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Neuroscience 145 (2007) 611–620

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HE INFLUENCE OF GROUP III METABOTROPIC GLUTAMATEECEPTOR STIMULATION BY (1S,3R,4S)-1-AMINOCYCLO-PENTANE-,3,4-TRICARBOXYLIC ACID ON THE PARKINSONIAN-LIKE AKINESIAND STRIATAL PROENKEPHALIN AND PRODYNORPHIN mRNA

XPRESSION IN RATS

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. KONIECZNY,a* J. WARDAS,a K. KUTER,a A. PILCb

ND K. OSSOWSKAa

Department of Neuropsychopharmacology, Institute of Pharmacol-gy, Polish Academy of Sciences, 12 Smetna Street, 31-343, Kraków,oland

Department of Neurobiology, Institute of Pharmacology, Polish Acad-my of Sciences, 12 Smetna Street, 31-343, Kraków, Poland

bstract—Group III metabotropic glutamate receptorsmGluRs) are widely distributed in the basal ganglia, espe-ially on the terminals of pathways which seem to be over-ctive in Parkinson’s disease. The aim of the present studyas to determine whether (1S,3R,4S)-1-aminocyclo-pentane-,3,4-tricarboxylic acid (ACPT-1), an agonist of group IIIGluRs, injected bilaterally into the globus pallidus (GP),

triatum or substantia nigra pars reticulata (SNr), can atten-ate the haloperidol-induced catalepsy in rats, and whetherhat effect was related to modulation of proenkephalinPENK) or prodynorphin (PDYN) mRNA expression in thetriatum. Administration of ACPT-1 (0.05–1.6 �g/0.5 �l/side)aused a dose-and-structure-dependent decrease in the hal-peridol (0.5 mg/kg i.p. or 1.5 mg/kg s.c.)-induced catalepsyhose order was as follows: GP>striatum>SNr. ACPT-1,iven alone to any of those structures, induced no catalepsy

n rats. Haloperidol (3�1.5 mg/kg s.c.) significantly increasedENK mRNA expression in the striatum, while PDYN mRNA

evels were not affected by that treatment. ACPT-1 (3�1.6 �g/.5 �l/side) injected into the striatum significantly attenuatedhe haloperidol-increased PENK mRNA expression, whereasdministration of that compound into the GP or SNr did notnfluence the haloperidol-increased striatal PENK mRNA lev-ls. Our results demonstrate that stimulation of group IIIGluRs in the striatum, GP or SNr exerts antiparkinsonian-

ike effects in rats. The anticataleptic effect of intrastriatallynjected ACPT-1 seems to correlate with diminished striatalENK mRNA expression. However, since the anticatalepticffect produced by intrapallidal and intranigral injection ofCPT-1 is not related to a simultaneous decrease in striatalENK mRNA levels, it is likely that a decrease in enkephaliniosynthesis is not a necessary condition to obtain an anti-arkinsonian effect. © 2006 IBRO. Published by Elsevier Ltd.ll rights reserved.

Corresponding author. Tel: �48-12-6623272; fax: �48-12-6374500.-mail address: koniecz@if-pan.krakow.pl (J. Konieczny).bbreviations: ACPT-1, (1S,3R,4S)-1-aminocyclopentane-1,3,4-tri-arboxylic acid; DA, dopamine; EPN, entopeduncular nucleus; GP,lobus pallidus; LSD, least significance difference; mGluRs, metabo-ropic glutamate receptors; NMDA, N-methyl-D–aspartate; OD, opticalensity; PD, Parkinson’s disease; PDYN, prodynorphin; PENK, proen-

lephalin; SNr, substantia nigra pars reticulata; STN, subthalamic nu-leus.

306-4522/07$30.00�0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2006.12.006

611

ey words: mGluRs, catalepsy, in situ hybridization, globusallidus, striatum, substantia nigra pars reticulata.

n the striatum, more than 90% of the output projectioneurons use GABA as their neurotransmitter (Lindefors,993). These neurons form two distinct subpopulations: atriatonigral (“direct”) and a striatopallidal (“indirect”) path-ay. The neurons projecting to the globus pallidus (GP)xpress mainly D2 dopamine (DA) receptors and synthe-ize the opioid peptide enkephalin which is co-localizedith GABA. The neurons innervating the substantia nigraars reticulata (SNr) and the entopeduncular nucleusEPN) express mainly D1 DA receptors and synthesize theeptide dynorphin (Gerfen and Young, 1988; Gerfen et al.,990). DA, which is tonically released from nigrostriatalopaminergic terminals, exerts differential control over thectivity of the two GABAergic projections and over theiosynthesis of opioid peptides. Thus, acting principally via

2 receptors, DA inhibits the striatopallidal pathway as wells striatal proenkephalin (PENK) mRNA expression; inontrast, while acting via D1 receptors, it stimulates thetriatonigral route and has a facilitatory influence on striatalrodynorphin (PDYN) mRNA level (Gerfen et al., 1991;ngber et al., 1992). It has been postulated that degener-tion of dopaminergic neurons in Parkinson’s disease (PD)

eads to a functional imbalance between the striatal effer-nts: disinhibition of striatopallidal neurons and inhibition oftriatonigral ones (Wichmann and DeLong, 1998). In lineith the above hypothesis, neurotoxic lesions of dopami-ergic neurons lead to increased levels of mRNA encodingENK and in the decreased levels of PDYN mRNA, in thetriatum (Gerfen et al., 1991; Reimer et al., 1992). There-ore it is suggested that any pharmacological manipulationhat decreases striatopallidal transmission may amelioratearkinsonian symptoms (Maneuf et al., 1994; Shiozaki etl., 1999; Chadha et al., 2000; Wardas et al., 2001).

Glutamate released from corticostriatal terminals acti-ates—via postsynaptically localized ionotropic glutamateeceptors—both striatopallidal and striatonigral neurons,nd has a stimulatory effect on the biosynthesis of opioideptides (Somers and Beckstead, 1992; Campbell andjorklund, 1994; Beckstead, 1995). The overactivity of thetriatopallidal projection results in the increased activity ofhe other excitatory pathways projecting from the subtha-

amic nucleus (STN) to the output basal ganglia struc-ved.

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J. Konieczny et al. / Neuroscience 145 (2007) 611–620612

ures—the SNr and EPN. Hence it seems that the loweringf the stimulatory influence of glutamate on the striatopal-

idal pathway or the blockade of glutamatergic transmis-ion at STN or SNr sites could be another potential targetf antiparkinsonian drugs. In fact, the antiparkinsonianffects of N-methyl-D-aspartate (NMDA) receptor antago-ists have been described in a number of animal studiesreviewed by Ossowska, 1994). Moreover, surgical inter-entions such as pallidotomy, subthalamotomy or STNeep brain stimulation are beneficial to the alleviation mo-or disturbances in parkinsonian patients (Gross et al.,999; Walter and Vitek, 2004).

The action of glutamate in the brain is mediated byoth ionotropic and metabotropic glutamate receptorsmGluRs). Eight subtypes of mGluRs have been clonedmGluR1-8) and subdivided into three groups on the basisf their sequence homology, pharmacological profile andignal transduction mechanism (reviewed by Schoepp,001). Group III mGluRs (mGluR4, 6, 7, 8) are localizedainly presynaptically at both glutamatergic and GABAergic

erminals (Kinoshita et al., 1998; Kosinski et al., 1999;radley et al., 1999b) where they are thought to inhibiteuronal transmission by decreasing glutamate and GABAelease, respectively (Pisani et al., 1997; Wittmann et al.,001; Marino et al., 2003; Valenti et al., 2003). Because ofhe poor penetration of the currently known group IIIGluR agonists through the blood–brain barrier, all these

ompounds have to be administered directly into the brain.ome recent studies have shown that agonists of group IIIGluRs may be effective in reversing parkinsonian-like

ymptoms in rats (Marino et al., 2003; Valenti et al., 2003;acInnes et al., 2004). However, there has been so far no

esearch aimed at comparing the ability of these com-ounds to evoke antiparkinsonian effects with the possibleimultaneous influence on striatopallidal and striatonigralutput neurons, measured by PENK and PDYN mRNAxpression, respectively.

ig. 1. Localization of cannula tips in (A) the GP, (B) striatum and (C) SNr on tlacement in one animal on one side.

Considering the attractive localization of group IIIGluRs on the terminals of pathways which seem to beveractive in PD, the aim of the present study was to deter-ine whether (1) (1S,3R,4S)-1-aminocyclopentane-1,3,4-tri-

arboxylic acid (ACPT-1), a group III mGluR agonist, admin-stered directly into the GP, striatum or SNr, was able toeverse the haloperidol-induced catalepsy in rats; (2) thenticataleptic effect of that compound may correspond to itsodulatory influence on the biosynthesis of opioid peptides

ocalized on the output striatal neurons, measured by PENKnd PDYN mRNA expression in the striatum.

EXPERIMENTAL PROCEDURES

nimals

he experiments were performed on male Wistar rats (250–50 g). The animals were kept in a well-ventilated room on anrtificial 12-h light/dark cycle (the light on from 7 a.m. to 7 p.m.) atroom temperature of 21–22 °C, with free access to food andater. All the experiments were carried out in compliance with thenimal Protection Act of August 21, 1997 (published in Poland’surrent Legislation Gazette [Dziennik Ustaw] no. 111/197, item24), and according to the National Institutes of Health Guide forhe Care and Use of Laboratory Animals, and were approved byhe Institute’s Local Bioethics Commission. All efforts were madeo minimize the number of animals used and their suffering.

nimal surgery and drug treatment

he rats were operated under pentobarbital anesthesia (Vet-utal; 30 mg/kg i.p.; Biowet, Pulawy, Poland). Two guide can-ulae (0.4 mm o.d., 11.9 mm long) were bilaterally implanted inhe GP, striatum or SNr. The experiment was performed ca. 1eek after stereotaxic surgery. Intrastructural injections of therug were made using Hamilton microsyringes connected, viaolyethylene tubing, to two inner cannulae (0.3 mm o.d.) whichrotruded by 0.7 mm from the guide cannulae. Cannulae tipsere placed in the GP (A��0.4 to �0.92 mm; L�2.4 –3.2 mm;�6.2–7.8 mm from the bregma), rostral striatum (A�1.7–.2 mm; L�1.4 –3.2 mm; H�5.0 –7.0 mm from the bregma) orNr (A��5.2 to �6.04 mm; L�1.4 –2.6 mm; H�7.6 –9.0 mm

he frontal sections of the rat brain. Each symbol (x) indicates cannula

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J. Konieczny et al. / Neuroscience 145 (2007) 611–620 613

rom the bregma) (Fig. 1) according to Paxinos and Watson1986). A striatal injection site was chosen on the basis of ourrevious studies in which that region of the striatum appearedo be the most sensitive to the actions of antiglutamatergicgents (i.e. NMDA receptor antagonists, mGluR1 antagonists)Lorenc-Koci et al., 1998; Ossowska et al., 2002).

The injections (0.5 �l/side) lasted 60 s, and the cannulae wereeft in place for an additional 60 s to allow diffusion. Localization ofll the cannulae tips was checked histologically on frozen frontallices of the brain after termination of the experiment.

The animals designed for the catalepsy test were divided intowo groups: the first group of rats was treated with haloperidol0.5 mg/kg/2 ml i.p.) and 60 min later with ACPT-1 (0.05 �g–.6 �g/0.5 �l) injected bilaterally into the GP, striatum or SNr; theecond group received haloperidol (1.5 mg/kg/2 ml s.c.) and 2 hater ACPT-1 (1.6 �g/0.5 �l) into the GP, striatum or SNr. Cata-epsy was evaluated twice: at 15 and 30 min after intracerebralnjection of ACPT-1. Control rats received physiological salinenstead of haloperidol or ACPT-1.

The animals used for an in situ hybridization study werereated with haloperidol (1.5 mg/kg/2 ml s.c.) three times at 3-hntervals (according to Noailles et al., 1996). ACPT-1 (1.6 �g/.5 �l) was administered bilaterally into the striatum, GP or SNr

hree times: at 2 h after each haloperidol injection. Control ratseceived physiological saline instead of haloperidol or ACPT-1.ne hour after the last intracerebral injection (3 h after the lastaloperidol administration) the rats were killed by decapitation.

rugs

aloperidol (Polfa, Warszawa, Poland, ampoules of 5 mg/1 ml)as diluted with distilled water to a concentration of 0.5 or 1.5 mg/g. ACPT-1 (Tocris Bioscience, Bristol, UK) was dissolved interile saline with the addition of a minimal amount of 0.1 M NaOHpH 6.0).

atalepsy

atalepsy was determined using a 9-cm cork test. For each testrat’s both forepaws were placed on the upper surface of the

ork (2.5�2.5 cm), and descent latency was measured threeimes (maximum duration, 300 s). The longest time of keepingt least one rat’s forepaw on the cork was accepted as aatalepsy score.

issue section preparation

he rats were killed by decapitation and their brains wereapidly removed, frozen in cold heptane (�70 °C) and stored at20 °C. Coronal sections (10 �m thick) were cut up using a

ryostat microtome, mounted on gelatin-coated slides andtored at �20 °C. The sections were warmed to a room tem-erature, postfixed with a buffered 4% paraformaldehyde,ashed with PBS, acetylated with a 0.25% acetic anhydrate in.1 M triethanoloamine and a 0.9% NaCl, dehydrated in in-reasing concentrations of ethanol, delipidated in chloroform,ehydrated in decreasing concentrations of ethanol and air-ried.

n situ hybridization of PENK and PDYN mRNA

48-mer synthetic oligonucleotide (30 pmol) was labeled with35S] �dATP (1250 Ci/mmol, MP Biomedicals, Heidelberg, Ger-any) in a 3=-tailing reaction using the terminal transferase en-

yme (Roche Diagnostics, Mannheim, Germany) at 37 °C for 1 h,o obtain a specific activity of about 1–4�105 cpm/�l. Radioactiverobes were purified using a phenol:chloroform standard protocol.he probes were complementary to the bases 436–483 of the

ENK gene mRNA sequence (GenBank accession number m

28263, gi:204025), or to the bases 854–901 of the PDYN geneRNA (GenBank accession number NM_019374, gi:31083335).equence homology with the other genes was verified using aenBank BLAST program.

The sections were incubated in a hybridization buffer (a0% formamide, a 10% dextran sulfate, 0.25 mg/ml tRNA, 0.5g/ml salmon shared and denatured sperm DNA, 1� Denhardt

olution, 4� SSC) for 20 h at 37 °C. The concentration of aadioactivity in the hybridization buffer was 5�106 cpm/ml.fter washing (3�20 min in 2� SSC at 42 °C, 1�15 min in 1�SC at a room temperature), the sections were dehydrated inthanol, air-dried and exposed to a Kodak Bio-Max MR filmSigma-Aldrich, Munich, Germany) for 4 weeks at 4 °C. Theutoradiograms were analyzed by a computer-assisted densi-ometry using an image analysis system (MCID, St. Catharines,ntario, Canada). Average optical density (OD) values werealculated. The means of the OD values were obtained byveraging the measurements of both sides of the brain perection in each brain structure. The level of PENK and PDYNRNAs was estimated in the dorsolateral and ventrolateral

triatum at two levels: rostral, A�1.6 –1.2 mm from the bregma;nd central, A�0.2 to �0.26 mm from the bregma, according toaxinos and Watson (1986) (Figs. 2, 3).

tatistics

statistical analysis of catalepsy was carried out by the Kruskal-allis and the Mann-Whitney U tests. For the statistical evaluation

f biochemical data, a two-way ANOVA was used (factor 1, hal-peridol; factor 2, ACPT-1), followed-when the F-ratio was signif-

cant (P�0.05)-by the least significance difference (LSD) post hocest.

RESULTS

he effect of intrastructural injection of ACPT-1 onhe haloperidol-induced catalepsy in rats

aloperidol administered both intraperitoneally (0.5 mg/kg)nd s.c. (1.5 mg/kg) induced strong catalepsy in rats,

ig. 2. Autoradiograms of frontal brain sections showing typical ex-mples of PENK mRNA expression in the central CP in four groups ofats: (1) control; (2) rats treated with haloperidol (3�1.5 mg/kg s.c.); (3)ats treated with ACPT-1 (3�1.6 �g/0.5 �g into the CP); (4) ratsreated jointly with haloperidol (3�1.5 mg/kg s.c.) and ACPT-13�1.6 �g/0.5 �g into the striatum). White outlines indicate the regionstudied. CP, striatum.

easured 75 and 90 min (Fig. 4), or 135 and 150 min later

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J. Konieczny et al. / Neuroscience 145 (2007) 611–620614

Fig. 5), respectively. ACPT-1 injected bilaterally into theP, striatum or SNr at 60 min after haloperidol significantlynd dose-dependently attenuated the haloperidol-inducedatalepsy at the time of either measurement (15 and 30in after its administration) (Figs. 4, 5).

Rats injected with the lower dose of haloperidol0.5 mg/kg) revealed the most pronounced effect ofCPT-1 after intrapallidal injection, much weaker after

ts injection into the striatum, and the weakest after itsdministration into the SNr. The differences in the struc-

ure-dependent effect of ACPT-1 were related to thefficacy of the doses used. After injection into the GP,CPT-1 (0.4 and 1.6 �g/0.5 �l) totally inhibited thealoperidol-induced catalepsy (Fig. 4A). The effect of its

ower dose (0.1 �g/0.5 �l) was slightly weaker, espe-ially during the second measurement, and the dose of.05 �g/0.5 �l only slightly and non-significantly de-reased catalepsy. ACPT-1 (0.4, 0.8 and 1.6 �g/0.5 �l)njected into the striatum significantly attenuated thealoperidol-induced catalepsy at the time of either mea-urement (Fig. 4B). However, only the highest dose ofCPT-1 (1.6 �g/0.5 �l) completely abolished catalepsy.he weakest effect of ACPT-1 was observed after its

njection to the SNr (Fig. 4C). Only the highest dose ofCPT-1 (1.6 �g/0.5 �l) reduced the haloperidol-inducedatalepsy, but its effect was weaker than that observedfter striatal or pallidal injection. A lower dose of ACPT-10.8 �g/0.5 �l) was ineffective. ACPT-1 (1.6 �g/0.5 �l)dministered alone to any of the examined structuresad no cataleptic effect in rats (Fig. 4).

In line with the above described results, ACPT-11.6 �g/0.5 �l) produced a structure-dependent anticata-eptic effect in rats injected with the higher dose of halo-eridol (1.5 mg/kg), the strongest effect being observedfter intrapallidal, a weaker one after intrastriatal and the

ig. 3. Autoradiograms of frontal brain sections showing typical ex-mples of PDYN mRNA expression in the rostral CP in four groups ofats: (1) control; (2) rats treated with haloperidol (3�1.5 mg/kg s.c.); (3)ats treated with ACPT-1 (3�1.6 �g/0.5 �g into the CP); (4) ratsreated jointly with haloperidol (3�1.5 mg/kg s.c.) and ACPT-13�1.6 �g/0.5 �g into the CP). Black outlines indicate the regionstudied. CP, striatum; NA, nucleus accumbens.

eakest after intranigral administration (Fig. 5).HH

he influence of haloperidol and ACPT-1 on PENKRNA expression in the striatum

s can be seen in Fig. 6, haloperidol administered threeimes at a dose of 1.5 mg/kg s.c. significantly increasedENK mRNA expression in the four examined regions of

he striatum. A two-way ANOVA showed a highly signifi-ant overall treatment effect of haloperidol in the dorsal and

ig. 4. The influence of ACPT-1 on the haloperidol (HAL)-inducedatalepsy in rats. HAL was injected at a dose of 0.5 mg/kg i.p. ACPT-1as administered bilaterally into (A) the GP, (B) striatum or (C) SNr atoses of 0.05–1.6 �g/0.5 �g/side 1 h after HAL injection. Catalepsyas evaluated at 15 and 30 min after intracerebral injection of ACPT-1.o cataleptic effect was observed for ACPT-1 injected alone (1.6 �g/.5 �g) into any of those structures. The results are shown as theean�S.E.M. The number of animals per group: (A) control, n�6;AL, n�8; HAL�ACPT-1 1.6 �g/0.5 �g, n�8; HAL�ACPT-1.4 �g/0.5 �g, n�4; HAL�ACPT-1 0.1 �g/0.5 �g, n�6;AL�ACPT-1 0.05 �g/0.5 �g, n�5; ACPT-1 1.6 �g/0.5 �g, n�; (B) control, n�6; HAL, n�8; HAL�ACPT-1 1.6 �g/0.5 �g, n�7;AL�ACPT-1 0.8 �g/0.5 �g, n�7; HAL�ACPT-1 0.4 �g/0.5 �g, n�8;CPT-1 1.6 �g/0.5 �g, n�6; (C) control, n�6; HAL, n�8; HAL�ACPT-1.6 �g/0.5 �g, n�6; HAL�ACPT-1 0.8 �g/0.5 �g, n�5; ACPT-1 1.6 �g/.5 �g, n�6. Statistics: the Kruskal-Wallis and the Mann-Whitney U

ests. Symbols indicate the significance of differences in the Mann-hitney U tests, * P�0.05, ** P�0.01, *** P�0.001 vs. control;P�0.05, ## P�0.01, ### P�0.001 vs. HAL; & P�0.05, && P�0.01 vs.

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J. Konieczny et al. / Neuroscience 145 (2007) 611–620 615

entral parts of the striatum at either level (rostral andentral): [for the striatal group: F(1,27–1,28)�46.67–70.00,�0.001; for the pallidal group: F(1,21)�12.05–31.69,�0.01–0.001; for the nigral group: F(1,24)�4.79–3.85, P�0.05–0.001].

ACPT-1 (1.6 �g/0.5 �l) injected bilaterally into theostral striatum significantly decreased the haloperidol ef-ect in all the striatal regions studied, but had no influencen the control PENK mRNA level when given alone to thattructure (Fig. 6A). A two-way ANOVA indicated a signifi-ant overall treatment effect of ACPT-1 [F(1,27–,28)�6.72–19.05, P�0.05–0.001]. After injection to theP, ACPT-1 did not reverse the haloperidol-induced in-rease in PENK mRNA expression; however, when in-ected alone to the GP, ACPT-1 significantly enhanced theormal PENK mRNA expression in the rostral striatumnd dorsal parts of the central striatum (Fig. 6B)

ig. 5. The influence of ACPT-1 on the haloperidol (HAL)-inducedatalepsy in rats. HAL was injected at a dose of 1.5 mg/kg s.c. ACPT-1as administered bilaterally into (A) the GP, (B) striatum or (C) SNr atdose of 1.6 �g/0.5 �g/side 2 h after HAL injection. Catalepsy was

valuated at 15 and 30 min after intracerebral injection of ACPT-1. Theesults are shown as the mean�S.E.M. The number of animals perroup: (A) HAL, n�6; HAL�ACPT-1, n�8; (B) HAL, n�7;AL�ACPT-1, n�7; (C) HAL, n�5; HAL�ACPT-1, n�7. Statistics:

he Mann-Whitney U tests. Symbols indicate the significance of differ-nces, * P�0.05 vs. HAL; ** P�0.01 vs. HAL.

F(1,21)�4.85–5.58, P�0.05]. Intranigral administration ofS*

CPT-1 influenced neither the haloperidol-increased norhe normal striatal PENK mRNA expression (Fig. 6C)F(1,24)�0.00–0.49, NS]. A two-way ANOVA showed aon-significant interaction between haloperidol and

ig. 6. The influence of ACPT-1 on normal or haloperidol (HAL)-in-reased PENK mRNA expression in the striatum. HAL was administeredhree times at a dose of 1.5 mg/kg s.c. ACPT-1 was administered threeimes bilaterally into (A) the striatum, (B) GP or (C) SNr at a dose of.6 �g/0.5 �g/side every 2 h after each HAL injection. PENK mRNAxpression was estimated in the dorsolateral and ventrolateral parts of theostral (A�1.6–1.2 mm from the bregma) and central striatum (A�0.2 to0.26 mm from the bregma, according to Paxinos and Watson, 1986).he results are presented as the mean�S.E.M. The number of animalser group: (A) control, n�6; ACPT-1, n�7–8; HAL, n�9; HAL�ACPT-1,�9; (B) control, n�7; ACPT-1, n�6; HAL, n�6; HAL�ACPT-1, n�6; (C)ontrol, n�8; ACPT-1, n�6; HAL, n�7; HAL�ACPT-1, n�7. Statistics: awo-way ANOVA, followed by the LSD test for post hoc comparisons.

ymbols indicate the significance of differences in the LSD test, * P�0.05,

* P�0.01 vs. control; # P�0.05, ## P�0.01 vs. HAL.

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J. Konieczny et al. / Neuroscience 145 (2007) 611–620616

CPT-1, when the latter compound was given to the GP orNr. However, a significant interaction between above-entioned factors was found in the ventral part of the

ostral striatum after intrastriatal administration of ACPT-1F(1,28)�4.16, P�0.05].

he influence of haloperidol and ACPT-1 on PDYNRNA expression in the striatum

n contrast to PENK mRNA expression, haloperidol failed tolter PDYN mRNA expression in any of the striatal regions

nvestigated (Fig. 7). ACPT-1 injected to the rostral striatumr SNr alone or jointly with haloperidol did not change PDYNRNA levels in any region of the striatum (Fig. 7A, C).owever, after injection to the GP, that compound givenlone or jointly with haloperidol increased PDYN mRNA ex-ression in either part of the rostral striatum (Fig. 7B). A

wo-way ANOVA indicated a significant effect of ACPT-1n the dorsal [F(1,18)�12.04, P�0.05] and ventralF(1,20)�22.36, P�0.05] parts of the rostral striatum. How-ver, no interaction between haloperidol and ACPT-1 was ob-erved at that level: [F(1,18)�0.50, NS] and [F(1,20)�0.21, NS]or the dorsal and ventral parts, respectively.

DISCUSSION

he neuroleptic-induced catalepsy has long been used asn animal model of extrapyramidal side-effects for screen-

ng antiparkinsonian drugs (Ossowska, 1994; Wadenbergt al., 2001). It is regarded as an animal equivalent ofkinesia, one of the symptoms appearing in the so-calledrug-induced Parkinsonism in humans, since it is reversedy commonly used antiparkinsonian drugs (Danysz et al.,994; Kobayashi et al., 1997; Maj et al., 1997; Moo-Puc etl., 2003). The present study demonstrates that adminis-ration of the group III mGluR agonist ACPT-1 directly intohe striatum, GP or SNr inhibits the haloperidol-inducedatalepsy in rats. These results are in line with some earliertudies in which the agonist of group III mGluRs L-SOP,njected directly to the GP, SNr or to the third ventricle,everses akinesia in reserpinized rats (MacInnes et al.,004). Moreover, Valenti et al. (2003) demonstrated that

.c.v. administration of another group III mGluR agonist,-AP4, produced antiparkinsonian effects in three differentodent models of PD.

In the present study the strongest effect of ACPT-1n the haloperidol-induced catalepsy was observed af-

er its injection into the GP. The effective doses ofntrapallidally administered ACPT-1 were about 4- and6-fold lower than in the case of intrastriatal and intrani-ral injection, respectively. The reason for the highestensitivity of pallidal group III mGluRs to ACPT-1 re-ains to be explained. Immunohistochemical and in situybridization studies have localized substantial group IIIGluRs, including mGluR4, 7 and 8 in the GP (Mizuno,998; Bradley et al., 1999b; Kosinski et al., 1999). Inarticular, mGluR4 is the most abundant in this structure,eing localized on both glutamatergic and GABAergic termi-als (Bradley et al., 1999b; Corti et al., 2002). Recent

ehavioral studies show that the allosteric potentiator of t

GluR4 PHCCC effectively decreases the reserpine-in-uced akinesia after both i.c.v. and systemic administra-ion to rodents (Marino et al., 2003; Battaglia et al., 2006).ence it seems that the activation of mGluR4 localized on

ig. 7. The influence of ACPT-1 on normal or haloperidol (HAL)-ncreased PDYN mRNA expression in the striatum. HAL was admin-stered three times at a dose of 1.5 mg/kg s.c. ACPT-1 was adminis-ered three times bilaterally into (A) the striatum, (B) GP or (C) SNr atdose of 1.6 �g/0.5 �g/side every 2 h after each HAL injection. PDYNRNA expression was estimated in the dorsolateral and ventrolateralart of the rostral (A�1.6–1.2 mm from the bregma) and centraltriatum (A�0.2 to �0.26 mm from the bregma, according to Paxinosnd Watson, 1986). The results are presented as the mean�S.E.M.he number of animals per group: (A) control, n�6–7; ACPT-1,�7–8; HAL, n�9; HAL�ACPT-1, n�8–9; (B) control, n�4–5;CPT-1, n�6; HAL, n�6; HAL�ACPT-1, n�5–8; (C) control, n�8;CPT-1, n�6; HAL, n�7; HAL�ACPT-1, n�7–8. Statistics: a two-ay ANOVA, followed by the LSD test for post hoc comparisons.ymbols indicate the significance of differences in the Mann-Whitneytest, * P�0.05 vs. control; # P�0.05 vs. HAL.

he striatopallidal pathway (which reduces GABA release

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n the GP) plays a prominent role in the pallidal effects ofCPT-1. The latter conclusion is supported by recent elec-

rophysiological data showing that PHCCC and L-AP4 di-inish inhibitory GABAergic transmission in the GP, actingt the level of striatopallidal terminals (Marino et al., 2003;atsui and Kita, 2003). Furthermore, the effect of L-AP4 isbsent in mGluR4 knockout mice (Valenti et al., 2003).

The density of mGluR7 in the GP is much lower thanhat of mGluR4; moreover, the potency of ACPT-1 atGluR7 (like that of the other group III mGluR agonists-SOP and L-AP4) is known to be considerably weaker

han that at other subtypes of group III mGluRs (Cartmellnd Schoepp, 2000; Moldrich et al., 2003). Therefore it isnlikely that this receptor may be of great importance tohe intrapallidal effects of ACPT-1. However, its involve-ent cannot be excluded, especially when higher doses of

his compound are used. It is also unlikely that mGluR8an mediate the intrapallidal effect of ACPT-1, since theensity of mGluR8 in basal ganglia structures is fairly lownd this receptor has not been found to participate in theodulation of a striatopallidal synapse (Valenti et al.,003). Moreover, (R,S)-3,4-DCPG [a mixed mGluR8gonist/AMPA antagonist] administered systemically in-uces catalepsy in mice which seems to be mediated viaGluR8 (Ossowska et al., 2004).

The high intrastriatal and intranigral doses of ACPT-1,ffectively alleviating haloperidol-induced catalepsy in ourxperiment, suggest, in turn, that these effects may beediated by mGluR7 rather than mGluR4. In contrast to

he fairly low density of mGluR7 in the GP, this receptorbounds in the striatum and SN (Bradley et al., 1999b;osinski et al., 1999; Messenger et al., 2002). In the stri-tum, ACPT-1 seems to act through activation of mGluR7

ocalized presynaptically on the glutamatergic synapse,hus decreasing glutamatergic tone at the corticostriatalynapse and attenuating transmission in cholinergic inter-eurons (Pisani et al., 1997; Bell et al., 2002). In the SNr,he anticataleptic effect of ACPT-1 may be mediated byGluR7 localized on glutamatergic subthalamonigral ter-inals (Bradley et al., 1999a), since L-AP4 has been

hown to inhibit glutamatergic transmission at this synapseWittmann et al., 2001). The weakest antiparkinsonian ef-ect of the intranigral injection of ACPT-1 compared with itsntrapallidal and intrastriatal effects is not surprising, iniew of the fact that group III mGluRs are also situated ontriatonigral GABAergic efferents (Bradley et al., 1999b;osinski et al., 1999); hence the activation of these recep-

ors counteracts the antiparkinsonian effect via SNr stim-lation (Wittmann et al., 2001).

The results of our study that reveal a haloperidol-nduced increase in striatal PENK mRNA expression are inine with the commonly known data indicating that thisompound directly influences striatal enkephalin biosyn-hesis by blocking D2 receptors (Tang et al., 1983; Morrist al., 1988; Angulo, 1992; Mavridis and Besson, 1999).ontrary to the above-reported effect, haloperidol does notffect striatal PDYN mRNA expression. However, recentata on the influence of haloperidol on PDYN mRNA and

eptide levels are somewhat controversial, since either no (

hanges (Peterson and Robertson, 1984; Morris et al.,988; Trujillo et al., 1990), or even an increase (Mavridisnd Besson, 1999) in the above parameters have beeneported after subchronic treatment with this neuroleptic.n the other hand, D1 agonists stimulate dynorphin bio-

ynthesis (Jiang et al., 1990; Gerfen et al., 1991; Engber etl., 1992), which is consistent with the commonly acceptedpinion that dopaminergic transmission increases the ac-ivity of the striatonigral pathway, mainly through D1 recep-ors. Haloperidol is considered to be a D2 receptor–prefer-ing DA antagonist, hence its ability to inhibit striatal D1

eceptors is poor. This may probably explain the lack of thenhibitory effect of haloperidol on PDYN mRNA expressionbserved in the present study.

In our in situ hybridization study, ACPT-1 injected intohe striatum (3�1.6 �g/0.5 �l) decreased the striatal PENKRNA level enhanced by haloperidol (3�1.5 mg/kg s.c.).ince that dose of ACPT-1 was also effective toward theatalepsy induced by both the lower (0.5 mg/kg i.p.) andhe higher dose of haloperidol (1.5 mg/kg s.c.), the anti-ataleptic effect of that compound may be related to theiminishing effect on enkephalin biosynthesis. As haseen mentioned above, the enhanced glutamatergic trans-ission in the striatum increases striatal PENK mRNAxpression, and group III mGluRs are found to inhibit theelease of glutamate from cortical efferents. Therefore theecrease in the levels of PENK mRNA induced by intra-triatal ACPT-1 most probably results from the diminishedlutamatergic tone in this structure.

In contrast to striatal administration, ACPT-1 in-ected to the GP— despite its strong anticataleptic ef-ect— did not attenuate the haloperidol-increased PENKRNA expression in the striatum. Furthermore, an in-

rease in PENK mRNA level was observed whenCPT-1 alone was injected to the GP. However, recenttudies show that, contrary to GABAergic transmission,veractive enkephalinergic transmission is not respon-ible for the generation of parkinsonian symptoms, since

ntrapallidal injection of the GABAA antagonist bicucul-ine (but not the opioid antagonist naloxone) has atrong antiparkinsonian effect in reserpine-treated ratsManeuf et al., 1994). In addition, naloxone decreaseshe effects of bicuculline (Maneuf et al., 1994), whicheems to indicate that activation rather than inhibition ofallidal opioid receptors mediates antiparkinsonian ef-

ects. In accordance with these findings, intrapallidalnjections of � or � receptor agonists cause an increasen locomotor activity, which in either case is reduced byaloxone (Dewar et al., 1985). Moreover, stimulation ofresynaptic � receptors in the GP leads to an increasedallidal neuronal activity (Stanford and Cooper, 1999).herefore it seems that an increase in enkephalin levelay be a compensatory mechanism through which en-

ephalin can attenuate the effects of increased GABAer-ic transmission. In fact, in an in vitro study enkephalinnalogs reduce GABA release from slices of rat GP

Dewar et al., 1987; Maneuf et al., 1994), and the opioid

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J. Konieczny et al. / Neuroscience 145 (2007) 611–620618

eceptor agonist DALA attenuates the K�-evoked GABAelease in the GP of MPTP-treated cats (Schroeder andchneider, 2002). Moreover, our results suggest that theecrease in GABAergic transmission at a pallidal syn-pse, probably related to the antiparkinsonian effects of

ntrapallidally injected ACPT-1, does not necessarilyorrelate with the concomitant decrease in enkephalin-rgic transmission; it even seems that the maintenancef the increases in enkephalinergic transmission may

ntensify the antiparkinsonian effect of ACPT-1.At present we can only speculate on a mechanism

esponsible for the observed increase in striatal PENKRNA level after intrapallidal injection of ACPT-1 alone or

ointly with haloperidol. In the former group, the decreasen GABA release induced by ACPT-1 may in turn, throughn increase in the activity of the GABAergic pallidostriatalrojection, inhibit striatal GABAergic interneurons and thuseduce their suppressing effect on the pallidostriatal path-ay and enkephalin biosynthesis (Bevan et al., 1998; Koosnd Tepper, 1999; Mörl et al., 2002). In the latter group, thenhancing effect of ACPT-1 on PENK mRNA expressionay be augmented by the effect of haloperidol, as haslready been reported above.

It seems that similar mechanisms are likely to con-ribute to the increase in striatal PDYN mRNA levelsnduced by intrapallidally administered ACPT-1, sincetriatal GABAergic interneurons are known to influenceoth the striatopallidal and the striatonigral pathwaysKawaguchi et al., 1995). Another explanation is that theecrease in pallidal GABA release induced by ACPT-1ay in turn reduce the activity of the SNr either directlyy activating the GABAergic projection to the SNr (Par-nt and Hazrati, 1995), or indirectly by decreasing thectivity of the glutamatergic subthalamonigral pathway.ike enkephalin in the striatopallidal pathway, dynorphin

n the striatonigral pathway is believed to exert an effectpposite to that of GABA, i.e. to stimulate SNr activityreviewed by Steiner and Gerfen, 1998); hence the in-rease in PDYN mRNA expression may be the mecha-ism that counterbalances the decreased activity of thistructure.

Intranigral administration of ACPT-1 neither influ-nced mRNA expression of either striatal neuropeptideor attenuated the haloperidol-induced increase inENK mRNA level in our study. It may thus be specu-

ated that intranigral injection of ACPT-1 stimulates notnly group III mGluRs located on glutamatergic termi-als, but also these situated on GABAergic striatonigralndings. This may decrease the inhibitory GABAergic

one in the SNr, which may be sufficient to counterbal-nce the decreased activity of the SNr, induced by thectivation of group III mGluRs localized on glutamatergic

erminals, no additional increase being observed in theeptide biosynthesis. On the other hand, the majority of

he neuronal impulses from the SNr are conveyed out-ide the basal ganglia—among others to the thalamus,uperior colliculus and pedunculopontine tegmental nu-leus. Hence the backward influence of the SNr on

triatal neurons seems to be limited.

CONCLUSION

n conclusion, our behavioral study demonstrates thattimulation of group III mGluRs within at least three struc-ures of the basal ganglia, e.g. the GP, striatum or SNr,xerts an antiparkinsonian effect in rats. mGluR4 localizedn striatopallidal GABAergic terminals and mGluR7 situ-ted on corticostriatal or subthalamonigral glutamatergicerminals may chiefly contribute to this effect. Moreover,ur in situ hybridization results suggest that the antiparkin-onian effect of ACPT-1 administered intrastriatally corre-

ates with the reversal of the increased striatal PENKRNA expression induced by the blockade of D2 recep-

ors. However, the reduction of elevated PENK mRNAevels in the striatum does not seem to be a necessaryondition of the antiparkinsonian effect of compounds de-reasing the activity of the striatopallidal pathway.

cknowledgments—This study was supported by a statutory fundrom the Institute of Pharmacology, Polish Academy of Sciences,raków, Poland.

The authors wish to express their thanks to Mrs. E. Smolak,As for her valuable help with the linguistic correction of theaper.

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(Accepted 1 December 2006)(Available online 16 January 2007)