Rostral medial preoptic area lesions' influence on female estrous processes and LHRH distribution

Physiology & Behavior, Vol. 27, pp. 855-861. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.

Rostral Medial Preoptic Area Lesions' Influence on Female Estrous Processes and L H R H Distribution I HERBERT

B. P O P O L O W , z J O A N C: K I N G a A N D A R N O L D A. G E R A L L

Department of Psychology, Tulane University, New Orleans, LA 70118 R e c e i v e d 20 S e p t e m b e r 1980 POPOLOW, H. B., J. C. KING AND A. A. GERALL. Rostral medial preoptic area lesions' influence on f e m a l e estrous processes and L H R H distribution. PHYSIOL. BEHAV. 27(5) 855-861, 1981.--The effect of bilateral electrolytic lesions placed in the rostrai medial preoptic area (MPOA) of the 26 day old rats was observed on each animal's onset of puberty, vaginal cyclicity, sexual receptivity and LHRH distribution. Lesions located in the lower two-thirds of the rostral MPOA induced persistent vaginal cornification and polyfollicular ovaries. Lesions limited to the dorsal third of the periventricular region of the rostral MPOA were associated with repetitive pseudopregnancy as indicated by repeated series of diestrous smears separated by one or two proestrous or estrous smears and ovaries dominated by corpora lutea. The age of onset of puberty was not altered by any of these lesions. Receptivity induced by estrogen alone or by estrogen followed by a low dosage of progesterone was lower in ovariectomized females with lesions than in control animals. Immunocytochemical identification of LHRH containing neurons using Sternberger's peroxidase-antiperoxidase method revealed LHRH reactive fibers in the MPOA in both lesioned and control animals. A greater reduction in immunopositive LHRH fibers was seen on the same side of the median eminence at the level of the tuberoinfundibular sulcus as that containing the larger damage in the MPOA.

Medial preoptic area

Estrous cyclicity

Estrous behavior

T H E purpose of the present study was to determine how lesions that primarily involve midline regions of the rostral MPOA affect the onset of puberty, cyclic ovulation and receptivity in rats. In addition, since L H R H perikarya are located in the MPOA and their axons terminate, at least in part, in the median eminence [3,15], the consequence of these lesions on the distribution of L H R H fibers in the median eminence was examined by immunocytochemistry. Precocious puberty has been reported to occur in rats subsequent to lesions in the anterior hypothalamus [9,22], amygdala [10], and stria terminalis [11]. Although a considerable proportion of stria terminalis fibers are distributed to the MPOA, data provided by Donovan and van der Werff ten Bosch [9] indicate that lesions in the MPOA have no effect on the onset of puberty. Electrically stimulating the MPOA evokes ovulatory surges of L H in proestrous rats [7] and isolating this area from the median eminence eliminates the cyclic release of LH in the otherwise intact female rat [17]. In most studies using rats, lesions in the MPOA and the neighboring organum vasculosum of the lamina terminalis (OVLT) disrupt vaginal cyclicity [14, 17, 29] and reduce the positive feedback effect on serum L H of sequentially administered estrogen and progesterone [12, 26, 29, 30]. An exception to these results is the finding obtained by Clemens, Smalstig, and

LHRH

Puberty

Sawyer [6] that ablation of a considerable portion of the MPOA induces repetitive pseudopregnancy in rats; a condition that should only occur in animals with fucntioning estrogen positive feedback and an intact L H surge mechanism. Evidence has been provided by Powers and Valenstein [24] and Nance, Christensen, Shryne and Gorski [23] that lesions in the MPOA increase the effectiveness of estrogen in inducing sexual receptivity. However, Singer [28] and Gray, S6dersten, Tallentire and D~ividson [14] have not found that the elicitation of estrous behavior is altered by MPOA lesions. Discrepancies among the f'mdings from the various studies are probably due to variance in lesion location and size and their resolution is likely to be facilitated when the functional characteristics of discrete regions within the MPOA are specified. METHOD

Upon arrival from the Camm Research Institute, groups of 4 male and 4 female 26 day old Sherman rats were placed together in 35 cm x 70 cm x 80 cm cages located in a 21 ° vivarium where they had free access to food and water. Lights were on from 0900-2100 hr. At 28--30 days of age, each of 13 females was anesthetized

~This research was supported by NICHD Grant 5F 32H005462-02 to HBP, HD 10505-01 to JCK and HD 00867 to AAG. ZNow at Gerontology Research Center, Baltimore City Hospitals, Baltimore MD. 3Now at Department of Anatomy, Tufts University School of Medicine, Boston, MA.

C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/110855-07502.00/0

856 with Equithesin and received two electrolytic lesions aimed at the rostral MPOA by passing a 0.4 mA anodal DC for 29 sec through a 0.013 mm diameter platinum-iridium electrode (Medwire Corp.) insulated with telfon except for approximately 0.6 mm at the tip. Stereotaxic coordinates were 0.6 mm anterior to Bregma, 0.6 mm lateral to the superior sagittal sinus directed at a 3° angle from each side, and 6.8 mm below dura. The same procedure was followed for 13 control animals, except that the electrode was lowered 3.0 mm and no current was applied. Males and females were segregated at 40 days of age. Starting at 30 days of age, each animal was inspected daily for vaginal opening in order to determine the onset of puberty. Beginning on the day of vaginal opening, smears were taken 6 days a week. Animals were classified persistent estrus when at least 10 consecutive smears contained only cornified cells. The criterion for repetitive pseudopregnancy was at least two 7-14 day periods of predominantly leukocytic smears, interrupted by 1 or 2 smear patterns containing cornified or nucleated epithelial cells. On the last day smears were taken, the ovaries were removed under ether anesthesia and immersed in Bouin's solution. Paraplast embedded sections were cut at 10 microns and stained with hematoxylin and eosin for histological examination. Sexual receptivity tests were initiated 8 days after ovariectomy. First, animals were injected IM with 3.0 /xg estradiol benzoate (EB)/kg b.wt. daily for 14 days and given mating tests every other day. Second, they were tested with estrogen and progesterone three weeks after the last estrogen alone test. The females were injected IM with 2.0/xg EB followed 40 hr later with either 100 or 400/xg progesterone and placed in the mating area 5 hr later. Prior to testing the following week, the progesterone dosage was reversed for each animal. Behavioral tests were conducted in semicircular arenas in which each female was mounted 10 times by a vigorous male. The proportion of mounts eliciting lordosis was converted to a lordosis quotient (LQ=lordoses/mounts × 100). For histological and immunocytochemical IICC) analyses, the animals were anesthetized with Equithesin and perfused intracardially with Bouin's solution under constant 90-100 mm Hg pressure. To maximize LHRH immunoreactivity, we injected each animal with 3.3 /xg EB for 3 days before perfusing it on the fourth day. The brains were extracted, trimmed at the top and sides, and immersed in Bouin's solution for 3 days before being dehydrated and embedded in Paraplast over a two day period. Serial sections cut coronally at 6/~ were mounted on slides in sets of three. Alternate slides through the lesion site were stained with toluidine blue and, in some cases, with cresyl violet and luxol fast blue. Lesion outlines were traced independently by three observers on atlas [20] diagrams of coronal sections of the brain. The remaining sections through the MPOA and median eminence were examined for LHRH according to the peroxidase antiperoxidase (PAP) immunocytochemical procedure described by King, Parsons, Erlandsen and Williams [ 18]. Antiserum No. 743 to synthetic LHRH and PAP were generously supplied by Dr. Akira Arimura and Dr. Ludwig Sternberger, respectively.

Data Analysis Analysis of results from vaginal smears and ovarian histology included 13 control animals and 12 animals with lesions. Behavioral data were based on 10 control females and

P()P()I.OW, KIN(I AND (;ERAi~f 10 females with lesions since 2 rats from each group died at ovariectomy and one animal was randomly selected 1¢, be omitted to produce equal n. Two-way repeated measure', analyses of variance were used to test for statistical reliability of behavioral results. The ICC analyses were based upon 8 rats from the control group and 8 from lhc group with lesions. RESULTS Neither the onset of puberty, as evidenced by vaginal opening, nor body weight differed significantly between the groups with MPOA-lesions and control operations. The mean number of days of age before vaginal opening was 37.7 and 38.2; the mean body weight at 72 days of age was 244.0 g and 231.4 g for the lesioned and control groups, respectively. Seven of the 12 lesioned females showed persistent vaginal cornification (PVC) while 5 exhibited repetitive pseudopregnancy (RPP). Ovaries from all PVC females were polyfollicular and without corpora lutea while those of RPP females were dominated by many large corpora lutea. All control rats evidenced 4-5 day estrous cycles and their ovaries had normal follicules and corpora lutea. The boundaries of the lesions varied among animals; generally they extended in the rostral-caudal plane of the lesion from the emergence of the preoptic recess to about 0.8 mm caudal, in the medial-lateral plane from the midline to about 0.8 mm lateral, and in the dorsal-ventral plane, fl'om the anterior commissure to the optic chiasm. The suprachiasmatic nuclei were spared in all rats. Composite outlines of the representative lesions superimposed on modified tracings of coronal sections through the preoptic area taken from the atlas of KBnig and Klippel 120] are shown in Fig. I as are photomicrographs of typical ventral and dorsal MPOA lesions in Fig. 2. A detailed analysis of the lesions revealed that in 5 of" the 7 PVC females, the OVLT was either completely destroyed or severely disrupted (Fig. la and lb). In the remaining 2 animals, the OVLT itself was spared, but the lesion extended in the midline ventrally to the optic chiasm immediately caudal to the plane of the OVLT. In 4 animals in which the lesion extended ventrally to the optic chiasm (Fig. 2a), virtually all of the vaginal smears contained only cornified cells. However, when the lesion spared tissue above the dorsal border of the optic chiasm (3 rats), the daily pattern of cornified smears was more frequently interrupted by brief periods of vaginal diestrus. Females demonstrating RPP, whether or not the vasculature associated with the OVLT was disrupted, had lesions located more dorsally (Fig. lc, Id and 2b) than those in females showing PVC. Only one RPP female had a lesion that approached the optic chiasm in the plane of the OVLT but it was localized more dorsally through most of the preoptic area. The degree of OVLT destruction or disruption was corroborated by the results of the immunocytochemistry. LHRH-positive processes were associated with capillaries of the OVLT in control-operated animals (Fig. 3a). No LHRH processes were apparent in this region in animals with MPOA lesions involving destruction of the vascular bed of the OVLT. LHRH immunoreactive processes were present in the median eminence (ME) capping the tuberoinfundibular sulcus in association with portal vessels and cells of the pars tuberalis (Fig. 3d). In control animals LHRH proc-

M E D I A L PREOPTIC A R E A L E S I O N S

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FIG. 1. Serial sections through the MPOA taken from KOnig and Klippel [20] showing typical ventral lesion (a and b) and dorsal lesion (c and d). (OVLT; organum vasculosum of the lamina terminalis; AC, anterior commissure; MPOA, medial preoptic area; OC, optic chiasm.)

esses was distributed symmetrically in the sulcus and stalk. Whenever a reduction in L H R H reactive fibers occurred in one of the lateral recesses, the extent of the lesion was greater in the ipsilateral MPOA (Figs. 3c and 3d). In 6 PVC animals examined extensively, L H R H processes appeared to be less numerous on one or both lateral recesses than in control animals. In 3 RPP animals, the number of stained L H R H fibers was also reduced on one or both sides of the recesses. However, the reduction was less extensive than in PVC animals. Finally, L H R H immunopositive L H R H perikarya were evident in the rostral preoptic area of both non-lesioned rats and lesioned rats outside the destroyed area. When tested for behavioral responsiveness to EB alone, all females with lesions, regardless of previous ovarian status, displayed diminished receptivity. Mean LQ scores of rats with lesions were lower than those of controls across tests (Fig. 4). Both groups displayed similar low LQ scores on the frist two tests but while the control group approached a high level of responding by the third test, the lesion group did not. Differences between groups, F(1,18)=28, p<0.01, and across tests, F(5,90)=23.7, p<0.01, were statistically significant as was the Groups x Tests interaction, F(5,90) = 395.2, p <0.01. Damage to the OVLT p e r s e was not requisite for diminished behavior since 3 animals with no OVLT damage had lower scores than control animals. While the control group showed high levels of responding to both dosages of progesterone, the group with lesions demonstrated a substantial increase in LQ from low to high

progesterone dosage (Fig. 5). At the high progesterone dosage, the groups responded comparably. These observations are supported by a significant Groups x Dosage interaction, F(1,18)=166, p<0.01. The only difference between the groups was at 100/xg progesterone which was less effective in leading to lordosis behavior in the lesioned animals. DISCUSSION

In agreement with Donovan and van der Werff ten Bosch [9], the MPOA lesions in this study did not induce precocious puberty. Also, the moderate loss of L H R H associated with the interruption of fibers coursing through the medial portion of the rostral MPOA did not alter the timing of puberty. Two L H R H immunoreactive fiber systems have been identified in the ME of the rat: a medial division that transverses the medial portion of the ME primarily adjacent to the third ventricle and a lateral division that follows the outer contours of the ME before turning medially and ventrally toward the infundibulum [19]. The lesions in the present study probably partially disturbed the L H R H contribution primarily in the medial division, leaving sufficient L H R H to enable the animals to have normal vaginal openings and one or more ovulations. The present study demonstrates that lesions restricted to the OVLT-MPOA region can disrupt ovarian cyclicity by either preventing ovulation or inducing repeated periods of pseudopregnancy. Lesions associated with PVC tended to include about two-thirds of the ventral periventricular re-

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gion, while those associated with RPP tended to be more dorsally localized. To the extent that can be ascertained from published descriptions of lesion location and size [2, 14, 17, 29, 30], the region whose destruction most likely leads to the absence of phasic LH release lies primarily adjacent to the lower lateral surfaces of the third ventricle. The area located immediately beneath the ventricle appears to be of lesser significance. In the present study, lesions producing PVC, while not always penetrating tissue lying medially beneath the ventricle consistently involved the lateral margins of this ventricle. Recently, Leedy [21] found that lesions restricted to this portion of the medial preoptic nucleus lying along the ventral surface of the third ventricle often did not lead to PVC. Lesions made by Wiegand, Terasawa, and Bridson [29] and Wiegand, Terasawa, Bridson and Goy [30] in the medial preoptic nucleus included neurons adjacent to the lateral surface of this ventricle and most of their animals developed PVC. Finally, lesions made by Gray e t a l . [14] that destroyed most of the MPOA but spared its periventricular regions did not alter the LH surge induced by injections of estrogen and progesterone or by mating.

The specific nature of the neural disruption that underlies the absence of phasic release of LH remains to be determined.Samson and McCann [26] identify separate causes for the absence of positive feedback of gonadal hormones on the LH surge in animals with OVLT and MPOA lesions. They suggest that lesions in the OVLT region destroy LHRH axons coursing medially and caudally to the median eminence, thereby reducing LHRH content below the amount requisite for an LH surge• However, MPOA lesions leave critical LHRH fibers intact but disrupt neurons synapsing on them that are responsive to the steroid hormone variations involved in phasic release of gonadotropins. In their study [26] MPOA lesions while eliminating steroid induced LH surge did not reduce significantly LHRH content in the median eminence. Immunocytochemical analysis in the present study also demonstrated that MPOA lesions reduce but do not eliminate LHRH immunoreactive processes in the median eminence. Thus, it is likely, as suggested by Samson and McCann [26], Kimura and Kawakami 1171 and Wiegand e t a l . [29,30] that the neurons destroyed in the part of the MPOA and disrupting the occurrence of the LH surge inter-

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act with L H R H neurons and do not themselves synthesize or transport this decapeptide. Lesions lirnited to the dorsal aspect of the MPOA were found in the present study and in those reported by Wiegand e t al. [30] and Brown-Grant, Murray, Raisman and Sood [4] to cause animals to become repetitively pseudopregnant. The rats manifested daily diestrous smears that were interrupted every 7-14 days by 1-2 days of estrous or proestrous

smears and their ovaries contained large numbers of corpora lutea. Clemens e t al. [6] who originally reported that MPOA lesions can lead to the RPP condition also showed that daily administration of the dopamine agonist, lergotrile mesylate, restored vaginal cyclicity in these animals. This demonstration indicates that dorsal MPOA lesions increase release of prolactin which maintains corpora lutea formed after each ovulation.

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FIG. 4. Lordosis quotient of control and MPOA-lesioned groups

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FIG. 5. Lordosis quotient of control and MPOA-lesioned animals during mating tests preceded by injections of EB and either 100/zg or 400/zg progesterone.

The diminution in LHRH immunoreactive material in processes within the median eminence of lesioned animals agrees with similar findings reported following transection of pathways connecting rostral hypothalamic structures with the medial basal hypothalamus [15,27]. By radioimmunoassay, Kalra [16] noted a reduction in medial basal hypothalamic levels of LHRH following frontal hypothalamic deafferentation. Samson and McCann [26] report significantly less LHRH in the median eminence after OVLT but not after MPOA lesions. These studies suggest that a considerable amount of LHRH is transported from rostrally located perikarya to the median eminence. The present study contributes to evidence of the presence of an ipsilateral projection of LHRH from the OVLT-MPOA region to the median eminence since a greater loss of LHRH-positive material was apparent on the side of greater lesion extent [5,15]. Females bearing lesions in the periventricular MPOA displayed a deficit in lordotic responding when injected daily with estrogen alone over a two week period. The synergistic effect of progesterone in facilitating receptivity was somewhat preserved since females with lesions showed approximately the same level of lordotic behavior as operated-

controls when given the high dosage of progesterone. However, no difference was observed between the animals with dorsal and ventral MPOA lesions. Nance et al. report that animals bearing dorsal lesions manifest greater lordotic tendencies than those having either ventral MPOA lesions or sham operations. Differing behavioral results have also been obtained in studies in which the site of damage within the MPOA is not specified. Powers and Valenstein [24] report that MPOA lesions lower the estrogen threshold for eliciting estrous behaviors and Rogers and Schneider [25] found that these lesions reduce mating behaviors on proestrous days and increase them on estrous days. However, Gray e t a / . [ 14] found the MPOA lesions had no significant effect on lordosis responses. Since the consequence of MPOA lesions on receptive behaviors varies considerably from one study to another, speculation as to which regions or physiological mechanisms within the MPOA are involved in a female sexual behavior must wait until greater consistency in results is obtained.

REFERENCES

1. Arendash, G. W. and R. V. Gallo. Regional differences in response to electrical stimulation within the medial preopticsuprachiasmatic region on blood luteinizing hormone levels in ovariectomized and ovariectomized, estrogen-primed rats. Endocrinology 104: 333-343, 1979. 2. Barraclough, C. A., S. Yrarrazaval and R. Hatton. A possible hypothalamic site of action of progesterone in the facilitation of ovulation in the rat. Endocrinology 75: 838-845, 1964.

3. Barry, J. Immunohistochemistry of luteinizing-hormonereleasing hormone-producing neurons of vertebrates. Int. Rev. Cytol. 60: 179-221, 1979. 4. Brown-Grant, K., M. A. F. Murray, G. Raisman and M. C. Sood. Reproductive fucntion in male and female rats following extra- and intra-hypothalamic lesions. Proc. R. Soc. Lond. B 198: 267-278, 1977.

MEDIAL PREOPTIC AREA LESIONS

5. Campbell, G. T. and J. A. Ramaley. Immunohistochemistry of hypothalamic GNRH following manipulation of sex steroid levels: evidence that androgens have divergent effects on GNRH stores and serum follicle stimulating levels. Biol. Reprod. 19: 620-627, 1978. 6. Clemens, J. A., E. B. Smalstig and B. D. Sawyer. Studies on the role of the preoptic area in the control of reproductive function in the rat. Endocrinology 99: 728--735, 1976. 7. Cramer, O. M. and C. A. Barraclough. Effect of electrical stimulation of the preoptic area on plasma LH concentrations in proestrous rats. Endocrinology 88:1175--1183, 1971. 8. Critchlow, V. and M. E. Bar-Sela. Control of the onset of puberty. In: Neuroendocrinology, edited by L. Martini and W. F. Ganong. New York: Academic Press, 1967. 9. Donovan, B. T. and J. J. van der Werff ten Bosch. The hypothalamus and sexual maturation in the rat. J. Physiol. 147: 78--92, 1959. 10. Elwers, M. and V. Critchlow. Precocious ovarian stimulation following hypothalamic and amygdaloid lesions in rats. Am. J. Physiol. 198: 381-385, 1960. 11. Elwers, M. and V. Critchlow. Precocious ovarian stimulation following interruption of stria terminalis. Am. J. Physiol. 201: 281-284, 1961. 12. Everett, J. W. Preoptic stimulative lesions and ovulation in the rat: Thresholds and LH-release time in late diestrus and proestrus. In: Major Problems in Neuroendocrinology, edited by E. Bajusz and C. Jasmin. Basel: Karger, 1964. 13. Gorski, R. A. The possible neural sites of hormonal facilitation of sexual behavior in the female rat. Psychoneuroendocrinology 1: 371-387, 1976. 14. Gray, G. D., P. Srdersten, D. Tallentire and J. M. Davidson. Effects of lesions in various structures of the suprachiasmaticpreoptic region on LH regulation and sexual behavior in female rats. Neuroendocrinology 25: 174-191, 1978. 15. Ibata, Y., K. Watanabe, H. Kinoshita, S. Kubo, Y. Sano, S. Sin, E. Hashimura and K. Imagawa. The localization of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence. Cell Tissue Res. 198: 381-395, 1979. 16. Kalra, S. P. Tissue levels of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus, and serum concentrations of gonadotropins following anterior hypothalamic deafferentation and estrogen treatment of the female rat. Endocrinology 99: 101-107, 1976. 17. Kimura, F. and M. Kawakami. Reanalysis of the preoptic afterents and efferents involved in the surge of HL, FSH and prolactin release in the proestrous rat. Neuroendocrinology 27: 74-85, 1978.

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18. King, J. C., J. A. Parsons, S. L. Erlandsen and T. H. Williams. Luteinizing hormone-releasing hormone (LH-RH) pathway of the rat hypothalamus revealed by the unlabelled antibody perioxidase anti-peroxidase method. Cell Tissue Res. 153:211217, 1974. 19. King, J. C., S. A. Tobet, F. L. Snavely and A. A. Arimura. The LHRH system in normal and neonatally androgenized female rats. Peptides 1: Suppl. 1, 85-100, 1980. 20. Krnig, J. F. R. and R. A. Klippel. The Rat Brain, a Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Baltimore: Williams and Wilkins, 1963. 21. Leedy, M. G. The effects of small preoptic area lesions on ovulation and receptivity in female rats. Unpublished doctoral dissertation, Tulane University, New Orleans, LA, 1980. 22. Meijs-Roelofs, H. M. A. and J. Moll. Differential effects of anterior and middle hypothalamic lesions on vaginal opening and cyclicity. Neuroendocrinology 9: 297-303, 1972. 23. Nance, D. M., L. W. Christensen, J. E. Shryne and R. A. Gorski. Modifications in gonadotropin control and reproductive behavior in the female rat by hypothalamic and preoptic lesions. Brain Res. Bull. 2: 307-312, 1977. 24. Powers, J. E. and E. S. Valenstein. Sexual receptivity: Facilitation by medial preoptic lesions in female rats. Science 175: 1003-1005, 1972. 25. Rogers, C. H. and V. M. Schneider. Inhibitory and facilitatory influences on mating in the female rat affected by lesions of the anterior hypothalamus or preoptic area. Psychoneuroendocrinology 4: 127-134, 1979. 26. Samson, W. K. and S. M. McCann. Effects of lesions in the organum vasculosum lamina terminalis on the hypothalamic distribution of luteinizing hormone-releasing hormone and gonadotropin secretion in the ovariectomized rat. Endocrinology 105: 939-946, 1979. 27. Srt~il6, G., S. Vigh, A. V. Schally, A. Arimura and B. Flerko. Immunohistological study of the origin of LH-RH-containing nerve fibers of the rat hypothalamus. Brain Res. 103: 597-602, 1976. 28. Singer, J. Hypothalamic control of male and female sexual behavior in female rats. J. comp. physiol. Psychol. 66: 738--742, 1968. 29. Wiegand, S. J., E. Terasawa and W. E. Bridson. Persistent estrus and blockade of progesterone-induced LH release follows lesions which do not damage the suprachiasmatic nucleus. Endocrinology 102: 1645-1648, 1978. 30. Wiegand, S. J., E. Terasawa, W. E. Bridson and R. W. Goy. Effects of discrete lesions of preoptic and suprachiasmatic structures in the female rat. Neuroendocrinology 31: 147-157, 1980.