昆虫神经生物学研究技术:用钴回填法对神经元染色

观察神经元的结构是昆虫神经生物学研究中的重要内容。钴盐回填法是对神经元染色的最常用的技术之一。作者借对与蝗虫后足胫节上 2个钟形感器有关的感觉神经元染色介绍了这一方法 ,包括材料的钴回填、着色、固定、脱水、澄清等。     

缺氧诱导体外培养大鼠海马神经元c-fos的表达及人重组白细胞介素-1β的影响

采用免疫组化方法,观察缺氧诱导体外培养大鼠海马神经元ｃ－ｆｏｓ的表达及人重组白细胞介素－１β（ｒｈＩＬ－１β）的影响。结果显示,缺氧后海马神经元中Ｆｏｓ染色阳性胞核的百分率随缺氧时间的延长而显著增加。图像分析的结果显示,缺氧后Ｆｏｓ染色阳性胞核的平均光密度亦随缺氧时间的延长而显著增加。经ｒｈＩＬ－１β孵育的神经元缺氧后Ｆｏｓ染色阳性胞核的百分率和Ｆｏｓ染色阳性胞核的平均光密度均明显低于对照组。本结果表明,缺氧能诱导体外培养海马神经元ｃ－ｆｏｓ表达,ｒｈＩＬ－１β能抑制缺氧神经元ｃ－ｆｏｓ表达。提示ｒｈＩＬ－１β对海马神经元缺氧损伤具有一定调控作用。     

缺氧诱导体外培养大鼠海马神经元c—fos的表达及人重组…

采用免疫组化方法,观察缺氧诱导体外培养大鼠海马神经元ｃ－ｆｏｓ的表达及人重组白细胞介素－１β（ｒｈｌＬ－１β）的影响,结果显示,缺氧后海马神经元中Ｆｏｓ染色了性胞核的百分率随缺氧时间的延长而显著增加。图像分析的结果显示,缺氧后Ｆｏｓ染色阳性胞核的平均光密度亦随缺氧时间的延长而显著增加。经ｒｈＩＬ－１β孵育的神经元缺氧后Ｆｏｓ染色阳性胞核的百分率和Ｆｏｓ染色阳性胞核的平均光密度均明显低于对照组。本结     

小清蛋白阳性神经元中 ErbB4 免疫荧光染色条件的优化北大核心

[目的]探究免疫荧光标记小清蛋白(parvalbumin,PV)阳性神经元中ErbB4的最佳实验条件。[方法]小鼠海马组织,PV和ErbB4做免疫荧光染色,通过激光共聚焦观察多聚甲醛后固定时间、抗原修复和一抗孵育时间对PV和ErbB4免疫荧光检测效果的影响。[结果]多聚甲醛后固定不同时间(8 h、48 h和240 h),一抗孵育40 h,结果显示,随着后固定时间的延长,PV和ErbB4的荧光强度和数量逐渐降低(P<0.05)。后固定240 h的脑组织,抗原修复后,PV和ErbB4荧光强度和数量显著升高(P<0.05)。一抗孵育12 h检测到PV和ErbB4荧光强度明显低于40 h(P<0.05),ErbB4阳性神经元的数量低于40 h(P<0.05)。[结论]PV阳性神经元中ErbB4的免疫荧光染色的最佳条件是后固定8 h、一抗孵育40 h。对于多聚甲醛后固定较久的组织,抗原修复能够提高其染色效果。     

小清蛋白阳性神经元中 ErbB4 免疫荧光染色条件的优化

[目的]探究免疫荧光标记小清蛋白(parvalbumin,PV)阳性神经元中ErbB4的最佳实验条件。[方法]小鼠海马组织,PV和ErbB4做免疫荧光染色,通过激光共聚焦观察多聚甲醛后固定时间、抗原修复和一抗孵育时间对PV和ErbB4免疫荧光检测效果的影响。[结果]多聚甲醛后固定不同时间(8 h、48 h和240 h),一抗孵育40 h,结果显示,随着后固定时间的延长,PV和ErbB4的荧光强度和数量逐渐降低(P0.05)。后固定240 h的脑组织,抗原修复后,PV和ErbB4荧光强度和数量显著升高(P0.05)。一抗孵育12 h检测到PV和ErbB4荧光强度明显低于40 h(P0.05),ErbB4阳性神经元的数量低于40 h(P0.05)。[结论]PV阳性神经元中ErbB4的免疫荧光染色的最佳条件是后固定8 h、一抗孵育40 h。对于多聚甲醛后固定较久的组织,抗原修复能够提高其染色效果。     

小清蛋白中ErbB4免疫荧光染色条件的优化

[目的]探究免疫荧光标记小清蛋白(parvalbumin,PV)阳性神经元中ErbB4的最佳实验条件。[方法]小鼠海马组织,PV和ErbB4做免疫荧光染色,通过激光共聚焦观察多聚甲醛后固定时间、抗原修复和一抗孵育时间对PV和ErbB4免疫荧光检测效果的影响。[结果]多聚甲醛后固定不同时间(8 h、48 h和240 h),一抗孵育40 h,结果显示,随着后固定时间的延长,PV和ErbB4的荧光强度和数量逐渐降低(P0.05)。后固定240 h的脑组织,抗原修复后,PV和ErbB4荧光强度和数量显著升高(P0.05)。一抗孵育12 h检测到PV和ErbB4荧光强度明显低于40 h(P0.05),ErbB4阳性神经元的数量低于40 h(P0.05)。[结论]PV阳性神经元中ErbB4的免疫荧光染色的最佳条件是后固定8 h、一抗孵育40 h。对于多聚甲醛后固定较久的组织,抗原修复能够提高其染色效果。     

Aβ25～35诱导大鼠记忆减退与星形胶质细胞及NOS阳性细胞变化

目的探讨Aβ25～35诱导模拟人类Alzheimer`s病(AD)的大鼠病理模型中星形胶质细胞变化与一氧化氮合酶神经元损伤引起的老年性记忆减退之间的关系.方法双侧海马内注射β-淀粉样多肽25～35片段(Aβ25～35)制作大鼠AD模型,注射一周后采用NOS组化染色、GFAP免疫组化染色及NOS组化和GFAP双重染色分析大鼠海马GFAP与NOS的表达.结果海马内注射Aβ25～35后出现海马星形胶质细胞增生、肥大、数目明显多于对照组(P<0.05),并出现一氧化氮阳性星形胶质细胞;海马一氧化氮神经元数量较对照组显著减少(P<0.05).结论 AD模型大鼠学习记忆功能低下与Aβ神经毒性导致NOS阳性神经元损伤、死亡直接相关,反应性星形胶质细胞参与Aβ导致NOS神经元细胞毒性损伤作用,间接导致学习记忆能力减退.     

Che-1通过抑制自噬减轻氧糖剥夺所致神经元损伤的研究

目的:研究Che-1蛋白对氧糖剥夺(Oxygen glucose deprivation, OGD)所致神经元损伤的保护作用及机制。方法:OGD处理神经元后,采用免疫荧光染色和免疫印迹法检测Che-1蛋白的表达;慢病毒转染神经元实现Che-1过表达,检测乳酸脱氢酶(Lactate dehydrogenase, LDH)释放量和流式细胞术检测神经元凋亡反映OGD所致神经元损伤程度,采用免疫荧光染色和免疫印迹法检测神经元自噬;使用自噬激动剂雷帕霉素(Rapamycin)处理神经元,并通过检测LDH释放量和流式细胞术研究自噬在Che-1保护作用中的作用。结果:免疫荧光结果显示,OGD后神经元Che-1蛋白表达明显增高;免疫印迹结果显示,OGD后6至48 h神经元Che-1蛋白表达明显增高;慢病毒转染过表达Che-1蛋白后,OGD所致神经元LDH释放量明显减低,且OGD所致神经元凋亡明显减少;过表达Che-1蛋白可显著减少OGD所致神经元Beclin1和LC3II的表达;自噬激动剂Rapamycin可逆转Che-1对OGD所致神经元损伤的保护作用。结论:过表达Che-1蛋白可通过抑制神经元自噬对OGD所致神经元损伤发挥保护作用。     

LPS诱导海马星形胶质细胞活化与活化的星形胶质细胞对海马神经元的影响

探讨脂多糖(Lipopolysaccharide,LPS)对长时间存活大鼠海马内星形胶质细胞的反应以及对神经元的影响。方法:本实验用10只健康成年雄性SD大鼠,海马CA3区注射LPS 10μ1．7和14d后,尼氏染色观察神经元的变化,免疫组织化学染色结合图像分析方法观察海马CA3区注射部位胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)、的表达变化。结果:脂多糖可促进海马星形胶质细胞的活化,但并不能引起海马区神经元的损伤。结论:星形胶质细胞在脑损伤后的脑内炎症反应起了一定的作用,但并不能引起神经元的损伤。     

扩散性抑制对脑缺血后海马迟发性神经元死亡的影响

目的为了研究阻断大鼠局灶性脑缺血诱导的扩散性抑制对同侧海马迟发性神经元死亡的影响。方法颈内动脉插线法制备大鼠大脑中动脉缺血再灌注模型,采用电生理学方法记录扩散性抑制波,尼氏染色和TUNEL染色检测海马迟发性神经元死亡;观察阻断局灶性脑缺血再灌注诱导的扩散性抑制对海马迟发性神经元死亡的影响。结果不给予SD阻断剂,大脑中动脉缺血模型有39%的动物出现海马迟发性神经元死亡;用MK-801阻断扩散性抑制后仅10%的动物出现海马迟发性神经元死亡,机率明显减小。结论局灶性脑缺血引起的海马迟发性神经元死亡可能与扩散性抑制由缺血区不断向远隔部位播散有关。     

Improved Methods for Cobalt Filling and Silver Intensification of Insect Motor Neurons

A Parafilm disk floating on saline and bearing a drop of cobalt solution in which the nerve stump is bathed provides a convenient and versatile method for in vitro or in vivo filling of neurons for cobalt sulfide staining. Silicone grease around the edge of the disk provides an effective seal around the nerve as it passes between the two solutions.

Using a modified developer, silver intensification of cobalt sulfide stained neurons may be done in the light at room temperature, and the time of optimum intensification may be observed under a dissecting microscope.     

Metal-catalyzed oxidation renders silver intensification selective. Applications for the histochemistry of diaminobenzidine and neurofibrillary changes

Physical developers can increase the visibility of end products of certain histochemical reactions, such as oxidative polymerization of diaminobenzidine and selective binding of complex silver iodide ions to Alzheimer's neurofibrillary changes. Unfortunately, this intensification by silver coating is generally superimposed on a nonspecific staining originating from the argyrophil III reaction, which also takes place when tissue sections are treated with physical developers. The present study reveals that the argyrophil III reaction can be suppressed when tissue sections are treated with certain metal ions and hydrogen peroxide before they are transferred to the physical developer. The selective intensification of Alzheimer's neurofibrillary changes requires a pre-treatment with lanthanum nitrate (10 mM/liter) and 3% hydrogen peroxide for 1 hr. The diaminobenzidine reaction can be selectively intensified when physical development is preceded by consecutive treatments with copper sulfate (10 mM/liter, pH 5, 10 min) and hydrogen peroxide (3%, pH 7, 10 min). In peroxidase histochemistry, this high-grade intensification may help to increase specificity and reduce the threshold of detectability in tracing neurons with horseradish peroxidase or in immunohistochemistry when the peroxidase-antiperoxidase method is used.     

Neuronal maps of the frontal ganglion of the cockroach, Periplaneta americana, prepared by heavy metal iontophoresis.

Neurons in whole mount preparations of the frontal ganglion (FG) of the cockroach, Periplaneta americana, were mapped with the aid of cobalt chloride staining and silver intensification techniques. Eighty-six neurons were counted in the FG after staining with reduced methylene blue. The cell size ranged between 20 to 35 microns in diameter. Of the somata located in the FG, 44 were found to contribute their fibers to the nervus recurrens, 26 to the right frontal commissure, 28 to the left frontal commissure, and 6 to the nervus connectivus. In addition, a few neurons presumably from the tritocerebral region also contribute their fibers in the formation of nervus connectivus. The present study has helped delineate the neuronal connections of the FG with the brain and neuroendocrine system (corpora cardiaca and corpora allata). This information will be useful in facilitating the positioning of microelectrodes in our future electrophysiological experiments.     

Evaluation of the total number of myenteric neurons in the developing chicken gut using cuprolinic blue histochemical staining and neurofilament immunocytochemistry

The aim of this study was to find an improved method with which to stain the entire population of myenteric neurons in the different segments of the developing chicken intestine. Histochemical staining with cuprolinic blue (quinolinic phthalocyanine) and immunostaining against neurofilament (NF) were performed on whole mounts prepared from intestinal segments of embryonic (day 19 of incubation) and hatched (1, 2, 4 and 7 days after hatching) chickens. Double labelling was performed to evaluate to what extent the two markers visualise the same nerve cell population. Cuprolinic blue stained neuronal somata highly selectively, whereas processes and glia cells were poorly labelled. The cuprolinic blue-positive neurons were uniform in shape. NF immunostaining revealed a morphologically highly variable neuron population. Double labelling with cuprolinic blue and NF resulted in an intensification of both stainings, allowing an accurate morphological classification of NF-stained myenteric neurons. Data obtained from the counting of cuprolinic blue-positive neurons were subjected to two-way ANOVA and the Tukey probe. The densities of ganglia and neurons were found to decrease, and the mean number of neurons per myenteric ganglion to increase, with different dynamics along the longitudinal axis of the gut during the examined time span. The variances in the number of NF-positive neurons were not homogeneous, and the data were therefore not suitable for ANOVA. Accordingly, only semiquantitative conclusions could be drawn.     

A Light Insensitive Method for Contrast Enhancement of Insect Neurons Filled with a Cobalt-Lysine Complex

Modified protocols for cobalt-filling and silver intensification of neurons in the larval and adult stages of the moth, Manduca sexta, have led to improved neuronal visualization and minimal background staining. In particular, long distance projecting multisegmental in-terneurons. originating in the pterothoracic or terminal abdominal ganglion, were best visualized when a cobalt:lysine complex was used to fill hemi-connectives for several days at 4 C. Ganglia closest to the placement of tracer, which became flooded with cobalt:lysine during the filling period. were removed from the insect. This step eliminated the artifactual filling of neurons that may have taken up the tracer from such pooled regions. This led to a more accurate assessment of whether a multisegmental interneuron projected through the full length of nerve cord to the original site of tracer placement. The protocol for light insensitive silver intensification of cobalt-filled neurons was modified to include an important pH adjustment. NaOH was used to alter the pH of the protective colloid, sodium tungstate, to 10.4 or greater in solution. Especially in larvae. our techniques produced intensely stained cobalt-filled neurons within ganglia that remained transparent and relatively free of nonspecific silver deposition.     

Intensification of Osmium Staining by p-Phenylenediamine: Paraffin and Epon Embedding; Lipid Granules in Renal Medulla

Tissue which had been fixed in 4% glutaraldehyde and postfixed in 2% OsO₄ was subsequently treated with p-phenylenediamine, either in the block prior to embedding in paraffin or Epon or, in the case of Epon-embedded material, after sectioning for light microscopy. The p-phenylenediamine was best used as a 0.8-1% solution in 70% alcohol. The p-phenylenediamine caused a very considerable intensification of staining of any cell components; this intensification of staining was particularly marked in the case of the lipid granules of renal medulla.     

Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry

Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. We now report a method for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers, and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over cobalt ion intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.     

A rapid and simple silver enhancement procedure for ultrastructural localization of the retrograde tracer WGAapoHRP-Au and its use in double-label studies with post-embedding immunocytochemistry

WGAapoHRP-Au is a colloidal gold conjugate of wheat germ agglutinin (WGA) coupled to enzymatically inactive (apo) horseradish peroxidase (HRP). This protein-gold complex has proven very useful for retrograde tracing studies in the nervous system (Basbaum and Menétrey: J Comp Neurol 261:306, 1987). To identify retrogradely labeled cells, the colloidal gold is made visible by silver intensification. As the tracer has no HRP enzymatic activity, it can be combined with HRP-based procedures (or with fluorescent methods) in a variety of multiple-label studies. Standard silver intensification procedures, however, are run at low pH and therefore are incompatible with good EM preservation; moreover, osmication of the tissue oxidizes the silver product, which is then lost in subsequent dehydration steps. This report describes a rapid and simple commercially available silver intensification procedure. The procedure is run at neutral pH and can be performed after osmication. The tracer is readily detected at the EM level and tissue preservation is excellent. This report also demonstrates how sections containing retrogradely labeled neurons can be stained with a post-embedding immunocytochemical method so that the transmitter content of synaptic inputs to these neurons can be identified.     

A Light Insensitive Method for Contrast Enhancement of Insect Neurons Filled with a Cobalt-Lysine Complex

Modified protocols for cobalt-filling and silver intensification of neurons in the larval and adult stages of the moth, Manduca sexta, have led to improved neuronal visualization and minimal background staining. In particular, long distance projecting multisegmental in-terneurons. originating in the pterothoracic or terminal abdominal ganglion, were best visualized when a cobalt:lysine complex was used to fill hemi-connectives for several days at 4 C. Ganglia closest to the placement of tracer, which became flooded with cobalt:lysine during the filling period. were removed from the insect. This step eliminated the artifactual filling of neurons that may have taken up the tracer from such pooled regions. This led to a more accurate assessment of whether a multisegmental interneuron projected through the full length of nerve cord to the original site of tracer placement. The protocol for light insensitive silver intensification of cobalt-filled neurons was modified to include an important pH adjustment. NaOH was used to alter the pH of the protective colloid, sodium tungstate, to 10.4 or greater in solution. Especially in larvae. our techniques produced intensely stained cobalt-filled neurons within ganglia that remained transparent and relatively free of nonspecific silver deposition.     

Trypan blue in vivo stains nigral dopaminergic neurons killed by 6-hydroxydopamine

Dopaminergic neurons in the substantia nigra killed by 6-hydroxydopamine were stained in vivo by intracerebral injections of trypan blue. Such staining appeared specific for dead neurons, although a proportion of these retained the ability to stain with Nissl dyes for at least 2 days. Neurons retained trypan blue in vivo for periods of up to 9 days. Trypan blue staining of some neurons outside the substantia nigra demonstrated the use of this dye in determining the degree of non-specific toxicity of 6-hydroxydopamine. Twenty-four hours after infusion of trypan blue almost no background staining was present and individually stained neurons were clearly visible. Thus the use of trypan blue may have a general application as a sensitive method for estimating discrete areas of toxin-induced neuronal death, and for estimating the degree of specificity of a toxin.