graft-derived recovery from 6-ohda lesions: specificity of ventral mesencephalic graft tissues

Exp Brain Res (1988) 71:411-424 Ex mental Bran Research �9 Springer-Verlag 1988

Graft-derived recovery from 6-OHDA lesions: specificity of ventral mesencephalic graft tissues

S.B. Dunnett 1, T.D. Hernandez 1, A. Summerfield 1, G.H. Jones 1, and G. Arbuthnott z

1 Department of Experimental Psychology, University of Cambridge, Downing St., Cambridge CB2 3EB, U.K. 2 M.R.C. Brain Metabolism Unit, Edinburgh, U.K.

Summary. A series of experiments have been conducted to assess the specificity of recovery from motor asymmetries that is provided by dopamine- rich grafts in the neostriatum of rats with unilateral dopamine-depleting lesions produced by injection of 6-hydroxydopamine into the ascending nigrostriatal pathway. Grafts of embryonic tissue taken from the substantia nigra (rich in dopamine neurons could provide a complete recovery of methamphetamine- induced rotation and a partial recovery of apomorphine-induced rotation, whereas no recovery was seen in rats with grafts of tissue rich in another monoamine (serotonin, dissected from the mesencephalic raphe) or of tissue appropriate to the target (dissected from the striatal eminence). 6-Hydroxy- dopamine lesions of dopamine cells in the grafts of recovered animals reinstated the initial lesion- induced asymmetry. Dopamine-rich grafts implanted into the intact neostriatum did not induce any "supernormal" asymmetry in the rats, but did provide a "prophylactic" protection against subsequent lesions of the intrinsic ipsilateral dopamine nigrostriatal system. Post-mortem biochemical assays indicated that the extent of dopamine depletion in the neostriatum of lesioned rats correlated highly with both methamphetamine and apomorphine turning rates. Similarly, both drug rotation tests correlated significantly with the extent of dopamine restoration in the dorsal striatum of rats with dopamine-rich grafts, the correlation being significantly higher for the methamphetamine than for the apomorphine test.

Key words: Neural transplantation - Dopamine - Rotation - Amphetamine - Apomorphine - Substan- tia nigra - Neostriatum

Offprint requests to: S.B. Dunnett (address see above)

In~oducfion

It is now well established that embryonic neural grafts can exert a functional effect on the host animal (Bj6rklund and Stenevi 1984). The most extensively studied model system has been recovery of rotation asymmetries following transplantation of catecholamine-rich central or peripheral tissues into rats with unilateral lesions of the ascending forebrain dopamine system. Thus, embryonic substantia nigra grafted to the deafferented neostriatum (either as a solid piece of tissue or as a dissociated cell suspension) can produce a substantial or complete recovery of rotation induced by peripheral injection of amphetamine or apomorphine, and the degree of recovery has been seen to correlate with both biochemical and histochemical indices of reinnervation (Bj6rklund et al. 1980, 1983b; Freed et al. 1980; Schmidt et al. 1982, 1983). However, adrenal medulla tissue implanted in the lateral ventricle adjacent to the deafferented neostriatum also has the capacity to reduce apomorphine-induced rotation, even in the absence of any evidence of fibre reinnervation of the host brain (Freed et al. 1981), support- ing the view that functional effects may be fully achieved by chronic diffuse secretion of catecholamines from the grafts (Freed et al. 1980). Indeed, in another model system it has been suggested that the grafts might do no more than provide an acute neuro- trophic influence over mechanisms of spontaneous recovery within the host brain (Kesslak et al. 1986; Dunnett et al. 1987).

Therefore, in the present series of experiments we have addressed a series of questions related to the specificity of the previously described effects of dopamine-rich nigral grafts on recovery from the motor asymmetries induced by unilateral nigrostriatal lesions:

412

i. Is the recovery dependen t upon the use of dopamine-r ich tissue? Previous studies have shown that nei ther embryon ic cortex or tectal tissues (Freed 1983; Freed et al. 1984), nor adult per ipheral nerve grafts (Freed et al. 1980, 1981) p roduce any demon- strable recovery. We here investigate whether two other embryonic sources of graft tissue, more closely related to the effective nigral tissue, might have any effects: these are serotonin-r ich raphe tissue to provide a bra ins tem source rich in m o n o a m i n e (but not dopamine) cells, and striatal eminence to provide a source that is fully appropr ia te to the target implantat ion site.

ii. Is the recovery dependen t upon the cont inued presence of dopamine cells in the graft? LeVere and LeVere (1985) have argued that one essential control procedure for any functional graft is the demonst ra- tion that removal of the graft reinstates the behavioural deficit. We have previously shown that aspirative removal of a solid nigral graft in a cortical cavity can reinstate rota t ional a symmet ry in recovered rats (Bj6rklund et al. 1980), but this control has not been provided for the m o r e powerful suspension graft procedure .

iii. A quest ion that has of ten been raised infor- mally, but not addressed specifically, is whether grafts implanted into an otherwise intact animal can exert " supernormal" effects? A related quest ion, since graf t- induced recovery generally takes a per iod of months in lesioned animals, is whether grafts in intact animals can provide an immedia te "prophylac-

tic" protect ion against subsequent lesion of the intrinsic system?

iv. Bo th amphe tamine- induced and apomorphine- induced rota t ion are widely used to provide in vivo assessment of the deve lopment of graft funct ion (Bj6rklund and Stenevi 1979; Per low et al. 1979; Dunne t t et al. 1981a, 1983; F reed et al. 1980, 1981). We wished to assess the extent to which these two behavioural indices systematically covary in grafted rats, since a relationship be tween pe r fo rmance on the two tests has been suggested following subdivision of the animals into subgroups on the basis of compensation of their amphe tamine rota t ion scores (Dunne t t et al. 1981a). We also wished to assess whether the two measures are equally adequate predictors of pos t -mor tem indices of graft viability and growth.

M e t h o d s

Subjects and experimental groups

112 young adult female Sprague-Dawley rats were housed in group cages of 6-8 rats and maintained on a natural light dark cycle with ad libitum access to food and water throughout. Graft tissue was taken from embryos (CRL 11.5-14 mm) obtained from pregnant mothers of the same outbred strain.

32 of the rats were randomly allocated to two groups, to provide unoperated controls (group C, n = 16), or to receive ventral mesencephalic grafts implanted into the otherwise intact striatum (group DAgr, n = 16).

The remaining 80 rats received unilateral lesions of the ascending nigrostriatal pathway, the completeness of which was

Table 1. Experimental groups

Group n NSP Graft Subsequent name 6-OHDA lesion tissue 6-OHDA lesion

1. C 16 - a. C/L 8* - b. C/C 8 -

2. DAgr 16 - a. DAgr/L 10 - b. DAgr/C 6 -

3. PL 20* (+)

4. L 10 +

5. L/DAgr 24* + a. L/DAgr/L 5 + b. L/DAgr/C 8 +

6. L/5HTgr 8* +

7. L/CPUgr 8* +

- NSP

ventral mesencephalon ventral meseneephalon NSP ventral mesencephalon

ventral mesencephalon ventral mesencephalon ventral mesencephalon

rnesencephalic raphe

striatal eminence

CPU

Abbreviations: NSP, nigrostriatal pathway; CPU, caudate-putamen (neostriatum); 6-OHDA, 6-hydroxydopamine * indicates that an animal died in the. course of the experiment, resulting in a reduction by 1 in the group size in later parts of the

experiment + indicates that these animals received NSP lesions at the start of the experiment

- indicates that the particular lesion or graft was not administered

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assessed by an amphetamine rotation test (see below). 20 of the rats did not reach the acceptance criterion and were considered to have partial lesions (group PL, n = 20). The remaining 60 rats were considered to have good lesions and were randomly allocated to 4 experimental groups: lesions alone (group L, n = 10); lesions plus dopamine-rich ventral mesencephalic grafts (group L/DAgr, n = 24); lesions plus serotonin-rich raphe grafts (group L/5HTgr, n = 8); or lesions plus striatal eminence grafts (group L/CPUgr, n = 8). The remaining 10 rats with good lesions were allocated to other pilot experiments.

Two supplementary experiments were conducted 18-20 weeks after the initial surgery.

In order to assess whether grafts in the intact forebrain might have a prophylactic effect against subsequent lesions, group C and DAgr were each subdivided to either receive long-term lesions of the nigrostriatal bundle (groups C/L, n = 8 and DAgr/L, n = 10), or to remain unoperated (groups C/C, n = 8 and DAgr/C, n = 6).

In order to assess whether the DA graft derived recovery was dependent on the ongoing efficacy of the grafted tissue, group L/ DAgr was subdivided. From 13 rats that were well compensated in the amphetamine rotation test (see below), 5 received lesions of the graft (group L/DAgr/L), for comparison with the remaining 8 rats (group L/DAgr/C).

Surgical procedures

Nigrostriatal lesions. 6-Hydroxydopamine HBr (Sigma, USA) was dissolved in 0.2 mg/ml ascorbate 0.9% saline at a concentration of 3 p~g/[~l calculated as the free base weight. Rats were anaesthetized with 0.3 ml/100 gm equithesin i.p., and the 6-OHDA solution was injected into two sites, in the ventral tegmental area and nigrostriatal bundle, respectively. Stereotaxic coordinates for the two sites were: ventral tegmental area, A = -4 .0 mm anterior to bregma, L = 0.8 mm on the right side lateral to the midline, V = 8.0 mm below dura, with the incisor bar set +3.4 mm above the interaural line, and nigrostriatal bundle A = -4 .4 ram, L = 1.1 ram, V = 7.8 ram, with the incisor bar set 2.3 mm below the interaural line. Injections were made via a 30 gauge stainless steel cannula connected to a microdrive pump, 2~I into the first site and 2.5 ~tl into the second site, at the rate of 1 ~l/min with a further 3 rain allowed for diffusion before retraction of the cannula.

Intrastriatal transplants'. Graft tissue was taken from donor embryos of crown-rump length 11.5-14 ram. Dopamine-rich tissue for the DAgr and L/DAgr groups was dissected from the floor of the mesencephalic flexure, containing the developing ceils of the substantia nigra and ventral tegmental area (Bj6rklund et al. 1983a). Serotonin-rich tissue for the L/5HTgr group was taken from a midline strip in the floor of the 4th ventricle between the mesencephalic and pontine flexures, containing the developing cells of the mesencephalic raphe (Foster et al. 1986). Caudate- putamen tissue for the CPUgr group was taken from the developing striatal eminence, approached from the dorsal surface by cut- ting through, and folding aside, the parietal cortex (Seiger 1985).

The tissue pieces from the 10-12 embryos of one pregnant mother were collected in sterile 0.6% glucose-enriched 0.9% saline, then incubated for 20 rain at 37 ~ C in a solution containing 0.1% trypsin (Sigma, grade II crude) in glucose-saline. Following incubation the pieces were washed by repeated replacement of glucose-saline to a final volume of 1 embryonic piece per 10 ~1, then mechanically dissociated by repeated aspiration through a fire-polished Pasteur pipette to form a dense suspension of dissociated cells. The cell suspension was drawn up into a 10 ~tl glass microsyringe (Scientific Glass Engineering, Ringwood, Australia) mounted directly onto the electrode carrier of the stereotaxic frame. Aliquots of graft tissue were then stereotaxi-

cally implanted into the right caudate putamen in three deposits at the following sites: A = 6 ram, L = 1.8 ram, V = 4.5 ram; A = 0.6 ram, L = 3.4 ram, V = 4.5 ram; and A = 1.8 mm, L = 2.6 ram, V = 4.5 ram, with the incisor bar set 2.3 mm below the interaural line. 3 ~tl was deposited at each site, injected over 3 rain with a further 3 rain allowed for diffusion before retraction of the syringe needle.

Neostriatal lesions. In order to lesion grafted dopaminergic cells in the L/DAgr/L group, 18 weeks after transplantation surgery 6- OHDA was injected into a site approximately midway between the 3 graft deposits. 4 p3 of 3 ~g/~tl 6-OHDA HBr (dissolved as for the nigrostriatal lesions) was stereotaxically injected over 4 rain at A = 1.2, L = 2.6, V = 4.5 with the incisor bar set -2.3 ram, followed by a further 3 rain for diffusion prior to retraction of the cannula.

Rotation tests

Rotation was assessed in a bank of 8 automated rotometer bowls modelled after the design of Ungerstedt and Arbuthnott (1970), and turns in either direction recorded by online connection to a CUBE System 10 microprocessor (Control Universal, Cambridge, U.K.).

Methamphetamine rotation. Each test was conducted over 90 min following 1 ml/kg injection i.p. of 5 mg/ml methylamphetamine HC1 (Macarthy's, Romford, Essex) dissolved in sterile 0.9% saline. All animals were initially tested 1 week following the nigrostriatal lesion surgery. Any lesioned animal that showed less than 7 net ipsilateral turns/rain was considered to have sustained only a partial lesion, and was assigned to group PL. Rats showing turning rates > 7 turns/rain were considered to have sustained good lesions (Bj6rklund et al. 1980) and were randomly allocated to lesion and graft groups. All animals then had four further tests at monthly intervals following transplantation surgery, or at the equivalent time points in non-grafted rats.

Apomorphine rotation. A single test was conducted several days after the final methamphetamine test. Rotation was monitored over 60 rain following 0.5 ml/kg injection s.c. of 0.1 mg/ml apomorphine HCI (Sigma, USA) dissolved in 0.01% ascorbate 0.9% saline.

Histochemistry

Animals were anaesthetized with 30 rag/0.5 ml sodium pentobar- bitone (Sagatal, May & Baker, Dagenham) i.p. and perfused through the heart with 200 ml 0.1 M phosphate buffer (pH = 7.4) containing 5000 units/litre heparin over 5-10 rain. This rinse was followed by a slower peffusion of 4% paraformaldehyde/0.05% glutaraldehyde in 300ml phosphate buffer at 4 ~ C over 30 rain. The brains were removed, post fixed in the same solution for 30 rain, and then transferred to 30% sucrose in phosphate buffered saline (PBS).

Coronal frozen sections were cut at 30 ~t thickness, and 6 parallel series were collected through the area of the neostriatum in 0.5 M PBS. Two series were mounted onto glass slides and stained for nissl substance with 1% thionin or for acetylcholines- terase by the thiocholine method (Koelle 1955) using 0.005% ethoproprazine as inhibitor of non-specific esterase and 0.25% silver nitrate to enhance the sulphide reaction product.

Three other series of sections were incubated in 20% normal goat serum for 2 h at room temperature, rinsed in PBS, and incubated overnight at 4 ~ C in primary polyclonal antibodies to

414

tyrosine hydroxylase (TH, 1 : 2000, Eugenetec, Allendale, USA), serotonin (5-HT, 1:2000, RIA(UK) Ltd), or somatostatin (1 : 10000, the kind gift of Dr. A.J. Harmer), in PBS containing 0.2% Triton X100 and 0.1% sodium azide. The final series was incubated overnight at 4 ~ C in a monoclonal antibody to choline acetyltransferase (CHAT, 1 : 40, the kind gift of Dr. F. Ecken- stein) in 100 mM Tris, 150 mM NaCI (pH = 7.4) containing 2% bovine serum albumen, 10% normal rabbit serum, 0.5% Triton X100 and 0.1% sodium azide. The ChAT sections were then rinsed and incubated for 1 h in biotinylated rabbit anti-rat IgG (Vector labs) in Tris buffer (as above, but with 0,2% Triton X100 and no azide). All sections were taken through the "Vector ABC" immunocytochemical method with diaminobenzedine as the chromogen. Sections were then mounted onto glass slides, dehy- drated and coverslipped in DPX.

Biochemistry

The animals were anaesthetized with ether, decapitated, and the brains rapidly removed. The brains were cut into 2 mm slabs in a dissection block, and 6 tissue samples were dissected on ice from each brain: the dorsal and ventral neostriatum rostral to the level of the anterior commissure and the nucleus accumbens from each hemisphere.

Brain tissue concentrations of dopamine and serotonin were measured by high performance liquid chromatography (HPLC) with electrical detection (Dooley et al. 1987; Mefford 1981). Chromatographic separation was accomplished using a stainless steel column (15 cm x 0.46 cm I.D.) packed with C18 5 ~tm reversed-phase Sperisorb ODS2 beads (Phase Separations Ltd, Clwyd, U.K.). The mobile phase consisted of 130 mM mono- chloroacetic acid, 0.4 mM 1-heptane sulphonic acid, 80 mM EDTA and 10% HPLC grade methanol, and was adjusted to pH = 3.0 with potassium hydroxide. The flow rate was 1 ml/min delivered by a Gilson model 302 pump fitted with a model 802 manometric module. Detection was achieved using a model LC-4B electrochemical detector (B.A.S., West Lafayette, USA) with a glassy carbon working electrode. The applied potential was 0.73 V versus an Ag/AgC1 reference electrode. Brain tissue samples were homogenised in 0.2 M perchloric acid, centrifuged and 50 ~tl of the supernatant injected directly onto the column using a WISP model 710B autosampler. External "standards were used for quantifica- tion.

R e s u l t s

Methamphetamine rotation

The ro t a t ion scores on the ini t ia l m e t h a m p h e t a m i n e test and on the four subsequen t m o n t h l y tests a re shown for all e x p e r i m e n t a l g roups in Fig. 1.

Efficacy of the lesions. Eigh ty ra ts r ece ived the ini t ia l 6 - O H D A les ion, and had a m e a n m e t h a m p h e t a m i n e ro ta t ion score of 847 + 49 turns on the ini t ia l 90 min test . Of these ra ts , 60 (75%) r e a c h e d the 7 turns / ra in cr i te r ion for accep tance and were r a n d o m l y a l loca ted to the var ious e x p e r i m e n t a l g roups , inc luding 10 ra ts in group L which r ece ived the ini t ia l les ion a lone . The r ema in ing 20 ra ts were c o n s i d e r e d as having only par t ia l lesions (g roup PL) , the p rogress of which was

Methamphetamine- induced ro ta t ion

1200-

B 800- -

~00-

._~ 0-

i ' I i i i

pre lmo. 2mo. 3mo. 4mo.

tests

G r ou p..~

e - - e C

o - - - o L

* - - * PL

* - - - * C/DAgr <>---<> L/DAgr

. . . . . L/5HTgr . . . . . ~ L/CPUgr

Fig. 1. Methamphetamine-induced rotation: each point shows the total net ipsilateral turns in each 90 min test following injection of 5 mg/kg methamphetamine i.p., tested prior to ("pre" test) and at four monthly intervals following transplantation in the seven groups of rats as specified in Table 1. The only rats which manifested compensation of the initial lesion-induced rotation bias were those with dopamine-rich ventral mesencephalic grafts (group L/DAgr). The time of the initial lesion and the transplantation surgeries are indicated by the open and filled arrows, respectively. The vertical bar indicates 2 standard errors of the difference in means derived from the interaction term of the analysis of variance

m o n i t o r e d behav iou ra l l y , a l though these rats were not used for add i t i ona l e x p e r i m e n t a l t r e a tmen t s .

The m e t h a m p h e t a m i n e ro t a t i on scores in g roup L were re la t ive ly s tab le across the four mon ths of test ing (see Fig. 1). By con t ras t , severa l an imals in group P L showed a p rogress ive inc rease in ro t a t i on a symmet ry over r e p e a t e d tests , and by the 4 m o n t h test 6 of the 20 P L rats had tu rn ing scores in excess of the ini t ial 7 tu rns /min accep t ance cr i te r ion .

Efficacy of the graft groups. The rats wi th grafts of se ro ton in- r ich t issue (group L / 5 H T g r ) or of s t r ia ta l t issue (group L / C P U g r ) i m p l a n t e d into the 6 - O H D A les ioned s t r i a tum bo th showed s imi lar high levels of m e t h a m p h e t a m i n e - i n d u c e d ro t a t i on on the int ia l test p r io r to t r a n sp l a n t a t i on and on the subsequen t four month ly tests , and d id no t differ at any po in t f rom the group L rats wi th les ions a lone (see Fig. 1). The rats with D A - r i c h grafts i m p l a n t e d in the l e s ioned s t r ia tum did not differ f rom group L on the ini t ial , p r e - t r ansp l an t a t i on tes t , bu t t hen showed a p rogres - sive r educ t ion in ro t a t i on a s y m m e t r y ove r the 4 months fo l lowing t r a n s p l a n t a t i o n (see Fig. 1). Ana ly - sis of va r iance con f i rmed the s ignif icance of these di f ferences (main effect of g roups , F(3 ,46) = 16.45; main effect of tes t , F(4 ,167) = 8.66; g roups • tes t in terac t ion . F(12,167) = 6.18; all p < 0.001). Thus , the c o m p e n s a t i o n of m e t h a m p h e t a m i n e ro t a t ion , tha t has been f r equen t ly r e p o r t e d fo l lowing grafts of

Do lesions of compensated grafts reinstate turning?

1200-

800-

400-

O-

o- - -o group k/OAgr/C

we 1too. 2too. 3too. 4mo. § §

u TR tests L2

Fig. 2. Methamphetamine-induced rotation prior to and following 6-OHDA lesion of dopamine-rich grafts in compensated animals. Lesion of the dopamine-rich grafts reinstated the initial rotation asymmetry (group L/DAgr/L), in contrast to the matched animals without subsequent lesion (group L/DAgr/C) which remained fully compensated. The times of initial nigrostriatal lesion and transplantation are indicated by the open and filled arrows, L1 and TR, respectively, and the time of the lesion of the graft is indicated by the second open arrow, L2, Vertical bars indicate s.e.m.

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Con DA grafts hove o "supernormol" effect ?

Con DA grafts hove o "prophylactic" effect ?

1200 -

~ 800-

"6 .-~ ~oo-

O-

L1 TR

* ~ * group C/L J [ - " - - ' - ~ /

I I I I I I I I

pre 1too. 2mo. 3too. 4mo. + l w k . . 2 w k +4wk tests LZ

Fig. 3. Methamphetamine-induced rotation in rats with dopamine- rich grafts implanted in the intact neostriatum. The grafts induced no rotation bias in the grafted rats (group C/DAgr), indicating the absence of a "supernormal" effect of the grafts. A subsequent lesion of the intrinsic nigrostriatal system (at the time indicated by the open arrow L2) induced strong rotation bias in the control rats (group C/L), whereas the grafts provided a "prophylactic" protection as revealed by a much reduced bias following the intrinsic lesion (group C/DAgr/L), Vertical bars indicate s.e.m.

DA-rich tissue, is replicated here but is not provided by grafts that are poor in DA ceils.

The degree of compensation of rotation asymmetry of the 23 L/DAgr rats surviving to the end of the experiment was categorized into four ranges, based on the rotation sources on the final 4 month test (see Bj6rklund et al. 1980):

Uncompensated, "U" rats, > 7 ipsilateral turns/min, n = 4, Partially compensated,"P" rats, 3-7 ipsilateral turns/rain, n = 3, Compensated, "C" rats, 0-3 ipsilateral turns/rain, n = 3. Overcompensated, "C+" rats, < 0 ipsilateral turns/rain, n = 13.

Thus, the disproportionate number of animals in group C+, in comparison to the numbers in groups C and P, confirm the previously reported tendancy of many animals with effective grafts to "overcompen- sate" (Herman et al. 1985) which in the present test is manifested by development of a substantial contralateral bias.

Effects of 6-OHDA lesion of the graft. In order to test whether removal of the grafts would reinstate the initial effects of the nigrostriatal bundle lesion, 5 of the 13 C+ L/DAgr group of rats received an additional 6-OHDA lesion of the grafts (subgroup L/ DAgr/L), and were compared on methamphetamine rotation with the remaining 8 C+ L/DAgr rats (subgroup L/DAgr/C) one and two weeks later. The rats for subgroup L/DAgr/L were selected to reflect a similar mean degree of "overcompensation" as subgroup L/DAgr/C on the 4 month test. As shown in

Fig. 2, the lesion of the graft immediately reinstated rotation under methamphetamine to a level that did not differ from that shown by the rats prior to transplantation, and the difference between the two subgroups was highly significant on the two tests 1 and 2 weeks after the subsequent lesion (F(1,13) = 29.39, p < 0.001). Thus, 6-OHDA lesions of DA-rich suspension grafts in compensated rats abolish their recovery on the methamphetamine rotation test and reinstate the deficit induced by the initial nigrostriatal lesion, akin to previous reports of aspirative removal of solid nigral grafts (Bj6rklund et al. 1980).

"Supernormal" effects of DA-rich grafts in the intact striatum? Rats with DA-rich grafts implanted in the intact striatum (group DAgr) showed no significant turning bias, and did not differ from the control rats without any lesion or graft (group C), on any methamphetamine rotation test (see Figs. 1 and 3). Thus, DA-rich grafts implanted into the intact striatum do not produce any detectable "supernormal" contralateral bias.

"Prophylactic" effects of DA-rich grafts in the intact striatum? After the 4 month methamphetamine test, 10 of the 16 DAgr rats and 8 of the 16 control rats received 6-OHDA lesions of the nigrostriatal bundle (subgroups DAgr/L and C/L, respectively), for comparison with the remaining rats of each group that received no subsequent lesion (subgroups DAgr/C

416

4o0.

300 2 2

200- _q P

o 100 u

Apomorphine-induced rotation

c c/ DAgr

$ L/ DAg r

/ L/ L/ 5HTgr CPUgr

PL

Fig. 4. Apomorphine-induced rotation: each bar shows the total net contralateral turns in each 60 min test following injection of 0.05 mg/kg apomorphine s.c., tested 4 months following transplantation in the seven groups of rats as specified in Table 1. The rats with dopamine-rich ventral mesencephalic grafts (group L/DAgr) showed lower rotation bias than the rats with comparable lesions alone (group L), whereas the rats with non-dopaminergic control grafts (groups L/5HTgr and L/CPUgr) did not differ from the lesion group. The rats with grafts into the intact neostriatum (group C/DAgr) showed no rotation bias and did not differ from the unoperated controls (group C). Vertical bars indicate s.e.m.

and C/C, respectively). As shown in Fig. 3, the lesion resulted in strong ipsilateral turning in the mature C/L controls 7 days later, which was markedly less in the DAgr/L rats with long-term grafts. In fact, using the same ranges that were used to characterize the L/DAgr group, the 10 DAgr/L rats could be subdivided into 2 U, 1 P, 1 C and 6 C+ cases. Analysis of variance indicated that the difference between groups was significant (F(3.28) = 5.86, p < 0.01), and Newman-Keuls comparisons indicated that group C/L had significantly higher scores than the other three groups (all p < 0.05), whereas group DAgr/L was not significantly greater than the two unlesioned C/C and DAgr/C groups (both p > 0.1). Thus, DA-rich grafts implanted in the intact striatum

do provide a significant "prophylactic" protection against the effects of subsequent damage in the intrinsic nigrostriatal DA system.

Apomorphine rotation

The rotation scores of each group on the single apomorphine test, conducted 1-2 weeks after the 4 month methamphetamine test, are shown in Fig. 4. Analysis of variance indicated a significant difference between groups (F(6,91) = 15.20, p < 0.001). Multiple Newman-Keuls comparisons indicated that the C and C/DAgr groups did not differ, that the PL and L/DAgr groups did not differ, and that the L, L/ 5HTgr and L/CPUgr groups did not differ, whereas all other pairwise comparisons were significant (at least p < 0.05). Thus, DA-rich grafts, but not grafts of non-DA cells, provide a substantial reduction of the striatal receptor "supersensitivity" that develops in response to nigrostriatal DA depletions.

Specific correlations were conducted on the relationship between apomorphine turning and methamphetamine turning on the 4 month test in the two largest sets of rats - the combined L and PL lesion groups (n = 29), and the L/DAgr group (n = 23). As shown in Fig. 5A, methamphetamine and apomorphine turning rates were very highly correlated within the combined lesion group (r = 0.749, t -- 5.89 with 27 df, p < 0.001). By contrast, as shown in Fig. 5B, the compensation of apomorphine turning was completely uncorrelated with the compensation of methamphetamine turning within the L/DAgr rats (r = 0.025, t = 0.11, ns).

Histochemical analysis

Four C+ rats from group L/DAgr, 4 rats from group L/5HTgr and all 7 surviving L/CPUgr rats were taken

gE

A �9 group L o

. / ogroupPL o ~ �9

i

-4 0 4 8 12 16 20

METHAMPHETAMINE ROTATION

B �9 group L/DAgr

( ips i la tera l turns x lO0)

16 I 20

Fig. 5A, B. Scatter plots of the relationship between turning rates on the 4 month amphetamine and apomorphine rotation tests in the combined lesion rats (A, group L and PL) and in the rats with dopamine-rich grafts (B, group L/ DAgr). The least squares linear regression line is also shown

417

Fig. 6A-I. Histochemistry: photo- micrographs of sections through grafted tissue in the neostriatum of rats from groups L/DAgr (A-E, G), L/5HTgr (F, H) and L/CPUgr (I), stained for TH immunoreactivity (A-C), 5-HT immunoreactivity (D-F), or AChE activity (G-I). A, B, D, E Are all of the same ventral mesencephalic graft. Note the TH positive neurons (A) and somewhat fewer 5-HT positive neurons (D) in adjacent sections, and similarly both TH positive (B) and 5.-HT positive fibres (E) growing into the host striatum. The 5-HT positive fibres soon disappear among the intact 5- HT innervation intrinsic to the host striatum surrounding the graft, whereas the TH positive fibres visible in B are entirely derived from graft outgrowth. C Shows the TH positive cells at higher magnification in a different ventral mesencephalic graft, and F shows 5-HT positive cells in a raphe graft at the same magnification. No darkly stained TH positive neurons were seen in any raphe graft. G, H, I Show the max- imum cross sectional areas of ventral mesencephalic, raphe and striatal grafts, respectively. Note the patches of AChE activity in small areas of the striatal grafts (I), which was absent in the other two types of grafted tissue (G, It). Calibration bars: A-F, 50 gm; G-I, 500 um

418

for histochemical analysis. Additionally, the 6 rats of group DAgr /C with DA-rich grafts in the intact striatum received a final lesion of intrinsic dopamine neurons by injection of 6 - O H D A into the nigrostriatal bundle after completion of all behavioural tests, and they were then also perfused for histochemical analysis 6 days later.

Surviving grafts were visible in all animals in the nissl and AChE stained sections (see Fig. 6G- I ) , although in some cases the grafts were quite small (e.g. Fig. 6H). In 3 L/5HT and 2 L/CPUgr rats, the grafts were not visible in the immunosta ined sections.

All animals of group L /DAgr and DAgr /C had grafts containing T H positive cells (Fig. 6A, C), and there were no detectable differences in the size of the grafts between the two groups. The grafts in all cases were seen to give rise to T H positive fibres growing

�9 into the host neostr iatum proximal to the grafts (Fig. 6B). Host striatal areas distal to the grafts were in all cases devoid of TH-posi t ive fibres on the side of the nigrostriatal lesions. All of the ventral mesencephalic grafts were also seen to contain 5-HT immunoreac- tive cells (Fig. 6D, E), but in smaller numbers than the T H positive cells in the same animals. 5-HT positive fibres were seen crossing the graft-host border (Fig. 6E), and although this appeared less impressive than the T H positive fibre ingrowth (compare with Fig. 6B), this is in part due to the higher background of intrinsic 5-HT positive fibres in the host striatum which survive the nigrostriatal lesion.

The raphe grafts of group L/5HTgr also survived and similarly gave rise to 5 -HT positive fibres growing into the host striatum (Fig. 6F). In contrast with the mesencephalic grafts, no T H positive neurons were seen in any of the raphe grafts.

Weak ChAT positive neurons were seen in the host striatum of most animals, although in some cases the staining was too faint to see any immunoreact ive cells at all. No clear cases of ChAT positive neurons were seen in any of the grafts. However , although the grafts in groups L/DAgr , DAgr /C and L/5HT were all AChE negative (Fig. 6G, H) , the striatal grafts showed patches of high A C h E activity (Fig. 61), suggesting that cholinergic cells may be contained in these grafts even though not detectable with the present ChAT antibody protocol.

A few, scattered, somatostat in immunoreact ive cells were seen both within the host striatum and within several of the grafts of each group.

Biochemical analysis

Biochemical assays were conducted on all surviving rats that were not taken for immunohistochemical

Table 2. Biochemical assays for Dopamine (ng/mg wet weight of tissue)

Group N. Accumbens Ventral CPU Dorsal CPU

Group C (n = 5) left 8.19 + 0.83 10.63 _+ 0.86 12.23 + 1.17 right 9.62 + 0.53 10.19 + 1.35 11.89 _+_ 1.07

Group L (n = 10) left 5.24 + 0.67 10.83 + 0.74 13.59 +_ 0.44 right 0.04 + 0.02 0.30 +__ 0.11 0.20 +_ 0.08

diff 1.06% 2.98% 1.41%

Group PL (n = 19) left 4.90 + 0.45 9.12 +_ 0.46 11.89 _+ 0.45 right 0.77 + 0.28 1.97 + 0.59 1.23 +_ 0.38

diff 12.94% ** 22.19% ** 9.62% **

Group L/DAgr (n = 14) left 5.48 + 0.38 10.62 + 0.54 12.19 + 0.69 right 0.06 + 0.02 0.25 __. 0.07 0.90 + 0.23

diff 1.07% 2.47% 7.25% **

Group L/DAgr/L (n = 5) left 5.91 + 0.27 8.67 _-+_ 1.32 13.76 + 0.71 right 0.04 +__ 0.04 0.33 + 0.19 0.12 + 0.09

cliff 0.68% 3.42% 0.84%

Group L/5HTgr (n = 3) left 8.88 + 0.59 13.25 +__ 0.19 13.57 + 1.13 right 0.85 + 0.47 0.43 + 0.35 0.16 + 0.11

diff 9.63% * 3.23% 1.18%

Group C/L (n = 7) left 7.94 + 0.45 10.15 _+ 0.54 14.10 + 0.69 right 0.30 + 0.27 0.08 _+ 0.05 0.03 + 0.02

diff 3.81% 0.78% 0.23%

Group DAgr/L (n = 10) left 5.53 + 0.61 9.37 + 0.65 11.11 + 0.88 right 0.09 + 0.04 1.32 _+ 0.45 1.81 + 0.45

diff 1.65% 14.08% * 16.31% **

The difference between the lesioned (right) and unlesioned (left) sides is significant (p < 0.01) for all three regions in all groups except group C Differences from lesion group L or C/L as appropriate: *, p < 0.05; **, p < 0.01 To correct for multiple comparisons, only differences p <0.01 are considered to be significant

analysis, with the exception that only 5 of the unoperated controls (group C) were processed. The numbers of animals of each group that underwent biochemical analysis are shown in Table 2. A full analysis of variance on all elements of Table 2 revealed few significant differences other than with the unoperated control group, in large part because of non-homogeneity due to greater variance on the right (control) sides and in groups C and PL than on the side of the lesions and grafts of animals that had initially sustained effective lesions. Therefore , explicit comparisons of interest have been made using Student 's t-tests, but requiring a more stringent criterion of p < 0.01 to allow for multiple comparisons.

.C

E 1 6 0 0

.E

800

0 O.

.c_ 6O0. E

0 (,0 .c 4 0 0

200

E C 8 0.

I I A methemphetamine B

=group L/DAgr �9 " �9 ogroupPL. .

o o o -

(r2='608) .4 (r~=.624)

1 i i i i I I C apom(r

\ .

i ~ � 9 ~ ~ �9 �9 o " - i o - . (r2=,360) . (r ='518) o O o oO %0 ' "

I 1 i i i = i I

0-4 I 2 4 10 20 40 .4 I 2 4 10 20 40

DOPAMINE CONCENTRATION (Log % intact side)

�9 group L -

D

419

Fig. 7A-D. Scatter plots of the relationship between turning rates on the 4 month amphetamine tests (A, B) and on the apomorphine rotation tests (C, D) with post mortem dopamine concentration in the dorsal striatum for the combined lesion rats (group L and PL in A and C) and in the rats with dopamine-rieh grafts (group L/ DAgr in B and D). Dopamine concentrations are expressed on a logarithmic scale. The least squares linear regression lines are shown in each panel, along with the estimate of the proportion of variance accounted for by the regression (r 2)

Dopamine depletions. The concentration of dopamine in the right and left striata, ipsilateral and contralateral to the nigrostriatal lesions (and grafts), are shown in Table 2. The lesions in group L produced depletions of 97-99% in the ipsilateral caudate-putamen and nucleus accumbens. The levels of depletion in the "partial" lesion group PL were considerably more variable, in some cases the depletions being > 99% but in other animals being only 70--80%, and in the worse case being only 31%.

The nigral grafts in rats with long-term lesions (group L/DAgr) produced a significant increase in dopamine concentration in the dorsal caudate-putamen (t(22) = 2.89), but not in the ventral caudate- putamen or nucleus accumbens (see Table 2). The five-fold increase in the dorsal caudate-putamen still only brought dopamine concentrations up to 7.2% of normal levels, but it should be noted that 9 of the 13 C+ rats were not included in this analysis as they had either been taken for immunocytochemistry or had received further lesions of the grafts (subgroup L/ DAgr/L). On the basis of behavioural recovery, these excluded rats would be expected to have the highest levels of dopamine in the dorsal caudate-putamen (see below).

Injection of 6-OHDA into the grafts (group L/ DAgr/L) was effective in reducing the concentration of dopamine in the dorsal caudate-putamen to a level that did not differ from the rats with lesions alone (group L) (t(13) = 0.28, ns).

The 3 rats with raphe grafts (group L/5HTgr) appeared to have less complete lesions in the nucleus accumbens than the other lesion groups. The difference from group L did not reach the criterion for significance (t( l l) = 2.95, 0.05 > p > 0.01). They did not show any increase in dopamine concentration in the vicinity of the grafts in the dorsal striatum above that seen in the rats with lesions alone, suggesting in agreement with the immunohistochemical observations that the dissection of embryonic raphe did not include substantial numbers of ventral mesencephalic dopamine neurons.

The rats that were initially part of the control group but which received nigrostriatal lesions 4 weeks prior to sacrifice (group C/L) appeared to have even greater depletions of dopamine in the dorsal and ventral striatum than the long-term lesion rats (group L), although this difference did not achieve significance in either site.

The unselected group of rats with nigral grafts implanted in the intact neostriatum, in which the intrinsic nigrostriatal system was lesioned 4 weeks prior to sacrifice (group DAgr/L), had mean dopamine concentrations 14-16% of normal levels in both dorsal and ventral caudate-putamen, which was significantly higher than that seen in the corre- sponding lesion control group C/L in the dorsal striatum (t(14) = 2.68, p < 0.01) but not in the ventral striatum, (t(14) = 2.50, 0.05 > p > 0.01).

420

Correlations with behaviour. The methamphetamine and apomorphine turning scores on the 4 month tests were correlated against the biochemical depletions in the caudate-putamen of each rat of groups L, PL and L/DAgr. The dopamine concentrations were subjected to logarithmic transformation in order to linearise the data and the scatter plots are shown in Fig. 7. Within the lesioned rats (groups L and PL combined), both methamphetamine and apomorphine rotation correlated highly with dopamine depletions in the dorsal caudate-putamen (r = -0.78, t(27) = -6.48, see Fig. 7A, and r = -0.72, t(27) = -5.42, see Fig. 7B, respectively, both p < 0.001). Within the graft group L/DAgr, methamphetamine rotation again correlated significantly with the level of dopamine depletion (r --- 0.79, t(12) = -4.52, p <0.001, see Fig. 7C), as also did apomorphine rotation (r -- -0.60, t(12) = -2.63, p < 0.05, see Fig. 7D), although in the latter case the relationship was not as strong. The slope of the linear regression in the combined lesion groups differed significantly from the slope in group L/DAgr for both the methamphetamine and the apomorphine tests (t(39) = -2.41 and-2 .18 , respectively, both p < 0.05).

Serotonin depletions. Parallel assays were conducted on all tissue samples for serotonin, with the exception of the rats from groups L, PL and L/DAgr. To provide comparable lesion data for group L, a separate group was added comprising of 6 rats with similar lesions, amphetamine rotation scores and survival times, but not included in the present behavioural experiments (group L'). Dopamine depletions in group L' did not differ from those observed in group L (Table 2). Serotonin levels in the groups that were successfully analysed are shown in Table 3.

The 6 -OHDA lesions induced moderate (25-70%) depletions in ipsilateral levels of serotonin in both the rats with long-term (group L') and short- term (group C/L) lesions. The differences between the lesion and intact side were significant (p < 0.01) in the nucleus accumbens and dorsal striatum but not in the ventral striatum of both groups L' and C/L (L': t(5) = 3.58, 8.38 and 1.87; C/L: t(6) = 5.03, 6.82, and 2.09, respectively).

The 3 rats with raphe grafts (group L/5HTgr) showed a dramatic increase in serotonin levels in the two striatal samples. Given the small group size, the difference between sides did not attain the stringent (p = 0.01) level of significance in the dorsal striatal sample (t(2) = 4.68, 0.05 > p > 0.01), but the increased serotonin concentration in the dorsal striatum was significantly higher than the decrease in the same area in group L' (t(7) = 8,71, p < 0.01).

Table 3. Biochemical assays for Serotonin (ng/mg wet weight of tissue)

Group N. Accumbens Ventral CPU Dorsal CPU

Group C (n = 5) left 0.840 + 0.073 0.480 _+ 0.012 0.252 _+ 0.031 right 0.716 + 0.032 0.528 +_ 0.059 0.246 _+ 0.037

Group L' (n = 6) left 0.587 +_ 0.273 0.545 _+ 0.012 0.489 + 0.026 right 0.273 + 0.056 0.376 + 0.074 0.154 + 0.025

diff 46.5% + 68.9% 31.2% ++

Group L/5HTgr (n = 3) left 1.020 + 0.168 0.559 _+ 0.027 0.296 _+ 0.016 right 0.775 + 0.104 0.884 + 0.133 1.080 + 0.169

diff 75.9% 158.2% * 364.3% +,**

Group C/L (n = 7) left 0.996 + 0.037 0.459 +_ 0.049 0.298 + 0.025 right 0.500 _+ 0.076 0.344 +_ 0.035 0.169 + 0.018

diff 50.2% ++ 74.8% + 56.5% ++

Group DAgr/L (n = 10) left 0.912 _+ 0.104 0.592 + 0.040 0.293 + 0.022 right 0.719 + 0.106 0.985 _+ 0.199 1.226 _+ 0.223

diff 78.9% 166.4% +,* 417.9%++,**

Differences from the unoperated side: +,p < 0.05; ++,p < 0.01 Differences from lesion group L' or C/L as appropriate: *, p < 0.05; **, p < 0.01 To correct for multiple comparisons, only differences p < 0.01 are considered to be significant

The nigral grafts implanted into the subsequently lesioned intact striatum (group DAgr/L) provided increases in serotonin concentration in the two striatal samples, which was several-fold higher than the levels seen in the intact (contralateral right) striatum (dorsal striatum, t(9) = 4.10, p < 0.01; ventral striatum, t(9) = 1.99, p < 0.05), and significantly different from the depletions in the short-term lesioned striatum without grafts (groups C/L vs. C/ DAgr/L: dorsal striatum, t(15) = 3.79, p < 0.01; ventral striatum, t(15) = 2.13, p < 0.05). This suggests the inclusion of some serotonergic cells of the mesencephalic raphe in the ventral mesencephalic nigral graft dissection, in parallel with the immunohistochemical observations of 5-HT positive neurons within the ventral mesencephalic grafts of groups L/DAgr and C/DAgr.

Discussion

The present experiments indicate that recovery from the motor asymmetry seen in rats with unilateral dopamine depletions can be induced by embryonic neural grafts and is dependent on the use of graft

tissue rich in dopaminergic cells. Several different experimental manipulations have been employed in reaching this conclusion.

Firstly, the use of alternate graft tissues indicates that significant compensation of both amphetamine- induced and apomorphine-induced rotation bias is only achieved with embryonic graft tissue derived from the ventral mesencephalon, containing the developing cells of the substantia nigra-ventral tegmental area complex (Olson and Seiger 1972; Specht et al. 1981). Freed and coworkers have previously shown that control tissues of embryonic neocortex, embryonic tectum or adult peripheral sciatic nerve do not provide any parallel recovery in apomorphine- induced rotation (Freed 1983; Freed et al. 1984). Both these types of graft tissue survive transplantation but are devoid of dopaminergic neurons. The present experiment has employed two further control tissues that might be considered more similar to the optimal dopamine-rich source. Non-dopaminergic monoamine cells were chosen from the serotonin- rich mesencephalic raphe nucleus in preference to noradrenergic neurons of the pontine locus coeruleus since the latter population of cells requires modification of the dissociation protocol to achieve viable cell suspensions (Bj6rklund et al. 1986), which would have introduced additional uncontrolled factors into the design. Striatal neurons were dissected from the developing striatal eminence as a source of graft tissue that is homotopic for the transplantation site in the host brain. Although both of these sources of graft tissue have been shown to be functionally effective in relevant behavioural tests for rats with lesions appropriate to the tissue source (Isacson et al. 1986; Luine et al. 1984), they were both without significant consequence to the rotation asymmetry caused by dopaminergic depletion. These observations suggest that use of dopamine-rich ventral mesencephalic tissue is critical for functional recovery from 6-OHDA induced motor asymmetry.

Secondly, lesion of dopaminergic neurons within ventral mesencephalic grafts reinstated the initial level of motor asymmetry as measured by methamphetamine-induced rotation tests. LeVere and LeVere (1985) have argued that the critical test for whether a graft is exerting a functional effect on the host animal is not whether the animals recover and the graft is seen to survive - such recovery might be attributable to a modification by the grafts of spontaneous sparing or regenerative processes in the host brain. Rather they argue that the critical test is whether removal of the graft subsequent to recovery would reintroduce the initial deficit. We have previously shown this to be the case with "solid" grafts of dopamine-rich ventral mesencephalic tissues into a

421

dorsal parietal cavity overlying the denervated neostriatum, aspirative removal of which reinstates amphetamine-induced rotation asymmetry in compensated rats (Bj6rklund et al. 1980). A similar result has been obtained in rats with xenografts of embryonic mouse ventral mesencephalon. When the host animals are treated daily with the immunosuppres- sant drug Cyclosporin A the grafts survive well and provide extensive compensation of amphetamine- induced rotation bias (Brundin et al. 1985). How- ever, if the drug treatment is terminated after 16-20 weeks, the grafts in the majority of animals undergo immune rejection and the compensation of the motor bias is abolished at the same time (Brundin et al. 1988). However, this latter study indicated that the rejection of established grafts is frequently accom- panied by extensive necrosis in the host striatum, which may influence the integrity of striatal output neurons irrespective of the health of the grafted dopamine neurons. The present results overcome this problem by demonstrating an equivalent aboli- tion of the functional recovery following a relatively selective 6-OHDA lesion of the grafted dopaminergic cells. Although these rats were all taken for biochemical confirmation of the dopamine denervation rather than for histology, the grafted tissue could be seen in all cases in the fresh brain at the time of dissection, suggesting that the toxin had not simply induced non-specific destruction of striatal tissue containing the graft. This observation then suggests that the recovery of motor asymmetry in the grafted rats is dependent upon the survival of dopaminergic cells within the grafts rather than the presence of other unspecified elements within the ventral mesencephalic tissue.

Thirdly, considerable variability was seen in the effectiveness of the lesions and the viability of the grafts within the lesion groups (L and PL) and the ventral mesencephalic graft group (L/DAgr), respectively, whether assessed by behavioural or biochemical indices. The behavioural indices covaried with the extent of dopamine depletion in the lesion groups and with the biochemically-determined extent of reinnervation in the dopamine-rich graft group. In both groups, the correlations were highest with dopamine concentration in the dorsal striatum (the area where the grafts were predominantly located, see Table 2), rather than in ventral regions or the whole striatal complex. It is the dorsal neostriatum that receives primary afferent input from motor areas of neocortex (Kelley et al. 1982), and it is the critical striatal region for reinnervation from dopamine-rich grafts to provide compensation on methamphetamine and apomorphine rotation tests (Dunnett et al. 1981b, 1983).

422

For both the combined lesion groups and the dopamine-rich graft group, the amphetamine-rotation test provided a higher correlation with the biochemical measure of depletion than did the apomorphine test. Although both measures have been employed to assess the completeness of nigrostriatal lesions and the efficacy of dopamine-rich grafts (Bj6rklund and Stenevi 1979; Bj6rklund et al. 1980; Dunnett et al. 1981a, 1983; Freed et al. 1980, 1981; Perlow et al. 1979), the present observations indicate that by accounting for greater variance, the amphetamine-induced rotation test might provide a somewhat more sensitive in vivo index of the biochemical depletions as assessed post mortem.

The correlation between the amphetamine and apomorphine scores in the combined lesion groups, by virtue of their covariation with the extent of nigrostriatal denervation, is unsurprising and accords with previous observations in groups with lesions of explicity different size (Hefti et al. 1980). Of greater surprise is the observation that recovery as determined by compensation on the amphetamine test was completely uncorrelated with apomorphine-rotation scores in the rats with ventral mesencephalic grafts. This suggests that the two measures might reflect quite different mechanisms of functional recovery. Indeed, we have previously found that the degree of compensation of methamphetamine-induced rotation is related to the extent of dopamine fibre ingrowth into the denervated neostriatum from solid nigral grafts, as assessed by catecholamine histofluores- cence (Bj6rklund et al. 1980) or biochemical assay (Schmidt et al. 1982), and the time-course of amphetamine-stimulated release of dopamine from graft-derived terminals in the neostriatum, determined by in vivo dialysis, is closely related to the time course of the behavioural rotation induced by the drug (Zetterstr6m et al. 1985). Conversely, the compensation of apomorphine-induced rotation is thought to reflect the reduction in dopamine receptor supersensitivity in the neostriatum ipsilateral to the lesion as a result of chronic spontaneous (as opposed to drug-induced) release of dopamine from the grafted dopaminergic neurons (Freed 1983; Freed et al. 1984). Such compensation is not dependent on graft-derived fibre reinnervation of the denervated host neostriatum since no clear relationship is seen between the extent of fibre ingrowth and recovery of apomorphine rotation (Freed et al. 1980, 1984), and effective recovery can be achieved on the apomorphine test with intraventricular nigra grafts that provide only limited fibre ingrowth (Freed et al. 1980). Indeed, intraventricular adrenal grafts which contain catecholamine-secreting chromaffin cells, but show no detectable reinnervation of the host brain

are equally effective (Freed et al. 1981, 1984). This reasoning would suggest that recovery on the methamphetamine tests is dependent on the concentration of dopamine stored in synaptic terminals reinnervating the host striatum and available for drug-stimulated release, whereas recovery on the apomorphine tests reflects spontaneous dopamine release, whether from dopamine neurons contained in the graft or from terminals in the host brain. The validity of such an interpretation cannot be resolved from the present biochemical assays which, as in other biochemical studies of suspension grafts (Schmidt et al. 1983), involve the total concentration of dopamine in the graft and in the host striatum together, nor do the assays distinguish between the presence of the transmitter in the cell cytoplasm, synaptic vesicles or extracellular compartments.

The present experiments included groups to assess the functional effects of dopamine-rich grafts implanted in the intact brain. The degree of survival, growth and reinnervation provided by neural implants can in many circumstances be promoted by lesions of intrinsic neural systems, and in some graft models the grafts only survive poorly in the absence of such denervation (Bj6rklund and Stenevi 1977, 1981; Gage and Bj6rklund 1986). However, grafts of other types, including ones involving monoaminergic neurons, have been seen to undergo substantial growth and reinnervation of the host brain even in the absence of an explicit denervating lesion (Azmitia et al. 1981; Schmidt et al. 1981). In particular, Schmidt et al. (1981) observed extensive ingrowth from grafted dopaminergic neurons into the intact neostriatum, making lesions of the intrinsic system only just prior to sacrifice in order to reveal graft-derived fibres in the striatum. Similarly, we have previously observed that when ventral mesencephalic cell suspensions were implanted bilaterally in the striatum of rats with unilateral lesions, substantial ingrowth was seen on the intact contralateral side, although this was not as extensive as on the lesioned side (Bj6rklund et al. 1983b). The present biochemical, immunohistochemical and behavioral results confirm the good survival and growth of ventral mesencephalic cell suspension in the intact neostriatum. However, this growth is not associated with "supernormal" effects, i.e. there was no evidence for the grafts inducing a motoric bias in the animals to the contralateral side. Nevertheless, this is not because the grafts were without functional capacity, since they provided substantial functional protection against the effects of a subsequent lesion of the intrinsic nigrostriatal pathway ipsilateral to the grafts. Rather, the "prophylactic" capacity revealed following the lesion indicates that the graft-derived

423

reinnervation is subject to similar compensatory mechanisms by which dopamine synthesis, turnover and release are regulated in the intact striatum for the maintenance of functional symmetry in the presence of disturbance (Stricker and Zigmond 1976).

In conclusion, functional recovery of drug- induced motor asymmetries by neural tissue grafts in rats with unilateral damage of the intrinsic nigrostriatal dopamine system is not a consequence of the non-specific stimulation by foetal tissues of growth or recovery mechanisms in the host brain. Rather, the functional effects of the grafts are dependent on the replacement of dopaminergic neurons into dop I amine-deficient sites. Dopamine-rich implants do not induce abnormal contralateral asymmetries in the intact brain and yet can provide protection against subsequent lesions, suggesting that the grafts do not simply secrete dopamine diffusely into the host brain but are subject to at least some local regulatory mechanisms similar to those that regulate intrinsic dopamine neurons. Parenthetically, although both methamphetamine-induced rotation and apomorphine-induced rotation provide convenient in vivo indices of the extent of dopamine denervation produced by the lesions and the extent of survival and growth of grafted dopaminergic neurons, the methamphetamine test is somewhat more sensitive.

Acknowledgements. The present study was funded by a grant from the Medical Research Council to SBD. TDH was supported by the Ford Foundation and GHJ by the Parkinson's Disease Society.

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Received November 23, 1987 / Accepted December 11, 1987

graft-derived recovery from 6-ohda lesions: specificity of ventral mesencephalic graft tissues

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