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Two types of lordosis-inhibiting systems in male rats: Dorsal raphe nucleus lesions and septal cuts
Physiology & Behavior, Vol. 56, No. 1, pp. 189-192, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All fights reserved 0031-9384/94 $6.00 + .00
Pergamon 0031-9384(94)E0056-A
Two Types of Lordosis-Inhibiting Systems in Male Rats: Dorsal Raphe Nucleus Lesions and Septal Cuts MASAKI KAKEYAMA AND KOREHITO YAMANOUCHI t
Neuroendocrinology, Department o f Basic Human Sciences, School of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359, Japan R e c e i v e d 8 June 1993 KAKEYAMA, M. AND K. YAMANOUCHI. Two types of lordosis-inhibiting systems in male rats: Dorsal raphe nucleus lesions and septal cuts. PHYSIOL BEHAV 56(1) 189-192, 1994.--To examine the functional relationships between the dorsal raphe nucleus and the septum in the inhibitory regulation of feminine sexual behavior in male rats, castrated male rats received destruction of the dorsal raphe nucleus (DRL), interruption of the septal outputs (ARD), or both DRL and ARD (DRL + ARD). All animals were treated with estradiol by using Silastic tubes, and feminine sexual behavior was observed every other day for 10 days. Most castrated control male rats did not show lordosis throughout the tests. In contrast, all of the males with DRL alone or ARD alone displayed lordosis, but the lordosis quotients (LQ) in these groups were lower than those of the female control group. On the other hand, DRL + ARD males showed higher LQs than the DRL or the ARD males, being comparable to control females. Thus, two types of strong inhibitory influence exist in the dorsal raphe nucleus and the septum, and resist the facilitation of feminine sexual behavior by estrogen in male rats. Furthermore, these inhibitory systems operate independently, because an additive effect of DRL and ARD in facilitating lordosis was clearly observed in DRL + ARD males. Lordosis
Dorsal raphe nucleus
Septum
Inhibitorysystem
Lesion
Male rat
be an another lordosis inhibitory center, with the serotonergic neural cells in this nucleus exerting the inhibitory influence. Serotonergic neurons in the dorsal raphe nucleus send their axons to forebrain nuclei that are known to be critical for lordosis regulation, such as the septum, preoptic area, ventromedial hypothalamus, and amygdala (2,14,19,22). In this study, to clarify functional relationships between the dorsal raphe nucleus and the septum, both the lesion of the dorsal raphe nucleus and the interruption of the septal outputs were performed in the same castrated male rat, and feminine sexual behavior was observed after treatment with estrogen.
IN male rats, strong inhibitory influences in the lordosis-regulating system have been reported to exist in the forebrain and the lower brain stem. The septum is thought to be one of the inhibitory centers for feminine sexual behavior in male rats, because destruction of the lateral septum (7,12) or interruption of the ventrolateral outputs (23-25) facilitated lordosis behavior. However, even if inhibition was reduced by surgical removal of the septum, the level of lordotic activity in male rats was not always the same as that in female rats (23,24). Similar phenomena have been observed in neonataUy androgenized female rats (6). Furthermore, longer exposure to an ovarian steroid is a prerequisite for facilitating lordosis in male rats with sept al lesions compared to female rats (12,13). There remains a possibility of the existence of inhibitory influences other than the septum. Recently, we reported that destruction of the dorsal raphe nucleus in the midbrain also facilitated lordosis in male rats, although the lordotic activity did not reach the female level (4). The dorsal raphe nucleus contains a large number of serotonergic neurons (14,19). Pharmacological studies indicate that serotonin plays an inhibitory role in lordosis-mediating systems in the male (5,9) and female rat (8,20,28). Because injection of the serotonin synthesis inhibitor, p-chlorophenylalanine, potentiated lordosis in estrogen-treated, castrated male rats, but not in male rats with the dorsal raphe nucleus lesion (5), the dorsal raphe nucleus may
METHOD
Male and female Wistar rats (240-280 g) were housed under conditions of controlled temperature (23-25*(2) and photoperiod (14:10 h, light:dark). Male rats were castrated and subjected to lesioning of the dorsal raphe nucleus (DRL) and/or transection of the ventral outputs of the septum (ARD: anterior roof deafferentation) under ether anesthesia. Eleven castrated males received DRL by a radiofrequency lesion generator (RGF-4A, Radionics Inc. Burlington, MA). Having set an incisor bar at 5 mm below the interaural line, an electrode (0.7 mm) was lowered stereotaxically 6.8 mm from the bregma level at a point 7.8 mm posterior to bregrna on the mid-
To whom requests for reprints should be addressed. 189
190
KAKEYAMA AND YAMANOUCHI
line. Current was applied and the temperature at the electrode tip was kept at 5 3 - 5 4 ° C for 1 min. Anterior roof deafferentation was performed in 10 males. An L-shaped Hal~sz knife, which has a horizontal blade of 2.5 mm, was lowered 7.0 m m from the bregma level at a point 1.5 m m anterior to bregma on the midline and rotated 180 ° anterohorizontally. In 11 male rats, both DRL and A R D were made simultaneously (DRL + ARD) [Fig. I(A)]. For comparison purposes, 13 castrated males without brain surgery (control group) and 12 ovariectomized females (female group) were prepared. Sham operations were not performed, because previous reports have shown no effects of sham DRL (4) and sham A R D (23,24). Four weeks after the surgery, all animals were implanted with two 3-cm-long Silastic tubes (1.57:3.18 mm, i.d.:o.d.; D o w C o m i n g No. 602-285) containing estradiol-17~ (F_~, Sigma). Be-
havioral tests were started 2 days after implantation of E2, and were carried out every other day for 10 days. For the behavioral tests, each experimental animal was placed in an observation cage with two vigorous males. The lordosis quotient (LQ; number of occurrences of lordosis + 10 mounts × 100) and the incidences of soliciting behavior (ear-wiggling and hopping) were recorded. At the end of the test, all animals were sacrificed and each brain was removed and fixed in 10% formalin solution. Subsequently, frozen sections stained with cresyl fast violet were made to determine the precise localization of the lesion and the cut. The differences between the mean LQs among groups were analyzed by using analysis of variance ( A N O V A ) followed by Scheffe's F-test, and the data regarding the incidence of lordosis and the soliciting behavior were evaluated by means of the chisquare test.
A
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'-.,;'-'.yMH
la
FIG. 1. (A) Schematic representation of the surgical cut and lesion in the midsagittal section of rat brain. The horizontal black bar just above the anterior commissure indicates anterior roof deafferentation (ARD). The dotted areas indicate the area of the dorsal raphe nucleus lesion (DRL). Abbreviations: ac, anterior commissure; AH, anterior hypothalamic nucleus; cc, corpus callosum; LS, lateral septum; POA, preoptic area; VMH, ventromedial hypothalamic nucleus; xscp, decussation of superior cerebellar peduncle. The drawings are modified from the stereotaxic atlas of Paxinos and Watson (15). Photomicrographs of representative frontal sections of the forebrain (B) and the midbrain (C) in a male with both DRL and ARD (No. 177, cresyl fast violet stain). Arrows indicate the ARD or the DRL.
LORDOSIS IN MALE RATS: DORSAL RAPHE N. AND SEPTUM
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[31P/BIdllr i l ~
Cont~ (13)
•
DRL (12)
•
ARD (10)
•
DRL+.A,RD (11)
0
Female-Control (12)
OrF'J
FIG. 2. The mean lordosis quotient in each group. Behavioral tests were started 2 days after implantation of Silastic tubes containing estradiol (E2) and were carried out every other day for 10 days. Numbers in parentheses are numbers of rats used. Groups: control; castrated males, DRL; dorsal raphe nucleus lesion, ARD; anterior roof deafferentation, DRL + ARD; both DRL and ARD, female; ovariectomized females. RESULTS
The mean LQ in each group is shown in Fig. 2. In the female group, the mean LQ increased from the first test (9.2 ___3.1) to the second test (77.5 _ 3.5), and reached 100 at the final test. In contrast, only three of 13 control males showed lordotic responses throughout the tests, with low LQ score, compared to other groups [F(4, 53) = 73.6, p < 0.05 in the last test]. The highest mean LQ value of this group was 13.8 --- 7.3 at the final test. In the DRL group, seven of 11 males displayed lordosis in the first test and all DRL males showed it in the second test and thereafter. The mean LQ of this group increased gradually, and reached a plateau at the fourth test. The highest LQ value in the DRL group was 69.1 _+ 5.3 in the final test, but was lower than that of the female group, F(1, 21) = 37.3, p < 0.001. In the ARD group, the mean LQ in the first test was comparable to that of the females. The mean LQs in this group increased gradually to the final test, but were lower than those of the female group, except for the final test. In the final test, the mean LQ (95.0 __.2.2) was significantlyhigher than that of the DRL group, F(1, 19) = 18.9, p < 0.001. All ARD males showed lordosis from the third to the final test. On the other hand, in the DRL + ARD group, all males displayed lordosis behavior throughout the tests, and the mean LQ in the first test (42.7 ___ 6.7) was higher than that of the other groups, F(4, 53) = 17.5,p < 0.001. The mean LQ of this group increased to 83.6 +- 5.9 in the second test. There were no statistically significant differences in mean LQ between DRL + ARD and female groups in all tests, except for the first test. In soliciting behavior, throughout the tests, three of 12 females showed ear-wiggling and two females displayed hopping. None of the control, ARD or DRL males, except one ARD, displayed soliciting behavior. In contrast, five of 12 DRL + ARD males showed ear-wiggling (p < 0.05, vs. control, DRL and ARD groups), and three DRL + ARD males displayed hopping behavior. In the behavioral tests, the locomotor activities did not seem to differ among groups, although there were no systematic analyses pursued in the present experiment. The precise locations of DRL and/or ARD were determined histologically. The DRL was located on the midline of the mid-
brain at the level of the locus ceruleus [Fig. I(C)]. The DRL damaged the dorsal raphe nucleus and the adjacent area, including a part of the ventral central gray. In most of the DRL males, the rostral tips of the dorsal raphe nucleus remained intact. The medial central gray was partially damaged by the insertion of the electrode. The ARD was located just above the anterior commissure and extended from the end of the anterior commissure to the middle level of the island of Calleja [Fig I(B)]. In some ARD and DRL + ARD males, a small part of the septum was damaged due to insertion of the Halfisz knife. DISCUSSION
In the present experiment, both the DRL and ARD facilitated lordosis in male rats. However, the levels of feminine sexual activity in these males with DRL or ARD were lower than those of the female rat. These results confirm the existence of an inhibitory influence on the lordosis-mediating system in the dorsal raphe nucleus (4,5) and the lateral septum (7,12,24,25) in male rats. Furthermore, ARD males showed maximum LQ, but the LQs in DRL males did not reach maximum level in the final test. In our previous report (4), LQs in DRL males reach a plateau at a lower level than in females. These facts suggest the possibility of different roles of inhibition in the regulation of lordosis between the dorsal raphe nucleus and the septum. Moreover, male rats with both DRL and ARD showed higher LQ than males with DRL alone or ARD alone, and the values of the mean LQ and the increasing pattern of LQ after the implantation of estrogen in this group was comparable to those of females. Soliciting behavior was also observed in DRL + ARD males but not in DRL or ARD males in the present experiment. This indicates that two different types of inhibitory systems resist the induction of feminine sexual behavior by estrogen and by tactile stimulation by males. These strong inhibitory mechanisms for lordosis in the brain are thought to develop during the neonatal period under the influence of androgen, because injection of androgen to neonatal females and castration in neonatal males resulted in suppression of lordosis and manifestation of lordosis in adulthood, respectively
192
KAKEYAMA AND YAMANOUCHI
(16). It has been reported that A R D recovered lordosis suppressed by the neonatal treatment of androgen in female rats (6). The dorsal raphe nucleus has been shown to contain large numbers of serotonergic neurons (14,19). The facilitatory effect of DRL on lordosis in male rats is a result of destruction of the serotonergic cells in the dorsal raphe nucleus, because injections of serotonin synthesis inhibitor, p-chlorophenylalanine, facilitated lordosis behavior in estrogen-primed, castrated male rats, but not in male rats with DRL (5). Serotonergic axons of the dorsal raphe nucleus terminate in the preoptic area, the septum, the amygdala, and the ventromedial hypothalamic nucleus (2,14,19,22). These areas in the forebraln have been reported to play an important role in regulating lordosis behavior (17,27). Because an additive effect of DRL and ARE) was observed in the present results, the septum is not regarded as being a possible functional focus of serotonergic neural fibers of the dorsal raphe nucleus in the regulation of lordosis in male rats. Rather, the ventral hypothalamus is one of the candidates of the focus, because the direct administration of serotonergic neurotoxin or receptor blocker into the ventromedial hypothalamic nucleus elicited lordosis in male (11) and female (1,3) rats. On the other hand, it has been suggested that the ventral output fibers of the septum play a role in carrying inhibitory signals
for lordosis, based on present and previous results for A R D (24,25). This septal inhibitory signal is thought to descend through the dorsal preoptic area and the medial forebrain bundle to the lower brain stem, based on results indicating that destruction of the dorsal preoptic area (6,18,21) or medial forebrain bundle (10,26) facilitated feminine sexual behavior. Although the terminating site of the septal lordosis-inhibitory signals is not yet known, the dorsal raphe nucleus can be excluded as a candidate, because both effects of the DRL and A R D were observed simultaneously. Thus, the lordosis-inhibiting system of the dorsal raphe nucleus involves an ascending serotonergic neural system from the lower brain stem to the forebrain. In contrast, the descending medial forebrain bundle is concerned with the septal inhibitory system. These two different types of inhibitory system operate independently of each other in male rat brain. Further experiments are needed to clarify the neural pathways and contact points of these inhibitory systems. ACKNOWLEDGEMENTS This study was supported by Grants in Aid to M.K. (053761) and K.Y. (05640756) from the Minstry of Education, Science, and Culture of Japan, and from Waseda University (93A-196).
REFERENCES 1. Aiello-Zaldivar, M.; Luine, V. N.; Frankfurt, M. 5,7-DHT facilitated lordosis: Effects of 5-HT agonist. Neuroreport 3:542-544; 1992. 2. Azmitia, E. C.; Segal, M. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. Comp. Neurol. 179:641-668; 1978. 3. Frankfurt, M.; Penner, K.; Azmitia, E.; Luine, V. Intrahypothalamic 5,7-dihydroxytryptamine: Temporal analysis of effects on 5-hydroxytryptamine content in brain nuclei and on facilitated lordosis behavior. Brain Res. 340:127-133; 1985. 4. Kakeyama, M.; Yamanouchi, K. Lordosis in male rats: The facilitatory effect of dorsal raphe nucleus lesions. Physiol. Behav. 51:181-184; 1992. 5. Kakeyama, M.; Yamanouchi, K. Female sexual behaviors in male rats with dorsal raphe nucleus lesions: Treatment with p-chlorophenylalanine. Brain Res. Bull. 30:705-709; 1993. 6. Kondo, Y.; Shineda, A.; Yamanouchi, K.; Arai, Y. Recovery of lordotic activity by dorsal deafferentation of the preoptic area in male and androgenized female rats. Physiol. Behav. 37:495-498; 1986. 7. 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. 8. Meyerson, B. J.; Lewander, T. Serotonin synthesis inhibition and estrous behavior in female rats. Life Sci. 9:661-671; 1970. 9. Meyerson, B. J.; Malmniis, C-O. Brain monoamines and sexual behaviour. In: Hutchison, J. B., ed.. Biological determinants of sexual behaviour. New York: Wiley; 1978:521-554. 10. Modianos, D. T.; Delia, H.; Pfaff, D. W. Lordosis in female rats following medial forebrain bundle lesions. Behav. Biol. 18:135141; 1976. ll. Moreines, J.; Kelton, M.; Luine, V. N.; Pfaff, D. W.; McEwen, B. S. Hypothalamic serotonin lesions unmask hormone responsiveness of lordosis behavior in adult male rats. Neuroendocrinology 47:453-458; 1988. 12. Nance, D. M.; Shryne, J.; Gorski, R. A. Facilitation of female sexual behavior in male rats by septal lesions: An interaction with estrogen. Horm. Behav. 6:289-299; 1975. 13. Nance, D. M.; Phelps, C.; Shryne, J. E.; Gorski, R. A. Alterations by estrogen and hypothyroidism in the effects of septal lesions on lordosis behavior of male rats. Brain Res. Bull. 2:49-53; 1977. 14. Parent, A.; Descarries, L.; Beaudet, A. Organization of ascending serotonin systems in the adult rat brain. A radioautographic study
15. 16. 17. 18. 19. 20. 21. 22. 23.
24. 25. 26. 27. 28.
after intraventricular administration of 3[H]5-hydroxytryptamine. Neuroscience 6:115-138; 1981. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. 2nd ed. New York: Academic Press; 1986. Pfaff, D. W.; Zigmond, R. E. Neonatal androgen effects on sexual and non-sexual behavior of adult rats tested under various hormone regimes. Neuroendocrinology 7:129-145; 1971. Pfaff, D. W. Estrogens and brain function. New York: SpringerVerlag; 1980. Rodoriguez-Sierra, J. F.; Terasawa, E. Lesions of the preoptic area facilitate lordosis behavior in male and female guinea pigs. Brain Res. 4:513-517; 1979. T0rk, I. Raphe nuclei and serotonin containing systems. In: Paxinos, G., ed. The rat nervous system, vol. 2, hindbrain and spinal cord. New York: Academic Press; 1985:43-78. Uphouse, L.; Caldarola-Pastuszka, M.; Droge, M. 8-OH-DPAT in the midbrain central gray inhibits lordosis behavior. Pharmacol. Biochem. Behav. 43:833-838; 1992. Van de Poll, N. E.; Van Dis, H. The effect of medial preoptic-anterior hypothalamic lesions on bisexual behavior of the male rat. Brain Res. Bull. 4:505-511; 1977. Vertes, R.P. A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat. J. Comp. Neurol. 313:643-668; 1991. Yamanouchi, K.; Arai, Y. Female lordosis pattern in the male rat induced by estrogen and progesterone: Effect of interruption of the dorsal inputs to the preoptic area and hypothalamus. Endocrinol. Jpn. 22:243-246; 1975. Yamanouchi, K.; Arai, Y. Lordosis behavior in male rats: Effect of deafferentation in the preoptic area and hypothalamus. J. Endocrinol. 76:381-382; 1978. Yamanouchi, K.; Arai, Y. Presence of a neural mechanism for the expression of female sexual behaviors in the male rat brain. Neuroendocrinology 40:393-397; 1985. Yamanouchi, K.; Arai, Y. Lordosis-inhibiting pathway in the lateral hypothalamus: Medial forebrain bundle (MFB). Zool. Sci. 6:141145; 1989. Yamanouchi, K.; Matsumoto, A.; Arai, Y. Neural and hormonal control of lordosis behavior in the rat. Zool. Sci. 2:617-627; 1985. Zemlan, F. P.; Ward, I. L.; Crowley, W. R.; Margules, D. L. Activation of lordotic responding in female rats by suppression of serotonergic activity. Science 179:1010-1011; 1973.
Pergamon 0031-9384(94)E0056-A
Two Types of Lordosis-Inhibiting Systems in Male Rats: Dorsal Raphe Nucleus Lesions and Septal Cuts MASAKI KAKEYAMA AND KOREHITO YAMANOUCHI t
Neuroendocrinology, Department o f Basic Human Sciences, School of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359, Japan R e c e i v e d 8 June 1993 KAKEYAMA, M. AND K. YAMANOUCHI. Two types of lordosis-inhibiting systems in male rats: Dorsal raphe nucleus lesions and septal cuts. PHYSIOL BEHAV 56(1) 189-192, 1994.--To examine the functional relationships between the dorsal raphe nucleus and the septum in the inhibitory regulation of feminine sexual behavior in male rats, castrated male rats received destruction of the dorsal raphe nucleus (DRL), interruption of the septal outputs (ARD), or both DRL and ARD (DRL + ARD). All animals were treated with estradiol by using Silastic tubes, and feminine sexual behavior was observed every other day for 10 days. Most castrated control male rats did not show lordosis throughout the tests. In contrast, all of the males with DRL alone or ARD alone displayed lordosis, but the lordosis quotients (LQ) in these groups were lower than those of the female control group. On the other hand, DRL + ARD males showed higher LQs than the DRL or the ARD males, being comparable to control females. Thus, two types of strong inhibitory influence exist in the dorsal raphe nucleus and the septum, and resist the facilitation of feminine sexual behavior by estrogen in male rats. Furthermore, these inhibitory systems operate independently, because an additive effect of DRL and ARD in facilitating lordosis was clearly observed in DRL + ARD males. Lordosis
Dorsal raphe nucleus
Septum
Inhibitorysystem
Lesion
Male rat
be an another lordosis inhibitory center, with the serotonergic neural cells in this nucleus exerting the inhibitory influence. Serotonergic neurons in the dorsal raphe nucleus send their axons to forebrain nuclei that are known to be critical for lordosis regulation, such as the septum, preoptic area, ventromedial hypothalamus, and amygdala (2,14,19,22). In this study, to clarify functional relationships between the dorsal raphe nucleus and the septum, both the lesion of the dorsal raphe nucleus and the interruption of the septal outputs were performed in the same castrated male rat, and feminine sexual behavior was observed after treatment with estrogen.
IN male rats, strong inhibitory influences in the lordosis-regulating system have been reported to exist in the forebrain and the lower brain stem. The septum is thought to be one of the inhibitory centers for feminine sexual behavior in male rats, because destruction of the lateral septum (7,12) or interruption of the ventrolateral outputs (23-25) facilitated lordosis behavior. However, even if inhibition was reduced by surgical removal of the septum, the level of lordotic activity in male rats was not always the same as that in female rats (23,24). Similar phenomena have been observed in neonataUy androgenized female rats (6). Furthermore, longer exposure to an ovarian steroid is a prerequisite for facilitating lordosis in male rats with sept al lesions compared to female rats (12,13). There remains a possibility of the existence of inhibitory influences other than the septum. Recently, we reported that destruction of the dorsal raphe nucleus in the midbrain also facilitated lordosis in male rats, although the lordotic activity did not reach the female level (4). The dorsal raphe nucleus contains a large number of serotonergic neurons (14,19). Pharmacological studies indicate that serotonin plays an inhibitory role in lordosis-mediating systems in the male (5,9) and female rat (8,20,28). Because injection of the serotonin synthesis inhibitor, p-chlorophenylalanine, potentiated lordosis in estrogen-treated, castrated male rats, but not in male rats with the dorsal raphe nucleus lesion (5), the dorsal raphe nucleus may
METHOD
Male and female Wistar rats (240-280 g) were housed under conditions of controlled temperature (23-25*(2) and photoperiod (14:10 h, light:dark). Male rats were castrated and subjected to lesioning of the dorsal raphe nucleus (DRL) and/or transection of the ventral outputs of the septum (ARD: anterior roof deafferentation) under ether anesthesia. Eleven castrated males received DRL by a radiofrequency lesion generator (RGF-4A, Radionics Inc. Burlington, MA). Having set an incisor bar at 5 mm below the interaural line, an electrode (0.7 mm) was lowered stereotaxically 6.8 mm from the bregma level at a point 7.8 mm posterior to bregrna on the mid-
To whom requests for reprints should be addressed. 189
190
KAKEYAMA AND YAMANOUCHI
line. Current was applied and the temperature at the electrode tip was kept at 5 3 - 5 4 ° C for 1 min. Anterior roof deafferentation was performed in 10 males. An L-shaped Hal~sz knife, which has a horizontal blade of 2.5 mm, was lowered 7.0 m m from the bregma level at a point 1.5 m m anterior to bregma on the midline and rotated 180 ° anterohorizontally. In 11 male rats, both DRL and A R D were made simultaneously (DRL + ARD) [Fig. I(A)]. For comparison purposes, 13 castrated males without brain surgery (control group) and 12 ovariectomized females (female group) were prepared. Sham operations were not performed, because previous reports have shown no effects of sham DRL (4) and sham A R D (23,24). Four weeks after the surgery, all animals were implanted with two 3-cm-long Silastic tubes (1.57:3.18 mm, i.d.:o.d.; D o w C o m i n g No. 602-285) containing estradiol-17~ (F_~, Sigma). Be-
havioral tests were started 2 days after implantation of E2, and were carried out every other day for 10 days. For the behavioral tests, each experimental animal was placed in an observation cage with two vigorous males. The lordosis quotient (LQ; number of occurrences of lordosis + 10 mounts × 100) and the incidences of soliciting behavior (ear-wiggling and hopping) were recorded. At the end of the test, all animals were sacrificed and each brain was removed and fixed in 10% formalin solution. Subsequently, frozen sections stained with cresyl fast violet were made to determine the precise localization of the lesion and the cut. The differences between the mean LQs among groups were analyzed by using analysis of variance ( A N O V A ) followed by Scheffe's F-test, and the data regarding the incidence of lordosis and the soliciting behavior were evaluated by means of the chisquare test.
A
ARD I
~
r /
."
'-.,;'-'.yMH
la
FIG. 1. (A) Schematic representation of the surgical cut and lesion in the midsagittal section of rat brain. The horizontal black bar just above the anterior commissure indicates anterior roof deafferentation (ARD). The dotted areas indicate the area of the dorsal raphe nucleus lesion (DRL). Abbreviations: ac, anterior commissure; AH, anterior hypothalamic nucleus; cc, corpus callosum; LS, lateral septum; POA, preoptic area; VMH, ventromedial hypothalamic nucleus; xscp, decussation of superior cerebellar peduncle. The drawings are modified from the stereotaxic atlas of Paxinos and Watson (15). Photomicrographs of representative frontal sections of the forebrain (B) and the midbrain (C) in a male with both DRL and ARD (No. 177, cresyl fast violet stain). Arrows indicate the ARD or the DRL.
LORDOSIS IN MALE RATS: DORSAL RAPHE N. AND SEPTUM
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Cont~ (13)
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DRL (12)
•
ARD (10)
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DRL+.A,RD (11)
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Female-Control (12)
OrF'J
FIG. 2. The mean lordosis quotient in each group. Behavioral tests were started 2 days after implantation of Silastic tubes containing estradiol (E2) and were carried out every other day for 10 days. Numbers in parentheses are numbers of rats used. Groups: control; castrated males, DRL; dorsal raphe nucleus lesion, ARD; anterior roof deafferentation, DRL + ARD; both DRL and ARD, female; ovariectomized females. RESULTS
The mean LQ in each group is shown in Fig. 2. In the female group, the mean LQ increased from the first test (9.2 ___3.1) to the second test (77.5 _ 3.5), and reached 100 at the final test. In contrast, only three of 13 control males showed lordotic responses throughout the tests, with low LQ score, compared to other groups [F(4, 53) = 73.6, p < 0.05 in the last test]. The highest mean LQ value of this group was 13.8 --- 7.3 at the final test. In the DRL group, seven of 11 males displayed lordosis in the first test and all DRL males showed it in the second test and thereafter. The mean LQ of this group increased gradually, and reached a plateau at the fourth test. The highest LQ value in the DRL group was 69.1 _+ 5.3 in the final test, but was lower than that of the female group, F(1, 21) = 37.3, p < 0.001. In the ARD group, the mean LQ in the first test was comparable to that of the females. The mean LQs in this group increased gradually to the final test, but were lower than those of the female group, except for the final test. In the final test, the mean LQ (95.0 __.2.2) was significantlyhigher than that of the DRL group, F(1, 19) = 18.9, p < 0.001. All ARD males showed lordosis from the third to the final test. On the other hand, in the DRL + ARD group, all males displayed lordosis behavior throughout the tests, and the mean LQ in the first test (42.7 ___ 6.7) was higher than that of the other groups, F(4, 53) = 17.5,p < 0.001. The mean LQ of this group increased to 83.6 +- 5.9 in the second test. There were no statistically significant differences in mean LQ between DRL + ARD and female groups in all tests, except for the first test. In soliciting behavior, throughout the tests, three of 12 females showed ear-wiggling and two females displayed hopping. None of the control, ARD or DRL males, except one ARD, displayed soliciting behavior. In contrast, five of 12 DRL + ARD males showed ear-wiggling (p < 0.05, vs. control, DRL and ARD groups), and three DRL + ARD males displayed hopping behavior. In the behavioral tests, the locomotor activities did not seem to differ among groups, although there were no systematic analyses pursued in the present experiment. The precise locations of DRL and/or ARD were determined histologically. The DRL was located on the midline of the mid-
brain at the level of the locus ceruleus [Fig. I(C)]. The DRL damaged the dorsal raphe nucleus and the adjacent area, including a part of the ventral central gray. In most of the DRL males, the rostral tips of the dorsal raphe nucleus remained intact. The medial central gray was partially damaged by the insertion of the electrode. The ARD was located just above the anterior commissure and extended from the end of the anterior commissure to the middle level of the island of Calleja [Fig I(B)]. In some ARD and DRL + ARD males, a small part of the septum was damaged due to insertion of the Halfisz knife. DISCUSSION
In the present experiment, both the DRL and ARD facilitated lordosis in male rats. However, the levels of feminine sexual activity in these males with DRL or ARD were lower than those of the female rat. These results confirm the existence of an inhibitory influence on the lordosis-mediating system in the dorsal raphe nucleus (4,5) and the lateral septum (7,12,24,25) in male rats. Furthermore, ARD males showed maximum LQ, but the LQs in DRL males did not reach maximum level in the final test. In our previous report (4), LQs in DRL males reach a plateau at a lower level than in females. These facts suggest the possibility of different roles of inhibition in the regulation of lordosis between the dorsal raphe nucleus and the septum. Moreover, male rats with both DRL and ARD showed higher LQ than males with DRL alone or ARD alone, and the values of the mean LQ and the increasing pattern of LQ after the implantation of estrogen in this group was comparable to those of females. Soliciting behavior was also observed in DRL + ARD males but not in DRL or ARD males in the present experiment. This indicates that two different types of inhibitory systems resist the induction of feminine sexual behavior by estrogen and by tactile stimulation by males. These strong inhibitory mechanisms for lordosis in the brain are thought to develop during the neonatal period under the influence of androgen, because injection of androgen to neonatal females and castration in neonatal males resulted in suppression of lordosis and manifestation of lordosis in adulthood, respectively
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(16). It has been reported that A R D recovered lordosis suppressed by the neonatal treatment of androgen in female rats (6). The dorsal raphe nucleus has been shown to contain large numbers of serotonergic neurons (14,19). The facilitatory effect of DRL on lordosis in male rats is a result of destruction of the serotonergic cells in the dorsal raphe nucleus, because injections of serotonin synthesis inhibitor, p-chlorophenylalanine, facilitated lordosis behavior in estrogen-primed, castrated male rats, but not in male rats with DRL (5). Serotonergic axons of the dorsal raphe nucleus terminate in the preoptic area, the septum, the amygdala, and the ventromedial hypothalamic nucleus (2,14,19,22). These areas in the forebraln have been reported to play an important role in regulating lordosis behavior (17,27). Because an additive effect of DRL and ARE) was observed in the present results, the septum is not regarded as being a possible functional focus of serotonergic neural fibers of the dorsal raphe nucleus in the regulation of lordosis in male rats. Rather, the ventral hypothalamus is one of the candidates of the focus, because the direct administration of serotonergic neurotoxin or receptor blocker into the ventromedial hypothalamic nucleus elicited lordosis in male (11) and female (1,3) rats. On the other hand, it has been suggested that the ventral output fibers of the septum play a role in carrying inhibitory signals
for lordosis, based on present and previous results for A R D (24,25). This septal inhibitory signal is thought to descend through the dorsal preoptic area and the medial forebrain bundle to the lower brain stem, based on results indicating that destruction of the dorsal preoptic area (6,18,21) or medial forebrain bundle (10,26) facilitated feminine sexual behavior. Although the terminating site of the septal lordosis-inhibitory signals is not yet known, the dorsal raphe nucleus can be excluded as a candidate, because both effects of the DRL and A R D were observed simultaneously. Thus, the lordosis-inhibiting system of the dorsal raphe nucleus involves an ascending serotonergic neural system from the lower brain stem to the forebrain. In contrast, the descending medial forebrain bundle is concerned with the septal inhibitory system. These two different types of inhibitory system operate independently of each other in male rat brain. Further experiments are needed to clarify the neural pathways and contact points of these inhibitory systems. ACKNOWLEDGEMENTS This study was supported by Grants in Aid to M.K. (053761) and K.Y. (05640756) from the Minstry of Education, Science, and Culture of Japan, and from Waseda University (93A-196).
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