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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 302:433–441, 2002
Vol. 302, No. 2
33266/995631
Printed in U.S.A.
Dopamine Modulates the Function of Group II and Group III
Metabotropic Glutamate Receptors in the Substantia Nigra
Pars Reticulata
MARION WITTMANN, MICHAEL J. MARINO, and P. JEFFREY CONN
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia (M.W., M.J.M., P.J.C.); Department of Neuroscience,
Merck Research Laboratories, West Point, Pennsylvania (M.J.M., P.J.C.); and Tierphysiologie, University of Tuebingen, Germany (M.W.)
Received February 6, 2002; accepted April 5, 2002
According to current models of basal ganglia (BG) function,
DA modulates BG output through differential regulation of
medium spiny striatal neurons. These output neurons of the
striatum form two separate but parallel descending pathways, the so-called direct and indirect pathways. The direct
pathway provides an inhibitory input to the GABA-containing projection neurons of the SNr and the entopeduncular
nucleus, the two principal output nuclei of the BG (Grofova et
This work was supported by grants from the National Institutes of Health,
the National Institute of Neurological Disorders and Stroke, the National
Parkinson’s Foundation, the Tourette’s Syndrome Association, National Alliance for Research on Schizophrenia and Depression, and the U.S. Army Medical Research and Material Command.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.033266.
III mGluR activation [L-(⫹)-2-amino-4-phosphonobutyric acid,
L-AP4, 500 ␮M], at STN-SNr synapses is significantly decreased. This effect could be mimicked in control slices by prior
bath application of haloperidol (20 ␮M) and R-(⫹)-7-chloro-8hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) (20 ␮M) but not sulpiride (50 ␮M). Furthermore, application of dopamine (100 ␮M) and (⫾)-6-chloro-7,8dyhydroxy-3allyl-1-phenyl-2,3,4,5-tetra-hydro-1Hbenzazepine (SKF82958) (1 ␮M) but not quinpirole (10 ␮M)
could rescue the group II mGluR effect in reserpinized slices.
The effect of group III mGluR activation (L-AP4, 100 ␮M) on
inhibitory synaptic transmission was also significantly reduced
in slices from reserpine-treated animals. This effect was mimicked by haloperidol (20 ␮M), SCH23390 (20 ␮M), and sulpiride
(50 ␮M) in control slices. Thus, in a Parkinsonian state, the loss
of nigral DA may add to the overall pathophysiological changes
in basal ganglia output.
al., 1982). The indirect pathway provides a disynaptic disinhibition of the glutamatergic neurons of the subthalamic
nucleus (STN), which in turn provides an excitatory input to
the BG output nuclei. The balance between excitation and
inhibition of BG output through these two pathways is important in the control of motor behavior (Wichmann and
DeLong, 1998). It is generally accepted that striatal DA decreases BG output through a D1 DA receptor-mediated excitation of the direct pathway and a D2 DA receptor-mediated
inhibition of the indirect pathway (Gerfen et al., 1990).
A growing body of literature suggests that extrastriatal DA
signaling may also modulate the output of the BG, thereby
contributing to the fine-tuning of motor control. In addition
to their nigro-striatal projection, neurons of the SNc also
synthesize, store, and release DA from their cell bodies and
ABBREVIATIONS: BG, basal ganglia; DA, dopamine; GABA, ␥-aminobutyric acid; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus;
SNc, substantia nigra pars compacta; PD, Parkinson’s disease; mGluR, metabotropic glutamate receptor; ACSF, artificial cerebrospinal fluid;
IPSC, inhibitory postsynaptic current; EPSC, excitatory postsynaptic current; ANOVA, analysis of variance; PK, protein kinase; LY354740,
(⫹)-2-aminobicyclo[3䡠1䡠0]-hexane-2,6-dicarboxylate monohydrate; SCH23390 hydrochloride, R-(⫹)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5tetrahydro-1H-3-benzazepine; SKF82958, (⫾)-6-chloro-7,8-dyhydroxy-3allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-benzazepine; L-AP4, L-(⫹)-2-amino-4phosphonobutyric acid.
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ABSTRACT
Recent findings have shown that dendritically released dopamine (DA) plays an important modulatory role in the substantia
nigra pars reticulata (SNr). It is therefore possible that the loss
of DA observed in Parkinson’s disease (PD) could hold important consequences for nigral function. Previously, we have
shown that activation of presynaptically localized group II
metabotropic glutamate receptors (mGluRs) inhibits excitatory
transmission at the subthalamic nucleus (STN)-SNr synapse
and that activation of presynaptically localized group III mGluRs
decreases excitatory and inhibitory transmission in the SNr. To
test the hypothesis that nigral DA can modulate mGluR function
in the SNr, we performed whole-cell patch-clamp recordings
from ␥-aminobutyric acidergic SNr neurons in slices obtained
from rats that were acutely reserpinized. In slices obtained from
reserpinized animals, the effect of group II mGluR activation by
the selective agonist (⫹)-2-aminobicyclo[3䡠1䡠0]-hexane-2,6-dicarboxylate monohydrate (LY354740) (100 nM), but not group
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Materials and Methods
Electrophysiology. All experiments involving rats were carried
out in accordance with the Guide for the Care and Use of Laboratory
Animals. Whole-cell patch-clamp recordings were obtained under
visual control as described previously (Bradley et al., 2000). Fifteento 18-day-old Sprague-Dawley rats were used for all patch-clamp
studies. After decapitation, brains were rapidly removed and submerged in an ice-cold sucrose buffer: 187 mM sucrose, 3 mM KCl, 1.9
mM MgSO4, 1.2 mM KH2PO4, 20 mM glucose, and 26 mM NaHCO3,
equilibrated with 95% O2, 5% CO2. Parasagittal slices (300 ␮m in
thickness) were made using a Vibraslicer (WPI, Sarasota, FL). Slices
were transferred to a holding chamber containing normal ACSF: 124
mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 2.0 mM
CaCl2, 20 mM glucose, and 26 mM NaHCO3, equilibrated with 95%
O2/5% CO2. In all experiments, 5 ␮M glutathione and 500 ␮M pyruvate were included in the sucrose buffer and holding chamber to
increase slice viability. Slices were transferred to the stage of a
Hoffman modulation contrast microscope (Olympus Optical, Tokyo,
Japan) and continually perfused with ACSF (⬃3 ml/min, 32°C).
Neurons in the SNr were visualized with a 40⫻ water immersion
lens. Patch electrodes were pulled from borosilicate glass on a vertical patch pipette puller (Narashige, Tokyo, Japan) and filled with
140 mM potassium gluconate, 10 mM HEPES, 10 mM NaCl, 0.6 mM
EGTA, 0.2 mM NaGTP, and 2 mM MgATP, pH adjusted to 7.4 with
0.5 N KOH. Electrode resistance was 3 to 7 M⍀.
GABA-ergic SNr neurons were identified according to previously
established electrophysiological criteria (Richards et al., 1997).
GABA-ergic neurons exhibited spontaneous repetitive firing, short
duration action potentials, little spike frequency adaptation, and a
lack of inward rectification, whereas dopaminergic neurons displayed no or low-frequency spontaneous firing, longer duration action potentials, strong spike frequency adaptation, and a pronounced
inward rectification. All of the data presented in these studies are
from neurons that fit the electrophysiological criteria of GABA-ergic
neurons.
Excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) were evoked with a bipolar tungsten stimulation electrode (0.4 –12.0 ␮A, every 30 s) placed within the cerebral
peduncle rostral to and outside of the SNr. EPSCs were recorded
from a holding potential of ⫺60 mV and bicuculline (20 ␮M) was bath
applied during all EPSC recordings to block inhibitory transmission.
IPSCs were recorded at a holding potential of ⫺50 mV and 6-cyano2,3-dihydroxy-7-nitroquinoxaline (10 –20 ␮M) and D(⫺)-2-amino-5phosphonopentanoic acid (10 –20 ␮M) were present in the bath to
block excitatory transmission. Kainate-evoked currents were recorded at a holding potential of ⫺60 mV, and tetrodotoxin (1 ␮M) was
present in the bath to block synaptic transmission. Kainate (100 ␮M)
was applied directly to the postsynaptic cell with a fast application
system (Alagarsamy et al., 1999).
Catecholamine Depletion. To deplete DA, reserpine (5 mg/kg
i.p.) was administered acutely to the rats 1 to 1.5 h before sacrifice,
and the brain slices were continually perfused with reserpine (10 –20
␮M) after the dissection. This acute treatment with reserpine induced a marked catalepsy in all animals (data not shown). The
degree of depletion of DA in the SNr and SNc induced by similar
treatment was described previously by histochemical and in vivo
studies (Bjorklund and Lindvall, 1975; Elverfors and Nissbrandt,
1991; Heeringa and Abercrombie, 1995).
Drugs and Drug Application. Drugs were made into stock
solutions of 1 to 100 mM and diluted to the desired concentration in
ACSF immediately before bath application to the slice. Antagonists
were applied at least 15 min before application of agonists. Dopamine hydrochloride and (⫺)-quinpirole hydrochloride stocks were
made in water containing sodium metabisulfite such that the final
solution applied to the slice contained 50 ␮M sodium metabisulfite.
Reserpine stocks were made in glacial acetic acid at 5000 times its
final concentration. Haloperidol hydrochloride stocks were made in 4
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dendrites (Bjorklund and Lindvall, 1975; Cheramy et al.,
1981; Rice et al., 1997). By its actions on D2 autoreceptors
located on SNc DA-ergic neurons, somatodendritically released DA can modulate SNc cell firing and subsequent DA
release in the striatum (Santiago and Westerink, 1991). Furthermore, it has been shown that the dendrites of DA neurons in the SNc extend ventrally into the SNr (Bjorklund and
Lindvall, 1975; Fallon and Loughlin, 1995) where they release DA (Geffen et al., 1976; Rice et al., 1997). This dendritically released DA is known to modulate GABA and glutamate release within the SNr (Abarca et al., 1995; Aceves et
al., 1995; Radnikow and Misgeld, 1998) and influence SNr
neurons (Martin and Waszczak, 1994; Huang and Walters,
1994; Martin and Waszczak, 1996).
Recent studies suggest that the loss of SNc dopaminergic
neurons observed in PD results in a loss of striatal DA and a
concomitant increase in activity in the indirect pathway relative to the direct pathway (Wichmann and DeLong, 1998).
The resulting increase in activity of glutamatergic neurons in
the STN ultimately leads to a net increase in synaptic excitation of GABA-ergic projection neurons in the output nuclei
and is believed to underlie the motor impairments characteristic of PD (DeLong, 1990). In addition to these effects on
striatal function, recent in vivo studies indicate that loss of
DA tone in the SNr also contributes to the symptoms of PD
(Mayorga et al., 1999).
We have previously reported that mGluRs modulate excitatory and inhibitory synaptic transmission in the SNr
(Bradley et al., 2000; Wittmann et al., 2001). Furthermore,
we have suggested that these receptors could provide possible novel therapeutic targets for the treatment of PD by
providing a method of pharmacologically reducing excitatory
drive through the indirect pathway. For example, we have
demonstrated that group II mGluRs are presynaptically localized on STN terminals in the SNr and that activation of
these receptors reduces excitatory synaptic transmission at
the STN-SNr synapse (Bradley et al., 2000). Therefore, in the
Parkinsonian state, an agonist of group II mGluRs could
selectively reduce the increased excitatory drive through the
indirect pathway. In contrast, we have shown that group III
mGluRs are presynaptically located on excitatory and inhibitory terminals projecting to SNr neurons and that activation
of these receptors decreases excitatory and inhibitory synaptic transmission in the SNr (Wittmann et al., 2001). This
suggests that the group III mGluRs might be a less useful
target for the treatment of PD because these effects on excitatory and inhibitory transmission would tend to cancel each
other.
DA can modulate the function of other G protein-coupled
receptors (Ferre et al., 1992; Garcia et al., 1997; Chen et al.,
2001), raising the interesting possibility that nigral DA can
modulate the function of mGluRs in the SNr. Because DA
depletion is the hallmark of PD, any alteration in mGluR
pharmacology in a DA-depleted state would have important
implications both for therapeutic targeting and our basic
understanding of how these receptors modulate information
flow through the BG. We therefore investigated the pharmacology of mGluR-mediated inhibition of synaptic transmission in the SNr of DA-depleted rats.
Dopamine Modulates mGluR Function in SNr
435
parts of 1 N sodium hydroxide and 6 parts of 8.5% lactic acid.
(S)-(⫺)-Sulpiride stocks were made in dimethyl sulfoxide at 5,000 to
10,000 its final concentration. L-AP4 stocks were made in 1 M
equivalents of sodium hydroxide. LY354740, SCH23390 hydrochloride, (⫾)-SKF82958 hydrobromide, (⫺)-bicuculline methiodide,
6-cyano-2,3-dihydroxy-7-nitroquinoxaline disodium salt, D-(⫺)-2-amino-5-phosphonopentanoic acid, and kainic acid stock solutions were
made in water. LY354740 was a gift from D. Schoepp and J. Monn
(Eli Lilly, Indianapolis, IN). Other drugs were obtained from SigmaAldrich (St. Louis, MO), Alexis (San Diego, CA), and Tocris Cookson
(Ballwin, MO).
Data Analysis. Values are expressed as mean ⫾ S.E.M. Statistical comparisons between two groups were performed by using a
Student’s t test. Statistical comparisons between several groups
were performed by one-way ANOVA followed by Tukey’s post hoc
comparisons.
Results
Fig. 1. In slices obtained from reserpinized animals, the suppression of
EPSCs induced by the group II mGluR-selective agonist LY354740 is
reduced in substantia nigra pars reticulata neurons. In control animals,
application of LY354740 (100 nM) significantly and reversibly decreases
EPSCs (n ⫽ 9; p ⬍ 0.01). A, example traces of evoked EPSCs in control
animals before (predug), during (LY354740), and after (washout) brief
bath application of LY354740. In reserpine-treated animals, this effect of
LY354740 is significantly reduced. B, example traces of evoked EPSCs in
reserpine-treated animals before, during, and after brief bath application
of LY354740 (100 nM). C, bar graph showing the average effect of the
selective group II mGluR agonist LY354740 (100 nM) in control animals
and reserpine-treated animals. Each column represents the mean
(⫾S.E.M.) of 9 and 12 experiments, respectively (ⴱⴱⴱ, p ⬍ 0.001; t test).
group II mGluRs on excitatory synaptic transmission at
STN-SNr synapses. To address this issue we investigated the
effect of bath application of the group II mGluR-selective
agonist LY354740 (100 nM) on kainate-evoked postsynaptic
currents by fast application of 100 ␮M kainate to the cell in
the presence of tetrodotoxin (1 ␮M) and the DA receptor
antagonist haloperidol (20 ␮M). We have shown previously
that in control animals, activation of group II mGluRs has no
effect on kainate-evoked currents (Bradley et al., 2000). Prior
bath application of haloperidol (20 ␮M) for 20 min had no
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Dopamine Modulates the Group II mGluR-Mediated
Inhibition of Excitatory Transmission in the Substantia Nigra Pars Reticulata. Previous studies have shown
that group II mGluRs are presynaptically localized on glutamatergic terminals in the SNr and that activation of these
receptors by group II mGluR-selective agonists decreases
excitatory transmission in this nucleus by a presynaptic
mechanism (Bradley et al., 2000). To determine whether
endogenous nigral DA modulates this effect, we performed
similar experiments in slices obtained from DA-depleted animals acutely treated with reserpine.
As described previously (Bradley et al., 2000), 3-min bath
application of 100 nM LY354740, a highly selective agonist of
group II mGluRs, produced a significant depression of EPSCs
at the STN-SNr synapse (54.5 ⫾ 4.5%; n ⫽ 9; p ⬍ 0.01; Figure
1, A and C). Interestingly, in slices obtained from DA-depleted animals the effect of LY354740 on EPSC amplitude
was significantly decreased (19.9 ⫾ 4.1% inhibition of EPSCs; n ⫽ 12; p ⬍ 0.005; Figure 1, B and C). To further test the
hypothesis that ambient DA modulates the function of presynaptic group II mGluRs at the STN-SNr synapse, we produced an acute block of DA receptors with selective DA
antagonists in control slices. All antagonists were applied at
least 20 to 30 min before bath application of LY354740. Bath
application of 10 to 20 ␮M haloperidol, which blocks both D1
and D2 receptors at this concentration, mimicked the effect of
DA depletion, significantly reducing the depression of EPSCs
induced by LY354740 (35.5 ⫾ 3.5%; n ⫽ 8; p ⬍ 0.01; Fig. 2, B
and E). This effect of haloperidol was mimicked by the selective D1 receptor antagonist SCH23390 (20 –50 ␮M) (30.4 ⫾
2.8% inhibition of EPSCs; n ⫽ 7; p ⬍ 0.01; Fig. 2, C and E),
but not by the D2-selective antagonist sulpiride (50 ␮M)
(45.7 ⫾ 4.3% inhibition of EPSCs; n ⫽ 6; p ⬎ 0.05; Fig. 2, D
and E). Interestingly, higher concentrations of either haloperidol or the D1-selective antagonist SCH23390 showed a
tendency to inhibit excitatory transmission (data not shown).
Taken together, these data suggest that D1-like DA receptor
activation controls presynaptic group II mGluR-mediated inhibition of excitatory transmission in the SNr. However, it is
conceivable that DA receptor blockade or reserpine treatment unmask a postsynaptic group II mGluR-mediated effect
that could counteract the presynaptic actions of group II
mGluRs at this synapse. This postsynaptic effect could therefore lead to an overall decrease of the inhibitory effect of
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Fig. 2. Prior bath application of DA receptor antagonists in slices obtained from control animals also decreases the effect of the group II
mGluR-selective agonist LY354740 on EPSCs in the SNr. A to D, example
traces of evoked EPSCs before (predrug) and during (LY354740) brief
bath application of LY354740 alone (A) or in the presence of selective and
nonselective DA antagonists (B–D). Prior bath application of the nonselective DA antagonist haloperidol (20 ␮M) and the D1-selective antagonist SCH23390 (20 ␮M) significantly decreases the effect LY354740 (100
nM) on EPSCs (B and C). Prior application of the D2-selective antagonist
sulpiride (50 ␮M) has no effect on the effect of LY354740 (D). E, bar graph
showing the average effect of nonselective and selective DA antagonists
on the effect induced by LY354740. Each column represents the mean
(⫾S.E.M.) of data collected from six to nine cells (ⴱⴱ, p ⬍ 0.01; ANOVA
and Tukey’s least significant difference).
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effect on the ability of LY354740 (100 nM) to influence the
amplitude of kainate-evoked currents (amplitude of kainateevoked current predrug, 243.3 ⫾ 79.1 pA; during application
of 100 nM LY354740, 221.5 ⫾ 53.6 pA; n ⫽ 3; p ⬎ 0.5, paired
t test).
If the effect of reserpine treatment on the group II mGluRmediated inhibition of transmission at the STN-SNr synapse
is due to a lack of activation of the D1 DA receptor, it should
be possible to rescue the mGluR-mediated modulation in
reserpinized animals by the application of DA, or selective D1
DA receptor agonists. We tested this hypothesis by bath
applying DA agonists for at least 30 min before the application of LY354740 in slices obtained from reserpinized animals. Interestingly, bath application of 100 ␮M DA restored
the effect of the group II mGluR agonist from 19.9 ⫾ 4.1 to
42.1 ⫾ 9.5% inhibition of EPSCs (n ⫽ 5; p ⬍ 0.05; Fig. 3, B
and E). Consistent with the antagonist pharmacology, this
effect was mimicked by the selective D1-like DA receptor
agonist SKF82958 (1 ␮M) (46.2 ⫾ 2.7% inhibition of EPSCs;
p ⬍ 0.05; n ⫽ 5; Fig. 3, C and E) but not by the D2-selective
agonist quinpirole (10 ␮M) (24.4 ⫾ 8.1% inhibition of EPSCs;
n ⫽ 4; p ⬎ 0.05; Fig. 3, D and E). In addition, we found no
potentiation of the effect of LY354740 (100 nM) on EPSCs in
control rats induced by prior DA application (data not
shown). Taken together, these data suggest that under normal conditions tonic activation of D1-like receptors by ambient DA maintains the group II mGluRs presynaptically localized at the STN-SNr synapse in an active state.
Dopamine Does Not Modulate the Group III mGluRMediated Inhibition of Excitatory Transmission in the
Substantia Nigra Pars Reticulata. Group III mGluRs are
also presynaptically localized at the STN-SNr synapse, and
activation of these receptors also decreases excitatory transmission in the SNr by a presynaptic mechanism (Wittmann
et al., 2001). We therefore investigated whether ambient DA
modulates the effect of group III mGluR-selective agonists on
EPSCs. As described previously, the selective group III
mGluR agonist L-AP4 (500 ␮M) reduces EPSCs at the STNSNr synapse in slices obtained from normal animals (57.1 ⫾
3.8%; n ⫽ 4; p ⬍ 0.05; Fig. 4, A and C). In contrast to the
effect of reserpine treatment on group II mGluR-mediated
inhibition of excitatory transmission, the effect of 500 ␮M
L-AP4 was not decreased in experiments performed in DAdepleted slices (68.5 ⫾ 5.2% inhibition of EPSCs; n ⫽ 5; p ⬎
0.05; Fig. 4, B and C).
Dopamine Modulates the Group III mGluR-Mediated
Decrease of Inhibitory Synaptic Transmission in the
Substantia Nigra Pars Reticulata. Group III mGluRs are
also found presynaptically localized on GABA-ergic terminals in the SNr, and activation of these receptors by the
group III mGluR-selective agonist L-AP4 inhibits IPSCs in
Dopamine Modulates mGluR Function in SNr
437
Discussion
We have shown previously that activation of mGluRs in
the SNr produces an inhibition of synaptic transmission at
both inhibitory and excitatory synapses. Activation of presynaptically localized group III mGluRs induces a decrease
in inhibitory transmission (Wittmann et al., 2001), whereas
activation of group II or group III mGluRs at the STN-SNr
synapse produces a marked inhibition of excitatory transmission (Bradley et al., 2000; Wittmann et al., 2001). The
present study provides evidence that ambient DA tonically
modulates these mGluR functions in the SNr. In particular,
DA depletion by reserpinization produces a marked decrease
in the ability of group II mGluR agonists to inhibit excitatory
transmission, and also in the ability of group III mGluR
agonists to decrease inhibitory transmission. Interestingly,
Fig. 3. Prior bath application of DA receptor agonists in slices obtained
from reserpine-treated animals can restore the effect of the group II
mGluR-selective agonist LY354740 on EPSCs in the SNr. A to D, example
traces of evoked EPSCs before (predrug) and during (LY354740) brief
bath application of LY354740 alone (A) or in the presence of selective and
nonselective DA receptor agonists (B–D). Prior bath application of DA or
the D1-selective agonist SKF82958 (1 ␮M) significantly increases the
effect LY354740 (100 nM) on EPSCs in reserpinized animals (B and C).
Prior application of the D2-selective agonist quinpirole (10 ␮M) has no
effect on the effect of LY354740 (D). E, bar graph showing the average
effect of nonselective and selective DA agonists on the effect induced by
LY354740. Each column represents the mean (⫾S.E.M.) of data collected
from 4 to 12 cells (ⴱ, p ⬍ 0.05; ANOVA and Tukey’s least significant
difference).
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GABA-ergic SNr neurons by a presynaptic mechanism (Wittmann et al., 2001). We therefore performed experiments to
determine whether this effect of L-AP4 on IPSCs is modulated by ambient DA. As described previously, brief bath
application of the group III mGluR-selective agonist L-AP4
(100 ␮M) significantly decreased IPSC amplitude (80.7 ⫾
3.8%; n ⫽ 8; p ⬍ 0.01; Fig. 5, A and C). Interestingly, this
effect of 100 ␮M L-AP4 was significantly decreased in slices
from DA-depleted animals (47.6 ⫾ 9.2% inhibition of IPSCs;
n ⫽ 6; p ⬍ 0.01; Fig. 5, B and C). To test whether acute
blockade of DA receptors has the same effect as DA depletion
we applied selective DA antagonists in control slices. Consistent with previous reports (Radnikow and Misgeld, 1998), we
found that D1-selective antagonists decrease the amplitude
of evoked IPSCs in the SNr (data not shown). We therefore
applied all antagonists for at least 30 min to ensure that a
stable baseline was reached. Haloperidol (20 ␮M) mimicked
the effect of DA depletion and significantly reduced the depression of IPSCs induced by L-AP4 (52.4 ⫾ 8.1% inhibition
of IPSCs; n ⫽ 5; p ⬍ 0.05; Fig. 6, B and E). Interestingly, the
effect of haloperidol was mimicked by both the D1-selective
antagonist SCH23390 (20 ␮M) (50.9 ⫾ 9.2% inhibition of
IPSCs; n ⫽ 7; p ⬍ 0.05; Fig. 6, C and E), and the D2-selective
antagonist sulpiride (20 ␮M) (48.2 ⫾ 7.7% inhibition of IPSCs; n ⫽ 8; p ⬍ 0.05; Fig. 6, D and E), suggesting that both
D1-like and D2-like receptors are involved in this modulation
of group III mGluR function.
Taken together, these data suggest that ambient DA modulates the function of presynaptically localized group II and
III mGluRs on excitatory and inhibitory terminals in the
SNr, respectively. Thus, in a Parkinsonian state, the loss of
this modulatory control of excitatory and inhibitory inputs to
the output neurons of the BG may add to the overall pathophysiological changes in the BG output.
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the group III mGluR-induced inhibition of excitatory transmission is not effected by the reserpine treatment, indicating
some level of receptor specificity. The effects of reserpine are
mimicked in slices from control animals by the application of
DA antagonists, and the mGluR-mediated effects are rescued
in slices from reserpinized animals by the application of DA
agonists. Therefore, tonic DA levels seem to be crucial in
maintaining the mGluRs in an active state, and interactions
Fig. 5. In slices obtained from reserpinized animals, the suppression of
IPSCs induced by the group III mGluR-selective agonist L-AP4 is reduced
in substantia nigra pars reticulata neurons. In control animals, application of L-AP4 (100 ␮M) significantly and reversibly decreases IPSCs (n ⫽
8; p ⬍ 0.01). A, example traces of evoked IPSCs in control animals before
(predrug), during (L-AP4), and after (washout) brief bath application of
L-AP4. In reserpine-treated animals, this effect of L-AP4 is reduced
significantly. B, example traces of evoked IPSCs in reserpine-treated
animals before, during, and after brief bath application of L-AP4 (100
␮M). C, bar graph showing the average effect of the selective group III
mGluR agonist L-AP4 (100 ␮M) in control animals and reserpine-treated
animals. Each column represents the mean (⫾S.E.M.) of eight and six
experiments, respectively (多多多, p ⬍ 0.01; t test).
between these two transmitter systems may play a crucial
role in the fine-tuning of synaptic function in the SNr.
The present findings add to a growing body of literature
suggesting that somatodendritically released DA is able to
directly influence BG output. Behavioral studies have shown
that local injection of DA agonists and antagonists into the
SNr modulates locomotor activity in normal animals (Kelly
et al., 1987; Trevitt et al., 2001) and in 6-hydroxy-dopaminelesioned rats (LaHoste and Marshall, 1990). In particular,
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Fig. 4. Application of the group III mGluR-selective agonist L-AP4 (500
␮M) significantly and reversibly decreases EPSCs in the SNr (n ⫽ 4; p ⬍
0.05). In contrast to the group II mGluR-mediated effect on EPSCs this
effect is not affected in slices obtained from reserpinized animals. A,
example traces of evoked EPSCs in control animals before (predug),
during (L-AP4), and after (washout) brief bath application of L-AP4 (500
␮M). In slices obtained from reserpine-treated animals, this effect of
L-AP4 is not reduced. B, example traces of evoked EPSCs in reserpinized
slices before, during, and after brief bath application of L-AP4 (500 ␮M).
C, bar graph showing the average effect of the selective group III mGluR
agonist L-AP4 (500 ␮M) in control animals and reserpine-treated animals. Each column represents the mean (⫾S.E.M.) of four and five experiments, respectively (p ⬎ 0.05; t test).
Dopamine Modulates mGluR Function in SNr
439
Fig. 6. Prior bath application of DA receptor antagonists in slices obtained from control animals also decreases the effect of the group III
mGluR-selective agonist L-AP4 on IPSCs in the SNr. A to D, example
traces of evoked IPSCs before (predrug) and during (L-AP4) brief bath
application of L-AP4 alone (A) or in the presence of selective and nonselective DA antagonists (B–D). Prior bath application of the nonselective
DA antagonist haloperidol (20 ␮M), the D1-selective antagonist
SCH23390 (20 ␮M), and the D2-selective antagonist sulpiride (20 ␮M)
significantly decreases the effect L-AP4 (100 ␮M) on IPSCs (B–D). E, bar
graph showing the average effect of nonselective and selective DA antagonists on the effect induced by L-AP4. Each column represents the mean
(⫾S.E.M.) of data collected from five to eight cells (ⴱ, p ⬍ 0.05; ANOVA
and Tukey’s least significant difference).
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the D1 DA receptor has been proposed as an important regulator of nigral function. Anatomical studies have shown that
there is a dense localization of D1 receptors in the SNr (Yung
et al., 1995). Activation of D1 receptors by local administration of agonists alters the firing activity of GABA-ergic SNr
neurons measured in vivo (Waszczak, 1990; Martin and
Waszczak, 1994). Activation of presynaptic D1 receptors at
the nigrostriatal synapse induces an increase in GABA release both in vivo (Rosales et al., 1997) and in vitro (Aceves et
al., 1995), and enhances inhibitory transmission (Radnikow
and Misgeld, 1998) (but see also Miyazaki and Lacey, 1998).
Furthermore, application of D1 antagonists has been shown
to decrease IPSCs in slices, suggesting that presynaptic D1
DA receptors are tonically activated (Radnikow and Misgeld,
1998). The D1 DA receptor has also been implicated in modulation of excitatory transmission in the SNr. In vivo microdialysis experiments have shown that activation of the D1
DA receptor produces an increase in nigral glutamate efflux
(Abarca et al., 1995), an effect likely mediated by the activation of receptors localized on STN terminals (Rosales et al.,
1997). In addition, our finding that application of the D1selective antagonist SCH23390 showed a tendency to decrease EPSCs in the SNr is in agreement with these findings.
Interestingly, activation of D1 receptors in the SNr has
been shown to have antiparkinsonian actions (Mayorga et al.,
1999) and has been proposed as a novel therapeutic treatment (Taylor et al., 1991; Williams et al., 1997). We previously suggested that agonists for group II mGluRs could
provide novel therapeutic targets for the treatment of PD
based on the findings that activation of presynaptic group II
mGluRs at STN-SNr synapses decreases glutamate release
and therefore could counteract the pathological increase in
BG outflow observed in this disease. However, the present
findings suggest that a loss of DA in the SNr leads to a
decrease in group II mGluR function. Presynaptically localized group II mGluRs have been hypothesized to act as autoreceptors, providing a feedback control over the release of
glutamate. Therefore, pathological loss of nigral DA may
exacerbate the hyperactivation of the BG output nuclei observed in PD. The effect of DA on group II mGluR function is
mediated by activation of D1 but not D2 receptors. Therefore,
activation of D1 receptors at the STN-SNr synapse may help
reduce transmission at this synapse by maintaining feedback
control of glutamate release by group II mGluRs.
It is of interest that the effects of DA depletion where
restricted to the group II mGluRs at the STN-SNr synapse,
yet effected group III mGluR function at inhibitory synapses.
This indicates that both group II and III mGluRs can be
modulated by the activation of DA receptors, yet some degree
of specificity must be imparted by differences in cellular
biochemistry. The basic mechanism by which activation of
DA receptors suppresses group II mGluR function on excita-
440
Wittmann et al.
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terminals is not presently understood. One possible mechanism is suggested by the recent findings that the function of
presynaptic mGluRs is tightly regulated by protein kinases.
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Address correspondence to: Dr. P. Jeffrey Conn, Senior Director, Neuroscience, Merck Research Laboratories, Merck & Co., Inc., 770 Sumneytown
Pike, P.O. Box 4, WP 46-300, West Point, PA 19486-0004. E-mail:
[email protected]
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