- Email: [email protected]
Effects of prepubertal handling on shock-induced fighting and ACTH in male and female rats
Physiology and Behavior, Vol. 14, pp. 413--420. Brain Research Publications Inc., 1975. Printed in the U.S.A.
Effects of Prepubertal Handling on Shock-Induced Fighting and ACTH in Male and Female Rats MARY S. ERSKINE 2, JUDITH M. STERN a AND SEYMOUR LEVINE 4
Department o f Psychiatry, Stanford University School o f Medicine, Stanford, California 94305
(Received 12 November 1974)
ERSKINE, M. S., J. M. STERN AND S. LEVINE. Effects ofprepubertal handling on shock-induced fighting and ACTH in male and female rats. PHYSIOL. BEHAV. 14(4) 413-420, 1975. - Rats which received either handling from Days 1-14 (EH) or 22-35 (LH) of life or no handling (NH) were tested in the shock-induced fighting situation in adulthood. Ten males and 10 females from each group were either paired for fighting (F), shocked singly (S), or received no shock (N) in the test chamber for 4.5 min in 3 sessions given 48 hr apart. Immediately after the third test session, trunk blood was obtained for determination of plasma ACTH concentrations. Males and females did not differ with respect to fighting frequency in response to shock, but males displayed the fighting posture substantially more than did females. Rats handled either preweaning (EH) or postweaning (LH) fought more often at the two lower shock intensities (0.5 and 1.3 mA) than did nonhandled controls (NH). The incidence of fighting positions did not differ as a function of prior handling. In all groups but one, the pattern of ACTH response to the different test conditions coincided with previous reports: higher levels of ACTH were found in response to shock alone than in response to fighting plus shock, and the lowest levels were found in the nonshocked controls. In the exceptional group, the NH males (but not females), there was a nondiscriminatory ACTH response to the S and F test conditions. The effects of prior handling are discussed in terms of an enhanced coping ability. The results point to the importance of comparing both pre- and postweaning groups when studying the handling phenomenon. Early experience
Handling
ACTH
Shock-induced fighting
BEHAVIORAL and physiological effects in adulthood of infantile handling in rats are well documented (see reviews by Denenberg [7], Denenberg and Zarrow [9] and Levine [ 19] ). The picture that has emerged is that laboratory rats receiving early experimenter stimulation have an enhanced ability to cope with novel or emotionally disturbing situations compared with their unmanipulated counterparts. Thus, when tested in adulthood, early-handled rats have been found to be less emotionally reactive, both behaviorally and physiologically, in novel, mildly aversive situations compared to nonhandled controls [22]. In highly aversive situations involving electric shock, behavior is less disrupted in handled animals, as indicated by enhanced avoidance conditioning [8, 14, 21] and water intake after deprivation [ 17 ]. In the shock-induced fighting situation, Conner et al. [4, 6] found lower plasma ACTH levels in paired fighting
Aggression
animals compared to animals shocked alone in the test chamber. They postulated [4] that the ability to express an organized behavior in response to aversive stimulation, resulting in lowered pituitary response to stress, represents a coping response. Similarly, rats allowed to avoid or escape s h o c k displayed increased weight gain and lowered frequency of gastric lesions when compared to yoked controls [30]. In the present experiment, therefore, earlyhandled rats and their controls were either fought with shock (paired animals), shocked alone (single animals), or placed alone in the chamber with no shock during the test session. A group comparison of the ACTH response of the animals receiving shock alone when in the test chamber was of interest since both increased [20] and decreased [1, 2] adrenocortical reactivity has been reported for earlyhandled rats in response to electric shock. Further, if the ability to fight indeed represents a coping response, then it
~This study was supported by Research Grant NICH&HD 02881 from the National Institutes of Health to S. L. Thanks are extended to Dr. Mary Dallman, Dept. of Physiology, U. C. Medical School, San Francisco for assaying the ACTH in these experiments, and further thanks to Mr. Martin Miner for his expert technical assistance. Present address: Department of Biobehavioral Sciences, Box U-154, University of Connecticut, Storrs, CT 06268. 3Present address: Department of Psychology, Rutgers College, Rutgers University, 88 College Avenue, New Brunswick, NJ 08903. 4Supported by USPHS Research Scientist Award K5-MH-19936 from the National Institute of Mental Health. 413
414
ERSKINE, STERN AND LEVINE
might be expected that the early-handled rats would fight more and secrete less ACTH in response to the shockinduced fighting situation when compared to nonhandled fought animals. Handling, as well as other forms of stimulation, has been shown to be effective in influencing adult behavior during several times both pre- and postweaning [7] and there is evidence that the adrenocortical response to stress may be modified in rats stimulated postweaning as well as preweaning [1,2]. Therefore, in addition to the preweaning early-handled (EH) and nonhandled (NH) rats, a third group of postweaning late-handled (LH) rats was included. Further, the present study compares the responses of males and females. Previously handled female rats, as well as males, have been found to be less reactive to adrenocortical activation [1,2]. However, behavioral measures of the effects of early handling have rarely tested both males and females on the same tasks.
METHOD
Apparatus The shock-induced fighting apparatus in adult testing consisted of a chamber (17.8 X 17.8 X 30.5 cm) with a grid floor and three metal walls [5]. The front wall consisted of 3.2 cm vertical grids on 12.7 mm centers allowing unobstructed observation of the animals during the test session. The test chambers were housed in commercial sound-attenuating chambers (Industrial Acoustics Corp.). Shock duration and intensity through a Grason-Stadler Shock Scrambler (Model No. 1064GS) was controlled by solid state circuitry.
Animals Animals were 180 rats obtained from 23 litters bred in our laboratory from Sprague-Dawley females and LongE v a n s males obtained from Simonsen Labs, Gilroy, California. Rats were housed in a room of constant temperature and humidity with noise kept to a minimum. An external source of food and water was available ad lib and light onset of the 12 hr light-dark cycle was 0800. Thirty males and 30 females were assigned to each of the EH, LH and NH treatment groups. Subgroups of 10 animals were randomly chosen for the three test conditions.
Procedure A. Infantile handling. Several days prior to birth, females were individually housed in 28 X 48 X 15 cm pans with wood shavings as bedding material. At birth, litters were divided randomly into three treatment groups: earlyhandled (EH, n=7), late-handled (LH, n=8) and nonhandled (NH, n=8). Litter size varied between 8 and 12 pups; litters of less than 8 pups were discarded from the experiment and large litters were culled to 12 pups on Day 1 in EH litters and at weaning in the LH and NH groups. Early-handled pups were removed from their cage and placed directly into 6 X 6 X 5 cm cardboard compartments for 3 min each day between Days 1 - 1 4 , the day of birth being Day 0. This handling procedure was carried out at 1 0 0 0 - 1 1 0 0 hr each day. No other manipulations of the EH litters, or those of groups LH and NH, occurred prior to weaning. At Day 21, pups were weaned by removing the mother from the cage; at this time, total litter weights were
recorded and bedding material changed. Lit and NH litters exceeding 12 pups were culled prior to weighing. Handling began on Day 22 in the LH groups and continued for 14 days. The procedure was similar to that used for the EH litters, except that the compartments used were 7 X 7 X 12 cm. EH and NH animals remained undisturbed from Days 2 2 - 3 5 . On Day 36 all animals were separated from littermates and were housed 2/cage with a rat of the same sex and group. They remained undisturbed in suspended metal cages (32 X 24 X 19~A cm) requiring no disruption for cleaning until testing in adulthood. Rats from each treatment group were housed at all times in separate racks within the same animal room. B. Adult testing. At approximately 95 days of age, animals from each of the three treatment groups were given three days of testing, each 48 hr apart. Ten males and 10 females from each treatment group received either: (1) three sessions of paired shock-induced fighting (F) after Ulrich and Azrin [29] ; (2) the same shock sequence as the F group but were prevented from exhibiting fighting behavior by being placed singly in the chamber (S); or (3) identical treatment as the S group but with no shock during the session (N). Testing occurred between 0830 and 1230 each day. Animals were tested during one of three consecutive test weeks and only one of the animals from each home cage was tested during a test week. During testing, littermates were at no time paired and pairing between litters was randomized. Since the colony and testing rooms were adjacent, handling was kept to a minimum just prior to testing. Each shock-induced fighting session consisted of the presentation of 90 shock and 90 no-shock trials of 0.5 sec duration with a 1.0 sec no-shock interval between trials, resulting in 180 discrete trials/4.5 rain session. Shocks were administered at each of the three intensities used in trains of 10 followed by 10 no-shock trials. The shock intensities and the order in which they were presented were as follows: 1.3, 2.5, 0.5, 1.3, 0.5, 2.5, 1.3, 2.5, 0.5 mA. The entire shock-induced fighting procedure has been described previously [ 5 ]. On each of the 180 trials in the fighting session, animals were scored for each of three possible behaviors according to criteria previously described [5,29]: (1) fight, consisting of both animals rearing up on their hind legs and making physical contact via slapping and biting movements toward the other; (2) position, consisting of rearing and facing each other in the stereotyped fighting posture but making no physical contact; and (3) no response, in which neither of the above occurred though physical contact could be made through random locomotor activity. Groups were run in a randomized order. Behavioral scoring was performed by an experienced observer; interobserver reliability in this situation in our laboratory has proven to be close to 100%. C. Blood sampling. Immediately following the last test session each animal was decapitated within 5-5V2 min after removal from the home cage and trunk blood collected for later plasma ACTH determination. This time interval has been found to be optimal for peak stress levels of ACTH [ I 1 ]. All blood was kept cold on ice until centrifugation at 4°C not more than aA hr later. Plasma from each Sex X Treatment X Test group was pooled randomly into two samples of approximately 25 cc. The plasma was frozen immediately on dry ice and stored at -20°C until assayed. Error at the time of pooling resulted in the elimination of 8 plasma pools from the study.
HANDLING, FIGHTING AND ACTH
415 (38.0 +- 1.6 g, mean + SE) rats were heavier than the combined weights of LH (30.0 +- 1.1 g) and NH (34.4 + 0.9 g) rats (t = 3.14, d f = 21, p<0.005). Among the two groups undisturbed until weaning, Lid animals were lighter than the NH controls (t = 3.05, d f = 13, p<0.01) which is probably attributable to the fact that there were more large litters in this group. Heavier body weights at weaning in the EH group confirms earlier findings [ 15,16 ]. The behavioral results of the paired rats in the shockinduced fighting procedure can be seen analyzed according to groups in Table 1 and Fig. 1 and analyzed according to sex in Table 2 and Fig. 2. No fights occurred in the absence of shock; data on position frequency, except for the analysis by shock intensity, represent all positions observed on both the shock and no-shock trials. EH and LH animals fought significantly more than NH rats (Fig. 1A; F(2,18) = 3.75, p = 0.04). There were no group differences in the incidence of positioning (Table 1). The frequency of fights attenuated over days (Fig. 1B; F(2,36) = 6.56, p = 0.004). The absence of a Treatment X Days interaction (F< 1) illustrates the consistent elevation of fight frequencies in the EH and LH groups. Fight frequency as a function of intensity can be seen in Fig. 1C. The effects of intensity were highly significant (F(2,18) = 491.04, p< 0.001), the highest frequency of fights occurring at the highest shock intensity. A significant treatment effect (F(2,9) = 4.63, p = 0.04) revealed increased frequency of fights in the EH and LH groups and a Treatment × Intensity interaction (F(4,18) = 6.82, p = 0.001) illustrates the increased responsiveness of the EH and LH groups at the low (0.5 mA) and intermediate (1.3 mA) shock intensities. Males displayed significantly more positions than females (Fig. 2A; F(1,18) = 12.31, p<0.002), but there were no sex differences in fighting frequency (Table 2).
D. A C T H assay. Plasma ACTH was measured b y radioimmunoassay using the method of Rees et al. [26]. In Dr. Dallman's Laboratory, sensitivity of that assay was approximately 20 pg and error within samples was approximately 6%. E. Statistical analyses. As a result of equipment failure, 3 pairs of F animals, each in a different group, received less than the total number of shock trials in one of the three sessions; all data from these pairs were omitted from analysis. A 3-way analysis of variance was used with n's reduced to 4 pairs for analysis of the Sex X Treatment X Days and Sex X Treatment X Intensities effects. Because means of zero were obtained in position frequency at the highest shock intensity in two of the groups, nonparametric statistics (Friedman Test, Mann-Whitney U) were used in these analyses. RESULTS Because of the need to disturb each litter as little as possible, the number of pups/litter was not constant. Though a one-way analysis of variance of Litter Size X Treatment group revealed no significant differences (F< 1), a significantly greater number of litters required culling on Day 21 in the LH group than on Day 1 in the EH or Day 21 in the NH group (5/8, 0/7, 0/8 respectively; Fisher Exact Probability: LH X EH, p = 0.037; LH × NH, p = 0.026). Sex distribution in the randomly assigned litters also showed no differences across treatment group (males, F(2,20)< 1; females, F(2,20) = 2.54, n.s.). Though not all animals from each litter were tested in adulthood, data on litter size, sex distribution and body weights at weaning reflect values obtained from each of the 23 litters. Body weight differences at weaning proved highly significant by treatment analysis (F(2,20) = 10.7 l, p< 0.001 ). EH 60
150 (a)
(b)
125
-
50
U. 100 U.
-
40
I
I
I
80,
(c)
I
I
I
cn 2
o >. cJ ztin . 7 5
30
b
t
... .......
o
UJ rr u= z < ILl :S
.°°.°.°......,
5O
""'°°"'°'°'°'t
20
2O !:':3:
25
10 ., ..-:-
:.::..: ...,. !:'f3.:: EH
LH
NH
0
I 1
I 2 SESSIONS
I 3
ol
,4
I
I
0.5
1.3
2.5
INTENSITY
(mA)
FIG. 1. Mean frequency of fights in the three treatment groups (males and females combined) summed across the three test sessions (a), during each session (b), and as a function of shock intensity (c) (± SEM).
8
8
8
EarLy-handled
Late-handled
Nonhandled
0.88 ± 0.44 10.12 + 2.17 21.62 ± 2.00
2.5
2.5
1.3
23.62 ± 1.50
1.3
0.5
3 . 5 0 ± 1.34 2 0 . 3 8 -+- 1 . 7 0
0.5
23.25 ± 1.84
2.5
3.50 ~ 1.30" 17.12 + 1.48
1.3
0.5
Session 1
1
2 1 . 3 8 ± 1.55
8 . 1 2 ÷ 1.61
0 . 1 2 -+ 0 . 1 2
20.00 + 2.83
13.00 + 3.09
1.75 -+ 0 . 9 0
23.12 + 1.74
17.25 ± 2.31
16.50 + 3.05
6.62 ± 2.34
0.62 ± 0.50
20.38 + 1.82
15.88 + 2.53
0.62 ± 0.32
22.00 + 2.08
13.25 ± 2.61
2.00 + 1.24
Session 3
(POSITIONS)ELICITED
3 . 0 0 + 1.21
Session 2
Fights
ON F1GHTS AND FIGHT1NG POSTURES
Shock Intensity (mA)
HANDLING
* M e a n ± SEM t n = 4 pairs o f m a l e s a n d 4 p a i r s o f f e m a l e s f r o m e a c h t r e a t m e n t g r o u p
N~
Group
EFFECTSOF
TABLE
0.00
1.88 + 0.81
13.50 + 2.96
0.50 ± 0.50
2.12 ± 1.49
1 7 . 5 0 +- 3 . 7 5
0.00
1.62 ± 1.62
15.00 + 3.41
FIGHTING
0.25 ± 0.16
4.25 ± 2.16
16.38 ÷ 4.45
1.00 ÷ 0.87
3 . 7 5 + 1.85
11.88 + 3.14
0.12 + 0.12
2.75 ± 1.70
16.43 + 3.01
Session 2
Positions
SHOCK-INDUCED
Session 1
DURING
0.00
5.62 + 2.31
1 2 . 1 2 -+ 4 . 3 4
0 . 7 5 +- 0 . 6 2
3.50 ~ 1.43
15.38 ± 3.84
1 . 8 8 + 1.61
5 . 5 0 -+ 2 . 2 0
18.50 + 2.24
Session 3
<
Z > Z
,-.]
4~
HANDLING, FIGHTING AND ACTH
417
15
(a)
(b)
75
I
I
(c)
I
I
I
o•
¢n 2 5 0 -
~~i
-
12
-
10
Z 9
k-
,~ 2 0 0 0 >
50-
I
o z
150 -
-
7
u.
100-
-
5
25-
z
\\ 0
o"
I
I
I
1
2
3
I 0.5
0
o ..... 1.3
o 2.5
I N T E N S I T Y (mA)
SESSIONS
FIG. 2. Mean frequency of positions in males and females (treatment groups combined) summed across the three test sessions (a), during each session (b), and as a function of shock intensity (c) (-+ SEM).
TABLE 2 EFFECTS OF SEX ON FIGHTS AND FIGHTING POSTURES (POSITIONS) ELICITED DURING SHOCK-INDUCED FIGHTING Fights Sex
Nt
Shock Intensity (mA)
Males
12
0.5
Females
12
Positions
Session 1
Session 2
Session 3
Session 1
Session 2
Session 3
1.50 ± 0.76*
0.92 ± 0.62
0.83 ± 0.44
19.25 ± 2.83
21.08 ± 2.27
20.58 -+ 2.93
1.3
15.67 ± 1.96
11.50 ± 2.20
10.08 ± 2.04
3.75 ± 1.31
6.83 +- 1.67
9.58 ± 1.14
2.5
24.42 +_ 1.33
24.00 ± 1.37
21.08 _+ 1.68
0.33 ± 0.33
0.83 ± 0.58
1.83 ± 1.10
0.5
3.75 ± 1.01
2.33 -+ 0.86
1.33 ± 0.82
11.42 -+ 2.08
10.42 ± 2.78
10.08 ± 2.02
1.3
16.08 -+ 1.91
14.08 ± 2.19
13.75 -+ 2.43
0.00
0.17 _+ 0.II
0.17 +- 0.11
2.5
21.25 ± 1.41
19.00 ± 1.72
18.17 ± 2.22
0.00
0.08 ± 0.08
0.00
*Mean ± SEM t n = 4 pairs from each treatment group
T h e r e was n o change in n u m b e r o f p o s i t i o n s o b s e r v e d across days in males and females. P o s i t i o n f r e q u e n c y was higher in males t h a n in females o n all test days (Fig. 2B) and at all s h o c k i n t e n s i t i e s (Fig. 2C; M a n n - W h i t n e y U; 0.5 mA, U = 21, p < 0 . 0 2 ; 1.3 m A , U = --6, p < 0 . 0 0 2 ; 2.5 m A , U = 22, p < 0 . 0 0 2 ) . T h e f r e q u e n c y o f p o s i t i o n s and s h o c k i n t e n s i t y were f o u n d t o be inversely related (X 2 r = 6.0, p = 0.028). Results o f the A C T H d e t e r m i n a t i o n s o b t a i n e d in all
animals p o s t t e s t i n g are p r e s e n t e d in Fig. 3. While statistical analysis is n o t a p p r o p r i a t e in view o f t h e small n u m b e r o f p o o l e d samples, p a t t e r n s o f r e s p o n s e can b e assessed. Within a particular test c o n d i t i o n , female ACTH c o n c e n t r a t i o n s w e r e higher t h a n t h o s e o f males and t h e l o w e s t A C T H levels were o b s e r v e d in the n o n s h o c k e d c o n t r o l s . In all groups e x c e p t t h e NH males, A C T H levels were a t t e n u a t e d in the f o u g h t animals w h e n c o m p a r e d t o t h o s e s h o c k e d singly in the c h a m b e r . The A C T H levels o f t h e NH males
418
ERSKINE, STERN AND LEVINE 600 FOUGHT
550
F"-I SHOCKED
~
500
CONTROL
450 400 E 0 0
350 300
.IIu <
250 200 150 100 50 0
EH
M, o, I
LH
I
NH
EH
I
LH
I
NH
o~
FIG. 3. Plasma ACTH concentration (PG/100 ml) obtained following testing, 5-51/2 min after removal from the home cage. Each dot represents an individual sample pool derived from 5 rats. were apparently neither attenuated in the fought animals when compared to EH and LH males, nor elevated in the shock condition when compared to all other groups. DISCUSSION
The present study shows that male and female rats receiving prepubertal handling, either pre- or postweaning, d i s p l a y e d increased shock-induced fighting as adults compared with rats not previously handled. The similarity of the EH and LH groups in frequency of overall fights and fights over days indicates that the effects of handling on the elicitation of shock-induced fighting are not determined exclusively in the early neonatal period. Further, while assessment of individual ACTH values would enable closer examination of treatment differences, male and female EH and LH rats evinced similar patterns of ACTH response. These data are consonant with previous reports [ 1,2] that modification of the adrenocortical response by handling is not accomplished solely during early postnatal life. Surprisingly, there have been no previous comparisons of prevs. postweaning prepubertal handling on a behavioral measure. Perhaps our finding is pertinent to shock-induced fighting only, but it is also possible that the behavioral and physiological effects of handling may be determined during a longer period of early (i. e. pre-adult) life than previously supposed [9 ]. In a study published after the completion of the present work, Thor e t al. [28] did not find a major effect of infantile handling on shock-elicited fighting in rats. The
divergence of our findings may be explained by several factors: (1) Thor e t al. used Long-Evans rats, while our rats were Long-Evans × Sprague-Dawley offspring; (2) Thor e t aL gave 10 daily test sessions, while our three sessions were given on alternate days; (3) Thor e t al. used a shock intensity of 2.0 mA throughout, while we used 0.5, 1.3 and 2.5 mA; and (4) relatively large amounts of handling occurred prior to the fighting sessions in the Thor e t aL study. Points (3) and (4) are probably the most important differences. Since in the present study the largest differences between handled and nonhandled rats occurred at 0.5 mA and 1.3 mA, with no difference at 2.5 mA, it is likely that the effect of early handling is masked at high shock intensities. In the Thor e t al. study, incidental handling during clearning of pan cages and determination of flinch and jump thresholds prior to the initial fighting test may have provided sufficient stimulation to overcome behavioral deficits seen in our nonhandled animals. If such incidental handling in adulthood does produce the same effects as early handling, then the hypothesis that the effects of handling are determined during an early organizational period [9] needs to be reconsidered. However, the effects of early handling on avoidance learning were not duplicated by adult handling [ 14]. Furthermore, some effects of early handling have been observed without the imposition of handling immediately prior to the adult measure [ 15,17], indicating that late handling is not always a confounding variable. Nevertheless, it is clear that the proper interpretation of each early handling effect requires the appropriate postweaning handled and nonhandled controls. An adult
HANDLING, FIGHTING AND ACTH
419
handled group in the present study, for example, may have clarified the difference between our results and those of Thor et al. [28]. The facilitative effect of prior handling on shockinduced fighting which we found in the present study may be interpreted several ways. One possible interpretation is that prior handling enhances aggressive behavior. This explanation is consistent with the increased aggression observed in handled [ 18] and socially-reared [ 12] male mice. However, the irritative nature [24] of the shockinduced fighting situation raises some questions as to the extent to which this test is solely a measure of aggression. On one hand, while conflicting reports appear in the literature of the presence of sex differences in other strains of rats in the shock-elicited fighting paradigm [5, 23, 25], no sex differences were found in fight frequency in this experim e n t . S i n c e sex is a variable powerfully affecting spontaneous agonistic behavior [24], the lack of sex differences suggests that shock-induced fighting may not be solely aggressive in nature. On the other hand, we observed marked sex differences in the number of fighting postures (positions) presented. The posturing response has been described as a defensive reaction to the fighting situation [27] or as an attack o r threatening posture [29]. The consistent elevation of positioning frequency in males over days and at each of the shock intensities indicates that the fighting posture is largely a male response pattern. If stereotyped fighting postures are a true aggressive component of the shock-induced fighting situation, then prior handling cannot be said to enhance aggression, since handling did not influence this behavior. We find an alternate explanation more attractive: The finding that EH and LH animals exhibit elevated fighting levels when compared to NH controls may be interpreted as an increase in the handled rat's ability to cope with aversive s t i m u l a t i o n in an appropriate manner. It has been hypothesized that the facilitation seen in avoidance conditioning in the early-handled animals is a result of a decrease in inappropriate random locomotor activity in response to shock [7]. If an important component of shock-induced fighting is reflexive, as has been suggested [29], then it is possible that a reduction of random activity would enable increased expression of the fighting response. This explanation is also consistent with the hypothesis that handled animals display lowered levels of emotional reactivity in novel situations [7]. Results of the ACTH determinations are only suggestive since each Group X Sex X Testing Condition subgroup is represented by only one or two values. However, three trends were apparent which lend credence to these data. First, the consistently higher ACTH values in females than in males are consonant with the greater pituitary-adrenal
activity of female than male rats [13]. Second, ACTH levels for rats placed in the test chamber but not shocked were consistently and considerably lower than those of rats receiving shock. Third, the earlier finding that fighting plus shock elicits lower ACTH secretion than shock alone [4,6] was replicated in all groups except the NH males. The present study, therefore, extends the results of Conner et al. [4, 6] on males to females. The major new finding from the ACTH data is that of a sex difference in the nonhandled groups with respect to the pituitary-adrenal responsiveness in the fighting situation. Although NH females fought less than handled females, all female groups evinced lower plasma ACTH when fought than when shocked alone. In contrast, in the NH males, decreased fighting was accompanied by a nondiscriminatory ACTH response to fighting and to shock alone, thus supporting our initial expectation that handling would, in fact, modify the pituitary-adrenal response to the shockinduced fighting situation. The differences in the ACTH response between NH males and females suggest that there may be a basic sex difference in coping. T h u s handling alters the capacity to discriminate between shock and fighting in the male, whereas the female appears to have this capacity without the necessity for early stimulation. NH shocked males showed reduced levels of ACTH when compared to the two handled groups, which is consistent with Levine's finding [20] that handled male rats have elevated plasma corticosterone in response to shock when compared to NH controls. NH fought males did not show reduced ACTH compared to the NH shocked response or to the EH and LH fought males. That the male pattern of ACTH release seen previously [4,6] was not evinced in the NH group can be explained in part by the fact that in the previous studies, males were received in the laboratory as adults and then handled extensively prior to testing. The sex difference in the pattern of ACTH in the NH groups revelas a dissociation between the behavioral and physiological response to the shock-induced fighting situation. While this dissociation may seem to confound the suggestion of a causal role for ACTH with respect to fighting in mice [3], Erskine and Levine [10] showed recently that shock-induced fighting in rats is not dependent upon fluctuations in pituitary-adrenal secretions. In summary, the results of the present experiment point out the importance of comparing both pre- and postweaning groups and males and females when studying the handling phenomenon. We found that handling either preor postweaning enhanced the adult fighting response to shock compared to nonhandled controls. Further, in the latter group, there was a sex difference in the physiological, but not behavioral, response to the fighting test situation.
REFERENCES 1. Ader, R. The effect of early experience on the adrenocortical response to different magnitudes of stimulation. Physiol. Behav. 5: 837-839, 1970. 2. Ader, R. and L. J. Grota. The effects of early experience on adrenocortical reactivity. Physiol. Behav. 4: 303-305, 1969. 3. Brain, P. F., N. W. Nowell and A. Wouters. Some relationships between adrenal functions and the effectiveness of a period of isolation in inducing intermale aggression in albino mice. Physiol. Behav. 6: 27-29, 1971.
4. Conner, R. L., J. Vemikos-Danellis and S. Levine. Stress, fighting and neuroendocrine function. Nature 234: 564-566, 1971. 5. Conner, R. L. and S. Levine. Hormonal influences on aggressive behavior. In: Aggressive Behaviour, edited by S. Garattini and E. B. Sigg. Amsterdam: Excerpta Medica, 1969. 6. Conner, R. L., S. Levine and J. Vernikos-Danellis. Shockinduced fighting and pituitary-adrenal activity. Proceedings 78th Annual Convention, APA, 1970. Pp. 201-202.
420 7. Denenberg, V. H. The effects of early experience. In: The Behaviour of Domestic Animals, edited by E. S. E. Hafez. London: Bailliere, Tindall & Cassell, 1969. 8. Denenberg, V. H. and G. G. Karas. Interactive effects of age and duration of infantile stimulation on adult learning. Psychol. Rep. 7: 313-322, 1960. 9. Denenberg, V. H. and M. X. Zarrow. Effects of handling in infancy upon adult behavior and adrenocortical acitivty: Suggestions for a neuroendocrine mechanism. In: Early Childhood, The Development of Self-Regulatory Mechanisms, edited by D. N. Walcher and D. L. Peters. New York: Academic, 1971. 10. Erskine, M. S. and S. Levine. Suppression of pituitary-adrenal activity and shock-induced fighting in rats. Physiol. Behav. 11: 787-790, 1973. 11. Hodges, J. R. and J. Vernikos. Circulating corticotropin in normal and adrenalectomized rats after stress. Acta Endocr. 30: 188-196, 1959. 12. King, J. A. and N. Gurney. Effect of early social experience on adult aggressive behavior in C57B1/10 mice. J. comp. physioL Psychol. 47: 326-330, 1954. 13. Kitay, J. I. Effects of estrogen and androgen on the adrenal cortex of the rat. In: Functions of the Adrenal Cortex, Vol. 2, edited by K. W. McKerns. New York: Appleton-CenturyCrofts, 1968. 14. Levine, S. A further study of infantile handling and adult avoidance learning. J. Pers. 25: 70-80, 1956. 15. Levine, S. Infantile experience and consummatory behavior in adulthood. J. comp. physiol. Psychol. 50: 609-612, 1957. 16. Levine, S. Infantile experience and resistance to physiological stress. Science 126: 405, 1957. 17. Levine, S. Noxious stimulation in infant and adult rats and consummatory behavior. J. comp. physioL Psychol. 51: 230-233, 1958.
E R S K I N E , S T E R N AND L E V I N E 18. Levine, S. Emotionality and aggressive behavior in the mouse as a function of infantile experience. J. Genet. Psychol. 94: 77-83, 1959. 19. Levine, S. The psychophysiological effects of infantile stimulation. In: Roots of Behavior, edited by E. L. Bliss. New York: Harper, 1962. 20. Levine, S. Plasma-free corticosteroid response to electric shock in rats stimulated in infancy. Science 135:795-796, 1962. 21. Levine, S., J. A. Chevalier and S. J. Korchin. The effects of early shock and handling on later avoidance learning. J. Pers. 24: 475-493, 1956. 22. Levine, S., G. C. Haltmeyer, G. Karas and V. H. Denenberg. Physiological and behavioral effects of infantile stimulation. Physiol. Behav. 2: 55-59, 1967. 23. Milligan, W. L., D. A. Powell and G. Borasio. Sexual variables and shock-elicited aggression. J. comp. physiol. Psychol. 83: 441-450, 1973. 24. Moyer, K. E. Kinds of aggression and their physiological basis. Communs Behav. Biol. 2: 6 5 - 8 7 , 1968. 25. Powell, D. A., J. F. Silverman and N. E. Schneiderman. The effects of sex and prior experience with fighting on shockinduced aggression. Communs Behav. Biol. 5 : 5 1 - 5 6 , 1970. 26. Rees, L. H., D. M. Cook, J. W. Kendall, C. F. Allen, R. M. Kramer, J. G. Ratcliffe and R. A. Knight. A radioimmunoassay for rat plasma ACTH. Endocrinology 89: 254-261, 1971. 27. Scott, J. P. Agonistic behavior of mice and rats: a review. Am. Zool. 6: 683-701. 28. Thor, D. H., W. B. Ghiselli and T. B. Ward. Infantile handling and sex differences in shock-elicited aggressive responding of hooded rats. Develop. Psychobiol. 7:'273-279, 1974. 29. Ulrich, R. E. and N. Azrin. Reflexive fighting in response to aversive stimulation. J. exp. Anal. Behav. 5 : 5 1 1 - 5 2 0 , 1962. 30. Weiss, J. M. Effects of coping responses on stress. J. comp. physiol. Psychol. 65:251 -260, 1968.
Effects of Prepubertal Handling on Shock-Induced Fighting and ACTH in Male and Female Rats MARY S. ERSKINE 2, JUDITH M. STERN a AND SEYMOUR LEVINE 4
Department o f Psychiatry, Stanford University School o f Medicine, Stanford, California 94305
(Received 12 November 1974)
ERSKINE, M. S., J. M. STERN AND S. LEVINE. Effects ofprepubertal handling on shock-induced fighting and ACTH in male and female rats. PHYSIOL. BEHAV. 14(4) 413-420, 1975. - Rats which received either handling from Days 1-14 (EH) or 22-35 (LH) of life or no handling (NH) were tested in the shock-induced fighting situation in adulthood. Ten males and 10 females from each group were either paired for fighting (F), shocked singly (S), or received no shock (N) in the test chamber for 4.5 min in 3 sessions given 48 hr apart. Immediately after the third test session, trunk blood was obtained for determination of plasma ACTH concentrations. Males and females did not differ with respect to fighting frequency in response to shock, but males displayed the fighting posture substantially more than did females. Rats handled either preweaning (EH) or postweaning (LH) fought more often at the two lower shock intensities (0.5 and 1.3 mA) than did nonhandled controls (NH). The incidence of fighting positions did not differ as a function of prior handling. In all groups but one, the pattern of ACTH response to the different test conditions coincided with previous reports: higher levels of ACTH were found in response to shock alone than in response to fighting plus shock, and the lowest levels were found in the nonshocked controls. In the exceptional group, the NH males (but not females), there was a nondiscriminatory ACTH response to the S and F test conditions. The effects of prior handling are discussed in terms of an enhanced coping ability. The results point to the importance of comparing both pre- and postweaning groups when studying the handling phenomenon. Early experience
Handling
ACTH
Shock-induced fighting
BEHAVIORAL and physiological effects in adulthood of infantile handling in rats are well documented (see reviews by Denenberg [7], Denenberg and Zarrow [9] and Levine [ 19] ). The picture that has emerged is that laboratory rats receiving early experimenter stimulation have an enhanced ability to cope with novel or emotionally disturbing situations compared with their unmanipulated counterparts. Thus, when tested in adulthood, early-handled rats have been found to be less emotionally reactive, both behaviorally and physiologically, in novel, mildly aversive situations compared to nonhandled controls [22]. In highly aversive situations involving electric shock, behavior is less disrupted in handled animals, as indicated by enhanced avoidance conditioning [8, 14, 21] and water intake after deprivation [ 17 ]. In the shock-induced fighting situation, Conner et al. [4, 6] found lower plasma ACTH levels in paired fighting
Aggression
animals compared to animals shocked alone in the test chamber. They postulated [4] that the ability to express an organized behavior in response to aversive stimulation, resulting in lowered pituitary response to stress, represents a coping response. Similarly, rats allowed to avoid or escape s h o c k displayed increased weight gain and lowered frequency of gastric lesions when compared to yoked controls [30]. In the present experiment, therefore, earlyhandled rats and their controls were either fought with shock (paired animals), shocked alone (single animals), or placed alone in the chamber with no shock during the test session. A group comparison of the ACTH response of the animals receiving shock alone when in the test chamber was of interest since both increased [20] and decreased [1, 2] adrenocortical reactivity has been reported for earlyhandled rats in response to electric shock. Further, if the ability to fight indeed represents a coping response, then it
~This study was supported by Research Grant NICH&HD 02881 from the National Institutes of Health to S. L. Thanks are extended to Dr. Mary Dallman, Dept. of Physiology, U. C. Medical School, San Francisco for assaying the ACTH in these experiments, and further thanks to Mr. Martin Miner for his expert technical assistance. Present address: Department of Biobehavioral Sciences, Box U-154, University of Connecticut, Storrs, CT 06268. 3Present address: Department of Psychology, Rutgers College, Rutgers University, 88 College Avenue, New Brunswick, NJ 08903. 4Supported by USPHS Research Scientist Award K5-MH-19936 from the National Institute of Mental Health. 413
414
ERSKINE, STERN AND LEVINE
might be expected that the early-handled rats would fight more and secrete less ACTH in response to the shockinduced fighting situation when compared to nonhandled fought animals. Handling, as well as other forms of stimulation, has been shown to be effective in influencing adult behavior during several times both pre- and postweaning [7] and there is evidence that the adrenocortical response to stress may be modified in rats stimulated postweaning as well as preweaning [1,2]. Therefore, in addition to the preweaning early-handled (EH) and nonhandled (NH) rats, a third group of postweaning late-handled (LH) rats was included. Further, the present study compares the responses of males and females. Previously handled female rats, as well as males, have been found to be less reactive to adrenocortical activation [1,2]. However, behavioral measures of the effects of early handling have rarely tested both males and females on the same tasks.
METHOD
Apparatus The shock-induced fighting apparatus in adult testing consisted of a chamber (17.8 X 17.8 X 30.5 cm) with a grid floor and three metal walls [5]. The front wall consisted of 3.2 cm vertical grids on 12.7 mm centers allowing unobstructed observation of the animals during the test session. The test chambers were housed in commercial sound-attenuating chambers (Industrial Acoustics Corp.). Shock duration and intensity through a Grason-Stadler Shock Scrambler (Model No. 1064GS) was controlled by solid state circuitry.
Animals Animals were 180 rats obtained from 23 litters bred in our laboratory from Sprague-Dawley females and LongE v a n s males obtained from Simonsen Labs, Gilroy, California. Rats were housed in a room of constant temperature and humidity with noise kept to a minimum. An external source of food and water was available ad lib and light onset of the 12 hr light-dark cycle was 0800. Thirty males and 30 females were assigned to each of the EH, LH and NH treatment groups. Subgroups of 10 animals were randomly chosen for the three test conditions.
Procedure A. Infantile handling. Several days prior to birth, females were individually housed in 28 X 48 X 15 cm pans with wood shavings as bedding material. At birth, litters were divided randomly into three treatment groups: earlyhandled (EH, n=7), late-handled (LH, n=8) and nonhandled (NH, n=8). Litter size varied between 8 and 12 pups; litters of less than 8 pups were discarded from the experiment and large litters were culled to 12 pups on Day 1 in EH litters and at weaning in the LH and NH groups. Early-handled pups were removed from their cage and placed directly into 6 X 6 X 5 cm cardboard compartments for 3 min each day between Days 1 - 1 4 , the day of birth being Day 0. This handling procedure was carried out at 1 0 0 0 - 1 1 0 0 hr each day. No other manipulations of the EH litters, or those of groups LH and NH, occurred prior to weaning. At Day 21, pups were weaned by removing the mother from the cage; at this time, total litter weights were
recorded and bedding material changed. Lit and NH litters exceeding 12 pups were culled prior to weighing. Handling began on Day 22 in the LH groups and continued for 14 days. The procedure was similar to that used for the EH litters, except that the compartments used were 7 X 7 X 12 cm. EH and NH animals remained undisturbed from Days 2 2 - 3 5 . On Day 36 all animals were separated from littermates and were housed 2/cage with a rat of the same sex and group. They remained undisturbed in suspended metal cages (32 X 24 X 19~A cm) requiring no disruption for cleaning until testing in adulthood. Rats from each treatment group were housed at all times in separate racks within the same animal room. B. Adult testing. At approximately 95 days of age, animals from each of the three treatment groups were given three days of testing, each 48 hr apart. Ten males and 10 females from each treatment group received either: (1) three sessions of paired shock-induced fighting (F) after Ulrich and Azrin [29] ; (2) the same shock sequence as the F group but were prevented from exhibiting fighting behavior by being placed singly in the chamber (S); or (3) identical treatment as the S group but with no shock during the session (N). Testing occurred between 0830 and 1230 each day. Animals were tested during one of three consecutive test weeks and only one of the animals from each home cage was tested during a test week. During testing, littermates were at no time paired and pairing between litters was randomized. Since the colony and testing rooms were adjacent, handling was kept to a minimum just prior to testing. Each shock-induced fighting session consisted of the presentation of 90 shock and 90 no-shock trials of 0.5 sec duration with a 1.0 sec no-shock interval between trials, resulting in 180 discrete trials/4.5 rain session. Shocks were administered at each of the three intensities used in trains of 10 followed by 10 no-shock trials. The shock intensities and the order in which they were presented were as follows: 1.3, 2.5, 0.5, 1.3, 0.5, 2.5, 1.3, 2.5, 0.5 mA. The entire shock-induced fighting procedure has been described previously [ 5 ]. On each of the 180 trials in the fighting session, animals were scored for each of three possible behaviors according to criteria previously described [5,29]: (1) fight, consisting of both animals rearing up on their hind legs and making physical contact via slapping and biting movements toward the other; (2) position, consisting of rearing and facing each other in the stereotyped fighting posture but making no physical contact; and (3) no response, in which neither of the above occurred though physical contact could be made through random locomotor activity. Groups were run in a randomized order. Behavioral scoring was performed by an experienced observer; interobserver reliability in this situation in our laboratory has proven to be close to 100%. C. Blood sampling. Immediately following the last test session each animal was decapitated within 5-5V2 min after removal from the home cage and trunk blood collected for later plasma ACTH determination. This time interval has been found to be optimal for peak stress levels of ACTH [ I 1 ]. All blood was kept cold on ice until centrifugation at 4°C not more than aA hr later. Plasma from each Sex X Treatment X Test group was pooled randomly into two samples of approximately 25 cc. The plasma was frozen immediately on dry ice and stored at -20°C until assayed. Error at the time of pooling resulted in the elimination of 8 plasma pools from the study.
HANDLING, FIGHTING AND ACTH
415 (38.0 +- 1.6 g, mean + SE) rats were heavier than the combined weights of LH (30.0 +- 1.1 g) and NH (34.4 + 0.9 g) rats (t = 3.14, d f = 21, p<0.005). Among the two groups undisturbed until weaning, Lid animals were lighter than the NH controls (t = 3.05, d f = 13, p<0.01) which is probably attributable to the fact that there were more large litters in this group. Heavier body weights at weaning in the EH group confirms earlier findings [ 15,16 ]. The behavioral results of the paired rats in the shockinduced fighting procedure can be seen analyzed according to groups in Table 1 and Fig. 1 and analyzed according to sex in Table 2 and Fig. 2. No fights occurred in the absence of shock; data on position frequency, except for the analysis by shock intensity, represent all positions observed on both the shock and no-shock trials. EH and LH animals fought significantly more than NH rats (Fig. 1A; F(2,18) = 3.75, p = 0.04). There were no group differences in the incidence of positioning (Table 1). The frequency of fights attenuated over days (Fig. 1B; F(2,36) = 6.56, p = 0.004). The absence of a Treatment X Days interaction (F< 1) illustrates the consistent elevation of fight frequencies in the EH and LH groups. Fight frequency as a function of intensity can be seen in Fig. 1C. The effects of intensity were highly significant (F(2,18) = 491.04, p< 0.001), the highest frequency of fights occurring at the highest shock intensity. A significant treatment effect (F(2,9) = 4.63, p = 0.04) revealed increased frequency of fights in the EH and LH groups and a Treatment × Intensity interaction (F(4,18) = 6.82, p = 0.001) illustrates the increased responsiveness of the EH and LH groups at the low (0.5 mA) and intermediate (1.3 mA) shock intensities. Males displayed significantly more positions than females (Fig. 2A; F(1,18) = 12.31, p<0.002), but there were no sex differences in fighting frequency (Table 2).
D. A C T H assay. Plasma ACTH was measured b y radioimmunoassay using the method of Rees et al. [26]. In Dr. Dallman's Laboratory, sensitivity of that assay was approximately 20 pg and error within samples was approximately 6%. E. Statistical analyses. As a result of equipment failure, 3 pairs of F animals, each in a different group, received less than the total number of shock trials in one of the three sessions; all data from these pairs were omitted from analysis. A 3-way analysis of variance was used with n's reduced to 4 pairs for analysis of the Sex X Treatment X Days and Sex X Treatment X Intensities effects. Because means of zero were obtained in position frequency at the highest shock intensity in two of the groups, nonparametric statistics (Friedman Test, Mann-Whitney U) were used in these analyses. RESULTS Because of the need to disturb each litter as little as possible, the number of pups/litter was not constant. Though a one-way analysis of variance of Litter Size X Treatment group revealed no significant differences (F< 1), a significantly greater number of litters required culling on Day 21 in the LH group than on Day 1 in the EH or Day 21 in the NH group (5/8, 0/7, 0/8 respectively; Fisher Exact Probability: LH X EH, p = 0.037; LH × NH, p = 0.026). Sex distribution in the randomly assigned litters also showed no differences across treatment group (males, F(2,20)< 1; females, F(2,20) = 2.54, n.s.). Though not all animals from each litter were tested in adulthood, data on litter size, sex distribution and body weights at weaning reflect values obtained from each of the 23 litters. Body weight differences at weaning proved highly significant by treatment analysis (F(2,20) = 10.7 l, p< 0.001 ). EH 60
150 (a)
(b)
125
-
50
U. 100 U.
-
40
I
I
I
80,
(c)
I
I
I
cn 2
o >. cJ ztin . 7 5
30
b
t
... .......
o
UJ rr u= z < ILl :S
.°°.°.°......,
5O
""'°°"'°'°'°'t
20
2O !:':3:
25
10 ., ..-:-
:.::..: ...,. !:'f3.:: EH
LH
NH
0
I 1
I 2 SESSIONS
I 3
ol
,4
I
I
0.5
1.3
2.5
INTENSITY
(mA)
FIG. 1. Mean frequency of fights in the three treatment groups (males and females combined) summed across the three test sessions (a), during each session (b), and as a function of shock intensity (c) (± SEM).
8
8
8
EarLy-handled
Late-handled
Nonhandled
0.88 ± 0.44 10.12 + 2.17 21.62 ± 2.00
2.5
2.5
1.3
23.62 ± 1.50
1.3
0.5
3 . 5 0 ± 1.34 2 0 . 3 8 -+- 1 . 7 0
0.5
23.25 ± 1.84
2.5
3.50 ~ 1.30" 17.12 + 1.48
1.3
0.5
Session 1
1
2 1 . 3 8 ± 1.55
8 . 1 2 ÷ 1.61
0 . 1 2 -+ 0 . 1 2
20.00 + 2.83
13.00 + 3.09
1.75 -+ 0 . 9 0
23.12 + 1.74
17.25 ± 2.31
16.50 + 3.05
6.62 ± 2.34
0.62 ± 0.50
20.38 + 1.82
15.88 + 2.53
0.62 ± 0.32
22.00 + 2.08
13.25 ± 2.61
2.00 + 1.24
Session 3
(POSITIONS)ELICITED
3 . 0 0 + 1.21
Session 2
Fights
ON F1GHTS AND FIGHT1NG POSTURES
Shock Intensity (mA)
HANDLING
* M e a n ± SEM t n = 4 pairs o f m a l e s a n d 4 p a i r s o f f e m a l e s f r o m e a c h t r e a t m e n t g r o u p
N~
Group
EFFECTSOF
TABLE
0.00
1.88 + 0.81
13.50 + 2.96
0.50 ± 0.50
2.12 ± 1.49
1 7 . 5 0 +- 3 . 7 5
0.00
1.62 ± 1.62
15.00 + 3.41
FIGHTING
0.25 ± 0.16
4.25 ± 2.16
16.38 ÷ 4.45
1.00 ÷ 0.87
3 . 7 5 + 1.85
11.88 + 3.14
0.12 + 0.12
2.75 ± 1.70
16.43 + 3.01
Session 2
Positions
SHOCK-INDUCED
Session 1
DURING
0.00
5.62 + 2.31
1 2 . 1 2 -+ 4 . 3 4
0 . 7 5 +- 0 . 6 2
3.50 ~ 1.43
15.38 ± 3.84
1 . 8 8 + 1.61
5 . 5 0 -+ 2 . 2 0
18.50 + 2.24
Session 3
<
Z > Z
,-.]
4~
HANDLING, FIGHTING AND ACTH
417
15
(a)
(b)
75
I
I
(c)
I
I
I
o•
¢n 2 5 0 -
~~i
-
12
-
10
Z 9
k-
,~ 2 0 0 0 >
50-
I
o z
150 -
-
7
u.
100-
-
5
25-
z
\\ 0
o"
I
I
I
1
2
3
I 0.5
0
o ..... 1.3
o 2.5
I N T E N S I T Y (mA)
SESSIONS
FIG. 2. Mean frequency of positions in males and females (treatment groups combined) summed across the three test sessions (a), during each session (b), and as a function of shock intensity (c) (-+ SEM).
TABLE 2 EFFECTS OF SEX ON FIGHTS AND FIGHTING POSTURES (POSITIONS) ELICITED DURING SHOCK-INDUCED FIGHTING Fights Sex
Nt
Shock Intensity (mA)
Males
12
0.5
Females
12
Positions
Session 1
Session 2
Session 3
Session 1
Session 2
Session 3
1.50 ± 0.76*
0.92 ± 0.62
0.83 ± 0.44
19.25 ± 2.83
21.08 ± 2.27
20.58 -+ 2.93
1.3
15.67 ± 1.96
11.50 ± 2.20
10.08 ± 2.04
3.75 ± 1.31
6.83 +- 1.67
9.58 ± 1.14
2.5
24.42 +_ 1.33
24.00 ± 1.37
21.08 _+ 1.68
0.33 ± 0.33
0.83 ± 0.58
1.83 ± 1.10
0.5
3.75 ± 1.01
2.33 -+ 0.86
1.33 ± 0.82
11.42 -+ 2.08
10.42 ± 2.78
10.08 ± 2.02
1.3
16.08 -+ 1.91
14.08 ± 2.19
13.75 -+ 2.43
0.00
0.17 _+ 0.II
0.17 +- 0.11
2.5
21.25 ± 1.41
19.00 ± 1.72
18.17 ± 2.22
0.00
0.08 ± 0.08
0.00
*Mean ± SEM t n = 4 pairs from each treatment group
T h e r e was n o change in n u m b e r o f p o s i t i o n s o b s e r v e d across days in males and females. P o s i t i o n f r e q u e n c y was higher in males t h a n in females o n all test days (Fig. 2B) and at all s h o c k i n t e n s i t i e s (Fig. 2C; M a n n - W h i t n e y U; 0.5 mA, U = 21, p < 0 . 0 2 ; 1.3 m A , U = --6, p < 0 . 0 0 2 ; 2.5 m A , U = 22, p < 0 . 0 0 2 ) . T h e f r e q u e n c y o f p o s i t i o n s and s h o c k i n t e n s i t y were f o u n d t o be inversely related (X 2 r = 6.0, p = 0.028). Results o f the A C T H d e t e r m i n a t i o n s o b t a i n e d in all
animals p o s t t e s t i n g are p r e s e n t e d in Fig. 3. While statistical analysis is n o t a p p r o p r i a t e in view o f t h e small n u m b e r o f p o o l e d samples, p a t t e r n s o f r e s p o n s e can b e assessed. Within a particular test c o n d i t i o n , female ACTH c o n c e n t r a t i o n s w e r e higher t h a n t h o s e o f males and t h e l o w e s t A C T H levels were o b s e r v e d in the n o n s h o c k e d c o n t r o l s . In all groups e x c e p t t h e NH males, A C T H levels were a t t e n u a t e d in the f o u g h t animals w h e n c o m p a r e d t o t h o s e s h o c k e d singly in the c h a m b e r . The A C T H levels o f t h e NH males
418
ERSKINE, STERN AND LEVINE 600 FOUGHT
550
F"-I SHOCKED
~
500
CONTROL
450 400 E 0 0
350 300
.IIu <
250 200 150 100 50 0
EH
M, o, I
LH
I
NH
EH
I
LH
I
NH
o~
FIG. 3. Plasma ACTH concentration (PG/100 ml) obtained following testing, 5-51/2 min after removal from the home cage. Each dot represents an individual sample pool derived from 5 rats. were apparently neither attenuated in the fought animals when compared to EH and LH males, nor elevated in the shock condition when compared to all other groups. DISCUSSION
The present study shows that male and female rats receiving prepubertal handling, either pre- or postweaning, d i s p l a y e d increased shock-induced fighting as adults compared with rats not previously handled. The similarity of the EH and LH groups in frequency of overall fights and fights over days indicates that the effects of handling on the elicitation of shock-induced fighting are not determined exclusively in the early neonatal period. Further, while assessment of individual ACTH values would enable closer examination of treatment differences, male and female EH and LH rats evinced similar patterns of ACTH response. These data are consonant with previous reports [ 1,2] that modification of the adrenocortical response by handling is not accomplished solely during early postnatal life. Surprisingly, there have been no previous comparisons of prevs. postweaning prepubertal handling on a behavioral measure. Perhaps our finding is pertinent to shock-induced fighting only, but it is also possible that the behavioral and physiological effects of handling may be determined during a longer period of early (i. e. pre-adult) life than previously supposed [9 ]. In a study published after the completion of the present work, Thor e t al. [28] did not find a major effect of infantile handling on shock-elicited fighting in rats. The
divergence of our findings may be explained by several factors: (1) Thor e t al. used Long-Evans rats, while our rats were Long-Evans × Sprague-Dawley offspring; (2) Thor e t aL gave 10 daily test sessions, while our three sessions were given on alternate days; (3) Thor e t al. used a shock intensity of 2.0 mA throughout, while we used 0.5, 1.3 and 2.5 mA; and (4) relatively large amounts of handling occurred prior to the fighting sessions in the Thor e t aL study. Points (3) and (4) are probably the most important differences. Since in the present study the largest differences between handled and nonhandled rats occurred at 0.5 mA and 1.3 mA, with no difference at 2.5 mA, it is likely that the effect of early handling is masked at high shock intensities. In the Thor e t al. study, incidental handling during clearning of pan cages and determination of flinch and jump thresholds prior to the initial fighting test may have provided sufficient stimulation to overcome behavioral deficits seen in our nonhandled animals. If such incidental handling in adulthood does produce the same effects as early handling, then the hypothesis that the effects of handling are determined during an early organizational period [9] needs to be reconsidered. However, the effects of early handling on avoidance learning were not duplicated by adult handling [ 14]. Furthermore, some effects of early handling have been observed without the imposition of handling immediately prior to the adult measure [ 15,17], indicating that late handling is not always a confounding variable. Nevertheless, it is clear that the proper interpretation of each early handling effect requires the appropriate postweaning handled and nonhandled controls. An adult
HANDLING, FIGHTING AND ACTH
419
handled group in the present study, for example, may have clarified the difference between our results and those of Thor et al. [28]. The facilitative effect of prior handling on shockinduced fighting which we found in the present study may be interpreted several ways. One possible interpretation is that prior handling enhances aggressive behavior. This explanation is consistent with the increased aggression observed in handled [ 18] and socially-reared [ 12] male mice. However, the irritative nature [24] of the shockinduced fighting situation raises some questions as to the extent to which this test is solely a measure of aggression. On one hand, while conflicting reports appear in the literature of the presence of sex differences in other strains of rats in the shock-elicited fighting paradigm [5, 23, 25], no sex differences were found in fight frequency in this experim e n t . S i n c e sex is a variable powerfully affecting spontaneous agonistic behavior [24], the lack of sex differences suggests that shock-induced fighting may not be solely aggressive in nature. On the other hand, we observed marked sex differences in the number of fighting postures (positions) presented. The posturing response has been described as a defensive reaction to the fighting situation [27] or as an attack o r threatening posture [29]. The consistent elevation of positioning frequency in males over days and at each of the shock intensities indicates that the fighting posture is largely a male response pattern. If stereotyped fighting postures are a true aggressive component of the shock-induced fighting situation, then prior handling cannot be said to enhance aggression, since handling did not influence this behavior. We find an alternate explanation more attractive: The finding that EH and LH animals exhibit elevated fighting levels when compared to NH controls may be interpreted as an increase in the handled rat's ability to cope with aversive s t i m u l a t i o n in an appropriate manner. It has been hypothesized that the facilitation seen in avoidance conditioning in the early-handled animals is a result of a decrease in inappropriate random locomotor activity in response to shock [7]. If an important component of shock-induced fighting is reflexive, as has been suggested [29], then it is possible that a reduction of random activity would enable increased expression of the fighting response. This explanation is also consistent with the hypothesis that handled animals display lowered levels of emotional reactivity in novel situations [7]. Results of the ACTH determinations are only suggestive since each Group X Sex X Testing Condition subgroup is represented by only one or two values. However, three trends were apparent which lend credence to these data. First, the consistently higher ACTH values in females than in males are consonant with the greater pituitary-adrenal
activity of female than male rats [13]. Second, ACTH levels for rats placed in the test chamber but not shocked were consistently and considerably lower than those of rats receiving shock. Third, the earlier finding that fighting plus shock elicits lower ACTH secretion than shock alone [4,6] was replicated in all groups except the NH males. The present study, therefore, extends the results of Conner et al. [4, 6] on males to females. The major new finding from the ACTH data is that of a sex difference in the nonhandled groups with respect to the pituitary-adrenal responsiveness in the fighting situation. Although NH females fought less than handled females, all female groups evinced lower plasma ACTH when fought than when shocked alone. In contrast, in the NH males, decreased fighting was accompanied by a nondiscriminatory ACTH response to fighting and to shock alone, thus supporting our initial expectation that handling would, in fact, modify the pituitary-adrenal response to the shockinduced fighting situation. The differences in the ACTH response between NH males and females suggest that there may be a basic sex difference in coping. T h u s handling alters the capacity to discriminate between shock and fighting in the male, whereas the female appears to have this capacity without the necessity for early stimulation. NH shocked males showed reduced levels of ACTH when compared to the two handled groups, which is consistent with Levine's finding [20] that handled male rats have elevated plasma corticosterone in response to shock when compared to NH controls. NH fought males did not show reduced ACTH compared to the NH shocked response or to the EH and LH fought males. That the male pattern of ACTH release seen previously [4,6] was not evinced in the NH group can be explained in part by the fact that in the previous studies, males were received in the laboratory as adults and then handled extensively prior to testing. The sex difference in the pattern of ACTH in the NH groups revelas a dissociation between the behavioral and physiological response to the shock-induced fighting situation. While this dissociation may seem to confound the suggestion of a causal role for ACTH with respect to fighting in mice [3], Erskine and Levine [10] showed recently that shock-induced fighting in rats is not dependent upon fluctuations in pituitary-adrenal secretions. In summary, the results of the present experiment point out the importance of comparing both pre- and postweaning groups and males and females when studying the handling phenomenon. We found that handling either preor postweaning enhanced the adult fighting response to shock compared to nonhandled controls. Further, in the latter group, there was a sex difference in the physiological, but not behavioral, response to the fighting test situation.
REFERENCES 1. Ader, R. The effect of early experience on the adrenocortical response to different magnitudes of stimulation. Physiol. Behav. 5: 837-839, 1970. 2. Ader, R. and L. J. Grota. The effects of early experience on adrenocortical reactivity. Physiol. Behav. 4: 303-305, 1969. 3. Brain, P. F., N. W. Nowell and A. Wouters. Some relationships between adrenal functions and the effectiveness of a period of isolation in inducing intermale aggression in albino mice. Physiol. Behav. 6: 27-29, 1971.
4. Conner, R. L., J. Vemikos-Danellis and S. Levine. Stress, fighting and neuroendocrine function. Nature 234: 564-566, 1971. 5. Conner, R. L. and S. Levine. Hormonal influences on aggressive behavior. In: Aggressive Behaviour, edited by S. Garattini and E. B. Sigg. Amsterdam: Excerpta Medica, 1969. 6. Conner, R. L., S. Levine and J. Vernikos-Danellis. Shockinduced fighting and pituitary-adrenal activity. Proceedings 78th Annual Convention, APA, 1970. Pp. 201-202.
420 7. Denenberg, V. H. The effects of early experience. In: The Behaviour of Domestic Animals, edited by E. S. E. Hafez. London: Bailliere, Tindall & Cassell, 1969. 8. Denenberg, V. H. and G. G. Karas. Interactive effects of age and duration of infantile stimulation on adult learning. Psychol. Rep. 7: 313-322, 1960. 9. Denenberg, V. H. and M. X. Zarrow. Effects of handling in infancy upon adult behavior and adrenocortical acitivty: Suggestions for a neuroendocrine mechanism. In: Early Childhood, The Development of Self-Regulatory Mechanisms, edited by D. N. Walcher and D. L. Peters. New York: Academic, 1971. 10. Erskine, M. S. and S. Levine. Suppression of pituitary-adrenal activity and shock-induced fighting in rats. Physiol. Behav. 11: 787-790, 1973. 11. Hodges, J. R. and J. Vernikos. Circulating corticotropin in normal and adrenalectomized rats after stress. Acta Endocr. 30: 188-196, 1959. 12. King, J. A. and N. Gurney. Effect of early social experience on adult aggressive behavior in C57B1/10 mice. J. comp. physioL Psychol. 47: 326-330, 1954. 13. Kitay, J. I. Effects of estrogen and androgen on the adrenal cortex of the rat. In: Functions of the Adrenal Cortex, Vol. 2, edited by K. W. McKerns. New York: Appleton-CenturyCrofts, 1968. 14. Levine, S. A further study of infantile handling and adult avoidance learning. J. Pers. 25: 70-80, 1956. 15. Levine, S. Infantile experience and consummatory behavior in adulthood. J. comp. physiol. Psychol. 50: 609-612, 1957. 16. Levine, S. Infantile experience and resistance to physiological stress. Science 126: 405, 1957. 17. Levine, S. Noxious stimulation in infant and adult rats and consummatory behavior. J. comp. physioL Psychol. 51: 230-233, 1958.
E R S K I N E , S T E R N AND L E V I N E 18. Levine, S. Emotionality and aggressive behavior in the mouse as a function of infantile experience. J. Genet. Psychol. 94: 77-83, 1959. 19. Levine, S. The psychophysiological effects of infantile stimulation. In: Roots of Behavior, edited by E. L. Bliss. New York: Harper, 1962. 20. Levine, S. Plasma-free corticosteroid response to electric shock in rats stimulated in infancy. Science 135:795-796, 1962. 21. Levine, S., J. A. Chevalier and S. J. Korchin. The effects of early shock and handling on later avoidance learning. J. Pers. 24: 475-493, 1956. 22. Levine, S., G. C. Haltmeyer, G. Karas and V. H. Denenberg. Physiological and behavioral effects of infantile stimulation. Physiol. Behav. 2: 55-59, 1967. 23. Milligan, W. L., D. A. Powell and G. Borasio. Sexual variables and shock-elicited aggression. J. comp. physiol. Psychol. 83: 441-450, 1973. 24. Moyer, K. E. Kinds of aggression and their physiological basis. Communs Behav. Biol. 2: 6 5 - 8 7 , 1968. 25. Powell, D. A., J. F. Silverman and N. E. Schneiderman. The effects of sex and prior experience with fighting on shockinduced aggression. Communs Behav. Biol. 5 : 5 1 - 5 6 , 1970. 26. Rees, L. H., D. M. Cook, J. W. Kendall, C. F. Allen, R. M. Kramer, J. G. Ratcliffe and R. A. Knight. A radioimmunoassay for rat plasma ACTH. Endocrinology 89: 254-261, 1971. 27. Scott, J. P. Agonistic behavior of mice and rats: a review. Am. Zool. 6: 683-701. 28. Thor, D. H., W. B. Ghiselli and T. B. Ward. Infantile handling and sex differences in shock-elicited aggressive responding of hooded rats. Develop. Psychobiol. 7:'273-279, 1974. 29. Ulrich, R. E. and N. Azrin. Reflexive fighting in response to aversive stimulation. J. exp. Anal. Behav. 5 : 5 1 1 - 5 2 0 , 1962. 30. Weiss, J. M. Effects of coping responses on stress. J. comp. physiol. Psychol. 65:251 -260, 1968.