Functional recovery of supersensitive dopamine receptors after intrastriatal grafts of fetal substantia nigra

EXPERIMENTAL

NEUROLOGY

111,

282-292

(1991)

Functional Recovery of Supersensitive Dopamine Receptors after lntrastriatal Grafts of Fetal Substantia Nigra TED M. DAWSON,*~’

VALINA

L. DAWSON,*~~*’

FRED H. GAGE,$

LISA J. FISHER,$

MARY A. HUNT,?+ AND JAMES K. WAMsLEYt’§*3 TDepartment of Pharmacology and Toxicology and the Western Institute of Neuropsychiatry, University of Utah Health Sciences Center, Lake City, Utah 84112; §Neuropsychiatric Research Institute, 700 First Avenue South, Fargo, North Dakota 58103; *Department Neurosciences, School of Medicine, University of California, San Diego, La Jolla, California 92037; and *Department of Neurology, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104

receptor plasticity may play a role in the functional recovery of substantia nigra transplanted animals and graft viability seems to be a prerequisite for behavioral recovery as well as receptor normalization. 0 1991

Interruption of the ascending dopamine neurons of the nigrostriatal pathway, by 6-hydroxydopamine (6OHDA) lesion in rats, produced a significant loss of the dopamine transport complexes labeled with the phencyclidine derivative [‘HIBTCP. This loss of dopamine innervation in the striatum was present at least 12 to 14 months after lesioning and was functionally manifested by ipsilateral rotation of the animals in response to amphetamine. In these same animals, in comparison to controls, there was a significant increase in the number (II,,) of [‘H]SCH 23390~labeled D-l receptors in the striatum (36.7%) and the substantia nigra (35.1%) and a 54.4% increase in the number (II,,) of [SH]sulpiridelabeled striatal D-2 receptors without an apparent change in affinity (&). Ten to twelve months after the transplantation of homologous fetal substantia nigra into the denervated striatum, there was a significant decrease in amphetamine-induced turning behavior. In these animals, there was an ingrowth of dopamine nerve terminals in the striatum as demonstrated by a return of tSH]BTCP binding. Accompanying this reinnervation was the normalization of D-l and D-2 receptors to control values in the striatum as well as the return of D-l receptors to prelesion densities in the substantia nigra. In a subgroup of transplanted rats, amphetamine continued to induce ipsilateral turning. In these animals both D-l and D-2 receptors remained supersensitive. These results support the hypothesis that the functional recovery of transplanted animals is due, in part, to reinnervation of the striatum. In addition, long-term alterations in receptor density may be related to the behavioral deficits that are associated with the 6-OHDA-lesioned rat. Furthermore, dopamine

Academic

Inc.

Fetal mesencephalic transplants which contain substantia nigra appear to reverse many of the behavioral dysfunctions observed in the 6-hydroxydopamine (6OHDA) rat model of Parkinson’s disease (PD) (4,524, 43). Although the mechanisms involved in such improvements are unclear, it is known that the grafts survive and provide functional reinnervation of the host by means of an outgrowth of fibers and the formation of synaptic arborizations (25,36,59). Dopamine (DA) neuronal grafts also actively secrete DA which has been shown to be under receptor-mediated autoregulatory control (52,67). In addition, there is an active DA-reuptake system that helps to maintain the control of synaptic levels of DA (58). This active but regulated release of DA has the potential to exert a functional effect on denervated supersensitive striatal DA receptors. As transplanted animals recover from their functional deficits, DA receptor densities and the DA-transport complex or DA-uptake site (DA-TC) densities should correlate with behavioral changes. We examined this possibility with semiquantitative autoradiographic techniques by measuring the densities of the subtypes of DA receptors (12, 32, 60, 66) and DA-TC sites with highly selective ligands in the caudate-putamen (CPU) and substantia nigra (SN) of the 6-OHDA-denervated rat with or without fetal mesencephalic transplants. MATERIALS

AND

METHODS

Lesions. DA cell bodies and ascending DA neurons of the nigrostriatal pathway were destroyed by 6-OHDA injection into the left medial forebrain bundle (MFB). 282

Inc. reserved.

Press,

INTRODUCTION

i Present address: Department of Neuroscience, WBSB 807, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. ’ Present address: NIDA/Addiction Research Center, P.O. Box 5180, Baltimore, MD 21224. ’ To whom reprint requests should be addressed at Neuropsychiatric Research Institute, 120 Eight Street South, Fargo, ND 58103.

0014-4886191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

Salt of

DOPAMINE

RECEPTORS

Female Sprague-Dawley rats (180-220 g) were anesthetized with a mixture of ketamine, Rompum, and acepromazine and then received a unilateral injection of 8 pg of 6-OHDA in the MFB (coordinates: AP = 4.4 mm from bregma, L = 1.1 mm, and V = -7.5 mm below dura). The 6-OHDA was dissolved in 2 ~1 of normal saline supplemented with 0.2 mg/ml ascorbic acid to retard oxidation. The animals were not pretreated with desipramine. The solution was injected at the rate of 1 pl/min. The needle was raised 2 mm and left in place for 2 min to allow for diffusion of 6-OHDA. In order to evaluate the completeness of the lesion, the animals were tested 7 to 10 days after the 6-OHDA lesion with a subcutaneous injection of amphetamine (5 mg/kg) (61, 62). Only animals exhibiting at least seven ipsilateral turns per minute over 60 min were used in subsequent experiments. Rotational testing with direct dopamine agonists was not performed. Transplantation. Eight of the successfully denervated rats were then grafted, 5 to 10 weeks following the 6-OHDA lesion, with substantia nigra cell suspensions prepared from 13- to 15-day gestational age rats. Rats were lesioned at different times and transplantation occurred when enough lesioned rats demonstrated greater than seven ipsilateral turns per minute to subcutaneous amphetamine (5 to 10 weeks after any specific lesion). The cells were placed into two different sites within the striatum. Nigral cell suspensions were prepared as described elsewhere (6). Rats previously lesioned with 6OHDA were anesthetized and placed in a stereotaxic apparatus. A 4-gl volume of suspension was injected into two different sites (approximately two fetal rats were used per host) within the denervated striatum (coordinates: AP = 0.7 mm from bregma, L = 2 mm, V = 4 mm and 5 mm below dura; AP = 1.5 mm from bregma, L = 3 mm, V = 4 and 5 mm below dura). At each site, the suspension was delivered in two separate 2-111deposits (1 jd/min) separated by 1 mm along the needle tract. The syringe was then raised 2 mm and left in place an additional 2 min to allow for diffusion of the cells. The needle was slowly removed and the wound sutured. Recovery was uneventful. Amphetamine-induced rotation was tested in the grafted animals approximately once every 2 months for a lo- to 12-month period following transplantation. The remaining 6-OHDA-lesioned rats did not receive a nigral cell suspension. A third group of rats, which did not receive any transplant, but were treated two to three times with subcutaneous amphetamine, was used as normal controls. Autoradiography. Ten to twelve months after transplantation, the animals were prepared for examination of [3H]SCH 23390 binding to D-l receptors (11, 13), [3H]sulpiride binding to D-2 receptors (26, 27), and [3H]BTCP binding to the DA-TC sites (19). One-half of the brains were cut coronally and the other brains were

AND TRANSPLANTATION

283

cut sagittally. D-l receptors were labeled by incubating slide-mounted tissue sections (10 pm) in a 50 mM TrisHCl buffer, pH 7.4, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl,, 1 mM MgCl,, and 1 nM [3H]SCH 23390 (60.4-66.0 Ci/mmol; DuPont-NEN, Boston, MA) for 30 min at 25°C. This was followed by 2 X 5 min rinses. Nonspecific binding was defined by the addition of 10 pm piflutixol. D-2 receptors were labeled with [3H]sulpiride by incubating tissue sections in a 0.17 M Tris-HCl buffer, pH 7.7 (120 mM NaCl, 5 mM KCl, 2 mM CaCl,, 1 mM MgCl,, 0.001% ascorbate), and 20 nM [3H]sulpiride (67.9-78.6 Ci/mmol; DuPont-NEN) for 20 min at 25°C. This was followed by 4 X 1 min rinses. Nonspecific binding was defined by 1 pm haloperidol. The DA-TC was labeled with [3H]BTCP by preincubating tissue sections in 50 mM Tris-HCl buffer, pH 7.0, containing 120 mM NaCl for 20 min at 4°C. This was followed by incubation in the same buffer for 1 h at 4°C with 10 nM [3H]BTCP (50.8 Ci/mmol; Research Products Int., Mount Prospect, IL). Nonspecific binding was defined by the addition of 1 pm GBR 12909 (Gist-Brocades, Delft, The Netherlands). Sections were then rinsed (4~ 5 min). Radiolabeled tissue sections were placed in X-ray cassettes with appropriate standards and juxtaposed to tritium-sensitive film for 2 to 10 weeks. The films were then developed and analyzed by computer-assisted microdensitometry (63). The data were quantitated by an IBM-based computer-assisted microdensitometer using Drexel’s XENIX-based image analysis system (DUMAS) (Drexel University, Philadelphia, PA) or a Leitz Orthoplan microscope (FRG) interfaced with a DADS-560 computerized analysis system (Stahl Research Laboratories, Rochester, NY), with reference to polymer-based tritium standards (Amersham, Arlington Heights, IL) and brain-mash standards (63). The CPU in the region adjacent to the graft and the SN was examined in all animals. The graft itself was not included in the quantitation. D-l and D-2 receptors were quantitated only in areas where [3H]BTCP binding was observed in transplanted animals in serial sections. Saturation studies were performed by incubating anatomically adjacent tissue sections with varying concentrations of [3H]SCH 23390 (0.25 to 4 nM) and [3H]sulpiride (15 to 40 nM). Scatchard analysis of saturation data was performed by using a weighted, modified version of the simplex algorithm (Ligand Binding Analysis Software, E.M.F. Software, Baltimore, MD) to fit the experimental binding data to a one-site model. Histology. Using the method of Koelle (34) with Naik (42) modification, acetylcholinesterase staining was performed. Ethapropazine was used as an inhibitor of nonspecific cholinesterase and sodium sulfide was used for visualization. Statistics. Overall significant F (Fisher’s ratio) values were determined and analyzed further via the

284

DAWSON NONCOMPENSATED GRAFTS

&d O I:% w

z

-2

-4 k< i$ f f -6 5u -8

COMPENSATED GRAFTS

FIG. 1. Rotation induced by subcutaneous administration of damphetamine (5 mg/kg) is shown prior to and subsequent to transplantation (10 to 12 months) of fetal substantia nigra (see text). Rotational behavior was conducted in automated rotameter bowls (5657) for a period of 60 min following administration of d-amphetamine. Rats were tested 7-10 days after 6-OHDA lesion and then approximately once every 2 months following transplantation. *P < 0.05, Student’s two-tailed t test for independent means.

Spjstvoll and Stoline modification of the Tukey HSD (honestly significant difference) for the purpose of clarifying the relationship between all possible pairs of lesion, compensated, noncompensated, and control groups. The Spjatvoll-Stoline modification was deemed the appropriate specific comparator because it allows pairwise comparison between means of groups with unequal sample size (33, 56). RESULTS

Two separate groups of transplanted animals could be identified based on their response to a subcutaneous injection of amphetamine (Fig. 1). Compensated rats were those that either ceased rotating or rotated solely in the contralateral direction. Noncompensated rats were those that continued to rotate in the ipsilateral direction at or above pretransplantation levels. In the 6-OHDA-lesioned animals which exhibited marked ipsilateral turning to subcutaneous amphetamine administration, the presynaptic DA-TC sites labeled with [3H]BTCP in the CPU were reduced by greater than 60% compared to controls (Figs. 2A and 3B). We found a significant increase of D-2 receptor density (57.7%) in the striatum ipsilateral to the 6OHDA lesion (Figs. 2C and 3H) and we observed a significant increase of D-l receptors (26.2%) in the CPU (Figs. 2B and 3E) and in the substantia nigra reticulata (SNR) (44.6%) (Fig. 2B). In the compensated animals, grafts could be identi-

ET

AL.

fied in the center of the striatum (Figs. 4 and 5). The graft and striatal regions adjoining the graft had a dense amount of [3H]BTCP binding (DA-TC sites) (Figs. 3C and 5B). In the majority of the animals the binding was confined to the immediate vicinity of the graft; however, in two animals [3H]BTCP binding extended beyond the vicinity of the graft as shown in Figs. 3C and 5B. Both striatal D-l (Figs. 2B and 3D) and D-2 (Figs. 2C and 31) receptor densities were returned to control values. [3H]SCH 23390 and [3H]sulpiride binding were not above background levels in the graft (Figs. 3F and 31). D-l receptors in the SNR were also returned to control values (Fig. 2B). Grafts could not be found in the noncompensated animals and the DA-TC sites and D-l and D-2 receptor densities (Figs. 2B and 2C) were similar to those of the nongrafted denervated rat. Further analysis of the D-l and D-2 receptor changes with saturation experiments and Scatchard analysis revealed that the observed alterations in both D-l and D-2 receptors were due to a change in receptor number (B,,) and not affinity (I&) (See Fig. 6 and Table 1). DISCUSSION

6-OHDA lesion of the nigrostriatal pathway was shown to cause a significant reduction in the DA-TC sites labeled with [3H]BTCP, and this reduction persisted throughout the length of the study. [3H]BTCP has been previously shown to label dopamine uptake sites (or dopamine transport complexes) with high affinity, as such it can be used as a marker of dopaminergic cell bodies and terminals (19). In these same animals, which had a successful ipsilateral lesion of the nigrostriatal dopaminergic pathway, there was a profound ipsilateral rotational response to administration of subcutaneous amphetamine. The induced rotational response can be used to monitor the extent of dopaminergic denervation. The amount of DA depletion has been shown to be highly correlated with the number of turns per minute (17) and it has previously been shown that if animals rotate greater than seven times per minute over 60 min, as in this study, there is greater than a 95% reduction of nigrostriatal DA (53). Thus, the 6OHDA-lesioned animals in this investigation had a marked reduction in nigrostriatal DA as demonstrated by the rotational response to amphetamine. Similar lesions have also been shown to cause a nearly complete loss of DA and its metabolites in the striatum (62,67). In contrast, the DA-TC sites were only reduced by 50 to 70%. Similar discrepancies between the loss of DA and DA-TC sites have been noted in Parkinson’s disease (PD) [see Seeman and Niznik (55)]. The discrepancy in the 95% reduction of DA content in the striatum and the 50 to 70% reduction in DA-TC sites has recently been resolved in PD [see Seeman and Niznik (55)]. This discrepancy may be due to 13H]BTCP

DOPAMINE

A

DOPAMINE

CONT

LESION

DOPAMINE 150

UPTAKE

SITES

COMP

D-l

RECEPTORS

NCOMP

RECEPTORS

, tt

+

:z 25

CONT

DOPAMINE

LESION

COMP

NCOMP

D-2 RECEPTORS

20.0 17.5 15.0 12.5 10.0 7.5 5.0 2.5 0.0 CONT

LESION

COMP

NCOMP

FIG. 2. Effects of 6-OHDA MFB lesion and fetal substantia nigra transplants on (A) 13H]BTCP labeling of the DA-TC sites, (B) [3H]SCH 23390 labeling of D-1 receptors, and (C) [3H]sulpiride labeling of D-2 receptors. The various treatment groups and binding conditions are as described in the text. Values represent the means f SEM. All animals were examined 10 to 1‘2 months post-transplantation or approximately 12 to 14 months post-6-OHDA lesion. *For DA-TC sites overall significant Fisher’s ratio was F(3,13) = 59.9, P < 0.01. The Spjetvoll-Stoline modified Tukey HSD (31, 51) clarifies these findings as follows: Controls (CONT), n = 5, were significantly different from the 6-OHDA (LESION), n = 4, compensated (COMP), n = 5, and noncompensated (NCOMP), n = 3, animals. The 6-OHDA animals were significantly different from the compensated animals and not significantly different from the noncompensated animals. The compensated animals were significantly different from the noncompensated animals. **For striatal D-l receptors, F(3,13) = 12.3, P < 0.01. The Spjetvoll-Stoline modified Tukey HSD (31,51) clarifies these findings as follows: Controls, n = 5, were significantly different (P < 0.05) from 6-OHDA, n = 4, and noncompensated, n = 3, animals and were not significantly different from compensated, n = 5, animals. The 6-OHDA group was significantly different (P c 0.05) from

AND

TRANSPLANTATION

285

binding to additional nondescript sites unrelated to the DA-TC sites (8,64). It has been postulated that denervation of the dopaminergic nigrostriatal pathway causes postsynaptic DA receptors to become supersensitive (2, 9, 23, 38, 47, 61, 62). Numerous studies have demonstrated postsynaptic D-2 receptor supersensitivity after 6-OHDA-induced DA denervation [see Hall et al. (28) for review]. Recent autoradiographic studies have shown that this upregulation is confined to the lateral sector of the caudateputamen (51) where D-2 receptors are concentrated and localized to cholinergic interneurons (12) and cortical glutaminergic nerve fibers (l&20). The supersensitivity of D-2 receptors, observed in this study, was shown to be due to a change in total receptor number (B,,,) and not affinity (&) and is in agreement with those studies previously cited. We were also able to show that the upregulation persisted for at least 12 to 14 months following 6-OHDA lesion. In contrast, no significant alteration in striatal D-l receptors, after lesion of the MFB or SN with 6-OHDA, has been observed by binding studies accomplished using a single concentration of [3H]SCH 23390 (l&21,51). D-l receptors, which are located postsynaptically on intrinsic striatal neurons (E&21) that are thought to contain GABA, substance P, or Leu-enkephalin (l), have been shown to exhibit plasticity in other experimental paradigms. They downregulate in response to chronic agonist therapy (39) and upregulate with neuroleptic blockade of DA receptors (40, 46). Recent homogenate studies have shown an upregulation of D-l receptors after MFB lesion with 6-OHDA when examining the total receptor number (B,,,) (7,45). D-l receptor supersensitivity has also been observed in behavioral studies (2) after 6-OHDA lesions. In addition, DA-stimulated adenylate cyclase activity (a D-l-mediated phenomenon) has been shown to increase after administration of the neurotoxin (31, 41). Therefore, the upregulation of

the compensated group and not significantly different from the noncompensated group. Compensated animals were significantly different (P < 0.05) from the noncompensated animals. +In the case of nigral D-l receptors, F(2,lO) = 15.8, P 4 0.01. The Spjotvoll-Stoline modified Tukey HSD (31, 51) clarifies these findings as follows. The control, n = 4, group was significantly different from the 6-OHDA group and was not significantly different from the compensated, n = 5, group. The 6-OHDA, n = 4, group was significantly different from the compensated group. One SN of the noncompensated transplanted group was analyzed and the result was similar to that of the 6-OHDA-lesioned animals (not shown). $For D-2 receptors, F(3,13) = 52.1, P < 0.01. The Spjetvoll-Stoline modified Tukey HSD (31,51) clarifies these findings as follows: Controls, n = 5, were significantly different from 6-OHDA, n = 4, and noncompensated, n = 3, animals and were not significantly different from compensated, n = 5, animals. The 6.OHDA group was significantly different from the compensated group and not significantly different from the noncompensated group. Compensated animals were significantly different from the noncompensated animals.

286

DAWSON

D’

‘.

* . . ‘

.

ET AL.

DOPAMINE

FIG. relatively

4.

Acetylcholinestera light staining pattern

RECEPTORS

se stain of a section of rat of the acetylcholinesterase

AND

brain through the caudate-putamen (CPU) in the transplant (T). Bar = 250 pm. Figure

D-l receptors observed in the present study confirms previous evidence for D-l receptor supersensitivity after 6-OHDA induced dopaminergic denervation. The upregulation of striatal D-l receptors was also shown to and be due to a change in B,,, and not Kd. Marshall co-workers (37) have recently shown a downregulation of striatal D-l receptors after 6-OHDA lesions. Why our results and those of others are at variance with results obtained by Marshall et al. (37) is unclear; however, it may be related to the fact that they performed 6-OHDA lesions of the ventral tegmental area, whereas the majority of the other studies performed 6-OHDA lesions of the MFB. The upregulation of both striatal D-l and D-2 receptors observed in the present study parallels that observed in untreated human PD in which it was shown that both striatal D-l and D-2 receptors are supersensitive (54). The profound extent of the upregulation of D-l and D-2 receptors may be related to the fact that the lesioned animals were examined at least 12 to 14

287

TRANSPLANTATION

of a compensated is adapted from

animal. Note the Dawson et al. (1 0).

months after the lesion. Creese et al. (9) has shown that the degree of upregulation of [3H]spiperone-labeled DA receptors is related to the time elapsing after the lesion. Also of note is that the supersensitivity of D-l and D-2 receptors persisted for an extended period, suggesting that the downregulation of DA receptors observed in other studies examining receptor changes in PD may be related to drug therapy as opposed to extent or duration of disease [see Pierot et al. (44) for review]. Following transplantation, the successfully grafted (compensated) animals had a gradual attenuation of the ipsilateral rotational response to amphetamine. At a period 10 to 12 months after transplantation the compensated animals either ceased rotating or rotated in a contralateral direction in response to subcutaneous amphetamine administration, whereas the noncompensated (nonviable grafts) animals continued to turn ipsilaterally. It has been shown that amphetamine-induced turning cannot only be used to determine the extent of DA denervation following 6-OHDA lesion, but it can be

FIG. 3. Autoradiograms of rat brain sections through the striatum labeled with [3H]BTCP (DA-TC sites) (A-C), [3H]SCH 23390 (D-l receptors) (D-F), and [3H]sulpiride (D-2 receptors) (G-I). Autoradiograms were photographed by using a Wild Photoautomat MPS45 camera system attached to a Wild Photomakroskop M400 microscope. Control (A, D, G), 6-OHDA-lesioned (B, E, H), and compensated animals (C, F, I) (see text) are depicted. Note the loss of the DA-TC sites in the 6-OHDA-lesioned (L) striatum depicted in B. Normalization of the DA-TC sites is shown in C in animals receiving fetal substantia nigra transplants (T). The actual graft is denoted by the arrow projecting from the T in all the figures. The small white arrows in C denote the areas of highest [sH]BTCP binding in the grafted striatum. Note the upregulation of D-l receptors in the 6-OHDA-lesioned (L) striatum depicted in E and the normalization of D-l receptors is shown in F in animals receiving fetal substantia nigra transplants (T). Note the upregulation of D-2 receptors in the 6-OHDA-lesioned (L) striatum depicted in H and the normalization of D-2 receptors is shown in I in animals receiving fetal substantia nigra transplants (T). D-l and D-2 receptor densities in the noncompensated animals were similar to those of the 6-OHDA-lesioned animals. Abbreviations: CPU, caudate-putamen; L, lesion; T, transplant. Bar = 250 uM.

288

FIG. 5. High power views tions: CPU, caudate-putamen;

DAWSON

of [3H]BTCP T, transplant;

ET

binding and acetylcholinesterase AC, anterior commissure. Bar

used to assess reinnervation. Compensated animals were found to have a dense amount of [3H]BTCP binding (DA-TC) in the graft and surrounding striatal regions. The reinnervation of the striatum, as indicated by the return of [3H]BTCP binding, was approximately 60 to 70% of control values which is in close agreement with results obtained from functional assays of DA uptake (30). DA uptake sites, as demonstrated autoradiographically by [3H]dopamine uptake (15), have also been shown to be at higher levels, compared to nontransplanted striatum, after transplantation of fetal nigra into the genetically DA-denervated striatum of the Weaver mouse. Similar changes, after transplantation of fetal nigra, have also been observed for other presynaptic catecholaminergic markers such as tyrosine hydroxylase staining and glyoxylic acid-induced histofluorescence of DA cell bodies and terminals (4, 5, 24, 25, 36, 43, 59). As expected, noncompensated animals had no evidence of reinnervation based on [3H]BTCP binding. Reinnervation of the host striatum by SN grafts and the resulting synaptic arborizations with appropriate host cells, such as giant aspiny (cholinergic cells with D-2 receptors) and spiny (GABAergic cells with D-l receptors) striatal neurons (25,36,59), might be expected to alter postsynaptic receptor densities. Not only were

AL.

staining of the transplant = 500 pm.

in compensated

animals.

Abbrevia-

postsynaptic receptor densities altered, but striatal D-l and D-2 receptor densities were normalized in the compensated animals. In the noncompensated animals (animals with nonviable grafts), D-l and D-2 receptor densities remained supersensitive. The normalization of DA receptor densities provides evidence that the graft not only makes synaptic contacts with appropriate host neurons, as demonstrated by prior EM studies (25, 36, 59), but it may integrate postsynaptically at the receptor level. Even though the nigral grafts only return approximately 60% of “normal” dopamine innervation (based on the dopamine uptake site data, Fig. l), a complete normalization of the postsynaptic receptors is occurring. These data suggest a powerful receptor plasticity in response to transplanted tissue. In addition, there is a known synergistic interaction between D-l and D-2 receptors (3) and it has been shown that D-l receptor activation is required for postsynaptic expression of D-2 effects (65). Therefore, normalization of both DA receptor subtypes may be required for full behavioral recovery following transplantation. Another major finding of the present study was the upregulation of denervated SNR D-l receptors and the subsequent normalization of these receptors after transplantation. The SNR is a projection area of the medium and small spiny neurons of the striatum which

DOPAMINE

RECEPTORS

AND

25

0

0

100

50

10

150

20

250

200

30

40

300

50

350

60

BOUND FIG. 6. Scatchard analysis obtained from the saturation data of the response of D-l (A) and D-2 (B) receptors to 6-OHDA lesion and fetal substantia nigra transplantation. Saturation data were generated by incubating serial tissue sections in varying concentrations of [3H]SCH 23390 (A) and [aH]sulpiride (B). These data are plotted as bound (fmol/mg tissue) versus bound/free [(fmol/mg tissuel/nM]. In the case of striatal D-l receptors: control (0) K,, = 2.9 nM, B,,, = 254.6 fmol/mg tissue; lesion 1M) Kd = 2.9 nM, B,,, = 340.9 fmol/mg tissue; and compensated (V) Kd = 2.8 nM, B,, = 255.1 fmob’mg tissue. For striatal D-2 receptors: control (0) Kd = 26.2 nM, B,, = 37.5 fmol/mg tissue; lesion (ml Kd = 26.0 nM, B,, = 58.6 fmol/mg tissue; and compensated (V) Kd = 25.9 nM, B,,, = 36.9 fmol/mg tissue. These Scatchard plots are representative data from one animal from each group presented in Table 1.

contain presynaptic D-l receptors on their nerve terminals (18, 21). As with striatal D-l receptors, SNR D-l receptors have been shown to upregulate after chronic neuroleptic therapy (40, 46) and downregulate after chronic agonist therapy (39). These data suggest that once a striatal neuron has altered its machinery to upor downregulate D-l receptors, it may not discriminate between dendritic or terminal locations to effect such changes. However, this may not always be the case, as we have demonstrated that changes in D-l receptors occurring at the soma may not accompany changes at

TRANSPLANTATION

289

terminal locations [see McCabe et al. (39)J. In addition, in Parkinson’s disease, nigral D-l receptors have been shown to be downregulated while striatal D-l receptors are upregulated in the same individuals (48). Nigral D-l receptors may be of major importance in control of motor movement in 6-OHDA-lesioned rats, since it has been shown that stimulation of supersensitive nigral D-l receptors, as well as striatal D-l receptors, is involved in the turning response induced by amphetamine administration (49). The normalization of D-l receptor density, in transplanted animals, at a site distant from the striatum suggests that more than just the tonic release of DA from the graft may be responsible for compensation. This normalization of nigral D-l receptor density may reflect a change in function of the striatonigral output neurons by the fetal transplant and it suggests that a functional integration with the host striatal circuity may be occurring (22, 57). The exact role that nigral D-l receptors may play in behavioral recovery after transplantation is not known. However, fetal substantia nigra grafts placed in the substantia nigra do not reverse rotational behavior (16, 50). Why animals with normalized DA receptor densities, after receiving the graft, rotate contralaterally following an injection of amphetamine is unknown. It was originally postulated that the contralateral rotation was due to a higher sensitivity of the grafted neurons than that of the intact nigrostriatal neurons to the releasing action of amphetamine (29). However, amphetamine-induced DA release from striata that has been successfully transplanted with fetal nigra has been shown to be, at most, at the same level as that of control striata (30, 67). From the data presented in this study, contralateral rotation is not due to amphetamine-induced release of DA acting on supersensitive receptors in the vicinity of the graft. It is unclear whether supersensitive DA receptors in striatal and other areas not reinnervated by the transplanted neural tissue may be responsible for amphetamine-induced contralateral rotations. Another possibility is that the lesion with 6-OHDA severs the nigral-striato-nigral feedback loop and disrupts feedback inhibition of an overdriven system. Thus, due to the lack of feedback inhibition in transplanted animals, amphetamine administration results in contralateral turning. In addition, changes in nondopamine striatalnigral transmitter systems may over- or undercompensate and be responsible for the behavioral observations. It has recently been shown that grafted human dopamine neurons survive and improve motor function in a patient severely affected with idiopathic PD (35). Restoration of DA synthesis and storage (i.e., graft viability) was demonstrated by positron emission tomography (PET) with L-[6-“Flfluorodopa, a tracer which can be used to assessstriatal presynaptic dopaminergic function. Ultimately the reinnervation of denervatedneostriatum in PD and the subsequent improvement of symp-

290

DAWSON

ET

AL.

TABLE1 Scatchard

Analysis D-l

of D-l

and

D-2

Receptors

D-2

CPU

SN

%ax (fmol/mg

tissue)

240.7 + 5.8

Control

(fmol/mg

Transplant

compensated

329.0 * 7.7* (n = 3) 243.6 f 6.8

Receptors CPU

B max tissue)

B(fmol/mg

tissue)

2.7 2

0.1

252.1 + 5.5 (n = 5)

2.7 + 0.2

37.7 f

1.0 (n = 5)

24.1 + 2.5

2.8 f

0.1

340.6 f 9.7**

3.0 ? 0.3

58.2 + 0.6***

25.1 + 1.3

(n = 5) 6-OHDA

Receptors”

2.6 k 0.2

(n = 4)

(n = 4) 255.4 + 10.9 (n = 4)

(n = 2)

3.2 ? 0.3

37.3 * 2.0

26.5 + 2.8

(n = 4)

a Saturation and Scatchard analysis of [aH]SCH 23390 (D-l) and [3H]sulpiride (D-2) binding in 6-OHDA MFB-lesioned and fetal SN-transplanted animals determined by semiquantitative autoradiography. These data indicate that the changes observed in the various treatment groups were due to a change in the number (B, ) of receptors without an apparent change in the affinity (Kd). * F(2, 9) = 64.6, P G 0.01. **F(2, 10) = 43.6, P < 0.01). ***F(2,9) = 36.3, P < 0.01. The Spjetvoll-Stoline modified Tukey HSD (31,51) was used to clarify the above findings for striatal D-l and D-2 and nigral D-l receptors as follows. The control groups were not significantly different from the compensated groups and were significantly different from the 6-OHDA groups. The compensated animals were significantly different from the 6-OHDA animals. No significant difference was observed for the Kd)s for striatal D-l and D-2 and nigral D-l receptors. Saturation studies on one animal from the noncompensated transplant group were performed and the results were similar to those of the 6-OHDA-lesioned animals (data not shown). Please see Fig. 6 for representative Scatchard plots.

toms by transplantation of fetal substantia nigra would be mediated via an interaction of DA produced by the graft with postsynaptic DA receptors. Perhaps it can be anticipated that if fetal substantia nigra transplaatation become a viable clinical treatment of idiopathic PD, not only will graft viability be determined by PET scanning with presynaptic markers for dopaminergic function, but integration of the graft with the host may also be assessed by examining D-l and D-2 receptors with PET.

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ACKNOWLEDGMENTS The authors thank Barbara Whiting for her excellent secretarial assistance. Time on the DUMAS image analyzer was generously supplied by Dr. Andrew Winokur. This research was supported by funds from the Public Health Service NS 22033, DA 05167, and the Schering Corp. (J. K. Wamsley), the Parkinson’s Disease Association and PEW Memorial Trust (F. H. Gage).

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