BACKGROUND:Studies have demonstrated that exogenous neurosteroid treatment prevents the development of morphine tolerance and dependence, and attenuates abstinence behavior in mice. However, there are few studies on whether the levels of endogenous neurosteroids can be changed by morphine dependence and withdrawal.OBJECTIVE:To investigate the levels of various neurosteroids in rat brain following morphine dependence and withdrawal. To evaluate the expressions of steroidogenic enzyme mRNAs and proteins. To identify the relationship between neurosteroids and morphine dependence at the whole animal behavior, neural biochemistry, and molecular levels.DESIGN, TIME AND SETTING:A randomized, controlled study. Experiments were performed at the Department of Pharmacology of Hebei Medical University and Department of Pharmacology of Beathune Intational Peace Hospital, China, from June 2004 to October 2007.MATERIALS:Morphine hydrochloride injection (Shenyang First Pharmaceutical Factory, China), naloxone hydrochloride (Hunan Yiqiao Pharmaceutical Co., China) and a gas chromatography-mass spectrometry system (Agilent, CA, USA) were used in this study.METHODS:Healthy adult Sprague Dawley rats were randomly divided into three groups:a morphine dependence group, morphine withdrawal group and control group (n = 20). The rats in the morphine dependence and morphine withdrawal groups were given increasing doses of morphine (5, 10, 15, 20, 30, 40 and 50 mg/kg, intraperitoneal) to create morphine dependence. The rats in the morphine withdrawal group were injected with 2 mg/kg naloxone to precipitate withdrawal 1 hour after the last morphine injection. Rats in the control group were treated with an equal volume of saline.MAIN OUTCOME MEASURES:Following morphine dependence and withdrawal, brain levels of the neurosteroids pregnenolone, progesterone and allopregnanolone were analyzed using gas chromatography-mass spectrometry. The mRNA expression of two key steroidogenic enzymes, P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), were determined in rat brain regions using reverse transcription-polymerase chain reaction. The distribution and expression of P450scc protein were visualized in brain regions associated with addiction by immunohistochemistry.RESULTS:In brain tissue from the morphine dependence group, the levels of pregnenolone and progesterone were decreased by 62% (P < 0.01) and 92% (P < 0.01) respectively, compared with the control group. In the morphine dependence group, the key steroidogenic enzyme P450scc mRNA was decreased in striatum (P < 0.05), while 3β-HSD mRNA was decreased in amygdala (P < 0.05), striatum (P < 0.05) and frontal cortex (P < 0.05) compared with the control group. Morphine withdrawal induced a significant increase in the neurosteroid levels compared with the control group (P < 0.01). However, there was no significant difference in the expressions of P450scc and 3β-HSD mRNAs between the morphine withdrawal and control groups (P > 0.05).CONCLUSION:The neurosteroid levels and expressions of steroidogenic enzymes changed similarly in morphine dependent rats, suggesting that the morphine dependence-induced decrease in neurosteroids might depend on local expression of steroidogenic enzymes in the central nervous system. However, the changes in neurosteroids in morphine withdrawal rats were not in accordance with the changes in the expression of steroidogenic enzymes, suggesting that the effects of morphine withdrawal on brain neurosteroid levels may not depend primarily on the local expression of steroidogenic enzymes in the central nervous system.