MPTP, MPDP+ and MPP+ cause decreases in dopamine content in mouse brain slices

Neuroscience Letters, 108 (1990) 213-218

213

Elsevier Scientific Publishers Ireland Ltd. NSL 06546

MPTP, M P D P + and M P P + cause decreases in dopamine content in mouse brain slices John A. Wilson 2, Thomas J. Doyle I and Yuen-Sum Lau 3 IParkinsons Disease Research Program," and Departments of 2Physiology and JPharmacology, Creighton University School of Medicine, Omaha, NE 68178 (U.S.A.)

(Received 12 June 1989; Revised version received 18 August 1989; Accepted 22 August 1989) Key words.

MPTP; MPDP+; MPP+; Brain slice; Dopamine content

MPTP causes a Parkinson's disease-like syndrome in which the dopamine content of the nigrostriatal system decreases. We have studied the relationship between physiological changes and dopamine content using a brain slice preparation developed for electrophysiological studies ofcorticostriate and nigrostriatal synaptic transmission. We report that MPTP, MPDP + and MPP + cause significant decreases in dopamine content of mouse brain slices. We also report that compounds (pargyline and GBR-12909) which block MPTP's toxicity in vivo and prevent non-reversible changes in synaptic transmission are not able to alter MPTP's ability to decrease slice dopamine contents. This indicates that the dopamine content in slices may not be causally related to the non-reversible decrease in synaptic transmission or in vivo neurotoxicity.

The discovery t h a t the d r u g i - m e t h y l - 4 - p h e n y l - 1 , 2 , 3 , 6 - t e t r a h y d r o p y r i d i n e ( M P T P ) can p r o d u c e P a r k i n s o n ' s disease like s y m p t o m s in m a n [9] caused an explosion o f interest in P a r k i n s o n ' s disease research. M P T P induces a P a r k i n s o n ' s like s y n d r o m e in n o n - h u m a n p r i m a t e s which resembles the i d i o p a t h i c disease in m a n . D o p a m i n e ( D A ) c o n t a i n i n g neurons, which project from the s u b s t a n t i a nigra p a r s c o m p a c t a to the n e o - s t r i a t u m , d e g e n e r a t e a n d n e o - s t r i a t a l D A levels decrease d r a m a t i c a l l y [2]. F u r t h e r research has shown that dogs [20] a n d mice [7] can also develop some o f the characteristics o f P a r k i n s o n ' s disease in response to M P T P injections. A c u t e decreases in D A levels a p p e a r to be caused in p a r t by a release o f D A from nigral neurons. M P T P - i n d u c e d release has been d e m o n s t r a t e d in b o t h mice [13] and rats [15]. M P P +, the second m e t a b o l i t e o f M P T P o x i d a t i o n , has also been shown to cause a release o f D A from rat c o r p u s s t r i a t u m f r a g m e n t s in vitro [4], in vivo using p u s h - p u l l c a n n u l a t i o n [16], o r using b r a i n dialysis [14]. The m a g n i t u d e o f the M P P + induced response is greater than t h a t p r o d u c e d by M P T P alone. U n f o r t u n a t e l y , studies o f M P T P a n d M P P + - i n d u c e d release have not r e p o r t e d the c o n c e n t r a t i o n o f D A r e m a i n i n g in the tissue after either M P T P or M P P +. A l s o the first m e t a b o l i t e o f Correspondence: J.A. Wilson, Department of Physiology, Creighton University School of Medicine, Omaha, NE 68178-0224, U.S.A.

0304-3940/90/$ 03.50 ~) 1990 Elsevier Scientific Publishers Ireland Ltd.

214 M P T P oxidation, M P D P ÷ was not tested. M P D P v is an extremely reactive metabolite of M PTP [17, 21] which has been proposed as a necessary compound for M P T P ' s specific effect [19]. M P D P + 's effect on DA content in the nigrostriatal system should be determined if we are to reach an understanding of the mechanism by which M P T P specifically damages the nigral neurons and causes a Parkinsonism-like syndrome. Mouse nigro-striatal brain slices undergo a non-reversible decrease in the amplitude of an extracellularly recorded synaptically mediated potential in response to both MPTP and M P D P + [18, 19]. The effect can be prevented by pargyline, a monoamine oxidase (MAO) inhibitor, or GBR-12909, a highly specific DA uptake blocker [12]. How well do the physiological findings correlate with other indicators of toxicity'? To address this question, we have used a brain slice preparation in which the physiological effects of MPTP, M P D P +, and MPP + have been studied. We found that MPTP and its metabolites cause a decrease in DA levels which are not prevented by agents which block M P T P ' s toxicity in vivo. Brain slices were made from C57BL mice [18]. Two 500/~m parasagittal slices containing the substantia nigra, neostriatum, internal capsule, and median forebrain bundle were obtained from each hemisphere. The more lateral slice from each hemisphere was used as it was anatomically less variable. The two lateral slices from an individual were assigned to different experimental conditions. Slices were maintained in artificial cerebrospinal fluid (ACSF) (124 mM NaC1, 3.3 mM KCI, 1.25 mM Na2HPO4, 2.4 mM CaC12 1.2 mM MgSO4, 26 m M NaHCO3, 10 mM glucose, and 100/~M ascorbic acid) which was continuously bubbled with a mixture of 95% 02-5% CO2. They were allowed to come to room temperature from O ~ C for at least 30 rain and then to equilibrate at 3 7 C in a water bath for at least another 30 rain. After equilibration at 37'C, slices were transferred to experimental solutions. The control solution was fresh ACSF. MPTP, M P D P +, or MPP + were added to the ACSF for 20 rain at a concentration of 300 ~M which is the maximum concentration obtained in mouse brain after injection of MPTP [10] and the concentration used for the physiological studies. If the slices were to be incubated in pargyline (100 itM) or GBR-12909 (100 nM), they were transferred to these solutions for 20 min. After this incubation, the paired control was maintained in pargyline or GBR-12909 for 20 more rain while the experimental slice was transferred to a solution containing pargyline or GBR-12909 with 300/tM M P T P for an additional 20 min. After treatments, slices were transferred to ACSF for a 20 rain wash. Each slice was suspended in 200 gl of cold 0.2 N perchloric acid, sonicated for 1 min, centrifuged at I 1,000 g for 15 rain at 4'~C, filtered through a 0.45 gm filter, and frozen for subsequent catecholamine analysis. A 15 /21 sample was injected into an H P L C (Waters) with a C18 reverse phase/~-Bondapack column ( 15 cm x 3.9 mm, Waters) and an electrochemical detector (Waters) at a reference voltage of 0.6 V. The mobile phase contained 100 mM Na-acetate, 20 mM citric acid, 100 mg/l Na-octyl-sulfate, 50 mg/l E D T A and 4% methanol (pH 4.1 ) and the flow rate was 2.0 ml/min. The output from the electrochemical detector was digitized and stored on an IBM PC-XT. Statistical analysis was done using the NCSS statistical package; P < 0.05 was considered as statistically significant.

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8 Fig. I. Comparison of treatments on brain D A content. Average values (plus 1 S.E.M.) are shown for control, MPTP-treated, MPDP+-treated and MPP+-treated slices. The different shadings indicate statistically significant differences from slices with different shadings, which in this case is all the different treatments.

Slices treated with M P T P contained significantly less DA than did control slices (control: n = 32, 45.6 + 2.2 ng/slice; M P T P treated: n = 24, 35.2 + 2.1 ng/slice). Those slices treated with M P T P contained significantly more DA than those treated with M P D P + (n = 26, 26.0 + 2.3 ng/slice) which in turn contained significantly more DA than those treated with MPP + (n = 25, 18.3 + 2.3 ng/slice) (Fig. 1). M P T P and its oxidative metabolites were therefore able to decrease the tissue level of DA in the nigrostriatal slice preparation. Furthermore, each metabolite of M P T P produced a progressively greater decrease in tissue DA levels. To determine whether or not the decreased DA content is correlated with the neurotoxic changes caused by MPTP, we treated slices with compounds which protect against MPTP's effects on synaptic transmission in vivo and in vitro [8, 12]. Pargyline and GBR-12909 were given both alone and in combination with MPTP. Application of pargyline alone caused no change in the DA contents of the slices (Fig. 2). When M P T P was added to solutions containing pargyline, DA levels in the tissue were significantly lower than the control levels and not significantly different from the decrease in DA levels caused by M P T P alone. Application of GBR-12909 alone also produced no change in the DA contents of the slices. When M P T P was added to the ACSF containing GBR-12909 the DA levels decreased significantly and were not different from those produced by M P T P alone (Fig. 2). When the effect of pargyline is compared with the effect of GBR-12909, no difference can be seen between the effects of either of these drugs. Therefore, conditions which prevent the non-reversible decrease in synaptic transmission in slices or block the toxicity of M P T P in vivo do not prevent the effect of M P T P in decreasing tissue DA levels. MPTP, M P D P +, and MPP + concentrations and application times which produce changes in synaptic transmission also cause decreases in DA contents. This finding is also in good agreement with predictions made on the basis of the studies which showed that M P T P and MPP + both cause release of DA [4, 13]. The decrease in tissue levels of DA indicates that the combination of the de novo synthesis and reup-

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Fig. 2. Comparison of treatments on brain DA contents. Average values (plus I S.E.M.) are shown for control and MPTP-treated, pargyline- and pargyline + MPTP-treated and GBR-12909- and GBR-12909 + MPTP-treated slices. The different cross hatchings indicate statistically significant differences. It can be seen that pargyline and GBR-12909 do not change DA content from what is found in control slices. Similarly MPTP plus pargyline- and MPTP plus GBR-12909-treated slices are not significantly different from MPTP-treated slices.

take are not sufficient to replace the DA which has been released by MPTP or MPP + . Hallman et al. [6] reported that DA concentration in the mouse neostriatum is lowest I h after an injection of MPTP and recovers during the 2nd-7th h after the injection. This in vivo finding supports the proposal that the release of DA caused by MPTP is so large that reuptake and synthesis can not replace the DA for a period of several hours. M P D P + also causes a depletion of DA from the tissue; therefore, it is logical to predict that M P D P + also causes a release of DA like that caused by MPTP and MPP +. M P D P + might therefore come into contact with DA in high concentrations in the synaptic clefts potentially reacting to form additional toxic substances [3]. The statistically different potencies of MPTP, M P D P + , and MPP + are of interest for they not only confirm earlier findings [4, 14, 16] but they also are similar to findings of others. Cultured neurons incubated in high concentrations of MPTP survive better than those incubated in lower concentrations of MPP + [11]. This occurs even though more MPP + may have been generated during the week the cultures were incubated in MPTP than was ever present in cultures which were placed directly in MPP + . This finding may indicate that MPP + causes different responses under different circumstances and that the presence of MPP + alone accounts for only a part of the specific neurotoxic response to MPTP. A similar argument is supported for the brain slice data [18, 19]. Direct application of MPP + to slices causes a greater decrease in D A levels than does application of MPTP or M P D P + ; whereas MPP + is only able to reversibly alter synaptic transmission. The fate of M P D P + is also a source of considerable controversy. M P D P + was thought to be converted to MPP + extremely rapidly [5] making its availability to slices questionable; however, a recent study by Wu et al. [22] has shown that 50 #M M P D P + has a half life of more than 4 h in mouse brain homogenates at pH 7.6. This finding is in sharp contrast with the work of Arora et al. [1]. Using rats they

217 f o u n d that 45 m i n after M P T P was given i n t r a p e r i t o n e a l l y only 16% o f the M P T P r e m a i n e d as M P T P , a n u n m e a s u r a b l e q u a n t i t y was present as M P D P +, a n d 44% of the M P T P had been metabolized to M P P +. The differences m a y be species related; however, it is quite clear that the m e t a b o l i s m o f M P T P is more complicated t h a n originally proposed. The finding that M P T P causes a decrease in slice D A levels in c o n d i t i o n s u n d e r which the in vivo neurotoxicity a n d in vitro physiological effect would have been blocked is puzzling. We f o u n d that there was n o statistical difference between the effect of M P T P alone or M P T P in the presence of pargyline or GBR-12909 on slice d o p a m i n e content. Therefore the depletion of D A in the slices appears to be independent o f the m e c h a n i s m responsible for neurotoxic, pathological changes. We t h a n k Mr. Chris Runice, Ms. A n n e Schulte, a n d Ms. K a r e n T r o b o u g h for their technical aid in this project. The P a r k i n s o n s Disease Research P r o g r a m is f u n d e d by a g r a n t from the Health F u t u r e s F o u n d a t i o n . 1 Arora, P.K., Riachi, N.J., Harik, S.I. and Sayre, L.M., Chemical oxidation of l-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) and its in vivo metabolism in rat brain and liver, Biochem. Biophys. Res. Commun., 152 (1988) 1339 1347. 2 Burns, R.S., Chiueh, C., Markey, S.P., Ebert, M.H., Jacobowitz, D.M. and Kopin, l.J., A primate model of parkinsonism: selectivedestruction of dopaminergic neurons in the pars compacta of the substantia nigra by MPTP, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 4546-4550. 3 Castagnoli, N. Jr., Chiba, K. and Trevor, A.J., Potential bioactivation pathways for the neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine, Life Sci., 36 (1985) 225- 230. 4 Chang, G.D. and Ramirez, V.D., The mechanism of action of MPTP and MPP ÷ on endogenous dopamine release from the rat corpus striatum superfused in vitro, Brain Res., 368 (1986) 134-140. 5 Chiba, K., Trevor, A.J. and Castagnoli, N., Jr., Metabolism of the neurotoxic tertiary amine, MPTP, by brain monoamine oxidase, Biochem. Biophys. Res. Commun., 120 (1984) 574-578. 6 Hallman, H., Lange, J., Olson, L., Stromberg, I. and Jonsson, G., Neurochemical and histochemical characterization of neurotoxic effects of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine on brain catecholamine neurons in the mouse, J. Neurochem., 44 (1985) 117 127. 7 Heikkila, R.E., Hess, A. and Duvoisin, R.C., Dopaminergic neurotoxicity of l-methyl-4-phenyl1,2,3,6-tetrahydropyridinein mice, Science,244 (1984) 1451 1453. 8 Heikkila, R.E., Manzino, L.M., Cabbat, F.S. and Duvoisin, R.C., Protection against dopaminergic toxicity of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine by monoamine oxidase inhibitors, Nature (Lond.), 311 (1984) 467~169. 9 Langston, J.W., Ballard, P., Tetrud, J.W. and Irwin, I., Chronic parkinsonism in humans due to a product of meperidine-analogsynthesis, Science,219 (I 9831979-980. 10 Markey, S.P., Johannessen, J.N., Chiueh, C.C., Burns, R.S. and Herkenham, M.A., Intraneuronal generation of a pyridinium metabolite may cause drug-induced parkinsonism, Nature (Lond.), 311 (1984) 464467. 11 Mytilineou, C. and Friedman, L., Studies on the metabolism and toxicity of l-methyl-4-phenyl-l,2,3,6tetrahydropyridine in cultures of embryonic rat mesencephalon,J. Neurochem,, 51 (1988) 751~755. 12 Pileblad, E. and Carlsson, A., Catacholamine-uptake inhibitors prevent the neurotoxicity of l-methyl4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) in mouse brain, Neuropharmacology, 24 (1985) 689-692. 13 Pileblad, E., Nissbrandt, H. and Carlsson, A., Biochemicaland functional evidencefor a marked dopamine releasing action of N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (NMPTP) in mouse brain, J. Neural Transm., 60 (1984) 199-203. 14 Rollema, H., de Vries, J.B., Damsma, G., Westerink, B.H.C., Kranenborg, G.L., Kuhr, W.G. and Horn, A.S., The use of in vivo brain dialysis of dopamine, acetylcholine, aminoacids and lactic acid

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