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Nuclear retention charactristics of [3H]estrogen by cells in four estrogen target regions of the rat brain
224
Brain Research, 229 (1981) 224-229 Elsevier/North-Holland Biomedical Press
Nuclear retention characteristics of PH]estrogen by cells in four estrogen target regions of the rat brain*
DONALD A. KEEFER Department of Anatomy, University of Virginia Medical School, Charlottesville, VA 22908 (U.S.A.) (Accepted August 27th, 1981) Key words: hypothalamus - - amygdala - - medial preoptic area - - estrogen - - estrogen receptor
Nuclear retention of radioactivity was studied in neural estrogen target cells 15 min-7 h after i.v. injection of [3H]estradiol. Maximal uptake occurred by 15 min. Cells of the medial preoptic nucleus retained the label longer than did those of the medial amygdaloid nucleus. Cells of the ventromedial nucleus and arcuate nucleus exhibited similar retention profiles which were intermediate between the medial preoptic and medial amygdaloidcells. These data are discussed in relation to observations that the duration of nuclear occupancy by estrogens is proportional to the magnitude of the cellular response. A number of laboratories have established that significant quantities of high affinity, estradiol (ED-specific, cytoplasmic receptors (Re) exist in the hypothalamus and amygdala6, 7. [3H]estrogen ([3H]E) is bound and retained by the nuclei of cells in circumscribed regions of the hypothalamus and amygdala; primarily the medial preoptic nucleus (MPO), arcuate nucleus (ARC), ventrolateral portion of the ventromedial nucleus (VLVM) and the medial amygdaloid nucleus (MA) 15,17. This genomic interaction results in an alteration either of sexually related behavior or of reproductive hormone levels 14. Studies of estrogen receptor dynamics in the uterus indicate a direct relationship between the duration of nuclear occupancy by the estrogen-receptor complex (ERe) and the magnitude of the response by the target cells 1,3. A recent study has demonstrated differences in the dynamics of [3H]E uptake between uterus and pituitary and possibly among individual cell types of these organs 10. The present study was undertaken to analyze the temporal pattern of [3H]E uptake in 4 major E target areas of the brain. Ovariectomized Sprague-Dawley rats, 85-95 g, were given i.v. injections of 0.54 /~g/100 g body weight [2, 4, 6, 7-~H]estradiol-17fl. Groups of 4 rats were sacrificed at intervals of 15 min, 1 h, 3 h and 7 h after injection. As a competition control, 2 of these rats were given injections of 100 × higher doses of cold estradioi prior to the isotope. One additional rat was injected with vehicle only and was processed for an autoradio-
* Portions of these data were presented at the Histochemistry Society meetings, Minneapolis, 1981. 0 0 0 6 - 8 9 9 3 / 8 1 / ~ / $ 0 2 . 7 5 © Elsevier/North-Holland Biomedical Press
225
Fig. 1. Autoradiograms of rat brain showing accumulation of [3H]estrogen by cell nuclei 15 min (A,E,F), 1 h (B), 3 h (C) and 7 h (D) after injection of [SH]estradiol. A-D, medial preoptic area. Notice that neuropil labeling decreases from 15 min to 7 h after injection. E, ventrolateral portion of ventromedial nucleus. F, medial amygdaloid nucleus. Magnification 460 ×. graphic control. At each time interval rats were decapitated and brains were frozen in rapidly vortexing liquid Freon-22 at - - 1 5 0 °C. Frozen sections 6/zm thick were cut at - - 3 0 °C and thaw mounted onto desiccated emulsion-coated slides (Kodak NTB-2). Every third frontal section was collected through the hypothalamus and adjacent amygdala. After exposure for 40.0 -I- 0.4 days at - - 2 0 °C in desiccator slide boxes the autoradiograms were photographically processed under carefully controlled conditions 9. Autoradiograms were stained with methyl green pyronin. The center of each of the 4 brain regions was identified by scanning each series of autoradiograms. For every cell that was analyzed the nuclear area was determined (by measuring nuclear dia-
226 meter) and the silver grain number counted at 1250 ×. Background silver grains were counted over an area of adjacent neuropil equal in size to the area of each nucleus. Silver grains were counted over nuclei of randomly chosen cells (labeled and unlabeled) from the center of each neural region until a total of 20 labeled cells had been counted from each of 2 adjacent autoradiograms from each region of every animal. As each individual cell was measured and its silver grain number determined these data were immediately processed by microcomputer using the Poisson distribution to classify each cell as either labeled or unlabeled 4. A cell was classified as labeled only when the Poisson formula indicated that the chance occurrance of such grain density per nuclear area was less than 0.01. Additionally, a 'labeling index' was determined for each neural region by dividing the number of labeled cells by the number of labeled plus unlabeled cells. The data were further analyzed by 2-way ANOVA. In all 4 neural regions maximal nuclear uptake of isotope by labeled cells occurred by 15 min after injection. At this early time interval background labeling in the neuropil was appreciably higher than at later times (Fig. 1). By using the Poisson distribution to determine the labeling status of each cell at every time interval the influence of this time-dependent variation in neuropil grain density was equalized. In frontal sections labeled neurons of the ARC and the adjacent VLVM often appeared to form a continuum. It is interesting to note, therefore, that the nuclear retention profiles of these 2 target areas were essentially identical over the time intervals examined, showing a gradual, steady decrease in labeling over the whole interval (Fig. 2). Labeled cells of the MPO retained maximal labeling throughout the first hour after injection. By contrast, however, labeled cells of the MA rapidly released the label between 15 min and 1 h. As a consequence, 15 min after injection labeled cells of the MPO and MA were almost equally labeled, but by 1 h after injection labeled MPO cells contained twice as much isotope as did the MA cells, i.e. the [3H]E was retained for a longer duration by MPO cells than by MA cells. Analysis of variance revealed
40
30 Z
~ 2o 0
10
1
2
3
4
S
6
7
Time [hours]
Fig. 2. Silver grain numbers (± S.E.M.) associated with labeled cells of medial preoptic area (MPO), ventrolateral portion of ventromedial nucleus (VLVM), arcuate nucleus (ARC) and medial amygdaloid nucleus (MA) of tile rat 15, 1 h, 3 h and 7 h after injection of [aH]estradiol.
227 1.0'
•
VLVM
.6,
"0
E := .4,
1
2
3
Time
4
5
6
7
[hoursl
Fig. 3. Labeling indices for cells in the MPO, VLVM, ARC and MA of the rat 15 min, 1 h, 3 h and 7 h after injection of [SH]estradiol. significant region (F = 48; df = 3,9; P < 0.05) and time (F = 26.3; df = 3,9; P < 0.005) effects. The region effects were primarily attributable to differences between MPO and MA cells since VLVM and ARC labeling was similar. The relative area under each curve in Fig. 2 gives an indication of the total amount of label retained in the nuclei of the labeled cells during this time interval. Between 15 min and 7 h after injection labeled cells in the MPO retained the greatest amount of isotope per cell. By comparison ARC cells retained only 74 ~ , VLVM cells only 71 ~ and MA cells only 56 ~ as much. Fig. 3 shows the portion of cells from the center of each neural region that were labeled (labeling index) at each time interval. In VLVM and MPO 80-95 of the cells were labeled at all time intervals examined. In the MA and ARC 90 ~ and 70 ~ of the cells were labeled, respectively, from 15 min-3 h but by 7 h only 50 ~ of them were still labeled. In the 2 animals receiving the competitive dose of cold Ee, nuclear labeling was reduced by more than 90 ~o. The present retention data confirm those derived from biochemical approaches while extending the limits of our topographical resolution. Accumulation and retention of nuclear ERe by cells in the hypothalamus has been shown to be maximal at 1 h and to decline by about 50 ~ by 7 h after administrationL In a recent study nuclear retention of Jail]estrogen by brain was shown to decline between 2 h and 4 h after injection while at 2 h the order of labeling was preoptic area > basal hypothalamus > amygdala 12. In addition to regional variation of nuclear retention within the brain, it also appears that nuclear retention of [aH]E varies among other target organs as well. Estrogen retention by the pituitary, for example, is maximal at 15 min after injection and declines rapidly thereafter while in uterine cells maximal nuclear retention does not occur until 1-3 h after injection 1°. The 4 neural regions examined in this study are involved in the control of sexual behavior and sex hormone levels. Lesions and electrical stimulation studies have revealed that neurons in the MPO suppress lordosis in the female rat, while those in the ventromedial nucleus (VMN) facilitate lordosis 14. Estrogen implants in the MPO 13 and VMN 15 are reported to facilitate lordotic behavior. Arcuate neurons project
228 p r i m a r i l y to the m e d i a n eminence a n d p r o b a b l y mediate releasing h o r m o n e secretion 11 while corticomedial a m y g d a l o i d neurons are b o t h inhibitory to g o n a d o t r o p h i n release a n d facilitatory to the p r e o v u l a t o r y L H surge 16. Even t h o u g h the physiochemical properties o f the Re, per se, m a y be similar for all target tissues a n d all neural regions 8, the mechanisms regulating the translocation, nuclear retention a n d r e m o v a l o f the E R e m a y vary significantly. Such t o p o g r a p h i c a l variations o f these regulatory mechanisms m a y be responsible, e.g. for our observation that M P O cells retain [3HIE for a longer d u r a t i o n t h a n d o M A cells. Since it has been d e m o n s t r a t e d in other target tissues that the m a g n i t u d e o f estrogen response is p r o p o r tional to the d u r a t i o n o f nuclear occupancy 1,3, it is possible that M P O cells m a y r e s p o n d m o r e strongly t h a n M A cells to similar levels o f estrogen or similarly m a y have lower thresholds o f response to estrogens. H o w these differences in response m a y be manifested is n o t yet known. I wish to t h a n k Mr. S. K. L a u for technical assistance. S u p p o r t e d by P H S G r a n t HD12173 a n d Research Career D e v e l o p m e n t A w a r d HD00243.
1 Anderson, J. N., Clark, J. H. and Peck, E. J., The relationship between nuclear receptor estrogen binding and uterotrophic responses, Biochem. Biophys. Res. Commun., 48 (1972) 1460-1468. 2 Anderson, J. N., Peck, E. J., Jr. and Clark, J. H., Nuclear receptor estrogen complex: accumulation, retention and localization in the hypothalamus and pituitary, Endocrinology, 93 (1973) 711-717. 3 Anderson, J. N., Peck, E. J. and Clark, J. H., Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei, Endocrinology, 96 (1975) 160-167. 4 Arnold, A. P., Quantitative analysis of steroid autoradiograms, J. Histochem. Cytochem., 29 (1981) 207-211. 5 Barfield, R. J. and Chen, J. J., Activation of estrous behavior in ovariectomized rats by intracerebral implants of estradiol benzoate, Endocrinology, 101 (1977) 1716-1725. 6 Eisenfeld, A. J., 3H-estradiol: in vitro binding to macromolecules from the rat hypothalamus, anterior pituitary and uterus, Endocrinology, 86 (1970) 1313-1318. 7 Ginsburg, M., Greenstein, B. B., MacLusky, N. J., Morris, I. D. and Thomas, P. J., An improved method for the study of high-affinity steroid binding: oestradiol binding in brain and pituitary, Steroids, 23 (1974) 773-792. 8 Kato, J., Characterization and function of steroid receptors in the hypothalamus and hypophysis. In Proceedings of the V International Congress of Endocrinology, Vol. 1, 1977, Excerpta Medica, pp. 12-17. 9 Keefer, D. A., Quantification of in vivo 3H-estrogen uptake by individual anterior pituitary cell types of male rat: a combined autoradiographic-immunocytochemical technique, J. Histochem. Cytochem., 2 (1981) 167-174. 10 Keefer, D, A., Dynamics of in situ estrogen uptake by nuclei of individual pituitary and uterine cell types, Horm. Metab. Res., in press. 11 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An autoradiographic study of the efferent connections of the ventromedial nucleus of the hypothalamus. J. comp. Neurol., 183 (1979) 785-816. 12 Lieberburg, I., MacLusky, N. and McEwen, B. S., Cytoplasmic and nuclear estradiol-17/3 binding in male and female rat brain: regional distribution, temporal aspects and metabolism, Brain Research, 193 (1980) 487-503. 13 Lisk, R. D., Diencephalic placement of estradiol and sexual receptivity in the female rat, Amer. J. PhysioL, 203 (1962) 493-496. 14 Pfaff, D. W., Estrogens and Brain Function: Neural Analysis of a Hormone-Controlled Marnmalian Reproductive Behavior, Springer-Verlag, New York, 1980.
229 15 Pfaff, D. and Keiner, M., Atlas of estradiol-concentrating cells in the central nervous system of the female rat, J. comp. Neurol., 151 (1973) 121-158. 16 Sawyer, C. H., Functions of the amygdala related to the feedback actions of gonadal steroid hormones. In B. E. Eleftheriou (Ed.), Ne~robiology of Amygdala, Plenum Press, New York, 1972 pp. 745-762. 17 Stumpf, W. E., Estrogen-neurons and estrogen-neuron systems in the periventricular brain, Arner. J. Anat., 129 (1970) 207-217.
Brain Research, 229 (1981) 224-229 Elsevier/North-Holland Biomedical Press
Nuclear retention characteristics of PH]estrogen by cells in four estrogen target regions of the rat brain*
DONALD A. KEEFER Department of Anatomy, University of Virginia Medical School, Charlottesville, VA 22908 (U.S.A.) (Accepted August 27th, 1981) Key words: hypothalamus - - amygdala - - medial preoptic area - - estrogen - - estrogen receptor
Nuclear retention of radioactivity was studied in neural estrogen target cells 15 min-7 h after i.v. injection of [3H]estradiol. Maximal uptake occurred by 15 min. Cells of the medial preoptic nucleus retained the label longer than did those of the medial amygdaloid nucleus. Cells of the ventromedial nucleus and arcuate nucleus exhibited similar retention profiles which were intermediate between the medial preoptic and medial amygdaloidcells. These data are discussed in relation to observations that the duration of nuclear occupancy by estrogens is proportional to the magnitude of the cellular response. A number of laboratories have established that significant quantities of high affinity, estradiol (ED-specific, cytoplasmic receptors (Re) exist in the hypothalamus and amygdala6, 7. [3H]estrogen ([3H]E) is bound and retained by the nuclei of cells in circumscribed regions of the hypothalamus and amygdala; primarily the medial preoptic nucleus (MPO), arcuate nucleus (ARC), ventrolateral portion of the ventromedial nucleus (VLVM) and the medial amygdaloid nucleus (MA) 15,17. This genomic interaction results in an alteration either of sexually related behavior or of reproductive hormone levels 14. Studies of estrogen receptor dynamics in the uterus indicate a direct relationship between the duration of nuclear occupancy by the estrogen-receptor complex (ERe) and the magnitude of the response by the target cells 1,3. A recent study has demonstrated differences in the dynamics of [3H]E uptake between uterus and pituitary and possibly among individual cell types of these organs 10. The present study was undertaken to analyze the temporal pattern of [3H]E uptake in 4 major E target areas of the brain. Ovariectomized Sprague-Dawley rats, 85-95 g, were given i.v. injections of 0.54 /~g/100 g body weight [2, 4, 6, 7-~H]estradiol-17fl. Groups of 4 rats were sacrificed at intervals of 15 min, 1 h, 3 h and 7 h after injection. As a competition control, 2 of these rats were given injections of 100 × higher doses of cold estradioi prior to the isotope. One additional rat was injected with vehicle only and was processed for an autoradio-
* Portions of these data were presented at the Histochemistry Society meetings, Minneapolis, 1981. 0 0 0 6 - 8 9 9 3 / 8 1 / ~ / $ 0 2 . 7 5 © Elsevier/North-Holland Biomedical Press
225
Fig. 1. Autoradiograms of rat brain showing accumulation of [3H]estrogen by cell nuclei 15 min (A,E,F), 1 h (B), 3 h (C) and 7 h (D) after injection of [SH]estradiol. A-D, medial preoptic area. Notice that neuropil labeling decreases from 15 min to 7 h after injection. E, ventrolateral portion of ventromedial nucleus. F, medial amygdaloid nucleus. Magnification 460 ×. graphic control. At each time interval rats were decapitated and brains were frozen in rapidly vortexing liquid Freon-22 at - - 1 5 0 °C. Frozen sections 6/zm thick were cut at - - 3 0 °C and thaw mounted onto desiccated emulsion-coated slides (Kodak NTB-2). Every third frontal section was collected through the hypothalamus and adjacent amygdala. After exposure for 40.0 -I- 0.4 days at - - 2 0 °C in desiccator slide boxes the autoradiograms were photographically processed under carefully controlled conditions 9. Autoradiograms were stained with methyl green pyronin. The center of each of the 4 brain regions was identified by scanning each series of autoradiograms. For every cell that was analyzed the nuclear area was determined (by measuring nuclear dia-
226 meter) and the silver grain number counted at 1250 ×. Background silver grains were counted over an area of adjacent neuropil equal in size to the area of each nucleus. Silver grains were counted over nuclei of randomly chosen cells (labeled and unlabeled) from the center of each neural region until a total of 20 labeled cells had been counted from each of 2 adjacent autoradiograms from each region of every animal. As each individual cell was measured and its silver grain number determined these data were immediately processed by microcomputer using the Poisson distribution to classify each cell as either labeled or unlabeled 4. A cell was classified as labeled only when the Poisson formula indicated that the chance occurrance of such grain density per nuclear area was less than 0.01. Additionally, a 'labeling index' was determined for each neural region by dividing the number of labeled cells by the number of labeled plus unlabeled cells. The data were further analyzed by 2-way ANOVA. In all 4 neural regions maximal nuclear uptake of isotope by labeled cells occurred by 15 min after injection. At this early time interval background labeling in the neuropil was appreciably higher than at later times (Fig. 1). By using the Poisson distribution to determine the labeling status of each cell at every time interval the influence of this time-dependent variation in neuropil grain density was equalized. In frontal sections labeled neurons of the ARC and the adjacent VLVM often appeared to form a continuum. It is interesting to note, therefore, that the nuclear retention profiles of these 2 target areas were essentially identical over the time intervals examined, showing a gradual, steady decrease in labeling over the whole interval (Fig. 2). Labeled cells of the MPO retained maximal labeling throughout the first hour after injection. By contrast, however, labeled cells of the MA rapidly released the label between 15 min and 1 h. As a consequence, 15 min after injection labeled cells of the MPO and MA were almost equally labeled, but by 1 h after injection labeled MPO cells contained twice as much isotope as did the MA cells, i.e. the [3H]E was retained for a longer duration by MPO cells than by MA cells. Analysis of variance revealed
40
30 Z
~ 2o 0
10
1
2
3
4
S
6
7
Time [hours]
Fig. 2. Silver grain numbers (± S.E.M.) associated with labeled cells of medial preoptic area (MPO), ventrolateral portion of ventromedial nucleus (VLVM), arcuate nucleus (ARC) and medial amygdaloid nucleus (MA) of tile rat 15, 1 h, 3 h and 7 h after injection of [aH]estradiol.
227 1.0'
•
VLVM
.6,
"0
E := .4,
1
2
3
Time
4
5
6
7
[hoursl
Fig. 3. Labeling indices for cells in the MPO, VLVM, ARC and MA of the rat 15 min, 1 h, 3 h and 7 h after injection of [SH]estradiol. significant region (F = 48; df = 3,9; P < 0.05) and time (F = 26.3; df = 3,9; P < 0.005) effects. The region effects were primarily attributable to differences between MPO and MA cells since VLVM and ARC labeling was similar. The relative area under each curve in Fig. 2 gives an indication of the total amount of label retained in the nuclei of the labeled cells during this time interval. Between 15 min and 7 h after injection labeled cells in the MPO retained the greatest amount of isotope per cell. By comparison ARC cells retained only 74 ~ , VLVM cells only 71 ~ and MA cells only 56 ~ as much. Fig. 3 shows the portion of cells from the center of each neural region that were labeled (labeling index) at each time interval. In VLVM and MPO 80-95 of the cells were labeled at all time intervals examined. In the MA and ARC 90 ~ and 70 ~ of the cells were labeled, respectively, from 15 min-3 h but by 7 h only 50 ~ of them were still labeled. In the 2 animals receiving the competitive dose of cold Ee, nuclear labeling was reduced by more than 90 ~o. The present retention data confirm those derived from biochemical approaches while extending the limits of our topographical resolution. Accumulation and retention of nuclear ERe by cells in the hypothalamus has been shown to be maximal at 1 h and to decline by about 50 ~ by 7 h after administrationL In a recent study nuclear retention of Jail]estrogen by brain was shown to decline between 2 h and 4 h after injection while at 2 h the order of labeling was preoptic area > basal hypothalamus > amygdala 12. In addition to regional variation of nuclear retention within the brain, it also appears that nuclear retention of [aH]E varies among other target organs as well. Estrogen retention by the pituitary, for example, is maximal at 15 min after injection and declines rapidly thereafter while in uterine cells maximal nuclear retention does not occur until 1-3 h after injection 1°. The 4 neural regions examined in this study are involved in the control of sexual behavior and sex hormone levels. Lesions and electrical stimulation studies have revealed that neurons in the MPO suppress lordosis in the female rat, while those in the ventromedial nucleus (VMN) facilitate lordosis 14. Estrogen implants in the MPO 13 and VMN 15 are reported to facilitate lordotic behavior. Arcuate neurons project
228 p r i m a r i l y to the m e d i a n eminence a n d p r o b a b l y mediate releasing h o r m o n e secretion 11 while corticomedial a m y g d a l o i d neurons are b o t h inhibitory to g o n a d o t r o p h i n release a n d facilitatory to the p r e o v u l a t o r y L H surge 16. Even t h o u g h the physiochemical properties o f the Re, per se, m a y be similar for all target tissues a n d all neural regions 8, the mechanisms regulating the translocation, nuclear retention a n d r e m o v a l o f the E R e m a y vary significantly. Such t o p o g r a p h i c a l variations o f these regulatory mechanisms m a y be responsible, e.g. for our observation that M P O cells retain [3HIE for a longer d u r a t i o n t h a n d o M A cells. Since it has been d e m o n s t r a t e d in other target tissues that the m a g n i t u d e o f estrogen response is p r o p o r tional to the d u r a t i o n o f nuclear occupancy 1,3, it is possible that M P O cells m a y r e s p o n d m o r e strongly t h a n M A cells to similar levels o f estrogen or similarly m a y have lower thresholds o f response to estrogens. H o w these differences in response m a y be manifested is n o t yet known. I wish to t h a n k Mr. S. K. L a u for technical assistance. S u p p o r t e d by P H S G r a n t HD12173 a n d Research Career D e v e l o p m e n t A w a r d HD00243.
1 Anderson, J. N., Clark, J. H. and Peck, E. J., The relationship between nuclear receptor estrogen binding and uterotrophic responses, Biochem. Biophys. Res. Commun., 48 (1972) 1460-1468. 2 Anderson, J. N., Peck, E. J., Jr. and Clark, J. H., Nuclear receptor estrogen complex: accumulation, retention and localization in the hypothalamus and pituitary, Endocrinology, 93 (1973) 711-717. 3 Anderson, J. N., Peck, E. J. and Clark, J. H., Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei, Endocrinology, 96 (1975) 160-167. 4 Arnold, A. P., Quantitative analysis of steroid autoradiograms, J. Histochem. Cytochem., 29 (1981) 207-211. 5 Barfield, R. J. and Chen, J. J., Activation of estrous behavior in ovariectomized rats by intracerebral implants of estradiol benzoate, Endocrinology, 101 (1977) 1716-1725. 6 Eisenfeld, A. J., 3H-estradiol: in vitro binding to macromolecules from the rat hypothalamus, anterior pituitary and uterus, Endocrinology, 86 (1970) 1313-1318. 7 Ginsburg, M., Greenstein, B. B., MacLusky, N. J., Morris, I. D. and Thomas, P. J., An improved method for the study of high-affinity steroid binding: oestradiol binding in brain and pituitary, Steroids, 23 (1974) 773-792. 8 Kato, J., Characterization and function of steroid receptors in the hypothalamus and hypophysis. In Proceedings of the V International Congress of Endocrinology, Vol. 1, 1977, Excerpta Medica, pp. 12-17. 9 Keefer, D. A., Quantification of in vivo 3H-estrogen uptake by individual anterior pituitary cell types of male rat: a combined autoradiographic-immunocytochemical technique, J. Histochem. Cytochem., 2 (1981) 167-174. 10 Keefer, D, A., Dynamics of in situ estrogen uptake by nuclei of individual pituitary and uterine cell types, Horm. Metab. Res., in press. 11 Krieger, M. S., Conrad, L. C. A. and Pfaff, D. W., An autoradiographic study of the efferent connections of the ventromedial nucleus of the hypothalamus. J. comp. Neurol., 183 (1979) 785-816. 12 Lieberburg, I., MacLusky, N. and McEwen, B. S., Cytoplasmic and nuclear estradiol-17/3 binding in male and female rat brain: regional distribution, temporal aspects and metabolism, Brain Research, 193 (1980) 487-503. 13 Lisk, R. D., Diencephalic placement of estradiol and sexual receptivity in the female rat, Amer. J. PhysioL, 203 (1962) 493-496. 14 Pfaff, D. W., Estrogens and Brain Function: Neural Analysis of a Hormone-Controlled Marnmalian Reproductive Behavior, Springer-Verlag, New York, 1980.
229 15 Pfaff, D. and Keiner, M., Atlas of estradiol-concentrating cells in the central nervous system of the female rat, J. comp. Neurol., 151 (1973) 121-158. 16 Sawyer, C. H., Functions of the amygdala related to the feedback actions of gonadal steroid hormones. In B. E. Eleftheriou (Ed.), Ne~robiology of Amygdala, Plenum Press, New York, 1972 pp. 745-762. 17 Stumpf, W. E., Estrogen-neurons and estrogen-neuron systems in the periventricular brain, Arner. J. Anat., 129 (1970) 207-217.