Differential effects on forelimb grasping behavior induced by fetal dopaminergic grafts in hemiparkinsonian rats

www.elsevier.com/locate/ynbdi Neurobiology of Disease 27 (2007) 24 – 35

Differential effects on forelimb grasping behavior induced by fetal dopaminergic grafts in hemiparkinsonian rats Alexander Klein, a,b,⁎ Gerlinde A. Metz, b Anna Papazoglou, a and Guido Nikkhah a Laboratory of Molecular Neurosurgery, Department of Stereotactic Neurosurgery, University Hospital Freiburg – Neurocentre, Breisacher Str. 64, D-79106 Freiburg, Germany b Canadian Centre for Behavioural Neuroscience, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4 a

Received 14 January 2007; revised 10 March 2007; accepted 29 March 2007 Available online 5 April 2007

Skilled forelimb movements depend on an intact dopaminergic (DA) neurotransmission and are substantially impaired in the unilateral rat model of Parkinson’s disease. It has remained unclear, however, to what extent reaching and grasping movements can be influenced by intrastriatal transplantation of fetal DA neurons. Here an extensive behavioral assessment of skilled forelimb movement patterns in hemiparkinsonian and DA-grafted rats was carried out. Good DA graft survival was accompanied by a compensation of drug-induced rotational asymmetries. Interestingly, skilled forelimb use was significantly improved in transplanted animals as compared to lesion-only animals in the staircase test. Qualitative analysis of single forelimb reaching movement components revealed dissociable patterns of graft effects: while some movement components in grafted animals improved, others remained unchanged or even deteriorated. These findings provide novel insights into the complex interactions of graft-derived restoration of DA neurotransmission and skilled forelimb behavior. © 2007 Elsevier Inc. All rights reserved. Keywords: Motor behavior; Neurodegeneration; Neuroregeneration; Parkinson’s disease; Skilled forelimb use; Transplantation; Staircase test; Single pellet reaching test; Restoration

Introduction Transplantation of fetal dopaminergic (DA) neurons in animal models of Parkinson’s disease (PD) has been extensively used as a tool to study the mechanisms of restoration of DAergic neurotransmission and its effects on sensorimotor behavior. The morphological and functional effects of DA grafts have been mainly studied in an animal model of PD which uses a unilateral infusion of 6-hydro⁎ Corresponding author. Laboratory of Molecular Neurosurgery, Department of Stereotactic Neurosurgery, University Hospital Freiburg– Neurocentre, Breisacher Str. 64, D-79106 Freiburg, Germany. Fax: +49 761 270 5021. E-mail address: [email protected] (A. Klein). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2007.03.010

xydopamine (6-OHDA) into the medial forebrain bundle in rats, causing a complete loss of the nigrostriatal DA pathway. This, in turn, results in characteristic sensorimotor behavioral deficits including drug-induced rotational asymmetries, impaired sensorimotor orientation, and stepping behavior as well as a substantial deterioration of skilled forelimb performance. These experimentally 6-OHDAinduced behavioral deficits share significant similarities with human patients affected by idiopathic PD. Experimental and clinical studies in PD have demonstrated that impaired sensorimotor functions can be improved by embryonic DA grafts (Wenning et al., 1997; Nikkhah et al., 1998a; Hagell et al., 1999; Rodter et al., 2000; Lindvall and Hagell, 2000; Dobrossy and Dunnett, 2004; Winkler et al., 2005). In animal models of PD, DA grafts lead to a functional restoration of simple motor behavior, e.g. drug-induced rotational asymmetry (Brundin et al., 1994), spontaneous circling, and sensorimotor neglect (Dunnett et al., 1983; Dunnett et al., 1987; Mandel et al., 1990; Nikkhah et al., 1993a; Bjorklund et al., 1994). In contrast, more complex behavior such as skilled forelimb performance (e.g. in the staircase test) has been proven to be more resistant to graft-induced recovery (Dunnett et al., 1987; Montoya et al., 1990; Mandel et al., 1990; Abrous et al., 1993; Nikkhah et al., 1993a). However a partial graft-induced recovery of skilled forelimb reaching in 6-OHDA-lesioned rats could be observed only under certain conditions, e.g. after extensive microtransplantation of intrastriatal grafts (Nikkhah et al., 1993a; Winkler et al., 1999) and after transplantation in animals that do not exhibit a strong hemispheric lateralization for paw use (Nikkhah et al., 2001). Importantly, the majority of previous transplantation studies examined paw reaching performance by using the staircase test (Montoya et al., 1991; Nikkhah et al., 1994a; Whishaw et al., 1997b). Although this test permits “quantitative” analysis of end point measurements (i.e. success or failure of the number of food pellets grasped and eaten), it lacks any “qualitative” analysis of the individual reaching components that may be affected differently by lesion and transplantation surgery (Kloth et al., 2006). In this context the single pellet reaching test (Whishaw and Pellis, 1990; Miklyaeva et al., 1994; Metz and Whishaw, 2000) has been shown to provide an

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excellent scoring system to examine individual limb and digit movements based on a frame-by-frame analysis of high-speed video recordings. This behavioral testing paradigm has been used in animals with 6-OHDA lesions (Miklyaeva et al., 1994; Whishaw et al., 2002) and to describe recovery mechanisms following behavioral therapy in hemiparkinsonian rats (Vergara-Aragon et al., 2003). Therefore, this study was undertaken to quantitatively and qualitatively evaluate the functional restorative impact of fetal DA grafts on skilled forelimb reaching movements in rats with unilateral 6-OHDA lesions. Methods Experimental design All animals were daily trained in the single pellet reaching test prior to lesion. Testing in the single pellet reaching test continued throughout the experiment (47 weeks, on 5 to 6 days/week) except for a recovery period of 2 days after each surgical manipulation. The rats were subdivided into three groups: (i) lesioned and transplanted rats (n = 13; tx), (ii) lesioned and sham-transplanted rats (n = 8; sham), and (iii) non-lesioned control animals (n = 8; con). The tx and sham animals received a unilateral 6-OHDA infusion into the medial forebrain bundle (MFB) contralateral to the preferred paw. Additionally, embryonic day 14 ventral mesencephalon-derived rat progenitor cells (E14 VM-derived cells) were transplanted into the DA-depleted striatum of the tx rats. Sham animals were grafted with cell culture medium without cells. In all animals, lesion and graft effects were evaluated by rotational behavior assessment 6 weeks after the 6-OHDA lesion (6 weeks post LX) and after the transplantation of E14 VM-derived cells (6 weeks post TX). Twice before the lesion (pre 1 and pre 2), twice after the lesion (L1 and L2) and three times after the transplantation (T1–T3) the reaching behavior was video-recorded (Fig. 1). In addition the rats performed the staircase test once after the lesion (post LX) and once after the transplantation (post TX). After the last testing day the animals were perfused and their brains underwent morphological analysis. All experiments were conducted and performed according to the guidelines of the local ethical board of the University of Freiburg, Germany. Subjects Subjects were 34 adult female Sprague-Dawley rats (Charles River, Sulzfeld, Germany), weighing 250 g ± 30 g at the beginning of the experiments. Rats were housed in standard cages in groups up to five animals in a temperature-controlled room (23 ± 0.3° C) on a 12-h light/12-h dark schedule. Each animal was fed with 12 g

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standard laboratory chow (Altromin, Lage, Germany) per day and water was provided ad libitum. Rats were weighed twice weekly in order to maintain body weight at 90% of baseline values. Surgery 6-OHDA lesion All rats (except the control rats) were anesthetized with ketamine (10%; 0.1 ml/kg bodyweight; Essex, München, Germany) and rompune (2%; 0.01 ml/kg bodyweight; Bayer, Leverkusen, Germany), and were stereotactically injected with 6-OHDA hydrobromide (3.6 μg 6-OHDA/μl in 0.1% L-ascorbic acid-saline; SigmaAldrich, Steinheim, Germany) into the MFB on the side contralateral to the preferred paw (Ungerstedt, 1968). Lesion coordinates were set according to bregma and dura in mm (Paxinos and Watson, 1997; Kirik et al., 1998): tract 1: 3 μl; tooth bar (TB) + 3.4, anterior/ posterior (AP) −4.0, lateral (lat) − 0.8, dorso-ventral (DV) − 8.0; tract 2: 2.5 μl; TB −2.3, AP − 4.4, lat − 1.2, DV − 7.8. The injection rate was 1 μl/min, and the cannula was left in place for 2 min before slowly retracting it. Transplantation All lesioned rats were anesthetized and stereotactically operated a second time. The tx rats were injected with a single cell suspension of E14 VM-derived cells into the DA-depleted striatum. The E14 rat embryos were dissected, and the VMs were mechanically and enzymatically digested in order to produce a single-cell suspension. This single cell suspension was transplanted using a glass capillary (Nikkhah et al., 1994b). Transplantation coordinates were set according to bregma and dura in mm: two tracts, four deposits (1 μl each): TB 0.0, AP + 1.0, lat − 2.5/− 3.3, DV − 5/− 4. The injection rate was 1 μl/min, and the glass capillary was slowly retracted after 30 seconds. The concentration of the cell suspension was 100,000 cells/μl so that each rat received 400,000 cells in total. The cell viability remained stable between 95% and 98% during the implantation procedure. Behavioral tests Three sensorimotor behavioral tests were performed: (1) the drug-induced rotation, (2) the staircase test for the analysis of reaching success, and (3) the single pellet reaching test for the detailed analysis of individual movement components of skilled forelimb reaching pattern. Drug-induced rotation Drug-induced rotational behavior was monitored in rotometer boxes 2 and 6 weeks post lesion and post transplantation as des-

Fig. 1. Time chart of all behavioral tests and surgeries. LX = unilateral 6-OHDA MFB lesion, TX = transplantation of E14 VM-derived cells, SP = single pellet reaching test, rot = drug-induced rotation, ST = staircase test in the free choice and forced choice modes, pre LX = behavioral assessment before the lesion (pre 1 + 2 = recordings of reaching behavior in the SP), post LX = behavioral assessment after the lesion (L1 + 2 = recordings of reaching behavior in the SP), post TX = behavioral assessment after the transplantation (T1–3 = recordings of reaching behavior in the SP), perfusion = sacrifice of experimental animals and morphological analysis of brains.

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cribed by Ungerstedt and Arbuthnott (1970, Fig. 3). Data of the 2 weeks’ drug-induced rotation are not shown. Apomorphineinduced rotation (apo) was tested for 40 min after subcutaneous injection of 1 ml/kg apomorphine solution (Sigma-Aldrich: 0.05 mg apomorphine + 0.2 mg L-ascorbate in 1 ml 0.9% saline). About 3 days later, amphetamine-induced rotation (amph) was tested for 90 min after intraperitoneal injection of 1 ml/kg amphetamine solution (Sigma-Aldrich: 2.5 mg D-amphetamine per 1.0 ml saline). Five animals were excluded from the study because they showed b 4.0 full body turns contralaterally to the lesioned side after apomorphine injection and b 6.0 full body turns ipsilaterally to the lesioned side after amphetamine injection. Apomorphine-induced rotation is presented as net rotation in negative values, and amphetamine-induced rotation is presented as net rotation in positive values. Staircase test The staircase test (Montoya et al., 1990) was used to evaluate skilled forelimb use to distinguish between the grasping performance of the unaffected (ipsilateral) and the impaired (contralateral) paw individually (Fig. 2A + B). A modified set-up of the staircase test was used as described previously by Nikkhah et al. (1998a,b). Individual rats were put in a box to reach for food pellets (BioServ, Frenchtown, NJ, USA) placed on two staircases with six steps each. They were able to grasp food pellets on the right staircase only with their right forepaw and on the left staircase only with their left forepaw. Right and left staircases were baited with ten pellets on each of the steps 2 to 5 (in total 40 pellets per staircase). After a period of 15 min the animals were removed from the staircase box and the remaining pellets were counted. Test performance was reflected by two parameters: pellets remained for pellets left on the steps they had originally been placed on, and pellets missed for pellets dropped elsewhere, i.e. on step 1 or 6 or outside the staircases in other parts of the staircase box. These results were used to

calculate other three parameters pellets eaten (40 − [pellets remained + pellets missed]), pellets taken (40 − pellets remained), and grasping success (in %; [pellets eaten / pellets taken] * 100). While the parameter pellets taken is considered to be a measure for general reaching activity and motivation, the parameter pellets eaten rather reflects the rats’ forelimb skills in reaching and grasping movements and is thus directly dependent on the rats’ sensorimotor performance status. Furthermore, the parameter pellets eaten expresses the ratio of the number of pellets eaten to the total number of pellets (40 pellets per staircase). The parameter grasping success shows the success of all attempts to grasp a pellet. A test session consisted of 9 days (d) within which the staircase test was repeated: the acquisition phase to the test set-up (data not shown) took place during d1–d3, and the free choice test was performed during d4–d6 when the steps of both staircases were filled with pellets. During the forced choice test the pellets were placed on the steps first of the side of the non-preferred paw and then of the side of the preferred paw. This test was carried out on d7–d9. During the forced choice test rats were tested twice 15 min for each side per day (data not shown). Single pellet reaching test This test (Fig. 2C + D) was used to analyze individual components of reaching and grasping patterns based on high-speed video recordings (Whishaw and Pellis, 1990; Whishaw et al., 1997a; Metz and Whishaw, 2000). Individual rats were put in a Plexiglas box (13 cm × 45 cm × 40 cm) and they were expected to extend their preferred paw to retrieve food pellets (BioServ). Attached to the front wall of the box was a shelf to hold single food pellets in an indentation that was located contralateral to the rats’ preferred paw. The rats were trained to extend their forelimb through a slit in the front wall to grasp and eat one pellet at a time. They were also trained to walk to the rear end of the box after each reach in order to readjust their body position before the next attempt to grasp a pellet. Rats that

Fig. 2. Photographs of the staircase test (A + B) and the single pellet reaching test (C + D). Note that the rats typically grasp the food pellets in the staircase box on both sides during the 15-min testing period and that the pellets taken and eaten are counted only afterwards (quantitative endpoint measurement). In the single pellet reaching test (C + D) the movement components for each single reach are recorded by high-speed video recordings and the performance of each movement component is rated afterwards based on a scoring system (qualitative endpoint measurement). See also the section on methods for detailed descriptions.

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showed severe catalepsy after 6-OHDA lesion were allowed to remain at the front of the box. Prior to testing and 6-OHDA lesion all the rats were trained for 16 weeks until they reached stable success rates in grasping food pellets. After lesion and transplantation the animals were trained five to six times per week. Two (pre 1) and one (pre 2) weeks before the lesion (=baseline performance), six (L1) and ten (L2) weeks after the 6-OHDA lesion, and two (T1), nine (T2) and twenty-one (T3) weeks after the transplantation the reaching behavior was recorded with a video camera with a shutter speed of 1000/s (Panasonic, NV-MX300). The tapes were analyzed frame by frame. A successful reach was recorded if a rat obtained a food pellet at its first attempt. Three successful reaches were scored for each animal. In case an animal was cataleptic, three reaching attempts were scored. The reaching movements were scored according to the scale as described by Metz and Whishaw (2000). Each reaching movement was divided into eleven movement components: 1. orient, 2. limb lift, 3. digits close, 4. aim, 5. advance, 6. digits

Fig. 3. Drug-induced rotation after apomorphine (A) and after amphetamine (B) injections 6 weeks post lesion and 6 weeks post transplantation. Data are presented as group means ± S.E.M.; p was set at b0.05 as level of significance: (€) indicates a significant difference between either the sham or the tx animals and the con animals. (#) marks a significant difference between the sham and the tx animals after transplantation (post TX). Note that post TX the rotational bias of tx animals was reduced after apomorphine challenge (A) and that the tx animals overcompensated to the ipsilateral side after amphetamine challenge (B) indicating good graft survival and graftderived functional recovery.

Fig. 4. TH immunohistochemistry of brain sections at the coordinate AP +1.0 of control (A), sham-transplanted (B), and transplanted (C) animals. Panel D displays the results of the quantitative assessment of graft integration and fiber outgrowth for the ipsilateral and contralateral striata. Data are presented as group means ± S.E.M.; p was set at b0.05 as level of significance: (€) indicates a significant difference between the ipsilateral and the contralateral striata for each group. (#) marks a significant difference on the ipsilateral side between the sham and the tx animals. cc = corpus callosum, str = striatum, g = graft.

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cold 0.1 M phosphate-buffered (pH 7.4) saline (PBS) and then with 4% paraformaldehyde (PFA; Sigma). After post-fixation overnight in PFA the brains were dehydrated in 30% sucrose. With a freezing microtome (Leica, Nussloch, Germany) the brains were cut in four series of coronal sections of 40 μm thickness each. One series was stained to detect tyrosine hydroxylase (TH; antibodies from SigmaAldrich) by using the free-floating TH-immunohistochemistry technique (for details see Winkler et al., 1996). Counting of grafted cells Within the graft, all cell bodies were counted under bright field illumination using a microscope with an X–Y motor stage (Leica). With the software “stereoinvestigator” (Microbrightfield, Magdeburg, Germany) a meander-like scan through each section of the graft was performed. The total number was estimated using Abercrombie’s (1946) formula. Fiber density At four coordinates within the striatum (AP + 1.7, + 1.0, + 0.5, − 0.4) the optical density of TH-positive immunoreactive fibers was analyzed as a measurement of graft integration and fiber outgrowth (Winkler et al., 1996). The non-lesioned side served as control side. With the exception of the graft core with its cell bodies the entire striatal area per section was evaluated under bright field illumination using an Olympus AX70 microscope (Hamburg, Germany) and a self-programmed macro in an analysis program (analySIS, Soft Imaging System, Münster, Germany). The optical density of the septum served as background and was subtracted from the densities of the contralateral and ipsilateral striata. The optical density of the contralateral side of a section was taken as 100% and compared to the data of the ipsilateral side. Data was collected from medial and lateral striata.

Fig. 5. Skilled forelimb performance in the staircase test after DA transplantation shown for the parameters pellets eaten (A) and pellets taken (B) during the free choice test. Data are presented as group means ± S.E.M.; p was set at b0.05 as level of significance: (€) indicates a significant difference between either the sham or the tx animals and the con animals. (#) marks a significant difference between the sham and the tx animals after the transplantation (post TX). Note that on the contralateral side tx animals develop a better performance in terms of pellets eaten than sham animals. The ipsilateral side with regard to the parameters pellets eaten and pellets taken remained unaffected by the DA grafts.

open, 7. pronation, 8. grasp, 9. supination 1, 10. supination 2, 11. release. These eleven components were subdivided into 35 subcomponents (Metz and Whishaw, 2000). Each sub-component was scored as a “normal movement” (1 point), an “abnormal movement” (0.5 point), or an “absent movement” (0 point). Thus, a higher score indicates a better reaching movement performance. Scores from three reaches per rat and group were averaged. Morphology Immunohistochemistry After 47 weeks (21 weeks after the transplantation) the rats were terminally anesthetized and transcardially perfused first with ice-

Graft volume In order to measure graft volume photographs of the entire graft were taken under bright field illumination with an Olympus AX70 microscope and analySIS software. The borders of the graft core (separated in a medial and lateral part) were marked in those sections in which cell bodies of the grafted DAergic neurons were found. Then the software calculated the area in μm2 within the marked region. The result of one section was multiplied by the original thickness of the section (40 μm) and by the number of series of coronal sections (4). The results of all sections were added together, and the total graft volume (μm3) was calculated.

Table 1 Skilled forelimb performance in the staircase test for the parameter success during the free choice test after the transplantation Grasping success (%)

Contralateral

Ipsilateral

Post LX

Post TX

Δ

Post LX

Post TX

Δ

con sham tx

84 49 46

96* 56 71*

12 7 25

84 67 75

97* 88* 92*

13 21 17

Data are presented as group means; p was set at b0.05 as level of significance: the asterisks (*) indicate significant differences between data before and after the transplantation for each group. While sham animals improved their skilled forelimb performance only on the ipsilateral side, tx and con animals improved on the ipsilateral and contralateral sides.

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Fig. 6. Skilled forelimb performance analyzed for the eleven movement components of a single grasping movement as assessed by the single pellet reaching test: (A) orient, (B) limb lift, (C) digits close, (D) aim, (E) advance, (F) digits open, (G) pronation, (H) grasp, (I) supination 1, (J) supination 2, and (K) release. Panel L displays the total score analysis as an overall view over the entire grasping movement (pooled data from panels A–K). Data are presented as group means ± S.E.M for each testing day (pre 1–T3). p was set at b0.05 as level of significance: the asterisks (*) indicate significant differences between sham and tx animals (asterisks in brackets mark a statistical trend which was defined as 0.5 b p b 0.1). Differences between the testing days L2 and T3 within the groups are marked separately: the circles (○) for con animals, the plus signs (+) for sham animals, and the pound signs (#) for tx animals. Panels A–K illustrate the most significant changes in reaching and grasping movement performance (i) between sham and tx animals after the transplantation (T1–T3) and (ii) between the last day of testing after the lesion (L2) and the last day of testing within each group after the transplantation (T3). Note that the analyses of the single-movement components revealed dissociable patterns of graft effects. The total score analysis is presented in panel L: there were no overall significant differences between the sham and the tx animals.

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Fig. 6 (continued).

Statistical analysis

Results

The data was subjected to one-factor ANOVA followed by Student–Newman–Keuls post-hoc test (StatView 4.5, Abacus Concepts Inc., Berkeley, USA) with p set at ≤0.05 as level of significance. Results are presented as means ± standard error of the mean (S.E.M.).

Drug-induced rotation Asymmetries in drug-induced behavior in all rats were measured 6 weeks after the 6-OHDA lesion and 6 weeks after the trans-

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plantation of E14 VM-derived cells (Fig. 3A + B). After 6-OHDA lesion the two lesioned groups (sham, tx) developed a strong rotational bias towards the contralateral side after apomorphine injection and towards the ipsilateral side after amphetamine injection (sham: −12.7 ± 2.10 apo, 13.2 ± 1.27 amph; tx: − 12.3 ± 1.10 apo, 15.5 ± 1.03 amph; sham/tx vs. con, p b 0.001). After transplantation the tx-group displayed a significant reduction of the apomorphineinduced rotational asymmetry (− 56.1%) and an overcompensation (+ 157.4%) with a net contralateral rotational response following a challenge with amphetamine (tx: − 5.4 ± 0.90 apo; − 8.9 ± 1.68 amph; sham/tx vs. con, p b 0.001; tx vs. sham, p b 0.001). 6-OHDA-induced DA depletion and graft-related reinnervation As shown by TH-immunohistochemistry there was a complete loss of striatal DA in 6-OHDA-lesioned and sham-transplanted rats ipsilateral to the lesion (Fig. 4B). In tx animals (Fig. 4C) there was a significant increase (176.6%) of DA fiber reinnervation in the striatum (Fig. 4D: sham 15.4 ± 1.38% ipsilateral; tx 43.6 ± 1.24% ipsilateral; sham/tx vs. con, p b 0.001; tx vs. sham, p b 0.001). Cell counting revealed 1631 ± 182 TH-positive cells per graft. Considering that 400,000 cells were injected into the striatum and that 8–10% of VM-derived cells are TH-positive (Nikkhah et al., 1993b), this represents a survival rate of 4%. Graft volume measured 0.72 ± 0.1 mm3. Staircase test Prior to transplantation (post LX) both lesioned groups (sham, tx) displayed severe impairments of contralateral skilled limb movements as found by quantitative endpoint measurements (82% reduction of pellets eaten and 72% reduction of pellets taken as compared to con animals [=baseline] during free choice and forced choice tests; data not shown). In addition, the results revealed moderate but still significant impairments on the ipsilateral side (22% reduction of pellets eaten compared to con animals during free choice and forced choice tests; sham/tx vs. con, p b 0.01). After the transplantation (post TX) the sham animals showed no change in reaching deficits for both forelimbs with regard to the parameter pellets eaten during the free choice test (Fig. 5A post TX: sham 8.8 ± 1.1 pellets contralateral, tx 13.0 ± 1.3 pellets contralateral, sham/tx vs. con, p b 0.001). In contrast, grafted (tx) rats improved in the number of pellets eaten with the contralateral paw as compared to sham animals (post TX: tx vs. sham, p b 0.001; 47.7% increase). The significant improvement in the number of pellets eaten for the contralateral forelimb failed to remain on a statistically significant level during the forced choice test (data not shown; post TX sham vs. tx, p = 0.68, not significant (n.s.)). There were no graft-related effects in the number of pellets taken neither in the free choice test (Fig. 5B) nor in the forced choice test (data not shown). Both groups (sham, tx) showed an impaired grasping performance on the contralateral side compared to control animals (sham: 16.2 ± 1.6 pellets contralateral; tx: 18.6 ± 1.7 pellets contralateral; sham/tx vs. con, p b 0.001). Table 1 displays the grasping success during the free choice test defined as [pellets eaten / pellets taken] * 100. The analysis revealed that sham rats were not able to improve on the contralateral (impaired) side after the (sham-)transplantation. In contrast, the tx animals improved their forelimb performance substantially from 46% to 71% after grafting (p b 0.01). The spontaneous improvement observed in con animals from prior (post LX) to after (post

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TX) the transplantation (around 13%) can be regarded as underlying natural learning effect. Single pellet reaching test Different from the staircase test this test analyzes the whole range of single movement components which are in use during each individual reaching and grasping movement by applying a scoring system to each movement component based on high-speed video recordings (qualitative evaluation). Figs. 6A–K show the average score of the eleven movement components for the testing days pre 1 to T3. The performance of the con animals never reached maximum score (Figs. 6A–L; average performance level was 85% which was equivalent to 29.7 of 35 points, see Fig. 6L). Nevertheless the con animals showed an improvement of motor performance from L2 to T3 (see Fig. 6L, (○) p = 0.03). The study of the eleven movement components (Figs. 6A–K) revealed significant impairments in both experimental groups (sham, tx) after the 6-OHDA lesion compared to the control group (con). The reaching and grasping deficits of the hemiparkinsonian rats in the individual grasping components varied between 30% and 79% of the performance of con animals on L2 (tx/sham vs. con, p b 0.01; Figs. 6A–K, significances not indicated). The total score (Fig. 6L) revealed that there was a lesion-induced reduction of the complete grasping performance of 60% on average (L2: sham/tx vs. con, p b 0.01). After the sham animals had received more training following sham-transplantation the total score analysis did not indicate any improvement in their grasping behavior (Fig. 6L on T3: sham vs. con: ~ 64% reduction of grasping performance, L2 vs. T3, n.s.). However, the eleven movement components performed by sham animals responded heterogeneously during the entire testing period (see Figs. 6A–K): the comparison between L2 and T3 revealed that the performance levels of the movement components limb lift, digits close, aim, and pronation improved (sham L2 vs. T3, p b 0.05), whereas those of the movement components orient, advance, digits open, and supination 1 did not change after more training. The

Table 2 Mean score analysis of single grasping movements in the single pellet reaching test on the last testing day T3: comparison of grasping performance between tx and sham animals Movement components

tx vs. sham (T3)

Orient Limb lift Digits close Aim Advance Digits open Pronation Grasp Supination 1 Supination 2 Release

− ○ − ○ − ○ − ○ ○ + +

Mean score

○

The (−) indicates that tx animals showed a significantly worse performance than sham animals. The (○) indicates that there were no differences between tx and sham animals. The (+) points out that tx animals benefited from the graft and developed a better grasping performance than sham animals.

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movement components grasp, supination 2, and release continued to deteriorate (sham L2 vs. T3, p b 0.01). In DA-grafted rats the overall qualitative aspects of forelimb performance were not significantly different from sham animals as displayed by the total score analysis (see Fig. 6L on T3: tx vs. con: 64% reduction of grasping performance of con animals; sham vs. tx, n.s.; L2 vs. T3, n.s.). Nevertheless, DA grafts clearly induced heterogeneous effects and dissociable patterns on the eleven reaching movement components (Table 2): while some movement components did not change (Fig. 6; limb lift, aim, digits open, grasp, supination 1; T3: sham vs. tx, n.s.), supination 2 (+ 18%) and release (+ 12%) improved significantly (Figs. 6 and 7; T3: sham vs. tx, p b 0.01). In the movement components orient (− 17%), digits close (−31%), advance (−16%), and pronation (−17%) grafted rats performed significantly worse than sham animals (sham vs. tx, p b 0.05). While analyzing the development of the individual grasping components for each group during the entire testing period (see in Figs. 6A–K) it became obvious that there were less time-depen-

dent changes in the tx animals than observed in the other two groups (con, sham): the movement components limb lift and aim improved (L2 vs. T3, p b 0.05), whereas the movement components orient, digits close, advance, digits open, pronation, supinations 1 and 2, and release did not significantly differ, and only the movement component grasp continued to deteriorate (L2 vs. T3, p b 0.01). Discussion In this study we investigated the ability of DA grafts to restore skilled forelimb performances in a rat model of PD utilizing complementary test systems that analyzed both quantitative (staircase test) and qualitative (single pellet reaching test) aspects of skilled reaching and grasping movements. Having received unilateral 6-OHDA lesion and DA transplantation the animals demonstrated a complete restoration of amphetamine-induced rotational asymmetry and a 50% reduction in apomorphine-induced rotational asymmetry. TH-immunohistochemistry revealed ample

Fig. 7. (A–F) Photographs of the last two movement components of a reaching movement with the contralateral (impaired) paw in the single pellet reaching test. The upper sequence displays the movement component supination 2 for the control, sham-transplanted (sham) and transplanted (tx) rats. The control rat (A) is aligned with the test box, its non-reaching paw is still on the ground, and its grasping paw is nearly fully supinated to allow the rat to release and eat the pellet. The sham rat (B) struggles to find its normal reaching position and leans for postural support on the side wall of the box. Both paws are raised; the non-grasping paw supports the rotational movement of the other paw, which is still in a vertical position with nearly open digits. The transplanted rat (C) uses a different strategy to supinate its paw: it puts both distal limbs on the ground to support the supinating movement of its grasping paw. The rat's body is appropriately aligned with the box. In the lower sequence of photographs, the movement component release is displayed for all three experimental groups. In panel D, the control rat raises its other forelimb to support the release and to eat the food pellet. The sham rat (E) still leans against the wall of the box, unable to supinate and fully close its paw. It loses the food pellet from the vertically oriented paw. The transplanted rat (F), once it has supinated with the help of the other paw, lifts up and turns towards the lesioned side and supports the grasping paw with the other paw and uses its mouth to open the paw and eat the pellet.

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graft survival (1631 TH-positive neurons/graft) and substantial graft-derived TH-positive fiber reinnervation of the striatum (44% of normal), which is within the range reported previously (Winkler et al., 2000; Brundin et al., 2000). The most salient findings of this study were the dissociable and different patterns of graft-induced functional recovery between quantitative and qualitative aspects of skilled forelimb movements. DA-transplanted animals exhibited signs of functional recovery in the staircase test, i.e. the number of pellets eaten was increased, although still significantly below normal levels. Additionally, the success rate for the contralateral-to-lesion forelimb rose from 46% to 71% after transplantation compared to 56% in sham animals. This confirms and extends previous observations that DA grafts can restore skilled forelimb use, at least partly under specific circumstances, e.g. (i) when a more extensive microtransplantation approach is used (Nikkhah et al., 1993a), (ii) when host animals do not exhibit a strong hemispheric lateralization for forepaw use (Nikkhah et al., 2001), and (iii) when additional GABAergic grafts are placed into the nigral target side (Winkler et al., 1999; for more extensive review see Winkler et al., 2000). However, this most commonly used form of the staircase test (Montoya et al., 1991; Nikkhah et al., 1998a; Nikkhah et al., 1998b) gives information only about the pellets eaten, the pellets taken and the grasping success and thus does not allow a more detailed analysis of indiõvidual movement components during the actual forelimb reaching and grasping performance. For this reason the single pellet reaching test, which enables the scoring of various movement components based on a frame-by-frame analysis of video recordings (Whishaw et al., 1993; Metz and Whishaw, 2000), was used in this study. This test has been extensively used to characterize unilateral (Miklyaeva et al., 1994; Vergara-Aragon et al., 2003) and bilateral (Faraji and Metz, 2007) 6-OHDA lesion-induced deficits of qualitative aspects of forelimb reaching movements in rats. In accordance with these previous findings the hemiparkinsonian animals in this study showed marked impairments in all individual movement components resulting in a performance level between 30% and 79% of that of normal control animals. Interestingly, the results obtained in DAgrafted rats clearly revealed a dissociable pattern of graft-induced alterations of movement components for the contralateral forelimb. Whereas nine of the eleven movement components of a single reach were either impaired (cf. orient, digits close, advance, and pronation), or unchanged (cf. limb lift, aim, digits open, grasp, and supination 1), the last two movement components (cf. supination 2 and release, see Fig. 7) demonstrated a graft-related functional improvement. Most importantly, the results derived from the individual scores of single movement components in this study provide the first evidence that various parts of skilled forelimb reaching movements can be differently affected by ectopically placed DA grafts. Previous observations have led to the hypothesis that complex and spontaneous sensorimotor behavior are more resistant to DA graft-induced functional improvements than simple behavioral elements of the DA-deficiency syndrome (for a more extensive review see Barker and Dunnett, 1999; Winkler et al., 2000). Numerous studies have demonstrated convincingly that drug-induced rotational asymmetries and simple sensorimotor orientation can be improved or even normalized by DA transplants (Isacson et al., 2003; Dobrossy and Dunnett, 2005) whereas skilled forelimb use and disengaged behavior most often failed to show any behavioral recovery following DA transplantation (Dunnett et al., 1987; Brundin et al., 1994; Winkler et al., 2000; Dobrossy et al., 2000).

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The results of this study provide novel insights into the mechanisms of skilled forelimb reaching movements underlying the restorative plasticity-induced behavioral effects after transplantation of DA neurons. These findings indicate that some movement components of skilled reaches may indeed be improved by the transplantation approach whereas other aspects fail to show any graft-related changes, or even deteriorate. It has been suggested that skilled limb use is mediated by separate movement subsystems which are associated with the nigrostriatal pathway, and which are therefore DA-dependent (Teitelbaum et al., 1983; Metz et al., 2003). It might be argued that posture and locomotion are produced by two different movement subsystems; thus a restoration of one system might leave the other unaffected. The results of the single pellet reaching test revealed that DA grafts had heterogeneous effects on skilled limb movement components, which might be explained by an incomplete rewiring of the neural circuit. It is expected that a more complete organotypic restoration of the striatum is necessary to restore the two movement subsystems as well as more components of the skilled forelimb movement pattern. Available data document that ectopically placed intrastriatal DA grafts can reinstate a tonic DA release, which is sufficient to normalize DA receptor supersensitivity in the vicinity of the graft (for review see Bjorklund, 1992), and thereby normalize e.g. the lesioninduced upregulation of mRNAs encoding for preproenkephalin (PPE) and the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67) throughout the striatum, whereas the lesioninduced downregulation of preprotachykinin mRNA remains unaffected (Winkler et al., 2003). The poor survival rate, the incomplete morphological and electrophysiological maturation of the DA neurons, and the ectopic graft placement concomitant with incomplete establishment of physiological afferent and efferent connections are major hurdles for a more complete graft-induced behavioral recovery (Bjorklund et al., 2003; Isacson et al., 2003). This may indicate that the first phases of skilled reaching movements are largely dependent on a full organotypic reconstruction of the nigrostriatal pathway, which at the present is only to be achieved in neonatal hosts (Nikkhah et al., 1995a, b; Bentlage et al., 1999) or in using bridge grafts (e.g. Wictorin et al., 1992; Wilby et al., 1999; Winkler et al., 2000). However, ectopic DA grafts can ameliorate some qualitative aspects of movement components during the final phase of skilled forelimb reaching, indicating that this part of reaching may be regulated by a more basic pattern of DA neurotransmission which can be reinstated by the current grafting protocol. The graft-induced mechanisms that underlie the dissociable patterns of behavioral recovery or impairment observed in skilled forelimb use under the two complimentary test conditions (staircase test and single pellet reaching test) are likely to involve testspecific compensatory strategies in addition to “true” recovery. In 6-OHDA-lesioned animals these can include adjusted patterns of forelimb reaching components, and head and forelimb positioning as well as differences in fore- and hindlimb supporting strategies, as illustrated for the staircase test by Whishaw et al. (1997b). Similarly, Miklyaeva et al. (1994) and Vergara-Aragon et al. (2003) have provided convincing evidence for compensatory strategies of 6-OHDA-lesioned rats in the single pellet reaching test, including postural adjustments of the tail and the body axis, and deviations of forelimb movement trajectories leading to functional improvements. Conversely, it could be postulated that regaining function does not necessarily mean regaining normal movement patterns. It seems likely that these compensatory adjustments have an

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influence on the degree of graft-induced behavioral changes, and it will be highly interesting for future studies to try to unravel the contribution of each of the processes governing functional impairment, compensation and recovery. The three complementary behavioral tests in this study vary in their complexity and in the way they challenge the motor skills of the animals (Cenci et al., 2002). Drug-induced rotation actually does not challenge voluntary motor performance of the animals but shows drug-induced movements and offers the possibility to measure changes in DA storage capacities and postsynaptic DA receptor sensitivity in vivo. In this test paradigm the animals not only displayed the most obvious recovery compared to the other tests, but they also showed overcompensation in amphetamineinduced rotation. The staircase test is a medium–complex motor test which offers – as previously described – a quantitative measurement of grasping behavior. The rats can “train” themselves within the 15-min testing period by unlimited grasping for food pellets and they probably utilize the staircase apparatus to develop compensatory movement strategies. In this test we observed a partial recovery of motor function. In contrast, we could not observe any significant overall recovery in grasping behavior in the single pellet reaching test which is the most complex and difficult one. Only the two movement components supination 2 and release showed significant improvements; they might have been the crucial movement components to help improve motor performance in the less complex staircase test. From these data we postulate that the opportunity to detect recovery after transplantation declines with increasing complexity of a motor task. Regaining (motor) function becomes not only a matter of regaining normal movement patterns but also a matter of the level of complexity of the applied motor tests. As forelimb reaching and grasping behavior in rats and humans is homologous (Whishaw et al., 2002), the rat model of PD may also have significant implications for current attempts to develop neuromodulatory, regenerative and restorative strategies for patients suffering from PD (Lang and Lozano, 1998). The results give evidence that graft-induced changes can lead to unwanted side effects or impairments as previously observed in some human parkinsonian patients who have received fetal nigral transplants (Winkler et al., 2005). The current clinical protocols of movement assessments in PD patients do not include qualitative aspects of grasping behavior but an overall summary of the movement performance (Unified Parkinson’s Disease Rating Scale, UPDRS). Therefore we strongly suggest that additional tests of grasping behavior should be included in clinical protocols in PD and other movement disorders before and after a therapeutical approach, such as neurotransplantation. In conclusion, by combining analyses of both qualitative and quantitative aspects of skilled forelimb use it becomes obvious that DA grafts induce dissociable patterns of functional improvements and impairments of specific movement components. These novel insights may enhance our understanding about the mechanisms underlying transplantation-induced functional effects in animal models of PD, and may also foster the development of clinically safe and effective restorative cell therapies. Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft (Ni-330), by the Neuroscience Graduate School Freiburg (DFG 843), by the Sonderforschungsbereich 505, the German Parkinson Foundation, and by the Alberta Heritage Foundation for

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