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.     

Neurogenesis in the retinal ganglion cell layer of the rat.

The present study has examined the birthdates of neurons in the retinal ganglion cell layer of the adult rat. Rat fetuses were exposed to tritiated thymidine in utero to label neurons departing the mitotic cycle at different gestational stages from embryonic days 12 through to 22. Upon reaching adulthood, rats were either given unilateral injections of horseradish peroxidase into target visual nuclei in order to discriminate (1) ganglion cells from displaced amacrine cells, (2) decussating from non-decussating ganglion cells, and (3) alpha cells from other ganglion cell types; or, their retinae were immunohistochemically processed to reveal the choline acetyltransferase-immunoreactive amacrine cells in the ganglion cell layer. Retinae were embedded flat in resin and cut en face to enable reconstruction of the distribution of labelled cells. Retinal sections were autoradiographically processed and then examined for neurons that were both tritium-positive and either horseradish peroxidase-positive or choline acetyltransferase-positive. Tritium-positive neurons in the ganglion cell layer were present in rats that had been exposed to tritiated thymidine on embryonic days E14-E22. Retinal ganglion cells were generated between E14 and E20, the ipsilaterally projecting ganglion cells ceasing their neurogenesis a full day before the contralaterally projecting ganglion cells. Alpha cells were generated from the very outset of retinal ganglion cell genesis, at E14, but completed their neurogenesis before the other cell types, by E17. Tritium-positive, horseradish peroxidase-negative neurons in the ganglion cell layer were present from E14 through to E22, and are interpreted as displaced amacrine cells. Choline acetyltransferase-positive displaced amacrine cells were generated between E16 and E20. Individual cell types showed a rough centroperipheral neurogenetic gradient, with the dorsal half of the retina slightly preceding the ventral half. These results demonstrate, first, that retinal ganglion cell genesis and displaced amacrine cell genesis overlap substantially in time. They do not occur sequentially, as has been commonly assumed. Second, they demonstrate that the alpha cell population of retinal ganglion cells and the choline acetyltransferase-immunoreactive population of displaced amacrine cells are each generated over a limited time during the periods of overall ganglion cell and displaced amacrine cell genesis, respectively. Third, they show that the very earliest ganglion cells to be generated in the temporal retina have exclusively uncrossed optic axons, while the later cells to be generated therein have an increasing propensity to navigate a crossed chiasmatic course.     

Genesis of neurons of the retinal ganglion cell layer in the opossum

Summary In this study, we have examined the genesis of neurons of the retinal ganglion cell layer of the opossum Didelphis marsupialis by [³H]-thymidine autoradiography. Our results suggest that most neurons surviving to adulthood are generated in postnatal life from day 1 to day 23. Cells are generated according to a coarse gradient from the retinal geometric center to the periphery. Regional analysis of soma size distributions in different cohorts suggest that this gradient is actually formed by two partially-overlapping, concentric waves of cell proliferation. Most medium and large ganglion cells are formed during the early wave, whereas most displaced amacrine cells and small ganglion cells are formed during the late wave. Our results confirm the appropriateness of the opossum as a model for studies of development of the mammalian visual system.     

Regrowth of transected retinal ganglion cell axons despite persistent astrogliosis in the lizard (Gallotia galloti)

We analysed the astroglia response that is concurrent with spontaneous axonal regrowth after optic nerve (ON) transection in the lizard Gallotia galloti. At different post-lesional time points (0.5, 1, 3, 6, 9 and 12 months) we used conventional electron microscopy and specific markers for astrocytes [glial fibrillary acidic protein (GFAP), vimentin (Vim), sex-determining region Y-box-9 (Sox9), paired box-2 (Pax2)¸ cluster differentiation-44 (CD44)] and for proliferating cells (PCNA). The experimental retina showed a limited glial response since the increase of gliofilaments was not significant when compared with controls, and proliferating cells were undetectable. Conversely, PCNA⁺ cells populated the regenerating ON, optic tract (OTr) and ventricular wall of both the hypothalamus and optic tectum (OT). Subpopulations of these PCNA⁺ cells were identified as GFAP⁺ and Vim⁺ reactive astrocytes and radial glia. Reactive astrocytes up-regulated Vim at 1 month post-lesion, and both Vim and GFAP at 12 months post-lesion in the ON-OTr, indicating long-term astrogliosis. They also expressed Pax2, Sox9 and CD44 in the ON, and Sox9 in the OTr. Concomitantly, persistent tissue cavities and disorganised regrowing fibre bundles reaching the OT were observed. Our ultrastructural data confirm abundant gliofilaments in reactive astrocytes joined by desmosomes. Remarkably, they also accumulated myelin debris and lipid droplets until late stages, indicating their participation in myelin removal. These data suggest that persistent mammalian-like astrogliosis in the adult lizard ON contributes to a permissive structural scaffold for long-term axonal regeneration and provides a useful model to study the molecular mechanisms involved in these beneficial neuron–glia interactions.     

Neuronal density in the human retinal ganglion cell layer from 16-77 years

Literature assessing whether or not neurons (retinal ganglion cells and displaced amacrine cells) are lost from the retinal ganglion cell layer in mammals with age is still controversial, some studies finding a decrease in cell density and others not. To date there have been no studies estimating the total number of neurons in the retinal ganglion cell layer of humans throughout life. Recent studies have concentrated on the macular region and examined cell densities, which are reported to decrease during aging. In a study of the human retinal pigment epithelium (RPE), we showed that, while RPE cell number does not change, cell density increases significantly in central temporal retina (macular region) as the retina ages. We speculated that the increase in density represents a "drawing together" of the retinal sheet to maintain high cell densities, in this region of the neural retina, in the face of presumed cell loss from the ganglion cell layer due to aging. Here, therefore, we have sampled the entire ganglion cell layer of the human retina and estimated total neuron numbers in 12 retinae aged from 16 to 77 years. Human retinae, fixed in formalin, were obtained from the Queensland Eye Bank and whole-mounted, ganglion cell layer uppermost. The total number of neurons was lower in the older than younger retinae and neuronal density was lower in most retinal regions in older retinae. Retinal area increased with age and neuronal density fell throughout the retina with a mean reduction of 0.53% per year. However, the percentage reduction in density was much lower for the macular region, with a value of 0.29% per year. It is possible that this lesser reduction in cell density in the macula is a result of the drawing together of the retinal sheet in this region as we speculated from RPE data.     

The control of retinal ganglion cell discharge by receptive field surrounds.

1. This paper describes the behaviour of the receptive field surround, and how surround signals combine with those from the centre to generate the discharge of the retinal ganglion cells of the cat. 2. A small test spot is flashed upon the middle of the receptive field of an on-centre X-cell, alone, or together with a concentric annulus of fixed luminance. The reduction in discharge brought about by the annulus is independent of spot luminance. From this it is inferred that centre and surround signals combine additively. 3. Knowing that the combination of signals is additive, the surround signal can be estimated by comparing the ganglion cell's response to diffuse illumination of its receptive field with that to an equiluminous spot which optimally stimulates the centre while encroaching minimally upon the periphery. 4. Application of this technique to X-cells shows that although the surround seems to have a threshold, it is at its most sensitive in the dark-adapted eye, and typically is only 0.3-0.5 log units less sensitive than the centre. 5. Centre and surround sensitivities are decreased from their dark-adapted levels by increasing background illumination, but the decline of surround sensitivity is initially less rapid than that of the centre. Thus with increasing light-adaptation the surround becomes relatively more sensitive. In the light-adapted eye centre and surround are about equally sensitive to diffuse illumination. 6. Although, in the dark-adapted eye, illumination of the receptive field periphery of an on-centre unit depresses firing, removal of that illumination produces no off-discharge. Off-discharges appear only when background illumination exceeds about 104 quanta (507)/deg 2 sec. This confirms Barlow & Levick (1969b). 7. In the dark-adapted eye surround latency is longer than that of the centre. With increasing background illumination the latency difference is reduced. 8. For X-cells, the rate of the maintained discharge depends to some extent on the balance of centre-surround antagonism. But this antagonism is not the major factor accounting for the relative constancy of mean rate at high background luminances, for the rate then can be almost independent of the size of a steady pot. 9. The mean rate of discharge of Y-cells seems to depend even less upon the balance of centre-surround antagonism. 10. Y-cell surrounds could not properly be isolated with the optimal spot-diffuse illumination technique, so detailed measurements of their behaviour were not made. However, the dark-adapted surround appear to be as sensitive as those of X-cells.     

The outer disinhibitory surround of the retinal ganglion cell receptive field

1. There is an outer disinhibitory zone surrounding the classical inhibitory surround of the retinal ganglion cell receptive field.2. The disinhibitory surround is strong and narrow in ;sustained' cells but weak and laterally spread in ;transient' cells.3. The disinhibitory surround can be demonstrated using a black spot as a probing stimulus as well as by a white spot, and is therefore not an artifact of scattered light.4. Stimulation with a light spot in the disinhibitory zone gives an increase in firing to ;stimulus on' in on-centre cells and to ;stimulus off' in off-centre cells.5. The disinhibitory surround may be revealed by plotting the latency of the first spike discharge following stimulation against position in the receptive field. The disinhibitory zone shows a decrease in latency to the centre-type stimulus.6. The disinhibitory surround may be revealed by plotting the threshold intensity of a spot against position in the receptive field. It is thus a feature of the sensitivity gradients of both ;transient' and ;sustained' cells.7. Using two spots, one at the centre of the receptive field and the other at varying distances from the receptive field centre, dynamic interactions between the centre, inhibitory and disinhibitory zones are demonstrated. A spot presented in the disinhibitory zone causes an enhancement of the centre response when flashing in phase with the centre spot, while it causes inhibition of the centre response when presented 180 degrees out of phase.8. A scheme for the anatomical basis of the disinhibitory surround is proposed, and the relation of disinhibition to the spatial transfer characteristics of the visual pathways is discussed.     

Neuronal density in the human retinal ganglion cell layer from 16–77 years

Literature assessing whether or not neurons (retinal ganglion cells and displaced amacrine cells) are lost from the retinal ganglion cell layer in mammals with age is still controversial, some studies finding a decrease in cell density and others not. To date there have been no studies estimating the total number of neurons in the retinal ganglion cell layer of humans throughout life. Recent studies have concentrated on the macular region and examined cell densities, which are reported to decrease during aging. In a study of the human retinal pigment epithelium (RPE), we showed that, while RPE cell number does not change, cell density increases significantly in central temporal retina (macular region) as the retina ages. We speculated that the increase in density represents a “drawing together” of the retinal sheet to maintain high cell densities, in this region of the neural retina, in the face of presumed cell loss from the ganglion cell layer due to aging. Here, therefore, we have sampled the entire ganglion cell layer of the human retina and estimated total neuron numbers in 12 retinae aged from 16 to 77 years. Human retinae, fixed in formalin, were obtained from the Queensland Eye Bank and whole‐mounted, ganglion cell layer uppermost. The total number of neurons was lower in the older than younger retinae and neuronal density was lower in most retinal regions in older retinae. Retinal area increased with age and neuronal density fell throughout the retina with a mean reduction of 0.53% per year. However, the percentage reduction in density was much lower for the macular region, with a value of 0.29% per year. It is possible that this lesser reduction in cell density in the macula is a result of the drawing together of the retinal sheet in this region as we speculated from RPE data. Anat Rec 260:124–131, 2000. © 2000 Wiley‐Liss, Inc.     

Qualitative and quantitative ultrastructural observations on retinal ganglion cell layer of rat after intraorbital optic nerve crush

Summary Rat retinal ganglion cell layer (GCL) was examined ultrastructurally 1–180 days after intraorbital crushing of one optic nerve. It was confirmed quantitatively that axotomized ganglion cells lost cisternal membranes of the rough endoplasmic reticulum (RER) and showed disintegration of Nissl bodies and ribosomal rosettes 3 days postoperatively. Between 60 and 180 days after neurotomy there was partial reversion of the RER towards normal. At postoperative intervals of 14–60 days, chromatin aggregation became conspicuous and some nuclei were prominently furrowed and contained electron-dense inclusions. Concurrently, profiles of dead ganglion cells were encountered. Mean mitochondrial area increased in axotomized neurons but mitochondrial density declined, while the Golgi apparatus, lamellar specializations of the RER and the size of nuclei did not change significantly. Cytoplasmic atrophy was profound, however. Small nerve cells of the GCL appeared morphologically distinct from ganglion cells and did not undergo appreciable alteration.A decline in neuronal density, approximating 35%, occurred between the third and seventh postoperative day and progressed slowly thereafter. Neuronal density was 32% of normal 180 days postoperatively. A temporary increase in glial density 3–28 days after operation was due to microglial hyperplasia. Müller cell and astrocytic processes hypertrophied, infiltrated nerve fibre bundles, and surrounded and intruded into neuronal somata. Bundles of unmyelinated small axons, invested by astrocytes and basal lamina, were present within the necrotic cavity of the lesioned nerve 28–90 days postoperatively and had cytologic features of regenerative axonal sprouts.We conclude that intraorbital optic nerve crush is followed by a noteworthy degree of regenerative axonal sprouting which occurs and persists against a background of slow but relentless decline in the retinal ganglion cell population. This slow decline follows a rapidly-sustained loss of approximately one-third of the axotomized retinal ganglion cells during the first postoperative week. Intraorbital, as opposed to intracranial, injury of the optic nerve appears, paradoxically, to induce both a greater degree of ganglion cell death and a greater amount of regenerative axonal sprouting. Cytologic changes in axotomized retinal ganglion cells resemble those described for other populations of mammalian intrinsic neurons subjected to like injury. However, they differ, especially with regard to patterns of nuclear, nucleolar and RER alteration, from changes observed in peripheral neurons of mammals and intrinsic neurons of submammalian vertebrates that successfully regenerate severed axons. The neuroglial response in the surround of retinal ganglion cells after optic nerve crush is characterized by hypertrophy of astrocytes and Müller cells and a transient, modest increase of microglia. The microglial reaction is clearly less pronounced than that which occurs in the surround of axotomized peripheral neurons of the rat. The data presented here provide qualitative and quantitative cytologic information against which any effects exerted on the axotomy response and optic nerve regeneration by growth-promoting agents may be assessed.     

The effects of remote retinal stimulation on the responses of cat retinal ganglion cells.

1. Action potentials were recorded from optic nerve fibres of lightly anaesthetized cats while parts of the retina remote from the receptive field were stimulated by a shifting grating. 2. Vigorous responses can be obtained under these conditions, confirming McIlwain (1966), Krüger & Fischer (1973), and others. 3. These 'shift responses' are not caused by fluctuations of stray light because (a) they cannot be reduced by deliberately increasing or decreasing the light falling on the receptive field synchronously with the shifting grating; (b) a steady adapting light applied to the receptive field does not raise the threshold for the responses, whereas adapting light on the peripheral retina does, and (c) the threshold for the responses is elevated more following bleaching adaptation of the periphery than following bleaching adaptation of the centre. 4. Shift responses are strong, of short latency, and brief in duration in brisk-transient (Y-type) neurones. With few exceptions they are weak but long-lasting in brisk-sustained (X-type) neurones. 5. Shift responses are unlike responses from the main receptive field in having a distinct threshold; the magnitude of the response to weak gratings is not simply proportional to contrast, as is the case with weak stimuli applied to the receptive field. 6. It is thought that the excitatory pathway may involve amacrine cells, and that this mechanism may be concerned with the detection of the shifts of the image that occur with saccadic eye movements.     

Quantal encoding of information in a retinal ganglion cell

A retinal ganglion cell receives information about a white-noise stimulus as a flickering pattern of glutamate quanta. The ganglion cell reencodes this information as brief bursts of one to six spikes separated by quiescent periods. When the stimulus is repeated, the number of spikes in a burst is highly reproducible (variance < mean) and spike timing is precise to within 10 ms, leading to an estimate that each spike encodes about 2 bits. To understand how the ganglion cell reencodes information, we studied the quantal patterns by repeating a white-noise stimulus and recording excitatory currents from a voltage-clamped, brisk-sustained ganglion cell. Quanta occurred in synchronous bursts of 3 to 65; the resulting postsynaptic currents summed to form excitatory postsynaptic currents (EPSCs). The number of quanta in an EPSC was only moderately reproducible (variance = mean), quantal timing was precise to within 14 ms, and each quantum encoded 0.1-0.4 bit. In conclusion, compared to a spike, a quantum has similar temporal precision, but is less reproducible and encodes less information. Summing multiple quanta into discrete EPSCs improves the reproducibility of the overall quantal pattern and contributes to the reproducibility of the spike train.     

Interspike interval analysis of retinal ganglion cell receptive fields

The interspike interval (ISI) preceding a retinal spike has a strong influence on whether retinal spikes will drive postsynaptic responses in the lateral geniculate nucleus (LGN). This ISI-based filtering of retinal spikes could, in principle, be used as a mechanism for processing visual information en route from retina to cortex; however, this form of processing has not been previously explored. Using a white noise stimulus and reverse correlation analysis, we compared the receptive fields associated with retinal spikes over a range of ISIs (0-120 ms). Results showed that, although the location and sign of retinal ganglion cell receptive fields are invariant to ISI, the size and amplitude of receptive fields vary with ISI. These results support the notion that ISI-based filtering of retinal spikes can serve as a mechanism for shaping receptive fields.     

Functional lamination in the ganglion cell layer of the macaque's retina

Close to the fovea of the primate retina the ganglion cell layer is at its maximal thickness and several layers of cells deep. In whole-mount preparations in which the ganglion cells had been retrogradely labelled to reveal the dendritic trees we have studied the distribution of the different ganglion cell types across the depth of the ganglion cell layer. The ganglion cells which project to the parvocellular layers (P ganglion cells) are found more vitread than those which project to the magnocellular layers (M ganglion cells). The cells which project to the midbrain lie in the outer part of the ganglion cell layer among the M cells and adjacent to the inner plexiform layer. Within the P and M classes of ganglion cell the On-centre cells lie more vitread than the Off-centre cells. These results are discussed with relation to the proportions of different cell types sampled with intraocular recordings from ganglion cells and the possible significance for the development of different types of ganglion cell.     

Development of normal retinal organization depends on Sonic hedgehog signaling from ganglion cells

The adult retina is organized into three cellular layers--an outer photoreceptor, a middle interneuron and an inner retinal ganglion cell (RGC) layer. Although the retinal pigment epithelium (RPE) and Müller cells are important in the establishment and maintenance of this organization, the signals involved are unknown. Here we show that Sonic hedgehog signaling from RGCs is required for the normal laminar organization in the vertebrate retina.     

视网膜干细胞移植对青光眼视网膜神经节细胞的保护

BACKGROUND:Stem cell transplantation is a new method for blinding eye disease. But there is a lack of research about the protective effect of retinal stem cell transplantation on retinal ganglion cells in glaucoma. OBJECTIVE:To explore the protective effect of retinal stem cell transplantation on retinal ganglion cells of rats with glaucoma. METHODS:Forty-five Sprague-Dawley rats were randomly divided into three groups (n=15 per group) including control, model and retinal stem cell transplantation groups. Rat models of glaucoma were prepared in the latter two groups, and at 7 days after modeling, rats in the three groups were given intravitreal injection of 1 mL retinal stem cells (5x10⁶ cells), the same amount of PBS, and no treatment, respectively. Subsequently, relative indicators were detected at 2 weeks after transplantation. RESULTS AND CONCLUSION:The expressions of brain-derived neurotrophic factor and insulin-like growth factor I protein as well as the number of retinal ganglion cells were the highest in the control group, followed by the retinal stem cell transplantation group model group, and the lowest in the model group (P < 0.05). The number of apoptotic retinal ganglion cells in model group was significantly higher than that of control group (P < 0.05), and which in the retinal stem cell transplantation group was significantly lower than that in the model group (P < 0.05), but higher than that in the control group (P < 0.05). These results suggest that retinal stem cell transplantation for rat glaucoma can exert a protective effect on retinal ganglion cells.     

Frog retinal ganglion cells projecting to the tectum layer F release acetylcholine as co-mediator

Neurons may release more than one classical neurotransmitter (co-mediator). It has been demonstrated in a recent study that a burst of action potentials in frog retina ganglion cells induces an after-burst increase (phasic potentiation) of the retinotectal transmission that lasts tens of seconds. This increase is mediated by presynaptic nicotinic acetylcholine receptors that are activated by the endogenous acetylcholine released during the burst of action potentials of the retinotectal fiber. The objective of the present study was to find out the origin of acetylcholine release. We show that reduction of the retinotectal transmission to the subthreshold level by application of moderate concentrations of kynurenic acid or CNQX had no effect on the phasic nicotinic potentiation of the retinotectal transmission. This demonstrates that the retinotectal terminals are the source of acetylcholine - responsible for the phasic potentiation of retinotectal transmission. The acetylcholine is thus co-released with glutamate.     

Spectral coding in cat retinal ganglion cell receptive fields

We have examined the spectral-coding properties of ganglion cell receptive fields in the cat retina. Two classes of spectral coding were found. The first class consists of cells in which color opponency is spatially local. That is, the opponent cone types cover the same (center or surround) region of the receptive field. The second class consists of cells that show color opponency only to large stimuli (relative to center diameter). Center and surround regions of cells of this class have different spectral sensitivities. Individually the regions are nonopponent. When both regions are stimulated, a spectral opponency is revealed. For example, we recorded from one unit in which the ON-center was mediated by the 556-nm cone type and the OFF-surround was mediated by both 450- and 556-nm cone types. Large-field, threshold-level stimulation in the short-wavelength end of the spectrum produced OFF-responses, while in the long-wavelength end produced ON-responses. For a small stimulus, cells of the second class could mediate spatial vision, largely unaffected by the chromatic properties of the stimulus. Cells of the second class (center/surround opponent) were more commonly encountered than cells of the first class (locally opponent). Color-opponent units of X, Y, and W types were all found.     

Rat retinal ganglion cells culture enriched with the magnetic cell sorter

The magnetic cell sorter (MACS) technique was applied to isolate retinal ganglion cells (RGCs) for culture. RGCs were labeled retrogradely with 1.1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). Subsequently retinal cell suspensions were incubated with biotinylated anti-rat Thy-1 antibody and MACS Streptavidin MicroBeads, and then applied onto the column in the magnetic fields. Cells attached on the column were flashed out without magnetism and plated on glass cover slips. RGCs were enriched to 31.0% of all cells with MACS from 0.55% before applying onto the magnetic column. Mean diameters of Dil-labeled cells were significantly larger than those of unlabeled cells. All cells with soma diameter over 11 microm were labeled. The number of viable RGCs were counted in the 10 fields of six cultures at a magnification of x200; the mean numbers on the 2nd, 7th and 14th culture-day were 53+/-3, 24+/-2 and 21+/-3, respectively (mean +/- SEM, n = 6). Thus, the MACS technique was confirmed to be useful for enrichment of RGCs and long-term study of cultured RGCs.