游离锌离子在小鼠视网膜的定位研究

目的研究游离锌离子在小鼠视网膜的定位分布。方法应用ZnSe金属自显影技术(AMG)检测硒酸钠注射40 m in后小鼠视网膜内的锌离子。结果注射硒酸钠40 m in后发现游离锌离子主要分布于小鼠视网膜的色素上皮细胞层、光感受器的内节、外核层、外网层、内核层、内网层和神经节细胞层。在色素上皮细胞层、光感受器的内节和内核层与内网层交界处AMG阳性反应最为明显,在光感受器外节和神经纤维层几乎没有AMG阳性反应产物。结论小鼠视网膜内锌离子,在视网膜神经元视觉信息的传导和形成过程中可能起着重要作用。     

脑红蛋白在绵羊视网膜中的分布

目的:探讨脑红蛋白在绵羊视网膜的分布特征。方法:利用免疫组织化学显色SP法,观察脑红蛋白在健康成年绵羊视网膜中的分布情况。结果:脑红蛋白在绵羊视网膜的视神经纤维层、内网状层、外网状层和光感受器内节段中有强阳性表达,在视网膜的内核层和节细胞层有弱阳性表达,在视网膜外核层、光感受器外节段和色素上皮层中未见有阳性表达,内界膜、外界膜和视神经中亦有脑红蛋白阳性表达。绵羊视网膜脑红蛋白阳性表达的细胞类型主要有节细胞、双极细胞和光感受器细胞,其中节细胞的阳性表达定位于细胞质,胞核中未见表达。结论;除外核层、光感受器外节段和色素上皮层外,脑红蛋白在绵羊视网膜其他各层中均有表达,提示脑红蛋白在维持视网膜中氧平衡状态时发挥重要作用。     

锌转运体ZNT1在人阿尔茨海默病大脑的定位

目的研究锌转运体-1(zinc transporter 1,ZNT1)在阿尔茨海默病(Alzheimer's Disease,AD)尸检大脑皮层内的定位分布,探讨ZNT1影响脑锌平衡从而参与AD发病的可能机制。方法应用免疫组织化学、免疫荧光和共聚焦激光扫描显微镜观察ZNT1蛋白和β-淀粉样蛋白(β-amyloid,Aβ)在AD患者大脑内的分布和二者之间的位置关系。结果ZNT1免疫阳性反应产物主要定位于AD大脑皮层老年斑内。几乎所有的Aβ老年斑内均有不同程度ZNT1表达。ZNT1和β-淀粉样蛋白免疫双标结果进一步证实ZNT1免疫产物与Aβ共存于AD患者的老年斑内。结论AD患者大脑皮质Aβ老年斑内有大量ZNT1蛋白表达,提示ZNT1可能参与AD大脑皮质Aβ老年斑的形成。     

细胞因子信号转导抑制因子SOCS-3在成年大鼠视网膜内的定位分布

目的 分析JAK-STAT信号转导途径抑制蛋白SOCS-3在大鼠视网膜内的基础表达和细胞内定位分布。方法 免疫细胞化学技术。结果 SOCS-3免疫阳性反应细胞广泛分布在视网膜节细胞层和内核层。在节细胞层，SOCS-3免疫阳性反应产物主要分布在节细胞核；在内核层，部分阳性细胞为MUller细胞。结论 SOCS-3在正常大鼠视网膜神经元及胶质细胞内有较为广泛的基础表达，并以核蛋白为其主要存在形式。     

鲫鱼、牛蛙、鸡和大鼠视网膜锌离子分布的光镜观察

本文应用neo-Timm染色技术研究了鲫鱼、牛蛙、鸡和大鼠视网膜内锌离子的分布状况。结果发现上述动物视网膜内均存在锌离子。锌离子位于视网膜光感受器的内段、外网层、双极细胞、无长突细胞和神经节细胞等处。鲟鱼视网膜的部分光感受器胞体锌离子染色阳性。此外，牛蛙、鸡和大鼠等动物视网膜内同层锌离子亦呈弥漫性着色。提示在较高等动物作为神经调质的锌离子对视网膜神经无视觉信号的传导与调制可能具有更为广泛的意义。     

牛蛙视网膜内γ-氨基丁酸免疫阳性反应的分布

用免疫组织化学ABC法．研究了GABA免疫阳性反应在牛蛙视网膜的分布。证明光感受器内段（主要是视锥细胞）呈棕褐色的GABA反应;在外核层未见GABA标记的胞体,但在靠近外网层处偶见GABA标记的终末;在内核层,大量无长突细胞呈GABA反应阳性,并可鉴别出胞体染色较深和淡的两个亚群,一些双极细胞和个别水平细胞的胞体及它们的突起呈较弱的GABA反应阳性,偶见双极细胞轴突终末以膨体紧密贴附在GABA标记的无长突细胞上。在节细胞层,一些神经节细胞和散在的移位无长突细胞呈GABA反应阳性。此外．外网层和内网层均呈GABA反应阳性。上述结果表明,GABA广泛分布于牛蛙视网膜的各层,提示它在视觉信号的传递过程中发挥着重要作用。     

ZnT1及游离锌离子在小鼠骺板软骨细胞的定位研究

目的研究锌转运体-1(Zinc transporter 1,ZnT1)和游离锌离子在小鼠骺板软骨细胞的定位分布,探讨ZnT1影响骺板软骨细胞锌离子代谢从而参与骨骼生长的可能机制。方法应用浸入式金属自显影技术(AMG)观察锌离子在小鼠肋骨骺板内的定位分布;应用免疫组织化学SABC法检测ZnT1在小鼠骺板软骨细胞的表达。结果金属自显影技术显示游离锌离子在小鼠肋骨骺板肥大带、增殖带和静止带三层区域结构内有不同程度的表达,其中肥大带软骨细胞层锌离子含量最高;ZnT1免疫阳性反应产物主要定位于软骨细胞膜附近,在小鼠肋骨骺板三层区域结构内有不同程度的表达,从肥大带到静止带,软骨细胞膜上的ZnT1免疫反应逐渐减弱。结论小鼠肋骨骺板内存在大量的游离锌离子和ZnT1蛋白,提示ZnT1可能参与锌离子在软骨细胞的转运和代谢,在骨的形成和发育过程中发挥作用。     

VEGF、bFGF在早期糖尿病大鼠视网膜的表达

应用免疫组化 ABC法观察生长因子 VEGF、b FGF在早期链脲佐菌素糖尿病大鼠视网膜的表达。结果如下 :随着病程进展 ,二者的表达呈逐渐增强趋势。病程 3月时 ,除了正常表达 VEGF的内核层及节细胞层部分细胞外 ,尚见血管阳性反应。6月时 ,阳性反应又出现于视杆内节和色素上皮细胞 ;b FGF于正常对照组只表达于外核层细胞 ,而糖尿病 3月组和 6月组阳性反应范围扩大至内核层和视网膜血管     

青光眼致病基因OPTN在大鼠光感受器细胞内的表达

目的观察OPTN在大鼠视网膜光感受器细胞内的表达及定位,探讨OPTN和NRL(neural retina leucine zipper)两蛋白相互的作用。方法制备正常Wistar大鼠的视网膜冷冻切片,用抗OPTN抗体、抗NRL抗体和抗CRX抗体分别进行多重免疫荧光染色。结果在外丛状层(OPL)、外核层(ONL)、光感受器细胞内节(IS)、神经节细胞层、内丛状层(IPL)和内核层(INL)中均观察到抗OPTN抗体不同强度的荧光信号。光感受器细胞内的荧光信号主要在细胞质内,而细胞核内未见染色,且具有视锥细胞特征的细胞未被抗OPTN抗体染色。结论 Optn(OPTN的大鼠同源基因)在除视锥细胞以外的光感受器细胞高表达。     

ZnT7及游离锌离子在小鼠室管膜和脉络丛细胞的分布

目的 研究锌转运体7（ZnT7）和游离锌离子在小鼠脊髓室管膜和脉络丛上皮细胞中的分布.方法 应用ZnSe金属自显影技术（AMG）检测硒酸钠注射1.5h后小鼠脊髓室管膜细胞及脑室脉络丛上皮细胞的游离锌离子;应用免疫组织化学SABC法检测小鼠脊髓室管膜细胞及脑室脉络丛上皮细胞中ZnT7的表达.结果 光镜下观察AMG染色的切片,小鼠脊髓室管膜细胞及脑室脉络丛上皮细胞中均有游离锌离子的分布;免疫组织化学结果表明,脊髓室管膜细胞及脉络丛上皮细胞中均有ZnT7的表达,且与游离锌离子分布区域基本一致.结论 锌离子可能在脊髓室管膜细胞及脉络丛上皮细胞内发挥重要作用,脊髓室管膜细胞及脉络丛上皮细胞可能在脑脊液锌转运过程中发挥重要的作用.     

On the synaptogenic sequence in the chick retina

Study of the developing chick retina with the electron microscope revealed that dyad ribbon synapses begin to form in the inner plexiform layer before synaptic ribbons begin to appear in photoreceptor terminals of the outer plexiform layer. This centrifugal (inner to outer) sequence of synaptogenesis in the predominantly cone retina of the chick differs from the centripetal sequence that has been reported for the predominantly rod retinas of the mouse and rat. This difference does not favor the hypothesis, suggested by others, that the photo-receptor may influence the maturation of inner retinal elements. The different patterns of synaptogenesis are discussed briefly with reference to anatomical differences between the retinas of different species.     

Histology of the ferret retina

The retina of the adult ferret, Mustelo furo, was studied with light and transmission electron microscopy to provide an anatomical basis for use of the ferret as a model for retinal research. The pigment epithelium is a simple cuboidal layer of cells characterized by a zone of basal folds, apical microvilli, and pigment granules at various stages of maturation. The distinction between rod and cone photoreceptor cells is based on their location, morphology, heterochromatin pattern and the electron density of their inner segments. The round, light-staining cone cell nuclei occupy the layer of perikarya along the apical border of the outer nuclear layer. The remainder of the outer nuclear layer consists of oblong, deeply-stained rod cell nuclei. Ribbon type synaptic complexes involving photoreceptor cell axons, horizontal cell processes, and bipolar cell dendrites characterize the outer plexiform layer. The inner nuclear layer is comprised of horizontal, bipolar, and amacrine cell perikarya as well as the perikarya of the Müller cells. The light-staining horizontal cell nuclei are prominent along the apical border of the inner nuclear layer. The light-staining amacrine cell nuclei form a more or less continuous layer along the basal border of the inner nuclear layer. Both conventional and ribbon-type synapses characterize the inner plexiform layer. The ganglion cells form a single cell layer. The optic fiber layer contains bundles of axons surrounded by Müller cell processes. Small blood vessels and capillaries are present in the basal portion of the retina throughout the region extending from the internal limiting membrane to the outer plexiform layer. The adult one-year-old retina is compared with the retina at the time of eye opening.     

Spatial relationships of connexin36, connexin57 and zonula occludens-1 in the outer plexiform layer of mouse retina

Horizontal cells form gap junctions with each other in mammalian retina, and lacZ reporter analyses have recently indicated that these cells express the Cx57 gene, which codes for the corresponding gap junctional protein. Using anti-connexin57 antibodies, we detected connexin57 protein in immunoblots of mouse retina, and found punctate immunolabeling of this connexin co-distributed with calbindin-positive horizontal cells in the retinal outer plexiform layer. Double immunofluorescence labeling was conducted to determine the spatial relationships of connexin36, connexin57, the gap junction-associated protein zonula occludens-1 and the photoreceptor ribbon synapse-associated protein bassoon in the outer plexiform layer. Connexin36 was substantially co-localized with zonula occludens-1 in the outer plexiform layer, and both of these proteins were frequently located in close spatial proximity to bassoon-positive ribbon synapses. Connexin57 was often found adjacent to, but not overlapping with, connexin36-positive and zonula occludens-1-positive puncta, and was also located adjacent to bassoon-positive ribbon synapses at rod spherules, and intermingled with such synapses at cone pedicles. These results suggest zonula occludens-1 interaction with connexin36 but not with Cx57 in the outer plexiform layer, and an absence of connexin57/connexin36 heterotypic gap junctional coupling in mouse retina. Further, an arrangement of synaptic contacts within rod spherules is suggested whereby gap junctions between horizontal cell terminals containing connexin57 occur in very close proximity to ribbon synapses formed by rod photoreceptors, as well as in close proximity to Cx36-containing gap junctions between rods and cones.     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

Dopaminergic transmission in the rat retina: evidence for volume transmission

The study was designed to determine whether dopaminergic neurotransmission in the retina can operate via volume transmission. In double immunolabelling experiments, a mismatch as well as a match was demonstrated in the rat retina between tyrosine hydroxylase (TH) and dopamine (DA) immunoreactive (ir) terminals and cell bodies and dopamine D2 receptor-like ir cell bodies and processes. The match regions were located in the inner nuclear and plexiform layers (D2 ir cell bodies plus processes). The mismatch regions were located in the ganglion cell layer, the outer plexiform layer, and the outer segment of the photoreceptor layer, where very few TH ir terminals can be found in relation to the D2 like ir processes. In similar experiments analyzing D1 receptor like ir processes versus TH ir nerve terminals, mainly a mismatch in their distribution could be demonstrated, with the D1 like ir processes present in the outer plexiform layer and the outer segment where a mismatch in D2 like receptors also exists. The demonstration of a mismatch between the localization of the TH terminal plexus and the dopamine D2 and D1 receptor subtypes in the outer plexiform layer, the outer segment and the ganglion cell layer (only D2 immunoreactivity (IR)) suggests that dopamine, mainly from the inner plexiform layer, may reach the D2 and D1 mismatch receptors via diffusion in the extracellular space. After injecting dopamine into the corpus vitreum, dopamine diffuses through the retina, and strong catecholamine (CA) fluorescence appears in the entire inner plexiform layer and the entire outer plexiform layer, representing the match and mismatch DA receptor areas, respectively. The DA is probably bound to D1 and D2 receptors in both plexiform layers, since the DA receptor antagonist chlorpromazine fully blocks the appearance of the DA fluorescence, while only a partial blockade is found after haloperidol treatment which mainly blocks D2 receptors. These results indicate that the amacrine and/or interplexiform DA cells, with sparse branches in the outer plexiform layer, can operate via volume transmission in the rat retina to influence the outer plexiform layer and the outer segment, as well as other layers of the rat retina such as the ganglion cell layer.     

Transient synaptic ribbons in the mammalian retina at unusual sites

In the vertebrate retina the presence of synaptic ribbons (SRs) is well documented in two sites only, viz., in photoreceptor axon terminals in the outer plexiform layer and in bipolar cell axons in the inner plexiform layer. The present paper reports the presence of non-photoreceptor SRs in the outer plexiform layer of cattle and mouse, where they were seen in small numbers in thin cell processes near cone pedicles of light-adapted animals. They were never seen near rod spherules. Quantitative data obtained in mice killed at different time-points revealed that the SRs under consideration increased in number during day time and were absent during the dark phase. Moreover, under high light intensity of 10000 lux they were more frequent in number compared to 100-lux-exposed animals. It is concluded that the cell processes revealing the temporary presence of SRs are processes of flat bipolar cells which may provide a feedback to cones during the light phase.     

Localization of primary cilia in mouse retina

The primary cilia are considered as “cellular antennae” that sense and interchange information with the extracellular environment. Nearly all mammalian cells have a single primary cilium. In the retina, the outer segment of the photoreceptor is known to be a specialized form of primary cilium, but studies on cilia in other layers of the retina are scarce. In this study, we investigated the expression of primary cilia in the whole thickness of the mouse retina using immunofluorescence with three different ciliary markers: Arl13b, acetylated α-tubulin and adenylyl cyclase III. Our results show positive reactions in the photoreceptor layer, outer plexiform layer and ganglion cell layer, which might suggest the possible presence of primary cilia in these areas, but we could not directly prove the strand-like shape of cilia in those areas. In the outer plexiform layer, all three markers showed intense staining along the neuronal synapses, which suggests that the neuronal processes themselves might share the features of cilia.