In rats, the behavioral profile of CCK-8 related peptides resembles that of antipsychotic agents

European Journal of Pharmacology, 93 (1983) 63-78 Elsevier

63

IN R A T S , T H E B E H A V I O R A L P R O F I L E O F CCK-8 R E L A T E D P E P T I D E S R E S E M B L E S T H A T O F ANTIPSYCHOTIC AGENTS JAN M. VAN REE *, ODILE GAFFORI and DAVID DE WIED Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands Received 12 January 1983, revised MS received 5 April 1983, accepted 31 May 1983

J.M. VAN REE, O. G A F F O R I and D. DE WIED, In rats, the behavioralprofile of CCK-8 relatedpeptides resembles that of antipsychotic agents, European J. Pharmacol. 93 (1983) 63-78. The action of some CCK-8 related peptides, desulphated CCK-8 (CCK-DS), the sulphated form of CCK-8 (CCK-8-S) and ceruletide was explored in a number of test procedures with rats, in which antipsychotic agents are active. Following injection into the nucleus accembens, all three peptides antagonized the hypolocomotion induced by low doses of apomorphine (10 ng). Ceruletide appeared to be the most potent in this respect (EDs0: approximately 5 pg). The increased locomotion observed following injection of relatively high doses of apomorphine (10 ~g) into the nucleus accumbens was antagonized by local pretreatment with CCK-8-S, but not with CCK-8-DS or ceruletide. None of these CCK-8 related peptides affected the stereotyped sniffing response elicited by treatment with apomorphine or amphetamine (10/ttg) given into the nucleus caudatus. Passive avoidance behavior was facilitated following subcutaneous administration of 10 #g of CCK-8-related peptides 1 h before the retention test. The same peptides given into the nucleus accumbens (0.3 pg) however attenuated passive avoidance behavior. Intraventricular injection with CCK-8-DS and CCK-8-S induced a positive effect in various 'grip tests'. Given subcutaneously, the CCK-8-related peptides decreased the rate of ambulation and rearing especially in the middle of the open field. These results indicate that CCK-8 related peptides, especially CCK-8-DS and ceruletide, exhibit behavioral effects that are similar to those observed following treatment with y-type endorphins and that resemble the effects of antipsychotic agents. Very low doses of CCK-8 related peptides exert behavioral effects following injection into the nucleus accumbens, indicating that this brain area is extremely sensitive to the action of these peptides. It is postulated that certain peptides which are either present in neurons (like CCK-8-related peptides) or generated by brain endorphin systems (like 7-type endorphins) control the activity of specific neurons of the mesolimbic dopaminergic pathways. This may be of relevance for the purported antipsychotic action of these peptides. Dopaminergic mesolimbic system Passive avoidance behavior Neuroleptic-like activity Ceruletide

CCK-8 related peptides Antipsychotic agents Nucleus caudatus

1. Introduction It has been d e m o n s t r a t e d that p e p t i d e s related to the gut h o r m o n e cholecystokinin ( C C K ) are present in the central nervous sytem ( D o c k r a y , 1976; V a n d e r h a e g h e n et al., 1975; Rehfeld, 1978; L a r s s o n a n d Rehfeld, 1979). I m m u n o c y t o c h e m i s t r y studies have identified C C K i m m u n o a c t i v -

* To whom all correspondence should be addressed. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.

Des-enkephalin-~,-endorphin Locomotor activity Nucleus accumbens

ity in nerve cell bodies as well as fibers (Innes et al., 1979; L o r e n et al., 1979; V a n d e r h a e g h e n et al., 1980). Evidence has been presented for a p o p u l a tion of mesencephalic d o p a m i n e r g i c neurons proj e c t i n g to the limbic forebrain including the nucleus accumbens, a n d also c o n t a i n i n g a C C K - l i k e p e p t i d e (HOkfelt et al., 1980; Skirboll et al., 1981). A l t h o u g h the exact role of C C K related p e p t i d e s in b r a i n function is still unclear, it has recently been suggested that these p e p t i d e s exhibit neuroleptic-like activity in rodents a n d an a n t i p s y c h o t i c

64 action in schizophrenic patients. Thus, CCK-octapeptide (CCK-8) and ceruletide (caerulein), a decapeptide homologous to CCK-8 and isolated from the frog's skin, produce catalepsy, sedation, and other behavioral effects in mice after peripheral injection, effects which are in some aspects comparable to those observed following treatment with neuroleptic drugs (Zetler, 1981). A single injection of a low dose of ceruletide resulted in a long-lasting antipsychotic effect in some schizophrenic patients resistant to neuroleptic therapy (Moroji et al., 1982). These studies, together with the observations dealing with the interaction between C C K related p e p t i d e s a n d b r a i n dopaminergic activity (Kov/lcs et al., 1981 ; Fuxe et al., 1980) suggest that C C K related peptides may somehow be involved in the pathogenesis of schizophrenia (H6kfelt et al., 1980). A similar hypothesis had been put forward for y-type endorphins (De Wied et al., 1978). Thus, non-opiate fragments of y-endorphin e.g. des-TyrLy-endorphin ( D T y E ) and des-enkephalin-y-endorphin (DETE) induce behavioral effects in rats, and these effects are comparable in some aspects to those induced in rats by neuroleptic drugs (De Wied et al., 1978, 1980; Van Ree et al., 1982b; Van Ree and De Wied, 1982a). These fragments also have an antipsychotic effect in a number of schizophrenic patients (Verhoeven et al., 1979; Van Ree et al., 1982c). We have argued that the most suitable animal tests predicting antipsychotic activity are those dealing with avoidance behavior, the grasping response and the behavioral effects induced by low doses of the dopamine agonist apomorphine (Van Ree and De Wied, 1982a). Thus, in the present series of experiments we have explored the activity of CCK-8 related peptides using these and a number of other relevant test procedures. We focussed particularly on the nucleus accumbens area as site of injection, since CCK-8 related peptides and dopamine coexist in terminals in that area (Skirboll et al., 1981; Studler et al., 1981). Also, mesolimbic dopamine systems have been implicated in schizophrenia (Crow, 1979) and ytype endorphins have been shown to interfere with certain dopaminergic systems in the nucleus accumbens (Van Ree et al., 1982a,d). The data revealed that in rats, C C K related peptides especially the

desulphated CCK-8 and ceruletide, have a behavioral profile that closely resembles that of antipsychotic agents including y-type endorphins.

2. Materials and methods 2.1. Animals and surgery Male Wistar rats of an inbred strain (TNO, Zeist, The Netherlands) weighing between 130 and 160 g were used. They were kept under standard conditions (room temperature 22 _+ I°C, light on from 5.00 a.m. till 7.00 p.m.), were housed in groups of 5 and received food and water ad libitum. For intraventricular injections polyethylene cannulae were implanted under H y p n o r m ® anaesthesia into one of the lateral ventricles and fixed to the skull as described previously (De Wied, 1976). For intracerebral injections, the rats were anesthetized with Hypnorm, secured in a stereotaxic instrument and stainless steel guide cannulae (0.6 m m outer dia, 0.3 m m inner dia) were implanted unilaterally or bilaterally (Van Ree and Wolterink, 1981; Van Ree, 1982). For implantation into the nucleus accumbens area the coordinates were 2.6 m m anterior to the bregma, 2.7 m m lateral to the midline, 6.1 m m below the dura at the point of penetration and the cannulae were inserted at an angle of 12 ° . For implantation into the nucleus caudatus the coordinates were 2.0 m m anterior to the bregma, 2.5 m m lateral to the midline, and 6.0 m m below the skull, according to Pellegrino and Cushman (1967) except that the level of the upper incisor bar was at the level of the interaural line. Operated animals were housed in single cages. Behavioral testing was performed no sooner than one week after operation. 2.2. Behavioral tests 2.2.1. Locomotor activity and sniffing For determining locomotor activity and sniffing two different test conditions were used (for details see Van Ree and Wolterink, 1981; Van Ree, 1982; Van Ree et al., 1982b). (A) Rectangular perspex observation cages (bottom 20 × 6.5 cm, height 32 cm). In this test cage, locomotor activity was mea-

65 sured by counting the number of crossings over the midline of the floor for 3 min. (B) Circular perspex test cages (diameter 19.5 cm, height 28.5 cm) with the bottom divided into 4 equal sections. In this small open field, locomotor activity (number of sections explored) and the duration (s) of (stereotyped) sniffing were measured for 3 or 4 min. Rats with cannulae into the nucleus accumbal area were injected twice. They were first treated with peptide or placebo and after 40 min with apomorphine or placebo. Following a low dose of apomorphine (10 ng) the rats were tested in the rectangular test case (A) 5 min after apomorphine treatment and subsequently 20 min after the last injection in the small open field (B) for 3 min. When a high dose of apomorphine (10 /~g) was injected, testing was performed in the small open field only 20 min after apomorphine treatment. The rats used in the experiments with a high dose of apomorphine were habituated to the test cage, by placing them twice in the small open field on the days before testing. Rats with cannulae into the nucleus caudatus were injected with peptide or placebo and were treated with apomorphine, amphetamine or placebo after 1 h. After 20 min the rats were placed in the small open field (B) and their behavior was observed for 4 min. In general, the rats were tested twice with an interval of at least one week between tests. Doses of apomorphine were selected on the basis of previous studies of dose-response relationships (Van Ree and Wolterink, 1981; Van Ree and De Wied, 1982b; Van Ree et al., 1982a).

2.2.2. Passive avoidance behavior Passive avoidance behavior was studied in a single step-through type passive avoidance situation (Ader et al., 1972). The apparatus consists of a dark box equipped with a grid floor and an illuminated, mesh-covered platform attached to the front center of the dark compartment. The rats were first adapted to the dark box for 120 s, then placed on the runway and allowed to enter the dark compartment (first trial). Three such trials were given the next day. Immediately after entering the dark compartment on the third trial, the rats received a single unavoidable scrambled footshock (duration 2 s shock intensity 0.25 mA

(s.c. treatment) or 0.5 mA (treatment into the nucleus accumbens)). Retention was tested twice, at 24 and 48 h after the learning trial. Latency to re-enter the dark compartment was recorded for a maximum of 300 s. Peptides and placebo were injected s.c. or unilaterally into the nucleus accumbens area 1 h prior to the first retention test.

2.2.3. Behavioral profile Rats equipped with an intraventricular cannula were inspected in an observation cage for spontaneous behavior (sedation, mobility, hyperactivity, grooming, rearing, stereotypy, wet shakes, vocalizing) appearance of the eyes (wide open, ptosis, exophthalmus), for reflexes (corneal reflex, righting reflex, reaching reflex, extending or sloping limbs), rigidity (stiffness of the tail and body and the bridge test) and for activity in 'grip tests' (holding on the edge of the case, to a vertical grid or to a pencil) as described before (De Wied et al., 1978). The items or activities were scored on a 0-2 scale based on the intensity and duration of the effect. Inspections were carried out 30, 60 and 120 min after the intraventricular injections of peptides or placebo. The rats were used only once. 2.2.4. Gross behavior Gross behavior was tested in a big, circular open field (diameter 75 cm) illuminated by a 75 W bulb suspended 1 m above the field centre, as described previously (Weijnen and Slangen, 1970). The floor of the open field was divided into 19 compartments, 12 of which were near the wall, and 7 inside the inner circle. Animals were observed for 3 min and the following items were recorded: ambulation middle (crossing compartments inside the inner circle of the open field), ambulation wall (crossing compartment near the wall of the open field), rearing middle, rearing wall, grooming and defecation. Peptides or placebo were injected s.c. and the animals were tested 1 h after injection. Each rat was used only once. 2.3. Drugs and injections The various peptides used were desulphated CCK-8 (CCK-8-DS, H-Asp-Tyr-Met-Gly-TrpMet-Asp-Phe-NH2), CCK-8 (referred to here as

66

CCK-8-S, H-Asp-Tyr (SO3H)-Met-Gly-Trp-MetAsp-Phe-NH2) (both donated by Dr. Gillessen, H o f m a n n La Roche, Basel, Switzerland), ceruletide (Glp-Gln-Asp-Tyr(SO 3H)-Thr-Gly-Trp-MetAsp-Phe-NH 2, Takus ®, Farmitalia, OPG, Utrecht, The Netherlands) and des-enkephalin-y-endorphin ( D E y E , /~-endorphin-(6-17), H-Thr-Ser-Glu-LysSer-Gln-Thr-Pro-Leu-Val-Thr-Leu-O H, donated by Dr. H.M. Greven, Organon International B.V., Oss, The Netherlands). Apomorphine (Apomorphine HC1) and amphetamine (Dexamphetamine sulphate) were obtained from OPG, Utrecht, The Netherlands. The peptides were stored dry and dissolved in saline immediately prior to use in a volume of 0.5 ml or 1 /~1 for s.c. or intraventricular/intracerebral injection respectively. Placebotreated rats received a similar volume of saline (0.9% NaCI). Intraventricular and intracerebral injections were performed using a Hamilton syringe with the needle inserted into the polyethylene or stainless steel guide cannula respectively.

3. Results

3.1. Localization of the injection sites The sites of injection aimed at the nucleus accumbens appeared to be in the middle and anterior part of the nucleus accumbens and bilateral in the case of two injection sites. In the nucleus caudatus the sites were found in the middle part (fig. 1). Data from rats with cannulae outside the nucleus accumbens or the nucleus caudatus were excluded from further analyses as were data from rats with the polyethylene cannula outside the ventricular space.

9.6

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2.4. Histology The injection sites were verified at the end of the experiment. The localization and the function of the intraventricular cannulae were inspected by injection of Evans blue. The location of the intracerebral injection sites were determined histologically. The rats were sacrificed and the brains fixed in 4% formalin. Serial sections with a thickness of 100/~m were cut on a cryostat. The injection sites were inspected microscopically using the atlas of Pellegrino and Cushman (1967).

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2.5. Statistical analysis The data from experiments dealing with the rectangular test case and the small and big open field were first analysed using a one way analysis of variance (ANOVA) and when the outcome revealed a statistically significant effect (P < 0.05), with Student's t-test. The data from experiments dealing with passive avoidance behavior and the behavioral profile were analysed by A N O V A (Kruskall-Wallis) and subsequently with MannWhitney U-tests.

Fig. 1. Position of the tip of the c a n n u l a e in the nucleus c a u d a t u s (O) a n d nucleus a c c u m b e n s ( × ) as revealed by histological evaluation. For each injection area 10 representative tips are indicated in the d r a w i n g s taken from Pellegrino and Cushm a n (1967); N A = nucleus a c c u m b e n s , C P = nucleus caudatus.

6-~

3.2. Interaction with apomorphine-induced behavioral changes following injection into the nucleus accumbens Injection of low doses of apomorphine (10 ng) into the nucleus accumbens resulted in a decrease of locomotor activity as tested in the rectangular test case as well as in the small open field, 5 and 20 min after injection respectively (fig. 2). This decrease was not accompanied by changes in the duration of sniffing as measured 20 min after injection (table 1). Pretreatment with CCK-8-S (10 ng) resulted in a statistically significant decrease of locomotor activity at 20 min after placebo injection, whereas pretreatment with CCK-DS (10 ng) and ceruletide (1 ng) did not significantly affect locomotor activity in placebo-treated rats (fig. 2). Pretreatment with similar doses of CCK-8-DS and ceruletide completely prevented the apomorphineinduced hypoactivity (fig. 2). The result was the same for CCK-8-S since there were no differences in locomotor activity of CCK-8-S pretreated rats injected with placebo or with apomorphine. A slight but significant increase in the duration of sniffing was observed in the rats pretreated with CCK-8-DS and ceruletide as compared to

placebo-pretreated controls (table 1). This effect was less pronounced with CCK-8-S and was hardly affected by apomorphine treatment. When dose-response relationships were tested it was found that, after injection into the nucleus accumbens, ceruletide was the most potent peptide with respect to antagonizing the apomorphine-induced hypoactivity (fig. 3). The EDs0 of ceruletide was approximately 5 pg and that of CCK-8-DS about 200 pg. It was not possible to obtain an EDs0 for CCK-8-S, because this peptide markedly decreased locomotor activity without the treatment with apomorphine (fig. 2). For comparison it is worth mention that the EDso for DE~,E, assessed as outlined for the CCK-8 related peptides, was approximately 50 pg (Van Ree et al., 1982a). Following a relatively high dose of apomorphine (10 /~g) injected into the nucleus accumbens locomotor activity was increased (fig. 4). The locomotor activity of the placebo-injected controls was lower than in the experiments dealing with low doses of apomorphine due to the habituation procedure as outlined in section 2.2.1. The duration of sniffing was not changed following 10 /~g of apomorphine injected into the nucleus accumbens (table 1). Pretreatment with CCK-8-DS and

TABLE 1 Duration of sniffing following injection with apomorphine a n d / o r CCK-8 related peptides into the nucleus accumbens, as assessed in a small open field. Treatment

Duration of sniffing

- 60 min

- 20 min

mean score + S.E.M.

Placebo a CCK-8-DS ( 10 ng) CCK-8-S (10 ng) Ceruletide (1 ng)

Placebo Placebo Placebo Placebo

15.9 _+2.5 27.2 _+4.6 d 22.3 _+3.0 29.5 _+6.9 d

(15) b (8) (7) (4)

Placebo CCK-8-DS (10 ng) CCK-8-S (10 ng) Ceruletide (1 ng)

Apomorphine Apomorphine Apomorphine Apomorphine

(10 (10 (10 (10

14.6 + 2.3 26.8 _+4.6 ~ 20.1 _+3.2 30.8 + 8.2 e

(16) (8) (9) (4)

Placebo Placebo CCK-8-DS (10 ng) CCK-8-S (10 ng) Ceruletide (10 ng)

Placebo c Apomorphine Apomorphine Apomorphine Apomorphine

(10/tg) (10 ~g) (10 ~g) (10/~ g)

ng) ng) ng) ng)

9.5 _+ 1.6 11.3 + 0.8 12.3 + 1.4 10.2 _+2.2 8.6 + 1.6

(8) (8) (8) (6) (7)

1 /~1 saline, b N u m b e r of rats in parentheses, c Rats were habituated to the test cage, see section 2.2.1. Different from rats treated with placebo instead of peptide (d p < 0.05; e p < 0.02).

68

score 16[-

APOMORPHINE

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: 1

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Fig. 2. The influence of pretreatment with CCK-8 related peptides on the apomorphine-induced hypomotility following bilateral injection into the nucleus accumbens. Locomotion was measured for 3 min at 5 min (rectangular testbox, A) and 20 min (small open field, B) after injection with placebo (1 /L1 saline) or apomorphine (10 ng). Animals were treated with placebo (plac.) (1 /~1 saline) or with desulphated CCK-8 (CCK8-DS, 10 ng), the sulphated form of CCK-8 (CCK-8-S, 10 ng) or ceruletide (CERU, 1 ng) 40 min before placebo or apomorphine. The mean locomotion score per treatment groups is presented. Vertical bars indicate S.E.M. The number of animals per group is shown in the columns. Different from placebo, placebo-treated rats (* P < 0.02, ** P < 0.001). Different from placebo, apomorphine-treated rats ( + P < 0.05, ÷+ P < 0.01, +++ P < 0.001).

ceruletide did not significantly affect the apomorphine-induced hyperactivity whether 1 ng or 10 ng of the CCK-8-related peptides had been injected (fig. 4). In contrast, pretreatment with CCK-8-S at both dose levels completely prevented the hyperactivity induced by apomorphine. This effect was probably not the result of decreased locomotor activity due to CCK-8-S itself since the activity after 10 ng of this peptide injected into the nucleus accumbens of rats habituated to the test cage was not significantly different from that of

ol

o~

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i

1~ o--o CCK-8~S

1c;o loJo

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Fig. 3. The influence of graded doses of CCK-8 related peptides on the apomorphine-induced hypomotility following bilateral injection into the nucleus accumbens, as assessed in a small open field 3 min, 20 min after injection with placebo (1 t~l saline) or different doses (pg) of desulphated CCK-8 (CCK8-DS, 8-12 animals per group), the sulphated form of CCK-8 (CCK-8-S, 6-10 animals per group) or ceruletide (CERU; 4-11 animals per group) 40 rain before placebo or apomorphine treatment. The mean locomotion score_+S.E.M. (vertical bars, shaded areas for placebo, placebo (n = 29) and placebo, apomorphine (n = 27) treated rats) is presented. The data for the highest dose of the peptides were taken from the experiment depicted in fig. 2.

placebo-treated controls (score of locomotor activity of placebo- and CCK-8-S-treated rats were (mean_+S.E.M.): 11.9+ 1.0 ( n = 8 ) and 8.2 4- 1.4 (n = 6) respectively). In apomorphine treated rats sniffing behavior was not affected by pretreatment with CCK-8 related peptides (table 1). 3. 3. Interaction with apomorphine or amphetamineinduced behavioral changes following injection into the nucleus caudatus Injection of 10 /~g of apomorphine into the nucleus caudatus did not affect the rate of locomotion of the animals, but increased the duration of sniffing (stereotyped sniffing) as reported earlier (Van Ree, 1982; Van Ree and De Wied, 1982b) (table 2; fig. 5). The same was found following the injection of amphetamine (10/~g) into the nucleus caudatus (table 2; fig. 5). Pretreatment with 10 ng

69 The stereotyped sniffing induced by apomorphine or amphetamine was also not changed by pretreatment with one of the peptides (fig. 5).

score

20

16

3.4. Passive avoidance behavior +

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Fig. 4. The influence of CCK-8 related peptides on the apomorphine-induced hyperactivity following bilateral injection into the nucleus accumbens, as assessed in a small open field for 3 min, 20 min after injection with placebo (plac., 1 ttl saline) or apomorphine (10 ~tg). Groups of animals were treated with placebo (1 ALl saline) or 1 ng (A) or 10 ng (B) of desulphated CCK-8 (CCK-DS), the sulphated form of CCK-8 (CCK-S) or ceruletide (CERU) 40 min before apomorphine. The mean locomotion score per treatment groups is presented. Vertical bars indicate S.E.M. The number of animals is shown in the columns. * Different from placebo, placebo-treated rats (P < 0.001)+different from placebo, apomorphine-treated rats (+ P < 0.005, ++ P < 0.001).

of CCK-8-DS, CCK-8-S or ceruletide affected neither the rate of locomotion nor the duration of sniffing in placebo-treated rats (table 2, fig. 5).

The s.c. administration of CCK-8-DS, CCK-8-S or ceruletide 1 h before the first retention test facilitated passive avoidance behavior (table 3). At the second retention test, 24 h later, the effect of the peptides was still visible. C C K - 8 - D S appeared to be the most potent of the three peptides, since as little as 3 # g induced a statistically significant facilitation of the avoidance response. D E 3 ' E administered via the same route exerted an opposite effect i.e., the passive avoidance response was attenuated (table 3, Gaffori and De Wied, 1982). The results were different when the peptides were injected into the nucleus accumbens which is a site sensitive to 3,-type endorphins with respect to attenuation of passive avoidance (Kov/tcs et al., 1982). A low dose of CCK-8-DS, CCK-8-S or ceruletide (0.3 pg) significantly attenuated passive avoidance responding (table 4). DE3,E induced a similar effect, although a 3 times higher dose of this peptide was needed. Higher doses of the peptides, except DE3,E, were less effective in attenuating the avoidance response. The range of effective dosages was smaller for ceruletide and CCK-8-S than for C C K - 8 - D S and DE3,E. The effect of treatment was still present at the second retention trial 24 h later and was most p r o n o u n c e d following treatment with DE3,E.

3.5. Behavioral profile Intraventricular injection of 20 /~g CCK-8-DS, CCK-8-S or D E y E did not change the state of reflexes, the appearance of the eyes, the rate of grooming and rearing. N o n e of the treated animals showed rigidity, hyperactivity, stereotypy or wet shakes (table 5). There was little sedation but especially CCK-8-S and D E 3 ' E induced a slight immobility. All three peptides had a positive effect in the various 'grip tests' (table 5). There were no marked differences in the effects of the three peptides in this respect.

70 TABLE 2 Locomotor activity of rats bilaterally injected into the nucleus caudatus with CCK-8 related peptides (10 ng) and subsequently with apomorphine ( 10 ~ g) or amphetamine ( 10/t g). Treatment

Locomotor activity

- 8 0 min

- 2 0 min

mean score_+ S.E.M.

Placebo ~ CCK-8-DS CCK-8-S Ceruletide

Placebo Placebo Placebo Placebo

37.2 ± 33.0 + 36.1 ± 33.2 ±

Placebo CCK-8-DS CCK-8-S Ceruletide

Apomorphine Apomorphine Apomorphine Apomorphine

31.7 ± 4.2 34.7 ± 2.8 32.5 _+6.4 34.3 ± 3.3

Placebo Placebo CCK-8-DS CCK-8-S Ceruletide

Placebo Amphetamine Amphetamine Amphetamine Amphetamine

31.1 + 37.7 ± 37.7 ± 43.6 ± 43.2 ±

2.9 2.9 3.7 1.5

(8) h (8) (11) (6) (8) (7) (6) (6)

2.8 2.1 3.5 2.0 2.9

(15) (16) (10) ( 11 ) (6)

a l /tl saline, b N u m b e r of rats in parentheses. sec

70

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15

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p

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Fig. 5. The influence of CCK-8 related peptides on the apomorphine- or amphetamine-induced stereotyped sniffing following bilateral injection into the nucleus caudatus, as assessed in a small open field for 4 min, 20 min after injection with placebo (1 /~1 saline), apomorphine (10 ~g) or amphetamine (10 ttg). Groups of animals were treated with placebo (1 p.1 saline) or 10 ng of desulphated CCK-8 (CCK-DS), the sulphated form of CCK-8 (CCK-S) or ceruletide (CERU) 1 h before placebo, apomorphine or amphetamine. The mean duration of sniffing (s) per treatment group is presented. The vertical bars indicate S.E.M. The number of animals is depicted in the columns. * Different from placebo, placebo-treated controls (P < 0.001).

TABLE 3 Effect CCK-8 related peptides and D E 7 E on retention of one trial learning passive avoidance response after subcutaneous injection 1 h before the first retention test. Treatment and dose per rat

Number of animals

Latency (median s) First retention test 24 h a

Second retention test 48 h

6 6 12

183 d (88-300) c 243 e (138-300) 86 (21--167)

77 (55 190) 212 d (59-300) 62 (18-107)

6 6 12

90 (28-300) 229 e (120--300) 86 ( 2 1 - 1 6 7 )

63 (60-300) 231 d (26--300) 62 (18-107)

6 6 12

75 (10-150) 150 d (112-300) 86 (21-167)

69 (40-300) 110 (16-161) 62 (18 107)

12 15 25

9 r (5- 15) 16 ~ (7- 22) 88 (61-287)

C C K - 8- D S

3 vg 10/~g Placebo b CCK-8-S

3 #g 10/~g Placebo Ceruletide

3/~g 10 # g Placebo DEyE

3/xg 10/xg Placebo

2 1 d ( 1 2 - 37) 4 9 ( 1 6 - 78) 75 (29-107)

a Hours after learning trial, b 0.5 ml saline, c Latency values are given as median in seconds, with the 25th and 75th percentile in parentheses. Different from placebo-treated rats (d p < 0.05; ~ P < 0.02; f P < 0.002). TABLE 4 Effect of graded doses of CCK-8 related peptides and D E T E on retention of one-trial learning passive avoidance response, following unilateral injection into the nucleus accumbens 1 h before the first retention test. Treatment and dose per rat

Number of animals

Latency (median s) First retention test 24 h a

Second retention test 48 h

6 6 6 6 6 6 18

4 6 ( 3 7 - 75) c 15 e (6- 34) 9 f (5- 12) 12 a (4- 24) 9 e (5- 24) 47 (22- 87) 78 (21 - 180)

3 7 ( 2 9 - 52) 11 (2- 43) 10 d (5- 31) 16 (3- 21) 2 0 ( 1 0 - 65) 40 (20- 62) 57 (27-180)

6 6 6 6 12

52 (30- 88) 33a (9- 41) 2 0 d ( 1 0 - 37) 53 (17- 77) 91 (31-110)

41 (36- 61) 30 (8- 52) 13 d (4- 24) 39 (16- 60) 67 (18- 71)

6 6 6 6

3 4 ( 2 9 - 75) 22d (6- 61) 69 (43- 72) 79 (23- 98)

18 (18- 65) 20 (5- 60) 56 (47-120) 57 (55- 90)

6 6 6 6 6 6 18

60 (22- 79) 43 (21- 62) 2 1 d ( l l - 47) 18d (6- 31) 10e (5- 27) 14 e (4- 24) 90 (30-138)

41 (12- 71) 35 (9- 70) 28 ( 8 - 39) 12d (7- 28) 5 a (3- 17) 6 d (4- 13) 59 (29-107)

CCK -8 -DS

0.1 pg 0.3pg 1 pg 3 pg 10 pg 30 pg Placebo b CCK-8-S

0.1 pg 0.3 pg 1 pg 3 pg Placebo Ceruletide

0.1 pg 0.3 pg 1 pg Placebo DETE

0.1 pg 0.3 pg 1 pg 3 pg 10 pg 30 pg Placebo

a Hours after learning trial, b 1 /~1 saline, c Latency values are given as median in seconds, with the 25th and 75th percentile in parentheses. Different from placebo-treated rats (d p < 0.05, ~ P < 0.02, f p < 0.002).

1 0 0 3d 3.5 e 5.5 ~ 0

0 0 0 0

20 ,ttg

1 ,al saline 0b 0 0

CCK-8-DS

Placebo

0

5d 4.5 d 6e

2.5 " 0 0

20/Lg

CCK-8-S

0

4d 4.5 d 5.5 "

2d 0 0

20/~g

DEy E

12 6 6 6 6

12 6 6 6 6

76.8±6.0 70.0±7.0 32-7± 2.0d 72.2±2.0 60-7± 2.7~

79.0±5.3 ~ 81.0±7.0 55.3+6.0 b 71.5±2.5 76.0±8.0

Wall

of animals

16.2±2.0 2-7+0.9d 1.7±0-9 d 3-8± I-1~ 4.8± 0-3d

12.8±1.5 8.3±0.9 4.7±1.1 d 12.5±1.2 7.8± 1.0

Middle

Ambulations (score)

Number

93.0±6.0 72.7±2.6 34.3±2.3 76.0±3.1 65.5+2-8

b d b d

91.8+5.8 89.3±7.9 60.0±6.9 c 83.8±1.6 83.8±0.8

Total

14.2+1.1 4.3±1.4 2.7±0.9 5.2+1.2 10.7±1.1

ll.8±l.0 11.8±1.6 10.0±1.2 13.5±2.5 tl.0+1.3

Wall

J d c b

Rearings (score)

Mean ± S.E.M. Different from p l a c e b o - t r e a t e d a n i m a l s ( b p < 0 . 0 5 ; ~ P < 0 . 0 1 : d p < 0 . 0 0 1 ) .

CCK-8-DS CCK-8-S Ceruletide DEyE

Placebo

0.5 ml Saline 10 #g 10 ,ug 10/~g 10/.tg

0.5 ml Saline 3/tg 3/~g 3/Lg 3 ~g

Placebo

CCK-8-DS CCK-8-S Ceruletide DEyE

Dose

Treatment

5.2± 1.2 0.2±0.2 0.2+0.2 0.2±0.2 2.5± 0.3

d d d c

3.7± 0.3 2.8± 0.8 0.2+ 0.2 ~ 3.0±0.8 2.5±0.5

Middle

19.2±2.0 4.5±1.4 2 .9 ±0 .9 5.4±1.2 13.2±1.2

d d d ~

15.5±1.8 14.7±2.2 10.2±1.3 b 16.5+1.8 13.5±1.8

Total

8±3.4 7.5±3.8 4 .2 +1 .8 5.2±1.7 10.0±1.8

7.5±3.6 8 .0 ±1 .8 9.5±3.3 8 .5 ±0 .7 6.0±2.0

(s)

Grooming

4.7±0.6 4.3±0.9 3.5±0.7 4 .1 ±0 .8 4 .3 ±0 .7

4 .2 ±1 .5 3.8±0.5 4.5±0.4 5.3+0.5 5.2±0.7

Boluses

The influence of CCK-8 related peptides and DE3,E on gross behavior of rats as assessed in the big open field for 3 min, 60 min after subcutaneous injection.

TABLE 6

Other activities tested were not changed (for details see De Wied et al., 1978). b Median score of 6 animals (maximum score 6 per animal). Different from placebo-treated rats (c p < 0.05; d p < 0.01: e p < 0.001).

Spontaneous behavior a Immobility LOSS of reflexes Rigidity Grasping response Edge cage Vertical grid Pencil Changes in appearance of the eyes

Dose

Treatment

Behavioral profile of rats treated with CCK-8 related peptides c,r DE~'E into the cerebral ventricle. Scoring was carried out at 30, 60 and 120 min after injection.

TABLE 5

73

3. 6. Open field behavior The rate of ambulation and rearing of rats injected s.c. with 3/~g of CCK-8-DS, ceruletide or DEyE 1 h before testing in an open field, was not different from that of placebo-treated controls (table 6). A higher dose of these peptides slightly decreased the rate of ambulation and more markedly the rate of rearing. CCK-8-S appeared to be more potent in this respect i.e. there was a significant decrease of the rate of ambulation and rearing following treatment with 3 /~g and profound effects were found after injection of 10 ~g (table 6). Interestingly, all the peptides induced a decrease in ambulation and rearing in the middle of the open field, suggesting that rats treated with the peptides spent relativey more time along the wall of the open field. None of the peptides significantly changed grooming behavior or the amount of boluses produced during the testing period.

4. Discussion

The present results show that CCK-8 related peptides, following their intracerebral injection, affect certain behavioral changes induced by apomorphine suggesting that these peptides interfere with brain dopaminergic systems. However, the effects of these peptides are specific with respect to both the peptide and the dopaminergic systems being investigated. Relatively high doses of apomorphine injected into the nucleus caudatus induced stereotyped sniffing without changing the locomotor activity of the animals, whereas similar injections into the nucleus accumbens resulted in hyperactivity but not in changes in sniffing behavior. This agrees well with the suggestion that the nigrostriatal and mesolimbic dopaminergic systems (the former with projections to the nucleus caudatus and the latter, to the nucleus accumbens) have a role in the apomorphine-induced stereotypy and hyperactivity respectively (Kelly et al., 1975; Kelly and Iversen, 1976; Pijnenburg et al., 1976; Costall et al., 1977). These effects of apomorphine are believed to be due to stimulation of postsynaptically located dopaminergic receptors. It was found that the 10 ng dose of CCK-8 related

peptides did not interfere with the stereotyped sniffing response following the injection of apomorphine or amphetamine into the nucleus caudatus. Amphetamine mimics the action of apomorphine probably by releasing endogenous dopamine. A similar amount of CCK-8-DS or ceruletide also failed to affect the hyperactivity evoked by high doses of apomorphine injected into the nucleus accumbens. However, CCK-8-S (1 and 10 ng) completely antagonized the apomorphineinduced hyperactivity, suggesting that this peptide, but not CCK-8-DS and ceruletide, interferes with the behavioral effects elicited by stimulation of the postsynaptically located dopamine receptor system in the nucleus accumbens. This is consistent with the finding that CCK-8-S, but not CCK-8-DS or ceruletide, induced hypoactivity when injected into the nucleus accumbens without additional treatment with apomorphine. Interestingly, another dopaminergic system present in the nucleus accumbens, and activated by low doses of apomorphine with decreased locomotor activity as the result, is influenced by all three CCK-8 related peptides. Thus, the hypoactivity induced by low doses of apomorphine injected into the nucleus accumbens was completely antagonized by local treatment with CCK-8-DS and more potently by ceruletide. CCK-8-S was less potent in this respect, and because of its effects per se, could only be tested at dose levels which partly antagonized the apomorphine-induced response. It has been argued before that the dopamine receptor system sensitive to low doses of apomorphine may be located presynaptically, and that its activation results in the decreased availability of dopamine for postsynaptic dopaminergic receptors and consequently in diminished dopamine transmission (Van Ree and Wolterink, 1981; Van Ree et al., 1982a,b). However, it cannot yet be excluded that we are dealing with two distinct dopaminergic pathways which have a different sensitivity to apomorphine at these postsynaptic dopamine receptor systems. Nevertheless, the present data show that the three CCK-8 related peptides tested have a common antagonistic action, albeit with different potencies, on the dopamine system activated by low doses of apomorphine, while only CCK-S interferes with the dopamine system activated by high doses of

74 apomorphine. A profile of action similar to that observed with CCK-DS and ceruletide had been found with "),-type endorphins. Thus, D E y E antagonized the hypoactivity induced by the injection of low doses of apomorphine into the nucleus accumbens (Van Ree et al., 1982a) but did not interfere with the hyperactivity and stereotyped sniffing evoked by high doses of apomorphine injected into the nucleus accumbens and nucleus caudatus respectively (Kirb,ly and Van Ree, 1982; Van Ree, unpublished data). Both classical and atypical neuroleptics e.g. haloperidol and sulpiride, at low dose levels (10-30 pg) effectively antagonized the hypoactivity induced by low doses of apomorphine when they were injected into the nucleus accumbens, while a similar dose of haloperidol but not of sulpiride was able to block the hyperactivity following similar treatment with high doses of apomorphine (Van Ree et al., 1982a; Kirfily and Van Ree, 1982). A much higher dose of haloperidol was needed for antagonizing the stereotyped sniffing response elicited by high doses of apomorphine injected into the nucleus caudatus (Van Ree, unpublished data). Thus, it may be concluded that with respect to dopaminergic systems in the nucleus accumbens, CCK-8-S has a profile similar to that of haloperidol, whereas CCK8-DS, ceruletide and DE~,E resemble sulpiride. While CCK-8-S at the dose level tested in our experiments did not interfere with the stereotyped sniffing response evoked by injection of high doses of apomorphine into the nucleus caudatus suggesting that this peptide does not affect postsynaptically located dopaminergic receptor systems in that area, the possibility of an action on these postsynaptic receptors cannot be excluded as yet. Such an action remains plausible, especially because much more haloperidol is needed to block the apomorphine-induced behavioural changes with injections into the nucleus caudatus than with injections into the nucleus accumbens. Unfortunately, there are only few other studies concerning the interaction of CCK-8 related peptides and brain dopaminergic systems and most of these studies deal with CCK-8-S only, which hampers any discussion of mode of interaction of these peptides with brain dopamine transmitters.

Apomorphine-induced stereotyped cage climbing behavior in mice was not affected by CCK-8-S but was enhanced by CCK-8-DS following systemic i n j e c t i o n ( K o v f i c s et al., 1981), while methamphetamine-induced hyperactivity was reduced by intraventricularly injected CCK-8-S, but not CCK-8-DS (Itoh and Katsuura, 1982). CCK8-DS, but not CCK-8-S facilitated the disappearance of striatal dopamine measured after synthesis inhibition in mice and both CCK-8-DS and CCK-8-S increased the in vitro release of [3 H]dopamine from striatal slices. On the basis of these studies, Kovfics et al. (1981) postulated that CCK-8 related peptides affect striatal dopaminergic neurotransmission at the presynaptic level and this postulate is consistent with our data obtained with injections into the nucleus accumbens. However, Fuxe et al. (1980) reported that CCK-8-S produced a reduction of dopamine turnover in discrete parts of the nucleus caudatus and in the anterior part of the nucleus accumbens, whereas no changes were observed in the posterior part of the nucleus accumbens and in the tuberculum olfactorium which contain dopamine terminals containing CCK-like immunoreactivity. Thus, CCK-like peptides may have a different action on nigrostriatal and mesolimbic dopaminergic system. Such a differential action was also evidenced by studies showing that there was a rather complex interaction of CCK-8 related peptides with [3H]spiroperidol and [3H]dopamine binding in vitro (Murphy and Schuster, 1982; Bhoola et al., 1982). Thus, more experiments should be focussed on the influence of both CCK-S and CCK-DS on the nigro-striatal and mesolimbic dopaminergic systems before definite conclusions can be drawn in this respect. The CCK-8 related peptides facilitated passive avoidance behavior when they were given s.c. 1 h before the retention test. This is in agreement with other studies showing that i.p. and intraventricularly administered CCK-8-DS as well as CCK-8-S facilitated the passive avoidance response when rats when treated immediately after the learning trial or 1 h before the retention test (Fekete et al.. 1981a; Kfid/tr et al., 1981). However, there was an opposite effect when the CCK-8 related peptides were injected directly into the nucleus accumbens,

75 in that low doses of CCK-8-DS, CCK-8-S and ceruletide attenuated passive avoidance behavior. A similar effect was found with the y-type endorphins, but these peptides still attenuated passive avoidance behavior when they were given systemically (De Wied et al., 1978; Greven and De Wied, 1980; Kov~cs et al., 1982; Gaffori and De Wied, 1982). This suggests that the CCK-8 related peptides not only mimic the action of y-type endorphins on passive avoidance behavior, but also interact with other brain structures in or outside the nucleus accumbens, resulting in the facilitation of passive avoidance behavior. It may be that low doses of CCK-8 related peptides attenuate, while higher doses facilitate passive avoidance behavior. Accordingly, K/td~r et al. (1981) reported that this behavior tended to be attenuated following intraventricular injection of a dose of both CCK-DS and CCK-8-S lower than that causing facilitation of passive avoidance behavior. Concerning the profile of the behavioral effects following intraventricular injection of CCK-8 related peptides, it was found that these peptides induced a positive effect in the various grip tests and that CCK-8-S induced a slight immobility. Such a behavioral profile is characteristic of y-type endorphins and neuroleptic drugs, including haloperidol (De Wied et al., 1978; Van Ree and De Wied, 1982a). The positive effects in the various grip tests may be related to the catalepsy that has been observed in mice following s.c. treatment with CCK-8-S and ceruletide (Zetler, 1981, 1982). We found that CCK-8 related peptides and DETE slightly reduced the rate of ambulation and rearing in an open field test after s.c. treatment. CCK-8-S was more active in this respect than the other peptides, an effect perhaps related to the finding that this peptide, but not CCK-8-DS and ceruletide decreased locomotor activity following their injection into the nucleus accumbens. All peptides increased the amount of time spent along the wall of the open field. This partly agrees with the observation that CCK-8-S but not CCK-8-DS reduced exploratory behavior in mice and rats and that CCK-8-S increased the amount of time the mice spent in the corners of a test arena (Crawley et al., 1981a,b). The rats treated with CCK-8 related peptides or DETE may have been avoiding

the center of the open field where the level of illumination was higher than near the wall. Decreased ambulation together with more or less avoiding of the central part of the open field had been found in rats whose dopaminergic systems in the nucleus accumbens were destroyed by injection of 6-OHDA (Simon, 1980). Thus, the effects of CCK-8 related peptides and DEyE, as found in the open field test, may be related to the interaction of these peptides with dopaminergic systems in the nucleus accumbens. The present data indicate that CCK-8 related peptides have behavioral effects that resemble in certain aspects the effects following treatment with antipsychotic agents. Thus, the hypoactivity induced by low doses of apomorphine is antagonized following injection with CCK-8 related peptides into the nucleus accumbens, as was found before with low doses of the neuroleptics haloperidol and sulpiride and the y-type endorphins (Van Ree et al., 1982a). Passive avoidance behavior is attenuated when CCK-8 related peptides are injected into the nucleus accumbens. A similar attenuation was seen previously with haloperidol given s.c. and following injection of the peptide D E y E either s.c. or into the nucleus accumbens (Kovfics and De Wied, 1978; Greven and De Wied, 1980; Kov~cs et al., 1982). Intraventricular treatment with CCK-8 related peptides induced a slight immobility and a marked effect in the various 'grip tests'. This grasping response, which is also observed following treatment with haloperidol, sulpiride and y-type endorphins might be conceived of as a physiological form of catalepsy (Van Ree and De Wied, 1982a). Notwithstanding the similarities, there are also differences between CCK-8 related peptides and neuroteptics e.g. the peptides did not affect the behavioral changes induced by apomorphine and amphetamine following injection into the nucleus caudatus, whereas haloperidol antagonized these behavioral changes. We have argued before that positive effects in the test procedures used in the present study (avoidance behavior, grasping response, apomorphineinduced hypo-activity), may predict an antipsychotic action. Although in the earlier studies we used extinction of active avoidance behavior rather than passive avoidance behavior as test procedure,

76

it has been shown that intraventricular administration of CCK-8-DS and CCK-8-S and peripheral treatment with CCK-8-S facilitate the extinction of active avoidance behavior, as was found following treatment with haloperidol and y-type endorphins (Cohen et al., 1982; Fekete et al., 1981b; De Wied et al., 1978, 1980; Kov~.cs and De Wied, 1978). Thus it may be postulated that, like y-type endorphins, CCK-8 reated peptides have inherent antipsychotic properties. In general, this possibility is consistent with the reported neuroleptic-like effects of these peptides in mice and their antipsychotic action in some schizophrenic patients (Zetler, 1981; Moroji et al., 1982). Differences between the various studies concerning animal behavior are present as well, which may possibly be related to the use of different species. It may be that certain peptides either present intraneuronally (like CCK related peptides) or originating from brain endocrine systems (like y-type endorphins as one of the products of the pro-opiomelanocortin system) control the activity of specific neurons of the mesolimbic dopaminergic pathways, and that disturbances in this homeostatic control will eventually lead to psychopathological symptoms characteristic of schizophrenic psychosis.

Acknowledgements The authors thank Tjitske P. Van der Woude, Emilie M.M. Bloemarts and Karien Janssen-Schwiebbe for their skilful technical assistance. This research was partly subsidized by the Stichting Pharmacologisch Studiefonds.

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