Forebrain Lordosis Inhibiting System and Serotonin Neuron in Female Rats: Effect of P-chloroamphetamine

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1 Endocrinol. Japon. 1982, 29 (4), Forebrain Lordosis Inhibiting System and Serotonin Neuron in Female Rats: Effect of P-chloroamphetamine KOREHITO YAMANOUCHI1, HIROSHI WATANABE2, REIKO OKADA3 AND YASUMASA ARAI Departments of Anatomy1, Obstetrics and Gynecology2 and Psychiatry3, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113 Abstract Relationship between forebrain lordosis inhibiting system and serotonergic mechanism was examined in estradiol benzoate (EB) and progesterone (P)-primed ovariectomized rats. In order to remove the forebrain inhibition of lordosis response, anterior roof deafferentation (ARD) which interrupts the dorsal inputs to the POA coming down anterior to the anterior commissure or medial preoptic area lesions (POA-L) were made. All experimental females pretreated with EB and P showed high levels of lordotic activity. Soon after the behavioral tests, the animals were injected with 10 mg/kg p-chloroamphetamine (PCA) intraperitoneally and tested again 30 min later. PCA strongly suppressed female sexual behavior. Regardless of the presence of ARD or POA-L, no PCA injected females showed lordosis. From these results, it can be said that serotonin may not directly act on the forebrain lordosis inhibiting system. Possible participation of serotonin in inhibiting lordosis behavior has been suggested by several investigators (see Meyerson & Malmnas, 1978; Crowley & Zemlan, 1981). Treatment with serotonin agonists suppressed lordosis in estrogen-progesterone primed ovariectomized rats (Everitt et al., 1974; Zemlan et al., 1977; Arai & Yamanouchi, 1979); conversely, serotonin antagonists facilitated lordosis behavior (Zemlan et al., 1973). From neuroanatomical evidence, most of the cell bodies of serotonergic neurons are found to be located in the lower brainstem (Dahlstrom & Fuxe, 1964; Parent et al., 1981). A number of their axons inervate the preoptic area (POA), the septum or the amygdala (Parent et al., 1981). On the other hand, there is evidence indicating that these preoptic and limbic areas exert an inhibitory influence on the display of lordosis, because destruction of the POA (Powers & Valenstein, 1972; Nance et al., 1977, Rodoriguez-Sierra & Terasawa, 1979), septum (Nance et al., 1975) or lateral amygdala (Masco & Carrer, 1980) and transection of the dorsal afferent fibers at the level of the anterior commissure (Yamanouchi & Arai, 1977) potentiated the display of lordosis in females. In the present study, in order to elucidate correlation between serotonergic neurons and the forebrain lordosis inhibiting system, the effects of p-chloroamphetamin (PCA, a serotonin agonist) on lordosis response were examined in estrogen-progesterone primed ovariectomized rats, in which the inhibitory influence was removed by POA lesion or dorsal deafferentation of the POA. Received June 24, 1982.

2 470 YAMANOUCHI et al. Endocrinel. Japon. August 1982 A B

3 Vol.29, No.4 LORDOSIS INHIBITING SYSTEM AND SEROTONIN 471 Materials and Methods Female Wistar rats ( g) housed under a control photoperiod (14/10 h, light/dark) and temperature (24 Ž) were castrated and subjected to the following brain surgery. For dorsal deafferentation of the POA, an L-shaped Halasz knife having a horizontal blade 2.5 mm long was lowered into the brain through the sagittal sinus to the level just above the anterior commissure and was rotated 180 anterohorizontally (anterior roof deafferentation, ARD, Fig. 1, see Yamanouchi & Arai, 1977). Bilateral medial POA lesions (POA-L) were made with a radiofrequency lesion generator (Radionics Inc., Balington, Massachusetts) (Fig. 1). For sham operation, the knife or electrode was lowered to the brain without further treatment. The sham group consisted of 5 ARD sham and 9 POA-L sham rats. A number of females castrated without brain surgery served as the control. Two or 3 week after the operations, behavioral tests were started. All ARD animals were treated with 2ƒÊg estradiol benzoate (EB, dissolved in 0.1 ml sesame oil) daily for 3 days and 0.5 mg progesterone (P, in 0.1 ml oil) on the fourth day 4-7 hrs before the behavioral test. Soon after the behavioral test, the animals were injected with 10 mg/kg DL-P-chloroamphetamine hydrochloride (PCA, Sigma, in saline) intraperitoneally and tested again 30 min later. In the animals with POA-L or POA-L sham and some of controls, two preliminary tests were performed before the PCA test described above to confirm the facilitatory effect of POA lesions on lordosis response. These animals were primed with low doses (0.2 and 0.5ƒÊg) of EB and 0.5 mg P and tested at two week intervals. In the behavioral test, each experimental female was placed in a plastic observation cage with two vigorous males. The ratio of lordosis response to 10 mounts times 100 (lordosis quotient, LQ) was recorded in each female. After the end of the behavioral tests, the precise localization of the cuts or lesions was determined histologically in each brain. The mean LQs were analyzed by the analysis of varianec (F-test) and then the Student's t-test or Chochran Cox method. For comparisson of incidence of lordosis, the x2 test with Yates' correction was used. Results In preliminary experiments to confirm the facilitatory effect of POA lesions (see Fig. 2), injections of 0.2ƒÊg EB for 3 days and P effectively induced lordosis in 6 out of 9 POA-L females, the mean LQ of the Fig. 2. Mean LQ and incidence of lordosis in the POA-L (POA lesion), sham and control groups in two preliminary tests. All females were treated with 0.2ƒÊg estradiol benzoate (EB) for 3 days and 0.5 mg progesterone in the first preliminary test. In the second preliminary test, the dose of EB was increased to 0.5ƒÊg. * P<0.05, vs control. **P<0.05, vs control and sham. Fig. 1. A. Schematic representation of anterior roof deafferentation (ARD) and POA lesions (POA-L) in midsagittal section. B. Representative frontal sections at the level of the POA and AH in a rat with POA-L. The number in each section show the distance (mm) from the level of interaural line. Abbreviations: AC, anterior commissure; AH, anterior hypothalamic nucleus; AL, anterior lobe of pituitary; AM, anteromeidal thalamic nucleus; ARC, arcuate nucleus; DBB, nucleus and diagonal band of Broca; FX, fornix; HPC, hippocampus; IP, interpeduncular nucleus; LS, lateral septal nucleus; MD, mediodorsal thalamic nucleus; MS, medial septal nucleus; OC, optic chiasma; PH, posterior hypothalamic nucleus; PL, posterior lobe of pituitary; POA, preoptic area, PV, paraventricular nucleus; RE nucleus reuniens; RH, nucleus rhomboideus thalami; SC, suprachiasmatic nucleus; VM, ventromecdial nucleus.

4 472 YAMANOUCHI et al. Endocrinol. Japon. August 1982 group being 40.0 }14.5. However, only 2 out of 8 females showed lordosis in the control group. The mean LQ (2.5 }1.6) was significantly lower than that of the POA-L group (P<0.05). When the does of EB was increased to 0.5ƒÊg, the mean LQs of the control and sham groups improved considerably (20.0 }11.5 and 44.0 Next, the effect of PCA on lordosis response was examined in POA-L or ARD females after EB-P priming. Daily injections with 2ƒÊg EB for 3 days and 0.5 mg P induced high scores of LQ in all of the 14 controls, 14 sham, 8 ARD and 9 POA-L females. The mean LQs of these group were 88.3 }3.9, 90.0 }3.5, 96.3 }3.8 and }0.0, respectively. Thirty minutes after the PCA injection, however, none of control, sham, ARD or POA-L females displayed lordosis. In the histological examinations (see Fig. 1), POA-L was found to extend from the anterior end of the medial preoptic area to the middle level of the anterior hypothalamic area. In 4 rats with POA-L, rostral periventricular POA seemed to be intact. The septum had no damage except the trace of the electrode. All cuts of ARD were located at the level of the anterior commissure. Discussion We have previously reported that the dorsal neural inputs to or through the POA which pass anterior to the anterior commissure exert a strong inhibitory effect on lordosis mediating system, based on the results that ARD effectively facilitated female sexual behavior in EB-P primed castrated female and male rats (Yamanouchi & Arai, 1977; 1978). Possible origins of this dorsal extrahypothalamic inhibitory influence are thought to be the septum, lateral amygdala and/or neocortex, because lesions of these areas potentiated lordosis behavior (Beach, 1944; Nance et al., 1975; Masco & Carrer, 1980). Consistent with the previous findings of Zemlan et al. (1977), PCA produced an immediate inhibition of lordosis response in EB-P primed ovariectomized rats. Furthermore, this inhibitory effect of PCA was evident regardless of the presence of ARD. PCA was also effective in suppressing lordsis in the femals bearing lesions in the POA. This area is thought to be one of the neural components sending lordosis inhibiting signals. Presumably, the dorsal afferents from the limbic area and the neural substrates in the POA may participate in forming the extrahypothalamic forebrain lordosis inhibiting system. Since the acute effect of PCA is to release serotonin from the synaptic vesicles and to inhibit its reuptake by a presynaptic site (Wong, et al., 1973), PCA acts as a serotonin agonist on the receptor. It is obvious therefore that the inhibition of lordosis following PCA administration in the present study is correlated with the serotonergic mechanism (Zemlan et al., 1977). However, serotonin does not seem to directly act on the extrahypothalamic forebrain lordosis inhibiting system, because PCA could effectively inhibit the display of lordosis even after the influence of the system had been removed by ARD or POA lesions. Rather, PCA may act on lordosis facilitating mechanisms such as the ventromedial hypothalamus (Mathews & Edwards, 1977; Barfield & Chen, 1977; Arai et al., 1978; Malsubury et al., 1977; Pfaff & Sakuma, 1979 a, b), the midbrain central gray (Sakuma & Pfaff, 1979 a, b) and the pontine periventricular gray (Yamanouchi & Arai, 1982). However, it is not clear which area is the real focus of serotonergic

5 Vol.29, No.4 LORDOSIS INHIBITING SYSTEM AND SEROTONIN 473 inhibition of lordosis response. Of these three areas, there is evidence indicating that serotonin may not directly be involved in the ventromedial hypothalamic mechanism, because the suppression of lordotic activity following lesions in this area could be reversed by reserpine treatment (Yamanouchi & Arai, 1979; Okada et al., 1980). Further study is needed to elucidate the neuroanatomical relationship between the lordosis mediating system and serotonergic system. Acknowledgements monoamines and sexual behavior. Wiley, New York. pp Nance, D. M., Shryne, N. J. and R. A. Gorski A. Ishizuka for his valuable assistance. (1975). Effects of septal lesion on behavioral sensitivity of female rats to gonadal hormones, Horm. This study was supported by Grants-in-Aid for Special Project Research ( ) awarded to Y. Behay. 6, A. and Encouragement of Young Scientists ( ) Nance, D. M., L. W. Christensen, J. E. Shryne and to K. Y. from the Ministry of Education, Science and Culture of Japan. References Arai, Y. and K. Yamanouchi (1979). Inhibition of lordosis by ergocornine in estrogen-progeseroneprimed ovariectomized rats. In Integrative Control Functions of the Brain, Vol.2 (Ito, M., N. Tsukahara, K. Kubota and K. Yagi eds.), Kodansha, Tokyo/Elsevier, Amsterdam. pp Arai, Y., K. Yamanouchi and A. Matsumoto (1978). Neuroendocrine regulation and sexual differentiation of lordosis behavior in rats. In Integrative control Functions of the Brain, Vol.1 (Ito, M., N. Tsukahara, K. Kubota and K. Yagi eds.), Kodansha, Tokyo/Elsevier, Amsterdam. pp Barfield, R. J. and J. J. Chen. (1977). Activation of estrous behavior in ovariectomized rats by intracerebral implants of estradiol benzoate. Endocrinoiogy 101, Beach, F. A. (1944). Effects of injury to the cerebral cortex upon sexually receptive behavior in the female rats. Psychosomat. Med. 6, Crowley, W. R. and F. P. Zemlan (1981). The neurochemical control of mating behavior. In Neuroendocrinology of Reproduction-Physiology and Behavior (Norman, A ed.), Plenum press, New York, pp Dhalstrom, A. and K. Fuxe (1964). Evidence for the existence of monoamine containing neurons in the central nervous system. Acta Physiol. Scand. Suppl. 232, Everitt, F. J., K. Fuxe and T. Hokfelt (1974). Inhibitory role of dapamine and 5-hydroxytryptamine in the sexual behaviour of female rats. Ear. J. Pharmacol. 29, Malsbury, C. W., L.-M. Kow and D. W. Pfaff (1977). Effects of medial hypothalamic lesions on the lordosis response and other behaviors in female golden hamster. Physiol. Behay. 19, Mathews, K. and Edwards (1977). Involvement of the ventromedial and anterior hypothalamic nuclei in the hormonal induction of receptivity in the female rat. Physiol. Behay. 19, Masco, D. H. and H. F. Carrer (1980). Sexual receptivity in female rats after lesion or stimulation in different amygdaloid nuclei. Phyiol. Behay. 24, Meyerson, B. J. and C.-O. Malmnas (1978). Brain R. A. Gorski (1977). Modifications in gonadotropin control and reproductive behavior in the female rat by hypothalamic and preoptic lesions. Brain Res. Bull. 2, Okada, R., H. Watanabe, K. Yamanouchi and Y. Arai (1980). Recovery of sexual receptivity in female rats with lesions of the ventromedial hypothalamus. Exp. Neurcl. 68, Parent, A., L. Descarries and A. Beaudet (1981). Organization of ascending serotonin systems in the adult rat brain. A radioautographic study intraventricular administration of [3H] 5 hydroxytryptamine. Neuroscience. 6, Pfaff, D. W. and Y. Sakuma (1979a). Facilitation of the lordosis reflex of female rats from the ventromedial nucleus of the hypothalamus. J. Physiol. (Lond.) 288, Pfaff, D. W. and Y. Sakuma (1979b). Deficit in the lordosis reflex of female rats caused by lesions in the ventromedial nuclues of the hypothalamus. J. Physiol. (Lond.) 288, Powers, B. and E. S. Valenstein (1972). Sexual receptivity: Facilitation by medial preoptic lesions in female rats. Science 175, Rodriguez-Sierra, J. F. and E. Terasawa (1979). Lesions of the preoptic area facilitate lordosis behavior in male and female guinea pigs. Brain Res. Bull. 4, Sakuma, Y. and D. W. Pfaff (1979a). Facilitation of female reproductive behavior from mesencephalic central grey in the rat. Am. J. Physiol. 237, R278-R284. Sakuma, Y. and D. W. Pfaff (1979b). Mesencephalic mechanisms for integration of female reproductive

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