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Olfactory bulbectomy decreases social behavior in male prairie voles (M. ochrogaster)
Physiology& Behavior,Vol. 55, No. 5, pp. 885-889, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA,All rightsreserved 0031-9384/94$6.00 + .00
Pergamon 0031-9384(93)E0044.Q
Olfactory Bulbectomy Decreases Social Behavior in Male Prairie Voles (M. ochrogaster) B R I A N K I R K P A T R I C K , *1 JESSIE R. WILLIAMS,i" B U R T O N M. S L O T N I C K ¢ A N D C. SUE C A R T E R t
*Maryland Psychiatric Research Center, Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD 21228, tDepartment of Zoology, University of Maryland, College Park, MD 20742, and ~cDepartment of Psychology, American University, Washington, DC 20016 Received 23 June 1993 KIRKPATRICK,B., J. R. WILLIAMS,B. M. SLOTNICKAND C. S. CARTER.Olfactorybulbectomydecreasessocialbehavior in maleprairie voles (M. ochrogaster). PHYSIOL BEHAV 55(5) 885-889, 1994.--Prairie voles (Microtusochrogaster)are social rodents that show characteristics of monogamy including high levels of social behavior and male parental care. Behavioral studies of prairie voles have implicated chemosignals in the control of various components of social behavior and reproduction. In the present study, the role of the olfactory system in male behavior was examined following surgical removal of the olfactory bulbs. Decreases in measures of sexual, paternal, and other social behaviors were observed following bilateral olfactory bulbectomy, but not unilateral or sham bulbectomy. Some bilaterally bulbectomized males also showed a stereotypy, but this probably did not account for the changes in social behaviors. Voles
Socialbehavior
Paternalbehavior
Olfactorybulbs
THE olfactory system has been implicated in sexual, aggressive, affiliative, and parental behaviors in a variety of rodents (8,12,14,25,27). However, the laboratory species most often used in these studies (rats, mice, and hamsters) are not highly social aside from maternal care. In contrast to more commonly studied laboratory rodents, prairie voles (Microtus ochrogaster) are highly social and monogamous (10). This species is remarkable for the degree of physical proximity maintained with selected conspecifics, both in the field and the laboratory. Virgin male prairie voles also are parental on first exposure to pups, retrieving pups that stray, licking pups, and huddling over them (20). In the present study, the effects of olfactory bulbectomy on paternal care, sexual behavior, and adult social contact were examined in adult male prairie voles. Studies of the behavioral effects of bulbectomy were planned as a first stage in a research program aimed at describing the role of the limbic system in social behaviors in prairie voles.
Mating
animals were housed with food and water available ad lib in a 14/10 h light/dark cycle. After weaning but prior to surgery, the males in this study were not exposed to pups or females aside from the behavioral tests described here.
Surgery Surgery was performed in one animal of each sibling pair. The other male of each pair was used as a stimulus animal. There were three experimental groups: bilateral offactory bulbectomy (n = 12), unilateral olfactory bulbectomy (n = 4), and sham surgery (n = 7). Animals were anesthetized with chloral hydrate and a bone flap was removed over the olfactory bulbs. In bilateral- and unilateral-bulbectomizedmales, aspiration of the olfactory bulbs was performed under direct visual guidance. In shamoperated males the bulbs were exposed then the experimental animal was returned to the cage with its brother. The animals were allowed 1 week of postsurgical recovery prior to the onset of behavioral testing.
METHOD
Animals
Behavioral Testing
Pairs of male prairie vole siblings bred in the University of Maryland College Park animal colony were housed together from birth and throughout the experiment. These animals were reared from wild stock originally captured near Urbana, IL. After weaning (approximately day 21), the male sibling pairs were housed separately from their parents and others siblings. Experimental manipulations began after the animals were 60 days of age. The
Animals underwent testing in three social paradigms in the following order: a two-chamber social test with the stimulus sibling, conducted both before and after surgery; a test for paternal behavior; and a test for sexual behavior. The two-chamber test was conducted 7 days after the surgery and the other tests within a few days. Behavioral scoring was conducted without knowledge of the animal's experimental treatment. After the comple-
1Requests for reprints should be addressed to Brian Kirkpatrick, M.D., Maryland Psychiatric Research Center, P.O. Box 21247, Baltimore, MD 21228. 885
886
KIRKPATRICK ET AL.
TABLE 1 SIMILARITYOF UNILATERALLYLESIONEDAND CONTROLANIMALS LesionedAnimals
Time* in brother's cage Prior to surgery Following surgery Entries into brother's cage Prior to surgery Following surgery Mated with familiar female Attacks on pups
Controls
Unilateral
Bilateral
7255 _+965 6821 _+ 1216
8915 + 367 9476 _+664
7198 _+ 1077 3621 _+ 1182
30 +_ 7 20 + 2 7/7 0/7
18 _+ 7 16 + 9 1/4 0/4
26 + 5 128 _+54 1/11 5/12
* Values are mean number of seconds ___SEM; maximum possible is 10,800. tion of behavioral testing, the animals were perfused with saline followed by formalin/saline. The brains were then examined. Two-Chamber Test. The experimental animal and his stimulus sibling were placed in a two-chamber apparatus comprised of two clear polycarbonate mouse cages (26.7 x 16.5 × 12.7 cm) connected by a cylindrical Plexiglas tunnel (approximately 7.5 X 13 cm). Each cage had food and water; lighting and bedding were the same in the two cages. The stimulus sibling was tethered so that he could move freely throughout his cage but could not enter the tunnel to the other cage. The surgical sibling was free to enter either cage, and was initially placed in the cage in which the stimulus animal was not tethered. The animals were videotaped by time-lapse photography (1:12 reduction, with a recording speed of 2.5 frames/s) for 3 h. The following behaviors were scored for the experimental animal: aggressive actions toward the stimulus animal; time in the stimulus male's cage; time in quiet physical contact with the stimulus male (social contact); frequency of autogrooming; and frequency of sniffing or grooming the stimulus sibling. In this paradigm, a distinctive motor abnormality (rapid pacing back and forth) was noted and was added to the scoring. Animals were considered abnormal and were not included in data analyses for this paradigm if, in the baseline testing period, they spent < 10% of the time in the brother's cage, or < 1% of the time in side-by-side contact with the brother. (Five animals, four in the lesion group and one in the sham group, were eliminated by this criterion, leaving the sample sizes noted above.) Paternal Behavior. A prairie vole pup, 2 - 4 days of age, was placed in a clean, small rodent cage (26.7 x 16.5 x 12.7 cm) containing a thin layer of wood chip bedding. The experimental animal was placed in the cage with the pup and observed for 15 rain. The following behaviors (of the adult) were scored: huddling over the pup; sniffing/grooming; side-by-side contact. Sexual Behavior. Sexually mature females were ovariectomized; after recovery, a SC pellet of 10 #g estradiol benzoate was implanted to induce estrus. Females were subsequently screened for lordosis by exposure to sexually experienced nonexperimental males, and then placed in a small rodent cage with wood chips, food, and water. Each experimental male was then placed with an estrous female for 24 h, and the behavior of both was monitored using time-lapse videotaping with a 1:12 reduction. Tapes were assessed for the presence or absence of mounting and intromission.
Statistical Analyses Statistical analyses involved two-tailed tests (where relevant); unless stated otherwise, these were repeated measure analyses of
variance (ANOVAs). When continuous variables were not normally distributed, the Mann-Whitney U statistic was used. Fisher's exact test was used for categorical variables. RESULTS Postmortem examination confirmed group assignment. Minor damage to the frontal pole was found in a few animals with lesions, but in all cases this was well anterior of the septum. Table 1 shows that relative to key measures of behavior, the unilateral and sham groups were similar to each other and very different from the bilaterally lesioned animals. Lesions did not extend into the septal area.
Two-Chamber Test Data for one animal from the baseline testing were lost due to technical problems in videotaping. Bilaterally lesioned animals showed an increase in motor activity as measured by the number of entries into the stimulus male's cage: the mean was 26 -+ 5 (SE) entries in the baseline testing period, compared to 128 _+ 54 following surgery. In contrast, the controls had 27 _+ 5 entries prior to surgery and 19 _+ 3 times following surgery (p < 0.004 for time by treatment interaction, log-transformed data). However, this measure was distorted somewhat by the presence of brief bouts of rapid, back-and-forth packing in three animals with bilateral lesions. In the lesioned animals, this pacing occurred for an average of 1% of the time in the two-chamber apparatus following surgery. Compared to controls, bilaterally lesioned males showed a decrease in the maintenance of physical proximity to their siblings. Bilaterally lesioned males spent approximately 67% of the testing period in the brother's cage in the presurgical test vs. 28% following surgery; for the controls, these percentages were, respectively, 72% and 71% (p < 0.03 for this time by treatment interaction; Fig. 1). In the presurgical test, bilaterally lesioned males spent approximately 41% of the testing period in quiet side-by-side contact with the sib, and 11% following surgery. For the controls, these percentages were, respectively, 52% and 51% (/9 = 0.06 for this time by treatment interaction; Fig. 2). When data from the one lesioned animal that exhibited rapid pacing for more than 2% of time in the two-chamber apparatus were excluded, a trend was still discernible in time in the brother's cage (p = 0.06 for group by time interaction). We considered the possibility that the apparent decrements in social behavior found in the animals with bilateral lesions could be attributed to the hyperactivity found in that group. We corn-
OLFACTORY BULBECTOMY IN MALE PRAIRIE VOLES
10000 9000 8000 7000 6000 5000 4000 A
3000-
w
2000 • 1000 • 0
i
i
pro
post
FIG. 1. Olfactory bulbectomy decreases quiet side-by-side contact with the brother. Three hours of testing. pared the social behaviors of bilaterally lesioned animals that showed the rapid pacing and those that did not (Table 2). In fact, bilaterally lesioned animals that displayed the rapid pacing appeared to be more social than bilaterally lesioned animals without the rapid pacing. (When the bilaterally lesioned animals are divided in this way, the cell sizes are small and these differences are not significantly different.) Thus, the change in social behavior does not appear to be explained by this change in motor behavior. There was no significant difference between experimental groups in the number of bouts of sniffing and grooming directed toward the experimental animal's sib in the two-chamber apparatus. However, in bilaterally lesioned animals the number of bouts of autogrooming declined from a pretest average of 10 to a postsurgical average of four bouts per test period, whereas controis had 7.8 prior to surgery and 8.7 after surgery (p < 0.05 for group by time interaction). Aggression toward the sib was not observed in either group. Parental Behavior
Because of attacks on the pups by lesioned animals (a rare occurrence among normal virgin male prairie voles), several triMs had to be terminated prematurely; consequently, the presence or absence of an attack was used to compare the groups. Five of 12 bilaterally lesioned animals attacked the pup, whereas none of the 11 controls did so (/9 < 0.04, Fisher's exact test; Table 3). These attacks usually occurred within the first 30 s. Within the first 10 rain of exposure, all of the control animals groomed the pup, and either huddled over it or rested next to it quietly.
887 exhibited changes in all of the domains of social and reproductive behavior that we examined. Significantly fewer of the bilaterally lesioned animals mated than did control animals, and several of the bilaterally lesioned males also attacked pups, a behavior that was not found in any of the control animals. Bilaterally lesioned males also spent less time in the stimulus male's cage in a freechoice paradigm, and less time in side-by-side contact with him. However, lesioned prairie voles did not actively avoid the stimulus sibling, explored the sibling's cage as much as the control animals did (as evidenced by the frequency of entries into his cage), and did not differ from controls in number of bouts of sniffing and grooming the sibling. Furthermore, lesioned animals did not show startle responses, nor did they run away from conspecifics. In the two-chamber paradigm, lesioned animals did not exhibit a significant change in sniff_ng and grooming the stimulus animal, but did autogroom less frequently. The loss of olfactory bulbs may remove one stimulus to autogrooming, as autogrooming aids rodents in the acquisition of chemosignals from conspecifics. Finally, an increase in pacing was shown by several of the animals with bilateral bulbectomy, but changes in social behavior were seen in lesioned animals that did not pace as well as in those that did so (Table 2). After weaning, the males lived without exposure to females until they underwent behavioral testing, and this condition may limit the generalizability of our results. However, the validity should not be undermined, because both lesioned and control animals were raised in the same environment. In addition, despite such rearing, many of the control males were able to mate successfully. Other studies have found changes in the social behavior of rodents following bulbectomy. For example, olfactory bulb removal is associated with alterations in sexual behavior in male (16) and in female (17,19) rats, male Syrian hamsters (28), and male guinea pigs (3). Bulbectomy also decreases mating in sexually inexperienced male rats, but not in experienced animals (16). The male prairie voles in the present study were sexually inexperienced, so we cannot eliminate the possibility that bulbectomy would have had less effect in sexually experienced animals. The reductions in male sexual behavior we found are consistent with findings in male guinea pigs (3) and rats (16). In the Syrian hamster, the vomeronasal organ and the main olfactory bulb appear to make separate contributions to the maintenance of normal
I0000 • lesioned 9000.
""
llk""
control
8000. 7000. e-
g
6O00 '
Sexual Behavior 5000-
One of 11 bilaterally lesioned animals mated with the estrous female (one lesioned animal died prior to this test); in contrast eight of 11 control males mated with the female ( p < 0.01, Fisher's exact test; Table 3). None of the males attacked the female, and those males that failed to mate usually did not sniff the female's anogenital area or mount her. v D I S C O S S I O N
Compared to animals with unilateral olfactory or sham surgery, male prairie voles that had undergone bilateral bulbectomy
.
|
i
pre
post
FIG. 2. Olfactory bulbectomy decreases time in brother's cage. Three hours of testing.
888
KIRKPATRICK ET AL.
TABLE 2 HYPERACTIVITY AND SOCIAL BEHAVIORS FOLLOWING SURGERY Lesioned
In two-chamberparadigm (means _ SEM) Time in brother's cage* Side-by-sidecontact* Bouts of autogrooming Sexual behavior Successfully mated Did not mate Paternal behavior Attacked pup Did not attack
With Pacing
Without Pacing
Sham
4017 __.2201 1855 _+ 1829 4.7 _+0.3
2307 _+ 1449 649 _ 543 3.5 +_0.6
7618 _ 937 5506 _ 1145 8.7 _+ 1.6
l 4
0 6
8 3
1 5
4 2
0 11
Values are mean _+ SEM. n = 3, 4, and 10 for data from the two-chamber paradigm, respectively. * In seconds (10,800 maximum possible).
male sexual behavior (26-28). Our design could not distinguish the contributions of these structures. Others have noted changes in parental behaviors or aggression following olfactory bulbectomy. In rats, the latency to the onset of parental behaviors is reduced following bulbectomy in both males and females (7,8). There also are reports of an increase in infanticide among female mice (21). In nonparturient rats, hormonal priming or repeated pup exposure over several days is usually required for the expression of parental behavior. In contrast, both male and female prairie voles typically retrieve and care for pups within minutes of their first exposure to pups (20,22). Increased mouse-killinghas been reported in rats (12,14) following olfactory bulb lesions, as well as an increase in aggression in gerbils (13) and an increase in play fighting in juvenile rats (2). However, bulbectomy has been reported to cause a decrease in aggression in guinea pigs and mice (3,21). Olfactory bulbectomized animals also showed abnormalities in social behavior in a nonparental, nonmating context in our study. We are not aware of comparable data in previous studies of the effects of bulbectomy. The alteration in motor behavior observed here is reminiscent of the hyperactivity previously noted in rats following olfactory bulbectomy (11,12). Our data suggest this motor abnormalitywas not the cause of the behavioral impairments described here. Bilaterally lesioned animals without the alteration in motor behavior also exhibited abnormalities in mating, paternal care, and the two-chamber paradigm with the stimulus sibling, and showed good motor coordination. For example, one bilaterally lesioned
TABLE 3 SEXUAL AND PARENTAL BEHAVIOR FOLLOWING OLFACTORY BULBECTOMY IN MALE PRAIRIE VOLES
Group
Pup Attacks
Mating
Bilateral lesion Unilateral lesion/sham
5/12 0/11 p < 0.04*
1/11 8/11 p < 0.03*
* Fisher's exact test.
animal that spent 27% of the test time pacing in the two-chamber apparatus did mate and attended to a pup normally. Finally, decreases in time in the brother's cage and time in side-by-side contact were found in the animals with bilateral bulbectomy whether or not they exhibited rapid pacing. The effects of olfactory bulbectomy in prairie voles are consistent with other studies implicating olfaction in rodent social and reproductive behaviors. For instance, a chemosignal from male urine is required to induce estrus in female prairie voles (4,6), and olfactory cues can suppress reproduction in female prairie voles (9). The role of olfactory stimuli in male prairie voles is less well described, but males also may require social stimulation for reproductive activation (5). Neurochemical or neurodegenerative changes located more centrally in the brain have been noted following olfactory bulbectomy. These include changes in norepinephrine concentrations in the telencephalon in the rat (15) as well as changes in serotonin (21), glutamic acid decarboxylase (24), and estrogen (19), and imipramine (1) binding in the amygdala. The effects observed here might be associated with similar changes. The vomeronasal system and amygdala have been implicated in a variety of behavioral changes that occur following olfactory bulbectomy. For example, Winans and coworkers found that lesions of the vomeronasal system and associated amygdalar lesions decrease sexual behaviors in male Syrian hamsters (18,27,28). Fleming and coworkers have also implicated the vomeronasal system and amygdala in maternal behavior in rats (8). In prairie voles we have found that lesions of the medial nucleus of the amygdala can reduce both parental and social behaviors (Kirkpatrick et al., submitted). Another, simpler interpretation of these results is that rodents rely primarily on olfaction for discrimination of conspecifics from other animals; following bulbectomy, they are unable to recognize other voles as conspecifics. One difficulty with such an explanation is the variance of behavioral effects found in the lesioned animals. Furthermore, vole pups produce ultrasounds and seem likely to play an important part in conspecific recognition, but paternal behavior was altered by olfactory bulbectomy alone. Whatever the mechanism, it is interesting to note that the present results contrast somewhat with results from parallel studies
O L F A C T O R Y B U L B E C T O M Y IN M A L E PRAIRIE VOLES
in female prairie voles. In females, olfactory bulbectomy did not result in an increase in aggression toward pups (25). This difference between males and females following CNS damage is consistent with a behavioral difference as well: prior to pregnancy, reproductively naive female prairie voles are more likely than males to direct aggression toward pups (23). Taken together, these observations raise the possibility that although the overt behaviors shown by males and females are very similar, the neu-
889
robiological underpinnings of pup care may differ between males and females. In summary, these studies implicate olfaction in the regulation of sexual and social behavior in male prairie voles. In conjunction with comparable studies of females and other research, the present study also suggests possible sex differences in the role of the olfactory system in the control of these behaviors. The mechanisms of these changes remain to be explored.
REFERENCES 1. Al-Khatib, I. M.; Fujiwara, M.; Kataoka, Y.; Ueki, S. Changes in [3H]-imipramine binding sites in relation to muricide following olfactory bulbectomy and the effect of nomifensine in brain of rats. Arch. Int. Pharmacodyn. Ther. 296:282-292; 1988. 2. Beatty, W. W.; Costello, IC B. Olfactory bulbectomy and play fighting in juvenile rats. Physiol. Behav. 30(4):525-528; 1983. 3. Beanchamp, G. IC; Magnus, J. G.; Shmunes, N. T.; Durham, T. Effects of olfactory bulbectomy on social behavior of male guinea pigs (Cavia porcellus). J. Comp. Physiol. Psychol. 91(2):336-346; 1977. 4. Carter, C. S.; Getz, L. L.; Gavish, L.; McDermott, J. L.; Arnold, P. Male-related pheromones and the activation of female reproduction in the prairie vole (Microtus ochrogaster). Biol. Reprod. 23(5):1038-1045; 1980. 5. Carter, C. S.; Getz, L. L.; Cohen-Parsons, M. Relationships between social organization and behavioral endocrinology in a monogamous mammal. Adv. Stud. Behav. 16:109-145; 1986. 6. Carter, C. S.; Wilt, D. M.; Schneider, J.; Harris, Z. L.; Volkening, D. Male stimuli are necessary for female sexual behavior and uterine growth in prairie vole (Microtus ochrogaster). Horm. Behav. 21(1):74-82; 1987. 7. Fleischer, S.; Kordower, J. H.; Kaplan, B.; Dicker, R.; Smerling, R.; Ilguer, J. Olfactory bulbectomy and gender differences in maternal behaviors of rats. Physiol. Behav. 26:957-959; 1981. 8. Fleming, A. S.; Gavarth, K.; Sarker, J. Effects of transections to the vomeronasal nerves or to the main olfactory bulbs on the initiation and long-term retention of maternal behavior in primiparous rats. Behav. Neural Biol. 57(3):177-188; 1992. 9. Getz, L. L.; Dluzen, D.; McDermott, J. L. Suppression of reproductive maturation in male-stimulated virgin female Microtus by a urinary chemosignal. Behav. Proc. 8:59-64; 1983. 10. Getz, L.L.; Carter, C.S. Social organization in Microtus ochrogaster populations. Biologist 62(1-4):56-69; 1980. 11. Gomita, Y.; Ueki, S. Effects of limbic lesions, especially of olfactory bulbectomy on simple conditioned avoidance response in rats. J. Pharmacobiodyn. 3(2):94-104; 1980. 12. Gomita, Y.; Ogawa, N.; Ueki, S. Effect of bilateral olfactory bub bectomy on discrimination avoidance conditioning in rats. Physiol. Behav. 32:1011-1016; 1984. 13. Hull, E. M.; Hamilton, K. I.; Engwall, D. B.; Rosselli, L. Effects of olfactory bulbectomy and peripheral deafferentation on reactions to crowding in gerbils (Meriones unguiculatus). J. Comp. Physiol. Psychol. 86(2):247-254; 1974. 14. Hull, E. M.; Homan, H. D. Olfactory bulbectomy, peripheral anosmia, and mouse killing and eating by rats. Behav. Biol. 14:481-488; 1975. 15. Iwasaki, K.; Fukiwara, M.; Ueki, S. In vivo changes in brain catecholamine release from rat hypothalamus following olfactory bulbectomy. Pharmacol. Biochem. Behav. 34(4):879-885; 1989.
16. Larsson, IC Sexual impairment of inexperienced male rats following pre and postpuberal olfactory bulbectomy. Physiol. Behav. 14(2):195-199; 1975. 17. Larsson, K. Transient changes in the female rat estrous cycle after olfactory bulbectomy or removal of the olfactory epithelium. Physiol. Behav. 18:261-265; 1977. 18. Lehman, M. N.; Winans, S. S.; Powers, L B. Medial nucleus of the amgydala mediates chemosensory control of male hamster sexual behavior. Science 210:557-560; 1980. 19. McGinnis, M. Y.; Lumia, A. R.; McEwen, B. S. Increased estrogen receptor binding in amygdala correlates with facilitation of feminine sexual behavior induced by olfactory bulbectomy. Brain Res. 334(1):19-25; 1985. 20. McGuire, B.; Novak, M. A comparison of the maternal behavior in the meadow vole (Microtus pennsylvanicus), prairie vole (M. ochrogaster), and pine vole (M. pinetorum). Anim. Behav. 32:11321141; 1984. 21. Neckers, L. M.; Zarrow, M. X.; Myers, M. M.; Denenberg, V. H. Influence of olfactory bulbectomy and the serotonergic system upon intermale aggression and maternal behavior in the mouse. Pharmacol. Biochem. Behav. 3(4):545-550; 1975. 22. Oliveras, D.; Novak, M. A comparison of paternal behavior in the meadow vole (Microtus pennsylvanicus), the pine vole (M. pinetorum), and the prairie vole (M. ochrogaster). Anita. Behav. 34:519526; 1986. 23. Roberts, R. L; Kearse, A. F.; Carter, C. S. Helper males attend more to pups than do females in prairie voles. Conf. Reprod. Behav. Abst. 24; 1992. 24. Tyler, J. L.; Gordon, J. H.; Groski, R. A. Effects of olfactory bulbectomy and estrogen on tyrosine hydroxylase and glutamic acid decarboxylase in the nigrostriatal and mesolhnbic dopamine systems of adult female rats. Pharmacol. Biochem. Behav. 11:549-552; 1979. 25. Williams, J. R.; Slotnick, B. M.; Kirkpatrick, B. W.; Carter, C. S. Olfactory bulb removal affects partner preference development and estrns induction in female prairie voles. Physiol. Behav. 52:635639; 1992. 26. Winans, S. S.; Lehman, M. N.; Powers, L B. Vomeronasal and olfactory CNS pathways which control male hamster mating behavior. In: Breipohl, W., ed. Olfaction and endocrine regulation. London: IRL Press Limited; 1982:23-34. 27. Winans, S. S.; Powers, J. B. Olfactory and vomeronasal deafferentation of male hamsters: Histological and behavioral analyses. Brain Res. 126:325-344; 1977. 28. Winans, S. S.; Powers, L B. Neonatal and two-stage olfactory bulbectomy: Effects on male hamster sexual behavior. Behav. Biol. 10:461-471; 1974.
Pergamon 0031-9384(93)E0044.Q
Olfactory Bulbectomy Decreases Social Behavior in Male Prairie Voles (M. ochrogaster) B R I A N K I R K P A T R I C K , *1 JESSIE R. WILLIAMS,i" B U R T O N M. S L O T N I C K ¢ A N D C. SUE C A R T E R t
*Maryland Psychiatric Research Center, Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD 21228, tDepartment of Zoology, University of Maryland, College Park, MD 20742, and ~cDepartment of Psychology, American University, Washington, DC 20016 Received 23 June 1993 KIRKPATRICK,B., J. R. WILLIAMS,B. M. SLOTNICKAND C. S. CARTER.Olfactorybulbectomydecreasessocialbehavior in maleprairie voles (M. ochrogaster). PHYSIOL BEHAV 55(5) 885-889, 1994.--Prairie voles (Microtusochrogaster)are social rodents that show characteristics of monogamy including high levels of social behavior and male parental care. Behavioral studies of prairie voles have implicated chemosignals in the control of various components of social behavior and reproduction. In the present study, the role of the olfactory system in male behavior was examined following surgical removal of the olfactory bulbs. Decreases in measures of sexual, paternal, and other social behaviors were observed following bilateral olfactory bulbectomy, but not unilateral or sham bulbectomy. Some bilaterally bulbectomized males also showed a stereotypy, but this probably did not account for the changes in social behaviors. Voles
Socialbehavior
Paternalbehavior
Olfactorybulbs
THE olfactory system has been implicated in sexual, aggressive, affiliative, and parental behaviors in a variety of rodents (8,12,14,25,27). However, the laboratory species most often used in these studies (rats, mice, and hamsters) are not highly social aside from maternal care. In contrast to more commonly studied laboratory rodents, prairie voles (Microtus ochrogaster) are highly social and monogamous (10). This species is remarkable for the degree of physical proximity maintained with selected conspecifics, both in the field and the laboratory. Virgin male prairie voles also are parental on first exposure to pups, retrieving pups that stray, licking pups, and huddling over them (20). In the present study, the effects of olfactory bulbectomy on paternal care, sexual behavior, and adult social contact were examined in adult male prairie voles. Studies of the behavioral effects of bulbectomy were planned as a first stage in a research program aimed at describing the role of the limbic system in social behaviors in prairie voles.
Mating
animals were housed with food and water available ad lib in a 14/10 h light/dark cycle. After weaning but prior to surgery, the males in this study were not exposed to pups or females aside from the behavioral tests described here.
Surgery Surgery was performed in one animal of each sibling pair. The other male of each pair was used as a stimulus animal. There were three experimental groups: bilateral offactory bulbectomy (n = 12), unilateral olfactory bulbectomy (n = 4), and sham surgery (n = 7). Animals were anesthetized with chloral hydrate and a bone flap was removed over the olfactory bulbs. In bilateral- and unilateral-bulbectomizedmales, aspiration of the olfactory bulbs was performed under direct visual guidance. In shamoperated males the bulbs were exposed then the experimental animal was returned to the cage with its brother. The animals were allowed 1 week of postsurgical recovery prior to the onset of behavioral testing.
METHOD
Animals
Behavioral Testing
Pairs of male prairie vole siblings bred in the University of Maryland College Park animal colony were housed together from birth and throughout the experiment. These animals were reared from wild stock originally captured near Urbana, IL. After weaning (approximately day 21), the male sibling pairs were housed separately from their parents and others siblings. Experimental manipulations began after the animals were 60 days of age. The
Animals underwent testing in three social paradigms in the following order: a two-chamber social test with the stimulus sibling, conducted both before and after surgery; a test for paternal behavior; and a test for sexual behavior. The two-chamber test was conducted 7 days after the surgery and the other tests within a few days. Behavioral scoring was conducted without knowledge of the animal's experimental treatment. After the comple-
1Requests for reprints should be addressed to Brian Kirkpatrick, M.D., Maryland Psychiatric Research Center, P.O. Box 21247, Baltimore, MD 21228. 885
886
KIRKPATRICK ET AL.
TABLE 1 SIMILARITYOF UNILATERALLYLESIONEDAND CONTROLANIMALS LesionedAnimals
Time* in brother's cage Prior to surgery Following surgery Entries into brother's cage Prior to surgery Following surgery Mated with familiar female Attacks on pups
Controls
Unilateral
Bilateral
7255 _+965 6821 _+ 1216
8915 + 367 9476 _+664
7198 _+ 1077 3621 _+ 1182
30 +_ 7 20 + 2 7/7 0/7
18 _+ 7 16 + 9 1/4 0/4
26 + 5 128 _+54 1/11 5/12
* Values are mean number of seconds ___SEM; maximum possible is 10,800. tion of behavioral testing, the animals were perfused with saline followed by formalin/saline. The brains were then examined. Two-Chamber Test. The experimental animal and his stimulus sibling were placed in a two-chamber apparatus comprised of two clear polycarbonate mouse cages (26.7 x 16.5 × 12.7 cm) connected by a cylindrical Plexiglas tunnel (approximately 7.5 X 13 cm). Each cage had food and water; lighting and bedding were the same in the two cages. The stimulus sibling was tethered so that he could move freely throughout his cage but could not enter the tunnel to the other cage. The surgical sibling was free to enter either cage, and was initially placed in the cage in which the stimulus animal was not tethered. The animals were videotaped by time-lapse photography (1:12 reduction, with a recording speed of 2.5 frames/s) for 3 h. The following behaviors were scored for the experimental animal: aggressive actions toward the stimulus animal; time in the stimulus male's cage; time in quiet physical contact with the stimulus male (social contact); frequency of autogrooming; and frequency of sniffing or grooming the stimulus sibling. In this paradigm, a distinctive motor abnormality (rapid pacing back and forth) was noted and was added to the scoring. Animals were considered abnormal and were not included in data analyses for this paradigm if, in the baseline testing period, they spent < 10% of the time in the brother's cage, or < 1% of the time in side-by-side contact with the brother. (Five animals, four in the lesion group and one in the sham group, were eliminated by this criterion, leaving the sample sizes noted above.) Paternal Behavior. A prairie vole pup, 2 - 4 days of age, was placed in a clean, small rodent cage (26.7 x 16.5 x 12.7 cm) containing a thin layer of wood chip bedding. The experimental animal was placed in the cage with the pup and observed for 15 rain. The following behaviors (of the adult) were scored: huddling over the pup; sniffing/grooming; side-by-side contact. Sexual Behavior. Sexually mature females were ovariectomized; after recovery, a SC pellet of 10 #g estradiol benzoate was implanted to induce estrus. Females were subsequently screened for lordosis by exposure to sexually experienced nonexperimental males, and then placed in a small rodent cage with wood chips, food, and water. Each experimental male was then placed with an estrous female for 24 h, and the behavior of both was monitored using time-lapse videotaping with a 1:12 reduction. Tapes were assessed for the presence or absence of mounting and intromission.
Statistical Analyses Statistical analyses involved two-tailed tests (where relevant); unless stated otherwise, these were repeated measure analyses of
variance (ANOVAs). When continuous variables were not normally distributed, the Mann-Whitney U statistic was used. Fisher's exact test was used for categorical variables. RESULTS Postmortem examination confirmed group assignment. Minor damage to the frontal pole was found in a few animals with lesions, but in all cases this was well anterior of the septum. Table 1 shows that relative to key measures of behavior, the unilateral and sham groups were similar to each other and very different from the bilaterally lesioned animals. Lesions did not extend into the septal area.
Two-Chamber Test Data for one animal from the baseline testing were lost due to technical problems in videotaping. Bilaterally lesioned animals showed an increase in motor activity as measured by the number of entries into the stimulus male's cage: the mean was 26 -+ 5 (SE) entries in the baseline testing period, compared to 128 _+ 54 following surgery. In contrast, the controls had 27 _+ 5 entries prior to surgery and 19 _+ 3 times following surgery (p < 0.004 for time by treatment interaction, log-transformed data). However, this measure was distorted somewhat by the presence of brief bouts of rapid, back-and-forth packing in three animals with bilateral lesions. In the lesioned animals, this pacing occurred for an average of 1% of the time in the two-chamber apparatus following surgery. Compared to controls, bilaterally lesioned males showed a decrease in the maintenance of physical proximity to their siblings. Bilaterally lesioned males spent approximately 67% of the testing period in the brother's cage in the presurgical test vs. 28% following surgery; for the controls, these percentages were, respectively, 72% and 71% (p < 0.03 for this time by treatment interaction; Fig. 1). In the presurgical test, bilaterally lesioned males spent approximately 41% of the testing period in quiet side-by-side contact with the sib, and 11% following surgery. For the controls, these percentages were, respectively, 52% and 51% (/9 = 0.06 for this time by treatment interaction; Fig. 2). When data from the one lesioned animal that exhibited rapid pacing for more than 2% of time in the two-chamber apparatus were excluded, a trend was still discernible in time in the brother's cage (p = 0.06 for group by time interaction). We considered the possibility that the apparent decrements in social behavior found in the animals with bilateral lesions could be attributed to the hyperactivity found in that group. We corn-
OLFACTORY BULBECTOMY IN MALE PRAIRIE VOLES
10000 9000 8000 7000 6000 5000 4000 A
3000-
w
2000 • 1000 • 0
i
i
pro
post
FIG. 1. Olfactory bulbectomy decreases quiet side-by-side contact with the brother. Three hours of testing. pared the social behaviors of bilaterally lesioned animals that showed the rapid pacing and those that did not (Table 2). In fact, bilaterally lesioned animals that displayed the rapid pacing appeared to be more social than bilaterally lesioned animals without the rapid pacing. (When the bilaterally lesioned animals are divided in this way, the cell sizes are small and these differences are not significantly different.) Thus, the change in social behavior does not appear to be explained by this change in motor behavior. There was no significant difference between experimental groups in the number of bouts of sniffing and grooming directed toward the experimental animal's sib in the two-chamber apparatus. However, in bilaterally lesioned animals the number of bouts of autogrooming declined from a pretest average of 10 to a postsurgical average of four bouts per test period, whereas controis had 7.8 prior to surgery and 8.7 after surgery (p < 0.05 for group by time interaction). Aggression toward the sib was not observed in either group. Parental Behavior
Because of attacks on the pups by lesioned animals (a rare occurrence among normal virgin male prairie voles), several triMs had to be terminated prematurely; consequently, the presence or absence of an attack was used to compare the groups. Five of 12 bilaterally lesioned animals attacked the pup, whereas none of the 11 controls did so (/9 < 0.04, Fisher's exact test; Table 3). These attacks usually occurred within the first 30 s. Within the first 10 rain of exposure, all of the control animals groomed the pup, and either huddled over it or rested next to it quietly.
887 exhibited changes in all of the domains of social and reproductive behavior that we examined. Significantly fewer of the bilaterally lesioned animals mated than did control animals, and several of the bilaterally lesioned males also attacked pups, a behavior that was not found in any of the control animals. Bilaterally lesioned males also spent less time in the stimulus male's cage in a freechoice paradigm, and less time in side-by-side contact with him. However, lesioned prairie voles did not actively avoid the stimulus sibling, explored the sibling's cage as much as the control animals did (as evidenced by the frequency of entries into his cage), and did not differ from controls in number of bouts of sniffing and grooming the sibling. Furthermore, lesioned animals did not show startle responses, nor did they run away from conspecifics. In the two-chamber paradigm, lesioned animals did not exhibit a significant change in sniff_ng and grooming the stimulus animal, but did autogroom less frequently. The loss of olfactory bulbs may remove one stimulus to autogrooming, as autogrooming aids rodents in the acquisition of chemosignals from conspecifics. Finally, an increase in pacing was shown by several of the animals with bilateral bulbectomy, but changes in social behavior were seen in lesioned animals that did not pace as well as in those that did so (Table 2). After weaning, the males lived without exposure to females until they underwent behavioral testing, and this condition may limit the generalizability of our results. However, the validity should not be undermined, because both lesioned and control animals were raised in the same environment. In addition, despite such rearing, many of the control males were able to mate successfully. Other studies have found changes in the social behavior of rodents following bulbectomy. For example, olfactory bulb removal is associated with alterations in sexual behavior in male (16) and in female (17,19) rats, male Syrian hamsters (28), and male guinea pigs (3). Bulbectomy also decreases mating in sexually inexperienced male rats, but not in experienced animals (16). The male prairie voles in the present study were sexually inexperienced, so we cannot eliminate the possibility that bulbectomy would have had less effect in sexually experienced animals. The reductions in male sexual behavior we found are consistent with findings in male guinea pigs (3) and rats (16). In the Syrian hamster, the vomeronasal organ and the main olfactory bulb appear to make separate contributions to the maintenance of normal
I0000 • lesioned 9000.
""
llk""
control
8000. 7000. e-
g
6O00 '
Sexual Behavior 5000-
One of 11 bilaterally lesioned animals mated with the estrous female (one lesioned animal died prior to this test); in contrast eight of 11 control males mated with the female ( p < 0.01, Fisher's exact test; Table 3). None of the males attacked the female, and those males that failed to mate usually did not sniff the female's anogenital area or mount her. v D I S C O S S I O N
Compared to animals with unilateral olfactory or sham surgery, male prairie voles that had undergone bilateral bulbectomy
.
|
i
pre
post
FIG. 2. Olfactory bulbectomy decreases time in brother's cage. Three hours of testing.
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KIRKPATRICK ET AL.
TABLE 2 HYPERACTIVITY AND SOCIAL BEHAVIORS FOLLOWING SURGERY Lesioned
In two-chamberparadigm (means _ SEM) Time in brother's cage* Side-by-sidecontact* Bouts of autogrooming Sexual behavior Successfully mated Did not mate Paternal behavior Attacked pup Did not attack
With Pacing
Without Pacing
Sham
4017 __.2201 1855 _+ 1829 4.7 _+0.3
2307 _+ 1449 649 _ 543 3.5 +_0.6
7618 _ 937 5506 _ 1145 8.7 _+ 1.6
l 4
0 6
8 3
1 5
4 2
0 11
Values are mean _+ SEM. n = 3, 4, and 10 for data from the two-chamber paradigm, respectively. * In seconds (10,800 maximum possible).
male sexual behavior (26-28). Our design could not distinguish the contributions of these structures. Others have noted changes in parental behaviors or aggression following olfactory bulbectomy. In rats, the latency to the onset of parental behaviors is reduced following bulbectomy in both males and females (7,8). There also are reports of an increase in infanticide among female mice (21). In nonparturient rats, hormonal priming or repeated pup exposure over several days is usually required for the expression of parental behavior. In contrast, both male and female prairie voles typically retrieve and care for pups within minutes of their first exposure to pups (20,22). Increased mouse-killinghas been reported in rats (12,14) following olfactory bulb lesions, as well as an increase in aggression in gerbils (13) and an increase in play fighting in juvenile rats (2). However, bulbectomy has been reported to cause a decrease in aggression in guinea pigs and mice (3,21). Olfactory bulbectomized animals also showed abnormalities in social behavior in a nonparental, nonmating context in our study. We are not aware of comparable data in previous studies of the effects of bulbectomy. The alteration in motor behavior observed here is reminiscent of the hyperactivity previously noted in rats following olfactory bulbectomy (11,12). Our data suggest this motor abnormalitywas not the cause of the behavioral impairments described here. Bilaterally lesioned animals without the alteration in motor behavior also exhibited abnormalities in mating, paternal care, and the two-chamber paradigm with the stimulus sibling, and showed good motor coordination. For example, one bilaterally lesioned
TABLE 3 SEXUAL AND PARENTAL BEHAVIOR FOLLOWING OLFACTORY BULBECTOMY IN MALE PRAIRIE VOLES
Group
Pup Attacks
Mating
Bilateral lesion Unilateral lesion/sham
5/12 0/11 p < 0.04*
1/11 8/11 p < 0.03*
* Fisher's exact test.
animal that spent 27% of the test time pacing in the two-chamber apparatus did mate and attended to a pup normally. Finally, decreases in time in the brother's cage and time in side-by-side contact were found in the animals with bilateral bulbectomy whether or not they exhibited rapid pacing. The effects of olfactory bulbectomy in prairie voles are consistent with other studies implicating olfaction in rodent social and reproductive behaviors. For instance, a chemosignal from male urine is required to induce estrus in female prairie voles (4,6), and olfactory cues can suppress reproduction in female prairie voles (9). The role of olfactory stimuli in male prairie voles is less well described, but males also may require social stimulation for reproductive activation (5). Neurochemical or neurodegenerative changes located more centrally in the brain have been noted following olfactory bulbectomy. These include changes in norepinephrine concentrations in the telencephalon in the rat (15) as well as changes in serotonin (21), glutamic acid decarboxylase (24), and estrogen (19), and imipramine (1) binding in the amygdala. The effects observed here might be associated with similar changes. The vomeronasal system and amygdala have been implicated in a variety of behavioral changes that occur following olfactory bulbectomy. For example, Winans and coworkers found that lesions of the vomeronasal system and associated amygdalar lesions decrease sexual behaviors in male Syrian hamsters (18,27,28). Fleming and coworkers have also implicated the vomeronasal system and amygdala in maternal behavior in rats (8). In prairie voles we have found that lesions of the medial nucleus of the amygdala can reduce both parental and social behaviors (Kirkpatrick et al., submitted). Another, simpler interpretation of these results is that rodents rely primarily on olfaction for discrimination of conspecifics from other animals; following bulbectomy, they are unable to recognize other voles as conspecifics. One difficulty with such an explanation is the variance of behavioral effects found in the lesioned animals. Furthermore, vole pups produce ultrasounds and seem likely to play an important part in conspecific recognition, but paternal behavior was altered by olfactory bulbectomy alone. Whatever the mechanism, it is interesting to note that the present results contrast somewhat with results from parallel studies
O L F A C T O R Y B U L B E C T O M Y IN M A L E PRAIRIE VOLES
in female prairie voles. In females, olfactory bulbectomy did not result in an increase in aggression toward pups (25). This difference between males and females following CNS damage is consistent with a behavioral difference as well: prior to pregnancy, reproductively naive female prairie voles are more likely than males to direct aggression toward pups (23). Taken together, these observations raise the possibility that although the overt behaviors shown by males and females are very similar, the neu-
889
robiological underpinnings of pup care may differ between males and females. In summary, these studies implicate olfaction in the regulation of sexual and social behavior in male prairie voles. In conjunction with comparable studies of females and other research, the present study also suggests possible sex differences in the role of the olfactory system in the control of these behaviors. The mechanisms of these changes remain to be explored.
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16. Larsson, IC Sexual impairment of inexperienced male rats following pre and postpuberal olfactory bulbectomy. Physiol. Behav. 14(2):195-199; 1975. 17. Larsson, K. Transient changes in the female rat estrous cycle after olfactory bulbectomy or removal of the olfactory epithelium. Physiol. Behav. 18:261-265; 1977. 18. Lehman, M. N.; Winans, S. S.; Powers, L B. Medial nucleus of the amgydala mediates chemosensory control of male hamster sexual behavior. Science 210:557-560; 1980. 19. McGinnis, M. Y.; Lumia, A. R.; McEwen, B. S. Increased estrogen receptor binding in amygdala correlates with facilitation of feminine sexual behavior induced by olfactory bulbectomy. Brain Res. 334(1):19-25; 1985. 20. McGuire, B.; Novak, M. A comparison of the maternal behavior in the meadow vole (Microtus pennsylvanicus), prairie vole (M. ochrogaster), and pine vole (M. pinetorum). Anim. Behav. 32:11321141; 1984. 21. Neckers, L. M.; Zarrow, M. X.; Myers, M. M.; Denenberg, V. H. Influence of olfactory bulbectomy and the serotonergic system upon intermale aggression and maternal behavior in the mouse. Pharmacol. Biochem. Behav. 3(4):545-550; 1975. 22. Oliveras, D.; Novak, M. A comparison of paternal behavior in the meadow vole (Microtus pennsylvanicus), the pine vole (M. pinetorum), and the prairie vole (M. ochrogaster). Anita. Behav. 34:519526; 1986. 23. Roberts, R. L; Kearse, A. F.; Carter, C. S. Helper males attend more to pups than do females in prairie voles. Conf. Reprod. Behav. Abst. 24; 1992. 24. Tyler, J. L.; Gordon, J. H.; Groski, R. A. Effects of olfactory bulbectomy and estrogen on tyrosine hydroxylase and glutamic acid decarboxylase in the nigrostriatal and mesolhnbic dopamine systems of adult female rats. Pharmacol. Biochem. Behav. 11:549-552; 1979. 25. Williams, J. R.; Slotnick, B. M.; Kirkpatrick, B. W.; Carter, C. S. Olfactory bulb removal affects partner preference development and estrns induction in female prairie voles. Physiol. Behav. 52:635639; 1992. 26. Winans, S. S.; Lehman, M. N.; Powers, L B. Vomeronasal and olfactory CNS pathways which control male hamster mating behavior. In: Breipohl, W., ed. Olfaction and endocrine regulation. London: IRL Press Limited; 1982:23-34. 27. Winans, S. S.; Powers, J. B. Olfactory and vomeronasal deafferentation of male hamsters: Histological and behavioral analyses. Brain Res. 126:325-344; 1977. 28. Winans, S. S.; Powers, L B. Neonatal and two-stage olfactory bulbectomy: Effects on male hamster sexual behavior. Behav. Biol. 10:461-471; 1974.