Physiology&Behavior.Vol. 51, pp. 939-943, 1992
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printedin the USA.
Lesions of the Medial Amygdala Produce Severe Impairment of Copulatory Behavior in Sexually Inexperienced Male Rats YASUHIKO KONDO
Department of Anatomy, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received 1 April 1991 KONDO, Y. Lesions of the media/amygdala producesevereimpairment of copulatory behavior in sexuallyinexperiencedmale rats. PHYSIOLBEHAV51(5) 939-943, 1992.--The effectsofamygdaloid lesionson masculinecopulatorybehaviorwere examined in male rats. Sexuallyinexperienced male rats were castrated and subjected to bilateral lesions in one of the followingareas: the medial amygdala, the cortical amygdala, or the basolateral amygdala. Three weeks later, all rats received implantation of silastic capsules containing testosterone. Then, four observations of copulatory behavior were carried out every 5 days following the implantation of testosterone. Rats with medial amygdala lesions showed a severe deficit of copulatory behavior, whereas rats with basolateral amygdala lesions showed no change in the performance of copulation. As for rats with cortical amygdala lesions, although their copulatory behavior was impaired, the effect was confined to a deficit in intromission and ejaculation responses. These findings suggest that the medial amygdala plays a critical role in regulating masculine sexual behavior in the rat. Copulatory behavior
Amygdalalesion
Limbicsystem
Male rat
METHOD
A considerable body of evidence suggests that the most important region involved in the copulatory behavior of male rats is the medial preoptic-anterior hypothalamic (mPOA-AH) continuum, and it has been reported that the destruction of this region markedly suppresses copulatory behavior (2,3,4,7,9,10,12,17). With reference to the mechanisms that may be involved, the corticomedial amygdala, which is a relay station between the olfactory-vomeronasal system and the mPOA-AH continuum, is thought to be an important part of the brain that regulates copulatory behavior. Earlier reports on monkeys and cats have indicated that extensive destruction of the temporal lobe, including the amygdala, resulted in hypersexuality. Similar experiments using male rodents, however, have failed to confirm the results seen in monkeys and cats. Further, basolateral amygdaloid lesions have been reported not to influence copulation in the rat (7,8) or in the hamster (14), whereas lesions in the corticomedial area of the amygdala have caused disturbances in the copulatory behavior of the male rat. Rats with corticomedial amygdaloid lesions have shown a prolonged latency of ejaculation (7,8). As for the hamster, it has been reported that lesions in the rostral corticomedial amygdala brought about a more severe suppression of copulatory behavior than lesions in the caudal corticomedial amygdala (14,16). The purpose of this experiment was to examine the effect of amygdaloid lesions on the copulatory behavior in sexually inexperienced male rats. Thus, three types of bilateral lesions were made in the amygdala--medial amygdaloid lesions, cortical amygdaloid lesions, and basolateral amygdaloid lesions--after which the resultant copulatory behavior was then studied.
Thirty-four sexually inexperienced male Wistar rats supplied by the Takasugi Animal Farm (Saitama, Japan) were used for this study. The rats, ranging from 220-250 g in weight, were housed two to a cage under a controlled photoperiod (light:dark = 14:10 h) and temperature (23-24°C). Food and water were given ad lib. All animals were orchidectomized and simultaneously received one of the following bilateral lesions: medial amygdaloid lesion (MAL), cortical amygdaloid lesion (CAL), basolateral amygdaloid lesion (BLAL), or a sham operation. The lesions were stereotaxically produced by a radiofrequency lesion generator (Radionics Inc., Burlington, MA). After intramuscular anesthetization with ketamine hydrochloride and xylazine hydrochloride, each rat was placed in a stereotaxic apparatus that had an incisor bar set at 3.3 mm below the interaural line. The stereotaxic coordinates for each of the lesions, relative to bregma, were as follows: for the MAL, anteroposteriorly (AP) -2.8 mm, mediolaterally (ML) +3.3 mm, and dorsoventrally (DV) +9.0 mm; for the CAL, AP -2.8 mm, ML +4.3 mm, and DV +9.8 mm; and for the BLAL, AP -3.3 mm, ML +5.0 mm, and DV +8.0 mm. Each radiofrequency lesion was produced by application of a current sufficient to raise the temperature at the tip of the electrode to 54°C for 1 min. The sham-operated animals received the same surgery, except for the application of the current. Three weeks later, after ether anesthetization, all animals received a subcutaneous implantation of two silastic capsules (5 cm in length, 1.57 x 3.18 mm, ID X OD; Dow-Corning, #602-
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285) containing testosterone (Sigma) (13). Masculine sexual behavior then was observed four times every 5 days after the implantation of testosterone. For the behavioral observations, each animal was placed into a transparent observation cage (60 × 50 × 40 cm) and allowed to become adapted to it for 5 rain. At the end of the adaptation period, a highly-estrous Wistar female rat that had been ovariectomized and primed with estrogen and progesterone was placed into the male rat's cage. Each observation period then lasted ['or 30 min, with the estrous female replaced every 10 min to prevent the male from becoming bored by exposure to the same female partner. Mount and intromission frequencies (MF and IF, respectively) for 5 min were calculated in accordance with the number of mount and intromission responses expressed for each 30-min observation period or until the first ejaculation occurred. The number of ejaculations during the 30 min, the latencies to mount, the intromission, and the ejaculation responses (the interval from the introduction of the estrous female to the first expression of the response during the 30 min observation) also were recorded. After completing all behavioral observations, the animals were sacrificed and each brain was histologically examined to determine the precise localization of the lesions. Results of the MF and IF for each observation were statistically analyzed by ANOVA with Duncan's test for individual group comparisons (25). The latencies to mount, intromission, and ejaculation were compared among the groups by M a n n Whitney's U-test. The incidence of these responses among the groups was compared by using Fisher's exact probability test. RESULTS
Sites ( f Lesions The largest and the smallest lesions of each experimental group are schematically shown in Fig. l. Most of the animals with large medial amygdaloid lesions (MAL) had sustained complete damage in the medial nucleus of the amygdala [for
I
classification of the amygdaloid nuclei, see I)e Olmos, Alheid, and Beltramino (5)]. Some of these lesions included the medial parts of the central nucleus, the basomedial nucleus, and/or the cortical nucleus of the amygdala. However, the most rostral and caudal parts of the medial nucleus remained still intact in a number of animals with MAL, In the CAL animals, the damage sustained was generally located in the center of the cortical nucleus of the amygdala and reached to the ventral surface of tile brain. These lesions did not always extend to the most rostral and caudal part of the nucleus, and some extended into the ventral portion of the basomedial nucleus. The damage sustained in animals with BLAI, occupied large portions of the basolateral nucleus and the lateral nucleus of the amygdala. However, some of these lesions did not extend into the most ventral part of the basolateral nucleus, but included the adjacent part of the central nucleus, the basomedial nucleus, and/or the dor~l endopiriform nucleus.
Masculine Copulatory Behavior The means of the MFs and IFs, and the number of ejaculation responses in this experiment are shown in Fig. 2. Mounting frequencies were subjected to a 4 × 4 ANOVA analysis (lesion sites, between-subjects, repetition of observations, within-subjects). Main effects were found to be significant ['or lesion sites, F(3, 30) =4.19, p < 0.05, repetition of observation, f:(3, 90) : 18.73, p < 0.001, and interaction effect of lesion site vs. repetition of observation was significant, F(9, 90) :-- 2.88, p < 0.01. For intromission frequencies significant main effects were tbund tbr lesion sites, F(3, 30) ~ 3.24, p < 0.05: repetition of observation, F(3, 90) ~- 8.95, p < 0.001: and the interaction effect of lesion site vs. repetition of observation was significant, FIg, 90) :-: 2.67, p < 0.01. The sexually inexperienced sham-operated controls showed a gradual increase in copulatory activity through the series of four behavioral observations (MF, F(3, 90) = 14.5. p < 0.001; IF, /;(3, 90) = 12.33, p < 0.001). In the fourth observation, all
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FIG. 1. Schematic representations of coronal sections with bilateral lesions in each group. The largest and the smallest lesions are indicated by hatched and dotted areas, respectively. Values in the figure are distances posterior from the bregma in each section: (left) the medial amygdaloid lesion (MAL). (center) the cortical amygdaloid lesion (CAL). (right) the basolateral amygdaloid lesion (BLAL).
AMYGDALA LESIONS AND COPULATION IN RATS
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ejaculation responses seemed to have been greatly affected by the CAL. Ejaculations appeared to be completely suppressed by the CAL. Lesions in the medial amygdala resulted in a very severe deficit of copulatory behavior. Out of eight MAL rats, only two attempted a small number of mounts during the fourth observation and none of the remaining MAL rats attempted mounting (Table 1). Further, their MFs and IFs were significantly lower than that of the controls (MF, p < 0.01; IF, p < 0.05; Duncan's test). When an estrous female partner was put into their cage, they showed minor interest for the first several minutes, and then no interest at all, and would often yawn as they stayed in a corner of the observation cage. Each female seemed to be regarded as a peculiar intruder not a sexual partner. (In Table 1, the latency of mount responses in the MAL rats appears to be shorter during the third test than that of the other three tests. This caused a few incidental mounts of some MAL animals that is not significant.) DISCUSSION
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DAYS AFTER IMPLANTATION OF TESTOSTERONE
FIG. 2. Copulatory performances in each group during four behavioral observations after implantation of testosterone: mount frequency (number of mounts per 5 min); intromission frequency (the same as mount frequency); and number of ejaculations (per 30 min). MAL, the medial amygdaloid lesion; CAL, the cortical amygdaloid lesion; BLAL, the basolateral amygdaloid lesion; and CONT, sham-operated controls. of these animals showed mount and intromission responses, and more than one-third of the animals achieved ejaculations (Table 1). The BLAL rats also showed an increase in masculine sexual behavior (MF, F(3, 90) = 8.30, p < 0.001; IF, F(3, 90) = 3.95, p < 0.05). No significant difference was seen between the controls and the BLAL rats in the mount, intromission, and ejaculation parameters. The CAL rats also showed a gradual increase in MF, F(3, 90) = 4.57, p < 0.05, but not in IF, [F(3, 90) < 1]. In the fourth observation, four-fifths of the CAL rats manifested mount behavior, and their latencies did not differ from that of the controls. The IFs of the CAL animals (p < 0.05 in Duncan's test) and their incidence of intromission (see Table 1) were significantly lower than those of the control rats, and their intromission and
Consistent with previous studies (7,8), no evidence was found that BLAL exerted an influence on copulatory behavior in male rats. Thus it appears likely that the basolateral nucleus of the amygdala is not directly involved in male copulatory behavior. In contrast, it should be noted that bilateral lesions in the medial amygdala yielded a complete suppression of copulatory behavior in the sexually inexperienced male rat. Informatively, the medial nucleus of the amygdala is known to be the region showing male-female differences of synaptic organization (22,23), nuclear volume (21), and the neuropeptide systems (6,18,20). In our study, CAL rats showed a relatively mild suppression of copulatory behavior, and most of the CAL males displayed mount behavior at the fourth observation, although their intromission frequency was very low and none had an ejaculation throughout the four observations. Earlier studies of lesions in the corticomedial amygdala of the rat have reported that an impairment in the copulatory behavior after the lesions was confined to ejaculatory response, and that the animals manifested normal mount activity (7,8). In these studies, however, sexually experienced male rats were used. Thus, a possible reason for differences seen in the copulatory behavior of these rats versus our rats may be the difference in the degree of previous sexual experience. There is evidence suggesting that the lack of sexual experience has an important influence on copulatory activity of male rats. In this regard, the disruption of copulatory behavior after removal of the vomeronasal organ has been reported to be severer in sexually inexperienced males than in experienced male hamsters (19) and rats (24). Furthermore, lesions in the sexually dimorphic nucleus of the medial preoptic area seem to have a greater inhibitory effect on copulatory behavior in sexually inexperienced male rats (4) than in sexually experienced males (l). Although no direct comparison of behavioral response between sexually experienced and inexperienced males was made in the present study, the medial amygdala may play a critical role in regulating masculine copulatory behavior. In hamsters, it has been reported that medial amygdaloid lesion, especially of the rostral part, produces a severe impairment of copulatory behavior (14), and the facilitatory influence of copulatory behavior may course through the nonstrial pathway, the ventral amygdalofugal pathway (l 5). As the corticomedial amygdala occupies a relatively large area, a single insertion of an electrode might not have inflicted a lesion large enough to destroy the entire corticomedial amyg-
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942
TABLE l MEDIAN LATENCIES AND INCIDENCES OF MASCULINF COPI, JLAIORY RESPONSES
Mount Group MAL CAL BLAL Control
lntromission
I~iaculation
1
2
3
4
I
2
3
4
475 3/8 1272 2/10 306 4/8 896 4/8
607 2•8* 691 4/10 42 6/8 136 7/8
111 3•8* 15 5/10 28 5/8 286 8/8
966 2•8+ 19 8/10 35§ 6/8 10 8/8
-0/8 1387 1/10 415 4/8§ 571 2/8
-0/8~ 837 3/10" 173 5/8§ 134 6/8
1741 118t 55 4/10" 60 5/8§ 357 8/8
1387 1/85 172 6/10 220 6/8§ 83 8/8
0f8
0t8
0/8
()/8
O/I 0 1054 1/8 -0/8
O/10 792 1/8 1321 2/8
(/l I 0 936 3/8 902 2/8
O/10 889 3/8 970 3/8
*+:~ Significant differences from control animals at * 5%, +0.1%, or $1% level. § Significant differences from animals with MAL at 5% level. Values are latency in min and incidence/number in group.
dala. Another possible explanation for the discrepancy between earlier results and those of our study may be that the effect of the lesion depends upon whether the focus of the lesion is shifted cortically or medially in the corticomedial amygdala. In fact, in the figures of some earlier studies (7,8), the corticomedial (the basomedial-corticomedial) lesions appear to be located more towards the cortical amygdala and/or the basomedial amygdala, while a large part of the medial amygdala appears to have remained intact. Thus, it is possible to assume that the similarities observed in the behavioral response between our CAL rats and the animals given corticomedial lesions in previous studies might be due to the location of the lesion. It is also of interest that our M A L rats showed a more remarkable suppression of copulatory behavior than did our CAL rats. This suggests that there is a functional difference between the medial and the cortical amygdala. Neuroanatomical findings have shown that the main olfactory bulb projects to both the medial and cortical amygdala, whereas the accessory olfactory bulb gives distinctive projections to only the medial amygdala. A very localized injection of wheat-germ agglutinin conjugated horseradish peroxidase ( W G A - H R P ) in the accessory olfactory
bulb has been reported to have caused anterograde labels exclusively in the molecular layer of the medial nucleus but not in the cortical nucleus of the amygdala (11 ). Generally, it is believed that the main olfactory bulb receives inputs from the nasal epithelium with reference to the general olfactory sense, whereas the accessory olfactory bulb receives the vomeronasal chemosensory inputs, especially pheromonal information. These findings may provide morphological support for our results showing that the medial amygdala plays a more critical role in regulating masculine sexual behavior than does the cortical amygdala in sexually inexperienced male rats. However, further studies are needed to test whether this is also true in sexually experienced male rats. ACKNOWLEDGEMENTS
The author thanks Dr. Y. Arai for his very kind and useful guidance in this study and in the writing of this article. This study was supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan, and also by a training grant and fellowship from the Japan Society for the Promotion of Science.
REFERENCES
1. Allendash, G.; Gorski, R. Effects of discrete lesions of the sexually dimorphic nucleus of the preoptic area or other medial preoptic regions on the sexual behavior of male rats. Brain Res. Bull. 10: 147-154; 1983. 2. Chen, J. J.; Bliss, D. K. Effects of sequential preoptic and mamillary lesions on male rat sexual behavior. J. Comp. Physiol. Psychol. 87: 841-847; 1974. 3. Christensen, L. W.; Nance, D. M.; Gorski, R. A. Effects of hypothalamic and preoptic lesions on reproductive behavior in male rats. Brain Res. Bull. 2:137-141; 1977. 4. De Jonge, F. H.; Louwerse, A. L.; Ooms, M. P.; Evers, P.; Endert, E.; van de Poll, N. E. Lesions of the SDN-POA inhibit sexual behavior of male Wistar rats. Brain Res. Bull. 23:483492; 1989. 5. De Olmos, J.; Alheid, G. F.; Beltramino, C. A. Amygdala. In: Paxinos, G., ed. The rat nervous system, vol. 1. Forebrain and midbrain. Sydney: Academic Press; 1985:223-334. 6. De Vries, G. J. Sex differences in neurotransmitter systems. J. Neuroendocrinol. 2:1-13; 1990. 7. Giantonio, G. W.; Lund, N. L.; GeraU, A. A. Effect of diencephalic and rhinencephalic lesions on the male rat's sexual behavior. J. Comp. Physiol. Psychol. 73:38-46; 1970.
8. Hams, V. S.; Sachs, B. D. Copulatory behavior in male rats following amygdaloid lesions. Brain Res. 86:514-518; 1975. 9. Heimer, L.; Larsson, K. Drastic changes in the mating behavior in male rats following lesions in the junction ofdiencephalon and mesencephalon. Experientia 20:460-461; 1964. 10. Heimer, L.; Larsson, K. Impairment of mating behavior in male rats following lesions in the preoptic-anterior hypothalamic continuum. Brain Res. 3:248-263; 1966/67. 11. Ichikawa, M. Synaptic reorganization in the medial amygdaloid nucleus after lesion of the accessory olfactory bulb of adult rat: 1. Quantitative and electron microscopic study of the recovery of synaptic density. Brain Res. 420:243-252; 1987. 12. Kondo, Y.; Shinoda, A.; Yamanouchi, K.; Arai, Y. Role of septum and preoptic area in regulating masculine and feminine sexual behavior in male rats. Horm. Behav. 24:421-434; 1990. 13. Kusaka, S.; Nagasawa, H.; Yamanouchi, K.; Arai, Y. Induction of male sexual behaviors by administration of testosterone using silastic tubes in castrated male and female rats. Zool. Sci. 6:1037-1040; 1989. 14. Lehman, M. N.; Winans, S. S. Vomeronasal and olfactory pathways to the amygdala controlling male hamster sexual behavior: Autoradiographic and behavioral analyses. Brain Res. 240:27-41; 1982.
A M Y G D A L A LESIONS A N D C O P U L A T I O N IN R A T S 15. Lehman, M. N.; Winans, S. S. Evidence for a ventral non-strial pathway from the amygdala to the bed nucleus of the stria terminalis in the male golden hamster. Brain Res. 268:139-146; 1983. 16. Lehman, M. N.; Winans, S. S.; Power, J. B. Medial nucleus of the amygdala mediates chemosensory control of male hamster sexual behavior. Science 210:557-560; 1980. 17. Lisk, R. D. Copulatory activity of the male rat following placement of preoptic-anterior hypothalamic lesions. Exp. Brain Res. 161:129136; 1968. 18. Malsbury, C. W.; McKay, K. A sex difference in the pattern of substance-P like immunoreactivity in the bed nucleus of the stria terminalis. Brain Res. 420:365-370; 1987. 19. Meredith, M. Vomeronasal organ removal before sexual experience impairs male hamster mating behavior. Physiol. Behav. 36:737-743; 1986. 20. Micevych, P. E.; Akesson, T.; Elde, R. Distribution of cholecystokinin-immunoreactive cell bodies in the male and female rat: II.
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21. 22. 23. 24. 25.
Bed nucleus of the stria terminalis and amygdala. J. Comp. Neurol. 269:381-391; 1988. Mizukami, S.; Nishizuka, M.; Arai, Y. Sexual difference in nuclear volume and its ontogeny in the rat amygdala. Exp. Neurol. 79:569575; 1983. Nishizuka, M.; Arai, Y. Sexual dimorphism in synaptic organization in the amygdala and its dependence on neonatal hormone environment. Brain Res. 212:31-38; 1981. Nishizuka, M.; Arai, Y. Male-female difference in the intra-amygdaloid input to the medial amygdala. Exp. Brain Res. 52:328-332; 1983. Saito, T. R.; Moltz, H. Copulatory behavior of sexually naive and sexually experienced male rats following removal of the vomeronasal organ. Physiol. Behav. 37:507-510; 1986. Winer, B. J. Statistical principles in experimental design. New York: McGraw-Hill; 1971.
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printedin the USA.
Lesions of the Medial Amygdala Produce Severe Impairment of Copulatory Behavior in Sexually Inexperienced Male Rats YASUHIKO KONDO
Department of Anatomy, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received 1 April 1991 KONDO, Y. Lesions of the media/amygdala producesevereimpairment of copulatory behavior in sexuallyinexperiencedmale rats. PHYSIOLBEHAV51(5) 939-943, 1992.--The effectsofamygdaloid lesionson masculinecopulatorybehaviorwere examined in male rats. Sexuallyinexperienced male rats were castrated and subjected to bilateral lesions in one of the followingareas: the medial amygdala, the cortical amygdala, or the basolateral amygdala. Three weeks later, all rats received implantation of silastic capsules containing testosterone. Then, four observations of copulatory behavior were carried out every 5 days following the implantation of testosterone. Rats with medial amygdala lesions showed a severe deficit of copulatory behavior, whereas rats with basolateral amygdala lesions showed no change in the performance of copulation. As for rats with cortical amygdala lesions, although their copulatory behavior was impaired, the effect was confined to a deficit in intromission and ejaculation responses. These findings suggest that the medial amygdala plays a critical role in regulating masculine sexual behavior in the rat. Copulatory behavior
Amygdalalesion
Limbicsystem
Male rat
METHOD
A considerable body of evidence suggests that the most important region involved in the copulatory behavior of male rats is the medial preoptic-anterior hypothalamic (mPOA-AH) continuum, and it has been reported that the destruction of this region markedly suppresses copulatory behavior (2,3,4,7,9,10,12,17). With reference to the mechanisms that may be involved, the corticomedial amygdala, which is a relay station between the olfactory-vomeronasal system and the mPOA-AH continuum, is thought to be an important part of the brain that regulates copulatory behavior. Earlier reports on monkeys and cats have indicated that extensive destruction of the temporal lobe, including the amygdala, resulted in hypersexuality. Similar experiments using male rodents, however, have failed to confirm the results seen in monkeys and cats. Further, basolateral amygdaloid lesions have been reported not to influence copulation in the rat (7,8) or in the hamster (14), whereas lesions in the corticomedial area of the amygdala have caused disturbances in the copulatory behavior of the male rat. Rats with corticomedial amygdaloid lesions have shown a prolonged latency of ejaculation (7,8). As for the hamster, it has been reported that lesions in the rostral corticomedial amygdala brought about a more severe suppression of copulatory behavior than lesions in the caudal corticomedial amygdala (14,16). The purpose of this experiment was to examine the effect of amygdaloid lesions on the copulatory behavior in sexually inexperienced male rats. Thus, three types of bilateral lesions were made in the amygdala--medial amygdaloid lesions, cortical amygdaloid lesions, and basolateral amygdaloid lesions--after which the resultant copulatory behavior was then studied.
Thirty-four sexually inexperienced male Wistar rats supplied by the Takasugi Animal Farm (Saitama, Japan) were used for this study. The rats, ranging from 220-250 g in weight, were housed two to a cage under a controlled photoperiod (light:dark = 14:10 h) and temperature (23-24°C). Food and water were given ad lib. All animals were orchidectomized and simultaneously received one of the following bilateral lesions: medial amygdaloid lesion (MAL), cortical amygdaloid lesion (CAL), basolateral amygdaloid lesion (BLAL), or a sham operation. The lesions were stereotaxically produced by a radiofrequency lesion generator (Radionics Inc., Burlington, MA). After intramuscular anesthetization with ketamine hydrochloride and xylazine hydrochloride, each rat was placed in a stereotaxic apparatus that had an incisor bar set at 3.3 mm below the interaural line. The stereotaxic coordinates for each of the lesions, relative to bregma, were as follows: for the MAL, anteroposteriorly (AP) -2.8 mm, mediolaterally (ML) +3.3 mm, and dorsoventrally (DV) +9.0 mm; for the CAL, AP -2.8 mm, ML +4.3 mm, and DV +9.8 mm; and for the BLAL, AP -3.3 mm, ML +5.0 mm, and DV +8.0 mm. Each radiofrequency lesion was produced by application of a current sufficient to raise the temperature at the tip of the electrode to 54°C for 1 min. The sham-operated animals received the same surgery, except for the application of the current. Three weeks later, after ether anesthetization, all animals received a subcutaneous implantation of two silastic capsules (5 cm in length, 1.57 x 3.18 mm, ID X OD; Dow-Corning, #602-
939
940
KON L)()
285) containing testosterone (Sigma) (13). Masculine sexual behavior then was observed four times every 5 days after the implantation of testosterone. For the behavioral observations, each animal was placed into a transparent observation cage (60 × 50 × 40 cm) and allowed to become adapted to it for 5 rain. At the end of the adaptation period, a highly-estrous Wistar female rat that had been ovariectomized and primed with estrogen and progesterone was placed into the male rat's cage. Each observation period then lasted ['or 30 min, with the estrous female replaced every 10 min to prevent the male from becoming bored by exposure to the same female partner. Mount and intromission frequencies (MF and IF, respectively) for 5 min were calculated in accordance with the number of mount and intromission responses expressed for each 30-min observation period or until the first ejaculation occurred. The number of ejaculations during the 30 min, the latencies to mount, the intromission, and the ejaculation responses (the interval from the introduction of the estrous female to the first expression of the response during the 30 min observation) also were recorded. After completing all behavioral observations, the animals were sacrificed and each brain was histologically examined to determine the precise localization of the lesions. Results of the MF and IF for each observation were statistically analyzed by ANOVA with Duncan's test for individual group comparisons (25). The latencies to mount, intromission, and ejaculation were compared among the groups by M a n n Whitney's U-test. The incidence of these responses among the groups was compared by using Fisher's exact probability test. RESULTS
Sites ( f Lesions The largest and the smallest lesions of each experimental group are schematically shown in Fig. l. Most of the animals with large medial amygdaloid lesions (MAL) had sustained complete damage in the medial nucleus of the amygdala [for
I
classification of the amygdaloid nuclei, see I)e Olmos, Alheid, and Beltramino (5)]. Some of these lesions included the medial parts of the central nucleus, the basomedial nucleus, and/or the cortical nucleus of the amygdala. However, the most rostral and caudal parts of the medial nucleus remained still intact in a number of animals with MAL, In the CAL animals, the damage sustained was generally located in the center of the cortical nucleus of the amygdala and reached to the ventral surface of tile brain. These lesions did not always extend to the most rostral and caudal part of the nucleus, and some extended into the ventral portion of the basomedial nucleus. The damage sustained in animals with BLAI, occupied large portions of the basolateral nucleus and the lateral nucleus of the amygdala. However, some of these lesions did not extend into the most ventral part of the basolateral nucleus, but included the adjacent part of the central nucleus, the basomedial nucleus, and/or the dor~l endopiriform nucleus.
Masculine Copulatory Behavior The means of the MFs and IFs, and the number of ejaculation responses in this experiment are shown in Fig. 2. Mounting frequencies were subjected to a 4 × 4 ANOVA analysis (lesion sites, between-subjects, repetition of observations, within-subjects). Main effects were found to be significant ['or lesion sites, F(3, 30) =4.19, p < 0.05, repetition of observation, f:(3, 90) : 18.73, p < 0.001, and interaction effect of lesion site vs. repetition of observation was significant, F(9, 90) :-- 2.88, p < 0.01. For intromission frequencies significant main effects were tbund tbr lesion sites, F(3, 30) ~ 3.24, p < 0.05: repetition of observation, F(3, 90) ~- 8.95, p < 0.001: and the interaction effect of lesion site vs. repetition of observation was significant, FIg, 90) :-: 2.67, p < 0.01. The sexually inexperienced sham-operated controls showed a gradual increase in copulatory activity through the series of four behavioral observations (MF, F(3, 90) = 14.5. p < 0.001; IF, /;(3, 90) = 12.33, p < 0.001). In the fourth observation, all
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FIG. 1. Schematic representations of coronal sections with bilateral lesions in each group. The largest and the smallest lesions are indicated by hatched and dotted areas, respectively. Values in the figure are distances posterior from the bregma in each section: (left) the medial amygdaloid lesion (MAL). (center) the cortical amygdaloid lesion (CAL). (right) the basolateral amygdaloid lesion (BLAL).
AMYGDALA LESIONS AND COPULATION IN RATS
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20
ejaculation responses seemed to have been greatly affected by the CAL. Ejaculations appeared to be completely suppressed by the CAL. Lesions in the medial amygdala resulted in a very severe deficit of copulatory behavior. Out of eight MAL rats, only two attempted a small number of mounts during the fourth observation and none of the remaining MAL rats attempted mounting (Table 1). Further, their MFs and IFs were significantly lower than that of the controls (MF, p < 0.01; IF, p < 0.05; Duncan's test). When an estrous female partner was put into their cage, they showed minor interest for the first several minutes, and then no interest at all, and would often yawn as they stayed in a corner of the observation cage. Each female seemed to be regarded as a peculiar intruder not a sexual partner. (In Table 1, the latency of mount responses in the MAL rats appears to be shorter during the third test than that of the other three tests. This caused a few incidental mounts of some MAL animals that is not significant.) DISCUSSION
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DAYS AFTER IMPLANTATION OF TESTOSTERONE
FIG. 2. Copulatory performances in each group during four behavioral observations after implantation of testosterone: mount frequency (number of mounts per 5 min); intromission frequency (the same as mount frequency); and number of ejaculations (per 30 min). MAL, the medial amygdaloid lesion; CAL, the cortical amygdaloid lesion; BLAL, the basolateral amygdaloid lesion; and CONT, sham-operated controls. of these animals showed mount and intromission responses, and more than one-third of the animals achieved ejaculations (Table 1). The BLAL rats also showed an increase in masculine sexual behavior (MF, F(3, 90) = 8.30, p < 0.001; IF, F(3, 90) = 3.95, p < 0.05). No significant difference was seen between the controls and the BLAL rats in the mount, intromission, and ejaculation parameters. The CAL rats also showed a gradual increase in MF, F(3, 90) = 4.57, p < 0.05, but not in IF, [F(3, 90) < 1]. In the fourth observation, four-fifths of the CAL rats manifested mount behavior, and their latencies did not differ from that of the controls. The IFs of the CAL animals (p < 0.05 in Duncan's test) and their incidence of intromission (see Table 1) were significantly lower than those of the control rats, and their intromission and
Consistent with previous studies (7,8), no evidence was found that BLAL exerted an influence on copulatory behavior in male rats. Thus it appears likely that the basolateral nucleus of the amygdala is not directly involved in male copulatory behavior. In contrast, it should be noted that bilateral lesions in the medial amygdala yielded a complete suppression of copulatory behavior in the sexually inexperienced male rat. Informatively, the medial nucleus of the amygdala is known to be the region showing male-female differences of synaptic organization (22,23), nuclear volume (21), and the neuropeptide systems (6,18,20). In our study, CAL rats showed a relatively mild suppression of copulatory behavior, and most of the CAL males displayed mount behavior at the fourth observation, although their intromission frequency was very low and none had an ejaculation throughout the four observations. Earlier studies of lesions in the corticomedial amygdala of the rat have reported that an impairment in the copulatory behavior after the lesions was confined to ejaculatory response, and that the animals manifested normal mount activity (7,8). In these studies, however, sexually experienced male rats were used. Thus, a possible reason for differences seen in the copulatory behavior of these rats versus our rats may be the difference in the degree of previous sexual experience. There is evidence suggesting that the lack of sexual experience has an important influence on copulatory activity of male rats. In this regard, the disruption of copulatory behavior after removal of the vomeronasal organ has been reported to be severer in sexually inexperienced males than in experienced male hamsters (19) and rats (24). Furthermore, lesions in the sexually dimorphic nucleus of the medial preoptic area seem to have a greater inhibitory effect on copulatory behavior in sexually inexperienced male rats (4) than in sexually experienced males (l). Although no direct comparison of behavioral response between sexually experienced and inexperienced males was made in the present study, the medial amygdala may play a critical role in regulating masculine copulatory behavior. In hamsters, it has been reported that medial amygdaloid lesion, especially of the rostral part, produces a severe impairment of copulatory behavior (14), and the facilitatory influence of copulatory behavior may course through the nonstrial pathway, the ventral amygdalofugal pathway (l 5). As the corticomedial amygdala occupies a relatively large area, a single insertion of an electrode might not have inflicted a lesion large enough to destroy the entire corticomedial amyg-
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942
TABLE l MEDIAN LATENCIES AND INCIDENCES OF MASCULINF COPI, JLAIORY RESPONSES
Mount Group MAL CAL BLAL Control
lntromission
I~iaculation
1
2
3
4
I
2
3
4
475 3/8 1272 2/10 306 4/8 896 4/8
607 2•8* 691 4/10 42 6/8 136 7/8
111 3•8* 15 5/10 28 5/8 286 8/8
966 2•8+ 19 8/10 35§ 6/8 10 8/8
-0/8 1387 1/10 415 4/8§ 571 2/8
-0/8~ 837 3/10" 173 5/8§ 134 6/8
1741 118t 55 4/10" 60 5/8§ 357 8/8
1387 1/85 172 6/10 220 6/8§ 83 8/8
0f8
0t8
0/8
()/8
O/I 0 1054 1/8 -0/8
O/10 792 1/8 1321 2/8
(/l I 0 936 3/8 902 2/8
O/10 889 3/8 970 3/8
*+:~ Significant differences from control animals at * 5%, +0.1%, or $1% level. § Significant differences from animals with MAL at 5% level. Values are latency in min and incidence/number in group.
dala. Another possible explanation for the discrepancy between earlier results and those of our study may be that the effect of the lesion depends upon whether the focus of the lesion is shifted cortically or medially in the corticomedial amygdala. In fact, in the figures of some earlier studies (7,8), the corticomedial (the basomedial-corticomedial) lesions appear to be located more towards the cortical amygdala and/or the basomedial amygdala, while a large part of the medial amygdala appears to have remained intact. Thus, it is possible to assume that the similarities observed in the behavioral response between our CAL rats and the animals given corticomedial lesions in previous studies might be due to the location of the lesion. It is also of interest that our M A L rats showed a more remarkable suppression of copulatory behavior than did our CAL rats. This suggests that there is a functional difference between the medial and the cortical amygdala. Neuroanatomical findings have shown that the main olfactory bulb projects to both the medial and cortical amygdala, whereas the accessory olfactory bulb gives distinctive projections to only the medial amygdala. A very localized injection of wheat-germ agglutinin conjugated horseradish peroxidase ( W G A - H R P ) in the accessory olfactory
bulb has been reported to have caused anterograde labels exclusively in the molecular layer of the medial nucleus but not in the cortical nucleus of the amygdala (11 ). Generally, it is believed that the main olfactory bulb receives inputs from the nasal epithelium with reference to the general olfactory sense, whereas the accessory olfactory bulb receives the vomeronasal chemosensory inputs, especially pheromonal information. These findings may provide morphological support for our results showing that the medial amygdala plays a more critical role in regulating masculine sexual behavior than does the cortical amygdala in sexually inexperienced male rats. However, further studies are needed to test whether this is also true in sexually experienced male rats. ACKNOWLEDGEMENTS
The author thanks Dr. Y. Arai for his very kind and useful guidance in this study and in the writing of this article. This study was supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan, and also by a training grant and fellowship from the Japan Society for the Promotion of Science.
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