小块神经组织整体孵育ABC法的探讨

本文介绍一种小块神经组织整体孵育的免疫细胞化学ABC法。组织块不切片而整体入一抗、二抗以及卵白素—生物素—过氧化物酶复合液中孵育,二氨基联苯胺—双氧水呈色,锇酸后固定,Epon包埋之后再行切片作光镜和电镜观察。依此程序以及改进的参数,对海兔侧神经节感觉神经元簇(平均直径1.0±0.2 mm)整体进行了5-羟色胺免疫组化反应。结果表明,侧神经节感觉神经元簇的浅、中、深层,光镜下均可见大量念珠状5-羟色胺免疫阳性反应纤维及其膨体。电镜下5-羟色胺免疫阳性反应物电子密度高,容易辨认。非免疫阳性反应的侧神经节感觉神经元的亚细胞结构亦清晰可见。此外,该法仍保持原法非特异性背底浅、免疫阳性反应物与背底对比明显以及保存时间长等特点。对于因组织小而不便先切片再进行免疫组化程序的,可试用此法。对1毫米左右厚的薄组织也可使用本法。     

组织化学染色与免疫电镜双标记方法在检测大鼠脑干前包钦格复合体细胞色素氧化酶活性中的应用北大核心CSCD

目的研究脑干前包钦格复合体(pre-B9t C)细胞色素氧化酶(COX)的活性变化。方法采用COX的组织化学染色与pre-B9t C的标记物神经激肽1受体(NK1R)纳米金-银加强免疫组织化学染色双标法,进行pre-B9t C特定神经核团尤其是不同亚细胞结构的COX活性检测,并进行COX活性的半定量分析。结果光镜下NK1R免疫反应性(NK1R-ir)产物主要分布于pre-B9t C神经细胞膜,清晰勾勒神经元轮廓,COX组织化学染色呈棕色,弥漫表达于NK1R-ir神经元胞质及突起内。电镜下NK1R-ir金颗粒主要分布于pre-B9t C神经元胞体和树突膜内表面,胞质内也可见金颗粒标记。pre-B9t C神经元不同亚细胞结构(胞体、轴突终末、树突)线粒体的形状和分布均有不同,在胞体和轴突终末线粒体多为圆形和椭圆形,在树突则以长杆状线粒体多见,胞体线粒体多为管泡状嵴,而板层嵴线粒体以轴突终末和树突多见。轴突终末内线粒体多聚集分布,树突内线粒体多近胞膜散在分布,但在突触部位,线粒体分布密集,且局部膨大接近突触后膜,这与突触的信息传递功能需要大量ATP产生密切相关。COX活性反应产物分布于线粒体嵴上,线粒体嵴电子密度不同表明COX活性不同。轴突终末和树突线粒体COX活性明显高于胞体。结论 pre-B9t C神经元不同亚细胞结构能量代谢不同,其中轴突终末和树突线粒体COX活性明显高于胞体,尤其突触部位线粒体分布密集,且表达高COX活性。将COX组织化学技术和免疫电镜技术相结合,为检测区域性COX活性,尤其是区域内不同亚细胞结构的COX活性提供了新方法。     

大鼠下边缘皮质-伏隔核投射神经元胞体受对称性突触支配

目的 :揭示向伏隔核壳部 (nucleusaccumbensshell,AcbSh)投射的下边缘皮质 (infralimbiccortex ,IL)神经元胞体上的突触体系 ,增加该神经通路中的突触学资料。方法 :注射HRP到大鼠的AcbSh ;以TMB法显示IL中被HRP逆行标记的神经元 ;光镜及电镜观察IL中标记神经元的形态、分布及胞体部的突触结构。结果 :含有HRP/TMB反应产物的神经元散在分布于IL中。神经元胞体呈三角形或圆锥体形锥体细胞的基本特征 ,直径约为 1 5μm× 2 5 μm。电镜可见神经元胞体仅接收 3～ 5个对称性突触。 结论 :向AcbSh投射的IL神经元通过对称性突触接受抑制性神经信息支配 ,提示在整个摄食行为神经通路中 ,IL支配AcbSh ,而自身又受抑制性控制。     

大鼠纹状体parvalbumin阳性中间神经元的超微结构

目的:研究纹状体parvalbumin (Parv)阳性中间神经元在神经通路上的突触连接.方法:利用免疫组织化学和神经示踪方法标记SD大鼠纹状体Parv及其相关神经元,光镜和电镜观察阳性神经元的结构和位置关系.结果:Parv阳性中间神经元中等大小,散在分布于纹状体,以背外侧居多.光镜免疫双标记显示Parv阳性中间神经元与皮质、丘脑和中脑黑质的传入轴突终末形成明显的形态位置上的邻近关系,其轴突终末则与纹状体不同类型投射神经元在光镜下也形成邻近关系.Parv阳性中间神经元的免疫电镜观察显示阳性产物主要游离于胞体、树突和轴突的胞质内.Parv阳性中间神经元的胞体和树突均接受大量的非对称型突触传入.Parv阳性轴突终末平均大小为(0.62±0.28)μm,可见其与纹状体神经元的树突、胞体和树突棘形成对称型突触,其中与树突形成的突触占69.64%,与胞体和树突棘形成的突触分别为26.78%和3.58%.结论:纹状体Parv阳性中间神经元形态学上与皮质、丘脑、黑质以及纹状体投射神经元形成突触连接,提示其可能在调节纹状体信息传入和输出过程中具有重要作用.     

钨酸钠做为稳定剂的新的高灵敏HRP-TMB法——Ⅰ、光镜研究

本文通过试管实验和在尾核—黑质投射模型上的比较研究,首次提出了一种以钨酸钠作为稳定剂的新的TMB成色法(TMB-ST法)用于HRP神经束路追踪术。与现确灵敏度最高的Mesulam的TMB法相比,TMB-ST法具有灵敏度更高,产物稳定,耐酒精、耐pH小于6.3的环境,无针状结晶和点状结晶,标记结构形态细腻,组织切片不皱缩等优点。此外,本文还对稳定剂在HRP-TMB组化反应中的作用作了较全面的讨论。     

正中隆起内NT能神经元的SP能神经支配的免疫电镜双标研究

应用包埋前免疫电镜PAP双标技术,对大鼠下丘脑正中隆起内的神经紧张素(neurotensin,NT)和P物质(substance P,SP)的分布进行了超微结构研究。先用DAB法显示SP免疫反应,然后用钼酸-TMB法显示NT免疫反应,再经DAB-氯化钴稳定后作免疫电镜包埋。电镜观察发现:在正中隆起内SP免疫反应呈电子密度高的颗粒状或絮状沉淀,广泛分布于轴突内的小清亮囊泡周围和基质内;NT免疫反应产物则为电子密度高的针状或块状,散在分布于胞体、树突和轴突内。含SP的轴突可接受免疫反应阴性轴突的传入性突触,也可与阴性树突形成传出性轴-树突触;含NT的树突和胞体均可接受阴性轴突的传入性突触。此外,SP阳性轴突末梢还可与NT阳性神经元的树突棘形成对称性轴-棘突触及与NT阳性轴突形成对称性轴-轴突触。实验结果表明:大鼠正中隆起内的NT能神经元接受SP能神经的支配,为下丘脑神经内分泌的突触调控提供了新的超微结构依据。     

包埋前免疫电镜双标技术在神经解剖学研究中的应用

目的　在超微结构水平观察两种神经递质在纤维终末内的共存或一种神经递质与其相应受体之间的关系。　方法　包埋前免疫电镜双重标记技术———酶标法和免疫金 银标记法相结合的方法。　结果　在免疫反应双重标记的纹状体切片上 ,电镜下观察到大量的SP样 (过氧化物酶免疫反应产物 )阳性终末和SP受体 (SPR ,免疫金 银标记颗粒 )样阳性神经元的胞体和树突 ,同时可见部分SP样阳性轴突终末分别与SPR样阳性神经元的胞体或树突形成对称性轴 体或轴 树突触联系。而在三叉神经脊束核尾侧亚核切片上 ,电镜下可观察到大量的两种囊泡膜谷氨酸转运体 ,即DNPI样 (过氧化物酶免疫反应产物 )和VGluT1样 (免疫金 银标记颗粒 )阳性轴突终末 ,同时还观察到DNPI样和VGluT1样双标的轴突终末与阴性树突形成非对称性突触。　结论　包埋前免疫电镜双重标记技术敏感性较高 ,组织的抗原性保存好 ,特别是在神经解剖学研究中 ,用于研究两种神经递质在同一个细胞或终末内的共存或分析神经递质与其相应受体之间的联系中有独到之处。     

大鼠室旁核内催产素免疫阳性细胞树突棘样结构在脱水状态下的改变

树突棘，室旁核，催产素，脱水，免疫组化，大鼠下丘脑内催产素大细胞神经分泌细胞与水盐代谢的调节有关，并伴有细胞形态的改变如细胞体积的增大及细胞膜与细胞膜间接触的增加和多突触的形成等。本文用免疫组化法发现在脱水状态下室旁核的催产素免疫阳性神经元上树突棘样结构有所增加。光镜观察并计算室旁核前大细胞亚核、内侧大细胞亚核及后大细胞亚核中含树突棘样结构的催产素神经元百分数变化，显示正常大鼠室旁核神经元树突棘样结构多位于树突干上，少数在胞体上。前大细胞亚核含树突棘样结构的细胞百分数为２１．２７％，内侧大细胞亚核为３０．２２％，后大细胞亚核为２０．２２％。轻度脱水大鼠室旁核含树突棘样结构细胞百分数显著增加（前大细胞亚核：２８．６５％，Ｐ＜０．０５；内侧大细胞亚核：３５．５３％，Ｐ＞０．０５；后大细胞亚核：３４．７８％，Ｐ＜０．０１）。细胞上树突棘样结构数也增加，其体积略为增大。重度脱水大鼠室旁核中树突棘样结构细胞百分数增加较小，仅后大细胞亚核内有显著变化（２７．１３％，Ｐ＜０．０５）。不同程度脱水组之间无显著变化。结果说明脱水可引起神经元膜结构的改变。树突棘样结构数变化是否与新突触的形成有直接关系？尚待进一步证实。     

大鼠外侧膝状体背核神经元超微结构定性与定量观察——HRP—TMB—ST电镜法

本文首次用HRP逆行标记并结合新的HRP电镜法(HRP—TMB—ST电镜法),对大鼠外侧膝状体背核(DLG)中间神经元与中继神经元的超微结构进行了定性与定量观察。结果表明,中间神经元胞体较中继神经元小,但胞核的体密度较大而多有较深的皱折,其细胞器相对稀疏但线粒体的体密度与中继神经元无差别。高尔基氏器数量在中间神经元明显比中继神经元少,可作为区别两类神经元的指标之一。     

三叉神经躯体传入成分向孤束核投射的间接途径—HRP跨节和逆行追踪技术的光镜和电镜研究

用HRP跨节及逆行追踪的光镜和电镜技术探索了三叉神经(TGN)躯体性传入成分向孤束核(NTS)的间接投射通路。HRP微量注射于闩平面的NTS后,逆行标记细胞出现于与TGN尾侧亚核背外部邻接的脊束内的间质核。同时,此核又是TGN的舌神经和眶下神经初级传入的投射部位。电镜观察发现,HRP标记的舌神经和眶下神经初级传入终末与间质核内神经元的树突形成较多的突触连接。有部分标记终末与向NTS投射的神经元的逆行标记树突和树突棘在间质核内形成突触连接。另外,本文研究证明,间质核和TGN尾侧亚核浅层的细胞构筑基本相同,且有些间质核的细胞团系由尾侧亚核浅层结构向脊束内突入而形成,故可将两者视为一个统一体。据此,本文作者认为,TGN和NTS之间存在着经间质核中继的两级神经元传入通路,经此通路传入的冲动应属于躯体性质。推测由两级神经元组成的这个通路可能是针刺TGN领域体表穴位调控孤束核内脏机能的结构基础。     

Immunohistochemical localization of creatine kinase BB-isoenzyme and tubulin in gerbil brain

Cellular distribution of creatine kinase BB-isoenzyme and tubulin was investigated immunohistochemically with tissue sections from various areas of the gerbil brain. Both the reaction for creatine kinase BB-isoenzyme and tubulin visualized nerve cell bodies, dendrites and axons. While the reaction for tubulin was similar between nerve cell bodies and their dendrites, the reaction for creatine kinase BB-isoenzyme tended to be more intense in dendrites. While the reaction for tubulin could be seen in nerve cell bodies of different sizes, the reaction for creatine kinase BB-isoenzyme was more intense in larger neurons. Both the reaction for creatine kinase BB-isoenzyme and tubulin visualized astrocytic cytoplasm but only in the corpus callosum and in the white matter of limited areas. Visualization of dendrites to their peripheries with the reaction for creatine kinase BB-isoenzyme may indicate participation of this isoenzyme in maintainance of high energy-phosphates locally within dendrites. A combination of the immunohistochemical procedure for creatine kinase BB-isoenzyme and tubulin may be very useful for demonstration of various pathophysiological states of the central nervous system which affect dendrites.     

大鼠中脑导水管周围灰质腹外侧区5-羟色胺样、P物质样和亮氨酸-脑啡肽样免疫反应阳性亚微结构的电镜观察

本文用免疫电镜技术研究了大鼠中脑导水管周围灰质腹外侧区内5-HT样、SP样和L-ENK样的免疫反应阳性亚微结构。5-HT样免疫反应阳性的胞体较多,常见5-HT样阳性树突与阴性轴突终末形成多为非对称性的轴-树突触;偶见阳性轴突终末与阴性树突以及阴性轴突终末与阳性胞体分别构成轴-树和轴-体突触.SP样阳性胞体数目较少,可见少量含多形性小泡的阴性轴突终末与之形成轴-体突触;由阴性轴突终末与阳性树突所形成的轴-树突触最常见;阳性轴突终末与阴性胞体和阳性树突分别构成轴-体突触和轴-树突触。L-ENK样阳性胞体数目也较少,L-ENK样阳性树突与阴性轴突终末所形成的轴-树突触最多见,可见L-ENK样阳性胞体与阴性轴突终末构成轴-体突触;偶见阳性轴突终末与阴性树突形成轴-树突触。上述各种突触均主要含圆形小泡,有时有少量扁平小泡、椭圆形小泡和颗粒囊泡。     

Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage--immunohistochemical investigation of dendritic damage

We investigated the neuronal distribution of microtubule-associated protein 2 in gerbil brain and monitored the progression of ischemic damage immunohistochemically by using this protein as a dendritic marker. The reaction for microtubule-associated protein 2 in normal gerbil brain clearly visualized neuronal soma and dendrites but other structures such as axonal bundles, glia and endothelial cells exhibited little immunoreactivity. In a reproducible gerbil model of unilateral cerebral ischemia, we could detect the ischemic lesions as early as 3 min after right common carotid occlusion at the subiculum-CA1 region of the ipsilateral hippocampus as faint loss of the reaction in the dendrites. After ischemia for 30 min, the ischemic lesions were clearly detected as loss of the reaction in the nerve cell bodies, dendrites and the neuropil in the hippocampus, cerebral cortex, thalamus and the caudoputamen. Although the mechanism for prompt disappearance of the immunohistochemical reaction for microtubule-associated protein 2 is not clear, the present investigation suggests that dendrites in the vulnerable regions may be quite susceptible to ischemic stress and that the immunohistochemical procedure for microtubule-associated protein 2 may be very useful for demonstration of dendritic damage in various pathophysiological states of the central nervous system.     

大鼠三叉神经感觉核簇内SP受体及三叉神经节内SP样阳性终末和SP受体分布的免疫组织化学研究

本文用免疫组织化学方法对三叉神经感觉核簇内的SP受体及三叉神经节内的SP样阳性终末和SP受体的分布状态进行了研究。三叉神经感觉主核内的SP受体样阳性胞体和树突主要见于其背侧部和腹侧部。三叉神经脊束核的各亚核内均可见到SP受体样阳性的结构:吻侧亚核的SP受体样阳性胞体和树突主要见于其中央部;极间亚核的周边部仅有少量SP受体样阳性的胞体和树突;尾侧亚核内SP受体样阳性的胞体和树突最密集,且主要位于Ⅰ、Ⅱ层。三叉神经节内可见SP样阳性终末呈丛状或环状包绕在阴性胞体周围,也可见到少量散在的SP受体样阳性的胞体。SP受体样免疫反应阳性产物位于阳性胞体和树突的膜上。     

大鼠三叉神经脊束核Ⅱ尾侧亚核层内CB样、PV样阳性结构的分布及其突触联系

本研究用免疫组织化学方法观察了 Calbindin D-2 8k( CB)样和 Parvalbumin ( PV)样胞体、纤维和终末在三叉神经脊束核尾侧亚核 ( Vc) 层内的分布及它们的突触联系。在光镜下观察到 CB样和 PV样阳性胞体、纤维和终末在 II层内侧带 ( IIi)最为密集 ,PV样阳性神经元的胞体稍大 ,但数量少于 CB样阳性神经元。在电镜下观察到 CB样或 PV样阳性结构主要形成下列 4种突触联系 :( 1)阳性轴突终末与阳性或阴性轴突终末形成对称性轴 -轴突触和少量非对称性轴 -轴突触 ;( 2 )阳性轴突终末与阳性树突形成非对称性和对称性轴 -树突触 ;( 3 ) CB样阳性轴突终末与阴性树突主要形成非对称性轴 -树突触 ,PV样阳性轴突终末与阴性树突主要形成对称性轴 -树突触 ;( 4 )阴性轴突终末与阳性树突形成非对称性和对称性轴 -树突触。另外还可见到 CB样或PV样阳性或阴性树突、轴突及终末与 CB样、PV样阳性或阴性的初级传入纤维终末形成 型和 II型突触小球。 型突触小球数量较多 ,有典型的扇贝样初级传入纤维终末和不均一的小泡 ,线粒体少 ;II型突触小球的初级传入纤维终末粗大而清亮 ,外观不规则 ,有均匀一致的小泡和丰富的线粒体。根据上述结果可以推知在面口部伤害性信息的传递和调控过程中 ,Vc II层神经元发挥着重要的作用     

Distribution of glutamic acid decarboxylase-immunoreactive structures in the barrel region of mouse somatosensory cortex

Neuronal structures in the barrel region of the mouse primary somatosensory cortex containing gamma-aminobutyric acid (GABA) were identified by an immunocytochemical method, using an antiserum to glutamic acid decarboxylase (GAD), the rate-limiting enzyme in the synthesis of GABA. GAD-positive cell bodies were found in all layers of the barrel cortex, but were more concentrated in the upper portion of layer II/III, and in layers IV and VI. Puncta, presumably axon terminals, were also distributed throughout the cortical layers; a high density of puncta occurs in layer IV, whereas a somewhat lower density characterizes layer VI. Based on the shapes of their somata and the distribution of their dendrites it was determined that all GAD-positive cell bodies were of the non-pyramidal type.     

Glutamatergic components of the retrosplenial granular cortex in the rat

The ultrastructural characteristics, distribution and synaptic relationships of identified, glutamate-enriched thalamocortical axon terminals and cell bodies in the retrosplenial granular cortex of adult rats is described and compared with GABA-containing terminals and cell bodies, using postembedding immunogold immunohistochemistry and transmission electron microscopy in animals with injections of cholera toxin- horseradish peroxidase (CT-HRP) into the anterior thalamic nuclei. Anterogradely labelled terminals, identified by semi-crystalline deposits of HRP reaction product, were approximately 1 microm in diameter, contained round, clear synaptic vesicles, and established asymmetric (Gray type I) synaptic contacts with dendritic spines and small dendrites, some containing HRP reaction product, identifying them as dendrites of corticothalamic projection neurons. The highest densities of immunogold particles following glutamate immunostaining were found over such axon terminals and over similar axon terminals devoid of HRP reaction product. In serial sections immunoreacted for GABA, these axon terminals were unlabelled, whereas other axon terminals, establishing symmetric (Gray type II) synapses were heavily labelled. Cell bodies of putative pyramidal neurons, containing retrograde HRP label, were numerous in layers V-VI; some were also present in layers I-III. Most were overlain by high densities of gold particles in glutamate but not in GABA immunoreacted sections. These findings provide evidence that the terminals of projection neurons make synaptic contact with dendrites and dendritic spines in the ipsilateral retrosplenial granular cortex and that their targets include the dendrites of presumptive glutamatergic corticothalamic projection neurons.     

电击足底对大鼠下丘脑视上核及室旁核加压素样细胞的影响

本实验用特异性兔抗精氨酸加压素血清对正常和受到不同时间电击足底刺激的雄性大鼠下丘脑组织切片作了免疫细胞化学染色,观察了视上核和室旁核内加压素样细胞的形态学变化,并以显微分光光度计和显微图象分析仪列这些细胞内加压素样物质的相对含量和细胞截面积的变化进行了研究,以期对这种应激条件下下丘脑加压素释放与否问题有一较直观的了解。结果显示,电击组与对照组相比,视上核和室旁核细胞内加压素样物质减少,细胞胞体缩小。早期下丘脑垂体束细串珠样纤维增多,后期减少。正中隆起内带加压素样纤维染色略变淡,外带内细串珠状加压素样纤维增加。视上核和室旁核某些加压素样细胞的树状突起的变化较为特殊。一些向第三脑室侧壁和脑底面神经胶质板投射的树突在电击组染色依旧很深,并出现了许多大的串珠样结构,提示这些树突有向第三脑室和蛛网膜下腔分泌加压素样物质的可能。上述结果说明电击足底刺激视上核和室旁核细胞的神经分泌机能增强,可能通过下丘脑垂体束向垂体后叶,经正中隆起外带门脉血管向垂体前叶以及通过树突向脑脊液中释放加压素样物质增加。     

大鼠红核脊髓束与脊髓小脑束起始细胞突触联系的电镜研究

采用神经元溃变和ＨＲＰ神经元逆行追踪结合的方法。电镜观察红核脊髓束终末与脊髓小脑束起始细胞的突触联系。结果表明红核脊髓束溃变终末与ＨＲＰ标记的脊髓小脑束起始神经元（脊髓边缘细胞和Ｃｌａｒｋｅ柱细胞）胞体及树突形成突触,但大部分溃变终扣则与非标记神经元构成突触。以轴－树触居多。溃变终扣主要呈电子致密型和清亮型两种。以致密型为主,内多含圆形小泡。实验提示红核脊髓束与脊髓小脑束起始细胞存在直接或间接的突     

Dendritic trees of neurones in the rat supraoptic nucleus

The cytoarchitecture of the rat supraoptic nucleus was studied using the Golgi-Cox method with a neutral red or Cresyl violet counterstain to allow the limits of the nucleus to be determined accurately. A small number of stained neurones were seen in each of the brains sectioned but they were unevenly distributed in the supraoptic nucleus. There were more cells posteriorly and close to the optic tract. A minority of cells studied (a total of 4) were round (diameter approximately 20 μm) and multipolar and were interpreted as interneurones. The majority of stained cells studied (60) had oval cell bodies (mean long diameter 22.7 μm in coronal sections) with a single fine axon directed dorsally and medially and 1–3 heavier dendrites passing ventrally. Both cell bodies and dendrites of the major cell type showed spines and both dendrites and axons had some varicosities.The anatomy of the larger cell group (the presumed neurosecretory cells) is thus consistent with their having a substantial synaptic input and the presence of long dendrites (up to 205 μm) implies that caution is necessary in the interpretation of experiments where cell activity is monitored while active materials are placed near the cell bodies.