Neurosteroids In Brain Research Articles

Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain

A new sample preparation method coupled to GC-MS analysis was developed and validated for quantification of sulfate esters of pregnenolone (PREG-S) and dehydroepiandrosterone (DHEA-S) in rat brain. Using a solid-phase extraction recycling protocol, the results show that little or no PREG-S and DHEA-S (<1 pmol/g) is present in rat and mouse brain. These data are in agreement with studies in which steroid sulfates were analyzed without deconjugation. We suggest that the discrepancies between analyses with and without deconjugation are caused by internal contamination of brain extract fractions, supposed to contain steroid sulfates, by lipoidal forms of PREG and DHEA (L-PREG and L-DHEA, respectively). These derivatives can be acylated very efficiently with heptafluorobutyric anhydride and triethylamine, and their levels in rodent brain (approximately 1 nmol/g) are much higher than those of their unconjugated counterparts. They are distinct from fatty acid esters, and preliminary data do not favor structures such as sulfolipids or sterol peroxides. Noncovalent interactions between steroids and proteolipidic elements, such as lipoproteins, could account for some experimental data. Given their abundance in rodent brain, the structural characterization and biological functions of L-PREG and L-DHEA in the central nervous system merit considerable attention.

Studies on neurosteroids XVII.: Analysis of stress-induced changes in neurosteroid levels in rat brains using liquid chromatography–electron capture atmospheric pressure chemical ionization-mass spectrometry

The analysis of stress-induced changes in the brain neurosteroid levels by liquid chromatography (LC)–electron capture atmospheric pressure chemical ionization-mass spectrometry (ECAPCI-MS) is described. In the present method, neurosteroids were derivatized with a highly electron-affinitive reagent, 2-nitro-4-trifluoromethylphenylhydrazine (NFPH), to convert them to the corresponding hydrazones. The derivatized steroids showed over a 20-fold higher sensitivity in ECAPCI-MS than intact steroids measured by positive atmospheric pressure chemical ionization (APCI)-MS. Application of this method to the analysis of rat brain samples confirmed the significant increase in the levels of pregnenolone (PREG), progesterone (PROG), 5α-dihydroprogesterone (DHPROG), allopregnanolone (3α-hydroxy-5α-pregn-20-one; AP), and epiallopregnanolone (3β-hydroxy-5α-pregn-20-one; EpiAP) in the fixated rats. The din stress, which we examined as a new short-term mental stress model, also elevated the brain neurosteroid levels. It is known that various types of stress lower the γ-aminobutyric acid type A (GABA A) receptor function and induce the neuronal overexcitation. The increase in the brain level of AP, a potent positive modulator of GABA A receptors, may be the defensive response against acute stress. The increase in the brain concentration of its precursors, PREG, PROG, and DHPROG, may be associated with the acceleration of the AP synthesis. Thus, the present studies suggest that changes in the brain levels of neurosteroids may play an important role in the homeostatic mechanisms that counteract the inhibitory effect of stress on the GABA A receptor function.

Influence of 17β-estradiol on the synthesis of reduced neurosteroids in the brain (in vivo) and in glioma cells (in vitro): possible relevance to mental disorders in women

Brain neurosteroids modulate gamma-aminobutyric acid type A (GABA A) receptor activity, thereby playing a role in mood disorders. Alterations in 17β-estradiol (E 2) and progesterone (P) are also known to play a significant role in psychopathology in women. The aim of the present study was to evaluate the synthesis of dihydroprogesterone (DHP), tetrahydroprogesterone (THP), and the activity of 5α-reductase (5αR) which regulates the reduction of P to DHP on exposure to supraphysiological levels of E 2 in vitro (C6 glioma cells) and in vivo (mouse brain). The results showed that supraphysiological levels of E 2 induced a decrease in the accumulation of both neurosteroids, probably by decreasing the activity of 5αR. We hypothesize that the high levels of E 2 in pregnancy attenuate the increase in the conversion of P to THP in the brain and that the ratio of E 2/P modulates the sedative effect of THP. This process may be relevant to psychopathological disorders that are ascribed to drastic alterations in estrogen levels, such as premenstrual syndrome, pregnancy-related mental disorders, and postpartum “blues”.

Brain Neurosteroids in Gender-Related Aggression Induced by Social Isolation

Genetic, environmental, or hormonal factors and their interactions have been implicated in the expression of gender-related aggressive behavior in humans. Several independent lines of evidence support the role of hormonal and environmental factors in the aggressive behavior of experimental animals. Social isolation (SI) for 2-4 weeks in male but not in female mice results in the expression of aggression to a same-sex intruder. Long-term treatment (3 weeks) with anabolic steroids during SI in female mice induces aggressive behavior toward a male intruder of a severity similar to that observed in socially isolated (SI) male mice. The induced aggression in male and female mice is associated with a decrease of brain allopreg-nanolone (Allo). In SI male mice, aggression can be prevented by treatment with L-methionine (MET), which has also been shown to decrease reelin and GAD67 mRNA expression and maintain normal brain Allo content. The histone deacetylase inhibitor valproic acid can reverse this process, suggesting that histone tail acetylation may reverse the action of MET. We conclude that during social isolation, aggression can be controlled either by preventing Allo downregulation (e.g., by treatment with MET) or by direct administration of Allo or of agents (e.g., fluoxetine) that upregulate brain Allo content in SI mice.

Neurosteroidogenesis: Relevance to Neurosteroid Actions in Brain and Modulation by Psychotropic Drugs

Neurosteroids--i.e., steroid produced in brain ex novo or through metabolism of precursors--affect neuronal and brain functions through genomic and nongenomic mechanisms, depending on their molecular structure. Among neurosteroids, 3alpha-hydroxylated, 5alpha-reduced metabolites of progesterone (3alpha-hydroxy,5alpha-pregnan-20one/3alpha,5alpha-THP) and deoxycorticosterone (3alpha,21-dihydroxy,5alpha-pregnan-20one/3alpha,5alpha-THDOC) are positive allosteric modulators of gamma-aminobutyric acid (GABA) action at GABAA receptors. In rodents, a reduction of their endogenous brain concentrations rapidly lowers the potency of GABA in eliciting GABAA receptor-mediated inhibitory postsynaptic currents. This effect is related to anxiety-like behavior, increased aggression, and a reduced sensitivity to the loss of righting reflex induced by GABAA receptor agonist or positive modulators. Conversely, enhancement of 3alpha,5alpha-THP or 3alpha,5alpha-THDOC brain content results in anxiolysis, sedation/hypnosis, anticonvulsant, and anesthetic action. Different classes of psychotropic drugs--i.e., antidepressants, selected atypical antipsychotics, ethanol, gamma-hydroxybutyric acid--increase neurosteroid concentrations in brain, and these increases may be relevant to their pharmacological actions. Drug-induced increases of neurosteroids in rodent brain are often associated with elevation of their plasma content, such that alterations of plasma steroid concentrations are assumed to reflect parallel changes in brain. Nevertheless, brain neurosteroid concentrations are uneven across various regions, and the dose-dependence of their response to a pharmacological challenge shows brain-regional differences as well. These observations are consistent with the present knowledge on the distribution of steroidogenic enzymes in brain--they show not only a brain region, but also a cell-specific expression that may spatially and temporally determine the local concentrations of specific neurosteroids, either produced ex novo or through metabolism of steroid precursors that reach the brain through blood.

3P218 最隣接熱力学計算法により最適化したプライマーを用いた新規神経調節因子である脳ニューロステロイド合成酵素の網羅的発現解析(神経・感覚 : 細胞・膜蛋白・分子)

3P218 最隣接熱力学計算法により最適化したプライマーを用いた新規神経調節因子である脳ニューロステロイド合成酵素の網羅的発現解析(神経・感覚 : 細胞・膜蛋白・分子)

Neurosteroid Biosynthesis in the Human Brain and Its Clinical Implications

This paper summarizes the current knowledge concerning the biosynthesis of neurosteroids in the human brain, the enzymes mediating these reactions, their localization, and the putative effects of neurosteroids. The presence of the steroidogenic enzymes cytochrome P450(SCC), aromatase, 5alpha-reductase, 3alpha-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase in the human brain has now been firmly established by molecular biological and biochemical studies. Their presence in the cerebral cortex and in the subcortical white matter indicates that various cell types, either neurons or glial cells, are involved in the biosynthesis of neuroactive steroids in the brain. The following functions are attributed to specific neurosteroids: modulation of GABA(A), N-methyl-d-aspartate (NMDA), nicotinic, muscarinic, serotonin (5-HT(3)), kainate, glycine and sigma receptors, neuroprotection and induction of neurite outgrowth, dendritic spines, and synaptogenesis. We still do not know whether and how the steroidogenic enzymes are involved in the pathophysiology of the nervous system. The first clinical investigations in humans produced evidence for an involvement of neuroactive steroids in conditions such as fatigue during pregnancy, premenstrual syndrome, postpartum depression, catamenial epilepsy, and depressive disorders. Further and improved knowledge of the biochemical pathways of neurosteroidogenesis and their actions on the brain may enable new perspectives in the understanding of the physiology of the human brain as well as in the pharmacological treatment of its disturbances.

Neurosteroids in rat brain: extraction, isolation, and analysis by nanoscale liquid chromatography-electrospray mass spectrometry.

A method designed for the analysis of sulfated neurosteroids and unconjugated ketonic neurosteroids in rat brain using nanoscale liquid chromatography-electrospray (nano-LC-ES) mass spectrometry is described. Neurosteroids in rat brain tissue were extracted, purified, and separated into two groups, neutral unconjugated steroids and steroid sulfates, by employing solid-phase partition, cation- and anion-exchange chromatography. The steroid sulfate fraction was analyzed by nano-LC-ES mass spectrometry. Contrary to expectations, the sulfates of pregnenolone and dehydroepiandrosterone (DHEA) were not detected. Internal standards, including pregnenolone sulfate, were recovered and the detection limit of the method was 0.3 ng/g of wet brain. Cholesterol sulfate was detected at a level of 1.2 microg/g of wet brain. The neutral unconjugated steroid fraction was derivatized with hydroxylamine hydrochloride to convert oxosteroids into their oximes. The oximes were isolated using cation-exchange chromatography and were analyzed by nano-LC-ES tandem mass spectrometry. The analyses of the neutral unconjugated steroid fraction confirmed the presence in rat brain of pregnenolone, pregnanolone isomers, progesterone, testosterone, and DHEA, which were characterized by their retention times, the mass of the protonated molecules, and characteristic fragment ions. The levels were estimated by addition of [3,4-(13)C(2)]-progesterone as an internal standard and found to be in a range of 0.04-20 ng/g.

Biosynthesis and Biological Actions of Neurosteroids in the Avian Brain

Biosynthesis and Biological Actions of Neurosteroids in the Avian Brain

Brain neurosteroids are 4th generation neuromessengers in the brain: Cell biophysical analysis of steroid signal transduction

This work reviews recent achievements made by our lab. on intercellular signal transduction related to the synthesis and action of steroids. These achievements were made possible by the high suitability of cytochrome P450, a key enzyme in signal transduction, to quantitative spectroscopic analysis. Both digital microscopic imaging, electrophysiology analysis and time–resolved protein rotation measurements were used: Real–time microscopic imaging was used to investigate Ca signaling, endosome trafficking and P450scc activity; Electrophysiology was necessary to examine synaptic neuron–neuron communication; Protein rotation was used to analyze molecular mechanisms of electron transfer in cytochrome P450 systems. Steroid hormones, including sex hormones, play an essential role in the homeostasis of the body, allowing the mediation of genomic processes via nuclear steroid hormone receptors. In a classical view, steroid hormones are modeled as being synthesized exclusively in peripheral organs such as the adrenal cortex and the gonads. They then reach the target tissues via blood circulation. Recently, however, steroids have been shown to be present in the brain. These brain neurosteroids have been demonstrated to act acutely (<30min) on neurotransmitter receptors located on the cell membranes in the hippocampus (the center for learning and memory), the amygdala (the center for emotional behavior), the retina and the olfactory bulb, and, as such, are likely to play an essential role in signal transduction processes related to learning, memory and emotional response. One example, the postsynaptic neuron activation circuit, consisting of neurosteroidogenic systems and the N-methyl-D-aspartate (NMDA) receptors of the hippocampus, was discovered in this laboratory. Although the physiological roles of these brain neurosteroids have not been sufficiently elucidated, it is

ラット海馬神経細胞における脳ニューロステロイド合成経路の解析

ラット海馬神経細胞における脳ニューロステロイド合成経路の解析

Influence of sleep disturbance on steroid 5α-reductase mRNA levels in rat brain

Sleep deprivation has been shown to affect the production of steroid hormones in peripheral steroidogenic organs, but little is known about the influence of sleep disturbance on the metabolism of steroid hormones in the brain. To elucidate a possible association of the sleep–wake cycle with brain neurosteroid metabolism, the influence of short-term sleep disturbance on the expression of mRNA encoding steroid 5α-reductase, the enzyme converting progesterone and other steroid hormones to their neuroactive 5α-reduced metabolites, was investigated. Rats were first subjected to non-selective disturbance of the sleep–wake cycle, and the expression of steroid 5α-reductase mRNA in rat hippocampus and brainstem was determined using a semi-quantitative one-step RT-PCR technique. Non-selective disturbance of the sleep–wake cycle resulted in the elevation of 5α-reductase mRNA levels in the brainstem, but not in the hippocampus, and the elevated mRNA expression returned to the basal levels after a short period of the sleep recovery. Further studies showed that selective REM sleep deprivation significantly elevated 5α-reductase mRNA levels in both hippocampus and brainstem, thus proposing the possibility that REM sleep reduction may largely contribute to the elevation of steroid 5α-reductase mRNA levels observed during short-term disturbance of the sleep–wake cycle. Since the enhancement of steroid 5α-reductase gene expression may result in the elevation of neuroactive 5α-reduced steroid production in the brainstem, the findings presented here provide further evidence for suggesting that neuroactive steroids may play a physiologically important role in the neuronal network for REM sleep initiation and maintenance.

Neurosteroids Enhance Spontaneous Glutamate Release in Hippocampal Neurons: POSSIBLE ROLE OF METABOTROPIC ς1-LIKE RECEPTORS

Pregnenolone sulfate (PREGS), one of the most abundantly produced neurosteroids in the mammalian brain, improves cognitive performance in rodents. The mechanism of this effect has been attributed to its allosteric modulatory actions on glutamate- and gamma-aminobutyric acid-gated ion channels. Here we report a novel effect of PREGS that could also mediate some of its actions in the nervous system. We found that PREGS induces a robust potentiation of the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in cultured hippocampal neurons. PREGS also decreased paired pulse facilitation of autaptic EPSCs evoked by depolarization, indicating that it modulates glutamate release probability presynaptically. PREGS potentiation of mEPSCs was mimicked by dehydroepiandrosterone sulfate and (+)-pentazocine but not by (-)-pentazocine, the synthetic (-)-enantiomer of PREGS or the inactive steroid isopregnanolone. The sigma receptor antagonists, haloperidol and BD-1063, blocked the effect of PREGS on mEPSCs, as did pertussis toxin and the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl) ester. These results suggest that PREGS increases spontaneous glutamate release via activation of a presynaptic G(i/o)-coupled sigma receptor and an elevation in intracellular Ca2+ levels. We postulate that presynaptic actions of neurosteroids have a role in the maturation and/or maintenance of synaptic networks and the processing of information in the central nervous system.

GABAergic Neurosteroid Modulation of Ethanol Actions

Summary: Systemic administration of ethanol elevates plasma and cerebral cortical GABAergic neuroactive steroids. The increase in neurosteroids is responsible for specific behavioural and electrophysiological actions of ethanol in rodents. This article recapitulates the current knowledge of the novel interaction between ethanol and neurosteroids and addresses the potential mechanism for ethanol-induced increase in brain neurosteroid levels. Ethanol-induced increase in the cortical neurosteroid content is modified by neurosteroid biosynthesis inhibitors and completely prevented by adrenalectomy in male rats. In line with this, adrenalectomy prevented the anticonvulsant and hypnotic effects of acute ethanol administration. It is speculated that acute ethanol administration might resemble acute stress and increase neuroactive steroids due to activation of hypothalamic-pituitary adrenal axis. Ethanol-induced increases in neuroactive steroids might be responsible for the anti-depressant, anxiolytic, spatial learning deficits and drug discriminatory actions in rodents. Thus ethanol-induced increases in neuroactive steroids represent a novel mechanism of ethanol's action, responsible for several pharmacological and behavioural actions of ethanol. The development of new therapeutic strategies for alcoholism may arise based on the novel interaction between ethanol and neurosteroids in the brain.

Neurosteroid metabolism in the human brain.

This review summarizes the current knowledge of the biosynthesis of neurosteroids in the human brain, the enzymes mediating these reactions, their localization and the putative effects of neurosteroids. Molecular biological and biochemical studies have now firmly established the presence of the steroidogenic enzymes cytochrome P450 cholesterol side-chain cleavage (P450SCC), aromatase, 5alpha-reductase, 3alpha-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase in human brain. The functions attributed to specific neurosteroids include modulation of gamma-aminobutyric acid A (GABAA), N-methyl-d-aspartate (NMDA), nicotinic, muscarinic, serotonin (5-HT3), kainate, glycine and sigma receptors, neuroprotection and induction of neurite outgrowth, dendritic spines and synaptogenesis. The first clinical investigations in humans produced evidence for an involvement of neuroactive steroids in conditions such as fatigue during pregnancy, premenstrual syndrome, post partum depression, catamenial epilepsy, depressive disorders and dementia disorders. Better knowledge of the biochemical pathways of neurosteroidogenesis and their actions on the brain seems to open new perspectives in the understanding of the physiology of the human brain as well as in the pharmacological treatment of its disturbances.

Identification of ST2A1 as a rat brain neurosteroid sulfotransferase mRNA

A hydroxysteroid sulfotransferase (ST2A1) was identified as a form mediating neurosteroid sulfation in rat brain. The sole expression among known rat ST2A forms was indicated by brain RT-PCR. All nucleotide sequences of seven ST2A cDNA clones isolated from brain matched completely with that of hepatic ST2A1. The recombinant ST2A1 protein mediated neurosteroid sulfation. These data strongly suggest a functional role of ST2A1 as a neurosteroid sulfotransferase in rat brain.

Expression and localization of cytochrome P450 17α-hydroxylase/c17,20-lyase in the avian brain

Steroids synthesized de novo from cholesterol in the brain are generally called neurosteroids. We have recently demonstrated, using biochemical and molecular biological methods, that certain structures in the quail brain possess cytochrome P450 side-chain cleavage enzyme ( P450scc) and 3β-hydroxysteroid dehydrogenase/Δ 5-Δ 4-isomerase (3β-HSD) and produce pregnenolone, pregnenolone sulfate and progesterone. To clarify the biosynthetic pathway of neurosteroids in the avian brain, therefore, we examined the expression of messenger RNA (mRNA) encoding for the enzyme cytochrome P450 17α-hydroxylase/c17,20-lyase ( P450 17α,lyase), which converts pregnenolone to dehydroepiandrosterone via 17α-hydroxypregnenolone or progesterone to androstenedione via 17α-hydroxyprogesterone. RT-PCR analysis followed by Southern hybridization indicated the expression of P450 17α,lyase mRNA in the brain of sexually mature birds without a clear-cut sex difference. Employing biochemical techniques combined with HPLC analysis, the conversion of progesterone to 17α-hydroxyprogesterone was also found in brain slices of the mature male. P450 17α,lyase mRNA was detected in various brain regions, but there was a clear regional difference in the expression. The expressions of P450 17α,lyase mRNA in the diencephalon and mesencephalon were significantly higher than those in the cerebrum and cerebellum, unlike 3β-HSD mRNA, which showed no regional difference in the expression. In situ hybridization revealed the cellular localization of P450 17α,lyase mRNA. The cells expressing P450 17α,lyase mRNA were detected several diencephalic and mesencephalic regions, such as the preoptic area, the anterior hypothalamus, the dorsolateral thalamus, the optic tectum and the ventral midbrain. The expression was also localized in the septum, the hyperstriatum accessorium, and the ventral portions of the archistriatum in the telencephalon. Cerebellar Purkinje cells also expressed P450 17α,lyase mRNA. These results suggest that the avian brain possesses P450 17α,lyase as well as P450scc and 3β-HSD in both sexes. The expression of P450 17α,lyase in the avian brain may be region-dependent.

Cataleptic effect of neurosteroid 3α-hydroxy-5α-pregnan-20-one in mice: modulation by serotonergic agents

The neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) induced catalepsy in mice is modified by dopaminergic, adenosinergic and GABAergic agents. In light of serotonergic agents being implicated in antipsychotic-induced catalepsy and their ability to increase brain neurosteroid content, the present study was undertaken to investigate the effect of various 5-HT agents on catalepsy induced by 3α,5α-THP in mice. Pretreatment with selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), 5-HT releaser, fenfluramine (10 mg/kg, i.p.), 5-HT 1A receptor agonist, 8-OH-DPAT (0.3 mg/kg, s.c.), 5-HT 1B/1C receptor agonist, TFMPP (3 mg/kg, i.p.), 5-HT 2A/1C receptor agonist, DOI (1.5 mg/kg, s.c.) and 5-HT 3 agonist, 2-methylserotonin (5 mg/kg, i.p.) potentiated the catalepsy induced by exogenous administration of 3α,5α-THP. Furthermore, FGIN 1–27, an MDR agonist that increases endogenous content of 3α,5α-THP although per se failed to exhibit any cataleptic effect but enhanced the cataleptic response in combination with these serotonergic agents. The potentiating action of 5-HT 1A, 5-HT 2A/1C or 5-HT 3 receptor agonist on 3α,5α-THP induced catalepsy was not blocked by prior administration of sub-effective dose (1 mg/kg, s.c.) of their respective receptor antagonists pindolol, ritanserin or ondansetron or by pretreatment with serotonergic neurotoxin 5,7-DHT (100 μg/mouse, i.c.v.). However this effect of different serotonergic agents was antagonized by the GABA A receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3α-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). The 5-HT agents enhance neurosteroid-induced catalepsy by increasing GABAergic tone, likely as a consequence of increased brain content of 3α,5α-THP.

Effects of CRH and ACTH administration on plasma and brain neurosteroid levels.

The 3alpha-hydroxy ring A-reduced metabolite of progesterone, 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) is among the most potent known ligands of the gamma aminobutyric acid (GABA) receptor, designated GABA-A, in the central nervous system. We determined by RIA serum levels of progesterone (PROG), 5-alpha-dihidroprogesterone (DHP) and allopregnanolone in male and female rats after corticotropin releasing hormone (CRH) and adrenocorticotropin hormone (ACTH) administration. Allopregnanolone was undetectable in plasma and brain of control males but detectable in plasma and brain of males injected with CRH and ACTH and of control and similarly treated females. Allopregnanolone increased in the plasma and brain after CRH and ACTH administration in all cases. The data demonstrate that the administration of CRH plus ACTH results in a rapid increase of the neuroactive steroid allopregnanolone in the brain of males and females to levels known to modulate GABA-A receptor function. Thus, stress could regulate neurosteroid biosynthesis via the hormones ACTH and CRH.

Quantification of Neurosteroids in Rat Plasma and Brain Following Swim Stress and Allopregnanolone Administration Using Negative Chemical Ionization Gas Chromatography/Mass Spectrometry

A simplified method for the quantitative analysis of neurosteroids in rat plasma and brain is described. The method uses negative chemical ionization gas chromatography/mass spectrometry and involves the synthesis of pentafluorobenzyloxime/trimethylsilyl ether derivatives with excellent chromatographic and electron-capturing properties. Deuterium-labeled analogs of the steroids of interest were synthesized and used as internal standards. The steroids (allopregnanolone, epiallopregnanolone, pregnenolone, testosterone, and dehydroepiandrosterone) were isolated from the plasma or brain matrix by a rapid and straightforward solid-phase extraction procedure. The mass spectrometer was operated in a selective ion monitoring mode, allowing for picograms of neurosteroids to be quantified from biological extracts. The method was linear (typical R2 = 0.999) over the concentration range (100 to 8000 pg from 0.3 ml plasma and 250 to 8000 pg from 100 mg brain tissue) with good precision and accuracy. In experimental protocols, the procedure was suitable for measuring concentrations of endogenous neurosteroids in rat plasma and brain. Significant elevations (P < 0.001) were observed in the frontal cortex for allopregnanolone and pregnenolone following a swim stress and for allopregnanolone and epiallopregnanolone following allopregnanolone injection (8 mg/kg, sc). The present method allows accurate determination of neurosteroids and will be helpful in elucidating the role of neurosteroids in health and disease.