Neuropeptide modulation of social and exploratory behaviors in laboratory rodents

Peptides. Vol. 2, Suppl. 1, pp. 123-129, 1981. Printed in the U.S.A.

Neuropeptide Modulation of Social and Exploratory Behaviors in Laboratory Rodents J. N . C R A W L E Y , 1 S. E. H A Y S , T. L . O ' D O N O H U E , AND F. K. GOODWlN

S. M. P A U L

National Institute o f Mental Health, Bethesda, M D 20205

CRAWLEY, J. N., S. E. HAYS, T. L. O'DONOHUE, S. M. PAUL AND F. K. GOODWIN. Neuropeptide modulation of social and exploratory behaviors in laboratory rodents. PEPTIDES 2: Suppl. 1, 123-129, 1981.--Neuropeptide influences on exploratory and social behaviors were investigated, using a video-monitored computer-assisted automated animal behavior analysis system. Cholecystokinin decreased exploratory tendencies in the dose range 0.1-5.0 /zg/kg IP and 0.5-5.0 /zg/IVT, indicating a peripheral mechanism in the CCK reduction of spontaneous behaviors. Neither arginine vasopressin nor alpha-melanocyte stimulating hormone changed parameters of exploratory and social behaviors, strengthening the possibility that their roles in increasing acquisition and retention of operant tasks are specific to neural mechanisms involved in memory and learning. Analysis of spontaneous exploratory and social behavior patterns appears to be a sensitive and effective tool for detecting changes in arousal and attention to environmental stimuli which may underlie more specific behavioral effects of brain neuropeptides. Cholecystokinin

Vasopressin

c~-Melanocyte stimulating hormone

THE past decade has witnessed an explosion in the number of brain peptides which may function as neurotransmitters [23]. Many of these peptides were well known as hormones in other body regions such as the gastrointestinal and urogenital tracts [19,27]. Discovery of a central nervous system location for over two dozen peptides raises the intriguing question, what behavioral functions might be mediated by each of these peptides in the mammalian brain? Behavioral roles are proposed for several classes of neuropeptides: pain sensitivity for the endorphins, enkephalins, and substance P [23]; learning and memory for vasopressin, oxytocin, adrenocorticotropic hormone and alpha-melanocyte stimulating hormone [2, 6, 10, 11, 18, 26, 29]; regulation of food intake for cholecystokinin, caerulein and bombesin [1, 3, 5, 9, 15, 22, 24, 25]; grooming and stereotyped scratching by ACTH, melatonin, alpha-melanocyte stimulating hormone, and bombesin [4, 13, 30]. The history of research into the behavioral functions of central monoamine neurotransmitters teaches us caution in the interpretation of apparently specific functions of neurotransmitters. Transmitters may change basic functions which secondarily influence more discrete behavior patterns. Norepinephrine and serotonin play major roles in mediating an animal's arousal, attention, and sensitivity to environmental stimuli [14,28]. An animal's level of arousal and attention to environmental stimuli underlies other behaviors. Alterations in a fundamental state could conceivably lead to numerous,

Exploratory behavior

Social behavior

more specific changes in behavior. Consequently, the study of the effects of a peptide on arousal and exploration is the first basic step in the evaluation of its behavioral functions. We are studying the influence of neuropeptides on some of the most common natural behaviors of laboratory rodents, exploratory activity and the initiation of social interactions. These studies utilize a complex computer-assisted videotracking animal behavior monitor which calculates eighteen parameters of exploratory and social behavior in mice and rats. The system has the capability of detecting sedation, hyperactivity, stereotyped motor activity, attention to test objects, approach, avoidance, distractability, habituation, and interactive tendencies toward another animal. In this communication, we report that cholecystokinin (CCK) inhibits exploration of a novel environment and initiation of social encounters, while arginine vasopressin (AVP) and alpha-melanocyte stimulating hormone (c~-MSH) do not change these spontaneous behaviors at doses shown to specifically increase memory and learning. METHOD Neuropeptides were administered either intraperitoneaily via 27 gauge needle or intraventricularly via 30 gauge stainless steel tubing inserted into the lateral ventricles through a previously implanted guide cannula, to conscious male Sprague Dawley rats (250-300 g) or male Swiss Webster

1Address reprint requests to J. N. Crawley, Clinical Psychobiology Branch, NIMH, Bldg. 10, Room 4S239, 9000 Rockville Pike, Bethesda, MD 20205.

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mice (20-25 g). CCK8 (mice: 0.1-5.0/xg/kg IP in 0.9% saline; rats: 0.5, 1.0, 5.0/zg/rat IP or IVT in 0.9% saline, gift of Dr. M. Ondetti, Squibb Laboratories, Princeton, N J), gastrin-17 unsulfated (100 /xg/kg IP in 0.9% saline, Calbiochem, La Jolla, CA), or 0.9% saline was injected immediately before testing, a - M S H (25 ng, 50 ng, 1.0 /xg IVT in 0.9% saline, Peninsula Laboratories, Belmont, CA) was injected 20 minutes before testing AVP (5 ng, 10 ng IVT; 5/zg SC in acidified 0.9% saline, United States Biochemical Corp, Cleveland, OH) was injected 60 minutes before the first test session. Testing was repeated one day and 4 days after the single AVP dose in this experimental group. Exploratory and social behaviors were monitored in five minute test sessions on a video-tracking animal behavior monitor and the data analyzed by the OMNI computer program which calculates the location of each animal in the test arena as a traveling X-Y coordinate. This system is a prototype, designed by Stanford Research Institute, and described in detail in [7]. The arena was enriched with objects normally attractive to rodents: (1) a cardboard loop; (2) a wire-mesh cage; and (3) a smear of cheese. A female conspecific moved freely in the arena with the male test animal in the AVP and a - M S H experiments, and was enclosed in a wire-mesh cage fastened in the center of the arena surface in the CCK experiments. Human observation of the video-monitor during the five minute test session allowed for recording of specific behavioral events not differentiated by the system, e.g. grooming. Group comparisons of peptide effects were made initially by analysis of variance. Significant differences were further analyzed by two-tailed group t-test statistics. Significance was defined at the level of p<0.01 for these experiments, as their multivariate nature requires stringency in eliminating the bias toward false positives.

RESULTS

CCK8 reduced exploratory and social behavior in mice and rats. Figure 1 illustrates the decrease in approaches toward a female mouse, investigative approaches to novel objects, and number of movements, with increases seen in length of pauses and time spent in the corners of the arena by male mice after intraperitoneal CCK8 in the range 0.1-5.0 /zg/kg. Figure 2 illustrates comparable decreases in approaches toward a female, investigation of objects, number of moves, and comparable increases in pause length and time spent in corners for male rats after intraperitoneal CCK8 at 5.0 /xg/kg and after intraventricular CCK8 at 1.0 and 5.0 /zg/rat. Distance per move was unchanged in all cases. The gait and posture of the CCK8-treated animals was normal throughout. Most noticeable was the propensity toward frequent, long pauses, in which the animal sat quietly, without sniffing or intention movements. These quiet pauses alternated with periods of normal exploratory activity. Acute treatment with arginine vasopressin did not change any of the parameters of exploratory and social behavior. Intraventricular AVP in doses of 5 ng and 10 ng/rat yielded values for movement, pauses, investigation, and encounters with the female which were not significantly different from those of intraventricular vehicle controls (data not shown). AVP 5 /xg/rat IP also did not change exploratory or social behavioral parameters as compared to saline controls either one hour, one day, or four days after treatment (Fig. 3). In the third test session, control animals showed significant re-

ductions in encounters with the female, investigative approaches, % time in corners, number of moves, and distance moved, and increases in pause duration (p<0.05 on each parameter, Day 4 compared to Day 1 saline group). These changes in control values probably reflect habituation to the arena and test objects, documented previously for mice in this behavior monitoring system [7]. The AVP-treated rats generally showed the same pattern of habituation on the third as compared to the first test session, with no significant differences from saline controls on Day 4 on any measures. Intraventricular treatment with a - M S H in the dose range 25-50 ng/rat, which has previously been shown to increase acquisition of a complex maze task [26], did not change the values for most parameters of exploratory and social behaviors (Table 1). Intraventricular treatment of rats with a - M S H at 1 ~g increased the total time spent grooming in the five minute test session (Table 1), in agreement with previous reports [4, 13, 30]. However, most of the other parameters of social and exploratory behaviors were not significantly changed at the p<0.01 level by 1/~g a-MSH. Normal exploration was interrupted for occasional grooming bouts of variable duration (2-30 seconds) in these a - M S H treated rats. The grooming bouts consisted of the rat sitting still in a corner of the arena, licking its body and scratching its head regions with both forepaws. The bouts of time spent grooming in the corners significantly increased the duration of pauses and the time spent in the corners and periphery of the arena. Grooming bouts appeared to alternate with bouts of exploratory investigation and initiations of social encounters. DISCUSSION

CCK dramatically reduced measures of exploratory and social behavior in mice and rats. Doses as low as 0.1/~g/kg IP in mice and 1.0/~g IP or IVT in rats abolished up to 8(F~ of spontaneous investigative approaches to novel environmental objects. These doses are well below the reported tranquilizing dose of 150/zg/kg [31]. The reduction in exploration in the present study occurred at CCK doses in mice which were the lowest doses that inhibit feeding behavior in fasted rats [1,22]. The CCK reduction in approach behavior to environmental objects demonstrated in this report may be partially responsible for the reduction in feeding previously reported [1, 3, 9, 22, 24, 25]. Central versus peripheral sites of action for neuropeptides are often discriminated by the dose required to obtain the same behavioral effect when the peptide is administered centrally versus peripherally. Figure 2 illustrates the similarity in CCK dose required intraventricularly and intraperitoneally to reduce exploratory behaviors. These results may indicate a lack of central specificity for CCK in mediating these behaviors. Controversy exists on the possibility that CCK may induce nausea or other unpleasant internal side effects [12,16]. In a recent report, the CCK-inhibition of feeding was blocked by bilateral lesioning of the subdiaphragmatic vagus nerve, which transmits sensory information from the gastrointestinal tract to the central nervous system [21]. The CCK effect of reducing feeding and exploration could conceivably be related to some aversive peripheral cues. Vasopressin has long been known as a pituitary hormone, with antidiuretic and pressor actions. Its localization in hypothalamic regions and its effect of facilitating retention of conditioned avoidance responses in rats and improved mem-

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FIG. 2. Cholecystokinin or saline was administered either intraventricularly or intraperitoneally to male rats immediately before testing. C C K s decreased interactions with a female, investigative approaches, and n u m b e r of moves. CCK~ increased duration o f p a u s e s and a m o u n t of time spent in the corners. CCK8 did not affect distance traversed. Intraventricular and intraperitoneal routes of administering 1.0 and 5.0 p~g C C K produced similar magnitudes of behavioral c h a n g e s , indicating lack of central specificity. Data are e x p r e s s e d as mean_+S.E.M. N = 7 for each group. *p<0.01, **p<0.001.

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TABLE 1 ALPHA-MELANOCYTE STIMULATING HORMONE (a-MSH) OR SALINE WAS ADMINISTERED INTRAVENTRICULARLY VIA PREVIOUSLY IMPLANTED CANNULA TO AWAKE RATS Intraventricular a-MSH Saline Number of moves Distance per move (cm) Duration per move (sec) Linearity per move Number of pauses Duration per pause (sec) Movement within pause Peripheral distance (cm) % Time in corners Number of encounters with female Time per encounter (sec) Centroid linearity Number of investigative approaches (loop + cheese + wire cage) Total groomingtime(sec)

49.0 34.9 3.59 0.74 49.0 2,6 1.3 6,91 8,91 18.2 0.99 0.68

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N = 7 for each group. Values are expressed as mean±S.E.M. No significant change in any exploratory parameters was seen with 25 ng or 50 ng. The high dose 1/.tg a-MSH, was effective in significantly increasing total amount of time spent grooming in the five minute test session (tp<0.01). The grooming bouts alternated with normal exploration resulting in significantly more total time in corners and near the periphery of the arena, and longer average duration of pauses (*p<0.01), as compared to saline controls by Student's t-test analysis.

ory in h u m a n s h a s b e e n well d o c u m e n t e d [11,29]. T h e A V P effect o n o p e r a n t t a s k s a p p e a r s n o t to c h a n g e g e n e r a l i z e d m o t o r activity as m e a s u r e d by total l o c o m o t i o n [29] o r by n u m b e r o f s q u a r e s c r o s s e d in a n o p e n field [20] a l t h o u g h s o m e i n c r e a s e s in foraging a n d g r o o m i n g activity h a v e b e e n r e p o r t e d at high i n t r a v e n t r i c u l a r d o s e s in mice [8]. O u r results c o n f i r m a n d e x t e n d the lack o f a c u t e A V P effects o n l o c o m o t o r a n d e x p l o r a t o r y b e h a v i o r s . N o c h a n g e was f o u n d e i t h e r i m m e d i a t e l y a f t e r a single A V P d o s e or up to four d a y s a f t e r w a r d s . This d o s e a n d t i m e s c h e d u l e r o u t i n e l y inhibits e x t i n c t i o n o f a o n e - w a y a c t i v e a v o i d a n c e task [29]. T h e ability of A V P to e n h a n c e m e m o r y d o e s not, t h e r e f o r e , a p p e a r to r e s u l t f r o m u n d e r l y i n g c h a n g e s in t h e a n i m a l ' s a t t e n t i o n to or i n t e r a c t i o n w i t h e n v i r o n m e n t a l stimuli e i t h e r d u r i n g or after their presentation. a - M S H h a s a v a r i e t y of a c t i o n s , including i n c r e a s i n g the a c q u i s i t i o n o f c o m p l e x m a z e t a s k s [2,26], i n d u c i n g e x c e s s i v e g r o o m i n g a n d s t r e t c h - y a w n b e h a v i o r s [4, 13, 17, 30], a n d p r o d u c i n g a high d e g r e e o f vigilance in h u m a n s [2]. T h e prese n t s t u d y i n v e s t i g a t e d the possibility t h a t t h e s e a - M S H effects m i g h t result f r o m a h e i g h t e n e d a t t e n t i o n to e x p l o r a t i o n o f e n v i r o n m e n t a l details at t h e l o w e r d o s e s w h i c h i n c r e a s e learning, a n d f r o m m o r e a t t e n t i o n to bodily stimuli at t h e h i g h e r w h i c h e n h a n c e g r o o m i n g d o s e s . T h e results o f T a b l e 1 i n d i c a t e t h a t low d o s e s o f a - M S H n e i t h e r i n c r e a s e n o r dec r e a s e e x p l o r a t i o n o f e n v i r o n m e n t a l stimuli. I n c r e a s e d g r o o m i n g at h i g h e r d o s e s a p p e a r s to b e a s e p a r a t e b e h a v i o r , w h i c h o c c u r s in b r i e f b o u t s , w i t h o u t i n t e r f e r i n g with n o r m a l p a t t e r n s o f e x p l o r a t o r y b e h a v i o r . A s with A V P , the a - M S H effects o n l e a r n i n g do not a p p e a r to result f r o m u n d e r l y i n g c h a n g e s in a r o u s a l or a t t e n t i o n to e n v i r o n m e n t a l stimuli. Rul-

ing o u t this possibility allows us to c o n c e n t r a t e o n m o r e specific m e c h a n i s m s o f a c t i o n o f t h e s e n e u r o p e p t i d e s in the neural processes of learning and memory. E x p l o r a t i o n o f a n o v e l e n v i r o n m e n t is o n e o f the m o s t c o m m o n , s p o n t a n e o u s a n d n a t u r a l activities o f mice a n d rats. O u r s y s t e m p r o v i d e s detailed a n a l y s e s o f a variety of computer-analyzed parameters from an automated tracking s y s t e m . T a b l e 1 illustrates s o m e o f t h e s e p a r a m e t e r s , including f r e q u e n c y a n d l e n g t h o f p a u s e s , f r e q u e n c y , d i s t a n c e a n d s p e e d o f m o v e m e n t , linearity or irregularity o f m o v e m e n t s , f r e q u e n c y a n d d u r a t i o n o f a p p r o a c h e s to n o v e l o b j e c t s , a n d a p p r o a c h e s a n d i n t e r a c t i o n s w i t h o t h e r animals. F r o m t h e s e t y p e s o f d a t a we c a n d e t e c t u n u s u a l b e h a v i o r a l states s u c h as h y p e r a c t i v i t y or s e d a t i o n , m o t o r d y s f u n c t i o n s o r u n c o o r d i n a t i o n , u n u s u a l s t e r e o t y p e d m o v e m e n t s , lack o f i n t e r e s t in n o v e l objects, slower or faster h a b i t u a t i o n to novelty, greater or lesser distractability of a t t e n t i o n from objects o f exploration. This s y s t e m a p p e a r s to be well suited for i n v e s t i g a t i o n s o f basic b e h a v i o r a l c h a n g e s p r o d u c e d by c e n t r a l a n d systemic administration of neuropeptides. In c o n c l u s i o n , o u r analysis o f basic s p o n t a n e o u s b e h a v iors in mice a n d rats i n d i c a t e s t h a t c h o l e c y s t o k i n i n p r o f o u n d l y r e d u c e s e x p l o r a t i o n a n d social i n t e r a c t i o n s . E i t h e r i n t r a v e n t r i c u l a r o r i n t r a p e r i t o n e a l r o u t e s o f CCKx a d m i n i s t r a t i o n w a s effective, suggesting s o m e p e r i p h e r a l c o n t r i b u t i o n . N e i t h e r A V P n o r c~-MSH significantly influe n c e d t h e n o r m a l p a t t e r n s o f e x p l o r a t o r y a n d social b e h a v iors in rats. T h e s e findings lend s u p p o r t to a specific role for v a s o p r e s s i n a n d m e l a n o c y t e - s t i m u l a t i n g h o r m o n e in m e m o r y a n d learning.

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