John L. Gerlach

Rat Brain Binds Adrenal Steroid Hormone: Radioautography of Hippocampus with Corticosterone

Tritiated corticosterone injected subcutaneously into adrenalectomized male rats 1 hour before killing produced intense labeling of the hippocampus in radioautograms prepared by a method that reduced or prevented diffusion of the radioactive material. The pyramidal neurons of the cornu ammonis and the granule neurons of the gyrus dentatus contained more radioactivity than did other regions of the brain; however, the intensity of labeling varied among adjacent neurons. The nuclei of many neurons were clearly labeled but radioactivity was relatively sparse in the cytoplasm, in the axons where they branch from cell bodies, and in adjacent neuropil.

Autoradiographic localization of estradiol-binding neurons in the rat hippocampal formation and entorhinal cortex

This study has examined the distribution of [3H]estradiol and [1 alpha,2 alpha-3H]testosterone uptake in the hippocampal formation and entorhinal cortex of male and female rats. In both males and females, [3H]estradiol-binding neurons in Ammons horn are located deep in stratum pyramidale and may correspond either to polymorphic interneurons or to early maturing pyramidal cells. Interneurons of strata oriens, lucidum and radiatum of Ammons horn and of stratum moleculare of the subiculum also bind [3H]estradiol, as do basket cell interneurons in the polymorphic, infragranular layer of the dentate gyrus. While no granule cells appear to accumulate [3H]estradiol, these cells may be affected transsynaptically by gonadal steroids via their afferent contacts with the entorhinal cortex, which, of the areas examined, contains the greatest number of [3H]estradiol-binding neurons. While relatively few neurons concentrate [3H]estradiol in the hippocampal formation, these are localized to specific subpopulations, which may enhance their functional significance. Because there is no significant nuclear accumulation of [3H]-alpha-testosterone in either the entorhinal cortex or hippocampal formation, it appears that aromatase enzyme activity is not a major contributor to estrogen receptor occupancy in adult rats.

Cells in regions of rhesus monkey brain and pituitary retain radioactive estradiol, corticosterone and cortisol differentially.

Gonadal and adrenal glucocorticoid hormones are biochemical messengers which influence a wide range of physiological and behavioral processes in all vertebrate orders. The availability of these and other steroid hormones, as well as the integrity of the processes they modulate, ultimately depends on the metabolic responsiveness of target brain cells, the nexus of neuroendocrine integration and regulation. Such target cells that are sensitive to estrogens and glucocorticoids have been localized in brains of rats by a variety of strategies, including cell fractionation and nuclear isolation ~, radioautographyl,14,~4, 36, enzyme studies7, 22, histofluorescence 12, implantation of hormones and recording of neuronal unit electrical activity26,3a, aS. The experiments we report here extend to female rhesus monkeys, for the first time to our knowledge, observations similar to those on rats 15,t7,24, made by cell fractionation and nuclear isolation (NI) as well as by radioautography (RA), of differential binding and retention of radioactivity from [aH]estradiol ([aH]E2), [aH]corticosterone (Jail]B) and [aH]cortisol ([aH]F) by cells in particular regions of the brain and pituitary. The three radioactive steroid hormones used in this study, [2,4,5,7-3H]E2 (95 Ci/mmole), [l,2,6,7-aH]B (84 Ci/mmole), and [1,2-aH]F (153 Ci/mmole), were obtained from New England Nuclear Corp. (Boston, Mass.). Each hormone was prepared for infusion by evaporating the stock solution to dryness under a stream of nitrogen, redissolving the residue in 1 ml absolute ethanol and, just before use, diluting with 19 ml isotonic saline. Young adult, cycling, female rhesus monkeys, Macaca mulatta (3.4-5.0 kg, feral born and obtained from Primate Research Laboratory, Long Island, N.Y.) were

Putative Glucocorticoid Receptors in Hippocampus and Other Regions of the Rat Brain

It has been known for some time from neuroendocrine and behavioral studies that the brain is both a master controller of endocrine function and a target for these endocrine secretions. There exist complex feedback interactions between endocrine secretions and the brain which not only control the secretion of hypothalamic and pituitary hormones but also influence neural activity underlying behavior. It is only quite recently that we have come to appreciate the role of the entire limbic brain, and not just the hypothalamus, in these endocrine-brain interactions.

Perinatal development of hypothalamic and cortical estrogen receptors in mouse brain: Methodological aspects

Binding of the estrogen, [3H]moxestrol, to fetal and neonatal mouse brain cytosol receptors was examined to determine the ontogeny of estrogen receptors in the hypothalamus and cerebral cortex from embryonic (E) day 15 to postnatal (P) day 18. Cytosol receptor assays were performed under exchange conditions at 25 degrees C for 4 h in order to measure receptors which had become occupied by estradiol during tissue homogenization. Scatchard analysis revealed high affinity (Kd = 0.4 nM) sites and was in good agreement with single point assays at 1 nM, which measured 70% of binding capacity. Binding was initially examined in the whole forebrain. Total binding of [3H]moxestrol in the forebrain increases between E15 and E18 and reaches adult levels at P9. The increase in binding relative to protein content peaks at P9 and then decreases, whereas the amount of binding relative to DNA content reaches a maximum between P12 and P15. The developmental time-course of the estrogen receptors was studied in the hypothalamus and 3 cortical regions. In the hypothalamus binding of [3H]moxestrol increases from P5 to P18 of the cortical areas. The cingulate cortex shows the highest amount of binding, increasing until P9 and then declining. In the other two cortical areas studied, the lateral and posterior cortex, binding expressed per mg DNA, is somewhat higher between P7 and P15 than in adults. When the binding is expressed per mg protein there is a sharp decline after P7, the magnitude of which is probably a result of a large increase in protein content relative to amount of receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for Glucocorticoid Target Cells in the Rat Optic Nerve. Hormone Binding and Glycerolphosphate Dehydrogenase Induction

Abstract: Biochemical evidence suggests that neuroglia are responsive to glucocorticoids, yet previous studies of glucocorticoid localization have typically failed to demonstrate significant uptake by neuroglial cells. To further investigate this problem, we measured glycerol‐3‐phosphate dehydrogenase (GPDH) activity and glucocorticoid receptor binding capacity in normal rat optic nerves and in those undergoing Wallerian (axonal) degeneration. Binding studies were also performed on hippocampus and anterior pituitary for comparison purposes. Normal optic nerve preparations possessed a high level of GPDH activity that was glucocorticoid‐inducible and that increased further following axonal degeneration. Antibody inactivation experiments demonstrated the presence of more enzyme molecules in the degenerating nerve preparations. Correlative immunocytochemical studies found GPDH‐positive reaction product only in morphologically identified oligodendrocytes, a result that is consistent with the previously reported localization of this enzyme in rat brain. Optic nerve cytosol fractions displayed substantial high‐affinity binding of both dexamethasone (DEX) and corticosterone (CORT) that, like GPDH, was elevated approximately twofold in degenerating nerves. Finally, in vivo accumulation of [3H]DEX and [3H]CORT by optic nerve and other myelinated tracts was examined using nuclear isolation and autoradiographic methods. Although neither steroid was found to be heavily concentrated by these tissues in vivo, a small preference for DEX was observed in the nuclear uptake experiments. These results are discussed in terms of the hypothesis that glial cells are targets for glucocorticoid hormones.

Biochemical and radioautographic analysis of estrogen-inducible progestin receptors in female ferret brain and pituitary: correlations with effects of progesterone on sexual behavior and gonadotropin-releasing hormone-stimulated secretion of luteinizing hormone.

Cytosolic binding molecules for the synthetic progestin [3H]R5020, were isolated in vitro from several brain regions including preoptic area/anterior hypothalamus, mediobasal hypothalamus, medial amygdala and parietal cortex as well as the pituitary gland of ovohysterectomized female ferrets. Binding of [3H]R5020 to cortical cytosols was saturable, of high affinity (apparent dissociation constant of 2.0 nM), and was steroid specific. Pretreatment of ferrets with a Silastic capsule containing estradiol caused significant increments in the concentration of cytosolic R5020 binding sites in hypothalamus and pituitary gland, but not in the other brain regions studied. Brains of additional ovohysterectomized ferrets, which had been primed with estradiol prior to receiving an i.v. injection of [3H]R5020, were processed radioautographically. Intense labeling of cells was seen in the medial and lateral preoptic area, in the lateral hypothalamus, and in the ventromedial and arcuate nuclei of the hypothalamus. Radioautograms from the brains of additional ovohysterectomized ferrets given an i.v. injection of [3H]estradiol revealed labeled cells in all of the above regions, in addition to the basolateral portion of the septum, the bed nucleus of the stria terminalis, and in the anterior amygdaloid area as well as the medial and cortical nuclei of the amygdala. This distribution of neural progestin and estrogen binding sites resembles those previously reported for these steroids in the rat, guinea pig, hamster and macaque. Functional studies showed that acute s.c. implantation of a Silastic capsule containing progesterone to ovohysterectomized ferrets, which had been primed with a low dosage of estradiol, failed to augment their sexual receptivity in limited tests with stimulus males given 4 and 8 h after progesterone treatment. This result contrasts with the well-established facilitatory effect of progesterone on sexual receptivity in rat and guinea pig. Chronic exposure to a progesterone capsule caused significant reductions in sexual receptivity in ovohysterectomized ferrets which were implanted concurrently with a second Silastic capsule containing a high dosage of estradiol. Similar effects of progesterone have been reported in rat and guinea pig, but not in the rhesus monkey. Thus species differences in the ability of progesterone to facilitate or inhibit sexual receptivity are not readily explained by species differences in the neural distribution of estrogen-induced progestin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Differentiation of embryonic hypothalamic transplants cultured on the choroidal pia in brains of adult rats.

SummaryHypothalamic tissue from 16 to 18-day fetal rats was transplanted onto the choroidal pia overlying the superior colliculus in adult female rats. After survival periods of 2 weeks to 19 months, brains containing transplants were processed for monoamine fluorescence histochemistry, immunohistochemistry for three neuropeptides (LHRH, somatostatin, neurophysin), or for autoradiography in ovariectomized hosts that received [3H] estradiol. Most of the transplants survived and retained or increased in size; 14 of 25 transplants examined by fluorescence histochemistry were found to contain median eminence-like structures. In almost all of the transplants that were stained for neuropeptides, beaded processes and occasional cell bodies were observed. Although immunoreactive fibers were found near blood vessels, no palisade arrangement typical of the normal median eminence was evident. Each of the hypothalamic transplants on which steroid autoradiography was performed contained clusters of estrophilic neurons, the intensity of labeling of which was comparable to that seen in the host hypothalamus. These results indicate that many characteristic morphological and chemical features of the hypothalamus, which are not evident in the 16 to 18-day fetus, are elaborated in transplants during the survival period in the host. Transplantation of fetal hypothalamus to adult choroidal pia thus appears to be a valuable approach for studying the factors, humoral or neural, that regulate the differentiation of this brain region.

Neural steroid hormone receptors

Abstract (1) Cell nuclear and cytoplasmic receptors for estrogens, androgens, and glucocorticoids have been identified in brains and pituitary glands of vertebrates. With respect to topography, estradiol (E 2 ) receptors are localized primarily in the hypophysiotrophic area and amygdala; 5-α-dihydrotestosterone (DHT) receptors are found in hypothalamus and limbic regions in smaller amounts and more uniformly distributed than those for estradiol; and corticosterone receptors are found in the hippocampal formation, septum, entorhinal cortex and amygdala. (2) Where information is available, mainly for estrogen receptors, their neural topography shows a remarkable constancy among vertebrates. The neural topography of estrogen and glucocorticoid receptors of rat and rhesus monkey will be compared. (3) A complicating factor in the study of androgens interacting with the brain is the conversion of testosterone (T) in neural tissue to both estrogenic and androgenic metabolites. Two of the products, E 2 and DHT, are recovered attached to cell nuclear receptors in the rat brain, whereas only DHT and T itself are found in pituitary cell nuclei. Evidence from other laboratories suggests that interactions of E 2 and DHT or T with intracellular receptors each subserve different behavioral and neuroendocrine functions in the rat. (4) The topography of estrogen receptors in the rat brain provides an excellent opportunity for studying estrogen action on brain chemistry. Estrogen effects on monoamine oxidase, choline acetylase, and glucose-6-phosphate dehydrogenase activity will be described. The overall importance of the action of steroid hormones on gene expression will be briefly discussed.

The role of androgen receptors in sexual differentiation of the brain: Effects of the testicular feminization (Tfm) gene on androgen metabolism, binding, and action in the mouse

The effects of the testicular feminization (Tfm) mutation on androgen metabolism, binding, and action were studied in the mouse. Cytosolic binding of [3H] dihydrotesterone (DHT) was reduced by approximately 90% in the brains, pituitaries, submaxillary glands, and kidneys of Tfin/Y males, as compared to wild-type male controls. Nuclear [3H] DHT binding was abolished in tissues from Tfm male animals. Implantation of testosterone-containing (T-containing) Silastic capsules elevated nuclear estrogen receptor (ERn) concentrations to a similar extent in the brains of all four genotypes studied (normal males, Tfm-affected males, normal females, and Tfm carrier females). Administration of T to normal males led to elevations of cytosolic progestin receptors (PRc) and monoamine oxidase (MAO) activity in the brain, and of alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) in the kidney. In Tfm-affected males, only the PRc response to T was observed, at a somewhat reduced level compared to that in normal male and female controls: no effect of T was observed with respect to either MAO in the brain, or ADH and G6PDH activities in the kidney. Estradiol (E2) administration increased the activity of pituitary G6PDH and lactic dehydrogenase (LDH) in all four genotypes. However, brain MAO activity was increased by E2only in normal and carrier females. The lack of an MAO response to E2 in Tfm-affected males is consistent with the hypothesis that the central nervous system of this genotype is at least partially masculinized, despite the loss of androgen receptor-mediated responses—most probably as a result of local conversion of T to E2 within the brain itself.