Studies on neurosteroids XV. Development of enzyme-linked immunosorbent assay for examining whether pregnenolone sulfate is a veritable neurosteroid

Higashi, Tatsuya; Daifu, Yuri; Ikeshima, Takehiro; Yagi, Takako; Shimada, Kazutake

doi:10.1016/s0731-7085(02)00534-4

Cited by 31 publications

(19 citation statements)

References 17 publications

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“…Two alternative approaches, enzyme-linked immunosorbent (ELISA) assays performed following separation of crossreactive steroids, and liquid or gas chromatography coupled with mass spectrometry or mass fragmentographic analyses, have provided revised and appreciably lower estimates of brain neurosteroid concentrations. Specifically, Cheney et al (1995) reported levels of ~ 1 ng/g for allopregnanolone in adult male rat brain homogenates, and levels of DHEAS and pregnenolone sulfate have been estimated to be either extremely low or below the level of detection in the adult male or unstaged female rat brain (Higashi et al, 2003;Liu et al, 2003;Liere et al, 2004). To my knowledge, comparable approaches have not yet been used for analysis of neurosteroids in the brain of cycling or pregnant females.…”

Section: Neurosteroid and Anabolic Androgenic Steroid Levels In The Cnsmentioning

confidence: 99%

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

Henderson

2007

Neuropharmacology

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The hypothalamus, the seat of neuroendocrine control, is exquisitely sensitive to gonadal steroids. For decades it has been known that androgens, estrogens and progestins, acting through nuclear hormone receptors, elicit both organizational and activational effects in the hypothalamus and basal forebrain that are essential for reproductive function. While changes in gene expression mediated by these classical hormone pathways are paramount in governing both sexual differentiation and the neural control of reproduction, it is also clear that steroids impart critical control of neuroendocrine functions through nongenomic mechanisms. Specifically, endogenous neurosteroid derivatives of deoxycorticosterone, progesterone and testosterone, as well and synthetic anabolic androgenic steroids that are self-administered as drugs of abuse, elicit acute effects via allosteric modulation of γ-aminobutyric acid type A receptors. GABAergic transmission within the hypothalamus and basal forebrain is a key regulator of pubertal onset, the expression of sexual behaviors, pregnancy and parturition. Summarized here are the known actions of steroid modulators on GABAergic transmission within the hypothalamus/basal forebrain, with a focus on the medial preoptic area and the supraoptic/paraventricular nuclei that are known to be central players in the control of reproduction.

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Section: Neurosteroid and Anabolic Androgenic Steroid Levels In The Cnsmentioning

confidence: 99%

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

Henderson

2007

Neuropharmacology

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“…8) Oasis HLB cartridges (60 mg: Waters Assoc., Milford, MA, U.S.A.) were successively washed with AcOEt (1 ml), EtOH (2 ml) and H 2 O (2 ml) prior to use.…”

Section: Materials and Reagentsmentioning

confidence: 99%

“…Three 25 ml aliquots of the sample were submitted for ELISA. 8) In the case where the regional levels of PREGS were measured, the brain tissue (50-200 mg) was homogenized in 1% AcOH/MeOH (1 ml) and then treated in the same way as described above.…”

Section: Materials and Reagentsmentioning

confidence: 99%

“…8) This ELISA is sensitive (limit of quantitation: 1 pg/well) and does not require solvolysis procedure. The present paper describes the application of the ELISA to the determination of PREGS in rat brains and some data concerning the source of the brain PREGS.…”

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“…7) Based on the results, we developed an enzyme-linked immunosorbent assay (ELISA) for PREGS using an antibody raised against 11a-hemiglutaryloxy-PREGS-bovine serum albumin conjugate. 8) This ELISA is sensitive (limit of quantitation: 1 pg/well) and does not require solvolysis procedure. The present paper describes the application of the ELISA to the determination of PREGS in rat brains and some data concerning the source of the brain PREGS.…”

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Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis

Higashi

Sugitani

Yagi

et al. 2003

Biological & Pharmaceutical Bulletin

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In the 1980s, Baulieu and co-workers demonstrated that some steroids, such as pregnenolone (PREG), dehydroepiandrosterone and their sulfates, are present in higher concentrations in the brain than in blood and are synthesized de novo in the central nervous system. 1) Such steroids are now universally referred to as neurosteroids. They act as modulators of several neurotransmitter receptors (g-aminobutyric acid A , N-methyl-D-aspartate and s 1 receptors) either as stimulators or inhibitors 1,2) and are involved in learning and memory performance.3) For these reasons, there is a considerable interest in determining the location and levels of different neurosteroids in the brain.The levels of PREG sulfate (PREGS) have conventionally been evaluated by measuring its genin formed after solvolysis by 5) or radioimmunoassay (RIA). 6) However, these methods have some shortcomings, such as requirement of complicated pretreatment and solvolysis steps and a decline in the recovery rate of the steroid. To overcome these problems, we employed liquid chromatography (LC)-electrospray ionization (ESI)-MS-MS to determine PREGS in rat brains without deconjugation, 7) but contrary to our expectations, its levels were less than one tenth of those previously reported (more than 5 ng/g tissue).4-6) One potential explanation for this discrepancy is that the LC-MS-MS method is more specific for brain samples than the GC-MS or RIA assay combined with solvolysis; the GC-MS may be detecting PREG derived from PREGS as well as that from other conjugated forms, such as sulfolipid conjugate, by solvolysis, and the RIA employs an antibody that reacts with other steroids having structures closely related to that of PREG. Although our LC-MS-MS method was specific and did not need a solvolysis procedure, it did not have sufficient sensitivity for the analysis of PREGS in the brain (limit of quantitation: ca. 160 pg/injection), and the obtained data using ca. 400 mg of tissue were not so reliable.7) Based on the results, we developed an enzyme-linked immunosorbent assay (ELISA) for PREGS using an antibody raised against 11a-hemiglutaryloxy-PREGS-bovine serum albumin conjugate. 8) This ELISA is sensitive (limit of quantitation: 1 pg/well) and does not require solvolysis procedure. The present paper describes the application of the ELISA to the determination of PREGS in rat brains and some data concerning the source of the brain PREGS. MATERIALS AND METHODS Materials and ReagentsStandard PREGS, PREG and all other reagents were those used in the previous study. 8)Oasis HLB cartridges (60 mg: Waters Assoc., Milford, MA, U.S.A.) were successively washed with AcOEt (1 ml), EtOH (2 ml) and H 2 O (2 ml) prior to use.Rats and Treatment Wistar strain rats (7 weeks old, male, 190-200 g) obtained from Japan S.L.C. (Hamamatsu, Japan) were used and euthanized by decapitation. Brain samples were collected from the rats assigned either to a control group (without injection, nϭ10), a group of animals receiving an intrapertioneal (i.p.) vehicle injection (5% EtOH/ s...

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The neurosteroids allopregnanolone and dehydroepiandrosterone modulate resting-state amygdala connectivity

et al. 2013

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The neurosteroids allopregnanolone and dehydroepiandrosterone (DHEA) are integral components of the stress response and exert positive modulatory effects on emotion in both human and animal studies. Though these antidepressant and anxiolytic effects have been well established, little research to date has examined their neural correlates, and no research has been conducted into the effects of neurosteroids on large-scale networks at rest. To investigate the neurosteroid impact on intrinsic connectivity networks, participants were administered 400 mg of pregnenolone (N = 16), 400 mg of DHEA (N = 14), or placebo (N = 15) and underwent 3T fMRI. Resting-state brain connectivity was measured using amygdala as a seed region. Compared to placebo, pregnenolone administration reduced connectivity between amygdala and dmPFC, between amygdala and precuneus, and between amygdala and hippocampus. DHEA reduced connectivity between amygdala and peri-amygdala and between amygdala and insula. Reductions in amygdala to precuneus connectivity were associated with less self-reported negative affect. These results demonstrate that neurosteroids modulate amygdala functional connectivity during resting-state, and may be a target for pharmacological intervention. Additionally, allopregnanolone and DHEA may shift the balance between salience network and default network, a finding that could provide insight into the neurocircuitry of anxiety psychopathology.

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Studies on neurosteroids XV. Development of enzyme-linked immunosorbent assay for examining whether pregnenolone sulfate is a veritable neurosteroid

Cited by 31 publications

References 17 publications

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

Studies on Neurosteroids XVI. Levels of Pregnenolone Sulfate in Rat Brains Determined by Enzyme-linked Immunosorbent Assay Not Requiring Solvolysis

The neurosteroids allopregnanolone and dehydroepiandrosterone modulate resting-state amygdala connectivity

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