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Influence of blinding and home-cage lighting on aggressive behaviors of laboratory rats
Physiology & Behavior, Vol. 25, pp. 217-226. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.
Influence of Blinding and Home-Cage Lighting on Aggressive Behaviors of Laboratory Rats D A V I D J. F O R D Y C E
A N D J O H N F. K N U T S O N
D e p a r t m e n t o f Psychology, The University o f Iowa, Iowa City, IA 52242 R e c e i v e d 25 O c t o b e r 1979 FORDYCE, D. J. AND J. F. KNUTSON. Influence of blinding and home-cage lighting on aggressive behaviors of laboratory rats. PHYSIOL. BEHAV. 25(2) 217-226, 1980.--Two experiments were conducted on the aggressive behavior of laboratory rats. In Experiment I, blinded and sighted subjects were raised with blinded or sighted cagemates in a colony maintained under a light-dark cycle. Although blinding affected the light synchronized rhythms in home-cage and shockinduced aggression, it did not affect the overall occurrence of these two behaviors. In Experiment 2, blinded and sighted subjects were raised with blinded or sighted cagemates in a colony maintained under continuous lighting (LL). The results of this experiment suggested that reduced aggression in rats from LL conditions is due to an LL induced state of the subject rather than social exposures during development. However, blinded rats reared with LL cagemates evidenced reduced shock-induced aggression, presumably due to reduced social experiences during development that were associated with being raised with LL rats. Shock-induced aggression
Home-cage aggression
T H E lighting conditions under which laboratory rats develop has been shown to influence aggression in adulthood [19,20]. Subjects raised under light-dark (LD) conditions displayed 24-hr periodic trends in home-cage [19] and shock-induced aggression [20] that were similar to the circadian rhythms reported for drinking behavior [31], eating behavior [37], and general activity [32]. Groups housed under continuous light (LL) did not manifest synchronized periodic trends in either home-cage or shock-induced fighting. The data of the Kane and Knutson [19] and Knutson et al. [20] studies also indicated that LD-reared groups generally aggress more than L L groups. Because Kane and Knutson [19] were able to demonstrate a significant correlational relationship between home-cage and shock-induced aggression in LD-reared subjects, the results of these studies led Kane and Knutson [19] to hypothesize that lighting cycles might control aggressive behavior in the home cage which may, in turn, determine performance in the shock-induced aggression paradigm. Support for the hypothesis that home-cage experience may influence shock-induced aggression was provided by recent studies by Kane [18] and Knutson, Kane, Schlosberg, Fordyce, and Simansky [21]. Intact rats raised with castrated cagemates since weaning displayed less shock-induced aggression in adulthood than rats raised with intact cagemates. These data suggest the possibility that reduced agonistic experiences that can result from living with castrated cagemates reduces later shock-induced aggression. It is possible that the tendency for less shock-induced aggression to be manifested by L L housed subjects [20] reflects reduced social experiences during development that are a consequence of the circadian asynchrony of L L rats.
Blinding
Light cycle
Social experience
Rats
That is, in the absence of the dominant Zeitgeber of a light cycle, rhythmic patterns in subjects maintained under constant illumination should be asynchronous and the probability of social interactions occurring among L L cagemates would be less than under LD conditions, where the rhythmic behavior patterns of cagemates are synchronized. Based on the work of Cairns [8] and Calhoun [9] on activity states and aggression, the asynchrony associated with constant lighting could be the factor that determines the reduced home-cage aggressive experiences of L L reared subjects [ 19] and that the reduced aggression during development results in less shock-induced aggression in adulthood. However, it is also possible that the lower rates of shock-induced aggression are a consequence of the momentary asynchrony of the subjects. That is, the asynchronous developmental history might not affect shock-induced aggression, but asynchrony between tested pair members could result in less aggression, since the performance of individual pair members contributes to the occurrence of shock-induced aggression [30]. EXPERIMENT 1 The purpose of this experiment was to determine whether behavioral synchrony at the time of testing or during development affected home-cage and shock-induced aggression. Since blinding can produce a free-running rhythm in some subjects through constant lighting conditions (DD) while sighted subjects in the same environment would be synchronized to an LD cycle, the use of sighted and blinded subjects reared with either blinded or sighted cagemates in a
1This research was supported in part by USPHS training grant No. MH05062. The assistance of Neal Kane and Kenneth E. Wahlstrand is gratefully acknowledged.
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/080217-10502.00/0
218
FORDYCE AND K N U T S O N
factorial design permits an assessment of the contribution of subject and cagemate rhythms to home-cage and shockinduced aggression. In addition to assessing the role of light controlled social asynchrony in aggression, this experiment would permit an assessment of the effect of an absence of visual experience on aggressive behavior. Several investigations have reported that blinding in adulthood has no effect on the shock-induced aggressive behavior of rats [7, 12, 14], but the impact of the absence of visual experience on shockinduced and home-cage aggression has not been reported. METHOD
Subjects Rats used in this experiment came from an original group of 193 experimentally naive male hooded rats from the colony of the Department of Psychology at The University of Iowa. From this group, 32 rats served as experimental subjects and 96 were designated as the cagemates of these subjects. Mortality throughout the course of the study included 21 sighted and 32 blinded rats. This difference was not statistically significant (X~=0.358, df= 1). Since the majority of deaths occurred within a few days of surgery, composition of the experimental cages was arranged after the high mortality period had passed. One cagemate from each of five cages distributed among groups died during the course of the experiment. Since adding a new rat to the cage could have an impact on the home-cage aggression [4], these cagemates were not replaced. The remaining rats of the original group served exclusively as targets in shock-induced aggression testing.
Apparatus Shock-induced aggression testing was conducted in a 22.68x29.21x 19.01 cm aggression test chamber constructed of 6.35 mm clear Plexiglas and a grid floor consisting of 2.38 mm diameter stainless steel rods spaced 1.27 cm center to center. To minimize the contribution of the target to variance in the experimental conditions, the restrained target procedure described by Follick and Knutson [13] was adopted. The aggression chamber was positioned behind an observation window in an Industrial Acoustics Corporation (New York, NY) 2.44x2.44x2.13 m sound-attenuating chamber. Shock was delivered to the grid floor by means of a tube-type constant current DC power supply scrambled through a BRS multiple relay scrambler. Shock to the tail of the target was generated by a tube-type DC power supply and transmitted through leads attached to the tail by modified alligator clips placed approximately 2.5 and 5 cm from the base of the tail. Shock sources did not share a common ground, precluding transfer of shock between members of a fighting pair. Vinyl tape covering the grids beneath the target and moleskin covering the feet of the target provided insulation from the subject-shock circuit. Shock to the target and to the subject was set at 2 mA, using a 22 K ohm resistor. This level of resistance is roughly equivalent to that which would be produced by an adult male rat receiving a 2 mA shock [10]. Shock frequency and duration were programmed using conventional electronic timers and relay circuitry, and shockinduced aggression data were recorded on electromechanical counters. Drinking in the home cages was sampled as a measure of cage synchrony using conventional contact relays, electronic timers, relay circuitry, and electromechanical counters. The
drinking tube, insulated with shrinkable tubing, served as one pole of the contact relay while the cage served as the other pole. Drinking was recorded in eight 3-hr bins within each 24-hr period. The recording equipment was located in another room to eliminate auditory cues and experimenter activity in the colony rooms.
Procedure In a series of four replications, rats were bred and pups whelped in a colony room maintained on an LD 12:12 lighting cycle (lights on 0700-1900). Since the aggressive behaviors of the laboratory rat begin to emerge at about 17 days of age [2,6], the procedures were scheduled to maximize social asynchrony during this early period. Rats begin to open their eyes at about 14 days of age [6], and the available evidence suggests that blinding at this age produces relatively immediate free running of circadian rhythms [33,37]. Based on these data, rat pups were blinded at 15 days of age, before most had opened their eyes. Since it has been reported that the maternal behavior of a lactating rat dam can act as a Zeitgeber entraining the behaviors of rat pups [25], pups were also weaned at 15 days of age. At weaning, the pups were randomly assigned to either a blinded or sham-operated condition. Rats assigned to the blinded condition were placed under ether anesthesia and submitted to bilateral enucleation. The eyelids, if closed, were separated with eye-dressing forceps and each globe was protruded with a small hemostat. A small curved scissor was used to sever the musculature and optic nerve. Following removal of the eye, the socket was packed with absorbable gelatin sponge coated with a 2% nitrofurazone topical antibiotic powder. Immediately following surgery, each rat was placed with 7 to 14 other pups in a 43 cm dia. recovery chamber. The entire surgical procedure required about 3.5 rain. Rats assigned to the sham-operated condition were submitted to ether anesthesia and the recovery treatment, but were not surgically manipulated. During surgery and recovery a 250 W infrared heat lamp was used to maintain the surface temperature at about 30°C. After surgery, all rat pups were transferred in the recovery chambers to a post-weaning room. The post-weaning room was maintained on the same LD light schedule as the breeding colony and the colony rooms used to house the rats of the experiment. A food mash of ground Teklad (Winfield, IA) pellets and diluted evaporated milk was available ad lib in shallow 30 ml saucers. During the first two days following surgery, each pup was fed this mixture twice per day by an experimenter placing a small portion of the mash on the tongue of the rat. At feeding times, the anal region of each rat was wiped clean of fecal material using a warm damp cloth. Feeding and cleaning sessions were distributed across the 12-hr light period to reduce the possibility of a regular laboratory routine entraining the rhythms of blinded pups. At 17 days of age, the rats were transferred from the recovery chambers and housed according to visual status in 24x40.6x 17.8 cm stainless steel and wire mesh cages. In addition to the mash, solid Teklad pellets were placed within each cage and water was supplied ad lib from an externally mounted water bottle. While the contents of the mash bowls were replaced twice per day on an aperiodic schedule during this period, the pups were neither hand fed nor cleaned. At 20 days of age the rats were rehoused in groups of four according to group membership in 24 ×40.6x 17.8 cm stainless steel and wire mesh cages; the external heat sources
INFLUENCE OF BLINDING were eliminated leaving the ambient room temperature at 22 ( _+ 2)°C. Blinded subjects were either housed with three blinded cagemates (Group BB) or three sighted cagemates (Group BS), and sighted subjects were housed with either three blinded cagemates (Group SB) or three sighted cagemates (Group SS). When the subjects reached 26 days of age, the cages were transferred from the post-weaning room to one of three experimental rooms where they were placed on racks. Cages were distributed among rooms so that each room housed cages from each of the four experimental conditions. Rats had free access to Teklad food pellets placed on the floor of the cage, and water was available from an externally mounted bottle. Daily maintenance activities within each room were randomly distributed between 0700 and 1700 to reduce the possibility of entrainment to a laboratory routine.
Home-Cage Aggression Observations Home-cage observations were conducted at two developmental ages, when the rats were 36-41 days of age and 65-70 days of age (Periods 1 and 2, respectively). Three days prior to the initiation of home-cage observations, the experimental subject within each cage was weighed and marked with black hair dye on the lateral surfaces of the body. To assess home-cage aggression, each cage was observed for 10 min at 0200, 0800, 1400, and 2000 ( +__30 min) on 2 consecutive days, yielding eight observations per developmental period. Only two cages within a room were observed within an observation time, with the order of observation within rooms randomized across the 2 days. The order in which the rooms were entered was also varied across days. During each observation, a 7.5 W red light was attached to the front of each cage by means of a metal clamp. This light provided illumination during observations in the dark, but was used during all observation times to insure the constancy of lighting, heat, and noise. McGuire, Rand, and Wurtman [26] have demonstrated that red light will not entrain the biological rhythms of rats. During the observations, trained observers recorded stereotypic dominance-submission encounters [15] as an index of home-cage aggression. An aggressive encounter was recorded when physical contact between two or more cagemates resulted in the positioning of one rat (the submissive member) on its back and the other (the dominant member) with its front paws on the submissive member's ventral or lateral surface. Another encounter involving the same rats was not scored unless a nonaggressive posture intervened. Observers distinguished between the encounters in which the experimental subject was dominant and encounters in which the subject was submissive. Dominance-submission encounters between cagemates that did not involve the experimental subject were also recorded. Observers were trained to exceed an inter-rater reliability criterion of 90%, and periodic assessments of reliability confirmed greater than 90% accuracy among observers.
Drinking Measures Drinking behavior provided an assessment of relative synchrony within cages at about the time when home-cage observations were conducted. Drinking in each experimental cage was recorded continuously for 2 consecutive days during each developmental period, with the drinking measure obtained no more than 8 days after the home-cage aggression observations.
219
Shock-Induced Aggression Testing Shock-induced aggression testing of experimental rats began when they were 95 to 96 days of age. The restrained target procedure eliminated target-initiated attack during testing. To eliminate the contribution of the target to possible periodic trends in shock-induced aggression, all targets had been blinded at 15 days of age. Unfamiliar targets were either selected from cages of blinded rats of identical age as those being tested or from the pool of blinded cagemates of this experiment. Because of target mortality between test sessions, four targets had to be replaced during the series of shock-induced aggression tests. Four shock-induced aggression test sessions were scheduled within 24 hr at approximately the same times that home cage observations had been conducted. The first aggression test session occurred at 1330, and the three subsequent sessions were scheduled at 1930, 0130, and 0730. During each aggression session, the harnessed rat was placed in the chamber and the subject was introduced immediately. Within 10-15 sec both the subject and the target were submitted to 100 shocks of 0.5 sec duration at an intershock interval of 3 sec, onset to onset. Trained observers, uninformed with respect to group membership, recorded aggressive behavior when the subject made physical contact with the target while exhibiting stereotypic boxing behavior in an upright posture. Biting of the target, regardless of the orientation of the subject, was also scored. A maximum of one aggressive response was scored per shock. Since successful avoidance of shock will attenuate the level of aggression, observers also recorded instances of shock avoidance. Observers had been trained to exceed an inter-rater reliability criterion of 90% for both aggression and avoidance behavior on a trial by trial (shock by shock) basis. Periodic reliability checks among observers confirmed greater than criterion reliability in recording both behaviors. To eliminate the contribution of avoidance to group differences or error variance, all analyses of aggressive behavior were based upon the percentage of shocks actually received by a subject that resulted in aggression. RESULTS
Home-Cage Synehrony The first analysis of home-cage aggression was conducted to assess synchrony in aggressive interactions. Based on the work of Kane and Knutson [19], synchronous aggressive interactions should be reflected in identifiable time-correlated or light-correlated periodic trends in home-cage aggression that follow the fundamental sine curve, the model that best approximates circadian rhythms. The regression analysis used incorporated a least squares procedure to develop periodic fitted curves based on the Fourier series, and then the curves were evaluated by analyses of variance according to the procedure developed by Bliss [5]. The relative amount of variance across observation sessions that can be accounted for by a fitted curve for an experimental group reflects the relative synchrony in behavior among cages in that group. Each of the analyzed home-cage aggression scores were based on the mean of the two 10-min observations taken during the same observation period on 2 consecutive days. Because the number of zero scores resulted in significant heterogeneity of variance, according to the procedure of Bartlett [36], the home-cage aggression analyses were corn-
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FIG. 2. The fitted curves based on the home-cage drinking behavior during Developmental Period Two from the groups of rats with either sighted subjects or sighted cagemates in Experiment 1. The curve follows the equation: ~-~ ( 2rrit 2~rit~ Y, = ao + ~ a~cos + bisin ~=,
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fundamental sine function formed the basis of the decision to exclude any analyses of higher order curves. During the first developmental period, significant time-correlated trends in home-cage drinking were evidenced for conditions BS and SS. With respect to the former, the significant fundamental sine function, F(2,42)=8.29, p<0.01, accounted for 20% of the total variance. For the SS group, the fitted fundamental sine curve, F(2,42)=37.41, p<0.01, accounted for 46% of the total variance in drinking. For both curves, the peaks were in the dark and the troughs in the lighted portion of the light cycle. At the second developmental period, LD synchronized time-correlated trends in cage drinking were manifested in all experimental conditions containing at least one sighted rat per cage. The fundamental sine function fitted to the drinking data of the cages of the SB group, F(2,42)=6.79, p<0.01, accounted for 15% of the total variance in drinking scores, and in the BS condition fundamental sine curve, F(2,49) = 16.54, p<0.01, accounted for 24% of the variance in drinking behavior. In the SS condition, the fundamental sine function, F(2,49)=49.00, p<0.001, accounted for 57% of the total variance in drinking behavior. Figure 2 shows the fitted curves for each of these experimental groups during Developmental Period Two. Although there are phase angle differences among these curves, and the amplitude of the curves increases with the number of sighted rats housed within a cage, there is a high degree of correspondence in the peaks and troughs of these curves. The analysis of the drinking data of the BB group also identified a statistically significant sinusoidal rhythm in drinking behavior, F(2,49)=12.95, p<0.01, during the second developmental period. This trend, accounting for 29% of the variance in drinking behavior, had a peak at 1600, a trough at 0400, and was not synchronized to the light cycle of the rooms. Since animal care was distributed throughout the
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light cycle, and the cages were distributed among three different rooms, the Zeitgeber of this synchronous group trend is unknown.
Home-Cage Aggression To assess the effect of blinding on the amount of aggression occurring in the home cages of the experimental subjects, an analysis of the total aggression occurring within cages was accomplished with a mixed analysis of variance, with cagemate and subject conditions as between subject factors and developmental period and observation time as within subject factors. The statistically significant cagemate conditionxdevelopmental period interaction, F(1,28)=5.30, p<0.05, is shown in Fig. 3. Tests of simple effects indicated that more aggression was manifested during Developmental Period Two relative to Developmental Period One in cages housing sighted cagemates (p<0.05). There was no significant difference between developmental periods in cages housing blinded cagemates. With respect to between-group comparisons, more aggression was observed in the group of cages housing blinded cagemates relative to those containing sighted cagemates at the first developmental period 09<0.05). The difference between groups at the second developmental period was not statistically significant. The statistically significant observation t i m e x d e v e l o p m e n t a l period, F(3,84)=3.49, p~<0.025, interaction and the statistically significant observation time x c a g e m a t e condition interaction, F(3,84)=8.01, p<0.001, was consistent with the differences among groups in synchrony assessed using regression analyses. To assess the impact of the experimental manipulations on the behavior of the experimental subjects, a series of analyses was conducted on only the subjects' home-cage aggression. That is, the scores for this analysis were the mean number of aggressive encounters observed at each observation time that involved the subject in an interaction with any of its cagemates. These scores, transformed with
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the 1o~ (X+ 1) transformation, were analyzed using a mixed analysis of variance with subject and cagemate condition as between subject factors and developmental period and observation time as within subject factors. An increase in transformed home-cage aggression scores from Developmental Period One to Developmental Period Two was statistically significant, F(1,28)=4.71, p<0.05. The statistically significant developmental period x observation time x experimental condition interaction, F(3,84)=3.81, p<0.025, was assessed through the regression analyses [4] to identify timecorrelated periodic trend following the fundamental sine curve. During the first developmental period, a significant time-correlated trend for subject home-cage aggression was obtained only in the SS condition, F(2,21)=4.44, p<0.05, yielding a fitted fundamental sine function with a peak during the dark period of the light cycle and a trough during the lighted segment. This periodic trend accounted for 23% of the total variance in transformed home-cage aggression displayed by the subjects. During the second developmental period, time-correlated trends for subject aggression were evidenced in both groups having sighted subjects (Groups SB and SS). The fitted curves for these groups, similar to that of Group SS during Developmental Period One, are shown in Fig. 4. In the SB condition, the fundamental sine curve, F(2,21)=9.84, p<0.01, accounted for 38% of the total variance in transformed subject aggression scores. In the SS condition, the
222
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fundamental sine function, F(2,21)=13.29, p<0.01, accounted for 67% of the total variance in the home-cage aggression of the subjects. Again, the peaks of these curves fell within the dark segment of the light cycle and the troughs were in the lighted segment. Subject Dominance To determine whether the experimental manipulation had any impact on the dominance of the subjects relative to their cagemates, subject dominance scores were analyzed. This score consisted of the proportion of aggressive encounters engaged by the subject within each developmental period in which it emerged as the dominant animal. The three subjects that were never observed in a dominance interaction were arbitrarily given the indeterminate dominance score of 0.5, a more conservative procedure than eliminating those subjects from the analysis. These data, analyzed with a mixed analysis of variance with subject and cagemate conditions as between subject factors and developmental period as a within subject factor, did not yield any statistically significant effects. Thus, the blinding of subjects or their cagemates had no significant effect on the relative dominance of subjects within their home cages. Shock-Induced Aggression Before analyzing the aggression data, the avoidance data were examined. Because only seven subjects, distributed among groups, avoided any shocks (3-11 shocks per session), the avoidance data were not analyzed. Consistent with other work in this laboratory [20,21], the aggression analysis was based on the percentage of shocks actually received by
the subject that evoked aggression. The first analysis of the shock-induced aggression data consisted of a three-factor analysis of variance with between subject factors of subject condition and cagemate condition and a within subjects factor of test sessions. Only the test session effect was significant, F(3,84)=22.49, p<0.001, reflecting an increase in shock-induced aggression across the four test sessions. To determine the effect of the experimental manipulations on time-correlated patterns of shock-induced aggression, planned regression analyses were employed on the shockinduced aggression data from each group. Based on the research of Knutson et al. [ 17], this regression analysis tested a composite model consisting of first order algebraic and fundamental sine terms according to the procedure of Bliss [4]. This composite trend reflects the joint contribution of repeated testing (linear term) and time-correlated periodicity (sine term) to shock-induced fighting. In the BB condition, only a statistically significant linear trend was identified, F( 1,21) = 22.62, p <0.0 l, accounting for 28% of the total variance in shock-induced aggression. Similarly, in the BS condition only the linear trend was statistically significant, F(1,21)= 12.75, p<0.01, accounting for 14% of the total variance. Within the SB condition the composite model could be fitted to the shock-induced aggression data. The statistically significant linear trend, F(1,21)=7.96, p<0.01, accounted for 15% of the total variance, and the statistically significant sine function, F(2,21)=8.03, p<0.05, accounted for an additional 2 ( ~ of the total variance. The composite fitted curve is shown in Fig. 5. Significant heterogeneity of variance, as assessed by the Bartlett procedure, characterized the trend analysis of the SS condition. A square root transformation of the shockinduced aggression scores resulted in adequately homogeneous variances. The composite model regression analysis applied to these transformed scores identified a statistically significant linear term, F(1,21)=13.34, p<0.01, that accounted for 25% of the variance in transformed shockinduced aggression scores, and the statistically significant fundamental sine function, F(2,21 ) = 4.03, p < 0.05, accounted for an additional 15% of the variance. Figure 6 shows the fitted curve based on the transformed shock-induced aggression data of the SS group. DISCUSSION With respect to the overall frequencies of home-cage and shock-induced aggression, there was relatively little difference in performance between blinded and sighted subjects. That is, only during the first developmental period was there a significant difference in frequencies of home-cage aggression, with cages housing blinded cagemates (Groups SB and BB) displaying more aggression than groups housing sighted cagemates (Groups SS and BS). However, the effect of blinding on the diurnal rhythmicities of aggression are striking. With respect to home-cage aggression, the analyses of the total cage and experimental subject aggression indicated that vision determined the sinusoidal rhythm in home-cage aggression, with increasingly synchronous rhythmicity as a function of age. Furthermore, the blinding procedure was effective in eliminating the rhythm in shock-induced aggression, and this rhythm was not influenced by the visual status of the cagemates with which the rats had been raised. The drinking data also suggest visual status determines the synchronous rhythmicity of the rats. That is, the differences in amplitude and phase angle of the sine curves fitted
INFLUENCE OF BLINDING
223 ing social asynchrony in the home cages, this asynchrony resulted in neither a suppression of shock-induced aggression nor a reduction of home-cage aggression. These data are not consistent with the hypothesis that the reduced aggressive behavior of rats maintained under L L conditions [ 19,20] is due to asynchrony, and the possibility exists that it is an L L induced state rather than asynchrony that results in less aggression in L L rats.
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Experiment 2 was conducted to determine whether an L L induced state or a more enduring influence of the social history of subjects maintained under 24-hr light determines the reduced frequency of shock-induced aggression in L L reared rats [20]. To accomplish this, Experiment 1 was systematically replicated under L L lighting conditions. Since the purpose of this experiment was to identify the determinants of reduced aggressive behavior among sighted rats maintained under 24-hr light, the procedures replicated those of experiments demonstrating the lowered frequency of aggression in L L rats [16,17].
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TEST SESSION FIG. 6. The fitted curve based on the square-root transformed shock-induced aggression data of the SS group of Experiment 1. This curve follows the equation: J-; ( 2~rit 2~rit~ Yl = a~; + a~X + ~] a ~ c o s - - + b~sin ~=; k k / where Yt is the derived value for time t, k is the number of observations, a(~ is the Y intercept, and a;, ai, and b~ are the least squares derived coefficients.
to the drinking data of the cages housing varying numbers of sighted animals reflects the degree of synchrony among cages as a function of the relative number of sighted rats per cage. The drinking rhythm in the BB group in Period Two is difficult to assess and could reflect a Type I error. Since these animals were distributed among three different rooms and since they were selected from different litters across replications, it seems unlikely that they could have been synchronized to any periodic laboratory routine. Furthermore, that is the only instance of synchronous rhythmicity in subjects maintained under a constant illumination condition in this laboratory [19, 20, 21]. Of course, it is also possible that this effect does not reflect a Type I error, and the blinding resulted in a dissociation of drinking and aggressive rhythms. Although the blinding procedure was effective in produc-
Thirty-six experimentally naive male hooded rats from the colony of the Department of Psychology at the University of Iowa served as experimental subjects. One hundred eight rats served as cagemates. Four subjects (3 blinded, 1 sighted) died during the course of the experiment and were excluded from all analyses. One subject from the BS group was also excluded as a result of equipment malfunction during shock-induced aggression testing. The final number of subjects per cell was: 7 each in Groups BB and BS, 8 in Group SB, and 9 in Group SS.
Apparatus The shock-induced aggression equipment used in Experiment 1 was used in this experiment.
Procedure In a series of four replications, rats were bred and pups whelped in a colony room maintained on the LD 12:12 lighting cycle of Experiment 1. At weaning (25-28 days of age) pups were randomly assigned to either a blinded or shamoperated condition. The surgical procedures were identical to those employed in Experiment 1, but the extended recovery period was eliminated. Following surgery, subjects and cagemates were randomly assigned to the experimental conditions and housed in 24x40.6x 17.8 cm stainless steel and wire mesh cages. These cages were placed on racks in an experimental room maintained under continuous light (LL). Rats had free access to Teklad food pellets placed on the cage floor and water was available from an externally mounted bottle. Maintenance tasks were distributed between 0700 and 1700 to eliminate synchronous activity in the laboratory room. Four 10-min home-cage observations were made between the ages of 35 and 75 days on each experimental cage. Subjects were dyed with black hair dye on the lateral surface 4 days before the first and third observations. Based on the results of Experiment 1 and previous work indicating asynchrony under L L conditions [19], no attempt was made to
224
FORDYCE A N D K N U T S O N
assess home-cage synchrony and rhythmicity. Hence, all home-cage observations took place between 1330 and 1530. The stereotypic dominance-submission encounter served as the index of home-cage aggression, and subject and cagemate behavior were recorded in a manner identical to Experiment 1. At 95 days of age, shock-induced aggression testing was conducted in a manner identical to that employed in Experiment 1.
1.0
W r," .8 0
Z 0
RESULTS W r~
Home-Cage Aggression The home-cage aggression data of this experiment were characterized by a large number of zero scores and severe heterogeneity of variance that could not be adequately eliminated with appropriate transformations. Since these data failed to meet the requisite assumptions for analysis of variance, parametric statistical analyses could not be completed. An alternative way of considering these home-cage aggression data is the proportion of cages from each experimental group in which the experimental subject within the cage engaged in some home-cage aggressive behavior during the observation sessions. Six of the seven experimental subjects in Group BB and seven of the eight experimental subjects in Group SB were observed engaging in agonistic behavior during the home-cage observations. Five of the seven subjects from the BS group were observed engaging in agonistic behavior in the home cage, and only three of the nine subjects from Group SS were observed in any aggressive exchanges. Even in those cages in which aggression was observed, there were few bouts per session in all but the BB condition. In the BB condition, a mean of 3.8 aggressive encounters per l0 min session was observed in those cages evidencing aggression. F o r the SB, BS, and SS conditions, 2.1, 1.8, and 1.5 bouts per session, respectively, were observed in those cages in which some aggression was observed. Thus, with the exception of the BB condition, very little home-cage aggression was observed during the course of this experiment.
Shock-Induced Aggression Because of heterogeneity of variance, the shock-induced aggression scores were submitted to a square-root transformation prior to analysis. The three-factor mixed analysis of variance on these transformed scores indicated a statistically significant effect of test sessions, F(3,81)=20.67, p<0.0001, and a marginally significant cagemate×subject interaction, F(1,27)=3.90, p<0.058. Figure 7 shows the mean transformed aggression scores of each group. Tests of simple effects indicated that the BS group displayed less shockinduced aggression than the BB condition, F(1,12)--5.29, p<0.05. The difference between the BB and SB groups only approached statistical significance, F(1,14) = 3.87, p <0.07. Thus, the cagemate condition did not influence sighted subjects reared under 24-hr light, however, being reared with sighted cagemates under L L conditions reduced the shockinduced aggression of blinded rats. DISCUSSION
Although the home-cage aggression data of Experiment 2 could not be submitted to parametric statistical analyses, the data do replicate previously reported effects of lighting on aggressive behavior. The data from the BB group of Experi-
~..[~--'~_ B L INDED SUBJECTS SIGHTED SUBJECTS
.6
.4
.2
!
BLINDED
!
SIGHTED
CAGEMATE CONDITION FIG. 7. The proportion of shocks presented to both members of a pair that induced aggression as a function of the visual status of the subjects and the visual status of the cagemates with which the subjects had been raised under 24-hr light in Experiment 2. ment 2 are consistent with the data of the BB group from Experiment 1, and the data from the SS group of Experiment 2 are consistent with those reported by Kane and Knutson [19]. Although this sample of home-cage aggression does not permit firm conclusions regarding social experience differences among groups, the pattern of home-cage behavior suggests that it is likely that the subjects from the BS condition experienced less aggression than the BB subjects, and that the SB subjects probably experienced more home-cage aggression than SS subjects. The cagemate x subject interaction in the shock-induced aggression data indicates that the visual status of the cagemates with which the subjects were raised had an effect on the shock-induced aggression of blinded subjects but no effect on the shock-induced aggression of sighted subjects. Since blinded cagemates did not increase the shock-induced aggression of the L L reared sighted rats, it is likely that an L L induced state rather than social experience during development determines the lower amount of shock-induced aggression in L L rats. However, the fact that BS subjects displayed less shock-induced aggression than BB subjects indicates that rearing with L L sighted cagemates can reduce later shock-induced aggression in blinded rats. The reduction of shock-induced aggression in blinded rats reared with sighted L L cagemates and the absence of a cagemate effect on L L sighted subjects is similar to the reduced shock-induced aggression of intact LD rats that were raised with castrated cagemates [18,21] and the lack of an effect of cagemate castration on intact L L reared rats [21]. This suggests that the L L rearing suppresses the aggression of these nocturnal experimental subjects to a level that is not further reduced by cagemates, but rearing with presumably less aggressive (LL or castrated) cagemates suppresses the aggression of rats exposed to darkness during development. GENERAL DISCUSSION These two experiments again demonstrate that the light-
I N F L U E N C E OF B L I N D I N G
225
ing conditions under which laboratory rats are reared and maintained can have a significant influence on the amount and pattern of aggressive behavior. Furthermore, these two experiments are consistent with the hypothesis that a difference in social synchrony is not the factor that determines the reported differences in aggression between LD and LL rats [19, 20, 21]. In recent years, the shock-induced aggression paradigm has been criticized for assessing behavior that may be unrelated to naturally occurring aggression [11,34]. The results of the present experiments, together with the results of the Kane [18], Kane and K n u t s o n [19], and K n u t s o n et al. [21] studies, provide evidence to suggest that shock-induced aggression is indeed related to other intraspecific aggressive behaviors. Visual status of the subjects and the lighting conditions under which they are maintained result in parallel patterns of aggressive behavior in both home-cage and shock-induced aggression. Furthermore, in addition to Kane and K n u t s o n ' s [19] indication that home-cage aggression and shock-induced aggression were significantly correlated, the Knutson et al. [21] and Kane [18] studies, together with the present experiments, indicate that the cagemates with which rats are reared can have an effect on shock-induced aggression. Such data suggest that social experiences during development can determine the frequency of occurrence of shock-induced aggression in adulthood. Fine-grain analyses of the social interaction of rats in their home cage similar to those conducted by Lehman and Adams [24] should now be
conducted to permit a process analysis of the specific aspects of social experience that determine the influence of cagemates on shock-induced aggression. Recent theorizing about the nature of shock-induced aggression [3] suggests that "defensive" experiences may be the important experiences. Because there was a difference in the timing of weaning between Experiments 1 and 2, and because the early weaning of Experiment 1 was not used in related experiments [18, 19, 20, 21], questions could be raised regarding the effects of early weaning on aggressive behavior and subtle differences in data between Experiment 1 and other experiments. Although the effects of premature weaning on aggression have not been investigated, it has been demonstrated that premature weaning can affect mortality, eye opening, heart rate, body temperature and respiration [16], androgen production [23], resting levels of ACTH [17], aversive conditioning [27,28], and gastric ulcers [1]. Physiological functioning of surviving rats appears to return to normal levels [16]. Furthermore, provision of an external heat source [16], a high fat diet [22, 27, 28], and tactile stimulation [35] increase survival and reduce the deleterious physiological and behavioral effects of premature weaning. Since the pups of Experiment 1 were provided an external heat source and a high fat diet and since they received tactile stimulation during cleaning and feeding, the effects of early weaning in Experiment 1 should have been minimized.
REFERENCES 1. Ader, R. Social factors affecting emotionality and resistance to disease in animals: III. Early weaning and susceptibility to gastric ulcers in the rat, a control for nutritional factors. J. comp. physiol. Psychol. 55: 600-602, 1962. 2. Baenninger, L. P. Comparison of behavioral development in socially isolated and grouped rats. Anim. Behav. 45: 312-323, 1967. 3. Blanchard, R. J. and D. C. Blanchard. Aggressive behavior in the rat. Behav. Biol. 21: 197-224, 1977. 4. Blanchard, R. J., D. C. Blanchard and L. K. Takahashi. Reflexive fighting in the albino rat: Aggressive or defensive behavior? Aggress. Behav. 3" 145-155, 1977. 5. Bliss, C. Statistics for Biologists, Vol. 2. New York: McGrawHill, 1970. 6. Bolles, R. C. and P. J. Woods. The ontogeny of behavior in the albino rat. Anita. Behav. 12: 427-441, 1964. 7. Bugbee, N. M. and E. S. Eichelman. Sensory alterations and aggressive behavior in the rat. Physiol. Behav. 8: 981-985, 1972. 8. Cairns, R. B. Fighting and punishment from a developmental perspective. In: Nebraska Symposium on Motivation, 1972, Vol. 21, edited by J. K. Cole and D. D. Jensen. Lincoln: University of Nebraska Press, 1973. 9. Calhoun, J. B. Disruption of behavioral states as a cause of aggression. In: Nebraska Symposium on Motivation, 1972, Vol. 21, edited by J. K. Cole and D. D. Jensen. Lincoln: University of Nebraska Press, 1973. 10. Campbell, B. and R. Teghtsoonian. Electrical and behavioral effects of different types of shock stimuli in the rat. J. comp. physiol. Psychol. 51: 185-192, 1958. 11. Denenberg, V. H. Developmental factors in aggression. In: Control of Aggression: Implications from Basic Research, edited by J. F. Knutson. Chicago: Aldine-Atherton, 1973. 12. Flory, R. K., R. E. Ulrich and P. C. Wolff. The effects of visual impairment on aggressive behavior. Psychol. Rec. 15: 185-190, 1965. 13. FoUick, M. J. and J. F. Knutson. Punishment of irritable aggression. Aggress. Behav. 4: 1-17, 1978.
14. Ghiselli, W. B. and D. A. Thor. Visual, tactual and olfactory deprivation effects on irritable fighting behavior of male hooded rats. Physiol. Psychol. 3: 47-50, 1975. 15. Grant, E. C. and J. H. MacKintosh. A comparison of the social postures of some common laboratory rodents. Behaviour 21: 246-259, 1963. 16. Hofer, M. A. Survival and recovery of physiologic functions after early maternal separation in rats. Physiol. Behav. 15: 475480, 1975. 17. Hromadov~, M., M. Macho, M. Alexandrov~i, K. ~;udowi and R. ~;tukovsk~,.The effect of premature weaning on the response of the rat adrenals to stress and ACTH. Physiologia bohemoslov. 21: 32%335, 1972. 18. Kane, N. L. The Effects of Social Experience on Aggressive Behavior in Rats. Unpublished doctoral dissertation, The University of Iowa, 1977. 19. Kane, N. L. and J. F. Knutson. Influence of colony lighting conditions on home-cage spontaneous aggression. J. comp. physiol. Psychol. 90: 88%897, 1976. 20. Knutson, J. F., M. T. Hynan and N. L. Kane. Influence of home-cage lighting conditions on shock-induced fighting. J. comp. physiol. Psychol. 90: 877-888, 1976. 21. Knutson, J. F., N. L. Kane, A. J. Schlosberg, D. J. Fordyce and K. J. Simansky. Influence of PCPA, shock level, and home-cage conditions on shock-induced aggression. Physiol. Behav. 23: 897-907, 1979. 22. Kraus, M., J. K~erek and M. Pop. The development of corticosterone production by the adrenal gland in normally and prematurely weaned rats. Physiologia bohemoslov. 16: 120-127, 1967. 23. K~erek, J. and V. Palate,. The effect of premature weaning on the development of androgenic activity in male rats, Physiologia bohemoslov. 16: 501-507, 1967. 24. Lehman, M. N. and D. B. Adams. A statistical and motivational analysis of the social behaviors of the male laboratory rat. Behaviour 61: 238-275, 1977.
226 25. Levin, R. and J. M. Stern. Maternal influences on the ontogeny of suckling and feeding rhythms in the rat. J. comp. physiol. Psychol. 89: 711-721, 1975. 26. McGuire, R. A., W. M. Rand and R. J. Wurtman. Entrainment of body temperature in rats: Effect of color and intensity of environmental light. Science 181: 956--957, 1973. 27. Nov~ikovti, V., O. Koldovsk~, J. Flatin, P. Hahn and V. Flandera. Conditioned reflex activity in male rats weaned normally or prematurely. Physiologia bohemoslov. 12:325-331, 1963. 28. Nov,'ikovfi, V., O. Koldovsk~, J. Flatin, P. Hahn and V. Flandera. The effect of premature weaning and high fat diet on retention of a memory trace in male rats. Physiologia bohemoslov. 12: 533-539, 1963. 29. Oswalt, G. L. and M. D. Koch. Temperature, handling, micturition, and the survival of early weaned rats. Anita. Learn. Behay. 3: 123-124, 1975. 30. Powell, D. A., J. Francis, M. J. Braman and N. Schneiderman. Frequency of attack in shock-elicited aggression as a function of the performance of individual rats. J. exp. Analysis Behav. 12: 817-823, 1969.
FORDYCE AND KNUTSON 31. Richter, C. P. Biological Clocks in Medicine and Psychiatry. Springfield, I11.: Charles C. Thomas, 1965. 32. Richter, C. P. Sleep and activity: Their relation to the 24-hour clock. Proc. Ass. Res. nerv. ment. Dis. 45: 8-29, 1967. 33. Richter, C. P. Inborn nature of the rat's 24-hour clock. J. comp. physiol. Psychol. 75: 1-4, 1971. 34. Rose, R. M., I. S. Bernstein, T. P. Gordon and S. F. Catlin. Androgens and aggression: A review of recent findings in primates. In: Primate Aggression, Territoriality, and Xenophobia: A Comparative Perspective, edited by R. L. Holloway. New York: Academic Press, 1974. 35. Thoman, E. B. and W. J. Arnold. Incubator rearing of infant rats without the mother: Effects on adult emotionality and learning, Devl Psychobiol. 1: 21%222, 1968. 36. Wirier, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1971. 37. Zucker, I. Light-dark rhythms in rat eating and drinking behavior. Physiol. Behav. 6: 115-126, 1971.
Influence of Blinding and Home-Cage Lighting on Aggressive Behaviors of Laboratory Rats D A V I D J. F O R D Y C E
A N D J O H N F. K N U T S O N
D e p a r t m e n t o f Psychology, The University o f Iowa, Iowa City, IA 52242 R e c e i v e d 25 O c t o b e r 1979 FORDYCE, D. J. AND J. F. KNUTSON. Influence of blinding and home-cage lighting on aggressive behaviors of laboratory rats. PHYSIOL. BEHAV. 25(2) 217-226, 1980.--Two experiments were conducted on the aggressive behavior of laboratory rats. In Experiment I, blinded and sighted subjects were raised with blinded or sighted cagemates in a colony maintained under a light-dark cycle. Although blinding affected the light synchronized rhythms in home-cage and shockinduced aggression, it did not affect the overall occurrence of these two behaviors. In Experiment 2, blinded and sighted subjects were raised with blinded or sighted cagemates in a colony maintained under continuous lighting (LL). The results of this experiment suggested that reduced aggression in rats from LL conditions is due to an LL induced state of the subject rather than social exposures during development. However, blinded rats reared with LL cagemates evidenced reduced shock-induced aggression, presumably due to reduced social experiences during development that were associated with being raised with LL rats. Shock-induced aggression
Home-cage aggression
T H E lighting conditions under which laboratory rats develop has been shown to influence aggression in adulthood [19,20]. Subjects raised under light-dark (LD) conditions displayed 24-hr periodic trends in home-cage [19] and shock-induced aggression [20] that were similar to the circadian rhythms reported for drinking behavior [31], eating behavior [37], and general activity [32]. Groups housed under continuous light (LL) did not manifest synchronized periodic trends in either home-cage or shock-induced fighting. The data of the Kane and Knutson [19] and Knutson et al. [20] studies also indicated that LD-reared groups generally aggress more than L L groups. Because Kane and Knutson [19] were able to demonstrate a significant correlational relationship between home-cage and shock-induced aggression in LD-reared subjects, the results of these studies led Kane and Knutson [19] to hypothesize that lighting cycles might control aggressive behavior in the home cage which may, in turn, determine performance in the shock-induced aggression paradigm. Support for the hypothesis that home-cage experience may influence shock-induced aggression was provided by recent studies by Kane [18] and Knutson, Kane, Schlosberg, Fordyce, and Simansky [21]. Intact rats raised with castrated cagemates since weaning displayed less shock-induced aggression in adulthood than rats raised with intact cagemates. These data suggest the possibility that reduced agonistic experiences that can result from living with castrated cagemates reduces later shock-induced aggression. It is possible that the tendency for less shock-induced aggression to be manifested by L L housed subjects [20] reflects reduced social experiences during development that are a consequence of the circadian asynchrony of L L rats.
Blinding
Light cycle
Social experience
Rats
That is, in the absence of the dominant Zeitgeber of a light cycle, rhythmic patterns in subjects maintained under constant illumination should be asynchronous and the probability of social interactions occurring among L L cagemates would be less than under LD conditions, where the rhythmic behavior patterns of cagemates are synchronized. Based on the work of Cairns [8] and Calhoun [9] on activity states and aggression, the asynchrony associated with constant lighting could be the factor that determines the reduced home-cage aggressive experiences of L L reared subjects [ 19] and that the reduced aggression during development results in less shock-induced aggression in adulthood. However, it is also possible that the lower rates of shock-induced aggression are a consequence of the momentary asynchrony of the subjects. That is, the asynchronous developmental history might not affect shock-induced aggression, but asynchrony between tested pair members could result in less aggression, since the performance of individual pair members contributes to the occurrence of shock-induced aggression [30]. EXPERIMENT 1 The purpose of this experiment was to determine whether behavioral synchrony at the time of testing or during development affected home-cage and shock-induced aggression. Since blinding can produce a free-running rhythm in some subjects through constant lighting conditions (DD) while sighted subjects in the same environment would be synchronized to an LD cycle, the use of sighted and blinded subjects reared with either blinded or sighted cagemates in a
1This research was supported in part by USPHS training grant No. MH05062. The assistance of Neal Kane and Kenneth E. Wahlstrand is gratefully acknowledged.
C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/080217-10502.00/0
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FORDYCE AND K N U T S O N
factorial design permits an assessment of the contribution of subject and cagemate rhythms to home-cage and shockinduced aggression. In addition to assessing the role of light controlled social asynchrony in aggression, this experiment would permit an assessment of the effect of an absence of visual experience on aggressive behavior. Several investigations have reported that blinding in adulthood has no effect on the shock-induced aggressive behavior of rats [7, 12, 14], but the impact of the absence of visual experience on shockinduced and home-cage aggression has not been reported. METHOD
Subjects Rats used in this experiment came from an original group of 193 experimentally naive male hooded rats from the colony of the Department of Psychology at The University of Iowa. From this group, 32 rats served as experimental subjects and 96 were designated as the cagemates of these subjects. Mortality throughout the course of the study included 21 sighted and 32 blinded rats. This difference was not statistically significant (X~=0.358, df= 1). Since the majority of deaths occurred within a few days of surgery, composition of the experimental cages was arranged after the high mortality period had passed. One cagemate from each of five cages distributed among groups died during the course of the experiment. Since adding a new rat to the cage could have an impact on the home-cage aggression [4], these cagemates were not replaced. The remaining rats of the original group served exclusively as targets in shock-induced aggression testing.
Apparatus Shock-induced aggression testing was conducted in a 22.68x29.21x 19.01 cm aggression test chamber constructed of 6.35 mm clear Plexiglas and a grid floor consisting of 2.38 mm diameter stainless steel rods spaced 1.27 cm center to center. To minimize the contribution of the target to variance in the experimental conditions, the restrained target procedure described by Follick and Knutson [13] was adopted. The aggression chamber was positioned behind an observation window in an Industrial Acoustics Corporation (New York, NY) 2.44x2.44x2.13 m sound-attenuating chamber. Shock was delivered to the grid floor by means of a tube-type constant current DC power supply scrambled through a BRS multiple relay scrambler. Shock to the tail of the target was generated by a tube-type DC power supply and transmitted through leads attached to the tail by modified alligator clips placed approximately 2.5 and 5 cm from the base of the tail. Shock sources did not share a common ground, precluding transfer of shock between members of a fighting pair. Vinyl tape covering the grids beneath the target and moleskin covering the feet of the target provided insulation from the subject-shock circuit. Shock to the target and to the subject was set at 2 mA, using a 22 K ohm resistor. This level of resistance is roughly equivalent to that which would be produced by an adult male rat receiving a 2 mA shock [10]. Shock frequency and duration were programmed using conventional electronic timers and relay circuitry, and shockinduced aggression data were recorded on electromechanical counters. Drinking in the home cages was sampled as a measure of cage synchrony using conventional contact relays, electronic timers, relay circuitry, and electromechanical counters. The
drinking tube, insulated with shrinkable tubing, served as one pole of the contact relay while the cage served as the other pole. Drinking was recorded in eight 3-hr bins within each 24-hr period. The recording equipment was located in another room to eliminate auditory cues and experimenter activity in the colony rooms.
Procedure In a series of four replications, rats were bred and pups whelped in a colony room maintained on an LD 12:12 lighting cycle (lights on 0700-1900). Since the aggressive behaviors of the laboratory rat begin to emerge at about 17 days of age [2,6], the procedures were scheduled to maximize social asynchrony during this early period. Rats begin to open their eyes at about 14 days of age [6], and the available evidence suggests that blinding at this age produces relatively immediate free running of circadian rhythms [33,37]. Based on these data, rat pups were blinded at 15 days of age, before most had opened their eyes. Since it has been reported that the maternal behavior of a lactating rat dam can act as a Zeitgeber entraining the behaviors of rat pups [25], pups were also weaned at 15 days of age. At weaning, the pups were randomly assigned to either a blinded or sham-operated condition. Rats assigned to the blinded condition were placed under ether anesthesia and submitted to bilateral enucleation. The eyelids, if closed, were separated with eye-dressing forceps and each globe was protruded with a small hemostat. A small curved scissor was used to sever the musculature and optic nerve. Following removal of the eye, the socket was packed with absorbable gelatin sponge coated with a 2% nitrofurazone topical antibiotic powder. Immediately following surgery, each rat was placed with 7 to 14 other pups in a 43 cm dia. recovery chamber. The entire surgical procedure required about 3.5 rain. Rats assigned to the sham-operated condition were submitted to ether anesthesia and the recovery treatment, but were not surgically manipulated. During surgery and recovery a 250 W infrared heat lamp was used to maintain the surface temperature at about 30°C. After surgery, all rat pups were transferred in the recovery chambers to a post-weaning room. The post-weaning room was maintained on the same LD light schedule as the breeding colony and the colony rooms used to house the rats of the experiment. A food mash of ground Teklad (Winfield, IA) pellets and diluted evaporated milk was available ad lib in shallow 30 ml saucers. During the first two days following surgery, each pup was fed this mixture twice per day by an experimenter placing a small portion of the mash on the tongue of the rat. At feeding times, the anal region of each rat was wiped clean of fecal material using a warm damp cloth. Feeding and cleaning sessions were distributed across the 12-hr light period to reduce the possibility of a regular laboratory routine entraining the rhythms of blinded pups. At 17 days of age, the rats were transferred from the recovery chambers and housed according to visual status in 24x40.6x 17.8 cm stainless steel and wire mesh cages. In addition to the mash, solid Teklad pellets were placed within each cage and water was supplied ad lib from an externally mounted water bottle. While the contents of the mash bowls were replaced twice per day on an aperiodic schedule during this period, the pups were neither hand fed nor cleaned. At 20 days of age the rats were rehoused in groups of four according to group membership in 24 ×40.6x 17.8 cm stainless steel and wire mesh cages; the external heat sources
INFLUENCE OF BLINDING were eliminated leaving the ambient room temperature at 22 ( _+ 2)°C. Blinded subjects were either housed with three blinded cagemates (Group BB) or three sighted cagemates (Group BS), and sighted subjects were housed with either three blinded cagemates (Group SB) or three sighted cagemates (Group SS). When the subjects reached 26 days of age, the cages were transferred from the post-weaning room to one of three experimental rooms where they were placed on racks. Cages were distributed among rooms so that each room housed cages from each of the four experimental conditions. Rats had free access to Teklad food pellets placed on the floor of the cage, and water was available from an externally mounted bottle. Daily maintenance activities within each room were randomly distributed between 0700 and 1700 to reduce the possibility of entrainment to a laboratory routine.
Home-Cage Aggression Observations Home-cage observations were conducted at two developmental ages, when the rats were 36-41 days of age and 65-70 days of age (Periods 1 and 2, respectively). Three days prior to the initiation of home-cage observations, the experimental subject within each cage was weighed and marked with black hair dye on the lateral surfaces of the body. To assess home-cage aggression, each cage was observed for 10 min at 0200, 0800, 1400, and 2000 ( +__30 min) on 2 consecutive days, yielding eight observations per developmental period. Only two cages within a room were observed within an observation time, with the order of observation within rooms randomized across the 2 days. The order in which the rooms were entered was also varied across days. During each observation, a 7.5 W red light was attached to the front of each cage by means of a metal clamp. This light provided illumination during observations in the dark, but was used during all observation times to insure the constancy of lighting, heat, and noise. McGuire, Rand, and Wurtman [26] have demonstrated that red light will not entrain the biological rhythms of rats. During the observations, trained observers recorded stereotypic dominance-submission encounters [15] as an index of home-cage aggression. An aggressive encounter was recorded when physical contact between two or more cagemates resulted in the positioning of one rat (the submissive member) on its back and the other (the dominant member) with its front paws on the submissive member's ventral or lateral surface. Another encounter involving the same rats was not scored unless a nonaggressive posture intervened. Observers distinguished between the encounters in which the experimental subject was dominant and encounters in which the subject was submissive. Dominance-submission encounters between cagemates that did not involve the experimental subject were also recorded. Observers were trained to exceed an inter-rater reliability criterion of 90%, and periodic assessments of reliability confirmed greater than 90% accuracy among observers.
Drinking Measures Drinking behavior provided an assessment of relative synchrony within cages at about the time when home-cage observations were conducted. Drinking in each experimental cage was recorded continuously for 2 consecutive days during each developmental period, with the drinking measure obtained no more than 8 days after the home-cage aggression observations.
219
Shock-Induced Aggression Testing Shock-induced aggression testing of experimental rats began when they were 95 to 96 days of age. The restrained target procedure eliminated target-initiated attack during testing. To eliminate the contribution of the target to possible periodic trends in shock-induced aggression, all targets had been blinded at 15 days of age. Unfamiliar targets were either selected from cages of blinded rats of identical age as those being tested or from the pool of blinded cagemates of this experiment. Because of target mortality between test sessions, four targets had to be replaced during the series of shock-induced aggression tests. Four shock-induced aggression test sessions were scheduled within 24 hr at approximately the same times that home cage observations had been conducted. The first aggression test session occurred at 1330, and the three subsequent sessions were scheduled at 1930, 0130, and 0730. During each aggression session, the harnessed rat was placed in the chamber and the subject was introduced immediately. Within 10-15 sec both the subject and the target were submitted to 100 shocks of 0.5 sec duration at an intershock interval of 3 sec, onset to onset. Trained observers, uninformed with respect to group membership, recorded aggressive behavior when the subject made physical contact with the target while exhibiting stereotypic boxing behavior in an upright posture. Biting of the target, regardless of the orientation of the subject, was also scored. A maximum of one aggressive response was scored per shock. Since successful avoidance of shock will attenuate the level of aggression, observers also recorded instances of shock avoidance. Observers had been trained to exceed an inter-rater reliability criterion of 90% for both aggression and avoidance behavior on a trial by trial (shock by shock) basis. Periodic reliability checks among observers confirmed greater than criterion reliability in recording both behaviors. To eliminate the contribution of avoidance to group differences or error variance, all analyses of aggressive behavior were based upon the percentage of shocks actually received by a subject that resulted in aggression. RESULTS
Home-Cage Synehrony The first analysis of home-cage aggression was conducted to assess synchrony in aggressive interactions. Based on the work of Kane and Knutson [19], synchronous aggressive interactions should be reflected in identifiable time-correlated or light-correlated periodic trends in home-cage aggression that follow the fundamental sine curve, the model that best approximates circadian rhythms. The regression analysis used incorporated a least squares procedure to develop periodic fitted curves based on the Fourier series, and then the curves were evaluated by analyses of variance according to the procedure developed by Bliss [5]. The relative amount of variance across observation sessions that can be accounted for by a fitted curve for an experimental group reflects the relative synchrony in behavior among cages in that group. Each of the analyzed home-cage aggression scores were based on the mean of the two 10-min observations taken during the same observation period on 2 consecutive days. Because the number of zero scores resulted in significant heterogeneity of variance, according to the procedure of Bartlett [36], the home-cage aggression analyses were corn-
220
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fundamental sine function formed the basis of the decision to exclude any analyses of higher order curves. During the first developmental period, significant time-correlated trends in home-cage drinking were evidenced for conditions BS and SS. With respect to the former, the significant fundamental sine function, F(2,42)=8.29, p<0.01, accounted for 20% of the total variance. For the SS group, the fitted fundamental sine curve, F(2,42)=37.41, p<0.01, accounted for 46% of the total variance in drinking. For both curves, the peaks were in the dark and the troughs in the lighted portion of the light cycle. At the second developmental period, LD synchronized time-correlated trends in cage drinking were manifested in all experimental conditions containing at least one sighted rat per cage. The fundamental sine function fitted to the drinking data of the cages of the SB group, F(2,42)=6.79, p<0.01, accounted for 15% of the total variance in drinking scores, and in the BS condition fundamental sine curve, F(2,49) = 16.54, p<0.01, accounted for 24% of the variance in drinking behavior. In the SS condition, the fundamental sine function, F(2,49)=49.00, p<0.001, accounted for 57% of the total variance in drinking behavior. Figure 2 shows the fitted curves for each of these experimental groups during Developmental Period Two. Although there are phase angle differences among these curves, and the amplitude of the curves increases with the number of sighted rats housed within a cage, there is a high degree of correspondence in the peaks and troughs of these curves. The analysis of the drinking data of the BB group also identified a statistically significant sinusoidal rhythm in drinking behavior, F(2,49)=12.95, p<0.01, during the second developmental period. This trend, accounting for 29% of the variance in drinking behavior, had a peak at 1600, a trough at 0400, and was not synchronized to the light cycle of the rooms. Since animal care was distributed throughout the
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light cycle, and the cages were distributed among three different rooms, the Zeitgeber of this synchronous group trend is unknown.
Home-Cage Aggression To assess the effect of blinding on the amount of aggression occurring in the home cages of the experimental subjects, an analysis of the total aggression occurring within cages was accomplished with a mixed analysis of variance, with cagemate and subject conditions as between subject factors and developmental period and observation time as within subject factors. The statistically significant cagemate conditionxdevelopmental period interaction, F(1,28)=5.30, p<0.05, is shown in Fig. 3. Tests of simple effects indicated that more aggression was manifested during Developmental Period Two relative to Developmental Period One in cages housing sighted cagemates (p<0.05). There was no significant difference between developmental periods in cages housing blinded cagemates. With respect to between-group comparisons, more aggression was observed in the group of cages housing blinded cagemates relative to those containing sighted cagemates at the first developmental period 09<0.05). The difference between groups at the second developmental period was not statistically significant. The statistically significant observation t i m e x d e v e l o p m e n t a l period, F(3,84)=3.49, p~<0.025, interaction and the statistically significant observation time x c a g e m a t e condition interaction, F(3,84)=8.01, p<0.001, was consistent with the differences among groups in synchrony assessed using regression analyses. To assess the impact of the experimental manipulations on the behavior of the experimental subjects, a series of analyses was conducted on only the subjects' home-cage aggression. That is, the scores for this analysis were the mean number of aggressive encounters observed at each observation time that involved the subject in an interaction with any of its cagemates. These scores, transformed with
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the 1o~ (X+ 1) transformation, were analyzed using a mixed analysis of variance with subject and cagemate condition as between subject factors and developmental period and observation time as within subject factors. An increase in transformed home-cage aggression scores from Developmental Period One to Developmental Period Two was statistically significant, F(1,28)=4.71, p<0.05. The statistically significant developmental period x observation time x experimental condition interaction, F(3,84)=3.81, p<0.025, was assessed through the regression analyses [4] to identify timecorrelated periodic trend following the fundamental sine curve. During the first developmental period, a significant time-correlated trend for subject home-cage aggression was obtained only in the SS condition, F(2,21)=4.44, p<0.05, yielding a fitted fundamental sine function with a peak during the dark period of the light cycle and a trough during the lighted segment. This periodic trend accounted for 23% of the total variance in transformed home-cage aggression displayed by the subjects. During the second developmental period, time-correlated trends for subject aggression were evidenced in both groups having sighted subjects (Groups SB and SS). The fitted curves for these groups, similar to that of Group SS during Developmental Period One, are shown in Fig. 4. In the SB condition, the fundamental sine curve, F(2,21)=9.84, p<0.01, accounted for 38% of the total variance in transformed subject aggression scores. In the SS condition, the
222
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fundamental sine function, F(2,21)=13.29, p<0.01, accounted for 67% of the total variance in the home-cage aggression of the subjects. Again, the peaks of these curves fell within the dark segment of the light cycle and the troughs were in the lighted segment. Subject Dominance To determine whether the experimental manipulation had any impact on the dominance of the subjects relative to their cagemates, subject dominance scores were analyzed. This score consisted of the proportion of aggressive encounters engaged by the subject within each developmental period in which it emerged as the dominant animal. The three subjects that were never observed in a dominance interaction were arbitrarily given the indeterminate dominance score of 0.5, a more conservative procedure than eliminating those subjects from the analysis. These data, analyzed with a mixed analysis of variance with subject and cagemate conditions as between subject factors and developmental period as a within subject factor, did not yield any statistically significant effects. Thus, the blinding of subjects or their cagemates had no significant effect on the relative dominance of subjects within their home cages. Shock-Induced Aggression Before analyzing the aggression data, the avoidance data were examined. Because only seven subjects, distributed among groups, avoided any shocks (3-11 shocks per session), the avoidance data were not analyzed. Consistent with other work in this laboratory [20,21], the aggression analysis was based on the percentage of shocks actually received by
the subject that evoked aggression. The first analysis of the shock-induced aggression data consisted of a three-factor analysis of variance with between subject factors of subject condition and cagemate condition and a within subjects factor of test sessions. Only the test session effect was significant, F(3,84)=22.49, p<0.001, reflecting an increase in shock-induced aggression across the four test sessions. To determine the effect of the experimental manipulations on time-correlated patterns of shock-induced aggression, planned regression analyses were employed on the shockinduced aggression data from each group. Based on the research of Knutson et al. [ 17], this regression analysis tested a composite model consisting of first order algebraic and fundamental sine terms according to the procedure of Bliss [4]. This composite trend reflects the joint contribution of repeated testing (linear term) and time-correlated periodicity (sine term) to shock-induced fighting. In the BB condition, only a statistically significant linear trend was identified, F( 1,21) = 22.62, p <0.0 l, accounting for 28% of the total variance in shock-induced aggression. Similarly, in the BS condition only the linear trend was statistically significant, F(1,21)= 12.75, p<0.01, accounting for 14% of the total variance. Within the SB condition the composite model could be fitted to the shock-induced aggression data. The statistically significant linear trend, F(1,21)=7.96, p<0.01, accounted for 15% of the total variance, and the statistically significant sine function, F(2,21)=8.03, p<0.05, accounted for an additional 2 ( ~ of the total variance. The composite fitted curve is shown in Fig. 5. Significant heterogeneity of variance, as assessed by the Bartlett procedure, characterized the trend analysis of the SS condition. A square root transformation of the shockinduced aggression scores resulted in adequately homogeneous variances. The composite model regression analysis applied to these transformed scores identified a statistically significant linear term, F(1,21)=13.34, p<0.01, that accounted for 25% of the variance in transformed shockinduced aggression scores, and the statistically significant fundamental sine function, F(2,21 ) = 4.03, p < 0.05, accounted for an additional 15% of the variance. Figure 6 shows the fitted curve based on the transformed shock-induced aggression data of the SS group. DISCUSSION With respect to the overall frequencies of home-cage and shock-induced aggression, there was relatively little difference in performance between blinded and sighted subjects. That is, only during the first developmental period was there a significant difference in frequencies of home-cage aggression, with cages housing blinded cagemates (Groups SB and BB) displaying more aggression than groups housing sighted cagemates (Groups SS and BS). However, the effect of blinding on the diurnal rhythmicities of aggression are striking. With respect to home-cage aggression, the analyses of the total cage and experimental subject aggression indicated that vision determined the sinusoidal rhythm in home-cage aggression, with increasingly synchronous rhythmicity as a function of age. Furthermore, the blinding procedure was effective in eliminating the rhythm in shock-induced aggression, and this rhythm was not influenced by the visual status of the cagemates with which the rats had been raised. The drinking data also suggest visual status determines the synchronous rhythmicity of the rats. That is, the differences in amplitude and phase angle of the sine curves fitted
INFLUENCE OF BLINDING
223 ing social asynchrony in the home cages, this asynchrony resulted in neither a suppression of shock-induced aggression nor a reduction of home-cage aggression. These data are not consistent with the hypothesis that the reduced aggressive behavior of rats maintained under L L conditions [ 19,20] is due to asynchrony, and the possibility exists that it is an L L induced state rather than asynchrony that results in less aggression in L L rats.
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Experiment 2 was conducted to determine whether an L L induced state or a more enduring influence of the social history of subjects maintained under 24-hr light determines the reduced frequency of shock-induced aggression in L L reared rats [20]. To accomplish this, Experiment 1 was systematically replicated under L L lighting conditions. Since the purpose of this experiment was to identify the determinants of reduced aggressive behavior among sighted rats maintained under 24-hr light, the procedures replicated those of experiments demonstrating the lowered frequency of aggression in L L rats [16,17].
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to the drinking data of the cages housing varying numbers of sighted animals reflects the degree of synchrony among cages as a function of the relative number of sighted rats per cage. The drinking rhythm in the BB group in Period Two is difficult to assess and could reflect a Type I error. Since these animals were distributed among three different rooms and since they were selected from different litters across replications, it seems unlikely that they could have been synchronized to any periodic laboratory routine. Furthermore, that is the only instance of synchronous rhythmicity in subjects maintained under a constant illumination condition in this laboratory [19, 20, 21]. Of course, it is also possible that this effect does not reflect a Type I error, and the blinding resulted in a dissociation of drinking and aggressive rhythms. Although the blinding procedure was effective in produc-
Thirty-six experimentally naive male hooded rats from the colony of the Department of Psychology at the University of Iowa served as experimental subjects. One hundred eight rats served as cagemates. Four subjects (3 blinded, 1 sighted) died during the course of the experiment and were excluded from all analyses. One subject from the BS group was also excluded as a result of equipment malfunction during shock-induced aggression testing. The final number of subjects per cell was: 7 each in Groups BB and BS, 8 in Group SB, and 9 in Group SS.
Apparatus The shock-induced aggression equipment used in Experiment 1 was used in this experiment.
Procedure In a series of four replications, rats were bred and pups whelped in a colony room maintained on the LD 12:12 lighting cycle of Experiment 1. At weaning (25-28 days of age) pups were randomly assigned to either a blinded or shamoperated condition. The surgical procedures were identical to those employed in Experiment 1, but the extended recovery period was eliminated. Following surgery, subjects and cagemates were randomly assigned to the experimental conditions and housed in 24x40.6x 17.8 cm stainless steel and wire mesh cages. These cages were placed on racks in an experimental room maintained under continuous light (LL). Rats had free access to Teklad food pellets placed on the cage floor and water was available from an externally mounted bottle. Maintenance tasks were distributed between 0700 and 1700 to eliminate synchronous activity in the laboratory room. Four 10-min home-cage observations were made between the ages of 35 and 75 days on each experimental cage. Subjects were dyed with black hair dye on the lateral surface 4 days before the first and third observations. Based on the results of Experiment 1 and previous work indicating asynchrony under L L conditions [19], no attempt was made to
224
FORDYCE A N D K N U T S O N
assess home-cage synchrony and rhythmicity. Hence, all home-cage observations took place between 1330 and 1530. The stereotypic dominance-submission encounter served as the index of home-cage aggression, and subject and cagemate behavior were recorded in a manner identical to Experiment 1. At 95 days of age, shock-induced aggression testing was conducted in a manner identical to that employed in Experiment 1.
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Home-Cage Aggression The home-cage aggression data of this experiment were characterized by a large number of zero scores and severe heterogeneity of variance that could not be adequately eliminated with appropriate transformations. Since these data failed to meet the requisite assumptions for analysis of variance, parametric statistical analyses could not be completed. An alternative way of considering these home-cage aggression data is the proportion of cages from each experimental group in which the experimental subject within the cage engaged in some home-cage aggressive behavior during the observation sessions. Six of the seven experimental subjects in Group BB and seven of the eight experimental subjects in Group SB were observed engaging in agonistic behavior during the home-cage observations. Five of the seven subjects from the BS group were observed engaging in agonistic behavior in the home cage, and only three of the nine subjects from Group SS were observed in any aggressive exchanges. Even in those cages in which aggression was observed, there were few bouts per session in all but the BB condition. In the BB condition, a mean of 3.8 aggressive encounters per l0 min session was observed in those cages evidencing aggression. F o r the SB, BS, and SS conditions, 2.1, 1.8, and 1.5 bouts per session, respectively, were observed in those cages in which some aggression was observed. Thus, with the exception of the BB condition, very little home-cage aggression was observed during the course of this experiment.
Shock-Induced Aggression Because of heterogeneity of variance, the shock-induced aggression scores were submitted to a square-root transformation prior to analysis. The three-factor mixed analysis of variance on these transformed scores indicated a statistically significant effect of test sessions, F(3,81)=20.67, p<0.0001, and a marginally significant cagemate×subject interaction, F(1,27)=3.90, p<0.058. Figure 7 shows the mean transformed aggression scores of each group. Tests of simple effects indicated that the BS group displayed less shockinduced aggression than the BB condition, F(1,12)--5.29, p<0.05. The difference between the BB and SB groups only approached statistical significance, F(1,14) = 3.87, p <0.07. Thus, the cagemate condition did not influence sighted subjects reared under 24-hr light, however, being reared with sighted cagemates under L L conditions reduced the shockinduced aggression of blinded rats. DISCUSSION
Although the home-cage aggression data of Experiment 2 could not be submitted to parametric statistical analyses, the data do replicate previously reported effects of lighting on aggressive behavior. The data from the BB group of Experi-
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CAGEMATE CONDITION FIG. 7. The proportion of shocks presented to both members of a pair that induced aggression as a function of the visual status of the subjects and the visual status of the cagemates with which the subjects had been raised under 24-hr light in Experiment 2. ment 2 are consistent with the data of the BB group from Experiment 1, and the data from the SS group of Experiment 2 are consistent with those reported by Kane and Knutson [19]. Although this sample of home-cage aggression does not permit firm conclusions regarding social experience differences among groups, the pattern of home-cage behavior suggests that it is likely that the subjects from the BS condition experienced less aggression than the BB subjects, and that the SB subjects probably experienced more home-cage aggression than SS subjects. The cagemate x subject interaction in the shock-induced aggression data indicates that the visual status of the cagemates with which the subjects were raised had an effect on the shock-induced aggression of blinded subjects but no effect on the shock-induced aggression of sighted subjects. Since blinded cagemates did not increase the shock-induced aggression of the L L reared sighted rats, it is likely that an L L induced state rather than social experience during development determines the lower amount of shock-induced aggression in L L rats. However, the fact that BS subjects displayed less shock-induced aggression than BB subjects indicates that rearing with L L sighted cagemates can reduce later shock-induced aggression in blinded rats. The reduction of shock-induced aggression in blinded rats reared with sighted L L cagemates and the absence of a cagemate effect on L L sighted subjects is similar to the reduced shock-induced aggression of intact LD rats that were raised with castrated cagemates [18,21] and the lack of an effect of cagemate castration on intact L L reared rats [21]. This suggests that the L L rearing suppresses the aggression of these nocturnal experimental subjects to a level that is not further reduced by cagemates, but rearing with presumably less aggressive (LL or castrated) cagemates suppresses the aggression of rats exposed to darkness during development. GENERAL DISCUSSION These two experiments again demonstrate that the light-
I N F L U E N C E OF B L I N D I N G
225
ing conditions under which laboratory rats are reared and maintained can have a significant influence on the amount and pattern of aggressive behavior. Furthermore, these two experiments are consistent with the hypothesis that a difference in social synchrony is not the factor that determines the reported differences in aggression between LD and LL rats [19, 20, 21]. In recent years, the shock-induced aggression paradigm has been criticized for assessing behavior that may be unrelated to naturally occurring aggression [11,34]. The results of the present experiments, together with the results of the Kane [18], Kane and K n u t s o n [19], and K n u t s o n et al. [21] studies, provide evidence to suggest that shock-induced aggression is indeed related to other intraspecific aggressive behaviors. Visual status of the subjects and the lighting conditions under which they are maintained result in parallel patterns of aggressive behavior in both home-cage and shock-induced aggression. Furthermore, in addition to Kane and K n u t s o n ' s [19] indication that home-cage aggression and shock-induced aggression were significantly correlated, the Knutson et al. [21] and Kane [18] studies, together with the present experiments, indicate that the cagemates with which rats are reared can have an effect on shock-induced aggression. Such data suggest that social experiences during development can determine the frequency of occurrence of shock-induced aggression in adulthood. Fine-grain analyses of the social interaction of rats in their home cage similar to those conducted by Lehman and Adams [24] should now be
conducted to permit a process analysis of the specific aspects of social experience that determine the influence of cagemates on shock-induced aggression. Recent theorizing about the nature of shock-induced aggression [3] suggests that "defensive" experiences may be the important experiences. Because there was a difference in the timing of weaning between Experiments 1 and 2, and because the early weaning of Experiment 1 was not used in related experiments [18, 19, 20, 21], questions could be raised regarding the effects of early weaning on aggressive behavior and subtle differences in data between Experiment 1 and other experiments. Although the effects of premature weaning on aggression have not been investigated, it has been demonstrated that premature weaning can affect mortality, eye opening, heart rate, body temperature and respiration [16], androgen production [23], resting levels of ACTH [17], aversive conditioning [27,28], and gastric ulcers [1]. Physiological functioning of surviving rats appears to return to normal levels [16]. Furthermore, provision of an external heat source [16], a high fat diet [22, 27, 28], and tactile stimulation [35] increase survival and reduce the deleterious physiological and behavioral effects of premature weaning. Since the pups of Experiment 1 were provided an external heat source and a high fat diet and since they received tactile stimulation during cleaning and feeding, the effects of early weaning in Experiment 1 should have been minimized.
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