大鼠视神经切断后视网膜双极细胞PKC-α和recoverin的表达

为了探讨视神经切断后视网膜内部是否存在突触可塑性改变,本实验采用大鼠视神经切断模型,通过免疫组织化学方法检测视神经切断后视网膜双极细胞PKC-α和recoverin的表达变化。结果显示:正常视网膜中,PKC-α和recoverin阳性产物主要见于视网膜内核层、内网层及节细胞层,另外外核层也可见少量recoverin阳性细胞。视神经切断后3d,大鼠视网膜内网层高倍镜下可见PKC-α和recoverin免疫阳性终末的数量开始增加,14d时增至最高,21d、28d呈现逐渐减少的趋势。本研究结果提示视神经切断后视网膜双极细胞与节细胞之间的突触可能存在早期增生,后期溃变的可塑性变化。     

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     

Ultramicroscopical immunolocalization of PAX6 in the adult chicken retina

Cell type-specific PAX6 protein expression was examined in all retinal layers of the normal chicken retina. The most intense PAX6 immunostaining was found in the ganglion cell and inner nuclear layers, and in lower amounts in the optic nerve fiber, the inner plexiform and the photoreceptor layers. PAX6 immunostaining was variable in terms of its subcellular localization, even within one cell. PAX6 immunostaining was mainly localized in nuclear heterochromatin of the ganglion cell and inner nuclear layers whereas in the outer nuclear layer, PAX6 immunostaining was only observed in the intercellular space and the cytoplasm. In photoreceptors, the myoid portion of the inner segment showed PAX6 immunostaining, but the ellipsoid portion and the outer segment did not. The ultrastructural distribution pattern of PAX6 in the adult chicken retina suggests that normal expression of PAX6 is variable even in subcellular structures in the same cell type.     

锌转运体ZNT7在小鼠视网膜的定位分布

目的研究锌转运体-7(zinc transporter 7,ZNT7)在小鼠视网膜的定位和分布。方法应用免疫组织化学技术检测CD-1小鼠视网膜内的ZNT7免疫反应产物的表达。结果ZNT7在小鼠视网膜内分布广泛,在神经节细胞和色素上皮细胞内ZNT7免疫阳性反应产物的表达最丰富,在无长突细胞和视神经纤维层中ZNT7免疫阳性反应产物为中等程度的表达,在内网层、外网层和光感受器外节中ZNT7免疫阳性染色较淡,在外核层和光感受器内节中ZNT7几乎没有表达。结论ZNT7可能在维持视网膜锌稳态过程中起到重要的作用。     

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。     

Is Thy-1 expressed only by ganglion cells and their axons in the retina and optic nerve?

Summary The distribution of Thy-1 in the retina and optic nerve has been examined immunohistochemically, and compared to that of the astrocytic marker glial fibrillary acidic protein. The axons and cell bodies of ganglion cells were found to be Thy-1 positive as were processes within the inner plexiform layer. Transection of the optic nerve in the neonatal rat results in the rapid degeneration of the ganglion cells but some Thy-1 staining remains in the inner plexiform layer. We have estimated using an immunoassay of normal and optic nerve transected retinae that about 70% of the Thy-1 in the retina is on ganglion cells and their axons and the remainder is on cells which contribute processes to the inner plexiform layer, presumably amacrine, bipolar or Müller cells.In the optic nerve the Thy-1 was found to be limited to the fascicles of optic nerve fibres and the intrafascicular spaces, containing astrocytes and their processes, were not stained. Axotomy of the adult nerve, which produced axonal degeneration and astrocytic proliferation, led to a loss of over 95% of the Thy-1 from the nerve. We found no evidence that the astrocytes of the retina or optic nerve were Thy-1 positive in normal animals or during degeneration.     

泛素蛋白酶系统在四氧嘧啶诱导的糖尿病大鼠视网膜中的表达

目的:建立泛素蛋白酶系统(UPS)在正常和8周糖尿病大鼠视网膜的表达图谱,探讨其在糖尿病视网膜病变(DR)发生发展中可能的分子机制。方法:在限制片段差异显示PCR(RFDD-PCR)技术建立正常和8周糖尿病大鼠视网膜基因表达谱的基础上,对差异片段进行生物信息学分析,筛选UPS DR相关基因,并以免疫组织化学、半定量RT-PCR技术进行验证。结果:RFDD-PCR结果显示,泛素蛋白酶系统DR相关基因UBS3A、PSMD8和PSMD11在糖尿病组表达上调。RT-PCR结果显示,糖尿病组UBS3A表达比正常组明显增高,PSMD8和PSMD11则仅在糖尿病组中表达。免疫组织化学结果显示,正常组UBE3A阳性免疫反应物见于内丛状层,外丛状层和锥、杆体细胞层,PSMD8和PSMD11未见阳性细胞;糖尿病组UBE3A、PSMD8和PSMD11阳性细胞明显增多,见于节细胞层、内核层和外核层。结论:UPS与DR发生发展有关。     

Nogo受体(N-20)在大鼠视神经损伤后视网膜的表达

观察Nogo受体(NgR)在Wistar大鼠视神经(ON)损伤后视网膜各层的表达变化及分布规律,为Nogo蛋白抑制中枢神经再生的理论提供形态学依据。本实验各组动物均采用眶内眼球后2 mm ON切断术,术后动物分别存活3 d和7 d后取视网膜固定、冰冻后做水平切片,用免疫组织化学的方法观察NgR的表达情况。结果显示:NgR在正常对照组视网膜内3层有表达;在切断ON后实验各组有强烈表达;各组在损伤ON和移植神经组织后存活3 d时也有强烈表达,至7 d时表达有所下降。以上结果表明:NgR在大鼠ON损伤后视网膜的表达位于节细胞层、神经纤维层和内网层;其表达程度与移植物的种类有关,并可随存活时间的延长而下降。     

Expression of Glutamate and GABA during the Process of Rat Retinal Synaptic Plasticity Induced by Acute High Intraocular Pressure

Acute high intraocular pressure (HIOP) can induce plastic changes of retinal synapses during which the expression of the presynaptic marker synaptophysin (SYN) has a distinct spatiotemporal pattern from the inner plexiform layer to the outer plexiform layer. We identified the types of neurotransmitters in the retina that participated in this process and determined the response of these neurotransmitters to HIOP induction. The model of acute HIOP was established by injecting normal saline into the anterior chamber of the rat eye. We found that the number of glutamate-positive cells increased successively from the inner part to the outer part of the retina (from the ganglion cell layer to the inner nuclear layer to the outer nuclear layer) after HIOP, which was similar to the spatiotemporal pattern of SYN expression (internally to externally) following HIOP. However, the distribution and intensity of GABA immunoreactivity in the retina did not change significantly at different survival time post injury and had no direct correlation with SYN expression. Our results suggested that the excitatory neurotransmitter glutamate might participate in the plastic process of retinal synapses following acute HIOP, but no evidence was found for the role of the inhibitory neurotransmitter GABA.     

Intermediate filament complement of the normal and gliotic canine retina.

Intermediate filament expression of various cell types in the adult canine normal and gliotic retina was determined by an immunoperoxidase method of using monoclonal antibodies on aldehyde-fixed tissues. In the normal retina, vimentin was present in astrocytes in the nerve fibre layer, horizontal cell processes, and Müller cell fibres from the internal limiting membrane to the outer nuclear layer. Neurofilamentous axons were noted in the nerve fibre, inner plexiform layer, and outer plexiform layer, although the degree of staining intensity varied among the three molecular weight neurofilament antisera used. Glial fibrillary acidic protein (GFAP) staining was confined to the nerve fibre and ganglion cell layer; this was interpreted as representing fibrous astrocytes. Astrocyte density varied according to retinal topography with an increased number around retinal blood vessels and in the peripapillary retina. Quantitative, but not qualitative differences in staining for vimentin and the neurofilaments were noted in degenerative, gliotic retinas. In common with several other mammalian species previously studied, the canine Müller cells accumulate or express GFAP under pathological conditions involving a gliotic response.     

Changes in NADPH diaphorase expression in the fish visual system during optic nerve regeneration and retinal development

The various functions of nitric oxide (NO) in the nervous system are not fully understood, including its role in neuronal regeneration. The goldfish can regenerate its optic nerve after transection, making it a useful model for studying central nervous regeneration in response to injury. Therefore, we have studied the pattern of NO expression in the retina and optic tectum after optic nerve transection, using NADPH diaphorase histochemistry. NO synthesis was transiently up-regulated in the ganglion cell bodies, peaking during the period when retinal axons reach the tectum, between 20–45 days after optic nerve transection. Enzyme activity in the tectum was transiently down-regulated and then returned to control levels at 60 days after optic nerve transection, during synaptic refinement. To compare NO expression in the developing and regenerating retina, we have looked at NO expression in the developing zebrafish retina. In the developing zebrafish retina the pattern of staining roughly followed the pattern of development with the inner plexiform layer and horizontal cells having the strongest pattern of staining. These results suggest that NO may be involved in the survival of ganglion cells in the regenerating retina, and that it plays a different role in the developing retina. In the tectum, NO may be involved in synaptic refinement.     

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.     

Somatostatin-like immunoreactivity in amacrine cells of the chicken retina

Somatostatin-like immunoreactivity was detected in chicken retina by radioimmunoassay. The levels of somatostatin-like immunoreactivity decreased after intra-ocular injection of kainic acid, but were not affected by destruction of the ganglion cells. By immunohistochemistry, somatostatinimmunoreactive amacrine cells were found in the inner nuclear layer. These cells were destroyed by kainic acid. At least some of the cells projected to all three sub-layers of the inner plexiform layer in which there were diffuse bands of fluorescence. Specific immunofluorescence was also detected at the level of the outer limiting membrane and the optic nerve fibre layer, but the outer nuclear and plexiform layers, horizontal, bipolar and ganglion cells did not show specific immunofluorescence.It is suggested that other amacrine cell sub-classes, defined in terms of their putative transmitter, may show specific patterns of cell body location and size, and terminal arborisation.     

Expression profile of heat shock protein 108 during retinal development in the chick

In the developing chick retina, heat shock protein 108 (HSP108), which exhibits transferrin binding activity, has been demonstrated at the mRNA level, while transferrin shows two expression peaks. Here, we investigated the expression profile of HSP108 in the developing chick retina at the protein level. The localization of HSP108 in embryonic days 15 (E15), E18, and postnatal day 2 (P2) chick retina was examined immunohistochemically using monoclonal antibody 9G10 specific for chick HSP108, while the expression levels of HSP108 in developing chick retina from E12 to P2 and adult were measured by Western blot analysis. HSP108 was expressed in the ganglion cell layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segments of photoreceptors and retinal pigment epithelium. Two peaks of HSP108 expression were found at around E13 and E18, respectively. Since the two HSP108 peaks appeared to be correlated with the transferrin expression peaks during retinal development, HSP108 may be associated with iron metabolism during the development of the retina.     

高血压大鼠视网膜组织Ca^2＋酸性磷酸酶组织化学的研究

目的：应用光镜定量酶组织化学方法对正常京都种大鼠（ＷＫＹ）和自发性高血压大鼠（ＳＨＲ）视网膜组织的Ｃａ＾２＋－酸性磷酸酶的分布和活性进行定量观察，结果：Ｃａ＾２＋酸性磷酸酶在ＷＫＹ视网膜组织的活性由强到弱依次为（Ｆ检验，Ｐ〈０．０５）；（１）杆锥细胞内节和外核层；（２）节细胞层；（３）内核层；（４）内网层和外网层；（５）杆锥细胞外节阴性，在ＳＨＲ视网膜组织中，各层Ｃａｓ＾２＋酸性磷酸酶活性下降，以     

高血压大鼠视网膜溶酶体酶组织化学研究

目的：探讨溶酶体酶在高血压视网膜网变发生过程中的作用。方法：应用光镜定量酶组织化学方法对ＷＫＹ大鼠和自发性高血压大鼠视网膜原酸性磷酸的分布和活性变化进行定量观察。结果：视网膜各层酸性磷酸酶活性岂强到弱依次是（Ｆ检验，Ｐ〈０．０５）；（１）色素上皮层；（２）视杆维层内节和外网层（两层间活性无显著性差异）；（３）内网层；（４）节细胞层和神经纤维层，（５）外核层和内核层（两层间活性无显著性差异）杆锥层外     

Differential localization of rat Eag1 and Eag2 potassium channels in the retina

Despite of their wide expression in the brain, the precise neurophysiological role of rat Eag1 (rEag1) and Eag2 (rEag2) K(+) channels remains elusive. Our previous studies in hippocampal pyramidal neurons demonstrate a somatodendritic localization of rEag1 and rEag2 channels, suggesting that the two channel isoforms may contribute to setting the membrane excitability of somas and dendrites. Here, we aim to further characterize the cellular and subcellular localization patterns of rEag1 and rEag2 proteins by studying their laminar distribution in the retina. Confocal microscopic analyses of immunofluorescence data revealed that rEag1 and rEag2 K(+) channels exhibit distinct cellular expression pattern in the retina. rEag1 immunoreactivity was most prominent in the outer half of the inner plexiform layer, whereas strong rEag2 immunostain was found in the outer and inner segments of photoreceptor cells, the outer plexiform layer, and the inner nuclear layer. These results suggest that rEag1 and rEag2 K(+) channels may play a significant role in the transmission of electrical signals along the retinal neuronal circuits. We also performed double-labeling experiments to demonstrate that rEag1 and rEag2 are predominantly expressed in the somatodendritic compartment of retinal neurons. In addition, we presented evidence suggesting that rEag1 channels may be expressed in the GABAergic amacrine cell. Finally, based on their different immunostaining patterns over the inner region of the retina, we propose that compared to rEag2, rEag1 expression encompasses a significantly broader range of the somatodendritic compartment of the retinal ganglion cell.     

Differential localization of rat Eag1 and Eag2 potassium channels in the retina

Despite of their wide expression in the brain, the precise neurophysiological role of rat Eag1 (rEag1) and Eag2 (rEag2) K⁺ channels remains elusive. Our previous studies in hippocampal pyramidal neurons demonstrate a somatodendritic localization of rEag1 and rEag2 channels, suggesting that the two channel isoforms may contribute to setting the membrane excitability of somas and dendrites. Here, we aim to further characterize the cellular and subcellular localization patterns of rEag1 and rEag2 proteins by studying their laminar distribution in the retina. Confocal microscopic analyses of immunofluorescence data revealed that rEag1 and rEag2 K⁺ channels exhibit distinct cellular expression pattern in the retina. rEag1 immunoreactivity was most prominent in the outer half of the inner plexiform layer, whereas strong rEag2 immunostain was found in the outer and inner segments of photoreceptor cells, the outer plexiform layer, and the inner nuclear layer. These results suggest that rEag1 and rEag2 K⁺ channels may play a significant role in the transmission of electrical signals along the retinal neuronal circuits. We also performed double-labeling experiments to demonstrate that rEag1 and rEag2 are predominantly expressed in the somatodendritic compartment of retinal neurons. In addition, we presented evidence suggesting that rEag1 channels may be expressed in the GABAergic amacrine cell. Finally, based on their different immunostaining patterns over the inner region of the retina, we propose that compared to rEag2, rEag1 expression encompasses a significantly broader range of the somatodendritic compartment of the retinal ganglion cell.     

大鼠视神经切断后视网膜外网层突触可塑性研究

应用闪光视网膜电图(fERG)、细胞色素c氧化酶(CO)组织化学染色及突触囊泡素免疫荧光染色方法检测视神经切断术后,视网膜外网层相关神经元的功能活动及突触可塑性变化。结果显示:fERGb波峰潜伏期在视神经损伤后均显著延长;b波振幅在损伤第3、5d组增高,此后逐渐下降并在7、14、21d组低于正常。外网层CO活性在视神经切断第3、5d上调,此后逐渐下降,至第14、21d低于正常。外网层突触囊泡素阳性颗粒在视神经切断第5d增多,此后逐渐下降,至第14、21d低于正常。本研究结果提示视神经切断后,视网膜内外网层神经元的功能在早期将出现一过性的增强,结构也有相应的代偿性改变,随后即发生跨神经元的逆行性溃变。