秋水仙素对大鼠前脑生长抑素mRNA表达的影响

本实验用地高辛标记生长抑素（ＳＯＭ）反意。ＲＮＡ探针原位杂交组织化学研究了秋水仙素对大鼠前脑ＳＯＭｍＲＮＡ表达的影响。结果表明秋水仙素对前脑各核区ＳＯＭｍＲＮＡ的表达有明显的影响。结果表明秋水仙素对前脑各核区ＳＤＭｍＲＮＡ的表达有明显的核区特异性：大脑新皮质各区,隔核和脚内核中ＳＯＭｍＲＮＡ阳性神经元数目及含量增加;海马复合体和下丘脑室周核和弓状核中ＳＯＭｍＲＮＡ阳性神经元数目及含量下降;嗅脑,尾壳核和丘脑等核区的则无明显变化、秋水仙素对ＳＯＭｍＲＮＡ表达的核区特异性对正确分析秋水仙素条件下获得的神经肽或神经递质的定位资料具有重要的指导意义。     

大鼠脊髓内生长抑素mRNA原位杂交的研究

采用地高辛标记生长抑素反意ＲＮＡ探针经原位杂交和显色后,光学显微镜下观察生长抑素ｍＲＮＡ在大鼠脊髓内的定位。结果显示：脊髓内含有大量呈紫蓝色的生长抑素ｍＲＮＡ阳性神经细胞,岍性磷酸酶反应产生     

青霉素诱发癫痫及耳穴电针后大鼠前脑生长抑素mRNA的变化

应用地高辛标记的生长抑素（ＳＳ）ＲＮＡ探针原位杂交法观察大鼠在青霉素诱发的癫痫及耳穴电针抑制癫痫发作后有关的脑区中ＳＳｍＲＮＡ含量的变化。结果显示,青霉素致痫后２４小时梨状皮质、额叶皮质、扣带回、隔外侧核、杏仁基底核海马ＣＡ１－ＣＡ４区和齿状回颗粒细胞层、多形层等脑区ＳＳｍＲＮＡ的表达显著增加,与正常对照组比较Ｐ＜００５。耳穴电针抑痫（８０１００ＨＺ,６ｍＡ）后额叶皮质、杏仁基底核、海马齿状回、前梨状皮质ＳＳｍＲＮＡ的表达明显减少,Ｐ＜００５。提示前脑结核中的生长抑素与青霉素致痫和耳针抑痫有关。     

地高辛标记反意RNA探针检测脑组织切片生长抑素mRNA

本实验采用含大鼠生长抑素基因的ｐＳＰ６５ｃＤＮＡ质位通过转化至噬菌体内大量扩增,经提取,纯化后,用限制性内切酶进行酶切使质枝线性化,并将其作为模板,用地高辛（ＤｉｇｏｘｉｇｅｎｉｎＤｉｇ）作为标记物,体外转录合成生长抑素反意ＲＮＡ（ｃＲＮＡ）探针。实验动物选用ｗｉｓｔａｒ新生大鼠。冰冻切片,端、间脑切片经杂交前用Ｄｉｇ－ＵＴＰ标记的ｃＲＮＡ探针杂交,杂交后用抗Ｄｉｇ－碱性磷酸酶复合物进行酶联免疫反应。Ｘ－磷酸盐－ＮＢＴ显色。结果显示新生大鼠脑内生长抑素ｍＲＮＡ神经元着紫蓝色。杂交反应物集中于核周的胞浆及短小的突起内。胞核不着色。胞体轮廓清晰,周围背底浅淡。结果表明Ｄｉｇ标记ｃＲＮＡ探针不仅具备非同位素标记探针的优点而且能快速和准确检测组织细胞内ｍＲＮＡ的表达。     

大鼠鼻粘膜肽能神经末梢分布的研究

用免疫组化技术（ＡＢＣ法）系统研究了大鼠鼻粘膜９种肽能神经末梢分布的特征,这９种神经肽分别是Ｐ物质（ｓｕｂｓｔａｎｃｅＰ,ＳＰ）,神经激肽Ａ（ｎｅｕｒｏｋｉｎｉｎＡ,ＮＫＡ）,神经激肽Ｂ（ｎｅｕｒｏｋｉｎｉｎＢ,ＮＫＢ）,降钙素基因相关肽（ｃａｌｃｉｔｏｎｉｎｇｅｎｅ－ｒｅｌａｔｅｄｐｅｐｔｉｄｅ,ＣＧＲＰ）,血管活性肠多肽（ｖａｓｏａｃｔｉｖｅｉｎｔｅｓｔｉｎａｌｐｏｌｙｐｅｐｔｉｄｅ,ＶＩＰ）,神经肽Ｙ（ｎｅｕｒｏｐｅｐｔｉｄｅＹ,ＮＰＹ）,甘丙肽（ｇａｌａｎｉｎ,ＧＡＬ）,生长抑素（ｓｏｍａｔｏｓｔａｔｉｎ,ＳＯＭ）及神经降压素（ｎｅｕｒｏｔｅｎｓｉｎ,ＮＴ）,同时选择与鼻粘膜神经肽（ＮＰ）作用密切相关的三叉神经节（ＴＧ）细胞进行上述ＮＰ的定位。用重组ＰＳＰ６５质粒（４００ＳＯＭｃＤＮＡ）制备ＳＯＭｍＲＮＡ单链探针,以地高辛精标记,在鼻粘膜及ＴＧ细胞进行ＳＯＭｍＲＮＡ的原位杂交组化研究。结果提示大鼠鼻粘膜有丰富的肽能神经末梢;ＴＧ细胞含有多种ＮＰ并且可以合成ＳＯＭ。该研究结果对重新认识鼻粘膜神经分布规律有一定意义。     

胃扩张刺激对大鼠大脑皮层及海马CCK mRNA表达的影响

胆囊收缩素（ｃｈｏｌｅｃｙｓｔｏｋｉｎｉｎ,ＣＣＫ）是脑肠肽中的一种,被认为是饱因子。本实验采用以地高辛标记的ＣＣＫ ｃＤＮＡ为探针的原位杂交和半定量ＲＴ－ＰＣＲ技术。用水囊扩张胃作为对胃壁的机械刺激模拟食物对胃的充盈作用,观察大鼠大脑皮层和海马内含ＣＣＫ神经元ＣＣＫ ｍＲＮＡ表达的变化情况。     

兴奋性氨基酸加强高原低氧大鼠下丘脑前生长抑素原mRNA的表达

目的和方法：应用大鼠高原低氧模型及原位杂交技术和氨基酸测定法,研究下丘脑前生长抑素原（ＰＰＳ）ｍＲＮＡ表达和谷氨酸（Ｇｌｕ）、天门冬氨酸（Ａｓｐ）含量的变化。结果：高原低氧组大鼠下丘脑Ｇｌｕ和Ａｓｐ的含量明显增多,室周核、室旁核、弓状核ＰＰＳ－ｍＲＮＡ阳性神经元数目显著增加;而ＮＭＤＡ受体拮抗抗剂氯铵酮,虽然对Ｇｌｕ和Ａｓｐ含量无明显影响,但可使高原低氧大鼠下丘脑ＰＰＳ－ｍＲＮＡ阳性神经元数目减少     

地高辛标记寡核苷酸探针检测大鼠胰岛素mRNA

本实验采用地高辛标记前胰岛素寡核苷酸探针原位杂交组织化学技术,研究了Ｗｉｓｔａｒ大鼠胰腺组织胰岛素基因的表达。实验用Ｗｉｓｔａｒ大鼠５只,胰腺经４％多聚甲醛灌注固定,并在同一固定液中后固定２４ｈ,常规石蜡包埋切片。结果表明,经前胰岛素寡核苷酸探针杂交的胰腺切片中,胰岛Ｂ细胞呈蓝色。杂交反应物位于Ｂ细胞的细胞质和部分核仁中,胞核无色。胰岛其它细胞及胰腺外分泌部腺泡细胞无杂交反应物。对照切片中均无阳性信号出现。结果表明地高辛标记寡核苷酸探针不仅具备非同位素标记探针的优点而且能精确检测特异性ｍＲＮＡ的表达。本研究方法操作便利,可靠精确,重复性好。     

人颌下腺cDNA文库的构建

本研究从新鲜的人颌下腺组织中提取总ＲＮＡ,从中分离出带ｐｏｌｙ（Ａ）尾的信使ＲＮＡ［ｐｏｌｙ（Ａ）〕ｍＲＮＡ］,并合成带ＮｏｔＩ位点的双链ｃＤＮＡ后,接上ＥｏｃＲＩ接头定向插入λｇｔ１１表达载体,经体外包装后转染Ｙ１０９０宿主菌,遂构建成人颌下腺ｃＤＮＡ文库。该文库有４．１×１０５个噬菌体,重组率为１００％,可供寡核苷酸探针和单克隆抗体两种方法筛选目的基因。     

人胎儿胸腺内bc1－2基因表达与分布

采用免疫酶组织化学与地高辛标记的单链ｃＤＮＡ探针原位杂交技术,研究了抗凋亡基因ｂｃｌ－２在人胎儿胸腺组织中的表达与分布。结果ｂｃｌ－２ｍＲＮＡ及其蛋白均优势定位于髓质区。提示胸腺中ｂｃｌ－２基因表达调控发生在转录水平;ｂｃｌ－２基因介导的细胞凋亡状态可能参与Ｔ淋巴细胞的成熟过程。     

The use ofin situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain

1. The application of in situ hybridization histochemistry to the study of neuropeptide gene expression in human brain postmortem tissues is reviewed. We focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia. 32P-labeled oligonucleotides were used as hybridization probes. 2. Autoradiography combined with computerized image analysis was used to visualize and quantify the hybridization signal. 3. Several criteria were considered in order to ascertain the specificity of the signal, including Northern analysis, use of heterologous probes, competition assays, and thermal stability of the hybrids. 4. In control human striatum high levels of hybridization signal were observed for somatostatin, neuropeptide Y, and preproenkephalin A mRNAs. In contrast, no detectable signal was observed with the cholecystokinin, arginine-vasopressin, and oxytocin probes in this area. In the hypothalamus high levels of oxytocin and arginine-vasopressin mRNAs were visualized in several nuclei. Preproenkephalin A and somatostatin mRNAs were also observed in this region, while cholecystokinin mRNA was not detected. 5. No significant correlations were found between the density of the hybridization signal and parameters such as postmortem delay, age, and gender in the population studied. 6. Finally, alterations of mRNA levels for some of these peptides were found in Parkinson's disease and Huntington's chorea striatal tissues. 7. These results show that in situ hybridization histochemistry can be used to examine at the microscopic level neuropeptide gene expression in postmortem materials.     

Simultaneous detection of neuropeptides and messenger RNA in the magnocellular hypothalamo-neurohypophysial system by a combination of non-radioactive in situ hybridization histochemistry and immunohistochemistry

A protocol was developed combining non-radioactive in situ hybridization histochemistry with enzyme based immunohistochemistry, detect the expression of mRNA in phenotypically defined neurons. Freefloating brain sections were hybridized with the oligonucleotide probes which have been 3-end labelled with biotin-11-dUTP. The hybridized probe was visualized by a combined avidin-biotin bridge method, anti-avidin immunohistochemistry, and horseradish peroxidase detection using diaminobenzidine as a substrate. The in situ hybridization step yielded a very stable reaction product enabling subsequent immunohistochemical reactions using horseradish peroxidase and benzidine dihydrochloride as a chromogen. Magnocellular neurons of the hypothalamo-neurophypophysial system synthesize either vasopressin or oxytocin; water deprivation and chronic saline ingestion are potent stimuli for the expression of both of the genes encoding these neuropeptides. A number of other neuropeptides with putative transmitter action are synthesized in magnocellular neurons during such stimulation. Experiments were performed to explore whether neuropeptide Y immunoreactivity is present within magnocellular vasopressin mRNA-expressing neurons of the hypothalamo-neurophypophysial system. The results clearly demonstrated that neuropeptide Y-immunoreactive elements were present within a number of magnocellular vasopressin mRNA-containing cells. In addition, immunohistochemical detection of the neuropeptides ocytocin and cholecystokinin was carried out on sections hybridized non-radioactively for vasopressin; as expected vasopressin mRNA did not co-exist with cholecystokinin, whereas a few oxytocin immunoreactive neurons in osmotically stimulated animals also contained vasopressin mRNA. The developed method makes possible the immunohistochemical detection of intracellular antigens with concomitant detection of intracellular mRNA.     

Characterization of hypothalamic neurons expressing a neuropeptide receptor, GALR2, using combined in situ hybridization-immunohistochemistry.

Our laboratory is interested in characterizing the neurotransmitter and hormonal phenotype of neurons in the rat hypothalamus expressing novel neuropeptide receptors of the neuropeptide Y and galanin families. In this review, we describe a technique combining nonradioactive in situ hybridization to detect mRNA and fluorescence immunohistochemistry to detect protein antigens. We examined paraffin sections of rat hypothalamus using confocal microscopy to determine whether mRNA for the galanin receptor, GALR2, was colocalized at the cellular level of resolution with somatostatin or tyrosine hydroxylase immunoreactivity. We found that many neurons in the hypothalamus expressed both GALR2 mRNA and either somatostatin or tyrosine hydroxylase immunoreactivity. The simultaneous detection of mRNA and protein immunoreactivity in individual neurons using the confocal microscope for visualization is an excellent tool for the analysis of newly characterized genes in the central nervous system.     

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.     

Topography and ontogeny of the neurons expressing vasopressin,oxytocin, and somatostatin genes in the rat brain: An analysis using radioactive and biotinylated oligonucleotides

1. The use of radioactive and biotinylated oligonucleotide probes has been optimized to detect and analyze by in situ hybridization, neurons expressing neuropeptide genes (vasopressin, oxytocin, somatostatin). 2. In situ hybridization was performed on cryostat-cut sections obtained from tissues perfused with 1% formaldehyde. Radioactive probes were labeled by tailing with 35S-dATP and revealed with autoradiography. Biotinylated probes were obtained either by the incorporation of 11-biotin dUTP or by the addition of biotinylated nucleotides to the oligonucleotide during its synthesis. Biotin was revealed with streptavidin alkaline phosphatase and the appropriate substrate. 3. In the adult rat brain, radioactive and biotinylated probes revealed peptidergic neurons. The biotinylated probes provided an optimal cellular and subcellular resolution with a sensitivity similar to that observed with radioactive probes. Staining was selectively restricted to the cytoplasm and to the proximal part of processes. 4. Biotinylated vasopressin probes with 10 biotins added demonstrated magnocellular neurons and parvocellular neurons in the suprachiasmatic nucleus and the bed nucleus stria terminalis. 5. Vasopressin gene expression was studied during ontogeny in the rat fetus and neonate. Vasopressin mRNA was first detectable at gestational day 16 in the supraoptic nucleus in neurons of neuroblastic appearance. An aspect similar to the one present in adult was found at gestational day 19 in magnocellular neurons and at day 3 postnatal in parvocellular neurons. 6. The results confirm that radioactive oligonucleotide probes are efficient tools to investigate neuropeptide gene expression by in situ hybridization and demonstrate that biotinylated oligonucleotides are very efficient and provide a much higher resolution than radioactive probes with a reasonable sensitivity.     

Characterization of Hypothalamic Neurons Expressing a Neuropeptide Receptor, GALR2, Using Combined in Situ Hybridization–Immunohistochemistry

Our laboratory is interested in characterizing the neurotransmitter and hormonal phenotype of neurons in the rat hypothalamus expressing novel neuropeptide receptors of the neuropeptide Y and galanin families. In this review, we describe a technique combining nonradioactive in situ hybridization to detect mRNA and fluorescence immunohistochemistry to detect protein antigens. We examined paraffin sections of rat hypothalamus using confocal microscopy to determine whether mRNA for the galanin receptor, GALR2, was colocalized at the cellular level of resolution with somatostatin or tyrosine hydroxylase immunoreactivity. We found that many neurons in the hypothalamus expressed both GALR2 mRNA and either somatostatin or tyrosine hydroxylase immunoreactivity. The simultaneous detection of mRNA and protein immunoreactivity in individual neurons using the confocal microscope for visualization is an excellent tool for the analysis of newly characterized genes in the central nervous system.