Modification of juvenile play and other social behaviour in the rat by neonatal progestins: Further studies

Physiology & Behavior, Vol. 33. pp. 217-219. Copyright©Pergamon Press Ltd., 1984. Printed in the U.S.A.

0031-9384/84 $3.00 + .00

Modification of Juvenile Play and Other Social Behaviour in the Rat by Neonatal Progestins: Further Studies L Y N D A I. A. B I R K E A N D D A W N S A D L E R

A n i m a l Behavior Research Group, Department o f Biology, The Open University Walton Hall, Milton Keynes, England R e c e i v e d 30 M a r c h 1983 BIRKE, L. I. A. AND D. SADLER. Modification of jm'enile ploy and other social behaviour in the rat by neonatal progestins: Further studies. PHYSIOL BEHAV 33(2) 217-219, 1984.--The effects of altering neonatal levels of progestins on the later development of social play behaviour was studied. Progestin levels were raised in experiment one by administering injections of either progesterone or medroxyprogesterone acetate. This indicated that exposure to either hormone led to reduced levels of social play in juvenile rats of both sexes, confirming earlier reports of lowered levels of play following medroxyprogesterone obtained via maternal milk. In Experiment 2, endogenous progestin levels were lowered by administration of the antiserum to progesterone. The prediction that this should result in raised levels of juvenile play was supported for males, but not for females. Females in Experiment 2 by contrast showed a decrease in play. Possible reasons for this sex difference in response to progesterone antiserum are discussed. Progesterone Medroxyprogesterone acetate Sexual differentiation

Rough-and-tumble play

WE have previously reported an effect of neonatal progestins on the subsequent development of social play, an experiment which formed part of a series aimed at investigating some effects of neonatal progestins on sexual differentiation. Specifically, neonatal exposure via maternal milk to the synthetic steroid, medroxyprogesterone acetate (MPA) resulted in decreased levels o f social play by both males and females during the prepubertal period [1]. Although MPA is known to be orally active [2] and to be secreted into rat milk [3] it remains possible that the effects of the hormone on play are not mediated directly by the hormone but by some maternal effect. The study reported here was designed to investigate this finding further by either raising (Experiment 1) or lowering pups progestin levels (Experiment 2). In these experiments, however, hormones were given directly to the pup, rather than via the mother. In Experiment 1 of the present study, both progesterone and medroxyprogesterone acetate were administered to pups in order to raise plasma progestin levels. In Experiment 2, pups were given the antiserum to progesterone, in order to lower endogenous progestin levels. On the basis of our previous experiments, it would be predicted that such lowering of progestin levels should result in an increase in later play behaviour in both sexes.

Progesterone antiserum

as described previously [1]. A split litter design was used to create matched pairs of litters receiving similar treatment, such that pups were divided equally as far as possible between pairs of mothers. Litters in which the sex ratio departed markedly from 1:1 were not used in the experiment. On the day following birth (day 2), the injection schedules began. In each litter, half the males and half the females were assigned at random to the experimental group, and half to the control group. Injections were given between 1500 and 1700 hr, after which the pups were returned to the dams.

INJECTION PROCEDURES

Experiment I Eighty offspring o f twelve litters were used in this experiment, divided into six litters receiving progesterone or the oil vehicle (group A), and six litters receiving MPA or vehicle (group B) as follows: on the day following birth (day 2), half the males and half the females in each group A litter were injected subcutaneously with 2.5 /zg progesterone in 0.01 ml arachis oil. The remaining pups comprised matched controls and were injected with 0.01 ml of the oil vehicle alone. These injections were repeated at 24 and 48 hr following the initial injection (days 3 and 4). The animals were then left with the dam until weaning. Half the males and half the females of group B litters were similarly injected on day 2 with MPA at a dose of 0.12 mg as a suspension in 0.01 ml o f 0.9% saline. The remaining animals were injected with 0.01 ml of the saline vehicle

GENERAL METHODS Offspring from litters of twenty nulliparous rats of Wistar-derived stock bred in the Animal House of the Open University were used. Litters were housed and maintained

217

218

BIRKE AND SADLER TABLE1 SUMMARY OF RESULTS FROM BEHAVIOURAL SAMPLING: EXPERIMENT I Behaviour Play-fight A: Progesterone Progesterone: males Control males Progesterone females Control females

3.5 12.2 1.75 6.25

B: MPA MPA males Control males MPA females Control females

5.1 _+ 1.9 9.7 ± 3.9* 1.7 ± 0.6 4.7 ± 2.0*

± 1.05 + 3.7+ ~_. 0.8 ± 2.5*

Social sniff

Rear

± +_ + +_

Groom

8.9 7.2 7.25 9.7

+ 2.8 _+ 2.0 ± 2.6 ± 3.6

21.1 21.2 23.6 19.0

6.4 6.2 8.5 6.8

4.7 7.3 6.3 9.2

_+ 2.1 ± 1.9 _+ 1.25 -+ 2.5

18.9 + 9.3 23.8 _+ 9.4 24.5_+ 10.3 24.3_+ 11.7

Pounce

5.4_+ 1.6 6.9 +_ 1.7 5.9 ± 2.6 5.7 + 2.2

2.7 2.6 1.4 2.5

± ~ ± ±

2.3 6.4 3.0 2.7

1.14 1.86 1.7 2.7

+~ 0.4 + 0.7 ~ 0.7 -~ 1.1

+_ 0.9 _+ 2.7 + 1.1 + 1.1

Explore

0.8 0.9 0.85 0.9

36.7 33.2 39.7 36.7

± ± ~ ~:

11.0 9.6 14.1 13.1/

25.3 35.1 41.3 35./)

:L 9.7 ~ 12.7 _~ 16.9 :~- 14.9

(Mean frequency ± s.e.m.). *=p~<0.05. +-p<0.01.

TABLE 2 SUMMARY OF RESULTS FROM BEHAVIOURAL SAMPLING: EXPERIMENT 2

Behaviour Play fight Anti-P males Control males Anti-P females Control females

9.4 5,6 2.2 5.13

± ± ± ±

1,45 1.15 0,8 1.25

Social sniff 9.2 10.5 12.4 8.3

± ± + ±

2.4 2.0 2.6 1.9

Rear 30,4 28.4 23.4 20.5

± 4.0 ± 3.1 ± ± 4.9

Groom 5.0 6.0 10.0 8.9

:_~ 1.5 -_~ 2.0 ± 1.5 -+ 2.5

Pounce 2.6 2.5 1.2 2.7

± ± ± ±

0.5 0.4 0.3 0.6

Explore 42.1 41.3 38.6 41.6

± 2.9 ± 5.5 + 8.2 +_ 9.5

(Mean frequency ± s.e.m.). * =p <0.05. t=0.05 > p>0.02. Sp <0.02.

alone. Since M P A is a l o n g - a c t i n g steroid, b e i n g r e l e a s e d slowly f r o m the i n j e c t i o n site, only this o n e s u b c u t a n e o u s injection was given. N o injections w e r e g i v e n o n s u b s e q u e n t days.

Experiment 2 E i g h t y - f o u r offspring o f t w e l v e litters w e r e used. T h e s e w e r e divided into t w o b a t c h e s , t e s t e d at different times. A s before, e a c h litter w a s divided into t w o halves. H a l f o f e a c h sex w e r e i n j e c t e d w i t h a s o l u t i o n o f the a n t i s e r u m to p r o g e s t e r o n e (anti-P), at a d o s e o f 0.025 m g d i s s o l v e d in 0.01 ml i s o t o n i c saline. T h e a n t i s e r u m w a s p u r c h a s e d f r o m S i g m a C h e m i c a l s Ltd, as t h e r a b b i t a n t i b o d y to p r o g e s t e r o n e 11-c~ h e m i s u c c i n a t e b o u n d to b o v i n e s e r u m a l b u m i n . T h e c o n t r o l a n i m a l s r e c e i v e d a similar i n j e c t i o n of 0.01 saline v e h i c l e alone. I n j e c t i o n s were g i v e n o n d a y s 2, 3 a n d 4. In pilot studies u s i n g identical d o s e a g e s o f t h e a n t i s e r u m o n d a y s 2, 3 a n d 4, p u p s h a d b e e n culled o n t h e a f t e r n o o n o f day 4 (following i n j e c t i o n 4 h r earlier) in o r d e r to d e t e r m i n e p l a s m a p r o g e s t e r o n e levels. T h e s e w e r e a n a l y s e d using r a d i o i m munoassay, which revealed that endogenous progesterone

levels w e r e r e d u c e d to a p p r o x i m a t e l y 60 p e r c e n t o f t h a t o f c o n t r o l s ( m e a n p r o g e s t e r o n e levels in p l a s m a f r o m p u p s w e r e : c o n t r o l f e m a l e s = 1 2 . 6 pg/100 ml plasma: antip r o g e s t e r o n e females = 7 . 9 4 pg/100 ml plasma. Control m a l e s = 6.3 pg/100 ml p l a s m a : a n t i p r o g e s t e r o n e m a l e s = 3 . 9 pg/100 ml plasma).

Behavioural Observations T h e o b s e r v a t i o n a l m e t h o d s used w e r e b r o a d l y similar to t h o s e d e s c r i b e d p r e v i o u s l y [ 1] e x c e p t t h a t all o b s e r v a t i o n s in this s t u d y w e r e c o n d u c t e d w h e n t h e a n i m a l s w e r e b e t w e e n 26 a n d 33 d a y s o f age. A n i m a l s w e r e t e s t e d in w i t h i n - g r o u p , s a m e - s e x pairing u s i n g a b e h a v i o u r a l s a m p l i n g m e t h o d . Since o b s e r v a t i o n s w e r e o f pairs o f a n i m a l s f r o m different litters, t e s t i n g was c a r r i e d o u t in a c l e a n t e s t i n g cage, unfamiliar to b o t h p a r t i c i p a n t s , b u t o f the s a m e t y p e as the s t a n d a r d s t o c k c a g e s in w h i c h the a n i m a l s w e r e n o r m a l l y m a i n t a i n e d . In o n e cage a n e x p e r i m e n t a l a n i m a l w a s placed a n d in a s e c o n d cage a c o n t r o l a n i m a l o f t h e s a m e sex f r o m the s a m e litter. T h e s e w e r e t h e n paired with a n i m a l s o f the s a m e sex f r o m the m a t c h e d litter to c r e a t e pairings of e x p e r -

MODIFICATION OF J U V E N I L E PLAY BY PROGESTINS imental animals in one cage and of control animals in the other. The behaviour of the animals in each pair was then sampled every 15 sec for a period of 10 min. The following behaviour patterns were noted: Play This consists of a number of components, including wrestling, boxing, and pinning (in which one individual pins the other down on its back). No attempt was made in behavioural sampling to separate the components. Social sniffing Sniffing at any part of the body of the animal. Exploring This was defined as active locomotion around the cage, usually accompanied by sniffing of the walls or floor. Rearing In which the animal rears up on its hindlegs. Pouncing Although this sometimes occurs as a component of play sequences, it also sometimes occurs on its own, and is commonly interpreted as a play initiation. Self-grooming In which any part of the body is cleaned. After testing, all animals were colour marked and returned to their home cages. The tests were repeated until all the males and all the females from each matched pair of litters had been tested. RESULTS AND DISCUSSION

Experiment 1 The results of this experiment are summarised in Table 1. Data were analysed using a t-test to compare scores obtained for MPA or progesterone animals with their controls. As some of the data were skewed, analyses were done on the log-transformed scores. The results show that the frequency of play observations was significantly greater in controls of both sexes than in experimentals (progesterone males vs. controls, p <0.01: progesterone females vs. controls p <0.05: MPA males vs. controls p<0.05: MPA females vs. controls p=0.05: No other significant differences were found. The results shown in Table 1, are very similar to those obtained in our previous study, using MPA administered orally via milk: that is, MPA animals of both sexes play less than controls. The data from the progesterone group also show similar effects on the frequency of play observations. In general, the results of this experiment support the hypothesis that raising neonatal progestin levels results in a decrease in play behaviour shown during the prepubertal period. Both MPA and progesterone have been shown to be effective in thus reducing the levels of play shown. In addition, these data confirm our earlier findings from experiments using MPA given orally, thus suggesting that the effect does not depend on the route of administration.

219 times, so these data were combined for further analyses. As predicted, the incidence of play behaviour exhibited by anti-P males is significantly greater than that shown for controis (o<0.02). No other significant differences emerged from males. However, for females, the differences in play behaviour were reversed: anti-P females showed significantly less play behaviour than did controls (p <0.02). Pouncing showed a similar effect; anti-P animals pounced less than controls (0.02

ACKNOWLEDGEMENTS

Experiment 2 The results of this experiment are summarised in Table 2. There were no noticeable differences between the results obtained from the two identical groups tested at different

We are very grateful to Neff Chalmers for assisting us with the computer analysis programmes used in these studies, and also to Christiane Holzhausen for kindly carrying out the progestero.ne radioimmunoassays.

REFERENCES

1. Birke, L. I. A. and D. Sadler. Progestin-induced changes in play behaviour of the prepubertal rat. Physiol Behav 30: 341-347, 1983. 2. Goldhiezer, J. W. Synthetic progestational steroids: their significance and use. Texas State J Med 57: 962-967, 1961.

3. Holzhausen, C., S. Murphy and L. I. A. Birke. Neonatal exposure to a progestin via milk alters subsequent LH cyclicity in the female rat. J Endocrinol I00: 149-154, 1984.