A behavioral pharmacological study on CCK-8 related peptides in mice

Neuropeptides

8:

273-286,

1986

A BEHAVIORAL PHARMACOLOGICAL PEPTIDES IN MICE

STUDY

ON

CCK-8

Takashi Moroji and Yoko Haglno Department of Psychopharmacology, Psychiatric Research Tokyo, ‘2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, Japan requests to TM)

RELATED

Institute of 156 (reprint

The effects of CCK-8, ceruletide, their non-sulfated forms and CCK-4 on locomotor activity and rearing in mice were examined. CCK-8 and ceruletide, but not their non-sulfated forms and CCK-4, significantly inhibited the behavioral parameters dose-dependently. The inhibitory effects of CCK-8 and ceruletide were similar, but ceruletide acted more slowly and its inhibitory effect continued much longer than CCK-8, suggesting a difference in stability with the chemical structure. The antagonistic effects of ceruletide on behavioral changes induced by DA agonists were also examined. Peripherally injected ceruletide antagonized both methylphenidate- and methamphetamineinduced hyperactivity in mice dose-dependently, whereas it had no dose-related antagonistic effect on methylphenidate-induced stereotyped behavior. Ceruletide also significantly inhibited apomorphineinduced hyperactivity when injected peripherally. However, no obvious dose-response relationship was observed in either intensity or duration of inhibitory action of ceruletide. These findings suggest that ceruletide does not interfere with dopaminergic transmission due to the blockade of postsynaptic DA receptors in the brain. In conclusion, peripheral injection of CCK-8 and ceruletide may affect directly or indirectly dopamine function producing behavioral changes that resemble those of neuroleptics in some respects. INTRODUCTION Cholecystokinin triacontatriapeptide (CCK-33) is a gastrointestinal hormone that produces gall bladder contraction and stimulates pancreatic enzyme secretion (1,2). Recently, CCK-like peptides have been found to be widely distributed throughout the mammalian brain (3-ll), being in especially high concentration within the limbic lobes (hippocampus, olfactory tubercle, amygdala and associated cortics) (7,9,10,11). The major CCK entity in the brain is the COOH-terminal octapeptide (CCK-8) (5,13,17), followed by the tetrapeptide (CCK-4) (5,13,17). The discovery of CCK-like peptides in the brain has raised interest in their physiological role in the brain function. Accumulating evidence suggests that CCK-like peptides play an important role as a neurotransmitter or neuromodulator (18-22).

273

Either immunohistochemical or neurochemical studies indicated that CCK-like peptides are present in a population of mesencephalic dopaminergic neurons projecting mainly to limbic regions in the forebrain including the nucleus accumbens (A-10) (23,24). Moreover, intraventricular administered COOH-fragments have been found to inhibit dopamine (DA) release in the nucleus accumbens-tuberculum olfactorium (25). These findings have been of particular interest in connection with the DA hypothesis of schizophrenia (26). Hokfelt et al. postulated an imbalance between DA and CCK-like peptides in the mesolimbic DA system as a component of disturbances underlying the disease process of schizophrenia (27). Centrally or peripherally administered CCK-like peptides induced behavioral changes in rodents, and these effects resemble those induced by neuroleptic drugs in some aspects (28-37). In fact, we previously found that a single intramuscular injection of ceruletide, a decapeptide chemically related to CCK-8, resulted in a long-lasting antipsychotic effects in some schizophrenic patients who responded poorly to standard neuroleptic therapy (38,39). Since then, several repeated studies performed as an open trial or double-blind placebocontrolled study have shown that CCK-like peptides, such as CCK-33, CCK-8 and ceruletide, are effective in treating a certain type of schizophrenia (40-49). These clinical studies, taken together with the findings obtained in animal experiments, suggest that CCK-like peptides may participate in the pathogenesis of schizophrenia and further that CCK-like peptides could be a new type of useful and effective antipsychotic drug. However, behavioral effects occurring in rodents after peripheral injection of CCK-like peptides have not been in good agreement. To clarify the behavioral profile after peripheral injection of CCK-like peptides, we have conducted several behavioral pharmacological studies. MATERIAL

AND METHOD

Male ddY strain mice weighing 25-33 g were used for all experiments. They were housed in group of 6 mice per cage in an animal room with controlled temperature and humidity, light on 8:00 a.m., off 8:00 p.m. All animals had accessed to commercial food and tap water ad libitum . The drugs used in the present study were as follows; CCK-8, its non-sulfated form (CCK-8-NS) and CCK-4 (Peptide Institute Inc., Osaka, Japan) ; ceruletide and its non-sulfated form (ceruletide-NS) (gift of Shionogi Co., Osaka, Japan) ; methylphenidate hydrochloride (Ciba-Geigy , Basel, Switzerland) ; methamphetamine hydrochloride (Dainippon Pharm. Co., Osaka, Japan); apomorphine hydrochloride (Sigma Chemical Co. , St. Louis, U.S. A. ) . Ceruletide-NS was dissolved in 1% sodium bicarbonate and was neutralized with 0.1% hydrochloric acid. CCK-4 was dissolved in disstilled water containing 0.01% cysteine and 2% sodium bicarbonate. Apomorphine hydrochloride was dissolved in physiological saline containing 0.1% ascorbic acid. Other drugs were dissolved in physiological saline. Methylphenidate and methamphetamine were injected intraperitoneally 0.p. > and other

274

drugs were injected subcutaneously (s.c. ) in a volume of 0.1 ml/10 g body weight. All solutions were freshly prepared just before use. Locomotor activity of mice was measured using MK-Animex (Muromachi Kikai Co. , Tokyo, Japan), a capacitive sensing activity monitor. The The number of times animal test cage was 28 x 39 x 30 acryl cage. lifted its forepaws completely from the ground, including a pause and upward lifting the head and trunk, was recorded as the rearing rate Each test session was 10 min long. Both locomotor activity (50). and rearing rate were determined 10, 40 and 70 min after the injection of CCK-8 and 30, 60 and 120 min after the administration of ceruletide. Behavior during the 10 min session was observed only once 10 min after the injection of CCK-4 and the non-sulfated form of CCK-8 or ceruletide. After each mouse was allowed 60 min to become acclimatized to the test cage, the effect of ceruletide on DA agonist-induced hyperThree different types of DA agonists such as activity was studied. methamphetamine, methylphenidate and apomorphine were injected (i.p.1 just after the injection of ceruletide, and locomotor activity was evaluated every 10 min for 80-180 min. Methylphenidate-induced stereotypy was assessed according to the method of Costa11 and Naylor (51) at 5 min intervals for 60 min after the intraperitoneal injection of 50 .mg/kg of methylphenidate. The mice were placed individually in wire-meshed cage (21 x 18 x 18)) and methylphenidate was administered 0.p.) 5 min after the injection of ceruletide. The date were analyzed by Each mouse was used only once. Student’s t-test (two-tailed) for comparison against the control group P values less than 0.05 treated the vehicle or physiological saline. were regarded as significant. RESULTS 1.

Effects of drugs on locomotor activity and rearing rate

CCK-8 caused a dose-related reduction in the rates of both locomotor activity and rearing at the dose of 50 and 100 ug/kg S.C. (Fig. 1). Ceruletide also reduced both behavioral parameters in a dosedependent manner at a dose of 25-200 u g/kg S.C. (Fig. 2). The inhibitory effect of ceruletide lasted longer than CCK-8, and its CCK-8-NS, ceruletide-NS duration was prolonged dose-dependently. and CCK-4 had no effect on the rates of locomotor activity and rearing at doses of up to 800 to 1600 p g/kg S.C. (Table 1). 2.

Effects of ceruletide on hyperactivity

induced by DA agonists

Ceruletide at doses ranging from 12.5 to 200 p g/kg (s.c.) dosedependently inhibited hyperactivity induced by intraperitoneal injecCeruletide had a tion of 5 mg/kg of methamphetamine (Fig. 3). similar effect on methylphenidate-induced hyperactivity at a dose of The duration of the inhibitory 100-400 pg/kg (s.c.1 (Fig. 4). 275

8

F40 \

I

30

0

-20 f aio,

Effects of CCK-8 on locomotor activity and rearing in mice Fig. 1. Subcutaneous injection of CCK-8 50 pg/kg (O), 100 ug/kg (A), 200 ug/kg ( n ), 400 ug/kg (0) and saline (0). Each point of CCK-8treated groups represents the mean + S. E. obtained from 12 mice. Control group consisted of 20 mice. Statistical analysis were based on the total counts for 10 min period (Student's t-test, two tailed).

AL A

I?

o-

O i

:,

P

‘ 0.06 pro.01

3zF:o *** ‘;:iz

Fig. 2. Effects of ceruletide on locomotor activity and rearing in mice Subcutaneous injection of ceruletide 12.5 ug/kg (O), 25 uglkg (0)s 50 ug/kg (A.), 100 xdkg (a), 200 &kg (~1, 400 pg/kg (0) and saline (0). Each point of ceruletide-treated groups represents the mean + S. E. obtained from 12 mice. Control group consisted of 44 mice. Statistical analysis were based on the total counts for 10 min period (Student's t-test, two-tailed).

276

Locomotor activity (counts/l0 min)

Dose (uglkg)

Drug Saline CLN-NS

Saline CCK-8-NS

Saline CCK-4

Rearing (counts/l0 min)

200 800

(12) ( 8) ( 8)

539.3 f 47.3 553.5 f 78.6 590.0 + 73.5

35.1 + 40.9 f 32.9 +

6.4 7.8 5.6

800 1600

(20) (12) (10)

737.9 f 45.3 699.9 + 61.3 692.2 + 50.3

55.9 + 51.8 2 48.1 f

6.2 9.6 7.0

800 1600

(10) (10) (10)

686.1 ? 62.6 698.9 + 72.8 679.8 f 81.8

65.1 ?: 10.0 62.8 f 7.9 62.1 2 11.5

Table 1. Effects of CCK-8-NS, ceruletide-NS and CCK-4 on locomotor activity and rearing in mice Each value represents the mean fi S. E. for the initial 10 min period after the subcutaneous injection of each compound. The value in parenthesis indicates the number of mice tested.

*

w

***

01-so ’

0

60

120

P

< 0.06 P eo.01 PCO.001

18Ollh

Fig. 3. Antagonistic effects of ceruletide on methamphetamine-induced hyperactivity in mice Methamphetamine 5 mglkg was injected intraperitoneally just after the subcutaneous injection of ceruletide 12.5 pg/kg (O), 50 pg/kg (A), Each point of ceruletide-treated 200 pg/kg ( n ) and saline (0). groups represents the mean _+ S. E. obtained from 10 mice. Control group consisted of 22 mice. Statistical analysis were based on the total counts for 10 min period (Student's t-test, two-tailed). Abbreviations: MAP; methamphetamine, SAL; saline, CLN; ceruletide.

277

Fig. 4. Antagonistic effects of ceruletide MPD+SALorCLN methylphenidateon induced hyperactivity in mice Methylphenidate 30 mglkg was injected intraperitoneally just after subcutaneous the injection of ceruletide 25 ug/kg ( 0 )t 100 ng/kg ( A ), 200 ng/kg (=), 400 ng/kg (0) and saline (0). Each point of ceruletide-treated groups represents the mean + S. E. obtained from 10 Control group mice. -50 60 0 12omln consisted of 24 mice. Statistical analysis were based on the total counts for 10 min period (Student's t-test, two-tailed). Abbreviation: MPD; methylphenidate.

APO + SAL or CLN I

!,

I ,

-50

0

30

60

90 min

Fig. 5. Antagonistic effects of ceruletide on apomorphine-induced hyperactivity in mice Apomorphine 3 mg/kg was injected subcutaneously just after the subcu(A), 200 taneous injection of ceruletide 12.5 ng/kg (O), 50 ug/kg Each point of ceruletide-treated groups ug/kg (m) and saline (0). represents the mean f S. E. obtained from 10 mice. Control group consisted of 20 mice. Statistical analysis were based on the total counts for 10 min period (Student's t-test, two-tailed). Abbreviation: APO; apomorphine.

278

was prolonged in proportion to the dosage employed, activity Apomorphine-induced hyperactivity was also suppressed by the injection of ceruletide at doses ranging from 12.5 to 200 p g/kg ( S.C. > (Fig. 5). However, no dose-response relationship was observed in either potency or duration of the antagonization by ceruletide of 50 and 200 pg/kg (s.c.1. 3.

Effect of ceruletide on methylphenidate-induced

stereotypy

Ceruletide attenuated methylphenidate-induced stereotyped behavior at doses larger than 100 ~.lg/kg (s.c.), but not completely (Fig. 6). This antagonistic action lacked dose-dependency.

_ CLN MPD 1

1

* ** ***

OL

’

-5

’ 0

15

30

45

P < 0.05 0.01 P e 0.001

P<

6owlhl

Antagonistic effects of ceruletide on methylphenidate-induced Fig. 6. stereotypy in mice Methylphenidate 50 mg/kg was injected intraperitoneally 5 min after the subcutaneous injection of ceruletide 100 ug/kg (O), 200 ug/kg (A >, 400 ug/kg (W) and saline (0). Stereotyped behaviors were assessed according to the method of Costa11 and Naylor at 5 min intervals for 60 min after the intraperitoneal injection of methylphenidate. Each point of ceruletide-treated groups represents the mean t S. E. obtained from 12 min. Control group consisted of 16 mice.

DISCUSSION The present study shows that the peripheral injection of CCK-8 and ceruletide induce a significant reduction in the rates of locomotor activity and rearing in mice in a dose-dependent manner. Moreover, ceruletide has been found to affect certain behavioral changes induced These findings are similar to those observed by by DA agonists. other investigators, suggesting that the peripheral injection of CCK-like peptides induce a behavioral profile resembling that of neuroleptics (29-31,33-35).

279

In a previous study, CCK-8-NS administered intraperitoneally failed to produce any reduction in the exploratory behavior of mice (52). This is consistent with our finding that CCK-8-NS did not affect locomotor activity and rearing of mice. In our experiment, moreover, ceruletide-NS also did not produce any behavioral changes in mice. Thus, the presence of O-sulfate on the tyrosine residue in the CCKmolecule seems to be necessary for the inhibitory effect of CCK-like peptides on at least locomotor activity and rearing when they are injected peripherally. Although the rate of ambulation and rearing of rats was not affected by the subcutaneous injection of CCK-8-NS and ceruletide at a dose level of 3 1~g, a higher dose (10 pg) of these CCK-like peptides reduced significantly the rate of ambulation and more markedly the rate of rearing (35). Spontaneous locomotor activity of rats was not affected by CCK-8 (34,361 but was increased CCK-8-NS following intraventricular administration. (34) by However, intraventricularly administered CCK-8, but not CCK-8-NS produced a significant immobility in rats at a dose level of 20 ~g Apomorphine-induced stereotypic cage-climbing behavior was (35). not affected by CCK-8 but enhanced by CCK-8-NS following intraventricular administration (32). Intraventricular administration of CCK-8 did not produce a significant alteration in amphetamine-induced Likewise, CCK-8 administered intraventricularly at locomotor activity. a dose of 5 or 25 ~g/lO l.11did not alter significantly either intensity of duration of various stereotypies induced by intraperitoneal injection However, methamphetamine-induced hyperof apomorphine (36). activity of rats was inhibited by intraventricular administration of CCK-8 but not of CCK-8-NS (34). In contrast to the findings cited CCK-8 and CCK-8-NS injected either centrally or peripherally above, effects on fear-motivated active and passive had almost equal avoidance, hunger-motivated conditioned feeding and the reward type These findings, electrical self-stimulation behavior of rats (53-55). indicate that the disparity between taken together with our results, the results obtained in behavioral pharmacology of CCK-like peptides and their non-sulfated forms is due to differences in experimental of CCK-like procedures employed (for example, route of administration used in the experiment) and experimental peptides ; DA agonists animals tested. Ceruletide was much more potent in suppressing rearing activity, prolonging hexobarbital-induced sleep, producing catalepsy and inhibiting stereotypic behavior induced by methylphenidate in mice Zetler postulated that the generally lower than CCK-8 (28,291. potency of CCK-8 is a consequence due to poor stability (29). Recently, however, van Ree et al demonstrated that CCK-8 inhibited ambulation and rearing of rats more strongly than ceruletide (35). In the present study, the inhibitory effects of CCK-8 and ceruletide on both locomotor activity and rearing were similar. However, the onset of action of ceruletide was slightly slower and its inhibitory affects continued much longer than CCK-8, although dependent on the dosage differences in experimental procedures employed. Consequently, employed (e.g. the time when the animal was pretreated with CCK-8 or ceruletide) and stability ascribable to the chemical structure between CCK-8 and ceruletide may be responsible for the conflicting results.

CCK-4 is also present in the brain (5,13,17). The COOH-terminal tetra-, hexa- and pentapeptides of CCK facilitated the extinction of active avoidance behavior and retention of passive avoidance behavior in rats (55). In the present study, however, CCK-4 did not produce a significant alteration in locomotor activity and rearing in mice. This is in good agreement with the finding that the minimum amino acid sequence necessary for reducing exploratory behavior was CCK-7 and that all smaller C- and N-terminal fragments of CCK-8 were biologically inactive as well as CCK-8-NS and CCK-7 (56). The binding sites in the brain may be different from those of CCK-8 (57,58). Methamphetamine-induced hyperactivity in rats was inhibited by intraventricular administration of CCK-8 (34). Peripherally injected CCK-8 or ceruletide also suppressed methylphenidate-induced stereotypic behavior in mice (29,30). In the present study, ceruletide inhibited both methamphetamineand methylphenidate-induced hyperactivity in mice in a dose-dependent manner, although it had no dose-related antagonistic ef feet methylphenidate-induced stereotyped behavior. Amphetamine an? methylphenidate cause DA release in the brain, consequently inducing various behavioral changes, whereas their biochemical effects appears to be different. Thus CCK-8 or ceruletide may influence presynaptic process in dopaminergic neurons. Recent single unit electrophysiological studies have shown that only those DA neurons projecting to limbic regions response to parenterally or iontophoretically administered CCK-8 (59) and parenteral administration of CCK-8 or ceruletide produces supersensitivity of the DA autoreceptor on neurons of the substantia nigra These findings support the above-mentioned assumption. (60). Apomorphine is known to be a direct-acting DA receptor stimulating agent. Furthermore, there is a biphasic response to apomorphine, that is, an autoreceptor-activating effect at lower doses, consequently limiting DA release and attenuating spontaneous locomotor activity, and a dose-dependent increase in stereotypic behavior reflecting postsynaptic receptor interaction at higher doses. Apomorphine-induced stereotypic behavior in rats was inhibited by peripheral injection of CCK-8 (33). As mentioned above, however, intraventricularly administered CCK-8 did not significantly alter either intensity or duration of apomorphine-induced stereotypies (36). In the present study, ceruletide significantly suppressed apomorphine-induced hyperactivity in mice. However, no dose-response relationship was observed in either intensity or duration of inhibitory action of ceruletide. These findings , taken together with the large doses required to inhibit apomorphine-induced stereotypic behavior in rats (331, suggest that CCK-8 or ceruletide does not interfere with dopaminergic transmission due to the blockade of postsynaptic DA receptors in the brain. This assumption is supported by the findings that the pretreatment with the injection of CCK-8, CCK-8-NS and ceruletide into the nucleus caudatus of rats did not antagonize the stereotyped sniffing response following the injection of apomorphine into the nucleus caudatus (35). CCK-8, However, but not CCK-8-NS or ceruletide, completely inhibited apomorphine-induced hyperactivity following the direct injection into the nucleus accumbens (35). Also, CCK-8 or ceruletide

281

reduced or abolished apomorphine-induced compulsive gnawing in mice sensitized by either scopolamine or trifluxiol, but did not antagonize the climbing activity (37). More recently, however, CCK-8, but not CCK-8-NS, has been found to potentiate the hyperlocomotor induced by DA when directly injected into the nucleus accumbens in conjunction with DA (61). Also, CCK-8 significantly increased apomorphine-induced stereotypy when injected into the nucleus CCK-8 did not affect apomorphine-induced accumbens, whereas stereotypy when injected into the nucleus caudatus. Thus, definite conclusions regarding effects of CCK-like peptides on postsynaptic DA receptors must await further studies. our behavioral pharmacological study on the central In conclusion, action of CCK-like peptides shows that the peripheral injection of CCK-8 and ceruletide, but not their non-sulfated forms, appears to affect dopamine function in the brain, consequently producing behavioral changes that resemble those induced by neuroleptics in some respects. ACKNOWLEDGEMENT We are greatly indebted to Shionogi Company (Osaka, Japan) for the kind gift of ceruletide and its non-sulfated form. This study was supported by a grant from the National Center for Nervous, Mental and- Muscular Disorders of the Ministry of Health and Welfare of Japan to T.M. REFERENCES 1.

Ivy, A.C. and Oldberg, E. (1928). Hormone mechanism for gall bladder contraction. Am. J. Physiol. 85: 381-383.

2.

Harper, A.A. and Raper, H.S. (1948). Pancreozymin, a stimulant of the secretion of pancreatic enzymes in extracts of the small intestine. J. Physiol. 102: 115-125.

3.

Vanderhaeghen, J.J., Signeau, J.C. and Gepts, W. (1975). New peptide in the vertebrate CNS reacting with antigastrin antibodies. Nature (London) 257: 604-605.

4.

Straus, E., Miiller, J.E., Choi, H.S., Paronetto, F. and Yalow, R.S. (1977). Immunohistochemical localization in rabbit brain of a peptide resembling the COOH-terminal octapeptide of cholecystokinin. Proc. Natl. Acad. Sci. USA 74: 3033-3035.

5.

Schneider, B.S., Monahan, J.W. and Hirsch, J. (1979). Brain cholecystokinin and nutritional status in rats and mice. J. Clin. Chem. 64: 1348-1356.

6.

Emson, P.C., Rehfeld, J.F., Langevin, H. and Rosser, M. (1980). Reduction in cholecystokinin-like immunoreactivity in the basal ganglia in Huntington's disease. Brain Res. 198: 497-500.

7.

Rehfeld, J.F. (1978). Immunochemical studies on cholecystokinin. II. Distribution and molecular heterogenity in the central nervous system and small intestine of man and hog. J. Biol. Chem. 253: 4022-4030.

282

8.

Innis, R.B., Correa, F.M.A., Uhl, G.R., Schneider, B. and Snyder, S.H. (1979). Cholecystokinin octapeptide-like immunoreactivity histochemical localisation in rat brain. Proc. Natl. Acad. Sci. USA 76: 521-525.

9.

Vanderhaeghen, J.J., Lotstra, F., De Mey, J. and Gilles, C. (1980). Immunohistochemical localization of cholecystokinin- and gastrin-like peptides in the brain and hypophysis of the rat. Proc. Natl. Acad. Sci. USA 77: 1190-1194.

10.

Beinfeld, M.C., Meyer, D.K., Eskey, R.L., Jensen, R.T. and Brownstein, M.J. (1981). The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay. Brain Res. 212: 51-57.

11.

Emson, P.C., Beinfeld, J.F. and Rosser, M.N. (1982). Distribution of cholecystokinin-like peptides in the human brain. J. Neurochem. 38: 1177-1179.

12.

Dockray, G.J. (1976). Immunochemical evidence of cholecystokinin-like peptides in brain. Nature (London) 264: 568-570.

13. Miiller,J.E., Straus, E. and Yalow, R.S. (1977). Cholecystokinin and its COOH-terminal octapeptide in the pig brain. Proc. Natl. Acad. Sci. USA 74: 3035-3037. 14. Dockray, G.J., Gregory, R.A., Hutchinson, J.B., Harris, J.I. and Bunswick, M.J. (1978). Isolation, structure and biological activity of two cholecystokinin octapeptides from sheep brain. Nature (London) 270: 359-361. 15.

Robberecht, P., Deschodt-Lanckman, M. and Vanderhaeghen, J.J. (1978). Demonstration of biological activity of brain gastrinlike peptidic material in human: its relationship with the COOHterminal octapeptide of cholecystokinin. Proc. Natl. Acad. Sci. USA 75: 524-528.

16.

Beinfeld, M.C. (1981). An HPLC and RIA analysis of the cholecystokinin peptides in rat brain. Neuropeptides 1: 203-209.

17. Rehfeld, J.F. and Golterman, R. (1979). Immunochemical evidence of cholecystokinin tetrapeptides in hog brain. J. Neurochem. 32: 1339-1341. 18.

Pinget, M., Straus, E. and Yalow, R.S. (1979). Release of cholecystokinin peptides from a synaptosome-enriched fraction of rat cerebral cortex. Life Sci. 25: 339-342.

19. Dodd, P.R., Edwardson, J.A. and Dockray, G.J. (1980). The depolarisation-induced release of cholecystokinin C-terminal octapeptide (CCK-8) from rat synaptosomes and brain slice. Reg. Peptides 1: 17-29. 20.

Emson, P.C., Lee, C.M. and Rehfeld, J.F. (1980). Cholecystokinin octapeptide: vesicular localization and calcium dependent release from rat brain in vitro. Life Sci. 26: 2157-2163.

21.

Goltermann, N.R., Stengaard-Pedersen, K., Rehfeld, J.F. and Christensen, N.J. (1981). Newly synthesized cholecystokinin in

283

subcellular fractions of the rat brain. 965.

J. Neurochem. 36: 959-

22.

Klaff, L.J., Hudson, A., Sheppard, M. and Tyler, M. (1981). In vitro release of cholecystokinin octapeptide-like immunoreactivity from rat brain synaptosomes. South Afr. Med. J. 59: 158-160.

23.

Hiikfelt,T., Rehfeld, J.F., Skirboll, L., Ivemark, B., Goldstein, M. and Markey, K. (1980). Evidence for coexistence of dopamine and CCK in mesolimbic neurons. Nature (London) 285: 476-478.

24.

Studler, J.M., Simon, H., Cesselin, F., Legrand, J.F., Glowinski, J. and Tassin, J.P. (1981). Biochemical investigation on the localization of the cholecystokinin octapeptide in dopaminergic neurons originating from the ventral tegmental area in the rat. Neuropeptides 2: 131-139.

25.

Fuxe, K., Andersson, K., Locatelli, V., Agnati, L.F., Hgkfelt, T ., Skirboll, L. and Mutt, V. (1980). Cholecystokinin peptides produce marked reduction of dopamine in discrete areas in the rat brain following intraventricular injection. Eur. J. Pharmacol. 67: 329-331.

26.

Meltzer, H.Y. and Stahl, S.M. (1976). The dopamine hypothesis of schizophrenia; a review. Schizophr, Bull. 2: 19-76.

27.

Hiikfelt, T., Johanssen, O., Ljungdahl, A., Lundberg, J.M. and Schultzberg, M. (1980). Peptidergic neurons. Nature (London) 284: 515-521.

28.

Zetler, G. (1980). Effects of cholecystokinin-like peptides on rearing activity and hexobarbital-induced sleep. Eur. J. Pharmacol 66: 137-139.

29.

Zetler, G. (1981). Central depressant effects of caerulein and cholecystokinin octapeptide (CCK-8) differ from those of diazepam and haloperidol. Neuropharmacology 20: 277-283.

30.

Zetler, G. (1981). Differential cataleptogenic and antistereotypic effects of caerulein and haloperidol. Neuropharmacology 20: 681-686.

31.

Itoh, S. and Katsuura, G. (1981). Suppressive action of cholecystokinin octapeptide on the behavioral effects of L-DOPA in the rat. Eur. J. Pharmacol. 75: 313-316.

32.

KOV&S, G.L., Szabo, G., Penke, B. and Telegdy, G. (1981). Effects of cholecystokinin octapeptide on striatal dopamine metabolism and on apomorphine-induced stereotypy cage-climbing in mice. Eur. J. Pharmacol. 69: 313-319.

33.

Cohen, S.L., Knight, M., Tamminga, C.A. and Chase, T.N. (1982). conditioned-avoidance Cholecystokinin-octapeptide effects on behavior, stereotypy and catalepsy. Eur. J. Pharmacol. 83: 213-222.

34.

Katsuura, G. and Itoh, S. (1982). Sedative action of cholecystokinin octapeptide on behavioral excitation by thyrotropinreleasing hormone and methamphetamine in rats. Jap. J. Physiol. 32: 83-91.

284

35.

Van Ree, J.M., Gaffori, 0. and De Wied, D. (1983). In rats, the behavioral profile of CCK-8 related peptides resembles that of antipsychotic agents. Eur. S. Pharmacol. 93: 63-78.

36.

Wider&, E., Kalivas, P.W., Lewis, M.H., Prange, Jr., A.J. and Breese, G.R. (1983). Influence of cholecystokinin on central monoaminergic pathways. Reg. Peptides 6: 99-109.

37.

Zetler, G. (1985). Antistereotypic effects of cholecystokinin octapeptide (CCK-8), ceruletide and related peptides on in apomorphine-induced gnawing sensitized mice. Neuropharmacology 24: 251-259.

38.

Moroji, T., Watanabe, N., Aoki, N. and Ttoh, S. (1982). Antipsychotic effects of ceruletide (caerulein) on chronic schizophrenia. Arch. Gen. Psychiatry 39: 485-486.

39.

Moroji, T., Watanabe, N., Aoki, N. and Itoh, S. (1982). Antipsychotic effects of caerulein, a decapeptide chemically related to cholecystokinin octapeptide, on schizophrenia. Int. Pharmacol. Psychiatry 17: 255-273.

40.

Itoh, H., Tanoue, G., Yagi, G., Tateyama, M., Kamisada, M., Fujii, Y., Takamiya, M. and Nakajima, S. (1982). Clinical study of the antipsychotic effects of caerulein: an open clinical trial in chronic schizophrenic patients. Keio J. Med. 31: 71-95.

41.

Nair, N.P.V., Bloom, D.M. and Nestoros, J.N. (1982). Cholecystokinin appears to be antipsychotic properties. Prog. NeuroPsychopharmacol. Biol. Psychiatr. 6: 509-512.

42.

Bloom, D.M., Nair, N.P.V. and Schwartz, G. (1983). CCK-8 in the treatment of chronic schizophrenia. Psychopharmacol. Bul. 19: 361-363.

43.

Nair, N.P.V., Bloom, D.M., Nestoros, J.N. and Schwartz, G. (1983). Therapeutic efficacy of cholecystokinin in neurolepticresistant schizophrenic subjects. Psychopharmacol. Bull. 19: 134-136.

44.

Albus, M., Ackenheil, M., Munch, H. and Naber, D. (1984). Ceruletide: a new drug for the treatment of schizophrenic patients? Arch. Gen. Psychiatry 41: 528.

45.

Jenkins, P.L. (1985). Is cholecystokinin an antipsychotic agent? J. Clin. Psychiatry 45: 317.

46.

Moroji, T., Itoh, K. and Itoh, K. (1985). Antipsychotic effects of ceruletide in chronic schizophrenia: an appraisal of the long-term intermittent medication of ceruletide in chronic schizophrenia. Ann. N. Y. Acad. Sci. 448: 518-534.

47.

Nair, N.P.V., Bloom, D.M., Lal, S., Debonnel, G., Schwartz, G. and Mosticyan, S. (1985). Clinical and neuroendocrine studies with cholecystokinin peptides peptides. Ann. N. Y. Acad. Sci. 448: 535-541.

48.

Van Ree, J.M., Verhoeven, W.M.A., Brouwer, G.J. and De Wied, D. (1984). Ceruletide resembles antipsychotics in rats and schizophrenic patients. Neuropsychobiology 12: 4-8.

285

49.

Verhoeven, W.M.A., Westenberg, H.G.M. and Van Ree, J.M. (1986). the antipsychotic properties of A comparative study on desenkephalin-y-endorphin and ceruletide in schizophrenic patients. Acta Psychiatr. Stand. 73: 337-347.

50.

Hsiao, S., Katsuura, G. and Itoh, S. (1984). Cholecystokinin tetrapeptide, proglumide and open-field behavior in rats. Life Sci. 34: 2165-2168.

51.

Costall, B. and Naylor, R.J. (1973). On the mode of action of apomorphine. Eur. J. Psychopharmacol. 21: 350-361.

52.

Crawley, J.N., Hays, S.E., Paul, S.M. and Goodwin, F.K. (1981). Cholecystokinin reduces exploratory behavior in mice. Physiol. Behav. 27: 407-411.

53.

Fekete, M., Penke, B. and Telegdy, G. (1981). Structure-activity studies concerning effect of cholecystokinin octapeptide on avoidance behavior in rats. Acta Physiol. Acad. Sci. Hung. 58: 197-200.

54.

Fekete, M., Rentsch, A., Schwartzberg, H. and Telegdy, G. (1983). Effect of cholecystokinin on self-stimulation behavior in rats. Eur. J. Pharmacol. 90: 77-32.

55.

Crawley, J.N., St-Pierre, S. and Gaudreau, P. (1984). Analysis of c- and N-terminal fragments of cholecystokinin octapeptide. J. Pharmacol. Exp. Ther. 230: 438-444.

56.

Salt, T.E. and Hill, R.G. (1982). The effects of C-terminal fragments of cholecystokinin on the firing of single neurons in the caudal tegmental nucleus of the rat. Neuropeptides 2: 301-306.

57.

Agnati, L.F., Fuxe, K., Benfenati, F., Gelani, M.F., Battistini, N ., Mutt, V., Cavicchioli, L., Galli, G. and Hgkfelt, T. (1983). Differential modulation by CCK-8 and CCK-4 of [3H]spiperone binding sites linked to dopamine and 5-hydroxy-tryptamine receptors in the brain of rats. Neurosci. Lett. 35: 179-183.

58.

Skirboll, L.R., Grace, A.A., Hommer, D.W., Rehfeld, J., Goldstein, H;ikfelt, T. and Bunney, B.S. (1981). Peptide-monoamine M ., coexistence: studies of actions of cholecystokinin-like peptide on the electrical activity of midbrain dopamine neurons. Neurosciences 6: 2111-2124.

59.

Hommer, D.W. and Skirboll, L.R. (1983). Cholecystokinin-like peptides potentiate apomorphine-induced inhibition of dopamine neurons. Eur. J. Pharmacol. 91: 151-152.

60.

Crawley, J.N. (1985). Cholecystokinin potentiation of dopaminemediated behaviors in the nucleus accumbens. Ann. N. Y. Acad. Sci. 448: 283-292.

Received 11/8/86 Accepted 25/8/86

286