Somatostatinergic neurones of the developing human and cat retinae

We have examined somatostatin-immunoreactive (S-IR) neurones in developing retinae of the human and cat. At 14 and 16 weeks' gestation (G14 and G16) in the human, S-IR cells were only found close to the putative fovea centralis, but by 18 weeks' gestation (G18), they were located in all retinal regions. By adulthood, the majority of S-IR cells were restricted to inferior retina. In the developing cat retina, two classes of S-IR cells were recognized. S1-IR cells were similar in morphology and distribution to adult cells: they had small round somata which were only found in inferior retina and gave rise to beaded processes which traversed the inner plexiform layer (IPL) and nerve fibre layer (NFL). S2-IR cells had larger somata located in the ganglion cell layer (GCL) and the label was compartmentalized within their cytoplasm. Most S2-IR cells had lost immunoreactivity by P (postnatal day) 25 and may have been alpha-ganglion cells transiently expressing somatostatin in association with their retention of plasticity into postnatal life.     

兔胎视网膜内P物质免疫反应神经元的发生

本文用免疫细胞化学ＡＢＣ法，研究了新西兰白兔１８、２２、２５、２６、２８和３０ｄ胎龄视网膜内Ｐ物质免疫反应（ＳＰＩＲ）神经元的发生。在胎龄１８和２２ｄ兔视网膜未见ＳＰＩＲ细胞体和纤维。在胎龄２５ｄ视网膜的节细胞层最先出现ＳＰＩＲ神经元，胞体浅染呈卵圆形，突起不明显，在神经纤维层偶见串珠状ＳＰＩＲ纤维，其平均细胞密度为１０４．６个细胞／ｍｍ￣２。到胎龄２６和２８ｄ时，在节细胞层的ＳＰＩＲ神经元的胞体渐深染，可见个别ＳＰＩＲ神经元发出粗而短的突起伸向内网层，平均细胞密度分别为３８７和７７９．５个细胞／ｍｍ２。到胎龄３０ｄ时ＳＰＩＲ神经元开始出现于内核层的内排细胞，但数量很少，胞体呈卵圆形，发出细突起伸入内同层，在节细胞层的ＳＰＩＲ神经元的突起分支增加。此时ＳＰＩＲ神经元平均细胞密度为３５７．４个细胞／ｍｍ￣２。     

Electron microscopy of glutamate decarboxylase (GAD) immunoreactivity in the inner plexiform layer of the rhesus monkey retina

Summary With indirect immunofluorescence, glutamate decarboxylase (GAD), the GABA synthesizing enzyme, was localized to cell bodies in the inner half of the inner nuclear layer and a few in the outer tier of the ganglion cell layer in the rhesus monkey retina. In the inner plexiform layer there were three strongly GAD-immunoreactive laminae separated by two less immunoreactive laminae. Electron microscopy demonstrated that the GAD was contained in amacrine cells and these GAD-immunoreactive amacrines were primarily pre- and postsynaptic to biopolar cell axon terminals. The GAD-containing processes possessed small synaptic vesicles and formed synapses that could be characterized as symmetrical. Large, dense-cored vesicles were often found in the cell bodies and synaptic processes of the GAD-immunoreactive amacrine cells. As the vast majority of the synaptic input and output of the GAD-containing amacrine cells was to and from bipolar cells and the strongest GAD-immunoreactivity correlated with the endings of bipolar cells that connect with a single cone, the functional effects of GABA in the primate retina are likely to be found in the responses of single cone pathways in the inner plexiform layer.     

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。     

NADPH-diaphorase positive amacrine cells in the retinae of the frog (Rana esculenta) and pigeon (Columbia livia)

The distribution of NADPH-diaphorase positive cells was examined histochemically in the retinae of the pigeon and frog. In the pigeon, three different types of amacrine cells were identified in the inner nuclear layer (INL) on the basis of cell body size and staining intensity. In the frog two types of NADPH-diaphorase positive amacrine cells have been demonstrated in the INL. Therefore, the NADPH-diaphorase method selectively stains several subtypes of amacrine cells in the retina of lower vertebrates. Although the actual function of NADPH-diaphorase activity is unknown, diaphorase histochemistry provides a convenient method for achieving Golgi-like images of amacrine cells in the retina.     

Glutamate receptor expression in the rat retina.

The expression of five genes (GluR A; B; C; D; GluR 5) encoding functional subunits of glutamate receptors was investigated in the rat retina using in situ hybridization with oligonucleotide probes. All five genes are expressed in the retina. All probes label cell bodies in the ganglion cell layer as well as somata in the inner third of the inner nuclear layer (INL), where the amacrine cells are located. In addition GluR 5, B and D, and to a lesser extent also GluR A are found in the middle and outer part of the INL, where bipolar and horizontal cells reside. Different subsets of retinal neurons may thus use glutamate receptors of different subunit composition.     

Indoleamine-accumulating cell death and endogenous glial cell reaction induced by 5,7-dihydroxytryptamine in the cat retina.

We investigated the patterns of degenerative changes of indoleamine-accumulating cells (IACs) induced by 5,7-dihydroxytryptamine (5,7-DHT, 100 microg), and the glial reaction to the neurodegenerative changes of IACs in the cat retina by using light-and electron-microscopy. The neurons accumulating 5,7-DHT in the cat retina were a few ganglion cells and displaced amacrine cells located in the ganglion cell layer (GCL), and some amacrine cells in the inner nuclear layer (INL). The cell density (per unit area, 1 mm2) of the 5,7-DHT accumulating cells in the GCL and INL was 910 and 134 cells, respectively. Most 5,7-DHT accumulating cells showed dark degeneration characterized by widening of the cellular organelles at early stage, and by darkening of the cytoplasm at a late stage. In addition, amacrine cells, showing a typical filamentous degeneration, were observed in a few cases. The degenerated neurons were phagocytosed by microglial cells and astrocytes. The immunoreactivity for glial fibrillary acidic protein (GFAP) in Muller cells was increased at early stage, but thereafter abruptly decreased. In a few cases, severe degenerative changes were observed in Miller cells. These results indicate that 5,7-DHT induces severe dark degeneration of IACs, and most degenerated cells could be eliminated by microglial cells and astrocytes in the cat retina.     

人视网膜星形胶质细胞发育及与血管前体细胞的关系

目的：研究人视网星菜胶质细胞的发育及与血管前体细胞的关系。材料和方法：收集１３４例发育各期胎儿视网膜和４例成人视网膜、石蜡包埋切片整装铺片，四种抗体免疫组分染色，光镜观察，结果：星形胶质细胞分为三种：（１）Ｓ－１００（＋）／胶质纤维酸性蛋白（ＧＦＡＰ）（＋）的双极形星形胶质细胞，视盘进入视网膜，与跟随其后的纤连蛋白（Ｆｎ）（＋）的血管前体细胞接触并相伴向锯齿缘迁移，足月后和成人此类星形胶质细胞主要     

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein in tanycytes of the mediobasal hypothalamus: distribution and relation to dopamine and luteinizing hormone-releasing hormone neurons and other glial elements

The distribution of a dopamine- and cyclic adenosine-3':5'-monophosphate (cAMP)-regulated phosphoprotein with an apparent molecular weight of 32,000 (DARPP-32) was investigated in the rat diencephalon and monkey hypothalamus by use of immunohistochemical techniques. In addition to single cells located peri- and paraventricularly in hypothalamus and thalamus in the rat, and ependymal cells, DARPP-32-immunoreactivity was found to be present in a subpopulation of ependymal tanycytes. These DARPP-32-positive tanycytes lined the walls and floor of the third ventricle, sending processes towards the arcuate nucleus, surrounding blood vessels in this nucleus, and continuing towards the median eminence, where they abutted on portal vessels. A second group of DARPP-32-positive tanycytes with cell bodies within the median eminence was also observed. Simultaneous labeling with antiserum against tyrosine hydroxylase, a presumptive marker for tuberoinfundibular dopamine neurons, revealed a close relation to DARPP-32-containing tanycytes in several anatomical locations. Thus, in the periventricular area DARPP-32-positive tanycytes ensheathed tyrosine hydroxylase-positive processes. These processes, presumably representing dopaminergic dendrites, virtually penetrated between the ependymal cells to the ventricular space and thus perhaps established direct contact with the cerebrospinal fluid. Tyrosine hydroxylase-terminals were also observed in close association with DARPP-32-immunoreactive tanycytes in the rat median eminence. However, in view of the density of DARPP-32-positive processes in the external layer of the median eminence, the DARPP-32 processes may be related to a number of other types of nerve endings, including luteinizing hormone-releasing hormone, as shown in this study. The close association of DARPP-32-immunoreactive processes with tyrosine hydroxylase- and luteinizing hormone-releasing hormone-immunoreactive nerve endings in the rat was directly visualized at the ultrastructural level using triple-labeling immunocytochemistry. Both the ultrastructural analysis and immunohistochemistry at the light microscopic level, comparing the distribution of DARPP-32 and glial fibrillary acidic protein, indicated the presence of two types of glial processes in the median eminence. The electron microscopic studies also suggested the presence of both DARPP-32-positive and DARPP-32-negative glial processes in the external layer of the median eminence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of GABA immunoreactivity in kainic acid-treated rabbit retina

Ischaemic retinal cell degeneration seems to involve both NMDA and non-NMDA receptor over stimulation. However, different retinal cell types differ largely in their susceptibility to excitatory amino acid induced neurotoxicity. We have investigated the vulnerability of GABAergic cells in the rabbit retina to the non-NMDA receptor agonist kainic acid (KA). The distribution of GABA immunoreactivity (GABA-IR) was examined in the central inferior retina at different survival times (5 h–6 days) following an intra-ocular injection of 140 nmol KA and compared to that of control and untreated retinas. In the normal retina, the majority of GABA-positive cells (79%) were located in the inner nuclear layer (INL), in one to four cell rows next to the inner plexiform layer (IPL), and in one cell row next to the outer plexiform layer (OPL). The remainder (21%) were found in the ganglion cell layer (GCL). Dense immunoreactivity was seen throughout the IPL. In the OPL, stained dots and occasional immunoreactive large processes could be seen. KA-exposed retinas processed for GABA immunocytochemistry 5 and 24 h after the injection showed an 85% reduction in the number of GABA immunoreactive cells. About the same degree of depletion was seen among GABA-IR cells located at different retinal levels. However, at these survival times, immunostaining was observed in three distinct bands in the IPL, indicating that the vulnerability to KA is not uniformly distributed among all GABAergic cells. At 48 h, an additional decrease in the number of labelled cells was noted, but immunoreactive cells were still found both in the INL and GCL. Even 6 days after KA treatment, a few stained cell bodies were seen in the INL next to the IPL, as well as a few processes in the IPL. The study shows that KA receptor overstimulation induces a marked depletion of the endogenous cellular GABA pools of the central rabbit retina, most likely as a result of GABAergic cell loss. However, a small population of GABAergic cells located in the INL appears to be less vulnerable to the toxic effects of 140 nmol KA.     

兔视网膜中P物质样免疫反应神经元的发育

本实验用免疫细胞化学ABC法,研究了成年、新生和生后兔视网膜中P物质(SP)样免疫反应神经元的定位和发育。结果表明,成年兔视网膜SP样免疫反应细胞胞体位于内核层和节细胞层,胞突分布在内网层的第1、3、5亚层,偶见于视神经纤维层。细胞密度以视纹最高,从视纹向背腹视网膜边缘区密度渐变小。在新生兔视网膜已有SP阳性胞体和胞突出现,胞体主要位于节细胞层,突起在内网层第5亚层,但未形成连续网层,在第1亚层很少,第3亚层未见SP阳性突起。SP阳性细胞密度从新生到生后第4天增加,生后第6天到第12天细胞密度渐下降。生后第12天SP阳性胞体主要位于内核层。生后第20天,SP阳性细胞的形态、密度与分布已接近成年水平。上述结果提示,在兔视网膜中SP样免疫反应胞体和突起在生前已出现,生后继续发育,到生后20天后其形态发育已接近成熟。     

Increased expression of ciliary neurotrophic factor receptor alpha mRNA in the ischemic rat retina

Using in situ hybridization, we investigated the expression of ciliary neurotrophic factor receptor ((CNTFRalpha) mRNA in the rat retina rendered ischemic by elevation of the intraocular pressure (IOP). The IOP was increased to 120 mmHg and maintained for 60 min. The rats were sacrificed on the day of reperfusion (DRP) 1, 3, 7, 14, and 28. In the normal retina, the signal for CNTFRalpha mRNA was present in retinal cells in the inner nuclear layer (INL) and in the ganglion cell layer (GCL). On DRP 1, numerous cells in the INL and GCL showed a CNTFRalpha mRNA signal. From DRP 3 onwards, CNTFRalpha mRNA appeared in photoreceptor cells located in the outer part of the outer nuclear layer. The signal in these cells increased up to DRP 14 and then decreased at DRP 28. Our findings suggest that cells expressing CNTFRalpha mRNA may resist the degenerative processes induced by ischemic insult in the rat retina.     

Synaptic organization of type 2 catecholamine amacrine cells in the rhesus monkey retina

Summary Two types of amacrine cell immunoreactive for tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway, are present in the retina of the rhesus monkey,Macaca mulatta. The well-known dopaminergic, or type 1 catecholamine amacrine cells have relatively large cell bodies almost exclusively in the inner nuclear layer with processes that densely arborize in the outermost stratum of the inner plexiform layer and fine, radially-oriented fibres in the inner nuclear layer. Type 2 catecholamine amacrine cells, in contrast, have smaller cell bodies in the inner nuclear layer, the inner plexiform layer and the ganglion cell layer, and have sparsely-branching processes ramifying in the centre of the inner plexiform layer. Although type 2 catecholamine cells are more numerous than type 1 catecholamine amacrines, type 2 cells contain less than one-third the amount of tyrosine hydrolase as the type 1 cells. Electron microscopy of retinal tissue immunoreacted for tyrosine hydrolase by the peroxidase-antiperoxidase method revealed synaptic input from amacrine cells at conventional synapses, and bipolar cells at ribbon synapses onto the type 2 catecholamine amacrine cells. Curiously, although the synaptic input is comparatively easily found, the output synapses, or synapses of the type 2 catecholamine amacrine cells onto other neuronal elements, are rarely found. Some synapses of the type 2 catecholamine cells onto non-immunoreactive amacrine cells have been identified, however. This unusual pattern of synaptic organization, with many identifiable input synapses but few morphologically characterizable output synapses, suggests a paracrine function for the dopamine released by the type 2 catecholamine amacrine cells in the primate retina.     

Neuronal and glial localization of homocysteate-like immunoreactivity in the rat retina

Summary To study the distribution ofl-homocysteate in the rat retina, specific polyclonal and monoclonal anti-homocysteate antibodies have been used in combination with a highly sensitive postembedding method for light microscopic immunocytochemistry. In central and peripheral retina, the most strongly immunoreactive cell bodies lay in the inner nuclear layer. They represented about 17% of the total neuronal cell population of the layer and were identified as bipolar cells (19–20% of cells in the outer half of the inner nuclear layer) and amacrine cells (15% of cells in the inner half of the inner nuclear layer). A third cell type showing heavy homocysteate-like immunoreactivity was identified as Müller glial cells. Characteristically, their descending processes formed three immunoreactive bands in the inner plexiform layer. Furthermore, the outer and inner limiting membranes as well as glia around and between ganglion cell axons and in the vicinity of blood vessels were labelled intensely. Photoreceptors and their terminals, and ganglion cells, were not immunostained. These findings indicate the presence of homocysteate in some bipolar and amacrine cells of the inner nuclear layer and support a role for this sulphur-containing excitatory amino acid as a neurotransmitter candidate in the retina.     

The birthdates of GABA-immunoreactive amacrine cells in the rat retina

The birthdates of GABAergic amacrine cells in the rat retina were investigated by immunocytochemistry using anti-GABA and anti-bromodeoxyuridine (BrdU) antisera. The ratio of co-localization of GABA to BrdU increased gradually from embryonic-day 13 (E13) and showed a peak value on E18 in the central retina and on E20 in the periphery. After birth, until postnatal-day 3 (P3), a few co-localized cells were observed in the inner nuclear layer (INL). However, in the peripheral retina, co-localized cells were observed in the INL and ganglion cell layer until P5. Our results suggest that the birthdates of GABA-immunoreactive cells vary, depending on cell-type and that there is a temporal lag in the GABA-immunoreactive cell production in the peripheral retina relative to the central retina. Received: 11 January 1999 / Accepted: 20 April 1999     

Cellular and subcellular localization of heme oxygenase-2 in monkey retina

Carbon monoxide (CO), an activator of soluble guanylate cyclase (SGC) and generated enzymatically by heme oxygenases (HO), is considered to function as an intra- and intercellular neuromodulator or neurotransmitter in the central and peripheral nervous systems. HO-2 is the constitutive isoform of HO and is more prevalent in nervous tissues than in the other peripheral tissues. Because previous studies have demonstrated different distributions of HO-2 in the retina depending on the species of animals, the aim of this study was to identify which cell types of the monkey retina express HO-2. The expression of HO-2 protein was examined in monkey retina by Western blot analysis. Immunoblottings from monkey homogenates revealed a single clear protein band with a molecular mass of 36 kDa that is corresponding to rat HO-2. Immunoreactivity of HO-2 was found in the perikarya of ganglion cells. Density of immunoreactive ganglion cells was higher in the central area of retina than in the peripheral retina, and somata of larger ganglion cells were stained more densely than smaller ones. In electron microscopy, immunoreactivity of HO-2 was localized on the membrane of the endoplasmic reticulum and the nuclear outer membrane of the ganglion cells. By contrast, inner plexiform layer, inner nuclear layer and outer nuclear layer were devoid of HO-2 immunoreactivity. cGMP were strongly localized in all of ganglion cells. Some cells contributed to the relatively faint cGMP staining were seen in the inner nuclear layer. In combination of HO-2 and cGMP immunocytochemistry, the overlap of co-localization of HO-2 and cGMP would suggest that HO-2 in the ganglion cells would serve as a source for CO generation and CO could serve as a gaseous signaling molecule modulator of neural activity in the retina of monkey.     

Co-localization of Shaker A-type K+ channel (Kv1.4) and AMPA-glutamate receptor (GluR4) immunoreactivities to dendrites of OFF-bipolar cells of goldfish retina

Immunocytochemical methods were used to determine the comparative distribution of Shaker Kv1.4 and Shal Kv4.2 A-type voltage-gated K⁺ channels and AMPA-type GluR4 glutamate receptors in the goldfish retina. Kv1.4-immunoreactivity (IR) was restricted to a very narrow band of bright puncta and filamentous processes in the outer plexiform layer (OPL), whereas GluR4-IR was found in radial processes of Müller cells in addition to a narrow band in the OPL. Kv4.2-IR was most prominent over cell bodies of horizontal cell, amacrine cells and ganglion cells, with very weak labeling over the synaptic terminal of cone photoreceptors. Double label experiments revealed complete co-localization of Kv1.4-IR and GluR4-IR in the OPL and showed that the Kv1.4 puncta in the OPL appeared enclosed by the Kv4.2-IR cone terminals. Electron microscopical immunocytochemistry showed that Kv1.4-IR and GluR4-IR were restricted to the dendrites of OFF-bipolar cells that innervated cone photoreceptor terminals and thin processes that coursed between the rod and cone terminals in the OPL. These data are consistent with other studies demonstrating the selective clustering of A-type voltage-gated K⁺ channels and ionotropic glutamate receptors. However, they differ from mammalian preparations in which Shal-like Kv4.2 rather than Shaker-like Kv1.4 co-localize postsynaptically with glutamate receptors.     

A system of corticotropin releasing factor-containing amacrine cells in the rat retina

Immunohistochemical processing of Long-Evans retina wholemounts using an antiserum directed against rat, human corticotropin releasing factor revealed a group of immunoreactive amacrine cells. Two subpopulations could be distinguished based primarily on the location of their cell bodies. One subpopulation had cell bodies situated along the junction of the inner nuclear layer and the inner plexiform layer. The other subpopulation had cell bodies in the ganglion cell layer. The latter was judged to be displaced amacrine cells since double-label experiments indicated that the pattern of corticotropin releasing factor-like immunoreactive staining in the ganglion cell layer did not coincide with that of ganglion cells labeled retrogradely with fluorogold. Corticotropin releasing factor-like immunoreactive amacrine cells on either side of the inner plexiform layer emitted processes which ramified extensively in sublamina 5 and, to a lesser degree, in sublamina 4. A minority of these cells also sent a single process to ramify in sublamina 1. Throughout the retina, corticotropin releasing factor-like immunoreactive cells were distributed relatively evenly, with a tendency to peak in the superior temporal region. Despite the anatomical classification into two subpopulations, it is proposed that the corticotropin releasing factor-like immunoreactive cells are functionally one system, influencing preferentially synaptic interactions associated with the inner half of the inner plexiform layer. The results of this study provide anatomical basis for further investigations of corticotropin releasing factor as a putative peptidergic neurotransmitter in the retina.     

ENK与CaBPs在PPE-GFP转基因小鼠视网膜细胞中的共存

目的:以PPE-GFP转基因小鼠为研究工具,观察绿色荧光蛋白(GFP)阳性的脑啡肽(ENK)能神经元与钙结合蛋白D28K(CB)、钙视网膜蛋白(CR)和小白蛋白(PV)等钙结合蛋白(CaBPs)成员在视网膜的分布及共存情况。方法:利用免疫组织化学和免疫荧光双标染色的方法。结果:GFP阳性的ENK能细胞主要分布在视网膜内核层内缘,少量分布在节细胞层。所有的GFP阳性细胞均与神经元标志物NSE共存,但不与星形胶质细胞标志物GFAP共存。GFP与CB、CR和PV均有部分共存,其中GFP/CB共存神经元占GFP阳性细胞的8.65%,占CB阳性细胞的5.84%;GFP/CR共存神经元占GFP阳性细胞的18.18%,占CR阳性细胞的14.28%,且共存细胞仅见于内核层;GFP/PV共存细胞占GFP阳性细胞的68.75%,占PV阳性细胞的91.67%,共存细胞主要位于内核层,少量见于节细胞层。结论:ENK能神经元在视网膜内具有板层特异性的分布特点和与钙结合蛋白成员有不同的共存模式,上述结果为深入研究小鼠视网膜ENK能神经元的功能意义提供了形态学依据。     

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。