratome 1000 plus; St. Louis, MO).
For the dissection and the cutting of brain slices, an ice-cold solution with
no sodium and high magnesium concentration was used. This solution
contained the following (in mM): 2.5 KCl, 1.25 NaH2PO4, 1 CaCl2, 7
MgCl2, 7 dextrose, 190 sucrose, 1 ascorbic acid, 3 pyruvic acid, and 28
NaHCO3. Brain slices were stored in artificial CSF containing the
following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2,
25 dextrose, 1 ascorbic acid, 3 pyruvic acid, and 25 NaHCO3. Both
solutions were constantly aerated with 95% CO2–5% O2 to maintain a
pH of 7.4 and O2 saturation. Measurements were done in storing solution
without the ascorbic acid and pyruvic acid. Recordings were made in
small, constantly perfused chambers (1–2 ml) at 30–32°C.
Calcium measurements in presynaptic structures. Presynaptic structures
were filled with the fluorescent calcium indicator fura-2 as described
Received Feb. 12, 2002; revised May 24, 2002; accepted May 29, 2002.
This work was supported by National Institutes of Health Grants NS33147 (P.S.)
and MH60230 (R.W.G.). We thank Dr. D. Johnston for helpful comments on this
manuscript.
Correspondence should be addressed to Dr. Peter Saggau, Baylor College of
Medicine, Neuroscience, 1 Baylor Plaza, Room S603, Houston, TX 77030. E-mail:
psaggau@bcm.tmc.edu.
Copyright © 2002 Society for Neuroscience 0270-6474/02/226885-06$15.00/0
The Journal of Neuroscience, August 15, 2002, 22(16):6885–6890

previously (Wu and Saggau, 1994a). In short, small amounts of the
membrane-permeant form of fura-2 (50
g of fura-2 AM, 5–10
l of
DMSO with 25% pluronic acid, and 50
l of extracellular solution
buffered to pH 7.3 with 10 mM HEPES) were pressure injected into the
SCC using a Picospritzer II (General Valve, Fairfield, NJ) with pipettes
of 2–3
m tip diameter (Fig. 1a). After being taken up by axons,
intracellular esterases cleaved the AM form to the membrane-
impermeant indicator, which filled the presynaptic terminals. One to 2 hr
after injection, brain slices were illuminated at 380 nm, in a small spot
(
150
m in diameter) in area CA1, 300–500
m away from the
injection site to avoid any contamination of the optical recordings by
accidental postsynaptic indicator loading. Fluorescence was detected
using a single photodiode connected to a low-noise current-to-voltage
converter and amplifier, and fractional changes (F/F ) were calculated.
CA3 axons were stimulated with bipolar tungsten electrodes placed in
the SCC tract, and extracellular field recordings were made using glass
microelectrodes filled with extracellular solution that were placed in the
middle of the optical recording spot (Fig. 1a).
Synaptic activity was evoked by current pulses (100–600
A for 200

sec) applied through the stimulation electrode. In every experiment, two
pulses with a 20 msec interval were given. The amplitude of F/F induced
by the second stimulus always exceeded that of the first stimulus, indicating
that the calcium indicator was not saturated during the first stimulus.
To verify the presynaptic origin of the observed calcium transients,
ionotropic glutamate receptor antagonists (20
M CNQX, 25
M D-APV,
or 50
M DL-APV) were added to the bath at the end of each experiment.
The antagonists always abolished synaptic transmission without affecting
the presynaptic fiber volley or the corresponding Ca 2 rise, indicating
that the Ca 2 signals were of presynaptic origin (Fig. 1b).
Field recordings were made using a model 5A amplifier (Getting
Instruments, San Diego, CA). Data were acquired using a 16 bit analog-
to-digital converter and processed with custom-made software. The same
software was used together with Microsoft (Seattle, WA) Excel to ana-
lyze the data.
Some of the experiments testing the effects of the mGluR5 antagonists
on synaptic transmission were performed on a separate setup at room
temperature in which we only measured the field EPSPs (fEPSPs). All
drugs used to determine receptor specificity were added 5 min before and
throughout the period of DHPG application. Stimulation and recording
electrodes were placed in hippocampal slice as described above. Record-
ings were made using a A310 Accupulser (World Precision Instruments,
Sarasota, FL), a low-pass Bessel Filter 4 pole amplifier (Warner Instru-
ments, Hamden, CT), an Axoclamp 2B, and pClamp 8.0 software (Axon
Instruments, Foster City, CA). The same program was used together
with Microsoft Excel to analyze the data.
Relative values reported throughout the text are with respect to
control situations. Statistical comparisons were made using the Student’s
t test, and differences are considered significant when p  0.05.
All drugs were purchased from Sigma (St. Louis, MO), except for
APV, DHPG, DNQX, 2-methyl-6-(phenylethynyl) pyridine (MPEP),
7-(hydroxyimino)cyclopropa()chromen-1a-carboxylate ethyl ester
(CPCCOEt), and LY367385, which were obtained from Tocris Cookson
(Ballwin, MO).
RESULTS
The role of mGluR5 and mGluR1 in DHPG-induced LTD
and acute depression of transmission in area CA1
Consistent with previous reports, we found that application of
DHPG (100
M, 20 min) produced a partially reversible depres-
sion of evoked fEPSPs (Fig. 2a). During application of DHPG,
Figure 1. Field potentials and corresponding calcium signals. a,
Membrane-permeant fura-2 AM was pressure injected into the SCC tract
and taken up by axons, in which intracellular esterases cleaved the AM
form to the membrane-impermeant calcium indicator, which filled the
presynaptic terminals. Fluorescence was recorded from a small spot as
shown. CA3 axons were stimulated with a bipolar electrode placed in the
SCC tract, and field recordings were made in area CA1. b, Bath applica-
tion of ionotropic GluR antagonists abolished fEPSPs, although presyn-
aptic volleys persisted. Simultaneously measured Ca 2 signals remained
unchanged, indicating their presynaptic origin.
Figure 2. mGluR5 activation is required for mGluR-LTD. a, Evoked
fEPSPs measured in stratum radiatum; stimulation artifacts are truncated.
The amplitude and slope of the fEPSPs were reduced during application
of DHPG (100
M) and recovered partially during washout, revealing a
DHPG-induced LTD. b, Slope of fEPSPs evoked at 1 min interstimulus
interval, as percentage of baseline values. In control experiments ( filled
circles), DHPG caused a strong acute depression of the fEPSPs followed by
LTD. The total response can be separated into two parts, a reversible
suppression by DHPG and the LTD. Application of the mGluR5 antagonist
MPEP at 1
M (open circles) or 10
M (open squares) did not abolish the
acute depression but prevented the induction of LTD. c, Application of the
mGluR1 antagonists LY367385 (10
M; open circles) or 10
M CPCCOEt
(open squares) strongly attenuated the acute depression but not LTD.
6886 J. Neurosci., August 15, 2002, 22(16):6885–6890 Faas et al. • Presynaptic Calcium Modulation by mGluR Activation