Presynaptic and postsynaptic localization of GABA(B) receptors in neurons of the rat retina

The recently cloned GABA(B) receptors were localized in rat retina using specific antisera. Immunolabelling was detected in the inner and outer plexiform layers (IPL, OPL), and in a number of cells in the inner nuclear layer and the ganglion cell layer. Double-labelling experiments for GABA (gamma-aminobutyric acid) and GABA(B) receptors, respectively, demonstrated a co-localization in horizontal cells and amacrine cells. Electron microscopy showed that GABA(B) receptors of the OPL were localized presynaptically in horizontal cell processes invaginating into photoreceptor terminals. In the IPL, GABA(B) receptors were present presynaptically in amacrine cells, as well as postsynaptically in amacrine and ganglion cells. The postnatal development of GABA(B) receptors was also studied, and immunoreactivity was observed well before morphological and synaptic differentiation of retinal neurons. The present results suggest a presynaptic (autoreceptor) as well as postsynaptic role for GABA(B) receptors. In addition, the extrasynaptic localization of GABA(B) receptors could indicate a paracrine function of GABA in the retina.     

The role in cell binding of a beta-bend within the triple helical region in collagen alpha 1 (I) chain: structural and biological evidence for conformational tautomerism on fiber surface

Dystrophin is a plasma membrane-associated cytoskeletal protein of the spectrin superfamily. The dystrophin cytoskeleton has been first characterized in muscle. Muscular 427 kDa dystrophin binds to subplasmalemmal actin filaments via its amino-terminal domain. The carboxy-terminus of dystrophin binds to a plasma membrane anchor, beta-dystroglycan, which is associated on the external side with the extracellular matrix receptor, alpha-dystroglycan, that binds to the basal lamina proteins laminin-1, laminin-2, and agrin. In the muscle, the dystroglycan complex is associated with the sarcoglycan complex that consists of several glycosylated, integral membrane proteins. The absence or functional deficiency of the dystrophin cytoskeleton is the cause of several types of muscular dystrophies including the lethal Duchenne muscular dystrophy (DMD), one of the most severe and most common genetic disorders of man. The dystrophin complex is believed to stabilize the plasma membrane during cycles of contraction and relaxation. Muscular dystrophin and several types of dystrophin variants are also present in extramuscular tissues, e.g. in distinct regions of the central nervous systems including the retina. Absence of dystrophin from these sites is believed to be responsible for some extramuscular symptoms of DMD, e.g. mental retardation and disturbances in retinal electrophysiology (reduced b-wave in electroretinograms). The reduced b-wave in electroretinograms indicated a disturbance of neurotransmission between photoreceptors and ON-bipolar cells. At least two different dystrophin variants are present in photoreceptor synaptic complexes. One of these dystrophins (Dp260) is virtually exclusively expressed in the retina. In the neuroretina, dystrophin is found in significant amounts in the invaginated photoreceptor synaptic complexes. At this location dystrophin colocalizes with dystroglycan. Agrin, an extracellular ligand of alpha-dystroglycan, is also present at this location whereas the proteins of the sarcoglycan complex appear to be absent in photoreceptor synaptic complexes. Dystrophin and dystroglycan are located distal from the ribbon-containing active synaptic zones where both proteins are restricted to the photoreceptor plasma membrane bordering on the lateral sides of the synaptic invagination. In addition, some neuronal profiles of the postsynaptic complex also contain dystrophin and beta-dystroglycan. These profiles appear to belong at least in part to projections of the photoreceptor terminals into the postsynaptic dendritic complex. In view of the abnormal neurotransmission between photoreceptors and ON-bipolar cells in DMD patients the dystrophin/beta-dystroglycan-containing projections of photoreceptor presynaptic terminals into the postsynaptic dendritic plexus might somehow modify the ON-bipolar pathway. Another retinal site associated with dystrophin/beta-dystropglycan is the plasma membrane of Müller cells where dystrophin/beta-dystroglycan appear to be present at particular high concentrations. At this location the dystrophin/dystroglycan complex may play a role in the attachment of the retina to the vitreous, and, under pathological conditions, in traction-induced retinal detachment.     

Hormonal correlates of environmentally induced metamorphosis in the Western spadefoot toad, Scaphiopus hammondii

An elaborate network of transmitter receptors, synapse associated proteins (SAPs), and cytoskeletal elements, generally known as the postsynaptic density, is involved with efficient synaptic signaling. The localization of the synapse associated protein SAP102 was studied in the rat retina by using immunocytochemical methods. Immunofluorescence for SAP102 was most prominent in the inner plexiform layer (IPL). It had a punctate appearance, suggesting a synaptic clustering of SAP102 in the IPL. Electron microscopy by use of pre-embedding immunocytochemistry showed that SAP102 is concentrated in the IPL in processes which are postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for SAP102. Double-labeling experiments were performed in order to find out whether SAP102 is involved with the clustering the N-methyl-D-aspartate (NMDA) receptor 2A subunit (NR2A). Only a fraction (approximately 23%) of the SAP102 clusters expressed NR2A, suggesting SAP102 is also associated with other subunits or receptors. Distinct SAP102 labeling was also present in horizontal cell processes in the outer plexiform layer (OPL), which are inserted as lateral elements into photoreceptor ribbon synapses (triads). The optic nerve fibre layer was also diffusely immunoreactive for SAP102. The postsynaptic aggregation of SAP102 at bipolar cell dyads and at photoreceptor triads suggests SAP102 is associated with the clustering of transmitter receptors. However, the labeling of the optic nerve fibre layer indicates additional functions of SAP102 in the retina.     

Contactin/F11 and tenascin-C co-expression in the chick retina correlates with formation of the synaptic plexiform layers

The neural immunoglobulin-like cell adhesion molecule contactin/F11 and the extracellular matrix glycoprotein tenascin-C are prominent molecules in the developing nervous system which interact in in vitro assays (Zisch et al., J. Cell Biol. 119, 203-213). To determine their potential role in neural development, the distribution of tenascin-C and contactin/F11 was examined in the developing chick retina. The onset of both tenascin-C and contactin/F11 expression coincides with the appearance of ganglion cell dendrides and neurites from bipolar and amacrine cells in the inner layer (IPL) at E8, and the extension of bipolar and horizontal cell processes in the outer plexiform layer (OPL) at E9. Contactin/F11 expression is co-ordinately upregulated with the TN190 and TN200 tenascin-C isoforms between embryonic day 8 (E8) and E17, while little, if any, of the TN220 isoform, which does not bind contactin/F11, is detected. In situ hybridization reveals that tenascin-C and contactin/F11 mRNAs are synthesized by different neuronal types. Tenascin-C mRNA probes hybridize to amacrine and displaced amacrine neurons, and horizontal neurons. In cultured retinal cells, tenascin-C is also present on process-bearing neurofilament-positive cells. Contactin/F11 mRNA is detected in bipolar cells or their precursors from E8-9, and later in horizontal and ganglion neurons. The highest levels and greatest overlap in the synaptic IPL and OPL are reached at E17, when the stratification of the retina is nearly complete. These results are consistent with a putative role for contactin/F11-tenascin-C interactions in the establishment of synaptic layers in the retina.     

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.     

Differences in the retinal GABA system among control, spastic mutant and retinal degeneration mutant mice

Immunocytochemical methods were used to compare the GABA system in control mice and two mutant strains: spastic which has reduced glycine receptors and retinal degeneration mutant in which the photoreceptors degenerate and reportedly have increased GABA and GAD levels. We found that the spastic mutant retina had reduced GABA-immunoreactivity (IR) in the proximal retina, reduced staining for GAD-1440 in the OPL, and reduced GABAA receptor staining in the OPL, compared to control. The retinal degeneration mutant retinas had enhanced GABA-IR throughout the retina, particularly in Müller cells, bipolar cells and IPL, and enhancement of GABAA receptor staining in the OPL, compared to control. The distributions of GABA-IR, GAD-1440-IR and GABAA receptor-IR in retinas of spastic mutant mice that also expressed the retinal degeneration phenotype resembled those found in retinas of mice that expressed only the retinal degeneration phenotype rather than those that expressed only the spastic mutation. No differences were observed among the conditions for GAD-65, GAD-67 or GABA-T. Our results with the spastic and retinal degeneration mutant mice demonstrate that attenuation in the glycinergic system and photoreceptor degeneration, respectively, is accompanied by alterations in different aspects of the GABA system, giving impetus for caution in the interpretation of experiments involving genetic manipulation of complex phenotypes.     

Synaptic inputs mediating bipolar cell responses in the tiger salamander retina

Postsynaptic receptors in bipolar cells were studied by focal application of glutamate and GABA to the outer and inner plexiform layers (OPL and IPL) under visual guidance in living retinal slices of the tiger salamander. Two different types of conductance change could be elicited in bipolar cells by applying glutamate to the OPL. In off-center cells, which had axon telodendria ramifying in the distal 55% of the IPL, glutamate elicited a conductance increase associated with a reversal potential near -5 mV. In on-center cells, which had telodendria stratified in the proximal 45% of the IPL, glutamate caused a conductance decrease associated with a reversal potential near -11 mV. These observations suggest that glutamate gates relatively nonspecific cation channels at synapses between photoreceptors and bipolar cell dendrites. Application of glutamate to the IPL elicited no conductance change in Co2+ Ringer's solution, but in normal Ringer's it generated a conductance increase associated with a reversal potential near the chloride equilibrium potential (ECl). These findings are consistent with the notion that glutamate receptors exist in GABAergic and/or glycinergic amacrine cells, and that glutamate in the IPL depolarizes these cells, causing GABA and/or glycine release and the opening of chloride channels in bipolar cell axon terminals. In Co2+ Ringer's, application of GABA at the OPL elicited no conductance changes in bipolar cells, suggesting that GABA receptors do not exist on bipolar cell dendrites. Applied at the IPL, GABA elicited large conductance increases associated with a reversal potential near ECl. Implications of these results for the functional circuitry of the tiger salamander retina are discussed.     

Comparison of immunolocalization patterns for the synaptic vesicle proteins p65 and synapsin I in macaque monkey retina

The distributions of the two synaptic vesicle proteins p65 [Matthew et al. (1981) J. Cell Biol., 91:257-269] and synapsin I [De Camilli et al. (1983) J. Cell Biol., 96:1337-1354] were compared in macaque monkey retina using pre-embedding immunocytochemistry for both light and electron microscopy. The monoclonal antibody AB-48 against p65 labeled ribbon-containing synaptic terminals of cone, rod, and bipolar cells as well as many conventional synapses of amacrine cells. In contrast, a polyclonal antiserum against synapsin I (SYN I) labeled many amacrine conventional synapses but no photoreceptor or bipolar ribbon synaptic terminals. Horizontal cell pre- and post-synaptic profiles in the outer plexiform layer were not labeled by either antibody. At the light microscopic level, the banding patterns in the inner plexiform layer also differed for the two antibodies, with four bands of AB-48 immunoreactivity in sublayers S1, S2, S4, and S5 but only three bands of SYN I immunoreactivity in S1, S3, and S5. SYN I also labeled varicose fibers in both the inner nuclear layer and the outer plexiform layer that are probably processes of dopaminergic and GABAergic interplexiform cells. Varicose fibers in the ganglion cell layer were labeled by both antibodies. These results provide the first electron microscopic immunocytochemical labeling for AB-48 and SYN I in intact retina and confirm that AB-48 labels both ribbon and conventional synaptic terminals, whereas SYN I labels only conventional synapses.     

A-type horizontal cells of the superior edge of the linear visual streak of the rabbit retina have oriented, elongated dendritic trees

The horizontal cells of the rabbit retina have been studied by light microscopy of Golgi-impregnated whole-mount retinas. The two types of horizontal cell of the rabbit retina are similar to the horizontal cells of the cat retina in most respects. However, the majority of the A-type horizontal cells of the rabbit have asymmetrical dendritic fields compared to the circular, symmetrical dendritic fields of this cell type in the cat. The A-type horizontal cells of the superior edge of the linear visual streak in the rabbit retina are the most strikingly asymmetric and most of them are elongated and oriented in a direction approximately parallel to the linear visual streak. Like H1 axon terminals of the turtle retina the oriented, elongated A-type horizontal cells of the rabbit visual streak region may play a role in the neurocircuitry which underlies orientation sensitive ganglion cells.     

Müller cells in the retina of the cane toad, Bufo marinus, express neuropeptide Y-like immunoreactivity

We have used light- and electron-microscopic immunohistochemistry to identify the presence of immunoreactivity to neuropeptide Y (NPY) within Müller cells in the retina of the cane toad, Bufo marinus. Müller cells containing NPY-like immunoreactivity (NPY-LI) were identified at the light-microscopic level by the coexistence with immunoreactivity to glial fibrillary acidic protein (GFAP) and at the ultrastructural level by their characteristic relationship to neuron cell bodies and processes. At the light-microscopic level, those cells which contained both NPY-LI and GFAP-LI usually had small cell bodies in the inner nuclear layer, while those cells which contained only NPY-LI were identified as large and small amacrine cells. The radially oriented primary processes in the inner plexiform layer and the vitreal end feet of GFAP-LI Müller cells also expressed NPY-LI. At the ultrastructural level, thin lamellar processes of Müller cells with NPY-LI enclosed some amacrine cell bodies in the inner nuclear layer and amacrine cell dendrites in the inner plexiform layer. These observations suggest that NPY-LI is localized in Müller cells in addition to two types of amacrine cells previously identified in the Bufo retina. This study provides the first evidence that glial elements in the vertebrate retina express NPY-LI.     

Modulation of ion channels in rod photoreceptors by nitric oxide

Subcellular compartments in the outer retina of the larval tiger salamander were identified as likely sites of production of nitric oxide (NO), a recently recognized intercellular messenger. NADPH diaphorase histochemistry and NO synthase immunocytochemistry labeled photoreceptor ellipsoids and the distal regions of bipolar and glial cells apposing photoreceptor inner segments, suggesting a role for NO in visual processing in the outer retina. We investigated the actions of NO on several rod photoreceptor ion channels. Application of the NO-generating compound S-nitrosocysteine increased Ca2+ channel current and a voltage-independent conductance, but had no affect on voltage-gated K+ or nonspecific cation currents. Given the steep relation between voltage-dependent Ca2+ influx and photoreceptor synaptic output, these results indicate that NO could modulate transmission of the photoresponse to second order cells.     

Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina

Synapse-associated proteins are the scaffold for the selective aggregation of ion channels at synapses; they provide the link to cytoskeletal elements and possibly are involved with the regulation of synaptic efficacy by electrical activity. The localization of the postsynaptic density protein PSD-95 was studied in different mammalian retinae (rat, monkey, and tree shrew) by using immunocytochemical methods. Immunofluorescence for PSD-95 was most prominent in the outer plexiform layer (OPL). The axon terminals of rods and cones, the rod spherules and cone pedicles, were strongly labeled. Electron microscopy, using preembedding immunocytochemistry, showed PSD-95 localized presynaptically within the photoreceptor terminals. Distinct PSD-95 labeling was also present in the inner plexiform layer (IPL). It had a punctate appearance suggesting the synaptic clustering of PSD-95 in the IPL. Electron microscopy showed that PSD-95 was concentrated in processes that were postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for PSD-95. Double-labeling experiments were performed for PSD-95 and for SAP 102 or PSD-93, respectively, two other members of the family of synapse-associated proteins. All three were found to be colocalized in the synaptic hot spots in the IPL. In the OPL, however, PSD-95 and PSD-93 were found presynaptically, whereas SAP 102 was located postsynaptically at photoreceptor synapses. Double-labeling experiments also were performed for PSD-95 and for the NR1 subunit of the NMDA receptor. They were found to be colocalized in synaptic hot spots in the IPL.     

C-reactive protein as an outcome predictor for maintenance hemodialysis patients

We produced the monoclonal antibody RT10F7, characterized its antigenic specificity and expression in the adult and developing retina, in cultured retinal cells and in other parts of the central nervous system. In metabolically-labelled retinal cultures RT10F7 immunoprecipitated a protein of approximately 36,000 mol. wt. In the adult, RT10F7 stained endfeet of Müller cells in the ganglion cell layer, four horizontal bands in the inner plexiform layer, and radial fibres in the outer plexiform layer which terminated at the outer limiting membrane. In the inner nuclear layer, most somata were underlined by Müller processes that wrapped around them, but some cell bodies were immunoreactive for RT10F7 in the cytoplasm. During development, postnatal day 21 was the first age at which the adult pattern of immunoreactivity was present, although a fourth band in the inner plexiform layer was less clear than for the adult. By 14 and eight days after birth, the pattern of RT10F7 immunoreactivity approximated that of the adult; however, only three bands and one band were present, respectively, in the inner plexiform layer. At earlier ages, postnatal days 4, 1 and embryonic ages 19 and 15, the monoclonal antibody stained Müller cell endfeet and radial fibres, from the inner plexiform layer through the neuroblastic layer to the outer limiting membrane. At these ages, the immunoreactivity was more prominent at the level of Müller cell endfeet. The monoclonal antibody stained glia in preparations of dissociated retinal cells maintained in culture but not astrocytes or oligodendrocytes from optic nerve cultures. In brain sections, tanycytes exhibited RT10F7 immunoreactivity. The monoclonal antibody RT10F7 recognized a specific cell type in the retina, the Müller cell. In the adult and developing retina, RT10F7 recognized an antigen that is present primarily in Müller cell processes. This feature allowed us to follow the maturation of the Müller cell and correlate it with developmental events in the retina. RT10F7 is a specific marker for Müller cells in vivo and in vitro and may be useful for studies of function of Müller cells after ablation or after injuries that are known to activate Müller cells.     

Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina

Adult rat hippocampus-derived neural progenitor cells (AHPC) show considerable adaptability following grafting to several brain regions. To evaluate the plasticity of AHPCs within the optic retina, retrovirally engineered AHPCs were grafted into the vitreous cavity of the adult and newborn rat eye. Within the adult eye, AHPCs formed a uniform nondisruptive lamina in intimate contact with the inner limiting membrane. Within 4 weeks of grafting to the developing eye, the AHPCs were well integrated into the retina and adopted the morphologies and positions of Müller, amacrine, bipolar, horizontal, photoreceptor, and astroglial cells. Although the cells expressed neuronal or glial markers, none acquired end-stage markers unique to retinal neurons. This suggests that the adult-derived stem cells can adapt to a wide variety of heterologous environments and express some but not all features of retinal cells when exposed to the cues present late in retinal development.     

N-methyl-D-aspartate receptors containing the NR2D subunit in the retina are selectively expressed in rod bipolar cells

N-methyl-D-aspartate receptor subunit messenger RNAs are widely expressed in the retina and several types of second and third order neurons are responsive to N-methyl-D-aspartate. Functional N-methyl-D-aspartate receptors are assembled from the NR1 subunit with at least one of the four NR2 subunit variants (NR2A-2D). We have analysed immunohistochemically the cellular distribution of N-methyl-D-aspartate receptors containing the NR2D subunit in the rat and rabbit retina. Using a subunit-specific NR2D antiserum, exclusively bipolar cells with somata localized close to the outer plexiform layer were labelled in both species. The axons were immunoreactive and arborized in the innermost inner plexiform layer. The morphology and localization of these cells, which were much more numerous in rat than in rabbit, suggested that they are rod bipolar cells. This was confirmed in both species by co-localization of the NR2D subunit immunoreactivity with protein kinase C-alpha, a selective marker for rod bipolar cells. At the subcellular level, a distinct polarization in the distribution of NR2D immunoreactivity was demonstrated by confocal laser scanning microscopy: staining was moderate in dendrites arborizing within the outer plexiform layer, intense at that pole of the soma facing the outer plexiform layer, and low in the portion of the soma embedded in the inner nuclear layer. Proximal axonal segments and axonal end-feet in the inner plexiform layer displayed the strongest NR2D subunit immunoreactivity. The axonal staining suggests that neurotransmission of the rod bipolar cells is modulated within the inner plexiform layer by N-methyl-D-aspartate receptors containing the NR2D subunit.     

Elevation of soluble IL-2 receptor and IL-4, and nonelevation of IFN-gamma in sera from patients with allergic asthma

This study is the first demonstration of glial fibrillary acidic protein (GFAP)-immunoreactivity in the retina of the lamprey Lampetra fluviatilis. This immunoreactivity is expressed on one hand, in radial processes and somata which belong to Müller cells and, on the other hand, in horizontal fibers in the intermediate plexus between horizontal cells. The tracing of these fibers to Müller cells or horizontal cells is discussed.     

Development of cell markers in subretinal rabbit retinal transplants

Retinas from embryonic rabbits at day E15 were transplanted to the subretinal space in adult rabbits. After survival times between 7 and 193 days, the rabbits were killed, and the transplants were processed for immunohistochemistry. The results show that subretinal transplants from embryonic rabbit retinas develop many, if not all, retinal neuronal types. The cells show approximately normal morphology and express a variety of cell-type-specific markers: photoreceptor cells express visual pigment proteins as identified by antibodies against rhodopsin (R2-15), color-specific cone pigments (COS-1, OS-2) and the cone specific antigen 50-1B11, rod bipolar cells express PKC, horizontal cells HPC-1 antigen and neurofilament 160 kDa, amacrine cells HPC-1 antigen, GABA and neurofilament 160 kDa, and glial cells express vimentin and glial fibrillary acidic protein. The high degree of rosette formation seen in many young grafts, diminishes with time; many transplant cells disappear, and the remaining cells present a less prominent formation of rosettes.     

Morphology and distribution of Müller cells in the retina of the toad Bufo marinus

We have previously shown that an antibody against neuron-specific enolase (NSE) selectively labels Müller cells (MCs) in the anuran retina (Wilhelm et al. 1992). In the present study the light- and electron-microscopic morphology of MCs and their distribution were described in the retina of the toad, Bufo marinus, using the above antibody. The somata of MCs were located in the proximal part of the inner nuclear layer and were interconnected with each other by their processes. The MCs were uniformly distributed across the retina with an average density of 1500 cells/mm2. Processes of MCs encircled the somata of photoreceptor cells isolating them from each other by glial sheath, except for those of the double cones. Some of the photoreceptor pedicles remained free of glial sheath. Electron-microscopic observations confirmed that MC processes provide an extensive scaffolding across the neural retina. At the outer border of the ganglion cell layer these processes formed a non-continuous sheath. The MC processes traversed through the ganglion cell layer and spread beneath it between the neuronal somata and the underlying optic axons. These processes formed a continuous inner limiting membrane separating the optic fibre layer from the vitreous tissue. Neither astrocytic nor oligodendrocytic elements were found in the optic fibre layer. The significance of the uniform MC distribution and the functional implications of the observed pattern of MC scaffolding are discussed.     

Nitric oxide modulates cGMP levels in neurons of the inner and outer retina in opposite ways

In the mammalian retina, neuronal nitric oxide synthase (NOS) is mainly localized in subpopulations of amacrine cells. One function of nitric oxide (NO) is to stimulate soluble guanylate cyclases which in turn synthesize cGMP. We used an antibody specific for cGMP to demonstrate cGMP-like immunoreactivity (cG-IR) in bovine, rat, and rabbit retinae and investigated the effects on cGMP levels of both exogenously applied NO and of endogenously released NO. We found that cGMP levels in inner and outer retina were controlled in opposite ways. In the presence of the NO-donors SNP, SIN-1 or SNAP, cG-IR was prominent in neurons of the inner retina, mainly in cone bipolar cells, some amacrine and ganglion cells. Retinae incubated in IBMX showed weak cG-IR in bipolar cells. Glutamate increased cG-IR in the inner retina, presumably by stimulating endogenous NO release, whereas NOS inhibitors or GABA and glycine decreased cG-IR in bipolar cells by reducing NO release. In somata, inner segments and spherules of rod photoreceptors the situation was reversed. cG-IR was undetectable in the presence of NO-donors or glutamate, was moderate in IBMX-treated retinae, but increased strongly in the presence of NOS inhibitors or GABA/glycine. We conclude that NO is released endogenously in the retina. In the presence of NO, cGMP levels are increased in neurons of the inner retina, but are decreased in rods.     

Localization of nitric oxide synthase, NADPH diaphorase and soluble guanylyl cyclase in adult rabbit retina

Nitric oxide (NO) acts as a neuronal messenger which activates soluble guanylyl cyclase (SGC) in neighboring cells and produces a wide range of physiological effects in the central nervous system (CNS). Using immunocytochemical and histochemical stains, we have characterized the NO/SGC system in the rabbit retina and to a lesser extent, in monkey retina. Based on staining patterns observed with an antibody to nitric oxide synthase (NOS) type I and a histochemical marker for NADPH diaphorase, a metabolic intermediate required for NOS activity, three major classes of neurons appear to generate NO in the rabbit retina. These include two subclasses of sparsely distributed wide field amacrine cells, rod and cone photoreceptors, and a subpopulation of ganglion cells. Equivalent cell populations were labeled in monkey retina. An antibody to SGC (tested only in rabbit retina), labeled large arrays of cone photoreceptors in the outer nuclear layer, both amacrine and bipolar cells in the inner nuclear layer (INL), as well as populations of neurons in the ganglion cell layer. These data suggest that the ability to generate NO is restricted to relatively few neurons in the inner retina and to photoreceptor cells in the outer retina; while presumptive target cells, containing pools of SGC, are widespread and form contiguous fields across the inner and outer nuclear layers (ONL) as well as the ganglion cell layer.