纹状体提取液增强体外培养的中脑黑质神经元的发育

制备新生２ｄ龄大鼠纹状体提取液及其不同分子量的组分，用快速自动化色微量分析法检测它们对体外培养的１４ｄ齿大鼠胚胎中脑黑质神经元的影响，结果表明，纹，状体提取液有支持中脑黑质神经元生存及增强其活性的作用，而且具有剂量依赖关系，其最适浓度为１．２５μｇ／ｍｌ。纹状体提取液还能促进中脑黑质神经元对＾３Ｈ－ＤＡ和＾３Ｈ－ＧＡＢＡ的摄取，同对照组相比，有显著性差异（Ｐ＜０．０１）。通过检测纹状体提取液小于１     

备用根大鼠脊髓后角组织神经营养活性物质的分离纯化和生 …

目的 进一步分离纯化备用根大鼠部分去传入纤维支配（手术侧）的脊髓后角组织提取液，以获得某种营养活性物质。方法 用Ｓｕｐｅｒｄｅｘ Ｇ－７５ ｐｒｅｐ ｇｒａｄｅ凝胶层析、高效液相色谱（ＨＰＬＣ）层析和细胞培养等技术分离和检测神经营养活性物质。结果 手术侧脊髓后角组织提取液经Ｓｕｐｅｒｄｅｘ Ｇ－７５ ｐｒｅｐ ｇｒａｄｅ凝胶层析和ＨＰＬＣ层析后，得到的Ａ峰洗脱液具有促进体外培养鸡胚背根节（ＤＲＧ     

帕金森病大鼠黑质和纹状体神经元的变化

本研究采用免疫细胞化学方法观察了由６－ＯＨＤＡ单侧微量注射制成的帕金森病大鼠黑质酪氨酸羟化酶样（多巴胺能）神经元及其纤维和纹状体头部Ｐ物质神经元及体部ＧＡＢＡ能神经元的变化，以进一步揭示黑质、纹状体间的功能联系。结果显示，６－ＯＨＤＡ注射侧黑质酪氨酸羟化酶样神经元及其纤维显著减少，注射侧纹状体面积、纹状体Ｐ物质神经元和ＧＡＢＡ能神经元数量均较正常对照侧明显减少。本研究表明黑质多巴胺能神经元的损毁，破坏了黑质纹状体间正常的递质运送，进而导致纹状体的改变。     

38 纹状体提取液增强体外培养的中脑黑质神经元的发育

<正>神经元的靶组织能释放可溶性化学因子(靶源性神经营养因子)增强神经元的存活和发育.纹状体是中脑黑质神经元主要的靶组织.我们制备新生2天龄SD大鼠纹状体提取液及其不同分子量的组分,用快速自动比色微量分析法检测它们对体外培养的14天龄SD大鼠胚胎中脑黑质神经元的影响.结果表明,纹状体提     

人胶质细胞源神经营养因子在大肠埃希菌中高效表达

胶质细胞源神经营养因子 (ＧＤＮＦ)是近几年来发现并克隆成功的一种新的神经营养因子 ,它对不同神经元的生长、发育过程都有重要的影响〔1 3〕。ＧＤＮＦ对中脑多巴胺能 (ＤＡ)神经元具有存活、支持作用 ,对损伤的ＤＡ能神经元具有修复作用〔4,5〕。ＧＤＮＦ除对ＤＡ能神经元有特异性营养作用外 ,还对胆碱能神经元、去甲肾上腺素能神经元、运动神经元及外周神经系统的各类感觉神经元、交感神经元等均有很强的营养作用。ＧＤＮＦ对 6 羟基多巴 (ＯＨＤＡ)及神经毒ＭＰＴＰ损伤的黑质纹状体多巴胺能神经元有保护及修复、再生的功能。甚至黑…     

不同剂量6-羟基多巴胺注射大鼠单侧前脑内侧束后对中脑多巴胺能神经元损伤作用的观察

将不同剂量的６－羟基多巴胺（６－ＯＨＤＡ）注入大鼠一侧黑质（ＳＮ）头端的前脑内侧束（ＭＦＢ），４７天后用酪氨酸羟化酶（ＴＨ）免疫细胞化学染色法，观察旋转鼠整个ＳＮ和腹侧被盖区（ＶＴＡ）不同切面上ＤＡ神经元的损伤程度，结果发现：损伤侧ＳＮ和ＶＴＡ的ＴＨ阳性神经元数在所有观察水平上均有明显下降，并从前囟后５．１ｍｍ开始呈现６－ＯＨＤＡ剂量依赖性。而下降率（与健侧相比）为ＳＮ致密部（ｓｎｃ）＞网状部和外侧部（ｓｎｒ＋ｓｎｌ）＞ＶＴＡ。同时还发现，ｓｎｃ的ＴＨ阳性细胞在黑质两端分布比率较高，而ＶＴＡ的相应细胞在黑质中部分布率较高。另外，在健侧前囟后４．８ｍｍ处出现非６－ＯＨＤＡ剂量依赖性的ＴＨ阳性神经元计数明显增加的现象。本文的结果提示：６－ＯＨＤＡ对中脑ＤＡ神经元的总体损伤程度取决于多种因素，如６－ＯＨＤＡ剂量、神经元对６－ＯＨＤＡ的敏感度以及敏感神经元的分布等。同时健侧中脑组织对邻侧发生的损伤并非毫无反应，一些交叉投射的纤维可能起着传递损伤信息的作用     

胚中脑黑质移植入震颤麻痹模型鼠脑内的实验研究

胚中脑黑质移植入震颤麻痹模型鼠脑内的实验研究朱道立（江苏省南通师专生物系）采用６－羟基多巴胺（６－ＯＨＤＡ）分２点注入ＳＤ鼠右侧中脑黑质内侧端，制成震颤麻痹模型，毁损后６～８周，用胚龄１４～１６天同种胚鼠中脑黑质细胞悬液植入模型鼠尾壳核。实验分实验对...     

大规模基因突变检测策略

研究新基因、遗传标记、遗传性疾病及肿瘤形成离不开对基因突变的检测。本文主要介绍用于大规模检测未知基因突变点的常规方法及最新策略。根据检测原理分为物理方法，包括常用的单链构象多态技术（ＳＳＣＰ）、变性梯度凝胶电泳（ＤＧＧＥ）和杂合双链分析（ＨＴＸ）等技术；裂解法包括ＲＮＡ酶裂解法（ＲＮａｓｅ）、化学错配裂解法（ＣＣＭ）和酶错配裂解法（ＥＭＣ）；其它方法则突出介绍了标志突变检测最新进展的毛细管电泳（ＣＧＥ）、变性高效液相色谱分析（ＤＨＰＬＣ）、碱基切割序列扫描（ＢＥＳＳＴ－ＳｃａｎＴＭ）和有发展潜力的ＤＮＡ芯片技术（ＤＮＡｃｈｉｐｓ）及相对准确、完美的测序法（Ｓｅｑｕｅｎｃｉｎｇ）。在简述各种方法原理的基础上，并说明所述策略在具体操作中的优缺点     

测定重组人粒细胞集落刺激因子生物学活性的MTT比色法

用粒细胞集落刺激因子（Ｇ－ＣＳＦ）的依赖细胞株（ＮＦＳ－６０细胞株）为靶细胞，以ＭＴＴ比色法测定重组人粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）的生物学活性，确定了在ＭＴＴ比色法中ＮＦＳ－６０细胞浓度为３×１０４／孔以及ｒｈＧ－ＣＳＦ浓度与ＮＦＳ－６０细胞有量效关系的范围为２ｎｇ／ｍｌ～７．８ｐｇ／ｍｌ，对几种溶解液进行了比较，选定１０％ＳＤＳ－０．０１ｍｏｌ／ＬＨＣｌ作为ＭＴＴ结晶的溶解液，对ＭＴＴ比色法的特异性、精密度和方法回收率进行了研究。     

阴离子交换FPLC制备级纯化单克隆抗体

作者采用ＤＥＡＥ－４０ＨＲ阴离子交换柱。在快速蛋白液相色谱系统（ＦＰＬＣ）建立了ＩｇＧ类单克隆抗体制备级纯化方法。该法是将ＭｃＡｂ腹水经５０％饱和硫酸铵盐析粗分离后，再用阴离子交换色谱纯化，一次上样量相当于８０～１００ｍｌ腹水。可得纯化ＭｃＡｂ５００～６００ｍｇ，得率为６～７ｍｇ／ｍｌ腹水，回收率为５７～６７％．一次纯化周期仅需４５ｍｉｎ。经ＳＤＳ－ＰＡＧＥ检测ＭｃＡｂ纯度为９５％，ＡＢＣ染色法测定ＭｃＡｂ活性为１：２００００（３．１３×１０－１０ｍｏｌ／Ｌ）．     

Inhibition of glutamate and acetylcholine release in behavioral improvement induced by electroacupuncture in parkinsonian rats

Prior evidence shows that acupuncture improves symptoms in both Parkinson's disease (PD) patients and animal models. We examined the effects of high-frequency (100 Hz) electroacupuncture (EA) on behavior in a rat PD model induced by medial forebrain bundle (MFB) transection. Neurotransmitters levels in the striatum were measured using in vivo microdialysis and high performance liquid chromatography (HPLC). High-frequency EA stimulation at Dazhui (GV14) and Baihui (GV20) acupoints decreased rotational behavior induced by apomorphine (APO) and improved motor coordination, protected axotomized dopaminergic neurons from degeneration in the substantia nigra (SN), it did not increase striatal dopamine (DA) levels. However, EA stimulation at acupoints significantly decreased the abnormally elevated glutamate (Glu) and acetylcholine (ACh) levels in the lesioned side of striatum. Moreover, the Glu levels correlated significantly with survival ratios of dopaminergic neurons in the SNc and rotational bahavior. These data suggested that behavioral alleviation with EA stimulation may be associated with modulation of neurotransmitters release, such as Glu and ACh in the striatum, rather than with DA restoration.     

Urocortin modulates dopaminergic neuronal survival via inhibition of glycogen synthase kinase-3β and histone deacetylase

Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that regulates stress responses. Although UCN is principally expressed in dopaminergic neurons in rat substantia nigra (SN), the function of UCN in modulating dopaminergic neuronal survival remains unclear. Using primary mesencephalic cultures, we demonstrated that dopaminergic neurons underwent spontaneous cell death when their age increased in culture. Treatment of mesencephalic cultures with UCN markedly prolonged the survival of dopaminergic neurons, whereas neutralization of UCN with anti-UCN antibody accelerated dopaminergic neurons degeneration. UCN increased intracellular cAMP levels followed by phosphorylating glycogen synthase kinase-3β (GSK-3β) on Ser9. Moreover, UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological approaches, we further demonstrated that inhibition of GSK-3β and HDAC contributes to UCN-mediated neuroprotection. These results suggest that dopaminergic neurons-derived UCN might be involved in an autocrine protective signaling mechanism.     

胚胎期脂多糖暴露可通过钟样受体4引起出生后多巴胺能神经元减少

目的 观察钟样受体4(TLR-4)在胚胎期脂多糖 (LPS) 暴露引起的出生后个体脑内多巴胺 (DA) 能神经元减少中的作用.方法 TLR-4 突变型C57BL/10ScNCr小鼠和野生型C57BL/10ScSn小鼠各15只,在妊娠期第10.5天给小鼠腹腔注射LPS或肽聚糖(PDG),出生后4月龄时收集大脑组织标本(n=5),通过免疫组织化学染色和体视学技术定量DA能神经元和小胶质细胞数量,高效液相色谱法测定DA及其代谢物水平,免疫荧光法结合流式细胞术测定TNF-α和IL-1β蛋白水平.结果 出生前接触过LPS的4月龄C57BL/10ScSn小鼠,与注射生理盐水的对照小鼠比较,黑质DA能神经元减少(25.3±2.1)%,纹状体DA含量降低(33.5±5.0)%,黑质小胶质细胞数量增加(294±24)%,黑质和纹状体TNF-α和IL-1β蛋白水平也明显增高;但是出生前接触过LPS的TLR-4突变型C57BL/10ScNCr小鼠脑内无相应变化.出生前接触过TLR-2配体PDG的4月龄C57BL/10ScNCr 和C57BL/10ScSn小鼠均出现脑内DA能神经元减少和免疫炎症改变.结论 胚胎期接触LPS可通过TLR-4引起出生后个体脑内DA能神经元减少.     

Dopaminergic mesencephalic systems and behavioral performance in very old rats

Morphologic and functional studies describing the impact of aging on mesencephalic dopaminergic (DA) neurons in laboratory animals are rather scanty and inconclusive. In rats, stereological studies characterizing age changes in the mesencephalic DA neurons have not been documented. In order to fill this information gap and to determine whether the very old rat may serve as a suitable animal model of Parkinson's disease, we performed a stereological assessment of the mesencephalic tyrosine hydroxylase immunoreactive (TH-ir) neurons in young-adult (4-6 months), old (22-24 months) and senile (30-32 months) Sprague-Dawley female rats. Morphometric analysis of the TH-ir neurons of the substantia nigra (SN) and ventral tegmental area (VTA) was performed using an appropriate image analysis system. Age changes in motor performance were assessed measuring the endurance of rats to hang from a wire mesh pole or to remain on a ramp set at different angles to the floor. Age changes in locomotion and exploratory activity were evaluated by the open field test. We observed a significant age-related reduction in TH-ir neuron numbers in the SN (17 and 33% reduction in old and senile rats, respectively compared with young counterparts) but not in the VTA. The size of the TH-ir cells increased significantly in both the SN and VTA of the senescent animals but TH labeling intensity fell. Motor, locomotor and exploratory performance deteriorated markedly in the old and senile rats as compared with young animals. These findings reveal the existence of a moderate but significant vulnerability of mesencephalic DA neurons to aging in rats. This phenomenon, which is particularly marked in the SN of very old rats, may contribute to the age-related decline in motor and exploratory performance recorded in this species.     

Host striatal projections into fetal ventral mesencephalic tissue grafted to the striatum of immature or adult rat

We have previously reported that few striatal axons from adult host brain innervate intrastriatal grafts of fetal ventral mesencephalic tissue. To see whether the immature rat brain would favor striatal innervation of the graft, unilateral implantation of fetal ventral mesencephalic tissue was carried out at 7 (P7), 14 (P14), or 60 (adults) days of age in neonatally dopamine- (DA)-lesioned and nonlesioned rats. Immunocytochemistry for tyrosine hydroxylase (TH), and/or dopamine- and adenosine 3′,5′-monophosphate-regulated phosphoprotein-32 (DARPP-32) was performed 2–6 months later. In the great majority of immature and in all adult recipients, the resulting graft consisted of a distinct intrastriatal mass of tissue surrounded by the host parenchyma. Most TH-immunopositive neurons were found within the confines of such grafts, although some were lying at short distances into the host striatal tissue, particularly in immature recipients. In a few immature recipients, there was, however, extensive intermingling of TH-positive neurons with the adjacent host brain tissue. In all recipients grafted at P7, P14, or as adults, the distinct, intra-parenchymal grafts contained moderate numbers of DARPP-32-positive processes, mainly at their periphery. These results indicate that the limited capacity of host striatal neurons to grow axons into transplanted fetal ventral mesencephalic tissue is not markedly different in young versus adult rats. A better integration of the ventral mesencephalic graft into the striatal circuitry of immature — as opposed to adult — recipients should therefore rely more on the higher tendency of DA neurons to become located into the host tissue following transplantation in young rats.     

Neuregulin-1 receptor tyrosine kinase ErbB4 is upregulated in midbrain dopaminergic neurons in Parkinson disease

Previously we demonstrated that systemically administered neuregulin-1-β1, a nerve growth and differentiation factor, passed the blood–brain barrier and accumulated in brain areas with expression of its receptor ErbB4. In substantia nigra (SN), neuregulin-1-β1 phosphorylated ErbB4 and protected dopaminergic neurons in a toxin-based mouse model of Parkinson disease (PD). We studied ErbB4 in the context of human midbrain dopaminergic degeneration in vivo and in vitro. Post-mortem ventral midbrain tissue sections of neuropsychiatric healthy individuals and PD patients (matched for age, gender and post-mortem delay) were immunostained for ErbB4. Cultured Lund human mesencephalic (LUHMES) post-mitotic dopaminergic neurons were treated with dopaminergic toxins and analyzed for ErbB4 expression. In control individuals, 85.0 ± 5.0% of dopaminergic neurons, containing cytoplasmic neuromelanin, expressed ErbB4 in the SN. In PD cases, the percentage of ErbB4-positive nigral dopaminergic neurons was increased to 94.9 ± 2.5%. The mean ErbB4 immunoreactivity of melanized neurons was higher in PD than controls. LUHMES neurons upregulated ErbB4 when exposed to toxins 1-methyl-4-phenylpyridinium and 6-hydroxydopamine. Increased rate of ErbB4-positive dopaminergic neurons in PD may either reflect a better survival of ErbB4-positive neurons or an increased expression of ErbB4 by remaining neurons to seek trophic support. Enhanced ErbB4 expression in human in vitro toxin-based PD models supports the latter interpretation. Thus, dopaminergic neurons in SN might be susceptible to neuregulin-1 treatment in PD.     

纹状体和中缝核外植块促进体外培养的中脑多巴胺神经元的发育

为了探讨神经元与其靶组织之间的相互作用及作用因素，建立了不同时期的纹状体外植场与１４ｄ大鼠胚胎中脑黑质神经元联合培养；１４ｄ大鼠胚胎中缝核外植块与黑质神经元联合培养。结果表明，生后２周、新生及１４ｄ大鼠胚胎纹状体外植块和１４ｄ大鼠胚胎中缝核外植块，对中脑黑质多巴胺神经元的形态发育有明显促进作用，其中包括长突起神经元数目和神经元突起长度的增加。上述３个时期的纹状体外植块，对中脑黑质多巴胺神经元的作用     

纹状体对体外培养中脑多巴胺神经元形态发育的影响

靶细胞在神经细胞的生长发育过程中起着重要的调节作用。本文通过胚胎腹侧中脑和纹状体联合培养研究纹状体对中脑多巴胺(DA)神经元形态发育的影响。纹状体(Str)和腹侧中脑(YMA)细胞悬液取自胚胎14天SD大鼠,联合培养3到14天后取出,用酪氨酸羟化酶(TH)免疫组化ABC法观察不同时期DA神经元的生长。与VMA单独培养相比,联合培养3天,发现细胞聚集程度较低,开始形成单层分布,培养7天TH阳性细胞数增加,多突起阳性细胞较易发现,细胞突起粗短,呈树枝状分枝。培养14天,TH阳性细胞数进一步增加,平均可达49个/25mm~2。本文对纹状体调节DA神经元生长发育的机制进行了讨论。     

Homeostatic regulation of excitatory synapses on striatal medium spiny neurons expressing the D2 dopamine receptor

Striatal medium spiny neurons (MSNs) are contacted by glutamatergic axon terminals originating from cortex, thalamus and other regions. The striatum is also innervated by dopaminergic (DAergic) terminals, some of which release glutamate as a co-transmitter. Despite evidence for functional DA release at birth in the striatum, the role of DA in the establishment of striatal circuitry is unclear. In light of recent work suggesting activity-dependent homeostatic regulation of glutamatergic terminals on MSNs expressing the D2 DA receptor (D2-MSNs), we used primary co-cultures to test the hypothesis that stimulation of DA and glutamate receptors regulates the homeostasis of glutamatergic synapses on MSNs. Co-culture of D2-MSNs with mesencephalic DA neurons or with cortical neurons produced an increase in spines and functional glutamate synapses expressing VGLUT2 or VGLUT1, respectively. The density of VGLUT2-positive terminals was reduced by the conditional knockout of this gene from DA neurons. In the presence of both mesencephalic and cortical neurons, the density of synapses reached the same total, compatible with the possibility of a homeostatic mechanism capping excitatory synaptic density. Blockade of D2 receptors increased the density of cortical and mesencephalic glutamatergic terminals, without changing MSN spine density or mEPSC frequency. Combined blockade of AMPA and NMDA glutamate receptors increased the density of cortical terminals and decreased that of mesencephalic VGLUT2-positive terminals, with no net change in total excitatory terminal density or in mEPSC frequency. These results suggest that DA and glutamate signaling regulate excitatory inputs to striatal D2-MSNs at both the pre- and postsynaptic level, under the influence of a homeostatic mechanism controlling functional output of the circuit.