Experimental Neurology 163, 85–97 (2000)doi:10.1006/exnr.1999.7341, available online at http://www.idealibrary.com on

Embryonic Donor Age and Dissection Influences Striatal GraftDevelopment and Functional Integration in a Rodent

Model of Huntington’s DiseaseColin Watts,*,†,1 Peter J. Brasted,* Dawn M. Eagle, and Stephen B. Dunnett*,‡

*MRC Cambridge Centre for Brain Repair, †Department of Neurosurgery, and ‡Department of Experimental Psychology,University of Cambridge, Cambridge, United Kingdom

Received March 10, 1999; accepted December 28, 1999

The method of embryonic dissection and the age ofthe donor material remain areas of controversy in thepreparation of striatal tissue for intrastriatal implan-tation. This study explores the relationship betweenthese two parameters with respect to the morphology,function, and physiological integration of the result-ant grafts. Tissue derived from embryos of 14 and 16days of gestation (CRL 10–11 and 14–15 mm, respec-tively) was prepared as whole, lateral, and medial gan-glionic eminence suspensions (WGE, LGE, and MGE,respectively). The embryonic material was implantedinto the excitotoxically lesioned striatum of host rats.Grafts derived from E14 LGE attenuated drug-inducedrotational bias whereas grafts from E14 MGE amelio-rated contralateral deficits in paw reaching. Sixmonths after grafting retrograde tracing of graft pro-jections to the globus pallidus was performed followedby electrical excitation of cortical afferent fibers.Grafts derived from E14 WGE had the largest volumeof striatum-like tissue and more striatal neurons com-pared to LGE from the same donor age. These resultssuggest that MGE tissue as well as LGE plays a role inthe structural and functional integration of striatalgrafts. © 2000 Academic Press

INTRODUCTION

The transplantation of embryonic striatal tissue inthe excitotoxically lesioned striatum of the adult rat, amodel of Huntington’s disease (HD), has provided auseful model for the study of graft development, graft–host connectivity, circuit reconstruction, and ameliora-tion of lesion-induced functional deficits (for reviewssee 1, 5, 33). These results, coupled with the results ofclinical trials in Parkinson’s disease (PD) (see, for ex-

1 To whom correspondence should be addressed at MRC Cam-bridge Centre for Brain Repair, University of Cambridge, ForvieSite, Robinson Way, Cambridge CB2 2PY, UK. Fax: (144) 1223331174. E-mail: cw209@cam.ac.uk.

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ample, 17), have led to early clinical trials in patientswith HD. While initial reports are encouraging (16, 18)the results in humans have yet to be fully realized.

Controversy still exists at a scientific level over var-ious aspects of transplantation in models of Hunting-ton’s disease (2, 28) including the issues of embryonicdissection and donor age. Recent case reports of fatal-ities in patients with PD due to the implantation andsubsequent growth of the wrong tissue (8, 19) highlightthe need for continued scientific rigor when applyingthis technology in the clinic.

The development of the subdissection of the lateralcomponent of the embryonic ganglionic eminence(LGE) and the high proportion of acetylcholinesterase(AChE) positive tissue they contained (4, 27) led toincreased interest in using this method of embryonicdissection to increase the amount of striatum-like tis-sue within the grafts. Correlation of the proportion ofstriatal tissue within the grafts with functional recov-ery (11, 23) strengthened the case for using LGE tis-sue.

However, the graft volumes reported by Pakzabanand co-workers were small indicating that the highproportion of striatum-like tissue may have been dueto exclusion of extraneous tissue rather than expansionof the striatal component per se. Increasing interest inthe role of the medial ganglionic eminence (MGE) instriatal neurogenesis (27) emphasized the need for adirect comparison of LGE grafts with grafts derivedfrom the whole ganglionic eminence (WGE).

The aim of this study was to investigate the potentialinteraction between donor age and embryonic dissec-tion on graft survival and development. In addition,the potential for physiological connectivity with thehost was explored to try to determine the effect ofdonor age and dissection on functional integration ofthe graft with the host. The relationship between graftmorphology and functional effects in simple and morecomplex motor tasks was also evaluated.

0014-4886/00 $35.00Copyright © 2000 by Academic Press

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METHODS

Experimental Design

Adult female Sprague–Dawley rats (Charles River,UK) of approximately 200 g at the start of each exper-iment were used. They were housed in group cages in anatural light–dark cycle with free access to food andwater throughout unless paw reaching studies werebeing conducted (q.v.).

Seventy animals were lesioned with quinolinic acidthen allocated to seven groups of 10 rats. Six groupsreceived implants of embryonic neural tissue fromWGE, LGE, or MGE from donors of 14 (E14) or 16(E16) days of gestation. One group were not grafted(see Table 1). On completion of behavioral testing trac-ing studies were performed and the animals were sac-rificed for graft analysis.

Lesioning of the Host Striatum

All surgical procedures were performed under halo-thane anesthesia (2%) with the animals positioned in asmall animal stereotaxic frame (Kopf surgical instru-ments). Unilateral quinolinic acid lesions were madeby the infusion of 0.5 ml, over 4 min, of quinolinic acid(Sigma) made up in 0.1 M phosphate buffer (PB; pH7.4) to a concentration of 360 nmol/ml. Infusions wereat two sites at the following coordinates: A 5 1.2 mm,L 5 2.6 mm, V 5 4.5 mm; and A 5 0.0 mm, L 5 3.4 mm,

5 4.5 mm; with anterior (A) measured from bregma,ateral (L) measured from the midline, vertical (V)

easured from the dura, and the incisor bar set 2.5m below the interaural line.

issue Preparation and Implantation

Time-mated rat embryos of appropriate gestationalge were removed from terminally anesthetized preg-ant Sprague–Dawley rats by caesarean section. Thembryonic brain was removed and transferred to a dishontaining Dulbecco’s modified Eagle mediumDMEM). The developing skull and underlying menin-es were removed and the lateral ventricle was opened

TABLE 1

Experimental Groups

Group Donor tissue CRL (mm) Cell number % Viable

1 E14 WGE 10–11 600,000 98.62 E14 LGE 10–11 400,000 97.23 E14 MGE 10–11 400,000 98.34 E16 WGE 14–15 1,200,000 975 E16 LGE 14–15 800,000 96.56 E16 MGE 14–15 600,000 95.47 Lesion — — —

Note. E14 and E16, days of gestation; WGE, LGE, and MGE,whole, lateral, and medial ganglionic eminence, respectively.

sing a parasaggital incision of the cortex to expose thetriatal primordium. The ganglionic eminence, con-aining the striatal primordium, can be identified as aentrolateral elevation separated into medial and lat-ral parts by a shallow groove from embryonic day 14nward.Dissection of the striatal anlage was performed us-

ng either the standard dissection (6), in which thehole ganglionic eminence was removed, or the selec-

ive dissection of lateral and/or medial components27).

The pooled embryonic neural tissue was washedhree times in DMEM then incubated in 0.1% bovinerypsin (Worthington) in DMEM at 37°C for 15 minnd then washed followed by further incubation 0.05%Nase in DMEM for 5 min. After additional washes inrafting medium the cells were then dissociated byrituration through fire-polished Pasteur pipettes ofrogressively smaller diameter. Cell number and ini-ial viability were assessed in a hemocytometer usingrypan blue dye exclusion. Only suspensions with aiability greater than 95% were implanted; the cellsere centrifuged at 1000 rpm for 5 min and resus-ended in the final volume of grafting medium.Two deposits of 1 ml each were made into the host

striatum in both lesioned and unlesioned animals atthe following coordinates: A 5 0.7 mm, L 5 2.9 mm,V 5 4.0 mm and 4.5 mm with A measured frombregma, L measured from the midline, V measuredfrom the dura, and the incisor bar set 2.5 mm below theinteraural line.

Processing of Tissue from Graft Experiments

Following terminal anesthesia with sodium pento-barbitone (Euthatal) each rat was perfused transcar-dially with phosphate-buffered saline (PBS; pH 7.4) for1 min, followed by 10% formalin in 0.1 M PBS for 4min. The brains were postfixed in 10% formalin for24 h at 14°C and then placed in 25% sucrose solutionor 48 h at 14°C. Sections were cut on a freezing

icrotome at 60 mm through the striatum and col-lected in Tris-buffered saline (TBS; pH 7.4).

One in six free-floating sections were processed fordopamine- and adenosine-regulated phosphoprotein of32 kDa (DARPP-32) immunohistochemistry and a sec-ond series for tyrosine hydroxylase (TH) immunohisto-chemistry. After quenching and washing in PBS thesections were blocked with normal goat serum (30 ml/ml) dissolved in 0.2% Triton X-100 in TBS (TXTBS) for1 h at room temperature. The sections were trans-ferred, unwashed, to the primary antibody solution(TH 1:3000, Jaques boy, Reims, France; DARPP-321:2,0000) with 1% normal goat serum in TXTBS for48 h at 14°C. Bound antibodies were visualized usingn avidin–biotin–peroxidase complex system (Dako

I

87DONOR AGE AND DISSECTION INFLUENCE GRAFT DEVELOPMENT AND FUNCTION

streptavidin ABC kit) with diaminobenzidine as chro-mogen.

Two additional series of mounted sections were alsoprocessed for AChE histochemistry (modified Koellemethod) and for the Nissl stain cresyl fast violet.

Postmortem detection of tracer within striatal graftsinvolved immunohistochemical identification using an-tibodies against fluorogold (FG) (Chemicon 1:5000).The projections were then quantified using optical den-sity measurements with the maximal level of stainingdefined by the optical density at the injection site.

Immunohistochemical detection of c-fos was per-formed using antibodies to c-fos (Chemicon 1:20,000).

Volume Determinations and Cell Quantification

Morphological evaluation of the volume of the grafts,lesions, and striatum was performed on every sixthsection using a computerized image processing system(Seescan Cambridge Systems). The margin of eachstructure was outlined manually on the video monitorand the surface area expressed in square millimeters.The volumes were then calculated by summing thesection areas and correcting for section thickness andfrequency. The grafts were further subdivided into ar-eas of dense AChE, TH, and DARPP-32 staining (patchor P-zones) and areas in which staining was weak orabsent (nonpatch or NP-zones) (12). Total P-zone vol-ume was determined from digitized images obtainedfrom camera lucida drawings of each section and vol-umes were calculated. Since the lesions and graftswere contained within the head of the neostriatum allstriatal volume measurements were truncated ana-tomically at the genu of the corpus callosum anteriorlyand the decussation of the anterior commissure poste-riorly. Striatal loss relative to the contralateral intactside was determined by the difference in striatal vol-umes between the lesioned and contralateral intactside.

TH fiber ingrowth to the graft was measured usingoptical density measurements. Maximal TH ingrowthwas defined by the optical density of the dorsomedialstriatum on the intact side (thereby avoiding internalcapsule fibers) and a background level of staining wasdefined by the optical density of the corpus callosum.

The total numbers of DARPP-32 cells in the graftsand in both intact and lesioned striatum were countedstereologically (Olympus, Denmark A/S) (13, 14).

Determination of Graft Connectivity

TH fiber ingrowth to the graft was measured usingoptical density measurements under conditions ofstandardized illumination (SeeScan Cambridge Sys-tems). Maximal TH ingrowth was defined by the opti-cal density of the dorsomedial striatum on the intactside (thereby avoiding internal capsule fibers) and a

background level of staining was defined by the opticaldensity of the corpus callosum.

Efferent projections from striatal grafts to the globuspallidus were identified in all animals using the retro-grade tracer FG (5% FG in 0.9% saline). The tracer wasinjected 6 days prior to termination of the experimentat the following coordinates relative to bregma: A 520.9 mm, L 5 23.0 mm, and V 5 26.0 mm with theincisor bar set at 23.3 mm below the interaural line.nfusions of 0.2 ml were made over 5 min after the

cannula had been lowered in to position and left for 5min. When the injection was complete the cannula wasleft in position for an additional 10 min to allow thetracer to disperse before the cannula was withdrawn.

Cortical Stimulation and the Activation of theImmediate Early Gene c-fos

Up-regulation of the immediate early gene c-foswithin the striatal grafts and the host globus palliduswas investigated following electrical excitation of affer-ent cortical projections. Cortical stimulation was per-formed on anesthetized Sprague–Dawley rats in whichthe right frontoparietal cortex had been exposed via asmall burr hole. The stimulating electrode (NE-100/50mm) (Clark Electromedical Instruments) was insertedjust below the pial surface of the exposed cortex at thefollowing coordinates relative to bregma: A 5 2.5 mm,L 5 21.0 mm, and V 5 21.0 mm relative to dura withthe incisor bar set at 23.3 mm below the interauralline. Current was delivered to the electrode from aGrass S11 dual output digital stimulator via twoPSIU6 photoelectric stimulus isolation units (GrassMedical Instruments). Symmetrical biphasic, 100-Hzcurrent pulses of 0.5 ms duration and a pulse delay of0.5 ms were delivered every 10 ms as 20-ms-long trainsat a frequency of one train every 200 ms. The voltagewas adjusted to produce limb and body movementswithout generalized convulsions. The stimulus wasthen maintained for 10 min after which the electrodewas withdrawn and the animal was recovered. Approx-imately 90 min after cortical stimulation the animalswere perfused in the normal manner and the tissuewas processed as described above.

Cortical stimulation was performed on all graftedanimals. Two animals in each group served as negativecontrols. These animals underwent exposure of thesensorimotor cortex and insertion of a stimulating elec-trode but no current was applied.

Drug-Induced Rotation

Unilateral lesions of the striatum allow the quanti-fication of a circling bias in response to the stimulantdrug amphetamine which acts presynaptically stimu-lating the release of dopamine.

The d-amphetamine isoform was used in these ex-periments due to its lower toxicity compared to met-

88 WATTS ET AL.

amphetamine. It was administered at a dose of 2.5mg/kg ip in 0.9% saline.

The animals were then placed in automated rotom-eter bowls after Ungerstedt and Arbuthnott (31). Thenumber of rotations per 10 min was collected over atest period of 90 min. The number of rotations to boththe left and the right was recorded for each 10-minperiod and then summated at the end of the test pe-riod. From these data the net bias to left or right wasdetermined.

Paw Reaching

Independent skilled forelimb use in lesioned andgrafted animals was analyzed using the “staircase”(21). The apparatus consists of a Perspex box with asmall clear Perspex platform at the front. On each sideof the platform a series of eight steps descends to thefloor of the chamber. Each step has a well in it in whichwere placed two food pellets (P. J. Noyes Company Inc.,Lancaster, UK).

Each animal was trained to retrieve these food pel-lets using its forepaws. Prior to testing each animalwas placed on a food deprivation regime of 10–12 g ofchow per animal per day sufficient to maintain bodyweight at 90% of normal. This was to maximize themotivational effort of retrieval. The apparatus is de-signed so that retrieval from any well below the firstcan only be achieved using the ipsilateral paw.

FIG. 1. A typical striatal graft. A typical graft illustrating thegross histological appearance on Nissl (A), AChE (B), DARPP-32 (C),and TH (D) staining. Note the patchy appearance against a relativelyunstained background. The patches comprise the striatum-like tis-sue within the graft. Magnification, 350; bar, 0.5 mm.

Testing took place 3 and 6 months after surgery.After 2 days as an initial period of familiarizationand training, in which wells were overfilled with foodpellets, the number of pellets was reduced to two perwell. The animals were left in the chamber for a testperiod of 10 min after which they were returned totheir home cages. At 3 months daily tests were per-formed until performance plateaued while at 6months daily tests were performed for an additional10 days. The number of pellets removed with eachpaw was determined from the total number missingon each side.

Statistical Analysis

Statistical evaluation was conducted by analysis ofvariance with Donor Age and Embryonic Dissectionas factors between groups with post hoc comparisonbetween groups using the Newman–Keuls test. Be-havioral results were analyzed with respect to donorage, embryonic dissection, and rotational bias foramphetamine-induced rotation and donor age, em-bryonic dissection, and side of surgery for paw reach-ing. Post hoc comparison between each grafted groupand the lesion only group was performed using Dun-nett’s test.

FIG. 2. LGE-derived grafts have a higher proportion of AChE-positive tissue. On gross examination a comparison of grafts of WGE(top row) and LGE (bottom row) revealed that E14 LGE graftsappeared noticeably smaller and had a greater proportion of AChE-positive tissue than their WGE counterparts (A and C, Nissl; B andD, AChE staining. Magnification, 350; bar, 0.5 mm).

89DONOR AGE AND DISSECTION INFLUENCE GRAFT DEVELOPMENT AND FUNCTION

RESULTS

General Appearance

The striatal lesions were similar to those describedelsewhere (29, 32). During the 6 months following le-sioning, the striatum underwent extensive atrophywith collapse of the neuropil and corresponding ven-tricular expansion. The atrophy was greatest at thehead of the caudate putamen and proceeded in a caudaldirection. Fibers of the internal capsule were preservedand constituted the bulk of the residual striatal vol-ume.

A typical graft is illustrated in Fig. 1. Nissl stainingof grafts from each group did not reveal evidence ofinflammatory or microglial cells within the grafts, atthe graft–host interface or within the residual hoststriatum. Large-bodied cells with centrally located nu-clei typical of large neurons were visible in abundancewithin all the grafts as were smaller cells more typicalof glia.

Analysis of Graft Morphology and Connectivity

Graft volume. Acetylcholinesterase histochemistrydemonstrated the typical patchy appearance of areas ofAChE positivity interspersed with areas of AChE neg-ativity that characterizes striatal grafts. This morphol-ogy was present in grafts derived from both WGE andLGE. Grafts derived from E14 lateral eminence tendedto have fewer AChE-negative regions but this was not

FIG. 3. MGE-derived grafts contain AChE-positive tissue. Graftsof donor age. There was no histological evidence of DARPP-32-positGrafts of MGE tissue from E14 donors (top row) and E16 donors (boand F). Magnification, 350; bar, 0.5 mm.

readily obvious in some grafts when compared to WGEgrafts of the same donor age (Fig. 2).

Grafts in E14 lateral and both medial groups werenoticeably smaller than grafts from other groups. Thedifference in size between E14 WGE and LGE graftswas more readily apparent than differences in patchzones. Grafts derived from medial eminence tissuewere visibly different from grafts derived from othergroups (Fig. 3). However, although typical patch-–non-patch compartmentalization of these grafts was notpresent there were areas of AChE positivity in bothE14 and E16 grafts. The distribution of cholinergicfibers within medial grafts from younger donors wasmore diffusely distributed compared to a more periph-eral orientation in older donor grafts (see Figs. 3A and3D). Fibers of passage were compressed around bothgrafts and did not penetrate the graft parenchyma. Inthe younger donor age MGE grafts the cholinergic tis-sue did not correspond to any DARPP-32 staining orTH staining which was completely absent in graftsfrom the younger donor age group (Figs. 3B and 3C). Ingrafts derived from the medial eminence of older em-bryos there was weak DARPP-32 and TH stainingaround the periphery of the grafts that appeared tocolocalize with the AChE staining (Figs. 3E and 3F).This observation was consistent in grafts from thesegroups.

Quantification of the graft volume with respect todonor age and dissection revealed that it was signifi-

ived from MGE contained AChE-positive tissue across the spectrummedium-spiny neurons or dopaminergic innervation in the grafts.

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cantly affected by both parameters (Fig. 5A; Age 3Dissection: F2,53 5 40.20, P , 0.001). There was nodifference in graft volume between donor ages for WGEand MGE dissections (Fig. 5A; E14 vs E16, t2,53 5 0.89nd 2.06, both nonsignificant). However, grafts derivedrom LGE were significantly larger from E16 donorsompared to E14 donors (Fig. 5A; E16 vs E14, t2,53 5

14.07, P , 0.01). Indeed the E16 LGE grafts werelarger than E16 WGE grafts (t2,53 5 4.77, P , 0.05).

Patch-zone volume. Grafts from both WGE andGE of both donor ages contained DARPP-32 patchesnd TH patches that corresponded closely with thereas of intense AChE positivity (e.g., Figs. 1B–1D).The volume of this striatum-like tissue was influ-

nced by both donor age and embryonic dissection (Fig.B; Age 3 Dissection: F2,53 5 16.13, P , 0.001). Theatch volume was greater in grafts derived fromounger donors in grafts of WGE tissue (E14 vs E16,2,53 5 4.58, P , 0.01) but from older donors in grafts ofGE tissue (E16 vs E14, t2,53 5 6.77, P , 0.01). Indeed,

of the standard donor tissue options the use of LGEmaterial from E14 donors resulted in grafts with thesmallest volume of striatal tissue (WGE vs LGE, t2,53 59.79, P , 0.01). Furthermore, the overall effect of dis-ection on patch volume favors WGE dissection irre-pective of donor age (F2,53 5 11.84, P , 0.001; WGE vGE, t2,53 5 5.82, P , 0.05). There was no difference inGE grafts with respect to age (t2,53 5 1.55, nonsignif-

icant).Proportion of striatum-like tissue. The proportion

of striatal tissue within the grafts was affected by bothembryonic dissection and donor age (Fig. 5C; Age 3Dissection: F2,53 5 123.97, P , 0.001). In grafts derivedfrom both WGE and LGE a higher proportion of stria-tal tissue was present in grafts of younger donor tissue(E14 vs E16, t2,53 5 5.29, P , 0.05 and t2,53 5 24.30, P ,0.01, respectively). Hence, E14 LGE grafts have thesmallest volume but the highest proportion of stria-tum-like tissue (Figs. 5B and 5C).

DARPP-32 cell count. The number of DARPP-32-positive medium spiny neurons within the grafts wasalso determined by an interaction between embryonicage and dissection (Fig. 5D; Age 3 Dissection: F2,53 532.08, P , 0.001). A similar number of DARPP-32-

ositive cells was present in grafts of WGE tissue fromoth donor ages (E16 vs E14, t2,53 5 1.88, nonsignifi-ant) but more cells were found in LGE grafts from E16onors compared to E14 donors (t2,53 5 17.38, P , 0.01).

This relationship also applied to DARPP-32-positivecells from MGE tissue (E16 vs E14, t2,53 5 5.90, P ,.05). More neurons were obtained using whole ratherhan lateral dissections from E14 donors (t2,53 5 9.14, P

, 0.01) and from lateral rather than whole dissectionswhen using E16 donors (t2,53 5 6.36, P , 0.01).

Graft connectivity. Projections to the globus palli-us were present in grafts of WGE and LGE tissue

rom both donor ages and were readily visualized.rafts of MGE tissue had visibly fewer FG-positive

ells within the grafts. The distribution of FG-positiveells within the grafts corresponded closely with theatch zones (compare Fig. 4A and Fig. 2D).Functional afferent cortical projections, as deter-ined by cortical stimulation, were more sparsely dis-

ributed and were identified in grafts derived fromGE and LGE of E14 donors only. Activation of the

mmediate early gene c-fos was identified in the nucleif cells arranged in clusters within the grafts. Theselusters appeared to correspond with areas ofARPP-32 positivity. Gene expression was also iden-

ified within the host parenchyma in small numbers.o activation was observed in nonpatch tissue and in

ontrol animals. Detailed observation revealed many,ut not all, of these cells to be exhibiting weak FGtaining (Fig. 4B). However, the number of cells withinhe grafts expressing c-fos was small.

There was no interaction between donor age andissection with respect to afferent dopaminergic inner-ation of the grafts (Fig. 6A; Age 3 Dissection: F2,53 5

3.16, P 5 0.051). However, dopaminergic innervationf grafts derived from older donor tissue was greaterhan younger donor tissue (Age: F1,53 5 6.75, P 5 0.012;

E16 vs E14, t2,53 5 3.68, P , 0.05) and greater forlateral dissection compared to whole or medial dissec-tions (Dissection: F2,53 5 14.13, P , 0.001; LGE vs

GE, t2,53 5 3.72, P , 0.05; and LGE vs MGE, t3,53 53.72, P , 0.01).

Efferent projections to the globus pallidus were de-termined by an interaction between donor age anddissection (Fig. 6B; Age 3 Dissection: F2,53 5 11.97, P ,0.001). There was no difference in projections fromgrafts of WGE from older or younger donors (E14 vsE16,1 t2,53 5 2.22, nonsignificant). However, LGEgrafts from E14 donors had more efferent projections tothe globus pallidus than E16 donors (E14 vs E16, t2,53 59.29, P , 0.01). LGE dissection was superior to WGEfor E14 donors but not for E16 donors (LGE vs WGE,t2,53 5 6.46, P , 0.05; and t2,53 5 0.61, nonsignificant,respectively) reflecting the higher proportion of patchzones within E14 LGE grafts. MGE-derived grafts hadsignificantly fewer efferent projections in grafts de-rived from both donor ages (post hoc comparisons all P, 0.05).

Analysis of Graft-Mediated Functional Recovery

Analysis of rotation bias revealed an interaction be-tween graft groups and the timing of the testing post-grafting (F24,252 5 1.83, P , 0.01). Post hoc comparisonof each transplanted group against the lesion onlygroup showed significant reduction in rotation bias forgrafts of E14 LGE tissue at 6 and 9 but not 12 weekspostgrafting (Fig. 7; t24,252 5 2.96 and 2.85 both P ,0.05; and t24,252 5 2.09, nonsignificant, respectively). No

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91DONOR AGE AND DISSECTION INFLUENCE GRAFT DEVELOPMENT AND FUNCTION

other group showed a significant reduction in rotationbias compared to the lesion only group. However, adownward trend was observed for the E14 WGE group(Fig. 7).

A different pattern of graft-mediated functional ef-fect was observed with more complex motor tests in-volving paw reaching and retrieval. The contralateralleft paw retrieved fewer pellets compared to the rightpaw (Fig. 8; Groups 3 Trials 3 Side F36,372 5 1.73, P 50.007). On the right side there was no difference be-tween groups in trials 3 and 6 months postgraftingcompared to the lesion only group (Dunnett’s post hoccomparisons all nonsignificant). Further analysis, re-

FIG. 4. Graft connectivity. (A) Efferent projections to the globuspallidus were identified in grafts of WGE and LGE tissue from bothdonor ages. These projections arose from areas of striatum-like tis-sue within the grafts. Arrows indicate graft–host boundary. Magni-fication, 3100; bar, 250 mm. (B) Some cells within E14 WGE and

GE grafts expressed c-fos (blue staining) in conjunction with weakuorogold labeling (brown stain) suggesting that physiological stim-lation of cortical afferent projections to striatal grafts may be ca-able of stimulating graft projection neurons.

stricted to the left paw, confirmed an interaction be-tween the graft groups and time over which trials wereconducted (F36,372 5 1.67, P , 0.01). Post hoc compari-on of each grafted group against the lesion only groupdentified the group grafted with medial ganglionicminence from E14 donors to be retrieving signifi-antly more pellets on the second and third week ofrials 3 months postgrafting and on both trials 6onths after grafting (Fig. 8; t36,372 5 2.54 and 2.51 at

3 months and 2.38 and 2.53 at 6 months all P , 0.05).No other graft group retrieved significantly more pel-lets compared to the ungrafted lesioned group.

DISCUSSION

This study has shown that the high proportion ofstriatum-like tissue in LGE grafts from E14 donors isdue to a reduction in nonstriatal tissue rather than anexpansion of the donor striatal neuronal population inthe host striatum. The largest volume of striatal tissuewas obtained from WGE grafts from E14 donors.

In this study there was a strong relationship be-tween amelioration of drug-induced motor deficits andthe volume of striatal tissue. No relationship was iden-tified between graft morphology and the ameliorationof more complex reaching skills which was obtained inanimals grafted with MGE tissue. These results sug-gest that different aspects of recovery of motor functionare subtended by different components of striatalgrafts and that both MGE- and LGE-derived compo-nents of striatal grafts may play important roles ingraft function.

General Appearance

The general appearance of the grafts from WGE andLGE groups was similar to many previous reports (forreviews see 1, 10). The appearance of MGE tissue wasalso consistent with previous reports in that there waslittle or no DARPP-32 positivity or TH staining butAChE staining was identified. In E14-derived graftscholinergic fibers were diffusely distributed through-out the grafts while in the E16-derived grafts theywere more peripherally arranged. These observationsare consistent with the origin of striatal cholinergicneurons from the MGE whereas striatal projectionneurons arise predominantly from LGE (25, 26).

Physiological electrical stimulation of the ipsilateralsensorimotor cortex resulted in the expression of theimmediate early gene c-fos in the nuclei of a limitedpopulation of cells within grafts derived from E14WGE and LGE donors. This observation suggests thatphysiological stimulation of cortical afferent projec-tions to striatal grafts is capable of stimulating graftprojection neurons. However, the number of cells in-volved was small which may reflect loss of corticalprojections to the grafts.

92 WATTS ET AL.

Graft Morphology and Connectivity

The interaction between donor age and embryonicdissection revealed no difference in graft volume be-tween E14 and E16 donors for WGE or MGE dissec-tions. However, grafts derived from LGE were sig-nificantly larger from E16 donors compared to theirE14 counterparts. This may in part reflect the dif-ference in size of the LGE between donors of thesegestational ages resulting in a larger number of cellsbeing implanted from one LGE from an E16 donor.This result would suggest that an equivalent LGEfrom E14 gan-glionic eminence is not able to compen-sate for the smaller quantity of tissue it containscompared to an LGE from an older donor. This im-plies that the ability of younger donor tissue to ex-pand in situ in the host neuropil may be limited. Itwas also found that grafts of E14 MGE tissue werelarger than E14 LGE further suggesting that LGEtissue alone has a limited capacity to expand. Thismay reflect the fact that LGE develops later thanMGE. In nigral grafts, only cells that have dividedprior to implantation express a TH phenotype in thegrafts (Sinclair and Dunnett, unpublished observa-tions). If a similar principle were to apply in striatalgrafts then very few striatal neurons would be ex-pected to have their fate determined and hence to

FIG. 5. Interrelationship between donor age and embryonic diswith respect to graft volume (A), volume of striatum-like tissue (BDARPP-32 cell count within the graft (D). The largest quantity of shighest proportion of striatum-like tissue was obtained from the smsimilar to those from WGE.

differentiate appropriately when taken from theLGE at the earlier time point.

Patch volume in grafts of LGE dissections from E14donors was smaller than the patch volume in graftsfrom the corresponding dissection from E16 donors. Itwas also smaller than the patch volume in grafts ofE14 WGE tissue. In contrast there was no difference inpatch volume between LGE and WGE grafts from E16donors which were both smaller than the volume ofstriatal tissue within E14 WGE grafts. These resultssuggest that the MGE may facilitate the developmentof the striatal component of the grafts in younger butnot older donor tissue. These data are consistent withthe elaboration of striatal interneurons from MGE be-tween E12.5 and E15.5 in transplantation studies re-ported elsewhere (25) and point to a significant role forMGE tissue in the development of striatal transplants.However, such an effect appears dependent on the ageof the donor tissue. Grafts of MGE tissue in this studywere relatively small, contained very little striatum-like tissue, had very few DARPP-32-positive striatalneurons, and developed limited afferent and efferentconnections. These results are also consistent with pre-vious studies (4, 26, 27).

The small size of the E14 LGE grafts resulted intheir having the highest proportion of striatal tissue,

ion. The relationship between donor age and embryonic dissectionhe proportion of striatum-like tissue within the graft (C), and thetum-like tissue was obtained from younger WGE donor tissue. Theer E14 LGE grafts. Conversely older LGE tissue gave rise to grafts

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93DONOR AGE AND DISSECTION INFLUENCE GRAFT DEVELOPMENT AND FUNCTION

approximately 80%, compared to 55 and 45% for E14and E16 WGE grafts, respectively. Taken in conjunc-tion with the volumetric analysis of the graft compart-ments these results indicate that the high proportion ofstriatum-like tissue in LGE grafts is due more to areduction of nonstriatal tissue in the graft than anexpansion of the striatal component. The volumes andproportions reported in this study are consistent withthose reported previously for specific dissections (9, 23,26, 27) and across different donor ages (10).

Quantification of the number of DARPP-32-positivestriatal neurons within the grafts supports this hy-pothesis. E14 LGE grafts have the lowest and E16 LGEgrafts the highest number of DARPP-32 projectionneurons. This result may reflect a larger number ofcommitted striatal precursor cells contained in the tis-sue from the older LGE tissue. These data also further

FIG. 6. Illustration of the afferent TH innervation of the grafts(A) and the efferent projections from the grafts (B) with respect toembryonic donor age and dissection. Donor age and dissection had nosignificant influence on dopaminergic innervation of striatal grafts.Efferent projections, determined by optical density measurements,were greatest from E14 LGE grafts reflecting their smaller densermorphology. (Note the different scales; the density of efferent pro-jections was less than the afferent fibers.)

support the hypothesis that the grafted striatal neuro-nal population undergoes only limited expansion insitu in the host neuropil.

The larger patch volume and higher DARPP-32 cellontent of WGE grafts compared to LGE grafts from14 donors suggests that MGE tissue promotes theurvival and development of the striatal component ofhese grafts. This effect was not present in grafts fromlder donors indicating that the beneficial effect ofGE tissue is donor age dependent. The timing of this

ffect coincides with the period of neurogenesis of stri-tal projection neurons (3, 4) and striatal cholinergicnterneurons (25). Thus the MGE may provide nonspe-ific trophic support of the developing neurons or mayrovide more specific developmental cues.The connectivity of the grafts in this study broadly

eflects their dominant neuronal population. Efferentrojections were greatest from E14 LGE grafts reflect-ng the high density of striatal DARPP-32 neuronsithin these small grafts. Projections from E15 LGEnd WGE grafts were similar. MGE grafts elaboratedignificantly fewer efferent projections and did not dif-er between donor ages. Grafts rich in projection neu-ons or their targets exhibit a high degree of connec-ivity whereas grafts potentially rich in striatal inter-eurons do not develop significant connections withistant targets in the host CNS. These results remainonsistent with the graft morphology of each group inhis study and in studies reported previously (26, 27);for reviews see 1, 33). Taken together the results ofhese studies suggest that WGE rather than LGE tis-ue from younger donors should be used for implanta-ion in rodent models of HD.

unctional Recovery

In this study drug-induced rotation bias was signif-cantly reduced in animals grafted with LGE tissuerom E14 animals (CRL 10–11 mm). This modest re-uction was not achieved by the other graft groups. Inhis study this group comprised relatively small graftsontaining small volumes of striatum-like tissue. How-ver, this small volume comprised the highest propor-ion of striatum-like P-zones of any group in this study.his concentration of striatal tissue was further re-ected in the greater density of projections to the glo-us pallidus (GP) from these grafts. These efferentonnections were not determined to be coming solelyrom the DARPP-32 cells within the grafts but sincehese grafts contained over 80% AChE-positive tissuet is reasonable to assume the majority of these projec-ions to the GP are from medium spiny striatal neu-ons.Graft-mediated reduction of drug-induced rotation

ias in rodent models of HD after unilateral placementf cell suspension grafts into rats previously lesionedith ibotenic acid (IA) has been documented previously

ia

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94 WATTS ET AL.

(7, 9). Similar results in animals unilaterally lesionedwith kainic acid (KA) have also been reported after theimplantation of solid pieces of embryonic striatal tissue(24). This study confirms the reproducibility of thisbehavioral paradigm in a unilateral quinolinic acidmodel of HD. The results lend further indirect supportthe concept of functional dopamine receptors (11, 20,30). However, an additional relationship to the volume

FIG. 7. Graft-mediated effect on drug-induced rotation. Only gnduced rotational bias at 3, 6, and 9 but not 12 weeks posttransplat time 0.

FIG. 8. Graft-mediated effect on paw –reaching. Grafts had no ef E14 MGE tissue gave rise to improved retrieval compared to lesionsignificant effect.

of striatal tissue within the grafts (9) was not repli-cated, emphasizing that it is likely to be connectivitywith the globus pallidus that is the important factor ingraft-mediated functional recovery of simple motor def-icits.

Graft-mediated amelioration of lesion-induced defi-cits in the more complex motor task of paw reachingand retrieval has been documented in four studies (7,

ts derived from E14 LGE significantly ameliorated amphetamine-Lesioning took place 4 weeks before grafting which was performed

t on ipsilateral retrieval compared to lesion only controls but graftsly controls on the contralateral side. No other graft group produced

rafnt.

ffecon

95DONOR AGE AND DISSECTION INFLUENCE GRAFT DEVELOPMENT AND FUNCTION

11, 22, 23). One report revealed no effect of striatalgrafts on retrieval (9). The interaction of donor age andembryonic dissection was not specifically addressed inthese studies. One study (23) using LGE tissue fromE14 embryos (CRL 12–13 mm) correlated paw reachingability with graft survival and morphology. Significantcorrelation with P-zone volume was reported and re-placement of approximately 50% of DARPP-32 neuronswas also calculated to be necessary to restore pawreaching ability to nearly normal. In the only otherstudy to correlate graft morphology with paw reachingFricker et al. (11) identified P-zone volume but notgraft volume to be strongly associated with recovery ofcontralateral reaching behavior. Paw reaching alsocorrelated strongly with functional activity of both D1

and D2 receptors as demonstrated by PET scans invivo. However, there was no correlation between PETsignal and patch volume. The present study also iden-tified improved retrieval in animals grafted withyounger donor tissue which also had enhanced bindingof dopaminergic ligands compared to older donors.These effects were mediated by WGE grafts with ap-proximately 40% P-zones (10, 11). Recovery in thestudy by Nakao and co-workers was mediated by LGEgrafts with approximately 65% P-zones (23). These pro-portions are comparable with those obtained usingWGE tissue in the present study and indicate thatgrafts with very high proportions of striatal tissue arenot an essential prerequisite for amelioration of defi-cits of complex motor function (2, 28).

The present study confirms the previous four reportsinasmuch as it records graft-mediated amelioration ofpaw reaching deficits. It is unusual in that it the firststudy to report this effect in animals grafted with MGEtissue. Indeed it is the first study to compare functionalrecovery in animals grafted with tissue from whole,lateral, and medial dissections of embryonic striatum.As reported in previous studies there was a significantdifference between ipsi- and contralateral sides withregard to the number of pellets retrieved. Paw reachingipsilateral to the grafted side (right paw) did not differsignificantly between any of the groups. By contrast, onthe contralateral side all the grafted groups retrievedmore pellets than the lesion only group in the last threetrials 3 months postgrafting and in both trials 6months postgrafting. However, only the effect of E14MGE grafts achieved statistical significance. One pos-sible reason for this is the fact that each well in thestaircase box was baited with only two pellets. Thismeant that retrieval of only one pellet would have asignificant impact on the number of pellets remainingin the wells making it more difficult to distinguish realfunctional effects. Previous studies have restricted thenumber of wells used (23) and baited the wells withmore pellets (11, 23). The paucity of striatum-like tis-sue within the grafts of this more successful group

raises the issue of the role of MGE tissue in graft-mediated functional recovery.

While it is generally accepted that the striatal LGEis the primary source of projection neurons, the genesisof striatal interneurons has only recently begun to beaddressed (25, 26). Striatal cholinergic neurons appearto develop predominantly from MGE tissue at an earlystage of striatal neurogenesis (25). Efferent projectionsfrom grafts of MGE tissue are sparse (25) reflecting theprecursor population of this tissue. The results of thisstudy support these observations and reveal thatgrafts predominantly composed of striatal cholinergicinterneurons are capable of mediating a functional ef-fect possibly by establishing local connections with thehost neuropil. Previous transplantation studies havesuggested that striatal cholinergic neurons are derivedfrom MGE between 12.5 and 15.5 days of gestation(25). In this study MGE donor tissue utilized from E14embryos would have contained striatal cholinergicneuronal precursors whereas donor tissue from E16embryos would not. This difference, reflected in thedifferent histological architecture of the MGE grafts,may partially explain why E14 but not E16 MGE graftshad a functional effect.

Taken together these data constitute growing evi-dence for a role for MGE tissue in the development ofthe normal striatum and in the functional efficacy ofstriatal transplants. The results of this study furthersuggest that different aspects of recovery of motorfunction may be subtended by different neuronal pop-ulations within striatal grafts. These observations mayalso partly explain the poorly defined relationship be-tween patch volume and motor recovery reported hereand elsewhere in primate models of HD (15).

SUMMARY

Early clinical trials of striatal transplantation inpatients with HD have begun. The surgical protocolsvary and both WGE and LGE tissue have been utilizedin different centers (Peschanski, personal communica-tion; 16).

This study provides the first comprehensive view ofissues that have been discussed on the basis of partialdata for several years and have direct relevance forclinical trials of cell replacement strategies in patientswith Huntington’s disease. Three principal resultshave been obtained that provide guides for clinicalapplication. First, age of donor tissue is important withyounger donors being preferred. Second, WGE andLGE grafts provide essentially equivalent behavioralchanges despite their disparity in size and volume ofstriatum-like tissue. Third, MGE tissue may have anas yet unidentified role in graft development and func-tion. Altogether, these data favor the concept of usingwhole ganglionic eminence grafts from younger donorsin patients; however, extrapolation to man is always

96 WATTS ET AL.

difficult. Nevertheless, these results highlight the needfor careful preclinical analysis of optimal sources, age,and dissection of donor tissue before clinical applica-tion of striatal transplantation in HD can be under-taken with any confidence of functional success.

ACKNOWLEDGMENTS

This work was supported by the MRC and the Peel Medical Re-search Trust. We thank Dr. Hemmings and Dr. Greengard for thegenerous donation of DARPP-32.

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