NO参与介导吗啡戒断大鼠脊髓神经元敏感化

运用Fos免疫组织化学、NADPH－d组织化学、F/NADPH－d双标、鞘内注射和反义寡核苷酸技术,观察吗啡戒断大鼠脊髓神经元活动变化及NO在其中的作用,结果发现：非吗啡依赖大鼠急性应用纳洛酮和吗啡依赖大鼠脊髓水平Fos-LI和NADPH－d阳性神经元表达与对照组相比无明显变化,二者也无Fos／NADPH－d双标神经元表达;吗啡依赖纳洛酮催促戒断大鼠脊髓Fos-LI、NADPH－d阳性神经元、纤维和终末表达明显增加,且出现Fos／NADPH－d双标神经元表达。Fos-LI和Fos／NADPH－d双标神经元呈现双侧脊髓全层分布,NADPH－d阳性神经元、纤维和终末主要位于双侧脊髓背角浅层。鞘内注射NOS抑制剂L－NA和nNOS反义寡核苷酸均明显降低吗啡依赖大鼠纳洛酮催促戒断症状评分,减少吗啡戒断大鼠脊髓Fos-LI表达。上述结果提示：NO参与介导吗啡戒断大鼠脊髓神经元敏感化。     

M受体对吗啡戒断大鼠脊髓和脑干NMDA受体基因表达及中脑导水管周围灰质中谷氨酸释放的调节

应用鞘内注射反义寡脱氧核苷酸技术和RT—PCR反应,观察毒蕈碱型乙酰胆碱受体(muscarinic acetylcholine receptor,M)对吗啡依赖大鼠脊髓和脑干NMDA受体NR1A和NR2A mRNA表达和中脑导水管周围灰质区(periaqueductal grey,PAG)中谷氨酸释放的影响。结果显示,吗啡依赖大鼠脊髓NR1A和NR2A mRNA表达明显升高,而脑干中NR1A和NR2A mRNA表达没有显著变化;注射纳洛酮后1h,吗啡戒断大鼠脊髓和脑干中NR1A和NR2A表达显著高于依赖组,经NMDA受体拮抗剂MK801(0．125mg／kg,i．p．)、M受体拮抗剂东莨菪碱(0．5mg／kg,i．p．)、M1受体拮抗剂呱伦西平(10mg/kg,i．p．)和NOS抑制剂L-NAME(10mg／kg,i．p．)处理后,脊髓和脑干中NR1A和NR2A基因表达都较戒断组明显减少。在纳洛酮激发前24h鞘内注射NR1A和M2受体的反义寡脱氧核苷酸(4μg／只),戒断症状评分值及脊髓和脑干的NR1A mRNA的表达均较对照组明显减少。吗啡依赖大鼠在纳洛酮注射前24h鞘内注射M2受体反义寡脱氧核苷酸(4μg/只),可以明显减少PAG内透析液中谷氨酸含量。上述结果提示：NMDA受体的基因表达和谷氨酸释放参与吗啡戒断过程,而这种表达受到M受体的调节。     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

脊髓水平nNOS和iNOS在吗啡戒断反应中的作用差异

目的:观察鞘内注射选择性一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)抑制剂对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响,以探讨nNOS和iNOS在吗啡依赖和戒断反应中的作用。方法:在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用nNOS抑制剂7-硝基吲哚(7-Ni)和iNOS抑制剂氨基胍(AG)对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响。结果:①鞘内注射7-Ni、AG可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,7-Ni组为16.2±3.99(P<0.01),AG组为22.94±4.0(P<0.05);戒断组TEA评分为13.5±2.55,7-Ni、AG组分别为7.5±2.56、10.5±2.71(P<0.05);②鞘内注射7-Ni、AG可减少脊髓背角Fos阳性神经元的数目,7-Ni、AG组为228.2±49.5、296.8±50.6,低于戒断组(380±71,P<0.05);③7-Ni、AG组nNOS和iNOS阳性神经元的数目分别为169±32、10.2±2.85,均低于戒断组(239±45,16.8±5.1,P<0.05),两给药组脊髓NOS蛋白的表达也显著减少。结论:nNOS和iNOS抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制nNOS和iNOS的表达,nNOS起主要作用而iNOS可能起辅助作用。     

脊髓水平iNOS在吗啡依赖大鼠戒断反应中的作用

目的:探讨脊髓水平诱导型一氧化氮合酶在吗啡依赖大鼠戒断反应中的作用。方法:健康雄性SD大鼠72只,体重200~250 g,吗啡剂量每次10 mg/kg,每日2次,隔日每次增加10 mg/kg,至第6天末次注射50 mg/kg,大鼠腹腔注射纳洛酮4 mg/kg建立吗啡依赖及戒断模型,在纳洛酮激发戒断前30 min鞘内注射iNOS特异性抑制剂氨基胍(AG)150μg。分为正常对照组、吗啡依赖组、吗啡戒断组、AG组。采用行为学(n=8)、免疫组织化学(n=6)和Western blot(n=4)方法观察鞘内应用iNOS特异性抑制剂氨基胍对吗啡依赖大鼠纳洛酮催促戒断反应和脊髓神经元iNOS表达的影响。结果:AG组戒断症状评分和戒断组促诱发痛评分均低于戒断组(P<0.05)。免疫组织化学和Western blot显示戒断组大鼠脊髓iNOS阳性神经元的数目和蛋白的表达增高,而AG组大鼠脊髓iNOS阳性神经元的数目和iNOS蛋白的表达低于戒断组(P<0.05)。结论:脊髓水平iNOS表达上调可能参与介导吗啡戒断反应。     

鞘内注射U0126对吗啡戒断大鼠脊髓神经元磷酸化CREB表达的影响

目的：研究鞘内注射细胞外信号调节激酶（ERK）抑制剂U0126对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓磷酸化cAMP反应元件结合蛋白（p-CREB）表达的影响。方法：建立大鼠吗啡依赖和戒断模型,分为正常对照组、吗啡依赖组、吗啡戒断组、U0126组、溶媒（DMSO）组,采用行为学（n=8）、免疫组织化学（n=6）和Western blot（n=4）方法观察鞘内应用U0126对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓p-CREB表达的影响。结果：①鞘内注射u0126可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28．6±4．89,U0126组为22．5±4．09（P〈0．05）;戒断组促诱发痛评分（TEAscore）为13．5±2．55,U0126组为10．0±2．76（P〈0．05）。②鞘内注射U0126可明显减少胸腰段脊髓背角p-CREB阳性神经元的数目,U0126组为287±54,低于戒断组（380±71,P〈0．05）。 ③westem blot结果显示：鞘内注射U0126明显抑制吗啡戒断期间脊髓p-CREB表达的增加。结论：鞘内注射U0126能明显抑制吗啡戒断大鼠脊髓神经元p-CREB的表达。     

鞘内注射NOS反义寡核苷酸抑制吗啡戒断大鼠脊髓和脑干NMDA1A受体mRNA表达的影响

运用逆转录－多聚酶联反应（RT－PCR）、鞘内注射和反义技术,研究脊髓水平一氧化氮（NO）对大鼠吗啡戒断反应和脊髓及脑干NMDA1A受体mRNA(NMDA1AR mRNA)表达的影响。结果表明,鞘内注射NOS反义寡核苷酸能明显减轻吗啡戒断反应,且脑型NOS（nNOS）反义寡苷酸的作用强于内皮型NOS(eNOS）反义寡核苷酸,吗啡依赖大鼠脊髓和脑干NMDA1AR mRNA表达增加,纳洛酮催促戒断,使其进一步增加;鞘内注射nNOS反义寡核苷酸,能明显抑制吗啡戒断大鼠脊髓和脑干NMDA1AR mRNA表达的增加;eNOS反义寡核苷酸也可抑制吗戒断大鼠脊髓NMDA1AR mRNA表达的增加,但作用弱于nNOS反义寡核苷酸,对脑干NMDA1AR mRNA表达无明显影响,上述结果提示：脊髓水平NO参与介导吗啡戒断反庆和NMDA受体表达的调控。     

MEK抑制剂抑制吗啡依赖及戒断大鼠脊髓神经元NOS的表达

目的：观察鞘内注射丝裂原活化蛋白激酶抑制剂U0126对吗啡依赖及戒断大鼠戒断症状和痛敏行为以及脊髓神经元NOS表达的影响。方法：采用吗啡依赖及戒断模型,分为正常对照组、依赖组、戒断组（戒断1h）、U0126组,分别作行为学评分（n=8）、免疫组织化学（n=6）和免疫印迹检测（n=4）。结果：①鞘内注射U0126可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28．6±4．89,U0126组为22．5±4．09（P〈0．05）;戒断组TEA评分为13．5±2．55,U0126组为10．0±2．76（P〈0．05）;②鞘内注射U0126可明显减少L5节段脊髓背角Fos阳性神经元的数目,U0126组为287±54,低于戒断组（380±71,P〈0．05）;③U0126组nNOS和iNOS阳性神经元的数目分别为180±32、10．8±2．8,均低于戒断组（239±45,16．8±5．1,P〈0．05）,两给药组脊髓NOS蛋白的表达也显著减少。结论：MEK抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制NOS的表达,表明ERK可能参与调控NOS的表达。     

脊髓水平细胞外信号调节激酶激活参与吗啡依赖的形成及戒断反应的表达

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察吗啡依赖及戒断大鼠脊髓神经元磷酸化细胞外信号调节激酶（phospho-extracellular signal-regulated kinase,pERK）表达的变化,及鞘内注射促分裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase,MEK）抑制剂U0126或ERK反义寡核苷酸对吗啡依赖大鼠纳洛酮催促戒断反应、触诱发痛及脊髓神经元pERK表达的影响,探讨脊髓水平pERK在介导吗啡依赖和戒断过程中的作用。结果显示：（1）在吗啡依赖形成过程中,大鼠脊髓胞浆与胞核非磷酸化ERK表达没有改变,但pERK表达逐渐增加,纳洛酮催促戒断后,仍有进一步增加的趋势,戒断1h后,其表达量明显下降,但仍高于对照组。（2）鞘内预先注射MEK抑制剂U0126或ERK反义寡核苷酸能明显抑制吗啡戒断反应和戒断引起的痛觉异常;与行为学结果一致,脊髓背角pERK阳性神经元表达与脊髓胞浆和胞核pERK表达也明显降低。上述结果提示,脊髓水平ERK激活和核转位参与吗啡依赖的形成及戒断反应的表达。     

cAMP反应元件结合蛋白介导磷酸化细胞外信号调节激酶在神经病理性痛形成中的作用

采用行为学、免疫组织化学和Western blot方法,观察鞘内注射细胞外信号调节激酶(extracellular signal-regulate kinase,ERK)信号转导通路阻滞剂对慢性压迫性损伤(chronic constriction injury,CCI)大鼠痛行为及脊髓背角内磷酸化cAMP反应元件结合蛋白(phosphorylated cAMP response-element binding protein,pCREB)和Fos表达变化的影响,探讨ERK／CREB转导通路在神经病理性疼痛中的作用。结果表明,CCI可明显增加双侧脊髓背角pCREB、损伤侧脊髓背角浅层Fos阳性神经元表达,以CCI后3与5d时尤为显著。鞘内沣射促分裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)阻滞剂U0126及ERK反义寡核苷酸在减轻大鼠痛行为的同时,能明显抑制双侧脊髓背角内pCREB的表达,同时,Fos阳性神经元的表达也明显减少。大鼠痛行为及脊髓背角pCREB和Fos的表达在时相上一致。上述结果提示pCREB参与pERK介导的神经病理性疼痛。     

鞘内注射M受体和GDNF反义寡脱氧核苷酸抑制吗啡戒断大鼠蓝斑c—Fos表达

The antisense approach and immunohistochemistry were used to study the effects of different muscarinic receptor (M) subtypes and glial cell derived neurotrophic factor (GDNF) on the scores of morphine-withdrawal syndrome and the expression of c-Fos in locus coeruleus (LC). Intrathecal injection of M2 receptor antisense oligonucleotides (M2AS-oligo) or GDNF antisense oligonucleotides (GDNFAS-oligo) decreased the scores of morphine withdrawal syndrome. The expression of c-Fos positive neurons in the LC increased in morphine-dependent rats and increased to a greater extent after the injection of naloxone (4mg/kg, ip) in morphine dependent rats. Intrathecal injection of M2AS-oligo or GDNFAS-oligo inhibited the increase of c-Fos expression in LC during morphine withdrawal, but there was no effect in case of M1AS-oligo. The results suggest that M2 receptor of spinal cord mediates the neural activation of LC during morphine withdrawal. And the interaction between neurons and glial cells may be involved in the ascending activation process.     

Role of PKC-alpha,gamma isoforms in regulation of c-Fos and TH expression after naloxone-induced morphine withdrawal in the hypothalamic PVN and medulla oblongata catecholaminergic cell groups

We previously demonstrated that morphine withdrawal induced hyperactivity of the hypothalamus-pituitary-adrenocortical axis by activation of noradrenergic pathways innervating the hypothalamic paraventricular nucleus (PVN), as evaluated by Fos expression and corticosterone release. The present study was designed to investigate the role of protein kinase C (PKC) in this process by estimating changes in PKCalpha and PKCgamma immunoreactivity, and whether pharmacological inhibition of PKC would attenuate morphine withdrawal-induced c-Fos expression and changes in tyrosine hydroxylase (TH) immunoreactivity levels in the PVN and nucleus tractus solitarius/ ventrolateral medulla (NTS/VLM). Dependence on morphine was induced in rats by 7 day s.c. implantation of morphine pellets. Morphine withdrawal was induced on day 8 by an injection of naloxone. The protein levels of PKCalpha and gamma were significantly down-regulated in the PVN and NTS/VLM from the morphine-withdrawn rats. Morphine withdrawal induced c-Fos expression in the PVN and NTS/VLM, indicating an activation of neurons in those nuclei. TH immunoreactivity was increased in the NTS/VLM after induction of morphine withdrawal, whereas there was a decrease in TH levels in the PVN. Infusion of calphostin C, a selective protein kinase C inhibitor, produced a reduction in the morphine withdrawal-induced c-Fos expression. Additionally, the changes in TH levels in the PVN and NTS/VLM were significantly modified by calphostin C. The present results suggest that activated PKC in the PVN and catecholaminergic brainstem cell groups may be critical for the activation of the hypothalamic-pituitary adrenocortical axis in response to morphine withdrawal.     

Role of Corticotropin-Releasing Factor (CRF) Receptor-1 on the Catecholaminergic Response to Morphine Withdrawal in the Nucleus Accumbens (NAc)

Stress induces the release of the peptide corticotropin-releasing factor (CRF) into the ventral tegmental area (VTA), and also increases dopamine (DA) levels in brain regions receiving dense VTA input. Since the role of stress in drug addiction is well established, the present study examined the possible involvement of CRF1 receptor in the interaction between morphine withdrawal and catecholaminergic pathways in the reward system. The effects of naloxone-precipitated morphine withdrawal on signs of withdrawal, hypothalamo-pituitary-adrenocortical (HPA) axis activity, dopamine (DA) and noradrenaline (NA) turnover in the nucleus accumbens (NAc) and activation of VTA dopaminergic neurons, were investigated in rats pretreated with vehicle or CP-154,526 (selective CRF1R antagonist). CP-154,526 attenuated the increases in body weight loss and suppressed some of withdrawal signs. Pretreatment with CRF1 receptor antagonist resulted in no significant modification of the increased NA turnover at NAc or plasma corticosterone levels that were seen during morphine withdrawal. However, blockade of CRF1 receptor significantly reduced morphine withdrawal-induced increases in plasma adrenocorticotropin (ACTH) levels, DA turnover and TH phosphorylation at Ser40 in the NAc. In addition, CP-154,526 reduced the number of TH containing neurons expressing c-Fos in the VTA after naloxone-precipitated morphine withdrawal. Altogether, these results support the idea that VTA dopaminergic neurons are activated in response to naloxone-precipitated morphine withdrawal and suggest that CRF1 receptors are involved in the activation of dopaminergic pathways which project to NAc.     

Roles for GFRalpha1 receptors in zebrafish enteric nervous system development

Components of the zebrafish GDNF receptor complex are expressed very early in the development of enteric nervous system precursors, and are already present as these cells begin to enter the gut and migrate caudally along its length. Both gfra1a and gfra1b as well as ret are expressed at this time, while gfra2 expression, the receptor component that binds the GDNF-related ligand neurturin, is not detected until the precursors have migrated along the gut. Gfra genes are also expressed in regions of the zebrafish brain and peripheral ganglia, expression domains conserved with other species. Enteric neurons are eliminated after injection with antisense morpholino oligonucleotides against ret or against both Gfra1 orthologs, but are not affected by antisense oligonucleotides against gfra2. Blocking GDNF signaling prevents migration of enteric neuron precursors, which remain positioned at the anterior end of the gut. Phenotypes induced by injection of antisense morpholinos against both Gfra orthologs can be rescued by introduction of mRNA for gfra1a or for gfra2, suggesting that GFRalpha1 and GFRalpha2 are functionally equivalent.