Expression of brain-derived neurotrophic factor in cholinergic and dopaminergic amacrine cells in the rat retina and the effects of constant light rearing

Brain-derived neurotrophic factor (BDNF) regulates many aspects of neuronal development, including survival, axonal and dendritic growth and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF in retinal development, the retinal cell types expressing BDNF remains poorly defined. The goal of the present study was to determine the localization of BDNF in the mammalian retina, with special focus on the subtypes of amacrine cells, and to characterize, at the cellular level, the effects of constant light exposure during early postnatal period on retinal expression of BDNF. Retinas from 3-week-old rats reared in a normal light cycle or constant light were subjected to double immunofluorescence staining using antibodies to BDNF and retinal cell markers. BDNF immunoreactivity was localized to ganglion cells, cholinergic amacrine cells and dopaminergic amacrine cells, but not to AII amacrine cells regardless of rearing conditions. Approximately 75% of BDNF-positive cells in the inner nuclear layer were cholinergic amacrine cells in animals reared in a normal lighting condition. While BDNF immunoreactivity in ganglion cells and cholinergic amacrine cells was significantly increased by constant light rearing, which in dopaminergic amacrine cells was apparently unaltered. The overall structure of the retina and the density of ganglion cells, cholinergic amacrine cells and AII amacrine cells were unaffected by rearing conditions, whereas the density of dopaminergic amacrine cells was significantly increased by constant light rearing. The present results indicate that cholinergic amacrine cells are the primary source of BDNF in the inner nuclear layer of the rat retina and provide the first evidence that cholinergic amacrine cells may be involved in the visual activity-dependent regulation of retinal development through the production of BDNF. The present data also suggest that the production or survival of dopaminergic amacrine cells is regulated by early visual experience.     

Localization ofSix4/AREC3 in the Developing Mouse Retina; Implications in Mammalian Retinal Development

TheSix4/AREC3 gene was originally isolated as a regulatory factor which bound to the positive regulatory region of the Na, K-ATPase α1 subunit. It is a murine homologue of theDrosophila sine oculis (so)gene, which is essential for the development of the entire insect visual system. In this study, we attempted to determine the localization of theSix4/AREC3 gene product in the developing mouse retina in order to examine its role in retinal cell differentiation. Immunohistochemistry with anti-SIX4/AREC3 and anti-Na, K-ATPase α1 subunit antisera was performed on developing mouse retinas, and immunoblotting analysis with anti-SIX4/AREC3 was also performed. The localization ofSix4-like immunoreactivity (Six4-LI) showed a temporally regulated pattern: During embryonic development,Six4-LI was found in the nuclei of cells located at the inner neuroblastic layer of the retina as early as on ED12, nearly corresponding to the onset of retinal cell differentiation. In the PD1 retina,Six4-LI was observed in the nuclei of the ganglion cells, and increased its intensity until PD4, and thereafter kept its intensity until PD7 whenSix4-LI was often found in the cytoplasm. On PD4, the presumptive amacrine cells found in the inner portion of the inner nuclear layer appeared to be immunostained in their nuclei. On PD7, the presumptive bipolar cells located in the outer portion were immunostained in the nuclei. After that,Six4-LI gradually decreased, and in the mature retina no detectableSix4-LI was observed in the nuclei. This pattern ofSix4-LI localization during retinal development seemed to correlate with retinal cell differentiation, but did not correlate with the distribution pattern of Na, K-ATPase α1 subunit protein-like immunoreactivity. These results suggest that theSix4gene may play a role in the differentiation of neural retinal cells during mouse retinal development, rather than regulating the expression of the Na, K-ATPase α1 subunit gene.     

Primary Cilia in Amacrine Cells in Retinal Development

PurposePrimary cilia are conserved organelles found in polarized cells within the eye that regulate cell growth, migration, and differentiation. Although the role of cilia in photoreceptors is well-studied, the formation of cilia in other retinal cell types has received little attention. In this study, we examined the ciliary profile focused on the inner nuclear layer of retinas in mice and rhesus macaque primates.MethodsRetinal sections or flatmounts from Arl13b-Cetn2 tg transgenic mice were immunostained for cell markers (Pax6, Sox9, Chx10, Calbindin, Calretinin, ChaT, GAD67, Prox1, TH, and vGluT3) and analyzed by confocal microscopy. Primate retinal sections were immunostained for ciliary and cell markers (Pax6 and Arl13b). Optical coherence tomography (OCT) and ERGs were used to assess visual function of Vift88 mice.ResultsDuring different stages of mouse postnatal eye development, we found that cilia are present in Pax6-positive amacrine cells, which were also observed in primate retinas. The cilia of subtypes of amacrine cells in mice were shown by immunostaining and electron microscopy. We also removed primary cilia from vGluT3 amacrine cells in mouse and found no significant vision defects. In addition, cilia were present in the outer limiting membrane, suggesting that a population of Müller glial cells forms cilia.ConclusionsWe report that several subpopulations of amacrine cells in inner nuclear layers of the retina form cilia during early retinal development in mice and primates.     

Calretinin immunoreactivity in the developing retina of sharks: Comparison with cell proliferation and GABAergic system markers

The calcium-binding protein calretinin (CR) has been widely used as a marker of neuronal differentiation. In the present study we analyzed the distribution of CR-immunoreactive (CR-ir) elements in the embryonic and postembryonic retina of two elasmobranchs, the lesser spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus). We compared the distribution of CR with that of a proliferation marker (the proliferating cell nuclear antigen, PCNA) in order to investigate the time course of CR expression during retinogenesis and explored the relationship between CR and glutamic acid decarboxylase (GAD), the synthesizing enzyme of the gamma-aminobutyric acid (GABA), which has been reported to play a role in shark retinogenesis. The earliest CR immunoreactivity was concurrently observed in subsets of: a) ganglion cells in the ganglion cell layer; b) displaced ganglion cells in the inner plexiform layer and inner part of the inner nuclear layer (INLi); c) amacrine cells in the INLi, and d) horizontal cells. This pattern of CR distribution is established in the developing retina from early stage 32, long after the appearance of a layered retinal organization in the inner retina, and coinciding with photoreceptor maturation in the outer retina. We also demonstrated that CR is expressed in postmitotic cells long after they have exited the cell cycle and in a subset of GABAergic horizontal cells. Overall our results provide insights into the differentiation patterns in the elasmobranch retina and supply further comparative data on the development of CR distribution in the retina of vertebrates. This study may help in understanding the possible involvement of CR in aspects of retinal morphogenesis.     

体外培养人胚胎来源视网膜干细胞的诱导分化

目的 探讨培养的人胚胎来源视网膜干细胞向视网膜终末细胞分化的可能性。方法 来自16～20周人胚胎的视网膜干细胞进行无血清体外培养,并分别进行有血清条件下体外诱导和用含视网膜色素上皮的眼杯模拟体内条件诱导的观察,采用免疫荧光法检测干细胞和视网膜终末细胞表面抗原的表达,采用实时荧光定量PCR法检测诱导前后细胞nestin基因在mRNA水平的表达差异。结果 从人胚胎视网膜神经感觉层分离出的视网膜干细胞,在体外诱导的条件下,可表达视网膜终末细胞标记PKCα、GFAP、Thy1,少数细胞表达nestin和MAP2;在模拟体内环境诱导后,则不仅表达上述细胞标记,而且rhodopsin和syntaxin表达阳性。实时荧光定量PcR法检测显示：诱导后细胞nestin基因表达量较诱导前细胞明显降低。结论 RPE可以促进体外培养的视网膜干细胞向视杆细胞和无长突细胞分化。(中华眼科杂志,2004,40：448-452)     

过氧化物酶体增生物激活受体γ在鼠眼球中的分布

背景过氧化物酶体增生物激活受体γ（PPAR-γ）是一类由配体激活的核转录因子,是潜在的抗炎、抗纤维增生、抗新生血管形成及神经保护因子,其在动物和人体组织中的生理病理功能是目前的研究热点之一,PPARγ与眼科疾病的研究受到关注。目的研究PPARγ在眼部不同组织细胞中的表达,为PPARγ激动剂在眼科疾病治疗中的应用提供参考依据。方法取SPF级C57BL／6J小鼠6只及SD大鼠1只,用质量分数3％水合氯醛麻醉处死后立即摘除眼球,采用Western blot法检测小鼠角膜、晶状体和视网膜组织中PPARγ蛋白的表达;采用免疫组织化学和免疫荧光化学法检测PPARγ在小鼠角膜、晶状体、视网膜、睫状体及视神经组织中的表达及定位。结果Western blot法检测表明,PPARγ在小鼠角膜、晶状体、视网膜中均呈阳性表达。免疫组织化学和免疫荧光化学法检测显示,PPARγ在角膜组织中主要表达于上皮层,以基底细胞染色最强,而角膜内皮及基质细胞上仅有弱表达。PPARγ在晶状体中主要表达于上皮细胞和浅皮质层;在视网膜组织中,PPARγ主要表达于视网膜节细胞层、内丛状层、外丛状层和内核层,此外PPARγ在SD大鼠睫状体组织中主要表达于无色素上皮。免疫荧光化学法检测显示,其在视网膜中与Muller细胞标志物谷氨酰胺合成酶（GS）共定位表达明显;PPARγ在视神经组织中的表达与星形胶质细胞标志物胶质纤维酸性蛋白（GFAP）共定位表达明显。结论PPARγ广泛分布于眼不同组织中并呈特异性表达,该结果为相关眼科疾病的靶向治疗提供了依据。     

Genetic targeting and physiological features of VGLUT3+ amacrine cells

Amacrine cells constitute a diverse class of interneurons that contribute to visual signal processing in the inner retina, but surprisingly, little is known about the physiology of most amacrine cell subtypes. Here, we have taken advantage of the sparse expression of vesicular glutamate transporter 3 (VGLUT3) in the mammalian retina to target the expression of yellow fluorescent protein (YFP) to a unique population of amacrine cells using a new transgenic mouse line. Electrophysiological recordings made from YFP-positive (VGLUT3+) amacrine cells provide the first functional data regarding the active membrane properties and synaptic connections of this recently identified cell type. We found that VGLUT3+ amacrine cells receive direct synaptic input from bipolar cells via both N-methyl-d-aspartate receptors (NMDARs) and non-NMDARs. Voltage-gated sodium channels amplified these excitatory inputs but repetitive spiking was never observed. VGLUT3+ amacrine cells responded transiently to both light increments (ON response) and decrements (OFF response); ON responses consisted exclusively of inhibitory inputs, while OFF responses comprised both excitatory and inhibitory components, although the inhibitory conductance was larger in amplitude and longer in time course. The physiological properties and anatomical features of the VGLUT3+ amacrine cells suggest that this bistratified interneuron may play a role in disinhibitory signaling and/or crossover inhibition between parallel pathways in the retina.     

AII amacrine cells express the MT1 melatonin receptor in human and macaque retina

AII amacrine cells are critical interneurons in the rod pathway of mammalian retina, active primarily in dim lighting conditions. Melatonin, a neuromodulator produced at night in the retina, is believed to induce retinal adaptation to dim lighting conditions in most vertebrate species examined to date, including humans. We hypothesized that melatonin may influence retinal light adaptation by acting on AII cells directly and thus investigated whether melatonin receptors were expressed in AII neurons. Postmortem nonpathological eyes from four human donors as well as two eyes from two Macaque Fasicularis monkeys were analyzed. Double immunocytochemistry was performed using an anti-MT(1) antibody and an antibody to calretinin, an AII marker. Analysis utilized confocal microscopy. A polyclonal anti-calretinin antibody labelled amacrine cells exhibiting the distinct AII morphology, in both human and macaque retina. MT(1) immunoreactivity in macaque retina was similar to human staining, in that horizontal, amacrine and ganglion cell bodies were stained, as were inner segments of photoreceptors. In human retina 86% of calretinin positive cells expressed the MT(1) receptor peripherally, whereas centrally, 78% colocalization was observed. In the macaque retina, 100% of AII amacrine cells expressed MT(1) immunoreactivity both centrally and peripherally. That virtually all AII neurons express the MT(1) receptor in both human and macaque retina, may provide the first evidence demonstrating a role for melatonin in AII regulation, furthering the hypothesis of melatonin function in retinal light adaptation.     

利用Cre/loxP系统定位腺苷A2A受体在小鼠视网膜上的分布

目的：运用Cre/loxP系统来定位腺苷A2A 受体（A2AR）在小鼠视网膜上的分布情况。方法：实验研究。将雄性B6.FVB（Cg）-Tg（Adora2a-cre KG139Gsat/Mmucd（简称A2A-cre）小鼠与雌性B6．129S6-Gt（ROSA）26Sortm9（CAG-tdTomato）Hze/J（简称Ai9）小鼠交配,对新生小鼠基因型进行鉴定,并选取5 只4 周龄A2A-cre+,Ai9 鼠,此小鼠表达的Cre酶能敲除Ai9 小鼠中的STOP序列使Tomato荧光蛋白表达。采用免疫荧光方法分别将Tomato红色荧光蛋白与不同类型的视网膜细胞抗体共标记,通过观察Tomato红色荧光蛋白和视网膜细胞特异性抗体的共定位结果,来明确A2AR在视网膜上的定位情况。结果：免疫荧光结果显示Tomato+细胞主要分布在视网膜神经节细胞层和内核层,外核层并没有发现。其中神经节细胞层中的Tomato+细胞少数为神经节细胞,多数为异位无长突细胞,而分布于内核层的Tomato+细胞主要为Müller细胞和无长突细胞。进一步对无长突细胞亚型分析发现,Tomato+无长突细胞多数为AⅡ型无长突细胞,而胆碱能无长突细胞和多巴胺能无长突细胞并无Tomato+表达。结论：在4 周龄A2A-cre+,Ai9 小鼠视网膜中,A2AR主要表达于神经节细胞、无长突细胞和Müller细胞中。     

Endothelial nitric oxide synthase is expressed in amacrine cells of developing human retinas

PURPOSE: To examine the expression and cellular distribution pattern of endothelial nitric oxide synthase (eNOS) in the developing human retina and to compare its expression with that in rats. METHODS: Expression of eNOS was examined by immunohistochemistry in retinas of humans ranging from 8.5 to 28 weeks of gestation (WG) and of rats. RESULTS: In the developing human retina, eNOS expression was first detected in the proximal margin of the neuroblastic layer in the incipient fovea-surrounding area at 12 WG. At 17 to 28 WG, eNOS-immunoreactive cells were located in the innermost part of the inner nuclear layer and in the ganglion cell layer, expanding to both temporal and nasal retinas and the processes projecting into the inner plexiform layer. These eNOS-positive cells coexpressed syntaxin and glutamate decarboxylase, and are probably GABAergic amacrine cells. The onset of eNOS expression in developing amacrine cells, however, preceded the invasion of retinal vasculature, long before vascular function involving these cells can be expected, suggesting that eNOS has a role not only in vasoregulation but also in retinal development. From 20 WG on, eNOS was also detected in the photoreceptors adjacent to the fovea. eNOS expression in amacrine cells and photoreceptors was observed in the central-to-peripheral and temporal-to-nasal gradients. However, in the developing rat retina, eNOS was expressed exclusively in the vascular endothelial cells. CONCLUSIONS: The results support that eNOS plays a role, not only in the regulation of vascular function but also in the process of retinal development in humans.     

OPA1, the disease gene for autosomal dominant optic atrophy, is specifically expressed in ganglion cells and intrinsic neurons of the retina

PURPOSE: Autosomal dominant optic atrophy is a hereditary disorder characterized by progressive loss of vision and caused by mutations in a dynamin-related gene, OPA1, which translates into a protein with a mitochondrial leader sequence. In this study the OPA1 gene and its protein were localized in the rat and mouse retina, and its rat orthologue, rOpa1, was identified. METHODS: The rOpa1 cDNA was isolated by using reverse transcribed cDNA from total RNA obtained from a rat retinal ganglion cell line. The spatial and temporal expression patterns of OPA1 and its gene product were investigated by RNA in situ hybridization and immunohistochemistry in mouse and rat retinas. To characterize further the OPA1-positive neurons, retinal ganglion cells were retrogradely labeled by an immunogold fluorescent tracer or double labeled with OPA1 and choline acetyltransferase or calbindin antibodies. RESULTS: Protein sequence alignment revealed a 96% identity between rat and human OPA1 mRNA. OPA1 expression was found as early as postnatal day 3 in the developing rodent retina. In the mature retina, the OPA1 gene and its protein were found not only in retinal ganglion cells, but also in starburst amacrine cells and horizontal cells, both of which are involved in lateral signal processing within the retina. However, OPA1 was absent from mitochondria rich nerve fibers and photoreceptor indicating a specific role for OPA1 in signal processing rather than in the requirement of mitochondrial energy supply in the retina. CONCLUSIONS: The data suggest an important and specific function of the OPA1 protein, not only in the optic nerve forming ganglion cells but also in the intrinsic signal processing of the inner retina.     

Cell differentiation in the retina of an epibenthonic teleost, the Tench (Tinca tinca, Linneo 1758)

Here we present a detailed study of the major events in the retinal histogenesis in a freshwater epibenthonic fish species, the Tench (Tinca tinca, Linneo 1758) during embryonic, prolarval, larval, and juvenile stages, using classical histological and immunohistological methods, providing a complete neurochemical characterization of retinal cells. We find a morphologically undifferentiated retina during embryonic stages and even at the hatching stage (postnatal day 0, P0). However, the emergence of the different retinal layers occurs in the first postnatal day (P1). Proliferating PCNA-positive cells are found in the retina of all postnatal individuals included in the present study, located in the circumferential germinal zone (CGZ), and in sparse cells dispersed throughout the inner nuclear layer (INL) and the outer nuclear layer (ONL). All neurochemical markers used start to express between P0 and P2. Anti-opsin, -α-protein kinase C, -α-tyrosine hydroxylase, -glutamine synthetase antibodies stain selectively different subpopulations of photoreceptor, bipolar, amacrine, and Müller cells respectively. Parvalbumin immunoreactivity is detected in amacrine and displaced amacrine cells. Several subpopulations of calretinin-positive ganglion, amacrine, and bipolar cells are detected in tench retina. Islet1 expression is confined to the nuclei of subpopulations of ganglion, amacrine, bipolar, and horizontal cells. All the maturational events described are first detected in the central retina and, as development progresses, they spread to the rest of the retina following a central-to-peripheral gradient. Therefore, tench postnatal retinal differentiation is a remarkable process not observed in the more common models of teleosts used in developmental biology.