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Clonidine and yohimbine separate the sedation and the ptosis caused by cholecystokinin octapeptide and ceruletide
European Journal of Pharmacology, 102 (1984) 333-340
333
Elsevier
CLONIDINE AND YOHIMBINE SEPARATE THE SEDATION AND THE PTOSIS CAUSED BY CHOLECYSTOKININ OCTAPEPTIDE AND CERULETIDE GERHARD ZETLER
lnstitut ff4r Pharmakologie der Medizinischen Hochschule L~beck, Ratzeburger A Ilee 160, D-2400 L~beck, Federal Republic of Germany Received 16 December 1983, revised MS received 13 March 1984, accepted 3 April 1984
G. ZETLER, Clonidine and yohimbine separate the sedation and the ptosis caused by cholecystokinin octapeptide and ceruletide, European J. Pharmacol. 102 (1984) 333-340. The central depressant effects of ceruletide (CER, 0.04 mg/kg s.c.) and cholecystokinin octapeptide (CCK-8, 0.25 mg/kg s.c.) were compared with those of clonidine (0.04 mg/kg s.c.). At doses that were nearly equipotent with respect to motor inhibition (catalepsy, reduction in ambulation and exploratory rearing), only the peptides produced ptosis. Yohimbine (1 mg/kg s.c., 30 min) antagonized the effect of clonidine but not of the peptides. Clonidine (0.07-0.2 mg/kg s.c., 30 min) antagonised the ptotic action of the peptides, and this effect was abolished by yohimbine (0.2-1 mg/kg i.p.) but resistant to haloperidol (0.05 and 0.15 mg/kg i.p.). These results separate the behavioural effects of the peptides from those of clonidine and also the ptotic effect of the peptides from their effect on motor activity. The antiptotic effect of clonidine may originate from activated adrenergic autoreceptors. Cholecystokinin octapeptide Yohimbine
Ceruletide Sedation
Clonidine Motility
1. Introduction
CCK-like peptides such as cholecystokinin octapeptide (CCK-8) and ceruletide (caerulein diethylammonium hydrate, CER) produce many central depressant effects in mice e.g. catalepsy, ptosis and inhibition of running movements and exploratory rearing activity (Zetler, 1980, 1981, 1983a). In rats tested for open field behaviour, the peptides decreased the rate of ambulation and rearing (Van Ree et al., 1983). The mechanism of these effects is not clear but the antiptotic action of low-dose apomorphine pointed to the involvement of presynaptic dopamine autoreceptors (Zetler, 1983b; the antirearing effect of CER was resistant to apomorphine). Another type of sedation, caused in rats and mice by clonidine, is specifically antagonized by the a2-adrenolytic drugs yohimbine and rauwolscine but is resistant to the a 1adrenolytic diastereoisomer of yohimbine, corynanthine (Florio et al., 1975; Delini-Stula et al., 1979; Drew et al., 1979; Clineschmidt et al., 1980; 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Rauwolscine
Apomorphine
Ptosis
Gower and Mariott, 1980; Sumners et al., 1981a; Timmermans et al., 1981; Ortmann et al., 1982). For these reasons the present study was designed to address the question of whether the depressant effects of CER and CCK-8 might have pharmacological properties similar to those of the clonidine effects.
2. Materials and methods
Male NMRI mice weighing about 25 g were kept in groups of 15 and tested (only once) at 22-23°C. Each mouse received first an intraperitoneal (i.p.) and 30 min later a subcutaneous (s.c.) injection of either 0.9% sodium chloride solution (saline) or a drug. Some experiments comprised a third treatment (s.c.). The time schedule of treatments is mentioned in the legends to figures. In the experiments on motor behaviour, the animals were put back in their home cage after
334
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Fig. 1. The effects of clonidine, CER and CCK-8 on the motor behaviour of mice were influenced differently by yohimbine, rauwolscine and corynanthine. Locomotor activity (ambulation, counts per 5 min), exploratory rearing activity (number of rearings per 5 min) and catalepsy (duration of immobility on the vertical rod) were observed in the same mouse. The columns show geometric means:f S.E.M. (number of mice per group indicated in columns). Each animal received two injections 30 min apart. The first treatment (i.p.) is mentioned in the first line below the columns (mg/kg): S, saline; Y, yohimbine (0.5); R, rauwolscine (0.5); C, corynanthine (0.5). The second treatment (s.c.) is indicated below each group of blocks and was either saline (SAL) or clonidine (CLON, 0.04), ceruletide (CER, 0.04) and CCK-8 (0.25). Asterisks indicate a statistically significant difference from the saline-treated control (first column of a given block): o p > 0.05; * P < 0.05; ** P < 0.01. The asterisk above the bracket refers to the difference between the s a m p l e ' S ' and the merged samples ' Y ' plus 'R'.
335 each treatment. Ten minutes after the last injection mice were placed individually on an illuminated glass disc (diameter 24 cm) with 20 photocells mounted under it. The number of crossings of a photocell was counted automatically for 5 rain and used as measure of locomotor activity. The number of rearings during these 5 min was observed and recorded. Thereafter the mouse was put on a vertical string-wrapped rod and the duration of akinesia (absence of running movements, catalepsy) observed for at least 1 min. In separate trials for ptosis, the mice were also kept in their home cages between the first and second injection. Immediately after the second injection, the animals were placed individually in glass cylinders and their palpebral opening scored every 5 min (Zetler, 1983b): score 2, eyes half-closed; score 4, eyes closed; scores 1 and 3, intermediate values. When an antiptotic EDs0 was to be determined, quantal responses were used and a ptosis score of less than 2 was taken as the condition for the verdict 'ptosis absent'. The drugs used were caerulein diethylammonium hydrate (ceruletide, CER, from Farmitalia Carlo Erba), cholecystokinin octapeptide, sulfated (CCK-8, Bachem), clonidine hydrochloride (Boehringer Ingelheim), yohimbine hydrochloride (Serva), rauwolscine hydrochloride (Carl Roth), corynanthine hydrochloride (Ega-Chemie), apomorphine hydrochloride (commercial), haloperidol (Janssen). All drugs were freshly dissolved in saline and administered in a constant volume (0.1 ml/10 g b.w.). Yohimbine, rauwolscine, and corynanthine were given i.p. since this was the route used by preceding workers (see Introduction). Means were compared by analysis of variance (ANOVA; Bartlett's test and Scheffe's test). Fourfold contingency tables were used for intergroup frequency comparisons. EDs0s were estimated with the method of Litchfield and Wilcoxon (1949). P < 0.05 was the condition for accepting statistical significance.
3. Results 3.1. Motor behaviour
Clonidine, CER and CCK-8, in the doses used, produced depressant effects of nearly equal strength (fig. 1). Yohimbine, rauwolscine and corynanthine were themselves ineffective except for a weak reduction of ambulation with corynanthine. Yohimbine and rauwolscine inhibited the effect of clonidine on ambulation and rearing; larger doses of yohimbine had no greater effect (not shown in fig. 1). Corynanthine antagonized clonidine only with respect to catalepsy; the catalepsy was not altered by yohimbine and rauwolscine. Most peptide effects were resistant to the alkaloids. The antirearing effect of CCK-8 was an exception having been somewhat reduced by yohimbine and rauwolscine. 3.2. Ptosis
Clonidine itself did not produce ptosis even at a dose of 0.2 mg/kg (results not shown) but it antagonized the ptosis induced by CER or CCK-8 (fig. 2). This effect was dose-dependent, which permitted the calculation of an antiptotic EDs0 (mg/kg, 95% confidence range): against CER, 0.14 (0.119-0.165); against CCK-8, 0.145 (0.114-0.184). It is noteworthy that these doses of clonidine were nearly 3 times that producing strong inhibition of motor behaviour (see fig. 1). Yohimbine, in doses between 0.5 and 2.0 mg/kg (i.p.), neither produced ptosis (agreement with the observations of Boissier et al., 1968, and Scatton et al., 1980) nor modified the ptotic effect of CER (results of these experiments not shown). However, yohimbine prevented the antiptotic effect of clonidine versus CER and CCK-8 (fig. 3, lower part). Corynanthine was inactive in this respect. The effect of yohimbine occurred over a limited dose range. The inverted U-shaped dose-effect curve is reminiscent of similar observations on the effect of yohimbine against clonidine-induced sedation (Delini-Stula et al., 1979; McKearney, 1983). The presentation of fig. 3 was necessary since larger doses of both yohimbine and corynanthine given together with clonidine pro-
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Fig. 2. Clonidine antagonized the ptosis induced by CER or CCK-8. Arithmetic means (and S.E.M.) of ptosis scores 20 min after the administration of either CER (0.04 mg/kg) or CCK-8 (0.25 mg/kg) are shown; 10 animals per column. The pretreatment (given i.p. 30 min before a peptide) is mentioned below the columns: S, saline; the numbers refer to doses of clonidine (mg/kg). Statistics (ANOVA) refer to the difference from control (S): © P > 0.05; * P < 0.05; ** P < 0.01.
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d u c e d ptosis (note the i n t e r r u p t e d lines in the u p p e r part of fig. 3). W h e n c o m b i n e d with clonidine, 2 m g / k g each of y o h i m b i n e or c o r y n a n t h i n e b r o u g h t a b o u t ptosis (score 2 or more) in 6 out of 10 or 7 out of 10 mice, respectively. Since u n treated mice never show ptosis u n d e r the conditions used b o t h results are statistically significant
(P < 0.02). The intricate drug interactions illustrated in fig. 3 m a d e it i m p o r t a n t to find out if c l o n i d i n e was c a p a b l e of i n t e r r u p t i n g a n existant C E R - i n d u c e d ptosis. Fig. 4 shows this to have b e e n the case. It is also evident that the p r e t r e a t m e n t with y o h i m b i n e completely p r e v e n t e d the a n t i p t o t i c action of c l o n i d i n e (the u n a l t e r e d c l o n i d i n e effect at 5 m i n after the a d m i n i s t r a t i o n of this drug m a y have b e e n the result of an i m m e d i a t e b u t transient competitive d i s p l a c e m e n t of y o h i m b i n e by clonid i n e at relevant structures). It can also be seen from fig. 4 that c o r y n a n t h i n e was n o t completely inactive (cf. the time p o i n t 20 min) b u t its antic l o n i d i n e action declined very rapidly (as early as after 25 min, the difference b e t w e e n the c l o n i d i n e effects with a n d without c o r y n a n t h i n e was n o longer statistically significant). The difference between y o h i m b i n e a n d c o r y n a n t h i n e at 20 m i n m a y suggest a less specific i n t e r a c t i o n of c o r y n a n t h i n e
=Y3 SCL=
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Fig. 3. Yohimbine prevented the antiptotic effect of clonidine. The columns show the arithmetic means of ptosis scores (and S.E.M.). Upper part: full lines, scores 20 min after the administration of a peptide; interrupted lines: scores immediately before the administration of a peptide. Lower part: corrected scores, i.e. peptide-induced changes in scores (differences between the two types of columns shown in the upper part); 10 animals per column. Numbers above a column indicate the number of animals having ptosis scores of 2 and more. Treatments as mentioned below columns. First line: first injection (i.p.), S, saline; Y1-Y4, yohimbine (mg/kg), 0.35 (Y1), 0.5 (Y2), 1.0 (Y3), 2.0 (Y4); corynanthine (mg/kg), 0.5 (C1), 1.0 (C2), 2,0 (C3). Second line: second injection (s.c., 30 rain later), S, saline; CL, clonidine (0.2 mg/kg). The peptides were given (s.c.) after a further 20 min: CER (0.04 mg/kg), CCK-8 (0.25 mg/kg). Only 'corrected' scores were used for statistical analyses and the differences are those from the second column of each block (S+CL). * P < 0.02; ** P < 0.002.
with the clonidine-sensitive mechanism. I n order to assess the specificity of the yohimb i n e - c l o n i d i n e interaction, we tested whether (a) y o h i m b i n e would alter the antiptotic effect of a p o m o r p h i n e a n d (b) haloperidol would reduce the antiptotic effect of clonidine. The rationale of these experiments was the possibility that yohimb i n e a n d haloperidol could be specific antagonists at presynaptic autoreceptors of noradrenergic a n d d o p a m i n e r g i c neurons, respectively. I n fact, low
337
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Fig. 4. Clonidine abolished the CER-induced ptosis and the interaction of yohimbine and corynanthine. Abscissa, time (rain) following the s.c. administration of CER (0.04 mg/kg). Ordinate, number of animals (out of I0 per curve) having ptosis scores of 3 or more. Arrow, s.c. administration of either saline (1) or clonidine, 0.2 mg/kg (©, zl, D). The mice received i.p. 30 min before the injection of CER either saline ( t , ©) or yohimbine, 1 m g / k g (zx), and corynanthine, 1 m g / k g (D). Statistics refer to the difference between two vertically adjacent points. The differences between the results obtained with yohimbine and corynanthine were statistically significant at all time points except 25 min.
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Fig. 5. Yohimbine antagonized the antiptotic effect of apomorphine. The means ( _+S.E.M.) of ptosis scores are shown. Each pair of columns refers to results from one group of 10 mice: first column, ptosis 10 min after CER; second column, ptosis 25 min after CER. Treatments as indicated below the columns: first line, first injection (i.p.); second line, second injection (CER, 30 min later, s.c.); third line, third injection (s.c., 10 rain after CER). Treatments (mg/kg): saline (S); CER, 0.04 (CE); apomorphine, 0.2 (A); yohimbine, 1.0 (Y). Symbols above columns refer to the difference from the corresponding column in the first block (S + CER + S): o p > 0.05; ** P < 0.01 (statistics: ANOVA).
doses of haloperidol (0.02-0.09 mg/kg) have been found to abolish the antiptotic effect of apomorphine versus CCK-like peptides (Zetler, 1983b). Fig. 5 shows that yohimbine eliminated the antiptotic effect of apomorphine. In contrast, haloperidol (0.05 and 0.15 mg/kg) did not alter the antiptotic effect of clonidine (0.2 mg/kg) versus CER (results not shown; same type of experiment as in fig. 3). It is noteworthy that the mice treated with haloperidol plus clonidine were nearly motionless but kept their eye fissures wide open. This behaviour was not altered by the subsequent administration of CER (0.04 mg/kg).
4. Discussion
It is appropriate to assume that clonidine, yohimbine and rauwolscine exerted their effects on motor behaviour by a selective influence on central adrenergic mechanisms via brain a2-autoreceptors (Hedler et al., 1981; Timmermans et al., 1981). The interactions of these drugs concerning ambulation and rearing (and the ineffectiveness of corynanthine) agree with what has been found by others, also with respect to the doses used (Florio et al., 1975; Delini-Stula et al., 1979; Drew et al., 1979; Clineschmidt et al., 1980; Gower and Marriott, 1980; Sumners et al., 1981b; Ortmann et al., 1982). Accordingly, the present results permit the following conclusions: first, the adrenergic system is more important for ambulation and rearing than for catalepsy; second, the adrenergic system cannot modulate the motor depressant actions of CCK-like peptides; third, the ptotic effect of CCK-like peptides can be antagonized by clonidine and thereby separated from the effects on motor behaviour (the latter could also be done with apomorphine; Zetler, 1983b). The antiptotic effect of clonidine was antagonized by yohimbine but not by corynanthine, which suggests that a2-adrenergic autoreceptors are involved (Hedler et al., 1981; Timmermans et al., 1981). A previous study led to the conclusion that the activation of dopamine autoreceptors is the cause of the antiptotic effect of apomorphine versus CCK-like peptides (Zetler, 1983b). Hence, the question arises as to how two different aminergic
338 mechanisms can result in the same net antiptotic effect. At the appropriate doses, as used in the present and previous experiments neither yohimbine nor haloperidol produced ptosis (yohimbine: own resuits, furthermore Boissier et al., 1968, and Scatton et al., 1980; haloperidol: Zetler, 1983b). Therefore, the antagonism of the antiptotic effect of clonidine by yohimbine as well as the antagonism of apomorphine by haloperidol cannot be functional by nature. Rather, it is appropriate to assume that both clonidine and apomorphine antagonized the ptosis indirectly via primary stimulation of their relevant receptors, i.e. presynaptic autoreceptors of the adrenergic and dopaminergic system. The view of an interaction of dopaminergic and CCKergic mechanisms has a broad experimental basis which has already been discussed (Zetler, 1983b; for more references see Agnati et al., 1983; Fuxe et al., 1983). The finding that CCK-like peptides (after systemic administration) can induce a supersensitivity of mesencephalic dopamine autoreceptors (Hommer and Skirboll, 1983) is most important in this respect. On the other hand, altered dopaminergic function in the brain (of mice and guinea-pigs) has been found to increase CCK-receptor binding in mesolimbic regions and frontal cortex (Chang et al., 1983); dopamine receptors modulate CCK release in rat neostriatal slices (Meyer and Krauss, 1983). Receptor-receptor interaction may be the mechanism of these dopaminergic/CCK-ergic interactions (Fuxe et al., 1983). In contrast, much less is known about a possible interconnection between CCK-ergic and noradrenergic structures or functions. It has been observed that i.c.v, administration of CCK-8 to rats increased the turnover of noradrenaline in the hypothalamus and amygdala but decreased it in the striatum (Fekete et al., 1981a), and altered the noradrenaline concentration in several brain parts (Fekete et al., 1981b). CCK antiserum given i.c.v. lowered the noradrenaline content of many brain areas (Khdhr et al., 1981). These findings cannot explain why the activation of adrenergic autoreceptors by clonidine was antiptotic. However, there are many reports of noradrenergic-dopaminergic
interaction and mutual modulation (Persson and Waldeck, 1970; Antelman and Caggiula, 1977; Sumners et al., 1981b), especially a stimulatory influence by noradrenaline neurons on dopamine neurons (And6n and Grabowska, 1976; Hedler et al., 1981; And6n et al., 1982). It may be speculated that clonidine antagonized the CER-induced ptosis indirectly, namely by first stimulating the noradrenaline autoreceptors, which reduced noradrenergic activity and thereby the stimulatory effect on dopaminergic neurons. The net result regarding ptosis would be the same as if the dopaminergic neuron were inhibited via activation of its autoreceptors by apomorphine. It is thus conceivable that haloperidol did not prevent the antiptotic action of clonidine, because the chain of events (a) started at adrenergic autoreceptors and (b) did not involve dopamine autoreceptors. The finding that yohimbine antagonized the antiptotic effect of apomorphine is a parallel of the reduction by yohimbine of the apomorphine-induced hypomotility of mice, which suggested the involvement of the noradrenergic system (Sumners et al., 1981b). In fact, according to experiments in vitro, adrenergic a2-receptors are operative in the regulation of dopaminergic transmission in the hypothalamus (Ueda et al., 1983), although not in the striatum (of the rat; Scatton et al., 1983). One may speculate that yohimbine increased the activity of the noradrenergic neurons (Hedler et al., 1981) and thereby enhanced the activity of the dopaminergic neurons (see above); this would eventually have nullified the apomorphine-induced reduction of dopaminergic activity. The activation of aminergic autoreceptors antagonized the ptotic but not the sedative effect of CCK-like peptides. Nerve terminal/cell body dopamine autoreceptors are present in the nigrostriatal/mesolimbic systems but absent in the mesocortical dopamine neurons (review: Bannon and Roth, 1983). Therefore, one could consider the possibility that CCK-like peptides produce ptosis via nigrostriatal/mesolimbic systems but produce sedation via the mesocortical system. This would apply provided results obtained with rats are valid for mice and vice versa which is questionable (Cools and Van Rossum, 1980; Wood et al., 1980).
339
Acknowledgements The author thanks Prof. R. de Castiglione (Farmitalia Carlo Erba/Milan) for the generous gift of ceruletide, Mrs. G. Schmidt for skilled technical assistance, Mrs. M.-L. Welge for graphical work and Mrs. G. Mundhenke for typing the manuscript.
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Elsevier
CLONIDINE AND YOHIMBINE SEPARATE THE SEDATION AND THE PTOSIS CAUSED BY CHOLECYSTOKININ OCTAPEPTIDE AND CERULETIDE GERHARD ZETLER
lnstitut ff4r Pharmakologie der Medizinischen Hochschule L~beck, Ratzeburger A Ilee 160, D-2400 L~beck, Federal Republic of Germany Received 16 December 1983, revised MS received 13 March 1984, accepted 3 April 1984
G. ZETLER, Clonidine and yohimbine separate the sedation and the ptosis caused by cholecystokinin octapeptide and ceruletide, European J. Pharmacol. 102 (1984) 333-340. The central depressant effects of ceruletide (CER, 0.04 mg/kg s.c.) and cholecystokinin octapeptide (CCK-8, 0.25 mg/kg s.c.) were compared with those of clonidine (0.04 mg/kg s.c.). At doses that were nearly equipotent with respect to motor inhibition (catalepsy, reduction in ambulation and exploratory rearing), only the peptides produced ptosis. Yohimbine (1 mg/kg s.c., 30 min) antagonized the effect of clonidine but not of the peptides. Clonidine (0.07-0.2 mg/kg s.c., 30 min) antagonised the ptotic action of the peptides, and this effect was abolished by yohimbine (0.2-1 mg/kg i.p.) but resistant to haloperidol (0.05 and 0.15 mg/kg i.p.). These results separate the behavioural effects of the peptides from those of clonidine and also the ptotic effect of the peptides from their effect on motor activity. The antiptotic effect of clonidine may originate from activated adrenergic autoreceptors. Cholecystokinin octapeptide Yohimbine
Ceruletide Sedation
Clonidine Motility
1. Introduction
CCK-like peptides such as cholecystokinin octapeptide (CCK-8) and ceruletide (caerulein diethylammonium hydrate, CER) produce many central depressant effects in mice e.g. catalepsy, ptosis and inhibition of running movements and exploratory rearing activity (Zetler, 1980, 1981, 1983a). In rats tested for open field behaviour, the peptides decreased the rate of ambulation and rearing (Van Ree et al., 1983). The mechanism of these effects is not clear but the antiptotic action of low-dose apomorphine pointed to the involvement of presynaptic dopamine autoreceptors (Zetler, 1983b; the antirearing effect of CER was resistant to apomorphine). Another type of sedation, caused in rats and mice by clonidine, is specifically antagonized by the a2-adrenolytic drugs yohimbine and rauwolscine but is resistant to the a 1adrenolytic diastereoisomer of yohimbine, corynanthine (Florio et al., 1975; Delini-Stula et al., 1979; Drew et al., 1979; Clineschmidt et al., 1980; 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Rauwolscine
Apomorphine
Ptosis
Gower and Mariott, 1980; Sumners et al., 1981a; Timmermans et al., 1981; Ortmann et al., 1982). For these reasons the present study was designed to address the question of whether the depressant effects of CER and CCK-8 might have pharmacological properties similar to those of the clonidine effects.
2. Materials and methods
Male NMRI mice weighing about 25 g were kept in groups of 15 and tested (only once) at 22-23°C. Each mouse received first an intraperitoneal (i.p.) and 30 min later a subcutaneous (s.c.) injection of either 0.9% sodium chloride solution (saline) or a drug. Some experiments comprised a third treatment (s.c.). The time schedule of treatments is mentioned in the legends to figures. In the experiments on motor behaviour, the animals were put back in their home cage after
334
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Fig. 1. The effects of clonidine, CER and CCK-8 on the motor behaviour of mice were influenced differently by yohimbine, rauwolscine and corynanthine. Locomotor activity (ambulation, counts per 5 min), exploratory rearing activity (number of rearings per 5 min) and catalepsy (duration of immobility on the vertical rod) were observed in the same mouse. The columns show geometric means:f S.E.M. (number of mice per group indicated in columns). Each animal received two injections 30 min apart. The first treatment (i.p.) is mentioned in the first line below the columns (mg/kg): S, saline; Y, yohimbine (0.5); R, rauwolscine (0.5); C, corynanthine (0.5). The second treatment (s.c.) is indicated below each group of blocks and was either saline (SAL) or clonidine (CLON, 0.04), ceruletide (CER, 0.04) and CCK-8 (0.25). Asterisks indicate a statistically significant difference from the saline-treated control (first column of a given block): o p > 0.05; * P < 0.05; ** P < 0.01. The asterisk above the bracket refers to the difference between the s a m p l e ' S ' and the merged samples ' Y ' plus 'R'.
335 each treatment. Ten minutes after the last injection mice were placed individually on an illuminated glass disc (diameter 24 cm) with 20 photocells mounted under it. The number of crossings of a photocell was counted automatically for 5 rain and used as measure of locomotor activity. The number of rearings during these 5 min was observed and recorded. Thereafter the mouse was put on a vertical string-wrapped rod and the duration of akinesia (absence of running movements, catalepsy) observed for at least 1 min. In separate trials for ptosis, the mice were also kept in their home cages between the first and second injection. Immediately after the second injection, the animals were placed individually in glass cylinders and their palpebral opening scored every 5 min (Zetler, 1983b): score 2, eyes half-closed; score 4, eyes closed; scores 1 and 3, intermediate values. When an antiptotic EDs0 was to be determined, quantal responses were used and a ptosis score of less than 2 was taken as the condition for the verdict 'ptosis absent'. The drugs used were caerulein diethylammonium hydrate (ceruletide, CER, from Farmitalia Carlo Erba), cholecystokinin octapeptide, sulfated (CCK-8, Bachem), clonidine hydrochloride (Boehringer Ingelheim), yohimbine hydrochloride (Serva), rauwolscine hydrochloride (Carl Roth), corynanthine hydrochloride (Ega-Chemie), apomorphine hydrochloride (commercial), haloperidol (Janssen). All drugs were freshly dissolved in saline and administered in a constant volume (0.1 ml/10 g b.w.). Yohimbine, rauwolscine, and corynanthine were given i.p. since this was the route used by preceding workers (see Introduction). Means were compared by analysis of variance (ANOVA; Bartlett's test and Scheffe's test). Fourfold contingency tables were used for intergroup frequency comparisons. EDs0s were estimated with the method of Litchfield and Wilcoxon (1949). P < 0.05 was the condition for accepting statistical significance.
3. Results 3.1. Motor behaviour
Clonidine, CER and CCK-8, in the doses used, produced depressant effects of nearly equal strength (fig. 1). Yohimbine, rauwolscine and corynanthine were themselves ineffective except for a weak reduction of ambulation with corynanthine. Yohimbine and rauwolscine inhibited the effect of clonidine on ambulation and rearing; larger doses of yohimbine had no greater effect (not shown in fig. 1). Corynanthine antagonized clonidine only with respect to catalepsy; the catalepsy was not altered by yohimbine and rauwolscine. Most peptide effects were resistant to the alkaloids. The antirearing effect of CCK-8 was an exception having been somewhat reduced by yohimbine and rauwolscine. 3.2. Ptosis
Clonidine itself did not produce ptosis even at a dose of 0.2 mg/kg (results not shown) but it antagonized the ptosis induced by CER or CCK-8 (fig. 2). This effect was dose-dependent, which permitted the calculation of an antiptotic EDs0 (mg/kg, 95% confidence range): against CER, 0.14 (0.119-0.165); against CCK-8, 0.145 (0.114-0.184). It is noteworthy that these doses of clonidine were nearly 3 times that producing strong inhibition of motor behaviour (see fig. 1). Yohimbine, in doses between 0.5 and 2.0 mg/kg (i.p.), neither produced ptosis (agreement with the observations of Boissier et al., 1968, and Scatton et al., 1980) nor modified the ptotic effect of CER (results of these experiments not shown). However, yohimbine prevented the antiptotic effect of clonidine versus CER and CCK-8 (fig. 3, lower part). Corynanthine was inactive in this respect. The effect of yohimbine occurred over a limited dose range. The inverted U-shaped dose-effect curve is reminiscent of similar observations on the effect of yohimbine against clonidine-induced sedation (Delini-Stula et al., 1979; McKearney, 1983). The presentation of fig. 3 was necessary since larger doses of both yohimbine and corynanthine given together with clonidine pro-
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Fig. 2. Clonidine antagonized the ptosis induced by CER or CCK-8. Arithmetic means (and S.E.M.) of ptosis scores 20 min after the administration of either CER (0.04 mg/kg) or CCK-8 (0.25 mg/kg) are shown; 10 animals per column. The pretreatment (given i.p. 30 min before a peptide) is mentioned below the columns: S, saline; the numbers refer to doses of clonidine (mg/kg). Statistics (ANOVA) refer to the difference from control (S): © P > 0.05; * P < 0.05; ** P < 0.01.
9
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d u c e d ptosis (note the i n t e r r u p t e d lines in the u p p e r part of fig. 3). W h e n c o m b i n e d with clonidine, 2 m g / k g each of y o h i m b i n e or c o r y n a n t h i n e b r o u g h t a b o u t ptosis (score 2 or more) in 6 out of 10 or 7 out of 10 mice, respectively. Since u n treated mice never show ptosis u n d e r the conditions used b o t h results are statistically significant
(P < 0.02). The intricate drug interactions illustrated in fig. 3 m a d e it i m p o r t a n t to find out if c l o n i d i n e was c a p a b l e of i n t e r r u p t i n g a n existant C E R - i n d u c e d ptosis. Fig. 4 shows this to have b e e n the case. It is also evident that the p r e t r e a t m e n t with y o h i m b i n e completely p r e v e n t e d the a n t i p t o t i c action of c l o n i d i n e (the u n a l t e r e d c l o n i d i n e effect at 5 m i n after the a d m i n i s t r a t i o n of this drug m a y have b e e n the result of an i m m e d i a t e b u t transient competitive d i s p l a c e m e n t of y o h i m b i n e by clonid i n e at relevant structures). It can also be seen from fig. 4 that c o r y n a n t h i n e was n o t completely inactive (cf. the time p o i n t 20 min) b u t its antic l o n i d i n e action declined very rapidly (as early as after 25 min, the difference b e t w e e n the c l o n i d i n e effects with a n d without c o r y n a n t h i n e was n o longer statistically significant). The difference between y o h i m b i n e a n d c o r y n a n t h i n e at 20 m i n m a y suggest a less specific i n t e r a c t i o n of c o r y n a n t h i n e
=Y3 SCL=
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Fig. 3. Yohimbine prevented the antiptotic effect of clonidine. The columns show the arithmetic means of ptosis scores (and S.E.M.). Upper part: full lines, scores 20 min after the administration of a peptide; interrupted lines: scores immediately before the administration of a peptide. Lower part: corrected scores, i.e. peptide-induced changes in scores (differences between the two types of columns shown in the upper part); 10 animals per column. Numbers above a column indicate the number of animals having ptosis scores of 2 and more. Treatments as mentioned below columns. First line: first injection (i.p.), S, saline; Y1-Y4, yohimbine (mg/kg), 0.35 (Y1), 0.5 (Y2), 1.0 (Y3), 2.0 (Y4); corynanthine (mg/kg), 0.5 (C1), 1.0 (C2), 2,0 (C3). Second line: second injection (s.c., 30 rain later), S, saline; CL, clonidine (0.2 mg/kg). The peptides were given (s.c.) after a further 20 min: CER (0.04 mg/kg), CCK-8 (0.25 mg/kg). Only 'corrected' scores were used for statistical analyses and the differences are those from the second column of each block (S+CL). * P < 0.02; ** P < 0.002.
with the clonidine-sensitive mechanism. I n order to assess the specificity of the yohimb i n e - c l o n i d i n e interaction, we tested whether (a) y o h i m b i n e would alter the antiptotic effect of a p o m o r p h i n e a n d (b) haloperidol would reduce the antiptotic effect of clonidine. The rationale of these experiments was the possibility that yohimb i n e a n d haloperidol could be specific antagonists at presynaptic autoreceptors of noradrenergic a n d d o p a m i n e r g i c neurons, respectively. I n fact, low
337
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Fig. 4. Clonidine abolished the CER-induced ptosis and the interaction of yohimbine and corynanthine. Abscissa, time (rain) following the s.c. administration of CER (0.04 mg/kg). Ordinate, number of animals (out of I0 per curve) having ptosis scores of 3 or more. Arrow, s.c. administration of either saline (1) or clonidine, 0.2 mg/kg (©, zl, D). The mice received i.p. 30 min before the injection of CER either saline ( t , ©) or yohimbine, 1 m g / k g (zx), and corynanthine, 1 m g / k g (D). Statistics refer to the difference between two vertically adjacent points. The differences between the results obtained with yohimbine and corynanthine were statistically significant at all time points except 25 min.
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Fig. 5. Yohimbine antagonized the antiptotic effect of apomorphine. The means ( _+S.E.M.) of ptosis scores are shown. Each pair of columns refers to results from one group of 10 mice: first column, ptosis 10 min after CER; second column, ptosis 25 min after CER. Treatments as indicated below the columns: first line, first injection (i.p.); second line, second injection (CER, 30 min later, s.c.); third line, third injection (s.c., 10 rain after CER). Treatments (mg/kg): saline (S); CER, 0.04 (CE); apomorphine, 0.2 (A); yohimbine, 1.0 (Y). Symbols above columns refer to the difference from the corresponding column in the first block (S + CER + S): o p > 0.05; ** P < 0.01 (statistics: ANOVA).
doses of haloperidol (0.02-0.09 mg/kg) have been found to abolish the antiptotic effect of apomorphine versus CCK-like peptides (Zetler, 1983b). Fig. 5 shows that yohimbine eliminated the antiptotic effect of apomorphine. In contrast, haloperidol (0.05 and 0.15 mg/kg) did not alter the antiptotic effect of clonidine (0.2 mg/kg) versus CER (results not shown; same type of experiment as in fig. 3). It is noteworthy that the mice treated with haloperidol plus clonidine were nearly motionless but kept their eye fissures wide open. This behaviour was not altered by the subsequent administration of CER (0.04 mg/kg).
4. Discussion
It is appropriate to assume that clonidine, yohimbine and rauwolscine exerted their effects on motor behaviour by a selective influence on central adrenergic mechanisms via brain a2-autoreceptors (Hedler et al., 1981; Timmermans et al., 1981). The interactions of these drugs concerning ambulation and rearing (and the ineffectiveness of corynanthine) agree with what has been found by others, also with respect to the doses used (Florio et al., 1975; Delini-Stula et al., 1979; Drew et al., 1979; Clineschmidt et al., 1980; Gower and Marriott, 1980; Sumners et al., 1981b; Ortmann et al., 1982). Accordingly, the present results permit the following conclusions: first, the adrenergic system is more important for ambulation and rearing than for catalepsy; second, the adrenergic system cannot modulate the motor depressant actions of CCK-like peptides; third, the ptotic effect of CCK-like peptides can be antagonized by clonidine and thereby separated from the effects on motor behaviour (the latter could also be done with apomorphine; Zetler, 1983b). The antiptotic effect of clonidine was antagonized by yohimbine but not by corynanthine, which suggests that a2-adrenergic autoreceptors are involved (Hedler et al., 1981; Timmermans et al., 1981). A previous study led to the conclusion that the activation of dopamine autoreceptors is the cause of the antiptotic effect of apomorphine versus CCK-like peptides (Zetler, 1983b). Hence, the question arises as to how two different aminergic
338 mechanisms can result in the same net antiptotic effect. At the appropriate doses, as used in the present and previous experiments neither yohimbine nor haloperidol produced ptosis (yohimbine: own resuits, furthermore Boissier et al., 1968, and Scatton et al., 1980; haloperidol: Zetler, 1983b). Therefore, the antagonism of the antiptotic effect of clonidine by yohimbine as well as the antagonism of apomorphine by haloperidol cannot be functional by nature. Rather, it is appropriate to assume that both clonidine and apomorphine antagonized the ptosis indirectly via primary stimulation of their relevant receptors, i.e. presynaptic autoreceptors of the adrenergic and dopaminergic system. The view of an interaction of dopaminergic and CCKergic mechanisms has a broad experimental basis which has already been discussed (Zetler, 1983b; for more references see Agnati et al., 1983; Fuxe et al., 1983). The finding that CCK-like peptides (after systemic administration) can induce a supersensitivity of mesencephalic dopamine autoreceptors (Hommer and Skirboll, 1983) is most important in this respect. On the other hand, altered dopaminergic function in the brain (of mice and guinea-pigs) has been found to increase CCK-receptor binding in mesolimbic regions and frontal cortex (Chang et al., 1983); dopamine receptors modulate CCK release in rat neostriatal slices (Meyer and Krauss, 1983). Receptor-receptor interaction may be the mechanism of these dopaminergic/CCK-ergic interactions (Fuxe et al., 1983). In contrast, much less is known about a possible interconnection between CCK-ergic and noradrenergic structures or functions. It has been observed that i.c.v, administration of CCK-8 to rats increased the turnover of noradrenaline in the hypothalamus and amygdala but decreased it in the striatum (Fekete et al., 1981a), and altered the noradrenaline concentration in several brain parts (Fekete et al., 1981b). CCK antiserum given i.c.v. lowered the noradrenaline content of many brain areas (Khdhr et al., 1981). These findings cannot explain why the activation of adrenergic autoreceptors by clonidine was antiptotic. However, there are many reports of noradrenergic-dopaminergic
interaction and mutual modulation (Persson and Waldeck, 1970; Antelman and Caggiula, 1977; Sumners et al., 1981b), especially a stimulatory influence by noradrenaline neurons on dopamine neurons (And6n and Grabowska, 1976; Hedler et al., 1981; And6n et al., 1982). It may be speculated that clonidine antagonized the CER-induced ptosis indirectly, namely by first stimulating the noradrenaline autoreceptors, which reduced noradrenergic activity and thereby the stimulatory effect on dopaminergic neurons. The net result regarding ptosis would be the same as if the dopaminergic neuron were inhibited via activation of its autoreceptors by apomorphine. It is thus conceivable that haloperidol did not prevent the antiptotic action of clonidine, because the chain of events (a) started at adrenergic autoreceptors and (b) did not involve dopamine autoreceptors. The finding that yohimbine antagonized the antiptotic effect of apomorphine is a parallel of the reduction by yohimbine of the apomorphine-induced hypomotility of mice, which suggested the involvement of the noradrenergic system (Sumners et al., 1981b). In fact, according to experiments in vitro, adrenergic a2-receptors are operative in the regulation of dopaminergic transmission in the hypothalamus (Ueda et al., 1983), although not in the striatum (of the rat; Scatton et al., 1983). One may speculate that yohimbine increased the activity of the noradrenergic neurons (Hedler et al., 1981) and thereby enhanced the activity of the dopaminergic neurons (see above); this would eventually have nullified the apomorphine-induced reduction of dopaminergic activity. The activation of aminergic autoreceptors antagonized the ptotic but not the sedative effect of CCK-like peptides. Nerve terminal/cell body dopamine autoreceptors are present in the nigrostriatal/mesolimbic systems but absent in the mesocortical dopamine neurons (review: Bannon and Roth, 1983). Therefore, one could consider the possibility that CCK-like peptides produce ptosis via nigrostriatal/mesolimbic systems but produce sedation via the mesocortical system. This would apply provided results obtained with rats are valid for mice and vice versa which is questionable (Cools and Van Rossum, 1980; Wood et al., 1980).
339
Acknowledgements The author thanks Prof. R. de Castiglione (Farmitalia Carlo Erba/Milan) for the generous gift of ceruletide, Mrs. G. Schmidt for skilled technical assistance, Mrs. M.-L. Welge for graphical work and Mrs. G. Mundhenke for typing the manuscript.
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