Introduction administration, a number of neurological complications

Brain Research 887 2000 110–117 www.elsevier.com locate bres Research report Time-course and dose–response study on the effects of chronic L -DOPA administration on striatal dopamine levels and dopamine transporter following MPTP toxicity a,b , b a a Francesco Fornai , Giuseppe Battaglia , Marco Gesi , Filippo S. Giorgi , b,c b b,c Francesco Orzi , Ferdinando Nicoletti , Stefano Ruggieri a Department of Human Morphology and Applied Biology , University of Pisa, Via Roma 55, I-56100 Pisa, Italy b I .R.C.C.S. I.N.M. Neuromed, Pozzilli IS, Italy c Department of Neurological Sciences , University ‘La Sapienza’, Roma, Italy Accepted 19 September 2000 Abstract Despite a long-lasting therapeutic use of L -DOPA in Parkinson’s disease, doubts still remain concerning the possibility that chronic L -DOPA might accelerate the progression of this movement disorder. To address this point, in the present study we examined the effects of chronic L -DOPA administration either in intact or MPTP-treated parkinsonian mice. We produced an intermediate striatal dopamine loss by administering a low dose of MPTP 30 mg kg; then, we treated mice chronically, for different time intervals, with a daily dose of L -DOPA 50 mg kg. In particular, to study the time-course of the effects of L -DOPA on the recovery of nigrostriatal dopamine axons, mice were sacrificed at 5, 30, 60, and 90 days after a daily L -DOPA administration. To evaluate presynaptic integrity of the nigrostriatal pathway we measured dopamine, metabolite levels, and dopamine uptake sites. In the same animals, we measured striatal serotonin levels and we analysed monoamine content in the olfactory bulb. Administration of MPTP produced a neurotoxic effect, which fully recovered in 2–3 months. Daily L -DOPA administration did not modify this recovery process. Additionally, there was no significant effect of L -DOPA in intact mice, despite a slight decrease in striatal dopamine levels at 5 and 30 days. However, this effect was neither worsened nor reproduced by administering higher doses of L -DOPA up to 400 mg kg for the same amount of time. These data rule out neurotoxic effects induced by prolonged L -DOPA administration, both in intact and MPTP-treated mice. Moreover, administration of L -DOPA does not change the recovery process which takes place after a nigrostriatal lesion.  2000 Elsevier Science B.V. All rights reserved. Theme : Disorders of the nervous system Topic : Degenerative disease: Parkinson’s Keywords : L -DOPA; MPTP; Recovery; Experimental parkinsonism; DAT

1. Introduction administration, a number of neurological complications

i.e., dyskinesia, tightly related to the ongoing therapy Since its introduction in the 1960s see Ref. [18], the occur [40,4]. It has been postulated that even the natural neurotransmitter precursor L -DOPA, via its decarboxylation course of the disease might be accelerated by chronic to dopamine DA, represents the most effective therapy in L -DOPA administration [15]. Thus, an open question still Parkinson’s disease PD [19]. There is concern, however, remains concerning L -DOPA-related symptoms: do they that the symptomatic relief induced by L -DOPA convoys a represent a functional and potentially reversible behavioral toxic effect by accelerating the neuronal loss which disorder, or do they reflect an actual progression of the normally takes place during the disease process [19]. disease, substantiated by accelerated neuron and or axon Indeed, there is no doubt that, after prolonged L -DOPA terminal loss promoted by L -DOPA? This latter hypothesis was suggested since the late 1970s [15], when it was demonstrated that L -DOPA produces Corresponding author. Tel.: 139-50-835-942; fax: 139-50-552-102. E-mail address : f.fornaidrugs.med.unipi.it F. Fornai. oxidative compounds like semiquinone and O-quinone 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 9 9 - 1 F . Fornai et al. Brain Research 887 2000 110 –117 111 derivatives which might be toxic for DA neurons. In line nigrostriatal DA terminals. In mice, in 2–3 months after with this, a number of in vitro and microinfusion studies the onset of a moderate striatal DA loss, a spontaneous carried out during the last decade, provided evidence for recovery of nigrostriatal DA axons takes place [12]. To L -DOPA- and or DA-induced neurotoxicity in non-cat- document the presynaptic integrity of the DA nigrostriatal echolamine [36,7,27] and catecholamine cell lines pathway we measured both DA, metabolite levels, and DA [22,29,39], in primary mesencephalic cell cultures uptake sites both in intact and MPTP-lesioned striatum, [23,24,30], and via direct microinfusion of DA within the injected chronically either with saline or L -DOPA. To striatum [10]. evaluate the site-specificity of the effects induced by Despite these results, obtained with in vitro and mi- MPTP and or L -DOPA we also analysed monoamine croinfusion studies, in vivo experiments consistently levels in the olfactory bulb. failed to demonstrate neurotoxic effects induced by L - DOPA on intact nigrostriatal DA system [17,31]. These results are in line with neuropathological studies 2. Materials and methods carried out in humans with a misdiagnosis of PD who, despite receiving a high daily dose of L -DOPA for several 2.1. Animals years, showed a normal mesencephalic histopathology [32,33]. Two hundred and sixty C57 Black male mice Charles However, it should be considered that during PD, River Calco, CO, Italy, 9 weeks old, were used for the exogenous L -DOPA is handled differently by the few study. Since previous experiments demonstrated that surviving DA neurones. For instance, during the pro- MPTP toxicity critically depends on age [3,8], all mice gression of the disease only scant striatal DA axons are used in the present work were 9 weeks old at the time of taking up the same amount of L -DOPA, which therefore, MPTP administration. Mice were housed with free access reaches a higher concentration in each nigrostriatal termi- to food and water and kept under environmentally con- nal. Moreover, in PD surviving DA axons undergo an trolled conditions 12-h light dark cycle with light on increased neurotransmitter metabolism [1,21] which leads between 07:00 and 19:00. In the present study, we to augmented rate of L -DOPA utilisation and, thereby, to evaluated the potential toxicity of L -DOPA: it is established heightened DA oxidation. These effects might converge in that DA neurotoxins i.e., amphetamines vary their effica- increasing oxidative stress within surviving DA terminals cy depending on housing conditions for a review, see Ref. accelerating the progression of PD [38]. [35]; therefore, we did not vary the number of animals per Indeed, a few in vivo experiments carried out in cage n510 and the size of the cages 38322 cm wide parkinsonian animals provided evidence for a neurotoxic and 15 cm high. effect of L -DOPA [5,28]; however, in other studies this Experiments were approved by Local Ethical Committee toxic effect was not observed [9,25]. In particular, in a and animals were treated in accordance with the Guide- recent elegant report, Murer et al. [25] demonstrated the lines for Animal Care and Use of the National Institutes of absence of L -DOPA toxicity, suggesting a trophic role of Health. L -DOPA administered for 6 months to rats carrying a partial striatal DA loss induced by 6-hydroxydopamine 2.2. Experimental design 6-OHDA. In the present study, we examined the effects of chronic 2.2.1. Time-course study L -DOPA administration in parkinsonian mice by using a In the time-course study, 160 C57 Black mice were different experimental approach. We administered L -DOPA divided into four groups each composed of 40 animals chronically to mice carrying a partial lesion of the nigros- receiving various treatments: Group A received an in- triatal DA pathway induced by the neurotoxin MPTP. The jection of saline and then again, it was administered daily experiment was performed in mice since, among rodents, with saline. Group B was administered saline once and this animal species is more sensitive to experimental than, chronically a daily dose of L -DOPA methyl ester Parkinsonism induced by DA neurotoxins [14]. In order to hydrochloride Research Biochemicals, RBI, Natick, MA; evaluate either potential deleterious, or protective effects 50 mg kg, free base. Group C was treated with a single induced by chronic L -DOPA, we produced an intermediate dose of MPTP hydrochloride RBI; 36 mg kg, corre- striatal DA loss by administering a low dose 30 mg kg sponding to 30 mg kg of MPTP, then it was administered of MPTP. Then, we challenged these animals with a daily with a daily dosage of saline. Group D was treated with dose of L -DOPA 50 mg kg. Mice were sacrificed at four MPTP and then received a daily chronic administration of different time intervals 5, 30, 60, and 90 days after L -DOPA. In all groups, repeated administration of L -DOPA MPTP administration. Apart from disclosing a potential or saline started 12 h following the single MPTP or saline effect depending on the time of exposure to L -DOPA, this injection. This time interval was chosen based on previous experimental protocol was designed to evaluate the time- studies to start chronic L -DOPA administration when 1 course of potential effects of L -DOPA on the recovery of MPTP and its toxic metabolite MPP were completely 112 F cleared from the striatum [11]. The time-course of the 1.5 ml of ice-cold buffer 50 mM Tris, 5 mM EDTA, 320 recovery process of striatal DA innervation following an mM sucrose; pH 7.5 and centrifuged at 10003g for 10 intermediate dose of MPTP was evaluated sacrificing mice min at 48C. The pellet was discarded and the supernatant from each group at four distinct time intervals. In par- centrifuged at maximum speed in a microfuge for 30 min ticular, the original four groups of mice were further at 48C. The pellet was re-suspended in 1.2 ml of binding divided into four subgroups each composed of 10 animals buffer 50 mM Tris, 300 mM NaCl, 5 mM, 0.1 ascorbic 3 which were sacrificed at 5 days, and 1, 2, and 3 months, acid; pH 7.9 by sonication. After addition of [ H]GBR respectively, after MPTP administration. 12935 DuPont NEN; final concentration 3 nM, 40–60 During chronic L -DOPA administration mice were Ci mmol, membrane preparations were incubated for 45 weighted once a week in order to maintain a constant min at 258C. Non-specific binding was determined in the amount of L -DOPA body weight. Injections were carried presence of DA 200 mM. Incubation was stopped by out intraperitoneally i.p., using a constant volume 10 centrifugation for 30 min. The pellet was washed twice ml kg. Mice were sacrificed at 40 h after the last L - with 1 ml of binding buffer and radioactivity was mea- DOPA saline injection and their brains were immediately sured by scintillation counting. removed. The left striatum and the olfactory bulb were dissected 2.5. Data analysis and processed for measuring monoamine levels, whereas the right striatum from the same animals was dissected and For monoamine assay, a standard curve was prepared processed for measuring presynaptic striatal DA uptake using known amounts of DA, NE, 5-HT and metabolites sites. Sigma, dissolved in 0.1 M perchloric acid containing a constant amount 10 ng ml of the internal standard 2.2.2. Dose–response study DBA, as used for tissue samples. The standard curve for Since in the time-course study see Section 3 we each compound DA, NE, 5-HT or metabolites was observed a slight decrease of striatal DA levels at early calculated using regression analysis of the peak areas for time intervals 5 days and 1 month after L -DOPA 50 known concentrations of each compound. For binding mg kg administration to intact mice, we explored this studies data are expressed as percentage of specific bind- effect in detail by increasing the dosage of L -DOPA. In this ing; the latter was calculated from the ratio between the additional experimental step, 100 C57 Black mice were cpm mg protein of specific binding, and the cpm mg divided into five groups each composed of 20 mice. Group protein of total binding. A received saline. Group B was administered with L -DOPA For NE, DA, 3,4-dihydroxyphenylacetic acid DOPAC, at 50 mg kg. Group C received L -DOPA at 100 mg kg. homovanillic acid HVA, 5-HT and 5-hydroxy-in- Group D was administered with L -DOPA at 200 mg kg. doleacetic acid 5-HIAA levels, results are expressed as Group E was injected with L -DOPA at 400 mg kg. From the mean6S.E.M. of eight to 10 animals per group. For each group, 10 animals were sacrificed at 5 days after a binding studies, results are expressed as the mean6S.E.M. daily saline L -DOPA administration, whereas 10 mice of eight to 10 animals per group. Effects of MPTP and or were sacrificed after 1 month of daily treatment. In these L -DOPA on monoamine levels in the striatum and olfactory mice we measured striatal monoamine levels. bulb as well as on striatal GBR 12935 binding sites, were evaluated using analysis of variance with Sheffe’s post-hoc 2.3. Assay of monoamines analysis. The striatum and the olfactory bulb were sonicated in 0.6 ml of ice-cold 0.1 M perchloric acid containing 10

3. Results