www.elsevier.com/locate/ynbdi

Neurobiology of Disease 27 (2007) 24–35

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

Alexander Klein,a,b,⁎ Gerlinde A. Metz,b Anna Papazoglou,a and Guido Nikkhaha

aLaboratory of Molecular Neurosurgery, Department of Stereotactic Neurosurgery, University Hospital Freiburg – Neurocentre, Breisacher Str. 64,D-79106 Freiburg, GermanybCanadian Centre for Behavioural Neuroscience, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4

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

Skilled forelimb movements depend on an intact dopaminergic (DA)neurotransmission and are substantially impaired in the unilateral ratmodel of Parkinson’s disease. It has remained unclear, however, to whatextent reaching and grasping movements can be influenced byintrastriatal transplantation of fetal DA neurons. Here an extensivebehavioral assessment of skilled forelimb movement patterns inhemiparkinsonian and DA-grafted rats was carried out. Good DAgraft survival was accompanied by a compensation of drug-inducedrotational asymmetries. Interestingly, skilled forelimb use was sig-nificantly improved in transplanted animals as compared to lesion-onlyanimals in the staircase test. Qualitative analysis of single forelimbreaching movement components revealed dissociable patterns of grafteffects: while somemovement components in grafted animals improved,others remained unchanged or even deteriorated. These findingsprovide novel insights into the complex interactions of graft-derivedrestoration of DA neurotransmission and skilled forelimb behavior.© 2007 Elsevier Inc. All rights reserved.

Keywords: Motor behavior; Neurodegeneration; Neuroregeneration; Par-kinson’s disease; Skilled forelimb use; Transplantation; Staircase test;Single pellet reaching test; Restoration

Introduction

Transplantation of fetal dopaminergic (DA) neurons in animalmodels of Parkinson’s disease (PD) has been extensively used as atool to study the mechanisms of restoration of DAergic neurotrans-mission and its effects on sensorimotor behavior. The morphologicaland functional effects of DA grafts have been mainly studied in ananimal model of PD which uses a unilateral infusion of 6-hydro-

⁎ Corresponding author. Laboratory of Molecular Neurosurgery, Depart-ment of Stereotactic Neurosurgery, University Hospital Freiburg–Neurocentre, Breisacher Str. 64, D-79106 Freiburg, Germany. Fax: +49761 270 5021.

E-mail address: alexander.klein@uniklinik-freiburg.de (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 includingdrug-induced rotational asymmetries, impaired sensorimotor orienta-tion, and stepping behavior as well as a substantial deterioration ofskilled forelimb performance. These experimentally 6-OHDA-induced behavioral deficits share significant similarities with humanpatients affected by idiopathic PD. Experimental and clinical studiesin PD have demonstrated that impaired sensorimotor functions can beimproved by embryonic DA grafts (Wenning et al., 1997; Nikkhah etal., 1998a; Hagell et al., 1999; Rodter et al., 2000; Lindvall andHagell, 2000; Dobrossy and Dunnett, 2004; Winkler et al., 2005).

In animal models of PD, DA grafts lead to a functional res-toration of simple motor behavior, e.g. drug-induced rotationalasymmetry (Brundin et al., 1994), spontaneous circling, and sen-sorimotor 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 per-formance (e.g. in the staircase test) has been proven to be moreresistant to graft-induced recovery (Dunnett et al., 1987; Montoya etal., 1990; Mandel et al., 1990; Abrous et al., 1993; Nikkhah et al.,1993a). However a partial graft-induced recovery of skilledforelimb reaching in 6-OHDA-lesioned rats could be observedonly under certain conditions, e.g. after extensive microtransplanta-tion of intrastriatal grafts (Nikkhah et al., 1993a; Winkler et al.,1999) and after transplantation in animals that do not exhibit astrong hemispheric lateralization for paw use (Nikkhah et al., 2001).

Importantly, the majority of previous transplantation studiesexamined 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 pointmeasurements (i.e. success or failure of the number of food pelletsgrasped and eaten), it lacks any “qualitative” analysis of theindividual reaching components that may be affected differently bylesion and transplantation surgery (Kloth et al., 2006). In this contextthe single pellet reaching test (Whishaw and Pellis, 1990; Miklyaevaet al., 1994; Metz andWhishaw, 2000) has been shown to provide an

25A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

excellent scoring system to examine individual limb and digitmovements based on a frame-by-frame analysis of high-speed videorecordings. This behavioral testing paradigm has been used inanimals with 6-OHDA lesions (Miklyaeva et al., 1994; Whishaw etal., 2002) and to describe recovery mechanisms following behavioraltherapy in hemiparkinsonian rats (Vergara-Aragon et al., 2003).

Therefore, this study was undertaken to quantitatively andqualitatively evaluate the functional restorative impact of fetal DAgrafts on skilled forelimb reaching movements in rats withunilateral 6-OHDA lesions.

Methods

Experimental design

All animals were daily trained in the single pellet reaching testprior to lesion. Testing in the single pellet reaching test continuedthroughout the experiment (47 weeks, on 5 to 6 days/week) exceptfor a recovery period of 2 days after each surgical manipulation. Therats were subdivided into three groups: (i) lesioned and transplantedrats (n=13; tx), (ii) lesioned and sham-transplanted rats (n=8;sham), and (iii) non-lesioned control animals (n=8; con). The txand sham animals received a unilateral 6-OHDA infusion into themedial forebrain bundle (MFB) contralateral to the preferred paw.Additionally, embryonic day 14 ventral mesencephalon-derived ratprogenitor cells (E14 VM-derived cells) were transplanted into theDA-depleted striatum of the tx rats. Sham animals were grafted withcell culture medium without cells. In all animals, lesion and grafteffects were evaluated by rotational behavior assessment 6 weeksafter the 6-OHDA lesion (6 weeks post LX) and after thetransplantation of E14 VM-derived cells (6 weeks post TX). Twicebefore the lesion (pre 1 and pre 2), twice after the lesion (L1 and L2)and three times after the transplantation (T1–T3) the reachingbehavior was video-recorded (Fig. 1). In addition the rats performedthe staircase test once after the lesion (post LX) and once after thetransplantation (post TX). After the last testing day the animalswere perfused and their brains underwent morphological analysis.All experiments were conducted and performed according to theguidelines of the local ethical board of the University of Freiburg,Germany.

Subjects

Subjects were 34 adult female Sprague-Dawley rats (CharlesRiver, Sulzfeld, Germany), weighing 250 g±30 g at the beginningof the experiments. Rats were housed in standard cages in groups upto five animals in a temperature-controlled room (23±0.3° C) on a12-h light/12-h dark schedule. Each animal was fed with 12 g

Fig. 1. Time chart of all behavioral tests and surgeries. LX=unilateral 6-OHDA Mreaching test, rot=drug-induced rotation, ST=staircase test in the free choice and1+2=recordings of reaching behavior in the SP), post LX=behavioral assessmenTX=behavioral assessment after the transplantation (T1–3=recordings of reachmorphological analysis of brains.

standard laboratory chow (Altromin, Lage, Germany) per day andwater was provided ad libitum. Rats were weighed twice weekly inorder to maintain body weight at 90% of baseline values.

Surgery

6-OHDA lesionAll rats (except the control rats) were anesthetized with ketamine

(10%; 0.1 ml/kg bodyweight; Essex, München, Germany) andrompune (2%; 0.01 ml/kg bodyweight; Bayer, Leverkusen, Ger-many), and were stereotactically injected with 6-OHDA hydro-bromide (3.6 μg 6-OHDA/μl in 0.1% L-ascorbic acid-saline; Sigma-Aldrich, Steinheim, Germany) into theMFB on the side contralateralto the preferred paw (Ungerstedt, 1968). Lesion coordinates were setaccording 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; tract2: 2.5 μl; TB −2.3, AP −4.4, lat −1.2, DV −7.8. The injection ratewas 1 μl/min, and the cannula was left in place for 2 min beforeslowly retracting it.

TransplantationAll lesioned rats were anesthetized and stereotactically operated

a second time. The tx rats were injected with a single cell suspensionof E14 VM-derived cells into the DA-depleted striatum. The E14 ratembryos were dissected, and the VMs were mechanically andenzymatically 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 setaccording to bregma and dura in mm: two tracts, four deposits (1 μleach): TB 0.0, AP +1.0, lat −2.5/−3.3, DV −5/−4. The injectionrate was 1 μl/min, and the glass capillary was slowly retracted after30 seconds. The concentration of the cell suspension was 100,000cells/μl so that each rat received 400,000 cells in total. The cellviability remained stable between 95% and 98% during theimplantation procedure.

Behavioral tests

Three sensorimotor behavioral tests were performed: (1) thedrug-induced rotation, (2) the staircase test for the analysis of rea-ching success, and (3) the single pellet reaching test for the detailedanalysis of individual movement components of skilled forelimbreaching pattern.

Drug-induced rotationDrug-induced rotational behavior was monitored in rotometer

boxes 2 and 6 weeks post lesion and post transplantation as des-

FB lesion, TX=transplantation of E14 VM-derived cells, SP=single pelletforced choice modes, pre LX=behavioral assessment before the lesion (pret after the lesion (L1+2=recordings of reaching behavior in the SP), posting behavior in the SP), perfusion=sacrifice of experimental animals and

26 A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

cribed by Ungerstedt and Arbuthnott (1970, Fig. 3). Data of the2 weeks’ drug-induced rotation are not shown. Apomorphine-induced rotation (apo) was tested for 40 min after subcutaneousinjection of 1 ml/kg apomorphine solution (Sigma-Aldrich: 0.05 mgapomorphine+0.2 mg L-ascorbate in 1 ml 0.9% saline). About3 days later, amphetamine-induced rotation (amph) was tested for90 min after intraperitoneal injection of 1 ml/kg amphetaminesolution (Sigma-Aldrich: 2.5 mg D-amphetamine per 1.0 ml saline).Five animals were excluded from the study because they showedb4.0 full body turns contralaterally to the lesioned side after apo-morphine injection and b6.0 full body turns ipsilaterally to thelesioned side after amphetamine injection. Apomorphine-inducedrotation is presented as net rotation in negative values, and amphe-tamine-induced rotation is presented as net rotation in positivevalues.

Staircase testThe staircase test (Montoya et al., 1990) was used to evaluate

skilled forelimb use to distinguish between the grasping perfor-mance of the unaffected (ipsilateral) and the impaired (contralateral)paw individually (Fig. 2A+B). A modified set-up of the staircasetest 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 withtheir right forepaw and on the left staircase only with their leftforepaw. Right and left staircases were baited with ten pellets oneach of the steps 2 to 5 (in total 40 pellets per staircase). After aperiod of 15 min the animals were removed from the staircase boxand the remaining pellets were counted. Test performance wasreflected by two parameters: pellets remained for pellets left on thesteps they had originally been placed on, and pellets missed forpellets dropped elsewhere, i.e. on step 1 or 6 or outside the stair-cases in other parts of the staircase box. These results were used to

Fig. 2. Photographs of the staircase test (A+B) and the single pellet reaching test (Cboth sides during the 15-min testing period and that the pellets taken and eaten arepellet reaching test (C+D) the movement components for each single reach are recocomponent is rated afterwards based on a scoring system (qualitative endpoint me

calculate other three parameters pellets eaten (40− [pelletsremained+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 forgeneral reaching activity and motivation, the parameter pelletseaten rather reflects the rats’ forelimb skills in reaching andgrasping movements and is thus directly dependent on the rats’sensorimotor performance status. Furthermore, the parameterpellets eaten expresses the ratio of the number of pellets eaten tothe total number of pellets (40 pellets per staircase). The parametergrasping success shows the success of all attempts to grasp a pellet.A test session consisted of 9 days (d) within which the staircase testwas repeated: the acquisition phase to the test set-up (data notshown) took place during d1–d3, and the free choice test wasperformed during d4–d6 when the steps of both staircases werefilled with pellets. During the forced choice test the pellets wereplaced on the steps first of the side of the non-preferred paw andthen of the side of the preferred paw. This test was carried out ond7–d9. During the forced choice test rats were tested twice 15 minfor each side per day (data not shown).

Single pellet reaching testThis test (Fig. 2C+D) was used to analyze individual com-

ponents of reaching and grasping patterns based on high-speed videorecordings (Whishaw and Pellis, 1990; Whishaw et al., 1997a; Metzand Whishaw, 2000). Individual rats were put in a Plexiglas box(13 cm×45 cm×40 cm) and they were expected to extend theirpreferred paw to retrieve food pellets (BioServ). Attached to thefront wall of the box was a shelf to hold single food pellets in anindentation that was located contralateral to the rats’ preferred paw.The rats were trained to extend their forelimb through a slit in thefront wall to grasp and eat one pellet at a time. Theywere also trainedto walk to the rear end of the box after each reach in order to readjusttheir body position before the next attempt to grasp a pellet. Rats that

+D). Note that the rats typically grasp the food pellets in the staircase box oncounted only afterwards (quantitative endpoint measurement). In the singlerded by high-speed video recordings and the performance of each movementasurement). See also the section on methods for detailed descriptions.

27A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

showed severe catalepsy after 6-OHDA lesion were allowed toremain at the front of the box. Prior to testing and 6-OHDA lesion allthe rats were trained for 16 weeks until they reached stable successrates in grasping food pellets. After lesion and transplantation theanimals 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 reachingbehavior was recorded with a video camera with a shutter speed of1000/s (Panasonic, NV-MX300). The tapes were analyzed frame byframe. A successful reach was recorded if a rat obtained a food pelletat its first attempt. Three successful reaches were scored for eachanimal. In case an animal was cataleptic, three reaching attemptswere scored. The reaching movements were scored according to thescale as described by Metz and Whishaw (2000). Each reachingmovement 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 arepresented as group means±S.E.M.; p was set at b0.05 as level of signi-ficance: (€) indicates a significant difference between either the sham or thetx animals and the con animals. (#) marks a significant difference betweenthe sham and the tx animals after transplantation (post TX). Note that postTX the rotational bias of tx animals was reduced after apomorphine chal-lenge (A) and that the tx animals overcompensated to the ipsilateral sideafter amphetamine challenge (B) indicating good graft survival and graft-derived functional recovery.

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

Fig. 5. Skilled forelimb performance in the staircase test after DA trans-plantation 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 dif-ference between either the sham or the tx animals and the con animals. (#)marks a significant difference between the sham and the tx animals after thetransplantation (post TX). Note that on the contralateral side tx animalsdevelop a better performance in terms of pellets eaten than sham animals.The ipsilateral side with regard to the parameters pellets eaten and pelletstaken remained unaffected by the DA grafts.

Table 1Skilled forelimb performance in the staircase test for the parameter successduring the free choice test after the transplantation

Graspingsuccess(%)

Contralateral Ipsilateral

Post LX Post TX Δ Post LX Post TX Δ

con 84 96* 12 84 97* 13sham 49 56 7 67 88* 21tx 46 71* 25 75 92* 17

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

28 A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

open, 7. pronation, 8. grasp, 9. supination 1, 10. supination 2, 11.release. These eleven components were subdivided into 35 sub-components (Metz and Whishaw, 2000). Each sub-component wasscored as a “normal movement” (1 point), an “abnormal movement”(0.5 point), or an “absent movement” (0 point). Thus, a higher scoreindicates a better reaching movement performance. Scores fromthree reaches per rat and group were averaged.

Morphology

ImmunohistochemistryAfter 47 weeks (21 weeks after the transplantation) the rats were

terminally anesthetized and transcardially perfused first with ice-

cold 0.1 M phosphate-buffered (pH 7.4) saline (PBS) and then with4% paraformaldehyde (PFA; Sigma). After post-fixation overnightin PFA the brains were dehydrated in 30% sucrose. With a freezingmicrotome (Leica, Nussloch, Germany) the brains were cut in fourseries of coronal sections of 40 μm thickness each. One series wasstained to detect tyrosine hydroxylase (TH; antibodies from Sigma-Aldrich) by using the free-floating TH-immunohistochemistrytechnique (for details see Winkler et al., 1996).

Counting of grafted cellsWithin 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, Magde-burg, Germany) a meander-like scan through each section of thegraft was performed. The total number was estimated using Aber-crombie’s (1946) formula.

Fiber densityAt four coordinates within the striatum (AP +1.7, +1.0, +0.5,

−0.4) the optical density of TH-positive immunoreactive fibers wasanalyzed 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 entirestriatal area per section was evaluated under bright field illumina-tion using an Olympus AX70 microscope (Hamburg, Germany) anda self-programmed macro in an analysis program (analySIS, SoftImaging System, Münster, Germany). The optical density of theseptum served as background and was subtracted from the densitiesof the contralateral and ipsilateral striata. The optical density of thecontralateral side of a section was taken as 100% and compared tothe data of the ipsilateral side. Data was collected from medial andlateral striata.

Graft volumeIn order to measure graft volume photographs of the entire graft

were taken under bright field illumination with an Olympus AX70microscope and analySIS software. The borders of the graft core(separated in a medial and lateral part) were marked in those sec-tions in which cell bodies of the grafted DAergic neurons werefound. Then the software calculated the area in μm2 within themarked region. The result of one section was multiplied by theoriginal thickness of the section (40 μm) and by the number ofseries of coronal sections (4). The results of all sections were addedtogether, and the total graft volume (μm3) was calculated.

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 groupmeans±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 txanimals (asterisks in brackets mark a statistical trend which was defined as 0.5bpb0.1). Differences between the testing days L2 and T3 within the groups aremarked separately: the circles (○) for con animals, the plus signs (+) for sham animals, and the pound signs (#) for tx animals. Panels A–K illustrate the mostsignificant changes in reaching and grasping movement performance (i) between sham and tx animals after the transplantation (T1–T3) and (ii) between the lastday 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-movementcomponents revealed dissociable patterns of graft effects. The total score analysis is presented in panel L: there were no overall significant differences betweenthe sham and the tx animals.

29A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

Fig. 6 (continued).

30 A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

Statistical analysis

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

Results

Drug-induced rotation

Asymmetries in drug-induced behavior in all rats were measured6 weeks after the 6-OHDA lesion and 6 weeks after the trans-

Table 2Mean score analysis of single grasping movements in the single pelletreaching test on the last testing day T3: comparison of grasping performancebetween 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 performancethan sham animals. The (○) indicates that there were no differences betweentx and sham animals. The (+) points out that tx animals benefited from thegraft and developed a better grasping performance than sham animals.

31A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

plantation of E14 VM-derived cells (Fig. 3A+B). After 6-OHDAlesion the two lesioned groups (sham, tx) developed a strongrotational bias towards the contralateral side after apomorphine in-jection 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, pb0.001). After transplantationthe tx-group displayed a significant reduction of the apomorphine-induced rotational asymmetry (−56.1%) and an overcompensation(+157.4%) with a net contralateral rotational response following achallenge with amphetamine (tx: −5.4±0.90 apo; −8.9±1.68 amph;sham/tx vs. con, pb0.001; tx vs. sham, pb0.001).

6-OHDA-induced DA depletion and graft-related reinnervation

As shown by TH-immunohistochemistry there was a completeloss of striatal DA in 6-OHDA-lesioned and sham-transplanted ratsipsilateral to the lesion (Fig. 4B). In tx animals (Fig. 4C) there was asignificant increase (176.6%) of DA fiber reinnervation in thestriatum (Fig. 4D: sham 15.4±1.38% ipsilateral; tx 43.6±1.24%ipsilateral; sham/tx vs. con, pb0.001; tx vs. sham, pb0.001).

Cell counting revealed 1631±182 TH-positive cells per graft.Considering that 400,000 cells were injected into the striatum andthat 8–10% of VM-derived cells are TH-positive (Nikkhah et al.,1993b), this represents a survival rate of 4%. Graft volumemeasured 0.72±0.1 mm3.

Staircase test

Prior to transplantation (post LX) both lesioned groups (sham,tx) displayed severe impairments of contralateral skilled limbmovements as found by quantitative endpoint measurements (82%reduction of pellets eaten and 72% reduction of pellets taken ascompared to con animals [=baseline] during free choice and forcedchoice tests; data not shown). In addition, the results revealedmoderate but still significant impairments on the ipsilateral side(22% reduction of pellets eaten compared to con animals duringfree choice and forced choice tests; sham/tx vs. con, pb0.01).

After the transplantation (post TX) the sham animals showed nochange in reaching deficits for both forelimbs with regard to theparameter pellets eaten during the free choice test (Fig. 5A postTX: sham 8.8±1.1 pellets contralateral, tx 13.0±1.3 pelletscontralateral, sham/tx vs. con, pb0.001). In contrast, grafted (tx)rats improved in the number of pellets eaten with the contralateralpaw as compared to sham animals (post TX: tx vs. sham, pb0.001;47.7% increase). The significant improvement in the number ofpellets eaten for the contralateral forelimb failed to remain on astatistically significant level during the forced choice test (data notshown; post TX sham vs. tx, p=0.68, not significant (n.s.)).

There were no graft-related effects in the number of pellets takenneither in the free choice test (Fig. 5B) nor in the forced choice test(data not shown). Both groups (sham, tx) showed an impairedgrasping performance on the contralateral side compared to controlanimals (sham: 16.2±1.6 pellets contralateral; tx: 18.6±1.7 pelletscontralateral; sham/tx vs. con, pb0.001).

Table 1 displays the grasping success during the free choice testdefined as [pellets eaten /pellets taken]*100. The analysis revealedthat sham rats were not able to improve on the contralateral (im-paired) side after the (sham-)transplantation. In contrast, the txanimals improved their forelimb performance substantially from46% to 71% after grafting (pb0.01). The spontaneous improve-ment observed in con animals from prior (post LX) to after (post

TX) the transplantation (around 13%) can be regarded as under-lying natural learning effect.

Single pellet reaching test

Different from the staircase test this test analyzes the wholerange of single movement components which are in use during eachindividual reaching and grasping movement by applying a scoringsystem to each movement component based on high-speed videorecordings (qualitative evaluation). Figs. 6A–K show the averagescore of the eleven movement components for the testing days pre 1to T3.

The performance of the con animals never reached maximumscore (Figs. 6A–L; average performance level was 85% which wasequivalent to 29.7 of 35 points, see Fig. 6L). Nevertheless the conanimals showed an improvement of motor performance from L2 toT3 (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 hemiparkinsonianrats in the individual grasping components varied between 30% and79% of the performance of con animals on L2 (tx/sham vs. con,pb0.01; Figs. 6A–K, significances not indicated). The total score(Fig. 6L) revealed that there was a lesion-induced reduction of thecomplete grasping performance of 60% on average (L2: sham/tx vs.con, pb0.01). After the sham animals had received more trainingfollowing sham-transplantation the total score analysis did notindicate any improvement in their grasping behavior (Fig. 6L onT3: sham vs. con: ~64% reduction of grasping performance, L2 vs.T3, n.s.).

However, the eleven movement components performed by shamanimals responded heterogeneously during the entire testing period(see Figs. 6A–K): the comparison between L2 and T3 revealed thatthe performance levels of the movement components limb lift, digitsclose, aim, and pronation improved (sham L2 vs. T3, pb0.05),whereas those of the movement components orient, advance, digitsopen, and supination 1 did not change after more training. The

32 A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

movement components grasp, supination 2, and release continuedto deteriorate (sham L2 vs. T3, pb0.01).

In DA-grafted rats the overall qualitative aspects of forelimbperformance were not significantly different from sham animals asdisplayed 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 hete-rogeneous effects and dissociable patterns on the eleven reachingmovement components (Table 2): while some movement compo-nents did not change (Fig. 6; limb lift, aim, digits open, grasp,supination 1; T3: sham vs. tx, n.s.), supination 2 (+18%) andrelease (+12%) improved significantly (Figs. 6 and 7; T3: sham vs.tx, pb0.01). In the movement components orient (−17%), digitsclose (−31%), advance (−16%), and pronation (−17%) grafted ratsperformed significantly worse than sham animals (sham vs. tx,pb0.05). While analyzing the development of the individual gras-ping components for each group during the entire testing period (seein Figs. 6A–K) it became obvious that there were less time-depen-

Fig. 7. (A–F) Photographs of the last two movement components of a reaching movThe upper sequence displays the movement component supination 2 for the controaligned with the test box, its non-reaching paw is still on the ground, and its graspingsham rat (B) struggles to find its normal reaching position and leans for postural supsupports the rotational movement of the other paw, which is still in a vertical positiosupinate its paw: it puts both distal limbs on the ground to support the supinating mbox. In the lower sequence of photographs, the movement component release is disother forelimb to support the release and to eat the food pellet. The sham rat (E) stillloses the food pellet from the vertically oriented paw. The transplanted rat (F), oncelesioned side and supports the grasping paw with the other paw and uses its mout

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, pb0.05), whereas the movement components orient,digits close, advance, digits open, pronation, supinations 1 and 2,and release did not significantly differ, and only the movementcomponent grasp continued to deteriorate (L2 vs. T3, pb0.01).

Discussion

In this study we investigated the ability of DA grafts to restoreskilled forelimb performances in a rat model of PD utilizingcomplementary test systems that analyzed both quantitative (stair-case test) and qualitative (single pellet reaching test) aspects ofskilled reaching and grasping movements. Having receivedunilateral 6-OHDA lesion and DA transplantation the animals de-monstrated a complete restoration of amphetamine-induced rota-tional asymmetry and a 50% reduction in apomorphine-inducedrotational asymmetry. TH-immunohistochemistry revealed ample

ement with the contralateral (impaired) paw in the single pellet reaching test.l, sham-transplanted (sham) and transplanted (tx) rats. The control rat (A) ispaw is nearly fully supinated to allow the rat to release and eat the pellet. Theport on the side wall of the box. Both paws are raised; the non-grasping pawn with nearly open digits. The transplanted rat (C) uses a different strategy toovement of its grasping paw. The rat's body is appropriately aligned with theplayed for all three experimental groups. In panel D, the control rat raises itsleans against the wall of the box, unable to supinate and fully close its paw. Itit has supinated with the help of the other paw, lifts up and turns towards theh to open the paw and eat the pellet.

33A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

graft survival (1631 TH-positive neurons/graft) and substantialgraft-derived TH-positive fiber reinnervation of the striatum (44%of normal), which is within the range reported previously (Winkleret al., 2000; Brundin et al., 2000).

The most salient findings of this study were the dissociable anddifferent patterns of graft-induced functional recovery betweenquantitative and qualitative aspects of skilled forelimb movements.DA-transplanted animals exhibited signs of functional recovery inthe staircase test, i.e. the number of pellets eaten was increased,although still significantly below normal levels. Additionally, thesuccess 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 canrestore skilled forelimb use, at least partly under specificcircumstances, e.g. (i) when a more extensive microtransplantationapproach is used (Nikkhah et al., 1993a), (ii) when host animals donot exhibit a strong hemispheric lateralization for forepaw use(Nikkhah et al., 2001), and (iii) when additional GABAergic graftsare placed into the nigral target side (Winkler et al., 1999; for moreextensive review see Winkler et al., 2000). However, this mostcommonly used form of the staircase test (Montoya et al., 1991;Nikkhah et al., 1998a; Nikkhah et al., 1998b) gives informationonly about the pellets eaten, the pellets taken and the graspingsuccess and thus does not allow a more detailed analysis of indiõ-vidual movement components during the actual forelimb reachingand grasping performance. For this reason the single pellet reachingtest, which enables the scoring of various movement componentsbased on a frame-by-frame analysis of video recordings (Whishawet al., 1993; Metz and Whishaw, 2000), was used in this study. Thistest has been extensively used to characterize unilateral (Miklyaevaet al., 1994; Vergara-Aragon et al., 2003) and bilateral (Faraji andMetz, 2007) 6-OHDA lesion-induced deficits of qualitative aspectsof forelimb reaching movements in rats. In accordance with theseprevious findings the hemiparkinsonian animals in this studyshowed marked impairments in all individual movement compo-nents resulting in a performance level between 30% and 79% of thatof normal control animals. Interestingly, the results obtained in DA-grafted rats clearly revealed a dissociable pattern of graft-inducedalterations of movement components for the contralateral forelimb.Whereas nine of the eleven movement components of a single reachwere either impaired (cf. orient, digits close, advance, and pro-nation), or unchanged (cf. limb lift, aim, digits open, grasp, andsupination 1), the last two movement components (cf. supination 2and release, see Fig. 7) demonstrated a graft-related functional im-provement. Most importantly, the results derived from the indi-vidual scores of single movement components in this study providethe first evidence that various parts of skilled forelimb reachingmovements can be differently affected by ectopically placed DAgrafts.

Previous observations have led to the hypothesis that complexand spontaneous sensorimotor behavior are more resistant to DAgraft-induced functional improvements than simple behavioralelements of the DA-deficiency syndrome (for a more extensivereview see Barker and Dunnett, 1999; Winkler et al., 2000). Nume-rous studies have demonstrated convincingly that drug-inducedrotational asymmetries and simple sensorimotor orientation can beimproved or even normalized by DA transplants (Isacson et al.,2003; Dobrossy and Dunnett, 2005) whereas skilled forelimb useand disengaged behavior most often failed to show any behavioralrecovery following DA transplantation (Dunnett et al., 1987;Brundin et al., 1994; Winkler et al., 2000; Dobrossy et al., 2000).

The results of this study provide novel insights into the mechanismsof skilled forelimb reaching movements underlying the restorativeplasticity-induced behavioral effects after transplantation of DAneurons. These findings indicate that some movement componentsof skilled reaches may indeed be improved by the transplantationapproach whereas other aspects fail to show any graft-relatedchanges, or even deteriorate. It has been suggested that skilled limbuse is mediated by separate movement subsystems which areassociated with the nigrostriatal pathway, and which are thereforeDA-dependent (Teitelbaum et al., 1983; Metz et al., 2003). It mightbe argued that posture and locomotion are produced by twodifferent movement subsystems; thus a restoration of one systemmight leave the other unaffected. The results of the single pelletreaching test revealed that DA grafts had heterogeneous effects onskilled limb movement components, which might be explained byan incomplete rewiring of the neural circuit. It is expected that amore complete organotypic restoration of the striatum is necessaryto restore the two movement subsystems as well as more com-ponents of the skilled forelimb movement pattern.

Available data document that ectopically placed intrastriatal DAgrafts can reinstate a tonic DA release, which is sufficient to nor-malize DA receptor supersensitivity in the vicinity of the graft (forreview see Bjorklund, 1992), and thereby normalize e.g. the lesion-induced upregulation of mRNAs encoding for preproenkephalin(PPE) and the GABA-synthesizing enzyme glutamic acid dec-arboxylase (GAD67) throughout the striatum, whereas the lesion-induced downregulation of preprotachykinin mRNA remainsunaffected (Winkler et al., 2003). The poor survival rate, the in-complete morphological and electrophysiological maturation of theDA neurons, and the ectopic graft placement concomitant with in-complete establishment of physiological afferent and efferentconnections are major hurdles for a more complete graft-inducedbehavioral recovery (Bjorklund et al., 2003; Isacson et al., 2003).This may indicate that the first phases of skilled reachingmovements are largely dependent on a full organotypic reconstruc-tion of the nigrostriatal pathway, which at the present is only to beachieved in neonatal hosts (Nikkhah et al., 1995a, b; Bentlage et al.,1999) or in using bridge grafts (e.g. Wictorin et al., 1992; Wilby etal., 1999; Winkler et al., 2000). However, ectopic DA grafts canameliorate some qualitative aspects of movement componentsduring the final phase of skilled forelimb reaching, indicating thatthis part of reaching may be regulated by a more basic pattern of DAneurotransmission which can be reinstated by the current graftingprotocol.

The graft-induced mechanisms that underlie the dissociablepatterns of behavioral recovery or impairment observed in skilledforelimb use under the two complimentary test conditions (stair-case test and single pellet reaching test) are likely to involve test-specific compensatory strategies in addition to “true” recovery. In6-OHDA-lesioned animals these can include adjusted patterns offorelimb reaching components, and head and forelimb positioningas 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 of6-OHDA-lesioned rats in the single pellet reaching test, includingpostural adjustments of the tail and the body axis, and deviations offorelimb movement trajectories leading to functional improve-ments. Conversely, it could be postulated that regaining functiondoes not necessarily mean regaining normal movement patterns. Itseems likely that these compensatory adjustments have an

34 A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

influence on the degree of graft-induced behavioral changes, and itwill be highly interesting for future studies to try to unravel thecontribution of each of the processes governing functionalimpairment, compensation and recovery.

The three complementary behavioral tests in this study vary intheir complexity and in the way they challenge the motor skills ofthe animals (Cenci et al., 2002). Drug-induced rotation actuallydoes not challenge voluntary motor performance of the animals butshows drug-induced movements and offers the possibility tomeasure changes in DA storage capacities and postsynaptic DAreceptor sensitivity in vivo. In this test paradigm the animals notonly displayed the most obvious recovery compared to the othertests, but they also showed overcompensation in amphetamine-induced rotation. The staircase test is a medium–complex motor testwhich offers – as previously described – a quantitative measure-ment of grasping behavior. The rats can “train” themselves withinthe 15-min testing period by unlimited grasping for food pellets andthey probably utilize the staircase apparatus to develop compensa-tory movement strategies. In this test we observed a partial recoveryof motor function. In contrast, we could not observe any significantoverall recovery in grasping behavior in the single pellet reachingtest which is the most complex and difficult one. Only the twomovement components supination 2 and release showed significantimprovements; they might have been the crucial movement com-ponents to help improve motor performance in the less complexstaircase test. From these data we postulate that the opportunity todetect recovery after transplantation declines with increasingcomplexity of a motor task. Regaining (motor) function becomesnot only a matter of regaining normal movement patterns but also amatter of the level of complexity of the applied motor tests.

As forelimb reaching and grasping behavior in rats and humansis homologous (Whishaw et al., 2002), the rat model of PDmay alsohave significant implications for current attempts to develop neuro-modulatory, regenerative and restorative strategies for patientssuffering from PD (Lang and Lozano, 1998). The results giveevidence that graft-induced changes can lead to unwanted sideeffects or impairments as previously observed in some humanparkinsonian patients who have received fetal nigral transplants(Winkler et al., 2005). The current clinical protocols of movementassessments in PD patients do not include qualitative aspects ofgrasping behavior but an overall summary of the movementperformance (Unified Parkinson’s Disease Rating Scale, UPDRS).Therefore we strongly suggest that additional tests of graspingbehavior should be included in clinical protocols in PD and othermovement disorders before and after a therapeutical approach, suchas neurotransplantation.

In conclusion, by combining analyses of both qualitative andquantitative aspects of skilled forelimb use it becomes obvious thatDA grafts induce dissociable patterns of functional improvementsand impairments of specific movement components. These novelinsights may enhance our understanding about the mechanismsunderlying transplantation-induced functional effects in animalmodels of PD, and may also foster the development of clinicallysafe and effective restorative cell therapies.

Acknowledgments

This study was supported by the Deutsche Forschungsgemein-schaft (Ni-330), by the Neuroscience Graduate School Freiburg(DFG 843), by the Sonderforschungsbereich 505, the GermanParkinson Foundation, and by the Alberta Heritage Foundation for

Medical Research (to G.A.M.). The authors want to thank EckartKlein for his valuable editorial assistance and Johanna Wessolleckfor her excellent technical assistance.

References

Abercrombie, M., 1946. Estimation of nuclear population from microtomesections. Anat. Rec. 239–247.

Abrous, D.N., Torres, E.M., Dunnett, S.B., 1993. Dopaminergic graftsimplanted into the neonatal or adult striatum: comparative effects onrotation and paw reaching deficits induced by subsequent unilateralnigrostriatal lesions in adulthood. Neuroscience 3, 657–668.

Barker, R.A., Dunnett, S.B., 1999. Nigral grafts in animal models ofParkinson’s disease, 4. Psychology Press, pp. 67–91.

Bentlage, C., Nikkhah, G., Cunningham, M.G., Bjorklund, A., 1999. Refor-mation of the nigrostriatal pathway by fetal dopaminergic micrograftsinto the substantia nigra is critically dependent on the age of the host. Exp.Neurol. 1, 177–190.

Bjorklund, A., 1992. Dopaminergic transplants in experimental parkinson-ism: cellular mechanisms of graft-induced functional recovery. Curr.Opin. Neurobiol. 5, 683–689.

Bjorklund, A., Dunnett, S.B., Nikkhah, G., 1994. Nigral transplants in therat parkinson model – functional limitations and strategies to enhancenigrostriatal reconstructions, 3. Raven Press, New York, pp. 47–69.

Bjorklund, A., Dunnett, S.B., Brundin, P., Stoessl, A.J., Freed, C.R., Breeze,R.E., Levivier, M., Peschanski, M., Studer, L., Barker, R., 2003. Neuraltransplantation for the treatment of Parkinson’s disease. Lancet Neurol.7, 437–445.

Brundin, P., Duan, W.M., Sauer, H., 1994. Functional effects of mesen-cephalic dopamine neurons and adrenal chromaffin cells grafted to therodent striatum, 2. Raven Press, New York, pp. 9–46.

Brundin, P., Karlsson, J., Emgard, M., Schierle, G.S., Hansson, O., Petersen,A., Castilho, R.F., 2000. Improving the survival of grafted dopaminergicneurons: a review over current approaches. Cell Transplant. 2, 179–195.

Cenci, M.A., Whishaw, I.Q., Schallert, T., 2002. Animal models of neuro-logical deficits: how relevant is the rat? Nat. Rev., Neurosci. 7, 574–579.

Dobrossy, M.D., Dunnett, S.B., 2004. Environmental enrichment affectsstriatal graft morphology and functional recovery. Eur. J. Neurosci. 1,159–168.

Dobrossy, M.D., Dunnett, S.B., 2005. Optimising plasticity: environmentaland training associated factors in transplant-mediated brain repair. Rev.Neurosci. 1, 1–21.

Dobrossy, M.D., Le Moal, M., Montaron, M.F., Abrous, N., 2000. Influenceof environment on the efficacy of intrastriatal dopaminergic grafts. Exp.Neurol. 1, 172–183.

Dunnett, S.B., Bjorklund, A., Schmidt, R.H., Stenevi, U., Iversen, S.D.,1983. Intracerebral grafting of neuronal cell suspensions: IV. Beha-vioural recovery in rats with unilateral 6-OHDA lesions followingimplantation of nigral cell suspensions in different forebrain sites. ActaPhysiol. Scand., Suppl. 29–37.

Dunnett, S.B., Whishaw, I.Q., Rogers, D.C., Jones, G.H., 1987. Dopamine-rich grafts ameliorate whole body motor asymmetry and sensory neglectbut not independent limb use in rats with 6-hydroxydopamine lesions.Brain Res. 1, 63–78.

Faraji, J., Metz, G.A., 2007. Sequential bilateral striatal lesions have additiveeffects on single skilled limb use in rats. Behav. Brain Res. 2, 195–204.

Hagell, P., Schrag, A., Piccini, P., Jahanshahi, M., Brown, R., Rehncrona, S.,Widner, H., Brundin, P., Rothwell, J.C., Odin, P., Wenning, G.K.,Morrish, P., Gustavii, B., Bjorklund, A., Brooks, D.J., Marsden, C.D.,Quinn, N.P., Lindvall, O., 1999. Sequential bilateral transplantation inParkinson’s disease: effects of the second graft. Brain 1121–1132.

Isacson, O., Bjorklund, L.M., Schumacher, J.M., 2003. Toward full resto-ration of synaptic and terminal function of the dopaminergic system inParkinson’s disease by stem cells. Ann. Neurol. S135–S146.

Kirik, D., Rosenblad, C., Bjorklund, A., 1998. Characterization of beha-vioral and neurodegenerative changes following partial lesions of the

35A. Klein et al. / Neurobiology of Disease 27 (2007) 24–35

nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopa-mine in the rat. Exp. Neurol. 2, 259–277.

Kloth, V., Klein, A., Loettrich, D., Nikkhah, G., 2006. Colour-coded pelletsincrease the sensitivity of the staircase test to differentiate skilledforelimb performances of control and 6-hydroxydopamine lesioned rats.Brain Res. Bull. 1, 68–80.

Lang, A.E., Lozano, A.M., 1998. Parkinson’s disease. Second of two parts.N. Engl. J. Med. 16, 1130–1143.

Lindvall, O., Hagell, P., 2000. Clinical observations after neural transplanta-tion in Parkinson’s disease. Prog. Brain Res. 299–320.

Mandel, R.J., Brundin, P., Bjorklund, A., 1990. The importance of graftplacement and task complexity for transplant-induced recovery ofsimple and complex sensorimotor deficits in dopamine denervated rats.Eur. J. Neurosci. 10, 888–894.

Metz, G.A., Whishaw, I.Q., 2000. Skilled reaching an action pattern:stability in rat (Rattus norvegicus) grasping movements as a function ofchanging food pellet size. Behav. Brain Res. 2, 111–122.

Metz, G.A., Gonzalez, C.L., Piecharka, D.M., Whishaw, I.Q., 2003. Acutealcohol administration improves skilled reaching success in intact butnot 6-OHDA dopamine depleted rats: a subsystems analysis of themotoric and anxiolytic effects of alcohol. Behav. Brain Res. 1–2,167–174.

Miklyaeva, E.I., Castaneda, E., Whishaw, I.Q., 1994. Skilled reachingdeficits in unilateral dopamine-depleted rats: impairments in movementand posture and compensatory adjustments. J. Neurosci. 11 (Pt. 2),7148–7158.

Montoya, C.P., Astell, S., Dunnett, S.B., 1990. Effects of nigral and striatalgrafts on skilled forelimb use in the rat. Prog. Brain 459–466.

Montoya, C.P., Campbell-Hope, L.J., Pemberton, K.D., Dunnett, S.B., 1991.The “staircase test”: a measure of independent forelimb reaching andgrasping abilities in rats. J. Neurosci. Methods 2–3, 219–228.

Nikkhah, G., Duan, W.M., Knappe, U., Jodicke, A., Bjorklund, A., 1993a.Restoration of complex sensorimotor behavior and skilled forelimb useby a modified nigral cell suspension transplantation approach in the ratParkinson model. Neuroscience 1, 33–43.

Nikkhah, G., Odin, P., Smits, A., Tingstrom, A., Othberg, A., Brundin, P.,Funa, K., Lindvall, O., 1993b. Platelet-derived growth factor promotessurvival of rat and human mesencephalic dopaminergic neurons inculture. Exp. Brain Res. 3, 516–523.

Nikkhah, G., Bentlage, C., Cunningham, M.G., Bjorklund, A., 1994a.Intranigral fetal dopamine grafts induce behavioral compensation in therat Parkinson model. J. Neurosci. 6, 3449–3461.

Nikkhah, G., Olsson, M., Eberhard, J., Bentlage, C., Cunningham, M.G.,Bjorklund, A., 1994b. A microtransplantation approach for cellsuspension grafting in the rat Parkinson model: a detailed account ofthe methodology. Neuroscience 1, 57–72.

Nikkhah, G., Cunningham,M.G., Cenci, M.A.,McKay, R.D., Bjorklund, A.,1995a. Dopaminergic microtransplants into the substantia nigra of neo-natal rats with bilateral 6-OHDA lesions: I. Evidence for anatomicalreconstruction of the nigrostriatal pathway. J. Neurosci. 5 (Pt. 1),3548–3561.

Nikkhah, G., Cunningham, M.G., McKay, R., Bjorklund, A., 1995b.Dopaminergic microtransplants into the substantia nigra of neonatal ratswith bilateral 6-OHDA lesions: II. Transplant-induced behavioralrecovery. J. Neurosci. 5 (Pt. 1), 3562–3570.

Nikkhah, G., Rosenthal, C., Falkenstein, G., Samii, M., 1998a. Dopaminer-gic graft-induced long-term recovery of complex sensorimotor behaviorsin a rat model of Parkinson’s disease. Zentralbl. Neurochir. 2, 97–103.

Nikkhah, G., Rosenthal, C., Hedrich, H.J., Samii, M., 1998b. Differences inacquisition and full performance in skilled forelimb use as measured bythe ‘staircase test’ in five rat strains. Behav. Brain Res. 1, 85–95.

Nikkhah, G., Falkenstein, G., Rosenthal, C., 2001. Restorative plasticity ofdopamine neuronal transplants depends on the degree of hemisphericdominance. J. Neurosci. 16, 6252–6263.

Paxinos, G., Watson, C., 1997. The Rat Brain in Stereotactic Coordinates.Academic Press, San Diego, CA.

Rodter, A., Winkler, C., Samii, M., Nikkhah, G., 2000. Complex senso-rimotor behavioral changes after terminal striatal 6-OHDA lesion andtransplantation of dopaminergic embryonic micrografts. Cell Transplant.2, 197–214.

Teitelbaum, P., Schallert, T., Whishaw, I.Q. 1983. ources of spontaneity inmotivated. 23–65.

Ungerstedt, U., 1968. 6-Hydroxy-dopamine induced degeneration of centralmonoamine neurons. Eur. J. Pharmacol. 1, 107–110.

Ungerstedt, U., Arbuthnott, G.W., 1970. Quantitative recording of rotationalbehavior in rats after 6-hydroxy-dopamine lesions of the nigrostriataldopamine system. Brain Res. 3, 485–493.

Vergara-Aragon, P., Gonzalez, C.L., Whishaw, I.Q., 2003. A novel skilled-reaching impairment in paw supination on the “good” side of the hemi-Parkinson rat improved with rehabilitation. J. Neurosci. 2, 579–586.

Wenning, G.K., Odin, P., Morrish, P., Rehncrona, S., Widner, H., Brundin,P., Rothwell, J.C., Brown, R., Gustavii, B., Hagell, P., Jahanshahi, M.,Sawle, G., Bjorklund, A., Brooks, D.J., Marsden, C.D., Quinn, N.P.,Lindvall, O., 1997. Short- and long-term survival and function ofunilateral intrastriatal dopaminergic grafts in Parkinson’s disease. Ann.Neurol. 1, 95–107.

Whishaw, I.Q., Pellis, S.M., 1990. The structure of skilled forelimb reachingin the rat: a proximally driven movement with a single distal rotatorycomponent. Behav. Brain Res. 1, 49–59.

Whishaw, I.Q., Pellis, S.M., Gorny, B., Kolb, B., Tetzlaff, W., 1993.Proximal and distal impairments in rat forelimb use in reaching followunilateral pyramidal tract lesions. Behav. Brain Res. 1, 59–76.

Whishaw, I.Q., Coles, B.L., Pellis, S.M., Miklyaeva, E.I., 1997a. Impair-ments and compensation in mouth and limb use in free feeding afterunilateral dopamine depletions in a rat analog of human Parkinson’sdisease. Behav. Brain Res. 1–2, 167–177.

Whishaw, I.Q., Woodward, N.C., Miklyaeva, E., Pellis, S.M., 1997b.Analysis of limb use by control rats and unilateral DA-depleted rats inthe Montoya staircase test: movements, impairments and compensatorystrategies. Behav. Brain Res. 1–2, 167–177.

Whishaw, I.Q., Suchowersky, O., Davis, L., Sarna, J., Metz, G.A., Pellis,S.M., 2002. Impairment of pronation, supination, and body co-ordinationin reach-to-grasp tasks in human Parkinson’s disease (PD) revealshomology to deficits in animal models. Behav. Brain Res. 2, 165–176.

Wictorin, K., Brundin, P., Sauer, H., Lindvall, O., Bjorklund, A., 1992. Longdistance directed axonal growth from human dopaminergic mesence-phalic neuroblasts implanted along the nigrostriatal pathway in6-hydroxydopamine lesioned adult rats. J. Comp. Neurol. 4, 475–494.

Wilby, M.J., Sinclair, S.R., Muir, E.M., Zietlow, R., Adcock, K.H., Horellou,P., Rogers, J.H., Dunnett, S.B., Fawcett, J.W., 1999. A glial cell line-derived neurotrophic factor-secreting clone of the Schwann cell lineSCTM41 enhances survival and fiber outgrowth from embryonicnigral neurons grafted to the striatum and to the lesioned substantianigra. J. Neurosci. 6, 2301–2312.

Winkler, C., Sauer, H., Lee, C.S., Bjorklund, A., 1996. Short-term GDNFtreatment provides long-term rescue of lesioned nigral dopaminergicneurons in a rat model of Parkinson’s disease. J. Neurosci. 22,7206–7215.

Winkler, C., Bentlage, C., Nikkhah, G., Samii, M., Bjorklund, A., 1999.Intranigral transplants of GABA-rich striatal tissue induce behavioralrecovery in the rat Parkinson model and promote the effects obtained byintrastriatal dopaminergic transplants. Exp. Neurol. 2, 165–186.

Winkler, C., Kirik, D., Bjorklund, A., Dunnett, S.B., 2000. Transplantationin the rat model of Parkinson’s disease: ectopic versus homotopic graftplacement. Prog. Brain 233–265.

Winkler, C., Bentlage, C., Cenci, M.A., Nikkhah, G., Bjorklund, A., 2003.Regulation of neuropeptide mRNA expression in the basal ganglia byintrastriatal and intranigral transplants in the rat Parkinson model.Neuroscience 4, 1063–1077.

Winkler, C., Kirik, D., Bjorklund, A., 2005. Cell transplantation inParkinson’s disease: how can we make it work? Trends Neurosci. 2,86–92.