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Dopamine independent rotational response to unilateral intranigral injection of serotonin
Life Sciences, Vol. 28, pp. 2595-2601 Printed in the U.S.A.
Pergamon Pres~
DOPAMINE INDEPENDENT ROTATIONAL RESPONSE TO UNILATERAL INTRANIGRAL INJECTION OF SEROTONIN C. Oberlander, P.F. Hunt, C. Dumont and J.R. Boissier Centre de Recherches Roussel-Uclaf, 93230 Romainville, France (Received in final form March 25, 1981)
Summary U n i l a t e r a l injections of 5-hydroxytryptamine (5-HT) into the pars r e t i c u l a t a of the substantia nigra of rats pretreated with a monoamine oxidase i n h i b i t o r induced a strong and longl a s t i n g c o n t r a l a t e r a l c i r c l i n g behaviour which was s e l e c t i v e l y increased as a function of time a f t e r degeneration of central 5-HT neurons with 5,7 dihydroxytryptamine. Rotations were not abolished a f t e r 6-hydroxydopamine lesion of n i g r o s t r i a t a l dopamine (DA) neurons, or a f t e r s t r i a t a l kainic acid lesions, but were on the contrary increased. I t is concluded that the cont r a l a t e r a l c i r c l i n g response to i n t r a n i g r a l 5-HT injection is caused by a s p e c i f i c stimulation of certain post-synaptic nigral 5-HT receptors susceptible to the development of denervation s u p e r s e n s i t i v i t y but does not require the p a r t i c i p a t i o n of n i g r o s t r i a t a l DA neurons. Pharmacological manipulations of serotoninergic processes or lesion of 5-hydroxytryptamine (5-HT) nuclei have provided good evidence that 5-HT exerts a functional i n h i b i t i o n of the n i g r o s t r i a t a l dopamine (DA) pathway of rats treated with e i t h e r DA agonists or antagonists. Thus 5-HT potentiates the c a t a l e p t i g e n i c action of DA antagonists (1,2,3) and i n h i b i t s stereotypies induced by DA agonists (4). S i m i l a r l y reduction of central 5-HT increases stereotypies (4,5) and decreases neuroleptic induced catalepsy ( 1 , 2 , 5 , 6 , 7 , 8 ) . Histochemical and electrophysiological studies have indicated that DA neurons at both the s t r i a t a l and nigral level receive terminals of 5-HT neurons o r i g i nating in mesencephalic raph~ nuclei (9,10,11) thus suggesting an i n t e r r e l a t i o n s h i p between 5-HT neurons and DA pathways. In an attempt to investigate possible i n t e r a c t i o n s between the two neuronal systems, we have made u n i l a t e ral i n j e c t i o n s of 5-HT into the substantia nigra (SN)*. On the basis of brain lesions or i n j e c t i o n techniques showing that u n i l a t e r a l a c t i v a t i o n of the n i g r o s t r i a t a l dopaminergic system in rats induces contralateral rotations while i n h i b i t i o n induces i p s i l a t e r a l ones (12) i t was assumed that a 5-HT induced i n h i b i t i o n of nigral DA c e l l bodies would result in i p s i l a t e r a l c i r c l i n g behavior. We report here experiments which show that i n t r a n i g r a l injections of 5-HT induce c o n t r a l a t e r a l c i r c l i n g behavior which is independent of an action on n i g r o s t r i a t a l DA neurons but is related to a specific post-synaptic 5-HT receptor stimulation as evidenced by denervation s u p e r s e n s i t i v i t y . * Preliminary results were presented at the International Society for Neurochemistry S a t e l l i t e Symposium, Athens 11-16 sept. 1979. See ref. (13). 0024-3205/81/232595-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2596
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Vol. 28, No. 23, 1981
M a t e r i a l s and methods
Male Sprague Dawley rats weighing 190-220g were anesthetized with sodium pentobarbital (brain lesion techniques) or ether (intranigral injection technique) and placed in a stereotaxic instrument (La Precision Cin~matographique). Compounds were unilaterally injected in the right side of the brain through a motor driven Hamilton syringe connected to a needle of 0.25 mm diameter according to the parameters indicated in table I. 6-hydroxydopamine hydrochloride (6-OHDA), 5,7-dihydroxytryptamine creatinine sulfate (5,7-HT) and muscimol were dissolved in Merle's multi-ionic solution for intracerebral injection. Just before injection, the solutions were brought to neutral pH with NaHCO3, except for those of 6-OHDA and 5,7-HT where ascorbic acid was added (1 and 0,1 mg/ml respectively). TABLE I BRAIN INJECTION PARAMETERS drugs
brain s t r u c t u r e
6-OHDA
medial f o r e b r a i n bundle
4.0
-2.8
1.0
8 (base)
4
280
5,7-HT
lateral ventricle
7.2
+1.4
1.3
150 (base)
20
280
kainic acid
striatum
8.2
-0.7
2.5
1.2
0.5
310
2.2
-2.8
2.0
variable
0.5
105
tested substantia nigra compounds pars r e t i c u l a t a
coordinates* A H L
dose ~g
volume ~I
duration sec.
* according to K~nig and Klippel (14). Circling behavior was assessed using an 8 compartment rotometer (15) connected to a microcomputer. Time-course responses were obtained directly off line and are expressed in mean number (~ S.E.M.) of turns per min. The position of the injecting needle and the extent of kainic acid lesion were verified by a photographic technique. Brains were fixed in formalin and frozen dry at -30"C on a microtome plate. Frontal slices (50 Nm) mounted on slides were used as negatives in an enlarger equipped with a point-source lamp and the images were projected (8 x enlargement) onto high contrast photographic paper. The photographs show fine details of brain structure and kainic acid lesioned areas have a characteristic opalescent appearance. Striatal DA levels in 6-OHDA lesioned rats were determined as previously described (16). The extent of 5,7-HT lesion in the presence or absence of nomifensine was estimated by measuring monoamine uptake into synaptosome preparations from lesioned and non lesioned rats. 5-HT uptake was measured in forebrain (17),DA uptake in the striatum and norepinephrine (NE) uptake in the hypothalamus, hippocampus and cortex (18). Statistical differences were calculated using a bilateral Student's t test or Dunnett's test on unpaired groups.
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Serotonin and the Substantia Nigra
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Results Effect of 5-HT in normal rats When injected into the SN of rats pretreated with the monoamine oxidase i n h i b i t o r nialamide (i00 mg/kg i . p . , 150 min before test) 5-HT induced a cont r a l a t e r a l c i r c l i n g behavior for which the threshold dose was 20 nmol (3.5 Ng) (see controls f i g . i ) . A potent and long l a s t i n g effect was observed with 50 or 70 nmol of 5-HT (see controls f i g . 3). In the absence of nialamide the 70 nmole dose of 5-HT only e l i c i t e d a weak c i r c l i n g response ( t o t a l of turns : 314 + 86 ; n = 7). In the following experiments a l l 5-HT injections were made afte~ nialamide pretreatment. Effect of 5-HT a f t e r 5,7-HT lesion In order to test the s p e c i f i c i t y of the response to 5-HT, experiments were carried out a f t e r denervation of central 5-HT receptors by means of i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n of 5,7-HT. Animals were pretreated with nomifensine in order to protect norepinephrine (NE) neurons, as already mentioned in the case of 6-OHDA lesions (19). The effect of 5,7-HT on central monoaminergic neurons was evaluated by measuring the rate of monoamine uptake into synaptosome preparations from various parts of the brain (table I I ) . TABLEAU I I EFFECT OF 5,7-HT LESION ON MONOAMINE UPTAKE in v i t r o uptake (% of non-lesioned controls) .
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no pretreatment time a f t e r 5,7-HT 5-HT forebrain
4d 34.2+2.3**
12d 16.0+1.1"*
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nomifensine pretreatment 4d 33.3+1.5"*
12d 18.8+1.0"*
DA striatum
88.5+4.3
92.0+6.3
Hypothalamus NE Hippocampus Cortex
22.7+1.7" 34.0¥2.3** 56.7¥2.0**
90.5+5.2 I11T 6 8~12
Synaptosome preparations w e r e incubated at 37" with the respective radioactive monoamines ( 5 H T , IO-7M ; DA, 5.10-8M ; NE, IO-7M) f o r 5 (5HT, DA) or I0 min (NE), during which time uptake was l i n e a r . The rate of uptake in preparations from lesioned rats (n=4 per group) was compared with that in non-lesioned controls (control values + SEM, pmoles/min/mg protein : 5HT, 1.9 + 0. i ; DA, 7.4 + i . i ; NE hypothal. 11.2 + 0.3, hippocamp. 7.1 + 0.3, cortex 5.3 -+ 0.8). S i g n i f i c a n t d i f f e ~ n c e from controls : * p - < 0.05, ** p < OTOI. For nomifensine pretreatment, the drug (I0 mg/kg, i . p . ) was given 30 min before 5,7-HT injection. 5-HT uptake was s i g n i f i c a n t l y decreased with respect to controls 4 days a f t e r i n j e c t i o n of 5,7-HT and f u r t h e r decreased at 12 days ( s i g n i f i c a n t l y d i f ferent from 4 day value, p < 0.01). The r e s u l t was the same whether the animals were pretreated with nomifensine or not. 5,7-HT did not affect DA uptake in the striatum but i t decreased NE uptake in the three brain areas tested. The e f f e c t of 5,7-HT on NE neurons was completely blocked by nomifensine pretreatment.
2598
Serotonin and the Substantia Nigra
~ 20,4E
I~tl Y (~1
I I
%LI~~//~
I ~ /I I,~/I /
• contRous5-Ht <> 4 DAYSAFTEr5,7-HT X 6 DAYSAFTER5,7-HT
Z~ g3~ I ~(14) . . . .
i
TIME(MIN)
. . . . . . . . . . . . 1 ~:~._ i
1 . . . . 200
I
. . . .
Vol. 28, No. 23, ]981
Figure i E f f e c t of u n i l a t e r a l i n t r a n i g r a l i n j e c t i o n of 5-HT (20 nmoles) at d i f f e r e n t times a f t e r l e s i o n w i t h 5,7-HT. S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s , p < 0.05 : on day 6 at 60 min, a day 12 at 120 min, p < 0 . 0 1 = on day 12 from 5 to 90 min. Numbers of animals are i n d i c a t e d in brackets.
I
3~JO
As 5-HT uptake decreases during the period from 4 to 12 days a f t e r 5,7-HT i n j e c t i o n , so the r o t a t i o n a l response to a 20 nmol dose of 5-HT increases w i t h approximately a 4 f o l d d i f f e r e n c e in the maximal rate of r o t a t i o n ( f i g . I ) . S u p e r s e n s i t i v i t y of 5-HT receptors in 5,7-HT lesioned rats was checked a f t e r each experiment by i . p . i n j e c t i o n of the 5-11T precursor, 5 - h y d r o x y t r y p t o p h a n . When s u p e r s e n s i t i v i t y has developed the s e r o t o n i n e r g i c syndrome (tremors, Straub t a i l , h i n d - l i m b abduction, forepaw t r e a d i n g , head weaving e t c . . . ) is observed at a dose of 25 mg/kg compared with 100-200 mg/kg i n non-lesioned r a t s . As shown in f i g u r e 2, the response to 5-HT in normal or lesioned rats was c l e a r l y dose-related and solvent i n j e c t i o n did not induce any r o t a t i o n s in e i t h e r group. In the lesioned group a c o n s i s t e n t c i r c l i n g response was obtained with a 5 nmol dose of 5-HT which was i n e f f e c t i v e in i n t a c t r a t s . In a f u r t h e r attempt to t e s t whether the increased response to 5-HT i n j e c t i o n s was r e l a t e d to s t i m u l a t i o n of s u p e r s e n s i t i v e 5-HT r e c e p t o r s , the indoleamine was replaced by muscimol, a potent GABA agonist which also induces c o n t r a l a t e r a l c i r c l i n g when i n j e c t e d i n t o the SN (20). 12 days a f t e r 5,THT l e s i o n , the response to I0 ng of muscimol was not modified ( t o t a l of t u r n s , c o n t r o l s / lesioned rat : 848 + 170/ 851 + 209 ; maximal i n t e n s i t y of r o t a t i o n s in t u r n s / min : 11.6 + 3 . 1 / - 1 1 . 1 + 2 . 9 ~ = 8). FITTING CONTROLS LESIONED
POLYNOMIAL
15
/j,,/'s/" i z
10
o
"t///'///
20
(7)
40
SIGN
CORRELATION COEFFICIENT
0.978**
0.986**
ORIGIN OF ORDINATES
0.14+0.25
1.8+0.8
NS
REGRESSION COEFFICIENT
0.13+0.1
0.29+0.04
**
SIGN : s i g n i f i c a n c e , * * p < 0.01 NS : not s i g n i f i c a n t Figure 2 Dose-response curves to u n i l a t e r a l i n t r a n i gral i n j e c t i o n s of 5-HT or solvent in normal rats ( 0 ) or 12 days a f t e r 5,7-HT l e s i o n ( 0 ) . Results are expressed in t u r n s per min at the maximal rate of r o t a t i o n s over a 30 min period. Dashed l i n e s show the polynomial f i t t i n g of the values from each group. Controls and treated rats were given the same s o l u t i o n adjusted to pH = 7 and changed a f t e r 4 i n j e c t i o n s .
Vol.
28, No. 23, 1981
Serotonin and the Substantia Nigra
2599
E f f e c t of 5-HT a f t e r 6-OHDA or k a i n i c acid l e s i o n . In order to determine whether or not the n i g r o s t r i a t a l DA system cont r i b u t e d to the expression of 5-HT induced r o t a t i o n s , lesions were made of nigrostriatal DA neurons or p o s t - s y n a p t i c DA receptors located on s t r i a t a l i n t e r n e u r o n s on the side of the n i g r a l 5-HT i n j e c t i o n . DA neurons were degenerated by 6-OHDA w h i l e s t r i a t a l interneurons and s t r i a t o - n i g r a l neurons were destroyed by k a i n i c acid i n j e c t i o n i n t o the s t r i a t u m (21). In 6-OHDA lesioned r a t s , 5-HT induced c i r c l i n g behaviour not only pers i s t e d a f t e r the l e s i o n but was even increased ( f i g . 3 ) . In these lesioned rats s t r i a t a l DA was reduced to undetectable l e v e l s ( < 0.2 ~g/g) on the lesioned side. A f t e r the k a i n i c acid l e s i o n the response to 5-HT i n j e c t i o n s was increased in a manner s i m i l a r to that in 6-OHDA lesioned rats ( f i g . 3). H i s t o l o g i c a l c o n t r o l s revealed a large but d i s c r e t e lesion of the s t r i a t u m w i t h minimal t i s s u e damage to the homolateral cerebral c o r t e x . Figure 3 E f f e c t of u n i l a t e r a l i n t r a n i g r a l i n j e c t i o n of 70 nmoles of 5-HT in c o n t r o l rats (A) or 18 days a f t e r n i g r o s t r i a t a l 6-OHDA l e s i o n ( ~ ) or 10 days a f t e r s t r i a t a l k a i n i c acid l e s i o n ( x ) . S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s : 6-OHDA, p < 0.01 at 5 and 90 to 150 min, p < 0.05 at I0 to 20 min. Kainic acid, p < 0.01 at 120 and 150 min, p < 0.05 at 25, 90 and 180 min.
kl 0"~ Z
16~o 20 0'
Discussion C o n t r a l a t e r a l c i r c l i n g behavior induced a f t e r u n i l a t e r a l i n j e c t i o n s of 5-HT i n t o the pars r e t i c u l a t a of the SN of nialamide pretreated rats was c h a r a c t e r i z e d by a strong, long l a s t i n g and dose r e l a t e d e f f e c t ; 5-HT a c t i o n was increased a f t e r 5,7-HT l e s i o n and f u r t h e r developed as a f u n c t i o n of time a f t e r l e s i o n . The s p e c i f i c i t y of the 5-HT e f f e c t is demonstrated by the fact t h a t c o n t r a l a t e r a l r o t a t i o n s caused by the GABA agonist muscimol, were not increased a f t e r 5,7-HT l e s i o n . Therefore i t is probable that p o s t - s y n a p t i c 5-HT receptors in the SN develop s u p e r s e n s i t i v i t y . These r e s u l t s c o r r e l a t e w i t h previous f i n d i n g s of a behavioral s u p e r s e n s i t i v i t y to systemic i n j e c t i o n s of 5 - h y d r o x y t r y p t o p h a n in 5,7-HT lesioned rats (22,23) and i n d i c a t e that the r o t a t i o n a l response to i n t r a n i g r a l 5-HT is caused by the s t i m u l a t i o n of posts y n a p t i c 5-HT r e c e p t o r s . I t is well e s t a b l l s h e d that a r e l a t i v e increase in dopaminergic a c t i v i t y on one side of the brain r e s u l t s in c o n t r a l a t e r a l c i r c l i n g behavior in the rat (24). I f 5-HT induced c i r c l i n g behavior were due to a modulation of dopaminergic a c t i v i t y in the SN, then one might conclude that 5-HT is e x e r t i n g an e x c i t a t o r y r a t h e r than i n h i b i t o r y action on n i g r o s t r i a t a l DA neurons. The hypothesis that 5-HT is modulating DA pathways is however c o n t r a d i c t e d by the persistence of r o t a t i o n s a f t e r d e s t r u c t i o n of the homolat e r a l s t r i a t a l DA neurons or DA r e c e p t o r s . This action of 5-HT is t h e r e f o r e e l i c i t e d independently of the n i g r o s t r i a t a l DA pathway. S u r p r i s i n g l y 6-OHDA or k a i n i c acid l e s i o n s even increased the r o t a t i o n a l response to i n t r a n i g r a l 5-HT. This observation suggests t h a t in a d d i t i o n to a non-DA mediated e f f e c t in the SN, 5-HT also exerted an i n h i b i t o r y a c t i o n on the n i g r o s t r i a t a l DA neurons. This would r e s u l t in i p s i l a t e r a l t u r n i n g which presumably is masked by the strong c o n t r a l a t e r a l motor asymetry and which would be suppressed when DA neurons or receptors are lesioned. This could p a r t l y e x p l a i n why in a
2600
Serotonin and the Substantia Nigra
Vol. 28, No. 23, 1981
recent publication i p s i l a t e r a l instead of contralateral rotations a f t e r i n t r a n i g r a l 5-HT were reported (25) while in our experimental conditions, not only 5-HT but other 5-HT agonists where able to provoke strong contralateral rotations (13). Our results are in agreement with previous findings showing that i n t r a n i g r a l i n j e c t i o n of the 5-HT synthesis i n h i b i t o r PCPA induced an i p s i l a t e r a l body asymetry (26). Unfortunately, we were unable to duplicate t h i s experiment, because s o l u b i l i s a t i o n of this compound in a minimal quantity of solvent was only possible at a non-physiological pH ( ) I i ) . In the l i g h t of recent evidence of non-DA mediated c i r c l i n g behavior and stereotypies induced by i n t r a n i g r a l muscimol (27,20) the SN is now considered as a relay between striatonigral neurons and output neurons necessary for the expression of behavior r e s u l t i n g from the stimulation of s t r i a t a l DA receptors (28). It is suggested that 5-HT could also be implicated in the function of a nigral output pathway in a manner which remains to be determined. This nigral action of 5-HT could p a r t l y explain why certain reports (29,30,31) are in apparent contradiction with the well documented hypothesis of an i n h i b i t o r y action of 5-HT on extrapyramidal dopaminergic symptoms. In p a r t i c u l a r , i t is interesting to note that a f t e r lesion of the 5-HT pathways ascending from the dorsal and medial nuclei of the raph~, a decrease rather than an increase in amphetamine or apomorphine induced stereotypies was observed (30). Moreover, quipazine, a 5-HT agonist, has even been shown (31) to exert an a n t i c a t a l e p t i c effect with respect to a dopamine blocker. Acknowledgments This work was carried out with the excellent technical assistance of Mrs Y. Demassey, A. Verdu and D. Massardier. References 1. C.J. CARTER and C.J. PYCOCK, Br. J. Pharmac., 60, 267P-268P, 1977. 2. L.D. FUENMAYORand M. VOGT, Br. J. Pharmac., 6T~, 309-318, 1979. 3. P.C. WALDMEIER and A.A. DELINI-STULA, Eur. J.--Pharmacol., 5_~5, 363-373, 1979. 4. W.J. WEINER, C. GOETZ and H.L. KLAWANS, Acta pharmac, et t o x . , 3___66, 155-160, 1975. 5. W. GUMULKA, W. KOSTOWSKI and A. CZLONKOWSKI, Pharmacology, 10, 363-372, 1973. 6. W. KOSTOWS~I, W. GUMULKAand A. CZLONKOWSKI, Brain Res., 48, 443-446, 1972. 7. C.J. CARTER and C.J. PYCOCK, Arch. Pharmacol., 304, 135-139, 1978. 8. B. COSTALL, D.H. FORTUNE, R.J. NAYLOR, C.D. MARSDENand C. PYCOCK, Neuropharmacology, 14, 859-868, 1975. 9. J.J. MILLER, T.L. RICHARDSON, H.C. FIBIGER and H. Mc LENNAN, Brain Res., 97, 133-138, 1975. I0. A. DRAY, T.J. GONYE, N.R. OAKLEY and T. TANNER, Brain Res., 113, 45-57, 1976. 11. D. VAN DER KOOY and T. HATTORI, Brain Res., 186, i - 7 , 1980. 12. U. UNGERSTEDT, L.L. BUTCHER, S.G. BUTCHER, N.E. ANDEN, and K. FUXE, Brain Res., 14, 461-471, 1969. 13. P. HUNT and C. OBERLANDER, Serotonin. Current Aspects of Neurochemistry and Function. B. Haber, Plenum. publishing corp., New York,in press,1980. 14. J.F.R. KONIG and R.A. KLIPPEL, The Rat Brain : A Stereotaxic Atlas Williams and Wilkins, Baltimore, 1963. 15. H. GUILLEUX and M. PETERFALVI J. Pharmacol. (Paris) ~, 63-74,1974.
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16. C. OBERLANDER, C. EUVRARD, C. DUMONTand J.R. BOISSIER, Eur. J. Pharmacol., 60, 163-170, 1979. 17. M.H. KANNENGIESSER, P.F. HUNT, and J.P. RAYNAUD, Biochem. Pharmac., 2__2, 73-84,1973. 18. P. HUNT, J.P. RAYNAUD, M. LEVEN and U. SCHACHT, Biochem. Pharmac., 28, 2011-2016, 1979. 19. R. SAMANIN, S. BERNASCONI and S. GARATTINI, Eur. J. Pharmacol., 34, 377-380, 1975. 20. C. OBERLANDER, C. DUMONTand J.R. BOISSIER, Eur. J. Pharmacol., 43, 389-390, 1977. 21. R. SCHWARCZand J.T. COYLE, Brain Res., 127, 235-249, 1977. 22. M.E. TRULSONand B.L. JACOBS, Annals o f ~ New York academy of sciences, 305, 497-509, 1978. 23. R.M. STEWART, J.H. GROWDON,D. CANClAN and R.J. BALDESSARINI, Neuropharmacol., 15, 449-455 (1976). 24. U. UNGERSTEDT, Acta physiol, scand., suppl. 367, 1-48, 1971. 25. T.A. JAMES and M.S. STARR, J. Pharm. PharmacoT., 32, 196-200, 1980. 26. T. TANNER, J. Pharm. Pharmac. 3__00,158-161, 1978. 27. J. SCHEEL-KRUGER, J. ARNT and G° MAGELUND, Neurosci. L e t t . , 4, 351-356, 1977. 28. G. DI CHIARA, M. MORELLI, M.L. PORCEDDUand G.L. GESSA. Life Sci., 23, 2045-2052, 1978. 29. H.M. CALIL, S. NAKANOand L.E. HOLLISTER, Psychopharmacology, 58, 115-116 1978. 30. B. COSTALL and R.J. NAYLOR, Eur. J. Pharmacol., 29, 206-222, 1974. 31. M. GRABOWSKA, L. ANTKIEWICZ and J. MICHALUK, J. Pharm. Pharmacol. 2_6_6, 74-76, 1974.
Pergamon Pres~
DOPAMINE INDEPENDENT ROTATIONAL RESPONSE TO UNILATERAL INTRANIGRAL INJECTION OF SEROTONIN C. Oberlander, P.F. Hunt, C. Dumont and J.R. Boissier Centre de Recherches Roussel-Uclaf, 93230 Romainville, France (Received in final form March 25, 1981)
Summary U n i l a t e r a l injections of 5-hydroxytryptamine (5-HT) into the pars r e t i c u l a t a of the substantia nigra of rats pretreated with a monoamine oxidase i n h i b i t o r induced a strong and longl a s t i n g c o n t r a l a t e r a l c i r c l i n g behaviour which was s e l e c t i v e l y increased as a function of time a f t e r degeneration of central 5-HT neurons with 5,7 dihydroxytryptamine. Rotations were not abolished a f t e r 6-hydroxydopamine lesion of n i g r o s t r i a t a l dopamine (DA) neurons, or a f t e r s t r i a t a l kainic acid lesions, but were on the contrary increased. I t is concluded that the cont r a l a t e r a l c i r c l i n g response to i n t r a n i g r a l 5-HT injection is caused by a s p e c i f i c stimulation of certain post-synaptic nigral 5-HT receptors susceptible to the development of denervation s u p e r s e n s i t i v i t y but does not require the p a r t i c i p a t i o n of n i g r o s t r i a t a l DA neurons. Pharmacological manipulations of serotoninergic processes or lesion of 5-hydroxytryptamine (5-HT) nuclei have provided good evidence that 5-HT exerts a functional i n h i b i t i o n of the n i g r o s t r i a t a l dopamine (DA) pathway of rats treated with e i t h e r DA agonists or antagonists. Thus 5-HT potentiates the c a t a l e p t i g e n i c action of DA antagonists (1,2,3) and i n h i b i t s stereotypies induced by DA agonists (4). S i m i l a r l y reduction of central 5-HT increases stereotypies (4,5) and decreases neuroleptic induced catalepsy ( 1 , 2 , 5 , 6 , 7 , 8 ) . Histochemical and electrophysiological studies have indicated that DA neurons at both the s t r i a t a l and nigral level receive terminals of 5-HT neurons o r i g i nating in mesencephalic raph~ nuclei (9,10,11) thus suggesting an i n t e r r e l a t i o n s h i p between 5-HT neurons and DA pathways. In an attempt to investigate possible i n t e r a c t i o n s between the two neuronal systems, we have made u n i l a t e ral i n j e c t i o n s of 5-HT into the substantia nigra (SN)*. On the basis of brain lesions or i n j e c t i o n techniques showing that u n i l a t e r a l a c t i v a t i o n of the n i g r o s t r i a t a l dopaminergic system in rats induces contralateral rotations while i n h i b i t i o n induces i p s i l a t e r a l ones (12) i t was assumed that a 5-HT induced i n h i b i t i o n of nigral DA c e l l bodies would result in i p s i l a t e r a l c i r c l i n g behavior. We report here experiments which show that i n t r a n i g r a l injections of 5-HT induce c o n t r a l a t e r a l c i r c l i n g behavior which is independent of an action on n i g r o s t r i a t a l DA neurons but is related to a specific post-synaptic 5-HT receptor stimulation as evidenced by denervation s u p e r s e n s i t i v i t y . * Preliminary results were presented at the International Society for Neurochemistry S a t e l l i t e Symposium, Athens 11-16 sept. 1979. See ref. (13). 0024-3205/81/232595-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2596
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Vol. 28, No. 23, 1981
M a t e r i a l s and methods
Male Sprague Dawley rats weighing 190-220g were anesthetized with sodium pentobarbital (brain lesion techniques) or ether (intranigral injection technique) and placed in a stereotaxic instrument (La Precision Cin~matographique). Compounds were unilaterally injected in the right side of the brain through a motor driven Hamilton syringe connected to a needle of 0.25 mm diameter according to the parameters indicated in table I. 6-hydroxydopamine hydrochloride (6-OHDA), 5,7-dihydroxytryptamine creatinine sulfate (5,7-HT) and muscimol were dissolved in Merle's multi-ionic solution for intracerebral injection. Just before injection, the solutions were brought to neutral pH with NaHCO3, except for those of 6-OHDA and 5,7-HT where ascorbic acid was added (1 and 0,1 mg/ml respectively). TABLE I BRAIN INJECTION PARAMETERS drugs
brain s t r u c t u r e
6-OHDA
medial f o r e b r a i n bundle
4.0
-2.8
1.0
8 (base)
4
280
5,7-HT
lateral ventricle
7.2
+1.4
1.3
150 (base)
20
280
kainic acid
striatum
8.2
-0.7
2.5
1.2
0.5
310
2.2
-2.8
2.0
variable
0.5
105
tested substantia nigra compounds pars r e t i c u l a t a
coordinates* A H L
dose ~g
volume ~I
duration sec.
* according to K~nig and Klippel (14). Circling behavior was assessed using an 8 compartment rotometer (15) connected to a microcomputer. Time-course responses were obtained directly off line and are expressed in mean number (~ S.E.M.) of turns per min. The position of the injecting needle and the extent of kainic acid lesion were verified by a photographic technique. Brains were fixed in formalin and frozen dry at -30"C on a microtome plate. Frontal slices (50 Nm) mounted on slides were used as negatives in an enlarger equipped with a point-source lamp and the images were projected (8 x enlargement) onto high contrast photographic paper. The photographs show fine details of brain structure and kainic acid lesioned areas have a characteristic opalescent appearance. Striatal DA levels in 6-OHDA lesioned rats were determined as previously described (16). The extent of 5,7-HT lesion in the presence or absence of nomifensine was estimated by measuring monoamine uptake into synaptosome preparations from lesioned and non lesioned rats. 5-HT uptake was measured in forebrain (17),DA uptake in the striatum and norepinephrine (NE) uptake in the hypothalamus, hippocampus and cortex (18). Statistical differences were calculated using a bilateral Student's t test or Dunnett's test on unpaired groups.
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Serotonin and the Substantia Nigra
2597
Results Effect of 5-HT in normal rats When injected into the SN of rats pretreated with the monoamine oxidase i n h i b i t o r nialamide (i00 mg/kg i . p . , 150 min before test) 5-HT induced a cont r a l a t e r a l c i r c l i n g behavior for which the threshold dose was 20 nmol (3.5 Ng) (see controls f i g . i ) . A potent and long l a s t i n g effect was observed with 50 or 70 nmol of 5-HT (see controls f i g . 3). In the absence of nialamide the 70 nmole dose of 5-HT only e l i c i t e d a weak c i r c l i n g response ( t o t a l of turns : 314 + 86 ; n = 7). In the following experiments a l l 5-HT injections were made afte~ nialamide pretreatment. Effect of 5-HT a f t e r 5,7-HT lesion In order to test the s p e c i f i c i t y of the response to 5-HT, experiments were carried out a f t e r denervation of central 5-HT receptors by means of i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n of 5,7-HT. Animals were pretreated with nomifensine in order to protect norepinephrine (NE) neurons, as already mentioned in the case of 6-OHDA lesions (19). The effect of 5,7-HT on central monoaminergic neurons was evaluated by measuring the rate of monoamine uptake into synaptosome preparations from various parts of the brain (table I I ) . TABLEAU I I EFFECT OF 5,7-HT LESION ON MONOAMINE UPTAKE in v i t r o uptake (% of non-lesioned controls) .
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.
no pretreatment time a f t e r 5,7-HT 5-HT forebrain
4d 34.2+2.3**
12d 16.0+1.1"*
.
.
.
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.
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nomifensine pretreatment 4d 33.3+1.5"*
12d 18.8+1.0"*
DA striatum
88.5+4.3
92.0+6.3
Hypothalamus NE Hippocampus Cortex
22.7+1.7" 34.0¥2.3** 56.7¥2.0**
90.5+5.2 I11T 6 8~12
Synaptosome preparations w e r e incubated at 37" with the respective radioactive monoamines ( 5 H T , IO-7M ; DA, 5.10-8M ; NE, IO-7M) f o r 5 (5HT, DA) or I0 min (NE), during which time uptake was l i n e a r . The rate of uptake in preparations from lesioned rats (n=4 per group) was compared with that in non-lesioned controls (control values + SEM, pmoles/min/mg protein : 5HT, 1.9 + 0. i ; DA, 7.4 + i . i ; NE hypothal. 11.2 + 0.3, hippocamp. 7.1 + 0.3, cortex 5.3 -+ 0.8). S i g n i f i c a n t d i f f e ~ n c e from controls : * p - < 0.05, ** p < OTOI. For nomifensine pretreatment, the drug (I0 mg/kg, i . p . ) was given 30 min before 5,7-HT injection. 5-HT uptake was s i g n i f i c a n t l y decreased with respect to controls 4 days a f t e r i n j e c t i o n of 5,7-HT and f u r t h e r decreased at 12 days ( s i g n i f i c a n t l y d i f ferent from 4 day value, p < 0.01). The r e s u l t was the same whether the animals were pretreated with nomifensine or not. 5,7-HT did not affect DA uptake in the striatum but i t decreased NE uptake in the three brain areas tested. The e f f e c t of 5,7-HT on NE neurons was completely blocked by nomifensine pretreatment.
2598
Serotonin and the Substantia Nigra
~ 20,4E
I~tl Y (~1
I I
%LI~~//~
I ~ /I I,~/I /
• contRous5-Ht <> 4 DAYSAFTEr5,7-HT X 6 DAYSAFTER5,7-HT
Z~ g3~ I ~(14) . . . .
i
TIME(MIN)
. . . . . . . . . . . . 1 ~:~._ i
1 . . . . 200
I
. . . .
Vol. 28, No. 23, ]981
Figure i E f f e c t of u n i l a t e r a l i n t r a n i g r a l i n j e c t i o n of 5-HT (20 nmoles) at d i f f e r e n t times a f t e r l e s i o n w i t h 5,7-HT. S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s , p < 0.05 : on day 6 at 60 min, a day 12 at 120 min, p < 0 . 0 1 = on day 12 from 5 to 90 min. Numbers of animals are i n d i c a t e d in brackets.
I
3~JO
As 5-HT uptake decreases during the period from 4 to 12 days a f t e r 5,7-HT i n j e c t i o n , so the r o t a t i o n a l response to a 20 nmol dose of 5-HT increases w i t h approximately a 4 f o l d d i f f e r e n c e in the maximal rate of r o t a t i o n ( f i g . I ) . S u p e r s e n s i t i v i t y of 5-HT receptors in 5,7-HT lesioned rats was checked a f t e r each experiment by i . p . i n j e c t i o n of the 5-11T precursor, 5 - h y d r o x y t r y p t o p h a n . When s u p e r s e n s i t i v i t y has developed the s e r o t o n i n e r g i c syndrome (tremors, Straub t a i l , h i n d - l i m b abduction, forepaw t r e a d i n g , head weaving e t c . . . ) is observed at a dose of 25 mg/kg compared with 100-200 mg/kg i n non-lesioned r a t s . As shown in f i g u r e 2, the response to 5-HT in normal or lesioned rats was c l e a r l y dose-related and solvent i n j e c t i o n did not induce any r o t a t i o n s in e i t h e r group. In the lesioned group a c o n s i s t e n t c i r c l i n g response was obtained with a 5 nmol dose of 5-HT which was i n e f f e c t i v e in i n t a c t r a t s . In a f u r t h e r attempt to t e s t whether the increased response to 5-HT i n j e c t i o n s was r e l a t e d to s t i m u l a t i o n of s u p e r s e n s i t i v e 5-HT r e c e p t o r s , the indoleamine was replaced by muscimol, a potent GABA agonist which also induces c o n t r a l a t e r a l c i r c l i n g when i n j e c t e d i n t o the SN (20). 12 days a f t e r 5,THT l e s i o n , the response to I0 ng of muscimol was not modified ( t o t a l of t u r n s , c o n t r o l s / lesioned rat : 848 + 170/ 851 + 209 ; maximal i n t e n s i t y of r o t a t i o n s in t u r n s / min : 11.6 + 3 . 1 / - 1 1 . 1 + 2 . 9 ~ = 8). FITTING CONTROLS LESIONED
POLYNOMIAL
15
/j,,/'s/" i z
10
o
"t///'///
20
(7)
40
SIGN
CORRELATION COEFFICIENT
0.978**
0.986**
ORIGIN OF ORDINATES
0.14+0.25
1.8+0.8
NS
REGRESSION COEFFICIENT
0.13+0.1
0.29+0.04
**
SIGN : s i g n i f i c a n c e , * * p < 0.01 NS : not s i g n i f i c a n t Figure 2 Dose-response curves to u n i l a t e r a l i n t r a n i gral i n j e c t i o n s of 5-HT or solvent in normal rats ( 0 ) or 12 days a f t e r 5,7-HT l e s i o n ( 0 ) . Results are expressed in t u r n s per min at the maximal rate of r o t a t i o n s over a 30 min period. Dashed l i n e s show the polynomial f i t t i n g of the values from each group. Controls and treated rats were given the same s o l u t i o n adjusted to pH = 7 and changed a f t e r 4 i n j e c t i o n s .
Vol.
28, No. 23, 1981
Serotonin and the Substantia Nigra
2599
E f f e c t of 5-HT a f t e r 6-OHDA or k a i n i c acid l e s i o n . In order to determine whether or not the n i g r o s t r i a t a l DA system cont r i b u t e d to the expression of 5-HT induced r o t a t i o n s , lesions were made of nigrostriatal DA neurons or p o s t - s y n a p t i c DA receptors located on s t r i a t a l i n t e r n e u r o n s on the side of the n i g r a l 5-HT i n j e c t i o n . DA neurons were degenerated by 6-OHDA w h i l e s t r i a t a l interneurons and s t r i a t o - n i g r a l neurons were destroyed by k a i n i c acid i n j e c t i o n i n t o the s t r i a t u m (21). In 6-OHDA lesioned r a t s , 5-HT induced c i r c l i n g behaviour not only pers i s t e d a f t e r the l e s i o n but was even increased ( f i g . 3 ) . In these lesioned rats s t r i a t a l DA was reduced to undetectable l e v e l s ( < 0.2 ~g/g) on the lesioned side. A f t e r the k a i n i c acid l e s i o n the response to 5-HT i n j e c t i o n s was increased in a manner s i m i l a r to that in 6-OHDA lesioned rats ( f i g . 3). H i s t o l o g i c a l c o n t r o l s revealed a large but d i s c r e t e lesion of the s t r i a t u m w i t h minimal t i s s u e damage to the homolateral cerebral c o r t e x . Figure 3 E f f e c t of u n i l a t e r a l i n t r a n i g r a l i n j e c t i o n of 70 nmoles of 5-HT in c o n t r o l rats (A) or 18 days a f t e r n i g r o s t r i a t a l 6-OHDA l e s i o n ( ~ ) or 10 days a f t e r s t r i a t a l k a i n i c acid l e s i o n ( x ) . S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s : 6-OHDA, p < 0.01 at 5 and 90 to 150 min, p < 0.05 at I0 to 20 min. Kainic acid, p < 0.01 at 120 and 150 min, p < 0.05 at 25, 90 and 180 min.
kl 0"~ Z
16~o 20 0'
Discussion C o n t r a l a t e r a l c i r c l i n g behavior induced a f t e r u n i l a t e r a l i n j e c t i o n s of 5-HT i n t o the pars r e t i c u l a t a of the SN of nialamide pretreated rats was c h a r a c t e r i z e d by a strong, long l a s t i n g and dose r e l a t e d e f f e c t ; 5-HT a c t i o n was increased a f t e r 5,7-HT l e s i o n and f u r t h e r developed as a f u n c t i o n of time a f t e r l e s i o n . The s p e c i f i c i t y of the 5-HT e f f e c t is demonstrated by the fact t h a t c o n t r a l a t e r a l r o t a t i o n s caused by the GABA agonist muscimol, were not increased a f t e r 5,7-HT l e s i o n . Therefore i t is probable that p o s t - s y n a p t i c 5-HT receptors in the SN develop s u p e r s e n s i t i v i t y . These r e s u l t s c o r r e l a t e w i t h previous f i n d i n g s of a behavioral s u p e r s e n s i t i v i t y to systemic i n j e c t i o n s of 5 - h y d r o x y t r y p t o p h a n in 5,7-HT lesioned rats (22,23) and i n d i c a t e that the r o t a t i o n a l response to i n t r a n i g r a l 5-HT is caused by the s t i m u l a t i o n of posts y n a p t i c 5-HT r e c e p t o r s . I t is well e s t a b l l s h e d that a r e l a t i v e increase in dopaminergic a c t i v i t y on one side of the brain r e s u l t s in c o n t r a l a t e r a l c i r c l i n g behavior in the rat (24). I f 5-HT induced c i r c l i n g behavior were due to a modulation of dopaminergic a c t i v i t y in the SN, then one might conclude that 5-HT is e x e r t i n g an e x c i t a t o r y r a t h e r than i n h i b i t o r y action on n i g r o s t r i a t a l DA neurons. The hypothesis that 5-HT is modulating DA pathways is however c o n t r a d i c t e d by the persistence of r o t a t i o n s a f t e r d e s t r u c t i o n of the homolat e r a l s t r i a t a l DA neurons or DA r e c e p t o r s . This action of 5-HT is t h e r e f o r e e l i c i t e d independently of the n i g r o s t r i a t a l DA pathway. S u r p r i s i n g l y 6-OHDA or k a i n i c acid l e s i o n s even increased the r o t a t i o n a l response to i n t r a n i g r a l 5-HT. This observation suggests t h a t in a d d i t i o n to a non-DA mediated e f f e c t in the SN, 5-HT also exerted an i n h i b i t o r y a c t i o n on the n i g r o s t r i a t a l DA neurons. This would r e s u l t in i p s i l a t e r a l t u r n i n g which presumably is masked by the strong c o n t r a l a t e r a l motor asymetry and which would be suppressed when DA neurons or receptors are lesioned. This could p a r t l y e x p l a i n why in a
2600
Serotonin and the Substantia Nigra
Vol. 28, No. 23, 1981
recent publication i p s i l a t e r a l instead of contralateral rotations a f t e r i n t r a n i g r a l 5-HT were reported (25) while in our experimental conditions, not only 5-HT but other 5-HT agonists where able to provoke strong contralateral rotations (13). Our results are in agreement with previous findings showing that i n t r a n i g r a l i n j e c t i o n of the 5-HT synthesis i n h i b i t o r PCPA induced an i p s i l a t e r a l body asymetry (26). Unfortunately, we were unable to duplicate t h i s experiment, because s o l u b i l i s a t i o n of this compound in a minimal quantity of solvent was only possible at a non-physiological pH ( ) I i ) . In the l i g h t of recent evidence of non-DA mediated c i r c l i n g behavior and stereotypies induced by i n t r a n i g r a l muscimol (27,20) the SN is now considered as a relay between striatonigral neurons and output neurons necessary for the expression of behavior r e s u l t i n g from the stimulation of s t r i a t a l DA receptors (28). It is suggested that 5-HT could also be implicated in the function of a nigral output pathway in a manner which remains to be determined. This nigral action of 5-HT could p a r t l y explain why certain reports (29,30,31) are in apparent contradiction with the well documented hypothesis of an i n h i b i t o r y action of 5-HT on extrapyramidal dopaminergic symptoms. In p a r t i c u l a r , i t is interesting to note that a f t e r lesion of the 5-HT pathways ascending from the dorsal and medial nuclei of the raph~, a decrease rather than an increase in amphetamine or apomorphine induced stereotypies was observed (30). Moreover, quipazine, a 5-HT agonist, has even been shown (31) to exert an a n t i c a t a l e p t i c effect with respect to a dopamine blocker. Acknowledgments This work was carried out with the excellent technical assistance of Mrs Y. Demassey, A. Verdu and D. Massardier. References 1. C.J. CARTER and C.J. PYCOCK, Br. J. Pharmac., 60, 267P-268P, 1977. 2. L.D. FUENMAYORand M. VOGT, Br. J. Pharmac., 6T~, 309-318, 1979. 3. P.C. WALDMEIER and A.A. DELINI-STULA, Eur. J.--Pharmacol., 5_~5, 363-373, 1979. 4. W.J. WEINER, C. GOETZ and H.L. KLAWANS, Acta pharmac, et t o x . , 3___66, 155-160, 1975. 5. W. GUMULKA, W. KOSTOWSKI and A. CZLONKOWSKI, Pharmacology, 10, 363-372, 1973. 6. W. KOSTOWS~I, W. GUMULKAand A. CZLONKOWSKI, Brain Res., 48, 443-446, 1972. 7. C.J. CARTER and C.J. PYCOCK, Arch. Pharmacol., 304, 135-139, 1978. 8. B. COSTALL, D.H. FORTUNE, R.J. NAYLOR, C.D. MARSDENand C. PYCOCK, Neuropharmacology, 14, 859-868, 1975. 9. J.J. MILLER, T.L. RICHARDSON, H.C. FIBIGER and H. Mc LENNAN, Brain Res., 97, 133-138, 1975. I0. A. DRAY, T.J. GONYE, N.R. OAKLEY and T. TANNER, Brain Res., 113, 45-57, 1976. 11. D. VAN DER KOOY and T. HATTORI, Brain Res., 186, i - 7 , 1980. 12. U. UNGERSTEDT, L.L. BUTCHER, S.G. BUTCHER, N.E. ANDEN, and K. FUXE, Brain Res., 14, 461-471, 1969. 13. P. HUNT and C. OBERLANDER, Serotonin. Current Aspects of Neurochemistry and Function. B. Haber, Plenum. publishing corp., New York,in press,1980. 14. J.F.R. KONIG and R.A. KLIPPEL, The Rat Brain : A Stereotaxic Atlas Williams and Wilkins, Baltimore, 1963. 15. H. GUILLEUX and M. PETERFALVI J. Pharmacol. (Paris) ~, 63-74,1974.
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16. C. OBERLANDER, C. EUVRARD, C. DUMONTand J.R. BOISSIER, Eur. J. Pharmacol., 60, 163-170, 1979. 17. M.H. KANNENGIESSER, P.F. HUNT, and J.P. RAYNAUD, Biochem. Pharmac., 2__2, 73-84,1973. 18. P. HUNT, J.P. RAYNAUD, M. LEVEN and U. SCHACHT, Biochem. Pharmac., 28, 2011-2016, 1979. 19. R. SAMANIN, S. BERNASCONI and S. GARATTINI, Eur. J. Pharmacol., 34, 377-380, 1975. 20. C. OBERLANDER, C. DUMONTand J.R. BOISSIER, Eur. J. Pharmacol., 43, 389-390, 1977. 21. R. SCHWARCZand J.T. COYLE, Brain Res., 127, 235-249, 1977. 22. M.E. TRULSONand B.L. JACOBS, Annals o f ~ New York academy of sciences, 305, 497-509, 1978. 23. R.M. STEWART, J.H. GROWDON,D. CANClAN and R.J. BALDESSARINI, Neuropharmacol., 15, 449-455 (1976). 24. U. UNGERSTEDT, Acta physiol, scand., suppl. 367, 1-48, 1971. 25. T.A. JAMES and M.S. STARR, J. Pharm. PharmacoT., 32, 196-200, 1980. 26. T. TANNER, J. Pharm. Pharmac. 3__00,158-161, 1978. 27. J. SCHEEL-KRUGER, J. ARNT and G° MAGELUND, Neurosci. L e t t . , 4, 351-356, 1977. 28. G. DI CHIARA, M. MORELLI, M.L. PORCEDDUand G.L. GESSA. Life Sci., 23, 2045-2052, 1978. 29. H.M. CALIL, S. NAKANOand L.E. HOLLISTER, Psychopharmacology, 58, 115-116 1978. 30. B. COSTALL and R.J. NAYLOR, Eur. J. Pharmacol., 29, 206-222, 1974. 31. M. GRABOWSKA, L. ANTKIEWICZ and J. MICHALUK, J. Pharm. Pharmacol. 2_6_6, 74-76, 1974.