Cholecystokinin accelerates the rate of habituation to a novel environment

Pharmacology Biochemist~ & Behavior, Vol. 20, pp. 23-27, 1984. © Ankho International Inc. Printed in the U.S.A.

Cholecystokinin Accelerates the Rate of Habituation to a N o v e l Environment J. N . C R A W L E Y

1

N e u r o b i o l o g y P r o g r a m , C R & D , E. 1. du P o n t de N e m o u r s & C o m p a n y , Glenolden, P A 19036 Received 5 January

1983

CRAWLEY, J. N. Cholecystokinin accelerates the rate of habituation to a novel environment. PHARMACOL BIOCHEM BEHAV 20(1) 23-27, 1984.--Cholecystokinin (CCK~), an octapeptide present in high concentrations in both gut and brain, has been proposed as a putative satiety signal in a variety of species. Exploratory and social behaviors in particular are inhibited by exogenously administered CCK,. One hypothesis for the mechanism by which CCK reduces feeding and exploration is that CCK~ is reducing arousal and attention to environmental stimuli. This possibility was tested by analyzing the rate of habituation to the novelty of objects placed in an unfamiliar arena. A video-tracking computer-assisted animal behavior monitor measured four parameters of exploratory behaviors in rats pretreated with intraperitoneal CCK.. During the first 30 minutes in the novel environment, CCK~-treated animals showed a reduced latency to cessation of exploration as compared to saline controls. In repeated five minute sessions on consecutive days, CCK~ treatment accelerated the decline in exploration over daily sessions. Pentobarbital, a known sedative, induced low levels of exploration throughout the repeated daily sessions, These data suggest that CCK, is inducing a more rapid rate of habituation to the novelty of environmental stimuli. An accelerated rate of habituation might underlie some of the satiety-related behavioral effects of this peptide. Cholecystokinin

Habituation

Exploration

CHOLECYSTOKININ-sulfated-octapeptide (CCK)inhibits total food intake in fasted rats, mice, sheep, pigs, m o n k e y s , and humans [4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. C C K has been p r o p o s e d as a signal for satiety, initiating the cornplete behavioral s e q u e n c e of satiety in rats [1]. W e have shown that C C K induces a constellation of behavioral changes which are consistent with satiety as an underlying behavioral state [2, 3, 4, 5]. These include reduction in exploration, social interactions, and investigation o f novel objects. The most characteristic behavioral pattern is the a p p e a r a n c e of long pauses of behavioral inactivity. T h e s e pauses are not associated with sleep episodes, as E E G recordings are normal [5]. N o m o t o r dysfunctions a c c o m p a n y these pauses, as l o c o m o t o r activity and co-ordination are normal o n c e m o v e m e n t s are initiated [3,4]. Rather, the animals appear to be either (A) behaviorally sedated, or (B) more quickly habituated to the novel e n v i o r n m e n t a l stimuli which normally elicit exploratory behaviors. If the sedation hypothesis is correct, then C C K - t r e a t e d animals will show consistently lower exploratory tendencies throughout an experiment. If the habituation hypothesis is correct, then exploration should start at a normal level and decline o v e r time, following the same pattern as control animals, but with a m o r e rapid time course. The predicted c u r v e s for these two models are illustrated in Fig. 1. The present study was undertaken to address the alternat i r e hypotheses of general behavioral sedation versus an accelerated rate of habituation. T w o paradigms were em-

ployed. The time course to cessation o f exploration in a single 30-minute session in a novel e n v i r o n m e n t was cornpared in rats treated with saline or C C K 1, 2.5, or 5 p.g/kg. Also, the rate of decline of exploratory parameters was compared o v e r c o n s e c u t i v e daily 5-minute sessions in the novel e n v i o r n m e n t in rats treated with saline, C C K 2.5 tzg/kg, C C K 5 /xg/kg, or pentobarbital, a standard sedative. Exploratory parameters w e r e analyzed by a video-tracking computer-assisted animal b e h a v i o r monitor [6]. The results of these two studies support the habituation rather than the sedation hypothesis as the more likely explanation for the ability of C C K to reduce spontaneous behaviors. METHOD Male S p r a g u e - D a w l e y rats, 250-300 g, housed in a ternperature and humidity controlled e n v i o r n m e n t on a 6 a.m.-6 p.m. lighting schedule, with food and w a t e r constantly available, were divided into groups o f t e n rats each. CCKx-sulfate (Bachem, T o r r a n c e , CA) was intraperitoneally administered immediately (less than one minute) before testing. L a t e n c y to the first period of inactivity of at least five minutes duration was r e c o r d e d for e a c h rat pretreated with either saline, C C K 1, 2.5 or 5 /~g/kg IP. In the second series of experiments, saline, C C K 2.5 or C C K 5 txg/kg IP was administered immediately before testing on five c o n s e c u t i v e days. Behavioral testing o v e r a five minute session e m p l o y e d a video-tracking c o m p u t e r - a s s i s t e d animal b e h a v i o r monitor

~Present address: Chief, Unit on Behavioral Neuropharmacology Clinical Neuroscience Branch. National Institute of Mental Health, Bethesda, MD 20205.

23

24

CRAWLEY

(SRI International, Menlo Park, CA). As previously described [6], the color channels in the videocamera follow the colored tape placed on the back of the animal. The location of the animal is converted to an X-Y co-ordinate which is sampled ten times per second. A V A X c o m p u t e r p r o g r a m then analyses the sequential location of the animal to yield parameters including number, duration, and variability within pauses, proximity to the corners and edges of the open field arena, number, duration, distance, and linearity of movements, and frequency, duration, and proximity of approaches to a labeled object. In the present experiment, each test animal was placed in a 60x60 cm arena lined with blotter paper, containing a color-labeled 7 x 7 × 11 cm wire mesh box. In the first experiment, each animal was allowed to remain in the arena for 30 minutes. Latency to the first period of inactivity of at least 5 minutes duration was recorded for each rat pretreated with either saline, CCK 1, 2.5 or 5/xg/kg IP immediately before testing. In the second experiment, each animal was allowed to remain in the arena for five minutes. Group 1 was administered saline immediately before testing on five consecutive days. Group 2 was administered CCK 2.5 /~g/kg IP immediately before testing on five consecutive days. Group 3 was administered CCK 5.0/~g/kg IP immediately before testing on five consecutive days. Group 4 was administered sodium pentobarbital (Nembutal, Abbott Laboratories, Chicago, IL), 25 mg/kg IP; 15 minutes before testing on four consecutive days. To test reversal of habituation, on day five, the wire mesh box was replaced by an open plastic cup, with saline and CCK drug treatment identical to days 1, 2, 3, and 4. Statistical analysis was performed by Analysis of Variance followed by a Newman-Keuls a posteriori test for significant differences of each treatment group on each test day. RESULTS

Latency to the first pause of at least five minutes duration within a single 30-minute session was reduced in CCKtreated rats (Fig. 2). One-tailed A N O V A for the four treatment groups yielded, F(3,36)=7.81, p<0.01. Newman-Keuls comparison of group means yielded p <0.01 for CCK 2.5 and CCK 5. CCK 2.5 and 5.0/xg/kg significantly reduced approaches to a novel object, reduced number of movements, reduced total distance traversed, and increased total pause time as compared to saline controls on the first and second day of testing (Figs. 3-6). One-way A N O V A of number of approaches, Day 1, yielded, F(2,27)=3.69, p <0.05, Newman-Keuls p < 0 . 0 5 for CCK 2.5 and CCK 5; Day 2, F(2,27)=5.80, p<0.01, Newman-Keuls p<0.05 for CCK 2.5 and CCK 5; Day 3, F(2,27)=2.46, non-significant; Day 4, F(2,27)=0.13, non-significant. Duration of pauses A N O V A for Day i, F(2,27)=4.03,p<0.05, N e w m a n - K e u l s p < 0 . 0 5 f o r CCK 2.5 and CCK 5; Day 2, F(2,27)=5.11, p<0.05, Newman-Keuls p<0.05 for CCK 5; Day 3, F(2,27)-0.28, non-signficant; Day 4, F(2,27)=0.04, nonsignificant. Total number of movements, Day 1, F(2,27)=5.23, p<0.05, Newman-Keuls p<0.05 for CCK 2.5 and CCK 5; Day 2, F(2,27)=5.88, p<0.01, Newman-Keuls p<0.05 for CCK 2.5 and CCK 5; Day 3, F(2,27)=0.03, non-signficiant; Day 4, F(2,27)=0.01 non-significant. Total distance traversed Day 1, F(2,27)=6.17, p<0.01, Newman-Keuls p<0.05 for CCK 2.5 and CCK 5; Day 2, F(2,27)-5.71, p<0.01, NewmanKeuls p<0.05 for CCK 5; Day 3, F(2,27)=0.09, non-

Hypothesis: ..hi,.....

Sedation

! s.....

s.....

T. . . . . .

T. . . .

FIG. 1. Alternate hypotheses for the role of cholecystokinin (CCK) in reducing spontaneous exploratory behaviors. A gradual reduction in exploration, at a rate exceeding control values, would support the explanation of an accelerated rate of habituation. A constant low level of exploration would support the explanation of a generalized state of behavioral sedation. Approaches to Novel Object

T ~

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FIG. 2. Latency to cessation of exploration was defined as the number of minutes until the rat paused for at least five minutes. Rats were pretreated with saline, CCK I/~g/kg, CCK 2.5 p.g/kg, or CCK 5 p~g/kgIP; immediately before placement in the arena environment, as in Figs. 3-6, but for a single 30 minute session.

significant; Day 4, F(2,27)-0.01, non-significant. Separate statistical analysis of exploratory parameters after pentobarbital treatment across the four test days yielded no significant effect of repeated daily testing on response to pentobarbital. Linearity within moves and grooming movements within pauses (Table 1) yielded no statistically significant differences between saline, CCK 2.5, and CCK 5 within any testing day. Replacement of the wire mesh object with a plastic cup on Day 5 increased exploratory parameters in all three treatment groups (Fig. 7). One-tailed paired t-test statistics yielded a decrease in total pause time on Day 5 as compared

CHOLECYSTOKININ

ACCELERATES HABITUATION

Pauses

300

25 Moves

60

--

• Saline ACCK5

40

200

~_

: CCK 2.5 ACCK 5 100

Day

Day

FIG. 3. Number of approaches to a novel object was defined as the total number of physical contacts, of at least 3 second duration, with a wire mesh box placed in the center of a 60×60 cm open field. Rats were pretreated with intraperitoneal saline, CCK 2.5/zg/kg, or CCK 5.0/zg/kg, immediately before, or sodium pentobarbital, 25 mg/kg, 15 minutes before the five-minute test session in the novel arena environment, on four consecutive days. N - 1 0 for each treatment group, *p <0.05.

FIG. 4. Total time spent in pauses was defined as the cumulative number of seconds in which the rat did not move outside an area of 5 cm radius for a minimum of 3 seconds. Rats were pretreated with saline, CCK 2.5/xg/kg, CCK 5/xg/kg, or pentobarbital 25 mg/kg, as in Fig. 2. N 10 for each treatment group, *p<0.05.

Habituation

Reversal

•

Sad,oe CCK25

ance

275

Traversed

1500

•

• Sa~lne :CCK25

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20

250

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Day 4

Day 5 New Objects

Day 4

Day 5 New Oblects

Day 4

Day 5 New Oblects

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I 2 Day

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Day 4

Day 5 New Objects

FIG. 5. Total number of moves was defined as the number of times the rat initiated a movement of greater than 5 cm distance. Rats were pretreated with saline, CCK 2.5/xg/kg, CCK 5/zg/kg, or pentobarbital 25 mg/kg IP, as in Fig. 2. N 10 for each treatment group, *p<0.05.

FIG. 6. Total distance traversed was defined as total cumulative cm moved in the five-minute test session. Rats were pretreated with saline, CCK 2.5/xg/kg, CCK 5/zg/kg, or pentobarbital 25 mg/kg, IP, as in Fig. 2. N = I 0 for each treatment group, *p<0.05.

to Day 4 in saline c o n t r o l s , t(9)=2.58, p < 0 . 0 5 ; in the C C K 2.5 group, t(9)=3.64, p < 0 . 0 5 ; and in the C C K 5 group, t (9) = 3.95, p <0.05. Total n u m b e r o f m o v e m e n t s i n c r e a s e d in all g r o u p s , saline, t(9)=4.13, p < 0 . 0 5 ; C C K 2.5, t(9)=2.48, p < 0 . 0 5 ; C C K 5 t(9)=3.52, p < 0 . 0 5 . Total d i s t a n c e t r a v e r s e d i n c r e a s e d in all g r o u p s , saline t(9)=4.81, p < 0 . 0 5 ; C C K 2.5, t(9)=2.33, p < 0 . 0 5 , C C K 5.0, t(9)=3.21, p < 0 . 0 5 . N u m b e r o f a p p r o a c h e s to the novel object i n c r e a s e d in all g r o u p s , saline, t(9)=3.88, p < 0 . 0 5 ; C C K 2.5, t(9)=2.95, p < 0 . 0 5 ; C C K 5, t(9)=3.26, p < 0 . 0 5 ,

DISCUSSION The o b s e r v e d gradual decline in e x p l o r a t o r y p a r a m e t e r s o v e r r e p e a t e d daily test s e s s i o n s in saline c o n t r o l s follows a normal rate o f h a b i t u a t i o n to a novel e n v i r o n m e n t . The data o f Figs. 2, 3, 4, 5, and 6 d e m o n s t r a t e that C C K shifts the time c o u r s e o f declining e x p l o r a t i o n to the left o f saline control values. E x p l o r a t i o n c u r v e s for p e n t o b a r b i t a l began at low values on the first day o f testing and r e m a i n e d low throughout the four c o n s e c u t i v e t e s t days. T r e n d s t o w a r d i n c r e a s e d

26

CRAWLEY

TABLE 1 NORMAL PARAMETERS WITHIN MOVES AND PAUSES

Linearity Within Moves

Movements Within Pauses

Day 1 Saline CCK 2.5 CCK 5

0.83 ± 0.01 0.82 ± 0.01 0.85 ± 0.02

1.23 + 0.2 1.05 ± 0.1 1.12 _+ 0. I

Day 2 Saline CCK 2.5 CCK 5

0.81 _+ 0.02 0.81 ± 0.04 0.80 _+ 0.2

1.23 + 0.1 1.13 ± 0.1 1.16 ÷ 0.1

Day 3 Saline CCK 2.5 CCK 5

0.76 ± 0.05 0.78 ± 0.04 0.83 ± 0.06

0.98 ± 0.1 0.98 ± 0.1 0.88 _+ 0.2

Day 4 Saline CCK 2.5

0.82 ± 0.05 0.71 ± 0.07

0.81 ± 0.2 0.84 ± 0.1

CCK 5

0.70 ± 0.12

1.03 + 0.1

Day 5 Saline CCK 2.5 CCK 5

0.73 + 0.03 0.76 _+ 0.06 0.79 _+ 0.02

1.17 + 0.1 0.94 _+ 0.1 1.21 ± 0.1

Linearity within moves was defined as the deviation from a straight line between two consecutive locations of the rat. This parameter indicates degree of locomotor co-ordination. Treatments were as in Fig. 2. N = 10 for each group, no significant differences by one-way ANOVA on any test day. Movements within pauses was defined as movements within the 5 cm radius. A low score on this parameter indicates extreme tonic immobility. A high score on this parameter indicates excessive grooming. Treatments were as in Fig. 2. N - 10 for each group, no significant differences by one-way ANOVA on any test day.

e x p l o r a t i o n o v e r time w i t h this s t a n d a r d s e d a t i v e are contrary to the C C K effect, and m a y reflect d e v e l o p i n g t o l e r a n c e to r e p e a t e d p e n t o b a r b i t a l t r e a t m e n t s . T h e s e results suggest that C C K is a c c e l e r a t i n g the rate o f h a b i t u a t i o n to the n o v e l t y o f the a r e n a e n v i r o n m e n t . The data o f Day 5, s h o w i n g that r e p l a c e m e n t o f a familiar o b j e c t by a n e w object i n c r e a s e s e x p l o r a t i o n in b o t h saline and C C K - t r e a t e d rats, indicates that C C K - t r e a t e d animals c a n r e s p o n d to a n o v e l t y challenge as well as s a l i n e - t r e a t e d animals. T h e s e results s u p p o r t the first h y p o t h e s i s illustrated in Fig. 1. C C K d o e s not a p p e a r to induce a s u s t a i n e d behavioral sedation. R a t h e r , the general e x p l o r a t o r y t e n d e n c i e s o f rats and mice a p p e a r to be r e d u c e d , [1, 2, 4] allowing a m o r e rapid rate o f habituation to a n o v e l e n v i r o n m e n t , T h e s e data do not a d d r e s s the issue o f p o s s i b l e a v e r s i v e internal s e n s a t i o n s i n d u c e d by i n t r a p e r i t o n e a l injections o f

30

Latency to Cessation of Exploration

/

f

0

(SaP,,ve/

25

CCKt~g k~

5

FIG. 7. Habituation reversal was defined as an increase in the four exploratory parameters when the novel object was changed from a wire mesh box to a plastic cup on the fifth consecutive day of testing for 5 minutes in the arena environment. All parameters were significantly changed in all treatment groups on Day 5 at the p<0.05 level by one-tailed paired t-test statistical analysis.

C C K . The d o s e s used in this study w e r e within the range w h i c h inhibits total food c o n s u m p t i o n in fasted rats. Therefore, an a c c e l e r a t e d rate o f habituation might underlie s o m e o f the satiety-related behavioral effects o f C C K . Alternatively, u n p l e a s a n t gastrointestinal s e n s a t i o n s could r e d u c e an a n i m a l ' s e x p l o r a t o r y t e n d e n c i e s in a novel e n v i r o n m e n t . E x p e r i m e n t s are in p r o g r e s s to c o m p a r e the effects o f k n o w n a v e r s i v e stimuli to C C K on the e x p l o r a t o r y p a r a m e t e r s emp l o y e d in o u r s y s t e m . A l t h o u g h the distinction b e t w e e n feelings o f satiation and feelings o f a v e r s i o n r e m a i n s c o n t r o v e r sial with r e s p e c t to C C K , the p r e s e n t study s e r v e s to eliminate the possibility that C C K is inhibiting s p o n t a n e o u s appetitive b e h a v i o r s t h r o u g h a non-specific behavioral sedation. It is i n t e r e s t i n g to speculate on the c a u s e s and c o n s e q u e n c e s o f a n a c c e l e r a t e d rate o f habituation to n o v e l t y a f t e r t r e a t m e n t with a p e p t i d e such as C C K . One m e c h a n i s m involved may be a r e d u c e d level o f arousal, or o f attention to e n v i o r n m e n t a l stimuli. Several h y p o p h y s e a l p e p t i d e s , such as A C T H , v a s o p r e s s i n , o x y t o c i n , and c~-melanocyte stimulating h o r m o n e , have b e e n implicated in learning and m e m o r y p r o c e s s e s . C C K might similarly induce c h a n g e s in rate o f learning or e x t i n c t i o n , b a s e d on its ability to alter s o m e s u b s t r a t e o f the habituation p r o c e s s . In c o n c l u s i o n the p r e s e n t study d e m o n s t r a t e s that C C K i n d u c e s a gradual d e c r e a s e in e x p l o r a t o r y b e h a v i o r w h o s e rate o f decline is g r e a t e r t h a n the gradual decline seen in s a l i n e - t r e a t e d c o n t r o l s . This finding s u p p o r t s a role for C C K in a c c e l e r a t e d habituation to n o v e l t y r a t h e r t h a n a m o r e global state o f behavioral sedation.

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CHOLECYSTOKININ ACCELERATES HABITUATION

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