BEHAVIORAL BIOLOGY 15, 317-331 (1975), Abstract No. 5112
Development of Nocturnal Behavior in Albino Rats 1
STATA NORTON, BRUCE CULVER, and P H Y L L I S M U L L E N I X
Department of Pharmacology, University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kansas 66103
When the activity of groups of rats is monitored in a residential maze equipped with photocells for recording passage through the corridors, rats can be shown to be more active in the 12-hr dark portion of a day than in the 12-hr light portion from an early age. Groups of rats weaned at 23 days of age show significantly greater activity at night than during the day but the maximum nocturnal/diurnal ratio is recorded in young adult rats 2-4 months old. Although females older than 5 weeks are more active than males both during the light and dark cycle, the nocturnal/diurnal activity ratios show the same trends in the two sexes. Males differ from females in the duration of their exploratory activity when they are first introduced into the maze. Females are consistently much more active during the second hour in the maze but the differences between the sexes in activity during the first hour are not as great. The development of nocturnal activity of groups of rats in these experiments corresponds generally with results obtained on isolated rats, implying that social interaction is not a primary cause of the nocturnal activity recorded in these experiments.
S o m e types o f brain damage cause rats to b e c o m e hyperactive only during certain p o r t i o n s o f the normal light-dark cycle ( C o s t a l et al., 1972; Culver and N o r t o n , 1974). T h e r e f o r e , if the activity of an animal is being measured in the light p o r t i o n o f the day, the results o f an e x p e r i m e n t involving brain damage m a y be different in a diurnal or a n o c t u r n a l animal. It is also i m p o r t a n t to consider the kind o f activity being measured since activity in a novel e n v i r o n m e n t ( e x p l o r a t o r y activity) m a y n o t respond to diurnal influences in the same way as activity in a h o m e cage. When, in addition, activity studies are made on y o u n g animals, the d e v e l o p m e n t o f day-night activity cycles in the m a t u r i n g animal b e c o m e s i m p o r t a n t . E x a m p l e s o f studies in w h i c h this p r o b l e m arises are in recent reports on the t o x i c o l o g i c effects o f 1This research was supported by United States Public Health Service Grants MH 17279, MH 43860, and HD 02528. 317 Copyright (~)1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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lead (Michaelson and Sauethoff, 1974; Silbergeld and Goldberg, 1974) and carbon monoxide exposure (Culver and Norton, 1974) in infant rodents. Both toxic events are described as producing hyperactive young animals which may serve as models for the hyperkinetic syndrome of childhood. The diurnal cycle of the rat has been studied experimentally for many years, and the adult rat is easily recognized as a nocturnal animal which spends most of the daylight hours asleep and is active most of the dark period of the day. A brief review of the early laboratory study of this phenomenon has been given by Levinson e t al. (1941). The methods used to detect the diurnal rhythm have been traditionally the running wheel (Slonaker, 1925) or activity cages which record body movement (Richter, 1922). With both methods the animals generally are housed individually during the period of study. When preweanling rats are studied, isolation of the animals for prolonged periods is impossible. The solution for this has been to study the young rats during portions of the day, up to 12hr in some cases, and to return them to the mother during the remainder of the day. Under these conditions, RiChter (1927) reported that the newborn rat showed continuous, rather than periodic, activity in an activity cage. Rhythmic activity appeared about the tenth day but the development of day-night cycles took much longer. At 60 days of age, the rat was 1.34 times more active during the night than the day in Richter's experiments (Richter, 1922). Old rats (600 days) were 2.2 times more active in the dark than the light. Man has been reported to have a day-to-night ratio close to one for the first 2 months of postnatal life, increasing to a ratio of 2 for the third and fourth months and to greater than 3 by the seventh month of life (F. G. Mullin, quoted by Kleitman, 1949). It appears that animals born at about the level of maturity of the rat and man show little regulation of activity in regard to the diurnal cycle in the neonatal period, and that the day-night cycle which characterizes the adult, either primarily diurnal or nocturnal, develops along with maturation of various other systems. The development of nocturnal behavior in the laboratory rat has been studied in a fragmentary way and only under conditions, such as isolation, which might be expected to alter the behavior of a social animal like the rat in unknown ways. Increasing recognition of interactions of day-night rhythms with other activities in adult rats makes it of interest to follow the development of nocturnal activity in the rat under social conditions. In the following experiments the development of nocturnal behavior in groups of rats was studied in a maze of dimensions which offered a method for evaluation of permanent groups of animals, allowing both diurnal and nocturnal activity to be recorded from the onset of walking in the young animals to maturity. It is proposed that the time course of increasing cyclic activity in the developing laboratory rat deserves study as a phenomenon which may affect many studies using young rats.
NOCTURNAL BEHAVIOR IN RATS
319
METHODS Two hundred rats, male and female Charles River (Sprague-Dawley derived) strain and their first generation offspring, were used in this study. Rats postweaning were housed in small groups in standard animal care facilities until they were studied in the environmental units. The basic environmental unit in which a group of rats was housed for study of activity was a runway maze around a central area with a raised dome (Fig. 1). Water was supplied at the end of one runway and a nest box or "burrow" was connected at the end of another runway. The remaining runways were loops with dual entrances to the central area. The runways and central area, excluding the nest box, covered an area 60 cm wide and 75 cm long. The total living area in each environmental unit was about 2240 cm 2. Each runway was 10 by 10 cm in cross section. The central area was roughly a cylinder 2 0 c m across by 2 2 c m high with six runway entrances. The WATER
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Fig. 1. Residential unit for groups of rats. Numerals identify nine areas and eight photocells (Numbers 1-8). Area 9, outlined by dotted line, is 22 cm high. Runways (areas 1-8) are 10 cm high. Nest box is 25 cm high.
320
NORTON, CULVERAND MULLENIX
detachable nest box was 20 cm square by 24 cm high. The walls of the unit were 20-gauge aluminum. The floor was expanded metal mesh except in the nest box which had a solid platform. Each runway was supplied with one or more photocell devices for recording each passage of a rat through the runway. The eight photocells in each unit were connected to counters. Cadmiun-selenium type photocells were used, activated by light from 6 V bulbs covered with red plastic lenses. All groups of rats appeared to be insensitive to the red light from these bulbs. The nest box was covered with a double thickness of red vinyl plastic. Light transmission of the plastic was determined in a Beckman Spectrophotometer and the double layer excluded most of the light longer than 600 riM. Activity of rats in the nest box could easily be observed through this cover since the visual sensitivity of the human eye is greater in the red range of visible light than the rat eye (Massof and Jones, 1972). The cover over the rest of the unit was colorless Plexiglas. In some experiments a double layer of red plastic vinyl sheeting was placed over the entire unit for 12 hr of the day to reverse the light-dark activity cycle of the rats and still allow the rats to be visible to an observer. In all experiments the rats were maintained on a 12-hr light-dark cycle. Except during the reversal, the light cycle was from 6:00 AM to 6:00 PM. Purina Rat Chow pellets were placed in the central area daily and water was supplied ad lib. by a watering tube with the reservoir bottle outside the unit. All maintenance of the unit (feeding, cleaning, etc.) was carried out daily from 9:00AM to 10:00 AM. Fresh nesting material (strips of paper) was placed in the maze daily. During this period all rats were removed from the maze except nursing mothers and their pups. The first 2 hr of the first day when a group of rats was placed in the maze were called the exploratory period. All other photocell counts per hour for diurnal and nocturnal activity of established groups of rats were taken after the first 24 hr in the maze. Diurnal activity of established groups was recorded from 10:00 AM to 6:00PM. Nocturnal activity was recorded from 6:00PM to 6:00 AM. Because the rats were removed from the maze from 9:00AM to 10:00AM each day this disturbance probably increased the diurnal hourly counts over the counts which would have been recorded for undisturbed rats. In interpretation of the data it is assumed here that any effects of the standard maintenance procedures were equivalent in both males and females of all ages. Six age groups of rats were studied: preweanling rats up to 3 weeks of age; weanling rats 3-5 weeks old; juveniles 5-8 weeks old; young adults 2-4 months old; mature adults 4-12 months old; and old adults 12-20 months of age. All groups consisted of four rats of the same sex, except for studies on nursing mothers in which the group contained the mother and four pups. From 10:05 to 10:15AM and again from 6:05 to 6:15PM each established group of adult rats was observed on 2 successive days. The red
NOCTURNAL BEHAVIOR IN RATS
321
light activating the photocells was adequate for the adapted human eye to identify the location and movements of the rats in the maze. The purpose of this was to compare the type of behavior involved in the initial burst of activity in the morning with the activity at the onset o f the dark cycle. Some groups were also checked for location of the rats every hour from 10:00 AM to 6:00 PM during the light cycle and again at the same clock times after the light cycle was reversed.
RESULTS
Diurnal activity of established groups. The term, established group, is used for any group of rats which was housed in one of the residential maze units for more than 24 hr. To illustrate the daily rhythm of activity, the 24-hr photocell record of an established group of young adult females is shown in Fig. 2. Most o f the activity occurs during the dark half of the daily cycle. Since the rats are removed from the maze unit for 1 hr each day (9:00-10:00tAM) during maintenance of the unit, there is a burst of activity when the rats are replaced in the unit. This initial period of activity may be an abbreviated form of the exploratory period which occurs on the first day when naive rats are placed in the maze. It may also be related to food carrying since food carrying activity around the maze is seen in the morning whether or not rats are removed from the maze. The diurnal and nocturnal activity of groups o f rats from weanling stage to old adult life is shown in Table 1. Weanlings have the most uniform
t o9 5 0 0
Fig. 2. Hourly photocell counts in a residential unit for an established group of four young adult female rats. Rats removed from unit at 9:00 AM and replaced at 10:00 AM. Lights on from 6:00 AM to 6:00 PM. Dark cycle 6:00 PM to 6:00 AM.
322
NORTON, CULVER AND MULLENIX TABLE 1 Average Hourly Photocell Activity of Established Groups of Four Rats Counts per hour _+SE
Composition of groups
Number of groups
Number of observation Diurnal days period
Nocturnal period
Ratio nocturnal-diurnal activity b
Weanlings (3-5 wk) Male Female
2 3
10 15
282 _+31 200 _+27
350 +- 19 286 +_16
1.24 1.43
7 7
21 21
95 a +_12 149 _+12
259 a _+20 457 _+22
2.68 3.07
Male Female
5 10
15 30
99a _+16 164 +_ 14
328a +_21 540 +_39
3.31 3.29
Mature adults (4-12 mo) Male Female
6 13
19 41
90a _+ 8 190 +_ 9
288a _+25 480 _+20
3.20 2.53
2 3
12 18
96_+12 130 + 11
174a_+ 2 369 +_14
1.81 2.84
Juveniles (5-8 wk) Male Female Young adults (2-4 mo)
Old adults (12-21 too) Male Female
astatistically significant difference between males and females of the same age. bThese ratios represent significant differences between nocturnal and diurnal activity for all groups (t test, P = < .05). d i s t r i b u t i o n o f n o c t u r n a l - d i u r n a l activity. T h e m a r k e d divergence in distributi'on o f a c t i v i t y is first seen in j u v e n i l e s a n d peaks in y o u n g adults. A f t e r t h e w e a n l i n g stage, m a l e rats s h o w less activity t h a n females o f the same age at all p e r i o d s o f t h e day. T h e m o s t active rats are y o u n g a d u l t females. A f t e r 12 m o n t h s o f age b o t h m a l e s a n d females d r o p in n o c t u r n a l activity. The r a t i o o f n o c t u r n a l to d i u r n a l a c t i v i t y is b e t w e e n 1 a n d 2 for t h e w e a n l i n g rats a n d over 3 f o r m o s t o f t h e o t h e r groups. Preweanling activity. T h e use o f t h e n e s t b o x b y t h e female for care o f the p u p s allowed t h e e x p e r i m e n t e r to r e c o r d e m e r g e n c e a n d early e x p l o r a t i o n o f t h e m a z e b y t h e pups. T h e n e s t b o x was a t t a c h e d so t h a t the level f r o m
323
NOCTURNAL BEHAVIOR IN RATS TABLE 2 Postnatal Activity of Mother and Rat Pups in Maze. Average Hourly Photocell Counts (± SE) in Three Experiments Postnatal day
Diurnal counts
Nocturnal counts
Ratio nocturnal/diurnal
Mother only (pups in nest box) 2 3 4 5 6
68 ± 11 64 ± 33 57 ± 10 48 ± 15
63 55 48 51 47
7 8 9 10
64 49 46 72
45± 43 ± 49 ± 52±
11 12 13 14
90 84 89 110
± ± ± ±
6 9 6 17
± 27 ± 17 ± 20 ± 10
85 79 94 115
± 15 -+ 11 ± 11 ± 12 ± 10 6 6 10 4
± 25 ± 18 ± 40 ± 38
Days 2-6 0.89
Days 7-10 0.79
Days 11-14 1.00
Mother and pups (emergence from nest box) 15 16 17 18
86 21 152 152
± 18 ± 18 ± 21 ± 21
108 139 136 137
± 37 ± 47 ± 19 ± 9
Days 15-18 1.08
19 20 21 22
169 186 183 174
± 25 ± 6 ± 31 ± 15
135 210 232 287
± 22 ± 14 ±20 ± 22
Days 19-22 1.21
23 24 25 26 27 28
189 251 180 132 245 164
± 19 ± 73 ± 54 -+39 ± 99 ± 32
Weanlings only (mother removed) 217 ± 51 284 ± 11 289 ± 37 320± 8 324 ± 45 317 ± 63
Days 23-28 1.50a
aThis ratio represents a significant difference between nocturnal and diurnal activity averaged for days 23-28 (t test, P = < .05). t h e n e s t b o x f l o o r to t h e r u n w a y f l o o r r e q u i r e d t h e p u p s to c l i m b u p a 5 - c m s t e p t o gain a c c e s s t o t h e r u n w a y . W a l k i n g o f p u p s w a s i d e n t i f i e d in t h e n e s t at l e a s t b y t h e t h i r t e e n t h d a y . E m e r g e n c e f r o m t h e n e s t b o x o c c u r r e d 2 d a y s later. D a t a o n a c t i v i t y o f t h r e e g r o u p s o f p r e w e a n l i n g rats a n d m o t h e r s w e r e o b t a i n e d while t h e y o u n g w e r e w i t h t h e m o t h e r ( T a b l e 2). B e f o r e t h e y o u n g
324
NORTON, CULVER AND MULLENIX
could leave the nest box, the daily activity of the mother alone was recorded. This period was the first 14 days after birth of the pups. The mother showed no significant diurnal-nocturnal rhythm during this period. The hourly photocell counts were not significantly different in the light and dark periods (Table 2). Mthough the young rats could climb up out of the nest box on the fifteenth day, their contribution to activity was minimal until the seventeenth day. About the twentieth day, a tendency to increasing nocturnal activity was noted in both the mother and the young, and this beginning preference for the dark cycle was seen in the young after removal of the mother. Activity in the maze by the young rats increased sharply on the day in which the mother was removed. The ratio of nocturnal to diurnal activity was 1.5 from the twenty-third to the twenty-eighth postnatal day. Exploratory activity. When a group of postweanling rats was first placed in a maze unit, there was an initial burst of activity which was greater than the activity on succeeding days. This period of activity lasted about 2 hr and was called the exploratory period. Females were more active than males during this period (Table 3). In the first hour of exploratory activity, old adult males were approximately one-third as active as females. Juvenile, young, and mature adult males were only slightly less active than females during the same period. The greatest divergence in male and female activity came during the second hour of exploratory activity when the males were much quieter than the females at all ages studied. Descriptive comments on activity. In the course of these experiments hourly observations were made which were not evaluated statistically. However, the observations were sufficiently consistent to illustrate the way in which the varied spaces of the maze were used by the rats. The nest box and areas 1-8 of the maze were all used for sleeping during the day by postweanling rats. Females of any age and young male rats tended to sleep huddled together. Old males were more isolated. Area 9 was very rarely used for sleeping by any rat and quiet huddling was never observed during the light cycle in area 9. Reversal of the diurnal cycle with reversed illumination has been shown to require 7-14 days (McGuire et al., 1973). A group of four adult animals was made active during the 9:00 AM to 9:00 PM period by making this the dark period of the day. Since the rat's eye is insensitive to red light (Massof and Jones, 1972), a red plastic cover on the maze was used to reverse their cycles and to allow observation of the rats with the room lights on. Reversal of the dark-light activity cycle required 8 days with the maze covered with red plastic (Fig. 3). During the reversed dark cycle various activities were observed. There was considerable social interaction among the four rats in the maze. In addition, grooming activities, feeding, and drinking were present in cycles throughout the dark period. No specific quantitation of the different activities
325
NOCTURNAL BEHAVIOR IN RATS TABLE 3 Exploratory Activity of Groups of Four Rats on the First Day in a Residential Maze
Composition of groups
Number of groups of four rats each
Average Photocell Counts -+ SE First hour in Second hour in residential maze residential maze
Weanlings (3-5 wk) Males
2
1197 _+ 122
465 ± 23
7 7
1171 a ± 222 1859 ± 193
390a ± 140 1105 ± 196
3 10
1430 ± 398 2326 ± 245
639 a ± 41 1979 ± 245
4 12
1553 ± 400 2204 _+ 152
302a ± 41 1610 ± 349
2 3
528 a-+ 16 1589 -+ 115
237a± 4 737 ± 156
Juveniles (5-8 wk) Males Females Young adults (2-4 mo) Males Females Mature adults (4-12 too) Males Females Old adults (12-21 mo) Males Females
aSignificant difference between males and females (P = < .05, Mann-Whitney U test) comparing same hour and age group. t h r o u g h o u t the dark cycle was a t t e m p t e d in these studies b u t the general appearance o f the activity differed f r o m the rapid e x p l o r a t o r y behavior which was characteristic o f the first i n t r o d u c t i o n o f rats to the maze unit. Congregation and aggressive kinds o f social behavior were seen in area 9. Areas 1-8 were used for running and chasing. This could be initiated by rats meeting in the corridors b u t the primary area for initiation o f these activities was area 9. S o m e t i m e s one rat w o u l d " d e f e n d " area 9 causing all other rats to run in the corridors and w o u l d block others f r o m entering the central area. Distribution of nocturnal activity. S o m e evidence o f the n o c t u r n a l utilization o f space can be gained by an analysis o f the p h o t o c e l l counts in the different areas o f the maze. Table 4 shows the distribution o f counts in the entrance to the nest b o x (area 1), the c o n t i n u o u s corridors (area 3, representing also areas 2 and 4 and area 7, representing also areas 6 and 8)
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NORTON, CULVER AND MULLENIX
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Fig. 3. Reversal of diurnal activity in an established group of four adult female rats in a residential unit with a red cover used to simulate darkness. Room lights on continuously. Red cover on 9:00 AM to 9:00 PM. a n d t h e b l i n d c o r r i d o r w i t h t h e w a t e r i n g t u b e ( a r e a 5). T h e n u r s i n g m o t h e r used areas 1 a n d 5 a b o u t e q u a l l y a n d areas 3 and 7 b o t h less f r e q u e n t l y , s h o w i n g a p r e f e r e n c e f o r a straight p a t h t h r o u g h area 9 t o the w a t e r source. G e n e r a l a m b u l a t i o n a r o u n d t h e t w o c o n t i n u o u s c o r r i d o r s was m u c h less t h a n t h a t o f n o n n u r s i n g a d u l t females. C o n v e r s e l y , n o n n u r s i n g a d u l t females used areas 3 a n d 7 s o m e w h a t m o r e t h a n areas 1 a n d 5. I n T a b l e 4, t h e c o m p a r i s o n o f c o u n t s f o r area 3 ( 6 7 -+ 5.1 c o u n t s p e r h o u r ) w i t h area 5 ( 4 9 -+ 3.7 c o u n t s TABLE 4 Distribution of Nocturnal Activity of Female Rats in Residential Maze Counts per hour _+SE Postnatal day
Number of groups
Area 1
One nursing mother before emergence of pups, days 2-14
1
18_+ 1.3a
8-+ 3.2 19_+ 1.7 a
5-+ 1.0
Mother and four pups after emergence, days 15-22
1
42_+7.5 ab
13_+ 1.4 24_+ 2.4 a
13_+2.7
Four young only after weaning, days 23-26
1
53-+2.3 a
29_+7.4
27-+8.3
Four adult females without young
7
62-+5.4
67-+ 8.1 49_+ 3.7
Area 3
aSignificantly different from areas 3 and 7, t test P = < .05. bSignificantly different from area 5, t test P = < .05.
Area 5
33+_ 10.7
Area 7
75+_7.9 b
NOCTURNAL BEHAVIORIN RATS
327
per hour) has a chance probability slightly more than 0.05, while the probability that use of area 7 (75 -+ 7.9 counts per hour) differs from area 5 is approximately 0.05. When the young rats could leave the nest box the highest counts occurred in area 1. Counts in areas 3, 5 and 7 became uniform shortly after weaning. DISCUSSION In the use of the residential maze in this study two hypotheses were. involved. One was that daily activity rhythms might develop differently in animals living in social groups than in animals living in isolation, and the second was that different spaces might be selectively used during the active (nocturnal) portion of the day if a complex environment was available to the rats. The first hypothesis was not substantiated by the data. Previous data in the literature obtained on isolated rats were essentially confirmed here in groups of rats, both in the ratio of nocturnal to diurnal activity and the time at which it arose during development. An exception to this was the failure to confirm Richter's (1922) observation that the ratio continued to increase up to old age. In the experiments here the peak ratio was reached in the mature adult and not in animals over 12 months old. This difference in results may be due to the social condition of testing, suggesting that the presence of a mature adult rat might increase the nocturnal activity of another mature rat. Another possible reason for the difference in results may be that Richter's results were influenced by feeding the rats at two different times of day (in the light or in the dark cycle) and then averaging the results. This experimental design would be expected to alter the behavioral patterns from those with ad lib. feeding. In the present experiments food was available during both the light and dark cycle. This avoided any pressure on growing rats to remain active during any particular portion of the light-dark cycle, thus allowing the maximum dark-light ratio to be developed much earlier than in the experiments by Richter (1922). It is possible that the nocturnal to diurnal ratio reported here is too low for all groups of rats. If the animals had not been removed from the maze for an hour every morning, the brief burst of activity which was seen on reintroduction of the rats would not have occurred and the total activity during the light cycle might have been less. Thus the ratio might be higher with a different experimental design. However, except for a modest increase in the ratio, there is no reason to expect that a more detailed examination of either the undisturbed group or different numbers of animals would produce any novel findings. Bolles and Woods (1964) reported that the preweanling rat does not show a light-dark cycle up to 3 weeks of age. The present experiments are in
328
NORTON, CULVER AND MULLENIX
agreement since the daily rhythm of activity developed in the weanling rats between 3 and 5 weeks of age but was not present or was poorly developed in younger rats. This activity thus predates the full maturation of the central nervous system (Friede, 1966). Zucker (1971) reported that a light-dark rhythm developed in food and water intake shortly after weaning. This corresponds to the increase in nocturnal activity observed here after weaning. After 5 weeks of age until they were old adults, the female rats were significantly more active than males. However, no clear changes in activity could be related to estms cycles in adult females in these experiments. This lack of cyclic estrus activity is to be expected under these environmental conditions since the techniques for displaying estrus activity require specific conditions such as isolation of the female, preferably from an early age, in a running wheel cage (Kennedy and Mitra, 1963). In a maze type environment, resembling the maze in these studies, Barnett and McEwan (1973) failed to find cyclic estrus activity in isolated mice. The second hypothesis proposed was that rats would utilize space in differing ways during the active portion of the day. The mothers with nursing young lost the greater nocturnal activity of the nonlactating adult and showed a very uniform distribution of activity throughout 24 hr. These results are of interest in comparison with the findings of Plaut in regard to maternal activity in a dual-chambered cage. Plaut (1974) found that nursing mothers spent more time with their pups during the day than during the night. If the dual-chamber used by Plant is comparable to the maze used in the present experiments then the activity of the mothers during the day must be kept equal to the nocturnal activity by an altered use of the period spent away from the young. For example, the mother could leave the young primarily to feed or drink during the day but might be relatively quiet in some area away from the nest box during the nocturnal period. This would account for the greater nocturnal time away reported by Plaut but not greater nocturnal activity reported here in nursing mothers. Alternatively, the two environmental conditions, the maze and the dual-chambers, may cause different behaviors on the part of nursing mothers. In a maze shaped like a cross with equal arms, called a plus maze by Barnett and McEwan (1973), mice did not utilize the four blind-ended corridors (containing food, water, balsa wood, or nothing) with equal frequency. The corridor with water was visited slightly less often than the empty corridor but the difference did not appear to be significant for virgin mice. Nursing mice, on the other hand, visited the empty corridor much less often. In the residential maze described here the animals had four different types of space to use, a darkened high area (nest box), a high central area (area 9), two continuous runways (areas 2, 3, 4, and 6, 7, 8), and a blind corridor with a watering bottle (area 5). No photocells were placed in the nest box or area 9. In retrospect, this was unfortunate since considerable social interaction was
NOCTURNAL BEHAVIOR IN RATS
329
seen in these areas, particularly area 9 from which there was access to all runways. The location of the photocells in areas 1-8 limited the type of recorded activity predominately to locomotion either social (such as chasing) or solitary (such as food carrying or walking). Rarely an animal might sit in front of a photocell and groom or move in a way to repeatedly break a light beam and cause counts. No preferential location in the corridors was seen in which this occurred and the impact of this circumstance on the activity counts is presumed to be small. A second drawback to the method is that the number of times an area is entered does not identify the amount of time spent in that area. Thus a rat causes only one count on entering and one on leaving regardless of time spent in the area. Minor preferences in use of space were found in adult rats. The distribution shown for groups of females (Table 4) was comparable to results for adult males. Most of these animals tended to use the continuous corridors slightly more than the blind-ended corridors during the nocturnal period. Observation of the rats after a brief period at the onset of the dark portion of the cycle agreed with the distribution of activity. However, the difference was not marked and was not present in all groups. A more detailed analysis would be needed to determine the significance of area use by adults. On the other hand, nursing mothers had a clear reversal of use of space from the nonnursing adult. Mothers localized most of their nocturnal activity away from the continuous corridors. This agrees with the findings of Barnett and McEwan (1973) for nursing mice. When the young rats left the nest box they initially concentrated their activity in area 1, as might be expected when locomotion was first attempted. At weaning the areas of the maze were about equally used by the young rats except that passage into the nest box was still high (Table 4). Exploratory activity as measured here on the first day in a residential maze differed in several respects from nocturnal activity. First, the amount of activity was greater during the first exploratory hour than during the maximum hourly nocturnal activity (Table 3 and Fig. 2). Second, males showed about the same amount of exploratory activity during the first hour as females, although the males were much more quiet during the second hour. Third, exploratory activity did not show the developmental pattern seen with light-dark activity. Exploratory activity of rats was affected by sex but not by age. These quantitative differences between the two types of activity lead to the speculation that if exploratory activity and nocturnal activity are quantitatively different, they may be qualitatively different as well. Direct observation of the rats reinforced the concept that qualitatively different activities were involved in the two types of activity. Different activities are also seen in natural environments, since the dark portion of the cycle is the period in which most feeding and drinking occurs, as well as much social interaction, for other nocturnal rodents such as Rattus rattus (Ewer, 1971). A direct comparison of the structure of exploratory and nocturnal behavior has not been made. The nature of one kind of exploratory behavior
330
NORTON, CULVER AND MULLENIX
and the effects of amphetamine on it have been examined in detail (Norton, 1973). It was found that rats show a distinct patterning in their behavior patterning and duration of behavior acts. It is likely, from the experiments reported here, that nocturnal activity does not have the same structure as exploratory activity and represents a different kind of activity, the detailed structure of which needs to be investigated. In particular, the early maturation of nocturnal activity in the rat makes it an interesting phenomenon for further studies relating to brain development. These experiments differ from previous descriptions of light-dark cycle activity primarily in that groups of four rats were monitored. While this design does not allow the activity of individuals to be compared during the dark and light cycles, it does add the dimension of social interaction which might affect the development of specific behaviors. Comparison of these results with reports on isolated animals shows considerable agreement betwen the results on isolated and grouped animals. This implies that social interaction is not a primary cause of nocturnal activity even though it may represent a large portion of this activity in grouped rats.
REFERENCES Barnett, S. A., and McEwan, I. M. (1973). Movements of virgin, pregnant and lactating mice in a residential maze. Physiol. Behav. 10, 741-746. Bolles, R. C., and Woods, P. J. (1964). The ontogeny of behaviour in the albino rat. Anita. Behav. 17,427-441. Costall, B., NaYlor, R. J., and Olley, J. E. (1972). On the involvement of the caudate-putamen, globus pallidus and substantia nigra with neuroleptic and cholinergic modification of locomotor activity. Neuropharmacology 11, 317-330. Culver, B., and Norton, S. (1974). Reversible hyperactivity in young rats after single exposure to carbon monoxide (CO). Pharmacologist 16, 208. Ewer, R. F. (1971). The biology and behaviour of a free-living population of black rats (Rattus rattus). Anim. Behav. Monogr. 4, 127-174. Friede, R. L. (1966). "Topographic Brain Chemistry." New York: Academic Press. Kennedy, G. C., and Mitra, J. (1963). Hypothalamic control of energy balance and the reproductive cycle in the rat. J. Physiol. 166, 395-407. Kleitman, N. (1949). Biological rhythms and cycles. Physiol. Rev. 29, 1-30. Levinson, L., Welsh, J. H., and Abramowitz, A. A. (1941). Effect of hypophysectomy on diurnal rhythm of spontaneous activity in the rat. Endocrinology 29, 41-46. Massof, R. W., and Jones, A. E. (1972). Electroretinographic evidence for a photopic system in the rat. Vision Res. 12, 1231-1239. McQuire, R. A., Rand, W. M., and Wartman, R. J. (1973). Entrainment of the body temperature rhythm in rats: effect of color and intensity of environmental light. Science 181, 956-957. Michaelson, I. A., and Sauerhoff, M. W. (1974). An improved model of lead-induced brain dysfunction in the suckling rat. ToxicoL Appl. Pharmacol. 28, 88-96. Norton, S. (1973). Amphetamine as a modei for hyperactivity in the rat. Physiol. Behav. 11, 181-186. Plant, S. M. (1974). Adult-litter relations in rats reared in single and dual-chambered cages. Develop. PsychobioL 7, 111-120.
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Richter, C. P. (1922). A behavioristic study of the activity of the rat. Comp. Psych. Monogr. 1, 1-55. Richter, C. P. (1927). Animal behavior and internal drive. Quart. Rev. Biol. 2, 307-343. Silbergeld, E. K., and Goldberg, A. M. (1974). A lead-induced behavioral dysfunction: an animal model of hyperactivity. Exp. Neurol. 42, 146-157. Slonaker, J. R. (1925). Analysis of daily activity of the albino rat. Amer. J. Physiol. 73, 485-503. Zucker, I. (1971). Light--dark rhythms in rat eating and drinking behavior. Physiol. Behav. 6, 116-126.
Development of Nocturnal Behavior in Albino Rats 1
STATA NORTON, BRUCE CULVER, and P H Y L L I S M U L L E N I X
Department of Pharmacology, University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kansas 66103
When the activity of groups of rats is monitored in a residential maze equipped with photocells for recording passage through the corridors, rats can be shown to be more active in the 12-hr dark portion of a day than in the 12-hr light portion from an early age. Groups of rats weaned at 23 days of age show significantly greater activity at night than during the day but the maximum nocturnal/diurnal ratio is recorded in young adult rats 2-4 months old. Although females older than 5 weeks are more active than males both during the light and dark cycle, the nocturnal/diurnal activity ratios show the same trends in the two sexes. Males differ from females in the duration of their exploratory activity when they are first introduced into the maze. Females are consistently much more active during the second hour in the maze but the differences between the sexes in activity during the first hour are not as great. The development of nocturnal activity of groups of rats in these experiments corresponds generally with results obtained on isolated rats, implying that social interaction is not a primary cause of the nocturnal activity recorded in these experiments.
S o m e types o f brain damage cause rats to b e c o m e hyperactive only during certain p o r t i o n s o f the normal light-dark cycle ( C o s t a l et al., 1972; Culver and N o r t o n , 1974). T h e r e f o r e , if the activity of an animal is being measured in the light p o r t i o n o f the day, the results o f an e x p e r i m e n t involving brain damage m a y be different in a diurnal or a n o c t u r n a l animal. It is also i m p o r t a n t to consider the kind o f activity being measured since activity in a novel e n v i r o n m e n t ( e x p l o r a t o r y activity) m a y n o t respond to diurnal influences in the same way as activity in a h o m e cage. When, in addition, activity studies are made on y o u n g animals, the d e v e l o p m e n t o f day-night activity cycles in the m a t u r i n g animal b e c o m e s i m p o r t a n t . E x a m p l e s o f studies in w h i c h this p r o b l e m arises are in recent reports on the t o x i c o l o g i c effects o f 1This research was supported by United States Public Health Service Grants MH 17279, MH 43860, and HD 02528. 317 Copyright (~)1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
318
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lead (Michaelson and Sauethoff, 1974; Silbergeld and Goldberg, 1974) and carbon monoxide exposure (Culver and Norton, 1974) in infant rodents. Both toxic events are described as producing hyperactive young animals which may serve as models for the hyperkinetic syndrome of childhood. The diurnal cycle of the rat has been studied experimentally for many years, and the adult rat is easily recognized as a nocturnal animal which spends most of the daylight hours asleep and is active most of the dark period of the day. A brief review of the early laboratory study of this phenomenon has been given by Levinson e t al. (1941). The methods used to detect the diurnal rhythm have been traditionally the running wheel (Slonaker, 1925) or activity cages which record body movement (Richter, 1922). With both methods the animals generally are housed individually during the period of study. When preweanling rats are studied, isolation of the animals for prolonged periods is impossible. The solution for this has been to study the young rats during portions of the day, up to 12hr in some cases, and to return them to the mother during the remainder of the day. Under these conditions, RiChter (1927) reported that the newborn rat showed continuous, rather than periodic, activity in an activity cage. Rhythmic activity appeared about the tenth day but the development of day-night cycles took much longer. At 60 days of age, the rat was 1.34 times more active during the night than the day in Richter's experiments (Richter, 1922). Old rats (600 days) were 2.2 times more active in the dark than the light. Man has been reported to have a day-to-night ratio close to one for the first 2 months of postnatal life, increasing to a ratio of 2 for the third and fourth months and to greater than 3 by the seventh month of life (F. G. Mullin, quoted by Kleitman, 1949). It appears that animals born at about the level of maturity of the rat and man show little regulation of activity in regard to the diurnal cycle in the neonatal period, and that the day-night cycle which characterizes the adult, either primarily diurnal or nocturnal, develops along with maturation of various other systems. The development of nocturnal behavior in the laboratory rat has been studied in a fragmentary way and only under conditions, such as isolation, which might be expected to alter the behavior of a social animal like the rat in unknown ways. Increasing recognition of interactions of day-night rhythms with other activities in adult rats makes it of interest to follow the development of nocturnal activity in the rat under social conditions. In the following experiments the development of nocturnal behavior in groups of rats was studied in a maze of dimensions which offered a method for evaluation of permanent groups of animals, allowing both diurnal and nocturnal activity to be recorded from the onset of walking in the young animals to maturity. It is proposed that the time course of increasing cyclic activity in the developing laboratory rat deserves study as a phenomenon which may affect many studies using young rats.
NOCTURNAL BEHAVIOR IN RATS
319
METHODS Two hundred rats, male and female Charles River (Sprague-Dawley derived) strain and their first generation offspring, were used in this study. Rats postweaning were housed in small groups in standard animal care facilities until they were studied in the environmental units. The basic environmental unit in which a group of rats was housed for study of activity was a runway maze around a central area with a raised dome (Fig. 1). Water was supplied at the end of one runway and a nest box or "burrow" was connected at the end of another runway. The remaining runways were loops with dual entrances to the central area. The runways and central area, excluding the nest box, covered an area 60 cm wide and 75 cm long. The total living area in each environmental unit was about 2240 cm 2. Each runway was 10 by 10 cm in cross section. The central area was roughly a cylinder 2 0 c m across by 2 2 c m high with six runway entrances. The WATER
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Fig. 1. Residential unit for groups of rats. Numerals identify nine areas and eight photocells (Numbers 1-8). Area 9, outlined by dotted line, is 22 cm high. Runways (areas 1-8) are 10 cm high. Nest box is 25 cm high.
320
NORTON, CULVERAND MULLENIX
detachable nest box was 20 cm square by 24 cm high. The walls of the unit were 20-gauge aluminum. The floor was expanded metal mesh except in the nest box which had a solid platform. Each runway was supplied with one or more photocell devices for recording each passage of a rat through the runway. The eight photocells in each unit were connected to counters. Cadmiun-selenium type photocells were used, activated by light from 6 V bulbs covered with red plastic lenses. All groups of rats appeared to be insensitive to the red light from these bulbs. The nest box was covered with a double thickness of red vinyl plastic. Light transmission of the plastic was determined in a Beckman Spectrophotometer and the double layer excluded most of the light longer than 600 riM. Activity of rats in the nest box could easily be observed through this cover since the visual sensitivity of the human eye is greater in the red range of visible light than the rat eye (Massof and Jones, 1972). The cover over the rest of the unit was colorless Plexiglas. In some experiments a double layer of red plastic vinyl sheeting was placed over the entire unit for 12 hr of the day to reverse the light-dark activity cycle of the rats and still allow the rats to be visible to an observer. In all experiments the rats were maintained on a 12-hr light-dark cycle. Except during the reversal, the light cycle was from 6:00 AM to 6:00 PM. Purina Rat Chow pellets were placed in the central area daily and water was supplied ad lib. by a watering tube with the reservoir bottle outside the unit. All maintenance of the unit (feeding, cleaning, etc.) was carried out daily from 9:00AM to 10:00 AM. Fresh nesting material (strips of paper) was placed in the maze daily. During this period all rats were removed from the maze except nursing mothers and their pups. The first 2 hr of the first day when a group of rats was placed in the maze were called the exploratory period. All other photocell counts per hour for diurnal and nocturnal activity of established groups of rats were taken after the first 24 hr in the maze. Diurnal activity of established groups was recorded from 10:00 AM to 6:00PM. Nocturnal activity was recorded from 6:00PM to 6:00 AM. Because the rats were removed from the maze from 9:00AM to 10:00AM each day this disturbance probably increased the diurnal hourly counts over the counts which would have been recorded for undisturbed rats. In interpretation of the data it is assumed here that any effects of the standard maintenance procedures were equivalent in both males and females of all ages. Six age groups of rats were studied: preweanling rats up to 3 weeks of age; weanling rats 3-5 weeks old; juveniles 5-8 weeks old; young adults 2-4 months old; mature adults 4-12 months old; and old adults 12-20 months of age. All groups consisted of four rats of the same sex, except for studies on nursing mothers in which the group contained the mother and four pups. From 10:05 to 10:15AM and again from 6:05 to 6:15PM each established group of adult rats was observed on 2 successive days. The red
NOCTURNAL BEHAVIOR IN RATS
321
light activating the photocells was adequate for the adapted human eye to identify the location and movements of the rats in the maze. The purpose of this was to compare the type of behavior involved in the initial burst of activity in the morning with the activity at the onset o f the dark cycle. Some groups were also checked for location of the rats every hour from 10:00 AM to 6:00 PM during the light cycle and again at the same clock times after the light cycle was reversed.
RESULTS
Diurnal activity of established groups. The term, established group, is used for any group of rats which was housed in one of the residential maze units for more than 24 hr. To illustrate the daily rhythm of activity, the 24-hr photocell record of an established group of young adult females is shown in Fig. 2. Most o f the activity occurs during the dark half of the daily cycle. Since the rats are removed from the maze unit for 1 hr each day (9:00-10:00tAM) during maintenance of the unit, there is a burst of activity when the rats are replaced in the unit. This initial period of activity may be an abbreviated form of the exploratory period which occurs on the first day when naive rats are placed in the maze. It may also be related to food carrying since food carrying activity around the maze is seen in the morning whether or not rats are removed from the maze. The diurnal and nocturnal activity of groups o f rats from weanling stage to old adult life is shown in Table 1. Weanlings have the most uniform
t o9 5 0 0
Fig. 2. Hourly photocell counts in a residential unit for an established group of four young adult female rats. Rats removed from unit at 9:00 AM and replaced at 10:00 AM. Lights on from 6:00 AM to 6:00 PM. Dark cycle 6:00 PM to 6:00 AM.
322
NORTON, CULVER AND MULLENIX TABLE 1 Average Hourly Photocell Activity of Established Groups of Four Rats Counts per hour _+SE
Composition of groups
Number of groups
Number of observation Diurnal days period
Nocturnal period
Ratio nocturnal-diurnal activity b
Weanlings (3-5 wk) Male Female
2 3
10 15
282 _+31 200 _+27
350 +- 19 286 +_16
1.24 1.43
7 7
21 21
95 a +_12 149 _+12
259 a _+20 457 _+22
2.68 3.07
Male Female
5 10
15 30
99a _+16 164 +_ 14
328a +_21 540 +_39
3.31 3.29
Mature adults (4-12 mo) Male Female
6 13
19 41
90a _+ 8 190 +_ 9
288a _+25 480 _+20
3.20 2.53
2 3
12 18
96_+12 130 + 11
174a_+ 2 369 +_14
1.81 2.84
Juveniles (5-8 wk) Male Female Young adults (2-4 mo)
Old adults (12-21 too) Male Female
astatistically significant difference between males and females of the same age. bThese ratios represent significant differences between nocturnal and diurnal activity for all groups (t test, P = < .05). d i s t r i b u t i o n o f n o c t u r n a l - d i u r n a l activity. T h e m a r k e d divergence in distributi'on o f a c t i v i t y is first seen in j u v e n i l e s a n d peaks in y o u n g adults. A f t e r t h e w e a n l i n g stage, m a l e rats s h o w less activity t h a n females o f the same age at all p e r i o d s o f t h e day. T h e m o s t active rats are y o u n g a d u l t females. A f t e r 12 m o n t h s o f age b o t h m a l e s a n d females d r o p in n o c t u r n a l activity. The r a t i o o f n o c t u r n a l to d i u r n a l a c t i v i t y is b e t w e e n 1 a n d 2 for t h e w e a n l i n g rats a n d over 3 f o r m o s t o f t h e o t h e r groups. Preweanling activity. T h e use o f t h e n e s t b o x b y t h e female for care o f the p u p s allowed t h e e x p e r i m e n t e r to r e c o r d e m e r g e n c e a n d early e x p l o r a t i o n o f t h e m a z e b y t h e pups. T h e n e s t b o x was a t t a c h e d so t h a t the level f r o m
323
NOCTURNAL BEHAVIOR IN RATS TABLE 2 Postnatal Activity of Mother and Rat Pups in Maze. Average Hourly Photocell Counts (± SE) in Three Experiments Postnatal day
Diurnal counts
Nocturnal counts
Ratio nocturnal/diurnal
Mother only (pups in nest box) 2 3 4 5 6
68 ± 11 64 ± 33 57 ± 10 48 ± 15
63 55 48 51 47
7 8 9 10
64 49 46 72
45± 43 ± 49 ± 52±
11 12 13 14
90 84 89 110
± ± ± ±
6 9 6 17
± 27 ± 17 ± 20 ± 10
85 79 94 115
± 15 -+ 11 ± 11 ± 12 ± 10 6 6 10 4
± 25 ± 18 ± 40 ± 38
Days 2-6 0.89
Days 7-10 0.79
Days 11-14 1.00
Mother and pups (emergence from nest box) 15 16 17 18
86 21 152 152
± 18 ± 18 ± 21 ± 21
108 139 136 137
± 37 ± 47 ± 19 ± 9
Days 15-18 1.08
19 20 21 22
169 186 183 174
± 25 ± 6 ± 31 ± 15
135 210 232 287
± 22 ± 14 ±20 ± 22
Days 19-22 1.21
23 24 25 26 27 28
189 251 180 132 245 164
± 19 ± 73 ± 54 -+39 ± 99 ± 32
Weanlings only (mother removed) 217 ± 51 284 ± 11 289 ± 37 320± 8 324 ± 45 317 ± 63
Days 23-28 1.50a
aThis ratio represents a significant difference between nocturnal and diurnal activity averaged for days 23-28 (t test, P = < .05). t h e n e s t b o x f l o o r to t h e r u n w a y f l o o r r e q u i r e d t h e p u p s to c l i m b u p a 5 - c m s t e p t o gain a c c e s s t o t h e r u n w a y . W a l k i n g o f p u p s w a s i d e n t i f i e d in t h e n e s t at l e a s t b y t h e t h i r t e e n t h d a y . E m e r g e n c e f r o m t h e n e s t b o x o c c u r r e d 2 d a y s later. D a t a o n a c t i v i t y o f t h r e e g r o u p s o f p r e w e a n l i n g rats a n d m o t h e r s w e r e o b t a i n e d while t h e y o u n g w e r e w i t h t h e m o t h e r ( T a b l e 2). B e f o r e t h e y o u n g
324
NORTON, CULVER AND MULLENIX
could leave the nest box, the daily activity of the mother alone was recorded. This period was the first 14 days after birth of the pups. The mother showed no significant diurnal-nocturnal rhythm during this period. The hourly photocell counts were not significantly different in the light and dark periods (Table 2). Mthough the young rats could climb up out of the nest box on the fifteenth day, their contribution to activity was minimal until the seventeenth day. About the twentieth day, a tendency to increasing nocturnal activity was noted in both the mother and the young, and this beginning preference for the dark cycle was seen in the young after removal of the mother. Activity in the maze by the young rats increased sharply on the day in which the mother was removed. The ratio of nocturnal to diurnal activity was 1.5 from the twenty-third to the twenty-eighth postnatal day. Exploratory activity. When a group of postweanling rats was first placed in a maze unit, there was an initial burst of activity which was greater than the activity on succeeding days. This period of activity lasted about 2 hr and was called the exploratory period. Females were more active than males during this period (Table 3). In the first hour of exploratory activity, old adult males were approximately one-third as active as females. Juvenile, young, and mature adult males were only slightly less active than females during the same period. The greatest divergence in male and female activity came during the second hour of exploratory activity when the males were much quieter than the females at all ages studied. Descriptive comments on activity. In the course of these experiments hourly observations were made which were not evaluated statistically. However, the observations were sufficiently consistent to illustrate the way in which the varied spaces of the maze were used by the rats. The nest box and areas 1-8 of the maze were all used for sleeping during the day by postweanling rats. Females of any age and young male rats tended to sleep huddled together. Old males were more isolated. Area 9 was very rarely used for sleeping by any rat and quiet huddling was never observed during the light cycle in area 9. Reversal of the diurnal cycle with reversed illumination has been shown to require 7-14 days (McGuire et al., 1973). A group of four adult animals was made active during the 9:00 AM to 9:00 PM period by making this the dark period of the day. Since the rat's eye is insensitive to red light (Massof and Jones, 1972), a red plastic cover on the maze was used to reverse their cycles and to allow observation of the rats with the room lights on. Reversal of the dark-light activity cycle required 8 days with the maze covered with red plastic (Fig. 3). During the reversed dark cycle various activities were observed. There was considerable social interaction among the four rats in the maze. In addition, grooming activities, feeding, and drinking were present in cycles throughout the dark period. No specific quantitation of the different activities
325
NOCTURNAL BEHAVIOR IN RATS TABLE 3 Exploratory Activity of Groups of Four Rats on the First Day in a Residential Maze
Composition of groups
Number of groups of four rats each
Average Photocell Counts -+ SE First hour in Second hour in residential maze residential maze
Weanlings (3-5 wk) Males
2
1197 _+ 122
465 ± 23
7 7
1171 a ± 222 1859 ± 193
390a ± 140 1105 ± 196
3 10
1430 ± 398 2326 ± 245
639 a ± 41 1979 ± 245
4 12
1553 ± 400 2204 _+ 152
302a ± 41 1610 ± 349
2 3
528 a-+ 16 1589 -+ 115
237a± 4 737 ± 156
Juveniles (5-8 wk) Males Females Young adults (2-4 mo) Males Females Mature adults (4-12 too) Males Females Old adults (12-21 mo) Males Females
aSignificant difference between males and females (P = < .05, Mann-Whitney U test) comparing same hour and age group. t h r o u g h o u t the dark cycle was a t t e m p t e d in these studies b u t the general appearance o f the activity differed f r o m the rapid e x p l o r a t o r y behavior which was characteristic o f the first i n t r o d u c t i o n o f rats to the maze unit. Congregation and aggressive kinds o f social behavior were seen in area 9. Areas 1-8 were used for running and chasing. This could be initiated by rats meeting in the corridors b u t the primary area for initiation o f these activities was area 9. S o m e t i m e s one rat w o u l d " d e f e n d " area 9 causing all other rats to run in the corridors and w o u l d block others f r o m entering the central area. Distribution of nocturnal activity. S o m e evidence o f the n o c t u r n a l utilization o f space can be gained by an analysis o f the p h o t o c e l l counts in the different areas o f the maze. Table 4 shows the distribution o f counts in the entrance to the nest b o x (area 1), the c o n t i n u o u s corridors (area 3, representing also areas 2 and 4 and area 7, representing also areas 6 and 8)
326
NORTON, CULVER AND MULLENIX
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Fig. 3. Reversal of diurnal activity in an established group of four adult female rats in a residential unit with a red cover used to simulate darkness. Room lights on continuously. Red cover on 9:00 AM to 9:00 PM. a n d t h e b l i n d c o r r i d o r w i t h t h e w a t e r i n g t u b e ( a r e a 5). T h e n u r s i n g m o t h e r used areas 1 a n d 5 a b o u t e q u a l l y a n d areas 3 and 7 b o t h less f r e q u e n t l y , s h o w i n g a p r e f e r e n c e f o r a straight p a t h t h r o u g h area 9 t o the w a t e r source. G e n e r a l a m b u l a t i o n a r o u n d t h e t w o c o n t i n u o u s c o r r i d o r s was m u c h less t h a n t h a t o f n o n n u r s i n g a d u l t females. C o n v e r s e l y , n o n n u r s i n g a d u l t females used areas 3 a n d 7 s o m e w h a t m o r e t h a n areas 1 a n d 5. I n T a b l e 4, t h e c o m p a r i s o n o f c o u n t s f o r area 3 ( 6 7 -+ 5.1 c o u n t s p e r h o u r ) w i t h area 5 ( 4 9 -+ 3.7 c o u n t s TABLE 4 Distribution of Nocturnal Activity of Female Rats in Residential Maze Counts per hour _+SE Postnatal day
Number of groups
Area 1
One nursing mother before emergence of pups, days 2-14
1
18_+ 1.3a
8-+ 3.2 19_+ 1.7 a
5-+ 1.0
Mother and four pups after emergence, days 15-22
1
42_+7.5 ab
13_+ 1.4 24_+ 2.4 a
13_+2.7
Four young only after weaning, days 23-26
1
53-+2.3 a
29_+7.4
27-+8.3
Four adult females without young
7
62-+5.4
67-+ 8.1 49_+ 3.7
Area 3
aSignificantly different from areas 3 and 7, t test P = < .05. bSignificantly different from area 5, t test P = < .05.
Area 5
33+_ 10.7
Area 7
75+_7.9 b
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per hour) has a chance probability slightly more than 0.05, while the probability that use of area 7 (75 -+ 7.9 counts per hour) differs from area 5 is approximately 0.05. When the young rats could leave the nest box the highest counts occurred in area 1. Counts in areas 3, 5 and 7 became uniform shortly after weaning. DISCUSSION In the use of the residential maze in this study two hypotheses were. involved. One was that daily activity rhythms might develop differently in animals living in social groups than in animals living in isolation, and the second was that different spaces might be selectively used during the active (nocturnal) portion of the day if a complex environment was available to the rats. The first hypothesis was not substantiated by the data. Previous data in the literature obtained on isolated rats were essentially confirmed here in groups of rats, both in the ratio of nocturnal to diurnal activity and the time at which it arose during development. An exception to this was the failure to confirm Richter's (1922) observation that the ratio continued to increase up to old age. In the experiments here the peak ratio was reached in the mature adult and not in animals over 12 months old. This difference in results may be due to the social condition of testing, suggesting that the presence of a mature adult rat might increase the nocturnal activity of another mature rat. Another possible reason for the difference in results may be that Richter's results were influenced by feeding the rats at two different times of day (in the light or in the dark cycle) and then averaging the results. This experimental design would be expected to alter the behavioral patterns from those with ad lib. feeding. In the present experiments food was available during both the light and dark cycle. This avoided any pressure on growing rats to remain active during any particular portion of the light-dark cycle, thus allowing the maximum dark-light ratio to be developed much earlier than in the experiments by Richter (1922). It is possible that the nocturnal to diurnal ratio reported here is too low for all groups of rats. If the animals had not been removed from the maze for an hour every morning, the brief burst of activity which was seen on reintroduction of the rats would not have occurred and the total activity during the light cycle might have been less. Thus the ratio might be higher with a different experimental design. However, except for a modest increase in the ratio, there is no reason to expect that a more detailed examination of either the undisturbed group or different numbers of animals would produce any novel findings. Bolles and Woods (1964) reported that the preweanling rat does not show a light-dark cycle up to 3 weeks of age. The present experiments are in
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agreement since the daily rhythm of activity developed in the weanling rats between 3 and 5 weeks of age but was not present or was poorly developed in younger rats. This activity thus predates the full maturation of the central nervous system (Friede, 1966). Zucker (1971) reported that a light-dark rhythm developed in food and water intake shortly after weaning. This corresponds to the increase in nocturnal activity observed here after weaning. After 5 weeks of age until they were old adults, the female rats were significantly more active than males. However, no clear changes in activity could be related to estms cycles in adult females in these experiments. This lack of cyclic estrus activity is to be expected under these environmental conditions since the techniques for displaying estrus activity require specific conditions such as isolation of the female, preferably from an early age, in a running wheel cage (Kennedy and Mitra, 1963). In a maze type environment, resembling the maze in these studies, Barnett and McEwan (1973) failed to find cyclic estrus activity in isolated mice. The second hypothesis proposed was that rats would utilize space in differing ways during the active portion of the day. The mothers with nursing young lost the greater nocturnal activity of the nonlactating adult and showed a very uniform distribution of activity throughout 24 hr. These results are of interest in comparison with the findings of Plaut in regard to maternal activity in a dual-chambered cage. Plaut (1974) found that nursing mothers spent more time with their pups during the day than during the night. If the dual-chamber used by Plant is comparable to the maze used in the present experiments then the activity of the mothers during the day must be kept equal to the nocturnal activity by an altered use of the period spent away from the young. For example, the mother could leave the young primarily to feed or drink during the day but might be relatively quiet in some area away from the nest box during the nocturnal period. This would account for the greater nocturnal time away reported by Plaut but not greater nocturnal activity reported here in nursing mothers. Alternatively, the two environmental conditions, the maze and the dual-chambers, may cause different behaviors on the part of nursing mothers. In a maze shaped like a cross with equal arms, called a plus maze by Barnett and McEwan (1973), mice did not utilize the four blind-ended corridors (containing food, water, balsa wood, or nothing) with equal frequency. The corridor with water was visited slightly less often than the empty corridor but the difference did not appear to be significant for virgin mice. Nursing mice, on the other hand, visited the empty corridor much less often. In the residential maze described here the animals had four different types of space to use, a darkened high area (nest box), a high central area (area 9), two continuous runways (areas 2, 3, 4, and 6, 7, 8), and a blind corridor with a watering bottle (area 5). No photocells were placed in the nest box or area 9. In retrospect, this was unfortunate since considerable social interaction was
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seen in these areas, particularly area 9 from which there was access to all runways. The location of the photocells in areas 1-8 limited the type of recorded activity predominately to locomotion either social (such as chasing) or solitary (such as food carrying or walking). Rarely an animal might sit in front of a photocell and groom or move in a way to repeatedly break a light beam and cause counts. No preferential location in the corridors was seen in which this occurred and the impact of this circumstance on the activity counts is presumed to be small. A second drawback to the method is that the number of times an area is entered does not identify the amount of time spent in that area. Thus a rat causes only one count on entering and one on leaving regardless of time spent in the area. Minor preferences in use of space were found in adult rats. The distribution shown for groups of females (Table 4) was comparable to results for adult males. Most of these animals tended to use the continuous corridors slightly more than the blind-ended corridors during the nocturnal period. Observation of the rats after a brief period at the onset of the dark portion of the cycle agreed with the distribution of activity. However, the difference was not marked and was not present in all groups. A more detailed analysis would be needed to determine the significance of area use by adults. On the other hand, nursing mothers had a clear reversal of use of space from the nonnursing adult. Mothers localized most of their nocturnal activity away from the continuous corridors. This agrees with the findings of Barnett and McEwan (1973) for nursing mice. When the young rats left the nest box they initially concentrated their activity in area 1, as might be expected when locomotion was first attempted. At weaning the areas of the maze were about equally used by the young rats except that passage into the nest box was still high (Table 4). Exploratory activity as measured here on the first day in a residential maze differed in several respects from nocturnal activity. First, the amount of activity was greater during the first exploratory hour than during the maximum hourly nocturnal activity (Table 3 and Fig. 2). Second, males showed about the same amount of exploratory activity during the first hour as females, although the males were much more quiet during the second hour. Third, exploratory activity did not show the developmental pattern seen with light-dark activity. Exploratory activity of rats was affected by sex but not by age. These quantitative differences between the two types of activity lead to the speculation that if exploratory activity and nocturnal activity are quantitatively different, they may be qualitatively different as well. Direct observation of the rats reinforced the concept that qualitatively different activities were involved in the two types of activity. Different activities are also seen in natural environments, since the dark portion of the cycle is the period in which most feeding and drinking occurs, as well as much social interaction, for other nocturnal rodents such as Rattus rattus (Ewer, 1971). A direct comparison of the structure of exploratory and nocturnal behavior has not been made. The nature of one kind of exploratory behavior
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and the effects of amphetamine on it have been examined in detail (Norton, 1973). It was found that rats show a distinct patterning in their behavior patterning and duration of behavior acts. It is likely, from the experiments reported here, that nocturnal activity does not have the same structure as exploratory activity and represents a different kind of activity, the detailed structure of which needs to be investigated. In particular, the early maturation of nocturnal activity in the rat makes it an interesting phenomenon for further studies relating to brain development. These experiments differ from previous descriptions of light-dark cycle activity primarily in that groups of four rats were monitored. While this design does not allow the activity of individuals to be compared during the dark and light cycles, it does add the dimension of social interaction which might affect the development of specific behaviors. Comparison of these results with reports on isolated animals shows considerable agreement betwen the results on isolated and grouped animals. This implies that social interaction is not a primary cause of nocturnal activity even though it may represent a large portion of this activity in grouped rats.
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Richter, C. P. (1922). A behavioristic study of the activity of the rat. Comp. Psych. Monogr. 1, 1-55. Richter, C. P. (1927). Animal behavior and internal drive. Quart. Rev. Biol. 2, 307-343. Silbergeld, E. K., and Goldberg, A. M. (1974). A lead-induced behavioral dysfunction: an animal model of hyperactivity. Exp. Neurol. 42, 146-157. Slonaker, J. R. (1925). Analysis of daily activity of the albino rat. Amer. J. Physiol. 73, 485-503. Zucker, I. (1971). Light--dark rhythms in rat eating and drinking behavior. Physiol. Behav. 6, 116-126.
