Neurosteroids In Brain Research Articles

Involvement of neurosteroids in the control of prolactin secretion in sheep under basal, stressful and pregnancy conditions

Prolactin (PRL) secretion by the anterior pituitary (AP) is responsive to changes in physiological conditions and many external factors that also affect brain neurosteroid levels. This study tested the hypothesis that neurosteroids can affect PRL secretion in sheep under basal, stressful and advanced pregnancy conditions. In Experiment 1, luteal-phase sheep were subjected to a three-day series of intracerebroventricular (icv.) control (n = 12) or allopregnanolone (AL, 4 × 15 μg/60 μL/30 min at 30-min intervals, n = 12) infusions. Acute stressful stimuli, isolation and partial movement restriction were applied on the third day of infusion to half of the animals in each group. In Experiment 2, pregnant sheep were subjected to a three-day series of icv. control (n = 6) or finasteride (4 × 25 μg/60 μL/30 min at 30-min intervals, n = 6) infusions during the 16th week of pregnancy. As a result, the relative abundance of PRL transcript increased in the AP of luteal-phase sheep treated with stress, AL and AL in combination with stress (P < 0.05 - P < 0.01) compared to controls. The level of PRL mRNA in stressed-AL-treated sheep was higher (P < 0.01) than in sheep only subjected to stress. The PRL protein content in the AP decreased in stressed-only sheep compared to controls (P < 0.05) and increased in stressed-AL-treated sheep compared to controls and other groups (P < 0.05 - P < 0.01). Plasma PRL concentration increased (P < 0.05 - P < 0.01) in stressed-only sheep compared to controls; AL infusion counteracted the stress-induced increase in PRL levels (P < 0.05 - P < 0.01) and had no effect in non-stressed animals. Inhibition of neurosteroid synthesis in the brain of pregnant sheep by finasteride caused transient increases (P < 0.05 - P < 0.001) in plasma PRL concentration compared to controls. In conclusion, the presented results indicate a bimodal effect of AL on PRL secretion in sheep: first at the molecular level – stimulation of PRL mRNA expression; second – inhibition of hormone release from pituitary lactotrophs. Both AL activities may involve various mechanisms regulating PRL secretion. In general, cerebral neurosteroids can affect the supply of pituitary PRL in the body under certain conditions, such as stress and pregnancy.

The novel dual-mechanism Kv7 potassium channel/TSPO receptor activator GRT-X is more effective than the Kv7 channel opener retigabine in the 6-Hz refractory seizure mouse model

Epilepsy, one of the most common and most disabling neurological disorders, is characterized by spontaneous recurrent seizures, often associated with structural brain alterations and cognitive and psychiatric comorbidities. In about 30% of patients, the seizures are resistant to current treatments; so more effective treatments are urgently needed. Among the ∼30 clinically approved antiseizure drugs, retigabine (ezogabine) is the only drug that acts as a positive allosteric modulator (or opener) of voltage-gated Kv7 potassium channels, which is particularly interesting for some genetic forms of epilepsy. Here we describe a novel dual-mode-of-action compound, GRT-X (N-[(3-fluorophenyl)-methyl]-1-(2-methoxyethyl)-4-methyl-2-oxo-(7-trifluoromethyl)-1H-quinoline-3-carboxylic acid amide) that activates both Kv7 potassium channels and the mitochondrial translocator protein 18 kDa (TSPO), leading to increased synthesis of brain neurosteroids. TSPO activators are known to exert anti-inflammatory, neuroprotective, anxiolytic, and antidepressive effects, which, together with an antiseizure effect (mediated by Kv7 channels), would be highly relevant for the treatment of epilepsy. This prompted us to compare the antiseizure efficacy of retigabine and GRT-X in six mouse and rat models of epileptic seizures, including the 6-Hz model of difficult-to-treat focal seizures. Furthermore, the tolerability of the two compounds was compared in mice and rats. Potency comparisons were based on both doses and peak plasma concentrations. Overall, GRT-X was more effective than retigabine in three of the six seizure models used here, the most important difference being the high efficacy in the 6-Hz (32 mA) seizure model in mice. Based on drug plasma levels, GRT-X was at least 30 times more potent than retigabine in the latter model. These data indicate that GRT-X is a highly interesting novel anti-seizure drug with a unique (first-in-class) dual-mode mechanism of action.

An LC-APCI+-MS/MS-based method for determining the concentration of neurosteroids in the brain of male mice with different gut microbiota

BackgroundAlthough the interaction between the gut microbiota and central nervous system (CNS) is well-known, the effects of gut microbiota on different brain regions remain obscure. New methodIn present study, we developed a simple and sensitive high-performance liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization in positive mode (LC-APCI+-MS/MS) for simultaneous detection of 12 analytes in the rodent’ brain with different housing conditions ResultsThe results showed that male mice in XZ group had significantly higher brain levels of dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), progesterone (P), corticosterone (CORT), aldosterone (ALD) and 11-dehydrocorticosterone (11-DHC) than those in SPF group. CORT level was higher in the left prefrontal cortex, whereas ALD and 11-DHC levels were higher in the left hypothalamus than in the right symmetrical areas in both groups. DHEA and CORT levels were highest in the striatum than in the prefrontal cortex, hippocampus, hypothalamus, regardless of the region and group (XZ and SPF). Comparison with existing methodsThese results demonstrated that the method developed in this study provides, for the first time, direct quantitation of neurosteroids in male mice brain. ConclusionsDHEA levels showed a left-lateralized pattern in the hippocampus and hypothalamus. Mice in the XZ group showed significantly elevated levels of CORT and/or its metabolites, ALD and 11-DHC in brain than mice in the SPF group. Insanitation living conditions increased more diverse gut microbiota.

Comorbidity: Chronic neurogenic pain affects malignant growth and changes balance of neurosteroids in brain of rats.

e23516 Background: Sex steroids in the brain regulate neurogenesis and the body's response to stress. Chronic neurogenic pain (CNP) and the tumor growth are stress factors that often accompany each other. The purpose of the study was to analyze levels of sex steroid hormones in white matter of the brain of rats with tumor development in presence of CNP. Methods: The study included white outbred male rats (n = 74). In the main groups, a CNP model was created by bilateral sciatic nerve ligation, and after 45 days, M1 sarcoma was transplanted subcutaneously (n = 11) or into the subclavian vein (n = 11). Two comparison groups (each n = 13) included sham operated animals with M1 sarcoma transplanted subcutaneously or into the subclavian vein. Control groups (each n = 13) included animals with CNP or sham operated rats. Levels of testosterone (T), estrone (E1), estradiol (E2), estriol (E3) and progesterone (P4) were measured by ELISA (Cusabio, China) in the brain tissues obtained on day 21 of the tumor growth. Results: Tumors transplanted subcutaneously with and without CNP grew in 100% of animals. Tumor volumes were 1.5 times (p&lt;0.05) greater in animals with CNP, compared with rats without CNP, while the survival in the groups was similar. Levels of all studied hormones, except for E1, in the brain tissue in subcutaneous sarcoma growth were lower in presence of CNP than without it: T and E3–on average by 1.4 times (p&lt;0.05), E2 and P4–by 3.5 times (p&lt;0.05). In rats with intravenous transplantation of M1, tumor nodes in the lungs were registered only in rats with CNP, and the survival of animals was 36 days shorter (p&lt;0.05) than in rats of the corresponding control group. Such specificity of selective neoplastic growth in the pulmonary tissue was combined with lower cerebral T and E3 levels than in the corresponding control–on average by 1.4 times (p&lt;0.05), E2–by 7.2 times, and higher levels of E1–by 1.3 (p&lt;0.05) and P4–by 2.0 times, compared to animals which did not develop the neoplastic process in the lungs without pain. Conclusions: The presence of CNP stimulates the growth of M1 sarcoma in standard subcutaneous inoculation and allows the development of tumors in the lung in intravenous inoculation. The specificity of malignant growth in presence of CNP is accompanied by changes in the brain levels of neurosteroids in rats.

Site of Action of Brain Neurosteroid Pregnenolone Sulfate at the N-Methyl-D-Aspartate Receptor.

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented.SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.

Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.

γ-Aminobutyric acid receptors (GABAARs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result from receptor dysfunction. A critical component of most native GABAARs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact anxiety, stress and depression, and for therapeutic drugs, such as general anesthetics. Understanding the basis for the modulation of GABAAR function requires high-resolution structures. Here we present the first atomic structures of a GABAAR chimera at 2.8-Å resolution, including those bound with potentiating and inhibitory neurosteroids. These structures define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable the exploitation of neurosteroids for therapeutic drug design to regulate GABAARs in neurological disorders.

Stress-induced changes of neurosteroid profiles in rat brain and plasma under immobilized condition

In this study, various neurosteroids in brain and plasma were simultaneously determined using liquid chromatography-tandem mass spectrometry and their profile changes in a stress-induced rats were investigated. The investigated neurosteroids are as follows: progesterone (P4), 5α-dihydroprogesterone (5α-DHP), 5β-dihydroprogesterone, estrone, androstenedione (AE), cortisol, cortisone, corticosterone (CORT), dehydroepiandrosterone (DHEA), pregnanolone (3α,5β-THP), allopregnanolone (ALLO), 11-deoxycorticosterone (DOC), 11-deoxycortisol, pregnenolone (PREG), and 5α/5β-tetrahydrodeoxycorticosterone (5α/5β-THDOC). Brain and plasma samples were processed using solid-phase extraction with methanol and acetic acid (99:1), and derivatized with a hydroxylamine reagent. Separation was achieved within 13min at a flow rate of 0.4mL/min with a C18 column (3.0×50mm, 2.7μm). The triple quadrupole mass spectrometer was operated in the positive electrospray ionization mode. Using this method, the neurosteroid level variation was quantitated and investigated in the brain and plasma upon immobilization stress in rats. As a result, AE, CORT, DOC, P4, 5α-DHP, 5α/5β-THDOC, DHEA, 3α,5β-THP, ALLO, and PREG levels were significantly altered in both the brain and plasma samples when stress was induced. These findings demonstrated that stress leads to the alteration of the GABAergic neurosteroid profile. The present results will be helpful for furthering an understanding of the role of neurosteroids in stressed conditions.

Differential efficacy of the TSPO ligands etifoxine and XBD-173 in two rodent models of Multiple Sclerosis

Neurosteroids such as progesterone and allopregnanolone have been shown to exert neuroprotective effects under a variety of pathological or insult conditions, and there is evidence that the neurosteroid system is perturbed in Multiple Sclerosis (MS) patients. Neurosteroids are synthesized in the central nervous system (CNS) through a series of metabolic transformations, beginning with a rate-limiting step of cholesterol transport through the outer mitochondrial membrane via the transporter translocator protein (TSPO). We examined the effects of etifoxine and XBD-173, two different brain penetrant TSPO agonists, for their ability to ameliorate clinical signs in two different experimental autoimmune encephalitis (EAE) models. Etifoxine, as previously reported, was efficacious in EAE, while XBD-173 was not. Surprisingly, XBD-173, but not etifoxine elevated relevant neurosteroids in brain of female rats and differed in its ability to exert anti-inflammatory and direct neuroprotective effects in vitro as compared to etifoxine. We conclude that the neurosteroid elevations produced in brain by XBD-173 are not sufficient to ameliorate EAE and suggest that etifoxine may have additional mechanisms of action that provide therapeutic benefit in this model system.

Methods for differential and quantitative analyses of brain neurosteroid levels by LC/MS/MS with ESI-enhancing and isotope-coded derivatization

The analysis of changes in the brain neurosteroid (NS) levels due to various stimuli can contribute to the elucidation of their physiological roles, and the discovery and development of new antipsychotic agents targeting neurosteroidogenesis. We developed methods for the differential and quantitative analyses of the brain levels of allopregnanolene (AP) and its precursor, pregnenolone (PREG), using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI–MS/MS) combined with derivatization using 2-hydrazino-1-methylpyridine (HMP) and its isotope-coded analogue, 2H3–HMP (d-HMP). For the differential analysis, the brain sample of an untreated rat was derivatized with HMP, while the brain sample of a treated (stressed or drug-administered) rat was derivatized with d-HMP. The two derivatives were mixed and then subjected to LC/ESI–MS/MS. The stress- and drug (clozapine and fluoxetine)-evoked increases in the brain AP and PREG levels were accurately analyzed by the developed method. It was also possible to determine the absolute concentrations of the brain steroids when a deuterium-coded moiety was introduced to the standard steroids of known amounts by the derivatization and the resulting derivatives were used as internal standards. The HMP-derivatization enabled the highly sensitive detection and the use of d-HMP significantly improved the assay precision [the intra- (n=5) and inter-assay (n=5) relative standard deviations did not exceed 13.7%] and accuracy (analytical recovery ranged from 98.7 to 106.7%).

Programming the brain and behaviour by early-life stress: a focus on neuroactive steroids.

Animal studies have amply demonstrated that stress exposure during pregnancy or in early postnatal life can adversely influence brain development and have long-term 'programming' effects on future brain function and behaviour. Furthermore, a growing body of evidence from human studies supports the hypothesis that some psychiatric disorders may have developmental origins. Here, the focus is on three adverse consequences of early-life stress: dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, heightened anxiety behaviour and cognitive impairments, with review of what is known about the underlying central mechanisms. Neuroactive steroids modulate neuronal activity and play a key role in neurodevelopment. Moreover they can negatively modulate activity of the HPA axis, exert anxiolytic actions and influence cognitive performance. Thus, neuroactive steroids may provide a link between early-life stress and the resultant adverse effects on the brain and behaviour. Here, a role for neuroactive steroids, in particular the 5α-reduced/3α-hydroxylated metabolites of progesterone, testosterone and deoxycorticosterone, is discussed in the context of early-life stress. Furthermore, the impact of early-life stress on the brain's capacity to generate neurosteroids is considered and the evidence for an ability of neuroactive steroids to over-write the negative effects of early-life stress on the brain and behaviour is examined. An enhanced understanding of the influence of early-life stress on brain neurosteroid systems could aid the identification of new targets for developing treatments for stress-related conditions in humans.

Expression levels of mRNA for neurosteroidogenic enzymes 17β-HSD, 5α-reductase, 3α-HSD and cytochrome P450 aromatase in the fetal wild type and SF-1 knockout mouse brain

The presence of steroidogenic enzymes in the brain suggests de novo synthesis of steroid hormones in the brain. The current study was designed to determine the developmental profiles of cytochrome p450 aromatase (cyp19), 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-reductase type I and 3α-hydroxysteroid dehydrogenase (3α-HSD) mRNA expression levels in the fetal mouse brain and potential influence of peripheral steroids, and the steroidogenic factor 1 (SF-1) gene on their expression. Brains were collected from WT and SF-1 knockout male and female fetuses at embryonic (E) days E12, E14, E16, and E18. Quantitative PCR analyses revealed age related increases in the expression levels of 17β-HSD and 5α-reductase. Differences between genotypes in the expression levels of 17β-HSD and 5α-reductase were detected on E14, with reduced levels of expression in SF-1 KO males and females for 17β-HSD and only between females for 5α-reductase. Expression of 3α-HSD mRNA did not differ significantly between sexes, age groups or genotypes with the exception of SF-1 KO males, which had an unexplained increase in mRNA for this enzyme on day E18. Expression of cyp19 was at the limit of detection and could not be analyzed effectively. There were no sex differences and, with the exception of small difference on E14 for 17β-HSD and 5α-reductase, no differences between genotypes. The results suggest that gonadal steroids do not influence the production of neurosteroids in the fetal brain, nor does SF-1 play a major role in the regulation of steroidogenic enzyme expression in the brain.

A role for picomolar concentrations of pregnenolone sulfate in synaptic activity-dependent Ca2+ signaling and CREB activation.

Fast excitatory synaptic transmission that is contingent upon N-methyl d-aspartate receptor (NMDAR) function contributes to core information flow in the central nervous system and to the plasticity of neural circuits that underlie cognition. Hypoactivity of excitatory NMDAR-mediated neurotransmission is hypothesized to underlie the pathophysiology of schizophrenia, including the associated cognitive deficits. The neurosteroid pregnenolone (PREG) and its metabolites pregnenolone sulfate (PregS) and allopregnanolone in serum are inversely associated with cognitive improvements after oral PREG therapy, raising the possibility that brain neurosteroid levels may be modulated therapeutically. PregS is derived from PREG, the precursor of all neurosteroids, via a single sulfation step and is present at low nanomolar concentrations in the central nervous system. PregS, but not PREG, augments long-term potentiation and cognitive performance in animal models of learning and memory. In this report, we communicate the first observation that PregS, but not PREG, is a potent (EC50 ∼2 pM) enhancer of intracellular Ca(2+) that is contingent upon neuronal activity, NMDAR-mediated synaptic activity, and L-type Ca(2+) channel activity. Low picomolar PregS similarly activates cAMP response element-binding protein (CREB) phosphorylation (within 10 minutes), an essential memory molecule, via an extracellular-signal-regulated kinase/mitogen-activated protein kinase signal transduction pathway. Taken together, the results are consistent with a novel biologic role for the neurosteroid PregS that acts at picomolar concentrations to intensify the intracellular response to glutamatergic signaling at synaptic but not extrasynaptic, NMDARs by differentially augmenting CREB activation. This provides a genomic signal transduction mechanism by which PregS could participate in memory consolidation of relevance to cognitive function.

Association between a common CYP17A1 haplotype and anxiety in female anorexia nervosa

Dehydroepiandrosterone (DHEA), the main brain neurosteroid, has been implicated in various psychiatric disorders especially those including gender differences. We studied genetic variability in the DHEA-producing enzyme CYP17A1 in relation to anorexia nervosa (AN) susceptibility and AN-related co-morbidities. We performed analysis of 100 Israeli AN family trios accounting for CYP17A1 haplotypes characteristic of populations of European origin and studied genotype-phenotype relationships using correlation analyses and transmission disequilibrium test. Although our analysis revealed no evidence of association between CYP17A1 and AN per se, it revealed an association between specific CYP17A1 haplotypes and AN co-morbidity, specifically anxiety. We found that a common CYP17A1 haplotype (H1) was associated with higher anxiety in AN patients (Clinical Global Impression; CGI-anxiety ≥4). Moreover, H1 homozygotes were at higher risk for expressing high CGI-anxiety levels (OR = 3.7), and H1 was preferentially transmitted to AN patients with high CGI-anxiety levels (P = 0. 037). We suggest that CYP17A1 H1 haplotype may contribute to genetic predisposition to higher CGI-anxiety levels in AN patients and that this predisposition may be mediated by reduced CYP17A1 enzymatic activity and corresponding lower DHEA production.

Neurosteroidogenic enzymes and their regulation in the early brain of the protogynous grouper Epinephelus coioides during gonadal sex differentiation

The regulatory role of neurosteroids in the early brain during gonadal sex differentiation is unclear. The aim of this study was to investigate the expression and cellular localization of key steroidogenic enzymes in the early brain of the protogynous orange-spotted grouper Epinephelus coioides and the temporal expressions has been correlated with gonadal sex differentiation. In this study, we showed that peak neurosteroidogenesis occurs in the early brain during gonadal sex differentiation. The temporal expressions of key enzymes, cyp11a1 (cytochrome P450 side chain cleavage), hsd3b1 (3β-hydroxysteroid dehydrogenase) and cyp17a1 (cytochrome P450c17) were studied at different developmental ages (from 90- to 150-dah: days after hatching) using quantitative real-time PCR (q-PCR). q-PCR analysis indicated that the transcript expressions of cyp11a1, hsd3b1 and cyp17a1 were increased in the brain around the period of gonadal sex differentiation. Further, in situ hybridization (ISH) analysis showed that cyp11a1, hsd3b1 and cyp17a1 transcripts were widely expressed in several discrete brain regions, especially the intense expression in the forebrain, with an overall similar expression pattern. High density in the cyp19a1b/Cyp19a1b expression was detected in radial glial cells. Thus, the expression of grouper cyp19a1b/Cyp19a1b is restricted to radial glial cells, suggesting estrogens can modulate their activity. Next, by combining Cyp19a1b immunohistochemistry (IHC) with florescence ISH (FISH) of cyp11a1, hsd3b1 and cyp17a1, we showed that sub-cellular localization of cyp11a1, hsd3b1 and cyp17a1 transcripts, in partial, appeared to be in Cyp19a1b radial glial cell soma. Further, exogenous estradiol (E2) increased the expression of cyp17a1 and cyp19a1b/Cyp19a1b in the brain of grouper. Consequently, our results illustrated that the locally synthesized E2 upregulated neurosteroidogenic enzymes in the early brain and suggest a role for these enzymes in the neurogenic process during gonadal sex differentiation.

Analysis of bioactive oxysterols in newborn mouse brain by LC/MS

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C(21)-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time.

Acute and chronic exposure of chick embryo to ethanol alters brain neurosteroid levels

Neurosteroids are modulators of neuronal function that may play important role in brain maturation. The aim of the present investigation was to study the effect of prenatal exposure to acute and chronic ethanol on brain progesterone, estradiol, and testosterone concentration on 10th and 15th days following egg incubation. Eggs were exposed to ethanol at 10% in chronic treatment and 70% in acute treatment. Progesterone, estradiol, and testosterone were assayed by radioimmunoassay method. It was shown that brain progesterone level was significantly decreased (P < 0.05) in chronic ethanol group on embryonic day 10, but it was significantly decreased (P < 0.05) in acute and chronic groups on embryonic day 15. Brain estradiol level was significantly increased (P < 0.05) in chronic ethanol group on embryonic day 10, and it was decreased (P < 0.05) in acute and chronic groups of ethanol on embryonic day 15. Brain testosterone was significantly increased (P < 0.05) in acute and chronic ethanol-exposed groups on embryonic days 10 and 15. Our observations suggest that ethanol may modulate neurosteroid synthesis in the brain.

P.4.004 Schizophrenia-like phenotypes induced by genetic over-expression of brain andenosine kinase - a potential novel antipsychotic target

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-HT1A receptor agonist, 8-OH-DPAT (0.3 mg/kg, s.c.), 5-HT1B/1C receptor agonist, TFMPP (3 mg/kg, i.p.), 5-HT2A/1C receptor agonist, DOI (1.5 mg/kg, s.c.) and 5-HT3 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-HT1A, 5-HT2A/1C or 5-HT3 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 GABAA 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.

Neurosteroids and Hepatic Encephalopathy: An Update on Possible Pathophysiologic Mechanisms

Cerebral complications of liver failure either due to chronic or acute manifestations lead to a neurological disorder known as Hepatic encephalopathy (HE). Neurosteroids, synthesized in the brain mainly by astrocytes but also in other brain cells independently from peripheral steroidal sources such as adrenal and gonads, are suggested to play a role in the pathogenesis of HE. The mechanisms by which neurosteroids affects brain function are not totally elucidated but may involve both genomic and non genomic effects. On one hand, neurosteroids bind and modulate different types of neuronal membrane receptors. While neurosteroid may affect directly postsynaptic receptors including GABA(A), 5-HT3, NMDA, glycine, and opioid receptors which have been involved in HE, neurosteroids effects through GABA(A) receptors may also compromise indirectly the function of neurons networking with GABAergic interneurons. On the other hand, some neurosteroids bind to intracellular receptors through which they also regulate gene expression, and there is substantial evidence confirming that expression of genes coding for key astrocytic and neuronal proteins are altered in HE. The mechanisms that trigger brain neurosteroid changes in HE are not yet well-known, but could involve (i) ammonia and manganese (in chronic HE)-induced translocator protein (TSPO) activation, (ii) neuroinflammation or (iii) blood-brain transfer of lipophylic neuroactive steroids. The present review summarizes evidence for the involvement of neurosteroids in HE and possible mechanisms for their altered brain production and central effects in human and experimental HE.

Allosteric modulation of the NMDA receptor by neurosteroids in rat brain and the impact of long term morphine administration

This study examined the allosteric modulation of the NMDA receptor by nanomolar concentrations of neurosteroids in rats treated long term with morphine. The neurosteroids dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate (PS) and pregnanolone sulfate (3α5βS) are important mediators in the central nervous system. They induce rapid responses by non-classical steroidal mechanisms, e.g. via interaction with the N-methyl- d-aspartate (NMDA) receptor, and are known to modify the binding of ifenprodil to the NMDA receptor subunit NR2B. The NMDA receptor is involved in several processes, including memory, learning, synaptic plasticity and neuronal development. Morphine, a μ-opioid receptor agonist, has an important role in the clinical treatment of pain. The main drawback of morphine treatment is the associated development of dependence and tolerance. The mechanisms behind these phenomena are still to be elucidated, but several reports suggest the involvement of the NMDA receptor. The results of the present study indicate that the allosteric modulation induced by the neurosteroids DHEAS, PS and 3α5βS was similar in all tested brain regions. This suggests that the NR2B receptor subunit behaves independently of its site of expression. Moreover, the NR2B subunit was up-regulated in the frontal cortex but not in the hippocampus or hypothalamus. It is concluded that morphine does not affect the neurosteroid modulatory effect on ifenprodil binding in the rat hippocampus or hypothalamus but does significantly affect both the expression of the NR2B subunit and the 3α5βS modulatory effect on ifenprodil binding in the frontal cortex. It is suggested that the observed effect of long term morphine on the properties of NR2B in the frontal cortex may be associated with the mechanism underlying the development of opiate dependence.

Discovery of a novel avian neurosteroid, 7α-hydroxypregnenolone, and its role in the regulation of the diurnal rhythm of locomotor activity in Japanese quail

The discovery of two novel avian neurosteroids in the quail brain, 7α- and 7β-hydroxypregnenolone is described. Intracerebroventricular administration of 7α-hydroxypregnenolone, but not 7β-hydroxypregnenolone was found to stimulate locomotor activity of male quail when spontaneous nocturnal activity is low. Diurnal changes in locomotor activity in male quail were found to be correlated with a diurnal change in the concentration of diencephalic 7α-hydroxypregnenolone. This correlation was a not seen in female quail which have a lower amplitude diurnal rhythm of locomotor activity and lower daytime concentrations of diencephalic 7α-hydroxypregnenolone. Treatment of male quail with melatonin was found to depress the synthesis of 7α-hydroxypregnenolone in the diencephalon. This is a previously undescribed role for melatonin in the regulation of neurosteroid synthesis in the brain of any vertebrate. We therefore deduced in male quail, that the nocturnal depression in locomotory activity is a consequence of a depression in diencephalic 7α-hydroxypregnenolone resulting from the inhibitory action of the nocturnal increase in melatonin. This observation may be of widespread significance for the molecular control of rhythmic locomotor activity in all vertebrates.