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Estrogen-concentrating hypothalamic and limbic neurons project to the medial preoptic nucleus
Brain Research, 451 (1988)381-385 Elsevier
381
BRE 22937
Estrogen-concentrating hypothalamic and Iimbic neurons project to the medial preoptic nucleus Thomas R. Akesson 1, Richard B. Simerly 2 and Paul E. Micevych 1 1Department of Anatomy and the Laboratory of Neuroendocrinology, Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) and 2Howard Hughes Medical Institute, Salk Institute, LaJolla, CA 92138 (U.S.A.)
(Accepted 1 March 1988) Key words: Tract tracing; Steroid autoradiography; Hypothalamus; Limbic system; Ventromedial nucleus; Medial amygdala; Reproduction
Estrogen-concentrating neurons that project to the medial preoptic nucleus of the male rat were found to be numerous in limbic and hypothalamic cell groups including the ventral part of the lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, the ventromedial nucleus, and the amygdalohippocampal zone. This steroid-sensitive circuitry is implicated in the activation of reproductive processes in the male.
The medial preoptic nucleus (MPN) has been shown to play a key role in steroid-regulated masculine sex behavior 22"24"41. Cells of the MPN concentrate both androgen and estrogen 19'25'32'35 and thus represent anatomical sites for gonadal steroid feedback, which is considered essential for reproductive function 10A3. While the principal circulating gonadal steroid in male rats is testosterone 33, the aromatization of testosterone to estradiol has been demonstrated to be an important step in the activation of male reproductive function 6'7'9'11'18'20,21,23,29,36. Indeed, numbers of estrogen receptors in the male hypothalamus and preoptic area increase in response to increasing levels of serum testosterone 19. Tract-tracing studies have shown that the MPN receives its strongest inputs from all of the major steroid-concentrating regions of the hypothalamus and limbic system, including the ventral part of the lateral septum (LSv), encapsulated part of the bed nucleus of the stria terminalis (BSTenc), posterodorsal medial nucleus of the amygdala (MeApd), ventrolateral part of the ventromedial hypothalamic nucleus (VMHvl), arcuate nu-
cleus (ARC), ventral premammillary nucleus (PMv), amygdalohippocampal zone (AHZ), and the midbrain periaqueductal gray 12.15,16,3°.37. The extensive connections between steroid sensitive brain regions have led to the hypothesis that steroid-accumulating neurons form an interconnected network that integrates autonomic and environmental signals necessary for gonadotropin release and the activation of reproductive behavior 12,14,30.37,39. The connectivity of estradiol-accumulating cells in the male central nervous system, however, remains to be determined. We have therefore characterized the distribution of estrogen-concentrating cells in the male rat that project to the MPN by combining the techniques of steroid autoradiography and retrograde tract-tracing. Part of this study has been reported elsewhere 3. Methods used were modified from Arnold 5 and Morrell and Pfaff27. Each adult male Sprague-Dawley rat (Simonson) received an iontophoretic injection of fluorogold (FG) (Fluorochrome, CO) through micropipettes (tip diameter = 20/~m) centered in the MPN by applying a 5-/~A positive current for 20 min. One week later, rats were castrated, allowed to sur-
Correspondence: T.R. Akesson, Department of Anatomy, UCLA Center for the Health Sciences, Los Angeles, CA 90024, U.S.A.
0006-8993/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Photomicrograph and line drawing illustrating location of fluorogold injection in the medial preoptic nucleus (MPN). ADP; anterodorsal preoptic nucleus; MePO, median preoptic nucleus; MPNc, m, 1, central, medial, lateral parts of the MPN; och, optic chiasm; PvPO, preoptic periventricular nucleus: V3. third ventricle, x90.
vive another week, and then infused with 150 ~Ci 2' [3H]estradio 1 (165 /.tCi/mM, New England Nuclear)/100 g b. wt. via the jugular vein over a 50min period. One h after infusions, rats were perfused transcardially with buffered 4% paraformaldehyde and brains were placed in buffered 20% sucrose for 1-3 days. Brains were then blocked, rapidly frozen in powdered dry ice,,and sectioned using a cryostat (Bright Instruments) in safelight conditions. Twenty#m-thick sections in the coronal plane were collected onto slides that had been previously coated with nuclear track emulsion (NTB 3, Kodak). Slides were stored at - 2 0 °C for 28 weeks and photodeveloped with D-19 and Kodafix (Kodak). Every 4th section from the decussation of the corpus callosum to the level of the mammillary bodies and every 10th section of the hindbrain through the PAG was analyzed. Using a 3 × background criterion to distinguish estrogen concentration, co-localization in each nucleus of interest was determined by counting all FG-, estrogen-, and doubly labeled cells in a 0.22-mm 2 area/nucleus/section. Areas sampled were selected from the region of each nucleus containing 4,6,7,16,17
the highest numbers of both FG- and estrogenlabeled cells. Dye-labeled, estrogen concentrating cells were identified using brightfield illumination of cellular-associated accumulations of silver grains and ultraviolet illumination of FG labeling (Fig. 2). Nomenclature was based on Simerly and Swanson 37. Since the analysis was restricted to regions of each
Fig. 2. Pliotomicrograph of estrogen-concentrating (E), fluorogold-labeled (FG), and doubly labeled neuronS in the ventrOlateral part of the ventromedial nucleus, x628.
383 TABLE I Proportions of estrogen-concentrating cells that project to the MPN as determined by combined steroid autoradiography and retrograde tract-tracing
Numbers of fluorogold-labeled (FG), estrogen-concentrating (E), and doubly labeled cells were sampled from a 0.22-mm2 area of each nucleus in 3 rats using the number of sections indicated. Abbreviations: LSv, ventromedial part of the ventrolateral septum; BSTenc, encapsulated part of the bed nucleus of the stria terminalis; MeA, medial nucleus of the amygdala; VMHvl, ventrolateral part of the hypothalamic ventromedial nucleus; ARC, arcuate nucleus; PMv, ventral premammillary nucleus; AHZ, amygdalohippocampal zone; PAG, periaqueductal gray. Nucleus
No. of sections analyzed
No. of FG cells sampled
No. of E cells sampled
No. of doubly labeled cells sampled
% FG cells concentrating E (~ + S.E.M.)
% E cells labeled by FG (:~ +_S.E.M.)
LSv BSTenc MeApd VMHvl ARC PMv AHZ PAG*
19 34 46 30 57 18 30 29
433 710 1031 1060 1234 577 1346 95
122 350 621 563 722 52 609 109
39 118 191 258 100 8 323 3
9.0 + 2.6 16.6 + 3.6 18.5 + 2.9 24.3 + 9.0 8.1 + 3.6 1.4 + 0.8 24.0 + 1.0 3.2 + 2.5
32.0 + 33.7 + 30.8 + 45.8 + 13.9 + 15.4 + 53.0 + 2.8 +
6.8 3.5 5.1 10.8 1.4 6.2 3.8 1.8
* Ventrolateral PAG, only 1 in 10 sections sampled; in all other nuclei 1 in 4 sections were sampled.
nucleus that were well represented by both FG- and estrogen-labeled cells, the numbers of FG-, estrogen-, and doubly-labeled cells (Table I) did not represent an overall nuclear composition. Within the areas that were sampled, the numbers of doubly labeled neurons in this study represent a conservative estimate of estrogen-concentrating cells projecting to the MPN because of the relatively thick sections used. Twenty-Mm sections facilitated cytoarchitectonic discrimination of brain regions analyzed, but because tritiated estradiol has a low emission energy, only the 1 - 4 Mm of tissue apposed to the emulsion could generate an autoradiographic signal 26. Thus, while FG-labeled cells were visualized throughout the depth of the section, only a fraction of the population of estrogen-positive cells could be identified. Overestimation of numbers of doubly labeled cells due to random coincidence of labels could be avoided by rejecting those cases where silver grains and FG were in different planes as determined by focusing through the section with a 40x objective. In general, the distribution of estrogen-concentrating cells in the male resembled that in the female rat 1'2'31'4°. Proportions of doubly-labeled neurons were particularly high in limbic structures surrounding the hypothalamus (LSv, BSTenc, M e A , A H Z ; Table I). Within the hypothalamus, the portion of estrogen-concentrating neurons that supplies the medi-
al MPN was largest in the VMHvl, and moderate in the A R C and PMv. In the P A G the proportion of estrogen-concentrating projection neurons was low (Table I). Our finding of an extensive, estrogen-receptive input to the MPN from limbic, hypothalamic, and brainstem sites underscores the importance of this region as an integrative locus mediating reproductive events 4'8. The chemical identity of these estrogen-accumulating neurons has not been elucidated. However, studies combining immunohistochemistry and steroid autoradiography in the female rat suggest possible candidates, which include dopamine 34, fl-endorphin 28, 7-aminobutyric acid ( G A B A ) 17, and substance p2. The latter finding that 24% of the estrogen-binding neurons of the VMHvl contain substance P, together with the present finding that 45% of VMHvl estrogen-binding neurons project to the MPN, suggest the possibility of a partial overlap. Moreover, in the same subdivision of the V M H , 2 6 - 3 6 % of estrogen-binding neurons project to the dorsal midbrain 27. Although the overlap of these subsets of estrogen-concentrating V M H v l neurons has not been determined, these findings together suggest that individual cells in the VMHvl may relay steroid sensitive signals to both the brainstem and the MPN. In summary, the diversity of steroid-receptive input to the MPN is consistent with the known corn-
384 plexity of sensory and e n d o c r i n e i n t e g r a t i o n asso-
tion of r e p r o d u c t i v e functions in the male.
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1 Akesson, T.R. and Micevych, P.E., Evidence of an absence of estrogen-concentration by CCK-immunoreactive neurons in the hypothalamus of the female rat, J. Neurobiol., 19 (1988) 3-16. 2 Akesson, T.R. and Micevych, P.E., Estrogen concentration by substance P-immunoreactive cells in the medial basal hypothalamus of the female rat, J. Neurosci. Res., in press. 3 Akesson, T.R., Simerly, R.B. and Micevych, P.E., Estradiol concentration by hypothalamic and limbic neurons which project to the medial preoptic nucleus, Soc. Neurosci. Abstr., 13 (1987) 1164. 4 Arendash, G.W. and Gorski, R.A., Effects of discrete lesions of the sexually dimorphic nucleus of the preoptic area or other medial preoptic regions on the sexual behavior of male rats, Brain Res. Bull., 10 (1983) 147-154. 5 Arnold, A., A technique for simultaneous steroid autoradiography and retrograde labeling of neurons, Brain Research, 192 (1980) 210-212. 6 Baum, M.J., Tobet, S.A., Starr, M.S. and Bradshaw, W.G., Implantation of dihydrotestosterone propionate into the lateral septum or medial amygdala facilitates copulation in castrated male rats given estradiol systemically, Horm. Behav., 16 (1982) 208-223. 7 Beyer, C., Morali, G., Naftolin, F., Larsson K. and PerezPalacios, G., Effect of some antiestrogens and aromatase inhibitors on androgen induced sexual behavior in castrated male rats, Horm. Behav., 7 (1976) 353-363. 8 Brackett, N.L. and Edwards, D.A., Medial preoptic connections with the midbrain tegmentum are essential for male sexual behavior, Physiol. Behav., 32 (1984) 79-84. 9 Chambers, K.C. and Phoenix, C.H., Testosterone is more effective than dihydrotestosterone plus estradiol in activating sexual behavior in old male rats, Neurobiol. Aging, 7 (1986) 127-132. 10 Christensen, L. and Clemens, L.G., Intrahypothalamic implants of testosterone or estradiol and resumption of masculine sexual behavior in tong term castrated male rats, Endocrinology, 95 (1974) 984-990. 11 Christensen, L.W. and Clemens, L.G., Blockade of testosterone-induced behavior in the male rat with intracranial application of the aromatization inhibitor, androst-l,4,6triene-3,17-dione, Endocrinology, 97 (1975) 1545-1551. 12 Cottingham, S.L. and Pfaff, D.W., Interconnections of steroid hormone-binding neurons: existence and implications. In D. Ganten and D.W. Pfaff (Eds.), Current Topics in Neuroendocrinology, Vol. 7, Springer, Berlin, 1986, pp. 223-249. 13 Davis, P.G. and Barfield, R.J., Activation of masculine sexual behavior by intracranial estradiol benzoate implants in male rats, Neuroendocrinology, 28 (1979) 217-227. 14 Dyer, R.G., MacLeod, N.K. and Ellendorff, F., Electrophysiological evidence for sexual dimorphism and synaptic
15
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27 28
convergence in the preoptic and anterior hypothalamic areas of the rat, Proc. R. Soc. Lond. B, 193 (1976) 421-440. Fahrbach, S.E., Morrell, J.I. and Pfaff, D.W., Estrogenconcentrating neurons in the medial preoptic area send axons to the ventral tegmental area and amygdala, Soc. Neurosci. Abstr., 9 (1983) 516. Fahrbach, S.E., Morrell, J.l. and Pfaff, D.W., Identification of medial preoptic neurons that concentrate estradiol and project to the midbrain in the rat, J. Comp. Neurol., 247 (1986) 364-382. Fliigge, G., Oertel, H. and Wuttke, W., Evidence for estrogen-receptive GABAergic neurons in the preoptic/anterior hypothalamic area of the rat brain, Neuroendocrinology, 43 (1986) 1-5. Gay, V.L. and Dever, N.W., Effects of testosterone propionate and estradiol benzoate alone or in combination on serum LH and FSH in orchidectomized rats, Endocrinology, 89 (1971) 161-168. Krey, L C . , Kamel, F. and McEwen, B.S., Parameters of neuroendocrine aromatization and estrogen receptor occupation in the male rat, Brain Research, 193 (1980) 277-283. Krey, C., MacLusky, N J . , Davis, P.A., Lieberburg, I. and Roy, E.J., Different intracellular mechanisms underlie testosterone's suppression of basal and stimulation of cyclic luteinizing hormone release in male and female rats, Endocrinology, 110 (1982) 2159-2167. Lieberburg, I. and McEwen, B.S., Brain cell nuclear retention of testosterone metabotites, 5a-dihydrotestosterone and estradiol-17,8 in adult rats, Endocrinology, 100 (1977) 588-597. Malsbury, C.W., Facilitation of male rat copulatory behavior by electrical stimulation of the medial preoptic area, Physiol. Behav., 7 (1971) 797-805. McEwen, B.S., Davis, P.G., Parsons, B. and Pfaff, D.W., The brain as a target for steroid hormone action, Annu. Rev. Neurosci., 2 (1979) 65-112. Merari, A. and Ginton, A., Characteristics of exaggerated sexual behavior induced by electrical stimulation of the medial preoptic area in male rats, Brain Research, 86 (1975) 97-108. Micevych, P.E. and Akesson, T.R., Differential distribution of estrogen and androgen concentrating cells in the rat medial preoptic area, Soc. Neurosci. Abstr., 13 (1987) 1164. Morrell, J.I. and Pfaff, D.W., Autoradiographic technique for steroid hormone localization. Application to the vertebrate brain. In N.T. Adler (Ed.), Neuroendocrinology of Reproduction, Plenum, New York, 1981, pp. 519-531. Morrell, J.I. and Pfaff, D . W , Characterization of estrogen-concentrating hypothalamic neurons by their axonal projections, Science, 217(1982) 1273-1276. Morrell, J.I., McGinty, F. and Pfaff, D.W., A subset of,8-
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endorphin- or dynorphin-containing neurons in the medial basal hypothalamus accumulates estradiol, Neuroendocrinology, 41 (1985) 417-426. Paup, D.C., Mennin, S.P. and Gorski, R.A., Androgenand estrogen-induced copulatory behavior and inhibition of luteinizing hormone (LH) secretion in the male rat, Horm. Behav., 6 (1975) 35-46. Pfaff, D.W. and Conrad, L.C.A., Hypothalamic neuroanatomy: steroid hormone binding and patterns of axonal projections, Int. Rev. Cytol., 54 (1978) 245-265. Pfaff, D.W. and Keiner, M., Atlas of estradiol-concentrating cells in the central nervous system of the female rat, J. Comp. Neurol., 151 (1973) 121-158. Rainbow, T.C., Parsons, B., MacLusky, N.J. and McEwen, B.S., Estradiol receptor levels in rat hypothalamic and limbic nuclei, J. Neurosci., 2 (1982) 1439-1445. Resko, J.A., Feder, H.H. and Goy, R.W., Androgen concentrations in plasma and testes of developing rats, J. Endocrinol., 40 (1968) 485-491. Sar, M., Estradiol is concentrated in tyrosine hydroxylasecontaining neurons of the hypothalamus, Science, 223 (1984) 938-940. Sar, M. and Stumpf, W.E., Distribution of androgen-concentrating neurons in rat brain. In W.E. Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger,
Basel, 1975, pp. 120-133. 36 Selmanoff, M.K., Brodkin, L.D., Weiner, R.I. and Siiteri, P.I., Aromatization and 5a- reduction of androgens in discrete hypothalamic and limbic regions of the male and female rat, Endocrinology, 101 (1977) 841-848. 37 Simerly, R.B. and Swanson, L.W., The organization of neural inputs to the medial preoptic nucleus of the rat, J. Comp. Neurol., 246 (1986) 312-342. 38 Simerly, R.B. and Swanson L.W., Projections of the medial preoptic nucleus: a PHA-L (Phaseolus vulgaris leucoagglutinin) anterograde-tract tracing study in the rat, J. Comp. Neurol., in press. 39 Stumpf, W.E. and Sar, M., Anatomical relationships between estrogen target sites and peptidergic-aminergic neurons: multiple activation of heterogeneous system (MAHS). In W. Wuttke and R. Horowski (Eds.), Gonadal Steroids and Brain Function, Springer, New York, 1981, pp. 18-28. 40 Stumpf, W.E., Sar, M. and Keefer, D.A., Atlas of estrogen target cells in rat brain. In W.E, Stumpf and L.D. Grant (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 104-119. 41 Van Dis, H. and Larson, K., Seminal discharge following intracranial electrical stimulation, Brain Research, 23 (1970) 381-386.
381
BRE 22937
Estrogen-concentrating hypothalamic and Iimbic neurons project to the medial preoptic nucleus Thomas R. Akesson 1, Richard B. Simerly 2 and Paul E. Micevych 1 1Department of Anatomy and the Laboratory of Neuroendocrinology, Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) and 2Howard Hughes Medical Institute, Salk Institute, LaJolla, CA 92138 (U.S.A.)
(Accepted 1 March 1988) Key words: Tract tracing; Steroid autoradiography; Hypothalamus; Limbic system; Ventromedial nucleus; Medial amygdala; Reproduction
Estrogen-concentrating neurons that project to the medial preoptic nucleus of the male rat were found to be numerous in limbic and hypothalamic cell groups including the ventral part of the lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, the ventromedial nucleus, and the amygdalohippocampal zone. This steroid-sensitive circuitry is implicated in the activation of reproductive processes in the male.
The medial preoptic nucleus (MPN) has been shown to play a key role in steroid-regulated masculine sex behavior 22"24"41. Cells of the MPN concentrate both androgen and estrogen 19'25'32'35 and thus represent anatomical sites for gonadal steroid feedback, which is considered essential for reproductive function 10A3. While the principal circulating gonadal steroid in male rats is testosterone 33, the aromatization of testosterone to estradiol has been demonstrated to be an important step in the activation of male reproductive function 6'7'9'11'18'20,21,23,29,36. Indeed, numbers of estrogen receptors in the male hypothalamus and preoptic area increase in response to increasing levels of serum testosterone 19. Tract-tracing studies have shown that the MPN receives its strongest inputs from all of the major steroid-concentrating regions of the hypothalamus and limbic system, including the ventral part of the lateral septum (LSv), encapsulated part of the bed nucleus of the stria terminalis (BSTenc), posterodorsal medial nucleus of the amygdala (MeApd), ventrolateral part of the ventromedial hypothalamic nucleus (VMHvl), arcuate nu-
cleus (ARC), ventral premammillary nucleus (PMv), amygdalohippocampal zone (AHZ), and the midbrain periaqueductal gray 12.15,16,3°.37. The extensive connections between steroid sensitive brain regions have led to the hypothesis that steroid-accumulating neurons form an interconnected network that integrates autonomic and environmental signals necessary for gonadotropin release and the activation of reproductive behavior 12,14,30.37,39. The connectivity of estradiol-accumulating cells in the male central nervous system, however, remains to be determined. We have therefore characterized the distribution of estrogen-concentrating cells in the male rat that project to the MPN by combining the techniques of steroid autoradiography and retrograde tract-tracing. Part of this study has been reported elsewhere 3. Methods used were modified from Arnold 5 and Morrell and Pfaff27. Each adult male Sprague-Dawley rat (Simonson) received an iontophoretic injection of fluorogold (FG) (Fluorochrome, CO) through micropipettes (tip diameter = 20/~m) centered in the MPN by applying a 5-/~A positive current for 20 min. One week later, rats were castrated, allowed to sur-
Correspondence: T.R. Akesson, Department of Anatomy, UCLA Center for the Health Sciences, Los Angeles, CA 90024, U.S.A.
0006-8993/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Photomicrograph and line drawing illustrating location of fluorogold injection in the medial preoptic nucleus (MPN). ADP; anterodorsal preoptic nucleus; MePO, median preoptic nucleus; MPNc, m, 1, central, medial, lateral parts of the MPN; och, optic chiasm; PvPO, preoptic periventricular nucleus: V3. third ventricle, x90.
vive another week, and then infused with 150 ~Ci 2' [3H]estradio 1 (165 /.tCi/mM, New England Nuclear)/100 g b. wt. via the jugular vein over a 50min period. One h after infusions, rats were perfused transcardially with buffered 4% paraformaldehyde and brains were placed in buffered 20% sucrose for 1-3 days. Brains were then blocked, rapidly frozen in powdered dry ice,,and sectioned using a cryostat (Bright Instruments) in safelight conditions. Twenty#m-thick sections in the coronal plane were collected onto slides that had been previously coated with nuclear track emulsion (NTB 3, Kodak). Slides were stored at - 2 0 °C for 28 weeks and photodeveloped with D-19 and Kodafix (Kodak). Every 4th section from the decussation of the corpus callosum to the level of the mammillary bodies and every 10th section of the hindbrain through the PAG was analyzed. Using a 3 × background criterion to distinguish estrogen concentration, co-localization in each nucleus of interest was determined by counting all FG-, estrogen-, and doubly labeled cells in a 0.22-mm 2 area/nucleus/section. Areas sampled were selected from the region of each nucleus containing 4,6,7,16,17
the highest numbers of both FG- and estrogenlabeled cells. Dye-labeled, estrogen concentrating cells were identified using brightfield illumination of cellular-associated accumulations of silver grains and ultraviolet illumination of FG labeling (Fig. 2). Nomenclature was based on Simerly and Swanson 37. Since the analysis was restricted to regions of each
Fig. 2. Pliotomicrograph of estrogen-concentrating (E), fluorogold-labeled (FG), and doubly labeled neuronS in the ventrOlateral part of the ventromedial nucleus, x628.
383 TABLE I Proportions of estrogen-concentrating cells that project to the MPN as determined by combined steroid autoradiography and retrograde tract-tracing
Numbers of fluorogold-labeled (FG), estrogen-concentrating (E), and doubly labeled cells were sampled from a 0.22-mm2 area of each nucleus in 3 rats using the number of sections indicated. Abbreviations: LSv, ventromedial part of the ventrolateral septum; BSTenc, encapsulated part of the bed nucleus of the stria terminalis; MeA, medial nucleus of the amygdala; VMHvl, ventrolateral part of the hypothalamic ventromedial nucleus; ARC, arcuate nucleus; PMv, ventral premammillary nucleus; AHZ, amygdalohippocampal zone; PAG, periaqueductal gray. Nucleus
No. of sections analyzed
No. of FG cells sampled
No. of E cells sampled
No. of doubly labeled cells sampled
% FG cells concentrating E (~ + S.E.M.)
% E cells labeled by FG (:~ +_S.E.M.)
LSv BSTenc MeApd VMHvl ARC PMv AHZ PAG*
19 34 46 30 57 18 30 29
433 710 1031 1060 1234 577 1346 95
122 350 621 563 722 52 609 109
39 118 191 258 100 8 323 3
9.0 + 2.6 16.6 + 3.6 18.5 + 2.9 24.3 + 9.0 8.1 + 3.6 1.4 + 0.8 24.0 + 1.0 3.2 + 2.5
32.0 + 33.7 + 30.8 + 45.8 + 13.9 + 15.4 + 53.0 + 2.8 +
6.8 3.5 5.1 10.8 1.4 6.2 3.8 1.8
* Ventrolateral PAG, only 1 in 10 sections sampled; in all other nuclei 1 in 4 sections were sampled.
nucleus that were well represented by both FG- and estrogen-labeled cells, the numbers of FG-, estrogen-, and doubly-labeled cells (Table I) did not represent an overall nuclear composition. Within the areas that were sampled, the numbers of doubly labeled neurons in this study represent a conservative estimate of estrogen-concentrating cells projecting to the MPN because of the relatively thick sections used. Twenty-Mm sections facilitated cytoarchitectonic discrimination of brain regions analyzed, but because tritiated estradiol has a low emission energy, only the 1 - 4 Mm of tissue apposed to the emulsion could generate an autoradiographic signal 26. Thus, while FG-labeled cells were visualized throughout the depth of the section, only a fraction of the population of estrogen-positive cells could be identified. Overestimation of numbers of doubly labeled cells due to random coincidence of labels could be avoided by rejecting those cases where silver grains and FG were in different planes as determined by focusing through the section with a 40x objective. In general, the distribution of estrogen-concentrating cells in the male resembled that in the female rat 1'2'31'4°. Proportions of doubly-labeled neurons were particularly high in limbic structures surrounding the hypothalamus (LSv, BSTenc, M e A , A H Z ; Table I). Within the hypothalamus, the portion of estrogen-concentrating neurons that supplies the medi-
al MPN was largest in the VMHvl, and moderate in the A R C and PMv. In the P A G the proportion of estrogen-concentrating projection neurons was low (Table I). Our finding of an extensive, estrogen-receptive input to the MPN from limbic, hypothalamic, and brainstem sites underscores the importance of this region as an integrative locus mediating reproductive events 4'8. The chemical identity of these estrogen-accumulating neurons has not been elucidated. However, studies combining immunohistochemistry and steroid autoradiography in the female rat suggest possible candidates, which include dopamine 34, fl-endorphin 28, 7-aminobutyric acid ( G A B A ) 17, and substance p2. The latter finding that 24% of the estrogen-binding neurons of the VMHvl contain substance P, together with the present finding that 45% of VMHvl estrogen-binding neurons project to the MPN, suggest the possibility of a partial overlap. Moreover, in the same subdivision of the V M H , 2 6 - 3 6 % of estrogen-binding neurons project to the dorsal midbrain 27. Although the overlap of these subsets of estrogen-concentrating V M H v l neurons has not been determined, these findings together suggest that individual cells in the VMHvl may relay steroid sensitive signals to both the brainstem and the MPN. In summary, the diversity of steroid-receptive input to the MPN is consistent with the known corn-
384 plexity of sensory and e n d o c r i n e i n t e g r a t i o n asso-
tion of r e p r o d u c t i v e functions in the male.
ciated with r e g u l a t i o n of r e p r o d u c t i v e processes. In particular,
the e x t e n s i v e localization of e s t r o g e n -
This r e s e a r c h
was s u p p o r t e d
by NS 21220 to
22869 to T . R . A . ,
and the H o w a r d
c o n c e n t r a t i n g cells that p r o j e c t to the M P N a g r e e s
P.E.M.,
with the p r o p o s e d role of a r o m a t i z a t i o n of testos-
H u g h e s M e d i c a l Institute (R. B. S.).
HD
t e r o n e in a circuitry which is i n v o l v e d in the activa-
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