Journal of NeurochemistryLippincott-Raven Publishers, Philadelphia© 1995 International Society for Neurochemistry

Clonidine Suppresses 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Reductions of Striatal Dopamine

and Tyrosine Hydroxylase Activity in Mice

Francesco Fornai, *Maria Grazia Alessandri, Flavia Fascetti,Francesca Vaglini, and Giovanni U. Corsini

Institute of Pharmacology, School of Medicine, and *IRCCS Stella Maris-INPE, University of Pisa, Pisa, Italy

Abstract: Recent findings have shown that excitatoryamino acid may be involved in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity . At the sametime, evidence is accumulating that the endogenous nor-adrenergic system plays a protective role in MPTP-in-duced striatal dopamine (DA) depletion and nigral dopa-minergic cell death . Recently, a2-adrenoceptors locatedon glutamatergic axons have been shown to inhibit gluta-mate overflow . In this study, we evaluated the effects ofan a 2-agonist (clonidine) and an a2-antagonist (yohim-bine) on MPTP-induced striatal DA depletion and tyrosinehydroxylase activity reduction . We show that clonidine isable to prevent the neurotoxicity of MPTP in mice . Toexert this effect, clonidine (0.5 mg/kg) must be adminis-tered at least twice (30 min before and 30 min afterMPTP) . Administration of another a2-agonist (detomi-dine, 0.3 mg/kg) attenuated the neurotoxicity induced byMPTP . We provide evidence that the protective effectobtained with clonidine was not due to decreased striatalcontent of 1-methyl-4-phenylpyridinium (MPP+) . We alsoshow that yohimbine, which is a classic a 2-adrenoceptorantagonist with low affinity for imidazoline receptors, pro-duced by itself an enhancement of MPTP toxicity andwas able to block the protective effect of clonidine. Thesedata raise the possibility that a2-adrenoceptor may mod-ulate the susceptibility of the nigrostriatal dopaminergicpathway to neurotoxicity . Key Words: Clonidine-De-tomidine-1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Parkinsonism-Yohimbine .J. Neurochem. 65, 704-709 (1995) .

The precise mechanism by which the dopaminergicneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-dine (MPTP) acts has been under intense investigationin an effort to gain insight into the etiology of idiopathicParkinson's disease as well as to discover new therapeu-tic strategies to treat or prevent this progressive neurode-generative disorder . Although the mechanism of actionof MPTP remains partially unknown, some crucial stepsappear to be well established . To exert its dopaminergicneurotoxicity, MPTP must be converted by mono-

704

amine oxidase type B to the toxic metabolite 1-methyl-4-phenylpyridinium (MPP+) (Heikkila et al ., 1984 ;Markey et al., 1984), which is accumulated intraneuro-nally by the high-affinity dopamine (DA) uptake system(Javitch and Snyder, 1985 ; Melamed et al ., 1985) . Al-though there is a general agreement among several au-thors regarding these steps, the toxic actions of MPP+are still not fully resolved .

Several studies have shown that MPP + causes directinhibition of mitochondrial complex I activity that im-pairs mitochondrial respiration (for review, see Son-salla and Nicklas, 1992) . Other authors focused onthe formation of free radicals or peroxides during theoxidative process of MPTP/MPP+ (Johannessen et al .,1986) . A third line of evidence suggests that the occu-pation of N-methyl-D-aspartate (NMDA) receptorscould be involved in MPTP toxicity . This is related tothe fact that NMDA antagonists may attenuate DAcell death produced by MPP + (Turski et al ., 1991) .However, findings concerning the role played by excit-atory amino acids (EAAs) on MPTP toxicity in rodentsare controversial (for review, see Tipton and Singer,1993) . Some authors have shown an involvement ofEAAs in the toxicity of MPP + injected directly intothe substantia nigra (Turski et al., 1991) or the striatum(Storey et al ., 1992) of the rat . Others could not con-firm these results (Sonsalla et al ., 1992) ; nor did theyobserve an involvement of EAAs after systemic injec-tion of MPTP in mice (Sonsalla et al ., 1989, 1992 ;Kupsch et al ., 1992) . Recently, we showed that inmonkeys dizocilpine reduces the DA depletion in the

Resubmitted manuscript received January 26, 1995 ; accepted Feb-ruary 13, 1995 .

Address correspondence and reprint requests to Dr. F. Fomai atInstitute of Pharmacology, School of Medicine, University of Pisa,Via Roma 55, 56100-I Pisa, Italy.

Abbreviations used: BPP', 1-butyl-4-phenylpyridinium ion ; DA,dopamine ; EAA, excitatory amino acid ; 5-HIAA, 5-hydroxyindole-acetic acid ; 5-HT, serotonin ; MPP+ , 1-methyl-4-phenylpyridinium ;MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; NMDA, N-methyl-D-aspartate ; TH, tyrosine hydroxylase .

striatum and the loss of DA perikarya in the substantianigra, as well as the development of behavioral parkin-sonism (Zuddas et al ., 1992) . Recently, these datahave been confirmed using the competitive NMDAantagonist CPP (3- [ (+) -2-carboxypiperazin-4-yl ] -propyl-l-phosphonic acid) in MPTP-treated marmo-sets (Lange et al ., 1993) . Because glutamate antago-nists produce serious side effects, alternative strategiesto prevent MPTP toxicity could involve the activationof receptors for other neurotransmitters that may havea role in limiting glutamate release .

Recently, the a2-adrenoceptor agonist clonidine (0.5mg/kg) has been reported to inhibit seizures (Stringerand Lothmann, 1991) . In line with this finding, cloni-dine activation of a2-adrenoceptor located on gluta-matergic axons has been shown to inhibit glutamateoverflow in several brain regions (Kamisaki et al .,1992, 1993) . In addition, recent studies clearly deline-ated a key role for the noradrenergic system in limitingthe progression of MPTP-induced parkinsonism inmonkeys (Mavridis et al ., 1991) and in mice (Bing etal ., 1992 ; Marien et al ., 1993) . Therefore, in the pres-ent study we evaluated the effects of a2-agonists (clon-idine and detomidine) and an a2-antagonist (yohim-bine) on MPTP-induced striatal DA depletion and ty-rosine hydroxylase (TH) activity reduction in C57/6Nblack mice . We also evaluated the time course of stria-tal MPP + after MPTP alone or after MPTP with cloni-dine at protective doses .

MATERIALS AND METHODS

AnimalsMale C57/6N black mice (Charles River, Calco, Italy),

8-9 weeks old, weighing 20-24 g, were kept under environ-mentally controlled conditions (12-h light/dark cycle withlights on between 0700 and 1900 h; ambient temperature21°C) with food and water ad libitum . Animals were treatedaccording to the National Institutes of Health Guide and theAmerican Physiological Association Guiding Principles inthe Care and Use of Animals .

Experimental procedureIn the first set of experiments, mice were treated intraperi-

toneally with a single dose of MPTP hydrochloride (Re-search Biochemicals, RBI, Natick, MA, U.S.A .) (36 mg/kg) . Clonidine (Sigma Chemical, St . Louis, MO, U.S.A .)was administered intraperitoneally at different doses (0.01,0.05, and 0.5 mg/kg) . When administered at 0.01 or 0.05mg/kg, clonidine was injected in triple doses (30 min beforeand 30 and 90 min after MPTP) ; whereas the highest dosageof clonidine (0.5 mg/kg) was injected according to the fol-lowing three different schedules : single dose, 30 min beforeMPTP; double dose, 30 min before and 30 min after MPTP;triple dose, 30 min before and 30 and 90 min after MPTP.The maximal dose of clonidine (0.5 mg/kg) was chosenbased on previous studies performed to investigate the occu-pation of a 2-adrenoceptors (Finberg and Kopin, 1987) aswell as studies on a2-adrenoceptor-mediated protection ofsoman toxicity by clonidine (0.1-1 mg/kg) (Buccafuscoand Aronstam, 1986) . Detomidine (Sigma, 0.3 mg/kg) wasadministered intraperitoneally, 30 min before MPTP. Yo-

CLONIDINE SUPPRESSES MPTP TOXICITY 705

himbine (Sigma) was administered intraperitoneally, at adose of 3 mg/kg, 30 min before MPTP. Control groupsreceived either saline, MPTP, yohimbine, detomidine, orclonidine alone at the same doses and times used for groupsadministered the combined treatments . In the last set of ex-periments, yohimbine (3 mg/kg) was administered in com-bination with clonidine (0 .5 mg/kg; triple dose, 30 min be-fore and 30 and 90 min after MPTP) 30 min before MPTP(30 mg/kg) . Seven days after treatment, animals were killedby cervical dislocation and their brains were removed . Theright striatum was dissected and assayed for DA, serotonin(5-HT), and metabolites, and the left striatum was removedand processed for the measurement of TH activity . In pilotstudies we did not observe any difference between the leftand the right striatum regarding monoamine levels or THactivity after the same treatments .

Another set of experiments was performed to evaluate theeffect of clonidine (at the dose most effective in preventingMPTP neurotoxicity) on the kinetics of MPP + (the toxicmetabolite of MPTP) . In this study, animals were treatedintraperitoneally with MPTP (30 mg/kg) and clonidine (0.5mg/kg) was administered intraperitoneally three times (30min before and 30 and 90 min after MPTP) . Control groupswere administered saline, MPTP, or clonidine at the samedoses and times used for the group administered the com-bined treatment. Animals were killed by cervical dislocationat different times (1, 2, 4, and 6 h) after treatment and theright striatum was dissected to study the time course ofstriatal MPP + concentration.Assay of DA, 5-HT, and metabolitesThe right striatum was sonicated and an aliquot of the

homogenate (50 /1) was assayed for protein (Lowry et al .,1951) . After centrifugation at 8,000 g for 10 min, 20 pl ofthe clear supernatant was injected into an HPLC systemwhere DA, 5-HT, and metabolites were analyzed as pre-viously described with modifications, to also assay 3-me-thoxytyramine in the same chromatographic run (Fornai etal ., 1993) . In brief, the HPLC system was coupled to an ESAcoulometric electrochemical detector . The applied potentialswere +0.32 and -0.35 V for oxidizing and reducing elec-trodes, respectively . Each compound was recorded at bothelectrodes and the amount ofeach neurotransmitter was mea-sured based on two different regression curves (the first forpeaks integrated by oxidizing electrode and the second forpeaks integrated by the reducing electrode) .Measurement of TH activityThe left striatum was homogenized in deionized water

and aliquots of the tissue homogenate were incubated withL-[' 4C]tyrosine (540 mCi/mmol) (Sigma) in a buffer . THactivity was measured radioenzymatically by quantifying theamount of L- ['4C] DOPA formed as described previously(Sonsalla et al ., 1987) .Assay of striatal MPP+ levels

The right striatum was homogenized in 0.6 ml of 0 .1 Mperchloric acid containing a known amount (200 pg/Eil) of1-butyl-4-phenylpyridinium ion (BPP+) used as the internalstandard . An aliquot of the homogenate (50 ,u1) was assayedfor protein (Lowry et al ., 1951) . After centrifugation at8,000 g for 10 min, 50 MI of the clear supernatant wasinjected into an HPLC coupled to a UV detector with wave-length set at 290 nm. The mobile phase consisted of 80%sodium acetate 0.1 M, 19.9% acetonitrile, and 0 .1 % triethyla-mine, pH 5.6 .

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TABLE 1 . Effects of clonidine (high doses), yohimbine,and MPTP on striatal DA levels and TH activity

MPTP hydrochloride was administered intraperitoneally at a doseof 36 mg/kg (corresponding to 30 mg/kg MPTP) . Clonidine (0 .5mg/kg) was administered in single, double, and triple dose, i .e .,alone (Clon 0 .5 X 1, Clon 0 .5 X 2, Clon 0 .5 X 3, respectively) or30 min before MPTP (MPTP + Clon 0 .5 X 1) ; 30 min before and30 min after MPTP (MPTP + Clon 0.5 X 2) ; 30 min before and 30and 90 min after MPTP (MPTP + Clon 0.5 X 3) . Yohimbine (3mg/kg) was injected intraperitoneally alone or 30 min before MPTP .Values are given as the means :t SEM of 6 to 10 mice per group,each assayed in duplicate .

° p < 0.05, compared with saline; by < 0.05, compared withMPTP.

Data analysisFor monoamine assays, a standard curve was prepared

using known amounts of DA, 5-HT, and their metabolites(Sigma), dissolved in 0 .1 M perchloric acid containing aconstant amount (10 pg/pl) of the internal standard (DBA),as used for tissue samples . The standard curve for each com-pound (monoamine or its metabolite) was calculated usingthe regression analysis of ratios of the peak areas (compoundarea/DBA area) for various concentrations of each com-pound recorded at the reducing electrode . An analogous re-gression analysis was performed for the oxidizing electrode .The correlation index for the standard curve ranged from0.996 to 1 .000 . For MPP + assay, a standard curve was pre-pared using known amounts of MPP+ (RBI) dissolved in0 .1 M perchloric acid containing 0.20 mg/ml of the internalstandard (BPP+ ) . The ratios of the peak areas for MPP +/BPP ` were measured and a regression curve was determinedusing various concentrations of MPP+ . For each study, re-sults are expressed as the mean ± SEM values from 6 to 10animals per group, each assayed in duplicate. Effects ofMPTP, clonidine, detomidine, and yohimbine on monoaminelevels and TH activity, as well as the effect of clonidineon MPP + striatal levels, were statistically evaluated usinganalysis of variance with Scheffé's post hoc analysis . Nullhypothesis was rejected when p < 0.05 .

RESULTS

As shown in Table 1, MPTP administration pro-duced a pronounced depletion in striatal DA concentra-tions at 7 days after treatment when compared withcontrols . Injections of clonidine alone (0.5 mg/kg) insingle or multiple injections (one to three) did notproduce any significant change in the levels of striatal

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F. FORNAI ET AL.

DA (Table 1) . Triple injections of clonidine at lowdoses (0.01 and 0.05 mg/kg) (Table 2) or a singleinjection of clonidine at the dose of 0.5 mg/kg (Table1) did not prevent the reductions in striatal dopaminelevels and TH activity . In contrast, double or tripleadministration of clonidine (0.5 mg/kg), before andafter MPTP injection, resulted in a pronounced protec-tion of striatal DA depletion induced by MPTP (Table1) . Detomidine (0.3 mg/kg) alone did not changestriatal DA levels compared with controls, whereaspretreatment with detomidine attenuated the reductionof striatal dopamine levels induced by MPTP (Ta-ble 2) .TH activity in the striatum showed the same trend

(Tables 1 and 2) . DA metabolites paralleled the effectof various treatments on striatal DA levels and THactivity, whereas 5-HT and 5-hydroxyindoleacetic acid(5-HIAA) did not differ significantly among variousgroups of animals (data not shown) . Striatal DA levelsof animals treated only with the a2-antagonist yohim-bine were similar to DA levels of saline controls (129.5± 3.0 and 125 .9 ± 5.8 ng/mg, respectively), whereasthe combination of administration of yohimbine 30min before the administration of MPTP produced anenhancement of MPTP toxicity (35.6 ± 1 .1 and 67.4± 3.2 ng/mg, respectively) (Table 1) . This enhance-ment was also documented by further reductions inTH activity (Table 1) and in the striatal levels of DAmetabolites . When yohimbine (3 mg/kg) was given30 min before MPTP (30 mg/kg), the protective effectof a triple dose of clonidine on MPTP toxicity wassuppressed completely (Fig . 1) . No noteworthy effectwas produced by yohimbine (alone, with MPTP, orwith clonidine + MPTP) on striatal 5-HT and 5-HIAAlevels (data not shown) .

TABLE 2 . Effects ofMPTP, clonidine (low doses), anddetomidine on striatal DA levels and TH activity

MPTP hydrochloride was administered intraperitoneally at a doseof 36 mg/kg (corresponding to 30 mg/kg MPTP). Clonidine (0.01and 0.05 mg/kg) was injected intraperitoneally in a triple dose, i .e.,alone (Clon 0 .01 X 3 and Clon 0.05 X 3, respectively) and 30 minbefore and 30 and 90 min after MPTP (MPTP + Clon 0.01 X 3 andMPTP + Clon 0.05 X 3, respectively) . Detomidine was injectedintraperitoneally at a dose of 0 .3 mg/kg alone (Detour) or 30 minbefore MPTP (MPTP + Detom) . Values are given as the means± SEM of 6 to 10 mice per group, each assayed in duplicate .

° p < 0.05, compared with saline; by < 0.05, compared withMPTP .

TreatmentDA (ng/mgof protein)

TH activity(nmol of

r .-DOPA/g/h)

Saline 117 .4 + 4.3 428 + 56Clon 0.01 X 3 110 .0 ± 7.4 405 ± 78Clon 0.05 X 3 111 .7 ± 5 .5 398 ± 60MPTP 48.4 +4.1 210 + 34MPTP + Clon 0.01 X 3 54 .9 + 5 .8° 225 ± 31MPTP + Clon 0.05 X 3 50 .5 + 3 .1' 232 ± 28Detom 114 .9 -±- 8 .5 408 ± 51MPTP + Detom 72 .9 + 4.36 304 ± 426

Treatment

SalineClon 0 .5 X 1

DA (ng/mg ofprotein)

125 .9 ± 5 .8129 .6 ± 3 .5

TH activity(nmol of L-DOPA/g/h)

421 ± 74447 ± 78

Clon0.5X2 119.0±5 .2 400±50Clon 0 .5 X 3 124 .8 ± 3 .0 410 ± 80MPTP 67 .4 ± 3 .2 ° 250 ± 45,MPTP + Clon 0.5 X 1 75 .2 ±4.9° 311 ± 36aMPTP + Clon 0 .5 X 2 104 .9 ± 3.76 378 ± 67'MPTP + Clon 0 .5 X 3 112 .4 ± 4.66 425 ± 856Yohimbine 129 .5 ± 3 .0 440 ± 81MPTP + yohimbine 35 .6 ± 1 .1 a,b 120 ± 19°.6

FIG. 1 . MPTP hydrochloride was administered intraperitoneallyat a dose of 36 mg/kg (corresponding to 30 mg/kg MPTP).Clonidine (0 .5 mg/kg) was administered in a triple dose 30 minbefore and 30 and 90 min after MPTP (CIon+MPTP) to obtainfull protection (see also Table 1) . Yohimbine (3 mg/kg) wasinjected 30 min before MPTP (Yoh+MPTP) to enhance the ef-fects of MPTP (see also Table 1) or in combination with a tripledose of clonidine anda single dose of MPTP (CIon+Yoh+MPTP)to antagonize the protective effect of clonidine on MPTP toxicity.Mice were killed 7 days after treatment. Data are the meansSEM of 10 mice per group, each assayed in duplicate. ap

< 0.05, compared with saline ; by < 0.05, compared with MPTP ;°p < 0.05, compared with clonidine + MPTP .

MPP + levelsMPTP administration (30 mg/kg i.p .) resulted in a

rapid increase of the striatal concentration of the toxicmetabolite (MPP + ) 1 h after MPTP injection (59.4± 5.1 ng/mg of protein) . This increase reached themaximal level 2 h after the injection (72.1 ± 6 ng/mgof protein) (Fig . 2) . Striatal levels of MPP + decreasedprogressively 4 and 6 h after the injection (10.2 ± 5.1and 3 .5 ± 0.2 ng/mg, respectively) . Combined cloni-dine administration at the dose (0.5 mg/kgx 3) that was most effective in preventing the bio-chemical effects of MPTP did not result in a significantchange in striatal concentration of the toxic metaboliteat 1 h, compared with animals treated with MPTP alone(63 .1 ± 5 and 59.4 ± 5.1 ng/mg of protein, respec-tively) . Furthermore, clonidine pretreatment did notchange striatal MPP + levels at the peak time, 2 h afterMPTP injection (73.5 -- 7 and 72.1 ± 6 ng/mg ofprotein) . Unexpectedly, clonidine administrationmarkedly delayed MPP + elimination from the stria-tum, because 4 and 6 h after administration MPP +levels were not changed significantly compared with1 h (Fig . 2) . Four hours after treatment striatal MPP +levels of animals administered with the combined treat-ment clonidine + MPTP were significantly higher thancontrols (61 -- 5 and 10 -- 2 ng/mg of protein, respec-tively) . The most noteworthy effect was observed 6 hafter treatment when the levels of MPP+ in animalstreated with MPTP alone were almost undetectable(3.5 ± 0.2 ng/mg of protein), whereas the levels ofthe toxic metabolite in animals treated with MPTP+ clonidine were still elevated (43 .7 ± 3 ng/mg ofprotein) (Fig. 2) .

CLONIDINE SUPPRESSES MPTP TOXICITY

DISCUSSION

707

Previous studies suggested a therapeutic role forclonidine in Parkinson's disease based on its pro-nounced behavioral effects when given in combinationwith atropine (Gourez-Mancilla et al ., 1991) orNMDA antagonists (Carlsson and Svensson, 1990) .Thus, clonidine has been shown to improve motordeficits in monoamine-depleted mice (Carlsson andSvensson, 1990) and in MPTP-treated monkeys al-ready exhibiting the behavioral symptoms of nigrostri-atal dopaminergic lesion (Gourez-Mancilla et al .,1991) .In the present study we show that relatively high

doses of clonidine prevent the neurotoxicity of MPTPin mice . To exert this effect, clonidine must be admin-istered at least twice (30 min before and 30 min afterMPTP) at the dose of 0.5 mg/kg, because the tripleadministration of clonidine in lower doses (0.01 and0.05 mg/kg) or a single dose of 0.5 mg/kg did notprotect .Given the nonlinear relationship between the de-

crease in striatal DA levels and the cell loss in the SN(Seniuk et al ., 1990), additional studies, using a widevariety of MPTP doses as well as TH immunohisto-chemistry and Nissl staining of the substantia nigra,are necessary to assess clonidine's ability to protectDA neurons . Nevertheless, the strength of the bio-chemical evidence (DA levels and TH activity) for aprotective role of clonidine shown here is indicativeof an overall protection . In this study we also showthat the protective role of clonidine on MPTP toxicityis not related to a diminished availability of the toxicmetabolite (MPP + ) in the striatum . In fact, the timeof appearance of MPP + in the striatum is the same inanimals treated with clonidine + MPTP or MPTPalone . The maximum amount of MPP+ is also similar

FIG. 2. MPTP hydrochloride was administered intraperitoneallyat a dose of 36 mg/kg (corresponding to 30 mg/kg MPTP) alone(MPTP) or in combination with a protective dose of clonidine(Clonidine+MPTP) (0 .5 mg/kg, 30 min before and 30 and 90min after MPTP). Animals were killed at 0, 1, 2, 4, and 6 hafter MPTP administration . Each value is given in nanograms permilligram of striatal protein and represents the mean ± SEMvalues of six to eight mice per group, each assayed in duplicate.*p < 0.05, compared with MPTP .

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in the two groups of animals . Paradoxically, the striatalclearance of MPP + is delayed in the group of ani-mals receiving the combined treatment (clonidine+ MPTP) . This is unusual for a substance that pro-vides protection and appears to represent an exceptionto the current belief that either increased concentrationor longer retention time of MPP + in the striatum isnecessarily related to increased DA toxicity . Thus, themechanisms of clonidine-induced protection of MPTPtoxicity do not appear to be related to a pharmacoki-netic influence . The lack of protective effect of lowerdoses (0.01 and 0.05 mg/kg X 3) of clonidine con-trasts with the ability of clonidine to induce sedationin mice with a dose-response curve ranging from 3to 300 leg/kg (Pichler and Kobinger, 1981) . Neverthe-less, Buccafusco and Aronstam (1986) have shown adose-response ranging from 0.1 to 1 .0 mg/kg for thea2-mediated protection of soman toxicity by clonidine .The reasons for such discrepancies are unknown ; i .e.,different strains of mice and/or different phenomena(soman toxicity, sedation, DA depletion by MPTP)analyzed might differ in their sensitivity to clonidine .

Several putative mechanisms of action need to beconsidered . Although clonidine has usually beenthought to exert its central effects as an a2-adrenocep-tor agonist, evidence is accumulating that part of itsactions is due to the stimulation of other receptors,especially the so-called imidazole-preferring receptors(Bousquet et al ., 1984 ; Tibrica et al ., 1991) . Indeed,the differences between low and high doses and be-tween single and repetitive administrations, shown inthis study, raise some doubts as to the kind of receptorthat is involved . Nevertheless, in this study we showedthat pretreatment with another a2-agonist (detomidine)was able to attenuate the reduction of striatal DA andTH activities induced by MPTP. Furthermore, yohim-bine, which is a classic selective a2-adrenoceptor an-tagonist with low affinity for imidazoline receptors(Feldman et al ., 1990), was able to suppress the pro-tective effect of clonidine on MPTP toxicity . Further-more, yohimbine produces an enhancement of MPTPtoxicity when administered before MPTP. Therefore,we suggest that the activation of a2-adrenoceptorsmight modulate MPTP toxicity . Other experiments, us-ing different types of a2-agonists (with selective affin-ity for various a2-adrenoceptor subtypes) are necessaryto fully address this issue . The enhancement of MPTPtoxicity by yohimbine raises the intriguing possibilitythat the noradrenergic system, through a2-adrenocep-tors, might modulate the susceptibility of the nigrostri-atal dopaminergic pathway to toxic lesions . In line withthis hypothesis are recent data showing that the lesionof the noradrenergic neurons of the locus ceruleus im-pairs the spontaneous recovery of MPTP-treated mon-keys (Mavridis et al ., 1991) and trice (Marien et al .,1993) . The mechanism of action of such a protectiverole of the noradrenergic system and whether the samemechanism is responsible for the effect described hereis presently unknown . Central a2-adrenoceptors have

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several distinct locations and produce many pharmaco-logical effects (Fornai et al ., 1990) . Although the brainarea responsible for the putative protective action ofclonidine at a2-adrenoceptors described here remainsunknown, it is likely to be nigral and not striatal be-cause the former brain region (unlike the striatum) isknown to receive a noradrenergic innervation from thelocus ceruleus . Although the glutamate-mediated hy-pothesis of MPTP toxicity is debated, it is importantto mention that clonidine, acting through a2-adreno-ceptors located on glutamatergic axons, is able to in-hibit glutamate overflow in several brain areas (Kami-saki et al ., 1992) . An analogous effect of a2-presynap-tic adrenoceptors located on cholinergic neurons isconsidered to be responsible for the protective effectof clonidine on soman toxicity (Buccafusco and Aron-stam, 1986) . Indeed, recent evidence indicates thatMPTP administration induces glutamate release (Car-boni et al ., 1990) . However, only a more direct ap-proach, i .e ., measuring the effects of clonidine onMPTP-induced glutamate overflow by using in vivomicrodialysis, would provide direct evidence for thishypothesis .

Another possible explanation of the clonidine/de-tomidine protective effect on MPTP toxicity shownhere relates to noradrenergic agonists increasingmRNA levels of fibroblast growth factor, which pre-vents MPTP toxicity (Otto and Unsicker, 1990), al-though this effect appears to be mediated by,8-adreno-ceptors (Follesa and Mocchetti, 1993) . Finally, analternative hypothesis must be considered . Thenoradrenergic system is responsible for the uptake ofa considerable amount of endogenously formed MPP +(Herkenham et al., 1991), and this could explain theincreased toxicity of MPTP in the absence of the nor-adrenergic system . Conversely, clonidine, which re-duces the firing rate of noradrenergic neurons (Agha-janian and VanderMaelen, 1982), could increase theamount of MPP + stored in noradrenergic terminals,making it unavailable to the synaptic cleft . This hy-pothesis could explain the paradoxical increased reten-tion time of striatal MPP+ in clonidine + MPTP-treated mice . This appears unlikely, however, giventhe ratio of noradrenergic terminals to dopaminergicterminals in the striatum .

In conclusion, our findings provide evidence for amodulatory effect of a2-adrenoceptors in experimentalparkinsonism and suggest the need for a more in-depthstudy of the relationship between the noradrenergicsystem, a2-adrenoceptors, and damage to the nigrostri-atal dopaminergic pathway .Acknowledgment: Dr. Kenneth Keller and David Dybdal

are gratefully acknowledged for reading the manuscript andfor helpful discussions . This research was supported by agrant from Ministero della Pubblica Istruzione (40-60%) .

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