Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Dopamine D1 and D2 Receptors and Uptake Sites: A Quantitative Autoradiographic Study

Journal of Neurochemistry Raven Press, Ltd., New York 0 I99 1 International Society for Neurochemistq

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Dopamine D1 and D2 Receptors and Uptake

Sites: A Quantitative Autoradiographic Study

Serge Przedborslu, Vladimir Kostic, Vernice Jackson-Lewis, Jean Lud Cadet, and Robert E. Burke

Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York, U.S.A

Abstract: The effect of a unilateral pennatal hypoxic-ischemic brain injury on dopamine D, and D2 receptors and uptake sites was investigated in rats by using in vitro quantitative binding autoradiography, 2-3 weeks after the insult. We observed significant decreases in the B,,, and K D for 13H]SCH 23390-labeled DI and in the B,,, for [3H]spiperone-labeled D2 receptors in the lesioned caudate-putamen in rats with moderate brain injury (visible loss in hemispheric volume ipsilateral to the injury) compared with the nonlesioned contralateral caudate-putamen or with control rats. Changes in [3H]SCH 23390 and [3H]spiperone binding predominated in the dorsolateral part of the lesioned caudate-putamen. Pro- nounced reduction in [3H]SCH 23390 binding was also observed in the substantia nigra pars reticulata on the side of the lesion. In contrast, we did not observe any significant

change in B,,, or KD for [3H]mazindol-labeled dopamine uptake sites. Similarly, no significant changes in the levels of dopamine or its metabolites were found on the side of the lesion. The observed reductions in striatal dopamine D1 and D2 receptors are a reflection of striatal cell loss induced by the hypoxic-ischemic injury. The absence of changes in [3H]mazindol binding or dopamine levels in the lesioned caudate-putamen indicates that the dopaminergic presynaptic structures are preserved. Key Words: Hypoxia-kchemia- Striat~m-Dystonia-[~H]SCH 23390-[3H]Spiperone- [3H]Mazindol. Przedborski S. et al. Effect of unilateral perinatal hypoxic-ischemic brain injury in the rat on dopamine DI and D2 receptors and uptake sites: A quantitative autoradiographic study. J. Neurochem. 57, I95 1 - 196 I ( 199 I).

The static encephalopathies of childhood are common disorders, affecting approximately one per 500 children (Paneth and Kiely, 1984). Neonatal hypoxic- ischemic injury remains one of the few identifiable and potentially treatable causes. A distinct type of cerebral palsy, the dyskinetic form, which is characterized predominantly by dystonia (Kyllerman et al., 1982), has a close relationship to adverse perinatal events (Hagberg and Hagberg, 1984). Like many other causes of dystonia, dystonic cerebral palsy is associated with pathology of the basal ganglia, particularly the striatum. A common neuropathological lesion seen is status marmoratus, a striatal lesion characterized by neuronal loss, abnormal myelination, and gliosis (Zeman and Whitlock, 1968; Carpenter, 1977). Little else is known of the alterations in the striatum in this lesion.

A unilateral rat model of perinatal hypoxia-ischemia, which reproduces some of the pathological fea- tures of status marmoratus (Rice et al., 198 1; Johnston, 1983; Burke and Karanas, 1990), provides a suitable paradigm for studying the effects of perinatal hypoxia- ischemia on the neurochemical anatomy of the basal ganglia in the developing brain (Johnston, 1983). Of particular interest in relation to disorders of motor control, such as dystonia, are changes in nigrostriatal dopaminergic systems, given the major role that these systems play (Wooten and Trugman, 1989). Johnston (1983) had shown in this model of unilateral hypoxia- ischemia that presynaptic biochemical markers of striatal dopaminergic systems, such as the activities of tyrosine hydroxylase (TH) and dopamine (DA) reup- take, are relatively spared. In addition, we have dem-

Received January 1 1, 199 1 ; revised manuscript received April 30, 199 I ; accepted May 1, 199 I .

Address correspondence and reprint requests to Dr. R. E. Burke at Black Building, Rm. 302, Columbia University, 630 West 168th Street, New York, NY 10032, U.S.A.

Abbreviations used: ANOVA, analysis of variance; CPu, caudate-

putamen; DA, dopamine; DMI, desmethylimipramine; DOPAC, 3,4- dihydroxyphenylacetic acid; GABA, y-aminobutyric acid; 5-HT, serotonin; HVA, homovanillic acid; MBP, myelin basic protein; NAc, nucleus accumbens; OTu, olfactory tubercle; SN, substantia nigra; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; TH, tyrosine hydroxylase.

1951

1952 S. PRZEDBORSKI ET AL.

onstrated morphologically that the nigrostriatal dopaminergic system is relatively spared because the density of striatal TH-positive neuropil is increased on the side of injury (Burke et al., 1991). It is difficult to predict the possible functional consequences of this sparing of dopaminergic systems because there has been no information available about effects on D A receptors. Therefore, to further clarify effects on striatal dopaminergic systems in this model, we have examined D A D1 and D2 receptor binding by use of quantitative autoradiography. Because significant anatomic alterations occur in this model, we have reexamined effects on the D A uptake sites, previously assessed in homogenates (Johnston, 1983) by means of autoradiography.

MATERIALS AND METHODS

Unilateral perinatal hypoxia-ischemia lesion Female rats (Sprague-Dawley) were 14- 16 days pregnant

on arrival from Charles River Laboratories (Wilmington, MA, U.S.A.). The day of delivery was defined as postnatal day 1. On day 7 or 8, the left carotid artery of the pups was ligated with 6-0 silk suture. After surgery, the pups were returned to the dam for 1-2 h and then exposed to humidified 8% oxygen at 37°C for 3 h, during which time pups were kept in a plastic bell jar immersed in a water bath at 37°C as described by Rice et al. (1981). This procedure was approved by the In- stitutional Animal Care and Use Committee of Columbia University (New York, NY, U.S.A.). For surgical control, pups underwent a sham ligation (sham control) in which the carotid was manipulated but not ligated, and then the pups were exposed to hypoxia. This procedure does not induce tissue damage in the brain. In addition, we also studied normal age-matched rats (normal control).

Tissue preparation At 3-4 weeks of age, rats were killed by decapitation, and

the brains were rapidly removed and rinsed in ice-cold saline. Each litter of rats contained both sham-operated and experimental rats, and a normal litter was killed concurrently. Thus, all conditions were matched for age at decapitation within the 3-4 week interval. The gross extent ofbrain injury in experimental rats was assessed as previously described (Burke and Karanas, 1990); brain injury was graded as mild if there was no visible loss in hemispheric volume on the side of the injury, and as moderate if there was a visible loss in hemispheric volume. Brains with cavitation on the side of the lesion were classified as severely injured and were excluded from further study. We have previously found that animals in the mild injury category show greater than 85% preservation of striatal cross-sectional area; those with moderate injury show less than 85% preservation (Burke and Karanas, 1990). Brains were classified before processing for binding autoradiography or biochemistry.

For receptor binding autoradiography studies, whole brains were frozen by slow immersion in isopentane cooled on dry ice and then kept at -80°C until sectioned. Coronal sections (1 2 pm) were cut in a cryostat at -2O"C, thaw-mounted onto gelatin-coated glass slides (2 sections per slide), dried at 4°C under vacuum for 2 h, and then kept in a sealed slide box at -20°C until use. Twenty-six rats were used for the autoradiographic studies. For saturation experiments, sections at the level of the mid portion of caudate-putamen complex (mid CPu) (interaural, 10.7-9.2 mm; Paxinos and Watson,

1982) from normal controls (n = 3) and moderate experimental pups (n = 3) were used. For regional distribution of binding sites experiments, sections at the level of rostra1 (interaural, 1 1.7- 1 1.2 mm), mid (as described above), and caudal (interaural, 8.7-7.7 mm) CPu and of substantia nigra (SN) (interaural, 4.2-3.2 mm) from normal controls (n = 5), sham controls (n = 5), and pups with both mild (n = 5) and moderate (n = 5) experimental injury were used. The radioligand binding assays were performed on adjacent sections of the same brain region from each rat.

For the determination of DA and its metabolites, 32 rats, as follows, were used: normal controls (n = lo), sham controls (n = 6), and pups with both mild (n = 9) and moderate (n = 7) injury. The brains were placed in a plexiglass rat brain matrix submerged in ice-cold saline. A 2-mm-thick coronal section containing the mid portion of the striatum was taken by cutting with razor blades through the matrix. The anterior plane of this thick section corresponded approximately to Paxinos and Watson's interaural plane 10.2 (Paxinos and Watson, 1982). The section was placed on a chilled glass plate and the striatum on each side was punched out with a 3-mm bore. The region of nucleus accumbens (NAc) was defined as a quadrant of tissue ventral and medial to the anterior commissure. Finally, the SN was dissected by hand from the midbrain. The tissue was immediately frozen on dry ice and kept at -80°C until assayed.

Autoradiography binding assays DA D1 receptors were labeled with [3H]SCH 23390 (71.3

Ci/mmol; NEN, MA, U.S.A.) according to a method previously described (Dawson et al., 1985) with minor modifications. The sections were preincubated for 10 min at 25°C in 50 mM Tris-HC1 (pH 7.4) containing 120 m M NaC1, 5 mM KCl, 2 mM CaC12, and 1 mM MgCl2, before the incubation (25"C, 60 min) in the same buffer with varying concentrations of [3H]SCH 23390 (0.15- 10 nM for saturation experiments; 1.25 nM for regional distribution studies). After the incubation, the sections were washed twice for 5 min in ice-cold buffer, dipped in ice-cold distilled water, and rapidly dried under a stream of cold air. Nonspecific binding was measured in the presence of 1 pM unlabeled R-(+)-SCH 23390 (RBI, MA, U.S.A.) and ranged from 0.3 to 18% of the total binding for the low (0.15 nM) to the high (10 nM) ligand concentration, respectively.

DA D2 receptors were labeled with [3H]spiperone (34.2 Ci/mmol; NEN) according to the method reported by Pa- lacios et al. (1 98 l ) with minor modifications. The sections were preincubated (25"C, 10 min) in 50 mM Tris-HC1 (pH 7.6) containing 120 m M NaCI, 5 mM KCI, 2 mM CaClz, 1 mM MgC12, and 0.00 1 % ascorbic acid, before being incubated (25"C, 60 min) in the same buffer with varying concentrations of [3H]spiperone (0.075-5 nM for saturation experiments; 0.6 nM for regional distribution studies). After the incubation, the sections were processed as described for [3H]SCH 23390 binding assay. Nonspecific binding was measured in the presence of 10 pM of unlabeled S-(-)-sulpiride (RBI) and ranged from 8 to 52% of the total binding for the low (0.075 nM) to the high (5 M) ligand concentration, respectively. Although [3H]spiperone binds to both the serotonin 5-HTz receptor and spirodecanone site (Palacios et al., 1981), the use of the highly selective D2 antagonist sulpiride to define nonspecific binding ensures that the specific binding includes only D2 receptors (List and Seeman, 1981).

DA uptake sites were labeled with [3H]mazindol (1 5.0 Ci/ mmol; NEN) according to the method described by Javitch

J. Neiri-ochrm., VoL 5 7, N o 6, 1991

EFFECT OF HYPOXIA-ISCHEMIA ON DA RECEPTORS 19.53

et al. (1985) with minor modifications. The sections were preincubated (4OC, 15 min) in 50 mM Tris-HC1 (pH 7.9) containing 120 mM NaCl and 5 mM KC1. They were then incubated (4"C, 60 min) in 50 mM Tris-HC1 (pH 7.9) containing 300 mM NaC1, 5 mM KCl, and 300 nM desmethylimipramine (DMI) with varying concentrations of [3H]mazindol (1-50 nM for saturation experiments; 15 nM for regional distribution studies). DMI was used to block the binding to norepinephrine uptake sites (Javitch et al., 1985). After the incubation, the sections were washed twice for 3 min in ice-cold buffer, dipped in ice-cold distilled water, and rapidly dried under a stream of cold air. Nonspecific binding was measured in the presence of 30 WM of benztropine me- sylate and ranged from 10 to 45% of the total binding for the low (1 nM) to the high (50 nM) ligand concentration, respectively.

Autoradiography and image analysis To assist in the performance and interpretation of receptor

autoradiographic studies of the striatum in this model, we have concurrently performed morphologic analyses of 36 rats with moderate lesions after perfusion fixation and immu- noperoxidase staining for glial fibrillary acidic protein, myelin basic protein (MBP), TH, and thionine Nissl staining (see Burke and Karanas, 1990; Burke et al., 199 1, for methods). In brief, we found that striata in moderate lesions were re- duced in size, but normal in their cross-sectional configura- tion. This decrease in the size of the striatum appeared pri- marily due to the loss of medium-sized striatal neurons; large neurons were relatively preserved (Burke and Karanas, 1990). The most consistent site of gliosis was the dorsolateral quadrant of the posterior striatal planes (Paxinos-Watson 8.7 and 8.2). Less often, gliosis was observed in the anterior planes, and mainly dorsally or laterally. It should be emphasized that neurons survived within the majority of these foci of gliosis. Occasionally, in the dorsal or lateral portion of the striatum, especially in the most caudal planes, extensive gliosis would occur such that abnormal aggregates of myelin bundles would form. Such aggregates were devoid of neurons and TH-positive fibers (Fig. 1; see also Burke et al., 199 1). Because such aggregates do not include these neural markers, they are unlikely to be functionally relevant striatal regions, so we excluded them in our autoradiographic studies. Because such aggregates could be defined by the absence of TH-positive fiber staining, we used the pattern of [3H]mazindol binding in adjacent sections as a guide. Regions devoid of binding were avoided in densitometric measurements over the striatum. It must be emphasized that such regions were not ne- crotic; they contained viable glial and myelin elements. Ne- crotic foci are small and rarely observed in moderate lesions. Among 36 brains, points of necrosis were observed in only three brains, all in single sections from plane 8.2 (Paxinos- Watson).

The possible differential absorption of the @-emission between the lesioned and nonlesioned side was assessed according to the method described by Kuhar and Unnerstall (1985). Brain sections were soaked in 50 mM Tris-HC1 (pH 7.4) containing 10 pA4 [3H]isoleucine (1 10.4 Ci/mmol, NEN) at room temperature for 75 min. Then they were dipped in ice-cold distilled water and rapidly dried under a stream of cold air. Dry sections were apposed to tritium-sensitive film (Hyperfilm, Amersham) for 12 days at 25°C. The film was then processed as for the binding assays (see below). We found that regions of pronounced @-emission quenching within the CPu corresponded to the foci of myelin aggregation (Fig. 1 C

FIG. 1. A: lrnmunoperoxidase staining for tyrosine hydroxylase (TH) in a coronal section of the striatum in a rat with moderate hypoxic-ischemic injury (see Burke et al., 1991, for methods). The arrows indicate a region devoid of staining in the dorsal striatum, subjacent to the external capsule (bar = 500 pm). 8: A higher magnification of the region marked by arrows in A. C: Irnmuno- peroxidase staining for myelin basic protein (MBP) in a section adjacent to the one shown in A. It can be seen that the ovoid regions devoid of TH-staining in A correspond to MBP-positive myelin bundles in C, marked by arrows. D: A higher magnification of the region marked by arrows in C. E Photomicrograph of a digitized coronal section of an autoradiograph for [3H]isoleucine binding (see Materials and Methods). Although this section is from a similar striatal plane as that shown in A and C, it is obtained from a different rat, processed for autoradiography. It can be seen that regions of significant &emission quenching are observed over my- elinated structures, including corpus callosurn (CC), external capsule (EC), anterior comrnissure (AC), and at a point of myelin aggregation subjacent to the external capsule (enclosed by white border).

J. Neurochem., Vol. 57, No. 6 . 1991

I954 S. PRZEDBORSKI ET AL.

and D), which were devoid of TH-positive fibers (Fig. 1A and B), as shown in Fig. 1E.

Dried labeled sections were placed in light-proof x-ray cas- settes along with tritium plastic standards ([ 3H]Micro-scales, Amersham) and were then apposed to tritium-sensitive film (Hyperfilm, Amersham) for 1-3 weeks at 4°C for [3H]mazindol and 1-3 or 2-4 weeks at 25°C for [3H]SCH 23390 and [3H]spiperone, respectively (Unnerstall et al., 1982). The films were then developed with D-19 developer (Kodak) and fixed with Kodak Rapid Fix. The optical density of the developed autoradiographic films was quantified using an RAS-3000 computerized image-analysis system (Amer- sham). Optical densities were converted to femtomoles of radioligand bound per milligram of tissue using a standard curve ([3H]Micro-scales). For each brain region of each rat and for each concentration of radioligand, readings from at least four separate sections were obtained and averaged for total and nonspecific binding. The regions of interest were defined according to the Paxinos and Watson (1982) rat brain atlas and included olfactory tubercle (OTu), NAc, CPu, SN pars compacta (SNC), and pars reticulata (SNR); mid CPu was subdivided along its greatest dorsoventral and medial- lateral axes into dorsolateral, dorsomedial, ventrolateral, and ventromedial quadrants. This procedure was used for both intact and lesioned mid CPu. Each region was outlined with a screen cursor driven by a hand-held mouse on a digitizer pad. After autoradiographic studies, representative sections were stained with cresyl violet for anatomical reference.

Measurement of dopamine and its metabolites DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and ho-

movanillic acid (HVA) were assayed by HPLC with electrochemical detection according to the method previously described (Reches et al., 1982). DA and metabolites were measured within a single chromatograph, using an electrochemical detector (BAS) with a glassy carbon electrode against an Ag/ AgCl reference electrode. Typical retention times were as follows: DA, 7.7 min; DOPAC, 10.4 min; HVA, 25.4 min. Cal- ibration for sample retention times was done before sample analyses, and recovery for all these compounds was 95-10096.

Data analysis Data from saturation experiments performed on the mid

CPu were transformed using the method of Scatchard, and the B,,, and K,, were estimated using unweighted linear- regression analysis. Then, both B,,, and KD values were sub- jected to one-way analysis of variance (ANOVA). ANOVA was also performed on data from regional distribution studies performed at single concentrations of the radioligands, and on measures of dopamine and its metabolites. The Scheffk F test was used for post-hoc comparison of the means. The null hypothesis was rejected at the 0.05 level. All values are expressed as the mean -t SEM.

RESULTS ('HJSCH 23390-labeled DA D, receptors

Moderate unilateral hypoxic-ischemic injury caused significant changes ( p < 0.01) in both striatal B,,, (502.0 k 6.4 fmol/mg of tissue) and KD (0.54 k 0.12 nM) for [3H]SCH 23390 on the lesioned side in comparison with the contralateral nonlesioned side (B,,, = 556.0 2 4.7 fmol/mg of tissue and KD = 0.29 & 0.02 nM) and normal controls (B,,, = left: 564.0 f 12.0, right: 568.3 -I- 6.1 fmol/mg of tissue, KD = left: 0.30

+_ 0.01, right: 0.29 t 0.01 nM) (Fig. 2A). There were no significant differences in B,, or KD values between either side of the brain of normal controls or in the nonlesioned side of rats with moderate brain injury (Fig. 2A). Both saturation and distribution studies re-

(D

I 0

x al

r

2 LL U c z 0 m

(D I 0

x al

T-

E LL

m

U c z 0

(D

I 0

x al

r

2 LL U z 0 m

2400

1800

1200

600

0

[3H]-SCH 23390 0 Normal Control, R. side

0 Normal Control, L. side

0 Moderate injury, Contr. side

Moderate injury, Ipsi. side

0 100 200 300 4 0 0 5 0 0 6 0 0

Bound (fmollmg tissue)

[ 3H] -S p i perone ~

0 Normal Control, R. side

1500

1000

500

0

0 Normal Control, L. side 4. 0 Moderate injury, Contr. side

Moderate injury. Ipsi. side

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 300


600 [ 3 HI -Ma2 i ndol 0 Normal Control, R. side

0 Normal Control, L. side 450 0 Moderate injury, Contr. side

Moderate injury, Ipsi. side

0 5 0 0 1 0 0 0 1500 2000


FIG. 2. Scatchard plot of the specific binding of [3H]SCH 23390 to dopamine D, receptors (A), [3H]spiperone to dopamine D2 receptors (B), and of [3H]mazindol to dopamine uptake sites (C) in coronal sections at the level of mid caudate-putamen of normal controls and rats with moderate brain injury. Each point represents the mean value obtained from three different rats per group, as described in Materials and Methods. R., right: L., left; Contr., contralateral; Ipsi., ipsilateral.

J. Neurochem., VCJI. 57. No. 6. 1991

EFFECT OF HYPOXIA-ISCHEMIA ON DA RECEPTORS 1955

vealed that the greatest decrease in [3H]SCH 23390 binding was in the dorsolateral quadrant (-24%) ofthe mid CPu, followed by the dorsomedial (- 19%) and the ventrolateral quadrants (- 16%), whereas no significant changes were observed in the ventromedial quadrant (Table 1 and Fig. 3). The decreases in binding were almost uniform in the anteroposterior dimension (rostral: - 14%, mid: - 17%, caudal: - 19%) (Table 1). A pronounced decrease in binding was also found in the SNR (-34%) (Table l), which predominated laterally (Fig. 3). A similar pattern of changes was observed on the lesioned side of rats with mild injury (n = 5) , although none of these changes achieved statistical sig- nificance (data not shown). In addition, no significant changes in binding concentrations were found in the OTu, NAc, and globus pallidus on the side of the lesion in rats with moderate injury (Table 1).

[3H]Spiperone-labeled DA D2 receptors Moderate unilateral hypoxic-ischemic injury re-

sulted in a significant decrease ( p < 0.01) in B,,, (213.67 f 19.27 fmol/mg of tissue) for [3H]spiperone on the lesioned side in comparison with the nonlesioned contralateral side (239.79 & 7.55 fmol/mg of tissue) and normal controls (left: 243.00 f 9.07, right: 242.0 f 8.1 fmol/mg of tissue) (Fig. 2B). There was not a significant change in KD on the injured side (0.3 1 f 0.03 nM; Fig. 2B) in comparison with the noninjured side (0.20 f 0.05 nM) and normal controls (left: 0.19 k 0.07, right: 0.16 k 0.07 nM). Again, there were no

differences in B,,, or KD values between either side of the brain of normal controls or in the nonlesioned side of rats with moderate brain injury (Fig. 2B). Data from saturation and regional distribution studies showed that the decreases in [3H]spiperone binding predominated in the dorsolateral (-24%), ventrolateral (- 19%), and dorsomedial (- 15%) quadrants of the mid CPu (Table 2 and Fig. 4). As for [3H]SCH 23390, reductions in [3H]spiperone binding were equally distributed in the anterior-posterior dimension of the CPu (rostral: - 14%, mid: -17%, caudal: -17%). Similar trends were observed on the lesioned side of rats with mild injury, but none of these changes were statistically significant (data not shown). There were no significant changes in [3H]spiperone binding concentrations in OTu, NAc, and SNC on the side of the lesion in rats with moderate injury (Table 2).

[3H]Mazindol-labeled DA uptake sites Saturation experiments showed that unilateral hyp-

oxic-ischemic injury did not cause any significant change in [3H]mazindol binding parameters on the side of the lesion in experimental rats with moderate injury (B,,, = 1,821.3 f 151.9 fmol/mg of tissue and KD = 4.34 rt 1.54 nM) in comparison with normal controls (B,,, = left: 1,887.0 f 148.4 and right: 1,826.0 k 152.7 fmol/mg of tissue; KD = left: 4.59 f 1.56 and right: 3.95 f 0.99 nM) (Fig. 2C). Experiments on regional distribution of this ligand did not disclose any significant change in experimental rats with mild or moderate

TABLE 1. Efect of unilateral perinatal hypoxia-ischeinia on the binding of 1.25 nM of ['HISCH 23390 to dopamine DI receptors

Specific binding (fmol per mg of tissue f SEM)

Normal control (n = 5) Sham control (n = 5 ) Moderate brain injury (n = 5 ) Brain areas Right Left Right Left Right Left Decrease (%)

OTu NAc CPU

Rostra1 Mid

Total DL DM VL VM

Caudal GP SNR

468.8 f 13.2 474.4 f 12.3

462.0 f 10.9

472.2 f 11.4 473.2 f 11.1 462.8 f 11.5 476.6 k 12.1 464.2 f 11.4 458.0 f 10.7 245.8 f 13.4 472.0 f 12.5

466.0 f 13.4 472.0 f 11.6

463.8 k 12.0

476.4 f 13.2 468.3 f 10.0 466.1 f 12.9 478.4 k 10.0 467.8 f 14.1 454.4 f 12.7 252.6 f 11.4 468.4 f 11.6

476.6 f 15.2 468.3 f 11.8

475.4 t 14.9

474.0 f 10.6 473.4 f 10.5 463.8 f 11.8 475.2 f 10.7 459.2 f 11.2 451.6 f 10.2 251.4 f 12.9 465.3 f 13.0

464.5 f 12.1 468.4 f 12.1

468.0 f 12.5

468.5 f 14.1 477.0 f 12.7 468.4 k 13.1 470.3 f 13.1 463.8 f 10.0 460.3 f 11.3 261.0 f 14.1 468.4 f 14.0

466.0 f 12.7 479.0 f 13.3

461.0 f 13.6

464.2 f 13.6 465.4 t 16.4 462.8 f 15.9 476.2 f 13.6 455.2 f 12.8 459.4 f 16.2 251.2 t 15.0 465.6 * 11.0

465.8 f 14.3 468.4 t 12.0

398.0 t 12.1 b,c,d

391.4 f 12.9e*C.d 358.2 f 14.1e,c3f 377.6 f 1 8.9b*c3d 402.4 S 12.7 b,d 425.6 f 18.8 372.6 t 13.2e.b'rf 250.8 f 16.3 309.0 t 10.98',"'

0 2

14

16 23 18 16 6

19 0

34

Because of the difference in KD between the lesioned side and the contralateral control (and normals), the differences shown are not strictly due to differences in the number of binding sites. At the concentration used ( I .25 nM), binding in the lesioned striatum is at 70% B,,,,,, whereas in the other conditions, it is at 80% B,,,. Nonspecific binding was defined by 1 pM unlabeled R-(+)-SCH 23390 and was subtracted from all readings. Values represent the mean f SEM. Differences between means were tested by one-way ANOVA, followed by a Scheffe post-hoc test. DL, dorsolateral; DM, dorsomedial; VL, ventrolateral; VM, ventromedial; GP, globus pallidus.

' p < 0.01; 'p < 0.001, compared with normal controls, sham controls, and nonlesioned contralateral side of rats with mild brain injury. dp < O.O1;/p < 0.001, compared with nonlesioned side of rat with moderate brain injury. g p i 0.05; ' p < 0.01, compared with lesioned side of rat with mild brain injury.

Side of the injury.

J. Neurochem.. Vol. 57, No. 6, 1991


FIG. 3. [3H]SCH 23390 (1.25 nM) binding in matched brain sections from normal controls (A and B) and rats with moderate brain injury (C and D). Pictures are representative color-coded transforms of original autoradiographs. Nonspecific binding (not shown), measured in the presence of 1 pM unlabeled R-(+)-SCH 23390, was similar in both groups of rats and represented 5% of total binding.

injury (data not shown). The preservation of [3H]mazindol binding on the side of the injury at the level of the CPu and SN is illustrated in Fig. 5.

Monoamine levels Levels of DA, DOPAC, and HVA in the CPu in

both the mild and moderate experimental injury conditions were not significantly different in comparison with normal or sham controls. On the side of injury, DA = 15.0 * 0.4 ng/mg of tissue for seven rats with moderate injury; on the opposite side, DA = 16.3 f 0.5. Normal controls (n = 10) had values of 15.9 2 0.4 and 16.3 t- 0.4 for left and right, respectively. In addition, there were no significant differences in DA, DOPAC, or HVA levels in the NAc or the SN of rats with mild or moderate injury compared with normal and sham controls (data not shown). Thus, hypoxic-ischemic injury did not cause an apparent change in steady-state DA levels, or DA turnover, in these structures.

DISCUSSION

We have found that unilateral perinatal hypoxic- ischemic injury in rats results in decreases in [3H]SCH 23390 and [’Hlspiperone binding on the side of the lesion. In contrast, no significant effects on

[3H]mazindol binding or on the levels of DA and its metabolites were observed 2-3 weeks after the insult.

[3H]Mazindol is a specific ligand for DA uptake sites when binding is performed in the presence of DMI (Javitch et al., 1985). Lesion studies indicate that [3H]mazindol binding is localized to dopaminergic neurons and their projections (Javitch et al., 1985; O’Dell and Marshall, 1988). The absence of changes in striatal [3H]mazindol binding on the injured side is in accordance with the previous biochemical data of Johnston (1 983), demonstrating preserved DA reup- take 2 weeks after the hypoxic-ischemic injury. Both the earlier finding of Johnston (1983) and our current results, however, are somewhat discrepant with our morphologic finding of an increased density of TH- positive fibers in the injured striatum (Burke et al., 199 1). It is possible that, although we have previously demonstrated an increased density of TH-positive neuropil, this is due strictly to shrinkage of the striatum, with compaction of fibers in passage. Alternatively, it is possible that there has been an increase in the number of dopaminergic terminals, but there are fewer uptake sites per terminal such that no change in uptake sites is observed at the population level. In either case, both morphologic and biochemical data indicate that do-


TABLE 2. Effect of unilateral perinatal hypoxia-ischemia on the binding of 0.6 nM of (3Hjspiperone to dopamine D2 receptors

Specific binding (fmol per mg of tissue f SEM)

Normal control (n = 5) Sham control (n = 5) Moderate brain injury (n = 5) Brain areas Right Left Right Left a Right Left Decrease (9%)

OTu NAc CPU

Rostral Mid

Total DL DM VL VM

Caudal SNC

189.5 k 15.5 178.0 f 12.8

190.3 f 14.2

179.0 f 11.2 184.8 f 12.0 180.3 f 11.3 213.3 f 11.7 141.0 k 10.5 148.8 f 12.8 102.3 f 14.9

191.5f 13.5 177.3 f 12.0

182.5 f 11.2

171.3 f 14.6 180.3 k 14.3 185.5 f 14.0 211.3 f 12.1 136.0 * 11.5 150.3 k 13.6 93.7 f 10.4

180.0 f 11.7 167.5 f 15.6

188.7 -t 15.6

177.9 * 17.5 195.0 * 10.5 178.2 +. 11.7 217.7 f 11.4 141.0 f 12.5 148.0 f 10.4 107.7 k 16.3

174.5 f 13.1 171.7 f 16.5

202.8 f 12.3

174.0 t 17.7 207.3 f 14.7 173.3 f 10.1 219.2 f 12.3 143.3 f 12.0 154.8 k 12.4 117.0 f 17.4

189.3 f 13.8 170.0 t 14.0

191.5 f 13.5

178.3 f 16.2 209.0 f 10.5 181.8 f 11.8 215.5 f 12.6 142.5 f 13.8 152.5 f 12.3 116.0f 11.1

192.5 f 14.3 168.3 f 13.2

165.1 f 12.1b3c*d

149.3 f 12.6',' 158.8 f l l . Y g ~ " ~ ' 154.7 f 12.9',' 175.0 f 13.66,kvd 135.8 k 10.3 124.3 f 10.66*d 112.0 2 14.3

0 1

14

16 24 15 19 4

19 3

Nonspecific binding was defined by 10 &S-(-)-sulpiride and was subtracted from all readings. Values represent the mean f SEM. Differences between means were tested by one-way ANOVA, followed by a Scheffk post-hoc test. DL, dorsolateral; DM, dorsomedial; VL, ventrolateral; VM, ventromedial.

Side of the injury. ' D < 0.05: b~ < 0.01: g~ < 0.001. comDared with normal control. sham control, and nonlesioned side of rats with mild brain iniurv. ,~ -~

< 0.05; "2. < 0.01;' 'p < 0.001, compared with nonlesioned side of rats with moderate brain injury. p < 0.05; 'p < 0.01, compared with lesioned side of rats with mild brain injury.

paminergic presynaptic structures in the striatum are preserved after hypoxic-ischemic injury. This conclusion is also supported by our demonstration that levels of endogenous dopamine are unchanged in the CPu after this injury.

Although [3H]SCH 23390 is a highly specific and selective ligand for D I receptors, pharmacological studies have suggested that it might also label serotonin 5-HT2 receptors in the frontal cortex in rats (Bischoff et al., 1986; Hess et al., 1986). In the striatum, however, there is no evidence for significant binding of [3H]SCH 23390 to 5-HT2 receptors (Bischoff et al., 1986; Hess et al., 1986; Savasta et al., 1986). [3H]Spiperone binds to both serotonin 5-HT2 receptors and spirodecanone sites (Palacios et al., 1981). The use of the highly selective D2 antagonist sulpiride to define nonspecific binding, however, ensures that the specific binding of [3H]spiperone includes only D2 receptors (List and Seeman, 1981). Thus, the observed changes in both [3H]SCH 23390 and [3H]spiperone binding on the side of the lesion can be interpreted as a change in D1 and Dz receptors, respectively.

Observed decreases in both [3H]SCH 23390 and [3H]spiperone binding within the injured striatum can be interpreted as either the consequence of a loss of neural elements that express DA receptors or as receptor downregulation due to increased release of DA after the insult. We are in favor of the former interpretation because no changes in striatal DA levels or [3H]mazindol-labeled DA-uptake sites were found on the side of the lesion. The greatest decreases in both [3H]SCH 23390 and [3H]spiperone bindings were ob-

served in the dorsolateral quadrant, which we and oth- ers have shown is the striatal region that undergoes the most extensive neuronal loss in this animal model (Johnston, 1983; Femero et al., 1988; Burke and Kar- anas, 1990). This topographic correspondence indicates a relationship between the extent of neuron loss and the degree of receptor binding changes.

Striatal shrinkage observed after hypoxic-ischemic injury has been shown associated with neuronal loss, gliosis, and abnormalities in myelin (Fig. 1A) (John- ston, 1983). Because tritiated ligands have been used in this study, the altered striatal myelin may raise the question of whether the observed decreases in autoradiographic optical densities for Dl and DZ binding assays might be related to an attenuation in 0-emission due to the increase in lipid content (Kuhar and Un- nerstall, 1985). Other than the regions of myelin aggregation, which we avoided (Fig. l), however, only minimal heterogeneity of quenching was observed on the side of the lesion after sections were soaked in high concentration of [3H]isoleucine, a nonvolatile radioligand that is distributed evenly throughout the tissue (Kuhar and Unnerstall, 1985). In addition, the lack of similar changes in [3H]mazindol-generated autoradiographs (Fig. 5) is strongly against this possibility.

In addition to the change in the B,,, for [3H]SCH 23390 in rats with moderate injury, we also observed a significant increase in KD. We are uncertain of the reason for this decrease in affinity. It may be due to a conformational alteration of the receptor secondary to the hypoxic-ischemic injury. It is unlikely that the observed increase in the value of the apparent KD is due

J Neurochem.. Vol. 57, No. 6, 1991


FIG. 4. [3H]Spiperone (0.6 nM) binding in matched brain sections from normal controls (A and B) and rats with moderate brain injury (C and D). Pictures are representative color-coded transforms of original autoradiographs. Nonspecific binding (not shown), measured in the presence of 10 p,M unlabeled S-(-)-sulpiride, was similar in both group of rats and represented 18% of total binding.

to an increase in the concentration of an endogenous ligand, such as DA, because (1 ) the sections were preincubated before being labeled, (2) no changes in KD were found for the other two ligands, and (3) no significant increase in DA level was observed on the side of the lesion.

Both DA receptor subtypes are found in high concentrations in the CPu, distributed in a high-to-low lateromedial gradient (Dubois et al., 1986; Beckstead et al., 1988; Wamsley et al., 1989). Benfenati et al. (1 989) have also shown that transient forebrain ischemia in adult rats selectively produces a pronounced decrease in [3H]SCH 23390-labeled D1 receptors in the lateral and particularly dorsolateral striatum of rats. The cellular distribution of the D1 receptors in the CPu is uncertain (Ariano et al., 1989), but there are data suggesting that most, if not all, D1 receptors in the basal ganglia are associated with cell bodies located in the striatum and nerve terminals in the entopeduncular nucleus and SNR (Spano et al., 1977; Ariano and Ufkes, 1983; Gehlert et al., 1987; Beckstead, 1988; Ar- iano et al., 1989, Monsma et al., 1990; Sunahara et al., 1990). It has been reported that the medium striatal spiny neurons of rats project to SNR and bear D, receptors (Ariano, 1987; Monsma et al., 1990; Sunahara

et al., 1990). We hypothesize that the reduction in striatal [3H]SCH 23390-labeled D1 receptors found in our study is associated with the loss of these striatal cell population(s) that possess DI receptors. This hypothesis is compatible with our morphologic obser- vations in this model, which show that predominantly medium-sized striatal neurons are lost; large cholinergic striatal interneurons are relatively spared (Burke and Karanas, 1990). In addition, this loss could explain the decrease in r3H]SCH 23390 binding (34%) in the SNR. This decrease in striatal D1 binding would provide the first biochemical evidence of loss of striatal medium- sized neurons in this model. Although morphologic analysis suggests that the medium-sized striatal neurons are predominantly affected, previous measurement of biochemical markers have not revealed changes. John- ston (1983) found no changes in the specific activity of striatal glutamate decarboxylase or y-aminobutyric acid (GABA) uptake (both markers of GABAergic neurons) at 3 weeks of age; Feniero et al. (1988) found no changes in levels of somatostatin, neuropeptide Y, dynorphin, or Leu-enkephalin at 2 weeks; and we have observed no changes in somatostatin, Met-enkephalin, or substance P at 3 weeks (Burke et al., 1989).

D2 receptors in CPu may be located on both corti-

J. Nettrochem.. Vol. 5 7. No. 6 , I991


FIG. 5. [3H]Mazindol (15 nM) binding in matched brain sections from normal controls (A and B) and rats with moderate brain injury (C and D). Pictures are representative dark-field transforms of original autoradiographs, thus, brighter areas represent greater binding. Nonspecific binding (not shown), measured in the presence of 30 pM benztropine, was similar in both group of rats and represented 22% of total binding. VTA, ventral tegmental area.

costriatal nerve terminals and on neurons intrinsic to the CPu (Minneman et al., 1978; Schwartz et al., 1978). Although more recent receptor binding autoradiography (Joyce and Marshall, 1987) and in situ hybridization evidence (Mengod et al., 1989) favors a D2 receptor location principally, if not entirely, on neurons whose cell bodies are located in the CPu, there nevertheless remains pharmacologic evidence for D2 receptors on corticostriatal afferents (Maura et al., 1988). In this model, the hypoxic-ischemic injury not only affected the striatum, but also the cortex. Thus, it is possible that the observed decrease in striatal D2 receptors is due to either striatal or cortical neuronal cell loss, or both. Because the pathology predominates at the level of the striatum and the changes in binding are similar to those seen after intrastriatal excitotoxin injection (Joyce and Marshall, 1987), however, we suspect that the loss is mainly due to intrinsic striatal neuron in- volvement. In the SN, D2 receptors predominate in the pars compacta (Boyson et al., 1986; Charuchinda et al., 1987; Wamsley et al., 1989) and are located on DA cell bodies and dendrites (Filloux et al., 1987, 1988). Unlike DI receptors, nigral D2 receptor concentrations

were unchanged after perinatal hypoxic-ischemic in-

In conclusion, the results obtained here reveal a reduction of striatal D1 and D2 receptors after perinatal hypoxic-ischemic injury in rats, 2-3 weeks after the insult. The observed reductions are a reflection of striatal cell loss induced by the injury. In contrast, [3H]mazindol binding was normal on the lesioned side, suggesting that the dopaminergic presynaptic structures are preserved after the unilateral hypoxic-ischemic injury. The functional consequences of these alterations in dopaminergic markers are unknown. If nigrostriatal DA release in the extracellular space is maintained at normal levels, then the loss of receptor sites may result in diminished dopaminergic influence on striatal elements. On the other hand, if there is an enhanced release of DA, a possibility made plausible by the presence of an increased density of TH-positive neuropil (Burke et al., 199 l), then the loss of receptors may be less important functionally. It is clear that alterations of dopaminergic transmission in the striatal complex have a major effect on sensorimotor integration and normal motor control (Wooten and Trugman, 1989).

jury.

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I960 S. PRZEDBORSKI ET AL.

Therefore, further characterization of dopaminergic alterations in this rat model may prove important for understanding the neural basis of motor disturbances, such as dystonia, which occur after hypoxic-ischemic injury in the developing striaturn.

Acknowledgment: This work was supported by National Institutes of Health grant NS 26836, United Cerebral Palsy Research and Education Foundation, Inc., and the Parkin- son's Disease Foundation (New York, NY, U.S.A.). Serge Przedborski is supported by the National Fund for Scientific Research (FNRS, Belgium) and the Parkinson's Disease Foundation. Vladimir Kostic is supported by a Fulbnght Scholarship.

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Documents

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Dopamine D1 and D2 Receptors and Uptake Sites: A Quantitative Autoradiographic Study