Sema6B,Sema6C,and Sema6D Expression and Function during Mammalian Retinal Development

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B^−/− or Sema6C^−/−; Sema6D^−/− retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization.     

The distribution and developmental regulation of NMDA receptor subunit proteins in the outer and inner retina of the rat

In order to investigate whether N-methyl-D-aspartate (NMDA) receptors with distinct pharmacological properties are differentially distributed within the retinal layers, the spatial distribution and temporal regulation of all NMDA receptor subunits was analyzed in parallel on the protein level in the rat retina during development. Immunohistochemistry was performed on retinal sections at different developmental ages between embryonic (E) days 20/21 and the adult stage using specific antibodies against NMDA subunits (NR1, NR2A-D). All NMDA subunits were expressed in the rat retina postnatally but showed different spatial patterns. In particular, and in contrast to previous in situ hybridization studies, labeling of NR2 subunits was observed in horizontal cell bodies and in the outer plexiform layer, indicating that functional NMDA receptors are expressed in this retinal cell type in the rat. Expression of NR2D was restricted to the inner retina and seemed to be involved in neurotransmission within the rod pathway. In the inner plexiform layer (IPL), distinct patterns of labeling were observed for different NMDA subunits. NR1 was found in two bands which can be related to the off- and on-signal pathways, whereas NR2A and NR2B were located in two bands within the off-sublaminae of the IPL. The antibody against NR2C was distributed throughout the whole IPL, and NR2D was expressed exclusively in the innermost part of the IPL where rod bipolar cell terminals terminate. Distinct bands of immunoreactivity in the IPL were observed only from P14 on. In conclusion, there are clear differences in the spatial distribution and temporal expression of NMDA receptor subtypes in the rodent retina. This indicates that specific retinal cells selectively express glutamate receptors composed of different subunit combinations and thus display different pharmacological and kinetic properties.     

Involvement of neuronal cell surface molecule B2 in the formation of retinal plexiform layers.

The B2 molecule is a 220 kd neuronal cell surface protein of Xenopus, recognized by monoclonal antibody B2 (MAb B2). Immunohistochemistry using MAb B2 revealed that the B2 molecule was expressed in both the inner and outer plexiform layers within the neural retina. During development of the neural retina, the B2 molecule first appeared at stages 35/36 in the newly formed plexiform layers. When embryonic eyes were cultured in the presence of anti-B2 antiserum (Fab fragments), the formation of the retinal plexiform layers was impeded. These data suggest that the cell surface molecule B2 plays a role in the development of retinal plexiform layers.     

An ultrastructural study of embryonic chick retinal neurons in culture

Summary The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study.At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, (i) outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and (ii) a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and (iii) a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form.Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated.These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.     

Immunocytochemical Evidence of the Localization of the Crumbs Homologue 3 Protein (CRB3) in the Developing and Mature Mouse Retina

CRB3 (Crumbs homologue 3), a member of the CRB protein family (homologous to the Drosophila Crumbs), is expressed in different epithelium-derived cell types in mammals, where it seems to be involved in regulating the establishment and stability of tight junctions and in ciliogenesis. This protein has been also detected in the retina, but little is known about its localization and function in this tissue. Our goal here was to perform an in-depth study of the presence of CRB3 protein in the mouse retina and to analyze its expression during photoreceptor ciliogenesis and the establishment of the plexiform retinal layers. Double immunofluorescence experiments for CRB3 and well-known markers for the different retinal cell types were performed to study the localization of the CRB3 protein. According to our results, CRB3 is present from postnatal day 0 (P0) until adulthood in the mouse retina. It is localized in the inner segments (IS) of photoreceptor cells, especially concentrated in the area where the connecting cilium is located, in their synaptic terminals in the outer plexiform layer (OPL), and in sub-populations of amacrine and bipolar cells in the inner plexiform layer (IPL).     

Celsr3 is required for normal development of GABA circuits in the inner retina

The identity of the specific molecules required for the process of retinal circuitry formation is largely unknown. Here we report a newly identified zebrafish mutant in which the absence of the atypical cadherin, Celsr3, leads to a specific defect in the development of GABAergic signaling in the inner retina. This mutant lacks an optokinetic response (OKR), the ability to visually track rotating illuminated stripes, and develops a super-normal b-wave in the electroretinogram (ERG). We find that celsr3 mRNA is abundant in the amacrine and ganglion cells of the retina, however its loss does not affect synaptic lamination within the inner plexiform layer (IPL) or amacrine cell number. We localize the ERG defect pharmacologically to a late-stage disruption in GABAergic modulation of ON-bipolar cell pathway and find that the DNQX-sensitive fast b1 component of the ERG is specifically affected in this mutant. Consistently, we find an increase in GABA receptors on mutant ON-bipolar terminals, providing a direct link between the observed physiological changes and alterations in GABA signaling components. Finally, using blastula transplantation, we show that the lack of an OKR is due, at least partially, to Celsr3-mediated defects within the brain. These findings support the previously postulated inner retina origin for the b1 component and reveal a new role for Celsr3 in the normal development of ON visual pathway circuitry in the inner retina.     

Transient requirement for ganglion cells during assembly of retinal synaptic layers

The inner plexiform layer (IPL) of the vertebrate retina comprises functionally specialized sublaminae, representing connections between bipolar, amacrine and ganglion cells with distinct visual functions. Developmental mechanisms that target neurites to the correct synaptic sublaminae are largely unknown. Using transgenic zebrafish expressing GFP in subsets of amacrine cells, we imaged IPL formation and sublamination in vivo and asked whether the major postsynaptic cells in this circuit, the ganglion cells, organize the presynaptic inputs. We found that in the lak/ath5 mutant retina, where ganglion cells are never born, formation of the IPL is delayed, with initial neurite outgrowth ectopically located and grossly disorganized. Over time, the majority of early neurite projection errors are corrected, and major ON and OFF sublaminae do form. However, focal regions of disarray persist where sublaminae do not form properly. Bipolar axons, which arrive later, are targeted correctly, except at places where amacrine stratification is disrupted. The lak mutant phenotype reveals that ganglion cells have a transient role organizing the earliest amacrine projections to the IPL. However, it also suggests that amacrine cells interact with each other during IPL formation; these interactions alone appear sufficient to form the IPL. Furthermore, our results suggest that amacrines may guide IPL sublamination by providing stratification cues for other cell types.     

The zebrafish Pard3 ortholog is required for separation of the eye fields and retinal lamination

The vertebrate retina develops from a sheet of neuroepithelial cells. Because adherens and tight junctions are critical for epithelial and neuronal differentiation in a variety of eukaryotic systems, we examined the role of Par-3, a PDZ scaffold protein that is critical in cellular membrane junction formation. We cloned the zebrafish Par-3 ortholog (pard3), which encodes two Pard3 proteins (150 and 180 kDa) that differ in their carboxyl-terminus. Immunohistochemistry revealed that Pard3 localized to the apical region of the retinal and brain neuroepithelium, partially overlapping the adherens junction-associated actin bundles. After retinal lamination, the Pard3 protein was restricted to the outer limiting membrane and the outer and inner plexiform layers in the retina. Reducing Pard3 expression with antisense morpholinos caused loss of the retinal pigmented epithelia, disruption of retinal lamination, and cell death in the ventral diencephalon, which resulted in cyclopia. Overexpressing Pard3 by injection of wild-type pard3 mRNA resulted in cyclopia and eyeless embryos. Thus, Pard3 plays a critical role in the origination and separation of zebrafish eye fields and retinal lamination.     

A multi-layer model of retinal oxygen supply and consumption helps explain the muted rise in inner retinal PO(2) during systemic hyperoxia

A multi-layer mathematical model of oxygen supply and consumption in the rat retina is described. The model takes advantage of the highly layered structure of the retina and the compartmentalisation of the available oxygen sources. The retina is divided into eight layers, each with a distinct oxygen consumption or supply rate. When applied to the available data from intraretinal oxygen measurements in the rat under normal physiological conditions, a close fit between the model and the data was achieved (r(2)=0.98+0.005, n=6). The model was then used to investigate recent evidence of oxygen regulating mechanisms in the rat retina during systemic hyperoxia. Fitting our model to the experimental data (r(2)=0.988+0.004, n=25) allowed the relative oxygen delivery or consumption of the key retinal layers to be determined. Two factors combine to produce the relative stability of inner retinal oxygen levels in hyperoxia. The retinal layer containing the outer plexiform layer/deep retinal capillaries, switches from a net source to a net consumer of oxygen, and the oxygen consumption of the outer region of the inner plexiform layer increases significantly. The model provides a useful tool for examining oxygen consumption and supply in all retinal layers, including for the first time, those layers within the normally perfused inner retina.     

Connexin 40 expression in bovine and rat retinas

Retinal neurons are extensively coupled through gap junction intercellular channels, but few connexin subtypes have been identified in mammalian retinal neurons. Based on previous findings that retinal gap junctional coupling is modulated by both dopamine and nitric oxide, presumably through connexin phosphorylation, we examined whether the connexin phosphoprotein subtype, connexin 40 (Cx40), was expressed in mammalian retinas. Immunostaining of rat and bovine retinas using Cx40-specific antibodies from two independent sources showed punctate staining between cells in the outer nuclear layer (ONL) and a sublayer of cells within the inner nuclear layer (INL). In addition, sparse punctate staining was detected in the ganglion cell/axon fiber layers (GCL/AFL). No punctate staining was observed in the outer (OS) or inner segment (IS) layers, and rarely in the outer plexiform layer (OPL) or inner plexiform layer (IPL). Double immunostaining of bovine retinas with antibodies to G(o), which stains bipolar cells, and to Cx40, showed little overlap, suggesting these bipolar cells do not express Cx40. Western blot analysis of alkaline-extracted bovine retinal membranes revealed Cx40 immunopositive bands of about 40 kD (monomer) and 80 kD (dimer). In both locations (monomer and dimer), the bands appeared as doublets, and their immunoreactivity was abolished when the antibody was pre-adsorbed with immunogenic Cx40 peptide. The doublet at 40 kD co-migrated with an immunopositive doublet present in heart membranes. Treatment with alkaline phosphatase altered the banding pattern of Cx40. The results suggest that the connexin phosphoprotein subtype, Cx40, is expressed within the neural layers of the mammalian retina.     

THE DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN VERTEBRATE RETINA1

Abstract— Choline acetyltransferase (ChAc) activity was determined in retinal layers from 10 vertebrates. In all animals, the highest activity was in the inner plexiform layer, intermediate activity in the inner nuclear and ganglion cell layers, and very low activity in the photoreceptor and outer plexiform layers and optic nerve. The pattern of distribution of enzyme activity within the inner nuclear layer corresponds quantitatively to the distribution of amacrine cells within that layer. A species difference of almost 90-fold was found between the lowest and highest values for ChAc activity in inner plexiform layer. The variation in enzyme activity found among homeotherms in inner nuclear and inner plexiform layers is related to the number of amacrine cell synapses in the inner plexiform layer. But the differences in enzyme activity are generally greater than those which have been found in numbers of amacrine cell synapses between species. The data suggest that cholinergic neurons in retina are to be found predominantly among the amacrine cell types and that not all amacrine cells will be found to be cholinergic.     

Studies of the developing chick retina using monoclonal antibody 8A2 that recognizes a novel set of gangliosides

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9-O-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab D1.1 which recognizes 9-O-acetyl GD3.     

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.     

Immunocytochemical localization of glycine and glycine receptors in the retina of the frog <Emphasis Type="Italic">Rana ridibunda</Emphasis>

Glycine and glycine receptors (GlyRs) were analyzed immunocytochemically in the retina of the frog Rana ridibunda. Glycine was localized to somata of glycinergic amacrine and interplexiform cells. Approximately 50% of the cells in the amacrine cell layer were found to be glycinergic. GlyRs of the inner plexiform layer (IPL) were localized to brightly fluorescent puncta, probably representing postsynaptic clusters of GlyRs. GlyR clusters were not evenly distributed across the IPL but showed patterns of stratification specific for the various GlyR subunits. Clusters containing the 1 subunit formed four narrow strata within the IPL. Clusters containing the 3 subunit were more abundant and covered the whole IPL, with a band of higher density in stratum 3. Clusters of GlyRs were also observed in the outer plexiform layer. Thus, several isoforms of synaptic GlyRs involved with different synapses and inhibitory circuits are present in the frog retina.This work was supported by the Deutsche Forschungsgemeinschaft SFB269/B4     

Differential Alterations in the Expression of Neurotransmitter Receptors in Inner Retina following Loss of Photoreceptors in rd1 Mouse

Loss of photoreceptors leads to significant remodeling in inner retina of rd1 mouse, a widely used model of retinal degeneration. Several morphological and physiological alterations occur in the second- and third-order retinal neurons. Synaptic activity in the excitatory bipolar cells and the predominantly inhibitory amacrine cells is enhanced. Retinal ganglion cells (RGCs) exhibit hyperactivity and aberrant spiking pattern, which adversely affects the quality of signals they can carry to the brain. To further understand the pathophysiology of retinal degeneration, and how it may lead to aberrant spiking in RGCs, we asked how loss of photoreceptors affects some of the neurotransmitter receptors in rd1 mouse. Using Western blotting, we measured the levels of several neurotransmitter receptors in adult rd1 mouse retina. We found significantly higher levels of AMPA, glycine and GABAa receptors, but lower levels of GABAc receptors in rd1 mouse than in wild-type. Since GABAa receptor is expressed in several retinal layers, we employed quantitative immunohistochemistry to measure GABAa receptor levels in specific retinal layers. We found that the levels of GABAa receptors in inner plexiform layer of wild-type and rd1 mice were similar, whereas those in outer plexiform layer and inner nuclear layer combined were higher in rd1 mouse. Specifically, we found that the number of GABAa-immunoreactive somas in the inner nuclear layer of rd1 mouse retina was significantly higher than in wild-type. These findings provide further insights into neurochemical remodeling in the inner retina of rd1 mouse, and how it might lead to oscillatory activity in RGCs.     

The receptor protein tyrosine phosphatase mu, PTPmu, regulates histogenesis of the chick retina

The formation of laminae within the retina requires the coordinate regulation of cell differentiation and migration. The cell adhesion molecule and member of the immunoglobulin superfamily, receptor protein tyrosine phosphatase Mu, PTPmu, is expressed in precursor and early, differentiated cells of the prelaminated retina, and later becomes restricted to the inner plexiform, ganglion cell, and optic fiber layers. Since the timing of PTPmu expression correlates with the peak period of retinal lamination, we examined whether this RPTP could be regulating cell adhesion and migration within the retina, and thus influencing retinal development. Chick retinal organ cultures were infected with herpes simplex viruses encoding either an antisense sequence to PTPmu, wild-type PTPmu, or a catalytically inactive mutant form of PTPmu, and homophilic adhesion was blocked by using a function-blocking antibody. All conditions that perturbed PTPmu dramatically disrupted retinal histogenesis. Our findings demonstrate that catalytic activity and adhesion mediated by PTPmu regulate lamination of the retina, emphasizing the importance of adhesion and signaling via receptor protein tyrosine phosphatases in the developing nervous system. To our knowledge, this is the first demonstration that an Ig superfamily RPTP regulates the lamination of any neural tissue.     

Control of neuronal morphology by the atypical cadherin Fat3

Neurons receive signals through dendrites that vary widely in number and organization, ranging from one primary dendrite to multiple complex dendritic trees. For example, retinal amacrine cells (ACs) project primary dendrites into a discrete synaptic layer called the inner plexiform layer (IPL) and only rarely extend processes into other retinal layers. Here, we show that the atypical cadherin Fat3 ensures that ACs develop this unipolar morphology. AC precursors are initially multipolar but lose neurites as they migrate through the neuroblastic layer. In fat3 mutants, pruning is unreliable and ACs elaborate two dendritic trees: one in the IPL and a second projecting away from the IPL that stratifies to form an additional synaptic layer. Since complex nervous systems are characterized by the addition of layers, these results demonstrate that mutations in a single gene can cause fundamental changes in circuit organization that may drive nervous system evolution.     

Development of retinal layers

A noticeable characteristic of nervous systems is the arrangement of synapses into distinct layers. Such laminae are fundamental for the spatial organisation of synaptic connections transmitting different kinds of information. A major example of this is the inner plexiform layer (IPL) of the vertebrate retina, which is subdivided into at least ten sublayers. Another noticeable characteristic of these retina layers is that neurons are displayed in the horizontal plane in a non-random array termed as mosaic patterning. Recent studies of vertebrate and invertebrate systems have identified molecules that mediate these interactions. Here, we review the last mechanisms and molecules mediating retinal layering.