An immunocytochemical marker for hamster retinal ganglion cells

Summary We examined the specificity and developmental time course of the labelling of retinal ganglion cells in Syrian hamsters by a monoclonal antibody AB5. In adult hamsters, AB5 selectively labelled somata in the ganglion cell layer, dendrites in the inner plexiform layer and axons in the nerve fibre layer. When retinal ganglion cells were retrogradely labelled with Dil prior to AB5 immunocytochemistry, all of the retrogradely labelled retinal ganglion cells in the ganglion cell layer were AB5 immunoreactive, indicating that AB5 labels all classes of ganglion cell in that layer. In retinae depleted of retinal ganglion cells by neonatal optic nerve transections, AB5 did not label any somata or processes, indicating that AB5 specifically labels retinal ganglion cells. During development, AB5 labelling first appeared as a weak staining of cell bodies in the ganglion cell layer on postnatal day 12 (P12; PO=first 24 h following birth) and acquired the staining pattern seen in the adult by postnatal day 14. From the onset of AB5 immunoreactivity, AB5-labelled somata of varying sizes were present across the entire retinal surface. Although AB5 labelled retinal ganglion cell axons in the nerve fibre layer of the retina it did not label the optic nerve or retinal ganglion cell axons in the brain at any age examined. AB5 labelling was also found to be compatible with bromodeoxyuridine immunocytochemistry and, therefore, useful for determining the time of generation of hamster retinal ganglion cells.     

Generation of retinal cells in the wallaby, Setonix brachyurus (quokka)

We have examined the generation of retinal cells in the wallaby, Setonix brachyurus (quokka). Animals received a single injection of tritiated thymidine between postnatal days 1-85 and retinae were examined at postnatal day 100. Retinae were sectioned, processed for autoradiography and stained with Cresyl Violet. Ganglion cells were labelled by injection of horseradish peroxidase into the optic tracts and primary visual centres. Other cells were classified according to their morphology and location. Retinal cell generation takes place in two phases. During the first phase, which concludes by postnatal day 30, cells destined to lie in all three cellular layers of the retina are produced. In the second phase, which starts by postnatal day 50, cell generation is almost entirely restricted to the inner and outer nuclear layers. Cells produced in the first phase are orthotopic and displaced ganglion cells, displaced and orthotopic amacrine cells, horizontal cells and cones. Glia in the ganglion cell layer, orthotopic amacrine cells, bipolar and horizontal cells. Muller glia, and rods are generated in the second phase. Cells became heavily labelled with tritiated thymidine in the central retina before postnatal day 7, over the entire retina (panretinal) by postnatal day 7 and from postnatal day 18, only in the periphery. The second phase of cell generation is initiated at P50, in a region extending from the optic nerve head to mid-temporal retina. Subsequently, cells are generated in annuli, centred on mid-temporal retina, which are seen at progressively more peripheral locations. Therefore, cell addition to the inner and outer nuclear layers continues for longer in peripheral than in mid-temporal retina. We suggest that such later differential cell addition to the inner and outer nuclear layers contributes to an asymmetric increase in retinal area. This non-uniform growth presumably results in more expansion of the ganglion cell layer peripherally than in mid-temporal retina and may play a role in establishing density gradients of ganglion cells.     

Specific transcellular carbocyanine-labelling of rat retinal microglia during injury-induced neuronal degeneration

The present work employed a new technique for labelling phagocytizing microglia in the axotomized retinal of adult rats. Transection axotomy was performed within the intraorbital segment of the optic nerve, and the fast-transported, vital fluorescent carbocyanine dyes DiI and 4Di-10ASP were deposited at the ocular stump of the nerve in order to retrogradely prelabel the ganglion cells which were destined to die. Optic nerve transection resulted in progressive degradation of ganglion cell axons, perikarya and dendrites within the retina and in release of fluorescent material which was then incorporated into cells identified as microglia but not into other cells of the retina. Incorporation of labelled material into microglia occurred only when the ganglion cells degenerated and not when the non-lesioned ganglion cells were labelled from the superior colliculus. Double-staining of microglia with both dyes helped to compare the pattern of labelling for each dye. After progression of ganglion cell degeneration, microglia displayed a staggered, bilaminated distribution within the ganglion cell layer and within the inner plexiform layer. Fluorescent microglia were not found within the deeper layers of the retina indicating that transneuronal degeneration and subsequent labelling of microglial cells do not occur. The results show that one major function of microglia within the ganglion cell and inner plexiform layers of the lesioned retina is to remove debris produced after degradation of neurons.     

Evidence for an amacrine cell system in the ganglion cell layer of the rat retina

In the ganglion cell layer of the rat retina approx 50% of the cells with the Nissl morphology of neurons survive optic nerve section in infant and adult rats and cannot be retrogradely labelled with horseradish peroxidase. The number of neurons which can be retrogradely labelled with horseradish peroxidase from subcortical visual centres is similar to the number of axons in the optic nerve, and it is concluded that the small neurons do not send an axon into the optic nerve. The dendritic tree of the cells which have axons was demonstrated by filling the cells with horseradish peroxidase from the optic nerve. The dendritic structure of the cells which survive optic nerve section was shown by injecting horseradish peroxidase into the retina or impregnating with the Golgi method the cells which survive optic nerve section. A variety of amacrine cells were found in the ganglion cell layer which form branches in the lower part of the inner plexiform layer.It can be concluded that amacrine cells form a substantial number of the neurons in the ganglion cell layer.     

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。     

Neuroretinal function is normal in early dementia of the Alzheimer type.

We recently demonstrated that retinal ganglion cell function, optic nerve head parameters and the retinal nerve fiber layer thickness are not altered in early dementia of the Alzheimer type (DAT). Our current objective was to assess whether the function of cells located more distally in the retina is also unaffected by the disease. We evaluated 23 individuals with early to moderate DAT and 23 healthy age-matched subjects, all displaying clinically normal visual function. Scotopic and photopic flash electroretinograms (fERGs) and oscillatory potentials (OPs) were recorded. The amplitude and latency of the retinal potentials did not differ between DAT and control subjects. Our current results showing normal fERGs and OPs in early DAT indicate that the underlying neurons giving rise to these signals are not impaired by the disease process. These data support and extend our recent findings suggesting that visual deficits in DAT do not stem from neuroretinal dysfunction.     

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.     

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。     

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。     

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.     

Immunohistochemical localization of phospholipase D1 in the retina of pigs

The expression of phospholipase D1 (PLD1) was examined in the retinas of pigs. Western blot analysis detected the expression of PLD1 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 2 years old adult pigs. Immunohistochemically, PLD1 was mainly immunostained in ganglion cell bodies in the ganglion cell layer, in some radial processes of Muller cells in the retinal layer and in the inner and outer segments of the rod and cone layer in newborn and adult pigs, but not in astrocytic bundles in nerve fiber layers. The immunoreactivity of PLD1 in the radial processes of Muller cells across the retinal layers was enhanced in adult pig retinas compared to those of newborn piglets. This was the first demonstration to show that PLD1 is constitutively expressed in the retina of pigs, implying that retinal PLD1 expression is enhanced in radial fibers of Muller cells with age. This finding suggests that PLD1 plays an important role in signal transduction of glial cells and neuronal cells in the retina.     

Somatostatin-like immunoreactive material in associational ganglion cells of human retina

The retinal ganglion cell is classically viewed as the output cell of the retina, sending a single axon via the optic nerve to synapse in visual relay nuclei of the brain. However, some ganglion cells, termed associational ganglion cells, have axons which do not leave the retina and presumably serve intraretinal communication. Using high-affinity and specific monoclonal antibodies to somatostatin-14 and the avidin-biotin-peroxidase immunohistochemical procedure, somatostatin-immunoreactive associational ganglion cells are specifically stained in human retinas obtained at necropsy. These cells are more numerous in the inferior than the superior retina; they have dendrites which ramify in the inner plexiform layer; and they have sparsely branching axons, many of which can be traced over 1 cm. These axons do not enter the optic nerve. They follow remarkably straight courses at the border of the inner plexiform layer and ganglion cell layer and thereby form a gridwork of fibers covering the entire retinal area. These observations verify the existence of associational ganglion cells in the human and establish somatostatin as a neurotransmitter or neuromodulator candidate for these neurons. The morphology of these cells suggests that they are involved in long-distance interactions within the retina.     

Growth hormone and its receptor in projection neurons of the chick visual system: retinofugal and tectobulbar tracts

Recent studies have shown the presence of growth hormone (GH) in the retinal ganglion cells (RGCs) of the neural retina in chick embryos at the end of the first trimester [embryonic day (E) 7] of the 21 day incubation period. In this study the presence of GH in fascicles of the optic fiber layer (OFL), formed by axons derived from the underlying RGCs, is shown. Immunoreactivity for GH is also traced through the optic nerve head, at the back of the eye, into the optic nerve, through the optic chiasm, into the optic tract and into the stratum opticum and the retinorecipient layer of the optic tectum, where the RGC axons synapse. The presence of GH immunoreactivity in the tectum occurs prior to synaptogenesis with RGC axons and thus reflects the local expression of the GH gene, especially as GH mRNA is also distributed within this tissue. The distribution of GH-immunoreactivity in the visual system of the E7 embryo is consistent with the distribution of the GH receptor (GHR), which is also expressed in the neural retina and tectum. The presence of a GH-responsive gene (GHRG-1) in these tissues also suggests that the visual system is not just a site of GH production but a site of GH action. These results support the possibility that GH acts as a local growth factor during early embryonic development of the visual system.     

Early expression of recoverin in a unique population of neurons in the human retina

 The calcium-binding protein recoverin has been reported as present in photoreceptors, cone bipolar cells and sparse cells in the ganglion cell layer in the adult retinae of various vertebrate species. The present study was undertaken to clarify the developmental pattern of recoverin-immunoreactive cells in the human retina with particular attention to the cells in the inner retinal layers. In the adult human retina, small populations of recoverin-containing cells are present in the ganglion cell and nerve fiber layers. However, the precursors of these cells are quite numerous on the inner and outer borders of the nerve fiber layer in the fetal retina. By 13 weeks of gestation these cells express recoverin very intensely. By 24 weeks they are mature-looking with relatively large soma sizes (mean=118 μm²) and appear round, oval or multipolar in shape, with varying numbers of short processes. There follows a noticeable reduction of the mean soma size, but little change in morphology and process number during the remaining gestational stages up to and after birth. The mean numerical density of the recoverin-positive cells in the fetal inner retinal layers is gradually reduced from the high level at 13 weeks until birth, when there is a great drop to the adult level. The recoverin-immunoreactive cells in the ganglion cell layer demonstrate distinctively different developmental and morphological features from the principle neurons and glial cells in the retina. They are probably the neurons derived from the marginal zone of the retinal primordium that reside in the inner and outer borders of the nerve fiber layer due to the invasion of ganglion cell axons. The expression of recoverin in the neurons may be significant in maintaining an inside-out and centro-peripheral gradient of calcium concentration in the premature retina, thereby playing a role in determining the polarity of the differentiating ganglion cells and the growth of their axons in a centrifugal spatiotemporal order. Accepted: 3 June 1996     

The distribution of displaced ganglion cells in the retina of the pigeon

Displaced ganglion cells in the pigeon's retina, at the inner margin of the inner nuclear layer, were labelled by retrograde axonal transport of horseradish peroxidase (HRP). Large HRP injections were made in order to fill all the retinal projection sites in the thalamus and midbrain. The distribution of labelled cells was studied in retinal whole mounts incubated with tetramethyl benzidine (TMB) substrate for HRP. A maximum of 5,300 HRP labelled displaced ganglion cells was found. They were concentrated in a band of retina centred on the horizontal meridian, with high density areas (of about 110 cells/mm2) near the area centralis and in the mid-temporal retina. This is a different distribution to that of ganglion and inner nuclear layer cells; these are concentrated in the area centralis and red field. The orientation of retinal maps was checked by ophthalmoscopic measurements of the angle of the pecten to the horizontal in alert pigeons; this was found to be approximately 70 degrees. The array of displaced ganglion cells, studied by nearest neighbour distributions, was irregular and nearly random, which is consistent with a system of low spatial acuity. In the central retina only the cell bodies and not the dendrites of small displaced ganglion cells (7.5 microns diameter) were labelled; towards the periphery large displaced ganglion cells (16 microns diameter) with 2-5 radially arranged primary dendrites were found.     

The development and the topographic organization of the retinal ganglion cell layer in Bufo marinus

Summary The number and distribution of neurons in the retinal ganglion cell layer were studied from the metamorphic climax to adulthood in the toad Bufo marinus. Retinal wholemounts stained with cresyl violet showed that total neuron numbers increased from 55,000 at metamorphic climax to about 950,000 in adult animals. During the same time the entire retinal area increased 46-fold from an average 3.4 mm² to 157 mm². The morphological character of the neurons and their density across the retina changed during development. In metamorphosing animals, the neurons of the ganglion cell layer had a uniform appearance and their density increased slightly from the centre to the dorsal ciliary margin. After metamorphosis a high neuron density area, the visual streak, evolved in the retinal centre, resulting in the formation of a 6 to 1 density gradient from the visual streak out to the dorsal and ventral retinal poles in adult animals. Optic fibre numbers in juvenile and adult optic nerves were estimated to be 330,000 and 745,000, respectively, corresponding to similar ganglion cell numbers. One optic nerve was sectioned in a few animals and 4 weeks later the number of intact neurons — assumed to be displaced amacrine cells (DA) — was estimated. They amounted to 80,000 in juvenile and 189,000 in adult animals or about 20% of the total neuron population of the retinal ganglion cell layer, the remaining 80% being GC. A 1.7 to 1 density gradient of DA from the visual streak out to the dorsal and ventral retinal periphery was established. These results show that the visual streak evolves after metamorphosis from an originally uniform neuron distribution of the retinal ganglion cell layer. The possible mechanisms of the formation of the visual streak are discussed.     

Immunohistochemical localization of heat shock protein 27 in the retina of pigs

The expression of heat shock protein 27 (HSP27) was examined in the retinas of pigs. Western blot analysis detected the expression of HSP27 in the retinas of 1-day-old piglets and showed that it was enhanced in the retinas of 6-month-old adult pigs. Immunohistochemically, HSP27 immunostaining was seen mainly in ganglion cell bodies in the ganglion cell layer, and in some processes of astrocytes in the innermost nerve fiber layer. In 1-day-old piglets, HSP27 was detected weakly in the inner plexiform, inner nuclear cell, outer plexiform, and rod and cone layers. The HSP27 immunoreactivity across the retinal layers was enhanced in the retinas of 6-month-old pigs compared with newborn piglets. The HSP27 immunoreactivity in the radial processes of Müller cells was particularly prominent in adult pig retinas. In summary, this finding suggests that HSP27 plays an important role in signal transduction of glial cells and neuronal cells in the retina.     

Spatial and temporal patterns of apoptosis during differentiation of the retina in the turtle

We investigated patterns of cell death in the turtle retina that could potentially be associated with the innervation of the optic tectum, and looked for mechanisms of retinal development that might be common to reptilian and homeotherm vertebrates. We used retinas of turtle embryos between the 23rd day of incubation (E23) (before the first optic fibres reach the optic tectum) and hatching (when all the optic fibres have established synaptic connections). Dying retinal neurons were identified in paraffin sections by the TUNEL technique, which specifically labels fragmented DNA. Apoptotic cells were found in the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL). Cell death in the GCL was intense between E29 and E47, and had disappeared by the day of hatching. In the INL, dead and dying cells were most abundant between E31 and E34, and progressively disappeared. The temporal pattern in the ONL was similar to the INL although the density was very low. In all the nuclear layers cell death spread from the dorso-temporal area of the central retina to the periphery. Additional dorsal to ventral and temporal to nasal gradients were distinguishable in a quantitative TUNEL analysis. The patterns of cell death observed in the developing turtle retina were thus similar to those found in birds and mammals. This process could be under the control of differentiation gradients in all the vertebrate classes.     

Direct and indirect retinal input into degenerated dorsal lateral geniculate nucleus after striate cortical removal in monkey: implications for residual vision

Summary We removed the striate cortex of one cerebral hemisphere in a macaque monkey, causing almost total retrograde degeneration of the corresponding dorsal lateral geniculate nucleus (dLGN) and extensive transneuronal degeneration of ganglion cells in the corresponding hemi-retina of each eye. The rare surviving geniculate projection neurons were retrogradely labelled by horseradish peroxidase (HRP) from extra-striate cortex and retinogeniculate terminals were labelled by an intraocular injection of HRP. Retinal terminals in the degenerated dLGN made synaptic contact exclusively with the dendrites of interneurons immunopositive for -aminobutyric acid (GABA) in both parvocellular and magnocellular regions of dLGN. As well as being postsynaptic to retinal terminals these vescicle-containing dendrites were pre- and postsynaptic to other similar dendrites, and presynaptic to relay cells. Surviving labelled projection neurons received retinal input indirectly, via both the GABA-immunopositive interneurons and GABA-immunonegative terminals characteristic of those from the superior colliculus. In the degenerated, as opposed to the normal dLGN, about 20% of retinal terminals were GABA-immunopositive and GABA-immunoreactivity was prominently elevated in the ganglion and amacrine cell layers of the degenerated half of the retina. The optic nerve also contained numerous GABA-immunopositive axons but very few such axons were found in a normal optic nerve processed in identical manner. The surviving pathways from the retina must underlie the visual abilities that survive striate cortical removal in monkeys and human patients and may involve the degenerated dLGN as well as the mid-brain.     

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