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Caffeine and copulatory experience: Interactive effects on social investigatory behavior
Physiology & Behavior, Vol. 36, pp. 707-711. Copyright©Pergamon Press Ltd., 1986. Printed in the U.S.A.
0031-9384/86 $3.00 + .00
Caffeine and Copulatory Experience: Interactive Effects on Social Investigatory Behavior D. H. T H O R A N D W. R. HOLLOWAY, JR. Edward R. J o h n s t o n e Training and R e s e a r c h Center, Bordentown, N J 08505 R e c e i v e d 12 J u l y 1985 THOR, D. H. AND W. R. HOLLOWAY, JR. Caffeine and copulatory experience: Interactive effects on social investigatory behavior. PHYSIOL BEHAV 36(4) 707-711, 1986.---Three experiments test the interaction of prior copulatory experience with acute caffeine exposure on social investigatory behavior of the male Norway rat, At a dosage of 20 mg/kg or greater, caffeine counteracts a decrease in social investigation attributed to copulatory experience. At a 10 mg/kg dosage, caffeine increases social investigatory behavior prior to sexual exposure but has no comparable effect after sexual exposure. The results are interpreted as confirming a long-term effect of copulatory exposure on social investigatory behavior and as describing a dose-related interaction of caffeine exposure with prior copulatory experience. Social investigation is viewed as a preliminary component of sexual behavior and the decrease in social investigation following copulatory experience as a gain in efficiency of social discrimination. Acute caffeine exposure apparently interferes with access or retrieval of reference information in long-term memory. Social investigation Long-term memory Rats Copulatory experience Drug-behavior interaction
Animal memory
S O C I A L investigation of a novel conspeciflc normally precedes other forms of social interaction (aggression, copulation, grooming). In the male Norway rat, such investigatory behavior is prolonged by endogenous or exogenous androgen exposure [12]. Perinatal androgenization of females also accentuates social investigatory behavior when exposed to androgen as an adult [14]. Furthermore, the gonadal condition of a male (intact or castrate) can interact with pharmacological effect on social investigatory behavior. F o r example, acute caffeine exposure prolongs social investigation by intact adult males, but castrate males and intact females are not comparably responsive to caffeine [6]. Prior social experience also affects the persistence of social investigatory behavior. Sexually experienced males engage in abbreviated social investigation of a novel conspecific (juvenile or castrate female adult) in comparison to sexually inexperienced males [13]. The latter finding was viewed as evidence for the long-term retention of information associated with initial copulatory experience and suggests that such experience can affect discrimination of social cues associated with female receptivity. Adult males have long been known to be influenced by copulatory experience in ways that alter their subsequent social behavior [1,2].
EXPERIMENT 1 In this experiment we examine the interaction of prior sexual experience and caffeine exposure on social investigatory behavior. We wished to determine the relative caffeine exposure effect on sexually experienced males that normally engage in less social investigatory behavior [13]. If
707
Social discrimination
caffeine interferes with reference to social cues linked to copulatory experience, the behavioral effect may be apparent in the comparison of social investigatory behavior by experienced and inexperienced males. On the other hand, if caffeine enhances the performance of a learned social discrimination, the behavioral effect may be to increase the disparity in social investigatory behavior between experienced and inexperienced males. No effect of caffeine on experienced males would suggest, counterintuitively, that the reported caffeine exposure effect on social investigatory behavior is limited to males with no copulatory experience. Method Subjects were 16 adult Long-Evans hooded rats 5 months of age. Eight males were sexually inexperienced and eight had been used as breeders with two 10-day exposures to adult females (each male housed with two females during each exposure). All males had been maintained in group cages of several males per cage prior to use in the present experiment. Stud males were known copulators (all exposed females produced normal litters). Social stimuli were juvenile Long-Evans hooded male rats 30-35 days of age at time of test. Exposure of sexually inexperienced males to castrate females as a procedural control was not incorporated since previous experiments have indicated no effect of such noncopulatory exposure to females on subsequent social investigatory behavior. Additionally, the postcopulatory effect on social investigation is demonstrable with juvenile social stimuli [13]. None of the male subjects had been exposed to juveniles as adults.
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FIG. 1. Mean social investigation time by sexually experienced and inexperienced male rats following administration of caffeine (20 mg/kg) or vehicle.
FIG. 2. Cumulative social investigation time over successive minutes of observation by sexually experienced and inexperienced male rats. Caffeine-treated groups did not differ at any time of observation and these data were collapsed.
Forty-eight hours prior to testing, each subject was placed in a clean polypropylene pan-type cage (41×51×22 cm) with wood chip litter and free access to food and water. All test observations were made in dim red light during the latter half of the normally dark phase of the 12:12 light-dark cycle (lights on at 2 p.m.). Frequency and duration measures were recorded on paper tape. Each subject was exposed to a male juvenile, in the subj e c t ' s home cage, after exposure to caffeine or saline vehicle. All subjects were tested after each treatment, and order of exposure was counterbalanced. Injection of the anyhydrous caffeine (Sigma, 20 mg/kg) or vehicle (0.9% NaC1) was subcutaneous in a volume of 2 ml/kg. All injections were given 30 min prior to test. Immediately prior to social exposure testing, the gross locomotor activity of each subject was recorded by counting the number of home-cage quadrants entered by the subject during a 2-min interval. After the activity measure, a novel juvenile male, 30-35 days of age, was placed in the home cage of the subject. (Each juvenile stimulus was used only once per day and when not in use as a social stimulus was maintained in a group cage with several other juveniles.) The behavior of the subject was then observed for a 10-min interval. Social investigation was recorded when the subject sniffed, followed or otherwise inspected the juvenile stimulus. Each interval of sustained investigative behavior was designated as an investigative bout, and the frequency and duration of such bouts were recorded on paper tape. Attempted mounts by the subject were also recorded. Upon completion of the test interval, the juvenile was removed and the subject remained isolated. Two days after the first test, each subject was tested again, in the same manner and at the same time of day, but following pretreatment with the alternate exposure to caffeine or vehicle.
Results
Caffeine administration increased social investigatory behavior, F(1,12)=65.41, p<0.001, and drug treatment interacted with sexual experience, F(1,12)=4.68, p<0.05 (see Fig. 1). The mean difference in social investigation by sexually experienced and nonexperienced males receiving only the vehicle was reliable, t(14)=2.85, p<0.01, confirming the original report of a long-term effect of copulatory experience, presumably mediated by associations formed during initial copulatory experience [13]. The result of the caffeine exposure was to increase mean social investigatory behavior of both treatment groups. Clearly, acute caffeine exposure counters the copulatory experience effect on persistence of social investigatory behavior when exposed to a novel juvenile conspecific. Caffeine exposure increased locomotor activity, F(1,12)= 131.21, p<0.001, and decreased mounting, F(1,12)=5.10, p<0.05. Means and variances are given in Table 1. Interactions of treatment with sexual experience were not reliable for activity and mounting measures. Experienced and nonexperienced male groups receiving caffeine did not differ reliably at any minute of observation and their data were thus collapsed for comparison with vehicle groups. One-way A N O V A s for each minute of observation indicated reliable mean differences (p<0.05) for minutes 2 through 9. During the initial minute of observation all groups engaged in comparably high levels of investigation and during the final minute of observation all groups engaged in comparably low levels of investigation. The caffeine treatment effect, as well as the copulatory experience effect, is readily apparent in the cumulative, group mean comparison described in Fig. 2. Mean frequency of investigative bouts was markedly
CAFFEINE AND COPULATORY EXPERIENCE
709
TABLE 1 LOCOMOTOR ACTIVITY, M O U N T I N G , AND SOCIAL I N V E S T I G A T I O N MEASURES OF S E X U A L L Y E X P E R I E N C E D AND N O N E X P E R I E N C E D M A L E RATS AFTER R E C E I V I N G C A F F E I N E OR V E H I C L E (MEAN - SE)
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E Non E
2.1 ± 0.9 1.8 ± 1.2
6.8 ± 1.5 2.5 ± 1.6
Investigation bout frequency
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32.6 ± 3.3 29.3 ± 2.8
20.1 ± 1.6 26.1 _+ 3. I
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FIG. 3. Mean social investigation time, time to criterion, activity and investigative bout frequency of sexually experienced male rats as a function of caffeine dosage.
greater with caffeine than with vehicle, F(1,12)=11.35, p<0.01. Sexual experience did not reliably affect bout frequency and the interaction of sexual experience with treatment was only marginally reliable, F(1,12) = 4.08, p <0.10. Mean bout frequency with vehicle was lower for sexually experienced than for sexually inexperienced males but the mean difference was not reliable. Means and variances are given in Table 1. Mean investigation time per investigative bout was not affected by dosage of caffeine, i.e., number of investigative episodes increased in parallel with total investigation time. An analysis of the present data confirms the finding of no reliable caffeine treatment effect on investigation time per investigative bout [5]. Similarly, the interaction of sexual experience with caffeine treatment was nonreliable in reference to the ratio scores. However, the mean ratio score for sexually nonexperienced males was reliably greater than that for sexually experienced males, F(1,12)=6.73, p<0.05. Means and variances are given in Table 1. EXPERIMENT 2
The results of Experiment 1 describe a substantial increase in the social investigatory behavior of sexually experienced male rats pretreated with 20 mg/kg of caffeine. In this experiment we examine the caffeine dose response curve in adult, sexually experienced male rats. Our purpose is to determine threshold dosage of caffeine that disinhibits social investigatory behavior in experienced males and to evaluate potential order effects in sequential exposures to caffeine.
prior to testing and throughout the testing period. Lab chow pellets and water were freely available throughout. Each subject was exposed to each dose of caffeine in ascending or descending order of magnitude; 5 males were assigned to each order. As in Experiment l, each subject was observed in its home cage, in dim red light, during the normally dark phase of the daily light-dark cycle. On daily tests, subjects were injected with anhydrous caffeine in saline vehicle. Dosages were 0, 5, 10, 20 and 40 mg/kg. Each injection was given 30 rain prior to test and each subject was observed for gross motor activity as described in Experiment 1. In this experiment our observation procedure was altered to conform to that of previously reported work [13]. Namely, we observed each subject to a criterion of neglect defined as 30 sec with no social behavior directed toward the social stimulus. Time to criterion (the interval from initial exposure to the stimulus to the end of the 30 consecutive seconds with no social behavior by the subject) has been defined as a measure of persistence in social investigation and has been used successfully by others in related work [11]. A hand-held panel of switches was used to record duration of social investigation, bouts of social investigation, and copulatory mount frequency on a paper tape recorder. Release of any switch closure activated a silent countdown timer that was automatically reset upon any subsequent switch closure that occurred within 30 sec. After the first 30-sec interval with no switch closure (criterion), a brief auditory cue was generated to signal termination of the observation interval. Results
Method Ten experimentally naive adult male Long-Evans hooded rats were used. Each male was approximately 5 months of age and had been used as a breeder with a 10-day exposure to four adult females. All males sired litters. Social stimuli were juvenile males 28-34 days of age at time of test. Each male subject was maintained in an individual cage for two days
Activity, social investigation time, time to criterion, and investigative bout frequency means for each dosage of caffeine exposure are given in Fig. 3. All measures of social investigation increased at the 20 mg/kg dose of caffeine. Caffeine-induced increase in locomotor activity was apparent at smaller dosages of 5 and 10 mg/kg. The data for each measure was analyzed by two-way re-
710
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FIG. 4. Interaction of caffeine (10 mg/kg) or vehicle administration and sexual experience in adult male rats. All subjects tested following caffeine and vehicle exposure before and after 5-day exposure to adult females.
peated measures A N O V A (Order x Dosage). All main effects and interactions of the order and mount frequency variables were nonsignificant. The main effect of caffeine dosage was reliable for total investigation time, time to criterion, investigative bout frequency and activity, Fs(4,32)= 12.04, 8.24, 8.46, and 26.72 (ps<0.001), respectively. Tukey HSD post hoc tests for paired mean comparisons indicated no reliable mean differences at 0, 5, and 10 mg/kg dosages on total investigation time, time to criterion, or investigative bout frequency. Similarly, no reliable mean differences were found on any of these measures between means at 20 and 40 mg/kg dosages. In each case, the higher dosages (20 and 40 mg/kg) differed reliably (p<0.05) from each of the lower dosages (0 and 5 mg/kg). Locomotor activity means differed reliably at lower dosages. Mean activity at dosages of 10, 20, and 40 mg/kg were all reliably greater (p<0.01) than vehicle control. A dosage of 10 mg/kg increased activity (p<0.01) over 5 mg/kg. Means at 20 and 40 mg/kg did not differ reliably, and means at 0 and 5 mg/kg did not differ reliably. These results, in addition to that of Experiment 1, describe a dose-related effect of acute caffeine exposure on social investigatory behavior of the sexually experienced adult male. At dosages of 10 mg/kg or less, there is no reliable increase over vehicle controls; at dosages of 20 mg/kg or greater there is a marked increase in persistence of investigatory behavior. EXPERIMENT 3 A caffeine dosage of 20 mg/kg disinhibits the effect of prior copulatory experience on social investigatory behavior (Experiment 1) and, in sexually experienced males, a 10 mg/kg caffeine dosage does not reliably increase social investigatory behavior (Experiment 2). Elsewhere, however, we have reported that, in sexually inexperienced adult (9
months old) males, a 10 mg/kg dosage of caffeine reliably increases social investigation [5]. 1"o further examine the interaction of caffeine exposure and sexual experience, we observed adult males following exposure to caffeine (10 mg/kg) and vehicle, before and after sustained exposure to adult females. Copulatory experience was expected to decrease the persistence of social investigatory behavior; caffeine exposure was expected to increase investigatory behavior, relative to vehicle, in precopulatory tests but to have little or no effect in postcopulatory tests. In effect, copulatory exposure to females was expected to counter the caffeine exposure effect of increased persistence in social investigatory behavior. Method
Subjects were 12 experimentally naive Long Evans male rats 4 months of age at the start of the experiment. Since weaning, all subjects were maintained in all-male groups of four per cage. Social stimuli were 28-32 day old male juveniles maintained since weaning in group cages. Each subject was individually housed two days prior to testing as described in Experiment 1. Social exposure tests to juvenile stimuli were made before and after each subject was housed with two nulliparous adult females for a 5-day interval. Social exposure tests were made as described in Experiment 2 following subcutaneous injection of vehicle or caffeine (10 mg/kg) on alternate days in counterbalanced order. Each subject was tested following each treatment before and after exposure to females. The second round of exposures was made commencing 6 days after removal of females from the home cage of each subject. Activity, mount frequency and social investigatory behaviors were observed, as described, to a 30-sec criterion of neglect. Restdl.v
Eight subjects impregnated both females and four subjects each impregnated one female. More than one copulatory series does not appear to proportionately decrease subsequent persistence in social investigatory behavior [13]. At present, it appears that sustained exposure (at least several days) to one or more females is necessary for an adult male to learn the social discrimination that is the basis of the observed difference in behavior between experienced and inexperienced males. The data were analyzed by a three-way A N O V A with repeated measures (Order x Pre-Post Copulatory Exposure Test x Caffeine-Vehicle Exposure). As in Experiment 2, all order effects were nonsignificant. Post hoc paired mean comparisons were made by Tukey HSD test. Means and variances for activity, social investigation time, time to criterion, and investigative bout frequency are given in Fig. 4. Copulatory exposure decreased each measure of investigatory behavior: Fs(1,10)=25.84, 10.78, 22.70 (ps<0.01). The effect of caffeine was to increase (p<0.01) all measures of investigation on precopulatory exposure and thus contribute to interactions (Pre-Post Copulatory Exposure Test z Caffeine-Vehicle Exposure); Fs(1,10)=8.87, 11.54, and 9.18 (ps<0.01). The main effect of caffeine was reliable, F(I, 10) = 12.24, p <0.01, for investigation bout frequency and marginally reliable (p<0.10) for investigation time and time to criterion. Caffeine exposure increased locomotor activity, F(I, 10) = 55.28, p <0.001, and interacted with sexual experience, F(1,10)=8.07, p<0.02. The main effect of sexual
CAFFEINE AND COPULATORY EXPERIENCE
711
experience was not reliable on the activity measure (as in Experiment l). Tukey HSD post hoc tests for paired mean comparisons indicated that caffeine exposure on precopulatory tests differed from saline exposure in its effect on investigation time (p<0.05), activity (p<0.01), bout frequency (p<0.01), and time to criterion (p<0.01). On postcopulatory tests, caffeine and saline exposure differed (p<0.01) only on the activity measure. Pre- and post-copulatory caffeine exposure differed (p<0.01) for investigation time, bout frequency, and time to criterion. Pre- and post-copulatory vehicle exposure differed for investigation time (p<0.01) and time to criterion
tp <0.05). The results of Experiment 3 confirm the effect of copulatory exposure on social investigatory behavior of the adult male rat as described elsewhere [13] and in the present series (Experiment 1). Exposure to 10 mg/kg of caffeine increased the persistence of social investigatory behavior in sexually inexperienced adult males but had no effect on sexually experienced adult males. In comparison, the activity measure suggests that the influence of copulatory experience on social investigatory behavior does not extend to the locomotor stimulant effect of caffeine. This interpretation is also supported by the results of Experiment 1.
GENERAL DISCUSSION Copulation decreases subsequent social investigatory behavior of young adult males when exposed to novel conspecifics, and the modification in social response is demonstrable by exposure to any ovariectomized female or juvenile. This robust effect suggests that sexual experience provides a reference for comparison of prior, sex-linked social cues with currently available social cues [8,10]. Such a reference capacity probably serves to increase the efficiency of social discrimination and thereby relieve males of the incumbency of expending surplus time and energy investigating conspecifics with minimal potential for reproductive engagement. The hormonal basis of sex-linked differences in social in-
vestigation [6, 12, 14] emphasizes the distinctive character of male behavior in premating interaction. Response to caffeine, for example, is contingent on gonadal testosterone; immature males, adult females, and gonadectomized adult males do not respond to caffeine with increased social investigation [10]. The present work further explicates a useful method for evaluating additional pharmacological interactions with long-term effects of prior copulatory experience. Threshold dosage of caffeine for increase in locomotor activity is lower than the threshold dosage for countering the effect of copulatory experience. This difference suggests that the effect of caffeine on social investigation is not simply a secondary effect of increased activity. Lack of order effects in Experiments 2 and 3 also suggest that caffeine effects on social investigation are temporary and reversible. All of these findings are consistent with other reports suggesting that caffeine effects on locomotor activity and social investigation are independent [5,6]. Furthermore, since caffeine defeats the effect of prior copulatory experience by an increase in behavior, this approach obviates the question-begging usually associated with blocking or inhibitory effects of drugs denoted by a decrease in behavior. Comparable directional effects are anticipated with other centrally active drugs capable of interfering with the accessing or retrieval of information in long-term memory. The sociosexual model herein outlined is founded on a long-term effect of initial copulatory experience. Presumably, social discrimination learning and social reference memory are intimately involved. Although we have purposively concentrated on the social investigatory behavior of males, there are certainly other aspects of social behavior that may be similarly affected [1, 3, 8, 9, 10]. This limitation, however, does not materially detract from the potential applicability of the model which may be capable of assessing neuroendocrinological as well as pharmacological interactions. Evidently, a major component of male social investigatory behavior is, in fact, sex-related, i.e., stimulusresponse contingencies during initial copulatory experience seem to provide a natural arrangement for gain in relevant social information that serves individual adaptation [4, 7, 9].
REFERENCES
1. Beach, F. A. Analysis of factors involved in the arousal, maintenance and manifestation of sexual excitement in male animals. Psychosom Med 4: 173-198, 1942. 2. Beach, F. A. Characteristics of masculine "sex drive." In: Nebraska Symposium on Motivation. edited by M. R. Jones. Lincoln, NE: University of Nebraska Press, 1956, pp. 1-31. 3. Carr, W, J. Phermonal sex attractants in the Norway rat. In: Advances in the Study of Communication and Affect: Nonverbal Communication, vol l, edited by L. Krames, P. Pliner and
T. Alloway. New York: Plenum Press, 1974. 4. File, S. E. The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J Neurosci Methods 2: 21%238, 1980. 5. Holloway, W. R., Jr. and D. H. Thor. Caffeine and social investigation in the adult male rat. Neurobehav Toxicol Teratol 5: 119--125, 1983. 6. Holloway, W. R., Jr. and D. H. Thor. Testosterone dependent effects of caffeine on social investigation by adult male rats. Physiol Behav 33: 95%964, 1984. 7. Landauer, M. R. and R. L. Balster. A new test for social investigation in mice: Effects of d-amphetamine. Psyehopharmaeology (Berlin) 78: 322-325, 1982.
8. Landauer, M. R., R. E. Wiese and W. J. Carr. Responses of sexually experienced and naive male rats to cues from receptive vs. nonreceptive females. Anita Learn Behav 5: 398-402, 1977. 9. Larsson, K. and E. H~d. Some aspects on behavioral teratology research. Scand J Psychol Suppl 1: 97-103, 1982. 10. Nyby, J. and G. Whitney. Experience affects behavioral responses to sex odors. In: Chemical Signals, edited by D. Miiller-Schwarze and R. M. Silverstein. New York: Plenum Press, 1980, pp. 173-192. 11. Sawyer, T. F., A. K. Hengehold and W. A. Perez. Chemosensory and hormonal mediation of social memory in male rats. Behav Neurosci 98: 908-913, 1984. 12. Thor, D. H. Testosterone and persistence of social investigation in laboratory rats. J Comp Physiol Psychol 94: 970--976, 1980. 13. Thor, D. H. and W. R. Holloway, Jr. Persistence of social investigatory behavior in the male rat: Evidence for long-term memory of initial copulatory experience. Anim Learn Behav 9: 561-565, 1981. 14. Thor, D. H., K. L. Wainwright and W. R. Hoiioway, Jr. Persistence of attention to a novel conspecific: Some developmental variables in laboratory rats. Dev Psychobiol 15: 1-8, 1982.
0031-9384/86 $3.00 + .00
Caffeine and Copulatory Experience: Interactive Effects on Social Investigatory Behavior D. H. T H O R A N D W. R. HOLLOWAY, JR. Edward R. J o h n s t o n e Training and R e s e a r c h Center, Bordentown, N J 08505 R e c e i v e d 12 J u l y 1985 THOR, D. H. AND W. R. HOLLOWAY, JR. Caffeine and copulatory experience: Interactive effects on social investigatory behavior. PHYSIOL BEHAV 36(4) 707-711, 1986.---Three experiments test the interaction of prior copulatory experience with acute caffeine exposure on social investigatory behavior of the male Norway rat, At a dosage of 20 mg/kg or greater, caffeine counteracts a decrease in social investigation attributed to copulatory experience. At a 10 mg/kg dosage, caffeine increases social investigatory behavior prior to sexual exposure but has no comparable effect after sexual exposure. The results are interpreted as confirming a long-term effect of copulatory exposure on social investigatory behavior and as describing a dose-related interaction of caffeine exposure with prior copulatory experience. Social investigation is viewed as a preliminary component of sexual behavior and the decrease in social investigation following copulatory experience as a gain in efficiency of social discrimination. Acute caffeine exposure apparently interferes with access or retrieval of reference information in long-term memory. Social investigation Long-term memory Rats Copulatory experience Drug-behavior interaction
Animal memory
S O C I A L investigation of a novel conspeciflc normally precedes other forms of social interaction (aggression, copulation, grooming). In the male Norway rat, such investigatory behavior is prolonged by endogenous or exogenous androgen exposure [12]. Perinatal androgenization of females also accentuates social investigatory behavior when exposed to androgen as an adult [14]. Furthermore, the gonadal condition of a male (intact or castrate) can interact with pharmacological effect on social investigatory behavior. F o r example, acute caffeine exposure prolongs social investigation by intact adult males, but castrate males and intact females are not comparably responsive to caffeine [6]. Prior social experience also affects the persistence of social investigatory behavior. Sexually experienced males engage in abbreviated social investigation of a novel conspecific (juvenile or castrate female adult) in comparison to sexually inexperienced males [13]. The latter finding was viewed as evidence for the long-term retention of information associated with initial copulatory experience and suggests that such experience can affect discrimination of social cues associated with female receptivity. Adult males have long been known to be influenced by copulatory experience in ways that alter their subsequent social behavior [1,2].
EXPERIMENT 1 In this experiment we examine the interaction of prior sexual experience and caffeine exposure on social investigatory behavior. We wished to determine the relative caffeine exposure effect on sexually experienced males that normally engage in less social investigatory behavior [13]. If
707
Social discrimination
caffeine interferes with reference to social cues linked to copulatory experience, the behavioral effect may be apparent in the comparison of social investigatory behavior by experienced and inexperienced males. On the other hand, if caffeine enhances the performance of a learned social discrimination, the behavioral effect may be to increase the disparity in social investigatory behavior between experienced and inexperienced males. No effect of caffeine on experienced males would suggest, counterintuitively, that the reported caffeine exposure effect on social investigatory behavior is limited to males with no copulatory experience. Method Subjects were 16 adult Long-Evans hooded rats 5 months of age. Eight males were sexually inexperienced and eight had been used as breeders with two 10-day exposures to adult females (each male housed with two females during each exposure). All males had been maintained in group cages of several males per cage prior to use in the present experiment. Stud males were known copulators (all exposed females produced normal litters). Social stimuli were juvenile Long-Evans hooded male rats 30-35 days of age at time of test. Exposure of sexually inexperienced males to castrate females as a procedural control was not incorporated since previous experiments have indicated no effect of such noncopulatory exposure to females on subsequent social investigatory behavior. Additionally, the postcopulatory effect on social investigation is demonstrable with juvenile social stimuli [13]. None of the male subjects had been exposed to juveniles as adults.
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FIG. 1. Mean social investigation time by sexually experienced and inexperienced male rats following administration of caffeine (20 mg/kg) or vehicle.
FIG. 2. Cumulative social investigation time over successive minutes of observation by sexually experienced and inexperienced male rats. Caffeine-treated groups did not differ at any time of observation and these data were collapsed.
Forty-eight hours prior to testing, each subject was placed in a clean polypropylene pan-type cage (41×51×22 cm) with wood chip litter and free access to food and water. All test observations were made in dim red light during the latter half of the normally dark phase of the 12:12 light-dark cycle (lights on at 2 p.m.). Frequency and duration measures were recorded on paper tape. Each subject was exposed to a male juvenile, in the subj e c t ' s home cage, after exposure to caffeine or saline vehicle. All subjects were tested after each treatment, and order of exposure was counterbalanced. Injection of the anyhydrous caffeine (Sigma, 20 mg/kg) or vehicle (0.9% NaC1) was subcutaneous in a volume of 2 ml/kg. All injections were given 30 min prior to test. Immediately prior to social exposure testing, the gross locomotor activity of each subject was recorded by counting the number of home-cage quadrants entered by the subject during a 2-min interval. After the activity measure, a novel juvenile male, 30-35 days of age, was placed in the home cage of the subject. (Each juvenile stimulus was used only once per day and when not in use as a social stimulus was maintained in a group cage with several other juveniles.) The behavior of the subject was then observed for a 10-min interval. Social investigation was recorded when the subject sniffed, followed or otherwise inspected the juvenile stimulus. Each interval of sustained investigative behavior was designated as an investigative bout, and the frequency and duration of such bouts were recorded on paper tape. Attempted mounts by the subject were also recorded. Upon completion of the test interval, the juvenile was removed and the subject remained isolated. Two days after the first test, each subject was tested again, in the same manner and at the same time of day, but following pretreatment with the alternate exposure to caffeine or vehicle.
Results
Caffeine administration increased social investigatory behavior, F(1,12)=65.41, p<0.001, and drug treatment interacted with sexual experience, F(1,12)=4.68, p<0.05 (see Fig. 1). The mean difference in social investigation by sexually experienced and nonexperienced males receiving only the vehicle was reliable, t(14)=2.85, p<0.01, confirming the original report of a long-term effect of copulatory experience, presumably mediated by associations formed during initial copulatory experience [13]. The result of the caffeine exposure was to increase mean social investigatory behavior of both treatment groups. Clearly, acute caffeine exposure counters the copulatory experience effect on persistence of social investigatory behavior when exposed to a novel juvenile conspecific. Caffeine exposure increased locomotor activity, F(1,12)= 131.21, p<0.001, and decreased mounting, F(1,12)=5.10, p<0.05. Means and variances are given in Table 1. Interactions of treatment with sexual experience were not reliable for activity and mounting measures. Experienced and nonexperienced male groups receiving caffeine did not differ reliably at any minute of observation and their data were thus collapsed for comparison with vehicle groups. One-way A N O V A s for each minute of observation indicated reliable mean differences (p<0.05) for minutes 2 through 9. During the initial minute of observation all groups engaged in comparably high levels of investigation and during the final minute of observation all groups engaged in comparably low levels of investigation. The caffeine treatment effect, as well as the copulatory experience effect, is readily apparent in the cumulative, group mean comparison described in Fig. 2. Mean frequency of investigative bouts was markedly
CAFFEINE AND COPULATORY EXPERIENCE
709
TABLE 1 LOCOMOTOR ACTIVITY, M O U N T I N G , AND SOCIAL I N V E S T I G A T I O N MEASURES OF S E X U A L L Y E X P E R I E N C E D AND N O N E X P E R I E N C E D M A L E RATS AFTER R E C E I V I N G C A F F E I N E OR V E H I C L E (MEAN - SE)
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E Non E
2.1 ± 0.9 1.8 ± 1.2
6.8 ± 1.5 2.5 ± 1.6
Investigation bout frequency
E Non E
32.6 ± 3.3 29.3 ± 2.8
20.1 ± 1.6 26.1 _+ 3. I
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FIG. 3. Mean social investigation time, time to criterion, activity and investigative bout frequency of sexually experienced male rats as a function of caffeine dosage.
greater with caffeine than with vehicle, F(1,12)=11.35, p<0.01. Sexual experience did not reliably affect bout frequency and the interaction of sexual experience with treatment was only marginally reliable, F(1,12) = 4.08, p <0.10. Mean bout frequency with vehicle was lower for sexually experienced than for sexually inexperienced males but the mean difference was not reliable. Means and variances are given in Table 1. Mean investigation time per investigative bout was not affected by dosage of caffeine, i.e., number of investigative episodes increased in parallel with total investigation time. An analysis of the present data confirms the finding of no reliable caffeine treatment effect on investigation time per investigative bout [5]. Similarly, the interaction of sexual experience with caffeine treatment was nonreliable in reference to the ratio scores. However, the mean ratio score for sexually nonexperienced males was reliably greater than that for sexually experienced males, F(1,12)=6.73, p<0.05. Means and variances are given in Table 1. EXPERIMENT 2
The results of Experiment 1 describe a substantial increase in the social investigatory behavior of sexually experienced male rats pretreated with 20 mg/kg of caffeine. In this experiment we examine the caffeine dose response curve in adult, sexually experienced male rats. Our purpose is to determine threshold dosage of caffeine that disinhibits social investigatory behavior in experienced males and to evaluate potential order effects in sequential exposures to caffeine.
prior to testing and throughout the testing period. Lab chow pellets and water were freely available throughout. Each subject was exposed to each dose of caffeine in ascending or descending order of magnitude; 5 males were assigned to each order. As in Experiment l, each subject was observed in its home cage, in dim red light, during the normally dark phase of the daily light-dark cycle. On daily tests, subjects were injected with anhydrous caffeine in saline vehicle. Dosages were 0, 5, 10, 20 and 40 mg/kg. Each injection was given 30 rain prior to test and each subject was observed for gross motor activity as described in Experiment 1. In this experiment our observation procedure was altered to conform to that of previously reported work [13]. Namely, we observed each subject to a criterion of neglect defined as 30 sec with no social behavior directed toward the social stimulus. Time to criterion (the interval from initial exposure to the stimulus to the end of the 30 consecutive seconds with no social behavior by the subject) has been defined as a measure of persistence in social investigation and has been used successfully by others in related work [11]. A hand-held panel of switches was used to record duration of social investigation, bouts of social investigation, and copulatory mount frequency on a paper tape recorder. Release of any switch closure activated a silent countdown timer that was automatically reset upon any subsequent switch closure that occurred within 30 sec. After the first 30-sec interval with no switch closure (criterion), a brief auditory cue was generated to signal termination of the observation interval. Results
Method Ten experimentally naive adult male Long-Evans hooded rats were used. Each male was approximately 5 months of age and had been used as a breeder with a 10-day exposure to four adult females. All males sired litters. Social stimuli were juvenile males 28-34 days of age at time of test. Each male subject was maintained in an individual cage for two days
Activity, social investigation time, time to criterion, and investigative bout frequency means for each dosage of caffeine exposure are given in Fig. 3. All measures of social investigation increased at the 20 mg/kg dose of caffeine. Caffeine-induced increase in locomotor activity was apparent at smaller dosages of 5 and 10 mg/kg. The data for each measure was analyzed by two-way re-
710
I H O R AND HOIA~OWAY
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FIG. 4. Interaction of caffeine (10 mg/kg) or vehicle administration and sexual experience in adult male rats. All subjects tested following caffeine and vehicle exposure before and after 5-day exposure to adult females.
peated measures A N O V A (Order x Dosage). All main effects and interactions of the order and mount frequency variables were nonsignificant. The main effect of caffeine dosage was reliable for total investigation time, time to criterion, investigative bout frequency and activity, Fs(4,32)= 12.04, 8.24, 8.46, and 26.72 (ps<0.001), respectively. Tukey HSD post hoc tests for paired mean comparisons indicated no reliable mean differences at 0, 5, and 10 mg/kg dosages on total investigation time, time to criterion, or investigative bout frequency. Similarly, no reliable mean differences were found on any of these measures between means at 20 and 40 mg/kg dosages. In each case, the higher dosages (20 and 40 mg/kg) differed reliably (p<0.05) from each of the lower dosages (0 and 5 mg/kg). Locomotor activity means differed reliably at lower dosages. Mean activity at dosages of 10, 20, and 40 mg/kg were all reliably greater (p<0.01) than vehicle control. A dosage of 10 mg/kg increased activity (p<0.01) over 5 mg/kg. Means at 20 and 40 mg/kg did not differ reliably, and means at 0 and 5 mg/kg did not differ reliably. These results, in addition to that of Experiment 1, describe a dose-related effect of acute caffeine exposure on social investigatory behavior of the sexually experienced adult male. At dosages of 10 mg/kg or less, there is no reliable increase over vehicle controls; at dosages of 20 mg/kg or greater there is a marked increase in persistence of investigatory behavior. EXPERIMENT 3 A caffeine dosage of 20 mg/kg disinhibits the effect of prior copulatory experience on social investigatory behavior (Experiment 1) and, in sexually experienced males, a 10 mg/kg caffeine dosage does not reliably increase social investigatory behavior (Experiment 2). Elsewhere, however, we have reported that, in sexually inexperienced adult (9
months old) males, a 10 mg/kg dosage of caffeine reliably increases social investigation [5]. 1"o further examine the interaction of caffeine exposure and sexual experience, we observed adult males following exposure to caffeine (10 mg/kg) and vehicle, before and after sustained exposure to adult females. Copulatory experience was expected to decrease the persistence of social investigatory behavior; caffeine exposure was expected to increase investigatory behavior, relative to vehicle, in precopulatory tests but to have little or no effect in postcopulatory tests. In effect, copulatory exposure to females was expected to counter the caffeine exposure effect of increased persistence in social investigatory behavior. Method
Subjects were 12 experimentally naive Long Evans male rats 4 months of age at the start of the experiment. Since weaning, all subjects were maintained in all-male groups of four per cage. Social stimuli were 28-32 day old male juveniles maintained since weaning in group cages. Each subject was individually housed two days prior to testing as described in Experiment 1. Social exposure tests to juvenile stimuli were made before and after each subject was housed with two nulliparous adult females for a 5-day interval. Social exposure tests were made as described in Experiment 2 following subcutaneous injection of vehicle or caffeine (10 mg/kg) on alternate days in counterbalanced order. Each subject was tested following each treatment before and after exposure to females. The second round of exposures was made commencing 6 days after removal of females from the home cage of each subject. Activity, mount frequency and social investigatory behaviors were observed, as described, to a 30-sec criterion of neglect. Restdl.v
Eight subjects impregnated both females and four subjects each impregnated one female. More than one copulatory series does not appear to proportionately decrease subsequent persistence in social investigatory behavior [13]. At present, it appears that sustained exposure (at least several days) to one or more females is necessary for an adult male to learn the social discrimination that is the basis of the observed difference in behavior between experienced and inexperienced males. The data were analyzed by a three-way A N O V A with repeated measures (Order x Pre-Post Copulatory Exposure Test x Caffeine-Vehicle Exposure). As in Experiment 2, all order effects were nonsignificant. Post hoc paired mean comparisons were made by Tukey HSD test. Means and variances for activity, social investigation time, time to criterion, and investigative bout frequency are given in Fig. 4. Copulatory exposure decreased each measure of investigatory behavior: Fs(1,10)=25.84, 10.78, 22.70 (ps<0.01). The effect of caffeine was to increase (p<0.01) all measures of investigation on precopulatory exposure and thus contribute to interactions (Pre-Post Copulatory Exposure Test z Caffeine-Vehicle Exposure); Fs(1,10)=8.87, 11.54, and 9.18 (ps<0.01). The main effect of caffeine was reliable, F(I, 10) = 12.24, p <0.01, for investigation bout frequency and marginally reliable (p<0.10) for investigation time and time to criterion. Caffeine exposure increased locomotor activity, F(I, 10) = 55.28, p <0.001, and interacted with sexual experience, F(1,10)=8.07, p<0.02. The main effect of sexual
CAFFEINE AND COPULATORY EXPERIENCE
711
experience was not reliable on the activity measure (as in Experiment l). Tukey HSD post hoc tests for paired mean comparisons indicated that caffeine exposure on precopulatory tests differed from saline exposure in its effect on investigation time (p<0.05), activity (p<0.01), bout frequency (p<0.01), and time to criterion (p<0.01). On postcopulatory tests, caffeine and saline exposure differed (p<0.01) only on the activity measure. Pre- and post-copulatory caffeine exposure differed (p<0.01) for investigation time, bout frequency, and time to criterion. Pre- and post-copulatory vehicle exposure differed for investigation time (p<0.01) and time to criterion
tp <0.05). The results of Experiment 3 confirm the effect of copulatory exposure on social investigatory behavior of the adult male rat as described elsewhere [13] and in the present series (Experiment 1). Exposure to 10 mg/kg of caffeine increased the persistence of social investigatory behavior in sexually inexperienced adult males but had no effect on sexually experienced adult males. In comparison, the activity measure suggests that the influence of copulatory experience on social investigatory behavior does not extend to the locomotor stimulant effect of caffeine. This interpretation is also supported by the results of Experiment 1.
GENERAL DISCUSSION Copulation decreases subsequent social investigatory behavior of young adult males when exposed to novel conspecifics, and the modification in social response is demonstrable by exposure to any ovariectomized female or juvenile. This robust effect suggests that sexual experience provides a reference for comparison of prior, sex-linked social cues with currently available social cues [8,10]. Such a reference capacity probably serves to increase the efficiency of social discrimination and thereby relieve males of the incumbency of expending surplus time and energy investigating conspecifics with minimal potential for reproductive engagement. The hormonal basis of sex-linked differences in social in-
vestigation [6, 12, 14] emphasizes the distinctive character of male behavior in premating interaction. Response to caffeine, for example, is contingent on gonadal testosterone; immature males, adult females, and gonadectomized adult males do not respond to caffeine with increased social investigation [10]. The present work further explicates a useful method for evaluating additional pharmacological interactions with long-term effects of prior copulatory experience. Threshold dosage of caffeine for increase in locomotor activity is lower than the threshold dosage for countering the effect of copulatory experience. This difference suggests that the effect of caffeine on social investigation is not simply a secondary effect of increased activity. Lack of order effects in Experiments 2 and 3 also suggest that caffeine effects on social investigation are temporary and reversible. All of these findings are consistent with other reports suggesting that caffeine effects on locomotor activity and social investigation are independent [5,6]. Furthermore, since caffeine defeats the effect of prior copulatory experience by an increase in behavior, this approach obviates the question-begging usually associated with blocking or inhibitory effects of drugs denoted by a decrease in behavior. Comparable directional effects are anticipated with other centrally active drugs capable of interfering with the accessing or retrieval of information in long-term memory. The sociosexual model herein outlined is founded on a long-term effect of initial copulatory experience. Presumably, social discrimination learning and social reference memory are intimately involved. Although we have purposively concentrated on the social investigatory behavior of males, there are certainly other aspects of social behavior that may be similarly affected [1, 3, 8, 9, 10]. This limitation, however, does not materially detract from the potential applicability of the model which may be capable of assessing neuroendocrinological as well as pharmacological interactions. Evidently, a major component of male social investigatory behavior is, in fact, sex-related, i.e., stimulusresponse contingencies during initial copulatory experience seem to provide a natural arrangement for gain in relevant social information that serves individual adaptation [4, 7, 9].
REFERENCES
1. Beach, F. A. Analysis of factors involved in the arousal, maintenance and manifestation of sexual excitement in male animals. Psychosom Med 4: 173-198, 1942. 2. Beach, F. A. Characteristics of masculine "sex drive." In: Nebraska Symposium on Motivation. edited by M. R. Jones. Lincoln, NE: University of Nebraska Press, 1956, pp. 1-31. 3. Carr, W, J. Phermonal sex attractants in the Norway rat. In: Advances in the Study of Communication and Affect: Nonverbal Communication, vol l, edited by L. Krames, P. Pliner and
T. Alloway. New York: Plenum Press, 1974. 4. File, S. E. The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J Neurosci Methods 2: 21%238, 1980. 5. Holloway, W. R., Jr. and D. H. Thor. Caffeine and social investigation in the adult male rat. Neurobehav Toxicol Teratol 5: 119--125, 1983. 6. Holloway, W. R., Jr. and D. H. Thor. Testosterone dependent effects of caffeine on social investigation by adult male rats. Physiol Behav 33: 95%964, 1984. 7. Landauer, M. R. and R. L. Balster. A new test for social investigation in mice: Effects of d-amphetamine. Psyehopharmaeology (Berlin) 78: 322-325, 1982.
8. Landauer, M. R., R. E. Wiese and W. J. Carr. Responses of sexually experienced and naive male rats to cues from receptive vs. nonreceptive females. Anita Learn Behav 5: 398-402, 1977. 9. Larsson, K. and E. H~d. Some aspects on behavioral teratology research. Scand J Psychol Suppl 1: 97-103, 1982. 10. Nyby, J. and G. Whitney. Experience affects behavioral responses to sex odors. In: Chemical Signals, edited by D. Miiller-Schwarze and R. M. Silverstein. New York: Plenum Press, 1980, pp. 173-192. 11. Sawyer, T. F., A. K. Hengehold and W. A. Perez. Chemosensory and hormonal mediation of social memory in male rats. Behav Neurosci 98: 908-913, 1984. 12. Thor, D. H. Testosterone and persistence of social investigation in laboratory rats. J Comp Physiol Psychol 94: 970--976, 1980. 13. Thor, D. H. and W. R. Holloway, Jr. Persistence of social investigatory behavior in the male rat: Evidence for long-term memory of initial copulatory experience. Anim Learn Behav 9: 561-565, 1981. 14. Thor, D. H., K. L. Wainwright and W. R. Hoiioway, Jr. Persistence of attention to a novel conspecific: Some developmental variables in laboratory rats. Dev Psychobiol 15: 1-8, 1982.