2J Inbred Mice

Physiology & Behavior, Vol. 67, No. 4, pp. 599–605, 1999 © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/99/$–see front matter

PII S0031-9384(99)00109-2

Maternal Behavior in Female C57BL/6J and DBA/2J Inbred Mice RICHARD E. BROWN,* W. BRUCE MATHIESON,† JENNIFER STAPLETON*† AND PAUL E. NEUMANN†1 Departments of *Psychology and †Anatomy & Neurobiology, Faculty of Medicine, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, N.S., Canada B3H 4H7 Received 18 November 1998; Accepted 22 May 1999 BROWN, R. E., W. B. MATHIESON, J. STAPLETON AND P. E. NEUMANN. Maternal behavior in female C57BL/6J and DBA/2J inbred mice. PHYSIOL BEHAV 67(4) 599–605, 1999.—Inbred strains of mice exhibit different patterns of maternal behavior, providing material for studies of genetic influences on the expression of maternal behavior. Beginning 1 day after birth, maternal behavior was recorded daily for 14 days in the first and second litters of C57BL/6J (B6) and DBA/2J (D2) mothers. D2 mice had higher pup survival than B6 mice, and pup survival was higher in both strains in second litters than in first litters. D2 mothers spent more time engaged in maternal behavior, especially resting with, crouching over, and nursing pups than B6 mothers with first litters, but not with second litters. Not all measures of maternal behavior were correlated with pup survival; with both litters, B6 mothers retrieved pups faster than D2 mothers. © 1999 Elsevier Science Inc. Maternal behavior

C57BL/6J mice

DBA/2J mice

that B6 mothers are better at retrieving pups than D2 mothers because they can hear the distress vocalizations of the pups outside of the nest and quickly locate them (5). Some of these behavioral differences may be related to neuroanatomic differences between B6 and D2 mice. The medial preoptic area (MPOA) of D2 mice has a pars compacta (MPOpc), which is absent in B6 mice (25). The MPOpc of D2 mice contains galanin-immunoreactive and nitric oxide-containing neurons (demonstrated by NADPH diaphorase activity) (15). The involvement of the MPOA in control of maternal behavior has been demonstrated in rats. Neuronal activity in the MPOA increases in female rats exhibiting maternal behavior (9,10). Lesions of the MPOA and anterior hypothalamus, or surgical interruption of their afferent and efferent connections, disrupt male and female maternal behavior in rats (12,13,16,18,20–24,26). Because of these neuroanatomic and maternal behavior differences between B6 and D2 mice, we performed a systematic analysis of maternal behaviors in B6 and D2 females with their first and second litters. This detailed analysis of maternal behavior in these parental strains is a prerequisite for the genetic study of the inheritance of the morphologic and behavioral traits in offspring of B6 and D2 mice.

MATERNAL behavior is regulated by complex interactions between the central nervous and endocrine systems, and sensory stimuli from the offspring and environment (31). The underlying biological bases of maternal behavior are also influenced by genetic factors. Mutant mice display various disorders of maternal behavior (3,34), and inbred strains of mice show different patterns of maternal behavior (4,8,35). In this article we compare maternal behavior in DBA/2J (D2) and C57BL/ 6J (B6) inbred mice, two strains that are extensively utilized in experiments on the genetic basis of behavior (6,29). A number of differences in maternal behavior have been reported in these two mouse strains. B6 females have larger first litters than D2 females (4,11,17,36), but D2 mothers have a higher survival rate in their first litters than B6 mothers (17). Pregnant D2 females build larger and more complete nests than pregnant B6 females (1,2). Adult male and nulliparous female B6 mice are more likely to show infanticide than D2 mice, which are more likely to ignore or retrieve pups (14, 32,33). B6 females are quicker than D2 females at locating, touching, and retrieving pups to the nest (5). B6 mothers can hear ultrasonic vocalizations emitted by their pups, but D2 mothers cannot hear in that frequency, and their pups emit few ultrasonic vocalizations (27). Thus, it has been proposed

1To

whom requests for reprints should be addressed. E-mail: [email protected]

599

600

BROWN ET AL.

FIG. 1. Cumulative percentage of pups surviving from birth to weaning in the first and second litters of DBA/2J (D2) and C57BL/6J (B6) females. FIG. 2. Mean pup weights (6SEM) in the first and second litters of DBA/2J and C57BL/6J females. MATERIALS AND METHODS

Subjects Pup survival was recorded in first and second litters of 25 female C57BL/6J (B6) mice and 24 female DBA/2J (D2) mice. Parental behavior was observed in 7 B6 and 9 D2 females. These mice were the descendants of mice purchased from The Jackson Laboratories (Bar Harbor, ME) and bred in the Animal Care Facility of the Faculty of Medicine, Dalhousie University. Females were housed singly in 28 3 12 3 16-cm Plexiglas cages with Micro-Barrier tops. The cages were lined with Pro-Chip shavings, and sterilized pine shavings were provided for nest material. The colony room was 21 6 28C, and kept on a 12:12-h reversed light/dark cycle, with lights on at 1900 h and off at 0700 h. Food (Prolab RMH 3000, PMI Nutrition International, Richmond, IN) and water were given ad lib. All procedures were carried out in accordance with the guidelines established by the Canadian Council on Animal Care, and were approved by the Dalhousie University Committee on Laboratory Animals.

Mating Protocol All mice were mated at 2–3 months of age, and again after their first litters were weaned. Males were removed from the cages after 2 weeks. Shortly before and after parturition, each cage was checked every 4 h, from 0900–2100 h, for newborn pups to establish the date of birth and litter size. Subsequently, litter size and weight were recorded each day at the same time (1000 h) until weaning. Behavioral Observations Behavioral observations began when pups were 1 day of age and continued for 14 consecutive days. Behavioral observations began 3 and 9 h after lights went out, and observations of the mothers were counterbalanced between the two sessions such that mothers observed during hour 4 of the dark cycle 1 day were observed during hour 10 on the next day and

TABLE 1 RESULTS OF THE ANALYSES OF VARIANCE OF MATERNAL BEHAVIORS WITH FIRST LITTERS A. Undisturbed Litter Mean (14 Days)

Behavior

Total periods of parental behavior Total periods of nonparental behavior Nest building Retrieving pups Crouching 1 nursing Sniffing pups Licking pups

B6 D2 B6 D2 B6 D2 B6 D2 B6 D2 B6 D2 B6 D2

*p , 0.05; †p , 0.01; ‡p , 0.001.

25.96 37.51 37.07 25.67 3.75 3.4 0.13 0.01 39.83 61.84 0.24 0.21 3.69 3.22

Strain F (1, 13)

Day F(13, 169)

B. Following Pup Removal and Replacement Strain 3 Day F (13 , 169)

7.24*

1.1

1.26

6.92*

1.26

1.22

,1.0

1.82*

,1.0

5.85*

2.06*

1.5

5.55*

1.55

1.55

,1.0

1.49

,1.0

,1.0

1.12

,1.0

Mean (14 Days)

39.67 39.12 21.54 20.66 21.45 14.06 0.66 0.32 33.94 45.42 0.91 1.93 7 8.52

Strain F (1, 13)

Day F(13, 169)

Strain 3 Day F (13, 169)

,1.0

,1.0

,1.0

,1.0

,1.0

,1.0

17.17†

9.73‡

,1.0

7.2*

1.3

2.36†

2.55

3.44‡

,1.0

9.51†

1.21

,1.0

1.68

3.67‡

,1.0

MATERNAL BEHAVIOR IN C57BL/6J AND DBA/2J MICE

601

FIG. 3. Maternal behavior with litter 1. Number of periods per day that undisturbed lactating female C57BL/6J and DBA/2J mice were observed (A) nest building, (B) retrieving pups, and (C) crouching over and nursing pups, and that they were observed (D) nest building, (E) retrieving pups, and (F) crouching over and nursing pups following pup removal for 1 h. The line graphs represent mean (6SEM) number of periods per day for each day, and bar graphs represent the mean (6SEM) per day over the entire 14-day period. *p , 0.05; **p , 0.01.

vice versa. During the observation periods in the dark phase of the daily cycle, the housing room was illuminated with red light. Mothers were observed with their litters for 10 min per day, and their behavior was scored every 10 s using a time sampling procedure (undisturbed behavior). The pups were then removed from their home cage. One hour later, four pups were placed at the end of the cage farthest away from the nest, and the remaining pups were placed in the nest. The observer then recorded the behavior of the mother every 10 s for a second 10-min period and the time (latency, in seconds) for the mother to retrieve each of the four pups to the nest. Seven parental behaviors and four nonparental behaviors

were recorded. The parental behaviors were: 1) nest building (carrying or pushing nest material towards nest or manipulating already incorporated nest material; 2) sniffing pups (touching pups with nose and sniffing); 3) licking pups (licking any part of pup’s body); 4) resting with pups (inactive, but in contact with a part of her body other than the tail to the body of at least one pup); 5) crouching over pups (assuming the arched-back nursing position over the pups); 6) nursing (sucking at least one pup); and 7) retrieving (carrying or pushing a pup to the nest by taking a part of the pup’s body into her mouth). The nonparental behaviors recorded were: 1) resting alone (inactivity without contacting any pups); 2) self-grooming (wiping, licking, or scratching of the dam’s own body); 3)

602

BROWN ET AL. TABLE 2 RESULTS OF THE ANALYSES OF VARIANCE OF MATERNAL BEHAVIORS WITH SECOND LITTERS A. Undisturbed Litter

Behavior

Total periods of parental behavior Total periods of nonparental behavior Nest building Retrieving pups Crouching 1 nursing Sniffing pups Licking pups

B6 D2 B6 D2 B6 D2 B6 D2 B6 D2 B6 D2 B6 D2

B. Following Pup Removal and Replacement Strain 3 Day F (13, 156)

Mean (14 Days)

Strain F (1, 12)

Day F(13, 156)

27.63 29.92 35.1 32.51 1.9 1.58 0.48 0 49.79 53.55 0.857 0.866 3.99 3.19

,1.0

2.13*

1.44

,1.0

1.98*

1.36

,1.0

3.55‡

3.43

Mean (14 Days)

,1.0

1.2

1.2

,1.0

1.8*

1.12

,1.0

1.35

1.03

,1.0

1.43

1.5

37.48 35.33 23.66 26.12 11.25 6.51 3.15 1.7 39.28 38.89 2.2 3.5 6.31 8.43

Strain F (1, 12)

Day F(13, 156)

Strain 3 Day F (13, 156)

,1.0

1.6

1.01

,1.0

2.24†

1.12 ,1.0

9.14*

9.64‡

27.12‡

14.18‡

4.46‡

10.27‡

2.17*

,1.0 4.99*

1.09

3.52

7.37‡

1.4 ,1.0

*p , 0.05; †p , 0.01; ‡p , 0.001.

feeding (ingestion of food or water); and 4) active (any activity such as climbing on bars, running or sniffing air, which was not assigned to another category). For each 10-s interval every behavioral occurrence was noted; thus, one or more behaviors could be scored in each interval. For analysis, the total number of observation periods (out of 60) in which one or more parental behaviors was recorded, and the total number of periods in which one or more nonparental behaviors occurred, were tabulated. The number of periods in which nest building, pup retrieval, sniffing, and licking pups were recorded was also compared. Finally, the sum of the scores for resting with pups, crouching over pups and nursing was used as a “crouching 1 nursing” score. Each of these behaviors and the latencies to retrieve each of the four pups, over the 14 days, were analyzed by split plot (between/ within) ANOVA.

RESULTS

Pup Survival B6 mice bore an average of 6.8 (163/24) pups in their first litters. At weaning, 64% (105/163) of these pups were alive (Fig. 1). All but three of the deaths occurred before postnatal day 3. D2 mice bore an average of 4.8 (121/25) pups in their first litters. At 5 days post partum, 87% (105/121) of these pups were alive, all of which survived to weaning. Thus, the B6 mice had larger litters (Mann–Whitney U-test, p , 0.005) and higher pup mortality, x2(1) 5 18.0, p 5 0.0002, than the D2 mice in their first litters. In their second litters, the B6 mice gave birth to an average of 8.8 (211/24) pups per litter. At weaning, 91% (192/211) of these pups were alive. All but two of the deaths occurred before postnatal day 3. D2 second litters had an average of 5.4 (135/25) pups. At postnatal day 3, 98% (132/135) of these pups were alive, all of which survived to weaning. Thus, B6 mice also had larger litters (Mann–Whitney U-test, p , 0.0001) and higher pup mortality, x2(1) 5 6.4, p 5 0.012, than the D2 mice in their second litters, and both strains had higher

pup survival in their second litters [B6: x2(1) 5 39.7, p 5 0.00001; D2: x2(1) 5 11.2, p 5 0.0008]. Pup Weight Gain D2 pups displayed greater weight gain than B6 pups in both first and second litters (Fig. 2), but the difference was only significant in the second litters (t-test, p , 0.01). Pup weight gain was greater in second litters than in first litters in both B6 and D2 mice, but only significantly so in D2 mice (t-test, p , 0.05). Maternal Behavior with Litter 1 With their first litters, D2 mothers showed significantly more periods of maternal behavior whereas B6 mothers displayed more periods of nonparental behavior (Table 1A). When individual parental behaviors were analyzed, however, B6 mothers displayed more of some of these behaviors than D2 mothers. There was no strain difference in periods of nest building, but there was a significant decline in nest building over days (Fig. 3A). B6 mothers showed significantly more pup retrieval than D2 mothers did. Although there was a significant day effect, this showed no specific trend (Fig. 3B). D2 females spent more time resting with, crouching over, and nursing pups than B6 mothers (Fig. 3C). There were no significant strain or day effects on the number of periods that D2 and B6 mothers were observed sniffing and licking pups. After the mothers were disturbed by having their pups removed for an hour and then replaced, there were no strain differences or day effects in the total number of periods of maternal or nonparental behavior with the first litter (Table 1B). B6 mice showed more periods of nest building and pup retrieval than D2 mice did (Fig. 3D and E). Nest building decreased over days, and pup retrieval showed significant strain 3 day interaction effects. There were no significant strain differences in the frequency of resting with, crouching over, and nursing pups, but their frequency increased over days (Fig. 3F). D2 mothers sniffed their pups more after they were returned than B6 mothers did, but there were no strain differ-

MATERNAL BEHAVIOR IN C57BL/6J AND DBA/2J MICE

603

FIG. 4. Maternal behavior with litter 2. Number of periods per day that undisturbed lactating female C57BL/6J and DBA/2J mice were observed (A) nest building, (B) retrieving pups, and (C) crouching over and nursing pups, and that they were observed (D) nest building, (E) retrieving pups, and (F) crouching over and nursing pups following pup removal for 1 h. The line graphs represent mean (6SEM) number of periods per day for each day, and bar graphs represent the mean (6SEM) per day over the entire 14-day period. *p , 0.05; ***p , 0.001.

ences in the number of periods that mothers were observed licking pups. Pup licking by both B6 and D2 mothers increased over days. Maternal Behavior with Litter 2 During the observations of the undisturbed second litters, there were no significant strain differences in the number of periods of parental or nonparental behaviors (Table 2A), nor were there any strain differences in nest building, crouching 1 nursing, sniffing, or licking pups (Fig. 4A, B, and C). B6 mothers showed more periods of pup retrieval than D6 mothers

did, but this was not significant. Parental behaviors, in particular nest building, decreased over days, as nonparental behaviors increased. There was also a significant day effect on crouching 1 nursing, but there was no specific trend. Following removal and replacement of the second litters, the total number of observation periods with parental behavior did not differ between strains (Table 2B). Nonparental behaviors of both strains decreased over days. B6 mothers were observed nest building more than D2 mothers were, and there was a decline in nest building over days (Fig. 4D). B6 mothers retrieved pups more frequently than D2 mothers, and the frequency of pup retrieval declined over days (Fig. 4E). There

604

BROWN ET AL.

was no strain difference in the number of periods that mothers spent resting with, crouching over, and nursing pups, but the frequency of these behaviors increased over days (Fig. 4F). D2 mothers spent more periods sniffing pups than B6 mothers did. Pup licking by both B6 and D2 mothers increased over days. Significant strain 3 day interaction effects were noted in pup retrieval and crouching 1 nursing. Pup Retrieval Tests With their first litters, B6 mothers retrieved the first three of the four pups to the nest significantly faster than D2 mothers (Table 3A). With their second litters, B6 mothers retrieved all four pups faster than D2 mothers (Table 3B). With both litters, the retrieval times increased for both strains as the pups aged (data not shown). DISCUSSION

Our results confirm that there are significant differences in maternal behavior between B6 and D2 strains of inbred mice. B6 litters were larger and had significantly higher pup mortality than D2 litters, with more pup deaths in the first than second litters, and most deaths occurring within the first 3 days after parturition. Pup weight gain during the experimental period was greater in D2 mice in both first and second litters. These results suggest delayed initiation of maternal behavior in B6 females; however, we do not know if pup death is due to infanticide, failure of mothers to lactate or to nurse, or failure of pups to suckle. Two test situations were used to measure maternal behavior: observation of undisturbed mothers, and observation of mothers aroused by the removal and replacement of their

pups. It is clear from comparing the before and after observations in Figs. 3 and 4 that removal and replacement of pups stimulated pup retrieval and nest building, and depressed crouching over and nursing pups. In both the undisturbed and disturbed conditions, however, the patterns of strain differences were the same. The B6 mothers showed more “self-activated” maternal behavior (nest building and pup retrieval) than D2 mothers. During the pup retrieval tests, the B6 mothers retrieved both the first and second litters of pups to the nest faster than D2 mothers. D2 mothers showed more resting with, crouching over, and nursing pups than B6 mothers with the first litter but not with the second litter. Many of these strain differences in behavior were observed during the first 5 days after parturition, which was the period of highest pup mortality, and during the rest of the observation days. Our results are consistent with results from other laboratories cited in the introduction, with the possible exception of nest building. Broida and Svare (1,2) reported that D2 females surpassed B6 females in pregnancy-induced nest building, as measured by the size and form of the nest, whereas we found that B6 females spent more time nest building after the removal and replacement of their pups. We are conducting further studies of nest building in B6 and D2 mice to better characterize the strain differences before and after parturition. The longer pup retrieval latencies of D2 mice contradict the generally held impression that shorter pup retrieval latencies in rodents represent better parental behavior. The better performance of B6 females in “active” maternal behavior (nest building and pup retrieval) and of D2 females in “passive” maternal behavior (resting with, crouching over, and nursing pups) parallels the differences between these strains in various learning tasks. B6 mice are better learners in active

TABLE 3 RESULTS OF THE ANALYSES OF VARIANCE OF RETRIEVAL LATENCIES A. Litter 1 Day F (13, 169)

Strain 3 Day F (13, 169)

Pup

Strain

Mean Latency (in seconds)

Strain F (1, 13)

First

B6 D2 B6 D2 B6 D2 B6 D2

46.56 248.01 76.06 293.33 112.7 378.73 324.41 458.65

27.77‡

7‡

3.47‡

37.27‡

7.7‡

2.26†

27.39‡

5.14‡

,1.0

2.84

3.23‡

,1.0

Pup

Strain

Mean Latency (in seconds)

Strain F (1, 12)

Day F (13, 156)

Strain 3 Day F (13, 156)

First

B6 D2 B6 D2 B6 D2 B6 D2

89.08 277.59 126.05 348.21 177.81 415.8 256.83 457.71

29.44‡

15.28‡

3.28‡

60.01‡

10.11‡

2.18*

39.04‡

12.05‡

2.73†

17.58‡

8.93‡

1.86*

Second Third Fourth B. Litter 2

Second Third Fourth

*p , 0.05; †p , 0.01; ‡p , 0.001.

MATERNAL BEHAVIOR IN C57BL/6J AND DBA/2J MICE avoidance paradigms (30) whereas D2 mice perform better in passive avoidance tests (28). B6 mice are also more active in open field exploration (7,35) and the forced swimming test (19). Thus, some of the differences in maternal behavior and learning performance between B6 and D2 mice may by due to differences in activity levels.

605 ACKNOWLEDGEMENTS

This research was supported by NSERC of Canada, Grant A7441 to R.E.B., and by MRC of Canada, Grant MA14218 to P.E.N. We would like to thank Allison Clarke, Darshaka Malaviarachchi, and Natasha Paul for help with behavioral observations.

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18. Nance, D. M.; Christensen, L. W.; Shryne, J. E.; Gorski, R. A.: Modifications in gonadotropin control and reproductive behavior in the female rat by hypothalamic and preoptic lesions. Brain Res. Bull. 2:307–312; 1977. 19. Nikulina, E. M.; Skrinskaya, J. A.; Popova, N. K.: Role of genotype and dopamine receptors in behavior of inbred mice in a forced swimming test. Psychopharmacology (Berlin) 105:525– 529; 1991. 20. Numan, M.: Maternal behavior. In: Knobil, E.; Neill, J. D., eds. The physiology of reproduction, 2nd ed. New York: Raven Press; 1994;221–302. 21. Numan, M.; Callahan, E. C.: The connections of the medial preoptic region and maternal behavior in the female rat. J. Comp. Physiol. Psychol. 87:746–759; 1980. 22. Numan, M.; Corodimas, K. P.; Numan, M. J.; Factor, E. M.; Piers, W. D.: Axon-sparing lesions of the preoptic region and substantia innominata disrupt maternal behavior in rats. Behav. Neurosci. 102:381–96; 1988. 23. Numan, M.; Rosenblatt, J. S.; Komisaruk, B. R.: Medial preoptic area and the onset of maternal behavior in the rat. J. Comp. Physiol. Psychol. 91:146–164; 1977. 24. Numan, M.; Sheehan, T. P.: Neuroanatomical circuitry for mammalian maternal behavior. Ann. NY Acad. Sci. 807:101–125; 1997. 25. Robinson, S. M.; Fox, T. O.; Sidman, R. L.: A genetic variant in the morphology of the medial preoptic area in mice. J. Neurogenet. 2:381–388; 1985. 26. Rosenblatt, J. S.; Hazelwood, S.; Poole, J.: Maternal behavior in male rats: Effects of medial preoptic area lesions and presence of maternal aggression. Horm. Behav. 30:201–215; 1996. 27. Roubertoux, P. L.; Martin, B.; Le Roy, I.; Beau, J.; Marchaland, C.; Perez–Diaz, F.; Cohen–Salmon, C.; Carlier, M.: Vocalizations in newborn mice: A genetic analysis. Behav. Genet. 26:427–437; 1996. 28. Sprott, R. L.: Passive-avoidance performance in mice: Evidence for a single locus inheritance. Behav. Biol. 11:231–237; 1974. 29. Sprott, R. L.: Behavioral characteristics of C57BL/6J, DBA/2 and B6D2F1 mice which are potentially useful for gerontological research. Exp. Aging Res. 1:313–323; 1975. 30. Stavnes, K. L.; Sprott, R. L.: Genetic analysis of active avoidance performance in mice. Psychol. Rep. 36:515–521; 1975. 31. Stern, J. M.: Maternal behavior: Sensory, hormonal, and neural determinants. In: Brush, F. R.; Levine, S., eds. Psychoendocrinology. New York: Academic Press; 1989:105–226. 32. Svare, B.; Broida, J.: Genotypic influences on infanticide in mice: Environmental, situational and experiential determinants. Physiol. Behav. 28:171–175; 1982. 33. Svare, B.; Kinsley, C.; Mann, M. A.; Broida, J.: Infanticide: Accounting for genetic variation in mice. Physiol. Behav. 33:137– 152; 1984. 34. Thomas, S. A.; Palmiter, R. D.: Impaired maternal behavior in mice lacking norepinephrine and epinephrine. Cell 91:584–592; 1997. 35. Thompson, W. R.: The inheritance of behaviour: Behavioural differences in fifteen strains. Can. J. Psychol. 7:145–155; 1953. 36. Verley, F. A.; Grahn, D.; Leslie, W. P.; Hamilton, K. F.: Sex ratio of mice as possible indicator of mutation rate for sex-linked lethals. J. Hered. 58:285–290; 1967.