Intranigral injections of SCH 23390 inhibit amphetamine-induced rotational behavior

56

Brain Reseatz'h. 623 (1993) 56-()4 .t~ 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19215

Intranigral injections of SCH 23390 inhibit amphetamine-induced rotational behavior David M. Yurek and Susan B. Hipkens Division of Neurosurgery and Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, KY 40536 (USA) (Accepted 20 April 1993)

Key words: Substantia nigra pars reticulata; Dopamine; Rodent; D1 receptor; Amphetamine; SCH 23390; Sulpiride; Apomorphine; Striatonigral pathway

Rats were given unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway and permanent indwelling cannula were surgically implanted into the non-lesioned side of the brain; cannula were used for direct injections of dopamine antagonists into the pars reticulata region of the non-lesioned substantia nigra. The selective D1 receptor antagonist, SCH 23390, was injected intranigrally at various concentrations (3.0, 1.5, 1.0, 0.6, or 0.3 mM) just prior to an intraperitoneal injection of amphetamine. SCH 23390 dose-dependently inhibited amphetamine-induced rotational behavior with the highest doses completely blocking rotational behavior in some animals. An intranigral injection of the selective D2 receptor antagonist, (-)-sulpiride (1.0 mM), did not produce a significant reduction in amphetamine-induced rotational behavior whereas an equivalent molar concentration of SCH 23390 (1.0 mM) produced a significant 62% reduction in amphetamine-induced rotational behavior. A concentration of SCH 23390 that produced a 50% reduction in rotational behavior when injected directly into the substantia nigra was unable to produce a significant reduction in rotational behavior when injected directly into the striatum. The effects of intranigral injections of SCH 23390 on apomorphine-induced rotational behavior were directly opposite to that observed for amphetamine-induced rotational behavior; contralateral rotational behavior increased relative to baseline measures. These data support the hypothesis that dopamine release in the midbrain may act as a neuromodulator of motor behavior, and that D1 receptors play a functional role in this process.

INTRODUCTION A n i m a l s with u n i l a t e r a l d e g e n e r a t i o n o f t h e nigrostriatal d o p a m i n e ( D A ) p a t h w a y show r o t a t i o n a l b e h a v ior w h e n t r e a t e d with a m p h e t a m i n e 3°'31, a n d t h e severity of d e g e n e r a t i o n , as m e a s u r e d by t h e p e r c e n t a g e o f striatal D A d e p l e t i o n , is positively c o r r e l a t e d with t h e n u m b e r o f r o t a t i o n s t h a t occur over a finite t i m e p e r i o d 15. A m p h e t a m i n e - i n d u c e d r o t a t i o n a l b e h a v i o r is b e l i e v e d to result f r o m an i m b a l a n c e in D A r e l e a s e b e t w e e n the l e s i o n e d a n d n o n - l e s i o n e d D A e r g i c p a t h ways, a n d as a c o n s e q u e n c e t h e n o n - l e s i o n e d D A e r g i c p a t h w a y affects i p s i l a t e r a l striatal o u t p u t p a t h w a y s t h a t a r e i n t e g r a t e d with m o t o r p a t h w a y s t h a t u l t i m a t e l y p r o d u c e a s y m m e t r i c a l l o c o m o t o r activity (rotations); in t h e case o f a m p h e t a m i n e t r e a t m e n t , t h e d i r e c t i o n o f l o c o m o t i o n is away f r o m the intact side o r t o w a r d the side of t h e lesion (ipsilateral). N i g r o s t r i a t a l D A e r g i c n e u r o n s of t h e n o n - l e s i o n e d p a t h w a y p r o j e c t exten-

sively to the i p s i l a t e r a l s t r i a t u m a n d r e l e a s e D A t h a t b i n d s to p o s t s y n a p t i c D A r e c e p t o r s o f several subtypes l o c a t e d on striatal n e u r o n s , a n d t h e s e n e u r o n s a r e involved in t h e m e d i a t i o n o f l o c o m o t o r b e h a v i o r including r o t a t i o n a l b e h a v i o r . In a d d i t i o n to striatal D A r e l e a s e , D A is r e l e a s e d within the s u b s t a n t i a nigra p a r s r e t i c u l a t a ( S N R ) a n d this p h e n o m e n o n has b e e n well d o c u m e n t e d . M o r p h o logical e v i d e n c e o f D A - c o n t a i n i n g s u b s t r a t e s in t h e d e n d r i t e s o f nigral n e u r o n s was c o n c u r r e n t l y r e p o r t e d by B j 6 r k l u n d a n d Lindvall 7 a n d S l a d e k a n d P a r n a v e l a s 28 in t h e rat a n d m o n k e y , respectively, a n d t h e d e n d r i t i c r e l e a s e o f D A was s u b s e q u e n t l y e s t a b l i s h e d a2'13'~4"2°. In vivo microdialysis studies have d e m o n s t r a t e d tonic D A r e l e a s e as well as p h a r m a c o l o g i c a l l y - i n d u c e d r e l e a s e of D A in t h e S N R 25'35. Physiological studies as well as r e c e p t o r a u t o r a d i o g r a p h i c studies o f D A r e c e p t o r s rev e a l e d D A r e c e p t o r s o f t h e D1 s u b t y p e in the SNR 2'6'9'11'22. Several n e u r o p h a r m a c o l o g i c a l studies

Correspondence: D.M. Yurek, Division of Neurosurgery, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536.

57 have provided evidence that dopamine released into the SNR has a direct effect on motor activity. For instance, direct injection of DA into the SNR resulted in increased locomotor activity and this effect was blocked with a DA antagonist 18. Similarly, unilateral injections of DA agonists into the SNR induced contralateral rotational behavior 19'21'29, suggesting that DAergic components in the substantia nigra are capable of initiating a motor response., Furthermore, animals with unilateral 6-OHDA lesions showed a modest reduction of levodopa-induced rotational behavior after intranigral injections of the D1 antagonist 24, SCH 23390, demonstrating that both nigral and striatal DA receptors act in concert for the expression of rotational behavior. Systemic injections of dopamine antagonists inhibit amphetamine-induced rotational behavior, with SCH 23390 demonstrating the lowest EDs0 when compared to EDsos for nonselective or selective D2 antagonists 3,5. SCH 23390's efficacy for blocking rotational behavior is examined in this study. Receptor binding studies have identified two brain sites with relatively high densities of D1 receptors: the substantia nigra and the striatum 2'6'9. In this study amphetamine-induced rotational behavior was measured after SCH 23390 was intracerebrally injected into the substantia nigra or striatum. MATERIALS AND METHODS

6-Hydroxydopamine lesions Male Sprague-Dawley rats (225-250 g, Harlan Farms) were given unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway. 6-hydroxydopamine hydrobromide (Sigma) was dissolved in 0.9% saline (containing 0.2% ascorbic acid) at a concentration of 2.0/~g//.d and stereotactically injected into the nigrostriatal pathway of anesthetized rats at a rate of 1.0 M / m i n for 3 rain. Each rat received two injections of 6-OHDA" one in the vicinity of the medial forebrain bundle (AP - 4.3, ML 1.2, DV - 7.5) and the other in the rostral pars compacta of the substantia nigra (AP -4.8, ML 1.5, DV -7.5); all coordinates reported in this study represent millimeter adjustments from bregma (AP, ML) and below the dural surface (DV) with the top of the skull in a flat position. Rotational behavior was monitored using the Videomex V image motion computer system (Columbus Instruments Inc.). Rodents were administered drug treatments and then individually placed inside opaque 16 inch diameter cylindrical chambers, which were situated directly beneath a video camera; multiple animals were monitored simultaneously. Videomex V system digitized the image of the ani-

mal and calculated the total number of 360° rotations that occurred during a ninety minute testing period s.

Cannulation methods Permanent indwelling guide cannulae were stereotactically implanted into the non-lesioned hemisphere of the brain at the same time the contralateral nigrostriatal DAergic pathway was lesioned with 6-OHDA. Guide cannula (22-gauge stainless steel, Plastics One) were stereotactically lowered into the brain and targeted for a site 3.0 mm dorsal to the SNR; coordinates: AP - 5.0, ML 2.3, DV - 4.8. Guide cannula were permanently affixed to the skull using anchor screws and dental acrylic, and a replaceable dummy cannula (28gauge, Plastics One) was inserted into the guide cannula immediately after surgery. Two types of injectors (28-gauge internal cannula, Plastics One) were designed to fit within the guide cannula and both were used for intracerebral injections. The first injector type was designed to penetrate into the brain parenchyma 0.5 mm beyond the tip of the guide cannula, this type of injector was used for negative placement injections. The second injector type was designed to extend 3.0 mm from the guide cannula tip and penetrate ventrally into the brain parenchyma of the SNR, and this injector type was used for positive placement injections. A second group of lesioned animals (Experiment 2) were given a permanently indwelling cannula that was strictly targeted for a negative site placement in the non-lesioned hemisphere and the coordinates for stereotactic placement of these cannulae were: AP - 5.2, ML 3.2, DV - 4.4. The first injector type was used to administered drug solutions into this site. A third group of lesioned animals (Experiment 3) received permanent indwelling cannula targeted for the non-lesioned striatum (AP + 0.6, ML 3.0, DV -5.0) or the non-lesioned substantia nigra (AP -5.0, ML 2.3, DV -4.8). The first injector type was used to administer drug solutions into these sites. A fourth group of lesioned animals (Experiment 4) were cannulated using the same procedures and coordinates as described in Experiment 1. These animals were subsequently pretreated with intracerebral injections (second injector type) into the non-lesioned substantia nigra with either 0.9% saline, (-)-sulpiride, or SCH 23390 ten minutes prior to an intraperitoneal injection of amphetamine. This group was used to compare the effects of selectively blocking D2 receptors vs. D1 receptors in the substantia nigra. A fifth group of lesioned animals (Experiment 5) were cannulated using the same procedures and coordinates as described in Experiment 1. These animals were pretreated with intracerebral injections (second injector type) of 0.9% saline or SCH 23390 into the non-lesioned substantia nigra ten minutes prior to an intraperitoneal injection of the direct DAergic agonist, apomorphine. Rotational behavior induced with apomorphine was quantified and compared for saline vs. SCH 23390 pretreatments.

Drug injections All intracerebral injections were made in unanesthetized, freelymoving animals. Injection cannula (28-gauge, Plastics One) were connected to a 1 ml syringe of a CMA/100 Microinjection pump (Carnegie Medicin) using PE 10 tubing as a conduit between the pump and injection cannula. R(+)-SCH 23390 hydrochloride (m.w. = 324.1, Research Biochemicals) was dissolved in 0.9% saline. S(-)-Sulpiride (m.w. = 341.4, Research Biochemicals) was dissolved in 5% lactic acid. D-amphetamine sulfate (Sigma) and apomorphine

TABLE I

SCH 23390 dosing schedule (l~g / 3 ~1 saline) Group

Day 0

Group

B1

Day 12 B2

Day 24 B3

Day 36 B4

Day 48

3.0

3.0

1.5

1.5

0.6

0.6

1.0

1.0

1 (n = 4) 2 (n = 4)

pos. neg.

neg.

pos.

neg.

pos.

neg.

pos.

neg.

B5

pos.

0.3

0.3 neg.

pos.

neg.

pos.

neg.

pos.

neg.

pos.

neg.

pos.

3 (n = 12)

pos.

-

pos.

-

pos.

-

pos.

-

pos.

-

58 hydrochloride (Sigma) were dissolved in 0.9% saline and administered intraperitoneally at a dose of 5.0 m g / k g and 0.2 mg/kg, respectively. All control injections were made with 0.9% saline except for the sulpiride control, for which 5% lactic acid was used. All intracerebral drug injections including control injections were delivered at a rate of 1.0/~l/min for 3 min.

Histological t,erification of cannulae Animals were perfused intracardially with ice-cold 0.9% saline and 4% paraformaldehyde. Brains were removed and and placed in 30% sucrose before being sliced into 35/xm sections. Sections were stained with cresyl violet and cannula tracks were identified. The ventral tip of each cannula track was noted and a plot of tip placements is shown in Fig. 1.

Experiment L Schedule of treatments Baseline values of amphetamine-induced rotational behavior were determined three weeks after animals received 6-OHDA lesions and animals were then divided into three groups; each group contained an equal representation of good rotators ( > 7 rotations/min) and moderate rotators (5-7 rotations/min) in order to balance the groups based upon their average rate of rotation for the 90 min testing period. All baseline scores were measured 10 min after an injection of 3 ~1 of 0.9% saline into the SNR. After initial baseline scores were obtained (B1), testing was performed every fourth day and various concentrations of SCH 23390 were dissolved into 0.9% saline and injected into positive or negative placement sites 10 min prior to an injection of amphetamine (5.0 mg/kg, i.p.); the schedule of SCH 23390 doses as given in Table 1 was used. Baseline amphetamine-induced rotational behavior was reassessed between each change of dose (B2, B3, B4, B5), which also conincided with every twelveth day of the experiment. A positive placement (pos.) was defined as a direct injection into the SNR. A negative placement (neg.) was defined as an injection into a site 2.5 mm dorsal to the SNR. The above schedule was used for groups 1 and 2 in order to determine carry-over effects related to the sequence in which animals received positive and negative placements.

Data analysis For Experiment 1, analysis of variance (ANOVA) with repeated measures was performed on baseline rotational data alone in order to determine whether ,or not baseline (control) rotational scores changed throughout the course of this experiment. As state above, animals in group 1 or 2 received intracerebral injections into positive or negative sites in different sequences in order to determine whether or not the sequence of injections contributed to differences in rotational scores, therefore a split-plot factorial design was utilized in this first analysis. The dataset included one between group factor (SEQUENCE) and one within group factor (BASELINE). The main effect of the factor S E Q U E N C E (F1.24 = 2.08, P > 0.20) was not statistically significant suggesting that the sequence of SCH 23390 injections into positive or negative placements did not have a significant effect on baseline scores. The main effect of the factor BASELINE (F4,24 = 1.39, P > 0.26) was not statistically significant, suggesting that baseline rotational scores did not change significantly throughout the treatment schedule. The S E Q U E N C E x BASELINE interaction term (F4,24 = 1.19, P > 0.33) was also statistically insignificant. Baseline data for group 3 were analyzed using a randomized block design with one level of BASELINE. Results of this analysis

A o o

Fig. 1. Diagram of coronal brain sections showing four different sites of drug injections. Cannula tracks were verified histologically. Each black dot designates the end of a cannula track, which also represents the approximate site for intracerebral drug injections; all sites were in the hemisphere contralateral to the side of the 6-OHDA lesion. A: sites for intrastriatal injections. Injector tip was target for the following coordinates: AP +0.6, ML 3.0, DV - 5 . 0 . B: positive and negative sites used in Experiment 1, groups I, II, III. Dots below double-headed arrow represent the sites for positive injections, which were targeted for a medial region of the substantia nigra pars reticulata (snr): AP - 5.0, ML 2.3, DV - 7 . 8 . Dots above double-headed arrow represent the sites for negative injections, which were targeted for a site 2.5 mm dorsal to the positive site and included areas ventral to the pretectal nucleus (npt) and portions of the medial geniculate nucleus (cgm): AP - 5.0, ML 2.3, DV -5.3. C: negative injection site: AP - 5 . 2 , ML 3.2, DV - 4 . 4 .

59 revealed that baseline scores did not change significantly throughout the treatment schedule for this group (F4,44 = 1.06, P > 0.37). Rotational data for groups 1 and 2 were combined and analyzed using a split-plot factorial design. The S E Q U E N C E of injections was designated as the between group effect. Two within group effects were analyzed: nested in each of the 5 levels of DOSE were three types of INJECTIONS (saline, drug-positive placement, or drugnegative placement). The main effects of DOSE (F6,24 = 10.8, P < 0.001) and of INJECTIONS (F2,12 = 44.3, P < 0.001) were statistically significant. The main effect of S E Q U E N C E (Ft, 6 = 0.80, P > 0.40) was not statistically significant, indicating that the sequence of injection placements did not influence rotational behavior. A statistically significant D O S E × INJECTION interaction (/'8,48 = 2.26, P < 0.05) was also detected; all other interaction terms were not statistically significant. Rotational data for group 3 were analyzed using a randomized block design. Rotational data was obtained at 5 levels of DOSE and nested within each dose level were two types of INJECTIONS (control or drug). The main effects of DOSE (F4,44 = 12.83, P < 0.001) and INJECTIONS ( F m l = 65.50, P < 0.001) were statistically significant. A statistically significant D O S E × I N J E C T I O N interaction (F4,44 = 8.43, P < 0.001) was also detected.

RESULTS

Experiment 1. Effect of intranigral SCH 23390 injections into the substantia nigra Fig. 2A shows the effects of injecting various doses of SCH 23390 or 0.9% saline directly into the substantia nigra, or injecting SCH 23390 into a negative site that was 2.5 mm dorsal to the substantia nigra. Analysis of variance was performed on these data; means were calculated for the total number of rotations that occurred over a ninety minute period. Mean rotational scores for animals pretreated with all doses of SCH

A

•

I.

S00

'°

-

~

SALINE I SCH-NEGATIVIE

I

800 .= ~

1

600

o~

SALINE I POSITIVE

400

rr ~, 400] ! 201] o.o

1000

_,

,

600

23390 injected into positive placements sites were found to be significantly lower than those observed for saline pretreatment when data for groups 1 and 2 were combined, and the same was also observed for group 3 animals that only received injections of SCH 23390 into a positive site (Fig. 2A and B). Mean rotational scores for animals pretreated with 1.0, 1.5, or 3.0 mM of SCH 23390 injected into a negative site were significantly lower than saline pretreated animals; mean rotational scores for animals pretreated with 0.6 or 0.3 mM of SCH 23390 into negative sites were not statistically different than scores for saline pretreatment. Except for the lowest dose of SCH 23390 (0.3 mM), mean rotational scores for all other doses of SCH 23390 injected into the positive site were significantly lower than those for the negative site. Saline scores did not change significantly throughout the course of this experiment as determined by the analysis mentioned in the Data Analysis section. The time course of amphetamine-induced rotational behavior was significantly altered by SCH 23390 pretreatment into positive sites (Fig. 3). Mean rotational scores for all doses of SCH 23390, except 0.3 mM, were significantly lower than saline scores at all time points. Fig. 4 shows that pretreatment with 0.3 mM of SCH 23390 significantly lowered amphetamine-induced rotational scores during the first 30 rain when compared to saline pretreatment, however, at later time points rotational scores for these two groups were not statistical different from each other.

200,0 ~:o

2:0

SCH23390(raM)

a.o

4:0

o.o

~:o

2:0

3:0

,*:o

SCH23390(mM)

Fig. 2. Inhibition of amphetamine-induced rotational behavior after intracerebral injections of saline or various doses of SCH 23390 into the non-lesionod substantia nigra. Each data point represents the mean value of total rotations that occurred over a 90 min period; error bars represent the standard error of mean. Unilaterally lesioned animals were pretreated with an intracerebral injection of either saline or one of the following five doses of SCH 23390: 0.3, 0.6, 1.0, 1.5, or 3.0 mM. Amphetamine (5.0 mg/kg, i.p.) was administered ten minutes after pretreatment and rotational behavior was quantified over a ninety minute period. (A) Experiment 1, groups 1 and 2 combined; n = 8 for each data point (B) Experiment 1, group 3 only; n = 12 for each data point. * P < 0.01 and * * P < 0.001 vs. saline mean; t p < 0.01 vs. SCH-negative mean.

60 '

q

8°°t

8

600 -4 6£ ~

~

_ ~

e

SALINE

,

0atom

N

06mM

~E

(~.

•

1 0raM

1:I.o

--

it

15mM

o

30raM

~

10

400 c

o_ 200 •

20

40

60

80

i

100

Time Post-Amphetamine (minutes)

0,

Fig. 3. Time course of amphetamine-induced rotational behavior following pretreatment with an intranigral injection of saline or various doses of SCH 23390. Animals with unilateral 6 - O H D A lesions were pretreated with an intranigral injection of either saline or one of the following five doses of SCH 23390: 0.3, 0.6, 1.0, 1.5, or 3.0 mM. A m p h e t a m i n e (5.0 m g / k g , i.p.) was administered ten minutes after pretreatment and rotational behavior was quantified every two minutes throughout a ninety minute observation period. Each data point represents the mean value (n = 12) of the total n u m b e r of rotations counted during each two minute interval. Analysis of variance revealed that for every SCH 23390 dose, except for 0.3 mM, rotational scores for SCH 23390 pretreated animals were significantly lower than saline scores at all time points.

Saline

SCH 23390

Fig. 5. A m p h e t a m i n e - i n d u c e d rotational behavior after pretreatment with saline or SCH 23390 into a second negative site. Six animals with unilateral 6 - O H D A lesions were pretreated with an intracerebral injection of saline or 1.0 m M SCH 23390 into a site bordering the medial and lateral geniculate nuclei (Fig. 1C). A m p h e t a m i n e (5.0 m g / k g , i.p.) was administered and rotational behavior was quantified over a ninety minute period. The left bar indicates the mean value_+ s.e.m, for total n u m b e r of rotations 90 min after saline pretreatment. O n e week later the same animals received a pretreatment injection of 1.0 m M SCH 23390, and the right bar indicates the mean value + s.e.m, for total n u m b e r of rotations that occurred after this pretreatment. Statistical analysis (paired t-test) of these data revealed that m e a n rotational scores for saline vs. SCH 23390 pretreatment were not statistical different, P > 0.43.

Experiment 2. Negative site injection of SCH 23390 A second group of animals were cannulated into a negative site that was different from the one used in Experiment 1 (Fig. 1C). These animals received injections of either 0.9% saline or SCH 23390 (1.0 mM) into this exclusive site. Fig. 5 shows that animals pretreated with an intracerebral injection of 1.0 mM SCH 23390 into this second negative site did not show a statistically significant reduction in amphetamine-induced rotational behavior when compared to their saline pretreatment scores (paired t-test, P > 0.43).

30

"6E r'r" 04

20

10.

•

a

'

SALINE .3 mM

20

40

60

80

I

I

100

Time Post-Amphetamine (minutes)

Fig. 4. Time course comparisons for saline versus 0.3 m M SCH 23390 pretreated animals from Fig. 3. Each data point represents the m e a n v a l u e + s . e . m . ( n = 1 2 ) for the total n u m b e r of rotations counted during each two minute interval. Statistcial analysis indicates that rotational scores for the 0.3 m M SCH 23390 pretreated animals were significantly lower than saline scores during the first 30 rain.

Experiment 3. SCH 23390 injection into the striatum us. substantia nigra Two groups of unilaterally lesioned animals were cannulated into the non-lesioned striatum or the nonlesioned substantia nigra. Baseline scores for amphetamine-induced rotational behavior were obtained three weeks after lesioning. One week later the animals were pretreated with 0.6 mM of SCH 23390 into the striatum or substantia nigra, and 10 min later these same animals received an intraperitoneal injection of amphetamine (5.0 mg/kg, i.p.). Fig. 6 shows that intrastriatal injections of 0.6 mM SCH 23390 did not significantly attentuate amphetamine-induced rotational behavior (paired t-test, P > 0.20), while the same molar concentration of SCH 23390, when injected intranigrally, produced a significant reduction in rotational scores (paired t-tests, P < 0.01).

Experiment 4. Intranigral injections of sulpiride ( - ) - S u l p i r i d e was injected intranigrally in order to observe the effect of selective D2 receptor blockade on amphetamine-induced rotational behavior. ( - ) - S u l p i ride was injected intracerebrally using the same methodology described for SCH 23390 injections in Experiment 1. Fig. 7 shows that pretreatment with an injection of 1.0 mM ( - ) - s u l p i r i d e did not show a significant reduction in amphetamine-induced rotational behavior when compared to previously measured

61

800t

1000

800

;aline ~CH 23390

O

600,

0~

600

--0 0~0~

~0

S~

~E ~v

400

400 c o

o 200

200

0

Slriaturn

Nigra

0

Fig. 6. Effect of intrastriatal SCH 23390 versus intranigral SCH 23390 on amphetamine-induced rotational behavior. Two groups of animals with unilateral 6-OHDA lesions were cannulated intrastriatally (n = 8, Fig. 1A) or intranigrally (n = 6, Fig. 1B) into the non-lesioned hemisphere. Both groups received pretreatment of intracerebral saline or 0.6 mM SCH 23390 injections, administered amphetamine (5.0 mg/kg, i.p.) ten minutes later, and then rotational behavior was quantified for a ninety minute period. Black bars indicate the mean value + S.E.M. for total number of rotations that occurred after receiving a pretreatment injection of saline. One week later the same animals received a pretreatment injection of 0.6 mM SCH 23390, and the stippled bars indicate the mean value + S.E.M. for total number of rotations that occurred after this treatment. Statistical analysis (paired t-tests) of these data revealed that rotational scores for saline vs. 0.6 mM SCH 23390 treatments were not statistical different for the intrastriatal group ( P > 0.20), while rotational scores for the 0.6 mM SCH 23390 treatment were significantly lower than saline scores for the intranigral group (* * P < 0.01).

1000.

Saline

SCH 23390

Fig. 8. Effect of intranigral SCH 23390 on apomorphine-induced rotational behavior. A group of six animals were given unilateral 6-OHDA lesions and screened for apomorphine-induced rotational behavior; baseline rotational scores induced with apomorphine (0.2 mg/kg, i.p.) alone were determined over a three week period. These same animals were then pretreated with an intranigral injection of SCH 23390 (1.0 mM) into the non-lesioned substantia nigra, and ten minutes later administered apomorphine. Bars represent mean values+s.e.m, for total number of rotations during a ninety minute observation period. Statistical analysis (paired t-test) revealed that rotational scores for animals pretereated with SCH 23390 (1.0 mM) were significantly higher than saline pretreatment scores (* P < 0.05).

baseline scores. When an equivalent molar concentration of SCH 23390 was injected into the same site, these same animals demonstrated a statistically significant 62% reduction in amphetamine-induced rotational when compared to their previously measured baseline scores.

800.

Experiment 5. Effect of intranigral injections of SCH 23390 on apomorphine-induced rotational behavior

O~ n-" o

600.

~E ~2

200.

0,

SALINE

SULPIRIDE (1.0 rnM)

SCH 23390 (1,0 rnM)

Fig. 7. Comparison of intranigral injections of ( - )-sulpiride or SCH 23390 on amphetamine-induced rotational behavior. A group of seven animals with unilateral 6-OHDA lesions were pretreated with intranigral injections of saline, sulpiride (1.0 raM), or SCH 23390 (1.0 raM) into the non-lesioned substantia nigra and ten minutes later administered amphetamine (5.0 mg/kg, i.p.); rotational scores were totaled during a ninety minute observation period. The animals were first pretreated with saline, one week later this same group was pretreated with SCH 23390 (1.0 mM), and one week later the same group was pretreated with sulpiride (1.0 mM). Bars represent the mean ±s.e.m. of rotational scores for each treatment. Analysis of variance with repeated measures detected a statistically significant effect of drug treatment (F2,12 = 16.2, P = 0.0004) and mean comparisons revealed that rotational scores for animals pretreated with with sulpiride (1.0 mM) were not significantly different from saline scores ( P > 0.96) while rotational scores for animals pretreated with SCH 23390 were significantly lower than saline scores ( * * P < 0.01).

Animals given unilateral 6-OHDA lesions were tested for rotational behavior induced with an injection of apomorphine (0.2 mg/kg, i.p.) and baseline rotational scores were obtained over a three week period (Fig. 8). Subsequently, animals were pretreated with an intranigral injection of 1.0 mM SCH 23390 into the non-lesioned substantia nigra and then tested again for apomorphine-induced rotational behavior. Animals pretreated with SCH 23390 showed a statistically significant increase in rotational scores when compared to their average baseline scores (paired t-test, P < 0.05). DISCUSSION

These data suggest that DAergic activity in the substantia nigra is an integral component for the expression of rotational behavior. Selective blockade of nigral D1 receptors with SCH 23390 dose-dependently inhibited amphetamine-induced rotational behavior whereas selective blockade of striatal D1 receptors did

62 not reduce amphetamine-induced rotational behavior. Past pharmacological studies have suggested that D1 and D2 agonists induce rotational behavior via stimulation of different output pathways ~6. Data from this study indicate that D1 mediated rotational behavior involves activity at receptors in the subtantia nigra, and those D1 receptors are most likely located on the terminals of striatonigral neurons. In Experiment 1 we found that injections of SCH 23390 into a negative site produced a significant reduction in amphetamine-induced rotational scores, and this phenomenon may be a result of using the same guide cannula for injections of SCH 23390 into both positive and negative sites. For instance, when the injector cannula was introduced into a positive site, it produced a cannula track directly into the substantia nigra, and it is conceivable that this cannula track may have facilitated the ventral diffusion of SCH 23390 into the substantia nigra even when drug injections were made into the negative site. On the other hand, when a second negative site was chosen that was caudal to the first negative placement, and when this site was used exclusively for negative injections of SCH 23390, it was found that 1.0 mM SCH 23390 was unable to significantly reduce amphetamine-induced rotational behavior. A third negative site injection, those injections of SCH 23390 into the striatum, also did not produce a significant reduction in amphetamine-induced rotational behavior; this latter site is important when one considers the abundance of D1 receptors found in this site. When these data are taken together they suggest that the focus of inhibition occurs at sites in or near the substantia nigra, and that SCH 23390 injections into other sites that are caudal, rostral, or dorsal to the positive placement tend to diminish its inhibiting effects on amphetamine-induced rotational behavior. What remains to be determined are the mechanisms by which intranigral injections of high doses of SCH 23390 were capable of completely inhibiting amphetamine-induced rotational behavior when striatal output pathways other than the striatonigral pathway were also affected by amphetamine stimulated D A release within the striatum. The classical striatal output pathways involve striatal efferents projecting to both the internal (GPi) and external (GPe) segements of the globus pallidus and these pathways are also involved with motor behavior, including rotational behavior. The complete inhibition of amphetamine-induced rotational behavior may be related to the distribution of D1 receptors in other brain sites that are known to modulate striatopallidal output. Besides the high density of D1 receptors found in the striatum and SNR, two other brain regions also contain moderate

densities of D1 receptors: the entopenduncular nucleus (EP), which is the rodent homologue of the GPi, and the subthalamic nucleus 2'6'9. It is unlikely that drug diffusion from the SNR to the EP was responsible for the inhibitory effects of SCH 23390 because the EP is anatomically located approximately 2.5 to 3.0 mm rostral to the SNR. When SCH 23390 was injected into a striatal site that was approximately 3.0 mm rostral to the central region of the EP, this treatment did not result in an inhibition of amphetamine-induced rotational behavior. The striatal and nigral injections sites used in this study were nearly equidistant from the EP, therefore if the diffusion of SCH 23390 from the site of injection to the EP was responsible for inhibition of amphetamine-induced rotational behavior, then injections of SCH 23390 into striatal or nigral sites should have produced similar results, which was not the case. Although this study does not definitively rule out the possibility that SCH 23390 was acting via D1 receptor blockade in the EP, it is unlikely that this was the case and this conclusion is based upon the various injection sites used in these experiments. The SNR is anatomically closer to the subthalamic nucleus (STN) than it is to the EP. The exact function of the STN is unclear, however, there is evidence that STN efferents modulate the output pathway of the GPi to the thalamus hI°. It is conceivable that intranigral injections of the higher doses of SCH 23390 diffused to the STN and modified striatopallidal output via activity of SCH 23390 in the subthalamic region, and therefore both striatonigral and striatopallidal pathways were modified by this treatment. Both the SNR and STN are in close proximity to each other, and this close anatomical relationship poses technical problems for studying intracerebral injections of functionally active doses of drugs into one site without affecting the other site. There is some debate to relevance and function of D1 receptors in the STN, and it is also important to note that one study reports negligible D1 binding in the STN 9. It remains a difficult task to determine whether the observed inhibition of amphetamine-induced rotational behavior is a result of SCH 23390's activity on the striatonigral pathway alone, or whether or not the combined activity of SCH 23390 in the STN and SNR produces this phenomenon. Nevertheless, this study demonstrates the significance of D1 receptor function in the midbrain. Although there is evidence that DA receptors in the SNR have picomolar affinities for SCH 233902, inhibition of amphetamine-induced rotational behavior required concentrations of SCH 23390 in a micromolar to millimolar range. The most likely explanation for the discrepancy between binding studies and this behavioral study takes into account the temporal effects of

63 receptor blockade. Unlike binding studies which measure ligand-receptor interactions under static conditions, rotational behavior elicited by amphetamine treatment requires a more dynamic and sustained neuropharmacological interaction between ligand and receptors. Drugs injected intracerebrally diffuse throughout the extracellular space, and are eliminated and cleared from the brain just as they are in other physiologic systems; it is conceivable that millimolar concentrations of SCH 23390 may be required in order to circumvent the problem of drug clearance in order to maintain a competitive concentration of SCH 23390 at D1 receptor sites throughout this ninety minute time period. This explanation is supported by observing the time course for the inhibition of rotational behavior at the lowest SCH 23390 dose (0.3 mM): at initial time points the effects of SCH 23390 are significantly lower than saline scores, however, at later time points the effects of SCH 23390 are not significantly different than saline values at corresponding time points. It is unclear if the observed effects of intranigral injections of SCH 23390 are strictly mediated by D1 receptors located on the terminals of striatonigral neurons, or whether another DA sensitive neuronal pathway is involved. Dopamine exerts an excitatory effect on striatonigral neurons that results in an increased release of GABA within the S N R 17'27 and behavioral studies have demonstrated that direct injections of GABA agonists into the substantia nigra elicit rotational behavior in a direction contralateral to the site of injection4. Several other electrophysiological and biochemical studies have demonstrated that DA release within the substantia nigra modulates GABAergic activity in the substantia n i g r a 23'27'32'33. It is unknown what effect blocking D1 receptors on the terminals of striatonigral neurons has on GABAergic activity in the substantia nigra, and it is conceivable that blockade of D1 receptors on these terminals alters GABA release and thus the effects of SCH 23390 on rotational behavior ultimately may be a function of GABA release within the substantia nigra. If this is the case, then our data would indicate that selective blockade of D1 nigral receptors diminishes GABA release because rotational behavior in a direction contralateral to the site of D1 receptor blockade is inhibited by this treatment. Another consideration is the effect that somatodendritic release of DA might have on nigrothalamic projections, and how SCH 23390 might exert its inhibitory effect via this pathway. There is no known evidence that D1 receptors exist on nigrothalamic neurons, however, there is evidence that iontophoretic application of DA onto neurons in the pars reticulata alters the electrophysiological response of nigrothala-

mic neurons projecting to the ventromedial nucleus of the thalamus 26. Although the blockade of D1 receptors in the nonlesioned substantia nigra inhibited amphetamine-induced rotational behavior, apomorphine-induced rotational behavior was augmented by SCH 23390 pretreatment. This may have been a result of selectively blocking striatal output from the non-lesioned striatum while the striatal output pathway from the lesioned striatum was spared from pharmacological challenge. Amphetamine-induced rotational behavior is a measure of DAergic activity mediated through the non-lesioned DAergic pathways. On the other hand, rotational behavior induced with apomorphine, a direct agonist, is a measure of imbalance between the up-regulated DAergic receptors on the lesioned side versus normosensitive DA receptors on the non-lesioned side, and thus pathways associated with the lesioned side predominate when a direct agonist is administered. In the case of apomorphine-induced rotational behavior, a greater imbalance of DAergic activity may result after selectively blocking the output of the non-lesioned striatum, and this may explain why we observed an increase in apomorphine-induced rotational behavior. HerreraMarschitz and Ungerstedt 16 suggested that apomorphine-induced rotational behavior is dependent upon the striatonigral pathway and their model would predict an augmentation of apomorphine-induced rotational behavior following inhibition of the contralateral striatonigral pathway, as was observed in this study. These data demonstrate that D1 receptors in the substantia nigra mediate rotational behavior, and also implicate the importance of DA release within the substantia nigra as a component of motor function. Studies which examine therapeutic strategies for restoring DAergic function after degeneration of the nigrostriatal pathway often focus on DAergic mechanisms within the striatum, however, DA restoration within the substantia nigra may be just as relevant. A recent neural grafting study demonstrated that DA grafts placed in proximity to the substantia nigra may facilitate the compensatory effects of intrastriatal DA grafts in animals with unilateral 6-OHDA lesions 34, and it is conceivable that this phenomenon may be related to DAergic modulation of the striatonigral pathway. Acknowledgements.

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