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Stimulus control of behavioral inhibition in the preweanling rat
Physiology & Behavior, Vol. 55, No. 4, pp, 717-721, 1994 Copyright © 1994 ElsevierScienceLtd Printed in the USA. All fights reserved 0031-9384/94 $6.00 + .00
Pergamon 0031-9384(93)E0029-P
Stimulus Control of Behavioral Inhibition in the Preweanling Rat LOREY
K. T A K A H A S H I 1
Department of Psychiatry, University of Wisconsin Medical School, and William S. Middleton Memorial Veterans Hospital, Madison, WI 53792
R e c e i v e d 30 A u g u s t 1993 TAKAHASHI, L. K. Stimuluscontrolof behavioral inhibitionin the preweanling rat. PHYSIOL BEHAV 55(4) 717-721, 1 9 9 4 . Previous studies demonstrate that 14-day-old rats reduce their emission of ultrasonic vocalizations and freeze when exposed to an unfamiliar adult male rat. This study sought to identify the stimulus characteristics of conspecific males that potentiate the display of behavioral inhibition. In Experiment 1, day 14 rats were isolated from the nest and exposed to either an unfamiliar prepubescent male rat or an unfamiliar adult male rat. Pups exposed to the unfamiliar adult male rat exhibited significantly elevated levels of freezing and reduced their emission of ultrasounds. In Experiment 2, pups were exposed to either a familiar or an unfamiliar adult male rat. Although several pups exposed to the familiar adult male rat exhibited freezing, pups tested with the unfamiliar adult rat showed a reliably higher duration of freezing and made fewer vocalizations. Results suggest that neither the unfamiliar factor nor cues associated with male adulthood are sufficient to account for the occurrence of behavioral inhibition when presented separately. However, the combination of unfamiliarity and adult male stimuli are highly potent stimulus features that elicit behavioral inhibition in preweanling rats. Preweanling rats Social isolation
Behavioral inhibition Conspecific odors
Ultrasonic vocalization
Freezing
Development
Conspecific threat
Although the adult Norway rat readily exhibits behavioral inhibition (4,5,11,13,20), neonatal altricial rats do not display behavioral inhibition until the end of the second postnatal week. In a series of studies, this laboratory has demonstrated that socially isolated 7- and 14-day-old rats emit a high number of ultrasonic vocalizations (44,45). Emission of ultrasounds by rat pups separated from the litter serves presumably to elicit and direct maternal responses (3,33,42). However, when day 14 rat pups are tested under social isolation conditions and exposed simultaneously to stimulus cues of an unfamiliar adult male rat, there is a pronounced cessation of ultrasounds accompanied by immobility posturing or freezing (45). In contrast, socially isolated day 7 rats continue to emit ultrasounds and fail to engage in freezing. It is important to emphasize that day 14 rats do not exhibit behavioral inhibition when exposed to the mother, attesting to the stimulus specificity nature of the response (44,45). This propensity to exhibit behavioral inhibition--cessation of vocalizations accompanied by f r e e z i n g - - i n older preweanling rats may be mediated by recently matured physiological systems (47). Several critical questions, however, concerning the general nature of the effective stimulus remain unanswered. These questions include whether the age of the male is a relevant factor and the importance of unfamiliarity. Therefore, Experiment 1 was designed to evaluate the effects of exposure to unfamiliar prepubescent rats on behavioral inhibition. Experiment 2 then determined whether
THE ability to reduce on-going behavioral activities and engage in defensive acts when alarmed or threatened is a characteristic evident in diverse vertebrate species. Foraging fish, birds, and mammals will immediately stop feeding and freeze or move to cover when danger or a predator is detected (15,16,27,30,39). Neotropical frogs that gather in breeding sites and advertise their presence to females by vocalizing exhibit an immediate cessation of calling when predatory bats are detected (49). Cessation of frog vocalizations significantly reduces the likelihood of bat predation. The threat-induced occurrence of freezing observed in diverse species also appears to facilitate survival (12,34,36). These responses elicited by threat are also a feature of many young. For example, young birds will reduce their vocalizations and inhibit their activity when alarmed (19,28), Domestic chicks exposed to stressful situations exhibit a reduction in behavioral movements and assume lying postures with eye closure (32). Peep vocalizations often produced by chicks are also reduced [(29), see also (22)]. Young deer will stop feeding in the presence of predator odors (31). Socially isolated infant rhesus monkeys exhibit a rapid cessation of locomotor activity and vocalizations and freeze when confronted with a human (24,25). In humans, some children are especially prone to become quiet and withdrawn in unfamiliar situations (23).
J Dr. Lorey K. Takahashi, Department of Psychiatry, University of Wisconsin Medical School, 600 Highland Ave., Madison, WI 53792-2475. 717
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exposure to familiar adult male rats potentiates behavioral inhibition. EXPERIMENT UNFAMILIAR
1: B E H A V I O R A L PREPUBESCENT
INHIBITORY AND ADULT
EFFECTS OF MALE RATS
Adult male rats exhibit infanticide (38,46). Furthermore, day 14 rats exposed to an anesthetized unfamiliar adult male rat from across a wire-mesh barrier exhibit behavioral inhibition, suggesting that odors of the unfamiliar adult rat is threatening. However, day 14 pups may exhibit behavioral inhibition in response to all unfamiliar conspecific odors rather than selectively to the odor of adult male rats. Therefore, the purpose of this study was to compare the behavioral inhibitory effects of unfamiliar odors produced by prepubescent and adult male rats.
Method Animals. Pups were offspring of Sprague-Dawley female rats ( 9 0 - 1 2 0 days old) derived from a stock obtained originally from Sasco Inc., Madison, WI. Rats were maintained on a 12 h light:dark cycle with lights on at 0600 h. After mating, female rats were housed singly in stainless steel hanging cages until day 20 of pregnancy when they were transferred to plastic breeding cages (31 × 22 × 18 cm) with wire-mesh tops. Each cage was provisioned with food, water, and a layer of wood shaving. Breeding cages were checked daily for the presence of pups (day of birth was designated postnatal day 0). Litters were left undisturbed except for routine cage cleaning that occurred every 5 days. A total of eight litters was tested. Naive stimulus male rats were housed in groups of three to five animals. The ages of prepubescent pups ranged from 24 to 30 days, whereas adult male rats were 60 to 70 days old. Apparatus. Tests were conducted in a Plexiglas enclosure (26.5 × 26.5 × 20 cm) housed in a temperature-controlled incubator with a glass front. The Plexiglas enclosure was subdivided by a wire-mesh partition positioned with the two ends attached to the midportion of two adjacent walls, thereby forming a triangular compartment consisting of 87.8 square cm of floor space. Its top was open except for the area above the triangular compartment, which housed the rat pup. The enclosure was placed on a cardboard floor that was changed after every test. The ultrasound detector was positioned directly above the Plexiglas enclosure. Ambient temperature in the incubator varied from 34 to 35°C. This temperature corresponds to the thermoneutral range for 14-day-old rats (10). Test procedure. At 14 days of age, three male pups were taken, whenever possible, from each litter. Each pup was assigned randomly to one of three conditions:
among day 14 pups result from differences in the stimulus rat's behavior. Anesthetized males were placed in the test chamber 3 to 5 rain prior to introduction of day 14 pups. All tests were 10 rain in duration and conducted in the first half of the light cycle. In each test, the number of ultrasonic vocalizations was recorded. Ultrasounds were detected with headphones attached to the socket of a Mini-2 bat detector (Bat Conservation International, Inc., Austin, TX) tuned to 4 0 - 4 5 kHz. The bat detector transforms the ultrasound into the audible range of humans. Each discrete ultrasound was recorded using a counter except those few that were produced during face washing. The duration (in s) of freezing was recorded with timers. Freezing was recorded whenever the pup assumed an immobile posture with the head in a stationary position and elevated above the floor (45). Immediately after behavioral testing, the pup's rectal temperature was measured using a microprobe (IT-21, Physitemp, Clifton, N J) attached to a BAT-12 digital thermometer (Physitemp), with a resolution of 0.1 °C. The probe was inserted into the rectum to a depth of 10 mm and held in position until the temperature stabilized. Rectal temperature measurements were obtained within a 10 s period. Rectal temperature was recorded because ultrasonic vocalizations are reported to vary with body temperature (2,7). Therefore, it is important to assess whether differences in vocalizations are associated with altered body temperature. Statistics. The nonparametric Kruskall-Wallis one-way A N O V A procedure was used to assess the overall significance of ultrasonic vocalization and freezing. Mann-Whitney U-tests were used for subsequent paired comparisons. A parametric oneway A N O V A was used to analyze the rectal temperature data.
Results Ultrasonic vocalization and freezing. Dramatic differences in the emission of ultrasounds and the exhibition of freezing were observed across the three stimulus conditions (Fig. 1). Day 14 pups showed a clear reduction in ultrasound production, H(2) = 8.46, p < .05, and a significant increase in freezing, H(2) = 17.71, p < .001, only when exposed to the unfamiliar adult male rat. In contrast, day 14 pups exposed to an unfamiliar prepubescent male rat emitted a high level of vocalizations that were similar in number to those produced in the control social isolation condition. Although three pups exhibited freezing in response to the unfamiliar prepubescent rat (range = 5 to 145 s), the overall duration of freezing did not differ significantly from the social
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The isolation condition serves as a control to evaluate the effects of conspecific exposure on behavioral inhibition. In the isolation condition, the day 14 pup was placed alone in the triangular compartment and the larger adjacent compartment was empty. In the isolation + prepubescent pup condition and the isolation + adult rat condition, the day 14 pup was placed in the triangular compartment and either the unfamiliar prepubescent male pup or the unfamiliar adult male rat was placed in the larger adjacent compartment. The prepubescent male and the adult male rat were anesthetized (sodium pentobarbital, 50 mg/kg, IP) prior to testing to eliminate any possibility that behavioral differences
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FIG. 1. Ultrasonic vocalizations (panel A) and freezing (panel B) shown by 14-day-old male rats during a 10 min test involving exposure to various stimuli. (*p < 0.05, **p < 0.01, significantly different from other two groups.)
POTENTIATION OF BEHAVIORAL INHIBITION IN RAT PUPS
isolation group. All pups exposed to the unfamiliar adult male rat engaged in freezing (range = 88 to 333 s). Rectal temperature. Rectal temperatures (mean group range = 36.3 to 36.8°C) did not differ significantly among the three groups of day 14 pups, F(2, 20) = 2.39,p > 0.05. It appears that differences in ultrasound production are not associated with differences in body temperature.
Discussion Because surviving close encounters with predators may occur only under very limited conditions, it was hypothesized that prey must utilize their species-specific defensive behavioral repertoire to avoid direct contact with threat (8). Within this theoretical framework, unfamiliar environmental stimuli should be especially potent elicitors of defensive reactions. In this study, however, day 14 rats exposed to unfamiliar prepubescent conspecific males did not exhibit behavioral inhibition. This result indicates clearly that not all unfamiliar conspecific stimulus cues are equally potent elicitors of behavioral inhibition. Day 14 rats must be able to differentiate the stimulus properties of an unfamiliar adult male rat from those of unfamiliar prepubescent rats, and act accordingly. Although the basis underlying these differences in stimulus properties is not clear, it is unlikely that visual cues such as differences in body size of prepubescent and adult stimulus rats are important factors. In this study, eye opening was not evident in 38% of tested pups nearly equally distributed across the three stimulus conditions. Furthermore, as demonstrated previously, behavioral inhibition occurs even when a solid partition, which prevents visual contact, is used to separate the pup from the stimulus animal (44). More likely, olfactory cues associated with male sexual maturation may be important in promoting the occurrence of behavioral inhibition. Studies indicate that adult male rats are less likely to direct agonistic behavior toward either adult castrated males or prepuberal males than toward intact conspecific males (17). Because unfamiliar conspecific male odors are important elicitors of agonistic behavior in rats (1), it is possible that hormone-induced changes in odor associated with male sexual maturation are salient stimuli that potentiate behavioral inhibition. EXPERIMENT 2: BEHAVIORAL INHIBITORY EFFECTS OF F A M I L I A R AND U N F A M I L I A R A D U L T MALE RATS
Although behavioral inhibition is often elicited by threatening stimuli, prey species also habituate to the presence of predators or learn when attack is unlikely to occur (39,41,48). Nonmodulated defensive reactions triggered in a reflex-like manner whenever stimuli associated with threat is perceived would be costly if the predator's presence poses no imminent danger because of interference with feeding, reproduction, and other regulatory activities (41). In this study, day 14 rat pups were tested for behavioral inhibition in the presence of familiar and unfamiliar adult male rats. This study answers the question of whether all adult male rats are equally effective stimuli in potentiating behavioral inhibition. Presumably, rat pups tested with familiar male rats will have habituated to their presence, and therefore, behavioral inhibition will be reduced.
Method Animals and pretest housing conditions. Rat pups were obtained from litters as described in Experiment 1. On day 20 of pregnancy, all female rats were placed in plastic breeding cages
719
(59 × 39 X 20 cm) along with a sexually experienced SpragueDawley male rat. Each cage contained food, water, and wood shavings. Pairing of adult male rats with pregnant rats several days prior to delivery did not appear to affect survival of pups. Breeding cages were checked for litters (n = 9) on a daily basis. Another group of sexually experienced male rats was housed singly in a different room and served as unfamiliar adult stimulus rats. Ages of the cohabiting male and the unfamiliar male ranged from 150 to 300 days. The adult males used in this study had sexual experiences 1 to 2 weeks prior to testing or housing with the pregnant female. Apparatus and test procedure. When pups were 14 days of age, the cohabiting adult male rat was removed from the nesting cage and anesthetized with sodium pentobarbital (50 mg/kg, IP). After injection, the adult rat was placed in a clean holding cage until it was fully anesthetized. The unfamiliar adult male rat was prepared similarly. Three male pups were taken from each litter. Each pup was assigned randomly to one of three test conditions: 1. Isolation condition (n = 9); 2. Isolation + familiar adult male condition (n = 9); and 3. Isolation + unfamiliar adult male condition (n = 9). Pups and adult male rats were placed in the test compartment as described in Experiment 1. Behavioral tests were 10 min in duration and conducted as indicated in Experiment 1. Statistics. The number of ultrasonic vocalizations, the duration of freezing, and the rectal temperatures of pups were analyzed in the same manner described in Experiment 1.
Resul~ Ultrasonic vocalization and freezing. As shown in Fig. 2, adult male rats are not equally effective in potentiating behavioral inhibition. Rather, day 14 rats show a selective decrease in ultrasonic vocalizations, H(2) = 12.92, p < 0.01, and a significant increase in freezing, H(2) = 20.04, p < 0.001, only when exposed to unfamiliar adult male rats. Several pups (n = 6) exhibited freezing (range = 10 to 59 s) when tested with the familiar adult male rat. However, no reliable differences in either ultrasound production or freezing duration were found between the social isolation and the familiar male group. All nine pups exposed to the unfamiliar male exhibited freezing, and one pup in the social isolation group froze for a total of 65 s. 150
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Rectal temperature. As in Experiment 1, no significant differences were found in rectal temperatures (mean group range = 35.9 to 36.3°C) among the three test conditions, F(2, 24) = 2.17, p > 0.05. As in Experiment 1, it appears that differences in behavioral inhibition cannot be linked unequivocally to differences in body temperature. Discussion
Cohabitation with an adult male rat from the day of birth significantly reduces the cohabiting male's effectiveness in eliciting behavioral inhibition. Cohabitation may habituate and simultaneously familiarize the pup to the adult male rat, thereby lessening its threatening impact. Furthermore, these rearing experiences with an adult male rat did not produce generalized effects, in that other adult male rats would also become less effective in potentiating behavioral inhibition. Rather, results of this study as well as those of Experiment 1 demonstrate that day 14 rats exhibit behavioral inhibition selectively to unfamiliar adult male rats. Although no significant behavioral differences were found between pups tested in social isolation and with the familiar adult male rat, a number of pups exposed to the latter situation did exhibit brief bouts of freezing. In previous studies using the mother rat as the stimulus animal, freezing rarely occurred and ultrasonic vocalizations did not differ from those produced in the social isolation test (45). The unfamiliar environment coupled with exposure to adult male rats may be prepotent factors underlying the elicitation of behavioral inhibition. Perhaps the duration or intensity of the behavioral inhibition pattern is then determined by other aspects such as experiential factors. Depending upon test conditions, studies indicate that a percentage (10 to 40%) of naive male rats does not exhibit infanticide of neonatal pups (9,35). In addition, studies conducted in mice suggest that pups exposed to urine odors of infanticidal males are more likely to exhibit agitation and avoidance responses than when tested with odors of parental males or noninfanticidal adult mice (14). In the present study, adult male rats were not selected on the basis of their potential to exhibit infanticide. Potential differences in male infanticidal behavior notwithstanding, current results underscore the potency of unfamiliar adult male stimuli in eliciting behavioral inhibition. The ultrasonic vocalization results of Experiment 2 appear to be consistent with studies performed on mice (14). In those studies, mouse pups exposed to urine of paternal male mice produced more ultrasonic vocalizations than when tested with urine of unfamiliar infanticidal male mice. The authors suggest that the chemical composition of the urine may differ between parental and dominant infanticidal males. However, it is also possible that habituation and familiarity with odors of the cohabiting adult male were important contributing factors underlying the behavioral response. It is noteworthy that pups exposed to the urine obtained from unfamiliar noninfanticidal male mice also reduced their ultrasound production in comparison to pups exposed to the urine of familiar parental males (14). These results appear to further support the hypothesis that unfamiliar adult conspecific
male odors are potent stimuli that trigger behavioral reactions. i.e.. cessation of vocalizations, in preweanling rodents. GENERAL DISCUSSION
Results of the two experiments suggest strongly that odors of unfamiliar adult male rats are highly effective stimuli that induce behavioral inhibition. However, additional investigations are required to determine whether all odors associated with threat will elicit behavioral inhibition. Previous studies indicate that preweanling rats do not consistently produce low rates of ultrasounds when exposed to odors of predators such as from minks and ferrets (26). However, these preweanling pups were tested between the ages of 3 to 9 days, a developmental period when behavioral inhibition has yet to fully emerge (44,45). That predatory odors do exert a pronounced influence on the behavior of rodents is demonstrated in field studies showing that traps, tainted with the odor of predators, are more likely to be avoided than clean traps or traps with nonpredator odors (43). These results are somewhat similar to those obtained in the laboratory showing that day 14 rats are more likely to avoid a compartment containing an unfamiliar adult male rat than an empty compartment (44). Studies conducted on adult rats indicate thai although cat odors are capable of facilitating freezing (20), stimulus movement, rather than odor, may be necessary and sufficient to induce freezing (6,13). In the present studies, freezing was potentiated in the presence of the unfamiliar adult male rat that was anesthetized to eliminate the possibility that the pup's behavior was significantly influenced by overt responses or auditory cues of the stimulus rat. These results may suggest that preweanling pups are especially responsive to olfactory stimuli. This increased responsiveness may be derived from the fact that when day 14 pups are tested, their olfactory experiences appear to be limited almost entirely to odors associated with the nest. Therefore, unfamiliar odors in the environment may be more salient to preweanling pups than adult rats that have already experienced a variety of odors prior to testing. Current results, as well as those reported previously (44,45), indicate a pronounced tendency of rat pups to reduce their emission of ultrasonic vocalizations and freeze when exposed to an unfamiliar adult male rat. However, behavioral inhibition may not be the only defensive response elicited when the maturing rat encounters a threatening stimulus. Numerous studies indicate that animals are equipped with an array of defensive behavioral responses. The predominant behavioral response(s) is dependent on a number of factors including but not limited to predator types (21,37,40), environmental circumstances (36,40), and previous experiences (41). Thus, the propensity of rat pups to exhibit behavioral inhibition when encountering unfamiliar conspecifics may reflect the consequence that promoted survival in situations mimicked by the current test environment. ACKNOWLEDGEMENTS The research was supported by NIMH grant MH-43986 and grant from the Wisconsin Alumni Research Foundation. The author thanks H. Kim for technical assistance.
REFERENCES 1. Alberts, J. R.; Galef, B. G., Jr. Olfactory cues and movement: Stimuli mediating intraspecific aggression in the wild Norway rat. J. Comp. Physiol. Psychol. 85:233-242; 1973. 2. Allin, J. T.; Banks, E. M. Effects of temperature on ultrasound production by infant albino rats. Dev. Psychobiol. 4:149-156; 1971.
3. Allin, J. T.; Banks, E. M. Functional aspects of ultrasound production in infant albino rats. Anim. Behav. 20:175-185; 1972. 4. Blanchard, R. J.; Blanchard, D. C. Antipredator defensive behaviors in a visible burrow system. J. Comp. Psychol. 103:70-82; 1989. 5. Blanchard, R. J.; Blanchard, D. C.: Agullana, R.: Weiss, S. M.
P O T E N T I A T I O N OF B E H A V I O R A L INHIBITION IN R A T PUPS
Twenty-two kHz alarm cries to presentation of a predator, by laboratory rats living in visible burrow systems. Physiol. Behav. 50:967972; 1991. 6. Blanchard, R. J.; Mast, M.; Blanchard, D. C. Stimulus control of defensive reactions in the albino rat. J. Comp. Physiol. Psychol. 88:81-88; 1975. 7. Blumberg, M. S.; Alberts, J. R. Ultrasonic vocalizations by rat pups in the cold: An acoustic by-product of laryngeal braking? Behav. Neurosci. 104:808-817; 1990. 8. Bolles, R. C. Species-specific defense reactions and avoidance learning. Psychol. Rev. 77:32-48; 1970. 9. Brown, R. E. Social and hormonal factors influencing infanticide and its suppression in adult male Long-Evans rats (Rattus norvegicus). J. Comp. Psychol. 100:155-161; 1986. 10. Conklin, P.; Heggeness, F. W. Maturation of temperature homeostasis in the rat. Am. J. Physiol. 220:333-336; 1971. 11. Courtney, R. J.; Reid, L. D.; Wasden, R. E. Suppression of running times by olfactory stimuli. Psychon. Sci. 12:315-316; 1968. 12. Curio, E. The ethology of predation. New York: Springer-Verlag; 1976. 13. Curti, M. W. Native fear responses of white rats to the presence of cats. Psychol. Monogr. 46:78-98; 1935. 14. Elwood, R. W.; Kennedy, H. F.; Blakely, H. M. Responses of infant mice to odors of urine from infanticidal, noninfanticidal, and paternal male mice. Dev. Psychobiol. 23:309-317; 1990. 15. Ficken, M. S. Acoustic characteristics of alarm calls associated with predation risk in chickadees. Anim. Behav. 39:400-401; 1990. 16. Ficken, M. S.; Witkin, S. R. Responses of black-capped chickadees to predators. Auk 94:156-157; 1977. 17. Flannelly, K. J.; Thor, D. H. Territorial aggression of the rat to males castrated at various ages. Physiol. Behav. 20:785-789; 1978. 18. Gaddis, P. Mixed flocks, accipiters, and antipredator behavior. Condor 82:348-349; 1980. 19. Greig-Smith, P. W. Parental investment in nest defence by stonechats (Saxicola torquata). Anim. Behav. 28:604-619; 1980. 20. Griffith, C. R. The behavior of white rats in the presence of cats. Psychobiology 2:19-28; 1920. 21. Hennessy, D. F.; Owings, D. H. Snake species discrimination and the role of olfactory cues in the snake-directed behavior of the California ground squirrel. Behaviour 65:115-124; 1978. 22. Jones, R. B.; Mills, A. D.; Faure, J.-M. Genetic and experiential manipulation of fear-related behavior in Japanese quail chicks (Coturniz coturnixjaponica). J. Comp. Psychol. 105:15-24; 1991. 23. Kagan, J.; Reznick, J. S.; Snidman, N. Biological bases of childhood shyness. Science 240:167-171; 1988. 24. Kalin, N. H.; Shelton, S. E. Defensive behaviors in infant rhesus monkeys: Environmental cues and neurochemical regulation. Science 240:167-171; 1989. 25. Kalin, N. H.; Shelton, S. E.; Takahashi, L. K. Defensive behaviors in infant rhesus monkeys: Ontogeny and context-dependent selective expression. Child Dev. 62:1175-1183; 1991. 26. Lyons, D. M.; Banks, E. M. Ultrasounds in neonatal rats: Novel, predator and conspecific odor cues. Dev. Psychobiol. 15:455-460; 1982. 27. Magurran, A. E.; Girling, S. L. Predator model recognition and response habituation in shoaling minnows. Anim. Behav. 34:510518; 1986.
721
28. Miller, D. B. Maternal vocal control of behavioral inhibition in mallard ducklings (Anas platyrhynchos). J. Comp. Physiol. Psychol. 94:606-623; 1980. 29. Montevecchi, W. A.; Gallup, G. G., Jr.; Dunlap, W. A. The peep vocalization in group reared chicks (Gallus domesticus): Its relation to fear. Anim. Behav., 21:116 - 123; 1973. 30. Morse, D. H. Interactions between tit flocks and sparrowhawks Accipiter nisus. Ibis 115:591-593; 1973. 31. Muller- Schwarze, D. Responses of young black-tailed deer to predator odors. J. Mammal. 53:393-394; 1972. 32. Murphy, L. B.; Wood-Gush, D. G. M. The interpretation of the behaviour of domestic fowl in strange environments. Biol. Behav. 3:39-61; 1978. 33. Noirot, E. Ultrasounds and maternal behavior in small rodents. Dev. Psychobiol. 5:371-387; 1972. 34. Palmer, W. Instinctive stillness in birds. Auk 26:23-36; 1909. 35. Paul, L.; Kupferschmidt, J. Killing of conspecific and mouse young by male rats. J. Comp. Physiol. Psychol. 88:755-763; 1975. 36. Rather, S. C. Comparative aspects of hypnosis. In: Gordon, J. E., ed. Handbook of clinical and experimental hypnosis. New York: Macmillian; 1967:550-587. 37. Robinson, S. R. Antipredator behaviour and predator recognition in Belding's ground squirrels. Anim. Behav. 28:840-852; 1980. 38. Rosenberg, K. M.; Denenberg, V. H.; Zarrow, M. X.; Frank, B. L. Effects of neonatal castration and testosterone on the rat's pup-killing behavior and activity. Physiol. Behav. 7:363-368; 1971. 39. Schaller, G. B. The Serengeti lion. Chicago: University of Chicago Press; 1972. 40. Seyfarth, R. M.; Cbeney, D. L.; Marler, P. Vervet monkey alarm call: Semantic communication in a free-ranging primate. Anim. Behav. 28:1070-1094; 1980. 41. Shalter, M. D. Predator-prey behavior and habituation. In: Peeke, H. V. S.; Petrinovich, L., eds. Habituation, sensitization, and behavior. New York: Academic Press; 1984:349-391. 42. Smotherman, W. P.; Bell, R. W.; Starzec, J.; Elias, J.; Zachman, T. A. Maternal responses to infant vocalizations and olfactory cues in rats and mice. Behav. Biol. 12:55-66; 1974. 43. Stoddart, D. M. Some responses of a free living community of rodents to the odors of predators. In: Muller-Schwarze, D.; Silverstein, R. M., eds. Chemical signals: Vertebrates and aquatic invertebrates. New York: Plenum Press; 1980:1-10. 44. Takahashi, L. K. Developmental expression of defensive responses during exposure to conspecific adults in preweanling rats (Rattus norvegicus). J. Comp. Psychol. 106:69-77; 1992. 45. Takahashi, L. K. Ontogeny of behavioral inhibition induced by unfamiliar adult male conspecifics in preweanling rats. Physiol. Behav. 52:493-498; 1992. 46. Takahashi, L. K.; Lore, R. K. Intermale and maternal aggression in adult rats tested at different ages. Physiol. Behav. 29:1013-1018; 1982. 47. Takahashi, L. K.; Rubin, W. W. Corticosteroid induction of threatinduced behavioral inhibition in preweanling rats. Behav. Neurosci. 107:860-866; 1993. 48. Thorpe, W. H. Learning and instinct in animals. London: Methuen; 1956. 49. Tuttle, M. D.; Ryan, M. J. Bat predation and the evolution of frog vocalizations in the neotropics. Science 214:677-678; 1981.
Pergamon 0031-9384(93)E0029-P
Stimulus Control of Behavioral Inhibition in the Preweanling Rat LOREY
K. T A K A H A S H I 1
Department of Psychiatry, University of Wisconsin Medical School, and William S. Middleton Memorial Veterans Hospital, Madison, WI 53792
R e c e i v e d 30 A u g u s t 1993 TAKAHASHI, L. K. Stimuluscontrolof behavioral inhibitionin the preweanling rat. PHYSIOL BEHAV 55(4) 717-721, 1 9 9 4 . Previous studies demonstrate that 14-day-old rats reduce their emission of ultrasonic vocalizations and freeze when exposed to an unfamiliar adult male rat. This study sought to identify the stimulus characteristics of conspecific males that potentiate the display of behavioral inhibition. In Experiment 1, day 14 rats were isolated from the nest and exposed to either an unfamiliar prepubescent male rat or an unfamiliar adult male rat. Pups exposed to the unfamiliar adult male rat exhibited significantly elevated levels of freezing and reduced their emission of ultrasounds. In Experiment 2, pups were exposed to either a familiar or an unfamiliar adult male rat. Although several pups exposed to the familiar adult male rat exhibited freezing, pups tested with the unfamiliar adult rat showed a reliably higher duration of freezing and made fewer vocalizations. Results suggest that neither the unfamiliar factor nor cues associated with male adulthood are sufficient to account for the occurrence of behavioral inhibition when presented separately. However, the combination of unfamiliarity and adult male stimuli are highly potent stimulus features that elicit behavioral inhibition in preweanling rats. Preweanling rats Social isolation
Behavioral inhibition Conspecific odors
Ultrasonic vocalization
Freezing
Development
Conspecific threat
Although the adult Norway rat readily exhibits behavioral inhibition (4,5,11,13,20), neonatal altricial rats do not display behavioral inhibition until the end of the second postnatal week. In a series of studies, this laboratory has demonstrated that socially isolated 7- and 14-day-old rats emit a high number of ultrasonic vocalizations (44,45). Emission of ultrasounds by rat pups separated from the litter serves presumably to elicit and direct maternal responses (3,33,42). However, when day 14 rat pups are tested under social isolation conditions and exposed simultaneously to stimulus cues of an unfamiliar adult male rat, there is a pronounced cessation of ultrasounds accompanied by immobility posturing or freezing (45). In contrast, socially isolated day 7 rats continue to emit ultrasounds and fail to engage in freezing. It is important to emphasize that day 14 rats do not exhibit behavioral inhibition when exposed to the mother, attesting to the stimulus specificity nature of the response (44,45). This propensity to exhibit behavioral inhibition--cessation of vocalizations accompanied by f r e e z i n g - - i n older preweanling rats may be mediated by recently matured physiological systems (47). Several critical questions, however, concerning the general nature of the effective stimulus remain unanswered. These questions include whether the age of the male is a relevant factor and the importance of unfamiliarity. Therefore, Experiment 1 was designed to evaluate the effects of exposure to unfamiliar prepubescent rats on behavioral inhibition. Experiment 2 then determined whether
THE ability to reduce on-going behavioral activities and engage in defensive acts when alarmed or threatened is a characteristic evident in diverse vertebrate species. Foraging fish, birds, and mammals will immediately stop feeding and freeze or move to cover when danger or a predator is detected (15,16,27,30,39). Neotropical frogs that gather in breeding sites and advertise their presence to females by vocalizing exhibit an immediate cessation of calling when predatory bats are detected (49). Cessation of frog vocalizations significantly reduces the likelihood of bat predation. The threat-induced occurrence of freezing observed in diverse species also appears to facilitate survival (12,34,36). These responses elicited by threat are also a feature of many young. For example, young birds will reduce their vocalizations and inhibit their activity when alarmed (19,28), Domestic chicks exposed to stressful situations exhibit a reduction in behavioral movements and assume lying postures with eye closure (32). Peep vocalizations often produced by chicks are also reduced [(29), see also (22)]. Young deer will stop feeding in the presence of predator odors (31). Socially isolated infant rhesus monkeys exhibit a rapid cessation of locomotor activity and vocalizations and freeze when confronted with a human (24,25). In humans, some children are especially prone to become quiet and withdrawn in unfamiliar situations (23).
J Dr. Lorey K. Takahashi, Department of Psychiatry, University of Wisconsin Medical School, 600 Highland Ave., Madison, WI 53792-2475. 717
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exposure to familiar adult male rats potentiates behavioral inhibition. EXPERIMENT UNFAMILIAR
1: B E H A V I O R A L PREPUBESCENT
INHIBITORY AND ADULT
EFFECTS OF MALE RATS
Adult male rats exhibit infanticide (38,46). Furthermore, day 14 rats exposed to an anesthetized unfamiliar adult male rat from across a wire-mesh barrier exhibit behavioral inhibition, suggesting that odors of the unfamiliar adult rat is threatening. However, day 14 pups may exhibit behavioral inhibition in response to all unfamiliar conspecific odors rather than selectively to the odor of adult male rats. Therefore, the purpose of this study was to compare the behavioral inhibitory effects of unfamiliar odors produced by prepubescent and adult male rats.
Method Animals. Pups were offspring of Sprague-Dawley female rats ( 9 0 - 1 2 0 days old) derived from a stock obtained originally from Sasco Inc., Madison, WI. Rats were maintained on a 12 h light:dark cycle with lights on at 0600 h. After mating, female rats were housed singly in stainless steel hanging cages until day 20 of pregnancy when they were transferred to plastic breeding cages (31 × 22 × 18 cm) with wire-mesh tops. Each cage was provisioned with food, water, and a layer of wood shaving. Breeding cages were checked daily for the presence of pups (day of birth was designated postnatal day 0). Litters were left undisturbed except for routine cage cleaning that occurred every 5 days. A total of eight litters was tested. Naive stimulus male rats were housed in groups of three to five animals. The ages of prepubescent pups ranged from 24 to 30 days, whereas adult male rats were 60 to 70 days old. Apparatus. Tests were conducted in a Plexiglas enclosure (26.5 × 26.5 × 20 cm) housed in a temperature-controlled incubator with a glass front. The Plexiglas enclosure was subdivided by a wire-mesh partition positioned with the two ends attached to the midportion of two adjacent walls, thereby forming a triangular compartment consisting of 87.8 square cm of floor space. Its top was open except for the area above the triangular compartment, which housed the rat pup. The enclosure was placed on a cardboard floor that was changed after every test. The ultrasound detector was positioned directly above the Plexiglas enclosure. Ambient temperature in the incubator varied from 34 to 35°C. This temperature corresponds to the thermoneutral range for 14-day-old rats (10). Test procedure. At 14 days of age, three male pups were taken, whenever possible, from each litter. Each pup was assigned randomly to one of three conditions:
among day 14 pups result from differences in the stimulus rat's behavior. Anesthetized males were placed in the test chamber 3 to 5 rain prior to introduction of day 14 pups. All tests were 10 rain in duration and conducted in the first half of the light cycle. In each test, the number of ultrasonic vocalizations was recorded. Ultrasounds were detected with headphones attached to the socket of a Mini-2 bat detector (Bat Conservation International, Inc., Austin, TX) tuned to 4 0 - 4 5 kHz. The bat detector transforms the ultrasound into the audible range of humans. Each discrete ultrasound was recorded using a counter except those few that were produced during face washing. The duration (in s) of freezing was recorded with timers. Freezing was recorded whenever the pup assumed an immobile posture with the head in a stationary position and elevated above the floor (45). Immediately after behavioral testing, the pup's rectal temperature was measured using a microprobe (IT-21, Physitemp, Clifton, N J) attached to a BAT-12 digital thermometer (Physitemp), with a resolution of 0.1 °C. The probe was inserted into the rectum to a depth of 10 mm and held in position until the temperature stabilized. Rectal temperature measurements were obtained within a 10 s period. Rectal temperature was recorded because ultrasonic vocalizations are reported to vary with body temperature (2,7). Therefore, it is important to assess whether differences in vocalizations are associated with altered body temperature. Statistics. The nonparametric Kruskall-Wallis one-way A N O V A procedure was used to assess the overall significance of ultrasonic vocalization and freezing. Mann-Whitney U-tests were used for subsequent paired comparisons. A parametric oneway A N O V A was used to analyze the rectal temperature data.
Results Ultrasonic vocalization and freezing. Dramatic differences in the emission of ultrasounds and the exhibition of freezing were observed across the three stimulus conditions (Fig. 1). Day 14 pups showed a clear reduction in ultrasound production, H(2) = 8.46, p < .05, and a significant increase in freezing, H(2) = 17.71, p < .001, only when exposed to the unfamiliar adult male rat. In contrast, day 14 pups exposed to an unfamiliar prepubescent male rat emitted a high level of vocalizations that were similar in number to those produced in the control social isolation condition. Although three pups exhibited freezing in response to the unfamiliar prepubescent rat (range = 5 to 145 s), the overall duration of freezing did not differ significantly from the social
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The isolation condition serves as a control to evaluate the effects of conspecific exposure on behavioral inhibition. In the isolation condition, the day 14 pup was placed alone in the triangular compartment and the larger adjacent compartment was empty. In the isolation + prepubescent pup condition and the isolation + adult rat condition, the day 14 pup was placed in the triangular compartment and either the unfamiliar prepubescent male pup or the unfamiliar adult male rat was placed in the larger adjacent compartment. The prepubescent male and the adult male rat were anesthetized (sodium pentobarbital, 50 mg/kg, IP) prior to testing to eliminate any possibility that behavioral differences
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FIG. 1. Ultrasonic vocalizations (panel A) and freezing (panel B) shown by 14-day-old male rats during a 10 min test involving exposure to various stimuli. (*p < 0.05, **p < 0.01, significantly different from other two groups.)
POTENTIATION OF BEHAVIORAL INHIBITION IN RAT PUPS
isolation group. All pups exposed to the unfamiliar adult male rat engaged in freezing (range = 88 to 333 s). Rectal temperature. Rectal temperatures (mean group range = 36.3 to 36.8°C) did not differ significantly among the three groups of day 14 pups, F(2, 20) = 2.39,p > 0.05. It appears that differences in ultrasound production are not associated with differences in body temperature.
Discussion Because surviving close encounters with predators may occur only under very limited conditions, it was hypothesized that prey must utilize their species-specific defensive behavioral repertoire to avoid direct contact with threat (8). Within this theoretical framework, unfamiliar environmental stimuli should be especially potent elicitors of defensive reactions. In this study, however, day 14 rats exposed to unfamiliar prepubescent conspecific males did not exhibit behavioral inhibition. This result indicates clearly that not all unfamiliar conspecific stimulus cues are equally potent elicitors of behavioral inhibition. Day 14 rats must be able to differentiate the stimulus properties of an unfamiliar adult male rat from those of unfamiliar prepubescent rats, and act accordingly. Although the basis underlying these differences in stimulus properties is not clear, it is unlikely that visual cues such as differences in body size of prepubescent and adult stimulus rats are important factors. In this study, eye opening was not evident in 38% of tested pups nearly equally distributed across the three stimulus conditions. Furthermore, as demonstrated previously, behavioral inhibition occurs even when a solid partition, which prevents visual contact, is used to separate the pup from the stimulus animal (44). More likely, olfactory cues associated with male sexual maturation may be important in promoting the occurrence of behavioral inhibition. Studies indicate that adult male rats are less likely to direct agonistic behavior toward either adult castrated males or prepuberal males than toward intact conspecific males (17). Because unfamiliar conspecific male odors are important elicitors of agonistic behavior in rats (1), it is possible that hormone-induced changes in odor associated with male sexual maturation are salient stimuli that potentiate behavioral inhibition. EXPERIMENT 2: BEHAVIORAL INHIBITORY EFFECTS OF F A M I L I A R AND U N F A M I L I A R A D U L T MALE RATS
Although behavioral inhibition is often elicited by threatening stimuli, prey species also habituate to the presence of predators or learn when attack is unlikely to occur (39,41,48). Nonmodulated defensive reactions triggered in a reflex-like manner whenever stimuli associated with threat is perceived would be costly if the predator's presence poses no imminent danger because of interference with feeding, reproduction, and other regulatory activities (41). In this study, day 14 rat pups were tested for behavioral inhibition in the presence of familiar and unfamiliar adult male rats. This study answers the question of whether all adult male rats are equally effective stimuli in potentiating behavioral inhibition. Presumably, rat pups tested with familiar male rats will have habituated to their presence, and therefore, behavioral inhibition will be reduced.
Method Animals and pretest housing conditions. Rat pups were obtained from litters as described in Experiment 1. On day 20 of pregnancy, all female rats were placed in plastic breeding cages
719
(59 × 39 X 20 cm) along with a sexually experienced SpragueDawley male rat. Each cage contained food, water, and wood shavings. Pairing of adult male rats with pregnant rats several days prior to delivery did not appear to affect survival of pups. Breeding cages were checked for litters (n = 9) on a daily basis. Another group of sexually experienced male rats was housed singly in a different room and served as unfamiliar adult stimulus rats. Ages of the cohabiting male and the unfamiliar male ranged from 150 to 300 days. The adult males used in this study had sexual experiences 1 to 2 weeks prior to testing or housing with the pregnant female. Apparatus and test procedure. When pups were 14 days of age, the cohabiting adult male rat was removed from the nesting cage and anesthetized with sodium pentobarbital (50 mg/kg, IP). After injection, the adult rat was placed in a clean holding cage until it was fully anesthetized. The unfamiliar adult male rat was prepared similarly. Three male pups were taken from each litter. Each pup was assigned randomly to one of three test conditions: 1. Isolation condition (n = 9); 2. Isolation + familiar adult male condition (n = 9); and 3. Isolation + unfamiliar adult male condition (n = 9). Pups and adult male rats were placed in the test compartment as described in Experiment 1. Behavioral tests were 10 min in duration and conducted as indicated in Experiment 1. Statistics. The number of ultrasonic vocalizations, the duration of freezing, and the rectal temperatures of pups were analyzed in the same manner described in Experiment 1.
Resul~ Ultrasonic vocalization and freezing. As shown in Fig. 2, adult male rats are not equally effective in potentiating behavioral inhibition. Rather, day 14 rats show a selective decrease in ultrasonic vocalizations, H(2) = 12.92, p < 0.01, and a significant increase in freezing, H(2) = 20.04, p < 0.001, only when exposed to unfamiliar adult male rats. Several pups (n = 6) exhibited freezing (range = 10 to 59 s) when tested with the familiar adult male rat. However, no reliable differences in either ultrasound production or freezing duration were found between the social isolation and the familiar male group. All nine pups exposed to the unfamiliar male exhibited freezing, and one pup in the social isolation group froze for a total of 65 s. 150
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720
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Rectal temperature. As in Experiment 1, no significant differences were found in rectal temperatures (mean group range = 35.9 to 36.3°C) among the three test conditions, F(2, 24) = 2.17, p > 0.05. As in Experiment 1, it appears that differences in behavioral inhibition cannot be linked unequivocally to differences in body temperature. Discussion
Cohabitation with an adult male rat from the day of birth significantly reduces the cohabiting male's effectiveness in eliciting behavioral inhibition. Cohabitation may habituate and simultaneously familiarize the pup to the adult male rat, thereby lessening its threatening impact. Furthermore, these rearing experiences with an adult male rat did not produce generalized effects, in that other adult male rats would also become less effective in potentiating behavioral inhibition. Rather, results of this study as well as those of Experiment 1 demonstrate that day 14 rats exhibit behavioral inhibition selectively to unfamiliar adult male rats. Although no significant behavioral differences were found between pups tested in social isolation and with the familiar adult male rat, a number of pups exposed to the latter situation did exhibit brief bouts of freezing. In previous studies using the mother rat as the stimulus animal, freezing rarely occurred and ultrasonic vocalizations did not differ from those produced in the social isolation test (45). The unfamiliar environment coupled with exposure to adult male rats may be prepotent factors underlying the elicitation of behavioral inhibition. Perhaps the duration or intensity of the behavioral inhibition pattern is then determined by other aspects such as experiential factors. Depending upon test conditions, studies indicate that a percentage (10 to 40%) of naive male rats does not exhibit infanticide of neonatal pups (9,35). In addition, studies conducted in mice suggest that pups exposed to urine odors of infanticidal males are more likely to exhibit agitation and avoidance responses than when tested with odors of parental males or noninfanticidal adult mice (14). In the present study, adult male rats were not selected on the basis of their potential to exhibit infanticide. Potential differences in male infanticidal behavior notwithstanding, current results underscore the potency of unfamiliar adult male stimuli in eliciting behavioral inhibition. The ultrasonic vocalization results of Experiment 2 appear to be consistent with studies performed on mice (14). In those studies, mouse pups exposed to urine of paternal male mice produced more ultrasonic vocalizations than when tested with urine of unfamiliar infanticidal male mice. The authors suggest that the chemical composition of the urine may differ between parental and dominant infanticidal males. However, it is also possible that habituation and familiarity with odors of the cohabiting adult male were important contributing factors underlying the behavioral response. It is noteworthy that pups exposed to the urine obtained from unfamiliar noninfanticidal male mice also reduced their ultrasound production in comparison to pups exposed to the urine of familiar parental males (14). These results appear to further support the hypothesis that unfamiliar adult conspecific
male odors are potent stimuli that trigger behavioral reactions. i.e.. cessation of vocalizations, in preweanling rodents. GENERAL DISCUSSION
Results of the two experiments suggest strongly that odors of unfamiliar adult male rats are highly effective stimuli that induce behavioral inhibition. However, additional investigations are required to determine whether all odors associated with threat will elicit behavioral inhibition. Previous studies indicate that preweanling rats do not consistently produce low rates of ultrasounds when exposed to odors of predators such as from minks and ferrets (26). However, these preweanling pups were tested between the ages of 3 to 9 days, a developmental period when behavioral inhibition has yet to fully emerge (44,45). That predatory odors do exert a pronounced influence on the behavior of rodents is demonstrated in field studies showing that traps, tainted with the odor of predators, are more likely to be avoided than clean traps or traps with nonpredator odors (43). These results are somewhat similar to those obtained in the laboratory showing that day 14 rats are more likely to avoid a compartment containing an unfamiliar adult male rat than an empty compartment (44). Studies conducted on adult rats indicate thai although cat odors are capable of facilitating freezing (20), stimulus movement, rather than odor, may be necessary and sufficient to induce freezing (6,13). In the present studies, freezing was potentiated in the presence of the unfamiliar adult male rat that was anesthetized to eliminate the possibility that the pup's behavior was significantly influenced by overt responses or auditory cues of the stimulus rat. These results may suggest that preweanling pups are especially responsive to olfactory stimuli. This increased responsiveness may be derived from the fact that when day 14 pups are tested, their olfactory experiences appear to be limited almost entirely to odors associated with the nest. Therefore, unfamiliar odors in the environment may be more salient to preweanling pups than adult rats that have already experienced a variety of odors prior to testing. Current results, as well as those reported previously (44,45), indicate a pronounced tendency of rat pups to reduce their emission of ultrasonic vocalizations and freeze when exposed to an unfamiliar adult male rat. However, behavioral inhibition may not be the only defensive response elicited when the maturing rat encounters a threatening stimulus. Numerous studies indicate that animals are equipped with an array of defensive behavioral responses. The predominant behavioral response(s) is dependent on a number of factors including but not limited to predator types (21,37,40), environmental circumstances (36,40), and previous experiences (41). Thus, the propensity of rat pups to exhibit behavioral inhibition when encountering unfamiliar conspecifics may reflect the consequence that promoted survival in situations mimicked by the current test environment. ACKNOWLEDGEMENTS The research was supported by NIMH grant MH-43986 and grant from the Wisconsin Alumni Research Foundation. The author thanks H. Kim for technical assistance.
REFERENCES 1. Alberts, J. R.; Galef, B. G., Jr. Olfactory cues and movement: Stimuli mediating intraspecific aggression in the wild Norway rat. J. Comp. Physiol. Psychol. 85:233-242; 1973. 2. Allin, J. T.; Banks, E. M. Effects of temperature on ultrasound production by infant albino rats. Dev. Psychobiol. 4:149-156; 1971.
3. Allin, J. T.; Banks, E. M. Functional aspects of ultrasound production in infant albino rats. Anim. Behav. 20:175-185; 1972. 4. Blanchard, R. J.; Blanchard, D. C. Antipredator defensive behaviors in a visible burrow system. J. Comp. Psychol. 103:70-82; 1989. 5. Blanchard, R. J.; Blanchard, D. C.: Agullana, R.: Weiss, S. M.
P O T E N T I A T I O N OF B E H A V I O R A L INHIBITION IN R A T PUPS
Twenty-two kHz alarm cries to presentation of a predator, by laboratory rats living in visible burrow systems. Physiol. Behav. 50:967972; 1991. 6. Blanchard, R. J.; Mast, M.; Blanchard, D. C. Stimulus control of defensive reactions in the albino rat. J. Comp. Physiol. Psychol. 88:81-88; 1975. 7. Blumberg, M. S.; Alberts, J. R. Ultrasonic vocalizations by rat pups in the cold: An acoustic by-product of laryngeal braking? Behav. Neurosci. 104:808-817; 1990. 8. Bolles, R. C. Species-specific defense reactions and avoidance learning. Psychol. Rev. 77:32-48; 1970. 9. Brown, R. E. Social and hormonal factors influencing infanticide and its suppression in adult male Long-Evans rats (Rattus norvegicus). J. Comp. Psychol. 100:155-161; 1986. 10. Conklin, P.; Heggeness, F. W. Maturation of temperature homeostasis in the rat. Am. J. Physiol. 220:333-336; 1971. 11. Courtney, R. J.; Reid, L. D.; Wasden, R. E. Suppression of running times by olfactory stimuli. Psychon. Sci. 12:315-316; 1968. 12. Curio, E. The ethology of predation. New York: Springer-Verlag; 1976. 13. Curti, M. W. Native fear responses of white rats to the presence of cats. Psychol. Monogr. 46:78-98; 1935. 14. Elwood, R. W.; Kennedy, H. F.; Blakely, H. M. Responses of infant mice to odors of urine from infanticidal, noninfanticidal, and paternal male mice. Dev. Psychobiol. 23:309-317; 1990. 15. Ficken, M. S. Acoustic characteristics of alarm calls associated with predation risk in chickadees. Anim. Behav. 39:400-401; 1990. 16. Ficken, M. S.; Witkin, S. R. Responses of black-capped chickadees to predators. Auk 94:156-157; 1977. 17. Flannelly, K. J.; Thor, D. H. Territorial aggression of the rat to males castrated at various ages. Physiol. Behav. 20:785-789; 1978. 18. Gaddis, P. Mixed flocks, accipiters, and antipredator behavior. Condor 82:348-349; 1980. 19. Greig-Smith, P. W. Parental investment in nest defence by stonechats (Saxicola torquata). Anim. Behav. 28:604-619; 1980. 20. Griffith, C. R. The behavior of white rats in the presence of cats. Psychobiology 2:19-28; 1920. 21. Hennessy, D. F.; Owings, D. H. Snake species discrimination and the role of olfactory cues in the snake-directed behavior of the California ground squirrel. Behaviour 65:115-124; 1978. 22. Jones, R. B.; Mills, A. D.; Faure, J.-M. Genetic and experiential manipulation of fear-related behavior in Japanese quail chicks (Coturniz coturnixjaponica). J. Comp. Psychol. 105:15-24; 1991. 23. Kagan, J.; Reznick, J. S.; Snidman, N. Biological bases of childhood shyness. Science 240:167-171; 1988. 24. Kalin, N. H.; Shelton, S. E. Defensive behaviors in infant rhesus monkeys: Environmental cues and neurochemical regulation. Science 240:167-171; 1989. 25. Kalin, N. H.; Shelton, S. E.; Takahashi, L. K. Defensive behaviors in infant rhesus monkeys: Ontogeny and context-dependent selective expression. Child Dev. 62:1175-1183; 1991. 26. Lyons, D. M.; Banks, E. M. Ultrasounds in neonatal rats: Novel, predator and conspecific odor cues. Dev. Psychobiol. 15:455-460; 1982. 27. Magurran, A. E.; Girling, S. L. Predator model recognition and response habituation in shoaling minnows. Anim. Behav. 34:510518; 1986.
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28. Miller, D. B. Maternal vocal control of behavioral inhibition in mallard ducklings (Anas platyrhynchos). J. Comp. Physiol. Psychol. 94:606-623; 1980. 29. Montevecchi, W. A.; Gallup, G. G., Jr.; Dunlap, W. A. The peep vocalization in group reared chicks (Gallus domesticus): Its relation to fear. Anim. Behav., 21:116 - 123; 1973. 30. Morse, D. H. Interactions between tit flocks and sparrowhawks Accipiter nisus. Ibis 115:591-593; 1973. 31. Muller- Schwarze, D. Responses of young black-tailed deer to predator odors. J. Mammal. 53:393-394; 1972. 32. Murphy, L. B.; Wood-Gush, D. G. M. The interpretation of the behaviour of domestic fowl in strange environments. Biol. Behav. 3:39-61; 1978. 33. Noirot, E. Ultrasounds and maternal behavior in small rodents. Dev. Psychobiol. 5:371-387; 1972. 34. Palmer, W. Instinctive stillness in birds. Auk 26:23-36; 1909. 35. Paul, L.; Kupferschmidt, J. Killing of conspecific and mouse young by male rats. J. Comp. Physiol. Psychol. 88:755-763; 1975. 36. Rather, S. C. Comparative aspects of hypnosis. In: Gordon, J. E., ed. Handbook of clinical and experimental hypnosis. New York: Macmillian; 1967:550-587. 37. Robinson, S. R. Antipredator behaviour and predator recognition in Belding's ground squirrels. Anim. Behav. 28:840-852; 1980. 38. Rosenberg, K. M.; Denenberg, V. H.; Zarrow, M. X.; Frank, B. L. Effects of neonatal castration and testosterone on the rat's pup-killing behavior and activity. Physiol. Behav. 7:363-368; 1971. 39. Schaller, G. B. The Serengeti lion. Chicago: University of Chicago Press; 1972. 40. Seyfarth, R. M.; Cbeney, D. L.; Marler, P. Vervet monkey alarm call: Semantic communication in a free-ranging primate. Anim. Behav. 28:1070-1094; 1980. 41. Shalter, M. D. Predator-prey behavior and habituation. In: Peeke, H. V. S.; Petrinovich, L., eds. Habituation, sensitization, and behavior. New York: Academic Press; 1984:349-391. 42. Smotherman, W. P.; Bell, R. W.; Starzec, J.; Elias, J.; Zachman, T. A. Maternal responses to infant vocalizations and olfactory cues in rats and mice. Behav. Biol. 12:55-66; 1974. 43. Stoddart, D. M. Some responses of a free living community of rodents to the odors of predators. In: Muller-Schwarze, D.; Silverstein, R. M., eds. Chemical signals: Vertebrates and aquatic invertebrates. New York: Plenum Press; 1980:1-10. 44. Takahashi, L. K. Developmental expression of defensive responses during exposure to conspecific adults in preweanling rats (Rattus norvegicus). J. Comp. Psychol. 106:69-77; 1992. 45. Takahashi, L. K. Ontogeny of behavioral inhibition induced by unfamiliar adult male conspecifics in preweanling rats. Physiol. Behav. 52:493-498; 1992. 46. Takahashi, L. K.; Lore, R. K. Intermale and maternal aggression in adult rats tested at different ages. Physiol. Behav. 29:1013-1018; 1982. 47. Takahashi, L. K.; Rubin, W. W. Corticosteroid induction of threatinduced behavioral inhibition in preweanling rats. Behav. Neurosci. 107:860-866; 1993. 48. Thorpe, W. H. Learning and instinct in animals. London: Methuen; 1956. 49. Tuttle, M. D.; Ryan, M. J. Bat predation and the evolution of frog vocalizations in the neotropics. Science 214:677-678; 1981.