Regional changes of striatal dopamine receptors following denervation by 6-hydroxydopamine and fetal mesencephalic grafts in the rat

Brain Research, 558 (1991) 251-263 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116948Q

251

BRES 16948

Regional changes of striatal dopamine receptors following denervation by 6-hydroxydopamine and fetal mesencephalic grafts in the rat C61ine Gagnon

1,2, Paul J. B6dard 3, Lise Rioux 3, Daniel Gaudin 3, Maria G. Martinoli 2, Georges Pelletier 2 and Th6r~se Di Paolo 1,2

1School of Pharmacy, Laval University, Quebec, Que. (Canada), 2Department of Molecular Endocrinology, CHILL Research Center, Laval University Medical Center, Ste-Foy, Que. (Canada) and 3Neurobiology Laboratory, L'Enfant-Jesus Hospital, Quebec, Que. (Canada) and Department of Pharmacology, Laval University, Faculty of Medicine, Quebec, Que. (Canada) (Accepted 23 April 1991)

Key words: Dopamine; Dopamine 1 receptor; Dopamine 2 receptor; Nigral graft; Striatum; 6-Hydroxydopamine

Young adult female rats received a 6-hydroxydopamine lesion in the left substantia nigra and, 3 weeks later, some of them were grafted with a cell suspension from the ventral mesencephalon of rat embryos (14-15 days old). Six months after transplantation, some grafted rats, following injection of amphetamine, had switched to turning only toward the intact side (type 1), whereas others turned toward the intact side only during the first half of the test (type 2). Levels of dopamine, dihydroxyphenylacetic acid and homovanillic acid were, respectively, 2%, 15% and 35% of the intact side in the denervated striatum of 6-hydroxydopamine rats. Dopamine concentrations were restored to 13% and 10% of the intact side in the grafted striatum of type 1 and type 2 animals, respectively. Levels of homovanillic acid were unchanged following grafts whereas those of dihydroxyphenylacetic acid increased by 209% and 247% in the grafted striatum of type 1 and type 2 animals, respectively. The ratios of dihydroxyphenylacetic acid/dopamine as well as homovanillic acid/dopamine were low in the intact striatum whereas they increased in the denervated striatum with or without graft. The tyrosine hydroxylase immunoreactivity decreased by about 80% in the denervated striatum of 6-hydroxydopamine rats. In type 1 rats, tyrosine hydroxylase immunoreactivity revealed that the graft was localized in the dorsomedial part of the denervated striatum, whereas in type 2 animals, it was also in the medial striatum but it overlapped the dorsal and ventral parts of it equally. D~ as well as 0 2 dopamine receptors were measured throughout the striatum (9.0-7.6 rostralcaudal coordinates), by autoradiography, using [3H]SCH 23390 (D 1 antagonist) and [3H]spiperone (D Eantagonist) binding. Supersensitive 0 2 receptors were normalized in the dorso- and ventromedial parts of the grafted striatum. D 2 receptor density was higher in type 2 than in type 1 rats, more specifically at 8.6-8.2 rostral-caudal coordinates, where the graft was. D 1 receptor supersensitivity was modest compared to D z receptors in the striatum of 6-hydroxydopamine rats and decreased following grafts. DA receptors changes in the striatum, following fetal mesencephalic grafts, may explain the behavioral recovery seen in grafted rats. INTRODUCTION

is responsible for the recovery 21, since adrenal medulla

Grafts of fetal mesencephalic d o p a m i n e ( D A ) neurons in the denervated striatum of 6-hydroxydopamine ( 6 - O H D A ) - l e s i o n e d rats can correct rotation asymmetries induced by peripheral injection of a m p h e t a m i n e or a p o m o r p h i n e 5'17. In addition, they also correct spontaneous m o t o r behavior in lesioned animals 12-14. The de-

tissue implanted in the lateral ventricle adjacent to the deafferented neostriatum was observed to reduce apomorphine-iffduced rotation even in the absence of any evidence of fiber reinnervation of the host brain. A n other study showed that a 50% decrease of circling to apomorphine was observed in unilateral 6 - O H D A rats which had received grafts of a controlled release silicone polymer containing D A 4. However, others suggest that

gree of recovery has b e e n seen to correlate with both biochemical and histochemical indices of reinnervation6' 7,22,47,48 Grafted n e u r o n s have b e e n shown to be spontaneously active in the host striatum, as demonstrated by electrophysiological a'19"43 and biochemical47'48'55 studies. However, a consensus on the functional effect of the graft has not b e e n reached. Some support the view that chronic diffuse secretion of catecholamines from the graft

the graft may provide a neurotrophic influence o n mechanisms of spontaneous recovery within the host brain 16, 30 O u r previous studies with unilateral 6 - O H D A - l e s i o n e d rats grafted with fetal mesencephalic D A n e u r o n s in the denervated striatum showed variable functional recovery 42. After the injection of a m p h e t a m i n e in these

Correspondence: T. Di Paolo, Department of Molecular Endocrinology, CHUL Research Center, 2705, Laurier Boulevard, Ste-Foy, Que., Canada G1V 4G2. Fax: (1) (418) 654-2735.

252 g r a f t e d r a t s , 4 1 % o f a n i m a l s s w i t c h e d t o t u r n i n g o n l y tow a r d s t h e i n t a c t side ( t y p e 1), 38% t u r n e d t o w a r d t h e i n t a c t side o n l y d u r i n g t h e first h a l f o f t h e t e s t ( t y p e 2),

V: 1.5; (2) A: +7.8, L: 3.0, V: 1.5; (3) A: +7.4, L: 2.5, V: 1.5. according to system A of Pellegrino's atlas). All graft suspensions were injected at a rate of 1 ~l/min, and the syringe was left in place for 2 min.

a n d 21% i n c r e a s e d t h e i r t o t a l n u m b e r o f i p s i v e r s i v e t u r n s in 90 m i n i n s p i t e o f t h e fact t h a t t h e y t u r n e d c o n t r a l a t e r a l l y d u r i n g t h e first 20 m i n o f t h e t e s t ( t y p e 3). W e found that there was a correlation between the number o f s u r v i v i n g g r a f t e d n e u r o n s a n d t h e g r o w t h o f t h e i r fibers into the host striatum, and the extent of recovery. T h e b a s a l l e v e l o f e x t r a c e l l u l a r D A , as m e a s u r e d b y m i crodialysis, was normalized by the graft, whereas those o f d i h y d r o x y p h e n y l a c e t i c acid ( D O P A C )

and homovan-

illic acid ( H V A ) r e m a i n e d m u c h l o w e r o n t h e l e s i o n e d side b e a r i n g t h e g r a f t . I n g r a f t e d r a t s w e also s h o w e d t h a t t h e c i r c l i n g t o a p o m o r p h i n e as well as c i r c l i n g t o C Y 208243 ( D 1 a g o n i s t ) a n d L Y 171555 ( D 2 a g o n i s t ) w e r e r e d u c e d 41. The purpose of the present study was to compare the modulation of D 1 and D 2 DA receptors, by autoradiogr a p h y u s i n g [ 3 H ] S C H 23390 ( D 1 a n t a g o n i s t ) a n d [3H]spipe r o n e b i n d i n g ( D 2 a n t a g o n i s t ) , in u n i l a t e r a l 6 - O H D A lesioned rats which received fetal mesencephalic grafts in t h e d e n e r v a t e d s t r i a t u m . C h a n g e s in D A r e c e p t o r s w e r e m e a s u r e d t h r o u g h o u t t h e s t r i a t u m f r o m 9 . 0 t o 7.6 rostral-caudal coordinates (according to the Atlas of Pelleg r i n o e t al. 38) t o s e e if t h e y w e r e h o m o g e n o u s o r if t h e y w e r e c o r r e l a t e d t o t h e l o c a l i z a t i o n o f t h e g r a f t . T h e ext e n t o f t h e g r a f t , its l o c a l i z a t i o n in t h e d e n e r v a t e d stria t u m as well as its d o p a m i n e r g i c a c t i v i t y w e r e m e a s u r e d .

MATERIALS AND METHODS

6-OHDA-lesion, graft surgery and rotation testing Young adult female Sprague-Dawley rats (Charles River C.D. strain obtained from Canadian Breeding Farms, St-Constant, Que.), weighing 200--250 g at the start of the experiment, were used in this study. They were housed two per cage under a 12 h light-12 h dark cycle with lights on at 07.00 h and ad libitum access to food and water. They received a unilateral 6-OHDA injection into the left nigro-striatal D A bundle (8/~g free base in 2/~1 of 0.9% saline ascorbate) (Sigma), under ketamine/xylazine-induced anesthesia, at the following coordinates (according to system A of the Atlas of PeUegrino et al.38), A: 3.6, L: 1.5, V: -3.0. The neurotoxin was delivered at a rate of 1/~l/min. The syringe was left in situ for a further minute after the injection of drug. Two weeks after the 6-OHDA lesion, all animals were tested with apomorphine (0.35 mg/kg, s.c.) (Merck Frosst) and only rats displaying a net contralateral rotation rate of I>5 turns/rain over 25 min were ineluded. These rats were tested with amphetamine (5 mg/kg, s.c.) over a 90 rain period, only animals with 9 turns or more per minute over 25 rain were used. One week later, some of them received nigral grafts. The amphetamine test was repeated six months after transplantation. Neural transplants were prepared from fetal ventral mesencephalic tissue according to the cell suspension method described by Bj6rklund et al. 7. Viability and concentration were assessed in an hemacymeter using Trypan blue as differential stain. Nine/zl of the suspension containing approximately 1.5 x 10 6 cells from 14 to 15days-old rat embryos was distributed in 3 sites in a triangular fashion in the center of the denervated striatum ((1) A: +8.2, L: 2.5,

Tissue preparation and autoradiography of DA receptors Six months after transplantation of nigral grafts, the 6-OHDAlesioned rats with or without grafts were sacrificed by decapitation, 72 h following the last circling test. The brains were rapidly removed, frozen and stored at -80 °C. For autoradiography, the brains were cut on a cryostat (-20 °C) in coronal sections (10/~m), thaw-mounted onto gelatin-coated slides. Slices were taken from the following rostral-caudal coordinates 9.0-7.6, according to system A of the Atlas of PeUegrino et al. 38. The coronal sections of rat striatum were preineubated, for 15 min at room temperature, in a phosphate buffer (81 mM Na2HPO 4, 19 mM KH2PO 4 and 2 mM MgCI 2, pH 7.40-7.45). They were then incubated (60 min at room temperature) with 1 nM of [3H]spiperone (D 2 antagonist; spec. act. 72.9 Ci/mmol) or 1 nM of [3H]SCH 23390 (D 1 antagonist; spec. act. 72.5 Ci/mmol). In the D 2 receptor assay, ketanserin (50 nM) was added to the incubation medium to block S~ serotonin receptors. Non-specific binding was determined in adjacent sections with the addition of 1/IM (+)-butaclamol (D2) or 1 ~M SCH 23390 (Dl). The sections were then rinsed for 15 min in the appropriate cold (4 °C) phosphate buffer and then in distilled water (10 s). The sections were dried overnight at room temperature and apposed to [3HI-sensitive film (Hyperfilm RPNI3, LKB) along with a set of tritium standards (3H-microscale, Amersham) for l 1 days at -20 °C. Films were developed with D-19 developer (Kodak) for 4 min, fixed into rapid fixer (Kodak) for 10 min and rinsed in water for 30 min before drying. The autoradiograms of the brain sections were analyzed with an image analyzer system (RAS-1000, Amersham). Quantitative values were obtained by direct autoradiography of [3H]-microscale (Amersham) with brain sections. The left and fight striata were divided into 4 parts (dorsomedial, dorsolateral, ventromedial and ventrolateral) (Fig. 1) and differences between groups (lesioned and grafted rats) were, in each case, examined: for separate rostral-caudal coordinates, for grouped coordinates and for the whole striatum. For separate coordinates, differences in the left lesioned striatum were examined by a covariance analysis with two factors (groups and rats within groups) taking the fight intact striatum as a covariate. By that means, we obtained for each coordinate an adjusted mean for the lesioned striatum (n = 2-15 slices). For grouped coordinates, the adjusted means of the individual coordinates were pooled and compared with a two factor analysis of variance ('group' as taken as a fixed factor and 'rat within group' as a random factor). For the whole striatum, all the individual adjusted means were pooled together and again analysed by a two factor ANOVA. The adjusted mean values for each rat were pooled for each coordinate, grouped coordinates and the whole striatum (n = 3-4 rats per group) to calculate an average density -+ S.E.M. DA receptor densities for the total striatum (9.07.6 rostral-caudal coordinates) in each region were also expressed in percent of intact value for each rat. The data were then pooled together (n = 3-4 rats). DA receptors from the intact and lesioned side of the stfiatum were compared with the paired Student's t-test (two-tail).

Catecholamine determination Caudate-putamen tissue from alternate coronal sections was dissected for catecholamine determination. DA, DOPAC and HVA were assayed by HPLC with electrochemical detection essentially as described by Di Paolo et al. 11. Each concentration value for the left lesioned striatum with or without grafts was expressed as a percent of the right intact striatum and pooled for each rat (n = 5-12 slices); the mean values obtained for each rat were then pooled together (n = 2-3 animals). For the determination of DOPAC/DA and HVA/DA ratios, concentration values were used. The multiple range test of Duncan-Kramer 31 was used for comparison of catecholamine levels as well as the log transformation

253 for the DOPAC/DA and HVA/DA ratios.

Tyrosine hydroxylase immunocytochemistry Tyrosine hydroxylase (TH) immunoeytochemistry was used to assess the extent of 6-OHDA-induced denervation and to localize striatal grafts. Caudate-putamen tissue from alternate coronal sections, stored at -80 °C were first fixed with 4% paraformaldehyde + 0.2% glutaraldehyde in phosphate buffer 0.05 M, pH 7.4, for 30 min at room temperature. TH immunocytochemistry was performed following the peroxidase-antiperoxidase (PAP) method of Sternberger et al. 5°. The preparation and specificity of the a-TH antiserum, purchased from Institute J. Boy S.A., have been already described 2"52. Briefly, slides were washed twice in Tris buffer 0.05 M + NaC1 0.9%, pH 7.6. They were then incubated in 0.3% H 2 0 2

for 30 min at room temperature, rinsed and incubated overnight with a a-TH antiserum diluted 1:1000 in "Iris buffer 0.05 M + BSA 0.1%. The following morning, slides were rinsed twice in Tris buffer and then exposed to peroxidase-labelled goat anti-rabbit Fab fragment (Hy clone, 1:500). Peroxidase activity was visualized with 3", 3"-diaminobenzidine and hydrogen peroxide. Quantitative values of optical density were obtained with an image analyzer system (RAS 1000, Amersham) from the brain slices. The left and fight striata were divided in 4 sections: dorsomedial, dorsolateral, ventromedial and ventrolateral (see Fig. 1). Each value of optical density in the left denervated striatum was expressed as a percent of the respective intact section of the right intact striatum and pooled to give a percent value (n = 1-4 animals and 3-23 slices per animal). The multiple range test of Duncan-Kramer 31 was used for comparison of TH immunoreactivity data.

RESULTS

Drug-induced rotation

ANTERIOR TO THE GRAFT (9.0-8.8)

In lesioned rats, the pattern of circling to amphetamine was strictly ipsiversive in all animals (Fig. 2). In some grafted animals, the circling to amphetamine switched completely from ipsiversive to contraversive, whereas in others, the switch was only partial. The grafted rats were therefore divided into 2 groups: type 1 (complete switch) and type 2 (incomplete switch) (Fig.

2). Catecholamine concentrations

AT THE LEVEL OF THE GRAFT (8.6-8.2)

POSTERIOR TO THE GRAFT (8.0-7.6)

Fig. 1. Diagram of coronal sections anterior, inside and posterior

to the graft in unilateral 6-OHDA rats which received nigral grafts in the denervated striatum. The left and fight striata were divided into four sections (DM, dorsomedial; DL, dorsolateral; VM, ventromedial; VL, ventrolateral) for the measurement of DA receptors as well as TH immunoreactivity.

D A levels decreased more than 98% in the denervated striatum of 6-OHDA-lesioned rats compared to the intact side (Fig. 3). Depletion of D O P A C (85%) and H V A (65%) were less extensive. Concentrations of D A were restored to 13% and 10% of the intact side in the grafted striatum of type 1 and type 2 rats, respectively. D O P A C and H V A levels were, respectively, 47% and 48% of the intact fight striatum in grafted type 1 animals, whereas they were 53% and 67% of the intact fight striatum in those of type 2. Levels of H V A were unchanged, whereas those of D O P A C increased by 209% and 247% in the grafted striatum of type 1 and type 2 rats, respectively, compared to the lesioned side of 6 - O H D A rats. In lesioned rats with or without grafts, the ratios D O P A C / D A and H V A / D A were low in the intact fight striatum ranging from 0.12 to 0.30, while these ratios increased in the left lesioned side of 6 - O H D A rats with values from 1.00 to 3.00 (Fig. 4). Values of D A , D O P A C and H V A for the intact fight striatum were, respectively, 59.7, 12.2 and 11.0 ng/mg of protein for 6 - O H D A rats; 48.8, 15.5 and 22.4 ng/mg of protein for type 1 grafted rats; and 42.9, 9.9 and 12.0 ng/mg of protein for type 2 grafted rats.

TH immunocytochemistry The T H immunocytochemistry method was used in order to assess the extent of 6 - O H D A denervation and

254 to localize striatal grafts (Fig. 5). The TH immunoreactivity decreased by about 80% in the denervated striatum of 6-OHDA rats compared to the intact side. This decrease was partly reversed by the grafts. Indeed, TH immunoreactivity was, respectively, 42% and 45% of the intact right striatum in type 1 and type 2 grafted rats. It represents a significant increase of about 105% and 119%, respectively, compared to the denervated striatum of 6-OHDA-lesioned rats. In type 1 rats, the graft was mostly localized in the dorsomedial part of the denervated striatum (Fig. 5). In this region, the T H immunoreactivity was 69% of the intact side, whereas in the ventromedial part, it was significantly less (44% of the intact side). In type 2 animals, the graft was also in the medial part of the striatum, but

TYPE I 80-

m

Before graft I After graft

¢U

d ~ 4o-

E ~

los! 90 ~

P-r

-~ n~L)
LESIONED GRAFTED, TYPE 1 GRAFTED, TYPE 2

i

75-

60 45 30 15

o~ o

DOPAC

DA

HVA

* P<0.05 AND ** P<0.01 VS LESIONED

Fig. 3. Dopamine (DA), dihydroxyphenylacetic acid ( D O P A C ) and homovanillic acid (HVA) levels in the left denervated striatum of unilateral 6-OHDA-lesioned rats with or without nigral grafts. Each individual left striatum concentration value was expresed as a % of the intact fight striatum and pooled for each rat (n = 5-12 slices). Data for each rat were then pooled together to calculate an average concentration value +- S.E.M. (n = 2-3 animals). Values given are % of the intact right striata. The multiple-range test of D u n c a n - K r a m e r was used for comparison of the data.

it extended in the ventral quadrant. Indeed, in the dorsoand ventromedial parts of the denervated striatum, the T H immunoreactivity was, respectively, 53% and 61% of the intact side. In the dorso- and ventrolaterat parts of

o.

C

o 0

<

-40-

._~ -80 -

[] LESIONED GRAFTED, TYPE GRAFTED, TYPE 2

-120

4'o

6'o

TIME (rain.) a. O TYPE II

d

E

-8°1 ~

i

40

,

e

or. g r e .

After graft

L

RIGHT STRIATA *P<0.05 AND**

o

fj

.p.

P<0.01

VS INTACT RIGHT STRIATA

~° ° ~ -40.

~

I ¢3 < "I-

-80 -120

LEFT STRIATA

2'0

4'O

~

8'0

,~0

TIME (min.) Fig. 2. Amphetamine-induced rotation: each point shows net (ipsilateral-contralateral) turns at 5 rain intervals during 90 min following injection of 5 mg/kg amphetamine s.c., tested before and at 6 months after graft. The vertical bar indicates S.E.M. (type 1, n = 6; type 2, n = 5). A t all time intervals tested, circling was significantly different (P < 0.01 two way A N O V A for repeated measures, Tukey's a postefiori test) in rats measured after as compared to before the graft for type 1 as well as type 2 grafted animals. In addition, circling was significantly different ( P < 0.05 two-way A N O V A for repeated measures, N e w m a n - K e u i s a posteriofi test) after graft in rats of type 1 compared to those of type 2.

RIGHT STRIATA

LEFT STRIATA

Fig. 4. D O P A C / D A and H V A / D A ratios in the right intact and left denervated striatum of unilateral ~ H D A - l e s i o ~ d rats with or without nigral grafts. For each rat, individual values o f metabolite concentrations in the left and right striata were divided b y the mean D A concentration of its r e s p e ~ v e striatum (n = 5-12 slices). Then data for each rat were pooled together to calculate a ratio value + S.E.M. (n = 2-3 animals). The multiple-range test of Duncan-Kramer was used on log (value x 100) for comparison of the data.

255 the denervated striatum of grafted rats, the T H imunoreactivity was the same as in lesioned animals. It is important to note that the graft was observed to be between 8.6 and 8.2 rostral-caudal coordinates, thus mostly in the anterior striatum. DA receptors D 2 receptors increased by 33% (P < 0.01) in the whole denervated striatum of 6-OHDA-lesioned rats compared to the intact side. In grafted rats, D 2 receptors were only 108% (P = 0.16) and 112% (P < 0.05) of the intact side in the whole striatum of type 1 and 2 animals, respectively, thus representing decreases of D 2 receptor density of 19% (P < 0.001) and 14% (P < 0.01) compared to the denervated striatum of 6-OHDA rats. D1 receptors were slightly increased after the lesion (12%) (P < 0.05). In grafted rats, D 1 receptor density was not significantly different between the intact and lesioned side. Indeed, in the grafted striatum, it was 107% (P = 0.277) and 105% (P = 0.250) of the intact side in, respectively, type 1 and type 2 animals. Thus, D 1 receptor density between the intact and denervated striatum was significantly different only for 6-OHDA non-grafted rats. To investigate the localization of the D A receptor changes, we have divided the left and right striata into 4 parts: dorsomedial, dorsolateral, ventromedial and ventrolateral (Fig. 6). D 2 receptors increased by 42%, 33%,

38% and 22% in, respectively, the dorsomedial, dorsolateral, ventromedial and ventrolateral parts of the left denervated striatum compared to the right intact side (Fig. 6). Following grafts, D 2 receptors decreased to 103% and 117% of the intact striatum in, respectively, type 1 and type 2 grafted animals, in the dorsomedial part of the striatum; to 116% and 125% in the dorsolateral part; to 104% and 103% in the ventromedial part and to 106% and 107% in the ventrolateral part. D 1 receptors increased by 8%, 10%, 12% and 20% in, respectively, the dorsomedial, dorsolateral, ventromedial and ventrolateral parts of the denervated striatum compared to the intact side (Fig. 6). D 1 receptors decreased to 100% and 106% of the intact striatum in, respectively,

* .~

120"

~ • 80

DORSOMEDIAL DORSOLATERAL VENTROMEDIAL VENTROLATERAL

's°t 90"

D-2

9O

1

m 20 U.

o

÷+

+

LESIONED

GRAFTED TYPE 1

GRAFTED TYPE 2

* P<0.05 AND ** P<0.01 VS INTACT STRIATA + Pc0.05,+ + P<0.01 AND ++ + P<0,001 VS LESIONED

U,I

o

+

1101

~ 60

°

DORSOMEDIAL DORSOLATERAL VENTROMEDIAL VENTROLATERAL

110"

13°1 [] D [] []

[] [] • []

D-1

130"

LESIONED

GRAFTED, TYPE 1

GRAFTED, TYPE 2

"x'P<0.05 AND ~'x-P<0.01 VS DORSOLATERAL ++P<0.01 VS VENTROMEDIAL ,~P<0.01 VS VENTROLATERAL Fig. 5. T H immunoreactivity in the left denervated striatum of unilateral 6-OHDA-lesioned rats with or without nigral grafts. The left and fight striata were divided into four regions (dorsomedial, dorsolateral, ventromedial and ventrolateral). Each individual value of optical density in each region of the left denervated striatum was expressed as a % of the respective intact fight striatum region and pooled to calculate an average density value --- S.E.M. (n = 3-23 slices). The multiple-range test of Duncan-Kramer was used for comparison of the data (n = 1-3 animals).

Fig. 6. Effect of a unilateral 6 - O H D A lesion as well as striatal grafts on [3H]SCH 23390 (1 nM) and [3H]spiperone (1 nM) binding to D z and D 2 receptors, respectively, in the fight intact and left lesioned striatum. The left and fight striata were divided into four sections (dorsomedial, dorsolateral, ventromedial and ventrolateral) and D t as well as D 2 receptors were measured from 7.6-9.0 eaudal-rostral coordinates. Differences in the left lesioned striatum were examined by a covariance analysis with two factors (groups and rats within groups) taking the fight intact striatum as a covadate. By that means, we obtained for each coordinate for each rat an adjusted mean for the lesioned striatum (n = 2-15 slices). The adjusted means of the individual coordinates were pooled and compared with a two factor analysis of variance ('group' was taken as a fixed factor and 'rats within group' as a random factor). Results given are % of respective fight intact striata region for each rat which are pooled together (n = 3-4 rats) to calculate an average value --- S.E.M. The Student's t-test was used to compare the intact and lesioned side and the covafiance analysis with two factors was used to compare the left stfiatum of lesioned and grafted rats.

256 the dorsomedial part of the grafted striatum of type 1 and type 2 rats; to 102% and 94% in the ventromedial part; to 113% and 110% in the dorsolateral part and to 113% and 107% in the ventrolateral part. We observed that O 1 a s well a s D E receptors were decreased, following grafts, mainly in the medial striatum. We have also measured D A receptors at each rostralcaudal coordinate from 9.0 to 7.6 for each striatal region that is anterior, in and posterior to the grafts which were mostly localized at 8.6 to 8.2 rostral-caudal coordinates (Fig. 7). D 2 D A receptor supersensitivity decreased significantly in the dorsomedial and ventromedial parts of the grafted striatum, while in the dorsolateral and ventrolateral parts, D E receptors were unaffected (Fig. 7). 0 2 receptor supersensitivity to be specifically more affected in the anterior part of the striatum (9.0-8.2 rostral-caudal coordinates). There were no significant dif-

ferences between type 1 and type 2 grafted animals, at any coordinate. After grouping striatal coordinates (Fig. 8), however, it appeared that in the striatal region of the graft (8.6-8.2 rostral-caudal coordinates, in the medial part of the striatum) D 2 receptor density was significantly different between type 1 and type 2 animals, type 2 rats being less normalized than type 1 animals. In the regions anterior (9.0-8.8) and posterior (8.0-7.6) to the graft, the D z receptors in both types of grafted rats were not different from each other. In contrast to D 2 receptors, the D x receptor density was not significantly different in the lesioned and grafted striatum whatever the rostral-caudal coordinate or the striatal region studied (Fig. 9). Striatal D 1 receptor density in type 1 and type 2 grafted rats was not different either for grouped coordinates (Fig. 10). D 2 D A receptor values for the intact side of 6 - O H D A -

DM

DL

150

140

t 100 I

i

I

50 INTACT LESIONED & P<0.05 VS INTACT

420

120

B-II LESIONED ~ GRAFTED, TYPE 1 0-~ GRAFTED, TYPE 2

~ ÷ +

1°°1 I 8O

500" *

&.-r.~ & [

~

INTACT LESIONED P<0,01 VS INTACT

500

340 400 260

•

300

uJ 180

2OO

VL

VM m

~_

~: 150

° ~-

-~ ~-

&&

1301

oil Io

''°°1 I

u. 450

INTACT LESIONED && P(O.Ol vs INTACT

350

&_,_&

7G INTACT LESIONED

550

& & P<0.01 VS INTACT

450

250 350 150 7.4

718

8.2

GRAFT GRAFT 8.6 910 2507.4 718 8.2 8.6 CAUDAL-ROSTRAL COORDINATES (mm) *P<0.051"H" P<0.01 &***P<0.00t VS LESIONED +P<0.05 & ++ P<0.01 VS GRAFTED, TYPE 1

910

Fig. 7. Effect of a unilateral 6-OHDA lesion as well as striatal grafts on [3H]spiperone (1 nM) binding t o D 2 receptors in the right intact and left lesioned striatum. The left and fight striata were divided into 4 sections (DM, dorsomedial; DL, dorsolateral; V M , v e n t r a l ; VL, ventrolateral) and D 2 receptors were measured from 7.6-9.0 caudal-rostral coordinates. Differences in the left lesioned striatum were examined by a covariance analysis with two factors (groups and rats within groups) taking the right intact striatum as a covariate; By that means, we obtained for each coordinate for each rat an adjusted mean for the lesioned striatum (n = 2-15 slices). Results Oven are the adjusted mean values for each rat for each region at each coordinate, pooled together (n = 3-4 rats) to calculate an average density value -+ S.E.M. The Student's t-test was used to compare the intact and lesioned side values of lesioned rats only, as shown in the insets.

257 DL

DM 450"

"1-

S00

¸

T

"I#÷

4OO

300

3OO 2OO

150 ¸ I&l

100

cn (n

0 LL

o o :E -a O :8

VM

[] LESIONED [] []

450"

VL

GRAFTED,TYPE 1 GRAFTED,TYPE2

"1-

U.

1

._.................=~ *,÷+

300

150

°1

:57;$5_1=~.

:7;::55_1====~.

100

:7;TzTzS_lm=~_

0

9.0-848

8.6-8.2

6.0-7.6

9.0-8.8

8.6-8.2

6.0-7.6

ROSTRAL-CAUDAL COORDINATES (ram) * P<0.05,** P<0.01 & * * * P<0.001 VS LESIONED + P,c0.06 &++P<0.01 VS GRAFTED, TYPE 1

Fig. 8. [3H]spiperone (1 nM) binding to D 2 receptors in the striatum anterior (9.0-8.8), inside (8.6-8.2) and posterior (8.0-7.6) to the nigral graft, in unilateral 6-OHDA rats. The left and fight striata were divided into four sections (DM, dorsomedial; DL, dorsolateral; VM, ventromedial; VL, ventrolateral) and D 2 receptors were measured in grouped coordinates. Differences in the left lesioned striatum were examined by a covariance analysis with two factors (groups and rats within groups) taking the right intact striatum as a covariate. By that means, we obtained for each coordinate for each rat an adjusted mean for the lesioned striatum. The adjusted means of the individual coordinates were pooled in three regions and compared with a two factor analysis of variance ('group' was taken as a fixed factor and 'rats within group' as a random factor). Results given are the adjusted mean values for each rat pooled together (n = 3-4 rats) to calculate an average density value -+ S.E.M.

lesioned rats with or without grafts were 240.1, 251.5 and 237.8 fmol/mg of tissue for, respectively, 6-OHDA, type 1 and type 2 animals in the dorsomedial part of the striatum; 326.6, 316.2 and 327.9 fmol/mg of tissue in the dorsolateral part; 228.3, 253.3 and 258.8 fmoi/mg of tissue in the ventromedial part and, finally, 379.0, 366.9 and 364.1 fmol/mg of tissue in the ventrolateral part. D DA receptor values for the intact side of 6-OHDA, type 1 and type 2 rats were, respectively, 696.9, 739.9 and 736.4 fmol/mg of tissue in the dorsomedial part of the striatum; 727.5, 730.6 and 759.8 fmol/mg of tissue in the dorsolateral part; 684.7, 704.1 and 730.2 in the ventromedial part, and 798.4, 821.9 and 859.8 fmol/mg of tissue in the ventrolateral part. DISCUSSION

Behavioral recovery following fetal mesencephalic grafts in the denervated striatum As w e 42 and several other groups 12'x5'2° have previ-

ously observed, our results show that fetal mesencephalic graft of D A neurons switch the circling to amphetamine from ipsiversive to contraversive. In this study, grafted rats were divided into 2 groups (type 1 and type 2 animals) depending on the degree of contraversive circling following amphetamine administration. Type 1 animals switched completely from ipsiversive to contraversive circling, whereas in type 2 animals, the switch was only partial. Dunnett et al. ~7 also described different degrees of recovery and showed that metamphetamine rotation correlated highly with D A depletion in the striatum of lesioned rats as well as grafted ones. Others have shown that the degree of compensation of metamphetamine-induced rotation is related to the extent of fiber ingrowth into the denervated striatum from solid grafts 6'48 as well as suspension grafts of fetal mesencephalic neurons 17. These studies suggest that the behavioral recovery following grafts depends on the reinnervation of the host striatum by the graft as well as its spontaneous D A activity.

258

&& P-

DM

~ ~< 100 ~2°I

100

_

9o

ae

80

NTAC

LF.~IONED

850 J/ ~

I

~/ I~

"

i

o

~

~

;e 1050"

..,L:_S.?NED

7SO

~'O GRAFTED,TYPE 1 o'oGRAFTED,TYPE 2

650'

,

550

GRAFT

500

19o

a0

~

700' 600

DL

~

I

I INTACT LESIONED

~

GRAFT

~ 1,o

8(; INTACT ~ D ~. P,0.05 VS INTACT

~e 1100

650•

~

.

INTACT & PxO.05 VS INTACT

800 700 GRAFT

7.4

718

8.2

GRAFT 8.6

910

6007 ,

7:.

s.~

"'e.8

91o

C A U D A L - R O S T R A L C O O R D I N A T E S (ram) * P < 0 . 0 5 VS LESIONED

Fig. 9.Effect of a unilateral 6-OHDA lesion as well as striatal grafts on [3H]SCH 23390 (1 nM) binding to D~ receptors in the right intact and left lesioned stdatum. The left and fight striata were divided into 4 sections (DM, dorsomedial; DL, dorsolateral; VM, ventronaedial; VL, ventrolateral) and D z receptors were measured from 7.6-9.0 caudal-rostral coordinates. Differences in the left lesioned stfiatum were examined by a ex)variance analysis with two factors (groups and rats within groups) taking the right intact striatum as a covaria~. By that means, we obtained for each coordinate for each rat an adjusted mean for the lesioned striatum (n = 2-15 slices). Results given are the adjusted mean values for each rat for each region at each coordinate pooled together (n = 3--4 rats) to calculate an average density value -S.E.M. The Student's t-test was used to compare the intact and lesioned side values of lesioned rats, as shown in the insets.

Biochemical indices of activity of striatal grafts In the present study, D A , D O P A C and H V A levels were, respectively, 2%, 15% and 35% of the intact fight striata in the d e n e r v a t e d side o f 6 - O H D A rats. A f t e r nigral grafts in the d e n e r v a t e d stfiatum, levels of D A were r e s t o r e d to 13% and 10% of the intact striatum in, respectively, type 1 and type 2 animals. O u r results are in a g r e e m e n t with those of D u n n e t t et al. 17 as well as Schmidt et al. 47 who showed that nigral grafts in the d e n e r v a t e d striatum of 6 - O H D A rats brought D A concentrations to 7.2% and 13-18% o f n o r m a l levels, respectively. These increases m a y a p p e a r relatively small, but it should be emphasized that in this study, D A levels were m e a s u r e d in the total stfiatum. We have, in a previous study, used the microdialysis technique 42 and shown that, at least in the a r e a o f the graft, levels o f D A were n o r m a l i z e d in the grafted striatum as o b s e r v e d by Z e t t e r s t r 6 m et al. 55 (40% o f normal) and Strecker et al. 51 (normal values). Following grafts, levels of H V A

were unchanged whereas those of D O P A C increased in the d e n e r v a t e d striatum of type 1 and type 2 rats, respectively, c o m p a r e d to the lesioned side of 6 - O H D A rats. D O P A C / D A as well as H V A / D A were increased in the d e n e r v a t e d striatum of 6 - O H D A - l e s i o n e d rats with or without grafts, c o m p a r e d to their intact fight striatum. T h e turnover of D A , as estimated with the D O P A C / D A and H V A / D A ratios, was increased in the d e n e r v a t e d striatum of lesioned rats to c o m p e n s a t e for the loss of D A neurons. This increased turnover was still present in the total grafted striatum. H o w e v e r , our previous study 42, using microdialysis, showed that the turnover of D A inside the graft a p p e a r e d decreased. Levels of D A , D O P A C and H V A were not different in the grafted striatum of type 1 rats c o m p a r e d t o those of type 2.

Localization of striatal grafts in the denervated striatum O u r results showed that following grafts, T H immu-

259 0M

o 6oo

DL

1: !! I ii i

iiiiiiiiii

8...

400

400"

200

~

200

O

'

~

O i

tE

,2oo t ~- ~

1000

"01 li ]

22

600 400

'

9.0-8.8

8.6-8.2

0

8.0-7.6

,

9.0-8.8

8.6-8.2

8.0-7.6

ROSTRAL-CAUDAL COORDINATES (mm) Fig. 10. [3H]SCH 23390 (1 nM) binding to D 1 receptors in the striatum anterior (9.0-8.8), inside (8.6-8.2) and posterior (8.0--7.6) to the nigral graft, in unilateral 6-OHDA rats. The left and fight striata were divided into four sections (DM, dorsomedial; DL, dorsolateral; VM, ventromedial; VL, ventrolateral) and D 1 receptors were measured in grouped coordinates. Differences in the left lesioned stfiatum were examined by a covariance analysis with two factors (groups and rats within groups) taking the fight intact striatum as a covafiate. By that means, we obtained for each coordinate for each rat an adjusted mean for the lesioned striatum. The adjusted means of the individual coordinates were pooled in three regions and compared with a two factor analysis of variance ('group' was taken as a fixed factor and 'rats within group' as a random factor). Results given are the adjusted mean values for each rat pooled together (n = 3-4 rats) to calculate an average density value -+ S.E.M.

noreactivity increased similarly in grafted type 1 and type 2 animals compared to the denervated striatum of 6-OHDA-lesioned rats. Our mesencephalic grafts were located in the medial striatum. Indeed, in grafted type 1 animals, the graft was mostly located in the dorsomedial part of the denervated striatum. For type 2 animals, however, the graft was also in the medial striatum, but it overlapped the dorsal and ventral part of it, equally. The localization of the graft may be important for behavioral recovery. Indeed, Dunnett et al. 13 have demonstrated that the dorsomedial striatum was the most important site for transplant-induced reduction of druginduced turning in unilaterally lesioned adult rats. In agreement with Dunnett, Joyce et al. 28 have also demonstrated that injections of D A into the dorsal and medial part of the striatum in normal rats resulted in a significantly greater duration of contralateral deviation than injections into ventral regions. Indeed, the different regions of the striatum seem to be associated with specific behaviors 9'18. It is well known that the dorsal striatum receives primary afferent input from motor areas of n e o -

cortex 29"34, whereas the ventral striatum is innervated by limbic cortical regions 25. We have observed that type 1 animals showed a more robust response to amphetamine administration than rats of type 2. It may hardly be explained by the slightly different localization of the graft because in our previous study we have observed that the localization of the graft was not different between type 1 and type 2 animals 42. Our previous results 42 showed that for type 1 grafted rats, there were more D A neurons which survived in the denervated striatum than for type 2 animals. Moreover, the reinnervation was more widespread and its density was more like the intact side in type 1 animals compared to rats of type 2, By contrast to our previous s t u d y 42, w e were not able to estimate the amount of grafted cell bodies by T H immunocytochemistry in the striatum; the grafted cells were indistinguishable due to a blurred reaction under the conditions used. Indeed in this study to perform T H immunocytochemistry, the tissues were fixed from the frozen brain slices as prepared for the receptor assays. However, the main objective of this study was to mea-

260 sure D A receptor changes and the T H immunocytochemistry was performed as an additional information to allow us to localize striatal grafts. The quantification of the degree of staining by measuring optical density using an image analyser was possible and was used to compare the relative staining between the different groups. Thus, with the present results, we cannot establish whether the localization of the graft or the number of surviving neurons associated to a higher degree of reinnervation was responsible for the different behavioral recovery seen in type 1 and type 2 animals as suggested in our previous study 42. It is probable that both of these factors are important.

Correlation between behavioral recovery and changes in DA receptors As previously shown by US 41 and o t h e r groups 10'37"45' 46.49, D2 D A receptors became supersensitive following 6 - O H D A lesions. By contrast to the studies of Savasta et al. 45"46, D 2 supersensitivity was seen in the medial striatum as well as the lateral striatum. This difference may be explained by the fact that our rats were lesioned for at least 6 months and theirs were killed only one month after the lesion. As expected, D 2 receptor supersensitivity decreased in the grafted striatum of type 1 and type 2 animals compared to the lesioned side of 6 - O H D A rats, thus restoring towards normal levels of D 2 D A receptors. Our results are in agreement with those of Freed et al. 2°. D~ D A receptors were slightly increased in the denervated striatum after the lesion. Our results are in agreement with those of Buonamici et al. 8 who showed an increase of D x receptor density 3 months following the 6 - O H D A lesion. In humans, Raisman et al. 4° have also demonstrated an increase of D 1 receptors in the putamen of parkinsonian patients. However, Marshall et al. 33 showed a decrease of striatal D1 binding eleven months following 6 - O H D A injections in rats, whereas Savasta et al. 46 have noted no change in D~ receptor density 1 month following a 6 - O H D A lesion. Since the graft is not homogeneously distributed in the striatum, D A receptor changes can also be expected to be heterogenous. Indeed, D 2 receptor supersensitivity decreased significantly in the dorsomedial and ventromedial part of the denervated striatum following nigral grafts, while the lateral parts were unaffected. Moreover, D 2 receptors were more affected in the anterior part of the striatum, where the graft was placed. D 2 D A receptor changes can thus be associated to the effect of the graft, since they were mostly observed in regions where the graft was localized. D A released spontaneously by the graft may cause a down regulation of postsynaptic D 2 receptors. This normalization of D 2 re-

ceptors may explain the decrease of circling to apomor.phine and LY 171555 (D 2 agonist) following grafts 4L, Differences in D 2 D A receptor changes between type 1 and type 2 grafted animals were seen only when striatal rostral-caudal coordinates were grouped into 3 regions: anterior (9.0-8.8), graft area (8.6-8.2) and posterior to the graft (8.0-7.6). D 2 receptor density was higher in type 2 than in type 1 rats, only at 8.6-8.2 rostral-caudal coordinates, in the medial part of the striaturn. Thus, D 2 receptors were more normalized for type 1 than for type 2 animals, strictly at the site of the graft. Differences in D A levels unlikely explain completely the greater normalization of D A receptors in type 1 rats. The extent of reinnervation by the graft is probably an important factor and it may also explain why type 1 animals had a greater overcompensation of circling to amphetamine than type 2 rats, despite the fact that DA receptor levels were higher in type 2 animals. The efficiency of the graft may depend on the integration of grafted neurons in the host striatum. In addition, our mesencephalic grafts do not only contain D A neurons, but also noradrenergic and serotoninergic neurons which are also affected by amphetamine. Thus the stimulation of these neurons may also affect the circling to amphetamine. D 1 receptor changes were small following the 6 - O H D A lesion compared to D 2 receptors, thus the normalization of these small D 1 receptor variations following grafts were difficult to measure. There was a normalization of D 1 receptor density following grafts from the increase due to the lesion. This normalization may explain why the circling to CY 208243 was diminished in grafted rats as compared to lesioned animals 41. In addition, the graft may affect D 1 receptors in other regions implicated in motor behaviors such as the entopeduncular nucleus or the substantia nigra pars reticulata. The extent of denervation supersensitivity after 6 - O H D A is different for D 1 compared to D 2 receptors in the rat striatum. The localization of DA receptors in different compartments of the striatum may explain variations in the regulation of these two types of D A receptors. In the striatum of carnivores and primates, D~ receptors are mostly located in the striosomes 36 whereas D e subtype as well as D A uptake sites are localized, to a greater extent, in the matrix a6'aT. On the other hand, this compartmental distribution is not as clear in the adult rat striatum in spite of the fact that amphetamine treatment can induce patchy anatomical distribution of transmitter-related compounds such as TH-immunoreactive 44 and dynorphin-immunoreactive patches 32. The 6 - O H D A lesion may produce more intense degeneration of fibers in the matrix than in the striosome as it is the case for N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine

261 (MPTP) 53"54 in dogs. Thus D t receptors would be less affected by the 6-OHDA-lesion than D 2 receptors. A n alternative explanation may be the increased turnover of D 1 receptors. Indeed, the half-life of D 1 receptors is about 34 h 24 compared to 94.2 h for D 2 receptors 39. D~ receptors may therefore adapt more rapidly to variation in D A neurotransmission than D 2 receptors without increasing their density. A n o t h e r variable to be considered in the adaption of D 1 and D 2 receptors to changes in neurotransmission is the pool of D~ receptors compared to D 2 receptors. Battaglia et al.3 have shown that the recovery of D t D A receptors is slower than D 1 receptor-mediated enzyme activity (adenylate cyclase) in the striatum, following N-ethoxycarbonyl-2-ethoxy- 1,2-dihydroquinoline ( E E D Q ) . Thus, not all D~ receptors are linked to the adenylate cyclase system and only the stimulation of a small proportion of the population may be necessary to bring a behavioral effect. In contrast, changes in the number of D 2 receptors correspond well with changes in apomorphine-induced stereotyped behavior 23. Meller et al. 35 have shown that there was a larger receptor reserve at pre- versus postsynaptic D 2 D A receptors and suggested that the up-regulation of postsynaptic D 2 receptors by 6 - O H D A lesion or chronic neuroleptic treatment may create a receptor reserve where none had existed before. The creation of this reserve may explain, in part, why partial D A agonists, ineffective at normosensitive receptors, can elicit substantial responses at supersensitive

REFERENCES 1 Arbuthnott, G., Dunnett, S.B. and MacLoed, N., Electrophysiological recording from nigral transplants in the rat, Neurosci. Lett., 47 (1985) 205-210. 2 Arluison, M., Dietl, M. and Thibault, J., Ultrastruetural morphology of dopaminergic nerve terminals and synapses in the striatum of the rat using tyrosine hydroxylase immunocytochemistry: a topographical study, Brain Research, 13 (1984) 269-285. 3 Battaglia, G., Norman, A.B., Hess, E.J. and Creese, I., Functional recovery of D-1 dopamine receptor-mediated stimulation of rat striatal adenylate cyclase activity following irreversible receptor modification by N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ): evidence for spare receptors, Neurosci. Lea., 69 (1986) 290-295. 4 Becker, J.B., Robinson, T.E., Barton, P., Sintov, A., Siden, R. and Levy, R.J., Sustained behavioral recovery from unilateral nigrostriatai damage produced by the controlled release of dopamine from a silicone polymer pellet placed into the denervated striatum, Brain Research, 508 (1990) 60-64. 5 Bj6rklund, A. and Stenevi, U., Intracerebral neural implants: neuronal replacement and reconstruction of damaged circuitries, Annu. Rev. Neurosci., 7 (1984) 279-308. 6 Bj6rklund, A., Dunnett, S.B., Stenevi, U., Lewis, M.E. and Iversen, S.D., Reinnervation of the denervated striatum by substantia nigra transplants: functional consequences as revealed by pharmacological and sensorimotor testing, Brain Research, 199 (1980) 307-333. 7 BjOrklund, A., Stenevi, U., Schmidt, R.H., Dunnett, S.B. and

sites. It is possible that D 1 supersensitivity was small compared to D 2 receptors because a large and sufficient amount of D 1 receptors is already available in the intact rat. In summary, the experiments reported here show that our grafts produced D A spontaneously. Indeed, D A levels were restored to 13% and 10% of the intact side in, respectively, type 1 and type 2 animals. In type 1 rats, the graft was strictly in the dorsomedial part of the grafted striatum whereas in type 2 animals, the graft overlapped the dorsomedial and ventromedial regions. Supersensitive D 2 receptors decreased following grafts and these changes are correlated with the localization of the graft (in the medial striatum). Normalization of D 2 receptors may explain the decrease of behavioral supersensitivity following administration of amphetamine. D receptors were less affected by the lesion than D 2 receptors in the denervated striatum. D1 receptors as D 2 receptors were normalized by the graft. In addition, D A receptor changes may occur in other structures related to motor behavior. Variations seen in the regulation of D 1 and D 2 receptors may be due to differences in localization, turnover or pool of these two D A receptors.

Acknowledgements. This research was supported by the Medical Research Council of Canada and the Parkinson Foundation of Canada. We wish to thank M. Michel Daigle and M. Laurent Gr6goire for technical assistance, and Mme Diane Leroux for help in the statistical analysis.

Gage, EH., Intracerebral grafting of neuronal cell suspensions. II. Survival and growth of nigral cell suspensions, Acta Physiol. Scand. Suppl. 522 (1983) 9-18. 8 Buonamici, M., Caccia, M., Carpentieri, M., Pegrassi, L., Rossi, A.C. and Di Chiara, G., D-1 receptor supersensitivity in the rat striatum after unilateral 6-hydroxydopamine lesions, Eur. J. Pharmacol., 126 (1986) 347-348. 9 Cameron, D.L. and Crocker, A.D., Localization of striatal dopamine receptor function by central injection of an irreversible receptor antagonist, Neuroscience, 32(3) (1989) 769-778. 10 Creese, I., Burt, D.R. and Snyder, S.H., Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity, Science, 197 (1977) 596-598. 11 Di Paolo, T., B6dard, P.J., Daigle, M. and Boucher, R., Longterm effect of MPTP on central and peripheral catecholamine and indolamine concentrations in monkeys, Brain Research, 379 (1986) 286-293. 12 Dunnett, S.B., Bj6rklund, A., Schmidt, R.H., Stenevi, U. and Iversen, S.D., Behavioral recovery in rats with unilateral 6-OHDA lesions following implantation of nigral cell suspensions, Acta Physiol. Scand., Suppl. 522 (1983a) 29-38. 13 Dunnett, S.B., BjOrldund, A., Schmidt, R.H., Stenevi, U. and Iversen, S.D., Intracerebral grafting of neuronal cell suspensions. IV. Behavioral recovery in rats with unilateral 6-OHDA lesions following implantation of nigral cell suspensions in different brain sites, Acta Physiol. Scand., Suppl. 522 (1983b) 2937. 14 Dunnett, S.B., BjOrldund, A., Schmidt, R.H., Stenevi, U. and Iversen, S.D., Intracerebral grafting of neuronal cell suspen-

262 sions. V. Behavioral recovery in rats with bilateral 6-OHDA lesions following implantation of nigral cell suspensions, Acta Physiol. Scand., Suppl. 522 (1983c) 39-47. 15 Dunnett, S.B., Bj6rklund, A., Stenevi, U. and Iversen, S.D., Behavioral recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal pathway. I. Unilateral lesions, Brain Research, 215 (1981) 147161. 16 Dunnett, S.B., Ryan, C.N., Levin, P.D. Reynoid, M. and Bunch, S.T., Functional consequences of embryonic neocortex transplanted to rats with prefrontal cortex lesions, Behav. Neurosci., 101 (1987) 489-503, 17 Dunnett, S.B., Hernandez, T.D., Summerfield, A., Jones, G.H. and Arbuthnott, G., Graft-derived recovery from 6-hydroxydopamine lesions: specificity of ventral mesencephalic graft tissues, Exp. Brain Res., 71 (1988) 411-424. 18 Fletcher, G.H. and Starr, M.S., Topography of dopamine behaviors mediated by D-1 and D-2 receptors revealed by intrastriatal injection of SKF 38393, lisuride and apomorphine in rats with a unilateral 6-hydroxydopamine-induced lesion, Neuroscience, 20 (1987) 589-597. 19 Forni, C., Brundin, P., Strecker, R.E., E1 Ganouni, S., BjSrklund, A. and Nieoullon, A., Time course of recovery of dopamine neuron activity during reinnervation of the denervated striatum by fetal mesencephalic grafts as assessed by in vivo voltammetry, Exp. Brain Res., 76 (1989) 75-87. 20 Freed, W.J., Ko, G.N., Neihoff, D.L., Kuhar, M.J., Hoffer, B.J., Olson, L., Cannon-Spoor, H.E., Morihisa, J.M. and Wyatt, R.J., Normalization of spiroperidol binding in the denervated rat striatum by homologous grafts of substantia nigra, Science, 222 (1983) 937-939. 21 Freed, W.J., Morihisa, J.M., Spoor, E., Holler, B.J., Oison, L., Seiger, A. and Wyatt, R.J., Transplanted adrenal chromaffin cells in rat brain reduce lesion-induced rotational behaviour, Science, 292 (1981) 351-352. 22 Freed, W.J., Perlow, M.J., Karoum, F., Sieger, A., Olson, L., Hoffer, B.J. and Wyatt, R.J., Restoration of dopaminergic function by grafting of fetal rat substantia nigra to the candate nucleus: long-term behavioral, biochemical and histochemical studies, Ann. Neurol., 8 (1980) 510-519. 23 Fukuchi, I., Fujita, N., Nakahiro, M., Saito, K. and Yoshida, H., D-2 dopamine receptor synthesis and turnover in rat striatum, Eur. J. Pharmacol., 127 (1986) 291-294. 24 Fuxe, K., Agnati, L.E, Pich, E.M., Meller, E. and Goldstein, M., Evidence for a fast receptor turnover of D-1 receptors in various forebrain regions of the rat, Neurosci. Lett., 81 (1987) 183-187. 25 Fuxe, K., Agnati, L.E, Kalia, M., Goldstein, M., Andersson, K. and H~-£strand, A., Dopaminergic systems in the brain and pituitary. In E. Fliickiger, E.E. Miiller and M.O. Thomer (Eds.), The Dopaminergic System, Springer, Berlin, 1985, pp, 11-25. 26 Graybiel, A.M. and Moratalla, R., Dopamine uptake sites in the striatum are distributed differentially in striosomes and matrix compartments, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 9020-9024. 27 Joyce, J.N., Sapp, D.W. and Marshall, J.F., Human striatal dopamine receptors are organized in compartments, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8002-8006. 28 Joyce, J.N., Davis, R.E. and Hartesveldt, C.V., Behavioral effects of unilateral dopamine injection into dorsal or ventral striatum, Eur. J. Pharmacol., 72 (1981) 1-10. 29 Kelley, A.E., Domesick, V.B. and Nauta, W.J.H., The amygdaio-striatal projection in the rat: an anatomical study by anterograde and retrograde tracing methods, Neuroscience, 7 (1982) 615-630. 30 Kesslak, J.P., Brown, L., Steichen, C. and Cotman, C.W., Adult and embryonic frontal cortex transplants after frontal cortex ablation enhance recovery on a reinforced alternation task, Exp. NeuroL, 94 (1986) 615-626. 31 Kramer, C.Y., Extension of multiple-range tests to group means

with unequal number of replications, Biometrics. 12 (1956) 307310. 32 Li, S., Sivam, S.P., McGinty, J.E, Jiang, H.K., Douglas, J., Calavetta, L. and Hong, J.S., Regulation of the metabolism of striatal dynorphin by the dopaminergic system, J. Pharmacol. Exp. Ther., 246 (1988) 403-408. 33 Marshall, J.E, Navarette, R. and Joyce, J.N., Decreased striatal D-1 binding density following mesotelencephalic 6-OHDA injections: an autoradiographic analysis, Brain Res.. 493 (1989) 247-257. 34 McGeorge, A.J. and Faull, L.M., The organisation of the projection from the cerebral cortex to the striatum in the rat, Neuroscience, 29(3) (1989) 503-537. 35 Meiler, E., Bohmaker, K., Namba, Y., Friedhoff, A.J. and Goldstein, M., Relationship between receptor occupancy and response at striatal dopamine autoreceptors, Mol. Pharmacol., 31 (1987) 592-598. 36 Murrin, L.C. and Zeng, W., Dopamine D-1 receptor development in the rat striatum: early localization in striosomes, Brain Research, 480 (1989) 170-177. 37 Neve, K.A., Altar, C.A., Wong, C.A. and Marshall, J.E, Quantitative analysis of 3H-spiroperidol binding to rat forebraln sections: plasticity of neostriatal dopamine receptors after nigrostriatal injury, Brain Research, 302 (1984) 9-18. 38 Pellegrino, J.J., Peilegfino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, 2nd edn., Plenum, New York, 1979. 39 Pich, E.M., Benfenati, F., Farabegoli, C., Fuxe, K., Meller, E., Aronsson, M., Goldstein, M. and Agnati, L.E, Chronic haloperidol affects striatal D-2 dopamine receptor reappearance after irreversible receptor blockade, Brain Research, 435 (1987) 147-152. 40 Raisman, R., Cash, R., Muberg, M., Javoy-Agid, F. and Agid, Y., Binding of [3H]-SCH 23390 to D-1 receptors in the putamen of control and parkinsonian subjects, Eur. J. Pharmacol., 113 (1985) 467-468. 41 Rioux, L., Gaudin, D., B6dard, P.J., Gr6goire, L., Gagnon, C. and Di Paolo, T., Decrease of lesion-induced dopaminergic supersensitivity in the rat by fetal nigral grafts, Soc. Neurosci. Abstr., 15 (1989) 1355. 42 Rioux, L., B6dard, P.J., Gr6goire, L., Di Paolo, T., Daigle, M. and Rouillard, C., Apparent supersensitivity to amphetamine of grafted foetal nigral neurons in the rat, Soc. Neurosci. Abstr., 14 (1988) 734. 43 Rose, G., Gerhardt, G., StrSmberg, I., Olson, L. and Hoffer, B., Monoamine release from dopamine-depleted rat caudate nucleus reinnervated by substantia nigra transplants: an in vivo electrochemical study, Brain Research, 341 (1985) 92-100. 44 Ryan, L.J., Martone, M.E., Linder, J.C. and Groves, P.M., Continuous amphetamine administration induces tyrosine hydroxylase immunoreactive patches in the adult rat neostriatum, Brain Res. Bull., 21 (1988) 133-137. 45 Savasta, M., Dubois, A., Feurstein, C., Manier, M. and Scatton, B., Denervation supersensitivity of striatal D-2 dopamine receptors is restricted to the ventro- and dorsolateral regions of the striatum, Neurosci. Lett., 74 (1987) 180-186. 46 Savasta, M., Dubois, A., Benavides, J. and Scatton, B., Different plasticity changes in D-1 and D-2 receptors in rat striatal subregions following impairment of dopaminergic transmission, Neurosci. Lett., 85 (1988) 119-124. 47 Schmidt, R.H., Bjttrklund, A., Stenevi, U., Dunnett, S.B. and Gage, EH., Intracerebral grafting of neuronal cell suspensions. III. Activity of intrastriatal nigral suspension implants as assessed by measurements of dopamine synthesis and metabolism, Acta Physiol. Scand., Supp. 522 (1983) 19-28; 48 Schmidt, R.H., lngvar, M., Lindvall, O., Stenevi, U. and Bj6rklund, A., Functional activity of substantia nigra grafts reinnervating the striatum: neurotransmitter metabolism and (lac)-2-deoxy-d-glucose autoradiography, J. Neurochem., 38 (1982) 737-748. 49 Staunton, D.A., Wolfe, B.B., Groves, P.M. and Molinoff, P.B.,

263 Dopamine receptor changes following destruction of the nigrostriatai pathway: lack of relationship to rotational behavior, Brain. Research, 211 (1981) 315-327. 50 Sternberger, L.A., Hardy, L.A. Jr., Cueulis, J. and Meyer, H.G., The unlabelled antibody enzyme method of immunocytochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase anti horseradish peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem., 18 (1970) 315-333. 51 Strecker, R.E., Sharp, T., Brundin, P., Zetterstr6m, T., Ungerstedt, U. and Bj6rklund, A., Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intraeerebral dialysis, Neuroscience, 22 (1987) 169-178. 52 Thibault, J., Vidal, D. and Gros, F., In vitro translation of mRNA from rat pheochromocytoma tumors, characterization of

tyrosine hydroxylase, Biochem. Biophys. Res. Commun., 99(3) (1981) 96-98. 53 Turner, B.H., Wilson, J.S., Me Kenzie, J.C. and Richtand, N., MPTP produces a pattern of nigrostriatal degeneration which coincides with the mosaic organization of the caudate nucleus, Brain Research, 473 (1988) 60-64. 54 Wilson, J.S., Turner, B.H., Morrow, G.D. and Hartman, P.J., MPTP produces a mosaic-like pattern of terminal degeneration in the caudate nucleus of dog, Brain Research, 423 (1987) 329332. 55 Zetterstr6m, T., Brundin, P., Gage, F.H., Sharp, T., Isacson, O., Dunnett, S.B., Ungerstedt, U. and Bj6rklund, A., In vivo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracerebral dialysis, Brain Research, 362 (1986) 344-349.