Brain, Vol. 123, No. 7, 1365-1379,
July 2000
© 2000 Oxford University Press
Embryonic ventral mesencephalic grafts improve levodopa-induced dyskinesia in a rat model of Parkinson's disease
1 Neurodegenerative Disorders Centre, Vancouver Hospital and Health Sciences Centre, Vancouver, Canada and 2 Wallenberg Neuroscience Center, University of Lund, Lund, Sweden
Correspondence to:
Dr Chong S. Lee, Neurodegenerative Disorders Centre, Purdy Pavilion, 2221 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5 E-mail: cslee{at}interchange.ubc.ca
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Abstract |
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We investigated the role of dopamine neurons in the manifestation of levodopa-induced dyskinesia in a rat model of Parkinson's disease. Daily treatment with a subthreshold dose of levodopa gradually induced abnormal involuntary movements (AIM) in 6-hydroxydopamine-lesioned rats, which included stereotypy and contraversive rotation. After 4 weeks of levodopa treatment, rats with mild and severe AIM were assigned to two treatment subgroups. The graft subgroup received embryonic ventral mesencephalic tissue into the striatum, whilst the sham-graft subgroup received vehicle only. Rats continued to receive levodopa treatment for 3 months post-graft. Brain sections at the level of the basal ganglia were processed for autoradiography using a ligand for dopamine transporter, and in situ hybridization histochemistry for mRNAs encoding postsynaptic markers. Levodopa-induced AIM significantly improved in grafted rats. The severity of AIM correlated inversely with the density of dopamine nerve terminals in the striatum (P < 0.001), with almost no AIM when the density of dopamine nerve terminals was >10–20% of normal. Embryonic dopamine neuronal grafts normalized not only mRNA expression for preproenkephalin (PPE) in the indirect pathway, but also mRNA expression for prodynorphin (PDyn) in the direct pathway, which was upregulated by levodopa treatment. AIM scores correlated linearly with expression of PPE mRNA in the indirect pathway (P < 0.001) and also with PDyn mRNA in the direct pathway (P < 0.001). We conclude that embryonic dopamine neuronal grafts may improve levodopa-induced dyskinesia by restoring altered activities of postsynaptic neurons, resulting not only from dopamine denervation, but also from levodopa therapy, provided that the density of striatal dopaminergic nerve terminals is restored above a `threshold' level.
Parkinson's disease; 6-OHDA; levodopa-induced dyskinesia; embryonic dopamine neuronal graft
AIM = abnormal involuntary movements; GAD = glutamic acid decarboxylase; ISHH = in situ hybridization histochemistry; 6-OHDA = 6-hydroxydopamine; PDyn = prodynorphin; PPE = preproenkephalin; VM = ventral mesencephalic
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Introduction |
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Various types of dyskinesias may manifest in patients with Parkinson's disease. Certain dyskinesias are provoked by low or intermediate levels of dopaminergic stimulation (Marsden et al., 1982
). However, the most common type of dyskinesia appears following administration of levodopa, in the form of chorea or dystonia, at peak dose or throughout the `on' time. This levodopa-induced, peak dose dyskinesia presents in 30–80% of patients who are treated with levodopa, and increases in frequency and severity with progression of disease and duration of treatment (Marsden et al., 1982; Nutt, 1990). Since the early stage of the levodopa era, it has been recognized that levodopa-induced dyskinesia generally occurs in patients with a good therapeutic response to levodopa (Cotzias et al., 1967); it appears only after chronic treatment with levodopa, not with the first dose of levodopa; and it seems to appear earlier and be worse on the more severely affected side in asymmetrically affected patients (Mones et al., 1971; Kempster et al., 1989; Nutt, 1990). These clinical observations, in agreement with animal studies (Bedard et al., 1986; Clarke et al., 1989), indicate that both chronic levodopa therapy and nigrostriatal dopaminergic denervation play an important role in the pathogenesis of levodopa-induced dyskinesia.
In an earlier experiment, we studied the effects of levodopa treatment in the rat with 6-hydroxydopamine (6-OHDA) lesions, in which the severity of dopaminergic denervation was static (Cenci et al., 1998). In this study, we explored the role of dopamine neurons in the pathogenesis of levodopa-induced dyskinesia using a grafting technique to increase the density of dopaminergic nerve terminals in the striatum. We report here our observations that suggest therapeutic effects of intrastriatal embryonic dopamine neuronal grafts on levodopa-induced dyskinesia.
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Material and methods |
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Female Sprague–Dawley rats, weighing 200–250 g at the time of lesion surgery, were given unilateral stereotaxic injections of 6-OHDA in the right ascending mesostriatal dopamine bundle. Two injections of 7.5 and 6 µg of free-base 6-OHDA (3 µg/µl in 0.02% ascorbate in saline) were given over 3–4 min under Equithesin anaesthesia [3 ml/kg body weight, intraperitoneally (i.p.)] at the following coordinates (in millimetres, relative to bregma and the dural surface): A = -4.4, L = 1.2, V = 7.8, tooth bar = -2.4; A = -4.0, L = 0.75, V = 8.0, tooth bar = +3.4. The toxin was infused at a rate of 1 µl/min, and the cannula was left in place for 3 min before being withdrawn. To determine lesion efficacy, rats were tested 1–2 weeks later with an automated rotometer following an injection of D-amphetamine (5 mg/kg, i.p.). Twenty-eight rats that displayed >7 ipsilateral rotations/min for 90 min were selected for the study.
Grafting
Pre-graft induction period
Six to eight weeks after 6-OHDA lesions, the rats started to receive 8 mg/kg of L-dopa methyl ester (Sigma, St Louis, Mo., USA), mixed with 15 mg/kg of benserazide (kindly provided by Hoffmann-La Roche, Basel, Switzland), i.p. twice daily for the first 3 weeks. In the fourth week, the dose of L-dopa methyl ester was increased to 12 mg/kg (plus 15 mg/kg of benserazide) to increase the number of rats with severe abnormal involuntary movements (AIM). Behavioural tests for AIM were carried out twice a week. At the end of the induction period, 23 rats that had developed mild to severe AIM were divided into two groups based on their final AIM scores. Group A included 13 rats with severe AIM (AIM score >20), and group B included 10 rats with mild AIM (AIM score 
20). The rats in each group were assigned to the graft and the sham-graft subgroups, at random, subject to approximately equal average AIM scores in the two subgroups.
Graft surgery
The rats assigned to the graft subgroup received grafts of developing ventral mesencephalic (VM) tissue, obtained from 14-day-old rat embryos (crown–rump length 11 mm) of the same strain in the form of a cell suspension (Bjorklund et al., 1983). Each recipient rat received two 2 µl deposits of the graft suspension into the dopamine-denervated striatum at the following coordinates (in millimetres, relative to bregma and the dural surface, and with the tooth bar set 2.3 mm below the interaural line): (i) A = 1.6, L = 3.2, V = 4.5; (ii) A = 0.6, L = 3.5, V = 4.5. The rats assigned to the sham-graft subgroup received cell-free vehicle only. The identification numbers of the rats were changed by altering their earmarks during the grafting surgery. The examiner of their behaviour was blinded to the new identification numbers until the end of the experiment.
Post-graft period
One week after the grafting surgery, treatment with levodopa was resumed with the same dose (12 mg/kg of L-dopa mesylate ester with 15 mg/kg of benserazide) but less frequently, twice weekly, for the following 12 weeks. The frequency of levodopa administration was reduced during the post-graft period to protect rats from developing excessive AIM, particularly masticatory dyskinesia, which could be self-mutilating. Behavioural measurements were carried out once a week. In the 13th week post-graft, rats were challenged by an increase in the dose or the frequency of the doses of levodopa. Rats in group B were given 24, 48, 96 and 192 mg/kg of levodopa (each mixed with 15 mg/kg of benserazide) every second day. Rats in group A were given 12 mg/kg of levodopa once daily during the 13th week. The rats were sacrificed for autoradiographic studies 3 days after the last dose of levodopa.
Behavioural measurements
AIM rating scale
Levodopa-induced AIM, which include stereotypy and contraversive rotation, were quantified using the rat AIM rating scale (Table 1). Stereotypy was classified into three subtypes based on the topographic distribution: limb dyskinesia, axial dystonia, and masticatory dyskinesia. Suppressibility of levodopa-induced AIM was tested by challenging the rat with novel sensory input, such as touching the rat or tilting the cage. The rats were assessed before injection of levodopa and every 35 min for 140 min after injection of levodopa. The AIM score was a sum of all measurements from sequential assessments after injection of levodopa. The maximum attainable AIM score was 64.
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Autoradiographic studies for ligand binding and in situ hybridization
Tissue preparation
Three days after the last dose of levodopa, the animals were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and killed by decapitation. The brains were removed, frozen immediately on dry ice and stored at -80°C. Serial sections (16 µm thick) through the basal ganglia were cut coronally on a cryostat and mounted onto chrome alum-treated glass slides (six sections per slide). The slide-mounted sections were stored at –20°C.
Dopamine transporter autoradiography
To assess the density of dopaminergic nerve terminals in the striatum, one series of sections was incubated with tritiated N-1-2-benzo(b)thiophenyl-cyclohexyl-piperidine ([3H]BTCP) that binds to plasma membrane dopamine transporter (Vignon et al., 1988; Filloux et al., 1989). The slide-mounted sections were first washed for 20 min at 4°C in 50mM Tris–HCl buffer, pH7.0, containing 200 mM NaCl, and subsequently incubated for 90 min in the same buffer containing 1 nM [3H]BTCP (specific activity 50 Ci/mmol; NEN DuPont, Boston, Mass., USA). Control sections were incubated with [3H]BTCP in the presence of 100 µM cocaine to determine the non-specific binding. At the end of the incubation, the slide-mounted sections were washed four times for 6 min each time in ice-cold 50 mM Tris–HCl/200 mM NaCl buffer, dried under a cold air stream and apposed to tritium-sensitive film (3H Hyperfilm, Amersham, Arlington Heights, Ill., USA) at 4°C for 6 weeks. The films were developed in Kodak D19, fixed and dried.
Preparation of probes for in situ hybridization histochemistry (ISHH)
The probes were oligodeoxyribonucleotides (Scandinavian Gene Synthesis, Köping, Sweden) complementary to nucleotides 322–360 of the cloned preproenkephalin (PPE) cDNA (Howells et al., 1984), also known as PPE-A (Duty and Brotchie, 1997), nucleotides 934–982 of the cloned prodynorphin (PDyn, or proenkephalin B) gene (Civelli et al., 1985), and nucleotides coding for amino acids 389–405 of feline glutamic acid decarboxylase (GAD) (Kobayashi et al., 1987). Oligonucleotides (0.2 µM) were labelled at the 3' end with 4 µM [
-35S]dATP (>37 TBq/mmol; Amersham) using 25–30 U of terminal deoxynucleotidyltransferase (TdT; Amersham) for 2 h at 37°C. The labelled probes were purified on NENSORB 20 nucleic acid purification cartridges (NEN DuPont) to specific activities of >109 c.p.m./µg. Specificity of hybridization was determined by competition experiments where the 35S-labelled probe was diluted with an excess of either the same unlabelled oligonucleotide or an unlabelled unrelated oligonucleotide. The specificity of the oligonucleotide probes was determined by Northern blot analysis, homology screens of the sequences in GenBank and comparisons of the anatomical distributions with previous studies attempting to localize the same mRNA transcripts.
Hybridization
All solutions and buffers were prepared with distilled water treated with diethyl-pyrocarbonate (Sigma) to inhibit RNase activity and then autoclaved. The slide-mounted sections were air dried and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 10 min, then rinsed three times in phosphate-buffered saline, 5 min each, before dehydration. The sections were dehydrated in a series of ascending concentrations of ethanol, and air dried. The slides were then incubated with the hybridization mixture, which comprised 50% formamide (deionized), 4x SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium citrate), 1x Denhardt's solution (0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 10 mg/ml of RNase-free bovine serum albumin), 1% sarcosyl, 10% dextran sulphate, 500 µg/ml sheared and denatured salmon sperm DNA, 200 mM dithiothreitol and 107 c.p.m./ml of 35S-labelled oligonucleotide probe. Approximately 50 µl of hybridization cocktail was added to each section. Slides were coverslipped with parafilm and incubated overnight (16–18 h) at 42°C in a humid chamber. After hybridization, the parafilm coverslips were floated off in 1x SSC at 55°C, and the sections were given four washes (15 min each) in 1x SSC at 55°C, plus a final wash beginning at 55°C and cooling to room temperature. The slides were then rinsed twice in distilled water, dehydrated in ethanol as above and exposed to autoradiographic film (Bmax, Amersham) at 20°C for 10–21 days. The films were developed in Kodak D-19 for 4 min and fixed.
Quantitative image analysis
Autoradiographic images were digitized and analysed by the image analysis program, Image (Wayne Rasband, NIMH). The optical density was converted to the intensity of radioactivity using 14C standards (Amersham) for ISHH or 3H standards (Amersham) for [3H]BTCP binding exposed on the same film (Miller, 1991). Striatal expression of [3H]BTCP binding or neuropeptide mRNA was analysed in 4–5 sections of the neostriatum, rostrocaudally at levels 10–18 according to the atlas of Paxinos and Watson (Paxinos and Watson, 1986).
Statistical analysis
In order to assess the effects of embryonic VM grafts during the post-graft period, the rat AIM scores were analysed on time and the treatment subgroup (grafted rats versus sham-grafted rats) in group A, and again in group B, using repeated measures analysis of variance. The last AIM scores during the pre-graft period were used as pre-graft baseline covariates. Effects of challenge tests (by higher or more frequent doses of levodopa) were evaluated by analysing AIM scores during the 13th week post-graft on the dose of levodopa and the treatment subgroup in group A, and again in group B, using repeated measures analysis of variance. The AIM scores in the 12th week post-graft were used as baseline covariates. To assess the effects of different doses of levodopa on the severity of AIM in the sham-grafted rats (in which the density of dopaminergic nerve terminals was not significantly different between groups A and B), the final post-graft AIM scores of group B (manifested by 192 mg/kg of levodopa) were compared with pre-graft baselines of the same group (manifested by 12 mg/kg of levodopa) by a paired t-test, and also with the final post-graft AIM scores of group A (manifested by 12 mg/kg of levodopa) by a two-sample t-test.
Signal intensity of [3H]BTCP binding or mRNA hybridization on the autoradiographic films was expressed as a ratio of the value in the right striatum or globus pallidus (lesioned side) to the value in the left striatum or globus pallidus (unlesioned control side), respectively. The logarithmic transform of the right/left ratio of [3H]BTCP binding was compared among four subgroups (grafted rats and sham-grafted rats in groups A and B) by analysis of variance, followed by Fisher's multiple comparisons. The logarithmic transforms of the right/left ratios of PPE, PDyn, GAD67 mRNAs in the striatum or GAD67 mRNA in the globus pallidus were analysed similarly. The relationship between AIM scores and a presynaptic marker was analysed by regression of AIM scores on the logarithm of [3H]BTCP binding. The logarithmic transformations were successful in symmetrizing the data in all cases. Relationships between AIM scores and postsynaptic markers were analysed by regression of AIM scores on the expression of mRNAs. The data were expressed as the mean ± standard deviation unless specified otherwise. Statistical significance level was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Characterization of levodopa-induced AIM in a rat model of Parkinson's disease
Pulsatile treatment with a subthreshold dose of levodopa gradually induced stereotypy or contraversive rotation in rats with unilateral 6-OHDA lesions, which were not observed in normal rats or in the 6-OHDA-lesioned rats that received vehicle only. Levodopa-induced stereotypy was observed only on the side contralateral to 6-OHDA lesions. Maneouvres for testing suppressibility of levodopa-induced movements evoked an arrest reaction in normal rats, but not in rats with severe levodopa-induced abnormal movements. Rats with mild to moderate levodopa-induced movements suppressed abnormal movements briefly, only to resume the same movements after pauses of variable length.
The motor pattern of each topographic subtype was highly stereotypic across individual rats as well as serial observations of the same rat. However, the proportion of each subtype was not uniform among individual rats. Furthermore, the manifestation of a motor pattern was influenced by the position of the rat: contraversive rotation was predominant in a quadrupedal position, whereas stereotypy was predominant in the bipedal sitting or standing position (Fig. 1). This mutually exclusive relationship of the two distinctive motor patterns was illustrated in the time–action curve of AIM subtypes, and of rotometer measurements (Fig. 2).
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Effects of embryonic dopamine neuronal grafts on levodopa-induced AIM
Pre-graft induction period
Among the 28 rats that received pulsatile treatment with levodopa, 23 developed mild to severe AIM by the end of the fourth week during the induction period. Five rats did not develop AIM. The onset of AIM was highly variable among individual rats (9.5 ± 7.9 days). The final AIM scores were also markedly variable. In group A, the mean final pre-graft AIM score was 40.9 ± 11.9 for the graft subgroup and 41.8 ± 10.2 for the sham-graft subgroup. There were no significant differences in the final pre-graft AIM score between the two treatment subgroups. In group B, the mean final pre-graft AIM score was 12.6 ± 3.8 for the graft subgroup and 14.0 ± 6.9 for the sham-graft subgroup, with no significant differences between the two treatment subgroups.
Post-graft period
Figure 3 illustrates the temporal courses of AIM scores in each treatment subgroup before and after grafting embryonic VM tissue. Repeated measures ANOVA indicated a highly significant time x treatment interaction [F(9,99) = 9.39, P < 0.0001 for subgroup x time]. In particular, specific time points were compared between the two subgroups, adjusting for multiple comparisons. In the rats with severe pre-graft AIM (group A), the AIM scores became increasingly and significantly different between the graft and sham-graft treatment subgroups from the 5th week post-graft onwards [F(1,11) = 8.09, P = 0.016 for subgroups; F(9,99) = 20.42, P < 0.0001 for time]. In the 12th week post-graft, the mean AIM score was 24.7% of pre-graft baseline in the grafted rats, and 73.8% of pre-graft baseline in the sham-grafted rats. In the rats with mild pre-graft AIM (group B), AIM scores were significantly reduced in both grafted and sham-grafted rats during the 12 weeks after grafting (P = 0.002). There were, however, no significant differences between the grafted and sham-grafted rats. Challenge tests with higher doses of levodopa during the 13th week post-graft (group B) increased AIM scores significantly in both grafted and sham-grafted rats (to 7.8 ± 5.3 and 19.6 ± 12.2, respectively, P = 0.007 for both subgroups), without significant differences between the two treatment subgroups. In the sham-grafted rats, the final post-graft AIM scores, which were manifested by 192 mg/kg of levodopa, were not significantly different from the final pre-graft AIM scores that were manifested by 12 mg/kg of levodopa (P = 0.64). In contrast to group B, daily administration of the same dose of levodopa (12 mg/kg) did not change AIM scores significantly in group A (9.0 ± 9.9 for grafted rats, 32.3 ± 10.8 for sham-grafted rats). The final post-graft AIM scores in the sham-grafted rats of group A were still significantly greater than those of group B (P = 0.049), despite the fact that the latter were manifested by a 16-fold higher dose of levodopa than the former.
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Effects of dopaminergic neuronal grafts on presynaptic and postsynaptic markers
Figure 4
illustrates autoradiographic images of [3H]BTCP binding and in situ hybridization for mRNA encoding PPE, GAD67 or PDyn in the striatal or pallidal sections. Results of quantitative analysis for presynaptic and postsynaptic markers are summarized in Table 2. In group A, the mean density of striatal [3H]BTCP binding was significantly greater in the grafted rats than in the sham-grafted rats (P < 0.001). Analysis of group B showed similar results (P < 0.001). [3H]BTCP binding in the sham-grafted rats did not show significant differences between groups A and B, contrary to the final post-graft AIM scores (P = 0.81). The mean expression of PPE mRNA was significantly less in the grafted rats than in the sham-grafted rats in group A (P < 0.001). Group B showed analogous results (P < 0.001). The mean expression of PDyn mRNA was significantly less in grafted rats than in sham-grafted rats (P < 0.001 for both groups A and B). The mean expression of GAD67 mRNA in the striatum (measured over the entire caudate nucleus and putamen, rostral to the globus pallidus) or GAD67 mRNA in the globus pallidus was also significantly less in the grafted rats compared with the sham-grafted rats (striatal GAD67 mRNA, P < 0.001 for both groups A and B; pallidal GAD67 mRNA, P = 0.009 for group A, P = 0.006 for group B).
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Relationship of the severity of levodopa-induced AIM to presynaptic or postsynaptic markers
In group A, the final post-graft AIM scores correlated inversely with the logarithm of [3H]BTCP binding (P < 0.001, Fig. 5A
). AIM scores were markedly reduced when [3H]BTCP binding was >10–20% of that of the control side. Analysis of group B showed similar findings (Fig. 5B). The magnitude of increase in AIM scores of group B during the challenge test correlated inversely with the logarithm of [3H]BTCP binding (P = 0.016, Fig. 5C).
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Expression of PPE mRNA correlated inversely with the logarithm of [3H]BTCP binding (P < 0.001, Fig. 6A
). PPE mRNA expression was normalized when [3H]BTCP binding was >10–20% of that of control. Analysis of PDyn mRNA and striatal GAD67 mRNA showed similar findings (P < 0.001 for both, Fig. 6B and C). Expression of GAD67 mRNA in the globus pallidus also correlated inversely with the logarithm of [3H]BTCP binding (P = 0.002, Fig. 6D).
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The final post-graft AIM scores in group A showed significant linear correlation with PPE mRNA, PDyn mRNA, striatal GAD67 mRNA and GAD67 mRNA in the globus pallidus (Fig. 7
). Analysis of the final post-graft AIM scores in group B also showed significant correlation with mRNA encoding PPE, PDyn, GAD67 in the striatum or GAD67 in the globus pallidus (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Levodopa-induced AIM of the rat
Pulsatile treatment with a low dose of levodopa gradually induced stereotypy and contraversive rotation in the 6-OHDA-lesioned rats. The involuntary nature of these levodopa-induced abnormal movements was inferred by their self-injuring tendency, and by their inability to suppress these abnormal movements when challenged by novel sensory stimuli (Walters et al., 1990
). Levodopa-induced stereotypy was classified into three subtypes based on the topographic distribution. Similar to levodopa-induced dyskinesia in patients with Parkinson's disease (Marsden et al., 1982; Hughes et al., 1994), the relative proportion of each topographic subtype varied considerably among individual rats, yet it was consistent through serial observations in the same rat. Earlier studies have shown that stereotypy is mediated primarily through the neostriatum and the globus pallidus, whereas the locomotor activity also involves the nucleus accumbens and ventral pallidum (Kelly et al., 1975; Scheel-Kruger, 1984; Sharp et al., 1987). Microinjection of a dopamine agonist into the ventrolateral striatum generates orofacial or limb stereotypy (Kelley et al., 1989; Amalric and Koob, 1993; Dickson et al., 1994). Thus, it seems that the basal ganglia are topographically organized in the rat, as has been observed in primates (DeLong et al., 1984; Alexander et al., 1986). Assessment of both topographic subtypes of stereotypy and locomotor activity, therefore, would enhance the sensitivity of detecting behavioural changes resulting from pathology in the neostriatum.
A rotometer has been used traditionally for measurement of drug-induced rotation in rats (Ungerstedt and Arbuthnott, 1970). We have demonstrated a competitive relationship between stereotypy and contraversive locomotor activity for the behavioural expression, as described previously (Koob et al., 1984; Rebec and Bashore, 1984; Sharp et al., 1987). Although rats are quadrupedal animals using both forelimbs and hindlimbs for locomotor activity, we observed that they use forelimbs for handling food in a bipedal sitting position. Hence, the forelimbs are involved in two different motor programmes, i.e. locomotor and feeding activities, which cannot be executed simultaneously. These behavioural characteristics of normal rats resemble the position-dependent appearance of stereotypy or locomotor activity in levodopa-treated rats with 6-OHDA lesions. Increased locomotor activity with contraversive directional bias in the rat with unilateral 6-OHDA lesions, which was induced by repetitive levodopa treatment, was expressed predominantly in the quadrupedal position, involving both forelimbs. In contrast, the limb dyskinesia was expressed predominantly in the bipedal sitting or standing position, involving only one forelimb on the side contralateral to the dopamine-denervated striatum. This position-dependent switching of the predominant motor pattern explains inconsistent, multiple peaks in the time–action curve of rotometer measurements in the rats that have both severe stereotypy and contraversive locomotor activity (as shown in Fig. 2C
). In order to measure the maximum intensity of stereotypy or locomotor activity, rats were forced into a bipedal sitting or standing position for expression of stereotypy, and into a quadrupedal position for expression of locomotor activity. This study shows that stereotypy and contraversive locomotor activity, induced by repetitive levodopa treatment, share the same characteristics as levodopa-induced dyskinesia in patients with Parkinson's disease. Both are abnormal involuntary movements, and appear during the course of levodopa treatment. Both show similar time courses of development: a peak-dose response after administration of levodopa, and gradual enhancement of the peak-dose response with repetitive levodopa treatment. Thus, we consider levodopa-induced stereotypy and contraversive locomotor activity to be a rat version of levodopa-induced, peak-dose dyskinesia.
Effects of embryonic dopamine neuronal grafts
On presynaptic markers
In the grafted rats, the mean density of dopaminergic nerve terminals measured by [3H]BTCP binding was 21.0% (range: 6.72–42.17%) of the contralateral control side, significantly greater than the 4.8% mean of the control in the sham-grafted rats. Earlier studies have shown that 6-OHDA lesions produce ~97–99% reduction of striatal tissue dopamine (Schmidt et al., 1982), and that embryonic VM grafts increased striatal tissue dopamine on average to 13–18% of normal, with the highest individual values reaching ~50% (Schmidt et al., 1983). Thus, the ~15% increase of dopaminergic nerve terminals by embryonic VM grafts in this study is comparable with previous results in the rat.
On postsynaptic markers
Nigrostriatal dopaminergic denervation upregulates expression of PPE mRNA and GAD67 mRNA, but not PDyn mRNA in the rat with 6-OHDA lesions (Normand et al., 1988; Gerfen et al., 1990; Li et al., 1990; Chesselet et al., 1993). Repetitive levodopa treatment upregulates PDyn mRNA (Cenci et al., 1998). Consistent with these earlier observations, the sham-grafted striatum showed upregulation of PPE mRNA, GAD67 mRNA and PDyn mRNA. In contrast, the grafted striatum showed near-normalized levels of PPE mRNA, GAD67 mRNA and PDyn mRNA, in agreement with previous reports (Cadet et al., 1991; Bal et al., 1993; Mendez et al., 1993; Roy et al., 1995; Cenci et al., 1997). Correlation analysis has shown that PPE mRNA was normalized when the density of dopaminergic nerve terminals was >10–20% of that of control. These observations are in keeping with previous studies in the rat with partial 6-OHDA lesions (Chritin et al., 1996). Levodopa-induced upregulation of PDyn mRNA was normalized in the grafted striatum when the density of dopaminergic nerve terminals was >10–20% of that of control. These observations indicate that upregulation of PDyn mRNA (which is induced by repetitive levodopa treatment) requires a certain level of dopamine terminal loss, as in the case of upregulation of PPE mRNA (that is induced by dopaminergic denervation). In the sham-grafted rats, expression of pallidal GAD67 mRNA was upregulated, in agreement with previous reports (Kincaid et al., 1992; Delfs et al., 1995). In the grafted rats, expression of pallidal GAD67 mRNA was near normal, suggesting that the grafted dopaminergic neurons in the striatum normalized upregulated expression of GAD67 mRNA in the globus pallidus. These observations are consistent with recent reports that intrastriatal VM grafts normalize neuronal activity not only in the target area, but also in remote areas, the globus pallidus and output nuclei of the basal ganglia (Nakao et al., 1998). Taken together, our data show that dopamine neuronal grafts normalize PPE mRNA and GAD67 mRNA in the indirect pathway and PDyn mRNA in the direct pathway if the density of dopaminergic nerve terminals is greater than a `threshold' level (which is 10–20% of normal, measured by [3H]BTCP).
On motor behaviours
AIM scores in the grafted rats began to fall during the second month post-graft. The delayed functional effects of dopaminergic neuronal grafts have been observed in the rat (Dunnett et al., 1983, 1987; Blunt et al., 1991) and in patients with Parkinson's disease (Lindvall et al., 1992; Kordower et al., 1995; Defer et al., 1996; Kopyov et al., 1996). There was also a modest, but significant reduction of AIM scores in the sham-grafted rats. This reduction may result from reduced frequency of levodopa administration during the post-graft period. Anatomical studies have not consistently shown evidence for graft-induced axonal sprouting in the host striatum (Kordower et al., 1995, 1998). The inverse correlation between the final AIM scores and the density of surviving dopaminergic nerve terminals suggests that dopaminergic neuronal grafts predominantly contributed to the improvement of levodopa-induced AIM during the post-graft period. Previous studies have also shown that the functional effects of grafts are dependent on the number of grafted dopamine neurons (Lindvall, 1994; Kopyov et al., 1997).
Factors influencing the severity of levodopa-induced AIM
Presynaptic dopaminergic nerve terminals
In the previous section, we have shown that the severity of AIM was inversely correlated with the density of surviving dopaminergic nerve terminals. Levodopa-induced AIM almost disappeared when the density of dopaminergic nerve terminals was >10–20% of that of control. These observations are consistent with the view that dopaminergic denervation is required for the manifestation of levodopa-induced dyskinesia, as has been observed in primates (Boyce et al., 1990) and in patients with Parkinson's disease (Nutt, 1990).
The dose of exogenous levodopa
Challenge tests (in group B) showed that higher doses of levodopa increased the severity of AIM in both sham-grafted and grafted rats. The magnitude of increased AIM scores correlated inversely with the density of surviving dopaminergic nerve terminals. Thus, our data suggest that the severity of levodopa-induced AIM is an outcome of interplay between the dose of levodopa and the density of surviving dopaminergic nerve terminals. These observations are consistent with the concept that dopaminergic neuronal loss lowers the threshold for the expression of levodopa-induced dyskinesia (Nutt, 1990). However, in sham-grafted rats, the final post-graft AIM scores remained significantly lower in group B (which received 192 mg/kg of levodopa) than in group A (which received 12 mg/kg of levodopa). As the density of dopaminergic nerve terminals was not significantly different between groups A and B, these observations suggest that the severity of levodopa-induced AIM is also determined by other factors in addition to the dose of levodopa and the density of dopaminergic nerve terminals.
Role of neurotransmitters in postsynaptic neurons
Consistent with our previous observations (Cenci et al., 1998) as well as those of others (Brotchie, 1999), the severity of AIM correlated with the expression of PPE mRNA in the indirect pathway, and also with the expression of PDyn mRNA in the direct pathway. The mRNAs are changed in a manner that precedes and is proportional to the changes in product peptide levels (Li et al., 1988; Gerfen et al., 1990; Engber et al., 1991), suggesting that expression of mRNA may reflect the rate of synthesis of corresponding product peptide. Enkephalin inhibits the release of excitatory amino acids from corticostriate fibres in the striatum (Jiang and North, 1992), and inhibits the release of GABA in the globus pallidus (Dewar et al., 1987; Maneuf et al., 1994). Thus, enkephalin in striatopallidal neurons simulates the action of D2 receptor agonists (Marin and Chase, 1995). In primates, injection of a GABA antagonist into the external globus pallidus induces dyskinesia in the monkey (Crossman et al., 1984; Matsumura et al., 1995). These observations suggest that the overactivity of enkephalin, which is induced by dopaminergic denervation, may be related to levodopa-induced dyskinesia. On the other hand, microinjection of dynorphin or a 
-opioid agonist into the substantia nigra reduces overactive glutamatergic transmission in the output nuclei of the basal ganglia (Maneuf et al., 1995) and suppresses neuronal activity in the substantia nigra pars reticulata (Lavin and Garcia-Munoz, 1986; Thompson and Walker, 1990). In the rat, microinjection of a -opioid agonist into the substantia nigra pars reticulata improves experimental parkinsonism, and induces contraversive rotation (Matsumoto et al., 1988). Upregulated expression of PDyn mRNA in the striatum correlated with the enhancement of the contraversive rotational response (Duty and Brotchie, 1997). These observations as well as ours suggest that the enhanced activity of dynorphin in the striatonigral neurons may contribute to the development of dyskinesia during levodopa treatment. Taken together, these observations suggest that levodopa-induced AIM are manifested by a synergistic action of neurotransmitters in the indirect and the direct pathways, which are upregulated by dopaminergic denervation and levodopa treatment; and that embryonic dopamine neuronal grafts may improve levodopa-induced AIM through normalization of postsynaptic neuronal activities that were altered by dopaminergic denervation and levodopa treatment. This view is consistent with clinical observations that levodopa-induced dyskinesia requires chronic levodopa therapy and dopaminergic denervation (Nutt, 1990).
In summary, using a novel quantitative rating scale for levodopa-induced AIM in the rat, we have demonstrated that embryonic VM grafts improved levodopa-induced AIM. Clinical observations, however, have not been consistent with regard to the therapeutic effects of foetal dopaminergic neuronal grafts on levodopa-induced dyskinesia (Peschanski et al., 1994; Kordower et al., 1998). This inconsistency may result from variable viability of the grafts derived from different transplantation techniques (Kordower et al., 1998; Lindvall, 1998). Indeed, post-graft improvement of the uptake rate constant (Ki) in [18F]DOPA/PET varies substantially in different studies, ranging from none to 100% of the pre-graft Ki (Lindvall et al., 1989, 1994; D. Eidelberg and J. Stoessl, personal communication). Observations in this rodent study suggest that the severity of levodopa-induced dyskinesia is determined by multiple factors: the density of presynaptic dopaminergic nerve terminals, the dose of levodopa and the activity of postsynaptic neurons in the direct and indirect pathways. Foetal dopamine neuronal grafts may improve levodopa-induced dyskinesia by normalization of the activity of neurotransmitters in postsynaptic neurons, provided that the density of striatal dopaminergic nerve terminals is restored above a `threshold' level.
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We wish to thank Alicja Flasch, Ulla Jarl and Gertrude Stridsberg for technical assistance, Edwin Mak for assistance in statistical analysis, and Ms Jennifer Norton and Mrs Susan Calne for assistance in the preparation of this manuscript. The study was supported by grants from the Pacific Parkinson Foundation (Vancouver), Swedish Medical Research Council (04X-3874) and the Göran Gustafsson Foundation.
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Received September 6, 1999. Revised January 13, 2000. Second revision on February 4, 2000. Accepted February 14, 2000.
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