RESUMO
This study was designed to compare the effects of several neuroactive steroids with varying patterns of modulation of gamma-aminobutyric acid (GABA)(A) and NMDA receptors on operant self-administration of ethanol or water. Once stable responding for 10% (w/v) ethanol was achieved, separate test sessions were conducted in which male Wistar rats were allowed to self-administer ethanol or water following pre-treatment with vehicle or one of the following neuroactive steroids: (3beta,5beta)-3-hydroxypregnan-20-one (epipregnanolone; 5, 10, 20 mg/kg; n=12), (3alpha,5beta)-20-oxo-pregnane-3-carboxylic acid (PCA; 10, 20, 30 mg/kg n=10), (3alpha,5beta)-3-hydroxypregnan-20-one hemisuccinate (pregnanolone hemisuccinate; 5, 10, 20 mg/kg; n=12) and (3alpha,5alpha)-3-hydroxyandrostan-17-one hemisuccinate (androsterone hemisuccinate; 5, 10, 20 mg/kg; n=11). The effect of the 3beta-epimer of PCA, (3beta,5beta)-20-oxo-pregnane-3-carboxylic acid (10, 20, 30 mg/kg; n=9), on ethanol self-administration was also examined. The compounds were administered using a Latin-square design 45 min prior to the weekly test sessions. The effects of the 30 mg/kg dose of the steroidal hemisuccinates on ethanol intake were also examined 5 min after administration of these drugs. Both epipregnanolone and PCA attenuated ethanol self-administration. However, neither of the hemisuccinate compounds significantly altered this behavior. The steroidal hemisuccinates (30 mg/kg; n=7) were also tested 5 min before behavior testing and had no effect on ethanol intake 5 min after administration. The 3beta-epimer of PCA also failed to alter ethanol intake. None of the test compounds altered water intake. In electrophysiological studies, the effects of PCA and androsterone hemisuccinate on evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (GABA(A)-IPSCs) was examined in brain slices of the amygdala. PCA had a stimulatory effect at concentrations of 5 and 25 muM. Androsterone hemisuccinate had no agonist activity. The ability of epipregnanolone and PCA to alter ethanol intake appears to be related to different inhibitory actions of these compounds on either GABA(A) or NMDA receptors, respectively. Thus, dual modulation of these systems by selected neuroactive steroids may offer potential for modifying the reinforcing effects of alcohol.
Assuntos
Etanol/administração & dosagem , Pregnanolona/farmacologia , Esteroides/farmacologia , Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/fisiologia , Androsterona/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Masculino , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Wistar , Receptores de GABA-A/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Autoadministração , Esteroides/químicaRESUMO
Among the pharmacological actions of gamma-hydroxybutyric acid (GHB), some may involve GABA(A) receptor-mediated mechanisms. GHB, however, fails to directly interact with sites for agonists and modulators on the GABA(A) receptor complex. We hypothesized that, in vivo, GHB may interfere with GABA(A) receptor function by altering the brain concentrations of the neurosteroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, AP) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), positive allosteric modulators of GABA-gated chloride currents. In male Wistar rats, GHB dose-dependently (75-1000 mg/kg, i.p.) increased AP, THDOC and their precursors pregnenolone and progesterone in brain cortex and hippocampus. The increases of AP (4-5 fold) and THDOC (3-4 fold) elicited by 300 mg/kg GHB peaked between 30 and 90 min and abated by 180 min. The selective GABA(B) receptor antagonist SCH 50911 (50 mg/kg, i.p.) prevented the action of GHB, while the GABA(B) receptor agonist baclofen (5-10 mg/kg) mimicked it. NCS-382 (50 mg/kg, i.p.), the purported selective antagonist of the GHB receptor, failed to antagonize GHB, but at 300 mg/kg increased brain cortical neurosteroids to the same extent as 300 mg/kg GHB; coadministration of GHB and NCS-382, however, failed to yield an additive effect. These results strongly suggest that GHB, via a GABA(B) receptor-mediated mechanism, increases the brain concentrations of neurosteroids, whose properties as amplifiers of the GABA-gated chloride conductances may play a role in the GABA(A) receptor-mediated pharmacological actions of GHB.
Assuntos
Desoxicorticosterona/biossíntese , Hidroxibutiratos/farmacologia , Pregnanolona/biossíntese , Receptores de GABA-B/fisiologia , Animais , Baclofeno/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Desoxicorticosterona/análogos & derivados , Desoxicorticosterona/sangue , Desoxicorticosterona/metabolismo , Relação Dose-Resposta a Droga , Agonistas dos Receptores de GABA-B , Antagonistas de Receptores de GABA-B , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Pregnanolona/sangue , Pregnanolona/metabolismo , Ratos , Ratos WistarRESUMO
The effects of oral contraceptives (OCs) on neurosteroid concentrations were evaluated in female rats and women. In rats, ethynylestradiol and levonorgestrel (0.030 and 0.125 mg, respectively, subcutaneously) administered daily for 6 weeks reduced the concentrations of pregnenolone (-41%) progesterone (-74%) and allopregnanolone (-79%) in the cerebral cortex; the plasma concentrations of these steroids were also reduced but by smaller extents. OC administration for 3 months also reduced the serum concentrations of pregnenolone, progesterone and allopregnanolone in women. Chronic administration of OCs in rats increased the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor gamma 2L and gamma 2S subunit mRNAs and the relative protein in the cerebral cortex, while the amounts of various alpha and beta subunit mRNAs were unaffected. Ovariectomy did not modify the effect of OCs administration on the concentrations of neurosteroids in the rat cerebral cortex (but not in the plasma) as well as on the GABA(A) receptor gene expression, suggesting a direct effect of OCs in brain. Finally, rats treated with OCs exhibited an anxiety-like behavior in the elevated plus-maze test. These results indicate that long-term treatment with OCs induced a persistent reduction in the concentrations of pregnenolone, progesterone and its GABA(A) receptor-active metabolite, allopregnanolone, both in rats and women. In rats this effect was associated with a plastic adaptation of GABA(A) receptor gene expression in the rat cerebral cortex.
Assuntos
Anticoncepcionais Orais Sintéticos/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de GABA-A/biossíntese , Receptores de GABA-A/genética , Adulto , Análise de Variância , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Injeções Subcutâneas , Pregnanolona/sangue , Pregnanolona/metabolismo , Pregnenolona/sangue , Pregnenolona/metabolismo , Progesterona/sangue , Progesterona/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
Here we summarize recent data from our laboratory pertaining to the effects of fluctuations in the brain concentrations of the progesterone (PROG) metabolite allopregnanolone (3alpha,5alpha-TH PROG) on the expression and function of gamma-aminobutyric acid type A (GABA(A)) receptors. The effects of long-term exposure to progesterone and of its sudden withdrawal on the activity of GABA(A) receptors and on the abundance of receptor subunit mRNAs were examined in cultured rat cerebellar granule cells and cortical neurons. The effects of a persistent reduction in the brain concentration of 3alpha,5alpha-TH PROG on GABA(A) receptor function and gene expression were examined in vivo in rats subjected to long-term administration of oral contraceptives. Our results demonstrate that long-lasting changes in the exposure of GABA(A) receptors to this PROG metabolite induce marked effects on receptor structure and function. These effects of 3alpha,5alpha-TH PROG appear to be mediated through modulation of GABA(A) receptor signaling mechanisms that control the expression of specific receptor subunit genes. Furthermore, the specific outcomes of such signaling appear to differ among neurons derived from different regions of the brain. Neuroactive steroids such as 3alpha,5alpha-TH PROG might thus exert differential actions on GABA(A) receptor plasticity in distinct neuronal cell populations, likely accounting for some of the physiological effects induced by these compounds.
Assuntos
Encéfalo/metabolismo , Plasticidade Neuronal/fisiologia , Pregnanolona/metabolismo , Progesterona/farmacologia , Receptores de GABA-A/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Pregnanolona/farmacologia , Ratos , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/genética , Transdução de Sinais/fisiologia , TempoRESUMO
GABAergic neurotransmission can be both positively and negatively modulated by steroids. The steroid effects are thought to be mediated by binding of steroids to specific sites on GABA(A) receptors. It appears that the receptor sites for positive and negative modulatory steroids are different. Thus far, the location and number of binding sites for steroids on these receptors have not been established. In this brief review, we concentrate largely on results from our own structure-activity studies. Novel analogues have been studied to further delineate the structural features required for compounds to modulate receptor function via steroid binding sites. Non-naturally occurring enantiomers of both positive and negative modulators have been studied to provide further evidence for the existence of specific steroid binding sites on the receptors.
Assuntos
Receptores de GABA-A/metabolismo , Esteroides/química , Esteroides/metabolismo , Animais , Sítios de Ligação/fisiologia , Humanos , Esteroides/farmacologia , Relação Estrutura-Atividade , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
The subunit composition of native gamma-aminobutyric acid type A (GABAA) receptors is an important determinant of the role of these receptors in the physiological and pharmacological modulation of neuronal excitability and associated behavior. GABAA receptors containing the alpha 1 subunit mediate the sedative-hypnotic effects of benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000), whereas the anxiolytic effects of these drugs are mediated by receptors that contain the alpha 2 subunit (Löw et al., 2000). In contrast, GABAA receptors containing the alpha 4 or alpha 6 subunits are insensitive to benzodiazepines (Barnard et al., 1998). Characterization of the functions of GABAA-receptors thus requires an understanding of the mechanisms by which the receptor subunit composition is regulated. The expression of specific GABAA-receptor subunit genes in neurons is affected by endogenous and pharmacological modulators of receptor function. The expression of GABAA-receptor subunit genes is thus regulated by neuroactive steroids both in vitro and in vivo. Such regulation occurs both during physiological conditions, such as pregnancy, and during pharmacologically induced conditions, such as pseudo-pregnancy and long-term treatment with steroid derivatives or anxiolytic-hypnotic drugs. Here, we summarize results obtained by our laboratory and by other groups pertaining to the effects of long-term exposure to, and subsequent withdrawal from, progesterone and its metabolite 3 alpha,5 alpha-tetrahydroprogesterone on both the expression of GABAA-receptor subunits and GABAA-receptor function.
Assuntos
Química Encefálica/efeitos dos fármacos , Progesterona/farmacologia , Receptores de GABA-A/genética , Síndrome de Abstinência a Substâncias/fisiopatologia , Animais , Expressão Gênica/efeitos dos fármacos , HumanosRESUMO
The discovery that the endogenous steroid derivatives 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, or 3 alpha,5 alpha-TH PROG) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, or 3 alpha,5 alpha-TH DOC) elicit marked anxiolytic and anti-stress effects and selectively facilitate gamma-aminobutyric acid (GABA)-mediated neurotransmission in the central nervous system (see Chapter 3) has provided new perspectives for our understanding of the physiology and neurobiology of stress and anxiety. Evidence indicating that various stressful conditions that downregulate GABAergic transmission and induce anxiety-like states (Biggio et al., 1990) also induce marked increases in the plasma and brain concentrations of these neuroactive steroids (Biggio et al., 1996, 2000) has led to the view that stress, neurosteroids, and the function of GABAA receptors are intimately related. Changes in the brain concentrations of neurosteroids may play an important role in the modulation of emotional state as well as in the homeostatic mechanisms that counteract the neuronal overexcitation elicited by acute stress. Indeed, neurosteroids not only interact directly with GABAA receptors but also regulate the expression of genes that encode subunits of this receptor complex. This chapter summarizes observations from our laboratories and others, suggesting that neurosteroids and GABAergic transmission are important contributors to the changes in emotional state induced by environmental stress.
Assuntos
Desoxicorticosterona/análogos & derivados , Desoxicorticosterona/fisiologia , Pregnanolona/fisiologia , Estresse Fisiológico/fisiopatologia , Animais , Química Encefálica/fisiologia , Humanos , Receptores de GABA-A/fisiologiaRESUMO
In this chapter, we review techniques used for the analysis of neurosteroids and discuss the advantages and disadvantages of each method. Because radioimmunoassay (RIA) procedures are well known, we focus more on the relatively recent mass spectrometric methods used for analyzing neurosteroids and their sulfates. We also discuss some promising methods that permit the detection of low levels of neurosteroids in small samples with a minimum number of sample pretreatment procedures. Lowering the limits of detection will enable a better understanding of the physiological function of neurosteroids and the mechanism(s) for neurosteroid regulation of brain function. Moreover, analyzing low levels of neurosteroids more efficiently will increase the throughput, which is important for clinical analysis. Initially, most neurosteroid analyses were performed by RIA. However, many analyses of neurosteroids are now performed by mass spectrometry. To date, the most sensitive, specific, and accurate method for the simultaneous analysis of several neurosteroids is the method of gas chromatography/electron capture/negative chemical ionization mass spectrometry. This method, with its many variants, is described in detail.
Assuntos
Desidroepiandrosterona/análise , Pregnenolona/análise , Animais , Cromatografia Gasosa-Espectrometria de Massas , Humanos , RadioimunoensaioRESUMO
RATIONALE: Recent preclinical and clinical studies have shown that selective serotonin re-uptake inhibitors modulate neurosteroid synthesis in an opposite manner. OBJECTIVES: The action of long-term administration of fluoxetine was investigated on the peripheral and central concentrations of 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-TH PROG) and 3alpha,5alpha-tetrahydrodeoxycorticosterone (of 3alpha,5alpha-TH DOC), progesterone, and pregnenolone in rats. We also investigated the effect of chronic treatment with fluoxetine on the foot-shock stress-induced increase in the plasma and brain concentrations of these steroids. METHODS: Fluoxetine was administered acutely (20 mg/kg) or chronically (10 mg/kg, once daily for 15 days). Steroids were extracted from plasma and brain, separated and purified by means of high-performance liquid chromatography, and quantified by means of radioimmunoassay. RESULTS: A single dose of fluoxetine (20 mg/kg, i.p.) induced in 20 min significant increases in the cerebral cortical and plasma concentrations of 3alpha,5alpha-TH PROG (+96% and +13%, respectively), 3alpha,5alpha-TH DOC (+129 and +31%, respectively), progesterone (+111 and +58%, respectively), and pregnenolone (+151 and +59%, respectively). In addition, the plasma concentration of corticosterone was also significantly increased (+24%) after acute administration of fluoxetine. In contrast, long-term administration of fluoxetine reduced the basal concentrations of these various steroids (ranging from -22 to -43%), measured 48 h after the last drug injection, in both brain and plasma. A challenge injection of fluoxetine (20 mg/kg, i.p.), however, was still able to increase the concentrations of steroids in both the brain and plasma of rats chronically treated with this drug. Acute foot-shock stress increased the cortical and plasma concentrations of steroids in rats chronically treated with fluoxetine to extents similar to those apparent in control rats. CONCLUSIONS: A repetitive increase in the brain concentrations of neuroactive steroids may contribute to the therapeutic action of fluoxetine.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Fluoxetina/administração & dosagem , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Esteroides/metabolismo , Animais , Córtex Cerebral/metabolismo , Corticosterona/sangue , Corticosterona/metabolismo , Esquema de Medicação , Injeções Intraperitoneais , Masculino , Pregnenolona/sangue , Pregnenolona/metabolismo , Progesterona/sangue , Progesterona/metabolismo , Ratos , Ratos Sprague-Dawley , Esteroides/sangue , Estresse Fisiológico/sangue , Estresse Fisiológico/metabolismoRESUMO
The extrapyramidal side effects of typical antipsychotics, which are induced to a markedly reduced extent by clozapine, have been linked to a dysfunction of central gamma-aminobutyric acid (GABA)-mediated neurotransmission. The effects of clozapine on the brain concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone, AP) and 3alpha,21-dihydroxy-5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), two potent and endogenous positive allosteric modulators of GABA-mediated chloride current intensities at GABA(A) receptors, were compared with those of the typical antipsychotic haloperidol. A single administration of clozapine (1.25-20 mg/kg, IP), but not of haloperidol (0.1 or 0.5 mg/kg, IP), induced dose- and time-dependent increases in the concentrations of progesterone, AP, and THDOC in the cerebral cortex and striatum of rats. Clozapine (at 10 mg/kg, but not at lower doses) also increased the concentrations of these steroids as well as that of corticosterone in plasma in intact rats, but failed to increase the cortical concentrations of AP and THDOC in adrenalectomized-orchidectomized rats. An acute challenge with clozapine (10 mg/kg), administered 48 h after the termination of daily treatment with the same dose for 19 days, still increased the cortical concentrations of progesterone, AP, and THDOC. These results suggest that the clozapine-induced increases in neuroactive steroid concentrations in the brain may contribute to the atypical pharmacological profile of this antipsychotic drug.
Assuntos
Antipsicóticos/farmacologia , Química Encefálica/efeitos dos fármacos , Clozapina/farmacologia , Desoxicorticosterona/análogos & derivados , Haloperidol/farmacologia , Neurotransmissores/metabolismo , Esteroides/metabolismo , Adrenalectomia , Animais , Desoxicorticosterona/metabolismo , Masculino , Orquiectomia , Progesterona/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Reduced pregnane neurosteroids such as allopregnanolone and pregnanolone are potent neuromodulators able to affect a number of membrane receptors, including gamma-aminobutyric acid (GABA)(A), N-methyl-D-aspartate (NMDA), 5-hydroxytryptamine (5-HT)(3), and sigma(1) receptors. The present study used a drug discrimination procedure to assess further the receptor effects of pregnanolone in vivo. Rats were trained to discriminate 5 mg/kg pregnanolone from saline in a two-lever operant task maintained by food reinforcement. The opiate agonist morphine and the negative GABA(A) modulator dehydroepiandrosterone sulfate did not substitute for pregnanolone. All of the GABA(A) positive modulators tested (allopregnanolone, epipregnanolone, androsterone, pentobarbital, midazolam, and zolpidem) dose dependently substituted for pregnanolone. The direct GABA-site agonists 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol and muscimol failed to substitute for pregnanolone. Ethanol and the sigma(1) receptor agonist SKF 10047 fully substituted for pregnanolone, and the NMDA antagonist MK-801 partially substituted for pregnanolone. The 5-HT(3) antagonist tropisetron did not substitute at any dose tested. The 5-HT(3) agonist SR 57227A reached full substitution, whereas the other 5-HT(3) agonist tested, m-chlorophenylbiguanide, produced partial substitution. These results suggest that positive GABA(A) modulation, but not direct agonism, confers a discriminative stimulus effect similar to pregnanolone. Additionally, antagonism of NMDA receptors and activation of 5-HT(3) and sigma(1) receptors modulate stimulus effects similar to the pregnanolone cue. Overall, the data suggest that pregnanolone produces discriminative stimulus effects representative of a wide-spectrum sedative hypnotic.
Assuntos
Discriminação Psicológica/efeitos dos fármacos , Pregnanolona/farmacologia , Animais , Relação Dose-Resposta a Droga , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Masculino , Ratos , Ratos Long-Evans , Receptores de GABA-A/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores sigma/agonistas , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
Pregnenolone sulphate (PREGS) has generated interest as one of the most potent memory-enhancing neurosteroids to be examined in rodent learning studies, with particular importance in the ageing process. The mechanism by which this endogenous steroid enhances memory formation is hypothesized to involve actions on glutamatergic and GABAergic systems. This hypothesis stems from findings that PREGS is a potent positive modulator of N-methyl-d-aspartate receptors (NMDARs) and a negative modulator of gamma-aminobutyric acid(A) receptors (GABA(A)Rs). Moreover, PREGS is able to reverse the amnesic-like effects of NMDAR and GABA(A)R ligands. To investigate this hypothesis, the present study in rats examined the memory-altering abilities of structural analogs of PREGS, which differ in their modulation of NMDAR and/or GABA(A)R function. The analogs tested were: 11-ketopregnenolone sulphate (an agent that is inactive at GABA(A)Rs and NMDARs), epipregnanolone ([3beta-hydroxy-5beta-pregnan-20-one] sulphate, an inhibitor of both GABA(A)Rs and NMDARs), and a newly synthesized (-) PREGS enantiomer (which is identical to PREGS in effects on GABA(A)Rs and NMDARs). The memory-enhancing effects of PREGS and its analogs were tested in the passive avoidance task using the model of scopolamine-induced amnesia. Both PREGS and its (-) enantiomer blocked the effects of scopolamine. The results show that, unlike PREGS, 11-ketopregnenolone sulphate and epipregnanolone sulphate failed to block the effect of scopolamine, suggesting that altering the modulation of NMDA receptors diminishes the memory-enhancing effects of PREGS. Moreover, enantioselectivity was demonstrated by the ability of natural PREGS to be an order of magnitude more effective than its synthetic enantiomer in reversing scopolamine-induced amnesia. These results identify a novel neuropharmacological site for the modulation of memory processes by neuroactive steroids.
Assuntos
Amnésia/tratamento farmacológico , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Memória/efeitos dos fármacos , Pregnanodiol/análogos & derivados , Pregnenolona/análogos & derivados , Receptores de GABA-A/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Amnésia/metabolismo , Amnésia/fisiopatologia , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/fisiologia , Encéfalo/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Memória/fisiologia , Antagonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Pregnanodiol/farmacologia , Pregnanolona/farmacologia , Pregnenolona/metabolismo , Pregnenolona/farmacologia , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Escopolamina/farmacologia , EstereoisomerismoRESUMO
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 R(2) = 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.
Assuntos
Química Encefálica , Cromatografia Gasosa-Espectrometria de Massas/métodos , Pregnanos/análise , Pregnanolona/administração & dosagem , Estresse Fisiológico/sangue , Animais , Desidroepiandrosterona/análise , Injeções Subcutâneas , Masculino , Pregnanos/isolamento & purificação , Pregnanolona/análise , Pregnanolona/isolamento & purificação , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Natação , Testosterona/análiseRESUMO
BACKGROUND: Alcohol preferring (P) and alcohol-nonpreferring (NP) rats have been shown to have differing behavioral and electrophysiological responses to drugs that are positive modulators of the gamma-aminobutyric acid type A (GABA-A) receptor complex, such as ethanol and benzodiazepines. The neuroactive steroid allopregnanolone is also a positive modulator of GABA-A receptors; therefore, we hypothesized that P and NP rats would respond differently to intraperitoneally administered allopregnanolone. METHODS: Male P and NP rats were implanted with screw electrodes that overlay the frontal and parietal cortices and with a depth electrode aimed at the amygdala. Allopregnanolone (0.0-10.0 mg/kg ip) was administered 10 min before recording the EEG. RESULTS: Allopregnanolone increased high-frequency power (8-32 Hz) in the cortex and amygdala of both P rats and NP rats. In addition, allopregnanolone increased the predominant frequency of the cortical EEG in the 8 to 16 Hz bandwidth, decreased the predominant frequency in the 32 to 50 Hz bandwidth, and increased EEG variability (16-50 Hz). The effects of allopregnanolone were qualitatively similar in P and NP rats. However, P rats were more sensitive to low doses of allopregnanolone in cortex, whereas NP rats responded to lower doses of allopregnanolone in the amygdala. CONCLUSIONS: These data indicate that P and NP rats differ in their sensitivity to the EEG effects of allopregnanolone in the cortex and amygdala, which suggests that differences in GABAergic systems between P and NP rats may contribute to some of the differences observed in their behavioral repertoire.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Tonsila do Cerebelo/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Eletroencefalografia/efeitos dos fármacos , Pregnanolona/farmacologia , Consumo de Bebidas Alcoólicas/fisiopatologia , Tonsila do Cerebelo/fisiologia , Animais , Córtex Cerebral/fisiologia , Masculino , RatosRESUMO
Although carbamazepine (CBZ) is used therapeutically in the treatment of various neurological and psychiatric conditions, its mechanism of action remains largely unknown. CBZ has now been shown to inhibit the binding of [(3)H]PK 11195 to peripheral benzodiazepine receptors (PBRs) in rat brain and ovary membranes in vitro with a potency (IC(50), approximately 60 microM) much lower than that of unlabeled PK 11195 (IC(50), approximately 2.0 nM). Administration of CBZ to rats induced dose (25 to 100 mg/kg, i.p.) and time (15 to 60 min) dependent increases in the concentrations of pregnenolone, progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone in both the cerebral cortex and plasma. CBZ also induced steroidogenesis in the brain of adrenalectomized-orchiectomized rats, suggesting that this effect is mediated in a manner independent of peripheral PBRs. The increase in brain concentrations of neuroactive steroids induced by a single injection of CBZ was associated with a marked protective effect against isoniazid-induced convulsions. In contrast, long-term administration of CBZ (50 mg/kg, twice a day for 30 days) induced tolerance to the anticonvulsant action of the drug. This same treatment, however, did not prevent the ability of a challenge dose of CBZ to stimulate steroidogenesis. These results indicate that CBZ-induced steroidogenesis might not be responsible for the anticonvulsant activity of this drug.
Assuntos
Anticonvulsivantes/farmacologia , Química Encefálica/efeitos dos fármacos , Carbamazepina/farmacologia , Esteroides/biossíntese , Animais , Antineoplásicos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Feminino , Injeções Intraperitoneais , Isoquinolinas/farmacologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The effects of social isolation on behavior, neuroactive steroid concentrations, and GABA(A) receptor function were investigated in rats. Animals isolated for 30 days immediately after weaning exhibited an anxiety-like behavioral profile in the elevated plus-maze and Vogel conflict tests. This behavior was associated with marked decreases in the cerebrocortical, hippocampal, and plasma concentrations of pregnenolone, progesterone, allopregnanolone, and allotetrahydrodeoxycorticosterone compared with those apparent for group-housed rats; in contrast, the plasma concentration of corticosterone was increased in the isolated animals. Acute footshock stress induced greater percentage increases in the cortical concentrations of neuroactive steroids in isolated rats than in group-housed rats. Social isolation also reduced brain GABA(A) receptor function, as evaluated by measuring both GABA-evoked Cl(-) currents in Xenopus oocytes expressing the rat receptors and tert-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding to rat brain membranes. Whereas the amplitude of GABA-induced Cl(-) currents did not differ significantly between group-housed and isolated animals, the potentiation of these currents by diazepam was reduced at cortical or hippocampal GABA(A) receptors from isolated rats compared with that apparent at receptors from group-housed animals. Moreover, the inhibitory effect of ethyl-beta-carboline-3-carboxylate, a negative allosteric modulator of GABA(A) receptors, on these currents was greater at cortical GABA(A) receptors from socially isolated animals than at those from group-housed rats. Finally, social isolation increased the extent of [(35)S]TBPS binding to both cortical and hippocampal membranes. The results further suggest a psychological role for neurosteroids and GABA(A) receptors in the modulation of emotional behavior and mood.
Assuntos
Encéfalo/fisiologia , Receptores de GABA-A/fisiologia , Isolamento Social , Esteroides/metabolismo , Sinaptossomos/fisiologia , Animais , Ansiedade , Córtex Cerebral/fisiologia , Conflito Psicológico , Corticosterona/sangue , Desidroepiandrosterona/metabolismo , Desoxicorticosterona/análogos & derivados , Desoxicorticosterona/metabolismo , Eletrochoque , Feminino , Hipocampo/fisiologia , Técnicas In Vitro , Masculino , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Pregnanolona/análogos & derivados , Pregnanolona/metabolismo , Pregnenolona/metabolismo , Progesterona/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Xenopus laevis , Ácido gama-Aminobutírico/farmacologiaRESUMO
The effects of caffeine, a naturally occurring stimulant, on the brain and plasma concentrations of neuroactive steroids were examined in the rat. A single intraperitoneal injection of caffeine induced dose- and time-dependent increases in the concentrations of pregnenolone, progesterone, and 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) in the cerebral cortex. The increases were significant at a caffeine dose of 25 mg/kg and greatest (+188, +388, and +71%, respectively) at a dose of 100 mg/kg in rats killed 30 min after caffeine administration. Caffeine also increased the plasma concentrations of pregnenolone and progesterone with a dose-response relation similar to that observed in the brain, whereas the caffeine-induced increase in the plasma concentration of allopregnanolone was maximal at a dose of 50 mg/kg. Caffeine increased the plasma concentration of corticosterone, but it had no effect on the brain or plasma concentrations of 3alpha, 21-dihydroxy-5alpha-pregnan-20-one and dehydroepiandrosterone. Moreover, the brain and plasma concentrations of pregnenolone, progesterone, and allopregnanolone were not affected by caffeine in adrenalectomized-orchiectomized rats. These results suggest that neuroactive steroids may modulate the stimulant and anxiogenic effects of caffeine.
Assuntos
Cafeína/farmacologia , Córtex Cerebral/efeitos dos fármacos , Pregnanolona/metabolismo , Pregnenolona/metabolismo , Progesterona/metabolismo , Animais , Córtex Cerebral/química , Relação Dose-Resposta a Droga , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/fisiologia , Masculino , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE: The central nervous system actions of allopregnanolone (3 alpha-hydroxy-5 alpha-pregnan-20-one) and ethanol are at least partially mediated by modulation of gamma-aminobutyric acid (GABA)-A receptors. Although ethanol and allopregnanolone have similar behavioral effects, their macro-electrophysiological profiles have not been directly compared. OBJECTIVE: The purpose of this study was to compare the effects of allopregnanolone and ethanol on the electroencephalogram (EEG) and event-related potentials (ERPs). METHODS: Male Wistar rats were implanted with cortical and amygdalar electrodes. The rats were then administered allopregnanolone (0.0-10 mg/kg), ethanol (0.0-1.0 g/kg), or a combination of the two before recording. RESULTS: Allopregnanolone and ethanol had similar effects on ERPs. When administered alone, both decreased cortical P1-N1 ERP amplitude by 25-50% and N1 amplitude in the amygdala by 75-80%. Combined administration of ethanol (0.50 g/kg) and allopregnanolone (5.0 mg/kg), doses which were ineffective alone, decreased N1 amplitude in the amygdala by 60%. Allopregnanolone and ethanol had dissimilar EEG effects. Allopregnanolone increased high frequency power in the cortex and amygdala by 25-30%. Ethanol decreased cortical and amygdalar power in the same high frequency bands by 25-45%. Allopregnanolone, but not ethanol, also shifted cortical frequency in the 32- to 50-Hz band. Combined administration of allopregnanolone and ethanol had no effect on EEG power but enhanced allopregnanolone's effect on cortical frequency. CONCLUSIONS: These data suggest that allopregnanolone's macro-electrophysiological profile resembles barbiturates and benzodiazepines more than ethanol. Further, the interactions of allopregnanolone and ethanol appear complex, with multiple effects observed (enhancement or reversal) depending on the neurophysiological variable assessed.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Etanol/farmacologia , Pregnanolona/farmacologia , Tonsila do Cerebelo/fisiologia , Animais , Córtex Cerebral/fisiologia , Eletroencefalografia/efeitos dos fármacos , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
Modulation of N-methyl-D-aspartate (NMDA) receptor function by a series of sulfated steroids and dicarboxylic acid ester analogs of pregnenolone sulfate and pregnanolone sulfate was investigated in cultured hippocampal neurons. The "bent" steroid ring structure associated with 5beta-stereochemistry favors receptor inhibition, whereas the more planar ring structure of the pregn-5-enes and 5alpha-pregnanes favors potentiation of NMDA-induced [Ca(2+)] increases and neuronal cell death. The nature of the negatively charged group attached to the steroid C3 position is important for both the neuroprotection afforded by pregnane steroids and the exacerbation of NMDA-induced neuronal death by pregn-5-enes. Dicarboxylic acid hemiesters of various lengths can substitute for the sulfate group of the positive modulator pregnenolone sulfate and the negative modulator pregnanolone sulfate. This result suggests that precise coordination with the oxygen atoms of the sulfate group is not critical for modulation and that the steroid recognition sites can accommodate bulky substituents at C3. The capacity of charged steroids to enhance or protect against NMDA-induced death of hippocampal neurons is strongly correlated with modulation of NMDA-induced Ca(2+) accumulation, indicating that direct enhancement or inhibition of NMDA receptor function is responsible for the proexcitotoxic or neuroprotective effects of these steroids.
Assuntos
Cálcio/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Esteroides/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Conformação Molecular , N-Metilaspartato/toxicidade , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologia , Relação Estrutura-AtividadeRESUMO
Virgin, ovariectomized rats exposed to 2 wk of sequential estradiol (E(2)) and progesterone (P) followed by P withdrawal have increased hypothalamic oxytocin (OT) mRNA and peptide levels relative to sham-treated animals. This increase is prevented if P is sustained. In the central nervous system, P is metabolized to the neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), which exerts effects by acting as a positive allosteric modulator of GABA(A) receptor/Cl(-)-channel complexes. In the present study, ovariectomized rats that received sequential E(2) and P for 2 wk followed by P withdrawal were administered allopregnanolone at the time of P withdrawal. Hypothalamic and plasma allopregnanolone concentrations, serum E(2) and P concentrations, and hypothalamic OT mRNA levels were measured at death. Steroid-induced increases in OT mRNA were attenuated in animals treated with allopregnanolone at the time of P withdrawal. The results suggest that allopregnanolone plays an important modulatory role in steroid-mediated increases in hypothalamic OT.