Characterization of green fluorescent protein-expressing retinal cells in CD 44-transgenic mice

Sensory information in the retina is transferred from rod and cone photoreceptors to higher visual centers via numerous parallel circuits that sample the photoreceptor mosaic independently. Each circuit consists of a unique combination of ganglion cell, bipolar and amacrine cell types. The morphology and physiological responses of many amacrine cells have been characterized. However, the synaptic connections and retinal circuits in which they participate are only rarely understood. A major problem that has prevented fuller characterization of retinal circuitry is the need for specific cellular markers for the more than 50 inner retinal cell types. One potential strategy for labeling cells is to use transgenic expression of a reporter gene in a specific cell type. In a recent study of cluster of differentiation 44 (CD44)-enhanced green fluorescent protein (EGFP) transgenic mice, we observed that the green fluorescent protein (GFP) was expressed in a population of amacrine and ganglion cells in the inner nuclear layer (INL) and the GCL. To characterize the morphology of the GFP-labeled cells, whole mount preparations of the retina were used for targeted iontophoretic injections of Lucifer Yellow and Neurobiotin. Furthermore, immunocytochemistry was used to characterize the antigenic properties of the cells. We found that many GFP-expressing cells were GABAergic and also expressed calretinin. In addition to the somatic staining, there was a strong GFP(+)-band located about 50-60% depth in the inner plexiform layer (IPL). Double labeling with an antibody to choline acetyltransferase (ChAT) revealed that the GFP-band was located at strata 3 inner retina. The best-labeled GFP-expressing cell type in the INL was a wide-field amacrine cell that ramified in stratum 3. The GFP-expressing cells in the GCL resemble the type B1, or possibly A2 ganglion cells. The CD44-EGFP mice should provide a valuable resource for electrophysiological and connectivity studies of amacrine cells in the mouse retina.     

大鼠视网膜神经元NOS与Parvalbumin的共存研究

用NADPH脱氢酶组化及Parvalbumin免疫组化双标记技术观察了正常大鼠视网膜一氧化氮合酶(NOS)与Parvalbumin(PV)的分布,结果显示NOS阳性神经元主要位于内核层内缘带第二列,少数位于节细胞层,胞体圆形/卵圆形,直径8～12μm,细胞一侧发出突起伸向内网层1、3、5亚层,以第3亚层最为明显,PV免疫反应(PV—Ⅰ)神经元位于内核层最内缘第一列,少数位于第二列、中间部及节细胞层,胞体卵圆形,直径6～10μm,由胞体一端发出突起伸向内网层第1、5亚层.神经纤维层可见PV～Ⅰ纤维.内核层内缘第二列可见少数双标阳性细胞,在它们的PV免疫反应胞质内散布有NOS颗粒.实验结果表明 NOS阳性神经元与PV～Ⅰ神经元均为无长突细胞,分属不同的亚型,少效PV～Ⅰ神经元属节细胞,个别双标细胞可能为另一种亚型的无长突细胞,提示NOS与PV在视觉信息传递中可能存在某些联系.     

Substance P-immunoreactive neurons in the retina of two lizards.

The dendritic morphology and retinal distribution of substance P(SP)-immunoreactive neurons was determined in two Australian lizard species Pogona vitticeps and Varanus gouldii, by using immunohistochemistry on retinal wholemounts and sectioned materials. In both species, two classes of SP-immunoreactive neurons were described in the inner nuclear layer (INL) and classified as amacrine cells (types A and B). Type A amacrine cells had large somata and wide-field, bistratified dendrites branching in sublaminas 1 and 5 of the inner plexiform layer (IPL). Their morphology and retinal distribution differed between the two species. Type B amacrine cells in both species had small somata and small-field dendritic branching. A population of SP-immunoreactive neurons with classical ganglion cell morphology were identified in the ganglion cell layer (GCL). Immunostained ganglion cells occurred in larger numbers of Varanus gouldii than in Pogona vitticeps. In both species type B SP cells were the most numerous and were estimated to be about 60,000-70,000. They were distributed non-uniformly with a high density band across the horizontal meridian of the retina, from where the density decreased towards the dorsal and ventral retinal margins. In both species type A amacrine cells occurred in small numbers distributed sparsely in the peripheral retina. The faint immunostaining of SP-immunoreactive neurons in the GCL, did not allow us to reliably determine their numbers and retinal distribution. The functional significance of SP-immunoreactive amacrine and ganglion cells in the lizard retina remains to be determined.     

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。     

The development of Kv4.2 expression in the retina

Throughout the brain, the potassium channel Kv4.2 regulates signal propagation in dendrites and action potential properties in subtypes of neurons. In adult rodents Kv4.2 is expressed predominantly in two bands in the inner plexiform layer (IPL) and in retinal ganglion cell (RGC) somas (Klumpp et al. [15]; Pinto and Klumpp [20]), suggesting a role regulating the activity of specific subtypes of RGCs. To understand the role of Kv4.2 in the regulation of the activity of RGCs during development we determined the developmental expression pattern of Kv4.2 immunoreactivity (Kv4.2-IR). At P4-6 Kv4.2-IR appeared diffusely throughout the IPL in cross-sectioned retinas. From postantal day 10 (P10) through adult there was an additional pair of brighter Kv4.2-IR bands between the ChAT bands that had a reticular pattern in flat-mounted retinas. Kv4.2-IR was not present in somas at P4–6, but appeared in ganglion cell layer (GCL) somas beginning at P10. The fraction of somas expressing Kv4.2 in the GCL was about 8% at P10–11, decreased to 5% at P20–21, then increased to 9% in adult retinas. The restriction of Kv4.2 expression to less than 10% of the GCL somas and the specificity of expression in the IPL suggest that Kv4.2 regulates activity in one or a few functional subtypes of RGCs. The pattern of Kv4.2-IR through postnatal development indicates that Kv4.2-mediated currents are important for development in a subset of RGCs, especially around P10 as the bipolar cells mature.     

Müller细胞反应性胶质化在急性高眼压致大鼠视网膜损伤中作用

 目的 观察Müller细胞反应性胶质化在急性高眼压(AOH)大鼠视网膜中变化及其抑制对视网膜损伤的影响。方法 建立大鼠AOH青光眼模型,分为正常对照(Ctrl)、AOH和AOH+玻璃体内注射胶质毒素α-氨基己二酸(AAA)后再灌注1、3和5d组,以及单纯AAA和AOH+PBS对照组。TUNEL染色检测细胞凋亡,GFAP免疫荧光染色反应Müller细胞反应性胶质化程度,Thy-1染色标记视网膜神经节细胞(RGCs)。结果 AOH可致大鼠视网膜内丛状层和内核层明显变薄、神经节细胞层内细胞排列紊乱和数量减少,并诱发Müller细胞反应性胶质化(GFAP表达增加)。同时,AAA抑制Müller细胞反应性胶质化可明显缓解AOH所致RGCs丢失和凋亡发生。结论 Müller细胞反应性胶质化参与AOH所致视网膜损伤,抑制其反应性胶质化可能是改善高眼压性青光眼视网膜病变的一种有效治疗方法。     

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.     

脑源性神经营养因子对高眼压后视网膜节细胞的保护作用

目的　观察大鼠眼高压所致视网膜损伤及脑源性神经营养因子 (BDNF)对节细胞的保护作用。方法　以生理盐水加压注入Wistar大鼠眼前房至动物视网膜电图 (ERG)b波消失的临界压并维持 90min ,制成大鼠急性高眼压模型。实验组加压前 2d玻璃体内注射BDNF(3μg/kg ,0 1μg/ μl) ,实验对照组注射等量小牛白蛋白 ,加压后存活 3d复查ERG后处死 ,美蓝染色后光镜下观察节细胞的形态改变并作细胞计数分析 ,测量视网膜内核层 (INL)及内网层外缘至内界膜 (IPL ILM)的厚度。ABC免疫组化法检测谷氨酸的表达变化。结果　视网膜高眼压后IPL -ILM厚度小于正常对照组 ;节细胞数目减少 ,部分节细胞呈现坏死特征。BDNF处理后视网膜病理改变有明显改善 ,与实验对照组比较节细胞存活数增加 ,IPL ILM厚度大于实验对照组。高眼压后视网膜内可见谷氨酸免疫反应阳性双极细胞 ,BDNF处理后其免疫反应性类似于正常对照组。 3d后视网膜电图b波在BDNF处理组为正常的 75 3% ,而实验对照组仅为正常的 11 4 % (P <0 0 5 )。结论　大鼠眼高压可导致视网膜节细胞死亡 ,BDNF对高压后节细胞具有明显的保护作用。兴奋性氨基酸对高眼压后节细胞的损害可被BDNF改善     

Lucifer yellow stains displaced amacrine cells of the chicken retina during embryonic development

We have used Lucifer Yellow for histological tracing of displaced amacrine cells within the ganglion cell layer (GCL) during the embryonic development of the chicken retina. Incubating whole eyes in the dye leads to bright staining of all displaced amacrine cells, whereas ganglion cells and glial cells are not stained. A subpopulation of cells of the inner part of the inner nuclear layer (INL) are also stained (for further details see ref. 13). Kainic acid, which is known to interfere with and kill amacrine cell systems, blocks the staining of these cells fully. This in addition to histological evidence confirms that the LY-stained cells in the GCL are displaced amacrine cells. Of the cells in the GCL, 23% (+/- 3%) are of the displaced amacrine type. Further, we find that the cytoarchitectural arrangement of these cells changes significantly during development.     

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.     

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。     

GABA_A和GABA_C受体各亚单位基因在鲫鱼视网膜内的分布──原位杂交组织化学法研究

应用原位杂交组织化学技术，利用同位素［￣（３５）Ｓ］－ｄＡＴＰ标记的寡核苷酸探针，在鲫鱼视网膜观察了含ＧＡＢＡＡ受体α１、α３、α４、α６，β１－３，γ１－２及ＧＡＢＡＣ受体ρ１亚单位ｍＲＮＡ的神经元分布。在外核层，所有测试的亚单位均无表达；而在内核层和神经节细胞层，除α４和γ２亚单位外，均有不同程度的表达。在不同区域标记神经元的数量和标记强度各不相同，α１亚单位广泛分布在内核层的远端、中部及神经节细胞层，呈强阳性；α３亚单位相对稀少，主要分布在内核层近端和神经节细胞层，呈中等阳性；α４和α６亚单位几乎无阳性表达，仅α６亚单位在神经节细胞层呈弱阳性。β１和β２亚单位在内核层及神经节细胞层呈中等阳性；β３亚单位主要分布在内核层，在神经节细胞层标记细胞较少，呈弱阳性。γ１亚单位分布在整个内核层，在神经节细胞层有零星阳性表达。ＧＡＢＡＣ受体主要分布在内核层，ρ１亚单位主要分布在内核层的远端及中间部分，呈强阳性，而在神经节细胞层表达相对较弱。这种独特的表达型式与其功能密切相关。     

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.     

The inner plexiform layer in the retina of the cat: electron microscopic observations

Summary Neural connections of cells ramifying in the inner plexiform layer of the cat retina have been studied by serial section electron microscopy. Flat cone bipolars and invaginating cone bipolars segregate their axon terminals to different sublaminae of the IPL (sublaminaa and sublaminab, respectively) where they relate to different subtypes of the same class of ganglion cell (a andb types respectively).Rod bipolar axon terminals end solely in sublaminab and synapse with amacrine cells (AI and AII). AI provides reciprocal synapses to clusters of rod bipolar axon terminals. The AII amacrine provides rod input toa type ganglion cells by means of chemical synapses and tob type ganglion cells through gap junctions with invaginating cone bipolar terminals.Amacrine cells exist which interconnect rod and cone bipolars, but some amacrines appear to be related specifically to neurons branching in particular sublaminae. Both large- and small-bodied ganglion cells have amacrine-dominated input while the medium-bodied ganglion cells with small dendritic trees have cone bipolar-dominated input.     

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。     

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.     

The generation and changing retinal distribution of displaced amacrine cells in Bufo marinus from metamorphosis to adult.

Summary The generation and retinal distribution of displaced amacrine cells (DAs) were studied from metamorphosis to adult in the cane toad Bufo marinus. Displaced amacrine cells were identified by inducing chromatolytic changes in ganglion cells (GCs) following optic nerve section. Cells that did not chromatolyse in the ganglion cell layer (GCL) of the retina were regarded as DAs. The number of DAs increased considerably from an estimated 10000 at metamorphosis to 211000 in the adult toad, and was accompanied by a substantial decrease of average cell density. In contrast to the reported 6:1 cell density gradient of all cells of the GCL in adult toad (Nguyen and Straznicky 1989) only a shallow 1.6:1 density gradient of DAs from the visual streak to the dorsal and ventral retinal margins was detected. Consequently, the incidence of DAs increased from 15% of all cells of the GCL in the visual streak to 30% in the dorsal and ventral peripheral retina. These results indicate that the ratio of the newly generated DAs and GCs at the ciliary margin must be changing during development. More GCs are generated before and around metamorphosis then DAs, in contrast to the relative increase of the percentage of DAs generated after metamorphosis. The possible control of the numbers of DAs in the GCL is discussed.     

Enhanced protein expressions of sortilin and p75NTR in retina of rat following elevated intraocular pressure-induced retinal ischemia

Elevated introcular pressure (IOP)-induced retinal neuron ischemic death includes an early phase of necrosis and prolonged phase of apoptosis. We used this ischemic model to observe the changes of sortilin and p75(NTR) protein expressions in rat retina. The results of Western blot analysis showed the expression of p75(NTR) at the band of 75 (mature form), 60 (non-glycosylated pieces) and 50 kDa (ectodomain shedding pieces), and the expression of sortilin at the 95 and 90 kDa (the mature form). The protein expressions of p75(NTR) (60 and 50 kDa pieces) and sortilin (90 kDa) increased significantly (p < 0.05) at days 3, 5 and 7 after retinal ischemia. This effect was also confirmed by immunofluorescence staining. Sortilin was primarily present in cell membrane of the ganglion cells layer (GCL) and large ganglion cell bodies by immunofluorescence labeling. There was little expression of p75(NTR) in the normal retina, while expression increased extensively in GCL, inner plexiform layer (IPL) and inner nuclear layer (INL) after retinal ischemia. p75(NTR) was shown to co-localize with neurofilament in the axons of neuronal cells by double-labeling. These results suggested that the protein expressions of 60 and 50 kDa forms of p75(NTR), and the 90 kDa mature form of sortilin increased in ischemia-induced retinal neuron of rats.     

Expression of somatostatin receptor subtype 5 in rat retinal amacrine cells

Somatostatin (SRIF), as a neuroactive peptide in the CNS, exerts its actions via five subtypes of specific receptors (ssts). In this work, the localization of sst(5) was studied immunocytochemically in rat retinal amacrine cells (ACs). Labeling for sst(5) was diffusely distributed throughout the full thickness of the inner plexiform layer (IPL) and formed two distinct fluorescence bands in the distal part of the IPL. Double labeling experiments showed that sst(5) was expressed in GABAergic ACs. It was further shown that labeling for sst(5) was observed in both dopaminergic and cholinergic ACs, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively. The immunostaining appeared mainly on the cell membranes and somatodendritic compartments of these ACs. For the cholinergic ACs, weak sst(5)-immunoreactivity was also observed in the processes terminating in the IPL. In contrast, no sst(5)-immunoreactivity was found in glycinergic AII ACs, stained by parvalbumin (PV). Furthermore, labeling for SRIF was co-localized with sst(5) in both dopaminergic and cholinergic ACs. These results suggest that sst(5) may serve as an autoreceptor or conventional receptor in retinal ACs.     

脑啡肽与生长抑素在鸡视网膜无长突细胞共存的免疫组织化学研究

本文介绍用免疫组织化学的单标和双标技术研究脑啡肽(ENK)和生长抑素(SOM)在鸡视网膜无长突细胞的定位和共存。单标的实验结果表明,一些SOM免疫反应阳性无长突细胞的形态、胞体在内核层的位置及其突起在内网层的分支式样与某些ENK免疫反应阳性无长突细胞相似,虽然其突起在内网层的第3、4亚层形成的丛网不象ENK免疫反应阳性突起那样丛密,在内网层的第5亚层也未见SOM免疫阳性突起。双标的实验结果表明,一些无长突细胞显示ENK和SOM两种免疫阳性反应,而另一些无长突细胞分别只显示ENK或SOM阳性免疫反应。文中还对视网膜神经多肽间或与经典神经递质的共存进行了讨论。