MASAZUMI KAWAKAMI, FUKUKO KIMURA AND TAKASHI HIGUCHI 2nd Department of Physiology, Yokohama City University School of Medicine, Yokohama

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1 Effects of Electrical Stimulation of the Brain on Gonadotropin Secretion in Male Rats MASAZUMI KAWAKAMI, FUKUKO KIMURA AND TAKASHI HIGUCHI 2nd Department of Physiology, Yokohama City University School of Medicine, Yokohama Synopsis Immature male rats (26-29 days and days of age) and mature male rats ( days of age) were electrically stimulated through chronically implanted electordes, and serum and pituitary concentrations of LH, FSH and prolactin were measured by radioimmunoassay. In mature male rats, stimulation of the medial basal prechiasmatic area (PVA), which showed a significant facilitatory effect on LH and prolactin release in the female, also induced an increase of serum LH and a relatively increased concentration of prolactin in serum. Stimulation of the medial preoptic area (MPO), medial and lateral parts of the amygdala (AMYG) induced a slight increase in serum concentration of LH. These results indicate that there is no sexual difference in the ability to induce LH release in the PVA and MPO. But the effect of AMYG stimulation is quite difficult to be explained, because it was observed in the present experiment that the stimulation in acute preparation had no effect on LH release. Dorsal hippocampus exerted an inhibitory effect on FSH secretion in immature rats. Immature male AMYG stimulation decreased pituitary concentrations of LH and FSH, in contrast to the immature female, which was indicative of the existence of sexual difference in the same area. All the brain areas examined except the PVA decreased serum concentration of prolactin following the stimulation in mature rats, but in immature rats all the areas examined showed no effect and rather facilitated it. Little information is available on the problems of gonadotropin control mechanisms of the brain in the male, in spite of many excellent works on those in the female. This may be due to the lack of easily recognizable event correlating with changes of gonadotropin secretion in the male. Nevertheless it seems requisit to know the brain mechanism controlling gonadotropin secretion in the male, because the female brain mechanisms responsible for cyclic gonadotropin secretion may be clarified, partly, in comparison with the mechanisms responsible for the acyclic secretion. Received for publication August 8, *This sudy was supported by grants from the Ministry of Education, Japan. Another interest of ours in this experiment was in the medial basal prechiasmatic area (PVA). Because our recent experiments have demonstrated that the PVA of the female rat is able to induce the release of LH and prolactin, when stimulated (Kawakami et al., 1973a). This function of the PVA seemed to resemble that of the arcuate nucleus-median eminence region. Morphologically, both areas face to the third ventricle and are named the circumventricular organ (Hofer, 1958, Vigh et al., 1963). There were some structural differences found in the ependymal cells lining the anterior hypothalamus and the tuber cinereum associated with sexual maturity and sex (Knowles and Kumar, 1969; Kumar, 1968). In the present experiment, attempts were made to elucidate the ability of various brain

2 areas, including the above PVA, to cause gonadotropin to release from the anterior pituitary in response to the electrical stimulation in immature and mature male rats. Materials and Methods A total of one hundred and twenty-six Wistar immature and mature rats from 26 to 29 and from 34 to 36 days of age, and mature male rats from 150 to 210 days of age were used for the experiment. All animals were maintained in a room illuminated daily from 5:00 to 19:00. Concentric bipolar electrodes made of stainless steel were inserted stereotaxically under pentobarbital anesthesia into the brain according to the atlas of Sherwood and Timiras (1970) in the case of immature rats and to the atlas of Albe-Fessard et al. (1966) in the case of mature rats. In immature rats the operation was performed one day before the stimulation experiment. Mature rats were usually allowed to recover from the surgical operations for at least 3 weeks. Electrical stimulation was consisted of monophasic square waves, whose frequency was 100 Hz and duration 0.1 ms, and was applied for 30min at 30 sec alternating with 30 sec rest period. The current was monitored with a cathode ray oscilloscope and the intensity was 100 pa for the immature rats and mature rats of Experiment 1. Mature rats of Experiment 2 were stimulated with 200 pa, but for the PVA stimulation (Fig. 1) 100 pa was employed. Only Experiment 3 was performed in the acute preparation, e. g., the rats were stimulated through acutely inserted electrodes under ether anesthesia with 500 pa in the case of the MPO and 200,uA in the case of m-amyg. Immediately after the stimulation, animals were anesthetized with ether and blood samples were collected from the femoral vein, then anterior pituitary glands were quickly removed after decapitation. Pituitary glands were weighed and homogenized with 2.0ml of phophate buffered saline of ph7.5. Blood samples were centrifuged and the sera were separated. All samples were stored at-20 Ž until the assay was performed. The radioimmunoassay procedure for LH, FSH and prolactin was based on the methods described by Niswender et al., (1968) and also on the procedure indicated by NIAMD instructions. The amounts of LH, FSH and prolactin were expressed in terms of NIH-LH-S1, NIH-FSH-Sl and prolactin IU, respectively. Abbreviations: ARC: arcuate nucleus, d-hpc: dorsal hippocampus, 1-AMYG:lateral part of amygdala, m-amyg:medial part of amygdala, ME: median eminence, MPO: medial preoptic area, PVA: medial basal prechiasmatic area and v-hpc: ventral hippocampus. Results 1. Electrical stimulation of the brain in immature male rats 26 to 29-day-old male As shown in table 1, pituitary concentra- Fig. 1 Sites of electrodes in the PVA.

3 Table 1. Pituitary and serum concentrations of LH, FSH and prolactin in immature and mature rats. tion of FSH in 26 to 29-day-old males was approximately two times as high as that in 34 or 36-day-old males and the serum concentration was relatively higher as well. In these males, sham controls bearing electrodes implanted into the d-hpc a day before had an increased concentration of pituitary FSH and a decreased concentration of serum FSH (Table 2). Electrical stimulation of the d-hpc produced a remarkable decrease in serum FSH concentration compared with that of sham controls, without changes in pituitary concentration. Serum concentration of prolactin was rather increased by the d-hpc stimulation. 34 or 35-day-old male The pituitary of the 34 or 36-day-old male contained almost equivalent concentration of LH to that of the female of the same age (Table 1). There was no difference in the serum concentration as well. FSH concentrations in pituitary and in serum were significantly higher than those of females of the same age. Sham control animals bearing electrodes implanted into the MPO, m-amyg and d-hpc a day before showed no significant alterations in the concentrations of pituitary LH and FSH compared with those of normal controls (Table 2). The serum LH concentration was low in the sham control animals with electrodes implanted into the MPO compared with that of normal controls, but FSH concentration was not different. Sham controls with electrodes into the m-amyg and the d-hpc did not differ in the serum concentrations of LH and FSH from those of normal controls. Electrical stimulation of the MPO produced no change in pituitary concentration of LH but produced an increase in pituitary concentration of FSH compared with that of sham controls (Table 2). Serum concentration of LH showed a tendency to increase following the MPO stimulation, while FSH in serum did not change compared with that of sham controls. The m-amyg stimulation produced decreases in LH and FSH concentrations in pituitary with a tendency to decrease serum concentrations. Pituitary and serum concentrations of LH were not changed

4 KAWAKAMI et al. Table 2. Effects of electrical stimulation of the brain on LH, FSH and prolactin in serum and pituitary gland in immature male rats. a: Mean d SE. *p<0.05 vs. control. **p<0.05 vs. sham control. by stimulation of the d-hpc, while the FSH concentration in pituitary showed a decrease and the serum concentration showed a tendency to decrease, although it was not statistically significant. In the 34 or 35-day-old males, pituitary concentration of prolactin was lower than in the 35- day-old females and serum concentration was slightly higher (Table 1). Prolactin concentration in pituitary was high in the sham controls with electrodes in the d-hpc, but other sham controls were not different in pituitary and serum concentrations of prolactin (Table 2). Stimulation of the MPO and the d-hpc produced neither decrease nor increase in serum and pituitary concentrations of prolactin, but stimulation of the m-amyg produced the increase of them. 2. Electrical stimulation of the brain in mature male rats Chronic experiment (Experiment 1 and 2) As shown in Table 1, pituitary of mature male rat contained almost twofold LH and ten times FSH concentration of mature female gland. Serum concentration of FSH in the male was also higher than that in the female. Electrical stimulation of the ARC at 100ƒÊA parameter did not induce any change in the serum and pituitary concentrations of LH and FSH (Table 3, Expt. 1). On the other hand, the same parameter stimulation of the PVA induced a significant increase of serum concentration of LH without change in the pituitary concentration (Table 3, Expt. 2). FSH in serum and pituitary was not affected by the stimulation. Stimulation of the MPO at the parameter

5 MALE BRAIN STIMULATION AND GONADOTROPIN Table 3. Effects of electrical stimulation of the brain on LH, FSH and prolactin in serum and pituitary gland in mature male rats. a: Mean }SE. *P<0.05 vs. control. 100ƒÊA (Table 3, Expt. 1) did not induce changes in both the pituitary and serum concentrations of LH and FSH. But the electrical stimulation at parameter 200ƒÊA (Expt. 2) induced an increase of serum concentration of LH, although no change was induced in the pituitary concentration. FSH in serum and pituitary gland was not affected by this stimulation. The m-amyg stimulation with 100ƒÊA increased both pituitary and serum concentrations of LH but not of FSH. This stimulatory effect due to the m-amyg stimulation was confirmed in Experiment 2, i. e., electrical stimulation of the m-amyg at 200ƒÊA parameter induced an increase of serum concentration of LH, as observed in Experiment 1, and the same stimulation also induced a decrease of serum and pituitary concentrations of FSH. Pituitary concentration of prolactin in male rats was only half as high as that in mature female rats (Table 1). Serum concentration was equivalent to that in the female. In the Experiment 1, electrical stimulation of the ARC, MPO, m-amyg and d-hpc decreased the serum prolactin concentration, and only the d-hpc stimulation was accompanied with the increase in pituitary concentration. But

6 KAWAKAMI et al. in the Experiment 2 in which higher parameter of 200ƒÊA was used both the MPO and m- AMYG stimulations did not induce significant change in prolactin. The 1-AMYG and v-hpc showed a tendency to decrease serum prolactin concentration following the stimulation. The PVA stimulation showed a tendency to increase serum prolactin concentration (Expt. 2). Acute experiment (Experiment 3) A high parameter stimulation of the MPO with 500ƒÊA under the acute conditions markedly increased serum concentrations of LH and FSH. LH concentration in the pituitary did not change but FSH concentration increased significantly after the "acute" stimulation. The m-amyg stimulation with parameter 200ƒÊA under the acute conditions did not induce any change in pituitary and serum LH, whereas it increased serum concentration of FSH. The same stimulation also decreased serum prolactin concentration. Discussion The male PVA exerted a facilitatory effect on LH release and somewhat increased prolactin release following electrical stimulation. These effects are quite similar to those in the female (Kawakami et al., 1973a). In contrast, electrical stimulation of the ARC with the same parameter as used in the PVA stimulation did not induce any signficant change in LH and FSH, whereas it inhibited prolactin release. Because the ARC stimulation under the similar experimental conditions in the cycling female rat on the day of proestrus was also ineffective to release LH, FSH and prolactin. (Kawakami et al. unpublished data), it appears that the PVA is more sensitive to the stimulation, inducing LH release than the ARC. Furthermore, there seems to be no sexual difference in the influence on gonadotropin secretion of the PVA, as well as of the ARC. It has been shown in both sexes that the MPO plays an important role in controlling the sexual behavior, as well as a remarkable sensitivity to steroid hormones (Soulairac and Soulairac, 1956, Lisk, 1960, Michael, 1962). In the female this area has been shown to play a fundamental role in producing cyclicity of gonadotropin release. Recently, Kawakami et al.(1971) demonstrated in the acute experiment that the area induced the LH release responding to electrical stimulation at any stage of the estrous cycle, even though the effective current was lowest on the day of proestrus. The present study showed that the male MPO could as well induce LH release in response to "acute" stimulation whose parameter was as high as that used for female stimulation on the days of diestrus and estrus. In addition, the same stimulation induced an increase of FSH, as did the female MPO in response to the "acute" stimulation. MPO stimulation through electrode chronically implanted with 200uA parameter also induced a slight increase of serum LH. That the MPO of neonatally androgenized or estrogenized females, which lost cyclic discharge of ovulatory hormone, could induce the release of a small amount of LH in response to the electrical stimulation was also demonstrated (Kawakami and Terasawa, 1972b; Kawakami et al., 1973b). These findings and others (Gorski and Barraclough, 1963; Everett et al., 1970) readily lead to the idea that there exists no sexual difference in the MPO itself at least in the ability to induce LH release. Comparison of electrophysiological phenomena, observed in our laboratory, between the female and male, indicates that the functional difference of the MPO concerning the cyclic discharge of LH may partly result from the manner of conduction to the basal hypothalamus, however functional or anatomical it may be, instead of the intrinsic property of the MPO itself. The stimulation of the m- or 1-AMYG in the chronic condition was effective to increase LH release, although the amount increased was very small. Contrary to that, the electrical

7 MALE BRAIN STIMULATION AND GONADOTROPIN stimulation of the m-amyg in the acute conditions under ether anesthesia was ineffective in inducing LH release. In the acute experiments, Velasco and Taleisnik (1969) observed no increase in plasma LH level after the electrochemical stimulation of the male m-amyg, and Arai (1971) noticed the refractoriness of the castrated male AMYG, either the medial or lateral part, to the electrochemical stimulation in inducing luteinization in transplanted ovary. Similarly, failure of stimulation of the m-amyg in inducing ovulation was demonstrated in androgenized persistent estrous rat (Terasawa, 1971; Arai, 1971). In turn, in the cycling female, the electrical stimulation of the m-amyg in the acute conditions under pentobarbital anesthesia in the afternoon of proestrus increased LH release (Kawakami et al., 1973c), and in persistent estrous rats due to constant illumination, the "acute" m-amyg stimulation was able to induce ovulation (Bunn and Everett, 1957; Velasco and Taleisnik, 1969). Therefore, judging from those experimental results obtained from acute preparations, it may be said that the male AMYG differs from the female one, being not capable of inducing LH release following the stimulation. On the other hand, according to the recent observation in our laboratory, the electrical stimulation of the female m-amyg in the chronic conditions on the day of proestrus was less effective to increase LH release. Comparison of the results in the male and female obtained from chronic preparations may indicate that the male AMYG somewhat differs from the female one in the effect on LH release, which was induced by the "chronic" stimulation. It may be speculated from the above findings that the effect of AMYG stimulation on LH release differs according to the experimental conditions, both in the female and the male, and the influence of experimental conditions on such effect differs between both sexes. Some kind of stress is known to inhibit facilitatory neurons or activate inhibitory neurons, and nembutal anesthesia does not necessarily inhibit nervous activity but sometimes facilitate it. Furthermore, there was a sex difference observed in the terminals of amygdaloid fibers in the MPO, although the entiated (Field and Raisman 1971). The electrical stimulation of the m-amyg under chronic preparation in both mature and immature males exerted rather inhibitory effects on FSH production and release. In immature males, the stimulation showed a tendency to decrease production and release of LH, as well. These results indicate that the male AMYG is probably concerned with the inhibitory control of FSH, and simultaneously, there is also some sex difference in the m-amyg of immature rats, because immature female AMYG facilitated the production of LH and FSH in response to the stimulation (Kawakami and Terasawa, 1972a). The HPC seems to participate mainly in the control of FSH secretion in both the male and female rats. In the mature females the stimulation of this area increased serum and pituitary concentrations of FSH on the day of estrus but decreased on the day of diestrus II (Kawakami et al., 1972). In 27-day-old female the HPC stimulation increased serum concentration of FSH (Kawakami and Terasawa, 1972a), but the stimulation inhibited it in 34- day-old female (Kawakami and Kimura, unpublished data). On the other hand, in the present experiment, the male HPC somehow exhibited an inhibitory effect on FSH release and production. Such difference of hippocampal influences on FSH according to the maturation process in both the male and female may reflect the state of hormonal environment, e.g., a remarkably high concentration of FSH in serum and constant circulation of testosterone in the male, in contrast to the fluctuations of gonadotropins and ovarian hormones in the female. In the mature male and female the existence of prolactin inhibiting mechanisms is now undoubtful, and almost all the brain areas

8 KAWAKAMI et al. participate in that mechanisms except for the PVA and ARC. It should be noteworthy that all the stimulated areas of the immature male rats in the present experiment did not decrease serum prolactin concentration and the m- AMYG even increased it. The lack of inhibition of prolactin secretion was also observed in the 27-day-old female rat (Kimura and Kawakami, unpublished data). Does it mean the immaturity of prolactin inhibiting neural mechanism, or the lack of some component of prolactin inhibintig factor in the immature rat? Besides, Krulich and Fawcett (1973) suggested the absence of responsiveness of the immature glands to prolactin inhibiting factor from the adult hypothalamic extract. Acknowledgments The authors wish to thank Drs. B. Tamaoki and K. Wakabayashi (National Institute of Radiological Sciences) for their great help in measuring LH, FSH and prolactin. We would like to thank the National Institutes of Health for supplying the materials for radioimmunoassay. References Albe-Fessard, D., Stutinsky and S. Libouban. Atals Stereotaxique du Diencephale du Rat Blanc, Editions du Centre National de la Recherche Scientifique, (1966). Arai, Y.(1971). Endocrinol. Japon. 18, 211. Bunn, J. P. and J. W. Everett (1957). Proc. Soc. Expt. Biol. Med. 96, 369. Everett, J. W., J. W. Holsinger, G. H. Zeilmaker, W. C. Redmond and D. L. Quinn (1970). Neuroendocrinol. 6, 65. Field, P. M. and G. Raisman (1971). Physiol. 218, 23. Gorski, R. A. and C. A. Barraclough (1963). Endocrinol. 73, 210. Hofer, H.(1958). Verh. dtsch. Zool. Ges. Frankfurt/M Kawakami, M., F. Kimura and T. Konno (1973a). Endocrinol. Japon. 20, 335. Kawakami, M., F. Kimura and K. Seto (1973b). Ibid. 20, 59. Kawakami, M., F. Kimura and K. Wakabayashi (1972). Ibid. 19, 85. Kawakami, M. and E. Terasawa (1972a). Ibid. 19, 335. Kawakami, M. and E. Terasawa (1972b). Ibid. 19, 349. Kawakami, M., E. Terasawa, F. Kimura and K. Wakabayashi (1973c). Neurondocrinol. 12, 1. Kawakami, M., E. Terasawa, K. Seto and K. Wakabayashi (1971). Endocrinol. Japon. 18, 13. Knowles, F. and Kumar, A. T. C. (1969). Philos. Trans.(B). 256, 357. Krulich, L. and C. P. Fawcett (1973). Program of the 55th Anual Meeting of the Endocrine Society, Chicago Kumar, A. T. C.(1968). Z. Zellforsch. 74, 28. Lisk, R. D.(1960). J. Exp. Zool. 145, 197. Michael, R. P.(1962). Proceeding of the Ist Intern. Congr. Hormonal Steroids, Milan, Vol.2, 457, Acad. Press, N. Y. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe and L. E. Reichert, Jr.(1968). Proc. Soc. Exp. Biol. Med. 128, 807. Sherwood, N. M. and P. S. Timiras. A. Stereotaxic Atlas of the Developing Rat Brain, University of California Press, Berkeley (1970). Soulairac, A. and M/L. Soulairac (1956). Ann. Endocrinol. 17, 731. Terasawa, E.(1971). Folia Endocrinol. Japon. 46, 1057 (in Japanese). Velasco, M. E. and S. Taleisnik (1969). Endocrinology 84, 132. Vigh, E., Aros, B., Wenger, T., Koritsanszky, S. and G. Cegledi (1963). Acta Biol. Acad. Hung. 13, 407.