Changes Of Neurosteroids In Brain Regions Of Morphine Dependent, Withdrawal And Relapsed Male Rats

Morphine dependence is a chronic, relapsing disorder in which compulsive drug-seeking and drug-taking behavior persists despite serious negative consequences. Continued using of morphine induces adaptive changes in the central nervous system that lead to tolerance, physical dependence, sensitization, craving, and relapse. Studies in the past decades suggest that morphine addiction involves not only the opioid receptors, but also the dopamine (DA) receptor system, amino acid receptor systems, 5-hydroxy tryptamine (5-HT) receptor system, and corticotropin-releasing factor (CRF) receptor system. The neuroanatomical basal for morphine physical dependence consists of locus coreuleus (LC), periaqueductal gray (PAG), hypothalamus, amygdala etc, and the neurochemical basal is the activation of noradranalin system. While the neuroanatomical basal for morphine psychical dependence consist of ventral tegmental area (VTA), nucleus accumbens (NAc), frontal cortex, hippocampus etc, and the neurochemical basal is the activation of dopamine neurons in mesolimibic system. Relapse to morphine induced by stress is mediated mainly by the CRF system in bed nucleus of stria terminalis (BNST). Despite numerous studies, the precise mechanisms underlying morphine addictive effects are still incompletely understood. Neurosteroids is a new group of neurotransmitters with wide effects. Neurosteroids included the steroids synthesized in central neural system (CNS), and the steroids secreted from steroidogenic gland but accumulated in the brain. Recent studies demonstrated that concomitant chronic administration of neurosteroids, allopregnanolone (AP), progesterone (PROG), pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), 5a-pregnan-3,20-dione, and deoxycorticosterone acetate, followed by morphine (10 mg·kg^-1) prevented the development of tolerance to the antinociceptive effect of morphine and suppressed the naloxone-precipitated withdrawal jumps. Pregnanolone produces discriminative stimulus effects in rats. AP dose-dependently increased the release of DA in the nucleus accumbens and doubled the DA response to morphine. These results suggest that neurosteroids PREG and AP not only have reinforcing effect themselves but also potentiate the reinforcing effect of morphine. Also studies indicate that morphine dependence, withdrawal and relapse were accompanied by the changes of neurosteroids levels in whole rat brain. Morphine dependece inhibites the expression of mRNAs of steroidogenic enzymes P450scc and 3β-HSD in related brain regions. This study was carried out to determine the changes of neurosteroids levels in related brain regions and plasma in morphine dependent, withdrawal and relapsed male rats and the changes of amino acid neurotransmitters levels in morphine dependent and withdrawal male rats. Methods 1 Animal models 1.1 Morphine physical dependent and withdrawal rat model Eighty male SD rats were designated to four groups randomly with twenty in each, including control group, withdrawal control group, dependent group, and withdrawal group. Morphine dependence was induced by seven consecutive days of pretreatment (thrice daily, ip) with increasing doses of morphine hydrochloride (at 5, 10, 15, 20, 30, 40, and 50 mg·kg^-1, respectively). Rats in control and withdrawal control groups received equal volumes of saline. Six hours after the last injection of morphine, rats in withdrawal control and withdrawal groups were injected with naloxone hydrochloride (2 mg·kg^-1, ip) and control and dependent group with saline. Withdrawal syndromes including jumping, wet-dog shaking, stretch, diarrhea,grooming etc, were observed for 15 min and scored. Weight loss was recorded 30 min after the naloxone precipitation. After that rats were rapidly euthanized by decapitation and trunk blood and brain regions were stored in -70 ℃for neurosteroids oramino acids detection. 1.2 Morphine psychical dependent rat model Twenty male SD rats were divided randomly to two groups with ten in each including control group and morphine group. Conditioned place preference (CPP) was induced by training with morphine (5 mg·kg^-1, ip) for ten consecutive days. Control group was trained with equal volume saline. CPP test was conducted 24 h after the last training. The time rats spent in the drug paired chamber in 10 min was recorded. After that rats were rapidly euthanized by decapitation and trunk blood and brain regions were stored in –70 ℃for neurosteroids detection. 1.3 Morphine relapse rat model Thirty male SD rats were designated to three groups randomly with ten in each, including control group, extinction group and relapse group. CPP was induced by morphine injection (ip) in extinction group and relapse group. The rats in control group received equal volumes saline. After training, seven days extinction was given. Randomly delivered foot shock was performed in control and relapse groups for 15 min. CPP tests were conducted right after training, extinction and 2 h after foot shock respectively. After that rats were rapidly euthanized by decapitation and trunk blood and brain regions were stored in –70 ℃for neurosteroids detection. 2 Extraction and quantification of neurosteroids 2.1 Extraction of neurosteroids 2.1.1 Extraction of neurosteroids from brain tissues Brain tissues was homogenized in phosphate buffered saline (PBS) and extracted with Ethyl Acetate/n-hexane (9/1, v/v) for disassociative neurosteroids and chloroform/2-butanol(1/1, v/v) for conjugated neurosteroids. The extractions were further purified by solid phase extraction using Oasis HLB cartridges. The conjugated neurosteroids were solvolyzed to disassociative form. Sample were then derivatized with 2-nitro-4-trifluoromethylphenylhydrazine (2NFPH) and stored for quantification.2.1.2 Extraction of neurosteroids from plasma Ethyl Acetate/n-hexane (9/1, v/v) was used for disassociative neurosteroids extraction. Acetonitrile was use for precipitation and supernate was subject to solid phase extraction for conjugated neurosteroids. The methods of solvolysis and derivatization were same to that of brain tissue. 2.2 Quantification of neurosteroids High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to quantify the neurosteroids in brain tissue and plasma. Gradient elution separation through a C18 analytical column was used with acetonitrile/water as mobile phase. Methyltestosterone (MT) and estrogen sulfate (ES) were the internal standards for disassociative and conjugated neurosteroids detection respectively. Negative atmosphere pressure chemical ionizer (APCI) was used for ionization. The ions selected in selected ion monitoring (SIM) were m/z 490.0 (DHEA ,DHEAS),m/z 518.0 (PREG,PREGS),m/z 520.0 (AP),m/z 504.0 (MT) and m/z 472.0 (ES) [M-H]-. The linear range was 0.015 ng² ng for DHEA, PREG, AP, DHEAS and PREGS. 3 Quantification of amino acids Brain tissues were homogenized in artificial cerebral spinal fluid (ACSF) and precipitated with methanol. The supernate was derivatized with O-phthaldialdehyde (OPA) for 10 min. Samples was subject to high performance liquid chromatography-electrochemical detection (HPLC-ECD) for glycine (GLY), glutamat (GLU) and γ-aminobutyric acid (GABA) with vanillin as internal standard. Mobile phase (0.05 mol·L^-1 acetate sodium, 0.05 mol·L^-1 citric acid, 1 μmol·L^-1 EDTA-2Na, 21% methanol v/v and 0.2% triethylamine v/v) was pumped through a 150 mm (5 μm) octadecasilane reversed-phase column. The potential setting of electrochemical detector is +0.8 V. The linear range was 10.24¹000 μg·L^-1 for GLY, GLU and GABA. Results 1 Changes of neurosteroids levels in brain regions and plasma of morphine physical dependent and withdrawal rats 1.1 Withdrawal syndromes of morphine dependent rats precipitated bynaloxone Compared with the withdrawal control group, the score of withdrawal syndrome increased by 105% (P<0.01) and the weight loss increased by 279% (P<0.01) in morphine withdrawal group. 1.2 Changes of neurosteroids levels in brain regions and plasma of morphine physical dependent rats Compared with control group, the DHEAS levels in nucleus accumbens of morphine dependent group decreased by 33% (P<0.05); the DHEA levels in amygdala and pituitary decreased by (P<0.01);and 24% (P<0.05) respectively; the DHEAS and PREGS levels in plasma increased by 381% (P<0.01) and 329% (P<0.01) respectively. 1.3 Changes of neurosteroids levels in brain regions and plasma of morphine withdrawal rats Compared with control group, the PREG and AP levels in nucleus accumbens of withdrawal group increased by 112% (P<0.01) and 44% (P<0.05) respectively; the PREG and AP levels in amygdals increased by 45% (P<0.05) and 42% (P<0.05) respectively; the PREG and AP levels in hypothalamus increased by 70% (P<0.05) and 53% (P<0.01) respectively; the PREG, AP, DEHAS and PREGS levels increased by 111% (P<0.01), 84% (P<0.01), 723% (P<0.01) and 389% (P<0.01) respectively while the DHEA level in plasma decreased by 42% (P<0.01). Compared with morphine dependent group, the PREG and AP levels in nucleus accumbens of withdrawal group increased by 105% (P<0.01) and 44% (P<0.05) respectively; the PREG and PREGS levels in amygdala increased by 60% (P<0.05) and 40% (P<0.05) respectively; the PREG and AP levels in hypothalamus increased by 89% (P<0.01) and 53% (P<0.01) respectively; the PREG and PREGS levels in plasma increased by 80% (P<0.05) and 99% (P<0.01) respectively while the DHEA level decreased by 31% (P<0.01). 2 Changes of neurosteroids levels in brain regions and plasma of morphine psychical dependent rats 2.1 Results of CPP trainingAfter CPP training, the time morphine group spent in drug paired chamber increase by 56% (P<0.01) compared with control group and increased by 116% (P<0.01) compared with that before CPP training. 2.2 Changes of neurosteroids levels in brain regions and plasma of morphine psychical dependent rats Compared with control group, the DHEA level in amygdala of morphine group decreased by 56% (P<0.05); the PREG level in hypothalamus decreased by 28% (P<0.05); the PREG level in pituitary increased by 28% (P<0.05); the DHEA level in plasma decreased by 44% (P<0.01). 3 Changes of neurosteroids levels in brain regions and plasma of morphine relapsed rats 3.1 Results of morphine relapse rat model After CPP training, the time extinction group and relapse group spent in drug paired chamber increase by 55% (P<0.01) and 50% (P<0.01) respectively compared the control group. After extinction, there was no significant difference among the three groups in the time spent in drug paired chamber (P>0.05). After foot shock, the time relapse group spent in drug paired chamber increased by 63% (P<0.01) and 44% (P<0.05) compared with control group and extinction group respectively. 3.2 Changes of neurosteroids levels in brain regions and plasma of morphine relapsed rats Compared with control group, the DHEA level in nucleus accumbens of morphine relapse group increased by 50% (P<0.05); the DHEAS level in amygdala decreased by 24% (P<0.01); the PREG and PREGS levels in hypothalamus increased by 36% (P<0.01) and 60% (P<0.05) respectively; the DHEAS level in plasma decreased by 33% (P<0.01). Compared with extionction group, the AP level in nucleus accumbens of relapse group increased by 75% (P<0.05); the PREGS level in hypothalamus increased by 60% (P<0.05) and the DHEAS level in pituitary increased by 12% (P<0.05)。4 Changes of amino acids levels in brain regions morphine withdrawal rats Compared with withdrawal control group, the GABA level in amygdala ofwithdrawal group decreased by 18% (P<0.01); the GLY level in hypothalamus increased by 18% (P<0.05). Compared with morphine physical dependent group, the GLU level in nucleus accumbens of withdrawal group increased by 45% (P<0.05); the GLY and GABA levels in amygdala decreased by 14% (P<0.05) and 19% (P<0.05) respectively. Conclusions The changes of neurosteroids levels in plasma and brain regions were different when morphine dependence, withdrawal and relapse were developed. This suggests that the changes of neurosteroids in central nervous system are independent of the systemic neurosteroids concentration. The changes of neurosteroids levels various in brain regions and neurosteroids in our experiments. This suggests that specific neurosteroid(s) in brain regions play important role in the development and regulation of morphine dependence, withdrawal and relapse. Similar changes exist among neurosteroids in brain regions, pituitary and plasma in our study. It could not be excluded that the hypothalamus-pituitary-adrenal axis (HPA axis) effects of morphine and foot shock play a role in the regulation of neurosteroids levels in brain regions, pituitary and plasma. When withdrawal was precipitated, nucleus accumbens was in excitatory state for the increase in GLU level and amygdala was in disinhibited state for the decrease of GABA and GLY levels. In morphine physical dependence and withdrawal animals, changes of neurosteroids and amino acids levels in nucleus and accumbens may different even opposite effects on NMDA and GABA_A receptors systems. These suggest that the role of neurosteroids in morphine physical dependence and withdrawal is mediated partly by their effects on NMDA and GABAA receptors systems. According to the effect on GABA_A and NMDA receptor systems, the decrease of nucleus accumbens DHEA level produces a physiologically...