Neil J. MacLusky

Steroid hormones affect limbic afterdischarge thresholds and kindling rates in adult female rats.

UNLABELLEDnCatamenial epileptics show particular vulnerability to seizures during menstruation and at the time of ovulation, when circulating estradiol (E(2))/progesterone (P(4)) ratios are high. The present study tested the hypothesis that alterations in neuronal excitability induced by E(2) and P(4) affect thresholds and the development of secondary generalization in kindled rats.nnnMETHODSnThe effects of endogenous hormones secreted during the estrous cycle, and of exogenous exposure to E(2) and P(4) after ovariectomy (OVX), with and without adrenalectomy (ADX), were tested. Kindling electrodes were implanted in the basolateral amygdala or dorsal hippocampus in adult female rats. The anticonvulsive effects of P(4) on amygdala kindled seizures were also determined in intact subjects.nnnRESULTSnIn intact females, afterdischarge thresholds (ADTs) in the amygdala were significantly lower (306+/-48 microA; peak to peak) at mid-day proestrus, just prior to ovulation, when serum E(2) is elevated. ADTs were more than twofold higher (808+/-95 microA) during metestrus, coincident with peak ovarian P(4) secretion. In OVX females, amygdala thresholds were lowest with E(2) replacement and highest with P(4) replacement. Hippocampal ADT was unaffected by hormone replacement after OVX. The rates of both amygdala and hippocampal kindling were significantly accelerated by E(2) and slowed by P(4). E(2) replacement significantly increased serum corticosterone (CORT) levels. In ADX rats, CORT replacement increased kindling rates, synergizing with the effects of E(2). In fully kindled animals, P(4) administration suppressed motor seizures in approximately 60% of cases.nnnCONCLUSIONSnE(2) lowers amygdala ADTs and facilitates kindling. This effect may involve both direct E(2) effects and indirect effects mediated via increased levels of circulating corticosterone. P(4) raises amygdala ADTs, slows kindling development and suppresses fully kindled seizures. Hence, P(4) may have potential therapeutic value for women with catamenial epilepsy.

Immunocytochemical detection of androgen receptor in human temporal cortex: Characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues

Immunocytochemical and biochemical studies have demonstrated the presence of androgen receptor protein in various regions of the rodent and non-human primate cortex. Localization of androgen receptor in the human brain has, however, not been studied as extensively, because of difficulties in obtaining suitable tissue samples. In the present study, we have localized androgen receptors in both frozen and paraffin-embedded temporal cortex from epileptic patients undergoing resection. Polyclonal antibodies were raised against fusion proteins containing fragments of the human androgen receptor protein. The antibodies were affinity-purified against the corresponding fusion protein. Immunoprecipitation and Western blotting using extracts from human cell lines demonstrated the specificity of the antibodies for the human androgen receptor and lack of cross-reactivity with other steroid hormone receptors. Immunocytochemistry was performed on frozen and paraffin sections of human temporal cortex and in paraffin-embedded benign hyperplastic prostates (BPH), as well as prostate and breast carcinomas, by the streptavidin-biotin-peroxidase method. Antigen-retrieval was performed in paraffin-embedded sections using microwave irradiation. Specific nuclear and cytoplasmic immunoreactivity for androgen receptor was detected in neurons, astrocytes, oligodendrocytes, and microglia cells of the temporal cortex. In contrast, only nuclear staining was observed in BPH, prostate and breast carcinomas. Immunoprecipitation of human temporal cortex lysate and subsequent Western blot analysis demonstrated the expression of a 98 kDa immunoreactive protein, slightly smaller than the reported molecular weight of the wild-type androgen receptor. These results provide further evidence for the expression of androgen receptor in the human temporal cortex. The use of these immunocytochemical techniques should enable the retrospective determination of possible changes in androgen receptor expression in a variety of archival paraffin-embedded tissues, including samples of the human central nervous system.

Cellular variations in estrogen receptor mRNA translation in the developing brain: evidence from combined [125I]estrogen autoradiography and non-isotopic in situ hybridization histochemistry.

The spatial distribution of cells in the adult rodent forebrain which express estrogen receptor mRNA, as shown by in situ hybridization histochemistry with isotopically-labeled probes, has been reported to overlap with regions that are known targets of estrogen and which bind estrogen. The extent to which detection of estrogen receptor mRNA within developing forebrain neurons of the postnatal day 10-12 female rat is accompanied by translation into estrogen binding sites was investigated by combining [125I]estrogen autoradiography with non-isotopic (digoxigenin) in situ hybridization, using a 48-base oligodeoxyribonucleotide probe encoding a sequence of the estrogen-binding domain of rat uterine estrogen receptor cDNA. Estrogen receptor mRNA and estrogen binding sites appeared to be restricted to neurons. No mRNA or binding was seen in ependymal cells. Cells expressing estrogen receptor mRNA were widely distributed in the developing rat forebrain and were found in brain regions generally corresponding to those previously shown in the adult, with the addition of some regions not previously described, such as the medial habenula and dorsal endopiriform nucleus. Although there was widespread overlapping of estrogen receptor mRNA expression with known estrogen binding sites, there were regional and cellular variations in the extent of receptor mRNA translation. This pattern was true for developing forebrain regions previously defined as estrogen receptor-containing (hypothalamus, preoptic area, medial and lateral septum, vertical and horizontal nuclei of the diagonal band, cerebral cortex, hippocampus and amygdala) as well as for regions heretofore not considered estrogen targets (the thalamus, dorsal endopiriform nucleus, claustrum, ventral pallidum/substantia innominata and the basal nucleus of Meynert) or characterized as estrogen-responsive in the adult without previously documented estrogen binding [caudate-putamen (striatum)]. While estrogen binding and receptor mRNA expression always co-localized, neurons expressing estrogen receptor mRNA did not always exhibit ligand binding and there was no clear-cut relationship between the intensity of the hybridization signal and estrogen binding. Little, however, is known about translational control of estrogen receptor expression in the brain. Localization of estrogen binding sites to regions not generally considered targets of estrogen would appear to reflect the greater sensitivity of the iodinated ligand than the tritiated estrogens more commonly used for autoradiography. Non-isotopic in situ hybridization histochemistry combined with [125I]estrogen autoradiography represents a very powerful tool with which to study regulation of estrogen receptor gene expression at the single cell level with an exceptional degree of cellular and anatomical resolution.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of Estrogen Receptor Concentrations in the Rat Brain: Effects of Sustained Androgen and Estrogen Exposure

To determine whether estrogen and androgens either alone or in combination downregulate estrogen receptors in the brain, ovariectomized/adrenalectomized female rats received one of the following four treatments: (1) one subcutaneously placed Silastic capsule containing 10% estradiol in cholesterol, (2) one capsule containing 10% estradiol and two capsules containing 100% 5 alpha-dihydrotestosterone (DHT), (3) two capsules containing DHT, or (4) empty Silastic capsules (control animals). Animals were killed 4 or 8 days after capsule insertion and the occupied, unoccupied and total estrogen receptor content in specific brain nuclei was determined by quantitative in vitro autoradiography. To determine if the effects of the androgen were reversible, DHT capsules were removed after 4 days from half of the estradiol+DHT-treated rats, and the animals were killed 4 days later. Estradiol downregulated estrogen receptor expression in the periventricular preoptic area, medial preoptic area, bed nucleus of the stria terminalis (BNST), arcuate nucleus (ARC), ventromedial nucleus (VMN), and medial and cortical amygdala, decreasing receptor content by 30-41% in animals treated for 4 days, and by 44-60% in animals treated for 8 days with estradiol alone. DHT treatment in combination with estradiol further decreased estrogen receptor content in the BNST, ARC and VMN, relative to the estradiol-only animals. DHT in the absence of estrogen was without effect. In animals in which the DHT capsules were removed after 4 days of exposure, allowing the estradiol to remain for a further 4 days, estrogen receptor levels were indistinguishable from those measured in control animals treated for 8 days with estradiol alone. These results demonstrate that sustained estrogen exposure downregulates levels of estrogen receptor in the brain and confirm that DHT synergizes with estrogen in inducing this response in some, but not all, target neuronal groups.

Neuroendocrine function and response to stress in mice with complete disruption of glucagon-like peptide-1 receptor signaling.

Glucagon-like peptide-1 (GLP-1), a potent regulator of glucose homeostasis, is also produced in the central nervous system, where GLP-1 has been implicated in the neuroendocrine control of hypothalamic-pituitary function, food intake, and the response to stress. The finding that intracerebroventricular GLP-1 stimulates LH, TSH, corticosterone, and vasopressin secretion in rats prompted us to assess the neuroendocrine consequences of disrupting GLP-1 signaling in mice in vivo. Male GLP-1 receptor knockout (GLP-1R−/−) mice exhibit reduced gonadal weights, and females exhibit a slight delay in the onset of puberty; however, male and female GLP-1R−/− animals reproduce successfully and respond appropriately to fluid restriction. Although adrenal weights are reduced in GLP-1R−/− mice, hypothalamic CRH gene expression and circulating levels of corticosterone, thyroid hormone, testosterone, estradiol, and progesterone are normal in the absence of GLP-1R−/− signaling. Intriguingly, GLP-1R−/− mice exhibit paradoxic...

Testosterone and its metabolites affect afterdischarge thresholds and the development of amygdala kindled seizures

UNLABELLEDnIn boys with epilepsy, pubertal increases in seizure frequency may be associated with rising androgen levels. The present study tested the hypothesis that testosterone (T) and/or its metabolites might affect amygdala seizure thresholds and the development of secondary generalization from amygdala foci (kindling). Afterdischarge thresholds and kindling rate were measured in gonadectomized (GDX) male rats, with or without T replacement therapy. Drugs that block either androgen or estradiol (E(2)) receptor-mediated responses were also tested.nnnMETHODSnKindling electrodes were implanted in the basolateral amygdala of adult male Wistar rats. In Experiment 1, subjects were GDX and implanted with a silastic capsule containing either: cholesterol (control); T; 5% E(2) in cholesterol; or 5alpha-dihydrotestosterone (DHT). In Experiment 2, intact subjects were treated with daily injections of vehicle (control); daily injections of flutamide (an androgen receptor antagonist); or Silastic implants containing 1,4,9-androstatriene 3,17-dione (ATD; an aromatase inhibitor).nnnRESULTSnIn Experiment 1, initial afterdischarge (AD) thresholds were significantly lowered by E(2) treatment, as compared to cholesterol controls, and remained low throughout the kindling paradigm. In T replaced males, AD threshold significantly decreased over the kindling period, a response that was not observed in DHT treated rats. Rates of kindling were significantly faster as a result of T, E(2) and DHT treatment, as compared to cholesterol controls. E(2) treated males kindled the fastest of all 3 groups. In Experiment 2, initial AD thresholds were significantly lowered by flutamide treatment, as compared to cholesterol controls, and remained low throughout the kindling paradigm. AD threshold significantly decreased over the kindling period in intact males, a response that was blocked by ATD treatment. Both flutamide and ATD significantly slowed the rate of kindling, as compared to intact controls. ATD had the most dramatic inhibitory effect on kindling rate.nnnCONCLUSIONSnIn males, T and its two metabolites, E(2) and DHT, all appear to enhance the development of amygdala-kindled seizures. E(2) has the most potent epileptogenic effect. Antagonism of E(2) mediated effects in the brain may have potential therapeutic value for males with epilepsy.

Limbic Seizures Alter Reproductive Function in the Female Rat

Summary: Purpose: Reproductive dysfunction and endocrine disorders are common among women with temporal lobe epilepsy. This study used the kindled rat model to test the hypothesis that limbic seizures directly contribute to reproductive dysfunction.

Sex and the developing brain : Suppression of neuronal estrogen sensitivity by developmental androgen exposure

The developmental effects of androgen play a central role in sexual differentiation of the mammalian central nervous system. The cellular mechanisms responsible for mediating these effects remain incompletely understood. A considerable amount of evidence has accumulated indicating that one of the earliest detectable events in the mechanism of sexual differentiation is a selective and permanent reduction in estrogen receptor concentrations in specific regions of the brain. Using quantitative autoradiographic methods, it has been possible to precisely map the regional distribution of estrogen receptors in the brains of male and female rats, as well as to study the development of sexual dimorphisms in receptor distribution. Despite previous data suggesting that the left and right sides of the brain may be differentially responsive to early androgen exposure, there is no significant right-left asymmetry in estrogen receptor distribution, in either sex. Significant sex differences in receptor density are, however, observed in several regions of the preoptic area, the bed nucleus of the stria terminalis and the ventromedial nucleus of the hypothalamus, particularly in its most rostral and caudal aspects. In the periventricular preoptic area of the female, highest estrogen receptor density occurs in the anteroventral periventricular region: binding in this region is reduced by approximately 50% in the male, as compared to the female. These data are consistent with the hypothesis that androgen-induced defeminization of feminine behavioral and neuroendocrine responses to estrogen may involve selective reductions in the estrogen sensitivity of critical components of the neural circuitry regulating these responses, mediated in part through a reduction in estrogen receptor biosynthesis.

Sexual differentiation of estrogen receptor concentrations in the rat brain: effects of neonatal testosterone exposure

This study tests the hypothesis that sex differences in estrogen receptor (ER) expression in the rat hypothalamus and preoptic area may at least partly result from androgen exposure during the immediate postnatal period. Male rats were castrated and female rats were injected with androgen, at either 15-30 min, 24 h or 10 days after birth. ER distribution in the brain was evaluated by in vitro autoradiography at 28 days of age. Males castrated immediately after birth exhibited higher ER levels in the preoptic area and the ventromedial and arcuate nuclei of the hypothalamus than either control males or males castrated on day 10. Females injected at birth with testosterone propionate exhibited reduced ER binding in the same brain regions. These data suggest that postnatal androgen secretion prior to postnatal day 10 permanently alters patterns of ER expression in the brain.

Partial and Generalized Seizures Affect Reproductive Physiology Differentially in the Male Rat

Summary: Purpose: Reproductive dysfunction and endocrine disorders occur frequently among men with epilepsy. This study tested the hypothesis that focal limbic seizures and generalized seizures may both contribute to reproductive dysfunction.