1Department of Physiology and 2Department of Pharmacology, Uniformed Services University, Bethesda, Maryland 20814-4799; and 3Department of Biology, Georgetown University, Washington, DC 20057-1229
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ABSTRACT |
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IV, Paul M. Lea,
Barbara Wroblewska,
John M. Sarvey, and
Joseph H. Neale.
-NAAG Rescues LTP From Blockade by NAAG in Rat Dentate Gyrus
via the Type 3 Metabotropic Glutamate Receptor.
J. Neurophysiol. 85: 1097-1106, 2001.
N-Acetylaspartylglutamate (NAAG) is an agonist at the type 3 metabotropic glutamate receptor (mGluR3), which is coupled to a Gi/o
protein. When activated, the mGluR3 receptor inhibits adenylyl cyclase
and reduces the cAMP-mediated second-messenger cascade. Long-term
potentiation (LTP) in the medial perforant path (MPP) of the
hippocampal dentate gyrus requires increases in cAMP. The presence of
mGluR3 receptors and NAAG in neurons of the dentate gyrus suggests that
this peptide transmitter may inhibit LTP in the dentate gyrus.
High-frequency stimulation (100 Hz; 2 s) of the MPP resulted in
LTP of extracellularly recorded excitatory postsynaptic potentials at
the MPP-granule cell synapse of rat hippocampal slices. Perfusion of
the slice with NAAG (50 and 200 µM) blocked LTP. Neither 50 nor 200 µM NAAG produced N-methyl-D-aspartate receptor
currents in the granule cells of the acute hippocampal slice. The group
II mGluR antagonist ethyl glutamate (100 µM) and a structural
analogue of NAAG,
-NAAG (100 µM), prevented the blockade of LTP by
NAAG. Paired-pulse depression of the excitatory postsynaptic potential
at 20- and 80-ms interpulse intervals (IPI) was not affected by NAAG or
-NAAG.
-NAAG did not affect inositol trisphosphate production
stimulated by the agonist glutamate in cells expressing the group I
mGluR1
or mGluR5.
-NAAG blocked the decrease in
forskolin-stimulated cAMP by the group II mGluR agonist
(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) but not the
group III mGluR agonist L(+)-2-amino-4-phosphonobutyric acid in
cerebellar granule cells. In cells transfected with mGluR3, but not
mGluR2,
-NAAG blocked forskolin-stimulated cAMP responses to
glutamate, NAAG, the nonspecific group I, II agonist
trans-ACPD, and the group II agonist DCG-IV. We conclude
that
-NAAG is a selective mGluR antagonist capable of
differentiating between mGluR2 and mGluR3 subtypes and that the mGluR3
receptor functions to regulate activity-dependent synaptic potentiation
in the hippocampus.
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INTRODUCTION |
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Since its discovery in
1964 (Curatolo 1964), the vertebrate neuropeptide,
N-acetylaspartylglutamate (NAAG) has been shown to be widely
distributed in neurons throughout the mammalian nervous system
(Anderson et al. 1986
; Forloni et al.
1987
; Moffett and Namboodiri 1995
;
Moffett et al. 1993
, 1994
; Renno et al.
1997
; Tieman and Tieman 1996
; Tieman et
al. 1987
, 1991
; Williamson and Neale
1988
). NAAG meets each of the traditional criteria for a neurotransmitter (for review, see Neale et al. 2000
).
NAAG is a selective agonist at the type 3 metabotropic glutamate
receptor (mGluR3) in neurons, glia, and transfected cells, where it has a potency similar to that of glutamate (Wroblewska et al. 1993
, 1997
, 1998
). The peptide is also a low potency agonist at the N-methyl-D-aspartate (NMDA) receptor
(Trombley and Westbrook 1990
; Valivullah et al.
1994
; Westbrook et al. 1986
). Additional data suggest that NAAG may act as a partial agonist at this receptor (Grunze et al. 1996
; Puttfarcken et al.
1993
). The inactivation of synaptically released NAAG is
achieved by a membrane-bound (Riveros and Orrego 1984
;
Robinson et al. 1987
) peptidase on the extracellular
face of glia (Cassidy and Neale 1993
). This glutamate carboxypeptidase II (GCP II) was found to be identical to prostate specific membrane antigen in humans (Carter et al. 1996
)
and has been cloned from rat nervous system libraries (Bzdega et
al. 1997
; Luthi-Carter et al. 1998
). GCP II
activity is inhibited by quisqualate (Ki = 2 µM), phosphate
(IC50 = 100 µM), sulfate
(IC50 = 1 mM), and
-NAAG
(Ki = 1 µM), a synthetic structural
analogue of NAAG in which the peptide bond is formed by the
-carboxyl group of aspartate (Robinson et al. 1987
;
Serval et al. 1990
). The distribution of both NAAG and
GCP II are altered in human degenerative diseases (see Coyle
1997
for review), although it is not possible from these data
to resolve primary from secondary degenerative tissue changes.
Acting at the mGluR3 receptor, NAAG causes a decrease in
forskolin-stimulated cAMP levels in neurons, glia, and cultured
mammalian cells stably expressing mGluR3 cDNAs (Wroblewska et
al. 1993, 1997
, 1998
). The mGluR3 is a member of the group II
metabotropic glutamate receptors, which have been implicated in
suppression of voltage-dependent calcium conductance in cerebellar
granule cells (Chavis et al. 1994
), neocortical neurons
(Sayer et al. 1992
), and amphibian olfactory neurons
(Bischofberger and Schild 1996
). The group II agonist,
DCG-IV, which also inhibits voltage-dependent calcium currents, has
been reported to suppress synaptic transmission to motor neurons in the
spinal cord (Ishida et al. 1993
), potassium-induced release of GABA in cortical cell cultures (Schaffhauser et al. 1998
; Zhao et al. 2001
), and transmission at the
mossy fiber-CA3 synapse in the hippocampus (Kamiya et al.
1996
) and a reciprocal synapse in the accessory olfactory tract
(Hayashi et al. 1993
). In cerebellar slices, exogenously
applied NAAG suppressed the excitatory response of Purkinje cell
dendrites to climbing fiber activation (Sekiguchi et al.
1989
). These data have led to the hypothesis that one function
of NAAG following synaptic release is to activate presynaptic
mGluR3 to inhibit subsequent transmitter release.
The rat hippocampal slice is a preparation richly endowed with
ionotropic and metabotopic glutamate receptors, including
mGluR3 (Shigemoto et al. 1997). NAAG is
concentrated in hippocampal interneurons (Anderson et al.
1986
; Moffett and Namboodiri 1995
;
Moffett et al. 1993
), and GCP II activity is
found throughout the hippocampus (Bzdega et al. 1997
;
Fuhrman et al. 1994
; Luthi-Carter et al. 1998
). Group II metabotropic glutamate receptors (mGluR2 and
mGluR3) have been localized to the suprapyramidal blade of the dentate gyrus in apparent association with the medial perforant path from entorhinal cortex to the midmolecular layer (Petralia et al.
1996
; Testa et al. 1994
). Inhibitory
interneurons and collaterals, located in the dentate gyrus, modulate
responses at the dendritic tree and cell body of the granule cells as
well as the presynaptic afferents at the medial perforant path-granule
cell synapse (Freund and Buzsaki 1996
).
Hippocampal LTP has been shown to require NMDA receptor activation
(Burgard et al. 1989; Harris and Cotman
1986
; Herron et al. 1986
) and an increase in
cAMP levels (Blitzer et al. 1995
, 1998
; Nguyen
and Kandel 1996
; Stanton and Sarvey 1985b
). In
the dentate gyrus, LTP also requires
-adrenergic receptor activation (Bramham et al. 1997
; Stanton and Sarvey
1985a
). Because NAAG decreases forskolin-stimulated cAMP levels
via the mGluR3 receptor (Wroblewska et al. 1993
, 1997
,
1998
) and NAAG and mGluR3 are present in the dentate gyrus, we
speculate that NAAG may have a modulatory role in synaptic plasticity
in the medial perforant path-granule cell (MPP-gc) synapse. Consistent
with a role for NAAG and mGluR3 in hippocampal plasticity, activation
of group II mGluRs has been reported to decrease excitatory
postsynaptic potentials (EPSPs) in the mid-molecular layer
(Kilbride et al. 1998
; Macek et al. 1996
), and the group II mGluR agonist, DCG- IV, blocks
induction of LTP in the dentate gyrus (Huang et al.
1997
). Additionally, DCG-IV and NAAG have been reported to
induce long-lasting depression in the medial perforant path of the
disinhibited dentate gyrus (Huang et al. 1999
), and NAAG
reduces LTP of inhibitory postsynaptic potentials in the recurrent
inhibitory circuit following alvear stimulation in CA1 (Grunze
et al. 1996
). In the course of testing the hypothesis that NAAG
affects synaptic plasticity in the hippocampus, we discovered that
-NAAG functions as a highly selective mGluR3 antagonist and that the
mGluR3 has a role in LTP.
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METHODS |
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Both - and
-isomers of NAAG and ATP were purchased from
Sigma. trans-1-aminocyclopentane-1,3-dicarboxylate
(trans-ACPD), (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), ethyl glutamate, L(+)-2-amino-4-phosphonobutyric acid (L-AP4), MK801, 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), and CGP-55845 were purchased from Tocris Cookson. Tissue culture reagents were obtained from Gibco and Biofluids. Leupeptin was obtained from Boehringer Mannheim, and QX-314 from Astra Pharmaceuticals.
Preparation of hippocampal slices
Male Sprague-Dawley rats (Taconic, Germantown, NY) weighing 80-210 g were anesthetized with ketamine hydrochloride (100 mg/kg ip) and decapitated. Experiments were conducted according to the principles set forth in the "Guide for Care and Use of Laboratory Animals," Institute of Animal Resources, National Research Council, National Institutes of Health Pub. No. 74-23. Transverse slices (400 µm) of hippocampus were prepared using a McIlwain tissue chopper. Slices were placed in a modified Oslo interface recording chamber at 32-34°C and perfused at a rate of 3 ml/min with artificial cerebrospinal fluid (ACSF) containing (in mM) 26 NaHCO3, 124 NaCl, 1.75 KCl, 1.25 mM KH2PO4, 1.3 MgSO4, 2.4 CaCl2, and 10 dextrose; pH was adjusted to 7.4 by bubbling with a 95% CO2-5% O2 gas mixture. Slices were allowed to equilibrate for at least 2 h before recordings were initiated.
Electrophysiology
Figure 1A is a schematic of the hippocampal slice showing the placement of the stimulating electrode in the medial perforant path (MPP) leading from the entorhinal cortex to the dentate gyrus. Recording electrodes were placed in the MPP and in the granule cell layer located along the suprapyramidal blade of the dentate gyrus. In subsequent figures, the initial negative slope of the EPSP, recorded in the MPP, is plotted as percent of baseline EPSP slope (mean ± SE).
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Stimuli were delivered to the MPP fibers through a 100-µm
diameter monopolar Teflon-insulated, stainless steel wire electrode, exposed only at the tip. Extracellular recordings were obtained using
glass micropipettes filled with 2 M NaCl, 2-6 M resistance. Recording electrodes were positioned a minimum of 500 µm from the
stimulating electrodes and lowered to a final depth of 80 µm into the
slice. Only slices showing complete abolition of the population spike,
recorded in the cell body layer at 20-ms interpulse intervals in a
paired-pulse paradigm, were selected for study. Isolation of medial
perforant-path responses was confirmed by paired-pulse depression of
the EPSP seen at an 80-ms interpulse interval using a current intensity
that elicited an EPSP that was just subthreshold for a reflected spike
(Bramham et al. 1997
; McNaughton 1980
).
Test stimuli were delivered to the mid-molecular layer of the dentate
gyrus every 30 s to evoke subthreshold EPSPs. After establishment
of a stable baseline recording, EPSPs and population spikes were
recorded extracellularly from the medial perforant path and the granule
cell body layer in the dentate gyrus. Application of pharmacological
agents was achieved by switching the chamber perfusion solution to ACSF
containing the drug.
Drugs were tested for possible effects on presynaptic release of
glutamate using a paired-pulse paradigm. Two subthreshold stimuli (10 µs) were given to the MPP at interpulse intervals of 20, 30, and 80 ms. The initial slope of the second EPSP was normalized to the slope of
the first EPSP of a pair (as shown in Bramham et al.
1997).
"Blind" whole cell patch-clamp recording was used to measure AMPA
and NMDA receptor-mediated current in granule cells of the acute
hippocampal slice (Blanton et al. 1989). Patch pipettes (4-8 M
) were pulled in two stages (Model p-80/PC Flaming-Brown Micropipette Puller; Sutter Instruments) and filled with a solution of:
(in mM) 140 CsF, 1 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES, 2 TEA, 5 Na2-phosphocreatine, 5 QX-314, 0.1 leupeptin, and
4 Na2-ATP; pH was adjusted to 7.35 with CsOH.
Osmolality was adjusted to 290 mOsmol/1,000 g with sucrose. During
patch experiments, 20 µM bicuculline and 50 nM CGP-55845 were added
to the ACSF. The whole cell configuration was established in
voltage-clamp mode approximately 10 min after establishing a seal in
the cell-attached configuration (Hamill et al. 1981
).
Current measurements were made at holding potentials of
80,
30, and
+20 mV (250-ms voltage steps from
80 to
30 and +20 mV; Axopatch 1D,
Digidata 1200 series interface, pClamp 8.0). The late EPSC component
(NMDA receptor current) was separated from the early EPSC component
(AMPA receptor current) (Hestrin et al. 1990
) by taking
current measurements approximately 40 ms after stimulus. Resting
membrane potential was also measured at the time of whole cell
formation. Cells with resting potentials near
80 mV were accepted for
data analysis. All other conditions of our preparations were the same
as those described during extracellular recording.
Cell cultures
Primary cultures of rat cerebellar granule cells were prepared
from 8-day-old Sprague-Dawley pups (Taconic, Germantown, NY) as
described previously (Gallo et al. 1982). Cells were
plated on the poly-L-lysine-coated dishes at the density
1.25 × 106 cells/ml and cultured in basal Eagle's medium
supplemented with 10% fetal bovine serum (heat inactivated), 2 mM
glutamine, 50 mg/ml gentamycin, and 25 mM KCl. To prevent proliferation
of the nonneuronal cells, cytosine arabinoside was added (10 µM) to
the culture 24 h after plating. Cells kept in vitro for 6-8 days
were used.
Transfected cell lines
Mammalian cell lines expressing metabotropic glutamate receptor
mRNAs were prepared as described previously (Wroblewska et al.
1997). Briefly, mGluR2 and mGluR3 cDNAs (kindly provided by Dr.
S. Nakanishi) were inserted into an EcoRI site of the
mammalian expression vector pcDNA3 (Invitrogen) containing a
neomycin-resistant gene. Mammalian cells (Chinese hamster ovary cells,
CHO, and baby hamster kidney cells, BHK) were transfected with these
constructs using the calcium phosphate method as described by
Chen and Okayama (1987)
. The BHK cell lines were
maintained in the Dulbecco's modified Eagle's medium (DMEM, Gibco)
with L-glutamine (2 mM), sodium pyruvate, 10% fetal bovine
serum (FBS, Gibco) and penicillin/streptomycin (pen/strep, Biofluids).
The CHO cells were maintained in the medium described previously
(Tanabe et al. 1992
). Gentamycin (G-418, Gibco) was used
to select stable neomycin-resistant cell lines expressing mGluR mRNAs.
Positive clones were identified by reverse transcription PCR reaction
with specific primers and the response to forskolin stimulated cAMP
formation as described previously (Wroblewska et al.
1997
).
Assays
IP3.
Cells were prepared and assayed for inositol trisphosphate
(IP3) using methods previously described (Wroblewska et
al. 1993).
CAMP.
Rat cerebellar granule cells, BHK-mGluR3 or CHO-mGluR2 stably
transfected cell lines, were inoculated on the 24-well plates and grown
for 6-8 days in the growing medium. Cells were preincubated for 10 min
in medium containing 1 µM MK801, 10 µM CNQX, and after 500 µM
3-isobutyl-1-methylxanthine (IBMX) for granule cells or PBS and
IBMX for cell lines. Cells were then incubated for 7 min with either
forskolin (10 µM) alone, forskolin + agonist, or forskolin + agonist + -NAAG (100 µM) as described previously (Wroblewska et al.
1993
, 1997
). The measurements of cAMP were performed with Amerlex cAMP 125I kit (Amersham). Curve fitting
of the data was performed using GraphPad Prism 2.0 (GraphPad Software).
Data analysis and statistics
LTP-EXTRACELLULAR RECORDINGS.
Responses were amplified, filtered (d.c.3 kHz), digitized at 20 kHz
(DAS-20 interface, Keithley Metrabyte, Taunton, MA), and stored for
analysis using the Labman data-acquisition analysis program (a gift of
Dr. T. Teyler, NeuroScientific Laboratories, Rootstown, OH). Measures
of synaptic efficacy were made using the initial slope of the EPSP.
Values were normalized to percentages of the mean baseline value. Data
are expressed as the means ± SE. Statistical analyses of drug
effects in hippocampal slices were carried out in Statview (Abacus
Concepts, Berkeley, CA) using a two-tailed paired t-test or
ANOVA plus a post hoc Bonferroni/Dunn test for multiple comparisons.
WHOLE CELL PATCH-CLAMP RECORDINGS. Responses were amplified, digitized at 20 kHz, and stored for analysis (Axopatch 1D, Digidata 1200 series interface, pClamp 8.0). Measurements of NMDA current were made when AMPA current subsided (approximately 40 ms).
BIOCHEMISTRY. Statistical analyses of drug effects in cultured cells were carried out in Sigma Plot using the Student's t-test. Data were fit to a sigmoidal log dose-response curve using Prism 2.0 (GraphPad Software). A probability of 0.05 was selected as the level of statistical significance for all data.
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RESULTS |
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NAAG blocks LTP
High-frequency stimulation (HFS; 100 Hz, 2 s) of the MPP
increased subthreshold EPSP slopes recorded in the MPP (Fig.
1B; see top trace in inset). Long-term
potentiation (LTP; approximately 120% baseline) was significant 120 min after HFS (Fig. 1B; n = 5;
P < 0.05; paired t-test). To test the
prediction that NAAG will block LTP in the dentate gyrus, we bath
perfused hippocampal slices with 200 µM NAAG for 20 min prior to
giving HFS (100 Hz, 2 s). NAAG perfusion had no detectable effect
on EPSP slope or amplitude during the acquisition of baseline
(see solid squares, bottom trace in Fig. 1B;
n = 4; P > 0.05; ANOVA). NAAG (200 µM) blocked the increase in slope and amplitude normally seen in
control LTP (n = 4; P > 0.05; paired
t-test; Fig. 1B; see bottom trace in
inset). To test the effects of a lower concentration of NAAG on LTP, the tissue was perfused with 50 µM NAAG for 20 min prior to
giving HFS (100 Hz, 2 s). As predicted, 50 µM NAAG prevented the
maintenance phase of LTP (n = 3; P > 0.05; paired t-test; Fig. 1C). The ability to
obtain posttetanic potentiation at a lower concentration of NAAG, but
not with 200 µM NAAG, suggested that the higher concentration of NAAG
was in some way affecting presynaptic release of transmitter. This was
supported by the findings of Macek et al. (1996), who
report that group II mGluR autoreceptors decrease EPSPs at the MPP-gc
synapse. In contrast, Huang et al. (1997)
reported that
NAAG acts at a postsynaptic site. In light of these previous findings,
further analysis of the effects of NAAG needed to be performed.
NAAG has no effect on NMDA or AMPA receptor current
To confirm that NAAG did not affect NMDA or AMPA receptor currents
in granule cells in the acute hippocampal slice, we used blind whole
cell patch-clamp recordings of MPP excitatory postsynaptic currents
(EPSCs). Neither 50 nor 200 µM NAAG had any effect on the NMDA
receptor-mediated slow EPSC or on the AMPA receptor-mediated fast EPSC
at any potential (n = 3; Fig.
2). To verify that NAAG itself does not
induce an inward current, potentially mediated through the ionotropic
glutamate receptors, currents were compared at 80,
30, and +20 mV
before eliciting an EPSC in the absence and presence of 50 and 200 µM
NAAG. Neither 50 nor 200 µM NAAG induced any current at any potential
(ANOVA; n = 3; P > 0.05; data not
shown). We conclude from these experiments that the ability of 200 µM
NAAG to inhibit the posttetanic potentiation seen with 50 µM NAAG is
not caused by NAAG acting as an agonist or antagonist at either NMDA or
AMPA receptors.
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Ethyl glutamate and NAAG rescue LTP from NAAG
To further characterize the NAAG-induced block of LTP, we tested the group II mGluR specific antagonist ethyl glutamate for its ability to antagonize the effects of NAAG. Ethyl glutamate prevented the blockade of LTP by NAAG (Fig. 3A; n = 3; P < 0.05; paired t-test). This supports the hypothesis that NAAG is acting through a group II mGluR, either mGluR2 or mGluR3.
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Although we previously demonstrated that the synthetic structural
isomer of NAAG, -NAAG, failed to act as an mGluR3 agonist (Wroblewska et al. 1993
, 1997
), it was not examined for
antagonist activity at the mGluRs. When tested in our LTP model,
-NAAG mimicked the effects of ethyl glutamate and prevented the
blockade of LTP by NAAG (Fig. 3B; n = 4;
P < 0.05; paired t-test). These data suggest that
-NAAG is a group II mGluR antagonist and that it rescues LTP at either the group II mGluR2 or mGluR3 subtype. Because we
have previously shown that NAAG is a specific agonist at the mGluR3
receptor, but not the mGluR2 receptor (Wroblewska et al. 1997
), we hypothesized that
-NAAG was acting as an
antagonist at the group II mGluR3 subtype.
While -NAAG is an inhibitor (Ki = 1 µM) of extracellular peptidase activity against NAAG (GCP II), this
peptidase activity is also inhibited by those concentrations of
PO
).
-NAAG has no effect on EPSPs
To characterize the response of the hippocampal slice to -NAAG,
100 µM
-NAAG was bath applied for 60 min without administering HFS. Our results showed that
-NAAG had no effect on MPP evoked EPSPs
(Fig. 3C).
-NAAG and NAAG have no effect on presynaptic transmitter release
There is evidence for both a presynaptic (Neale et al.
2000) and postsynaptic (Huang et al. 1999
) site
of action for NAAG presumably through the group II mGluR3 subtype. We
utilized a typical paired-pulse paradigm (McNaughton
1982
) to test the hypothesis that NAAG and
-NAAG act at
presynaptic mGluR3 to affect transmitter release (Neale et al.
2000
) and that this mechanism may underlie the peptide's
action on LTP. Two subthreshold stimuli (10 µs) were given to the MPP
with interpulse intervals (IPIs) of 20 and 80 ms. Neither exposure to
50 µM NAAG (n = 3; P > 0.05; paired t-test) nor exposure to100 µM
-NAAG (n = 5; P > 0.05; paired t-test) affected
paired-pulse depression of granule cell EPSP slopes recorded during
paired-pulse paradigms (Fig. 3D). If necessary, stimulus
intensity was adjusted prior to the paired-pulse paradigms to ensure
that the EPSP caused by the first 10-µs stimulus of each pair matched
that measured during baseline recordings. While it remains possible
that NAAG is acting at presynaptic GluR3 to suppress LTP following
high-frequency stimulation, the peptide's failure to influence
paired-pulse depression is not supportive of this view.
-NAAG affects group II but not group I or III mGluRs
The ability of -NAAG to rescue LTP from blockade by NAAG,
together with our previous data demonstrating that NAAG is a selective mGluR3 agonist (Wroblewska et al. 1997
), suggests that
-NAAG may be an mGluR3 antagonist. To test this hypothesis, we
examined the activity of
-NAAG on cells expressing group I (mGluR1
and 5), group II (mGluR2 and 3), and group III receptors.
Glutamate stimulates IP3 formation in CHO cells stably expressing
mGluR1 or mGluR5 (Wroblewska et al. 1997). Dose-response studies using up to 100 µM glutamate on mGluR1
expressing cells and up to 30 µM glutamate on mGluR5 expressing cells gave
IC50 values of 15 and 5 µM, respectively.
Parallel glutamate dose-response assays in the presence of 100 µM
-NAAG were not significantly different from those obtained in the
absence of this peptide. When these cell lines were stimulated with
glutamate at these IC50 values (Fig.
4), 1-300 µM
-NAAG was found to be
without a significant effect. We conclude that
-NAAG does not act as a group I mGluR agonist or antagonist.
|
We tested the effects of -NAAG on both group II and group III mGluRs
in cerebellar granule cells in culture (Fig.
5A). We used glutamate (groups
II and III), trans-ACPD (group II), or L-AP4 (group III) to
activate mGluRs in these cells. We have previously shown that group II
and III mGluR activation in cerebellar granule cells results in
substantial decreases in forskolin-induced cAMP levels
(Wroblewska et al. 1993
). We found that
-NAAG (100 µM) blocked the ability of glutamate and trans-ACPD to
decrease cAMP levels induced by forskolin. In contrast,
-NAAG failed
to block the ability of L-AP4 to decrease cAMP via the group III mGluRs (Fig. 5A).
|
To verify the efficacy of -NAAG at group II mGluRs, increasing
concentrations of
-NAAG (10-100 µM) were applied to cerebellar granule cells that were treated with forskolin (10 µM) and DCG-IV (3 µM), an mGluR2 and mGluR3 agonist (Fig. 5B). Both 30 and
100 µM
-NAAG were found to block the action of DCG-IV in these
neurons (Fig. 5B; n = 6; P < 0.05; Student's t-test). We conclude from these results
that
-NAAG is an antagonist at the group II mGluRs but not at the
group I or group III mGluRs.
-NAAG antagonizes mGluR3, but not mGluR2, receptors
We have shown previously that NAAG decreases forskolin-stimulated
cAMP levels in cerebellar granule cells in culture (Wroblewska et al. 1993). Moreover, using cell lines expressing single
subtypes of metabotropic glutamate receptors, we have shown that NAAG
selectively activates the group II metabotropic glutamate receptor
subtype mGluR3 (Wroblewska et al. 1997
). To determine if
one or both of the two group II mGluR subtypes (mGluR2 or mGluR3) is
antagonized by
-NAAG, we tested for specificity using similar methods.
When tested on CHO cells stably transfected with mGluR2, -NAAG had
no significant effect on either DCG-IV (Fig.
6A)- or trans-ACPD (Fig. 6B)-mediated decreases in forskolin-stimulated cAMP.
In the same mGluR2-expressing cell line, up to 300 µM NAAG failed to
significantly affect forskolin-stimulated cAMP levels (102 ± 4.6% of forskolin stimulation; data not shown).
|
Cerebellar granule cells, which express group I-III mGluRs, were used
to test the specificity of -NAAG for the native mGluR3 receptor
subtype. At 10 µM and higher, NAAG significantly inhibited the
forskolin-stimulated increase in cAMP levels, while
-NAAG (100 µM)
blocked the effect of all concentrations of NAAG (Fig. 7A).
|
To further confirm the ability of -NAAG to block the effects of NAAG
via the mGluR3 receptor, a BHK-mGluR3 cell line was stimulated with
increasing concentrations of NAAG in the absence and presence of 100 µM
-NAAG (Fig. 7B). As little as 3 µM NAAG significantly inhibited the forskolin-stimulated cAMP levels, while
-NAAG blocked the effect of NAAG. Taken together these data provide
the first evidence that
-NAAG is a selective mGluR3 antagonist.
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DISCUSSION |
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The data presented in this paper demonstrate that NAAG blocks LTP
of extracellularly recorded EPSPs at the MPP-gc synapse, 50 and 200 µM NAAG does not affect NMDA receptor current in granule cells of the
acute hippocampal slice, -NAAG and ethyl glutamate relieve the
blockade of LTP by NAAG,
-NAAG alone does not affect MPP-gc EPSPs,
NAAG and
-NAAG do not affect paired-pulse depression of the MPP, and
-NAAG is the first compound to be identified as a selective mGluR3 antagonist.
The peptide neurotransmitter, NAAG, is a selective agonist for mGluR3
in neurons (Wroblewska et al. 1993, 1997
) and glia
(Wroblewska et al. 1998
). This receptor is coupled to a
G protein that mediates a reduction in cytoplasmic levels of cAMP. We
have reported the presence of NAAG in interneurons in the dentate gyrus
as well as the presence of extracellular peptidase activity against
NAAG in this tissue (Anderson et al. 1986
; Bzdega
et al. 1997
; Fuhrman et al. 1994
; Moffett
et al. 1993
, 1995
). Additionally, group II receptors (mGluR2/3)
have been demonstrated in neurons and glia in the dentate gyrus
(Petralia et al. 1996
). We previously observed that LTP
in the dentate gyrus requires norepinephrine, acting on a
-adrenergic receptor, which stimulates adenylyl cyclase and produces
an increase in cAMP (Stanton and Sarvey 1985b
). These results, coupled with the fact that the mGluR3 receptor inhibits adenylyl cyclase, suggests that the mGluR3 receptor may regulate the
induction of LTP in this region of the hippocampus. Our results militate in favor of this hypothesis.
We found that, similar to the group II selective mGluR antagonist ethyl
glutamate, the synthetic -isomer of NAAG,
-NAAG, blocked the
action of NAAG on LTP. The only previously reported action of
-NAAG
was as a nonhydrolyzable inhibitor of nervous system GCP II activity
(Serval et al. 1990
). However, in the ACSF used to
perfuse the in vitro hippocampal slice preparation in this study, GCP
II was inhibited by the concentrations of phosphate and sulfate used in
the perfusion medium (Robinson et al. 1987
). Inhibition
of GCP II should enhance rather than diminish the actions of NAAG.
Because NAAG is a known agonist at the mGluR3 receptor and
-NAAG
reversed the effects of NAAG, we speculated that
-NAAG may act as an
mGluR3 receptor antagonist.
In testing the efficacy of -NAAG as an mGluR antagonist, we found
that it did not affect glutamate stimulation of group I mGluRs
expressed in CHO cells. Similarly
-NAAG had no effect on the
stimulation of cerebellar granule cell group III mGluRs by L-AP4. In
contrast,
-NAAG antagonized the action of trans-ACPD on
group II mGluRs (mGluR2 and mGluR3) and the action of NAAG on mGluR3 in
cerebellar granule cells.
To confirm that -NAAG discriminates mGluR3 from mGluR2, we examined
its effect in cell lines transfected with either mGluR2 or mGluR3.
Again the selectivity of NAAG was demonstrated as up to 300 µM NAAG
failed to activate mGluR2 and 30 µM NAAG maximally activated mGluR3
expressed in transfected cells. With similar selectivity,
-NAAG
antagonized the mGluR3 but not mGluR2 receptors in these cells. These
data support the conclusion that
-NAAG is an mGluR3 selective
antagonist and that induction of LTP in the MPP of the dentate gyrus
can be regulated by mGluR3.
-NAAG is the first subtype selective
mGluR3 antagonist to be identified.
Beyond functioning as an mGluR3 agonist, NAAG clearly has been
demonstrated to act as a low-potency agonist at NMDA receptors (Sekiguchi et al. 1992; Trombley and Westbrook
1990
; Westbrook et al. 1986
). In contrast,
Sekiguchi et al. (1989)
found that 5 µM NAAG decreased
the depolarization induced by both NMDA and quisqualate in oocytes that
had been injected with rat brain mRNA. These results led to speculation
that the partial inhibition of NMDA-induced transmitter release that
was observed following 200 µM NAAG application may be mediated by the
peptide acting as an antagonist at this receptor (Puttfarcken et
al. 1993
). Binding studies suggest that the peptide's affinity
for rat brain NMDA receptors is 30-fold less than glutamate
(Valivullah et al. 1994
). Given NAAG's low potency as
an NMDA agonist in physiological studies, a potentially more
parsimonious explanation of the findings of Sekiguchi et al.
(1989)
, and Puttfarcken et al. (1993)
is the possibility that NAAG may function as a partial agonist at this receptor. Additionally, it seems likely that NAAG interacts
differentially with NMDA receptor subtypes expressed at various
synapses (Benke et al. 1995
; Hess et al.
1999
; Monyer et al. 1994
; Wenzel et al. 1995
).
We have shown previously that MPP EPSP amplitude and area are decreased
by NMDA receptor antagonists (Dahl et al. 1990). Neither effect was seen during NAAG perfusion in our study. Additionally, we
find here that NAAG has no significant effect on NMDA receptor currents
in the granule cells of the acute hippocampal slice. These data support
our hypothesis that NAAG is affecting LTP via the mGluR3 receptor
rather than acting as an agonist or partial agonist at the NMDA receptor.
The location of the mGluR3 receptors that NAAG activates to block LTP
remains to be defined. An antibody that reacts with both mGluR2 and
mGluR3 has been used to identify receptors on both pre- and
postsynaptic membranes (Petralia et al. 1996;
Shigemoto et al. 1997
; Testa et al.
1994
). The activation of group II receptors decreases
voltage-dependent calcium currents (Bischofberger and Schild
1996
; Chavis et al. 1994
; Sayer et al.
1992
) and group II mGluR agonists have been shown to suppress
synaptic release in several systems (Hayashi et al.
1993
; Ishida et al. 1993
; Poncer et al.
1995
; Schaffhauser et al. 1998
; Vignes et
al. 1995
). We recently found that NAAG acting via presynaptic
mGluR3 receptors reduces depolarization-induced release of GABA from
cortical neurons and that this action is blocked by ethyl glutamate and
-NAAG (Zhao et al. 2001
). In the present study,
however, neither NAAG (50 or 200 µM) alone nor
-NAAG (100 µM)
alone affected the EPSPs evoked by single or paired-pulse stimulation
of the MPP. Since the paired-pulse paradigms reflect the efficacy of
presynaptic transmitter release, these data do not support a
presynaptic action for NAAG or
-NAAG at the MPP-gc synapse. It
remains possible, however, that NAAG acting via mGluR3 may render
presynaptic targets insensitive to the potentiating effects of
high-frequency stimulation.
Huang et al. (1999) reported that NAAG and DCG-IV induce
long-lasting depression in the dentate gyrus following MPP stimulation. In their hippocampal slice preparation, NAAG had no detectable effect
on paired-pulse depression but did cause a significant increase in MPP
evoked EPSPs. In contrast, NAAG had no effect on either EPSPs or
paired-pulse depression in our experiments. These contrasting findings
may be related to differences in methodological approach that include
submerged versus interface chambers, picrotoxin disinhibited versus
naturally inhibited slices, and age differences in the animals (40- to
80-g vs. 80- to 210-g rats).
The relative contributions of endogenous glutamate and NAAG to the
activation of mGluR3 are unknown. While our discovery of -NAAG's
antagonist properties may permit detection of endogenous ligand
activation of the mGluR3 receptor, discrimination between the actions
of endogenous glutamate and NAAG will be more complex. The development
and application of inhibitors (Nan et al. 2000
) of the
extracellular peptidase activity that hydrolyzes NAAG will contribute
to answering this question in the hippocampus and elsewhere in the
nervous system.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Brian M. Cox for helpful discussions concerning receptor binding.
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-38080 and NS-23865 and by Uniformed Services University of the Health Sciences (USUHS) Grants RO75EJ and TO75FU. The opinions and assertions contained herein are the private opinions of the authors and are not to be construed as official or reflecting the views of the USUHS or the U.S. Department of Defense.
Present address of P. M. Lea: Dept. of Neuroscience, Georgetown University, Washington, DC 20007-2197.
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FOOTNOTES |
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Address for reprint requests: J. M. Sarvey, Dept. of Pharmacology, Uniformed Services University, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799 (E-mail: jsarvey{at}usuhs.mil).
Received 26 July 2000; accepted in final form 9 November 2000.
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REFERENCES |
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