Localization of Cells Retaining 3H-estradiol in the Forebrain of Rabbits ' CONWELL H. ANDERSON Department of Anatomy, University of Illinois at the Medical Center, 1853 W. Polk Street, Chicago, Illinois 60680 ABSTRACT Ovariectomized rabbits received "-estradiol via an ear vein and were killed one hour later. Autoradiograms were prepared and exposed up to six months. Labeled cells, as indicated by many silver grains over the nucleus of a neuron, were found in many nucIei of the brain. Thus, the bed nucleus of the stria terminalis had labeled cell bodies. The stria terminalis leads into the medial preoptic area where great numbers of cells concentrated the estrogen. Farther into the hypothalamus the labeled cells were numerous in the ventromedial and arcuate nuclei. Other locations with labeled cells were the lateral septa1 nucleus and nucleus accumbens septi, the periventricular preoptic nucleus, anterior hypothalamic nucleus, nucleus supraopticus diffusus, posterior hypothalamic nucleus, and premammillary nucleus. The labeled cells could be followed into the central gray surrounding the aqueduct of Sylvius. The amygdaloid nuclei, and in particular the medial amygdaloid nucleus, had labeled cells as did the most ventral posterior part of the hippocampal cortex. The results are discussed in comparison with those in the rat. and with reference to physiologic data. Several studies have confirmed the autoradiographic localization of tritiated estradiol in cells in the brain of the rat (Stumpf, '68; Pfaff, '68; Anderson and Greenwald, '69; Attramadal, '70). These studies can be correlated in part with reports on hormone secretions and behavior after lesions or steroid implants. The implantation studies provide localized concentrations of hormone in the brain so that physiological end points can be observed and related to a hormone-brain interaction. These studies have demonstrated the need to consider a series of interconnected structures in the brain that are related to complex behavioral and physiologic functions (Rodgers and Law, '68). In this study, the rabbit was investigated because, in some respects, it has served as a contrast to the rat in the control of reproduction. Thus, the rabbit is a reflex ovulator, and the area for control of reproductive behavior appears to be in the posterior hypothalamus (Palka and Sawyer, '66). Because of the species differences, it was felt worthwhile to study the location of neurons which concentrate estradiol in the rabbit. ANAT.REC.,181: 287-292. MATERIALS AND METHODS New Zealand white rabbits (from Scientific Small Animals) weighing 4-5 pounds and approximately two months of age were housed under 14 :10 light: dark, with constant temperature. A sample of estradiol, 2,4,6,7-tritiated (New England Nuclear, 106 C/mM) was chromatographed on ITLC (Hexane:Ethyl acetate, 10:2) to assure its purity. One week after ovariectomy, the radioactive estrogen, 100 pc, was injected via an ear vein in a 20% EtOH in normal saline solution. One hour after the injection, the rabbit was killed with an overdose of Nembutal (1.V.) and quickly decapitated. The brain was removed from the skull, frozen over liquid nitrogen and stored in liquid nitrogen until sectioning. For the autoradiography, acid cleaned slides were coated with Kodak NTB-3 emulsion. Sections cut at 4-6 at - 18" to - 20°C, were then picked up on a precoated slide. The sections melted onto the slide and dried quickly. The slides were stored with drierite at 4°C for six months. Received March 8, "74. Accepted Julv . . 15,. '74. 1 This work has been supported by a grant from the NationaI Institute of Child Health and Human Development, NIH, HD-5865. 287 288 CONWELL H. ANDERSON Dektol developer ( 2 min. ) and Kodak acid fixer (5 min.) were used to process the film. The sections were stained with hematoxylin. Positive and negative chemography in the autoradiograms were checked by appropriate exposure of film to non-radioactive tissues and are not a problem. Similar experiments were performed with 2,4,6,7-3H estrone in ovariectomized rabbits (New England Nuclear, 106 C/mM). The sections were systematically scanned at 400X to identify and locate labeled cells. A cell was considered labeled if at least 4-5 silver grains were over the nucleus. The atlases of Sawyer et al. ('54), Urban and Richard ('72) and Fifkovh and MarGala ('67) were used to identify nuclear groups. Numbers of labeled cells in autoradiograms of hypothalamic and preoptic areas were determined in one rabbit to gain an impression of the distribution of labeled cells. RESULTS Cells concentrating radioactivity were found in all amygdaloid nuclei, but subjectively, the medial amygdaloid nucleus had the greatest number. Labeled cells could be seen in the bed nucleus of the stria terminalis. This tract projects into the medial preoptic area where the number of labeled cells per unit area was quite large (figs. 2, 4 ) . The ventrally placed periventricular nucleus of the preoptic area, beside the supraoptic recess of the third ventricle, also had many labeled cells (fig. 2 ) . In the hypothalamus, many labeled cells were found in the arcuate nucleus imme- Fig. 1 Representative autoradiogram showing cells labeled with tritiated-estradiol. Arcuate nucleus. x 635. diately above the median eminence (fig. 1). A few labeled cells at the ventral lateral border of the arcuate nucleus were in the median eminence. Immediately behind the optic chiasma the nucleus supraopticus diffusus had numerous labeled cells. This area was just above the median eminence (fig. 2). Scattered labeled cells were also found in the anterior hypothalamic nucleus and adjacent periventricular nucleus as well as a few throughout the entire ventromedial nucleus. Between the arcuate nucleus and the ventral border of the ventromedial nucleus many labeled cells were found. The major concentration of cells in the ventromedial nucleus was on the ventral surface and continued up the lateral border to the area immediately below and medial to the fornix, thus forming an arc of labeled cells (fig. 2). Between ventromedial and mammillary nuclei, cells of the premammillary nucleus also accumulated estrogen. A few labeled cells were found in the posterior hypothalamic nucleus and these sparsely distributed cells could be followed into the central gray of the midbrain (which was as far caudally as sections were cut). There were also labeled cells in the lateral septa1 nucleus and the nucleus accumbens septi. In the most ventral extent of the hippocampal formation (posterior hippocampus), and probably restricted to the CA1 area of Lorente de N6 ('34), there were many labeled cells in a row of large neurons (fig. 3). Furthermore, the cells retaining estradiol tended to be in the pyramidal layer and placed near the stratum oriens. Figure 4 shows the numbers of labeled cells in several sections throughout the preoptic area and hypothalamus of one brain. The distribution of the labeled cells was quite similar to what has been reported for rats (Anderson and Greenwald, '69), except the numbers of cells immediately behind the optic chiasma does not drop near zero due to the numerous cells in the nucleus supraopticus diffusus. Posterior to the pituitary stalk the majority of labeled cells were those in the arcuate nucleus. The injection of tritiated estrone did not result in heavily labeled cells in the amygdala or hypothalamus. However, a very few labeled cells were found in the posterior 289 'H-ESTRADIOL IN THE BRAIN OF RABBITS Fig. 2 Coronal secCions of rabbit hypothalamus. Dots indicate locations of labeled cells, The lateral ventricles and third ventricle are shown in cross hatching. Abbreviations: A, arcuate nucleus; AC, anterior ccmmissure; CC, corpus callosum; DHA, dorsal hypothalamic area; DMH, dorsomedial hypothalamic nucleus; F, fornix; FF, fimbria of fornix; HIP, hippocampus; LHA, lateral hypothalamic area; LPO, lateral preoptic area; MPO, medial preoptic area; OC, optic chiasm; OT, optic tract; PHA, posterior hypothalamic area; PMA, premammillary area; PPO, preoptic periventricular nucleus; PV, paraventricular nucleus; SCH,suprachiasmatic nucleus; SO, supraoptic nucleus; SOD, supraopticus diffusus nucleus; SP, septum; VMH, ventromedial hypothalamic nucleus. 57 450 R -. I MICRONS Fig. 3 Ventral hippocampus. Inked black dots show the location of a row of labeled cells on the deep side of the pyramidal layer. x 13. hypothalamic area. These cells were not numerous and the assurance of the identification deserves further support. DISCUSSION In in vitro studies, hypothalamic tissue Fig. 4 Number of labeled cells in cross sections of rabbit hypothalamus, closed circles. Number of cells in arcuate nucleus only, shown with Xs. Animal killed 1 hour after IV injection of 3H-estradiol. Abbreviations: AC, anterior commissure; OC, optic chiasm. from rabbits did not metabolize 'H-estradiol to estrone or estriol (Chader and Villee, '70). This corresponds with reports in other species in which in vivo experiments found the majority of the radioactivity detected was 'H-estradiol (Wade and Feder, '72). Thus, the author assumes that at 290 CONWELL H. ANDERSON least the majority of radioactivity detected in this experiment was associated with estradiol. In a series of papers on the rabbit, the area posterior to the hypophyseal stalk was found important for the control of estrous behavior, for the reflex secretion of ovulating hormone( s ) and for feedback inhibition of LH by estrogen (Sawyer, ’59; Davidson and Sawyer, ’61; Kanematsu and Sawyer, ’63; Palka and Sawyer, ’66). It was anticipated from the contrast of these findings with those in rats, where these “centers” are more anteriorly localized, that there might also be some difference in the pattern of cells which accumulate estradiol, However, with the exception of species differences in shapes and dimensions of nuclei, the cells that retain estrogen were in virtually the same locations as in the rat. That is, a “system” of neurons that bind estrogen forms a continuum from amygdaloid nuclei through cells in the bed nucleus of the stria terminalis to medial preoptic and hypothalamic nuclei. Palka and Sawyer (’66), using implants, localized the most estrogen sensitive area for induction of sexual behavior in the posterior ventromedial nucleus - premammillary area, up to 1 mm from midline but not in midline or third ventricle. This same area had numerous, heavily labeled cells which may be important in the neuronal circuits eliciting the estrous response. Dorsal to this area there were a few labeled cells in the hypothalamic posterior and dorsomedial nuclei, but the number of cells was not great and they were sparsely distributed. As was previously reported for the rat, there were cells in the hippocampus which accumulate estradiol (Stumpf and Sar, ’69). The cells show a very restricted distribution which appears to correlate well with the CA1 area of the ventral (posterior) hippocampus (Lorente de N6, ’34). The posterior part of field CA1 projects to sites different from those to which the anterior part projects. The posterior CA1 field has connections to septal nuclei and diagonal band nuclei, while the anterior CA1 field does not project to these structures (Raisman et al., ’66). The labeled hippocampal cells were large and appear to correspond to the deep pyramidal cells of Lorente de N6 (’34), but may possibly be basket cells as described by Raisman et al. (’65, p. 967). Basket cells form inhibitory synapses with pyramidal cells (Anderson et al., ’63), particularly in the superficial layer of pyramids and seem to have less input to the deep pyramids (Lorente de N6, ’34). Labeled cells of the hippocampus and septal nuclei form a second “system” of labeled neurons. Gallo et al. (’71), in the rat, have demonstrated effects of ventral hippocampal and subiculum stimulation on gonadotropin secretion and multiple unit activity in the arcuate nucleus. They also found, in multiple unit recording experiments in the ventral hippocampus-subiculum, that injected LH would reduce recorded activity in one-half of the sites explored (Gallo et al., ’72). The labeled cells in the present experiment may be slightly displaced laterally from the subiculum-LH responsive area identified by these authors. According to Nauta (’56) and Raisman et al. (’66), the medial corticohypothalamic tract originates exclusively from the caudal onethird of the rat subiculum. This tract projects to the arcuate nucleus of the hypothalamus . The biochemistry of binding and specific cellular effects of estrogen have been most clarified in the uterus (Jensen and DeSombre, ’73). In brain, the localization by a cell may result in an excitatory or inhibitory influence on that cell, and that cell, via a short axon may have local actions or, via long axons, may act upon distant neural structures. 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