Physiology & Behavior, Vol. 18, pp. 119 123. Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.
Loss of Socialized Patterns of Behavior in Mouse Colonies Following Daily Sleep Disturbance During Maturation' F L O R A M. C. W A T S O N A N D J A M E S P. H E N R Y
University o f Southern California, School o f Medicine, Department o f Physiology Los Angeles, CA 90007
(Received 30 July 1975) WATSON, F. M. C. AND J. P. HENRY. Loss of socialized patterns of behavior in mouse eolonies fbllowing daily sleep disturbance during maturation. PHYSIOL. BEHAV. 18(1) 119-123, 1977.-- An ethologically-orientcd experimental design has been evolved to test the hypothesis that in the developing animal, rapid eye movement (REM) sleep may be concerned with integrating inherited aspects of attachment and territorial behavior with higher cognitive processes. Isolated weaned mice were daily submitted to 3 - 5 hr of treadmill sleep deprivation throughout maturation from 28 days until they were adult at 4 months. In some (controls) the treadmill preceded, and in others (experimental), it followed a 2 V2 hr socialization period during which the males and females interacted in a complex population cage. The adult responses of the various groups were tested in an open field by measuring their activity and elevation of plasma corticosterone. The capacity for organized social interaction was evaluated by observing blood pressure and other responses to two months of continuous exposure to colony life in a population cage. Mice that were routinely put on the treadmill immediately after socialization behaved as though they had been isolated throughout their development. Those that experienced the treadmill before socialization developed a normal hierarchy despite 21 Y2 hr of isolation between the daily socialization experiences. There was a statistically significant difference between the open field activity, the open field corticosterone value, and the blood pressure of the control and the experimental groups. REM sleep
Sleep deprivation
Socialization
Social interaction
IN A detailed n e u r o p h y s i o l o g i c a l analysis of the f u n c t i o n o f dreaming, J o u v e t [7] has a t t a c h e d great i m p o r t a n c e to the high level of rapid eye m o v e m e n t ( R E M ) in u t e r o and i m m e d i a t e l y after birth. He p o s t u l a t e d t h a t since cellular growth a n d d i f f e r e n t i a t i o n c o n t i n u e while the y o u n g animal is developing its final p a t t e r n s of behavior, d r e a m i n g or the REM phase m a y be i m p o r t a n t in the o r g a n i z a t i o n of complicated behavioral sequences or instincts, such as defense of territory, h u n t i n g , a n d mating. The f u n c t i o n of d r e a m i n g would be to s t i m u l a t e n e u r o n s t h a t c o m m a n d a n e t w o r k responsible for p r o g r a m m i n g p a t t e r n s of b e h a v i o r characteristic of the species' r e s p o n s e to i n c o m i n g stimuli derived from the e x p e r i e n c e s o f the individual. He suggested t h a t such p r o g r a m m i n g would n o t be likely to occur during wakefulness w h e n the c o m m a n d n e u r o n s were r e s p o n d i n g to external stimuli: rather, the a l t e r a t i o n of synapses t h r o u g h learning in response to the needs of genetic prog r a m m m g m i g h t occur d u r i n g REM sleep w h e n the c o m m a n d n e u r o n s are less engaged by the e n v i r o n m e n t . R e c e n t work is c o m p a t i b l e with this t h e o r y . Lucero [10] observed t h a t the d u r a t i o n of REM sleep increases after a learning experience. The o b s e r v a t i o n was c o n f i r m e d and e x t e n d e d in several o t h e r studies by various groups [3,
Blood pressure
5, 9, 13, 14, 16]. If REM is p r e v e n t e d for 2 3 h o u r s i m m e d i a t e l y after the experience, there is a deficit in learning. K i t a h a m a , Valatx, and J o u v e t [81 s h o w e d that the REM-suppressing s u b s t a n c e a l p h a m e t h y l - D O P A slows maze learning, and P e a r l m a n and Becker 113] f o u n d a similar effect w h e n i m i p r a m i n e or c h t o r d i a z e p o x i d e was used during bar pressing. O f special interest from the poinl of view of social situations, the latter a u t h o r s also r e p o r t e d t h a t REM d e p r i v a t i o n p r e v e n t e d a rat from assimilating lhe significance o f the skilled bar-pressing b e h a v i o r of a con> panion. G r e e n b e r g and P e a r h n a n [4] reviewed these and o t h e r studies p o i n t i n g to the adaptive role of REM sleep in light of Seligman's r e c e n t critique of the generality of the laws of learning 115]. Seligman has p o i n t e d o u t t h a t while animals are prepared by virtue of e v o l u t i o n a r y history to make some associations, they are in fact u n p r e p a r e d or even c o n t r a p r e p a r e d for others. He predicled t h a t successful avoidance will be rapidly learned only after those evenls relating to the n a t u r a l species-specific repertoire of the organism. G r e e n b e r g and Pearlman [41 suggest that such rapidly learned h a b i t u a l reactions for which the animal "'i.~ p r e p a r e d " in Seligman's sense will be s h o w n to be R I M
' This research was supported by the National Institutes of Health Grant No. MH 19441. 119
120
WVI'SON AND I:tt-NR'~
i n d e p e n d e n t . On t h e o t h e r h a n d , activities involving the a c c u m u l a t i o n of u n u s u a l i n f o r m a t i o n w o u l d be likely to require REM sleep for o p t i m a l c o n s o l i d a t i o n into long-term memories. The q u e s t i o n arises w h e t h e r the learning involved in t h e a c q u i s i t i o n of territorial and m a t e r n a l behavior n e e d e d by a hierarchically o r g a n i z e d c o l o n y also falls i n t o t h e REM d e p e n d e n t category. T h e peer i n t e r a c t i o n of animals has b e e n s h o w n t~) be essential to the d e v e l o p m e n t of n o r m a l social b e h a v i o r Mason [ 11 ] has s h o w n t h a t r e s p o n s e s to social cues were poorly e s t a b l i s h e d in socially deprived m o n k e y s . W a t s o n Henry, a n d H a l t m e y e r [18] f o u n d that w h e n socially de prived mice were e x p o s e d to o t h e r mice for 30 rain ol social i n t e r a c t i o n , t h e y had a m u c h higher plasma corficos t e r o n e r e p o n s e t h a n group-reared mice. They were also less active in t h e o p e n field, F u r t h e r m o r e . w h e n these formerl3 isolated animals were p u t i n t o a c o m p l e x p o p u l a t i o n cage, t h e y failed to a d a p t socially to c h r o n i c psyehosocial slimu l a t i o n , w i t h resulting high levels of aggression and hypertension. T h u s r e p e a t e d social i n t e r a c t i o n after weaning enables t h e mice to acquire c e r t a i n b e h a v i o r p a t t e r n s lhat are valuable d u r i n g a d u l t h o o d . These p a t t e r n s mcrease the t o l e r a n c e of e x t e r n a l stimuli a n d m o d i f y adult activities so as to decrease the i n t e n s i t y of social s t i m u l a t i o n arisin~ within the group. As J o u v e t [7] a n d G r e e n b e r g and P e a r l m a n {4] suggest. d r e a m i n g or REM sleep m a y be i m p o r t a n t to the developing central n e r v o u s system as it integrates a n d r e p r o g r a m s e m o t i o n a l l y a r o u s i n g e x p e r i e n c e s i n t o fresh social behavioral patterns. If REM does i n d e e d provide an o p p o r t u n i t y for the a d a p t a t i o n of i n h e r i t e d species-specific r e p e r t o i r e s ~o a p p r o p r i a t e e x t e r n a l cues, then animals t h a t were repeatedly deprived of all sleep (and t h e r e f o r e of REM sleep l following socialization might n o t e x h i b i t the full effect ot early socialization as adults. Thus, t h e objective of this s t u d y was to d e t e r m i n e w h e t h e r t h e socialization of CBA mice c o u l d be i n h i b i t e d b y s y s t e m a t i c a l l y f o l l o w i n g each p e r i o d of i n t e r a c t i o n w i t h a few h o u r s of sleep deprivatiol~ on a r o t a t i n g treadmill t h r o u g h o u t m a t u r a t i o n .
Animals l'he CBA agouti mice used m [hJ~ .qudy were originall3 o b t a i n e d from tile J a c k s o n Memorial L a b o r a t o r y , Bar t t a r b o r , Maine and have been bred ~md raised in our labo r a t o r y since 1955.
Proccdz#¢ T w e n t y p r e g n a n t CBA mice were placed in pairs m 2,S 17 ~ 12 cm nest b o x e s and were aItowed to deliver. I h e litters were r e d u c e d to 8 pups al b i r t h and were m~t d~sturbed f u r t h e r u n t i l t h e y were weaned at 2 t days o f a g e . . \ t this time. the males were numbereat by ear pm~ching and were individually placed m V2-1iter jars c o n t a i n i n g w o o d shavings. A p a p e r towel was taped a r o u n d each jar Ic, eliminate visual c o n t a c t with o t h e r mice. F o o d and water were supplied as n e e d e d , and the shavings were changed once u week. Twelve males were assigned al r a n d o m to each of the 5 t r e a t m e n t c o m b i n a t i o n s s h o w n in Fig. !. The [re;ttm e n t s were initiated o n e week after w e a n i n g and c o n t i n u e d until the mice were 4 m o n t h s old. T h e isolated mice were n o t d i s t u r b e d excepl w h e n their jars were cleaned once a week. but the mice assigned t,, socmlization were r e m o v e d f r o m their .tars and placed toT 2!& hr in an i n t e r c o m m u n i c a t i n g o-box p o p u l a t i o n cage (Fig. 2t where they could explore anti engage in social a c t i v i t y t 5 ] . This system is c o n s t r u c t e d o f Plexiglas boxes measuring 23 - 11 11 cm which are c o n n e c t e d m t o a circle by flexible plastic tubes J3.8 cm i d.'). The cenlral h e x a g o n holds f o o d a n d w a t e r and is c o n n e c t e d to each box hy t u b e s {3.3 cm i. d.) The cage wits designed to p t o m o l e social i n t e r a c t i o n , for the n a r r o w tubes c o n n e c t i n g Ihc boxes and the central feeding and water s t a t i o n forced lhc animals into c o n f r o n t a t i o n . This socialization p r o c e d u r e was done in the m o r n i n g , a 12 h o u r light-dark cycle t 7 a m . 7 p.m. ) was m a i n t a i n e d for h o u s i n g and testing areas l ' h e treadmill e x p e r i e n c e consisted of placing the mice on a d r u m . "7 em in d i a m e t e r , r o t a t i n g at: 2 r p m (Fig. 3) this
METHOI)
The e x p e r i m e n t a l design as s h o w n in Fig. 1 can be considered as a 2 × 2 factorial in which t h e t r e a t m e n t comb i n a t i o n s are: isolation (1), i s o l a t i o n with 2 V: h o u r s ol socialization (I-Soc), isolation w i t h t r e a d m i l l e x p e r i e n c e (I-TM), isolation w i t h socialization followed b y t r e a d m i l l e x p e r i e n c e prior to s o c i a l i z a t i o n (I-Soc-TM). and an a d d i t i o n a l group t h a t received treadmill e x p e r i e n c e prior to socialization (I-TM-Soc).
[
I
t
,~ I -
I
TREADMILL EXPERIENCE
I - TM
FOOD ~ _ ~_ _ ~_[ J~
J
(SOC) SOCIALIZATION
(I)
NO SOCIALIZATION NO iTREADMILL EXPERIENCE
L._~J
I
',\
1 SOC
B::i::~i~::ii~!~i~J I
I - TM - SOC
F",,~.:i~iiiii~::ii~!l
I - SOC- TM
I
FIG. 1. A 2 × 2 factorial design of the experiment. 1 = isolation, 'FM = treadmill experience, Soc = socialization.
5 0 cm FIG. 2. A 6 box intercommunicating population Cage with central feeding and watering area.
BEHAVIOR AND SLEEP DISTURBANCE
121
FIG. 3. The treadmill with a 7 cm dia. rotating drum, black Plexiglas partitions, and motor. Rotation rate was 2 rpm. treadmill was p a r t i t i o n e d so t h a t the mice r e m a i n e d in isolation while walking. Ice was placed on the floor of the c o m p a r t m e n t b e n e a t h the treadmill to ensure t h a t mice kept m o v i n g c o n t i n u o u s l y . F r o m t h e first week on the treadmill u n t i l they were 2 m o n t h s old, the exercise gradually increased, s t a r t i n g w i t h 3 and e n d i n g with 5 hours. The mice were r e t u r n e d to isolation after each daily t r e a t m e n t c o m b i n a t i o n , and the treadmill and socialization p r o c e d u r e s were c o n t i n u e d 5 days a week until the animals were 4 m o n t h s old. Each animal was t h e n tested in an open field a p p a r a t u s [ 1 8 ] , and the n u m b e r of squares traversed during a 2 rain interval was r e c o r d e d . The open field a p p a r a t u s consisted of a light b r o w n , circular p l y w o o d base 76 cm in d i a m e t e r , s u r r o u n d e d by a 76 cm wall. T h i n black lines were used to mark off sixteen 13 cm squares. Blood was collected by m e a n s of a r e t r o - o r b i t a l p u n c t u r e 15 min after each a n i m a l ' s initial e x p o s u r e to the o p e n field. The b l o o d samples were c e n t r i f u g e d and the plasma r e m o v e d and frozen for c o r t i c o s t e r o n e assay, using a f l u o r o m e t r i c t e c h n i q u e [ 17 ]. The final phase of the e x p e r i m e n t was then c o m m e n c e d . Six males from each of the 5 t r e a t m e n t comb i n a t i o n s were assigned to separate 6 b o x i n t e r c o m m u n i cating p o p u l a t i o n cages for 2 m o n t h s to i n t e r a c t socially a m o n g themselves as well as w i t h 12 females t h a t were assigned to each group (Fig. 2). During this period of socialization, the systolic b l o o d pressure was m e a s u r e d (4 times) once every 2 weeks by a tail p l e t h y s m o g r a p h y m e t h o d . The data for each animal were t h e n pooled and averaged for final statistical analysis. The work of Ely and Henry [21 and of Watson e t a l . [18] has established t h a t blood pressure is a reliable measure o f social i n t e r a c t i o n . The observations, r o u t i n e l y m a d e during animal h u s b a n d r y , of aggressive b e h a v i o r in the mice and the n u m b e r of scars inflicted on the males r e m a i n anecdotal. T h e i r trend did, however, s u p p o r t t h a t established b y the b l o o d pressure data. ]-he statistical analyses, based on an analysis of
variance b y t h e N e w m a n - K e u l s procedure, c o m p a r e d the three socialized groups and were carried o u t on the b l o o d pressure, c o r t i c o s t e r o n e , and o p e n field data. RESULTS The principal findings are s u m m a r i z e d in Figs. 4 and 5. Socialized mice traversed significantly more squares d u r i n g open field testing t h a n isolated mice, F ( 1 , 4 4 ) --- 2.222, p < 0 . 0 1 . There was a significant i n t e r a c t i o n b e t w e e n trcadm i l l e x p e r i e n c e and socialization, I7(l,44) = 2v.70, p < 0 . 0 0 1 t r e a d m i l l e x p e r i e n c e p r o d u c e d increased a c t i v i b in the I-TM group. Moreover, the N e w m a n - K e u l s test I0-
~ug% -25 W Z 0 nw
tm laJ 0,3 03
o o
-20 0 nO
6
03 LLI •15
g
c. Although the I-TM had no sociul[zatum expe~ieucc. Ihc} had been picked up and forced dait> !(~ walk on lhc Ireadmill. Ihus. when faced with l h e h fh-,~. ? rain d opca lield c×posure, ~l mighl have been a Less ovetwhelnlit)g novel ex. perience lhan Io a group that had l~ev.'r ventllred out ~I Iheti glass_jars. We may now ask w h y the I-IM ~verc m o r e acll'~e !h,l!t lhe |-Soc-TM despite having the ~ame level o f arousal a> measured by c o r t i c o s t e r o n e . The o p e n field explorali(m b?, tile I-Soc-TM might have been inl/ihitcd ax a result t,I !heu extra periods o f social interacti(m, l'hcy had n o t learned social a d j u s t m e n t during these pert
Loss of Socialized Patterns of Behavior in Mouse Colonies Following Daily Sleep Disturbance During Maturation' F L O R A M. C. W A T S O N A N D J A M E S P. H E N R Y
University o f Southern California, School o f Medicine, Department o f Physiology Los Angeles, CA 90007
(Received 30 July 1975) WATSON, F. M. C. AND J. P. HENRY. Loss of socialized patterns of behavior in mouse eolonies fbllowing daily sleep disturbance during maturation. PHYSIOL. BEHAV. 18(1) 119-123, 1977.-- An ethologically-orientcd experimental design has been evolved to test the hypothesis that in the developing animal, rapid eye movement (REM) sleep may be concerned with integrating inherited aspects of attachment and territorial behavior with higher cognitive processes. Isolated weaned mice were daily submitted to 3 - 5 hr of treadmill sleep deprivation throughout maturation from 28 days until they were adult at 4 months. In some (controls) the treadmill preceded, and in others (experimental), it followed a 2 V2 hr socialization period during which the males and females interacted in a complex population cage. The adult responses of the various groups were tested in an open field by measuring their activity and elevation of plasma corticosterone. The capacity for organized social interaction was evaluated by observing blood pressure and other responses to two months of continuous exposure to colony life in a population cage. Mice that were routinely put on the treadmill immediately after socialization behaved as though they had been isolated throughout their development. Those that experienced the treadmill before socialization developed a normal hierarchy despite 21 Y2 hr of isolation between the daily socialization experiences. There was a statistically significant difference between the open field activity, the open field corticosterone value, and the blood pressure of the control and the experimental groups. REM sleep
Sleep deprivation
Socialization
Social interaction
IN A detailed n e u r o p h y s i o l o g i c a l analysis of the f u n c t i o n o f dreaming, J o u v e t [7] has a t t a c h e d great i m p o r t a n c e to the high level of rapid eye m o v e m e n t ( R E M ) in u t e r o and i m m e d i a t e l y after birth. He p o s t u l a t e d t h a t since cellular growth a n d d i f f e r e n t i a t i o n c o n t i n u e while the y o u n g animal is developing its final p a t t e r n s of behavior, d r e a m i n g or the REM phase m a y be i m p o r t a n t in the o r g a n i z a t i o n of complicated behavioral sequences or instincts, such as defense of territory, h u n t i n g , a n d mating. The f u n c t i o n of d r e a m i n g would be to s t i m u l a t e n e u r o n s t h a t c o m m a n d a n e t w o r k responsible for p r o g r a m m i n g p a t t e r n s of b e h a v i o r characteristic of the species' r e s p o n s e to i n c o m i n g stimuli derived from the e x p e r i e n c e s o f the individual. He suggested t h a t such p r o g r a m m i n g would n o t be likely to occur during wakefulness w h e n the c o m m a n d n e u r o n s were r e s p o n d i n g to external stimuli: rather, the a l t e r a t i o n of synapses t h r o u g h learning in response to the needs of genetic prog r a m m m g m i g h t occur d u r i n g REM sleep w h e n the c o m m a n d n e u r o n s are less engaged by the e n v i r o n m e n t . R e c e n t work is c o m p a t i b l e with this t h e o r y . Lucero [10] observed t h a t the d u r a t i o n of REM sleep increases after a learning experience. The o b s e r v a t i o n was c o n f i r m e d and e x t e n d e d in several o t h e r studies by various groups [3,
Blood pressure
5, 9, 13, 14, 16]. If REM is p r e v e n t e d for 2 3 h o u r s i m m e d i a t e l y after the experience, there is a deficit in learning. K i t a h a m a , Valatx, and J o u v e t [81 s h o w e d that the REM-suppressing s u b s t a n c e a l p h a m e t h y l - D O P A slows maze learning, and P e a r l m a n and Becker 113] f o u n d a similar effect w h e n i m i p r a m i n e or c h t o r d i a z e p o x i d e was used during bar pressing. O f special interest from the poinl of view of social situations, the latter a u t h o r s also r e p o r t e d t h a t REM d e p r i v a t i o n p r e v e n t e d a rat from assimilating lhe significance o f the skilled bar-pressing b e h a v i o r of a con> panion. G r e e n b e r g and P e a r h n a n [4] reviewed these and o t h e r studies p o i n t i n g to the adaptive role of REM sleep in light of Seligman's r e c e n t critique of the generality of the laws of learning 115]. Seligman has p o i n t e d o u t t h a t while animals are prepared by virtue of e v o l u t i o n a r y history to make some associations, they are in fact u n p r e p a r e d or even c o n t r a p r e p a r e d for others. He predicled t h a t successful avoidance will be rapidly learned only after those evenls relating to the n a t u r a l species-specific repertoire of the organism. G r e e n b e r g and Pearlman [41 suggest that such rapidly learned h a b i t u a l reactions for which the animal "'i.~ p r e p a r e d " in Seligman's sense will be s h o w n to be R I M
' This research was supported by the National Institutes of Health Grant No. MH 19441. 119
120
WVI'SON AND I:tt-NR'~
i n d e p e n d e n t . On t h e o t h e r h a n d , activities involving the a c c u m u l a t i o n of u n u s u a l i n f o r m a t i o n w o u l d be likely to require REM sleep for o p t i m a l c o n s o l i d a t i o n into long-term memories. The q u e s t i o n arises w h e t h e r the learning involved in t h e a c q u i s i t i o n of territorial and m a t e r n a l behavior n e e d e d by a hierarchically o r g a n i z e d c o l o n y also falls i n t o t h e REM d e p e n d e n t category. T h e peer i n t e r a c t i o n of animals has b e e n s h o w n t~) be essential to the d e v e l o p m e n t of n o r m a l social b e h a v i o r Mason [ 11 ] has s h o w n t h a t r e s p o n s e s to social cues were poorly e s t a b l i s h e d in socially deprived m o n k e y s . W a t s o n Henry, a n d H a l t m e y e r [18] f o u n d that w h e n socially de prived mice were e x p o s e d to o t h e r mice for 30 rain ol social i n t e r a c t i o n , t h e y had a m u c h higher plasma corficos t e r o n e r e p o n s e t h a n group-reared mice. They were also less active in t h e o p e n field, F u r t h e r m o r e . w h e n these formerl3 isolated animals were p u t i n t o a c o m p l e x p o p u l a t i o n cage, t h e y failed to a d a p t socially to c h r o n i c psyehosocial slimu l a t i o n , w i t h resulting high levels of aggression and hypertension. T h u s r e p e a t e d social i n t e r a c t i o n after weaning enables t h e mice to acquire c e r t a i n b e h a v i o r p a t t e r n s lhat are valuable d u r i n g a d u l t h o o d . These p a t t e r n s mcrease the t o l e r a n c e of e x t e r n a l stimuli a n d m o d i f y adult activities so as to decrease the i n t e n s i t y of social s t i m u l a t i o n arisin~ within the group. As J o u v e t [7] a n d G r e e n b e r g and P e a r l m a n {4] suggest. d r e a m i n g or REM sleep m a y be i m p o r t a n t to the developing central n e r v o u s system as it integrates a n d r e p r o g r a m s e m o t i o n a l l y a r o u s i n g e x p e r i e n c e s i n t o fresh social behavioral patterns. If REM does i n d e e d provide an o p p o r t u n i t y for the a d a p t a t i o n of i n h e r i t e d species-specific r e p e r t o i r e s ~o a p p r o p r i a t e e x t e r n a l cues, then animals t h a t were repeatedly deprived of all sleep (and t h e r e f o r e of REM sleep l following socialization might n o t e x h i b i t the full effect ot early socialization as adults. Thus, t h e objective of this s t u d y was to d e t e r m i n e w h e t h e r t h e socialization of CBA mice c o u l d be i n h i b i t e d b y s y s t e m a t i c a l l y f o l l o w i n g each p e r i o d of i n t e r a c t i o n w i t h a few h o u r s of sleep deprivatiol~ on a r o t a t i n g treadmill t h r o u g h o u t m a t u r a t i o n .
Animals l'he CBA agouti mice used m [hJ~ .qudy were originall3 o b t a i n e d from tile J a c k s o n Memorial L a b o r a t o r y , Bar t t a r b o r , Maine and have been bred ~md raised in our labo r a t o r y since 1955.
Proccdz#¢ T w e n t y p r e g n a n t CBA mice were placed in pairs m 2,S 17 ~ 12 cm nest b o x e s and were aItowed to deliver. I h e litters were r e d u c e d to 8 pups al b i r t h and were m~t d~sturbed f u r t h e r u n t i l t h e y were weaned at 2 t days o f a g e . . \ t this time. the males were numbereat by ear pm~ching and were individually placed m V2-1iter jars c o n t a i n i n g w o o d shavings. A p a p e r towel was taped a r o u n d each jar Ic, eliminate visual c o n t a c t with o t h e r mice. F o o d and water were supplied as n e e d e d , and the shavings were changed once u week. Twelve males were assigned al r a n d o m to each of the 5 t r e a t m e n t c o m b i n a t i o n s s h o w n in Fig. !. The [re;ttm e n t s were initiated o n e week after w e a n i n g and c o n t i n u e d until the mice were 4 m o n t h s old. T h e isolated mice were n o t d i s t u r b e d excepl w h e n their jars were cleaned once a week. but the mice assigned t,, socmlization were r e m o v e d f r o m their .tars and placed toT 2!& hr in an i n t e r c o m m u n i c a t i n g o-box p o p u l a t i o n cage (Fig. 2t where they could explore anti engage in social a c t i v i t y t 5 ] . This system is c o n s t r u c t e d o f Plexiglas boxes measuring 23 - 11 11 cm which are c o n n e c t e d m t o a circle by flexible plastic tubes J3.8 cm i d.'). The cenlral h e x a g o n holds f o o d a n d w a t e r and is c o n n e c t e d to each box hy t u b e s {3.3 cm i. d.) The cage wits designed to p t o m o l e social i n t e r a c t i o n , for the n a r r o w tubes c o n n e c t i n g Ihc boxes and the central feeding and water s t a t i o n forced lhc animals into c o n f r o n t a t i o n . This socialization p r o c e d u r e was done in the m o r n i n g , a 12 h o u r light-dark cycle t 7 a m . 7 p.m. ) was m a i n t a i n e d for h o u s i n g and testing areas l ' h e treadmill e x p e r i e n c e consisted of placing the mice on a d r u m . "7 em in d i a m e t e r , r o t a t i n g at: 2 r p m (Fig. 3) this
METHOI)
The e x p e r i m e n t a l design as s h o w n in Fig. 1 can be considered as a 2 × 2 factorial in which t h e t r e a t m e n t comb i n a t i o n s are: isolation (1), i s o l a t i o n with 2 V: h o u r s ol socialization (I-Soc), isolation w i t h t r e a d m i l l e x p e r i e n c e (I-TM), isolation w i t h socialization followed b y t r e a d m i l l e x p e r i e n c e prior to s o c i a l i z a t i o n (I-Soc-TM). and an a d d i t i o n a l group t h a t received treadmill e x p e r i e n c e prior to socialization (I-TM-Soc).
[
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TREADMILL EXPERIENCE
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FIG. 1. A 2 × 2 factorial design of the experiment. 1 = isolation, 'FM = treadmill experience, Soc = socialization.
5 0 cm FIG. 2. A 6 box intercommunicating population Cage with central feeding and watering area.
BEHAVIOR AND SLEEP DISTURBANCE
121
FIG. 3. The treadmill with a 7 cm dia. rotating drum, black Plexiglas partitions, and motor. Rotation rate was 2 rpm. treadmill was p a r t i t i o n e d so t h a t the mice r e m a i n e d in isolation while walking. Ice was placed on the floor of the c o m p a r t m e n t b e n e a t h the treadmill to ensure t h a t mice kept m o v i n g c o n t i n u o u s l y . F r o m t h e first week on the treadmill u n t i l they were 2 m o n t h s old, the exercise gradually increased, s t a r t i n g w i t h 3 and e n d i n g with 5 hours. The mice were r e t u r n e d to isolation after each daily t r e a t m e n t c o m b i n a t i o n , and the treadmill and socialization p r o c e d u r e s were c o n t i n u e d 5 days a week until the animals were 4 m o n t h s old. Each animal was t h e n tested in an open field a p p a r a t u s [ 1 8 ] , and the n u m b e r of squares traversed during a 2 rain interval was r e c o r d e d . The open field a p p a r a t u s consisted of a light b r o w n , circular p l y w o o d base 76 cm in d i a m e t e r , s u r r o u n d e d by a 76 cm wall. T h i n black lines were used to mark off sixteen 13 cm squares. Blood was collected by m e a n s of a r e t r o - o r b i t a l p u n c t u r e 15 min after each a n i m a l ' s initial e x p o s u r e to the o p e n field. The b l o o d samples were c e n t r i f u g e d and the plasma r e m o v e d and frozen for c o r t i c o s t e r o n e assay, using a f l u o r o m e t r i c t e c h n i q u e [ 17 ]. The final phase of the e x p e r i m e n t was then c o m m e n c e d . Six males from each of the 5 t r e a t m e n t comb i n a t i o n s were assigned to separate 6 b o x i n t e r c o m m u n i cating p o p u l a t i o n cages for 2 m o n t h s to i n t e r a c t socially a m o n g themselves as well as w i t h 12 females t h a t were assigned to each group (Fig. 2). During this period of socialization, the systolic b l o o d pressure was m e a s u r e d (4 times) once every 2 weeks by a tail p l e t h y s m o g r a p h y m e t h o d . The data for each animal were t h e n pooled and averaged for final statistical analysis. The work of Ely and Henry [21 and of Watson e t a l . [18] has established t h a t blood pressure is a reliable measure o f social i n t e r a c t i o n . The observations, r o u t i n e l y m a d e during animal h u s b a n d r y , of aggressive b e h a v i o r in the mice and the n u m b e r of scars inflicted on the males r e m a i n anecdotal. T h e i r trend did, however, s u p p o r t t h a t established b y the b l o o d pressure data. ]-he statistical analyses, based on an analysis of
variance b y t h e N e w m a n - K e u l s procedure, c o m p a r e d the three socialized groups and were carried o u t on the b l o o d pressure, c o r t i c o s t e r o n e , and o p e n field data. RESULTS The principal findings are s u m m a r i z e d in Figs. 4 and 5. Socialized mice traversed significantly more squares d u r i n g open field testing t h a n isolated mice, F ( 1 , 4 4 ) --- 2.222, p < 0 . 0 1 . There was a significant i n t e r a c t i o n b e t w e e n trcadm i l l e x p e r i e n c e and socialization, I7(l,44) = 2v.70, p < 0 . 0 0 1 t r e a d m i l l e x p e r i e n c e p r o d u c e d increased a c t i v i b in the I-TM group. Moreover, the N e w m a n - K e u l s test I0-
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c. Although the I-TM had no sociul[zatum expe~ieucc. Ihc} had been picked up and forced dait> !(~ walk on lhc Ireadmill. Ihus. when faced with l h e h fh-,~. ? rain d opca lield c×posure, ~l mighl have been a Less ovetwhelnlit)g novel ex. perience lhan Io a group that had l~ev.'r ventllred out ~I Iheti glass_jars. We may now ask w h y the I-IM ~verc m o r e acll'~e !h,l!t lhe |-Soc-TM despite having the ~ame level o f arousal a> measured by c o r t i c o s t e r o n e . The o p e n field explorali(m b?, tile I-Soc-TM might have been inl/ihitcd ax a result t,I !heu extra periods o f social interacti(m, l'hcy had n o t learned social a d j u s t m e n t during these pert
