Structure analysis of spontaneous behavior during the estrous cycle of the rat

Physiology & Behavior, Vol. 27, pp. 723-726. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.

Structure Analysis of Spontaneous Behavior During the Estrous Cycle of the Rat I PHYLLIS

MULLENIX

Department o f Neuropathology, Harvard Medical School and Department of Psychiatry, Children's Hospital Medical Center, Boston, MA 02115 R e c e i v e d 27 O c t o b e r 1980 MULLENIX, P. Structure analysis o f spontaneous behavior during the estrous cycle o f the rat. PHYSIOL. BEHAV. 27(4)

723-726, 1981.--The structure of spontaneous motor behavior during estrous and diestrous stages of the adult female rat's reproductive cycle was analyzed using time-lapse photography. Repeated measures of the frequency, duration, sequences and distribution of fifteen different motor acts were taken for each female in diestrus and again during estrus. In contrast to findings of previous studies, no increase in general activity was observed in correlation with the physiological condition of estrus. Furthermore, consideration of factors including the individual, environment and estrous cycle stage demonstrated that the individual was most important, the environment next and estrous cycle stage least for influencing behavior sequence consistency. Consequently, these results, from a more complete, quantitative analysis of female spontaneous behavior, do not support the widely accepted belief that female rats' motor activity varies with estrus to preclude their use in studies of behavioral toxicity. Estrus

Diestrus

Spontaneous behavior

Time-lapse photographic analysis

IT is widely accepted that female rodents exhibit increased general activity during estrus [1, 5, 7, 18, 26, 28]. In fact, many behaviors, such as feeding [8], distractability [4], conditioned avoidance [11], light avoidance [12] and aggressiveness [15], have been reported to vary with estrous cycle stage. Because of this variability, females are often considered less suitable than males as subjects in studies of behavioral toxicity. However, a toxic substance's full potential to affect behavior may not be recognized unless both sexes are considered. Rather than excluding female subjects, factors contributing to their behavioral variability should be more carefully examined to facilitate detection of behavioral toxicity. Variation in activity level with estrous cycle stage is examined in this study. In contrast to the many studies finding an association between increased activity level and estrus, a few failed to demonstrate any such correlation [3,6], whereas others found it depended upon the activity monitoring device [9,10] or the selection of behavioral acts recorded [14]. Outcome variability due to monitoring technique is commonplace in activity studies. The effects of food deprivation [27,29], amphetamine [17], septal lesions [20], globus pallidus lesions [23] and lead [16] are all known to vary with activity monitoring technique. The most frequently used activity devices, including the running wheel, photocell beams, shuttle boxes and others, determine only frequency counts of one or two locomotor behaviors such as walking or run-

ning. Open field studies generally are limited to counts of squares traversed within a short time interval. Observers of rat behavior, however, have recognized that activity consists of multiple behavioral acts [2,13] occurring spontaneously with highly structured frequency, duration, sequence [22] and distribution over time [24]. Therefore, by design most techniques are not capable of measuring the complex structure of rodent activity. In this study of the relationship between estrous cycle physiological changes and female spontaneous behavior, a monitoring technique is used which is capable of measuring the various parameters comprising the complex structure of activity. Variation in activity level with estrous cycle stage can then be examined without the influence of restricted measurement capability. METHOD

Animals Sixteen Charles River, Sprague-Dawley derived, virgin females at four months of age were housed in groups of four in standard (28 x 21.5 x 15 cm) plastic cages containing hardwood bedding. They were maintained on a 12-hour light (6:00 a.m. to 6:00 p.m.), 12-hour dark (6:00 p.m. to 6:00 a.m.) cycle and received Purina rat chow and water ad lib. Vaginal smears were taken by isotonic saline lavage using a previ-

1This research was supported by NICHD Program Project # HD 08945.

C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/100723-04502.00/0

724 ously described technique [30], and estrous cycle stage was checked each morning between 9:00 and 10:00 a.m. Estrous cycles were checked for at least fifteen days in order to select females with the most consistent 4 or 5 day cycles and lordosis responses to manual stimulation. Five additional cycles were checked in ten consistently cycling females before analyzing their behavior. Each selected female was tested twice during a single cycle, once during estrus and again during diestrus when leukocytes were the predominant vaginal cell type. Because of repeated testing, each female was adapted to the test environment 15 minutes daily for three weeks prior to beginning behavioral analyses.

Time-Lapse Photographic Behavioral Analysis After checking estrous cycle stage, one estrous and one diestrous female from separately housed groups were placed on either side of a Plexiglas box divided by a clear center panel. The center panel had 1 cm holes to allow the females to see and smell each other and not be completely isolated during test periods. The box, 30×23 ×24 cm high, had a wire mesh floor and was located inside a sound-attenuated chamber with a clear plastic door allowing photography of animal movements. Inside the chamber where temperature was maintained at 24 to 26°C, a fan provided ventilation and a masking noise, and two 15-W fluorescent tubes located 49 cm above the box provided light for the photography. Using the technique described by Norton [22], each pair of rats was filmed for 15 minutes using an Arriflex 16 mm camera and time-lapse photography at one frame per second. The 900 frames were analyzed individually, and one of the following body positions was recorded for each rat: standing (ST), walking (WA), sitting (SI), rearing (RE), or lying down (LD). In addition, one of the following behaviors when present was recorded for each frame: scratching (SC), grooming (GR), washing face (WF), head bobbing (BO), turning (TU), pawing (PW), sniffing (SN), head turning (HT), smelling (SM), or looking (LO). Reliability for identification of these fifteen behavioral acts by one or more observers was previously demonstrated [25]. Because the rats were identified on film by random numbers only and assignment of estrous and diestrous rats to either side of the observation box was random, behavior could be scored blind without reference to estrous cycle stage. Data obtained from the fdms were processed using various Fortran computer programs which determined the frequency, duration, sequences [22] and distribution [24] of the behavioral acts: (1) Frequency. Single behavior item frequency was determined from the total number of frames each behavioral act appeared during the 15-minute test period. In addition, because considerable variation can occur between individual animals in the frequency they perform any one behavior [19], total frequencies for the Grooming (including behaviors SI, WF, GR, SC and LD), Exploratory (WA, RE, PW, SN, TU, and BO) and Attention (ST, SM, HT and LO) clusters were determined to detect more subtle estrous and diestrous frequency differences. (2) Duration. The mean duration of each behavioral act was determined from the mean number of consecutive frames that a behavior continued, once initiated. (3) Distribution. To determine if a behavior occurred (a) randomly throughout the test period; (b) maximally clustered, such as occurring repetitively each time initiated; or (c) maximally dispersed, occurring once between intermit-

MULLENIX TABLE 1 TOTAL OCCURRENCESOF FEMALEBEHAVIORALACTS (MEAN_+SE*) Behavior Item

Diestrus

Estrust

ST WA SI RE LD SC GR WF BO TU PW SN HT SM LO

679 _+ 50 30 _+ 6 126 _+ 46 65 -+ 24 0 2 _+ 1 33 _+ 14 56 _+ 13 17_+ 4 27_+ 5 6_+ 2 23 _+ 7 67_+ 11 194 + 36 73 _+ 17

716 _+ 36 26 _+ 6 110 _+ 34 48 _+ 9 0 1_+ 1 25 _+ 12 58 _+ 14 16 + 3 21 _+ 4 6_+ 3 26 + 7 88 + 8 177 + 33 90 _+ 19

*N = 10 and df=9 for each behavior item. tCalculated paired t values for all behavior items ranged from 0.18 to 1.81, p>0.10.

tent delays, an index of dispersion was calculated for each behavior with a total frequency greater than 0.01 during the 15-minute test period. In contrast to the Norton study [24] where indices were calculated for animal groups, indices were calculated here for individual females to determine between-rat variability. (4) Sequences. To compare the similarity of behavior act linkage by estrous and diestrous rats, sequential analyses were performed by using the Chi Square test for observed versus expected frequencies of behavior pairs. Correlation coefficients were calculated for ranked Chi Square values for all possible pairs of behavior occurring within 5 second intervals, a time span from which maximum correlations can be obtained [22]. Behavior pairs that occurred less than 5 times in any experimental condition were eliminated from the analysis. Various statistical comparisons were made of behavior act sequencing. First, each female's behavior act linkage during estrus was compared to that she performed during diestrus. This comparison with the individual being the same was labeled Group 1 for convenience. A similar estrousdiestrous sequence comparison was made for Group 2, but the estrous and diestrous sequences were performed by different females who were photographed simultaneously so their environment was consistent. Finally, behavior sequences performed by different females photographed at different times were compared, only estrous cycle stage remained consistent. Diestrous females' behavior linkage was compared to that of other diestrous females in Group 3, and estrous females' behavior linkage was compared to that of other estrous females in Group 4.

Statistical Analyses Each female's estrous and diestrous behavior frequencies, durations and distributions were compared using paired t-tests. The Wilcoxon signed rank test also was applied to

GENERAL ACTIVITY DURING ESTRUS

725

TABLE 2 TOTAL FREQUENCY FOR BEHAVIOR CLUSTERS (MEAN±SE)*

TABLE 3 DURATION OF FEMALE BEHAVIORAL ACTS (MEAN-+SE*)

Cluster

Diestrus

Estrust

Behavior Item

Diestrus

Estrus

Grooming Exploratory Attention

217 --- 63 170 --- 41 1013 -+ 66

194 +- 51 144 -+ 22 1071 -+ 56

ST WA SI RE LD SC GR WF BO TU PW SN HT SM LO

49.5 _+ 27.9 1.6_+ 0.1 13.8 - 3.3 3.8 _+ 0.7 0 1.4 -+ 0.5 3.4_+ 0.9 5.9-+- 1.0 1.2-+ 0.1 1.4 +_ 0.1 1.5 -+ 0.2 2.0 -+ 0.3 1.1 _+ 0.03 3.2 _+ 0.5 2.3 -+ 0.3

27.5 _+ 6.5 1.4_+ 0.1t 15.4 -+ 3.3 3.8 - 0.4 0 1.0 -+ 0.3 3 . 6 - 1.3 8.8_+ 2.1 1.1 - 0.02 1.2 -+ 0.04 1.4 _ 0.5 2.7 _+ 0.4 1.2 -+ 0.04 3.0 -+ 0.4 2.0 _+ 0.2

*N=I0 and df=9 for each cluster. *Paired t-test comparisons; paired t values ranged from 0.63 to 1.20, p >0.20.

TABLE 4 INDEX OF DISPERSION: OBSERVED FREQUENCY/EXPECTED FREQUENCY (MEAN-+SE*) Behavior Item Sitting Washing Face Walking Turning Rearing Head Turning Smelling Standing Looking

Diestrus 0.329 0.270 0.688 0.730 0.413 0.874 0.661 0.869 0.639

- 0.053 _+ 0.026 _+ 0.049 -+ 0.080 _+ 0.067 - 0.031 _+ 0.072 - 0.029 -+ 0.117

Estrust 0.302 0.255 0.654 0.714 0.346 0.893 0.615 0.881 0.657

_+ 0.054 -+ 0.032 _ 0.047 _+ 0.054 _+ 0.041 _+ 0.031 _+ 0.046 _+ 0.024 -+ 0.041

*N= 10 and df=9 for each behavior item. *Paired t-test comparisons; p>0.10 for all behavior items.

e a c h c o m p a r i s o n b e c a u s e o f small sample size and o c c a sional wide data variation. An analysis o f v a r i a n c e was used to d e t e r m i n e significant differences b e t w e e n average correlation coefficients for group b e h a v i o r s e q u e n c e c o m p a r i s o n s . S c h e f f e ' s m e t h o d o f multiple c o m p a r i s o n s was applied to individual groups in the e v e n t o f significant group-related F ratios.

*N=I0 and df=9 for each behavior item. tp<0.05, paired t-test (p>0.05 for all other behavior items).

TABLE 5 CORRELATION COEFFICIENTS: COMPARISON OF FACTORS INFLUENCING BEHAVIOR SEQUENCE

Group

Consistent Factor

1 2 3 4

Individual Environment Diestrus Estrus

Correlation Coefficient (Mean_+ SE) 0.807 0.744 0.635 0.592

_+ 0.044 _+ 0.044 _+ 0.047 _+ 0.042

N* 30 24 29 23

*N = average number of behavior pairs ranked for each correlation coefficient.

RESULTS

Frequency, Duration and Distribution of Behavioral Acts The m e a n f r e q u e n c y o f e a c h behavioral act (Table 1), as well as the total f r e q u e n c y for each b e h a v i o r cluster (Table 2) did not significantly change b e t w e e n the estrous and diestrous stages of the sexual cycle. The duration (Table 3) and distribution (Table 4) o f m o s t behavioral acts likewise did not v a r y b e t w e e n stages. F o r all o f these comparisons, application o f the W i l c o x o n signed rank test did not give different results than those obtained with paired t-tests.

Sequences of Behavioral Acts Correlation coefficients for b e h a v i o r s e q u e n c e comparisons varied significantly b e t w e e n groups, F(3,32)=5.1, p < 0 . 0 1 (Table 5). The highest m e a n correlation coefficient was obtained w h e n the individual was the consistent factor. T h e r e f o r e , b e h a v i o r s e q u e n c e structure was m o s t consistent w h e n it was p e r f o r m e d by the same female, regardless o f her estrous cycle stage o r e n v i r o n m e n t while photographed. The next m o s t important influence on b e h a v i o r s e q u e n c e was e n v i r o n m e n t , and the least was stage of estrous cycle.

DISCUSSION Analysis of female spontaneous b e h a v i o r in this study did not reveal heightened general activity during estrus. H y p e r a c t i v i t y has been d e m o n s t r a t e d to include increased f r e q u e n c i e s o f e x p l o r a t o r y behaviors (WA, BO, T U , R E , P W and SN), d e c r e a s e d frequencies o f grooming behaviors (GR, SI, W F and SC), shorter duration o f m o s t behaviors e x c e p t for perhaps walking and m o r e r a n d o m behavioral seq u e n c e and distribution [22, 23, 24, 25]. N o n e o f these changes w e r e o b s e r v e d to correlate with estrous vaginal cornification here. In fact, m e a s u r e m e n t s o f b e h a v i o r seq u e n c e structure r e v e a l e d that estrous cycle stage was of lesser importance to s e q u e n c e c o n s i s t e n c y than either individual or e n v i r o n m e n t a l influences. The females tested in this study w e r e at the age w h e n close to peak differences b e t w e e n estrous and diestrous activity levels w e r e to be e x p e c t e d [26]. Yet, improving measu r e m e n t capability, from f r e q u e n c y counts o f one or two b e h a v i o r s to f r e q u e n c y , duration, s e q u e n c e and distribution determinations o f m a n y behaviors, did not d e m o n s t r a t e

726

MULLENIX

previously reported estrous activity increases. O t h e r factors may have contributed to the contradictory findings. First, the time-lapse photographic technique used here allowed a p e r m a n e n t record to be made o f e a c h f e m a l e ' s behavior. Recording b e h a v i o r from film allowed verification o f observed behaviors, second-by-second analysis of m o v e m e n t s and determination o f the a m o u n t of b e h a v i o r lost b e t w e e n observations [21]. Verification o f r e c o r d e d b e h a v i o r and unr e c o r d e d b e h a v i o r loss was not possible using direct observational techniques e m p l o y e d in previous studies [5, 10, 14]. Second, the 15 minute test period in this study was long c o m p a r e d to those in o t h e r studies finding heightened activity during estrus [5, 7, 18]. Martin and B/ittig [18] found that the greatest increases in m a z e estrous activity o c c u r r e d during the first 1.5 minutes of testing then disappeared by six minutes. T h e y suggested that Barnett and M c E w a n [3] did not detect estrous activity changes in the m a z e because they tested for m u c h longer periods. Third, in contrast to previous studies, the influence of stress was minimized here. Exposure to light during the nocturnal period [18], constant or sudden noise [5,7], electric shock [12] and complete isolation [5, 10, 28] were some o f the laboratory stresses avoided in this study to p r e v e n t induction o f artificial behavioral p h e n o m e n a not likely e x p r e s s e d by the wild rat. R e v e r s e d lighting cycles were also avoided because a period of days is required to reverse an animal's light-dark cycle [23], possi-

bly creating another laboratory stress. Gray [12] did not find differences b e t w e e n estrous and nonestrous open field and running wheel activity in mice under unstressful situations, but under stressful conditions such as light and electric shock, differences b e c a m e apparent. A final difference possibly contributing to contradictory findings is that behavioral m e a s u r e m e n t s in prior studies [14, 18, 28] were made generally during the animal's nocturnal period and not during the diurnal period as in this study. It might appear that estrous activity increases are a nocturnal p h e n o m e n o n only, requiring nocturnal or r e v e r s e d lighting m e a s u r e m e n t s . H o w e v e r , this is not a likely possibility because increased activity levels during estrus have been reported to o c c u r during the diurnal period as well [5], while not all nocturnal m e a s u r e m e n t s have d e t e c t e d it [6]. In addition, the more sensitive time-lapse photographic technique used in this study can detect hyperactivity during the diurnal period, when o t h e r techniques detect it during the nocturnal period only [23]. In conclusion, reducing stress, lengthening test periods and increasing monitoring technique sensitivity and reliability appear to alleviate estrous related changes in spontaneous behavior. Variation in activity level with estrous cycle stage, therefore, does not appear to be a constant behavioral p h e n o m e n o n precluding the use of females in studies of behavioral toxicity.

REFERENCES

I. Ball, J. The female sex cycle as a factor in learning in the rat. Am. J. Physiol. 78: 533-536, 1926. 2. Barnett, S. The Rat: A Study in Behavior. Chicago, I11: Aldine Publishing Co., 1963. 3. Barnett, S. A. and I. M. McEwan. Movements of virgin, pregnant and lactating mice in a residential maze. Physiol. Behav. 10: 741-746, 1973. 4. Birke, L. I. A., R. J. Andrew and S. M. Best. Distractibility changes during the oestrous cycle of the rat. Anim. Behav. 27: 597-601, 1979. 5. Birke, L. I. A. and J. Archer. Open-field behavior of oestrous and dioestrous rats: evidence against an 'emotionality' interpretation. Anita. Behav. 23: 509-512, 1975. 6. Bolles, R. C. A failure to find evidence of the estrous cycle in the rat's activity level. Psychol. Rep. 12: 530, 1963. 7. Burke, A. W. and P. L. Broadhurst. Behavioral correlates of the oestrous cycle in the rat. Nature 209: 223-224, 1966. 8. Drewett, R. F. Oestrous and dioestrous components of the ovarian inhibition on hunger in the rat. Anita. Behav. 21: 772-780, 1973. 9. Finger, F. W. Estrous activity as a function of measuring device. J. comp. physiol. Psychol. 54: 524-526, 1961. 10. Finger, F. W. Estrus and general activity in the rat. J. comp. physiol. Psychol. 68: 461-466, 1%9. 11. Gray, P. Effect of the estrous cycle on conditioned avoidance in mice. Hormones Behav. 8: 235-241, 1977. 12. Gray, P. Correlation between estrus and reduced light avoidance in mice. Hormones Behav. 10: 277-284, 1978. 13. Gross, C. G. General Activity. In: Analysis o f Behaviorul Change, edited by L. Weiskrantz, New York: Harper and Row, 1968, pp. 91-106. 14. Guttman, R., I. Lieblich and R. Gross. Behavioral correlates of estrous cycle stages in laboratory mice. Behav. Biol. 13: 127132, 1975. 15. Hyde, J. S. and T. F. Sawyer. Estrous cycle fluctuations in aggressiveness of house mice. Hormones Behav. 9: 290-295, 1977. 16. Kostas, J., D. J. McFarland and W. G. Drew. Lead-induced hyperactivity. Chronic exposure during the neonatal period in the rat. Pharmacology 14: 435-442, 1976.

17. Marriott, A. S. The effects of amphetamine, caffeine and methylphenidate on the locomotor activity of rats in an unfamiliar environment. Int. J. Neuropharm. 7: 487-491, 1968. 18. Martin, J. R. and K. B/ittig. Exploratory behaviour of rats at oestrus. Anita. Behav. 28: 900-905, 1980. 19. Mullenix, P. Effect of lead on spontaneous behavior. In: Clinical Implications o f Low Level Lead Exposure, edited by H. Needleman. New York: Raven Press, 1980, pp. 211-220. 20. Nielson, H. C. , A. H. Mclver and R. S. Boswell. Effect of septal lesions on learning, emotionality, activity, and exploratory behavior in rats. Expl. Neurol. ll: 147-157, 1965. 21. Norton, S. Photographic analysis of rat behavior before and after amygdaloid lesions. Brain Res. 18: 477-490, 1970. 22. Norton, S. Amphetamine as a model for hyperactivity in the rat. Physiol Behav. 11: 181-186, 1973. 23. Norton, S. Amphetamine as a model for hyperactivity in the rat. Physiol. Behav. 11: 181-186, 1973. 24. Norton, S. The structure of behavior of rats during morphineinduced hyperactivity. Communs. Psychopharmac. 1: 333-341, 1977. 25. Norton, S., P. Mullenix and B. Culver. Comparison of the structure of hyperactive behavior in rats after brain damage from x-irradiation, carbon monoxide and pallidal lesions. Brain Res. 116: 49-67, 1976. 26. Slonaker, J. R. The effect of pubescence, oestruation and menopause on the voluntary activity in the albino rat. Am. J. Physiol. 68: 294--315, 1924. 27. Treichler, F. R. and J. F. Hall. The relationship between deprivation weight loss and several measures of activity. J. comp. physiol. Psychol. 55: 346-349, 1%2. 28. Wang, G. H. The relation between "spontaneous" activity and oestrous cycle in the white rat. Comp. Psychol. Monogr. 2: 1-27, 1923. 29. Weasner, M. H., F. W. Finger and L. S. Reid. Activity changes under food deprivation as a function of recording device. J. comp. physiol. Psychol. 53: 470-474, 1%0. 30. Zarrow, M., J. Yochim and J. McCarthy. The estrogens. In: Experimental Endocrinology. A Sourcebook o f Basic Techniques, edited by M. Zarrow, New York: Academic Press, 1964, pp. 17-64.