Striatal metabotropic glutamate receptor function following experimental parkinsonism and chronic...

Striatal metabotropic glutamate receptor functionfollowing experimental parkinsonism and chroniclevodopa treatment

Barbara Picconi,1,2 Antonio Pisani,1,2 Diego Centonze,1,2 Giuseppe Battaglia,4 Marianna Storto,4

Ferdinando Nicoletti,3,4 Giorgio Bernardi1,2 and Paolo Calabresi1,2

1Clinica Neurologica, Dipartimento di Neuroscienze,

UniversitaÁ di Roma Tor Vergata, 2I.R.C.C.S. Fondazione

`Santa Lucia', 3Dipartimento di Fisiologia Umana e

Farmacologia, UniversitaÁ di Roma La Sapienza, Roma and4I.N.M. Neuromed, Pozzilli, Isernia, Italy

Correspondence to: P. Calabresi, Clinica Neurologica,

Dipartimento di Neuroscienze, UniversitaÁ di Roma `Tor

Vergata', Via Montpelier 1, Rome 00133, Italy

E-mail: [email protected]

SummaryExcessive activation of ionotropic glutamate receptorsin the striatum contributes to the pathophysiology ofmotor symptoms in Parkinson's disease. Metabotropicglutamate (mGlu) receptors regulate striatal excitatorysynaptic transmission, and adaptive changes in theirfunction might occur following dopaminergic denerva-tion and chronic levodopa-treatment (L-DOPA).Corticostriatal glutamatergic transmission was exam-ined in striatal slices obtained from rats unilaterallydenervated with the dopaminergic neurotoxin, 6-hydroxy-dopamine (6-OHDA), and from denervated rats chron-ically treated with L-DOPA plus benserazide (25 + 6.25mg/kg, intraperitoneally, twice daily for 21 days).Selective agonists of mGlu2 and -3 receptor subtypes[compounds LY379268 and (2S,2¢R,3¢R)-2-(2¢,3¢-[3H]-dicarboxycyclopropyl)glycine ([3H]DCG-IV)] exhibited amuch greater potency in depressing excitatory transmis-sion and corticostriatal synapses in slices prepared from6-OHDA-lesioned animals. Dopaminergic denervationaffected neither the ability of L-(+)-2-amino-4-phospho-nobutyric acid (L-AP4; a selective agonist of mGlu4, -6,

-7 and -8 receptors) to inhibit corticostriatal transmis-

sion, nor the ability of (S)-3,5-dihydroxyphenylglycine

(3,5-DHPG; a selective agonist of mGlu1 and -5 recep-

tors) to potentiate responses mediated by N-methyl-D-

aspartate (NMDA) receptor activation in striatal neu-

rones. The increased responsiveness to mGlu2/3 recep-

tor agonists was no longer detected in slices from

6-OHDA-lesioned animals chronically treated with

L-DOPA. 6-OHDA-induced denervation also led to an

increased expression of striatal mGlu2/3 receptor

proteins and to a >2-fold increase in the maximal dens-

ity (Bmax) of [3H]DCG-IV binding sites. These increases

were again reversed by chronic treatment with L-

DOPA. No changes in the expression of mGlu4 recep-

tors or the ai1 and ai2 subunits of the Gi proteins were

induced by any of the treatments. We suggest that an

enhanced sensitivity of pre-synaptic inhibitory mGlu2/3

receptors might represent an adaptive change triggered

by dopaminergic denervation, which can be reversed by

L-DOPA treatment.

Keywords: basal ganglia; dopamine; excitatory synaptic transmission; glutamate synaptic transmission

Abbreviations: DCG-IV = (2S,2¢R,3¢R)-2-(2¢,3¢-dicarboxycyclopropyl)glycine; 3,5-DHPG = (S)-3,5-

dihydroxyphenylglycine; L-AP4 = L-(+)-2-amino-4-phosphonobutyric acid; L-DOPA = levodopa; EPSC = excitatory post-

synaptic current; EPSP = excitatory post-synaptic potential; 6-OHDA = 6-hydroxydopamine; NMDA = N-methyl-D-

aspartate; RMP = resting membrane potential

IntroductionThe main pathological feature of Parkinson's disease is the

degeneration of dopamine-containing nigrostriatal neurones.

The irreversible loss of the dopamine-mediated control of

striatal function leads to the motor symptoms observed in this

disorder: bradykinesia, tremor and rigidity (Obeso et al.,

2000). The striatum also receives a massive glutamatergic

innervation arising from most cortical areas (Graybiel, 1990;

Smith and Bolam, 1990; Calabresi et al., 1996). The

contribution of the descending glutamatergic corticostriatal

pathway to the motor disorders associated with Parkinson's

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disease and the dyskinesias observed following treatment

with levodopa (L-DOPA) has been of increasing interest in

recent years (Calabresi et al., 2000; Chase and Oh, 2000). An

experimental lesion of the ascending nigrostriatal dopamine

pathway by 6-hydroxydopamine (6-OHDA) mimics parkin-

sonian pathology (Papa et al., 1994) and results in an

increased glutamatergic transmission in the striatum, as

determined by electron microscopy, in vivo microdialysis or

electrophysiology (Lindefors and Ungerstedt, 1990;

Calabresi et al., 1993; Ingham et al., 1998; Meshul et al.,

1999). Accordingly, blockade of ionotropic glutamate

receptors ameliorates Parkinson's disease symptoms and

reverses the 6-OHDA-induced changes in striatal glutamate

immunolabelling (Papa et al., 1995; Chase and Oh, 2000;

Robinson et al., 2001).

Metabotropic glutamate (mGlu) receptors, which are

coupled to G proteins, modulate corticostriatal transmission

and control motor behaviour (reviewed by Pisani et al., 1998).

To date, eight mGlu receptor subtypes (designated mGlu1 to

mGlu8) have been cloned from mammalian brain. These

mGlu receptors are classi®ed into three main groups on the

basis of sequence homology, pharmacological pro®le, and

coupling to second messenger systems (Pin and Duvoisin,

1995; De Blasi et al., 2001). The recent availability of

subtype-selective ligands (Schoepp et al., 1999) allows a

detailed analysis of how individual mGlu receptor subtypes

(or groups of subtypes) are involved in physiology and

pathology. Interestingly, selective activation of different

mGlu receptor subtypes differentially affects excitatory

transmission at corticostriatal synapses (Lovinger and

McCool, 1995; Pisani et al., 1997a, b, 2001). Thus, we

decided to examine the action of these agonists on excitatory

synaptic transmission in striatal slices obtained from 6-

OHDA lesioned rats and from denervated animals receving a

chronic L-DOPA treatment. To our knowledge, this study

represents the ®rst characterization of the adaptive changes of

mGlu receptors following experimental parkinsonism and

chronic L-DOPA treatment.

MethodsPreparation and maintenance of brain slicesCorticostriatal slices were prepared from adult male Wistar

rats (3±4 months old). The preparation and maintenance of

coronal slices have been described previously (Calabresi

et al., 1993; Pisani et al., 1997a, b). Brie¯y, corticostriatal

coronal slices (200±300 mm) were prepared from tissue

blocks of the brain using a vibratome. A single slice was

transferred to a recording chamber and submerged in a

continuously ¯owing Krebs solution (35°C, 2±3 ml/min)

gassed with 95% O2/5% CO2. The control solution comprised

126 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 1.2 mM

NaH2PO4, 2.4 mM CaCl2, 11 mM glucose and 25 mM

NaHCO3.

Intracellular recordingsIntracellular recording electrodes were ®lled with 2 M KCl

(30±60 MW). Signals were recorded with the use of an

Axoclamp 2A ampli®er, displayed on a separate oscilloscope

and stored on a digital system. The characteristics of action

potentials and of current±voltage curves in different experi-

mental conditions were studied using a fast chart recorder and

a digital system (pClamp 8; Axon Instruments, Foster City,

CA, USA). For synaptic stimulation, bipolar electrodes were

used. These stimulating electrodes were located either in the

cortical areas close to the recording electrode or in the white

matter between the cortex and the striatum in order to activate

corticostriatal ®bres. To study paired-pulse facilitation, the

intensity of stimulation was set to evoke corticostriatal

excitatory post-synaptic potentials (EPSPs) of 5±8 mV

amplitude, and the interstimulus interval was 60 ms. In

addition, some experiments were performed in voltage-clamp

in the whole-cell con®guration. Electrodes (4±5 MW) were

®lled with a solution containing 125 mM K+-gluconate, 10

mM NaCl, 1.0 mM CaCl2, 2.0 mM MgCl2, 0.5 mM 1,2-bis (2-

aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA),

19 mM N-(2-hydroxyethyl)-piperazine-N-s-ethanesulfonic

acid (HEPES), 0.3 mM guanosine triphosphate (GTP) and

1.0 mM Mg-adenosine triphosphate (Mg-ATP), adjusted to

pH 7.3 with KOH. Striatal spiny neurones were clamped at ±

80 to ±85 mV, close to their resting membrane potential

(RMP), and were recorded using an Axopatch 1D ampli®er

and Clampex 8.1 software. To evoke excitatory post-synaptic

currents (EPSCs), bipolar electrodes were placed on corticos-

triatal ®bres. Stimuli were delivered at 0.1 Hz, with an

interstimulus interval of 60 ms.

About 50% of the recordings were obtained in the

presence of 50 mM picrotoxin in order to rule out a

possible contamination of the EPSPs by depolarizing

potentials mediated by g-aminobutyric acid A-receptors.

Since these experiments gave similar results to those

obtained in the absence of this drug, all the data were pooled

together.

Data analysis and drug applicationsQuantitative data on modi®cations of EPSPs are expressed

as a percentage of the controls, the latter representing the

mean of responses recorded during a stable period (15±30

min) before the drug application. Values given in the text

and ®gures are mean 6 standard error of the mean

(SEM) of changes in the respective cell populations.

Wilcoxon's test or Student's t-test (for paired and

unpaired observations) were used to compare the means,

and ANOVA (analysis of variance) was used when

multiple comparisons were made against a single control

group. The concentration±response curves shown in the

®gures and the EC50 were obtained using Kaleidagraph

3.0 software running on a Power Macintosh. Drugs were

applied by dissolving them in saline to the desired ®nal

2636 B. Picconi et al.


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concentration. LY379268 was kindly provided by Dr D.D.

Schoepp and Dr A.E. Kingston (Eli Lilly, Indianapolis

IN, USA). All other drugs were from Tocris.

Preparation of 6-OHDA denervated rats andchronic L-DOPA treatmentTo obtain unilateral nigrostriatal lesions, rats (1±2 months

old, anaesthetized with 45 mg/kg of pentobarbitone intraper-

itoneally) were injected with 6-OHDA (8 mg/4 ml of saline

containing 0.1% ascorbic acid) via a Hamilton syringe

through a cannula inserted just rostral to the substantia

nigra using stereotaxic coordinates (Paxinos and Watson,

1986). Sham-operated (control) rats were injected with saline

+ 0.1% ascorbic acid alone. Twenty days later, the rats were

tested with a 0.05 mg/kg subcutaneous injection of apomor-

phine, and contralateral turns were counted for 1 h. Only

those rats that consistently made at least 400 contralateral

turns were used for the electrophysiological recordings

performed 2 months after the lesion. In some cases, 6-

OHDA-lesioned rats were anaesthetized with diethyl ether

and brain dissection con®rmed that the nigrostriatal pathway

was lesioned. This was established by the observation of a

>95% loss of dopamine neurones in the substantia nigra pars

compacta and the almost complete absence of dopamine

terminals in the striatum. This was monitored by using a

monoclonal antibody for tyrosine-hydroxylase. A group of 6-

OHDA-lesioned rats received L-DOPA/benserazide (25/6.25

mg/kg) twice daily intraperitoneally for 3 weeks. Benserazide

was given to prevent decarboxylation of L-DOPA in the

periphery, as commonly used in clinical practice. As reported

previously (Papa et al., 1994, 1995), the magnitude of the

rotational response to L-DOPA more than doubled during the

®rst week of treatment, but essentially remained constant

thereafter.

Western blot analysisStriata were removed and stored at ±80°C for 20 days. On the

day of the experiment, tissue was homogenized at 4°C in ice-

cold sodium dodecyl sulfate (SDS) lysis buffer containing 1

mM phenylmethylsulfonyl ¯uoride, pH 7.4, with a motor-

driven Te¯on-glass homogenizer (1700 r.p.m.). Five micro-

litres were used for protein determinations. Thirty-®ve

micrograms of proteins were resuspended in SDS±bromo-

phenol blue reducing buffer containing 20 mM dithiothreitol.

Western blot analysis was carried out using 8% SDS

polyacrylamide gels; gels were run on a minigel apparatus

(Mini Protean II Cell; Bio-Rad, Italy) and electroblotted on

ImmunBlot PVDF Membrane (Bio-Rad) for 1 h using a semi-

dry electroblotting system (Trans-blot system SD; Bio-Rad).

Filters were then blocked overnight in TTBS buffer (100 mM

Tris±HCl, 0.9% NaCl, 0.1% Tween 20, pH 7.4) containing

5% non-fat dry milk. Blots for mGlu receptors were

incubated for 1 h at room temperature with primary

polyclonal antibodies (1 mg/ml) against mGlu2/3 or mGlu4

receptors, or against the ai1 and ai2 subunits of the G

proteins (Upstate Biotechnology, Lake Placid, USA). Blots

were washed three times with TTBS buffer and then

incubated for 1 h with secondary peroxidase-coupled

anti-rabbit antibodies (diluted 1:5000 with TTBS).

Immunostaining was revealed by enhanced chemilumin-

escence (Amersham, Milan, Italy).

Measurement of [3H]DCG-IV binding in striatalmembranesFor binding studies, striata were stored at ±80°C for 20 days

and then (2S,2¢R,3¢R)-2-(2¢,3¢-[3H]-dicarboxycyclopropyl)-

glycine ([3H]DCG-IV]) binding was measured in striatal

homogenates, essentially as described by Mutel et al. (1998).

In brief, tissue was homogenized in 25 volumes of 50 mM

Tris±HCl (pH 7.1) and homogenates were centrifuged at

48 000 g for 10 min. The pellet was resuspended, incubated at

37°C for 10 min, and then centrifuged again at 48 000 g. The

resulting pellet was stored frozen at ±80°C. On the day of the

experiment, membranes were thawed and washed three times

in the assay buffer (50 mM Tris-HCl, pH 7.4, containing 2

mM MgCl2) by centrifugation at 48 000 g. The ®nal pellet

was resuspended in assay buffer. Aliquots of the ®nal

suspensions (100 mg of proteins) were transferred to test

tubes containing 1 ml of 3±1000 nM [3H]DCG-IV

(Amersham, speci®c activity 18.3 Ci/mmol). Samples were

incubated for 1 h at room temperature. At the end of the

incubation, membranes were ®ltered onto Whatmann GF/C

glass ®bre ®lters and washed three times with ice-cold

binding buffer. Non-speci®c binding was de®ned as the

binding left in the presence of 10 mM LY379268 (mGlu2/3

receptor agonist).

ResultsIntrinsic and synaptic properties of striatalneuronesPresumed striatal spiny neurones were recorded intracellu-

larly in slices from control (sham-operated) rats (n = 31), 6-

OHDA-lesioned rats (n = 33) and 6-OHDA-lesioned rats

chronically treated with L-DOPA (25 + 6.25 mg/kg,

twice daily for 21 days) (n = 32). Striatal spiny neurones

recorded from the three experimental groups had similar

intrinsic membrane properties: high RMP (sham: ±85 63 mV; 6-OHDA-lesioned: ±84 6 3 mV; 6-OHDA-lesioned

plus L-DOPA: ±84 6 4 mV; P >0.05) and action potential

discharge with little adaptation during depolarizing current

pulses (data not shown). These electrophysiological proper-

ties were similar to those reported previously by our group

and by others for medium spiny neurones of the striatum (Kita

et al., 1984; Jiang and North, 1991; Calabresi et al., 1993).

To evoke corticostriatal EPSPs, the stimulating

electrode was placed close to the recording electrode in the

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white matter between the cortex and the striatum. At the

RMP, these EPSPs were not affected by the N-methyl-D-

aspartate (NMDA) receptor antagonist, MK-801 [(5S,10R)-

(+)-5-methyl-10,11-dehydro-5H-dibenzo[a,d]cyclohepta-5,10-

imine maleate] (30 mM; n = 6 for each group), while they were

fully abolished by the a-amino-3-hydroxy-5-methyl-isoxa-

zole-4-propionate (AMPA) receptor antagonist, CNQX (6-

cyano-7-nitroquinoxaline 2,3-dione) (10 mM; n = 6 for each

group).

Effects of group II mGlu receptor activation oncorticostriatal transmission in brain slices fromcontrol and treated ratsWe examined the possible differential role of group II mGlu

(mGlu2 and mGlu3) receptors in the modulation of synaptic

transmission at corticostriatal synapses in control rats, 6-

OHDA-lesioned rats, and 6-OHDA-lesioned rats treated with

L-DOPA. In most of the experiments we used LY379268, a

potent and selective mGlu 2/3 receptor agonist (Monn et al.,

1999). As shown in Fig. 1A and C, this agonist produced a

dose-dependent inhibition of EPSPs evoked by corticostriatal

stimulation in all three experimental groups. However, the

potency of this compound in inhibiting the EPSPs was much

higher in 6-OHDA-lesioned slices (n = 13; EC50 0.042 mM)

than in slices obtained from sham-operated rats (n = 12; EC50

0.37 mM; P < 0.001). Conversely, slices obtained from 6-

OHDA-lesioned rats with chronic L-DOPA treatment showed

a pharmacological sensitivity to LY379268 similar to that

observed in sham-operated animals (n = 13; EC50 0.32 mM; P

> 0.05). Similar results were obtained with DCG-IV, a drug

that selectively activates mGlu2/3 receptors at concentrations

lower than 2±5 mM (Schoepp et al., 1999) (Fig. 1B). In order

Fig. 1 Differential sensitivity of pre-synaptic group II mGlu receptors in normal (sham-operated), 6-OHDA-denervated and 6-OHDA-denervated plus L-DOPA-treated rats. (A) The graph shows the dose±response curve obtained for the inhibitory effects of LY379268 inthe three experimental animal groups. (B) The dose±response curve obtained for the inhibitory effects of DCG-IV in the threeexperimental animal groups. (C) The electrophysiological traces are corticostriatal EPSPs recorded before (left) and during (right) theapplication of LY379268, a group II mGlu receptor agonist, in sham (a), 6-OHDA-lesioned (b) and 6-OHDA-lesioned plus L-DOPA-treated rats (c). The dotted lines represent the RMP of the recorded neurones (±85 mV). (D) Electrophysiological traces of corticostriatalEPSCs recorded from 6-OHDA-lesioned rats, before (above) and after (below) the application of 0.1 mM LY379268. The neurone wasclamped at ±80 mV. (E) Histogram showing that LY379268 signi®cantly increased paired-pulse facilitation in the three experimentalgroups (**P < 0.01 compared with the pre-drug condition), suggesting a pre-synaptic mechanism of action (see text). Each data point wasobtained from at least four single experiments.



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to investigate whether the depression of EPSPs by group II

mGlu receptor agonists was dependent on pre- or post-

synaptic sites of action, we measured synaptic responses to a

pair of stimuli before and during the applications of

LY379268. In these experiments, the interstimulus interval

was 60 ms. Paired-pulse modi®cation of neurotransmission

has been studied extensively and is attributed to a pre-

synaptic change in release probability (Manabe et al., 1993;

Schulz et al., 1994). An increase in the ratio of the second

pulse response to the ®rst pulse response (EPSP2/EPSP1)

indicates a decrease in the release probability. The decrease in

transmitter release is consistent with the observations that

manipulations depressing transmitter release usually increase

the magnitude of this ratio also at corticostriatal synapses

(Calabresi et al., 1997). Interestingly, 0.1±1 mM LY379268

and 1 mM DCG-IV (n = 5 for each drug and experimental

group in current±clamp experiments; n = 4 for each drug and

experimental group in voltage±clamp experiments; P < 0.01

for all) induced a decrease in the amplitude of EPSCs and

EPSPs that was coupled to a clear increase in the

EPSP2:EPSP1 ratio (or EPSC2:EPSC1 ratio) in all the

experimental groups (Fig. 1D and E). LY379268 and DCG-

IV, at the concentrations tested in this study, did not affect

RMP and input resistance of the recorded neurones (n = 12 for

each experimental condition and each drug; P > 0.05; data not

shown).

Effects of group III mGlu receptor activationon corticostriatal transmissionWe examined the action of l-(+)-2-amino-4-phosphorobu-

tyric acid (L-AP4), a selective agonist acting at group III

mGlu receptors (Pisani et al., 1997a), on corticostriatal

EPSPs in slices obtained from the three groups of

animals. As shown in Fig. 2A and B, L-AP4 induced a

dose-related inhibitory effect on the EPSP amplitude. The

EC50 calculated for this effect was similar in the three

groups of experiments (P > 0.05; n = 12 for each

experimental condition): 11.8 mM in slices from sham-

operated rats, 12 mM in slices from 6-OHDA-lesioned

rats, and 11.4 mM in slices from 6-OHDA-lesioned rats

treated with L-DOPA. Also, for L-AP4 (30 mM; n = 4

for each experimental condition), the reduction of the

EPSP amplitude was associated with a signi®cant (P <

0.01) increase in the paired-pulse facilitation, suggesting a

pre-synaptic mechanism of action in all three experimen-

tal groups (Fig. 2C). L-AP4, at the concentration tested in

this study, did not affect RMP and input resistance (n =

10 for each experimental condition; P > 0.05; data not

shown).

Effects of group I mGlu receptor activation onNMDA responses in striatal neuronesWe also investigated the effect of 3,5-DHPG, a mGlu

receptor agonist selectively acting at group I (mGlu1 and

mGluR5) (Pisani et al., 1997b, 2001) on the EPSP amplitude

at the various concentrations used. As reported in Fig. 3A,

this agonist failed to affect EPSP amplitude both in sham-

operated rats and in 6-OHDA-lesioned animals (n = 5 for each

experimental condition; P > 0.05). Accordingly, no effect on

Fig. 2 Similar sensitivity of pre-synaptic group III mGlu receptorsin normal (sham-operated), 6-OHDA-denervated and 6-OHDA-denervated plus L-DOPA-treated rats. (A) Electrophysiologicaltraces of corticostriatal EPSPs recorded before (left) and during(right) the application of L-AP4, a group III mGlu receptoragonist, in control (a), 6-OHDA-lesioned (b) and 6-OHDA-lesioned plus L-DOPA-treated rats (c). The dotted lines representthe RMP of the recorded neurones (±85 mV). (B) The dose±response curve obtained for the inhibitory effects of L-AP4 in thethree experimental animal groups. (C) Histogram showing that L-AP4 signi®cantly increased paired-pulse facilitation in the threeexperimental groups (**P < 0.01).



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EPSP amplitude was detected in 6-OHDA-lesioned rats

treated with L-DOPA either (n = 6; P > 0.05; data not shown).

Since activation of group I mGlu receptors by 3,5-DHPG has

been reported to enhance NMDA-mediated membrane

depolarization in striatal spiny neurones (Pisani et al.,

1997b, 2001), we also tested whether this facilitatory effect

could express some pharmacological changes in 6-OHDA-

lesioned rats compared with sham-operated animals. As

shown in Fig. 3B, the EC50 measured for this effect was

similar in the two groups: 44.8 mM in sham-operated rats (n =

10) and 40.2 mM in 6-OHDA-lesioned animals (n = 11; P >

0.05). Also, in 6-OHDA-lesioned rats treated with L-DOPA,

the EC50 for the facilitatory action was similar (42.3 mM; n =

7; P > 0.05; data not shown). 3,5-DHPG, at the concentrations

tested in this study, did not affect RMP and input resistance (n

= 11 for each experimental condition; P > 0.05; data not

shown).

Expression of mGlu2/3 receptor protein, mGlu4receptor protein, and Gai1 and Gai2 proteins incontrol and treated ratsImmunoblots with mGlu2/3 antibodies revealed two faint 100

kDa bands, corresponding to receptor monomers, and a

higher molecular weight band, corresponding to receptor

dimers. The higher band was more heavily labelled in the

striatum of all groups of animals. Expression of mGlu2/3

receptors was up-regulated in the denervated striatum (left) of

rats unilaterally infused with 6-OHDA. No changes were seen

in the contralateral striatum as compared with the left or right

striata of control rats. L-DOPA treatment had no effect on

striatal mGlu2/3 receptor expression in control rats, but it

brought the expression to control levels in the denervated

striatum of 6-OHDA-treated rats (Fig. 4). No changes were

detected in the expression levels of mGlu4 receptors, or the

Fig. 3 Activation of group I mGlu receptors does not alter the amplitude of corticostriatal EPSPs, but enhances NMDA-mediatedmembrane depolarization to a similar extent as in normal (sham-operated) and 6-OHDA-denervated rats. (A) The graph to the left showsthe lack of effect of 3,5-DHPG, a group I mGlu receptor agonist, on EPSP amplitude both in control and dopamine-denervated striatalneurones. Traces on the right show EPSPs recorded before (left) and during (right) the application of this agonist in control (a) anddopamine-denervated (b) brain slices. (B) The graph to the left shows that the 3,5-DHPG-induced potentiation of NMDA-mediatedresponses is unaltered after dopamine denervation of striatal neurones. Traces to the right are examples of NMDA-induced membranedepolarization before (left) and during (right) the application of 3,5-DHPG in control (a) and 6-OHDA-denervated slices (b). The RMPwas ±86 mV in (a) and ±84 mV in (b).



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ai1 and ai2 subunits of the G proteins in any group of animals

(Fig. 4).

[3H]DCG-IV binding in striatal membranesfrom control and treated ratsSaturation analysis of [3H]DCG-IV binding in striatal mem-

branes of sham rats revealed a Bmax value of 1043 + 182 fmol/

mg protein, and an apparent KD value of 212 + 58 nM, in

agreement with values reported in rat cortical homogenates

(Mutel et al., 1998) (Fig. 5). The Bmax of [3H]DCG-IV

binding was increased by >2-fold in the denervated striatum

of 6-OHDA-treated rats, whereas no substantial changes were

observed in the contralateral striatum. L-DOPA treatment in

intact rats did not change the maximal density of [3H]DCG-

IV binding sites, although it slightly increased the binding

af®nity. L-DOPA treatment in 6-OHDA-injected animals

prevented the increase in the Bmax of [3H]DCG-IV binding in

the denervated striatum (Table 1).

DiscussionThe corticostriatal projection represents the major excitatory

glutamatergic input to the striatum (Smith and Bolam, 1990).

Glutamatergic ®bres also arise from the thalamus (Lapper and

Fig. 4 Expression of mGlu2/3 and mGlu4 receptor proteins, andGai1 and Gai2 proteins in the striatum of sham, 6-OHDA-lesionedand 6-OHDA-lesioned plus L-DOPA-treated rats. Representativeimmunoblots are shown in (A). Densitometric analysis of lesioned(SX) and contralateral (DX) slices is shown in (B). Values aremeans + SEM of four individual samples. *P < 0.05 (one-wayANOVA and Fisher's PLSD, probability at the least signi®cantdifference) compared with all other values.

Fig. 5 [3H]DCG-IV binding in membranes prepared from ratstriata. Scatchard analysis of the saturation isotherm from arepresentative experiment. The saturation curve was constructedusing six points, for a range of concentrations between 3 nM and1 mM.

Table 1 [3H]DCG-IV binding [means (+SEM)] in striatalmembrane from sham, 6-OHDA-lesioned and 6-OHDA-lesioned plus L-DOPA-treated rats

Bmax (fmol/mg protein) KD (nM)

UnlesionedSaline 1043 (182) 212 (58)L-DOPA 1064 (49) 98 (33)

6-OHDAIpsilateral striatum 2335 (323*) 325 (87)Contralateral striatum 1407 (215) 185 (49)

6-OHDA + L-DOPAIpsilateral striatum 1233 (205) 192 (61)Contralateral striatum 995 (145) 95 (42)

Values are means (+SEM) of three individual determinations.Each binding assay was performed in duplicate.*P < 0.05 (one-way ANOVA plus Fisher's PLSD) compared withall other Bmax values.



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Bolam, 1992). Interestingly, however, it has been reported

that while thalamostriatal ®bres preferentially target choli-

nergic interneurones, corticostriatal inputs mainly impinge on

spiny neurones, which represent the neuronal subtype

recorded for this study.

An enhanced glutamatergic transmission in the basal

ganglia and, in particular, in the striatum is thought to be

involved in the expression of motor de®cits in Parkinson's

disease (Chase and Oh, 2000; Greenamyre, 2001). Thus,

blocking glutamate receptors has been a major challenge in

the treatment of this pathology (Papa et al., 1995; Rouse et al.,

2000; Robinson et al., 2001). Recent studies have focused on

mGlu receptors as targets for drugs of potential use in

Parkinson's disease (Rouse et al., 2000; Bruno et al., 2001).

These receptors modulate excitatory synaptic transmission by

at least two mechanisms: (i) by regulating the activity of

voltage-sensitive ion channels or ionotropic glutamate

receptors (particularly NMDA receptors) at post-synaptic

sites; and (ii) by facilitating or inhibiting the release of

glutamate from afferent ®bres (reviewed by Pin and

Duvoisin, 1995). In addition, mGlu receptors regulate

processes of neurodegeneration and neuroprotection, and

they have been implicated in the pathophysiology of

neuronal degeneration in Parkinson's disease (Bruno et al.,

2001). Hence, appropriate drugs interacting with mGlu

receptors might relieve motor symptoms and, at the same

time, delay the ongoing neuronal degeneration of Parkinson's

disease.

mGlu receptors are coupled to G proteins and form a

family of at least eight subtypes, subdivided into three groups

(Pin and Duvoisin, 1995; De Blasi et al., 2001). Group I

includes mGlu1 and mGlu5 receptors, which are coupled to

polyphosphoinositide hydrolysis. Group II includes mGlu2

and mGlu3 receptors, which are negatively coupled to

adenylyl-cyclase via a Gi protein. Group III mGlu receptors

(mGlu4, -6, -7 and -8) are also coupled to a Gi protein in

heterologous expression systems, although native mGlu6

receptors are coupled to a cGMP phosphodiesterase in the

`ON' bipolar cells of the retina (reviewed by De Blasi et al.,

2001). Both group II and group III mGlu receptors are pre-

synaptically localized and their activation inhibits glutamate

release (reviewed by Pin and Duvoisin, 1995). Accordingly,

we found that selective agonists of group II (LY379268,

DCG-IV) and group III (L-AP4) mGlu receptors decrease

excitatory synaptic transmission at the corticostriatal pathway

via a pre-synaptic mechanism. In contrast, selective acti-

vation of group I mGlu receptors with 3,5-DHPG did not

affect corticostriatal EPSPs, while it signi®cantly enhanced

post-synaptic responses to NMDA. We found that 6-OHDA-

induced denervation of the nigrostriatal dopaminergic path-

way selectively potentiated responses mediated by group II

mGlu receptors without affecting responses mediated by

group I or group III mGlu receptors.

Tang and colleagues found that paired-pulse facilitation

is affected in denervated striata as a result of altered

synaptic plasticity at glutamatergic synapses (Tang et al.,

2001). Conversely, we did not ®nd substantial modi®cations

of paired-pulse facilitation following 6-OHDA treatment.

Several reasons might account for this discrepancy, in

particular the different age of the animals. Tang and

colleagues, in fact, used young rats (up to 1 month old) to

study paired-pulse facilitation under control conditions and

following intrastriatal 6-OHDA injection, while our experi-

ments were performed in adult rats (3±4 months), recorded 2

months after intranigral injection of 6-OHDA. It is therefore

conceivable that the absence of paired-pulse facilitation

described by Tang and colleagues in young 6-OHDA treated

rats recovers with time after the lesion (Tang et al., 2001). In

addition, it should be noted that the absent increase in paired-

pulse facilitation described in young animals treated with 6-

OHDA re¯ects the absence of corticostriatal long-term

depression observed in these animals. Consistently, this

form of synaptic plasticity is pre-synaptic in young animals

(Choi and Lovinger, 1997), and therefore accounts for the

increase in paired-pulse facilitation observed during devel-

opment, but is post-synaptic in adults animals (Calabresi

et al., 1999), and therefore it is not expected to affect paired-

pulse facilitation.

Among other receptors present on corticostriatal terminals

and coupled to Gi proteins, only the activity of D2 receptors is

up-regulated in response to 6-OHDA denervation, whereas

the activity of g-aminobutyric acid B-receptors or m2/4

muscarinic receptors remains unchanged (Calabresi et al.,

1993). This suggests that dopaminergic denervation induces

changes in the expression and/or regulatory properties of

mGlu2/3 receptors (see Alagarsamy et al., 2001; De Blasi

et al., 2001) rather than changes in molecules that lie

downstream in the signal propagation and are therefore

common to all receptors coupled to Gi proteins. In our study,

6-OHDA lesions led to an increased expression of mGlu2/3

receptors without detectable changes in mGlu4 receptors.

Although an increase in mGlu2/3 receptor density is in line

with the enhanced responsiveness to LY379268 and DCG-IV,

it is hard to explain why we detected an increase in the

potency, but did not see the expected increase in ef®cacy, of

mGlu2/3 receptor agonists. Possible explanations include a

greater receptor reserve in denervated animals (as suggested

by the lack of changes in the alpha subunits of Gi proteins) or

the contribution of other pools of mGlu2/3 receptors (such as

post-synaptic or glial receptors) to the overall increase in

receptor number. The examination of mGlu2/3 receptor

expression at the cellular or subcellular level may help to

solve this caveat.

Remarkably, all changes induced by dopaminergic

denervation were reversed by a chronic treatment with L-

DOPA, which is standard drug in the treatment of Parkinson's

disease. Although L-DOPA does not protect or rescue

nigrostriatal dopaminergic ®bres (see Camp et al., 2000;

Ishida et al., 2000 and references therein), it partially restores

striatal dopaminergic transmission and exerts a trophic effect,

repairing the ultrastructural changes in the corticostriatal

pathway caused by dopaminergic denervation (Ingham et al.,



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1998). The normalization of the responsiveness of pre-

synaptic mGlu2/3 receptors represents a novel example

of the adaptive changes induced by L-DOPA in

experimental animal models of parkinsonism (Chase and

Oh, 2000).

The present ®ndings have several implications. It has

been hypothesized that selective agonists of group II

mGlu receptors are bene®cial in Parkinson's disease

because they reduce the hyperactivity of corticostriatal

®bres and subthalamic excitatory ®bres that develops in

response to dopaminergic denervation (Rouse et al., 2000;

Messenger and Duty, 2001). Accordingly, intracerebro-

ventricular injection of mGlu2/3 receptor agonists reduces

akinesia in reserpine-treated rats (Dawson et al., 2000;

Messenger and Duty, 2001) The increased potency of

LY379268 and DCG-IV found in 6-OHDA-lesioned rats

may re¯ect the existence of a compensatory mechanism

aimed at reducing the excessive activity of the corticos-

triatal pathway, and allows the prediction that, in

parkinsonian patients, group II mGlu receptor agonists

are effective at low doses. This may considerably

improve the risk-to-bene®t ratio associated with the use

of these drugs in Parkinson's disease.

The restorative effects of L-DOPA suggest that the

activity of corticostriatal mGlu2/3 receptors is under the

control of nigrostriatal dopaminergic transmission and

that, for this reason, mGlu2/3 receptor agonists might

have a lesser chance of success when given in combin-

ation with L-DOPA. One could predict an optimal

response to mGlu2/3 receptor agonists either before the

beginning of L-DOPA treatment or, alternatively, when L-

DOPA loses its ef®cacy and patients spend most of their

time in the `off' phase. If this prediction is correct, an

appropriate use of mGlu2/3 receptor agonists might help

to resolve the motor ¯uctuations that are typical of the

long-term L-DOPA syndrome. The potential use of

mGlu2/3 receptor agonists in the treatment of

Parkinson's disease is further supported by the recent

evidence that LY379268 protects nigral dopaminergic

neurones against 6-OHDA toxicity (O'Neill et al., 2001).

It is worth noting that an alternative strategy in the

treatment of Parkinson's disease might be also represented by

the use of group I selective antagonists. Accordingly, it has

been reported that a mGlu5 receptor antagonist reverses the

akinesia in 6-OHDA-lesioned rats (Amalric et al., 2001;

Spooren et al., 2001). In line with this ®nding, we have

demonstrated that activation of group I mGlu receptors (and

in particular mGlu5 receptors) facilitates striatal NMDA

responses in normal (Pisani et al., 1997b, 2001) as well as

denervated animals (present study).

Future pharmacological studies are required to investigate

the possibility that new drugs, combining both agonistic

effects on group II mGlu receptors and antagonistic properties

on group I mGlu receptors, may represent an ideal approach

in the treatment of Parkinson's disease.

AcknowledgementsWe wish to thank Dr A. E. Kingston (Eli Lilly,

Indianapolis IN, USA) for providing LY379268. This

work was supported by a Telethon (E. 729) grant

and a CNR (Invecchiamento) and a CNR-MIUR

(Neurobiotecnologie) grant to P.C. The study was also

supported by a MIUR/CNR (Ministero dell'lstruzione,

UniversitaÁ Ricerca/Consiglio Superiore delle Ricerche)

(legge 95/95) grant to G.B., a MURST/CNR Neuroscience

grant to P.C. and a grant from the National Research Council

Biotechnology Project to P.C.

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Received April 17, 2002. Revised June 19, 2002.

Accepted June 22, 2002



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