| Neurosteroids are endogenous modulators of neuronal functions responsible for many biological and pathophysiological effects. The neurosteroid pregnane (AP) is a potent positive modulator of GABAA receptors, and shows anxiolytic, sedative/hypnotic, anticonvulsant functions. Recent studies have demonstrated that the development of tolerance and dependence of morphine can be inhibited by concomitant chronic administration of neurosteroids such as AP, PREGS. Moreover, they can influence the form of conditioned position preference and have preference or aversion actions. AP also can decrease the drinking volume of ethanol in dependent rat, but increase in nondependent rat. Ethanol dependence and withdrawal decreased the levels of neurosteroids in frontal cortex, hippocampus , cerebellum and blood, and altered the functional properties of GABAA receptor and its sensitization to neurosteroids, which resulted in the adaptation in GABAA receptor. Therefore, it is possible that changes in endogenous neurosteroid levels during chronic ethanol exposure and/or withdrawal might affect GABAA receptor function, which may underlie the development of ethanol dependence. However, the mechanisms that account for alterations in GABAA receptor function following chronic ethanol administration have not been clarified in details. The mechanisms responsible for adaptation of GABAA receptors to chronic ethanol exposure may involve in ethanol-induced changes in gene expression, subcellular localization, synaptic localization, receptor phosphorylation, neurosteroids, and/or changes in GABAA receptor subunit composition. These results suggested that endogenous neurosteroids might be involved in the development of ethanol dependence through reward circuit. Endogenous neurosteroids might also play an important role in the complex interactions between ethanol and GABAA receptors. Now the pathway of neurosteroid synthesis and metabolism in the brain tissue has been clarified basically. The study suggested that ethanol may influence other steroidogenic enzymes involved in steroid biosynthesis and metabolism. Effects of acute EtOH administration on the StAR, 3β-HSD and 3α-HSD enzymes activity have been demonstrated, whereas limited data are available on the regulation of neurosteroidogenic enzymes by chronic EtOH exposure and withdrawal. one study reported that chronic EtOH exposure reduced 3β-HSD and 5α-reductase expression levels in rat brain regoins. Although chronic ethanol administration changeded the levels of neurosteroids in several brain regions, it is still unknown whether the changes of neurosteroids levels are related with the mRNAs expression of steroidogenic enzymes. It is also unknown whether chronic ethanol administration affected the expressions of other steroidogenic enzymes in brain regoins. The identification of neurosteroid modulation on ethanol action may lead to important progress in the development of ethanol dependence and the strategy of novel therapeutics for alcoholism. In this study, a method for the determination of dehydroepiandrosterone (DHEA), pregnenolone (PREG), allopregnanolone (AP), pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulfate (DHEAS) in several rat brain regions or plasma using liquid-chromatography-mass spectrometry (LC-MS) was developed. The levels of neurosteroids were investigated in ethanol dependence and withdrawal rats using LC-MS. Expression of the polypeptide for P450scc and GABAA receptor α1 subunit in the rat brain regions were examined using immunohistochemical staining analysis. The principal results were as follows: 1. Quantification of unconjugated neurosteroids in rat brain tissue and plasma by liquid chromatography-mass spectrometryA LC-MS method for determination of unconjugated neurosteroids in several rat brain regions or plasma was developed. Methyltestosterone(MT) was chosen as the internal standard. Neurosteroids were isolated in a two-step procedure using ethyl acetate/hexane(9/1,v/v) in the first step to extract the unconjugated steroids (PREG, DHEA and AP), and this steroid fractions were further purified by SPE. All steroids were derivatized with 2NFPH and analyzed by LC-MS using selected-ion monitoring. LC-MS was performed using a Agilent 1100 liquid chromatograph-mass spectrometry, and APCI was employed. A Zorbax SB C18 column was used at a flow rate of 1ml/min at 40℃. The mobile phase consisted of acetonitrile and distilled water(degrade elution). Unconjugated steroids form PREG-2NFP-, DHEA-2NFP-, AP-2NFP-and MT-2NFP-by derivatization with 2NFPH. The ions selected for PREG-2NFP-, DHEA-2NFP-, AP-2NFP-and MT-2NFP-are m/z 518, m/z 490, m/z 520 and m/z 504, respectively, based on the relative intensity of the respective target ion in the corresponding mass spectra. The detection limits for PREG-2NFP-, DHEA-2NFP-and AP-2NFP-was 2.5pg, 1.6pg and 1.2pg respectively. The results of the reproducibility, linearity and accuracy experiments indicate that the procedure is reliable for quantification of trace amounts of neurosteroids in brain tissue by LC-MS. Thus, this methodology should be suitable for determining the levels of different neurosteroids in rat brain tissues or plasma. 2. Quantification of sulfated neurosteroids in rat brain tissue and plasma by liquid chromatography-mass spectrometry A method has been established by LC-MS, to detect two sulfated neurosteroids in rat brain tissues or plasma. Estrogen sulfate(ES) was chosen as the internal standard. Neurosteroids were isolated in a two-step procedure using chloroform/2-butanol (50/50,v/v) in the first step to extract sulfated steroids (PREGS and DHEAS) from the water phase of brain, and the plasma was deposited with acetonitrile(1/2,v/v), then the steroid fractions were further purified by SPE and the sulfated steroids were solvolyzed. All steroids werederivatized with 2NFPH and analyzed by LC-MS using selected-ion monitoring. LC-MS was performed using a Agilent 1100 liquid chromatograph-mass spectrometry, and APCI was employed. Sulfated steroids after solvolysis form PREG-2NFP-, DHEA-2NFP-and ES-2NFP-. The ions selected for PREG-2NFP-, DHEA-2NFP-and ES-2NFP-are m/z 518, m/z 490 and m/z 472.1, respectively, based on the relative intensity of the respective target ion in the corresponding mass spectra. The detection limits for DHEA-2NFP-and PREG-2NFP-was 4.0pg and 4.7pg respectively. Good linearity and accuracy were observed for each steroid. The procedure was suitable for quantitative analysis of the sulfated steroids in rat brain regions and plasma. 3. Effects of ethanol dependence and withdrawal on the levels of neurosteroids in rat brain regions Ethanol-treated rats were administrated gradually to 6% ethanol(w/v) in the drinking water within a 1w. (day 1 0% ethanol, day 2,3 1.5% ethanol, day 4,5 3.0% ethanol, day 6,7 4.5% ethanol, the later 6% ethanol) and then were maintained on this ethanol concentration for 6w. The drinking dose of ethanol is about 6.0-8.2g/kg/d. our experiment has determined the concentrations of neurosteroid in the nucleus accumbens, amygdala, frontal cortex, hippocampus, striatum, hypothalamus and cerebellum. Compared with the control group, chronic ethanol administrations resulted in a marked decrease in the levels of AP (P<0.05) in male rat nucleus accumbens and frontal cortex; The levels of AP decreased by 41% (P=0.051) in amygdala; But there was significantly increased the levels of AP in cerebellum (P<0.01). In contrast, ethanol withdrawal(6h,24h) resulted in a increase on the levels of AP in the reward circuit, there was significant effects on the levels of AP in nucleus accumbens (P<0.01, vs dependent group). There were no significant effects of chronic ethanol administration and withdrawal on the levels of AP in hypothalamus, hippocampus and striatum(vs control group or dependent group).Compared with the control group, chronic ethanol treament significantly decreased the levels of PREG and DHEA in nucleus accumbens(P<0.05); Although there was no effect of withdrawal(6h and 24h) on the levels of PREG in reward circuit, there was significant increase on the levels of DHEA in amygdala(P<0.01), and decrease in nucleus accumbens (P<0.05); Chronic ethanol treament significantly increased the levels of DHEA in hippocampus(P<0.05); Ethanol withdrawal(24h) induced a significant increase in the concentrations of PREG in hippocampus and cerebellum (P<0.01, P<0.05). Compared with dependent group, ethanol withdrawal(6h) induced a significant decrease in the concentrations of DHEA in hippocampus (P<0.05) , and increase in the concentrations of PREG in cerebellum (P<0.05); Ethanol withdrawal(24h) resulted in a significant increase on the DHEA and PREG levels in amygdala(P<0.05); Ethanol withdrawal(24h) significantly increased cerebellum and hippocampus PREG levels (P<0.05), and decreased hypothalamus DHEA levels (P<0.05). Compared with the control group, chronic ethanol treament significantly decreased the levels of PREGS and DHEAS in nucleus accumbens (P<0.05,P<0.01), PREGS in frontal cortex(P<0.01); Ethanol withdrawal(6h) induced a significant decrease in the concentrations of DHEAS in frontal cortex(P<0.05), and decrease in the concentrations of PREGS in nucleus accumbens, amygdala, frontal cortex, hippocampus and hypothalamus (P<0.01, P<0.05); Ethanol withdrawal(24h) resulted in a significant decrease in the DHEAS levels of in nucleus accumbens, amygdala and cerebellum (P<0.01), but increase in the DHEAS level in frontal cortex and striatum (P<0.01), ethanol withdrawal(24h) induced a significant decrease in the concentrations of PREGS in nucleus accumbens, amygdala, frontal cortex, hippocampus, and striatum(P<0.01). Our date showed that ethanol dependence and withdrawal affected the levels of neurosteroids in rat brain. These results suggest that endogenous neurosteroids in brain might be related to the development of ethanol dependence and withdrawal. 4. Effects of ethanol dependence and withdrawal on the levels of neurosteroids in rat plasma Compared with control group, chronic ethanol administrations resulted in a insignificant decrease in the concentrations of PREG, PREGS and DHEAS by 23%, 44% and 9% (P>0.05) in the plasma, respectively, there was no significant change observed in the concentrations of AP and DHEA; Ethanol withdrawal(6h) induced a significant increase in the concentrations of AP by 109%, PREG by 50%(P<0.05); Ethanol withdrawal(24h) induced a significant increase in the concentration of PREG by 42%(P<0.01); In contrast, the concentration of DHEAS decreased by 70%(P<0.01). There was no significant change in the concentration of the other tested neurostroids. Compared with dependent group, ethanol withdrawal(6h) induced a significant increase in the concentrations of PREG by 95% (P<0.01), AP by 114%(P<0.05), but there were no significant effects of withdrawal(6h) on the concentrations of DHEA, PREGS and DHEAS. Furthermore, ethanol withdrawal(24h) induced a significant increase in the concentrations of PREG and AP by 85%(P<0.01), 85%(P<0.05) respectively, and a significant decrease in the concentrations of DHEAS by 67% compared with dependent group, but there were no significant effects of withdrawal(24h) on the concentrations of DHEA and PREGS. The analytical results suggested that there was no significant correlation of the neurosteroids values between the brain regions and the plasma in all rats examined. 5. Effects of ethanol dependence and withdrawal on the expression of GABAA receptor α1 subunit and steroidogenic enzymes P450scc polypeptide in rat brain. Immunohistochemical staining results showed that GABAA receptor α1 subunit protein were expressed in all tested brain regions including the nucleus accumbens, amygdala, frontal cortex, ventral tegmental area(VTA), hippocampus, striatum and hypothalamus. Compared with control group, chronic ethanol administration significantly inhibited the expression ofGABAA receptor α1 subunit protein in the nucleus accumbens, striatum and hippocampus CA3 region; There was a decrease of GABAA receptor α1 subunit protein in the frontal cortex, VTA, hypothalamus and hippocampus (CA1,CA2 region). There was no significant difference between withdrawal and dependence group. The results suggest that the alteration in GABAA receptor gene expression underlies the change of functional properties of GABAA receptor in ethanol-dependent rats and those undergoing ethanol withdrawal. Compared with the control group, chronic ethanol treatment significantly inhibited the expression of P450scc protein in the amygdala and hippocampus(CA1,CA3 region), There was no significant difference in other brain regions; Ethanol withdrawal(24h) resulted in a significant increase the expression of P450scc protein in the frontal cortex and VTA. Compared with the dependence group, Ethanol withdrawal(24h) resulted in a marked increase the expression of P450scc protein in the frontal cortex and hippocampus(CA1 region). The results suggest that the changes of brain neurosteroids levels by chronic ethanol treatment might mainly depend on the activity of steroidogenic enzymes in the CNS. In this study, we developed and optimized a selective procedure using SPE, specific derivatisation, and LC-MS to quantify very small amounts of different neurosteroids. The validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue and plasma by liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry was established. MT and ES were chosen as the internal standard for the determination of unconjugated neurosteroids and sulfated neurosteroids, respectively. Good linearity, reproducibility and accuracy were observed for each steroid. The procedure was suitable for quantitative analysis of the unconjugated neurosteroids and sulfated steroids in rat brain regions and plasma.
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