Brain Research, 167 (1979) 355-365 © Elsevier/North-Holland Biomedical Press

355

DISSOCIATION BETWEEN T H E PRESYNAPT1C DOPAMINE-SENSITIVE A D E N Y L A T E CYCLASE AND [~H]SPIPERONE B I N D I N G SITES IN RAT SUBSTANTIA N I G R A

M. QUIK*, P. C. EMSON and E. JOYCE MRC Neuroehemical Pharmacology Unit, Department of Pharmacology, Medical School, Cambridge and ( E.J.) Department of Psyehology, University of Cambridge, Cambridge (Great Britain)

(Accepted August 24th, 1978)

SUMMARY [3H]Spiperone binding sites and the dopamine-sensitive adenylate cyclase were measured in rat substantia nigra (s. nigra) 7 or 14 days after various lesions. Hemisections, which resulted in a 6 6 ~ decline in tyrosine hydroxylase and cyclic nucleotide phosphodiesterase and a 73 ~ decrease in glutamate decarboxylase, led to a 50 ~ decrease in [3H]spiperone binding and to the almost complete disappearance of the dopamine-sensitive adenylate cyclase from the s. nigra on the lesioned side. 6Hydroxydopamine injection into the s. nigra, which depleted tyrosine hydroxylase activity within the s. nigra by 8 5 ~ , while leaving phosphodiesterase unaffected, resulted in a 40 ~ decrease in [ZH]spiperone binding but no change in the dopaminesensitive adenylate cyclase, lntrastriatal injections of kainic acid did not alter tyrosine hydroxylase activity in the s. nigra, but decreased both glutamate decarboxylase (54 ~ ) and phosphodiesterase (68 ~); [3H]spiperone binding was unaffected by this lesion while the dopamine-sensitive adenylate cyclase was greatly reduced (50-75 ~). These results suggest that within the s. nigra the dopamine receptor binding sites as defined using [3H]spiperone are located on dopamine neurones while the dopamine-sensitive adenylate cyclase is located presynaptically on striatonigral nerve terminals.

INTRODUCTION The dopamine-sensitive adenylate cyclase which exists in homogenates of dopamine-rich areas of brain such as striatum is thought to represent a CNS * Present address: Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond St., Montreal, Quebec, H3G 1Y6 Canada.

356 dopamine receptor4,1z, 17,2v. The substantia nigra (s. nigra) has been shown to contain a dopamine-sensitive adenylate cyclase with characteristics similar to the striatal enzyme 18,33,'~9,'~1. To determine the cellular location of the dopamine-sens itivc adenytate cyclase in rat s. nigra, lesion experiments involving iniection o f 6-hydroxydopamine (6-OHDA) into the s. nigra or medial forebrain bundle, hemisections, selective destruction of striatonigral and pallidonigral projections, and intrastriatal kainic acid injections have been used :~2,'~4,:3~;. The ,esults of these experiments have demonstrated that the dopamine-sensitive adenylate cyclase is localized presynaptically on the terminals of descending afferents to the s. nigra. The binding of radiolabelled agonists or antagonists can also be used:to identify and characterize possible transmitter receptors. [3H]Spiperone, a potent butyrophenone neuroleptic drug, is one of the ligands currently used to selectively label CNS dopamine receptors because of its high affinity for the dopamine receptor and low non-specific binding 7,s,2z. In order to investigate the relationship between the dopamine-sensitive adenylate cyclase and the dopamine receptor binding sites defined by [3H]spiperone, a series of different lesions were made within the striatonigral complex in rat brain. These lesions include 6-OHDA injection into the s. nigra, kainic acid injection into the striatum and hemisections of the striatonigral pathway. M ETHO DS

Stereotactic surgery Male Sprague-Dawley rats weighing approximately 300 g [280-340 g) were anaesthetized with Equithesin (0.8-1.0 rot) and placed in a stereotaxic frame (David Kopf). Coronal hemisections were performed unilaterally using a tungsten wire microknife as described by Paxinos and Bindra ~°. The knife cuts were made at the level of the posterior hypothalamus and severed both the medial forebrain bundle and the internal capsule. 6-OHDA injections (8 #g 6-OHDA/2/~1 saline-containing ascorbic acid 1 mg/ml) were made into the s. nigra at AP 3.0 mm, L 2.0 ram, D 7.8 mm according to the atlas of Pellegrmo and Cushman ~1. Rats which received 6-OHDA injections were pretreated with desmethylimipramine (15 mg/kg) 30 rain before. An additional group of rats received kainic acid (3 #g kainic acid/2 #1 phosphate-buffered saline) into the caudate putamenat AP 2.5 mm, L 2.5 mm. D 5.5 mm31. Rats were killed after 7 or 14 days survival. The brains were rapidly removed, placed on ice and the control and lesioned s. nigra were dissected as described by Jessell TM. The dissection of the s. nigra from 400 ,urn thick brain slices included both pars compacta and reticutata regions, but excluded the A8 dopamine system in the mesencephalic reticular formation and the AI0 system of the ventral tegmental area. Each s, nigra weighed approximately 5 mg (wet weight).

[aH ] Spiperone binding Right or left s. nigra (representing lesioned and control sides, respectively) were pooled from 6 rats and homogenized in 50 vol. 50 mM Tris.HC1 buffer (pH 7.7),

357 Membranes were then prepared as previously described 7,s,29-. Incubation tubes containing 50 #1 [3H]spiperone (Radiochemical Centre: specific activity 26 Ci/mmol) to give a final concentration as indicated in the figure legends, 50 #1 dopamine (300/~M final concentration) or distilled H20 and 900/zl homogenate (equivalent to approximately 7 mg wet weight of original tissue) were incubated for 10 min at 37 °C. This proved to be the optimal time for maximal specific binding to s. nigra. Membranes were then collected by filtration and washed twice with 5 ml 50 mM Tris, pH 7.7; more extensive washing decreased specific binding. The bound radioactivity was then determinedV,S,zz, 3~. Specific binding was defined as the difference between ['~H]spiperone binding in the absence and presence of 300 /zM DA and represented approximately 45 ~ of the total binding.

Measurement of adenylate eyclase and phosphodiesterase Adenylate cyclase determinations were carried out by the method of Kebabian et al. iv. Right (lesioned) or left (control) s. nigra tissue from 3 rats was pooled and homogenized in 25 vol. 2.0 mM Tris.maleate buffer (pH 7.4) containing 2.0 mM EGTA. Twenty-five microlitre aliquots of this homogenate were added to assay tubes containing 125 ~1 of buffer consisting of 80 mM Tris.maleate (pH 7.4), 2 mM MgSO4, 10 mM theophylline and 0.2 mM EGTA plus dopamine as indicated. The tubes were incubated with shaking at 30 °C for 2.5 rain and then transferred to a boiling water bath, followed by centrifugation to sediment the denatured protein. Ten microlitre aliquots of the supernatant solution were taken for analysis of cyclic AMP by the method of Brown et al. 3. The method of Thompson and Appleman 40 was used for the measurement of cyclic nucleotide phosphodiesterase activity. The original 2 ~ homogenate from the binding assay was further diluted 1 in 400 in 50 mM Tris.HCl (pH 7.7) containing 0.1 ~ Triton X~ 100; 50/~1 of the homogenate was then added to incubation tubes (final volume 100/zl) containing 40 mM Tris.HCl (pH 8.0), 5 mM MgClz, [3H]cAMP(27.5 Ci/mmol from NEN; 10 nCi per assay tube) and 1 #M non-radioactive cAMP.

Determination of glutamate decarboxylase and tyrosine hydroxylase Tyrosine hydroxylase was measured by the method of Hendry and Iversen 14 in a final volume of l0/A substrate mix and l0 #1 homogenate (2 ~ homogenate used for [3H]spiperone binding). Tetrahydrobiopterin (1 mM) was used as cofactor and the final concentration of L-[3H]tyrosine was 5 ! #M. Glutamate decarboxylase was assayed using the method of Albers and Brady z as modified by Fonnum et al. 10. The incubation medium contained (final concentrations): 1.3 mM e-[14C]l-glutamate (55 mCi/mmol, Amersham); 4.17 mM L-glutamate; 50 mM potassium phosphate buffer (pH 6.5); 200/zM pyridoxal-5-phosphate; 3.9 mM dithiothreitol and 0.25 ~ Triton X100. Reactions were started by the addition of 2/zl substrate to 2/zl homogenate. The reaction was carried out for 1 h at 37 °C and stopped by the addition of 0.2 M sulphuric acid. The 14CO2 evolved was collected in 50 #I NCS (Packard). The NCS solution was transferred to a scintillation vial containing 2 ml ethanol and l0 ml toluene scintillant.

358 TABLE ! The effect of lesions on tyrosine hydroxylase, glutamate decarboxylase, and phosphodies te'rase activities in the s. nigra

Rats were lesioned and, after the indicated time intervals, killed and the enzyme activities determined as described in Methods. Values are expressed as per cent of control ; control values for tyrosine hydroxylase ranged from 150 to 200 pmol/h/mg prot., for glutamate decarboxylase from 190 to 280 nmo] h mg prot. and for phosphodiesterase from 4.8 to 6.7 nmol/min/mg prot. The numbers in parenthesis indicate the number of experiments, each involving pooled tissue from 3 to 6 rats. Significance of differences from control: * P < 0.001. Lesion

Hemisection 6-OHDA Kainic acid Kainic acid

Days alter lesion

7 7 7 14

Enzyme activities (per cent controi Tyrosine hydroxylase

Glatamate decarboxvlase

Phosphodiesterase rcA MP

34z 3(10)* 15 _-r 6 (5J* 85 ~ 9 (14) 101 ~ 9 (9)

27 ~ 313)*

34-r 2~7)* 82 i 8 (5) 34 ~- 3 (9) * 32 4 (9)*

46 t 5 t31* -

P r o t e i n was d e t e r m i n e d by the m e t h o d o f L o w r y et al. 24. Statistical corn parisons were m a d e by S t u d e n t ' s t-test. P values o f less than 0.05 were considered significant. RESULTS The efjkct o f lesions on tyrosine hydroxylase, glutamate decarboxylase and cyclic nucleotide phosphodiesterase in rat s. nigra

T o o b t a i n i n f o r m a t i o n on the effectiveness o f the various lesions, the activities o f several m a r k e r enzymes were determined in c o n t r o l a n d lesioned tissue (Table I). Hemisections, which lead to r e t r o g r a d e and a n t e r o g r a d e d e g e n e r a t i o n o f s. nigra fibres, decreased the p r e s y n a p t i c enzymes glutamic acid d e c a r b o x y l a s e T M a n d cyclic nucleotide p h o s p h o d i e s t e r a s e ~8 by 77 ~,, and 66 ~ , respectively; tyrosine h y d r o x y l a s e H declined to 34 ~ o f control indicating t h a t these lesions also d a m a g e d d o p a m i n e r g i c n e u r o n e s within the s. nigra. 6 - O H D A injections into the s. nigra, which d e s t r o y the d o p a m i n e cell bodies 2a, decreased tyrosine h y d r o x y l a s e by 8 5 ~ : however, the p r e s y n a p t i c enzyme p h o s p h o d i e s t e r a s e located on s t r i a t o n i g r a l nerve terminals was n o t significantly affected. I n t r a s t r i a t a l injections o f kainic acid, which leave d o p a m i n e cell b o d i e s z,lz,zS,~6,36,37 intact, did not alter tyrosine h y d r o x y l a s e activity, while b o t h g l u t a m a t e d e c a r b o x y l a s e a n d cyclic n u c l e o t i d e p h o s p h o d i e s t e r a s e were greatly reduced (by 54 ~o-68 ~ ) . Binding o f [ZH]spiperone to control s. nigra membranes The characteristics o f [3H]spiperone binding to rat s. n i g r a are d e p i c t e d in Fig. 1. Non-specific binding, m e a s u r e d in the presence o f 300 ~ M D A , increased l i n e a r l y up to a c o n c e n t r a t i o n o f 2 n M [ZH]spiperone (Fig. 1A). A t a c o n c e n t r a t i o n o f 0.5 n M . which was routinely used in the experiments, the non-specific c o m p o n e n t c o n s t i t u t e d

359 B ~o 30~-

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-.4" 1.0

0 rn

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2.0

{3H-SPIPERONE)nM

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2~ fro/ ~l.~f~

C

• Total ~ non-specific

110 (~H-SPIPERONE)nM

210 o=

20 40 BOUND (fmoles/mg prot)

Fig. 1. Saturation of [3H]spiperonebinding in rat s. nigra. A : increasingconcentrations of [3H]spiperone were incubated with s. nigra membranes (5 mg tissue/•l) in the presence (non-specific binding) and absence (total binding) of 300/~M dopamine. B: specificbinding was determined as total minus nonspecificbinding. Data shown are the pooled results of two experiments done in triplicate. C : Scatchard analysis of specific [3H]spiperonebinding. about 45 ~ of total binding. Specific binding was saturable and plateaued at about I 2 nM (Fig. I B). Scatchard analysis of the data indicated the presence of two populations of binding sites (Fig. 1C), a high affinity component with a dissociation constant (Ka) of 0.4 nM and a maximal number of binding sites of 18 fmol/mg prot. (Bronx) and a low affinity binding site with a Ka of 3.9 nM and a Bmax of 70 fmol/mg prot. The inhibition of [3H]spiperone binding in rat s. nigra by various drugs is shown in Fig. 2. Dopamine and 2-amino-6,7-dihydroxytetralin (ADTN) displace the radio]igand to a similar degree (ICs0s of 10 #M and 3.9 #M, respectively) while 5-hydroxytryptamine was substantially less potent with an ICs0 of 170 #M. The ( + ) isomer of butaclamol was the most potent displacer (IC50 0.008 #M), in marked contrast to the inactive enantiomer ( 7 butaclamol which displaced [3H]spiperone only weakly.

The effect of lesions on [3H]spiperone binding and the dopamine-sensitive adenylate cyclase in rat s. nigra Hemisections (Fig. 3), which produce both a loss of presynaptic afferents to the s. nigra and also damage dopamine cell bodies within the s. nigra due to retrograde degeneration, resulted in depletions of both [3H]spiperone binding ( 5 0 ~ ) and the dopamine-sensitive adenylate cyclase (80~). Intranigral 6-OHDA injection, which selectively destroys dopamine cell bodies, resulted in a decrease in [3H]spiperone binding (36~) with no associated alteration in the dopamine-sensitive adenylate cyclase (Fig. 4). lntrastriatal kainic acid injection, on the other hand, which selectively destroys the presynaptic striatonigral inputs to the s. nigra did not affect [~H]spiperone binding after 7 or 14 days, but did result in a 50 % and 77 ~ reduction in the dopamine-sensitive adenylate cyclase, respectively (Fig. 5).

360 100

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~' DA

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,

,

,

,

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100

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O3

so

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10 3

[ ] (÷) Butaclamol



(-)Buta clamol

~~ =\ []%.[] \ i 10 8

I 10-6

I 10 4

{DRUG)

Fig. 2. Effect of drugs on specific binding of [3H]spiperone in rat s. nigra homogenates. Incubations were carried out as described in Methods. Tubes contained 0.50 nM [3H]spiperone. Each point represents the mean of 2-8 determinations from 1 to 4 separate experiments. The results are expressed as per cent of specific [SH]spiperone binding, defined as that displaced by 300 .uM dopamine. D A , dopamine; 5-HT, 5-hydroxytryptamine; A D T N , 6,7-dihydroxy-2-aminotetralin. Control values were 13.8 :i: 1.6 (n = 8) fmol/mg prot. At 0.5 nM [aH]spiperone, total cpm (after subtraction 0fthe filter blank) was 443 z 34 (N -- 8) and the blank cpm was 228 d: 12.

12E1

HEMISECTION

~s .9 s.

E

3H-SPIPERONE BINDING

ADENYLATE CYCLASE

Fig. 3. The effect of hemisections on [aH]spiperone binding and dopamine-stimutated adenytatecyclase in rat s. nigra. Seven days after a unilateral knife cut lesion, ratswere killed and the binding and enzyme assays determined as described in Methods. Each bar represents the mean :E S.E,M. o f 4 and 7 determinations for the dopamine-sensitive adenylate cyclase and [aI'I]spiperone binding, respectively; each determination involved pooled tissue from 4 to 6 rats. For the adenylate cyclase assay, basal values for control (open bar) and lesion fhatched bar) rats were 50.9 ± 4.6(4) and 40.9 _-E3.4 (4)pmot/mgprot./ min, respectively. At 0.5 nM [aH]spiperone, the control value for total cpm (after subtraction of the filter blank) was 479 ± 32 (N = 6) andthe blank cpm was 154 4- 21. Significanceofdifferences from control: * * P < 0.001 ; * P < 0.01.

361 6OHDA

"@o

P_~. o

12

60

,

.o

o~ c

3H-SPIPERONE

BINDING

ADENYLATE

CYCLASE

Fig. 4. The effect of unilateral 6-hydroxydopamine (6-OHDA) lesions on [3H]spiperone binding and dopamine-stimulated adenylate cyclase in rat s. nigra. Seven days after a unilateral 6-OHDA injection into the s. nigra, rats were killed and the binding and enzyme assays determined. Each bar represents the mean ± S.E.M. of 3 and 9 determinations for the dopamine-sensitive adenylate cyclase and [3H]spiperone binding, respectively; each determination involved pooled tissue from 4 to 6 rats. For the adenylate cyclase assay, basal values for control (open bar) and lesion (hatched bar) rats were 56.7 ± 5.4 (3) and 57.8 ± 4.6 (3) pmol/mg prot./min, respectively. At 0.5 nM [3H]spiperone, the control value for total cpm (after subtraction of the filter blank) was 699 + 54 (N 8) and the blank cpm was 395 _-t: 33. Significance of difference from control: * P < 0.05.

KAINIC ACID LESION 7 DAYS

Q.

12f

t40

Dissociation between the presynaptic dopamine-sensitive adenylate cyclase and [3H]spiperone binding sites in rat substantia nigra.

Brain Research, 167 (1979) 355-365 © Elsevier/North-Holland Biomedical Press 355 DISSOCIATION BETWEEN T H E PRESYNAPT1C DOPAMINE-SENSITIVE A D E N Y...
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