Introduction

Parkinson disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and an associated decline in striatal dopamine. The cardinal symptoms of PD include tremor at rest, slowness of movement, stiffness, and postural instability. Several genes that cause familial forms of PD when mutated or overexpressed have been identified ¹. Three different missense mutations in the -synuclein (approved symbol PARK1) gene and a heterozygous triplication of a genomic region encompassing the -synuclein locus have been linked to the development of early onset dominant PD ^2,3,4,5. Loss-of-function mutations in the parkin (approved symbol PARK2) gene are responsible for autosomal-recessive juvenile parkinsonism (AR-JP) ^6,7. Parkin is a ubiquitin protein ligase, and AR-JP-associated mutations abolish or reduce the ubiquitin ligase activity of parkin ⁸, resulting in the accumulation of certain parkin substrates in the brain ^9,10. Finally, deletions and point mutations in the DJ-1 gene (approved symbol PARK7) are linked to another recessively inherited early onset form of PD in at least two families ^11,12. DJ-1 functions in antioxidant defense but its homology to other proteins suggests that it may also act as a chaperone ^13,14. Although inherited and sporadic PD may have different causes, they likely intersect in common pathways ^15,16. Current evidence suggests that mitochondrial complex I inhibition may be the central cause of sporadic (idiopathic) PD and that complex I deficiency may cause -synuclein aggregation, which contributes to the demise of dopamine neurons ^15,16.

The observation that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces an irreversible and severe parkinsonian syndrome in humans characterized by all of the features of PD ^17,18 led to the development of MPTP-induced mouse and nonhuman primate models of PD ^16,19. MPTP is converted by astrocytes to MPP⁺, which is taken up into dopaminergic neurons via the dopamine transporter. Within dopamine neurons, MPP⁺ inhibits mitochondrial complex I activity and, in primates, induces the formation of Lewy body-like inclusions ¹⁶. Thus, MPTP selectively kills dopamine neurons and reproduces the major neuropathological and biochemical deficiencies of idiopathic PD ^16,19. The neurotoxicity of MPTP is mediated by the induction of both mitochondrial dysfunction and free radical production ¹⁶, and chronic application of MPTP in mice induces apoptosis ^20,21. Recently, it has been shown that activation of the proapoptotic c-jun N-terminal kinase (JNK) pathway, cyclooxygenase-2, and caspases is important for MPTP-induced dopamine neuron loss ^22,23,24,25. In agreement with this, viral transduction of dopamine neurons with JNK-interacting protein-1 (JIP-1) or an apoptosis protease activating factor-1 (APAF-1) dominant negative inhibitor was shown to inhibit MPTP-induced neuron loss ^26,27.

In this work we investigated whether recombinant adeno-associated virus (rAAV)-mediated expression of heat-shock protein 70 (Hsp70) (approved gene symbol HSPA1A) can protect dopamine neurons from degeneration induced by MPTP in mice. Hsp70 appeared as an attractive candidate because it acts as a chaperone to assist the proper folding of nascent proteins and prevents protein aggregation under adverse conditions, including oxidative stress ^28,29. In addition, Hsp70 inhibits several proapoptotic factors ^30,31,32, and it was shown to be a potent suppressor of neuronal loss in Drosophila models of polyglutamine disease and PD ^33,34,35.

Results

Transduction of mouse dopaminergic neurons

We transduced nigral dopaminergic neurons of mice unilaterally by injection of a rAAV encoding HA-tagged Hsp70 (HA-Hsp70). We injected control mice with a rAAV carrying an identical enhanced green fluorescent protein (EGFP) expression cassette. We confirmed expression of the virus-derived gene products in a subset of animals by native fluorescence microscopy (Figs. 1A–1C) and immunohistochemistry with an anti-HA antibody (Figs. 1D–1F).

Figure 1.

Expression of EGFP and HA-Hsp70 in the substantia nigra of rAAV-injected mice. Expression of (A) EGFP and (D) HA-Hsp70 in tyrosine hydroxylase (TH)-positive neurons (B and E) of the substantia nigra pars compacta is shown. (C and F) Overlays. EGFP was detected in the FITC channel (without antibodies). HA-Hsp70 was detected by immunohistochemistry with an anti-HA antibody. No transgene expression was observed in the uninjected hemisphere or in the absence of the primary antibody (data not shown).

Full figure and legend (285K)

Hsp70 inhibits nigrostriatal degeneration in the MPTP mouse model of PD

Five weeks after virus delivery, we lesioned the dopaminergic system of mice by chronic application of MPTP on 4 consecutive days. A third group of age-matched control mice received neither virus nor MPTP (normal mice). In the AAV-EGFP group, treatment with MPTP resulted in the loss of about 37% of the dopaminergic neurons in both uninjected and virus-injected substantia nigra, compared to normal mice (Figs. 2A, 2D, and 2E). Likewise, the uninjected substantia nigra of MPTP-treated AAV-Hsp70 animals on average contained 40% fewer dopamine neurons than that of normal mice (Fig. 2A). In contrast, only 16% of the dopamine neurons degenerated in the AAV-Hsp70-injected nigral hemisphere after MPTP treatment (P < 0.0001) (Figs. 2A, 2D, and 2F). MPTP injection reduced the striatal dopamine levels by 73% in AAV-EGFP-injected mice relative to normal animals, and there were no differences in the dopamine concentrations between uninjected and virus-injected striatal hemispheres in AAV-EGFP mice (Fig. 2B). In contrast, striatal dopamine levels were significantly higher in the rAAV-injected hemisphere compared to the uninjected side in the AAV-Hsp70 group (P = 0.0011) (Fig. 2B). Finally, we immunostained striatal sections with an antibody to tyrosine hydroxylase (TH) and determined TH fiber density. In the AAV-Hsp70 group, TH fiber density in the virus-injected side was significantly less reduced than in the contralateral uninjected hemisphere after MPTP treatment (P < 0.0001) (Figs. 2C and 2G). In contrast, we observed no difference in the TH fiber density between the two striatal hemispheres in the AAV-EGFP group (Figs. 2C and 2G). Collectively, these results show that nigral expression of Hsp70 confers significant protection against MPTP-induced nigrostriatal degeneration.

Figure 2.

HA-Hsp70 inhibits MPTP-induced nigrostriatal degeneration in mice. Mice were injected above the right substantia nigra with the indicated rAAV vector and 5 weeks later treated with MPTP (chronic regimen). Animals were sacrificed and analyzed 9 days after the last MPTP injection. Normal control mice were age matched to the MPTP-treated mice. (A) Number of tyrosine hydroxylase (TH)-positive neurons in sections of the substantia nigra (mean SEM). Normal, 4 mice, n = 13 sections; AAV-EGFP, 5 mice, n = 16 sections; AAV-Hsp70, 12 mice, n = 37 sections. (B) Striatal dopamine levels expressed in ng/mg wet tissue (mean SEM). Normal, n = 6 mice; AAV-EGFP, n = 11 mice; AAV-Hsp70, n = 12 mice. (C) Percentage TH fiber density (mean SD). Normal, 4 mice, n = 14 sections; AAV-EGFP, 6 mice, n = 18 sections; AAV-Hsp70, 7 mice, n = 23 sections. (D–F) Pictures of DAB-stained TH-positive neurons of a (D) normal, (E) MPTP-treated, AAV-EGFP-injected, and (F) MPTP-treated, AAV-Hsp70-injected substantia nigra. (G) Illustration of TH fiber protection in the virus-injected striatal hemisphere of an AAV-Hsp70-injected mouse (stained with an antibody to TH).

Full figure and legend (106K)

Unilateral nigral Hsp70 expression results in amphetamine-induced turning behavior in MPTP-lesioned mice

Amphetamine induces presynaptic dopamine release and inhibits dopamine reuptake by the dopamine transporter. The amount of dopamine released is dependent on the number of nigral dopaminergic neurons, and unilateral nigral lesions induce locomotor activity toward the lesioned brain side ³⁶. We therefore tested whether the protection of dopamine neurons in the AAV-Hsp70-treated (right) hemisphere resulted in drug-induced turning behavior. Compared to AAV-EGFP-injected mice, animals of the AAV-Hsp70 group showed increased general turning biased toward the uninjected (left) side (Fig. 3). We observed no overall difference between the two groups when on-spot rotations were monitored, although one animal of the Hsp70 group clearly showed on-spot rotations toward the left side (Fig. 3). However, this is in agreement with previous studies, which have shown that significant drug-induced and spontaneous rotations will appear only in animals in which the nigral dopamine neurons and striatal dopamine levels are reduced by more than 50–60% ^37,38. In the AAV-Hsp70 mice, the difference between the two hemispheres amounted to 28% (TH neuron number) and 24% (striatal dopamine). Thus, the more subtle turning behavior observed in our studies is consistent with a less severe imbalance in the dopamine levels between the two hemispheres.

Figure 3.

Amphetamine-induced behavior. Behavioral analysis showing box plots of amphetamine-induced on-spot rotations and general turning bias during locomotion. Boxes indicate the 25th and 75th, whiskers the 10th and 90th percentiles. AAV-Hsp70 and AAV-EGFP groups were compared using the Mann–Whitney U test. The injection of amphetamine induced marked locomotion in all animals, interrupted by short rests and episodes of lingering. By direct observation during the experiment, on-spot rotations were noted in only one animal of the Hsp70 group, which earned a score of 2.08 rounds per minute (RPM) (black dot). Quantitative analysis of on-spot rotations was in agreement with direct observation and revealed no significant group difference (ns). Evaluation of general turning bias during locomotion showed a significant bias toward left turns in the AAV-Hsp70 group, while the AAV-EGFP group showed no turning bias. Overall locomotor activity was slightly higher in the AAV-Hsp70 group but this did not reach statistical significance (data not shown).

Full figure and legend (37K)

Hsp70 Inhibits MPTP-induced apoptosis in the substantia nigra

Chronic application of MPTP induces dopaminergic neuron death by apoptosis ^20,21. Hsp70 can inhibit both caspase-dependent and caspase-independent apoptosis by blocking the functions of APAF-1 and apoptosis-inducing factor (AIF), and the recruitment of procaspase-9 to a functional apoptosome ^30,31,32. To study whether the protective effect of Hsp70 was related to its antiapoptotic activity, we stained sections of the substantia nigra for apoptotic cells using the TUNEL assay. Typical apoptotic profiles, similar to those observed by others ²¹, are shown in Fig. 4A. Compared to normal control mice, sections of MPTP-treated AAV-EGFP-injected mice contained a 4.7-fold increased number of apoptotic nuclei in both hemispheres (Fig. 4B). A similar increase in apoptosis has been observed previously in chronically MPTP-treated mice ²¹. In the AAV-Hsp70 group, apoptosis was increased 4.4-fold in the uninjected substantia nigra, but only 3-fold in the rAAV-injected substantia nigra (P < 0.0001) (Fig. 4B). Transient overexpression of HA-Hsp70 in human dopaminergic SH-SY5Y cells did not result in increased levels of the antiapoptotic molecules Bcl-2 and Bcl-XL (data not shown). Thus, we conclude that Hsp70 inhibits MPTP-induced apoptosis in the substantia nigra, most likely at a step downstream of cytochrome c release (see also Discussion).

Figure 4.

Hsp70 inhibits MPTP-induced apoptosis. Mice were sacrificed and analyzed 9 days after the last MPTP injection. (A) TUNEL-stained apoptotic nuclei in the substantia nigra of MPTP-treated mice. Sections fr om normal mice treated with DNase I served as a positive control. (B) Quantitation of apoptotic nuclei (mean SEM) in the substantia nigra (identified by the presence of the MTN). Normal, 3 mice, n = 9 sections; AAV-EGFP, 4 mice, n = 12 sections; AAV-Hsp70, 12 mice, n = 36 sections.

Full figure and legend (65K)

Discussion

Mitochondrial complex I deficiency, oxidative stress, and proteasomal dysfunction have been implicated in the pathogenesis of idiopathic PD ^{15,39,40,41,42}. Recently, the identification and characterization of genes linked to inherited forms of PD shed some light on the affected cellular pathways. Collectively, these studies show that aggregation of -synuclein as a consequence of mutation or overexpression, and insufficient degradation of specific neuronal proteins due to mutational inactivation of a ubiquitin protein ligase termed parkin, underlie the development of dominant and recessive, early onset familial PD, respectively ^1,9,39,43,44. In addition, mutations in the gene encoding DJ-1, which protects cells against oxidative stress ¹³, are linked to PD in two kindreds ^11,12. Overall, these findings suggest that, although the causes may be different, idiopathic and inherited PD may share common downstream pathological pathways.

In this work we studied whether rAAV-mediated expression of Hsp70 may protect dopamine neurons from degeneration induced by MPTP. In mice and primates, MPTP causes selective dopamine neuron loss and reproduces many of the neuropathological and biochemical deficiencies of idiopathic human PD ^16,19. Previously, it had been shown that Hsp70 inhibits degeneration of cerebellar Purkinje cells and improves rotarod performance in SCA1 transgenic mice that overexpress ataxin-1 with an expanded polyglutamine tail ⁴⁵. Likewise, neurodegeneration in a Drosophila model of Machado-Joseph disease was suppressed by overexpression of Hsp70 and exacerbated in flies with a mutant form of hsc4, the major constitutively expressed fly hsp70 ³⁴. In a P-element insertion screen for dominant genetic modifiers of a degenerative eye phenotype caused in flies by the overexpression of a polypeptide composed of 127 glutamine residues (Q127) (a general model for triplet-repeat diseases), two suppressor genes encoding chaperones with J domains were identified, one being homologous to human Hsp40/HDJ1 ⁴⁶. With relevance to PD, -synuclein toxicity and dopamine neuron loss were suppressed by Hsp70 in -synuclein-transgenic flies, while interference with endogenous chaperone activity accelerated -synuclein toxicity ³³. Finally, polymorphisms in the promoter of the Hsp70-1 gene that reduce the expression of a luciferase reporter gene in transfection assays are significantly associated with PD in a Taiwanese population, suggesting that reduced Hsp70-1 expression may increase the risk of developing PD ⁴⁷.

Here we report that adeno-associated virus-mediated Hsp70 gene transfer to dopaminergic neurons significantly protected the mouse nigrostriatal system against MPTP-induced neuron and dopamine loss. Compared to animals of the EGFP group, Hsp70 mice showed 33% fewer apoptotic figures, TH neuron loss was reduced by 43%, and the degeneration of striatal TH fibers was inhibited by 46% (Figs. 2A, 2C, and 4B). The number of apoptotic cells detected depends on the time passed since MPTP treatment ^16,48. This may explain the slightly larger protective effect of Hsp70 at the level of TH neurons and TH fiber density compared to apoptosis inhibition. Alternatively, part of the protection afforded by Hsp70 may be mediated by apoptosis-independent mechanisms. Unexpectedly, animals of the Hsp70 group showed higher levels of dopamine in the uninjected hemisphere compared to AAV-EGFP mice. This cannot be due to different treatments, since all mice were injected with the same batch and preparation of MPTP. A few virus-transduced TH neurons were occasionally observed in the uninjected hemisphere (data not shown), presumably by virus leakage during injection to the contralateral side. Although unlikely, more neurons may have been transduced by HA-Hsp70 at levels undetectable by HA immunohistochemistry but still sufficient to confer increased protection against MPTP.

Behavioral analyses showed that mice of the AAV-Hsp70 group displayed amphetamine-induced turning behavior biased toward the left uninjected side, suggesting that Hsp70 also preserved the ability of dopamine neurons to release dopamine upon administration of amphetamine. However, with the exception of one animal in the Hsp70 group, no on-spot rotations were observed. Based on previous studies ^37,38, we believe that residual dopamine levels in the uninjected hemisphere dampened on-spot circling but could not prevent a more subtle amphetamine-induced locomotor side bias. Taken together, the histological, biochemical, and behavioral data show that Hsp70 confers significant protection against MPTP-induced nigrostriatal degeneration.

Chronic application of MPTP induces apoptosis ^20,21. Expression of the proapoptotic molecules Fas and Bax are upregulated by MPTP administration, and both Bax- and Fas-deficient mice show markedly attenuated MPTP sensitivity ^21,49. In addition, several caspases are activated by MPTP ^24,25. It has previously been shown that Hsp70 can inhibit both caspase-dependent and caspase-independent apoptosis by blocking the functions of APAF-1 and AIF and the recruitment of procaspase-9 to a functional apoptosome ^30,31,32. We therefore investigated whether apoptosis was altered in the AAV-Hsp70-expressing substantia nigra. Our results show that apoptosis is reduced by 32% in the Hsp70-transduced substantia nigra compared to the uninjected hemisphere of the same animals and by 33% relative to both hemispheres of the AAV-EGFP group. Our experiments do not address whether Hsp70 inhibits apoptosis directly or indirectly via protection against oxidative stress ^50,51,52, which is a key mediator of apoptosis in the MPTP model of PD ^16,53. However, inhibition of apoptosis by Hsp70 most likely occurs downstream of cytochrome c release, because overexpression of Hsp70 failed to increase the levels of Bcl-2 and Bcl-XL, at least in transfected cells (data not shown). We thus favor the idea that Hsp70 inhibits the effector phase of apoptosis at the level or downstream of the apoptosome. Consistent with this model, it was recently shown that blocking apoptosis by viral vector-mediated expression of a dominant negative inhibitor of APAF-1 (DN-APAF-1) inhibits MPTP-induced dopamine neuron loss ²⁷. Because Hsp70 can inhibit APAF-1 ^30,31, the mechanisms by which Hsp70 and DN-APAF-1 inhibit dopamine neuron loss may be similar.

Recently, it has been shown that c-Jun is activated in dopaminergic neurons from PD patients and in MPTP-treated mice. In mice, c-Jun activation involves the c-Jun N-terminal kinases JNK-2 and JNK-3, and cyclooxygenase-2 is an indispensable target of JNK for MPTP-induced apoptosis ^22,23. Consistent with these findings, viral vector-mediated transduction of dopamine neurons with JIP-1 inhibited MPTP-induced neuron loss ²⁶. Interestingly, constitutive overexpression of Hsp70 has been shown to inhibit stress-induced apoptosis at a step downstream of the activation of JNK ^54,55, suggesting an additional mechanism by which Hsp70 may protect dopamine neurons. Alternatively, chaperones may preserve proteasome function under oxidative stress ⁵⁶.

Finally, MPTP administration leads to the accumulation and nitration of -synuclein in the cytosol of substantia nigra dopaminergic neurons ^57,58, and oxidative nitration has been shown to enhance the ability of -synuclein to misfold and aggregate ⁵⁹. Moreover, -synuclein-null mice are resistant to MPTP ⁶⁰. Although there is no evidence of Lewy bodies in MPTP-treated mice, toxic soluble misfolded -synuclein may be present and Hsp70 may protect dopaminergic neurons against these species ^33,61.

Whatever the exact mechanism(s), our results show for the first time that overexpression of Hsp70 is protective in a relevant mammalian model of sporadic PD and suggest that increasing chaperone activity may be beneficial for the treatment of idiopathic PD in humans.

Materials and methods

Plasmid constructions

The rAAV-2 vector pAAV2-CBA-WPRE was constructed as described ⁶⁵. To construct pAAV2-CBA-Hsp70-WPRE, the coding sequence of human Hsp70 (Accession No. BC002453) was amplified by RT-PCR with primers 5'-ACGAATTCTTATGGCCAAAGCCGCGGCGATC-3' and 5'-GCGAATTCCCTAATCCACCTCCTCAATGG-3' and inserted into the PmlI site of pAAV2-CBA-WPRE. pAAV2-CBA-EGFP-WPRE was generated by insertion of the SmaI/HpaI EGFP fragment derived from pEGFP-N1 (Clontech, Palo Alto, CA, USA) into the PmlI site of pAAV2-CBA-WPRE.

Generation and purification of rAAV

High-titer, helper virus-free rAAV-2 was generated with plasmid pDG ⁶² and purified as described ⁶³. The rAAV stocks were sterile-filtered (0.2 m) and titrated by slot-blot hybridization. Titers (genomes/ml) were 3.0 10¹¹ for AAV-CBA-HA-Hsp70-WPRE and 1.0 10¹² for AAV-CBA-EGFP-WPRE. Viruses were stored at -80°C as aliquots and kept on ice during stereotactic injections.

Animals and stereotactic surgery

All animals were housed according to institutional and national guidelines. Anesthetized male C57BL/6 mice (8 weeks of age; 28 3 g) were stereotactically injected with 1.5 l rAAV dorsal to the right substantia nigra pars compacta (SNc) (AP -3.0 mm, ML +1.6 mm, DV -4.1 mm, relative to bregma) at a speed of 0.25 l/min using an sp200i syringe pump (World Precision Instruments, Boston, MA, USA) and a 10-l Hamilton syringe fitted with a 31-gauge steel cannula. The cannula was left in place for 1 min before and 3 min after each injection.

Immunohistochemistry

Freshly isolated brains were fixed for 48 h in 4% paraformaldehyde and incubated for 48–72 h in 30% sucrose. HA-Hsp70 was detected in 20-m-thick coronal cryosections with 1:500 diluted mouse anti-HA antibody (12CA5; Roche Applied Science, Basel, Switzerland) followed by incubation with 1:400 diluted FITC-conjugated donkey anti-mouse IgG (Milan Analytica AG, La Roche, Switzerland). TH was detected with 1:600 diluted sheep anti-TH antibody (Pel-Freeze Biologicals, Rogers, AR, USA), followed by incubation with 1:500 diluted Cy3-conjugated donkey anti-sheep IgG (Milan Analytica AG). EGFP fluorescence was detected directly (without antibodies) using a FITC filter set.

Quantification of TH neurons and TH fiber density

Dopaminergic neurons were detected in 3% H₂O₂/10% methanol-quenched 20-m-thick coronal sections by immunohistochemistry with 1:600 diluted sheep anti-TH antibody (Pel-Freeze Biologicals) and 1:1000 diluted biotinylated donkey-anti-sheep IgG (Milan Analytica AG). TH-positive neurons were visualized by 3,3'-diaminobenzidene (DAB) staining using the ABC Vector kit (Vector Laboratories, Burlingame, CA, USA). DAB-stained, TH-positive neurons in substantia nigra were counted in 20-m-thick coronal sections containing the medial terminal nucleus of the accessory optical tract (MTN) to distinguish between dopaminergic neurons of the SNc and the ventral tegmental area. Counting was carried out at 400 magnification (40 lens) using the Neurolucida Software (MicroBrightField, Inc., Colchester, VT, USA). Optical densities of the TH-immunoreactive fibers in the striatum were measured using the NIH 1.62 Image software (Research Services Branch, National Institute of Mental Health, NIH, Bethesda, MD, USA). For each animal, the optical density was measured in three to four different striatal sections. Optical density readings were corrected by subtraction of nonspecific background density, determined in adjacent sections that were incubated without the primary TH antibody. Analyses of TH neuron number and TH fiber density were carried out with coded slides by a "blinded" experimenter.

Tissue preparation and quantitation of striatal dopamine

Mice were killed by cervical dislocation. Their brains were rapidly removed and placed on an ice-cold plate for dissection of the neostriatum. Left and right striata were placed into preweighed 1.5-ml microcentrifuge tubes. Tissues were homogenized by sonication for 15 s in 0.5 ml ice-cold 6% trichloroacetic acid, and the homogenates were clarified by centrifugation for 15 min at 15,000g at 4°C. The cleared homogenates were passed through a 0.2-m filter into new tubes and kept on ice until analysis. Dopamine levels were analyzed by a blinded experimenter using reverse-phase ion-pair chromatography combined with electrochemical detection under isocratic conditions as described ⁶⁰. Data were calculated relative to an external standard calibration.

MPTP injections and amphetamine-induced behavior

Five weeks after rAAV delivery, mice were injected ip with MPTP-HCl (Sigma) in phosphate-buffered saline at a dose of 20 mg/kg (free base) at 24-h intervals on 4 consecutive days. Seven days after the last MPTP injection, mice were injected ip with D-amphetamine sulfate (Sigma) (2.0 mg/kg body weight in 0.9% NaCl). Rotational behavior was analyzed in 8 AAV-Hsp70-injected and 11 AAV-EGFP-injected animals. Immediately after the injection, animals were placed into a 50 50-cm-square arena with 37-cm-high aluminum side walls and their movements were recorded between 15 and 30 min after the injection. Floor and side walls of the arena were painted black, allowing us to video-track (EthoVision 3.0; Noldus Information Technology, Wageningen, The Netherlands) at 12.5 Hz and 288 364 pixel spatial resolution a white mark placed proximally on the tail of each mouse. During on-spot rotational behavior, this tail mark drew circles with a radius of 5–8 cm. Raw data were further analyzed with the public domain software Wintrack ⁶⁴. To reduce noise and to eliminate redundant points, data were down-sampled using a minimal distance of 2.5 cm between subsequent points as criterion. Filtered paths were then subdivided into straight segments/rests and consistently left- (positive) or right-bent (negative) curves covering a distance of at least 10 cm. Rotations were defined as curves making at least one full revolution with a radius of 8 cm or less. Net turning scores were obtained by calculating the signed sum of all curves or rotations, respectively.

Apoptosis assay

Apoptotic cells were detected in 3% H₂O₂/10% methanol-quenched 20-m-thick coronal cryosections containing the MTN, using the TUNEL in situ cell death detection kit (Roche Applied Science). DNase I-treated sections served as positive controls and sections incubated in the absence of terminal transferase were used as negative controls.

Stastistical analyses

TH neuron numbers, striatal dopamine levels, TH fiber densities, and numbers of apoptotic cells were evaluated statistically by ANOVA and Fisher's probable least-squares difference test. Behavior was analyzed with the Mann–Whitney U test.

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Acknowledgements

We thank Lucja Kierat (Children's Hospital, University of Zurich) for HPLC analysis of mouse striatal dopamine levels, Fritz Ochsenbein for preparing the figures, and Jean-Charles Paterna for critically reading the manuscript. This work was supported by grants from the Swiss National Science Foundation to H.B. and H.P.L. and by the National Center for Competence in Research on Neural Plasticity and Repair.