Optic Fiber Layer Research Articles

C-Raf Regulates Cell Survival and Retinal Ganglion Cell Morphogenesis during Neurogenesis

The signaling cascade Ras/Raf/mitogen-activated protein kinases modulates cell proliferation, differentiation, and survival, all key cellular processes during neural development. To better define the in vivo role of Raf during chick retinal neurogenesis, we interfered with Raf-dependent signaling during days 4.5 to 7.5 of embryonic development by expressing a dominant negative mutant of c-Raf (DeltaRaf), which blocks Ras-dependent Raf activation, and by overexpressing wild-type c-Raf. DeltaRaf expression induced an increase in cell death by apoptosis, whereas it did not affect overall cell proliferation and differentiation. In parallel, the number of Islet-1/2-positive and TUJ1-positive retinal ganglion cells were diminished in their definitive layer, whereas there was an increase in the number of mislocated Islet-1/2-positive cells. This disturbed morphogenesis correlated with a disruption of the optic fiber layer. Conversely, c-Raf overexpression caused moderate opposite effects on apoptosis. These results frame in vivo early neurogenesis processes in which c-Raf is essential.

Expression of chondroitin sulfate proteoglycans in the chiasm of mouse embryos.

Chondroitin sulfate (CS) proteoglycans have been implicated as molecules that are involved in axon guidance in the developing neural pathways. The spatiotemporal expression of CS was investigated in the developing retinofugal pathway in mouse embryos by using the CS-56 antibody. Immunoreactive CS was detected in inner regions of the retina as early as embryonic day 11 (E11). Its expression in subsequent stages of development followed a centrifugal, receding gradient that appeared to correlate with the sequence of axogenesis in the retina. In the chiasm, immunoreactive CS was expressed at E12, before the arrival of retinal axons. When the retinal axons navigated in the chiasm at E13-E14, immunoreactive CS remained at a low level in the optic fiber layer of the chiasm but was observed prominently in the caudal parts of the ventral diencephalon. This pattern followed closely the array of stage-specific-embryonic-antigen-1-positive neurons in the ventral diencephalon, with a V-shaped configuration that bordered the posterior boundary of the retinal axons, and a rostral raphe extension that ran across the decussating axons in the chiasm. Thus, the CS epitope is implicated in patterning the course of early retinal axons and in regulating axon divergence in the chiasm. At the lateral region of the chiasm, where the retinal axons cross the midline and approach the optic tract, a CS-immunopositive region coincided with the region in which active sorting of dorsal retinal axons from ventral retinal axons occurs. Moreover, at the threshold of the optic tract, the immunoreactive CS was restricted only to the deep part of the optic fiber layer, suggesting an inhibitory role of the CS epitope in repelling newly arrived axons to superficial regions of the optic tract during the development of chronotopic order at this part of the retinofugal pathway.

The developmental study on lamination of the optic tectum in relation to the retinotectal projection in chicks and chick embryos.

The tectal lamination was investigated in the central part of the chick embryonic tectum. Two and 5 layers were observed above the neuroepithelium (NE) on embryonic day 6 (E6) and E8, respectively. Optic fibers extended on the surface of the tectum by E8. On E10-11, the outer tectum was composed of 2 layers, that is, a fibrous layer forming the optic fiber layer on the tectal surface and a cellular layer showing the gradient of cell density. In the inner tectum, the lamination was almost completed. On E12-13, the outer tectal layers, which showed the gradient of cell density, was divided into dark and light cellular layers. The dark cellular layer was divided into 2 layers on E14-15 and further into 4 layers (layer C-F in chick) on E18. On the other hand, the light cellular layer did not change until E18, but finally, it was divided into 2 layers (layer A and B in chick) by E20. Optic fibers reached the bottom of the outer tectum by E14 showing different densities of terminals. Stratification by optic fibers was going to step into the final stage on E18. On E20, laminations according to cytoarchitectural features and the optic fiber terminals were substantially completed. In the tectum affected by destruction of the contralateral embryonic eye (E4), some cellular layers were incompletely discriminated by differences of cell density.

Expression, distribution and ultrastructural localization of the synapse-organizing molecule agrin in the mature avian retina.

At the vertebrate neuromuscular junction the extracellular matrix molecule agrin is responsible for the formation, maintenance and regeneration of most if not all postsynaptic specializations. Several agrin isoforms are generated by alternative splicing which differ in their function and which are all expressed in the CNS. To analyse the role of agrin in the CNS, we investigated the expression and ultrastructural localization of agrin in the posthatched chick retina. In situ hybridization revealed the presence of agrin mRNA in all cellular layers of the mature retina, indicating that most if not all major retinal cell types synthesize agrin. Pan-specific as well as isoform-specific antiagrin antisera stained the optic fibre layer and the outer plexiform layer. However, only the pan-specific antiserum additionally stained the inner limiting membrane. Immunoelectron microscopy showed that in the optic fibre layer agrin was associated with ganglion cell axons and that at least part of this agrin corresponds to a neuronal isoform of agrin. In the outer plexiform layer, agrin was localized in the cleft between the photoreceptor terminals and the invaginating horizontal and bipolar cell dendrites. In the synapse-containing inner plexiform layer both antisera revealed punctate immunoreactivity. This staining corresponded to agrin concentrated in the synaptic cleft of conventional synapses as determined by preembedding immunoelectron microscopy. Agrin is thus concentrated at mature interneuronal synapses as it is at the neuromuscular junction, consistent with a role of agrin during formation and/or maintenance of synapses in the CNS.

Fine structure of the retina of black bass, Micropterus salmoides (Centrarchidae, Teleostei).

The structure of light- and dark-adapted retina of the black bass, Micropterus salmoides has been studied by light and electron microscopy. This retina lacks blood vessels at all levels. The optic fiber layer is divided into fascicles by the processes of Müller cells and the ganglion cell layer is represented by a single row of voluminous cells. The inner nuclear layer consists of two layers of horizontal cells and bipolar, amacrine and interplexiform cells. In the outer plexiform layer we observed the synaptic terminals of photoreceptor cells, rod spherules and cone pedicles and terminal processes of bipolar and horizontal cells. The spherules have a single synaptic ribbon and the pedicles possess multiple synaptic ribbons. Morphologically, we have identified three types of photoreceptors: rods, single cones and equal double cones which undergo retinomotor movements in response to changes in light conditions. The cones are arranged in a square mosaic whereas the rods are dispersed between the cones.

Laminar restriction of retinal macrophagic response to optic nerve axotomy in the rat

Following optic nerve transection, most of the retinal ganglion cells die. Their debris is promptly cleared by phagocytic cells. It is currently not known to what extent peripherally derived macrophages contribute to this activity. Using antibodies OX42 and ED-1, phagocytic cells were labeled in the retinas of optic nerve lesioned adult rats. To distinguish whether the cells were reactive microglial or macrophagic in origin, blood-borne monocytes were labeled with fluorescent microspheres while in the systemic circulation. Macrophages invaded the retina, but only in the nerve fiber layer, sparing the ganglion cell and other layers. These macrophages engulfed only the axonal debris from dying ganglion cells, not their degenerating cell bodies. These results indicate that although peripherally derived monocytic cells are recruited into the retrogradely degenerating retina, their role in clearing debris is limited to the optic fiber layer.

Immunochemical localization of calretinin in the retina of the turbot (Psetta maxima) during development.

The expression of the calcium-binding protein calretinin was analysed by immunohistochemistry techniques in the retina of turbot (Psetta maxima) from embryonic to juvenile stages. Calretinin immunoreactivity was first detected in retinae from newly hatched larvae, in which the anlage of the inner plexiform layer and a subset of amacrine and ganglion cells displayed a faint immunolabelling. First appearance of photoreceptors during larval life coincided with an increase in the intensity of the labelling. During subsequent larval development, the expression of calretinin affected distinctive retinal components. The inner plexiform layer, optic fiber layer, and a population of amacrine and ganglion cells were invariably labelled. Occasional bipolar cells were labelled at the end of the larval period. By metamorphosis, calretinin is sequentially expressed in horizontal cells, and bipolar immunoreactive cells become numerous. The pattern of calretinin immunoreactivity of the inner plexiform layer changes from the larval to juvenile period. In all cases, calretinin immunoreactivity exhibited variations between the peripheral retina, which contains the most recently differentiated retinal components, and the remainder of the differentiated retina. Our results suggest that the progressive expression of calretinin in the turbot retina appears associated with some degree of neuronal differentiation. Once the definitive pattern of calretinin immunoreactivity is established in the turbot retina, both similarities and differences with the calretinin location in the retina of other vertebrates can be demonstrated.

Up-regulation of protein kinase C in regenerating optic nerve fibers of goldfish: immunohistochemistry and kinase activity assay.

Protein kinase C (PKC) activation has been associated with synaptic plasticity in many projections, and manipulating PKC in the retinotectal projection strongly affects the activity-driven sharpening of the retinotopic map. This study examined levels of PKC in the regenerating retinotectal projection via immunostaining and assay of activity. A polyclonal antibody to the conserved C2 (Ca2+ binding) domain of classical PKC isozymes (anti-panPKC) recognized a single band at 79-80 kD on Western blots of goldfish brain. It stained one class of retinal bipolar cells and the ganglion cells in normal retina, as shown previously. Strong staining was not present in the optic fiber layer of retina or in optic nerve, optic tract, or terminal zone in tectum, with the exception of a single fascicle of optic nerve fibers that by their location and by L1 (E587) staining were identified as those arising from newly added ganglion cells at the retinal margin. Normal tectal sections showed dark staining of a subclass of type XIV neuron with somas at the top of the periventricular layer and an apical dendrite ascending to stratum opticum. In regenerating retina, swollen ganglion cells stained darkly and stained axons were seen in the optic fiber layer. In regenerating optic nerve (2-11 weeks postcrush), all fascicles of optic fibers stained darkly for both PKC and L1(E587). At 5 weeks postcrush, PKC staining could also be seen in the medial and lateral optic tracts and stratum opticum at the front half of the tectum and very lightly over the terminal zones. PKC activity was measured in homogenized tissues dissected from a series of fish with unilateral nerve crush from 1 to 5 weeks previously. Activity levels stimulated by phorbols and Ca2+ were measured by phosphorylation of a specific peptide and referred to levels measured in the opposite control side. Regeneration did not increase overall PKC activity in retina or tectum, but in optic nerve there was an 80% rise after the first week. The increased activity verifies that the increased staining in nerve represented an up-regulation of functional PKC during nerve regeneration.

Disruption of the retinal basal lamina during early embryonic development leads to a retraction of vitreal end feet, an increased number of ganglion cells, and aberrant axonal outgrowth

Bacterial collagenase was injected into the vitreous of the eye of chick and quail embryos. Immunocytochemical and ultrastructural studies revealed that the collagenase dissolved the retinal basal lamina of the injected eye. The basal lamina disruption was first detectable 1 hour after enzyme injection and was complete within 3 hours. With further development, the retinal basal lamina was not reestablished; newly developing neuroepithelium in the peripheral retina, however, generated an intact basal lamina. Western blot analysis showed that Clostridial collagenase degraded various collagens but spared noncollagenous proteins. Basal lamina disruption of embryonic day 3 to 6 retinae led to the retraction of the end feet of the neuroepithelial cells, caused an increase in the number of Islet-1+ cells (most likely ganglion cells), an increase in the thickness of the optic fiber layer, and aberrant growth of optic axons on their way toward the optic disc. None of these changes were observed when retinal basal laminae were disrupted at later stages of development. The present data demonstrate that the retinal basal lamina, by anchoring the neuroepithelial cells to the pial surface of the retina, has an important function in the development of the normal cytoarchitecture of this structure. It is proposed that the altered extracellular environment in the vitreal part of the retina, resulting in the retraction of the neuroepithelial end feet, is responsible for the increased number of Islet-1+ cells and the aberrant axonal navigation.

Different cell surface areas of polarized radial glia having opposite effects on axonal outgrowth.

During neuronal development neurites are likely to be specifically guided to their targets. Within the chicken retina, ganglion cell axons are extended exclusively into the optic fibre layer, but not into the outer retina. We investigated, whether radial glial cells having endfeet at the optic fibre layer and somata in the outer retina, might be involved in neurite guidance. In order to analyse distinct cell surface areas, endfeet and somata of these glial cells were purified. Glial endfeet were isolated from flat mounted retina by a specific detachment procedure. Glial somata were purified by negative selection using a monoclonal antibody/complement mediated cytolysis of all non-glial cells. Retinal tissue strips were explanted either onto pure glial endfeet or onto glial somata. As revealed by scanning and fluorescence microscopy, essentially no ganglion cell axons were evident on glial somata, whereas axonal outgrowth was abundant on glial endfeet. However, when glial somata were heat treated and employed thereafter as the substratum, axon extension was significantly increased. Time-lapse video recording studies indicated that purified cell membranes of glial somata but not of endfeet induced collapse of growth cones. Collapsing activity was destroyed by heat treatment of glial membranes. The collapsing activity of retinal glia was found to be specific for retinal ganglion cell neurites, because growth cones from dorsal root ganglia remained unaffected. Employing four different kinase inhibitors revealed that the investigated protein kinase types were unlikely to be involved in the collapse reaction. The data show for the first time that radial glial cells are functionally polarized having permissive endfeet and inhibitory somata with regard to outgrowing axons. This finding underscores the pivotal role of radial glia in structuring developing nervous systems.

Morphology of human retinal ganglion cells with intraretinal axon collaterals.

Ganglion cells with intraretinal axon collaterals have been described in monkey (Usai et al., 1991), cat (Dacey, 1985), and turtle (Gardiner & Dacey, 1988) retina. Using intracellular injection of horseradish peroxidase and Neurobiotin in in vitro whole-mount preparations of human retina, we filled over 1000 ganglion cells, 19 of which had intraretinal axon collaterals and wide-field, spiny dendritic trees stratifying in the inner half of the inner plexiform layer. The axons were smooth and thin (approximately 2 microm) and gave off thin (<1 microm), bouton-studded terminal collaterals that extended vertically to terminate in the outer half of the inner plexiform layer. Terminal collaterals were typically 3-300 microm in length, though sometimes as long as 700 microm, and were present in clusters, or as single branched or unbranched varicose processes with round or somewhat flattened lobular terminal boutons 1-2 microm in diameter. Some cells had a single axon whereas other cells had a primary axon that gave rise to 2-4 axon branches. Axons were located either in the optic fiber layer or just beneath it in the ganglion cell layer, or near the border of the ganglion cell layer and the inner plexiform layer. This study shows that in the human retina, intraretinal axon collaterals are associated with a morphologically distinct ganglion cell type. The synaptic connections and functional role of these cells are not yet known. Since distinct ganglion cell types with intraretinal axon collaterals have also been found in monkey, cat, and turtle, this cell type may be common to all vertebrate retinas.

Distribution, size and number of axons in the optic pathway of ground squirrels.

The present study has examined the distribution of axons of differing sizes in the optic pathway of the ground squirrel. Axon diameters were measured from electron micrographs at various locations across sections of the optic nerve and tract, and total distributions and numbers were estimated. In both the nerve and tract, roughly 1.2 million optic axons were present. The population of optic axons had a unimodal size distribution, peaking at 0.9 microm in diameter and having an extended tail toward larger diameters. Local axon diameter distributions in the optic tract indicated distinct (though partially overlapping) axon diameter classes, including one of fine sizes peaking at 0.8-0.9 microm, a second of medium sizes peaking around 1.7-1.8 microm, and a third composed of the larger fibers with diameters up to 4.8 microm. The fine-caliber axons were found at all locations in the tract, and were the only axons present immediately adjacent to the pia, while the medium- and coarse-caliber axons were found at deeper locations. Curiously, the larger axons were found primarily in the medial parts of the tract, where axons from the dorsal retina normally course. A similarly restricted distribution of the larger axons was observed in the dorsotemporal parts of the optic nerve, suggesting that this difference in the tract may relate to an asymmetric distribution of ganglion cells on the retina giving rise to these axons. Measurements of axonal size taken within the optic fiber layer in dorsal and ventral parts of the retina confirmed this asymmetry, consistent with previous demonstrations of soma size differences in the dorsal versus ventral retina. The partial segregation of axons by size in the optic tract of the ground squirrel then reflects both the asymmetric distribution of retinal ganglion cell classes and the chronotopic reordering of optic axons that occurs within the chiasmatic region.

SOMATOSTATIN-LIKE IMMUNOREACTIVE CELLS IN THE GROUND SQUIRREL RETINA

Immunocytochemical techniques were employed to locate somatostatin (SS)-containing cells in the retina of the 13-lined ground squirrel (Spermophilus tridecemlineatus). In normal retinas immunostain was limited to neuronal processes, yet distinctly labeled somata were detected in retinas of animals pretreated with colchicine. Labeled cell bodies were located in the outermost and innermost portions of the inner nuclear layer (INL) and in the ganglion cell layer (GCL). The largest population of SS-like immunoreactive neurons was found in the innermost INL. These cells were identified as small and medium sized amacrine cells whose soma diameters ranged from 4 to 14μm. A smaller population of immunoreactive cells was observed in the outermost region of the INL. These cells, presumptive horizontal cells, were found mainly in peripheral regions of the retina. Immunoreactive cells in the GCL were of two types: displaced amacrines, and retinal ganglion cells. SS-positive axons in the optic fiber layer suggest that some of the immunoreactive GCL neurons were ganglion cells, and it is our opinion that these cells belong to a class of associational ganglion cells previously identified in other species.

Basic fibroblast growth factor, its high- and low-affinity receptors, and their relationship to form-deprivation myopia in the chick

Form deprivation myopia in chickens is a widely accepted model to study visually-regulated postnatal ocular growth. Recently we showed that basic fibroblast growth factor-2 provides a “stop” signal for the growing eye. To understand further its action, we have localized basic fibroblast growth factor-2 and its low- and high-affinity receptors in the chicken eye, and determined the localization of basic fibroblast growth factor receptors in the inner plexiform layer with respect to that of neurotransmitter systems known to play a role in form-deprivation myopia. By immunocytochemistry and in situ hybridization, two complementary methods, we found that nearly all cells in the retina, and scleral chondrocytes, contain basic fibroblast growth factor-2 protein and messenger RNA as well as high-affinity basic fibroblast growth factor receptor protein and messenger RNA. Immunocytochemical localization of basic fibroblast growth factor-2 binding sites (a high resolution alternative to autoradiography), combined with N-glycanase and heparitinase treatment or heparin competition, revealed additional binding sites in specific synaptic layers of the inner plexiform layer and low-affinity binding sites in the choroid and optic fibre layer. Some binding sites in the synaptic layers were found to co-stratify with neurites of dopamine-, vasoactive intestinal polypeptide- or enkephalin-containing amacrine cells, suggesting that basic fibroblast growth factor-2 could modulate synaptic transmission to or from these cells. Form deprivation did not affect the levels of basic fibroblast growth factor receptor-1 messenger RNA in retina/retinal pigment epithelium/choroid (Northern blotting), but it abolished the decrease in amount of extractable basic fibroblast growth factor normally observed in the dark (Western blotting). The results are discussed with respect to previous findings on basic fibroblast growth factor-2 and basic fibroblast growth factor receptor-1 localization in the avian and other vertebrate eyes, and their relevance to form-deprivation myopia. The widespread distribution of basic fibroblast growth factor-2 and its receptor makes it impossible to predict which cells might mediate the action of basic fibroblast growth factor-2 in form-deprivation myopia. However, the alteration in amounts of extractable retinal basic fibroblast growth factor-2 in form-deprived, dark-adapted retinas, in which basic fibroblast growth factor-2 probably serves as a “stop” signal for ocular growth, is consistent with a role for basic fibroblast growth factor-2 in the regulation of ocular growth.

Developmental shift of synaptic vesicle protein 2 from axons to terminals in the primary visual projection of the hamster

Synaptic vesicle protein 2 is an integral synaptic vesicle membrane glycoprotein which is present in all synapses for which it has been examined. We used an anti-synaptic vesicle protein 2 monoclonal antibody to examine synaptic vesicle protein 2 localization in the developing hamster retinofugal pathway. From postnatal day 0 to day 1, a period of elongation of retinal ganglion cell axons to their central targets, fiber fascicles in the optic tract over the lateral geniculate nucleus were intensely synaptic vesicle protein 2-immunoreactive. Adjacent to the optic tract, single fibers could be seen. We also observed a marked immunostaining in growth cones and fiber fascicles in retinal explants in culture. By postnatal day 2, the staining of single fibers had ended, and by postnatal day 5, during the formation of terminal arbors, numerous fine puncta of synaptic vesicle protein 2 immunoreactivity were distributed within the neuropil of the lateral geniculate nucleus. In the adult, the optic tract was devoid of synaptic vesicle protein 2 staining, while the neuropil contained distinct immunoreactive profiles, particularly in the outer shell of the lateral geniculate. These synaptic vesicle protein 2-positive profiles closely resembled the grape-like clusters and large swellings of two known retinal axon terminal types. Eye removal resulted in the rapid disappearance of these synaptic vesicle protein 2-labelled terminal profiles contralateral to the enucleation. A similar pattern of synaptic vesicle protein 2 immunoreactivity was observed in the superior colliculus. From postnatal day 0 to day 2, retinal fiber fascicles in the stratum griseum superficiale/stratum opticum were darkly stained for synaptic vesicle protein 2. By postnatal day 5, the immunoreactivity shifted to the neuropil and from postnatal day 6 onwards, the synaptic vesicle protein 2 immunoreactivity was more intense in the stratum griseum superficiale than in the optic fiber layer. This study demonstrates dense synaptic vesicle protein 2-labelling of elongating axons both in vivo and in vitro. However, coincident with the transition from retinal ganglion cell axon elongation to terminal arborization, synaptic vesicle protein 2 is progressively restricted to synaptic terminals and becomes undetectable in axons. This study is the first to document an axonal localization of synaptic vesicle protein 2 during development and raises the question as to its role during axonal elongation.

Formation of synaptic specializations in the inner plexiform layer of the developing chick retina.

The development of synapse-like specializations was investigated in the inner plexiform layer of the developing chick retina by using light and electron microscopy. Six monoclonal antibodies, directed against glycine and gamma-aminobutyric acid (GABA)A receptor subunits, the intracellular receptor-associated protein gephyrin, synaptotagmin, and synaptophysin were used to determine the initial appearance and distribution of their antigens. Synaptophysin and synaptotagmin immunoreactivity was detected in the retina concurrent with the formation of the inner plexiform layer at embryonic day 7. This early appearance before synaptic differentiation, together with the transient expression of synaptotagmin immunoreactivity in the synapse-free optic fiber layer, suggests that in the developing central nervous system (CNS) these proteins are not confined to synapses. The first immunofluorescence signal detected with specific antibodies against the beta 2 and beta 3-subunits of the GABAA receptor, the glycine receptor, and gephyrin appeared at embryonic day 12. In contrast, the alpha 1-subunit of the adult-type glycine receptor heteromeric complex was detectable only at later stages of development, after embryonic day 16, suggesting a change in the subunit composition of some glycine receptor complexes. The staining was clearly punctate, indicating the clustering of the alpha 1-subunit at synapses. Electron microscopic investigation revealed the first postsynaptic densities and active zones in the inner plexiform layer of the retina at embryonic day 12. These results reveal different patterns of development for the investigated pre- and postsynaptic proteins and indicate a parallel appearance of gephyrin, glycine receptor, and the beta 2 and beta 3-subunits of the GABAA receptor with the first synaptic specializations in the inner plexiform layer of the developing chick retina.

Multiple γ‐aminobutyric acid plasma membrane transporters GAT‐1, GAT‐2, GAT‐3 in the rat retina

γ-Aminobutyric acid (GABA) plasma membrane transporters (GATs) influence synaptic neurotransmission by high-affinity uptake and release of GABA. The distribution and cellular localization of GAT-1, GAT-2, and GAT-3 in the rat retina have been evaluated by using affinity-purified polyclonal antibodies directed to the C terminus of each of these GAT subtypes. Small GAT-1-immunoreactive cell bodies were located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL), and processes were distributed to all laminae of the interplexiform layer (IPL). Varicose processes were in the optic fiber layer (OFL) and the outer plexiform layer (OPL). Weak GAT-1 immunostaining surrounded cells in the INL and GCL, and it was found in the OFL and OPL and in numerous processes in the outer nuclear layer (ONL) that ended at the outer limiting membrane. GAT-1 is therefore strongly expressed by amacrine, displaced amacrine, and interplexiform cells and weakly expressed by Müller cells. GAT-2 immunostaining was observed in the retina pigment epithelium and the nonpigmented ciliary epithelium. GAT-3 immunoreactivity was distributed to the OFL, to all laminae of the IPL, GCL and INL, and to processes in the ONL that ended at the outer limiting membrane. Small GAT-3-immunoreactive cell bodies were in the proximal INL and GCL. GAT-3 is therefore strongly expressed by Müller cells, and by some amacrine and displaced amacrine cells. Together, these observations demonstrate a heterologous distribution of GATs in the retina. These transporters are likely to take up GABA from, and perhaps release GABA into, the synaptic cleft and extracellular space. This suggests that GATs regulate GABA levels in these areas and thus influence synaptic neurotransmission. © 1996 Wiley-Liss, Inc.

Multiple gamma-Aminobutyric acid plasma membrane transporters (GAT-1, GAT-2, GAT-3) in the rat retina.

gamma-Aminobutyric acid (GABA) plasma membrane transporters (GATs) influence synaptic neurotransmission by high-affinity uptake and release of GABA. The distribution and cellular localization of GAT-1, GAT-2, and GAT-3 in the rat retina have been evaluated by using affinity-purified polyclonal antibodies directed to the C terminus of each of these GAT subtypes. Small GAT-1-immunoreactive cell bodies were located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL), and processes were distributed to all laminae of the interplexiform layer (IPL). Varicose processes were in the optic fiber layer (OFL) and the outer plexiform layer (OPL). Weak GAT-1 immunostaining surrounded cells in the INL and GCL, and it was found in the OFL and OPL and in numerous processes in the outer nuclear layer (ONL) that ended at the outer limiting membrane. GAT-1 is therefore strongly expressed by amacrine, displaced amacrine, and interplexiform cells and weakly expressed by Müller cells. GAT-2 immunostaining was observed in the retina pigment epithelium and the nonpigmented ciliary epithelium. GAT-3 immunoreactivity was distributed to the OFL, to all laminae of the IPL, GCL and INL, and to processes in the ONL that ended at the outer limiting membrane. Small GAT-3-immunoreactive cell bodies were in the proximal INL and GCL. GAT-3 is therefore strongly expressed by Müller cells, and by some amacrine and displaced amacrine cells. Together, these observations demonstrate a heterologous distribution of GATs in the retina. These transporters are likely to take up GABA from, and perhaps release GABA into, the synaptic cleft and extracellular space. This suggests that GATs regulate GABA levels in these areas and thus influence synaptic neurotransmission.

Morphology of retinal ganglion cells in a new world monkey, the marmoset Callithrix jacchus.

We studied the morphology of retinal ganglion cells in a diurnal New World primate, the marmoset Callithrix jacchus. This species is of interest as a model for primate vision because it has good behavioural visual acuity, and the retina and subcortical visual pathways are very similar to those of Old World monkeys and humans. Ganglion cells were labelled by placing small crystals of the carbocyanin dye DiI into the optic fibre layer, or by intracellular injection of neurobiotin. Two main classes of ganglion cell were labelled. We call these Group A cells and Group B cells: they are respectively homologous to parasol and midget cell classes. Group A and Group B cells show similar patterns of dye coupling, dendritic stratification and dendritic field size as their counterparts in Old World monkeys and humans. A third group of cells, which we call Group C, is morphologically heterogeneous. Examples corresponding to wide-field ganglion cell types described in Old World primates were encountered. One subgroup of C cells has a morphology very similar to that of the small bistratified (blue-on) cell described in macaque retina, suggesting that this functional pathway is common to all primates. As for other New World monkeys, the marmoset shows a sex-linked polymorphism of cone pigment expression, such that all males are dichromats and the majority of females are trichromats. No systematic differences in Group B cells were seen between male and female retinas, suggesting that trichromacy is not accompanied by specific changes in ganglion cell morphology.

The developing avian retina expresses agrin isoforms during synaptogenesis.

The localization, isoform pattern, and mRNA distribution of the synapse-organizing molecule agrin was investigated in the developing avian retina. Injection of anti-agrin Fab fragments into the vitreous humor of chick eyes of embryonic days 3 to 20, a procedure that labels only extracellular agrin, reveals staining in the inner and outer plexiform layers before, during, and after the period of synapse formation. The labeling in these layers changes from a diffuse to a punctate pattern at the time when synapses form. At all stages investigated, the inner limiting membrane (a basal lamina that separates vitreous from neural retina) is intensely labeled, as are the axonal processes of retinal ganglion cells in the optic fiber layer and in the optic nerve, although the staining intensity declines after embryonic day 10 in both retina and optic nerve. In culture, axons of retinal ganglion cells also express agrin-like immunoreactivity on their surfaces. Polymerase chain reaction analysis reveals that several different agrin isoforms are expressed in the developing neural retina. In situ hybridization studies show that agrin isoforms are expressed in the ganglion cell and inner nuclear layers, correlating well with the staining for agrin protein in the optic fiber and plexiform layers. The expression of mRNA coding for several agrin isoforms and the presence of extracellular agrin in the synapse-containing layers during the period of synapse formation is consistent with the idea that agrin isoforms might play a role during synapse formation in the central nervous system.