Further analysis of the effects of cholecystokinin octapeptides on avoidance behaviour in rats

European Journal of Pharmacology, 98 (1984) 79-91 Elsevier

79

FURTHER ANALYSIS OF THE EFFECTS OF CHOLECYSTOKININ OCTAPEPTIDES ON AVOIDANCE BEHAVIOUR IN RATS M.~TY,~S FEKETE 1., ./~GNES LENGYEL I, BI~LAHEGED(JS 1, BOTOND PENKE 2 M/kRTA ZARANDY 2, G.~BOR K. TOTH 2 and GYULA TELEGDY 1 I Department of Pathophysiology, University Medical School, Szeged, P.O. Box 531, Hungary, and 2 Department of Medical Chemistry, University Medical School, Szeged, Hungary

Received 13 July 1983, revised MS received 25 October 1983, accepted 1 November 1983

M. FEKETE, ,~. LENGYEL, B. HEGED(JS, B. PENKE, M. ZAIL~NDY, G.K. TOTH and G. TELEGDY, Further analysis of the effects of cholecystokinin octapeptides on avoidance behaviour in rats, European J. Pharmacol. 98 (1984) 79-91. Experiments were performed to examine the acute effects of cholecytokinin octapeptides and fragments on the active and passive avoidance behaviour of rats following peripheral and central administration. Both the sulphated (CCK-8-SE) and non-sulphated cholecystokinin octapeptide (CCK-8-NS) and also the COOH-terminal tetra-, penta-, hexa- and heptapeptides of cholecystokifiin octapeptide facilitated the extinction of active avoidance behaviour and retention of passive avoidance behaviour. This latter effect of cholecystokinin octapeptides was reversed by anxiolytic chlordiazepoxide pretreatment, showing that in these test situations cholecystokinin octapeptides are able to modify fear-motivation or arousal of the animals; their effect is at least partly similar to that of the neuroleptic substance haloperidol. Subcutaneous treatment with CCK-8-SE or CCK-8-NS appeared to be 3-10 times more effective than intraperitoneal treatment. Following intracerebroventricular administration, 100-300 times lower doses were needed to cause a behavioural effect similar to that after subcutaneous injection. Microinjection of CCK-8-SE or CCK-8-NS in the fmol dose range into the nucleus accumbens facilitated the extinction of active avoidance behaviour and attenuated the retention of passive avoidance behaviour, while microinjection of these peptides into the central amygdaloid nucleus caused opposite effects on these behavioural tests. It seems that the neuroleptic-like effects of cholecystokinin octapeptides are mediated through the nucleus accumbens, and the opposite action (non neuroleptic-like) through the central amygdaloid nucleus. Peripheral and central administration Passive avoidance behaviour

Central amygdaloid nucleus Active avoidance behaviour

1. Introduction Previous experiments showed that cholecystokinin octapeptides (CCK-8), injected peripherally or intracerebroventricularly (i.c.v.), impaired the acquisition and facilitated the extinction of active avoidance behaviour (Fekete et al., 1981e, 1982; Cohen et al., 1982). However, these peptides facilitated the retention of the passive avoidance * To whom all correspondence should be addressed: Department of Pathophysiology, University Medical School, Szeged, P.O. Box 531, H-6701 Hungary. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.

Cholecystokinin octapeptides Nucleus accumbens

behaviour of rats (Fekete et al., 1981c,d; K~d~r et ~d., 1981; Telegdy et al., 1982). These effects have been found seemingly contradictory. The aim of the present experiments was to analyse further these effects of C C K - 8 on fearmotivated avoidance behaviour, and to c o m p a r e the actions of C C K - 8 with those of a neuroleptic substance, haloperidol. In addition, it was investigated whether the minor tranquillizer chlordiazepoxide which is k n o w n to decrease behavioural arousal (Sansone et al., 1981; Sahgal and Wright, 1983), was able to modify the behavioural effects of CCK-8. The effects of subcutaneously, in-

80 traperitoneally and intracerebroventricularly administered C C K - 8 and its fragments were also compared. Finally, the site and mode of action of C C K - 8 were studied following intracerebral microinjections into the different brain nuclei which had been shown to contain significant amounts of C C K (for review see E m s o n and Marley, 1983).

2. Materials and methods

the dorsomedial hypothalamic nucleus (AP = 2.0; L = 0.5; D = 7.5), the supraoptic nucleus (AP = 0.0; L = 1.0; D = 7.5), the paraventricular nucleus (AP---1.0; L = 0.5; D = 6.5) and the nucleus accumbens (AP = - 1.5; L = 1.0; D = 5.5) according to the atlas of Fifkov~ and Marsala (1962). Placement of the i.c. cannula was examined histologically. The brains were removed and placed in 10% formalin solution after the experiments had been completed. The localization of the cannula was determined on 200 ffm thick frozen sections.

2.1. Animals Male Sprague-Dawley (CFY) rats of an inbred strain (LATI, G6d6116, Hungary), weighing 160180 g, were used. The animals were housed 5 or 6 per cage at r o o m temperature (20-21°C). All animals had access to commercial food (LATI, GOd6116, H u n g a r y ) and tap water ad libitum. The animals were kept under a standard illumination schedule (lights on between 6 a.m. and 6 p.m.). All observations were started between 8 a.m. and 1 p.m.

2.2. Surgery For intracerebroventricular (i.c.v.) administration of peptides, a stainless steel cannula was implanted into the lateral cerebral ventricle under pentobarbital anaesthesia (40 m g / k g N e m b u t a l ®, Ceva, Neuilly-sur-Seine, France) intraperitoneally (i.p.) as described by Fekete et al. (1980b). The rats were allowed to recover for 7 days. The correct positioning of the cannula was checked individually by injection of methylene blue after the experiments had been completed. F o r intracerebral (i.c.) a d m i n i s t r a t i o n of peptides, a stainless steel cannula was implanted stereotaxically under pentobarbital anaesthesia (40 m g / k g N e m b u t a l ®, CEVA, Neuilly-sur-Seine, France, i.p.). The tip of the cannula was aimed at the ventral tegmental area (AP = 4.0; L = 0.3; D = 9.0), the dorsal raphe nucleus (AP = 6.5; L = 0.0; D = 7.5), the central amygdaloid nucleus (AP = 1.0; L = 3.0; D = 7.5), the dentate gyrus of the h i p p o c a m p u s (AP = 3.0; L = 2.0; D = 3.0), the stria terminalis (AP -- 1.0; L = 2.0; D --- 4.0), the caudate nucleus (AP = - 1 . 0 ; L = 2.5; D = 5.0),

2. 3. Active avoidance behaviour Active avoidance behaviour was studied in a bench-jumping situation. The procedure was slightly different from that described previously (Fekete et al., 1981e) and the experimental protocol was similar to that of Van Wimersma Greidanus and De Wied (1971). Rats were conditioned to avoid the unconditioned stimulus (US) of an electric footshock (0.7 mA, AC) by j u m p i n g onto a plexiglass bench (13 x 9 cm) located 7 cm above the floor, on one side of the box (45 x 25 x 45 cm). The conditioned stimulus (CS) was a light signal (45 W bulb). The US was applied if an avoidance response had not occurred within 5 s after the onset of the CS. The CS remained on during presentation of the US (maximum 15 s). Ten acquisition trials were given daily, with a mean intertrial interval of 60 s (range 50-70 s). Acquisition training for 3 days was followed by extinction sessions on day 4. In other experiments 4 days of acquisition was used to make the rats more resistant to extinction, and the extinction sessions were run on day 5. Ten non-reinforced trials were presented per session, in which the CS was terminated immediately after the rat had j u m p e d onto the bench within 5 s (positive response, conditioned avoidance response, C A R ) or after 5 s in the absense of avoidance. Those animals which made 8 or more avoidances during the first extinction session on day 4 (or 5) were used for further experimentation. The rats received peptide, haloperidol or vehicle 30 s after completion of the first extinction session, and two more extinction sessions were run 2 and 4 h after the first one.

81

2.4. Passive avoidance behaviour Animals were trained in a step-through type one-trial learning passive avoidance test (Ader et al., 1972). The experimental apparatus consisted of an illuminated platform attached to a large, dark compartment equipped with a grid floor. After habituation to the dark compartment (2 min) rats were placed on the platform and allowed to enter the dark compartment; since rats prefer dark to light, they normally entered within 15 s. On the next day, after three more trials (intertrial interval 5 min), an unavoidable scrambled footshock (0.75 mA, 2 s) was delivered through the grid floor of the dark compartment (learning trial). Rats were removed from the shock box 10 s after the termination of the shock. Passive avoidance latencies were tested 24 h after the learning trial (retention test); the rat was placed on the platform and the latency to enter the dark compartment was measured up to a maximum of 300 s. Treatment with peptide, haloperidol or vehicle was given 1 h before the retention test.

CCK-5-8 ( T r p - M e t - A s p - P h e - N H 2 ; M W 598), CCK-6-8 (Met-Asp-Phe-NH2; MW 412), CCK-7-8 (Asp-Phe-NH2; MW 280), CCK-2-4-SE (Tyr(SO3H)-Met-Gly-OH; MW 451) and CCK-1-4-NS (Asp-Tyr-Met-Gly-OH; MW 485) were dissolved in 0.9% saline. The synthesis, yield, purity of these peptides have been reported elsewhere (Penke et al., 1979, 1981, 1983; Fekete et al., 1981a). Peripheral injections were given s.c. or i.p. in a volume of 2.5 ml/kg. Control animals received the same volume of 0.9% saline. For i.c.v, or i.c. injection, the above-mentioned peptides were dissolved in 0.9% saline containing 0.5% bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, U.S.A.) to prevent adhesion of small amounts of injected peptides. All i.c.v, injections were given in a volume of 2 /~1, while i.c. injections were given in a volume of 1/tl with a microinjection (Mikrometerspritze, Hormuth-Vetter) to conscious, freely moving rats. Control animals received the same volume of vehicle.

2.7. Haloperidol treatment 2.5. Open-field activity The locomotor activity of the animals was studied in a rectangular open-field box measuring 60 × 60 cm, consisting of 36 squares measuring 10 × 10 cm each. The apparatus was illuminated by a 60 W bulb. Open-field exploratory activity was scored by counting the number of squares crossed (ambulation), rearing (at wall and middle), grooming and defecation during a 5 rain session (Fekete et al., 1981e). Animals were treated s.c. with CCK-8-SE, CCK-8-NS or haloperidol 1 h before being tested.

2.6. Peptides For peripheral injection, CCK-8-SE (AspTyr(SO3 H ) - M e t - G l y - T r p - M e t - A s p - P h e - N H 2; CCK-1-8-SE; MW 1145), CCK-8-NS (Asp-TyrMet-Gly-Trp-Met-Asp-Phe-NH 2; CCK-1-8-NS; MW 1064), CCK-2-8-SE (Tyr(SO3H)-Met-GlyTrp-Met-Asp-Phe-NH2; MW 1030), CCK-3-8 (Met-Gly-Trp-Met-Asp-Phe-NH2; MW 786); CCK-4-8 (Gly-Trp-Met-Asp-Phe-NH2; MW 655),

Haloperidol (haloperidol, Gedeon Richter Pharmaceutical Co., Budapest, Hungary; MW 376) was dissolved in 0.9 saline and injected in a volume of 2.5 m l / k g s.c.

2.8 Chlordiazepoxide pretreatment Chlordiazepoxide (Elenium, Polfa, Warsaw, Poland; MW 300) was dissolved in 0.9% saline and injected in a volume of 2.5 m l / k g s.c. 30 s after completion of the first extinction session of active avoidance behaviour and 70 min before the retention test of passive avoidance behaviour (i.e. 10 min before the peptide treatment). Control animals received the same volume of 0.9% saline.

2.9. Statistical analysis Statistical comparisons of the data relating to active avoidance behaviour and open-field activity were performed by analysis of variance. A level of P < 0.05 was accepted as indicating a statistically significant effect. This analysis was followed by

82 TABLE 1 Effect of subcutaneously administered C C K octapeptides and haloperidol on the rate of extinction of active avoidance behaviour of rats (4 day acquisition). Treatment

n

Number of avoidances Oh

2h

4h

10 10 11

9,3_ 0.2 a 9.0___0.2 9.6 ___0.2

7.2 + 0.7 5.9_+0.9 ~' 8.5 _+0.2

5.6 _+ 1.0 3 4.0_+0.8 3 8.7 _+0.3

11 10 10

9.0__+0.2 9.0 ___0.2 9.3 _+0.3

6.4-+0.8 d 4.5 -+ 0.9 d 8,8 _+0.3

4.7_+0.9 d 3.3 _+0.7 d 7.9 _+0,6

10 9 10

9.0_+0.2 9.0 _+0.3 9.4 _+0.2

6,4_+0.7 ~ 5.3 -4-_0.8 ~ 8.7 _+0.3

4.1 _+0.9 d 3.0 _+0.6 d 7.6 _4-0.5

CCK - 8 - SE

40 n m o l / k g I20 n m o l / k g Saline CCK - 8 - NS

40 n m o l / k g 120 n m o l / k g Saline Haloperidol

40 n m o l / k g 120 n m o l / k g Saline

Mean _+S.E.M. b p < 0.05 vs. saline-treated rats. ~ P < 0.01 vs. saline-treated rats. d p < 0.005 vs. saline-treated rats.

the a posteriori test of Student-Newman-Keuls (Sokal and Rohlf, 1969). The non-parametric ranking tests of KruskalWallis and Mann-Whitney (Weber, 1979) were used for statistical analysis of passive avoidance behaviour data. A probability level of 0.05 or less was accepted as level of significance.

ble 2). However, treatment with the same amounts of haloperidol dose dependently reduced the avoidance latency of the rats. Whereas a 120 n m o l / k g dose of haloperidol produced a significant decrease in ambulation (P < 0.05), CCK-8-SE and CCK-8-NS produced no change in the ambula-

TA B LE 2

3. Results 3. I. Avoidance behaviour and open-field actioity

Effect of subcutaneously administered CCK octapeptides and haloperidol on the retention of passive avoidance behaviour of tilts. Treatment

As can be seen from table 1, s.c. treatment with CCK-8-SE, CCK-8-NS or haloperidol immediately after the first extinction session facilitated extinction of the bench-jumping avoidance response 2 and 4 h later in a dose-dependent fashion. In these experiments acquisition training for 4 days was followed by extinction sessions on day 5; this experimental design was used to make the rats more resistant to extinction. We failed to find any significant effect of these substances when acquisition training for 3 days was followed by extinction sessions on day 4 (data not shown). Preretention treatment with CCK-8-SE and CCK-8-NS before the retention test~ 24 h after the learning trial, significantly facilitated passive avoidance behaviour in a dose-dependent manner (ta-

n

Median latency in seconds (25th and 75th percentile)

10 11 10 11

78 115 152 243

(20-178) (22-201) (114-300) a (148-300) b

10 10 10 10 11

67 100 120 194 64

(43-137) (16-189) (103-300) a (125-300) b (21-139)

CCK - 8- SE

4 12 40 120

nmol/kg nmol/kg nmol/kg nmol/kg

CCK- 8- NS

4 nmol/kg 12 n m o l / k g 40 n m o l / k g 120 n m o l / k g Saline Haloperidol

40 n m o l / k g 120 n m o l / k g Saline

8 9 8

27 (12- 31) b 10 (5- 14) ~ 85 (49-121)

P < 0.05 vs. saline-treated rats. b p < 0.01 vs. saline-treated rats. c p < 0.001 vs. saline-treated rats.

83

A CCK-8-SE

CARs ~0"

CCK-8-NS

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~

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~

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6

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09 % NoCI

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zoo.

300

CHLOROlAZ EPOXIDE (5/aMOL IKG. SC)

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nr 0

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ma

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Fig, 1. (A) Effects of chlordiazepoxide pretreatment on the (left panel) CCK-8-SE- and (right panel) CCK-8-NS-induced facilitation of extinction of active avoidance behaviour (4-day acquisition). • • Saline + saline; C) (3 chlordiazepoxide (5 ~ m o l / k g s.c.) + saline; • • saline + CCK-8-SE or CCK-8-NS (40 n m o l / k g s.c.); zx zx chlordiazepoxide (5/~ m o l / k g s.c.)+ CCK-8-SE or CCK-8-NS (40 n m o l / k g s.c.). (B) Effects of chlordiazepoxide pretreatment on the CCK-8-SE and CCK-8-NS-induced changes in passive avoidance behaviour. • P < 0.05 vs. control.

tion, rearing, grooming and defecation of the animals in the open-field test situation (data not shown). 3.2. Avoidance behaviour: chlordiazepoxide pretreatment

The effective dose of chlordiazepoxide was first determined. Chlordiazepoxide in doses of from

0.15 to 500 /Lmol/kg injected s.c. 1 h before the retention test of the passive avoidance behaviour, showed a typical U-shaped dose-response curve. While 1.5, 5.0 and 15.0 /xmol/kg doses of chlordiazepoxide attenuated the passive avoidance latency, 150 and 500/xmol/kg doses enhanced it (data not shown). A 5 F m o l / k g dose of chlordiazepoxide was used in further experiments since this dose was sufficient to decrease the behavioural

84

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CCK-I-B-NS

•

0

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40 12040o

CCK-5-8

,

0

•

•

40 120 400

CCK-6-8

CCK-2-8-SE

,

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,

CCK-3-8

CCK-4-8

0 40 120400

0 40 120 400 NMOL /KG, SC

,

0 40 120 400

CCK-7-8

CCK-I-Z.-NS CCK-2-4-SE J

•

w

10"

) •

0 U4660100

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C) 40 I:~04;0

•

,

+

0 40 120 400 NMOL tKG,SC.

IASP ..2TYR 3MET_4GLY5TRP_6MET-7ASp-SpHE-NH2 503 H I

Fig. 2. Effects of graded doses of cholecystokinin octapeptides or their fragments on the extinction of active avoidance behaviour (4 day acquisition). Points represent the total number of avoidance responses in the 2 and 4 h extinction sessions (n = 6-10 per group; S.E.M. are between 0.5 and 1.3).

arousal of the animals but was too low to cause sedation. Treatment with chlordiazepoxide itself in a 5 /zmol/kg dose failed to cause a significant change in the extinction of active avoidance behaviour (fig. 1A) while chlordiazepoxide pretreatment 10 min prior to the CCK-8-SE or CCK-8-NS (40 n m o l / k g s.c.) treatment significantly enhanced the facilitatory effect of these peptides 2 h (P < 0.05) and 4 h (P < 0.01) later. CCK-8-SE and CCK-8-NS dose dependently facilitated passive avoidance behaviour (P < 0.01) in 0.9% saline-pretreated rats, while in chlordiazepoxide-pretreated animals these peptides attenuated the retention of passive avoidance behaviour (P < 0.05) (fig. 1B). 3.3. Active avoidance." structure-activity studies Given s.c. CCK-8-SE, CCK-8-NS, CCK-2-8-SE, CCK-3-8, CCK-4-8 or CCK-5-8 dose dependently decreased the number of avoidance responses in the extinction of active avoidance behaviour (fig.

2), but treatment with the same of 3-10 times higher amounts of CCK-6-8, CCK-7-8, CCK-1-4NS or CCK-2-4-SE was ineffective. 3.4. Avoidance behaviour." systemic routes of administration Both s.c. and i.p. injections of CCK-1-8-SE, CCK-1-8-NS or CCK-5-8 reduced avoidance in the active avoidance test situation, and there were differences only in the effective doses (fig. 3A): 40, 120 and 297 n m o l / k g doses of CCK-1-8-SE or CCK-5-8 and also 120 and 297 n m o l / k g doses of CCK-1-8-NS, administered s.c., significantly (P < 0.05) facilitated the extinction of active avoidance behaviour 2 and 4 h later. We failed to find any significant effect after the i.p. administration of 120 and 297 n m o l / k g doses of these same peptides. However, a 400 n m o l / k g dose of CCK-1-8-SE, CCK-1-8-NS or CCK-5-8 significantly (P < 0.05) facilitated the extinction of the active avoidance behaviour 2 and 4 h later. On a molar basis, s.c.

85

A

ACTIVEAVOD I ANCEBEHAVIOUR EXTN I CTO IN N ITRAPERT IONEAL SUBCUTANEOUS CA~I~ 6

i

CCK-II.SE

i H~

S

CCK-14-NS

o

;

i

i

i ,.,R

k ~R

B PASSIVE AVOIDANCE BEHAVIOUR INTRAPERtTONEAL

SUBCUTANEOUS $

$

~

C,300-

3OO'

Z W

l,iJ ~t J ZOO' Z ,,1

5 zooZ

~Ioo. ~E

1o0

0

t.

12

t.o

',20

NMOLIKG

o

12

t.o

12o

NMOLIKG

Fig. 3. (A) Effects of graded doses of s.c. or i.p. injected sulphated cholecystokinin octapeptide (CCK-1-8-SE), non-sulphated cholecystokinin octapeptide (CCK-1-8-NS) or C O O H terminal tetrapeptide of these peptides (CCK-5-8) on the extinction of active avoidance behaviour (4 day acquisition), x - x control; © © 40 n m o l / k g ; • • 120 n m o l / k g ; t, zx 297 nmol/kg; • • 400 n m o l / k g , n = 6-7; S.E.M. are between 0.2 and 1.0. (B) Effects of graded doses of s.c. or i.p. injected CCK-8-SE ([]) or CCK-8-NS (~) on the retention of passive avoidance behaviour. [] control.

treatment appeared to be 3-10 times more effective than i.p. treatment. A similar result was found with regard to passive avoidance behaviour. Both s.c. and i.p. admin-

istration of CCK-8-SE or CCK-8-NS facilitated the passive avoidance behaviour of the rats (fig. 3B). These peptides, administered s.c. in 40-120 n m o l / k g doses 1 h before the retention test, sig-

86

CCK-I-8-SE

CARs

CCK-1-8-NS

CCK-5-8

CCK- 6-8

CCK-2-L- SE

10"

6

\ 2

1

°

2 4HR

I

i

0 2 ~HR

0 2 ~HR

0 ½ ~HR

0 ½ ~HR

Fig. 4. Effects of graded doses of cholecystokinin octapeptides or their fragments, administered intracerebroventricularly, on the extinction of active avoidance behaviour of rats (4-day acquisition). × x control; O © 24 pmol; • • 80 pmol; zx ,', 240 pmol. S.EM. are between 0.2 and 0.7. n = 6-9 per group.

nificantly increased the avoidance latencies (P < 0.05 and P < 0.01). A 120 n m o l / k g dose of CCK8-SE or CCK-8-NS given i.p. significantly facilitated (P < 0.05) passive avoidance behaviour. On a molar basis, s.c. treatment appeared to be 3 times more effective than i.p. treatment.

s

300>-

t.) z uJ p/Ix

x

//\N <~

200

I

\\

Z~ \N. //\N

II\\

//\\

100"

ll\"

lIX\

N 0

x

Z~

/ / \\ //~.\ \\

z~ 10 10 8

11 I0 i

24

The i.c.v, administration of CCK-1-8-SE, CCK1-8-NS or CCK-5-8 in 24 pmol (P < 0.05) and 80 pmol (P < 0.01) doses facilitated the extinction of bench-jumping active avoidance behaviour 2 and 4 h later (fig. 4). However, 24-240 pmol doses of CCK-6-8 or CCK-2-4-SE caused no significant effect on the extinction of active avoidance behaviour. Preretention treatment with CCK-8-SE or CCK-8-NS in 8 and 24 pmol doses 1 h before the retention test significantly (P < 0.01) facilitated the passive avoidance behaviour of the rats in a dosedependent manner (fig. 5). 3. 6. A uoidance behauiour: i.c. administration

/ ¢ \ \

uJ

3.5. Auoidance behaviour." i.c.u, administration

PMOL

ICV.

Fig. 5. Effects of graded doses of CCK-8-SE I~t or CCK-8-NS [] injected intracerebroventricularly, on the retention of passive avoidance behaviour. [] control.

CCK-8-SE or CCK-8-NS 24 and 80 fmol injected into the ventral tegmental area, the dorsal raphe nucleus, the dentate gyrus of the hippocampus, the stria terminalis, the caudate nucleus, the dorsomedial h y p o t h a l a m i c nucleus, the supraoptic nucleus or the paraventricular nucleus, failed to induce any significant effect on the extinction of the active avoidance behaviour (with either the 3 day or 4 day acquisition design) or the retention of the passive avoidance behaviour of the rats (data not shown).

87

A

B NUCLEUS ACCUMBENS NUCLEUS ACCUMBENS CARs

10' s

300"

\\

6'

r

~"~

(7)

>. (J z hi

171

~"

O

24

80

FMOL

Fig. 6, (A) Effects of graded doses of CCK-8-SE (© (3 8 fmol; • • 24 fmol) or CCK-8-NS (r, zx 8 fmol; • • 24 fmol), injected into the nucleus accumbens, on the extinction of active avoidance behaviour (4 day acquisition). × - x control. (B) Effects of graded doses of CCK-8-SE [] or CCK-8-NS ~, injected into the nucleus accumbens, on the retention of passive avoidance behaviour. [] control. Typical localizations of the tip of the cannula are shown in the coronal brain section according to the atlas of Fifkovfi and Marsala (1962).

A

B CENTRAL AMYGDALOID

CENTRAL AMYGOAL010 NUCLEUS

NUCLEUS

CARs

10-

8-

300(7)

>U

z

Lfl

6

200. z hi

J

6

(8) (8) ('7)

~

~, HR

:E 100

0

8

99i 24

FMOL

Fig. 7. (A) Effects of graded doses of CCK-8-SE (C) O 24 fmol; • • 80 fmol) or CCK-g-NS (A zx 24 fmol; • • 80 fmol) injected into the central amygdaloid nucleus, on the extinction of the active avoidance behaviour (3 day acquisition). × × control. (B) Effects of graded doses of CCK-8-SE [] or CCK-8-NS [] injected into the central amygdaloid nucleus, on the retention of the passive avoidance behaviour. [] control. Typical localizations of the tip of the cannula are shown in the coronal brain section according to the atlas of Fifkov~t and Marsala (1962).

88 An 8 fmol dose of CCK-8-SE or CCK-8-NS, injected into the nucleus accumbens in the 4 h extinction session (P < 0.05) or a 24 fmol dose of these peptides in the 2 and 4 h extinction sessions (P < 0.01), significantly decreased the number of avoidances when the animals were trained in a 4 day acquisition design (fig. 6A). We failed to find any significant effect of 24-80 fmol doses of CCK8-SE or CCK-8-NS when acquisition training for 3 days was followed by extinction sessions on day 4 (data not shown). Preretention treatment 1 h before the retention test with 24 or 80 fmol doses of CCK-8-SE or CCK-8-NS, injected into the nucleus accumbens, dose dependently attenuated the passive avoidance retention of the animals (P < 0.01) (fig. 6B). Administration of an 80 fmol dose of CCK-8-SE or CCK-8-NS into the central amygdaloid nucleus delayed the extinction of the bench-jumping avoidance response 2 and 4 h later (P < 0.01) when the animals were trained in a 3 day acquisition (fig. 7A). A 24 fmol dose of these peptides caused only a slight tendency to delay the extinction. However, the administration of 24-80 fmol doses of CCK-8SE or CCK-8-NS into the central amygdaloid nucleus was absolutely ineffective on extinction when the animals were trained in a 4 day acquisition to make them more resistant to extinction (data not shown). As can be seen from fig. 7B, 8-24 fmol doses of CCK-8-SE or CCK-8-NS, injected into the central amygdaloid nucleus, dose dependently facilitated passive avoidance behaviour (P < 0.01).

4. Discussion

As found in previous studies, systemic and i.c.v. administration of CCK-8-SE or CCK-8-NS facilitated the extinction of active avoidance behaviour .(Fekete et al., 1981e, 1982; Cohen et al., 1982) and the retention of passive avoidance behaviour (Fekete et al., 1981c,d; Kfidfir et al., 1981). Using a wide dose range, Kfidfir et al. (1981) have demonstrated that CCK-8-SE and CCK-8-NS show a typical inverted U-shaped dose-response curve. Small amounts of these peptides facilitated retention while higher doses attenuated it, showing that

in this test situation their effect is at least partly similar to that of the neuroleptic substance haloperidol. This neuroleptic-like effect of CCK-8 was shown earlier in other behavioural tests by Zetler (1981) and Cohen et al. (1982). In the present experiments CCK-8 was found to induce a neuroleptic-like effect on active avoidance behaviour (De Wied et al., 1978; Arnt, 1982, 1983; Van Ree and De Wied, 1982a) and, following chlordiazepoxide pretreatment, on passive avoidance behaviour too. It seems that CCK-8 exhibits a neuroleptic-like effect, but we have to consider another action of CCK-8 (non neuroleptic-like), which is easily overcome by chlordiazepoxide pretreatment. With the present doses of CCK-8-SE or CCK8-NS 1 h after s.c. injection we failed to find any effect on the motor activity of the rats in the open-field test. It thus seems unlikely that the peptides reduced motor activity in the rats causing facilitated extinction of the active avoidance behaviour and retention of the passive avoidance behavior. A similar negative result was reported earlier for the motor activity of rats following peripheral or central administration of CCK-8 (Fekete et al., 1981e, 1982; Rojas-Ramirez et al., 1982). Thus it seems more likely that CCK-8 is able to enhance the behavioural arousal a n d / o r fear-motivation of the animals (non neurolepticlike effect), and furthermore to evoke a neuroleptic-like effect, causing facilitated extinction and retention. When this increased arousal is inhibited by chlordiazepoxide pretreatment, the neuroleptic-like effect of CCK-8-SE or CCK-8-NS is overcome. The anatomical substrate of this dual effect has also been shown by intracerebral microinjections. CCK-8 caused a neuroleptic-like effect on both active and passive avoidance behaviour when it was injected into the nucleus accumbens, whereas it showed an opposite (non neuroleptic-like) action after central amygdaloid nucleus microinjection. The dual effect (neuroleptic-like and non neuroleptic-like) of CCK-8 on fear-motivated behaviour might explain at least in part why relatively high amounts of peripherally injected peptides are needed to induce a behavioural effect. However, the injection of 12SI-CCK-8 into the ear vein of rabbits resulted in no detectable radioactivity in the cerebrospinal fluid (Passaro et al., 1982), fur-

89

thermore ~25I-CCK revealed no significant brain retention after single circulatory passage following arterial injection in the rat (Oldendorf, 1981), suggesting that the blood-brain barrier is impermeable to the peptide. However these studies do not preclude a direct brain effect mediated through regions of the brain which normally lack a bloodbrain barrier. Nevertheless, this impermeability might be an other reason for the high dose of CCK-8 used, though the smaller fragments of CCK-8 can pass the blood-brain barrier more easily. In this respect, it is interesting that the COOH-terminal tetrapeptide of CCK-8 (CCK-5-8) possesses the full information to induce behavioural effects on the extinction of active avoidance behaviour and the retention of passive avoidance behaviour (Fekete et al., 1981d; Telegdy et al., 1982). However, Cohen et al. (1982) showed that a 297 n m o l / k g dose of CCK-5-8 administered i.p. failed to induce any effect on avoidance behaviour. We have replicated their dose in our test design and the samedose of CCK-5-8 was ineffective in our hands also. However, in our experiments animals were treated 2 or 4 h before the extinction session, while Cohen et al. tested their animals 15 min after peptide injection. On the other hand, we have shown that s.c. treatment was 3-10 times more effective than i.p. treatment. It is possible that absorption, degradation, etc. are different with these two types of peripheral administration. The present study clearly indicates that, on i.c.v, administration, 300 times lower doses of CCK-8 were needed to induce the same efect as that due to peripheral injection, on both active and passive avoidance behaviour. This finding shows that these peptides interact with central nervous mechanisms rather than with peripheral ones. CCK-8-SE or CCK-8-NS, injected in the fmole dose range into the nucleus accumbens, dose dependently facilitated the extinction of active avoidance behaviour and attenuated passive avoidance behaviour, while these peptides caused an opposite effect when administered into the central amygdaloid nucleus. Injection into other brain regions containing appreciable amounts of CCK-8 did not show similar effects. On i.c. administration of CCK-8-SE or CCK-8-NS, 1000 times lower doses

caused the same effect as that obtained on i.c.v. injection. It has been reported that CCK-containing neurons and axons are located in the dopamine cell group of the ventral tegmental area (Vanderhaeghen et al., 1980; Beinfeld et al., 1981), and it has been shown that CCK and dopamine coexist in a subpopulation of these mesolimbic neurons (H0kfelt et al., 1980b; Studler et al., 1981). A decreased dopamine turnover has been found in the septal and amygdaloid areas (Fekete et al., 1980a, 1981b; Fuxe et al., 1980; Vasar et al., 1982) following CCK-8 treatment. Furthermore endogenous dopamine is able to stimulate the secretion of cholecystokinin from slices of rat caudate-putamen (Meyer and Krauss, 1983). It has also been shown that the dopaminergic system plays a critical role in conditioned avoidance behaviour (Jackson et al., 1977; De Wied et al., 1978; Van Ree et al., 1982; Van Ree and De Wied, 1981b, 1982; Kov/lcs et al., 1982). Chang et al. (1983) reported an increase in brain 125I-CCK receptor binding following chronic haloperidol treatment, intracisternal 6-hydroxydopamine or ventral tegmental lesion. It was further suggested that CCK may play a role in various disorders, including schizophrenia, perhaps by modulating the release of dopamine (H6kfelt et al., 1980a,b). It could be that CCK-8 exhibits neuroleptic-like properties, together with other neuropeptides ('t-type endorphins) (De Wied et al., 1978; De Wied, 1979; Van Ree and De Wied, 1981a,b). In this context it is interesting that Roberts et al. (1982) and Edwardson and McDermott (1982) showed that the CCK concentration in the limbic system revealed disease-related changes in schizophrenic patients. However, Moroji et al. (1982), Nair et al. (1982, 1983) demonstrated both the therapeutic efficacy of cholecystokinin and of caeruletide (a decapeptide chemically related to CCK-8) in neurolepticresistant schizophrenic subjects and an antipsychotic effect of CCK.

Acknowledgements This research was supported by a grant from the Hungarian Ministry of Health (No. 16/4-10/502). The skilful technical

9o assistance of Mrs. Maria Fekete and Miss Anna Nemes is gratefully acknowledged.

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