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Olfactory-mediated choice behavior in mice: Developmental and genetic aspects
BEHAVIORAL BIOLOGY 19, 324-332 (1977), Abstract No. 6170
Olfactory-Mediated Choice Behavior in Mice" Developmental and Genetic Aspects 1 MARVIN A . KOSK[, L I N D A K . DIXON, AND N E L L FAHRION
University o f Colorado at Denver, 1100 14th Street, Denver, Colorado 80202 Two experiments investigating the developmental and genetic aspects of an olfactory-guided choice behavior were completed. In the first experiment, the age at which a mouse pup responded to a container scented by the mother's odor was determined. Random-bred Swiss-Webster pups were tested from 3 to 12 days of age. Pups 9 to 12 days of age spent significantly more time exploring a container odorized by the scent of the mother than 3-day-old pups. In the second experiment, differences in olfactory-guided choice behavior among genetically different groups of mice were investigated. Significant differences in the development of the behavior were found when the T(5;13)264Ca translocation group carrying the W~ marker was compared with three other groups: one carrying the marker only and two nonmarker control groups. It is possible that the difference is due to the position of the translocated material, since no differences were found between the marker group and the two nonmarker control groups.
Behavioral studies investigating neonatal olfactory-guided behavioral development in mice have been lacking, although a behavioral response of mice to odors associated with stress has been reported (Carr, Martorano, and Krames, 1970). Rat pups discriminate and prefer home cage shaving odor to other odors as early as 12 days of age (Gregory and Pfaff, 1971). Home cage odors were preferred by hamster pups at 7 to 8 days of age; this preference declined at 9 days of age (Devor and Schneider, 1974). These authors attributed the decline in preference for home cage odor to a rise in exploratory and play behavior as well as to a lengthening of the olfactory tether. Leon and Moltz (1972) demonstrated that lactating female rats emit a pheromone to which preweanling pups are attracted. Their results indicated that the emission of the pheromone coincided with the age at which the pups are first attracted to the pheromone. Fox (1965) carried out an extensive study of the behavioral development of the mouse. His investigation of the development of sensory faculties did not include olfaction. Fuller and Wimer (1966) point out that the primary receptors for the mouse are olfactory and tactual. Since the mouse cerebral volume consists largely of olfactory bulb, and since behavioral studies often control for olfactory variables, an investigation of We wish to thank Dr. Janis W. Driscoll and Dr. James R. Wilson for their helpful comments during the preparation of this manuscript. 324 Copyright (~) 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0091-6773
OLFACTORY-MEDIATED CHOICE BEHAVIOR
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the development of behavior guided by olfaction should provide useful information. In this paper we: (1) examine and describe the development of an olfactory-guided choice behavior in a random-bred strain of mouse pups, and (2) compare the development of this behavior in genetically different groups of mice. EXPERIMENT 1
Method Subjects. Ninety-nine mouse pups (48 males, 51 females) born in 10 litters to adult multiparous female Swiss-Webster mice (Simonsen Labs, Calif.) were utilized in this study. When pregnancy of females was confirmed, they were transferred to polypropylene cages 13 × 29 x 9 cm with 1.27-cm galvanized wire-mesh lids. The solid bottom floor was covered with SAN-I-CEL bedding material. Water and Purina Mouse Chow were provided ad libitum. The cages of pregnant mice were checked at 8:00 AM daily for the presence of new litters. Two days after parturition, litters were culled to 10, and the individual pups were weighed. Litter sizes before culling ranged from 9 to 17 pups. Testing of mouse pups began on the third day. The animal colony was temperature controlled and maintained on a 12 Light/12 Dark cycle, with light onset at 3:00 AM. Testing was conducted during the light cycle. Apparatus. The test apparatus consisted of polypropylene cages modified to an interior bedding area of 13 x 16.5 cm. In the middle of each lengthwise wall was a circular opening 8.2 cm in diameter and 1 cm from the floor. The 1-cm depth from the bottom of the circular opening to the apparatus floor accommodated fresh bedding material. The 8.2-cm diameter opening accommodated the scented and nonscented containers. These containers were constructed of 7.9-cm (outside diameter) cardboard tubes, 7.4 cm long, closed at one end with ~-in. wire mesh and ½ in. of compressed insulation-grade Fiberglass. The interior of each container was 7.5 cm in diameter and 6.7 cm long. Procedure. Mouse pups were tested on the third through twelfth days after litter detection, since observation had indicated that mouse pups are capable of moving several centimeters by Day 3, although cage exploration is not a dominant behavior until several days later, Five of the ten testing days were randomly selected for testing of the ten pups per litter, except that no 2 of the 5 days were the same. A maximum of 10 pups was tested at each age. On each of the 5 testing days, 2 of the 10 pups were tested in separate apparatuses. Each pup was tested only once during the entire experiment. The pup was tested, weighed, sexed, earmarked with fingernail clippers for identification in order not to retest pups, and returned to the home cage. Sex of pups was rechecked on the thirteenth day.
326
KOSKI, DIXON, AND FAHRION
During the light cycle of each testing day, the mouse mother was removed from the home cage and housed for 30 min in two joined scenting containers. During this period the cardboard containers became imbued with the urine and feces of the mother. It is a basic assumption that approach tendencies to these odors will reflect the onset of an olfactoryguided choice behavior. The number of days that the pups' mother had been lactating coincided with the age of the mouse pup; therefore, the extent of the mother's influence in the olfactory-guided choice behavior of the pup is not measured in this experiment. After the 30-min container scenting period, each of the two mouse pups was placed in the middle of the interior bedding area of its test apparatus for a 3-rain period. In each of the apparatuses, one of the circular containers was scented with the mother's odor; the other container was a nonscented control. Scented and nonscented containers were randomly placed in the lateral positions in the testing apparatus. The pup's test-starting point was always between the two containers. The amount of time that each pup spent exploring (at least a nose inserted into) each container was recorded by means of a timer.
Results Ninety-nine scores were obtained for exploration of the scented and nonscented containers from the 99 mouse pups tested. The 10 scores for each day are represented by 10 mouse pups (except for Day 4, in which only nine pups were tested)from five different litters. In order to eliminate zero scores, 0.25 was added to all scores. All scores were then transformed to a logarithmic function as suggested by Myers (1972, p. 77). Homogeneity of variance was achieved (c = 0.147). For simplification of analysis, a mean score for Day 4 was used for the missing score on that day. Analysis of variance revealed that exploration of the scented container changed significantly as a function of time [F (9, 99) = 3.88, P < 0.001]. Exploration of a nonscented container did not change significantly over time. To determine the age at which mice began to explore the scented container, planned orthogonal contrasts (Myers, 1972) between Day 3 and all other days were carried out. These tests indicated that olfaction significantly affected a choice behavior at 10 days of age IF (1, 9) = 7.96, P < 0.025). Figure 1 illustrates the mean percentage of time in exploration of scented and nonscented containers. EXPERIMENT 2
Method Subjects. One hundred seventy mice were used in this experiment, 87 from a translocation stock consisting of 37 translocation mice containing a dominant mutant marker (W~) and 50 of their nontranslocated
OLFACTORY-MEDIATED CHOICE BEHAVIOR 6C
Scented Container --
(n
327
- Non-scented Container
'7_50
o=
(J
"4 40 i,i
._~ 50 I.-
"6 20
>= 0
3
4
5
6
7
8
9
I0
II
12
Age in Days
FIG. 1. Mean percentage of time in exploration of scented and nonscented containers by Swiss-Webster mice.
nonmarked siblings, and 83 from a nontranslocation marker stock consisting of 42 mice with the W ~ marker and 41 of their nonmarked siblings. Foundation populations for this study were purchased from the Jackson Laboratories in Bar Harbor, Maine; these animals produced the animals tested in this study. The translocation stock consisted of 9 T(5;13)264Ca mice whose genotype was designated B6CBAF2-T264 Wv/++(N22 or N23) and 11 homozygous nonaffected normal siblings. The stock was maintained by breeding translocation mice to the Fj offspring of a cross between C57BL/6J and CBA animals. The offspring of such a cross consist of both translocation pups and nontranslocation pups. The animals we received were from the twenty-second and twenty-third successive crosses. These mice were chosen for a large scale developmental and adult behavior study because we wished to examine the behavioral properties of a translocation stock. Moreover, for this translocation stock easily obtainable control groups were available about which there were behavioral reports in the literature (i.e., Thiessen, Owen, and Whitsett, 1970 on W V marker). The generation of the T264 translocation is discussed by Carter, Lyon, and Phillips (1955). The T264Ca translocation was induced by X-irradiation in mature sperm carried in adult male mice. Males were of the CWX stock carrying marker W v and were mated to females of the CBA stock. Fertility was reduced in translocation heterozygotes. These authors also showed that the position of the translocation break for the T264 translocation relative to other markers in the linkage group is the lx-WV-chromosome break. The recombination rate between Wv and the
328
KOSKI, DIXON, AND FAHRION
translocation is 4%. The W v marker is known to produce a slight macrotic anemia; W v homozygotes have a more severe anemic condition, and many die shortly after birth. To maintain the translocation stock, a nonmarker animal from the translocation stock was mated with a translocated animal carrying the marker. Males and females were paired monogamously with the criterion of no brother-sister mating. The animals which were to constitute the marker stock came from six C57BL/6J-W~ animals and six CBA animals purchased from Jackson Laboratories. To obtain an F1 population, crosses were made between the C57BL animals carrying the marker and the CBA animals. Offspring from the F1 population were the tested marker stock. A nonmarker animal from the F1 was mated to another F~ animal which had the mutant marker, with the criterion of no brother-sister mating. All of the tested animals were the offspring of these crosses, and each litter consisted of markered and nonmarkered animals. All of the litters in the marker stock were culled to match the litter sizes of the translocation stock on the day after litter detection. During breeding, animals were kept in small polypropylene cages (29 x 13 × 9 cm high) containing about 3 cm of aspen woodshavings for bedding. The cage tops were constructed of 1.27-cm wire mesh. Standard Purina Mouse Chow and tap water were available ad libitum. The light cycle was 12 Light/12 Dark, with light onset at 10:00 AN. The day after litters were detected, parents and offspring were transferred to polypropylene animal cages (29.21 × 19.05 x 13.96 cm high) in which was placed about 3 cm of aspen woodshavings for bedding. Procedure. All of the 170 mouse pups used in this experiment were tested on a series of developmental parameters daily for 20 consecutive days following litter detection (Koski, 1975). The battery of developmental tests was adapted from Fox (1965), McClearn, Wilson, and Meredith (1970), and Wahlsten (1974). The developmental tests, observations, and measures consisted of 24 measures of spontaneous behavior, reflexologic responses, and standard developmental parameters. The olfactory-guided choice behavior described in this paper was a part of the developmental test battery, except that the olfactory test was an independent measure and not a repeated measure as were the rest of the developmental parameters. After being developmentally tested, each pup was isolated in a holding cage with aspen bedding material for at least 5 min prior to testing in the olfactory apparatus. The test apparatus for the testing of mouse pups was the same as that described in Expt i. Since Expt 1 indicated that the most rapid development of an olfactory-guided choice behavior occurred during Days 8 through 12, the mice in this experiment were tested only on those days. A testing day was randomly assigned to each pup in a litter. One or two pups from the same litter were tested on a
OLFACTORY-MEDIATED CHOICE BEHAVIOR
329
testing day depending on the random assignment. Each pup was tested only once during the entire experiment. The remainder of the testing procedure was the same as that described for E x p t 1, except that individual identification of the pups in this experiment was accomplished by carbol fuchsin stain markings on each animal's back before it was returned to the litter. These markings were renewed daily beginning with the first day of developmental testing. Results
The data were transformed to a logarithmic function as described in E x p t 1. H o m o g e n e i t y of variance was achieved (c = 0.327). The four groups of animals tested in this experiment were a translocation group with a mutant marker, a nontranslocation group with a mutant marker, and control groups of animals consisting of n o n m a r k e r siblings in each of the two groups. To determine if the two nonmarker sibling control groups differed, a 2 x 5 (control group condition by age) factorial analysis of variance was computed using scented container exploration time as the dependent variable. The main effect for age was significant [F (4, 81) = 8.46, P < 0.001]. There was no significant effect IF (1, 81) = 0.01, P > 0.75] due to the group from which the control animals were obtained, nor were there any interaction effects IF (4, 81) = 0.88, P > 0.50]. Accordingly, the two groups of nonmarker siblings were pooled into one control group for further analysis. The average percentage of time spent exploring a scented container is shown in Fig. 2. In a 3 x 5 (genetic condition by age) factorial analysis of variance for the time spent in exploration o f an odorized container, the main effects for genetic condition and days were significant IF (2, 155) -- 4.87, P < 0.01, and F (4, 155) = 8.15, P < 0.001, respectively]. A planned contrast between the translocation group and the control group was significant [F (1, 126) = 8.97, P < 0.005]. A simple effects test was computed to determine the age at which genetic condition differences existed. There was a significant difference at Day 10 between the translocation group and the control group IF (1, 15) = 5.57, P < 0.05]. The difference between the marker and control groups at Day 10 was nonsignificant IF (1, 25) = 0.12, P > 0.75].
DISCUSSION The development of olfactory-guided choice behavior can be related to the morphological changes which occur during mouse development. The eyes, nose, and ears of the mouse pup are not open at birth and are not functional until several days after birth. The development of behaviors associated with vision and hearing has been related to the morphological differentiation of the eyes and ears (Fox, 1965; McClearn, et al., 1970;
330
KOSKI, DIXON, AND FAHRION 70 c~
+ Translocafions o Markers • Siblings
~ 5O o 40
it. 3O
~ 2o
0 8
FIG. 2.
9 I0 Age in Days
II
12
Odorized container exploration for genetically different groups of mice.
Wahlston, 1974). In general, the morphological development begins before the onset of such behavioral responses as the visual placing response, the ear twitch, and the auditory startle. In our study, microscopic examination revealed that the nostrils of mouse pups are fully formed with the presence of mucosa by 5 to 8 days of age; a significant olfactory response is observable shortly thereafter. The development of olfaction in mouse pups can be compared to studies of hamsters (Devor and Schneider, 1974) and rats (Gregory and Pfaff, 1971) which show an age-dependent change in behavior associated with olfaction. In these studies, as in ours, the behavior of the pups was not independent of the influence of the odor of the lactating mother. It is not our intention to compare directly the behavioral responses of animals in Expts 1 and 2. These are genetically different groups of animals, and the experiments were conducted separately. However, a cursory comparison of Figs. 1 and 2 will reveal that the development of an olfactory-guided choice behavior in the two experiments was quite similar. There is a striking difference in the onset and development of olfactory-guided choice behavior between translocation animals and all other groups (nontranslocation siblings, marker animals, and nonmarker siblings). The animals in this experiment were part of an extensive developmental study; the handling and stress which are an inherent part of the developmental testing may have had some effect on the behavior of these animals. By postulating a genotype-developmental testing interac-
OLFACTORY-MEDIATED CHOICE BEHAVIOR
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tion for the development of this behavior, one could argue that the genetic groups were modified differentially. However, whatever conditions were affecting the behavior, the genotype of the mouse pup was intricately involved. Leon and Moltz (1972) have shown that a pheromone emitted by a lactating mother of the litter might explain the pup's attraction to the odor of a mother. These investigators measured responses on alternate days (omitting Days 11, 13, and 15) and were concerned primarily with the role of the lactating rat mother in her pup's development of olfaction. It may be that the lactating mouse also emits a pheromone before Day 10, but this would not explain the differences that occurred due to genetic condition, since translocation mice and their sibling controls were tested with the same mother. In this study, we were concerned primarily with measuring the development of an olfactory-guided choice behavior in the mouse pups and the genetic influences that might affect that behavior. Since lactating females of the translocation stock consisted of both translocated and nontranslocated animals, and both translocated and nontranslocated offspring were tested using odors of both genotypes of mothers, the differences found could not be due to differences in genotypes of lactating mothers. Thus, the differences between the translocation and other groups on Day 10 may have reflected differences in their sensitivity to the mother's cues regardless of the genotype of the mother. Although it might be argued that the difference between the nontranslocation and the translocation marker mice was due to different genetic backgrounds, it might also be argued that the difference was due to a position effect of the translocated material, since the two sibling control groups responded the same in the behavioral test. Ropartz (1968) has elaborated on the role ofolfaction and aggression. In this study we have provided a link between olfactory-guided choice behavior and genetic condition. Thus, the translocation stock used in this study, in addition to being useful for the study of the lactating mother's influence on her pup's behavior, would also be useful for studying the relationships among olfaction, aggression, and genetic condition. REFERENCES Carr, W. J., Martorano, R. D., and Krames, L. (1970). Responses of mice to odors associated with stress. J. Comp. Physiol. Psychol. 71, 223-228. Carter, T. C., Lyon, M. R., and Phillips, R. J. S. (1955). Gene-tagged chromosome translocations in eleven stocks of mice. J. Genet. 53, 154-166. Devor, M., and Schneider, G. E. (1974). Attraction to home cage odor in hamster pups: Specificity and changes with age. Behav. Biol. 10, 211-221. Fox, W. M. (1965). Reflex-ontogeny and behavioral development of the mouse. Anirn. Behav. 13, 234-241. Fuller, J. L., and Wimer, R. E. (1966). Neural, sensory, and motor functions. In E. L. Green (Ed.), "Biology of the Laboratory Mouse," pp. 609-628. New York: McGraw-Hill.
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Gregory, E. H., and Pfaff, D. W. (1971). Development of olfactory-guided behavior in infant rats. Physiol. Behav. 6, 573-576. Koski, M. A. (1975). "Behavioral Effects of The T(5;13)264Ca Translocation in Mice," 107 pp. Unpublished Masters Thesis. University of Colorado, Boulder. Leon, M., and Moltz, H. (1972). The development of the pheromonal bond in the albino rat. Physiol. Behav. 8, 683-686. McClearn, G, E., Wilson, J. R., and Meredith, W. (1970). The use of isogenic and heterogenic mouse stocks in behavior research. In G. Lindzey and D. D. Thiessen (Eds.), "Contributions to Behavior-Genetic Analysis--The Mouse as a Prototype." New York: Appleton-Century-Crofts. Myers, J. L. (1972). "Fundamentals of Experimental Design," 2nd ed. Boston: AUyn and Bacon. Ropartz, P. (1968). The relation between olfactory stimulation and aggressive behavior in mice. Anim. Behav. 16, 97-100. Thiessen, D. D., Owen, K., and Whitsett, M. (1970). Chromosome mapping of behavioral activities. In G. Lindzey and D. D. Thiessen (Eds.), "Contributions to BehaviorGenetic Analysis-The Mouse as a Prototype." New York: Appleton-Century-Crofts. Wahlsten, D. (1974). A developmental time scale for postnatal changes in brain and behavior of B6D2F2 mice. Brain Res. 72, 251-264.
Olfactory-Mediated Choice Behavior in Mice" Developmental and Genetic Aspects 1 MARVIN A . KOSK[, L I N D A K . DIXON, AND N E L L FAHRION
University o f Colorado at Denver, 1100 14th Street, Denver, Colorado 80202 Two experiments investigating the developmental and genetic aspects of an olfactory-guided choice behavior were completed. In the first experiment, the age at which a mouse pup responded to a container scented by the mother's odor was determined. Random-bred Swiss-Webster pups were tested from 3 to 12 days of age. Pups 9 to 12 days of age spent significantly more time exploring a container odorized by the scent of the mother than 3-day-old pups. In the second experiment, differences in olfactory-guided choice behavior among genetically different groups of mice were investigated. Significant differences in the development of the behavior were found when the T(5;13)264Ca translocation group carrying the W~ marker was compared with three other groups: one carrying the marker only and two nonmarker control groups. It is possible that the difference is due to the position of the translocated material, since no differences were found between the marker group and the two nonmarker control groups.
Behavioral studies investigating neonatal olfactory-guided behavioral development in mice have been lacking, although a behavioral response of mice to odors associated with stress has been reported (Carr, Martorano, and Krames, 1970). Rat pups discriminate and prefer home cage shaving odor to other odors as early as 12 days of age (Gregory and Pfaff, 1971). Home cage odors were preferred by hamster pups at 7 to 8 days of age; this preference declined at 9 days of age (Devor and Schneider, 1974). These authors attributed the decline in preference for home cage odor to a rise in exploratory and play behavior as well as to a lengthening of the olfactory tether. Leon and Moltz (1972) demonstrated that lactating female rats emit a pheromone to which preweanling pups are attracted. Their results indicated that the emission of the pheromone coincided with the age at which the pups are first attracted to the pheromone. Fox (1965) carried out an extensive study of the behavioral development of the mouse. His investigation of the development of sensory faculties did not include olfaction. Fuller and Wimer (1966) point out that the primary receptors for the mouse are olfactory and tactual. Since the mouse cerebral volume consists largely of olfactory bulb, and since behavioral studies often control for olfactory variables, an investigation of We wish to thank Dr. Janis W. Driscoll and Dr. James R. Wilson for their helpful comments during the preparation of this manuscript. 324 Copyright (~) 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0091-6773
OLFACTORY-MEDIATED CHOICE BEHAVIOR
325
the development of behavior guided by olfaction should provide useful information. In this paper we: (1) examine and describe the development of an olfactory-guided choice behavior in a random-bred strain of mouse pups, and (2) compare the development of this behavior in genetically different groups of mice. EXPERIMENT 1
Method Subjects. Ninety-nine mouse pups (48 males, 51 females) born in 10 litters to adult multiparous female Swiss-Webster mice (Simonsen Labs, Calif.) were utilized in this study. When pregnancy of females was confirmed, they were transferred to polypropylene cages 13 × 29 x 9 cm with 1.27-cm galvanized wire-mesh lids. The solid bottom floor was covered with SAN-I-CEL bedding material. Water and Purina Mouse Chow were provided ad libitum. The cages of pregnant mice were checked at 8:00 AM daily for the presence of new litters. Two days after parturition, litters were culled to 10, and the individual pups were weighed. Litter sizes before culling ranged from 9 to 17 pups. Testing of mouse pups began on the third day. The animal colony was temperature controlled and maintained on a 12 Light/12 Dark cycle, with light onset at 3:00 AM. Testing was conducted during the light cycle. Apparatus. The test apparatus consisted of polypropylene cages modified to an interior bedding area of 13 x 16.5 cm. In the middle of each lengthwise wall was a circular opening 8.2 cm in diameter and 1 cm from the floor. The 1-cm depth from the bottom of the circular opening to the apparatus floor accommodated fresh bedding material. The 8.2-cm diameter opening accommodated the scented and nonscented containers. These containers were constructed of 7.9-cm (outside diameter) cardboard tubes, 7.4 cm long, closed at one end with ~-in. wire mesh and ½ in. of compressed insulation-grade Fiberglass. The interior of each container was 7.5 cm in diameter and 6.7 cm long. Procedure. Mouse pups were tested on the third through twelfth days after litter detection, since observation had indicated that mouse pups are capable of moving several centimeters by Day 3, although cage exploration is not a dominant behavior until several days later, Five of the ten testing days were randomly selected for testing of the ten pups per litter, except that no 2 of the 5 days were the same. A maximum of 10 pups was tested at each age. On each of the 5 testing days, 2 of the 10 pups were tested in separate apparatuses. Each pup was tested only once during the entire experiment. The pup was tested, weighed, sexed, earmarked with fingernail clippers for identification in order not to retest pups, and returned to the home cage. Sex of pups was rechecked on the thirteenth day.
326
KOSKI, DIXON, AND FAHRION
During the light cycle of each testing day, the mouse mother was removed from the home cage and housed for 30 min in two joined scenting containers. During this period the cardboard containers became imbued with the urine and feces of the mother. It is a basic assumption that approach tendencies to these odors will reflect the onset of an olfactoryguided choice behavior. The number of days that the pups' mother had been lactating coincided with the age of the mouse pup; therefore, the extent of the mother's influence in the olfactory-guided choice behavior of the pup is not measured in this experiment. After the 30-min container scenting period, each of the two mouse pups was placed in the middle of the interior bedding area of its test apparatus for a 3-rain period. In each of the apparatuses, one of the circular containers was scented with the mother's odor; the other container was a nonscented control. Scented and nonscented containers were randomly placed in the lateral positions in the testing apparatus. The pup's test-starting point was always between the two containers. The amount of time that each pup spent exploring (at least a nose inserted into) each container was recorded by means of a timer.
Results Ninety-nine scores were obtained for exploration of the scented and nonscented containers from the 99 mouse pups tested. The 10 scores for each day are represented by 10 mouse pups (except for Day 4, in which only nine pups were tested)from five different litters. In order to eliminate zero scores, 0.25 was added to all scores. All scores were then transformed to a logarithmic function as suggested by Myers (1972, p. 77). Homogeneity of variance was achieved (c = 0.147). For simplification of analysis, a mean score for Day 4 was used for the missing score on that day. Analysis of variance revealed that exploration of the scented container changed significantly as a function of time [F (9, 99) = 3.88, P < 0.001]. Exploration of a nonscented container did not change significantly over time. To determine the age at which mice began to explore the scented container, planned orthogonal contrasts (Myers, 1972) between Day 3 and all other days were carried out. These tests indicated that olfaction significantly affected a choice behavior at 10 days of age IF (1, 9) = 7.96, P < 0.025). Figure 1 illustrates the mean percentage of time in exploration of scented and nonscented containers. EXPERIMENT 2
Method Subjects. One hundred seventy mice were used in this experiment, 87 from a translocation stock consisting of 37 translocation mice containing a dominant mutant marker (W~) and 50 of their nontranslocated
OLFACTORY-MEDIATED CHOICE BEHAVIOR 6C
Scented Container --
(n
327
- Non-scented Container
'7_50
o=
(J
"4 40 i,i
._~ 50 I.-
"6 20
>= 0
3
4
5
6
7
8
9
I0
II
12
Age in Days
FIG. 1. Mean percentage of time in exploration of scented and nonscented containers by Swiss-Webster mice.
nonmarked siblings, and 83 from a nontranslocation marker stock consisting of 42 mice with the W ~ marker and 41 of their nonmarked siblings. Foundation populations for this study were purchased from the Jackson Laboratories in Bar Harbor, Maine; these animals produced the animals tested in this study. The translocation stock consisted of 9 T(5;13)264Ca mice whose genotype was designated B6CBAF2-T264 Wv/++(N22 or N23) and 11 homozygous nonaffected normal siblings. The stock was maintained by breeding translocation mice to the Fj offspring of a cross between C57BL/6J and CBA animals. The offspring of such a cross consist of both translocation pups and nontranslocation pups. The animals we received were from the twenty-second and twenty-third successive crosses. These mice were chosen for a large scale developmental and adult behavior study because we wished to examine the behavioral properties of a translocation stock. Moreover, for this translocation stock easily obtainable control groups were available about which there were behavioral reports in the literature (i.e., Thiessen, Owen, and Whitsett, 1970 on W V marker). The generation of the T264 translocation is discussed by Carter, Lyon, and Phillips (1955). The T264Ca translocation was induced by X-irradiation in mature sperm carried in adult male mice. Males were of the CWX stock carrying marker W v and were mated to females of the CBA stock. Fertility was reduced in translocation heterozygotes. These authors also showed that the position of the translocation break for the T264 translocation relative to other markers in the linkage group is the lx-WV-chromosome break. The recombination rate between Wv and the
328
KOSKI, DIXON, AND FAHRION
translocation is 4%. The W v marker is known to produce a slight macrotic anemia; W v homozygotes have a more severe anemic condition, and many die shortly after birth. To maintain the translocation stock, a nonmarker animal from the translocation stock was mated with a translocated animal carrying the marker. Males and females were paired monogamously with the criterion of no brother-sister mating. The animals which were to constitute the marker stock came from six C57BL/6J-W~ animals and six CBA animals purchased from Jackson Laboratories. To obtain an F1 population, crosses were made between the C57BL animals carrying the marker and the CBA animals. Offspring from the F1 population were the tested marker stock. A nonmarker animal from the F1 was mated to another F~ animal which had the mutant marker, with the criterion of no brother-sister mating. All of the tested animals were the offspring of these crosses, and each litter consisted of markered and nonmarkered animals. All of the litters in the marker stock were culled to match the litter sizes of the translocation stock on the day after litter detection. During breeding, animals were kept in small polypropylene cages (29 x 13 × 9 cm high) containing about 3 cm of aspen woodshavings for bedding. The cage tops were constructed of 1.27-cm wire mesh. Standard Purina Mouse Chow and tap water were available ad libitum. The light cycle was 12 Light/12 Dark, with light onset at 10:00 AN. The day after litters were detected, parents and offspring were transferred to polypropylene animal cages (29.21 × 19.05 x 13.96 cm high) in which was placed about 3 cm of aspen woodshavings for bedding. Procedure. All of the 170 mouse pups used in this experiment were tested on a series of developmental parameters daily for 20 consecutive days following litter detection (Koski, 1975). The battery of developmental tests was adapted from Fox (1965), McClearn, Wilson, and Meredith (1970), and Wahlsten (1974). The developmental tests, observations, and measures consisted of 24 measures of spontaneous behavior, reflexologic responses, and standard developmental parameters. The olfactory-guided choice behavior described in this paper was a part of the developmental test battery, except that the olfactory test was an independent measure and not a repeated measure as were the rest of the developmental parameters. After being developmentally tested, each pup was isolated in a holding cage with aspen bedding material for at least 5 min prior to testing in the olfactory apparatus. The test apparatus for the testing of mouse pups was the same as that described in Expt i. Since Expt 1 indicated that the most rapid development of an olfactory-guided choice behavior occurred during Days 8 through 12, the mice in this experiment were tested only on those days. A testing day was randomly assigned to each pup in a litter. One or two pups from the same litter were tested on a
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testing day depending on the random assignment. Each pup was tested only once during the entire experiment. The remainder of the testing procedure was the same as that described for E x p t 1, except that individual identification of the pups in this experiment was accomplished by carbol fuchsin stain markings on each animal's back before it was returned to the litter. These markings were renewed daily beginning with the first day of developmental testing. Results
The data were transformed to a logarithmic function as described in E x p t 1. H o m o g e n e i t y of variance was achieved (c = 0.327). The four groups of animals tested in this experiment were a translocation group with a mutant marker, a nontranslocation group with a mutant marker, and control groups of animals consisting of n o n m a r k e r siblings in each of the two groups. To determine if the two nonmarker sibling control groups differed, a 2 x 5 (control group condition by age) factorial analysis of variance was computed using scented container exploration time as the dependent variable. The main effect for age was significant [F (4, 81) = 8.46, P < 0.001]. There was no significant effect IF (1, 81) = 0.01, P > 0.75] due to the group from which the control animals were obtained, nor were there any interaction effects IF (4, 81) = 0.88, P > 0.50]. Accordingly, the two groups of nonmarker siblings were pooled into one control group for further analysis. The average percentage of time spent exploring a scented container is shown in Fig. 2. In a 3 x 5 (genetic condition by age) factorial analysis of variance for the time spent in exploration o f an odorized container, the main effects for genetic condition and days were significant IF (2, 155) -- 4.87, P < 0.01, and F (4, 155) = 8.15, P < 0.001, respectively]. A planned contrast between the translocation group and the control group was significant [F (1, 126) = 8.97, P < 0.005]. A simple effects test was computed to determine the age at which genetic condition differences existed. There was a significant difference at Day 10 between the translocation group and the control group IF (1, 15) = 5.57, P < 0.05]. The difference between the marker and control groups at Day 10 was nonsignificant IF (1, 25) = 0.12, P > 0.75].
DISCUSSION The development of olfactory-guided choice behavior can be related to the morphological changes which occur during mouse development. The eyes, nose, and ears of the mouse pup are not open at birth and are not functional until several days after birth. The development of behaviors associated with vision and hearing has been related to the morphological differentiation of the eyes and ears (Fox, 1965; McClearn, et al., 1970;
330
KOSKI, DIXON, AND FAHRION 70 c~
+ Translocafions o Markers • Siblings
~ 5O o 40
it. 3O
~ 2o
0 8
FIG. 2.
9 I0 Age in Days
II
12
Odorized container exploration for genetically different groups of mice.
Wahlston, 1974). In general, the morphological development begins before the onset of such behavioral responses as the visual placing response, the ear twitch, and the auditory startle. In our study, microscopic examination revealed that the nostrils of mouse pups are fully formed with the presence of mucosa by 5 to 8 days of age; a significant olfactory response is observable shortly thereafter. The development of olfaction in mouse pups can be compared to studies of hamsters (Devor and Schneider, 1974) and rats (Gregory and Pfaff, 1971) which show an age-dependent change in behavior associated with olfaction. In these studies, as in ours, the behavior of the pups was not independent of the influence of the odor of the lactating mother. It is not our intention to compare directly the behavioral responses of animals in Expts 1 and 2. These are genetically different groups of animals, and the experiments were conducted separately. However, a cursory comparison of Figs. 1 and 2 will reveal that the development of an olfactory-guided choice behavior in the two experiments was quite similar. There is a striking difference in the onset and development of olfactory-guided choice behavior between translocation animals and all other groups (nontranslocation siblings, marker animals, and nonmarker siblings). The animals in this experiment were part of an extensive developmental study; the handling and stress which are an inherent part of the developmental testing may have had some effect on the behavior of these animals. By postulating a genotype-developmental testing interac-
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tion for the development of this behavior, one could argue that the genetic groups were modified differentially. However, whatever conditions were affecting the behavior, the genotype of the mouse pup was intricately involved. Leon and Moltz (1972) have shown that a pheromone emitted by a lactating mother of the litter might explain the pup's attraction to the odor of a mother. These investigators measured responses on alternate days (omitting Days 11, 13, and 15) and were concerned primarily with the role of the lactating rat mother in her pup's development of olfaction. It may be that the lactating mouse also emits a pheromone before Day 10, but this would not explain the differences that occurred due to genetic condition, since translocation mice and their sibling controls were tested with the same mother. In this study, we were concerned primarily with measuring the development of an olfactory-guided choice behavior in the mouse pups and the genetic influences that might affect that behavior. Since lactating females of the translocation stock consisted of both translocated and nontranslocated animals, and both translocated and nontranslocated offspring were tested using odors of both genotypes of mothers, the differences found could not be due to differences in genotypes of lactating mothers. Thus, the differences between the translocation and other groups on Day 10 may have reflected differences in their sensitivity to the mother's cues regardless of the genotype of the mother. Although it might be argued that the difference between the nontranslocation and the translocation marker mice was due to different genetic backgrounds, it might also be argued that the difference was due to a position effect of the translocated material, since the two sibling control groups responded the same in the behavioral test. Ropartz (1968) has elaborated on the role ofolfaction and aggression. In this study we have provided a link between olfactory-guided choice behavior and genetic condition. Thus, the translocation stock used in this study, in addition to being useful for the study of the lactating mother's influence on her pup's behavior, would also be useful for studying the relationships among olfaction, aggression, and genetic condition. REFERENCES Carr, W. J., Martorano, R. D., and Krames, L. (1970). Responses of mice to odors associated with stress. J. Comp. Physiol. Psychol. 71, 223-228. Carter, T. C., Lyon, M. R., and Phillips, R. J. S. (1955). Gene-tagged chromosome translocations in eleven stocks of mice. J. Genet. 53, 154-166. Devor, M., and Schneider, G. E. (1974). Attraction to home cage odor in hamster pups: Specificity and changes with age. Behav. Biol. 10, 211-221. Fox, W. M. (1965). Reflex-ontogeny and behavioral development of the mouse. Anirn. Behav. 13, 234-241. Fuller, J. L., and Wimer, R. E. (1966). Neural, sensory, and motor functions. In E. L. Green (Ed.), "Biology of the Laboratory Mouse," pp. 609-628. New York: McGraw-Hill.
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Gregory, E. H., and Pfaff, D. W. (1971). Development of olfactory-guided behavior in infant rats. Physiol. Behav. 6, 573-576. Koski, M. A. (1975). "Behavioral Effects of The T(5;13)264Ca Translocation in Mice," 107 pp. Unpublished Masters Thesis. University of Colorado, Boulder. Leon, M., and Moltz, H. (1972). The development of the pheromonal bond in the albino rat. Physiol. Behav. 8, 683-686. McClearn, G, E., Wilson, J. R., and Meredith, W. (1970). The use of isogenic and heterogenic mouse stocks in behavior research. In G. Lindzey and D. D. Thiessen (Eds.), "Contributions to Behavior-Genetic Analysis--The Mouse as a Prototype." New York: Appleton-Century-Crofts. Myers, J. L. (1972). "Fundamentals of Experimental Design," 2nd ed. Boston: AUyn and Bacon. Ropartz, P. (1968). The relation between olfactory stimulation and aggressive behavior in mice. Anim. Behav. 16, 97-100. Thiessen, D. D., Owen, K., and Whitsett, M. (1970). Chromosome mapping of behavioral activities. In G. Lindzey and D. D. Thiessen (Eds.), "Contributions to BehaviorGenetic Analysis-The Mouse as a Prototype." New York: Appleton-Century-Crofts. Wahlsten, D. (1974). A developmental time scale for postnatal changes in brain and behavior of B6D2F2 mice. Brain Res. 72, 251-264.