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Behavioral and biochemical changes following acute administration of MPTP and MPP+
Life Sciences, Vol. 40, pp. 1309-1318 Printed in the U.S.A.
Pergamon Journal
BEHAVIORAL AND BIOCHEMICAL CHANGES FOLLOWING ACUTE ADMINISTRATION OF ~ T P AND MPP+ Takeshi Tadano, Nobunori Satoh, Ichiro Sakuma, Toshihiro Hatsumura, Kensuke Kisara, Yuichiro Arai* and IIiroyasu Kinemuchi* Department
of Pharmacology, Tohoku College of Pharmacy, 4-4-1, Komatsushima, Sendai 983, *Department of Pharmacology, School of Medicine, Showa University, 1-5-8, Hatanodai, Shinagawa-Ku, Tokyo 142, Japan (Received in final form January 5, 1987)
S urnma ry.
The acute effects of 1 -methy 1 -4-phenyl - I ,2,3,6tetrahydropyridine (MPTP) a n d l - m e t h y l - 4 - p h e n y l p y r i d i n i u m ion (IvlPP÷) on m o u s e l o c o m o t o r activity and s t r i a t a l dopamine (DA) and 5-hydroxytryptamine (5-HT) levels were investigated. A single dose of either MPTP ( 1 0 - 3 0 m g / k g , i . p . ) o r MPP÷ ( 5 - 2 0 ug/mouse, i.c.v.) decreased locomotor activity 10-40 min after injection: this locomotor effect was significantly suppressed by e i t h e r pretreatment with nomifensine or l-deprenyl alone, or by the combinat ion of desmethyl imipramine and 6hydroxydopamine. Pretreatment with clorgyline did not suppress this behavior, and a single dose of haloperidol enhanced the effect. The s t r i a t a l levels of DA, 3-methoxytyramine an d 5-HT increased in parallel with the decrease in locomotor activity caused by MPTP o r MPP+. In c o n t r a s t , levels of 3,4dihydroxyphenylacet ic acid, homovani I 1 ic acid and 5hydroxyindoleacetic acid were decreased by i n j e c t i o n of either MPTP o r MPP÷. P o s s i b l e mechanism(s) of the behavioral and biochemical changes caused by t h e a c u t e actions o f MPTP a n d MPP+ with respect to their neurotoxic effects on the nigrostriatal DA s y s t e m a r e d i s c u s s e d . MPTP produces irreversible neurological damage to nigrostraital dopamine (DA) n e u r o n s in primates, including human, similar to the well-established changes in patients with Parkinson)s disease (for review, s e e 1,2). The n e u r o t o x i c effects o f MPTP a r e d e r i v e d from its oxidized product, MPP+, which is formed in a reaction catalyzed by t h e 3 f o r m of monoamine oxidase (ivlAO, EC 1 . 4 . 3 . 4 ) (MAO-B), via the intermediate, i - m e t hy 1 - 4 - p h e n y I - 2 , 3 - d i h y d r o p y r i d i n i um i o n (MPDP+) ( 1 , 2 ) . The high uptake of MPP+ by DA neurons is another crucial step in the expression of its neurotoxicity (3,4). Thus, MPTP i s n o t intrinsically neurotoxic and its
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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Acute MPTP and MPP+ Effects
Vol. 40, No. 13, 1987
neurotoxicity depends mainly on its biotransformation to the active metabolite MPP+, s i n c e p r e t r e a t m e n t with selective MAO-B inhibitors, which decrease the production o f MPP+, p r e v e n t s the neurotoxicity (5,6). Although little information is available on its precise mechanism, reports have been accumulating that indicate a species difference in susceptibility and behavioral response t o MPTP, w i t h p r i m a t e s being the most sensitive. Repeated higher doses and longer durat ion of treatment of rodents with MPTP c a u s e t h e DA neurotoxicity, as judged by marked reductions in locomotor activity, striatal DA a n d i t s m a j o r m e t a b o l i t e levels, 3,4dihydroxyphenylacetic a c i d (DOPAC) a n d h o m o v a n i l l i c a c i d (HVA) (1,2,7-9). These results suggest that the action o f MPTP o n small laboratory animals might provide a useful model for studying the etiology and the processes of neuronal degeneration in patients with Parkinson's disease. This animal model may also offer an opportunity to screen antiparkinsonian drugs and, experimentally, to determine the side-effects o f l o n g t e r m DA p r e c u r s o r and agonist therapy. To our knowledge, the acute effects of the toxic metabolite, MPP+, on the behavioral system a n d DA m e t a b o l i s m in the mouse nigrostriatal system have not yet been fully established, despite many neurotoxicity reports after repeated MIaTP injections (1,2). Thus, possible relations between these acute changes and the neurotoxicity o f MPTP a n d MPP+ t o w a r d s DA neurons need to be resolved to clarify the initial step(s) of the toxicity and its resulting degeneration o f DA n e u r o n s . The present study was undertaken to elucidate the acute behavioral effects caused by either compound injected systemically or intracerebroventricularly (i.c.v.), and to clarify parallelism between the behavioral changes and monoamine metabolism in the mouse striatum. Materials Chemicals
and Methods
and Drugs
Clorgyline HCI a n d I - d e p r e n y l i-t(31 w e r e k i n d l y provided by May & Baker Ltd., Dagenham, England, and Dr. J. Knol l, Semmelweis University of Medicine, Budapest, Hungary, respectively. MPTP HCI w a s p u r c h a s e d from Aldrich Chemical Co., Milwaukee, Wis., U.S.A.; MPP+ i o d i d e from Research Biochemicals Inc. Ohio, U.S.A.; and nomi fensine HCI from Hoechst, Frankfurt, West Germany. Desmethylimipramine HCI f r o m Ciba-Geigy, Basel, Switzerland and 6-hydroxydopamine HCl f r o m Sigma Chemicals, St. Louis, MO, U . S . A . T h e o t h e r chemicals used were of the highest grade commercially available. The MPP+ solution was administered i.c.v, in a volume of l0 ul/mouse, since, in general, charged compounds cannot easily pass through the blood-brain-barrier. Other solutions used in this study were intraperitoneally (i.p.) injected. All drugs used for i.c.v, injection were dissolved in Ringer's solution. For i.p. injection, they were dissolved in saline. The d o s e s of all compounds are expressed as their respective salts. Pretreatment with nomifensine, l-deprenyl or clorgyline was performed l hr prior t o MPTP o r MPP+ i n j e c t i o n ; that with haloperidol w a s 30 m i n , a n d d e s m e t h y l i m i p r a m i n e (DMI) p l u s 6 -
Vol. 40, No. 13, 1987
hydroxydopamine injection. Animals
Acute MPTP and MPP+ Effects
(6-OHDA)
and m e a s u r e m e n t
of
was
performed
locomotor
7 days
1311
prior
to
the
activity
In t h i s study, male albino m i c e (ddY s t r a i n ) weighing 22-25 g were used. M i c e w e r e h o u s e d in s u s p e n d e d p l a s t i c cages with free access to food and water in a colony room that was illuminated from 10:00 to 18:00 hr and maintained at 22~2;C. The locomotor activity (ambulation) of mice was determined by the tilting cage method reported previously (10). A handmade apparatus assembled from two round plastic, commercially available basins ( 2 5 cm i n d i a m e t e r ) put one upon the other was used to measure the ambulatory activity. Any s l i g h t tilt of the activity c a g e c a u s e d by m o v e m e n t o f t h e a n i m a l s was detected by three micro-switches attached on t h e b r i m o f t h e o u t e r basin, and t h e s e activated an e l e c t r o m a g n e t i c counter. In t h e p r e s e n t study, 10 a c t i v i t y cages of same type were used simultaneously. Recording the number of counts indidicating locomotor activity started to immediately upon placing mice treated with a drug(s) or vehicle in the cages, and was accumulated in successive 10 min intervals throughout the observation periods. Determinations
of
amines
and
their
metabolites
in
striatum
In o r d e r to determine the levels o f DA a n d 5 - H T a n d t h e i r metabolites, mice were sacrificed 30 m i n a f t e r the injection of either MPTP o r MPP+. The m o u s e s t r i a t a were then dissected out on an ice-cold glass plate, homogenized and, after deproteinization, assayed f o r DA, 3 , 4 - d i h y d r o x y p h e n y l a c e t i c acid (DOPAC), 3-methoxytyramine (3-MT), h o m o v a n i 11 i c a c i d (HVA), 5 - H T and 5 - h y d r o x y i n d o l e a c e t i c acid ( 5 - H I A A ) by h i g h performance liquid chromatography with electrochemical detection (11). Statistical comparisons of the data were estimated by a m i x e d t w o f a c t o r ANOVA w i t h D u n n e t t t s test. Resu I t s Effects
o f MPTP a n d MPP+ on m o u s e
locomotor
activity
Fig. 1 shows the time-course of mouse locomotor activity caused by MPTP ( 1 0 , 20 a n d 30 m g / k g , i . p . ) o r MPP+ {5, 10 a n d 20 ug/mouse, i.c.v.). The single injection of MPTP significantly decreased locomotor activity after 1 0 - 4 0 m i n in a dose dependent manner. Total counts of locomotor activity for the first 30 m i n w e r e 5 3 . 4 + 1 1 ( m e a n s + S . E . M . ) (-44%) for a dose o f 10 m g / k g , 3 7 . 6 + 7 ( - 6 1 % ) f o r 20 m g / k g a n d 1 7 . 7 . 2 (-82%) for 30 m g / k g , compared to 96.2-12 for saline control. This decrease in locomotor activity disappeared 50 m i n a f t e r the injection (Fig. 1). T h e i . c . v , administration of a single dose o f MPP+ a l s o dose-dependently and s i g n i f i c a n t l y decreased the total counts of locomotor activity for 30 m i n : Ringer control, 64.5; -55.5% by 5 pg; -72.2% by 10 u g ; -93.5% by 20 u g / m o u s e o f MPP+. As s h o w n in Fig. 1, i n c o n t r a s t to MPTP, 5 0 - 7 0 m i n a f t e r t h e MPP+ i n j e c t i o n (10 an d 20 u g / m o u s e ) , the locomotor activity increased significantly compared to that during the first 1 0 - 4 0 m i n and a l s o to that of the controls.
1312
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Acute MPTP and MPP+ Effects
60
Vol. 40, No. 13, 1987
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Fig. 1. E f f e c t of single injection o f MPTP o r MPP+ o n m o u s e locomotor activity. I.eft panel shows locomotor activity (counts/10 min) during periods 10-120 rnin after different d o s e s o f MPTP ( i . p . ) {A) o r MPP+ ( i . c . v . ) (13) R i g h t p a n e l shows the total locomotor activity (counts/30 min). Vertical bars s h o w t h e S.E.M. * ; p < 0 . 0 5 , **;p<0.01 difference from Saline or Ringer control. This decreased locomotor activity by MPTP ( 1 0 a n d 20 mg/kg) was prevented by p r e t r e a t m e n t with nomifensine (5 mg/kg), but the prevention was incomplete in the case of the h i g h d o s e o f MPTP ( 3 0 m g / k g ) ( F i g . 2 ) . T h u s , t h e ED 50 v a l u e o f MPTP for reducing locomotor activity b y 50% a f t e r this pretreatment was higher <27 m g / k g (14.95-48.78 mg/kg, a confidence limit)> than that without nomifensine pretreatment <12.5 mg/kg (7.98-19.58 mg/kg, a confidence limit),. As s h o w n i n F i g . 2, t h i s prevention by n o m i f e n s i n e also occurred for MPP+ (5 a n d 10 u g / m o u s e , i.c.v.). However, a s f o u n d f o r MPTP, this effect was absent for the highest d o s e o f MPP+ ( 2 0 ug/mouse) used in this study. T h e ED 50 v a l u e s f o r t h e MPP+ alone and the nomifensine pretreatment were estimated t o be 4.6 ug/mouse (2.54-8.34 ug/mouse) and 11.7 ug/mouse (7.64-17.92 ug/mouse), respectively. Pretreatment with 5 mg/kg l-deprenyl, a selective MAO-B inhibitor (12), significantly and selectively prevented the decrease of locomotor activity induced by MPP÷ (20
Vol. 40, No. 13, 1987
Acute MPTP and MPP+ Effects
1313
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F i g . 2. E f f e c t of pretreatment with nomifensine (5 m g / k g ) o n mouse locomotor activity caused by MPTP o r MPP+. L e f t panel shows total locomotor activity (counts) during first 30 min of measurement after combined pretreatment with nomifensine plus MPTP ( i . p . ) (A) o r MPP+ ( i . c . v . ) (B). In this study, nomifensine (i.p.) was pretreated 1 hr prior to injection of MPTP ( 1 0 , 20 a n d 30 m g / k g ) or MPP+ ( 5 , 10 a n d 20 u g / m o u s e ) . Vertical b a r s show t h e S.E.M. * ; p < 0 . 0 5 , **;p<0.01 difference f r o m MPTP o r MPP+ alone. Right panel shows correlation b e t w e e n p o t e n c y o f MPTP o r MPP+ in reducing locomotor activity and pretreatment with nomifensine. Ordinate: percent reduction of locomotor activity. ug/mouse), as well as by MPTP ( 3 0 m g / k g ) , more than nomifensine pretreatment (Fig. 3A). In cntrast, pretreatment with a selective tv'~O-A i n h i b i t o r , clorgyline (12), failed to antagonize the decreased locomotor activity. 1"o s e l e c t i v e l y denervate DA n e u r o n s , mice were first pretreated w i t h DMI (5 mg/kg, i.p.) and t h e n 6 - O H D A ( 5 0 p g / m o u s e , i.c.v.) prior to MPP+ ( 2 0 u g / m o u s e , i.c.v.) injection. After 7 days of this combined pretreatment, mice were injected with the MPP+ and locomotor activity was d e t e r m i n e d . This combined pretreatment slightly, but significantly, prevented the decrease in locomotor activity induced by MPP+ (Fig. 3B). When haloperidol (0.25 mg/kg, i.p.) was given prior to MPP+ ( 2 0 u g / m o u s e , i.c.v.), the decreased locomotor activity caused b y MPP+ w a s s i g n i f i c a n t l y enhanced (p<0.01) (data not shown).
1314
Acute MPTP and MPP+ Effects
Depreny~,O.Mpp
f D~xqmyH-MPTP
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Vol. 40, No. 13, 1987
(s)
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Fig. 3. E f f e c t of pretreatment with l-deprenyl (5 m g / k g ) or desmethylimipramine (25 mg/kg) plus 6-hydroxydopamine (50 ~g/mouse) on locomotor activity caused by MPTP o r MPP+. Left and middle panels shows effect of pretreatment with ldeprenyl {A) o r by t h e c o m b i n a t i o n of desmethylimipramine plus 6-hydroxydopamine (B) on l o c o m o t o r activity c a u s e d by MPTP or MPP+ during first 30 mln of measurement, l-Deprenyl was pretreated 30 m i n p r i o r to injection o f MPTP ( 1 0 , 20 a n d 30 mg/kg, i . p . ) o r MPP+ ( 5 , 10 a n d 20 ~ g / m o u s e , i.c.v.). T h e MAO inhibitor did not significantly affect total counts f o r 30 m i n . Desmethyl imipramine (i.p.) was pretreated 30 m i n b e f o r e injection of 6-hydroxydopamine (i.c.v.), and 6-hydroxydopamine (i.c.v.) was pretreated 7 days before i.c.v, injection o f MPP+ (20 9g/mouse, i.c.v.). Vertical bars show S.E.M. *;p<0.05, **;p<0.01 difference f r o m MPTP o r MPP+. # ; p < 0 . 0 1 d i f f e r n c e from desmethylimipramine, 6-hydroxydopamine plus Ringer.
TABLE I Effect of MPTP (30 mg/kg, i.p.) and MPP+ (20 ug/mouse, i.c.v.) on the DA, 5-HT and their Metabolites in the Mouse Striatum (>g/gram of tissue) DA Control 7043.6+205 MPTP
DOPAC 2952.4+176
3-MT 1004.8+197
HVA 1400.6+109
5-HT
5-HIAA
503.3+25
624.5+23
9078.6+377** 1028.4+188"* 3254.2+265** 1172.2+102" 753.2+36* 434.4+23* DA
DOPAC
3-MT
Control 7731.2+221
2946.2+123
1152.7+143
MPP+
2258.4+110
2206.6+125"
8071.4+354"
HVA
5-HT
5-HIAA
1407.4+98
469.2+23
732.3+23
1314.2+]02
793.8+46* 495.4+15"
MPTP (i.p.) and MPP+ (i.c.v.) were given 30 min before mice were killed. The results are expressed as the mean+S.E.M, for i0 animals in each group.
*; p~9.O5, **; p
d i f ~ e r , : n c e from S a l i n ~ o r Ringer c o n t r o l .
Vol. 40, No. 13, 1987
Levels
o f DA a n d
Acute MPTP and MPP+ Effects
5-HT a n d
their
metabolites
1315
in striatum
As s h o w n i n T a b l e 1, 30 m g / k g MPTP p r o d u c e d a slight elevation o f DA l e v e l s ( a b o u t 30%) a n d a great elevation of 3-MT (about 230%) in the striatum 30 min after the injection, whereas, DOPAC a n d HVA, t h e m a j o r DA m e t a b o l i t e s , decreased by 6 5 . 2 % a n d 1 6 . 3 % , respectively. Striatal 5-HT levels increased 49.7% b u t 5 - H I A A , t h e m a j o r 5-HT m e t a b o l i t e , decreased a b o u t 30%. As f o u n d f o r MPTP, i . c . v , administration o f MPP+ (20 u g / m o u s e ) a l s o c a u s e d s i m i l a r effects on t h e l e v e l s of amines and their metabolites (DA: + 4 0 . 8 % , 3 - M T : + 9 1 . 4 % , DOPAC: - 2 3 . 3 % , HVA: - 6 . 6 % , 5-HT: +69.0% and 5-HIAA: -32.4%, respectively) (Table 1). Discussion The present data show that a single d o s e o f MPTP o r MPP+ injected into mice rapidly and dose-dependently decrease the early stage of locomotor activity. This result was in contrast to the report of an acute increase in the locomotor activity after MPTP i n j e c t i o n (13). The d i f f e r e n c e between the two acute locomotor effects found in earlier (13) and the present work may be due to the different MPTP d o s e s injected and/or the measurement period of the locomotion: in the earlier study (13), mice were injected with a higher dose (60 mg/kg) and the start of measurement of locomotion was later (30 min after injection) than doses (10-30 mg/kg) and the start of measurement in the present stuty. In the present study, we estimated total counts of locomotion o v e r 30 m i n , s i n c e t h i s period included a peak in behavioral c h a n g e s . T h e MPTP d o s e s used here did not cause any locomotor changes after this time. The unusual dose-response relationships between low and high d o s e s o f MPTP on l o c o m o t o r activity noted earlier (14) a n d i t s activation ( 1 3 ) m i g h t be a t l e a s t in part due to non-specific action, rather than action o n DA n e u r o n s , of high doses of MPTP. H o w e v e r , w h e n MPP+, instead o f MPTP, w a s g i v e n ( i . c . v . ) , a second phase of increased locomotor activity was detected following the first decrease p h a s e ( F i g . 1). At p r e s e n t , the mechanism for this increase in locomotion is unclear. MPP+ f o r m e d f r o m MPTP i s h i g h l y taken u p b y DA n e u r o n s (3,4) and this mechanism is responsible for selectivity of the neurotoxin. Nomi fensine, a DA u p t a k e inhibitor (15), antagonized this effect c a u s e d b y MPTP a s w e l l a s MPP+. T h i s protective effect o f DA u p t a k e inhibitors was also evident in decrease o f DA l e v e l s in mouse neostriatum after repeated d o s e s o f MPTP ( 1 6 - 1 8 ) . A s i m i l a r antagonism for the locomotor effect o f MPTP w a s a l s o f o u n d h e r e by p r e t r e a t m e n t with ldeprenyl but not with clorgyline. Taken together, the present results suggest that the behavioral change after MPTP m i g h t be predominantly mediated ?~two cruci~ step~ similar to its neurotoxicity: 1) o x i d a i n t o MPP+ MAO-B ( 1 , 2 ) a n d 2) a high uptake of MPP+ into striatal DA; n e u r o n s (3,4). Participation o f DA n e r v e t e r m i n a l s as t a r g e t s f o r i . c . v . MPP+ into the acute decreased locomotor activity a l s o s e e m s to be likely, s i n c e t h e MPP+ a t t h e i n i t i a l stage caused a locomotor
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Acute MPTP and MPP+ Effects
Vol. 40, No. 13, 1987
effect similar to that by MPTP, and both effects were prevented by pretreatment with nomifensine. The n e c e s s i t y o f DA n e u r o n s and/or postsynaptic DA r e c e p t o r ( s ) for locomotion induced b y MPP+ i s s u p p o r t e d by e v i d e n c e that the decreased locomotive effect was suppressed by selective chemical denervation o f DA n e u r o n s b y DMi f o l l o w e d by 6 - O H D A , but enhanced by a s i n g l e haloperidol injection, treatment that causes hypersensitivity of the receptor (19). The a b i l i t y of Ideprenyl pretreatment to prevent decreased locomotor activity by MPP+, as w e l l as t h a t by MPTP, i n d i c a t e that this selective MAO-B inhibitor has an additional protective effect beyond the inhibition of the formation o f MPP+ f r o m M P T P . T h i s w a s f o u n d f o r DA n e u r o n s of cultured rat embryonic midbrain (20). This effect of l-deprenyl on m o u s e locomotion might be i n p a r t d u e t o a c t i v a t i o n by t h i s MAO i n h i b i t o r o f DA and/or noradrenaline neurons by releasing DA a n d / o r noradrenaline at the nerve terminals through biotransformation of this inhibitor to d-methamphetamine (21). In t h e p r e s e n t study, b o t h MPTP a n d MPP+ s i m i l a r l y produced changes in levels of mouse striatal DA a n d 5 - H T , a s w e l l as their metabolites. Among these level changes, increase i n DA was slight, but there was a great increase in i t s O - m e t h y l a t e d metabolite, 3-MT ( a b o u t +230 a n d +92%, r e s p e c t i v e l y ) (Table I). The present results after MPTP w e r e c o m p a t i b l e with the acute changes i n DA ( 2 2 ) and 3-MT levels (23). On the other hand, both MPTP a n d MPP+ m a r k e d l y decreased DOPAC l e v e l s , but the change i n HVA w a s s l i g h t , indicating inhibition of striatal MAO a c t i v i t y and/or o f DA r e u p t a k e into the terminals. MAO l o c a l i z e s in n e u r o n s as w e l l as in o t h e r cells. Of t h e t w o forms o f MAO ( 1 2 ) , MAO-A p r e d o m i n a t e s in neurons a n d MAO-B predominates in cells other than neurons (see, e.g. 24). The DOPAC r e d u c t i o n a n d t h e 3-MT e l e v a t i o n m i g h t be r e s u l t s of both inhibition o f MAO-A i n D A - n e u r o n s , that is mainly responsible f o r DA o x i d a t i o n in rodent brains (25), and from DA release by MPP+ ( 2 6 ) , either injected o r f o r m e d f r o m MPTP. T h e f o r m e r possibility is consistent with earlier findings t h a t b o t h MPTP a n d MPP+ i n h i b i t MAO-A a n d -B a c t i v i t i e s with a higher MAO-A selectivity (27-29). Many reports indicate decreases i n DA, DOPAC a n d HVA, a f t e r repeated administration of MPTP ( 1 , 2 ) . However, acute effects o f MPTP on c h a n g e s in t h e s e a n i m a l s an d the metabolites greatly depended on t h e t i m e o f m e a s u r e m e n t after the injection (23). In t h e p r e s e n t study, after treatment with either MPTP o r MPP+, t h e DOPAC:DA r a t i o was low, but that of 3-MT:DA was very high. This confirms inhibition o f MAO a n d DA r e l e a s e , together with probable inhibition o f DA reuptake (3,4). Additional support for this hypothesis comes from the findings that, in v i t r o , MPP+ a s w e l l a s MPTP r e l e a s e d DA f r o m the striatum (30). These actions consequently result in increased 3-MT a n d d e c r e a s e d DOPAC a n d HVA. Although many possibilities have been pointed out for the initiation of degeneration of nigrostriatal DA n e u r o n s , the precise mechanism(s) is not yet clear. The corpus striatum as w e l l as t h e s u b s t a n t i a nigra receive S-FIT n e u r o n p r o j e c t i o n s originating in the midbrain raphe nuclei (31). Many reports have indicated interaction between DA a n d 5 - H T i n t h e b r a i n (e.g. 32), but the exact role of 5-HT in DA-mediated behavior is not well understood. A 5-HT elevation, but a reduction of its metabolite, S-HIAA after a single injection o f MPTP o r
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Acute HPTP and HPP+ E f f e c t s
1317
MPP+ m a y e l i c i t an effect similar to that f o u n d i n DA n e u r o n s . Together with the finding t h a t b o t h DA a n d 5 - H T n e u r o n s highly t a k e u p MPP+ ( 1 7 ) , o u r p r e s e n t locomotor effects induced by MPTP and MPP+ might be related, at least in part, to a c h a n g e i n 5-HT m e t a b o l i s m . References
1. J.W. LANGSTON, T r e n d s in Neurosci. 8 79-83 (1985). 2. J.W. LAN~STON, T r e n d s in P h a r m a c o l . Sci. 6 375-378 (1985). 3. J . A . J A V I T C H a n d S . H . SNYDER, E u r . J . P h a r m a c o l . 106 4 5 5 456 ( 1 9 8 4 ) . 4. K. C H I B A , A . J . TREVOR a n d N. C A S T ~ L I Jr., Biochem. Biophys. R e s . Commun. 128 1 2 2 8 - 1 2 3 2 ( 1 9 8 5 ) . 5. J . W . LANGSTON, I . I R W I N , E . B . LAAK3STON a n d L.S. FORN~, Science 225 1 4 8 0 - 1 4 8 2 { 1 9 8 4 ) . 6. R.W. FULLER a n d S . K . HEIV~ICK-LUECKE, L i f e S c i . 37 1 0 8 9 I096 (1985). 7. R . E . H E I K K I L A , A. HESS a n d R.C. DUVOISIN, S c i e n c e 224 1 4 5 1 1453 ( 1 9 8 4 ) . 8. H. HALLMAN, L. OLSON a n d G. JONSSON, E u r . J. P h a r m a c o l . 97 133-136 (1984). 9. L.R. STERANKA, L.N. P O L I T E , K.W. PERRY a n d R.W. FULLER, R e s . Commun. S u b . A b u s e , 4 3 1 5 - 3 1 9 ( 1 9 8 5 ) . I 0 . T. H A Y A S H I , K. OHASHI a n d S. T ~ O R O , J a p a n . J. P h a r m a c o ] . 30 9 3 - I 0 0 (1980). I I . K. I S H I K A W A a n d J . L . MCGAUC.~, J. C h r o m a t o g r . 164 4 4 1 - 4 4 9 (1982). 12. H. K I N E M U C H I , C . J . FOWLER a n d K.F. TIPTON, M o n o a m i n e Oxidase and Disease, p.53, Academic Press, New Y o r k (1982). 13. E. P I L E B L A D , H. N I S S B R A N D T a n d A. CARLSSON, J. N e u r . T r a s m . 60 1 9 9 - 2 0 3 ( 1 9 8 4 ) . 14. A. SAHGAL, L . S . ANDREWS, J . A . B I G G I N S , J.M. CANDY, J.A. EDWARDSON, A . B . KEITH; J~D. TURNER a n d C. WRIGHT, N e u r o s c i . Lett. 48 1 7 9 - 1 8 4 ( 1 9 8 4 ) . 15. J. ~ I S T O , E u r . J. P h a r m a c o l . 42 1 0 1 - 1 0 6 (1977). 16. E. P I L E B L A D a n d A. C_ARLSSON, N e u r o p h a r m a c o l . 7 689-692 (1985). 17 R . E . H E I K K I L A , S . K . YOUNGSTER, L. MANZINO, F . S . CABBAT a n d R . C . DUVOISIN, J . N e u r o c b e m . 44 3 1 0 - 3 1 3 ( 1 9 8 5 ) . 18 E. SUNDSTROM a n d G. JONSSON, E u r . J. P h a r m a c o l . If0 293-299 (1985). 19 M . P . MARTRES, J. C O S T E N T I N , M. ~ Y , H. MARCAIS, P. PROTAIS a n d J . C . SCHWRTZ, B r a i n R e s . 136 3 1 9 - 3 3 7 ( 1 9 7 7 ) . 20 C. M Y T I L I N E O U a n d G. COHEN, J. N e u r o c h e m . 45 1 9 5 1 - 1 9 5 3 (1985). 21 M. DA PRADA, H.H. KELLER, R. KETTLER, R. SCHAFNER, M. P I E R I , W . P . BURKARD, A. KORN a n d W . E . HAEFELY, M o n o a m i n e Oxidase: Bassic and Clinical Frontiers, p.183, Excerpta Medica, Amsterdam (1982). 2 2 . T . L . PERRY, V.W. YONG, K. J O N E S , R.A. WALL, R.M. CLAVIER, J . G . FOULKS a n d J . M . WRIGHT, N e u r o s c i . Lett. 58 3 2 1 - 3 2 6 ¢1985). 2 3 . E. P I L E B L A D , B. FORNSTEDT, D. CLARK a n d A. CARLSSON, J. Pharm. Pbarmacol. 37 7 0 7 - 7 1 2 ( 1 9 8 5 ) .
1318
Acute MPTP and MPP+ Effects
Vol. 40, No. 13, 1987
24. L° ORELAND, Y. A R A I , A. STENSTROM a n d C . J . FOWLER, M o d e r n Problems of Pharma¢opsychiatry, v o l . 1 9 , p°246, K a r g e r , Basel (19831. 25. T. KATO, B. DONG, Ko I S H I I a n d H. KINEMUCHI, J. N e u r o , ¢ h e m . 46 1 2 7 7 - 1 2 8 2 11986). 26. C . J . SCHMIDT, L.A. MATSUDA a n d J.W. G I B E , E u r . J . Pharma¢ol. 103 2 5 5 - 2 6 0 (19841. 27. H. KINEMUCHI, Y. ARAI a n d Y. T O Y O ~ H I ~ , N e ~ r ~ ¢ ~ . Le~. ~8 195-200 (1985). 28. H. KINEMUCHI, Y. A R ~ I , ¥ . TOYOSHIMA, T. TAdI~CqlD:a ~ d K. KISARA, N e u r o p s y c h o v h ~ r m a c m , l.ogy o~ The T r a c e ~ i ~ e s , p.16f, The Humana. P r e s s , N ~ . ) e ~ ~ ~8S)!, 29. R.W. FULLER $m,d S . K . EE/~t~CIK-LUEC'IKE, J . P h a r m a ¢ o l . ex~. T h e r . 2 3 2 ~9@-7~E ~ 8 5 ) . ~ 0 . G.D. CH/~d~ a ~ d V . D . R / ~ d ~ I R E Z , B r a i n R e s . 3 6 8 1 3 4 - 1 4 0 r i g a 6 ) . 3'r. N d ~ N P ~ . , M.. ~,__~ARC~A-~g..INI3~, G.W. ~ a n d D. ECCL--, F u r . J. P h a r m a c o l . 57 2 9 5 - 3 0 5 119791. 3 2 . R. SAMANFN a n d S. G A R A T F I N I , L i f e S c i . 17 1 2 0 1 - 1 2 1 0 1 1 9 7 5 ) .
Pergamon Journal
BEHAVIORAL AND BIOCHEMICAL CHANGES FOLLOWING ACUTE ADMINISTRATION OF ~ T P AND MPP+ Takeshi Tadano, Nobunori Satoh, Ichiro Sakuma, Toshihiro Hatsumura, Kensuke Kisara, Yuichiro Arai* and IIiroyasu Kinemuchi* Department
of Pharmacology, Tohoku College of Pharmacy, 4-4-1, Komatsushima, Sendai 983, *Department of Pharmacology, School of Medicine, Showa University, 1-5-8, Hatanodai, Shinagawa-Ku, Tokyo 142, Japan (Received in final form January 5, 1987)
S urnma ry.
The acute effects of 1 -methy 1 -4-phenyl - I ,2,3,6tetrahydropyridine (MPTP) a n d l - m e t h y l - 4 - p h e n y l p y r i d i n i u m ion (IvlPP÷) on m o u s e l o c o m o t o r activity and s t r i a t a l dopamine (DA) and 5-hydroxytryptamine (5-HT) levels were investigated. A single dose of either MPTP ( 1 0 - 3 0 m g / k g , i . p . ) o r MPP÷ ( 5 - 2 0 ug/mouse, i.c.v.) decreased locomotor activity 10-40 min after injection: this locomotor effect was significantly suppressed by e i t h e r pretreatment with nomifensine or l-deprenyl alone, or by the combinat ion of desmethyl imipramine and 6hydroxydopamine. Pretreatment with clorgyline did not suppress this behavior, and a single dose of haloperidol enhanced the effect. The s t r i a t a l levels of DA, 3-methoxytyramine an d 5-HT increased in parallel with the decrease in locomotor activity caused by MPTP o r MPP+. In c o n t r a s t , levels of 3,4dihydroxyphenylacet ic acid, homovani I 1 ic acid and 5hydroxyindoleacetic acid were decreased by i n j e c t i o n of either MPTP o r MPP÷. P o s s i b l e mechanism(s) of the behavioral and biochemical changes caused by t h e a c u t e actions o f MPTP a n d MPP+ with respect to their neurotoxic effects on the nigrostriatal DA s y s t e m a r e d i s c u s s e d . MPTP produces irreversible neurological damage to nigrostraital dopamine (DA) n e u r o n s in primates, including human, similar to the well-established changes in patients with Parkinson)s disease (for review, s e e 1,2). The n e u r o t o x i c effects o f MPTP a r e d e r i v e d from its oxidized product, MPP+, which is formed in a reaction catalyzed by t h e 3 f o r m of monoamine oxidase (ivlAO, EC 1 . 4 . 3 . 4 ) (MAO-B), via the intermediate, i - m e t hy 1 - 4 - p h e n y I - 2 , 3 - d i h y d r o p y r i d i n i um i o n (MPDP+) ( 1 , 2 ) . The high uptake of MPP+ by DA neurons is another crucial step in the expression of its neurotoxicity (3,4). Thus, MPTP i s n o t intrinsically neurotoxic and its
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1310
Acute MPTP and MPP+ Effects
Vol. 40, No. 13, 1987
neurotoxicity depends mainly on its biotransformation to the active metabolite MPP+, s i n c e p r e t r e a t m e n t with selective MAO-B inhibitors, which decrease the production o f MPP+, p r e v e n t s the neurotoxicity (5,6). Although little information is available on its precise mechanism, reports have been accumulating that indicate a species difference in susceptibility and behavioral response t o MPTP, w i t h p r i m a t e s being the most sensitive. Repeated higher doses and longer durat ion of treatment of rodents with MPTP c a u s e t h e DA neurotoxicity, as judged by marked reductions in locomotor activity, striatal DA a n d i t s m a j o r m e t a b o l i t e levels, 3,4dihydroxyphenylacetic a c i d (DOPAC) a n d h o m o v a n i l l i c a c i d (HVA) (1,2,7-9). These results suggest that the action o f MPTP o n small laboratory animals might provide a useful model for studying the etiology and the processes of neuronal degeneration in patients with Parkinson's disease. This animal model may also offer an opportunity to screen antiparkinsonian drugs and, experimentally, to determine the side-effects o f l o n g t e r m DA p r e c u r s o r and agonist therapy. To our knowledge, the acute effects of the toxic metabolite, MPP+, on the behavioral system a n d DA m e t a b o l i s m in the mouse nigrostriatal system have not yet been fully established, despite many neurotoxicity reports after repeated MIaTP injections (1,2). Thus, possible relations between these acute changes and the neurotoxicity o f MPTP a n d MPP+ t o w a r d s DA neurons need to be resolved to clarify the initial step(s) of the toxicity and its resulting degeneration o f DA n e u r o n s . The present study was undertaken to elucidate the acute behavioral effects caused by either compound injected systemically or intracerebroventricularly (i.c.v.), and to clarify parallelism between the behavioral changes and monoamine metabolism in the mouse striatum. Materials Chemicals
and Methods
and Drugs
Clorgyline HCI a n d I - d e p r e n y l i-t(31 w e r e k i n d l y provided by May & Baker Ltd., Dagenham, England, and Dr. J. Knol l, Semmelweis University of Medicine, Budapest, Hungary, respectively. MPTP HCI w a s p u r c h a s e d from Aldrich Chemical Co., Milwaukee, Wis., U.S.A.; MPP+ i o d i d e from Research Biochemicals Inc. Ohio, U.S.A.; and nomi fensine HCI from Hoechst, Frankfurt, West Germany. Desmethylimipramine HCI f r o m Ciba-Geigy, Basel, Switzerland and 6-hydroxydopamine HCl f r o m Sigma Chemicals, St. Louis, MO, U . S . A . T h e o t h e r chemicals used were of the highest grade commercially available. The MPP+ solution was administered i.c.v, in a volume of l0 ul/mouse, since, in general, charged compounds cannot easily pass through the blood-brain-barrier. Other solutions used in this study were intraperitoneally (i.p.) injected. All drugs used for i.c.v, injection were dissolved in Ringer's solution. For i.p. injection, they were dissolved in saline. The d o s e s of all compounds are expressed as their respective salts. Pretreatment with nomifensine, l-deprenyl or clorgyline was performed l hr prior t o MPTP o r MPP+ i n j e c t i o n ; that with haloperidol w a s 30 m i n , a n d d e s m e t h y l i m i p r a m i n e (DMI) p l u s 6 -
Vol. 40, No. 13, 1987
hydroxydopamine injection. Animals
Acute MPTP and MPP+ Effects
(6-OHDA)
and m e a s u r e m e n t
of
was
performed
locomotor
7 days
1311
prior
to
the
activity
In t h i s study, male albino m i c e (ddY s t r a i n ) weighing 22-25 g were used. M i c e w e r e h o u s e d in s u s p e n d e d p l a s t i c cages with free access to food and water in a colony room that was illuminated from 10:00 to 18:00 hr and maintained at 22~2;C. The locomotor activity (ambulation) of mice was determined by the tilting cage method reported previously (10). A handmade apparatus assembled from two round plastic, commercially available basins ( 2 5 cm i n d i a m e t e r ) put one upon the other was used to measure the ambulatory activity. Any s l i g h t tilt of the activity c a g e c a u s e d by m o v e m e n t o f t h e a n i m a l s was detected by three micro-switches attached on t h e b r i m o f t h e o u t e r basin, and t h e s e activated an e l e c t r o m a g n e t i c counter. In t h e p r e s e n t study, 10 a c t i v i t y cages of same type were used simultaneously. Recording the number of counts indidicating locomotor activity started to immediately upon placing mice treated with a drug(s) or vehicle in the cages, and was accumulated in successive 10 min intervals throughout the observation periods. Determinations
of
amines
and
their
metabolites
in
striatum
In o r d e r to determine the levels o f DA a n d 5 - H T a n d t h e i r metabolites, mice were sacrificed 30 m i n a f t e r the injection of either MPTP o r MPP+. The m o u s e s t r i a t a were then dissected out on an ice-cold glass plate, homogenized and, after deproteinization, assayed f o r DA, 3 , 4 - d i h y d r o x y p h e n y l a c e t i c acid (DOPAC), 3-methoxytyramine (3-MT), h o m o v a n i 11 i c a c i d (HVA), 5 - H T and 5 - h y d r o x y i n d o l e a c e t i c acid ( 5 - H I A A ) by h i g h performance liquid chromatography with electrochemical detection (11). Statistical comparisons of the data were estimated by a m i x e d t w o f a c t o r ANOVA w i t h D u n n e t t t s test. Resu I t s Effects
o f MPTP a n d MPP+ on m o u s e
locomotor
activity
Fig. 1 shows the time-course of mouse locomotor activity caused by MPTP ( 1 0 , 20 a n d 30 m g / k g , i . p . ) o r MPP+ {5, 10 a n d 20 ug/mouse, i.c.v.). The single injection of MPTP significantly decreased locomotor activity after 1 0 - 4 0 m i n in a dose dependent manner. Total counts of locomotor activity for the first 30 m i n w e r e 5 3 . 4 + 1 1 ( m e a n s + S . E . M . ) (-44%) for a dose o f 10 m g / k g , 3 7 . 6 + 7 ( - 6 1 % ) f o r 20 m g / k g a n d 1 7 . 7 . 2 (-82%) for 30 m g / k g , compared to 96.2-12 for saline control. This decrease in locomotor activity disappeared 50 m i n a f t e r the injection (Fig. 1). T h e i . c . v , administration of a single dose o f MPP+ a l s o dose-dependently and s i g n i f i c a n t l y decreased the total counts of locomotor activity for 30 m i n : Ringer control, 64.5; -55.5% by 5 pg; -72.2% by 10 u g ; -93.5% by 20 u g / m o u s e o f MPP+. As s h o w n in Fig. 1, i n c o n t r a s t to MPTP, 5 0 - 7 0 m i n a f t e r t h e MPP+ i n j e c t i o n (10 an d 20 u g / m o u s e ) , the locomotor activity increased significantly compared to that during the first 1 0 - 4 0 m i n and a l s o to that of the controls.
1312
E
Acute MPTP and MPP+ Effects
60
Vol. 40, No. 13, 1987
100 (A) MPTP
¢~ I \ ~
~ 40
e lOmg/kg ~" 20mglkg * 30mg/kg ~ saline ~
-¢ 80 E o ~o 60
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40 ~
20 20
.
. o
~
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I
t
10 20
30
t
30
I
40
t
50
L
J
L
i
•
t
J
0
60 70 80 90 100 110 120 (rain)
(B)
MPP.~ 5.0ug -- lOng - 20pg
/ k I \
20
60
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40
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o
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Fig. 1. E f f e c t of single injection o f MPTP o r MPP+ o n m o u s e locomotor activity. I.eft panel shows locomotor activity (counts/10 min) during periods 10-120 rnin after different d o s e s o f MPTP ( i . p . ) {A) o r MPP+ ( i . c . v . ) (13) R i g h t p a n e l shows the total locomotor activity (counts/30 min). Vertical bars s h o w t h e S.E.M. * ; p < 0 . 0 5 , **;p<0.01 difference from Saline or Ringer control. This decreased locomotor activity by MPTP ( 1 0 a n d 20 mg/kg) was prevented by p r e t r e a t m e n t with nomifensine (5 mg/kg), but the prevention was incomplete in the case of the h i g h d o s e o f MPTP ( 3 0 m g / k g ) ( F i g . 2 ) . T h u s , t h e ED 50 v a l u e o f MPTP for reducing locomotor activity b y 50% a f t e r this pretreatment was higher <27 m g / k g (14.95-48.78 mg/kg, a confidence limit)> than that without nomifensine pretreatment <12.5 mg/kg (7.98-19.58 mg/kg, a confidence limit),. As s h o w n i n F i g . 2, t h i s prevention by n o m i f e n s i n e also occurred for MPP+ (5 a n d 10 u g / m o u s e , i.c.v.). However, a s f o u n d f o r MPTP, this effect was absent for the highest d o s e o f MPP+ ( 2 0 ug/mouse) used in this study. T h e ED 50 v a l u e s f o r t h e MPP+ alone and the nomifensine pretreatment were estimated t o be 4.6 ug/mouse (2.54-8.34 ug/mouse) and 11.7 ug/mouse (7.64-17.92 ug/mouse), respectively. Pretreatment with 5 mg/kg l-deprenyl, a selective MAO-B inhibitor (12), significantly and selectively prevented the decrease of locomotor activity induced by MPP÷ (20
Vol. 40, No. 13, 1987
Acute MPTP and MPP+ Effects
1313
Neml~r,anwt.~w1~ 120
I'
.
e0
7O 5O
|
3O
10 0
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10
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30 mQ/kg
N ~ te ns l ne , I -M P P
~ ;5
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20
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F i g . 2. E f f e c t of pretreatment with nomifensine (5 m g / k g ) o n mouse locomotor activity caused by MPTP o r MPP+. L e f t panel shows total locomotor activity (counts) during first 30 min of measurement after combined pretreatment with nomifensine plus MPTP ( i . p . ) (A) o r MPP+ ( i . c . v . ) (B). In this study, nomifensine (i.p.) was pretreated 1 hr prior to injection of MPTP ( 1 0 , 20 a n d 30 m g / k g ) or MPP+ ( 5 , 10 a n d 20 u g / m o u s e ) . Vertical b a r s show t h e S.E.M. * ; p < 0 . 0 5 , **;p<0.01 difference f r o m MPTP o r MPP+ alone. Right panel shows correlation b e t w e e n p o t e n c y o f MPTP o r MPP+ in reducing locomotor activity and pretreatment with nomifensine. Ordinate: percent reduction of locomotor activity. ug/mouse), as well as by MPTP ( 3 0 m g / k g ) , more than nomifensine pretreatment (Fig. 3A). In cntrast, pretreatment with a selective tv'~O-A i n h i b i t o r , clorgyline (12), failed to antagonize the decreased locomotor activity. 1"o s e l e c t i v e l y denervate DA n e u r o n s , mice were first pretreated w i t h DMI (5 mg/kg, i.p.) and t h e n 6 - O H D A ( 5 0 p g / m o u s e , i.c.v.) prior to MPP+ ( 2 0 u g / m o u s e , i.c.v.) injection. After 7 days of this combined pretreatment, mice were injected with the MPP+ and locomotor activity was d e t e r m i n e d . This combined pretreatment slightly, but significantly, prevented the decrease in locomotor activity induced by MPP+ (Fig. 3B). When haloperidol (0.25 mg/kg, i.p.) was given prior to MPP+ ( 2 0 u g / m o u s e , i.c.v.), the decreased locomotor activity caused b y MPP+ w a s s i g n i f i c a n t l y enhanced (p<0.01) (data not shown).
1314
Acute MPTP and MPP+ Effects
Depreny~,O.Mpp
f D~xqmyH-MPTP
i
Vol. 40, No. 13, 1987
(s)
17
60
l
a
~ 8o
50
i "I
I
3~
~ 4o
4o
i. iio
~o
±
a
15
~ 2o 0
9
10
oi
,11
Fig. 3. E f f e c t of pretreatment with l-deprenyl (5 m g / k g ) or desmethylimipramine (25 mg/kg) plus 6-hydroxydopamine (50 ~g/mouse) on locomotor activity caused by MPTP o r MPP+. Left and middle panels shows effect of pretreatment with ldeprenyl {A) o r by t h e c o m b i n a t i o n of desmethylimipramine plus 6-hydroxydopamine (B) on l o c o m o t o r activity c a u s e d by MPTP or MPP+ during first 30 mln of measurement, l-Deprenyl was pretreated 30 m i n p r i o r to injection o f MPTP ( 1 0 , 20 a n d 30 mg/kg, i . p . ) o r MPP+ ( 5 , 10 a n d 20 ~ g / m o u s e , i.c.v.). T h e MAO inhibitor did not significantly affect total counts f o r 30 m i n . Desmethyl imipramine (i.p.) was pretreated 30 m i n b e f o r e injection of 6-hydroxydopamine (i.c.v.), and 6-hydroxydopamine (i.c.v.) was pretreated 7 days before i.c.v, injection o f MPP+ (20 9g/mouse, i.c.v.). Vertical bars show S.E.M. *;p<0.05, **;p<0.01 difference f r o m MPTP o r MPP+. # ; p < 0 . 0 1 d i f f e r n c e from desmethylimipramine, 6-hydroxydopamine plus Ringer.
TABLE I Effect of MPTP (30 mg/kg, i.p.) and MPP+ (20 ug/mouse, i.c.v.) on the DA, 5-HT and their Metabolites in the Mouse Striatum (>g/gram of tissue) DA Control 7043.6+205 MPTP
DOPAC 2952.4+176
3-MT 1004.8+197
HVA 1400.6+109
5-HT
5-HIAA
503.3+25
624.5+23
9078.6+377** 1028.4+188"* 3254.2+265** 1172.2+102" 753.2+36* 434.4+23* DA
DOPAC
3-MT
Control 7731.2+221
2946.2+123
1152.7+143
MPP+
2258.4+110
2206.6+125"
8071.4+354"
HVA
5-HT
5-HIAA
1407.4+98
469.2+23
732.3+23
1314.2+]02
793.8+46* 495.4+15"
MPTP (i.p.) and MPP+ (i.c.v.) were given 30 min before mice were killed. The results are expressed as the mean+S.E.M, for i0 animals in each group.
*; p~9.O5, **; p
d i f ~ e r , : n c e from S a l i n ~ o r Ringer c o n t r o l .
Vol. 40, No. 13, 1987
Levels
o f DA a n d
Acute MPTP and MPP+ Effects
5-HT a n d
their
metabolites
1315
in striatum
As s h o w n i n T a b l e 1, 30 m g / k g MPTP p r o d u c e d a slight elevation o f DA l e v e l s ( a b o u t 30%) a n d a great elevation of 3-MT (about 230%) in the striatum 30 min after the injection, whereas, DOPAC a n d HVA, t h e m a j o r DA m e t a b o l i t e s , decreased by 6 5 . 2 % a n d 1 6 . 3 % , respectively. Striatal 5-HT levels increased 49.7% b u t 5 - H I A A , t h e m a j o r 5-HT m e t a b o l i t e , decreased a b o u t 30%. As f o u n d f o r MPTP, i . c . v , administration o f MPP+ (20 u g / m o u s e ) a l s o c a u s e d s i m i l a r effects on t h e l e v e l s of amines and their metabolites (DA: + 4 0 . 8 % , 3 - M T : + 9 1 . 4 % , DOPAC: - 2 3 . 3 % , HVA: - 6 . 6 % , 5-HT: +69.0% and 5-HIAA: -32.4%, respectively) (Table 1). Discussion The present data show that a single d o s e o f MPTP o r MPP+ injected into mice rapidly and dose-dependently decrease the early stage of locomotor activity. This result was in contrast to the report of an acute increase in the locomotor activity after MPTP i n j e c t i o n (13). The d i f f e r e n c e between the two acute locomotor effects found in earlier (13) and the present work may be due to the different MPTP d o s e s injected and/or the measurement period of the locomotion: in the earlier study (13), mice were injected with a higher dose (60 mg/kg) and the start of measurement of locomotion was later (30 min after injection) than doses (10-30 mg/kg) and the start of measurement in the present stuty. In the present study, we estimated total counts of locomotion o v e r 30 m i n , s i n c e t h i s period included a peak in behavioral c h a n g e s . T h e MPTP d o s e s used here did not cause any locomotor changes after this time. The unusual dose-response relationships between low and high d o s e s o f MPTP on l o c o m o t o r activity noted earlier (14) a n d i t s activation ( 1 3 ) m i g h t be a t l e a s t in part due to non-specific action, rather than action o n DA n e u r o n s , of high doses of MPTP. H o w e v e r , w h e n MPP+, instead o f MPTP, w a s g i v e n ( i . c . v . ) , a second phase of increased locomotor activity was detected following the first decrease p h a s e ( F i g . 1). At p r e s e n t , the mechanism for this increase in locomotion is unclear. MPP+ f o r m e d f r o m MPTP i s h i g h l y taken u p b y DA n e u r o n s (3,4) and this mechanism is responsible for selectivity of the neurotoxin. Nomi fensine, a DA u p t a k e inhibitor (15), antagonized this effect c a u s e d b y MPTP a s w e l l a s MPP+. T h i s protective effect o f DA u p t a k e inhibitors was also evident in decrease o f DA l e v e l s in mouse neostriatum after repeated d o s e s o f MPTP ( 1 6 - 1 8 ) . A s i m i l a r antagonism for the locomotor effect o f MPTP w a s a l s o f o u n d h e r e by p r e t r e a t m e n t with ldeprenyl but not with clorgyline. Taken together, the present results suggest that the behavioral change after MPTP m i g h t be predominantly mediated ?~two cruci~ step~ similar to its neurotoxicity: 1) o x i d a i n t o MPP+ MAO-B ( 1 , 2 ) a n d 2) a high uptake of MPP+ into striatal DA; n e u r o n s (3,4). Participation o f DA n e r v e t e r m i n a l s as t a r g e t s f o r i . c . v . MPP+ into the acute decreased locomotor activity a l s o s e e m s to be likely, s i n c e t h e MPP+ a t t h e i n i t i a l stage caused a locomotor
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Vol. 40, No. 13, 1987
effect similar to that by MPTP, and both effects were prevented by pretreatment with nomifensine. The n e c e s s i t y o f DA n e u r o n s and/or postsynaptic DA r e c e p t o r ( s ) for locomotion induced b y MPP+ i s s u p p o r t e d by e v i d e n c e that the decreased locomotive effect was suppressed by selective chemical denervation o f DA n e u r o n s b y DMi f o l l o w e d by 6 - O H D A , but enhanced by a s i n g l e haloperidol injection, treatment that causes hypersensitivity of the receptor (19). The a b i l i t y of Ideprenyl pretreatment to prevent decreased locomotor activity by MPP+, as w e l l as t h a t by MPTP, i n d i c a t e that this selective MAO-B inhibitor has an additional protective effect beyond the inhibition of the formation o f MPP+ f r o m M P T P . T h i s w a s f o u n d f o r DA n e u r o n s of cultured rat embryonic midbrain (20). This effect of l-deprenyl on m o u s e locomotion might be i n p a r t d u e t o a c t i v a t i o n by t h i s MAO i n h i b i t o r o f DA and/or noradrenaline neurons by releasing DA a n d / o r noradrenaline at the nerve terminals through biotransformation of this inhibitor to d-methamphetamine (21). In t h e p r e s e n t study, b o t h MPTP a n d MPP+ s i m i l a r l y produced changes in levels of mouse striatal DA a n d 5 - H T , a s w e l l as their metabolites. Among these level changes, increase i n DA was slight, but there was a great increase in i t s O - m e t h y l a t e d metabolite, 3-MT ( a b o u t +230 a n d +92%, r e s p e c t i v e l y ) (Table I). The present results after MPTP w e r e c o m p a t i b l e with the acute changes i n DA ( 2 2 ) and 3-MT levels (23). On the other hand, both MPTP a n d MPP+ m a r k e d l y decreased DOPAC l e v e l s , but the change i n HVA w a s s l i g h t , indicating inhibition of striatal MAO a c t i v i t y and/or o f DA r e u p t a k e into the terminals. MAO l o c a l i z e s in n e u r o n s as w e l l as in o t h e r cells. Of t h e t w o forms o f MAO ( 1 2 ) , MAO-A p r e d o m i n a t e s in neurons a n d MAO-B predominates in cells other than neurons (see, e.g. 24). The DOPAC r e d u c t i o n a n d t h e 3-MT e l e v a t i o n m i g h t be r e s u l t s of both inhibition o f MAO-A i n D A - n e u r o n s , that is mainly responsible f o r DA o x i d a t i o n in rodent brains (25), and from DA release by MPP+ ( 2 6 ) , either injected o r f o r m e d f r o m MPTP. T h e f o r m e r possibility is consistent with earlier findings t h a t b o t h MPTP a n d MPP+ i n h i b i t MAO-A a n d -B a c t i v i t i e s with a higher MAO-A selectivity (27-29). Many reports indicate decreases i n DA, DOPAC a n d HVA, a f t e r repeated administration of MPTP ( 1 , 2 ) . However, acute effects o f MPTP on c h a n g e s in t h e s e a n i m a l s an d the metabolites greatly depended on t h e t i m e o f m e a s u r e m e n t after the injection (23). In t h e p r e s e n t study, after treatment with either MPTP o r MPP+, t h e DOPAC:DA r a t i o was low, but that of 3-MT:DA was very high. This confirms inhibition o f MAO a n d DA r e l e a s e , together with probable inhibition o f DA reuptake (3,4). Additional support for this hypothesis comes from the findings that, in v i t r o , MPP+ a s w e l l a s MPTP r e l e a s e d DA f r o m the striatum (30). These actions consequently result in increased 3-MT a n d d e c r e a s e d DOPAC a n d HVA. Although many possibilities have been pointed out for the initiation of degeneration of nigrostriatal DA n e u r o n s , the precise mechanism(s) is not yet clear. The corpus striatum as w e l l as t h e s u b s t a n t i a nigra receive S-FIT n e u r o n p r o j e c t i o n s originating in the midbrain raphe nuclei (31). Many reports have indicated interaction between DA a n d 5 - H T i n t h e b r a i n (e.g. 32), but the exact role of 5-HT in DA-mediated behavior is not well understood. A 5-HT elevation, but a reduction of its metabolite, S-HIAA after a single injection o f MPTP o r
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MPP+ m a y e l i c i t an effect similar to that f o u n d i n DA n e u r o n s . Together with the finding t h a t b o t h DA a n d 5 - H T n e u r o n s highly t a k e u p MPP+ ( 1 7 ) , o u r p r e s e n t locomotor effects induced by MPTP and MPP+ might be related, at least in part, to a c h a n g e i n 5-HT m e t a b o l i s m . References
1. J.W. LANGSTON, T r e n d s in Neurosci. 8 79-83 (1985). 2. J.W. LAN~STON, T r e n d s in P h a r m a c o l . Sci. 6 375-378 (1985). 3. J . A . J A V I T C H a n d S . H . SNYDER, E u r . J . P h a r m a c o l . 106 4 5 5 456 ( 1 9 8 4 ) . 4. K. C H I B A , A . J . TREVOR a n d N. C A S T ~ L I Jr., Biochem. Biophys. R e s . Commun. 128 1 2 2 8 - 1 2 3 2 ( 1 9 8 5 ) . 5. J . W . LANGSTON, I . I R W I N , E . B . LAAK3STON a n d L.S. FORN~, Science 225 1 4 8 0 - 1 4 8 2 { 1 9 8 4 ) . 6. R.W. FULLER a n d S . K . HEIV~ICK-LUECKE, L i f e S c i . 37 1 0 8 9 I096 (1985). 7. R . E . H E I K K I L A , A. HESS a n d R.C. DUVOISIN, S c i e n c e 224 1 4 5 1 1453 ( 1 9 8 4 ) . 8. H. HALLMAN, L. OLSON a n d G. JONSSON, E u r . J. P h a r m a c o l . 97 133-136 (1984). 9. L.R. STERANKA, L.N. P O L I T E , K.W. PERRY a n d R.W. FULLER, R e s . Commun. S u b . A b u s e , 4 3 1 5 - 3 1 9 ( 1 9 8 5 ) . I 0 . T. H A Y A S H I , K. OHASHI a n d S. T ~ O R O , J a p a n . J. P h a r m a c o ] . 30 9 3 - I 0 0 (1980). I I . K. I S H I K A W A a n d J . L . MCGAUC.~, J. C h r o m a t o g r . 164 4 4 1 - 4 4 9 (1982). 12. H. K I N E M U C H I , C . J . FOWLER a n d K.F. TIPTON, M o n o a m i n e Oxidase and Disease, p.53, Academic Press, New Y o r k (1982). 13. E. P I L E B L A D , H. N I S S B R A N D T a n d A. CARLSSON, J. N e u r . T r a s m . 60 1 9 9 - 2 0 3 ( 1 9 8 4 ) . 14. A. SAHGAL, L . S . ANDREWS, J . A . B I G G I N S , J.M. CANDY, J.A. EDWARDSON, A . B . KEITH; J~D. TURNER a n d C. WRIGHT, N e u r o s c i . Lett. 48 1 7 9 - 1 8 4 ( 1 9 8 4 ) . 15. J. ~ I S T O , E u r . J. P h a r m a c o l . 42 1 0 1 - 1 0 6 (1977). 16. E. P I L E B L A D a n d A. C_ARLSSON, N e u r o p h a r m a c o l . 7 689-692 (1985). 17 R . E . H E I K K I L A , S . K . YOUNGSTER, L. MANZINO, F . S . CABBAT a n d R . C . DUVOISIN, J . N e u r o c b e m . 44 3 1 0 - 3 1 3 ( 1 9 8 5 ) . 18 E. SUNDSTROM a n d G. JONSSON, E u r . J. P h a r m a c o l . If0 293-299 (1985). 19 M . P . MARTRES, J. C O S T E N T I N , M. ~ Y , H. MARCAIS, P. PROTAIS a n d J . C . SCHWRTZ, B r a i n R e s . 136 3 1 9 - 3 3 7 ( 1 9 7 7 ) . 20 C. M Y T I L I N E O U a n d G. COHEN, J. N e u r o c h e m . 45 1 9 5 1 - 1 9 5 3 (1985). 21 M. DA PRADA, H.H. KELLER, R. KETTLER, R. SCHAFNER, M. P I E R I , W . P . BURKARD, A. KORN a n d W . E . HAEFELY, M o n o a m i n e Oxidase: Bassic and Clinical Frontiers, p.183, Excerpta Medica, Amsterdam (1982). 2 2 . T . L . PERRY, V.W. YONG, K. J O N E S , R.A. WALL, R.M. CLAVIER, J . G . FOULKS a n d J . M . WRIGHT, N e u r o s c i . Lett. 58 3 2 1 - 3 2 6 ¢1985). 2 3 . E. P I L E B L A D , B. FORNSTEDT, D. CLARK a n d A. CARLSSON, J. Pharm. Pbarmacol. 37 7 0 7 - 7 1 2 ( 1 9 8 5 ) .
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24. L° ORELAND, Y. A R A I , A. STENSTROM a n d C . J . FOWLER, M o d e r n Problems of Pharma¢opsychiatry, v o l . 1 9 , p°246, K a r g e r , Basel (19831. 25. T. KATO, B. DONG, Ko I S H I I a n d H. KINEMUCHI, J. N e u r o , ¢ h e m . 46 1 2 7 7 - 1 2 8 2 11986). 26. C . J . SCHMIDT, L.A. MATSUDA a n d J.W. G I B E , E u r . J . Pharma¢ol. 103 2 5 5 - 2 6 0 (19841. 27. H. KINEMUCHI, Y. ARAI a n d Y. T O Y O ~ H I ~ , N e ~ r ~ ¢ ~ . Le~. ~8 195-200 (1985). 28. H. KINEMUCHI, Y. A R ~ I , ¥ . TOYOSHIMA, T. TAdI~CqlD:a ~ d K. KISARA, N e u r o p s y c h o v h ~ r m a c m , l.ogy o~ The T r a c e ~ i ~ e s , p.16f, The Humana. P r e s s , N ~ . ) e ~ ~ ~8S)!, 29. R.W. FULLER $m,d S . K . EE/~t~CIK-LUEC'IKE, J . P h a r m a ¢ o l . ex~. T h e r . 2 3 2 ~9@-7~E ~ 8 5 ) . ~ 0 . G.D. CH/~d~ a ~ d V . D . R / ~ d ~ I R E Z , B r a i n R e s . 3 6 8 1 3 4 - 1 4 0 r i g a 6 ) . 3'r. N d ~ N P ~ . , M.. ~,__~ARC~A-~g..INI3~, G.W. ~ a n d D. ECCL--, F u r . J. P h a r m a c o l . 57 2 9 5 - 3 0 5 119791. 3 2 . R. SAMANFN a n d S. G A R A T F I N I , L i f e S c i . 17 1 2 0 1 - 1 2 1 0 1 1 9 7 5 ) .