注射热损伤大鼠纹状mRNA的爪蟾卵母细胞对多巴胺的反应

将从下沉大鼠和热损伤大鼠的中枢纹状体提取的ｐｏｌｙ（Ａ）＾＋ｍＲＮＡ,注入非洲爪蟾卵母细胞表达。用电生理方法检测多巴胺诱发的膜电位和电流的变化,分析热损伤对中枢多巴胺受体表达的影响。结果表明,注射大鼠纹状体ｍＲＮＡ后,卵母细胞的静息电位与注射 前没有变化,但多巴胺能诱发膜电流。经验证,此受体电流的主要载流离子是Ｃ１＾－。注射热务大鼠纹状体ｍＲＮＡ的卵母细胞对多巴胺反应的敏感性降低,与正常大鼠组相比     

淀粉样β蛋白和自由基对扑赡卵母细胞表达的大鼠脑谷氨酸受体功能的影响

目的探讨淀粉样β蛋白(Aβ1-40)和自由基对爪蟾卵母细胞表达的大鼠脑谷氨酸受体(GluR)功能的影响.方法采用Promega试剂盒提取3月龄大鼠脑组织总RNA和mRNA,并将50nl(50ng)的mRNA显微注射到每个爪蟾卵母细胞.注射后的卵母细胞在(19±1)℃条件下以改良的巴氏液孵育进行受体表达.这些表达的受体的激活电流采用双电极电压钳位技术记录.超氧阴离子自由基(SAFRs)和Aβ1-40于记录前12h、24h、96h分别加入孵育液.结果爪赡卵母细胞可以表达出M型ACh、谷氨酸、多巴胺、GABA和5-羟色胺受体.Aβ-40对表达的GluR有抑制效应,其程度依作用时间和浓度而异.20nmol/LAβ1-40作用24h对表达的GluR功能无影响.与SAFRs共同孵育时,20nmol/LAβ1-40作用12h即对表达的GluR功能有明显影响,60nmol/LAβ1-40作用12h即对表达的GluR有明显抑制效应(降低21%,P＜0.05),而60nmol/LAβ-40作用24h下降达52%.维生素E对这些效应有拮抗作用.结论自由基和Aβ对GluR具有抑制效应,该效应能被维生素E拮抗.Aβ可能通过抑制受体功能在AD病理生理学中起作用.     

大鼠纹状体脑片热损伤模型的建立

热损伤是环境医学领域的重要课题。据报道 ,脑内多巴胺受体与热损伤有密切关系。由于多巴胺受体主要集中分布于纹状体 ,为此 ,本实验建立了离体大鼠纹状体脑片胞外记录模型 ,以观察热损伤对离体大鼠纹状体脑片电位的影响。1　材料与方法将成年大鼠 (Ｗｉｓｔａｒ)在乙醚麻醉下断头取脑 ,分离出纹状体 ,于 4℃供氧条件下用国产震动切片机将其切成厚约 35 0 μｍ的脑片 ,并迅速置于 32℃恒温供氧条件下的人工脑脊液 (ＡＣＳＦ)中孵育 1ｈ。ＡＣＳＦ液成份 (ｍｍｏｌ/Ｌ) :ＮａＣｌ 1 2 0 .0 ,ＫＣｌ 3.3,ＫＨ2 ＰＯ4 1 2 ,ＮａＨＣＯ3 2 6 .…     

热损伤对大鼠纹状体神经元凋亡的影响

目的:探讨热损伤对原代培养的大鼠纹状体神经元凋亡的影响.方法:对原代培养的大鼠纹状体神经元进行43℃热损伤40 min后,用共聚焦激光扫描显微镜(LSCM)观察神经元细胞内Ca2 浓度的变化、神经元线粒体膜电位的变化,TUNEL法检测热损伤前后纹状体神经元凋亡的变化.结果:热损伤使纹状体神经元内Ca2 浓度明显升高,线粒体膜电位明显降低(P＜0.01);热损伤后纹状体神经元凋亡增多.结论:热损伤可能通过增加细胞内钙离子浓度、降低细胞线粒体膜电位而诱发大鼠纹状体原代培养神经元凋亡.     

红藻氨酸和GABA受体在中华大蟾蜍卵母细胞的表达

本文报道了利用中华大蟾蜍卵母细胞作为外源性膜蛋白的表达及其功能特性研究的模式系统。将大鼠脑的mRNA微量注入蟾蜍卵母细胞(每个卵母细胞注射50ng),在19℃下经48h以上培养后,由外源mRNA表达的大鼠脑的红藻氨酸和γ-氨基丁酸受体被整合到了卵母细胞膜上。红藻氨酸(5×10~(-5)mol/L)和γ-氨基丁酸(10~(-4)mol/L)所诱导的膜电流分别达到294.0±6.4nA(n=5)和309.5±4.9nA(n=4)。红藻氨酸浓度在10~(-3)mol/L时,其诱导的膜电流达最大值。进而,注射mRNA的卵母细胞,~(36)Cl~-流入速度比对照组高一倍多。这些结果表明,中华大蟾蜍卵母细胞,如同爪蟾卵母细胞一样,能表达具有功能的外源膜蛋白(受体蛋白和离子运输蛋白)。     

D2DR在纹状体突触可塑性及运动障碍中枢调控中的作用

多巴胺Ⅱ型受体在大脑基底神经节纹状体区域表达丰富,可反馈性调节突触前多巴胺合成并介导细胞信号转导。纹状体神经元突触可塑性受多巴胺Ⅱ型受体介导的cAMP/PKA和PLC信号通路调节,也是自主运动控制的神经基础。在运动性疲劳及以帕金森病为代表的运动功能障碍的中枢疾病中,多巴胺Ⅱ型受体通过平衡基底神经节直接通路和间接通路发挥重要作用。本文对多巴胺Ⅱ型受体在纹状体神经元突触可塑性和运动功能障碍中枢调控中的作用进行综述,为相关疾病的靶向干预和治疗提供理论基础。     

强啡肽对纹状体内D_1多巴胺受体反应的调节

用6-羟多巴胺破坏黑质纹状体通路,使大鼠多巴胺耗竭后,应用原位杂交组织化学方法测量D1多巴胺受体对即早基因c－fos和zif268诱导反应,分析强啡肽对突触前、后调节作用。先用D1多巴胺受体激动剂SKF－38393反复处理动物,促进纹状体内强啡肽表达,在伏隔核强啡肽表达增加,同时伴随着即早基因c-fos和zif268的减少．在纹状体的背部和两侧,强啡肽表达虽大量增加,而D1多巴胺受体反应仍然维持原水平．在中央纹状体区,即早基因的表达处于中间水平。结果提示,纹状体内强啡肽起着调节多巴胺输入到纹状体黑质神经元的作用,包括突触前、后位置;并且调节作用在纹状体的腹、背侧区是不同的     

神经生长因子对正常和放射线-化学复合损伤小鼠造血调节因子及其受体影响研究

目的 探究神经生长因子(NGF)对正常和放射线-化学复合损伤小鼠造血调节因子及其受体的影响.方法 用实时荧光定量PCR和酶联免疫吸附检测注射NGF后正常和经60Coγ射线照射+腹腔注射环磷酰胺(放射线-化学复合损伤,放-化复合损伤)小鼠肾脏红细胞生成素(Epo)、脾脏红细胞生成素受体(EpoR)、骨髓细胞粒-巨噬系集落刺激因子(GM-CSF) mRNA表达量和血清Epo、GM-CSF、白细胞介素-3(IL-3)浓度变化.结果 注射NGF后正常小鼠脾脏EpoR mRNA表达、放-化复合损伤小鼠血清GM-CSF、IL-3显著高于注射生理盐水的对照组.结论 NGF可以改变小鼠的造血调节因子及其受体水平,但对正常和放-化复合损伤机体,其作用各不相同.     

过表达PINK1抵抗鱼藤酮引起多巴胺神经元损伤的研究

目的:明确在C57BL/6小鼠纹状体过表达野生型的人源帕金森相关蛋白PINK1能否减轻由侧脑室注射鱼藤酮引起多巴胺神经元损伤。方法:通过向C57BL/6小鼠(雄性,7周龄,18~20g)左侧纹状体中注射带有GFP人源野生型PINK1及突变体PINK1^G309D的慢病毒包装颗粒,两周后向小鼠左侧侧脑室中定位注射鱼藤酮,通过蛋白质印迹,免疫组化和行为学的方法检测PINK1对鱼藤酮引起多巴胺神经元损伤的影响。结果:蛋白质印迹和免疫组化的实验都证明了在C57BL/6小鼠纹状体过表达野生型的PINK1对于鱼藤酮引起多巴胺能神经元的减少有明显的抑制作用(P<0.01),但对鱼藤酮引起的行为学损伤没有明显的改善作用。     

大鼠纹状体GABA受体的表达及年龄性变化

目的观察大鼠纹状体GABA受体的表达特征及增龄性变化。方法应用免疫组织化学及图像分析,Western blot观察大鼠纹状体GABARa受体的表达及年龄相关性变化。结果随着年龄的增加,大鼠纹状体GABAR阳性神经元及纤维明显增加,神经纤维排列紊乱,GABA免疫反应明显增强。结论老龄大鼠纹状体神经元GABAR的表达增加可能是一种有规律的增龄性变化。老年机体的某些抑制性精神症状可能与之有关。     

Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex

The ontogenesis of mRNAs coding for GABA and glycine receptors in the cerebral cortex of the rat was examined by extracting poly(A)+ mRNA from the brains of embryonic, postnatal or adult rats and injecting it into Xenopus oocytes. The ability of a messenger to express functional receptors was then assayed by measuring the membrane currents elicited by the agonists. The size of the GABA-induced current increased progressively with age, being undetectable in oocytes injected with mRNA from embryonic day 15 and reaching a maximum in oocytes injected with mRNA from postnatal day 30. In contrast, the glycine-induced response was negligible in oocytes injected with mRNA from the cerebral hemispheres of embryos 15 days old; it increased sharply to a maximum with newborn animals and then decreased with age to become very small with mRNA from adult cortex. GABA and glycine receptors induced by mRNA from the cerebral cortex of all ages are associated with chloride channels.     

Expression of Functional Bradykinin Receptors in Xenopus Oocytes

mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108-15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 microM), with an initial inward current (10-20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 microM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 microM). mRNA from both NG108-15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately five-fold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108-15 cells.     

Responses to GABA, glycine and beta-alanine induced in Xenopus oocytes by messenger RNA from chick and rat brain

Poly (A)+ messenger RNA (mRNA) was extracted from rat and chick brains, and injected into oocytes of Xenopus laevis. This led to the expression of receptors that evoked membrane currents in response to gamma-aminobutyric acid (GABA), glycine and beta-alanine. These currents all inverted at about the chloride equilibrium potential in the oocyte, and showed a marked rectification at negative potentials. Oocytes injected with mRNA from chick optic lobe gave large responses to GABA and beta-alanine, but small responses to glycine. In contrast, one fraction of mRNA from rat cerebral cortex (obtained by sucrose density gradient centrifugation) caused oocytes to develop sensitivity to GABA, glycine and beta-alanine, but very little to GABA. The pharmacological properties of the three amino acid responses also differed. Barbiturate and benzodiazepines potentiated the responses to GABA and beta-alanine, but not to glycine. Strychnine reduced the responses to glycine and beta-alanine, but not to GABA, whereas bicuculline reduced the responses to GABA and beta-alanine, but not to glycine. We conclude that different species of mRNA code for receptors to GABA and glycine, and possibly also for separate beta-alanine receptors.     

Changes in messenger RNAs coding for neurotransmitter receptors and voltage-operated channels in the developing rat cerebral cortex.

The ontogenetic development of poly(A)+ mRNAs coding for receptors to several neurotransmitters (kainate, glutamate, acetylcholine, and serotonin) and voltage-operated channels (sodium and calcium) was studied by isolating total poly(A)+ mRNA from the brains of rats at various developmental stages and injecting it into Xenopus oocytes. The oocytes translated the foreign mRNA and incorporated functional receptor/ion channel complexes into the cell membrane. Thus, recording of induced membrane currents in voltage-clamped oocytes gave a measure of the relative amounts of the different messengers. Responses induced by kainate, glutamate, acetylcholine, and serotonin all increased with age and reached a maximum in oocytes injected with mRNA from adult cortex. Messenger RNAs for the earliest ages examined, Embryonic Days 15 and 18, expressed little or no response to kainate, glutamate, or acetylcholine, while 50-70% of the adult response was reached by Postnatal Day 10. In contrast, the serotonin-induced response was already comparatively large (16% of the adult level) in oocytes injected with mRNA from Embryonic Day 15 brain and increased postnatally to adult levels. The expression of voltage-dependent sodium and calcium channels was small in oocytes injected with mRNA from embryonic animals and increased postnatally to reach a maximum in oocytes injected with mRNA from adult animals.     

Expression of Olfactory Receptors in Xenopus Oocytes

The rat olfactory epithelium and the amino acid-sensitive catfish olfactory system have been used as models to study the molecular mechanisms of olfactory transduction. Here we report the functional expression of rat and catfish olfactory receptors in Xenopus oocytes injected with mRNA isolated from the respective tissues. Application of odor ligands to injected oocytes, monitored by two-electrode voltage clamp, activates stimulus-dependent transmembrane currents that reverse direction at about the chloride equilibrium potential. The currents show characteristic secondary oscillations that are presumed to reflect underlying Ca2+ oscillations. Similar ligand-activated membrane currents induced in oocytes after injection of other mRNAs have been shown to be due to activation of endogenous Ca(2+)-activated chloride channels. In summary, our results demonstrate the usefulness of the Xenopus oocyte expression system for cloning and characterization of olfactory receptors in both fish and mammalian species.     

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.