MPTP potentiates 3-nitropropionic acid-induced striatal damage in mice: reference to striatonigral degeneration

Experimental Neurology 185 (2004) 47 – 62 www.elsevier.com/locate/yexnr

MPTP potentiates 3-nitropropionic acid-induced striatal damage in mice: reference to striatonigral degeneration P.O. Fernagut, 1 E. Diguet,1 B. Bioulac, and F. Tison * Physiologie et Physiopathologie de la Signalisation Cellulaire, UMR-CNRS 5543, Universite´ Victor Segalen Bordeaux2, 33076 Bordeaux cedex, France Received 6 May 2003; revised 16 September 2003; accepted 26 September 2003

Abstract Striatonigral degeneration (SND) is a parkinsonian disorder due to the combined degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and striatal output neurons. The aims of this study were to explore (1) the behavioral and histopathological consequences of combined MPTP plus 3-nitropropionic acid (3-NP) intoxication in C57/Bl6 mice and (2) its ability to reproduce the neuropathological hallmarks of SND. 3-NP was administered i.p. every 12 h (total dose = 450 mg/kg in 9 days) and MPTP i.p. at 10 mg/(kg day) (total dose = 90 mg/kg in 9 days). Four groups of mice (n = 10) were compared: control, 3-NP alone, MPTP alone, MPTP + 3-NP. Mice intoxicated with 3-NP and MPTP + 3-NP developed motor symptoms, including hindlimb dystonia and clasping, truncal dystonia and impaired balance adjustments. The severity of motor disorder was worse and lasted longer in MPTP + 3-NP-treated mice compared to 3-NP alone, MPTP alone and controls. 3-NP and MPTP + 3-NP-treated mice also displayed altered gait patterns, impaired motor performance on the pole test, rotarod and traversing a beam tasks and activity parameters. Several of these sensorimotor deficits were also more severe and lasted longer in MPTP + 3-NP-treated mice. Histology demonstrated increased neuronal loss along with astrocytic activation (glial fibrillary acid protein, GFAP) and a higher incidence of circumscribed striatal lateral lesions in MPTP + 3-NP-treated mice compared to 3-NP. Neuronal loss and astrocytic activation were increased in the lateral part of the striatum in 3-NP-intoxicated mice while observed both in the medial and lateral part in MPTP + 3-NP-intoxicated mice. There was also a significant loss of SNc dopaminergic neurons and striatal terminals, similar to that in MPTP-treated mice. Altogether, these results suggest that MPTP potentiates striatal damage and behavioral impairments induced by 3-NP intoxication in mice and constitutes a useful model of the motor disorder and its histopathological correlates in SND. D 2003 Elsevier Inc. All rights reserved. Keywords: MPTP; 3-NP; Striatonigral degeneration; Motor behavior; Astrocytic activation; Mouse

Introduction The substantia nigra pars compacta (SNc) dopaminergic neurons and the striatum are prone to neurodegeneration of Parkinson’s disease (PD) and Huntington’s disease (HD), respectively (Beal, 2001; Brouillet et al., 1999). They display particular sensitivity to the various forms of energy compromise that can lead to neuronal loss and secondary excitotoxic damage: the striatum is sensitive to hypoxia, ischemia or inhibition of mitochondrial complex II (Gould and Gustine, 1982); and SNc displays an almost specific

* Corresponding author. Laboratoire de Neurophysiologie, Universite´ Victor Segalen Bordeaux2, UMR-CNRS 5543, Bat 2a, Zone Nord, 146 rue Le´o Saignat, 33076 Bordeaux cedex, France. Fax: +33-05-56-90-14-21. E-mail address: [email protected] (F. Tison). 1 These two authors contributed equally to this work. 0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2003.09.014

sensitivity to the inhibition of mitochondrial complex I by MPTP (Nicklas et al., 1985; Scotcher et al., 1990). A unique situation of combined degeneration of the SNc and the striatum occurs in striatonigral degeneration (SND), the main clinical presentation of the sporadic adult-onset human neurodegenerative disease known as multiple system atrophy (MSA). Parkinsonism unresponsive to dopaminergic drugs is the core feature of SND or MSA (Tison et al., 1995) and is due to such a combined degeneration with a distribution of neuronal loss roughly similar to that observed in HD and PD (Fearnley and Lees, 1990). Striatal neuronal loss affects both striatal output pathways and is assumed to be partly responsible for the dopaminergic unresponsiveness of the parkinsonian syndrome of SND or MSA (Tison et al., 1995). However, a specific cytopathology distributed in the cortico –subcortical motor loops is also found in this disease and consists in cytoplasmic or nuclear glial or neuronal inclusions containing alpha-syn-

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uclein aggregates (Lantos, 1998; Papp et al., 1989). Previous attempts to develop experimental equivalents of SND, either in the rat following a ‘‘double-toxin – doublelesion’’ approach by using 6-OHDA and quinolinic acid (QA) stereotaxic injections (Ghorayeb et al., 2001; Scherfler et al., 2000; Wenning et al., 1996) or in mice by systemic sequential 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 3-nitropropionic acid (3-NP) (Stefanova et al., 2003), have encountered the drawback of ‘‘negative’’ interactions between nigral and striatal degeneration. Indeed, prior lesioning of the SNc induced a reduction in the subsequent striatal sensitivity to excitotoxicity (kainate, QA) or mitochondrial complex II inhibition (3-NP, malonate), as previously reported (Buisson et al., 1991; Chapman et al., 1989; Maragos et al., 1998). Several lines of evidence suggest that dopamine can modulate neuronal loss in the nigrostriatal pathway (Jakel and Maragos, 2000). For example, we have previously shown that elevated dopaminergic transmission as observed in dopamine transporter (DAT) knockout mice increases the effects of 3-NP (Fernagut et al., 2002a). On the other hand, prior striatal degeneration induced by either QA in the rat or 3-NP in the mouse partially protects against subsequent nigral degeneration through as yet unidentified mechanisms (Scherfler et al., 2000; Stefanova et al., 2003; Venero et al., 1995). The complex interactions induced during combined nigral and striatal degeneration thus remain to be fully characterized. The aim of the present study was to assess for the first time the effects of simultaneous systemic administration of MPTP with 3NP on the motor behavioral and histopathological outcome based on our previous experience using 3-NP-induced motor impairment and striatal damage correlates in mice (Fernagut et al., 2002c). The issue was whether simultaneous MPTP + 3-NP systemic intoxication would avoid the negative interactions in the nigrostriatal degenerative processes, and whether it would provide sensorimotor deficits and histopathological correlates suitable for a systemic mouse model of SND or MSA.

MPTP plus 3-NP intoxication MPTP and 3-NP were administered during a 9-day intoxication period. Mice were weighed before each injection. MPTP (Sigma, France) was dissolved in 0.9% NaCl and injected daily (at noon, between the two 3-NP injections) with a constant dose of 10 mg/kg (total cumulated dose: 90 mg/kg in 9 days) using a 200 Al Hamilton syringe. 3-NP (Sigma, France) was prepared in 0.1 M phosphatebuffered saline (PBS), final pH = 7.4 as previously described (Fernagut et al., 2002c). The solution was filtered (Millipore, 0.22 Am) and kept at +4jC until use. Mice received two daily i.p. injections 12 h apart (8:00 a.m. and 8:00 p.m.) according to the following regimen: 10 mg/kg  4, 20 mg/kg  4, 30 mg/kg  4, 40 mg/kg  4 and 50 mg/ kg  1 (total cumulated dose: 450 mg/kg in 9 days). The concentration of the solution was adjusted to keep the injected volume stable (250 Al using a 500 Al Hamilton syringe) as doses increased. Clinical semiquantitative motor disorder assessment Motor symptoms were rated before each injection during intoxication and daily until the end of the experiment (day 30) using a previously validated motor scale including the following five items: global locomotor activity, hindlimb clasping, hindlimb dystonia, truncal dystonia and balance adjustment following postural challenge (Fernagut et al., 2002c). Each item was rated on a three-level scale of severity (absent, slight to moderate, severe) to obtain a total score ranging from 0 to 10. Standardized motor and sensorimotor integration tests The following motor and sensorimotor integration tests were performed the week before intoxication (baseline), then during the first, second and third week following the end of intoxication. Rotarod

Experimental procedures Animals Forty C57Bl/6 adult male mice aged 16 weeks (Iffa Credo, France) were used in accordance with European Community Council Directives (86/609/EEC) for animal care and use. All efforts were made to minimize the number of animals used and their suffering. Animals were raised and housed under a 12-h light cycle with food and water available ad libitum. Four experimental groups composed of randomly allocated littermates were constituted: control (PBS), 3-NP, MPTP and MPTP + 3-NP. Animals were handled and habituated to the experimenter for 1 week before any behavioral assessment.

Animals were placed on a stationary rod and were trained to stay on it as it rotated at 5 rpm. Usually, four to six training sessions on consecutive days (each constituted by a maximum of 10 trials) were needed to achieve the maximal performance established at 180 s (Fernagut et al., 2002a,c). The mean time spent on the rotating rod during a 10-trial session was kept as the variable. Rotarod testing was performed at baseline then at week 1 (day 5), 2 (day 12) and 3 (day 18) post-intoxication. Pole test The pole test was performed according to Matsuura et al. (1997) with minor modifications (Fernagut et al., 2002c).

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The mouse was placed head upward on the top of a vertical wooden rough-surfaced pole (diameter: 1 cm, height: 50 cm). Each mouse was habituated to the apparatus on the day prior to testing, then allowed to descend five times. The total time until the mouse reached the floor with its four paws was recorded (T-total) as well as the time needed for the mouse to turn completely head downward (T-turn). For each session of five descents, the best performance was kept for the T-turn and T-total. If the mouse was unable to turn completely downwards, fell or slipped down, the default value of 120 s was taken into account. The pole test was performed at baseline, then at week 1 (day 7), 2 (day 11) and 3 (day 19) post-intoxication. Stride lengths Stride lengths were analyzed with the method adapted from D’Hooge et al. (1999) by wetting the animals’ forepaws then hindpaws with black pencil ink and letting them trot on a strip of paper (4.5 cm wide, 42 cm long) down a brightly lit runway towards a dark goal box (Fernagut et al., 2002b). Forelimb stride lengths were first measured for all the animals, then hindlimb stride lengths on a new strip of paper, once the inked forelimb paws had dried. Mice were habituated to the experimenter and to the apparatus (total of three runs) for 3 days before starting the experiments. The measurements were made after three training runs. Stride length was measured manually as the distance between two paw prints. The three longest stride lengths (corresponding to maximal velocity) were measured from each run. Paw prints made at the beginning (7 cm) and end (7 cm) of the run were excluded because of velocity changes. Runs in which the mice made stops or obvious deceleration observed by the experimenter were excluded from analysis. Stride lengths were measured at baseline and at day 7 postintoxication. Measurements were made for each limb, then the mean length for both forelimbs and for both hindlimbs were used as variables. Traversing a beam Motor coordination and balance were assessed with the method adapted from Carter et al. (1999) by measuring the ability of the mice to traverse a narrow beam to reach a dark goal box. The beams consisted of two different strips of wood (each measuring 50 cm long, one was 1.6 cm and the other 0.9 cm square cross-section) placed horizontally 50 cm above the floor. During training, three daily sessions of three trials (nine crossings) were performed using the 1.6 cm square large beam. Mice were then tested at baseline, then at week 1 (day 5), 2 (day 8) and 3 (day 19) post-intoxication using the 0.9 cm square beam. Mice were allowed to perform three consecutive trials. The number of sideslips was recorded on each trial and the mean number of sideslips during a three-trial session was kept as the variable.

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Open field spontaneous activity Mice were placed in the center of an open field (44  44  32 cm) under weak red light (40 W) illumination at the same time of the day (4 p.m.). On each side of the open field, two frames placed at 1and 5-cm height with 16 photocell beams per side ensured movement detection. The computer defined grid lines that divided the open field into four ‘‘side and corner regions’’, each line being 10 cm from the wall and the ‘‘central region’’ measuring 576 cm2 (Actitrack, Panlab, S.L, Barcelona, Spain) (Dulawa et al., 1999). The tracking, traveled distance, mean velocity (Vmean), maximal velocity (Vmax), number of rearings, time spent and number of visits to the central compartment were calculated during a single 5-min session (Gahtan et al., 1998). To avoid a reduction in locomotor activity due to habituation to the open field, open field spontaneous activity was recorded only at baseline and at week 1 post-intoxication. Histopathology Tissue processing Animals were perfused at week 4 post-intoxication under pentobarbital overdose with 25 ml 0.9% NaCl, followed by 200 ml of 4% paraformaldehyde (PFA) pH = 7.4. Brains were quickly removed and stored for 24 h in 4% PFA at 4jC, then in a 10% sucrose/0.1 M PBS pH 7.4 solution until they sank. Brains were frozen in isopentane at 40jC and stored at 80jC. Four adjacent 10-Am-thick cryostat sections were collected every 100 Am throughout the striatum and 20 Am free-floating sections collected in between in PBS-sodium azide 0.2% for immunohistochemistry. Twenty-micrometer-thick free-floating sections were collected at three rostrocaudal levels of the SNc (Franklin and Paxinos, 1997): rostral (bregma = 2.92 mm), midnigral (bregma = 3.16 mm) and caudal (bregma = 3.80 mm) in PBS-sodium azide 0.2% for tyrosine hydroxylase (TH) immunohistochemistry. Immunohistochemistry Tyrosine hydroxylase (TH) immunohistochemistry (rabbit polyclonal antibody, Jacques Boy, France, 1:2000 overnight, diluted in 0.1 M PBS pH = 7.4, 0.1% Triton X-100, 2% BSAc, 4jC), NeuN immunohistochemistry (mouse monoclonal, Chemicon Intl. Inc., Temecula, CA, diluted 1:1000, 48 h diluted in 0.1 M PBS pH = 7.4, 0.1% Triton X100, 2% BSAc, at room temperature), calbindin-D-28 K immunohistochemistry (mouse monoclonal antibody, Sigma, France, diluted 1:1000 overnight, 0.1 M PBS pH = 7.4, 0.1% Triton X-100, 2% BSAc, 4jC) and glial fibrillary acid protein analysis (GFAP) (DAKO, rabbit anti-GFAP, 1: 400, overnight, 4jC) were performed as previously described (Fernagut et al., 2002a,c). Immunoreactions were revealed

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using the Vectastain ABC Elite System (Vector, Burlingham, CA). Morphometric analysis and cell counts The general procedure is summarized and illustrated in Fig. 1.

The extent of the lesion within the striatum was mapped on cresyl violet Nissl-stained sections as described elsewhere (Fernagut et al., 2002c). For striatal surface analysis, four anatomical levels of the striatum were defined from rostral to caudal (level 1 = bregma +1.18 mm, 2 = +0.98 mm, 3 = +0.26 mm, 4 = 0.22 mm), according to the mouse brain atlas of Franklin and Paxinos’s (1997). The outer

Fig. 1. Schematic representation of the design of the histopathological study illustrating the determination of striatal surface (A) and an illustrative Nissl-stained striatal section (B, scale bar = 150 Am). Neurons are recognized by their round, oval or polygonal shape and at least one emanating process (arrow: medium spiny neuron; double arrow: large inter-neuron), and astroglial cells by their small, round and hyperchromatic nuclei (*). Counting levels in the SNc are illustrated on tyrosine-hydroxylase (TH)-stained sections: rostral (C, bregma = 2.92 mm), medial (D, bregma = 3.16 mm) and caudal (E, bregma = 3.80 mm). Scale bar = 500 Am. cp: cerebral peduncle, ml: medial longitudinal fasciculus, 3n: third cranial nerve.

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border of the striatum was defined by the lateral ventricle medially, the corpus callosum dorsally and laterally, and ventrally by a line drawn between the two anterior commissures. Anatomical landmarks (aspect, size and situation of the anterior commissures, corpus callosum, septum, lateral ventricles, striatum, nucleus accumbens-Nac- and pallidum) were used to ensure that levels studied were similar within and between groups as previously proposed (Fernagut et al., 2002a,c). The estimated striatal surface was the mean on each side of two adjacent 10-Am-thick sections randomly picked within the anatomical levels of interest. Additionally, the total volume of the striatum was calculated according to the principle of Cavalieri (volume = s1d1 + s2d2,. . . + sndn, s = surface, d = distance between two sections) (Rosen and Harry, 1990). Neuronal profiles were then counted on striatal sections selected at the anterior midstriatal level 2 (bregma = +0.98 mm), a striatal level where we have previously shown maximal striatal damage following 3-NP subacute intoxication (Fernagut et al., 2002c). To assess the differential effect of 3-NP and MPTP + 3-NP at this striatal level, the striatum was divided into four equal subregions (DL: dorsolateral, VL: ventrolateral, DM: dorsomedial, VM: ventromedial) and cell counting was performed using 0.2-mm2 areas (20 objective) randomly placed within each of the four quadrants. The estimated neuronal numerical density (Nv) was the mean count of ‘‘top’’ profiles entirely within the counting area on two couples of 10-Am-thick adjacent sections, in the determined dissector volume (Vdis) (Nv = A‘‘tops’’/AVdis, Vdis = area  section height). The estimated absolute number of neurons (N) per striatal section and per animal was the mean numerical density in each striatal section according to the striatal section volume (Vref) (N = Nv  Vdis) (Coggeshall, 1992). Neurons were identified as the largest cells in the field, with typical morphological features including a round, oval or polygonal shape, depending on their nature (striatal output or interneurons), and at least one emanating process, while the microglial cell profiles were distinguished by their small, round and hyperchromatic nuclei (Ghorayeb et al., 2001; Roberts et al., 1993) (Fig. 1B). Neurons were also additionally identified by their NeuN immunoreactivity on 20-Am free-floating sections. Medium spiny output neurons were identified on 20-Am-thick free-floating sections by their calbindin-D-28 K immunoreactivity. Counts were only performed on cresyl violet sections. The extent of substantia pars compacta (SNc) cell loss was estimated on adjacent 20-Am-thick free-floating THstained sections at the three rostrocaudal levels mentioned above. All positive neurons lying dorsally to a line drawn between the tips of the cerebellar peduncle and outside a vertical line passing through the accessory optical tract (mid-nigral level) or the medial tip of the medial lemniscus (caudal and rostral levels) were counted on each side and on three adjacent sections. The estimated number of neurons was those seen in a ‘‘pick’’ reference section and not in an

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adjacent 20-Am ‘‘look-up’’ section (Coggeshall, 1992). The final estimation of the number of neuronal profiles per animal was the mean of neurons sampled on both sides of the section and on three different couples of adjacent sections. Cell loss in the SNc was also indirectly estimated by the relative TH-immunostaining intensity between the dorsal striatum and the adjacent sensorimotor cortex determined as optical densities and by subtracting the nonspecific staining from the total staining (relative striatal TH OD = (striatal TH OD non-specific striatal OD)/(cortical TH OD non-specific cortical OD), following a method adapted from Georgievska et al. (2002) and Robinson et al. (2003). This was performed on 20-Am free-floating THstained striatal section at level 2 (bregma = +0.98 mm), handled in a single experiment with simultaneous incubation and revelation times for all four groups using a striatal control section with primary antibody omitted (nonspecific staining). Relative ODs were measured in the right and left dorsolateral striatum, a major area of input of SNc dopaminergic fibers. All the surface, volume measurements density measures and cell counts were performed by an investigator blind for the mouse intoxication paradigm using computer-assisted image analysis (Biocom Visioscan and Densirag v2.0, Les Ullis, France). Succinate dehydrogenase (SDH) histochemistry 3-NP-induced SDH inhibition was assessed according to Brouillet et al. (1998). Twenty-four 16-week-old male C57Bl/6 mice received either a PBS, 3-NP (50 mg/kg) or 3-NP (50 mg/kg) immediately followed by MPTP (10 mg/ kg) injection (n = 6 in each group) and were sacrificed by cervical dislocation 2 h after, a delay sufficient to observe any stable inhibition of SDH (Brouillet et al., 1998). Briefly, 20-Am-thick sections collected throughout the striatum were first incubated (10 min) in 0.1 M PBS (37jC), then washed in PBS and immersed in the reaction medium (0.3 mM nitroblue tetrazolinium, Sigma-France, 0.05 mM succinic acid, Sigma-France, in 0.05 M phosphate buffer at 37jC) for 30 min. To determine nonspecific staining, adjacent sections were incubated in the same reaction medium without succinic acid. After incubation, sections were post-fixed (10 min) in 2% PFA and washed in deionized water. Mean SDH activity was determined as optical densities by subtracting nonspecific staining from total staining by using an image analysis system (Biocom, Densirag v2.0, Les Ullis, France). Data analysis All data are expressed as mean values F SEM. For all statistical tests performed, a probability level of 5% was considered significant. For behavioral motor tests, comparisons with baseline performances within groups were performed using either a paired t test or a Wilcoxon matched

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paired test, depending on the distribution of the parameter studied (normality test). For behavioral and histopathological comparisons between groups, one-way ANOVA with the post hoc Bonferroni method for multiple comparisons was performed.

Results Lethality Two out of 10 (20%) 3-NP and 4 out of 10 (40%) MPTP + 3-NP-intoxicated mice died during the intoxication, while there was no lethality in MPTP and saline-treated mice. In all cases, death occurred after the last injection at 50 mg/kg. There was a significant difference in lethality between groups (Chi-square global = 8.63, 3 degrees of freedom, P < 0.01). This was due to a statistical difference between PBS and MPTP groups compared with the 3-NP and MPTP + 3-NP groups (Fisher exact test, P = 0.01). However, there was no demonstrable statistical difference between 3-NP alone and MPTP + 3-NP groups. Motor symptoms monitoring The time course of the motor symptoms in 3-NP and MPTP + 3-NP groups using the motor symptom scale

demonstrated that no significant motor or behavioral signs appeared until the dose of 40 mg/kg of 3-NP was reached. The earliest motor sign observed was motor slowness, then transient followed by permanent hindlimb dystonia, then hindlimb clasping, followed by truncal dystonia and finally global postural abnormalities with balance impairment (Fig. 2). However, the delay of onset and severity of motor symptoms were somewhat different between 3-NP and MPTP + 3-NP groups, because the motor score was significantly different from baseline after the second injection at 40 mg/kg in MPTP + 3NP-intoxicated mice and after the fourth injection at 40 mg/kg in 3-NP-intoxicated mice. Motor impairment worsened during the incremental increase in intoxication from 40 to 50 mg/kg and was maximal on the day following the end of intoxication (Fig. 3A). The motor symptoms as rated using the scale decreased progressively during the post-intoxication period but were significantly superior in MPTP + 3-NP-treated mice compared to 3-NP alone on day 1 (6.7 F 0.28 vs. 4.7 F 0.67, P < 0.05), day 4 (4.28 F 0.42 vs. 3 F 0.42, P < 0.05), day 11 (4.14 F 0.26 vs. 3 F 0.42, P < 0.05) and day 18 (2.85 F 0.26 vs. 1.87 F 0.29, P < 0.05) post-intoxication. In MPTP + 3-NP-intoxicated mice, hindlimb clasping was occasionally associated with trunk rotation and forelimb clasping (Fig. 2B). At the end of the post-intoxication period (day 21 post-intoxication), 3-

Fig. 2. Motor and postural abnormalities in MPTP + 3-NP-intoxicated mice. (A) Truncal dystonia (arrow), (B) Hindlimb + forelimb clasping (arrows). Note the rotation of the trunk when the mouse is hanged by the tail. (C) Hindlimb dystonia (arrow). (D) Disruption of postural adjustments (sideslip, arrow).

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Fig. 3. (A) Changes in motor behavioral score during and after MPTP, 3-NP or MPTP + 3-NP intoxication. Statistical significance: #P < 0.05, ##P < 0.01 (MPTP + 3-NP compared to 3-NP alone). During the intoxication phase, the motor scores of MPTP + 3-NP or 3-NP-intoxicated mice became significantly different from baseline condition as from days 7 to 8, respectively. Mice were rated before each injection during intoxication, then daily during the postintoxication period. (B, C, D, E) Motor and sensorimotor integration tests performed at baseline and after intoxication. Statistical significance: *P < 0.05, **P < 0.01 (vs. baseline), #P < 0.05 (3-NP vs. MPTP + 3-NP). (B) Mean time spent on the rotarod (5 rpm). (C) Time spent to turn downward on the pole test (T-Turn). (D) Difference between fore- and hindlimb stride length. (E) Mean number of sideslips on the beam traversing task.

NP and MPTP + 3-NP-intoxicated mice displayed intermittent hindlimb clasping and slight-to-moderate increased hindlimb space, thus providing a global score between 1 and 2 but without alteration of gait or general locomotor activity.

Motor and sensorimotor tests Rotarod At the end of the training procedure, there was no significant difference in the mean time spent on the rotating

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Table 1 Open field locomotor activity parameters and stride length in PBS, MPTP, 3-NP and MPTP + 3-NP-intoxicated mice performed at baseline and at week 1 postintoxication, featuring maximal velocity (Vmax), mean velocity (Vmean), distance traveled, number of rearings, hindlimb stride length and fore – hindlimb difference PBS Vmax (cm/s)

Baseline Post-intoxication Baseline Post-intoxication Baseline Post-intoxication Baseline Post-intoxication

Vmean (cm/s) Distance (cm) Rearings

35.46 32.72 8.95 7.07 2682 2121 33.5 27.6

3-NP F F F F F F F F

1.2 1.0 0.4 0.3 125.6 86.9 3.9 1.9

MPTP

34.43 F 17.03 F 7.82 F 2.11 F 2347 F 634.8 F 29.9 F 2.88 F

2.1 0.9** 0.6 0.4** 167.3 124.7** 2.5 0.8***

32.73 28.70 7.46 5.42 2242 1625 32.9 26.4

F F F F F F F F

MPTP + 3-NP 1.9 1.7* 0.6 0.4*** 181.1 120.8*** 3.6 2.8*

32.18 F 0.9 11.41 F 1.4*** 7.39 F 0.4 1.41 F 0.2*** 2215 F 111.5 424.5 F 74.8*** 31.55 F 2.8 0.14 F 0.1***

* P < 0.05 vs. baseline condition. ** P < 0.01 vs. baseline condition. *** P < 0.0001 vs. baseline condition.

10.22 s, T-Total: 74.3 F 21.54 s at day 11, P < 0.05). There was no significant difference compared to baseline concerning either the T-Turn or T-Total at week 3 postintoxication in 3-NP-intoxicated mice, while this difference persisted in MPTP + 3-NP-intoxicated mice (Fig. 3C).

rod among the four groups. During the post-intoxication period, there was a significant reduction in the mean time spent on the rod in 3-NP and MPTP + 3-NP-intoxicated mice ( P < 0.05, compared to baseline performance levels and also compared to PBS or MPTP-injected mice). Motor impairment on the rotarod at week 1 post-intoxication was significantly greater in MPTP + 3-NP-intoxicated mice compared to 3-NP alone (31.73 F 6.96 vs. 125.8 F 21.6 s, P < 0.01). At day 18 post-intoxication, there was no significant difference among the four groups concerning the mean time spent on the rotating rod, although the level of performance of 3NP intoxicated mice was slightly but significantly lower in 3NP-intoxicated mice compared to their baseline performance level, which was maximal (137.6 F 15.43 vs. 180 F 0 s) (Fig. 3B).

Stride lengths Before intoxication, there was no significant difference among the four groups concerning forelimb and hindlimb stride length. At day 7 post-intoxication, there was a significant reduction in hindlimb stride length in 3-NP intoxicated mice ( 5.2%, P < 0.05) and in MPTP + 3NP-intoxicated mice ( 8.3%, P < 0.01). The difference between forelimb and hindlimb stride length at day 7 postintoxication was significantly increased compared to baseline in 3-NP (0.62 F 0.18 vs. 0.15 F 0.08 cm, P < 0.05), MPTP (0.59 F 0.2 vs. 0.2 F 0.08 cm, P < 0.05) and 3-NP + MPTP-intoxicated mice (0.55 F 0.24 vs. 0.09 F 0.08 cm, P < 0.05) (Fig. 3D).

Pole test Before intoxication, there was no significant difference either in T-Turn or T-Total among the four groups. At day 7 post-intoxication, there was a significant increase in T-Turn and T-Total in 3-NP and MPTP + 3-NP-intoxicated mice (T-Turn: 39.6 F 9.9 and 44.6 F 9.9 s, respectively, P < 0.01; T-Total: 79.1 F 19.9 and 88 F 20.6 s, respectively, P < 0.01). On subsequent testing days, 3-NP and MPTP + 3-NPintoxicated mice improved gradually (T-Turn: 38.3 F

Traversing a beam Baseline performance levels were not different among the four groups. At day 5 post-intoxication, there was a significant decrease in the number of sideslips in control mice (0.06 F 0.04 vs. 0.8 F 0.16, P < 0.01) and a significant increase in 3-NP (3.9 F 1.1 vs. 1.1 F 0.2, P <

Table 2 Histopathology Control 3

Striatal volume (mm ) Striatal neuronal density (neurons/mm2) Absolute number of neurons GFAP + cells (per mm2) Absolute number of GFAP + cells

4.48 1632 5432 89.22 261

F F F F F

3-NP 0.07 41 210 11.3 32

3.63 1622 3816 404.6 1111

MPTP F F F F F

0.18** 64 307** 20.2** 64**

3.97 1369 4745 370 1046

F F F F F

3-NP + MPTP 0.06* 72.6* 222 50.2** 124**

3.69 F 1184 F 3510 F 624.4 F 1698 F

0.21** 82.8* 336**,*** 34.8**,***,**** 67**,***,****

Striatal volume in mm3 calculated between bregma +0.98 and +0.22 mm, striatal neuronal density and absolute number of striatal neurons determined at bregma +0.98 mm. Density and absolute number of GFAP-positive astrocytes at bregma +0.98 mm. * P < 0.05 vs. control. ** P < 0.001 vs. control. *** P < 0.001 vs. MPTP. **** P < 0.001 vs. 3-NP.

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0.05) and MPTP + 3-NP-intoxicated mice (7.2 F 1.3 vs. 0.7 F 0.5, P < 0.01) compared to their respective baseline performance levels. The number of sideslips in MPTP + 3NP-intoxicated mice was also significantly different from that of 3-NP-intoxicated mice ( P < 0.05). On subsequent testing days (days 8 and 19 post-intoxication), the number of sideslips in 3-NP intoxicated mice was not different from baseline (2.4 F 0.74 and 1.75 F 0.52), while it was still significantly elevated in MPTP + 3-NP-intoxicated mice (4.7 F 1.4 and 3.5 F 0.9, P < 0.05) and reduced in control mice (0.13 F 0.07 and 0.16 F 0.05, P < 0.05) (Fig. 3E).

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Open field activity Open field activity demonstrated a significant reduction in the number of rearings on the first week postintoxication compared to baseline in MPTP, 3-NP and MPTP + 3-NP-intoxicated mice. In MPTP + 3-NP-intoxicated mice, rearings were also significantly reduced compared to 3-NP alone ( P < 0.01). Distance traveled was also reduced in 3-NP ( P < 0.01), MPTP ( P < 0.0001) and MPTP + 3-NP-treated mice ( P < 0.0001). Accordingly, mean velocity (Vmean) and maximal velocity (Vmax) were reduced in 3-NP, MPTP and MPTP + 3-NPintoxicated mice (Table 1).

Fig. 4. (A, B, C, D) Cresyl violet staining (scale bar = 500 Am) and (E, F, G, H) NeuN staining (scale bar = 200 Am) at bregma + 0.98 mm. Arrowhead indicates circumscribed striatal lesion in 3-NP and MPTP + 3-NP-intoxicated mice with staining artifacts. Note the striatal atrophy in the MPTP + 3-NP striatum with dorsolateral neuronal loss (H). (I, J, K, L) GFAP-immunoreactive astrocytes in the striatum. Note the potentiation of astrocytic activation in the MPTP + 3-NP intoxicated mice (H). Scale bar = 60 Am. (M, N, O, P) Calbindin-D-28K immunoreactive cell bodies in the dorsolateral striatum illustrating the loss of medium spiny neurons in 3-NP and MPTP + 3-NP-intoxicated mice. Scale bar = 20 Am.

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Histopathology Histopathological results are displayed in Tables 2 and 3 and illustrated in Figs. 4 and 5.

compared to saline). Co-administration of MPTP and 3-NP did not modify the degree of such SDH inhibition ( 22.2%, P < 0.001 compared to saline).

Effects of MPTP on 3-NP-induced SDH inhibition

Frequency of striatal lesions

Injection of a single dose of 3-NP (50 mg/kg) induced a significant reduction in SDH activity ( 21.7%, P < 0.001

In 3-NP-intoxicated mice, three out of the eight surviving animals (37.5%) had a visible striatal lesion compared to

Fig. 5. (A) Distribution of neuronal loss in the striatum of 3-NP, MPTP and MPTP + 3-NP-intoxicated mice. DL: dorsolateral quadrant, VL: ventrolateral quadrant, DM: dorsomedial quadrant, VM: ventromedial quadrant. (B) Distribution of astrocytic activation (GFAP) in the striatum of 3-NP, MPTP and MPTP + 3-NP-intoxicated mice. Statistical significance: *P < 0.05, **P < 0.01 vs. PBS. #P < 0.01 vs. MPTP. ##P < 0.01 vs. 3-NP.

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five out of the six surviving MPTP + 3-NP-treated mice (83.3%, ns) (Fig. 4). No apparent extrastriatal damage was observed. Striatal volume and 3-NP-induced striatal lesions Following 3-NP or MPTP + 3-NP-intoxication, there was a significant reduction in the striatal volume estimated between bregma +1.18 mm and bregma 0.22 mm (3-NP: 19.7%, P < 0.001 and 3-NP + MPTP: 17.7%, P < 0.01). In MPTP-treated mice, there was also a slight but significant decrease in striatal volume ( 10.5%, P < 0.05).

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more marked than that induced by 3-NP or MPTP alone. Further analysis revealed a differential distribution of GFAP-immunoreactive astrocytes in the striatum among MPTP, 3-NP and MPTP + 3-NP-intoxicated mice. MPTP induced a homogeneous increase of the density of GFAPpositive astrocytes throughout the striatum while 3-NP induced a preferential increase in the lateral part of the striatum (DL and VL, P < 0.05, Fig. 5B). When MPTP and 3-NP were co-administered, GFAP-positive reactive astrocytes were homogeneously distributed throughout the striatum and were significantly more numerous than those induced by MPTP or 3-NP alone for all the striatal quadrants studied.

Neuronal loss in the striatum 3-NP intoxication induced a significant neuronal loss at the anterior – mid-striatal level (bregma = +0.98 mm), considering either the mean numerical density or the absolute number of neurons on cresyl violet sections (respectively 16.1%, P < 0.05 and 31%, P < 0.001). Neuronal loss affected the NeuN-positive and calbindin-positive neuronal population (Fig. 4). Co-administration of 3-NP and MPTP induced a more pronounced reduction in the neuronal density and the absolute number of neurons (respectively 27.4%, P < 0.001 and 39%, P < 0.001). In MPTPintoxicated mice, there was no significant modification of the mean neuronal density compared to vehicle-injected animals ( 0.6%) (Table 2). Further analysis of the distribution of mid-striatal neuronal loss revealed a differential distribution of neuronal loss between 3-NP and MPTP + 3NP. Neuronal density was found to be significantly reduced in the medial part of the striatum (DM and VM) in MPTP + 3-NP compared with 3-NP-intoxicated mice (DM: 253.5 F 11 vs. 304.1 F 8.9, VM: 260.2 F 13.8 vs. 308.8 F 9, P < 0.01 each) (Fig. 5A). Astrocytic activation in the striatum Intoxication with MPTP or 3-NP induced a significant increase in the number of GFAP-immunoreactive astrocytes at the mid-striatal level 2 (Fig. 4). When both neurotoxins were co-administered, astrocytic activation was significantly

Neuronal loss in the SNc and loss of striatal dopaminergic terminals Results are indicated in Table 3. In MPTP and MPTP + 3-NP-intoxicated mice, there was a significant neuronal loss in the SNc compared to PBS-treated mice (MPTP = 19%, P < 0.05; MPTP + 3-NP = 26.5%, P < 0.01). There was no significant difference in cell loss between MPTP and MPTP + 3-NP groups. Cell loss predominated in the mid and caudal levels of the SNc in 3-NP, MPTP and MPTP + 3-NP groups. According to the results obtained in the SNc, there was a significant ( P < 0.001) decrease in the relative striatal TH staining intensity in the MPTP ( 25.3%) and MPTP + 3-NP groups ( 27.2%) compared to controls. There was no difference between MPTP and MPTP + 3-NP groups.

Discussion The present study investigated the behavioral and histopathological consequences of a combined systemic intoxication with MPTP and 3-NP in the mouse and aimed at determining whether and how the co-administration of MPTP with 3-NP would modify 3-NP-induced motor disorders and striatal damage in C57Bl/6 mice. Several previous studies have demonstrated the occurrence of complex interactions between striatal and nigral degeneration during attempts at combined striatal and nigral degeneration using

Table 3 Cell counts in the substantia nigra pars compacta (SNc) on tyrosine-hydroxylase (TH)-stained sections and estimation of relative TH staining intensity in the dorsal striatum (see Experimental procedures—Morphometric analysis and cell counts) SNc

PBS (control)

Mean number TH+ cells

Number

Rostral level(bregma Middle level(bregma Caudal level(bregma Total Striatum or cortex Mean relative OD

248.9 292.9 178 251.8

2.92 mm) 3.16 mm) 3.80 mm)

* P < 0.05 vs. control.

F F F F

3-NP Relative loss (%)

16.9 9.5 8.4 12.0

1.492 F 0.04

– – – –

MPTP

Number 256.4 249.6 154 239.5

F F F F

14.9 17.9 5.9 11.5

1.573 F 0.06

Relative loss (%)

Number

– ( 14.8%) ( 13.5%) ( 4.9%)

223.3 231.5 128.1 203.9

F F F F

MPTP + 3-NP

20.2 12.4 9.6 11.1

1.114 F 0.03

Relative loss (%)

Number

Relative loss (%)

( ( ( (

10.3%) 21%)* 28%)* 19%)*

206.0 F 23.48 204.6 F 21.9 118.7 F 8.57 185.2 F 15.0

( ( ( (

( 25.3%)*

1.087 F 0.02

( 27.2%)*

17.2%) 30.2%)* 33.3%)* 26.5%)*

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sequential administration of two neurotoxins, either injected locally or systemically. These interactions led to changes in the subsequent sensitivity to the second neurotoxic administered: that is, prior lesioning of the striatum with quinolinic acid (QA) protected SNc neurons against the neurotoxic effects of 6-OHDA. This effect is thought, but not fully demonstrated, to be due to neurotrophic factors (GDNF, BDNF) released by activated microglia (Ghorayeb et al., 2001; Scherfler et al., 2000; Teismann et al., 2003; Venero et al., 1995). Similarly, prior lesioning of the SNc exerts a protective effect upon striatal degeneration induced by excitotoxins (QA) or mitochondrial complex II inhibitors (3-NP, malonate) (Maragos et al., 1998; Scherfler et al., 2000). It is admitted that mitochondrial complex I inhibition by MPTP results in a relatively specific cell death in dopaminergic neurons of the SNc (due to the central role of the dopamine transporter) and that mitochondrial complex II (SDH) inhibition leads to degeneration of striatal outflow pathways. The combined systemic administration of mitochondrial complex I (MPTP) and II (3-NP) inhibitors has not yet been studied. An initial experimental approach proposed either the systemic administration of MPTP then 3-NP or 3-NP then MPTP sequentially in C57Bl/6 mice to reproduce a combined nigral and striatal degeneration mimicking the core pathology of the human disease SND (Stefanova et al., 2003). Although combined nigral and striatal degeneration was observed, the results demonstrated that primary MPTP-induced nigral lesions attenuated secondary 3-NP striatal toxicity and that reciprocally, primary 3-NP-induced striatal lesions reduced subsequent nigral MPTP toxicity (Stefanova et al., 2003). Thus, we sought to determine whether a simultaneous administration paradigm could address the following questions: (1) whether a combined mitochondrial compromise would modify the neurotoxic potency of each toxin; (2) whether this would result in any consecutive modifications (potentiation, protection) in striatal or nigral sensitivity; and (3) whether this new MPTP + 3-NP intoxication paradigm might constitute a more suitable model of MSA-P or SND. To address these issues, we studied the time course and severity of motor disorders and their histopathological correlates after 3-NP, MPTP or MPTP + 3-NP intoxication compared to controls. Time course and severity of the motor disorders 3-NP-induced motor symptoms developed once the dose of 40 mg/kg was reached, with a characteristic pattern including bradykinesia, hindlimb clasping, hindlimb dystonia, truncal dystonia and loss of balance control as previously reported (Fernagut et al., 2002c). However, forelimb clasping and dystonic rotations of the trunk were also observable in the most severely affected MPTP + 3-NP mice. While MPTP did not induce any new or unexpected motor symptoms, its co-administration with 3-NP shortened the onset of motor symptoms and increased the severity of these signs at the end of the intoxication phase. During the

post-intoxication period, the motor symptoms of MPTP + 3NP-intoxicated mice tended to be more severe than those of 3-NP-intoxicated mice. The motor symptoms lasted 3 weeks after the end of intoxication. This 3-week progressive recovery phase following 3-NP or MPTP + 3-NP intoxication thus appears critical for the evaluation of motor disorders and for testing the efficacy of future therapeutic strategies. This limited time window might be extended in the future with more chronic intoxication paradigms to provide a more prolonged motor disorder. In a previous experiment, we showed a significant correlation between the motor score of 3-NP-intoxicated mice and striatal damage but not with that of the SNc (Fernagut et al., 2002c). The present results indicate that MPTP potentiated 3-NP-induced motor symptoms as rated by the clinical motor rating scale. This potentiation of 3-NP-induced motor disorder was also demonstrated on standardized motor and sensorimotor integration tests in MPTP + 3-NP-intoxicated mice. They displayed a significantly more severe deterioration in performance on the rotarod, pole test and the beam traversing test. Such motor behavioral tests have been shown to be sensitive to striatal dysfunction, both in 3-NP intoxicated mice and in R6/2 transgenic mice (Carter et al., 1999; Fernagut et al., 2002b,c). Such impaired motor performances are related to motor slowness, hindlimb incoordination with dystonia and impaired balance control. MPTP alone induced subtle alteration on the beam walking task at day 5 post-intoxication and on activity parameters (distance, velocity, rearings). Since performance levels on the pole test have been correlated with the intensity of striatal dopamine depletion (Matsuura et al., 1997), the lack of alteration on the pole test in our experiment in MPTP-intoxicated mice may be due to the low MPTP intoxication schedule. The induction of behavioral impairments in the mouse following MPTP intoxication greatly depends upon the intoxication regimen and subsequent intensity of nigrostriatal denervation. It often requires highly challenging testing situations to unmask significant deficits (Sedelis et al., 2001). In the absence of available data concerning the combined use of MPTP and 3-NP, we deliberately used a moderate MPTP exposure to avoid potentially unacceptable lethality. Indeed, 40% of the MPTP + 3-NP-intoxicated animals died at the end of the intoxication paradigm (50 mg/kg), compared to 20% using 3-NP alone. This lethality rate may constitute a limitation of the model, which requires a large number of animals to reach a significant power of analysis. Altogether, the behavioral data suggest that the more pronounced motor impairment in MPTP + 3-NP-intoxicated mice might be due to a potentiation of 3-NP-induced striatal dysfunction by MPTP, provided that 3-NP in itself did not potentiate the neuronal loss in the SNc. Histopathological correlates SDH histochemistry demonstrated that MPTP did not interfere with 3-NP-induced brain SDH inhibition, and thus

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with 3-NP brain bioavailability. Subacute intoxication by 3NP or MPTP + 3-NP induced a significant reduction in the striatal volume ( 19.7% and 17.7%, respectively) along with neuronal degeneration and the occurrence of circumscribed striatal lesions. Although the reduction of striatal surface or volume can be observed throughout the striatum, it predominates along with striatal lesions and cell loss in the lateral part of the anterior and mid-striatum of 3-NP or MPTP + 3-NP intoxicated mice (Fernagut et al., 2002c). This is consistent with previous reports in 3-NP-intoxicated rat or mice (Fernagut et al., 2002c). We thus focused our cell loss analysis and related astrocytic activation at the level of the maximal striatal damage, (i.e., mid-anterior striatum). We also previously demonstrated that there was a clear-cut correlation among striatal surface or volume reduction, cell loss as determined on cresyl violet sections and impaired motor behavior (Fernagut et al., 2002c). As in our previous experiments, cresyl violet sections were used for the purpose of cell counting on thin cryostat adjacent sections where neuronal profiles can be differentiated from astroglial cells (Fernagut et al., 2002c; Ghorayeb et al., 2001). We however verified that cell loss concerned the neuronal NeuN-positive population and, like others, we confirm that the cells mainly affected are calbindin-positive outflow neurons (Blum et al., 2001a,b). Interestingly, we demonstrated a specific distribution of striatal neuronal loss and astrocytic activation in MPTP, 3-NP and MPTP + 3-NPintoxicated mice that has never been demonstrated in detail before. 3-NP intoxication induced a significant neuronal loss in the lateral part of the striatum associated with a circumscribed striatal lesion in 37.5% of surviving mice. When MPTP was co-administered with 3-NP, the magnitude of cell loss in the medial part of the mid-striatum was enhanced compared to 3-NP alone ( P < 0.05). Neuronal loss in the lateral part of the striatum and estimation of the absolute number of neurons were not found to be significantly different at the mid-striatal level between 3-NP and MPTP + 3-NP intoxicated mice, albeit the frequency of circumscribed lesions was superior in this latter group (83.3% vs. 37.5% in the 3-NP group). MPTP-induced potentiation of 3-NP induced striatal damage was also found at the level of astrocytic activation in the striatal level examined. MPTP induced a homogeneous GFAP expression throughout the medio-lateral striatum, as previously described (Francis et al., 1995; Reinhard et al., 1988), while 3-NP-induced astrocytic activation was significantly more marked in the lateral part of the striatum and particularly dense near the lesion. However, the overall numerical density of GFAP-immunoreactive astrocytes in the midstriatum of 3-NP or MPTP-intoxicated mice was similar. Interestingly, co-administration of the two neurotoxins induced a significantly increased astrocytic activation compared to that induced by MPTP or 3-NP alone, but in that case with a homogeneous medio-lateral striatal distribution. In the 3-NP rat model of HD, increased striatal GFAP expression has been found to be correlated with behavioral

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impairments (Teunissen et al., 2001). Several other results also show that the time course of reactive astrocyte formation parallels that, and is a consequence, of cell loss, particularly in the SNc (Teismann et al., 2003; Wu et al., 2002). This suggests that in our experiment, MPTP-induced striatal astrocytic activation might have accompanied a more pronounced striatal dysfunction and motor impairment in the MPTP + 3-NP group compared to 3-NP alone and that it was associated with more severe mid-striatal damage (neuronal loss). Administration of 3-NP induces ultrastructural changes in astrocytes a few hours after injection (Nishino et al., 1997). Moreover, GFAP-positive astrocytes are detectable for several weeks following 3-NP intoxication (Teunissen et al., 2001). Following MPTP administration, GFAP-reactive astrocytes are detectable from 24 h, then increase greatly and remain up-regulated for at least 6 weeks (Francis et al., 1995). This time course of astrocytic activation following MPTP or 3-NP intoxication thus involves early expression of GFAP and is sustained for several weeks. Recent evidence in microglial cell cultures indicates that 3-NP also activates microglia with production of reactive oxygen species (ROS) and subsequent glial cell death, suggesting that not only activated, but also damaged, microglia may also contribute to the neurodegenerative process (Ryu et al., 2003). This issue was not assessed in the present study. At the level of the SNc, there was a significant, albeit limited dopaminergic neuronal loss in MPTP and MPTP + 3-NP groups. This cell loss predominates in the mid- and caudal substantia nigra. Although cell loss was mild-to-moderate, it could also be demonstrated in view of the relative TH-immunoreactivity within the dorsal and anterior striatum. We have previously shown that systemic intoxication with 3-NP induces a moderate ( 20%) but dose-dependent loss of SNc neurons (TH and Nissl stains) at the mid-striatal level as well as a loss of DAT binding in the striatum (Fernagut et al., 2002c). In this experiment, a slight ( 15%) neuronal loss in the SNc of 3NP-intoxicated mice was found at the mid and caudal level, but this was no more significant. Therefore, this issue will need to be further assessed to determine whether mesencephalic dopaminergic neurons display an intrinsic vulnerability to complex II inhibition, as suggested by in vitro studies (Zeevalk et al., 1995), or is a consequence of a nonspecific retrograde degeneration occurring within the circumscribed striatal lesion, as suggested by loss of DATbinding within the circumscribed striatal lesion (Fernagut et al., 2002c). A detailed mapping of neuronal loss using a stereological cell counting method throughout the SNc is necessary to complement the present study. MPTP induced a significant (about 20%) neuronal loss in the SNc predominating in the mid (21%) and rostral level (28%), with a concomitant loss of 25% of relative striatal TH staining sections. Since MPTP toxicity is highly dependent on the cumulated dose administered and more importantly on the administration schedule (Gerlach and Riederer, 1996), this moderate extent of MPTP-induced neuronal death in the

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SNc is in accordance with the intoxication regimen used in this study with low daily doses (10 mg/(kg 24 h), i.p.), a regimen chosen to prevent unacceptable lethality in the absence of available information on the combined use of MPTP + 3-NP. The magnitude of neuronal loss in the SNc of MPTP + 3-NP-intoxicated mice tended to be greater ( 30% at the mid-and caudal levels) compared to MPTP. This suggests that combined administration of 3-NP did not induce a ‘‘negative’’ effect towards MPTP neurotoxicity on dopaminergic neurons in the SNc. However, this difference was found to be slight and the relative loss of striatal THimmunoreactivity proved to be almost similar. Again, further experiments using stereological cell counting and mapping throughout the SNc will aim at determining whether it is the same cells that are prone to die following bioenergetic failure induced by MPTP (mitochondrial complex I) or 3-NP (mitochondrial complex II). Further experiments will also be needed to increase the extent of SNc cell death by using more frequent MPTP administration. How can MPTP potentiate 3-NP-induced striatal damage? Among the numerous factors that might be involved in the interactions between nigral and striatal degeneration, dopamine is of particular interest because modifications in the nigrostriatal dopaminergic transmission have been shown to modulate striatal cell loss. Increased nigrostriatal dopamine transmission or administration of methamphetamine increases striatal sensitivity to 3-NP (Fernagut et al., 2002a; Reynolds et al., 1998). In vitro studies have demonstrated that inhibition of mitochondrial complex II with 3NP decreases DAT function (Maragos et al., 2002) and that administration of dopamine to cultured striatal neurons potentiates cell death induced by methylmalonate (McLaughlin et al., 1998). Moreover, in vivo studies have shown that inhibition of mitochondrial complex II with malonate induces a transient but massive release of dopamine in the striatum (Moy et al., 2000) and that generation of reactive oxygen species (ROS) in the striatum following malonate is dependent on dopamine release (Ferger et al., 1999). Systemic administration of 3-NP also induces an increase in striatal dopamine release lasting for several hours (Nishino et al., 1997). Interestingly, several studies have shown that administration of MPTP or its active metabolite MPP+ rapidly induces a massive release of dopamine in the striatum (Obata, 2002; Ozaki et al., 1987; Rollema et al., 1986). This suggests that in our experiment, MPTP-induced dopamine release might have increased 3NP-induced striatal dysfunction and neuronal loss. On the other hand, metabolic compromise arising from combined inhibition of complex I or II induces the production of ROS from various sources, including mitochondria, cytosolic calcium or activated microglia (Blum et al., 2001a,b; Teismann et al., 2003). Since MPTP and 3-NP both induce the production of ROS in the striatum (Ali et al., 1994; Beal et al., 1995), oxidative stress may have been

enhanced in the MPTP + 3-NP group. Finally, although some controversy still persists, there is more evidence that reactive microglia exerts a more deleterious than protective effect in neurodegenerative diseases (Teismann et al., 2003). Clinical relevance The results of the present study demonstrate that a paradigm of combined administration of MPTP and 3-NP is a reliable strategy to overcome the ‘‘negative’’ interactions between striatal and nigral sides of degeneration observed in previous attempts at inducing a double striatal and nigral lesion modelling SND. The MPTP-induced potentiation of 3-NP-induced motor impairment and striatal damage demonstrated in this study is relevant to many clinical and neuropathological aspects of SND. Parkinsonism in SND is severe and rapidly worsens as the disease progresses, although the relative contribution of striatal and nigral degeneration to the motor disorders is unclear (Tison et al., 1995; Wenning et al., 1997). Our present results agree with previous experimental studies and clinical findings suggesting that the combined striatal and nigral degeneration significantly increases the intensity or the duration of motor disorders (Ghorayeb et al., 2001; Tison et al., 1995; Wenning et al., 1994). In SND, striatal degeneration predominates in the dorsolateral part of the putamen along with microglial activation, a distribution similar to that found in HD or in 3-NP and MPTP + 3-NP-intoxicated mice. Striatal and nigral degenerations in SND are thought to occur simultaneously (Fearnley and Lees, 1990; Tison et al., 1995). Our results indicate that a combined nigral and striatal degeneration is able to increase significantly the extent of striatal damage. This raises the question whether the degeneration of nigrostriatal dopaminergic neurons in the human disease can influence the progression of the striatal degeneration. Cytopathologic hallmarks of SND such as glial cytoplasmic inclusions with alpha-synuclein aggregates, which are thought to play an important role in the pathogenesis of SND (Burn and Jaros, 2001), were not found in our study. Recently, transgenic mice were generated with the expression of human alpha-synuclein in oligodendrocytes, but they displayed neither neuronal loss nor motor impairment (Kahle et al., 2002). Further experiments assessing the sensitivity of these transgenic mice to MPTP or 3-NP may help to elucidate the potential pathogenic role of oligodendrocytes and alpha-synuclein. As opposed to PD or HD, the existence of mitochondrial complex deficiencies has to date been controversial in MSA (Blin et al., 1994; Gu et al., 1997). Despite the lack of a demonstrated bionergetic defect in the SNc of SND or MSA (Burn and Jaros, 2001; Gu et al., 1997), the similar distribution of nigral and striatal neuronal loss in human SND compared to PD and HD suggests that different pathogenic mechanisms with or without proven subsequent bionergetic failures can affect the same neuronal populations in the basal ganglia.

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Conclusion We have characterized for the first time the behavioral and histopathological outcomes of a combined nigral and striatal degeneration in mice using MPTP and 3-NP and have demonstrated that this combined degeneration in the nigrostriatal system potentiates striatal damage and motor impairment. This strategy of combined intoxication is in accordance with the physiopathological hypothesis in SND, suggesting that nigral and striatal degeneration occurs simultaneously, and that this combined degeneration accounts for the severity of the motor disorder. This paradigm of combined MPTP + 3-NP intoxication avoids ‘‘negative’’ interactions between nigral and striatal degeneration that have limited the exploration of previous animal models of SND. Furthermore, such systemic intoxication paradigms will enable more chronic administration of the neurotoxins, which have been proven to be important for the relevance of experimental models, especially in HD (Brouillet et al., 1999). The behavioral impairments following MPTP + 3NP and their histopathological correlates provide a basis for future studies on the pathophysiological mechanisms underlying parkinsonism in SND and for the emergence of therapeutic strategies. Acknowledgments Elsa Diguet is a recipient of a France Parkinson grant. We would like to thank Dr. Emmanuel Brouillet for his help with SDH histochemistry. References Ali, S.F., David, S.N., Newport, G.D., Cadet, J.L., Slikker Jr., W., 1994. MPTP-induced oxidative stress and neurotoxicity are age-dependent: evidence from measures of reactive oxygen species and striatal dopamine levels. Synapse 18, 27 – 34. Beal, M.F., 2001. Experimental models of Parkinson’s disease. Nat. Rev., Neurosci. 2, 325 – 334. Beal, M.F., Ferrante, R.J., Henshaw, R., Matthews, R.T., Chan, P.H., Kowall, N.W., Epstein, C.J., Schulz, J.B., 1995. 3-Nitropropionic acid neurotoxicity is attenuated in copper/zinc superoxide dismutase transgenic mice. J. Neurochem. 65, 919 – 922. Blin, O., Desnuelle, C., Rascol, O., Borg, M., Peyro Saint Paul, H., Azulay, J.P., Bille, F., Figarella, D., Coulom, F., Pellissier, J.F., et al., 1994. Mitochondrial respiratory failure in skeletal muscle from patients with Parkinson’s disease and multiple system atrophy. J. Neurol. Sci. 125, 95 – 101. Blum, D., Gall, D., Cuvelier, L., Schiffmann, S.N., 2001a. Topological analysis of striatal lesions induced by 3-nitropropionic acid in the Lewis rat. NeuroReport 12, 1769 – 1772. Blum, D., Torch, S., Lambeng, N., Nissou, M., Benabid, A.L., Sadoul, R., Verna, J.M., 2001b. Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Prog. Neurobiol. 65, 135 – 172. Brouillet, E., Guyot, M.C., Mittoux, V., Altairac, S., Conde, F., Palfi, S., Hantraye, P., 1998. Partial inhibition of brain succinate dehydrogenase by 3-nitropropionic acid is sufficient to initiate striatal degeneration in rat. J. Neurochem. 70, 794 – 805.

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