A developmentally regulated microsomal protein specific for the pigment epithelium of the vertebrate retina

In the vertebrate retina, the retinal pigment epithelium (RPE) performs specific functions critical to the normal process of vision. Although some of these functions are well documented, molecular data are still scarce. Using the RPE-specific monoclonal antibody RPE9, raised against human RPE cells, we have identified a novel 65 kD protein, conserved in mammals, birds, and frogs. This RPE-specific protein was found to be nonglycosylated. It was most effectively solubilized in the presence of detergent suggesting that it is associated with the RPE cell membranes. Its partitioning in the detergent phase of Triton X-114 and its solubilization in 0.75 M and 1.0 M KCl suggest that it interacts with the membrane either through a polypeptide anchor or charged amino acids. Cell fractionation by differential solubilization and differential centrifugation demonstrated that the protein was preferentially associated with the microsomal membrane fraction, where it is the major protein. Developmental expression of this 65 kD protein was examined in neonatal rats. Morphologically well-differentiated RPE cells did not express the 65 kD protein at birth. However, expression was detectable at postnatal day 4, that is, one to two days before the photoreceptors develop their outer segments, suggesting that the expression of the 65 kD protein may be coordinated with other developmental events in the intact retina. This is further supported by the fact that RPE cells in confluent culture lose the expression of this protein within two weeks, while they maintain their characteristic epithelial morphology. Because of its specificity, its evolutionary conservation, and its timing of expression, it is possible that this protein may be involved in one of the key roles of RPE and as such is an important molecular marker for RPE differentiation. © 1993 Wiley-Liss, Inc.     

Expression of voltage-dependent potassium channels in the developing visual system of Xenopus laevis

Accumulating evidence suggests that voltage-dependent potassium (Kv) channels have important and varied roles in the development of neuronal and non-neuronal cell types. They have been implicated in processes such as proliferation, cell adhesion, migration, neurite outgrowth, and axon guidance. In this study, we used antibodies against several electrically active Kv channel alpha-subunits (Kv1-4) to describe the spatial and temporal expression patterns of Kv channel subunits in Xenopus laevis retinal ganglion cell (RGC) somata, axons, and growth cones. We found that RGCs express Kv1.3-, Kv1.5-, Kv3.4-, and Kv4.2-like subunits. Each subunit displayed unique cellular and subcellular distributions. Moreover, the expression patterns changed considerably over the major period of Xenopus retinal cell genesis and differentiation. Weak or no immunoreactivity was observed with antibodies against Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv3.2 subunits in RGCs or other retinal cell types. In support of our previous pharmacologic evidence implicating Kv channels in RGC axon outgrowth, we found that Kv1.5-, Kv3.4-, and Kv4.2-like proteins, but not Kv1.3-like subunits, are abundantly expressed in RGC growth cones.     

Presynaptic and postsynaptic localization of GABAB 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 (γ-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.     

Normal chiasmatic routing of uncrossed projections from the ventrotemporal retina in albino Xenopus frogs

Albino mammals lacking melanin in the embryonic retinal pigment epithelium (RPE) have abnormal retinal decussation patterns at the optic chiasm: their uncrossed projections are smaller and arise from fewer, more peripheral temporal retinal ganglion cells than in con-specific wild-types. To determine whether these abnormalities generalize to nonmammalian mutants, we used anterograde and retrograde labeling methods to compare the distribution of retinal projections to the thalamus in adult normal and albino Xenopus frogs. In both pigmentation phenotypes, crossed retinal terminations covered approximately 80% of the neuropil of Bellonci (nB) and corpus geniculatum thalamicum (cgt) and uncrossed inputs occupied, respectively, approximately 75% and 25% of these two main visual centers. In the wild-type frogs and in the albinos, ganglion cells giving rise to the crossed projections were distributed throughout the retina, whereas ipsilaterally projecting cells were confined to a specific ventrotemporal retinal division. This region comprised approximately 40% of the total retinal area, was bordered by a well-defined line of decussation, and contained an average of approximately 3,000 ipsilaterally projecting ganglion cells of equivalent soma sizes in the two pigmentation phenotypes. In summary, we found no evidence of chiasmatic misrouting in the uncrossed retinothalamic projections of albino Xenopus, even though these pathways are substantial in normal frogs and share features in common with mammalian retinogeniculate projections. Our findings suggest that congenital RPE melanin deficiency results in major defects in the development of the retina and its central projections only in mammals.     

The optokinetic nystagmus and ocular pigmentation of hypopigmented mouse mutants

We tested the optokinetic nystagmus (OKN) reflex of various hypopigmented mutant mice and ultrastructurally examined the pigmentation of various ocular structures in these mutants. Using electron microscopy we examined the pigmentation of the choroid and retinal pigment epithelium (RPE) and measured the numerical density, volume density, and distribution of RPE melanosomes of mice with the following phenotypes: (1) wild type, (2) mutants that have abnormal or no OKN in response to horizontally moving, full-field stimulation, and (3) other mutants that have normal OKN but reduced choroidal pigmentation. We also measured the OKN of all these mice in response to horizontally moving stimuli that were restricted to the nasal or to the temporal retina. We found that in the mutants with normal OKN the numerical density of melanosomes in the RPE was within the range found for wild type, while the numerical density was reduced for the mutants with abnormal OKN. For one mutant with normal RPE pigmentation and normal OKN, the choroidal pigmentation was nearly absent. For the genotypes with abnormal OKN the volume density of the RPE melanosomes and percent apical melanosomes were sometimes greater and sometimes less than normal. The OKN patterns of these mice fell into the following categories: (1) wild type; (2) field-restriction dependent OKN with small following movements but no OKN in response to full-field stimulation, normal OKN in response to stimulation of the nasal retina, and OKN of reversed direction in response to stimulation of the temporal retina; (3) oblique with slow oblique following movements and reduced numbers of OKNs with oblique quick phases in response to horizontally moving, full-field stimulation, nearly normal OKN in response to stimulation of the nasal retina, and OKN of reversed direction in response to stimulation of the temporal retina. The horizontal component of the oblique response to full-field stimulation was in the same direction for the two eyes, but the vertical component was in the opposite direction. (4) Slow, small amplitude, with no or very small following movements in response to full-field stimulation, following movements in response to stimulation of the nasal retina and reversed "following" movements in response to stimulation of the temporal retina but few or no quick phases of the OKN for any stimulus condition. These results show that a variety of abnormalities of the OKN occur for hypopigmentation mutants of the mouse.(ABSTRACT TRUNCATED AT 400 WORDS)     

Concanavalin A inhibits nicotinic acetylcholine receptor function in cultured chick ciliary ganglion neurons

The effects of various lectins and toxins on neuronal nicotinic acetylcholine receptor function have been studied in primary cultures of chick ciliary ganglion neurons. Neuronal response to acetylcholine receptor activation was measured by a cation flux method at 4 degrees C in a high potassium-low sodium medium designed to stabilize membrane potential near zero, with acetylcholine as the agonist and cesium-137 as the tracer ion. Exposure to 1 mM acetylcholine for 30 s produced a 5-10-fold stimulation of cesium-137 influx. Acetylcholine-stimulated influx was inhibited more than 95% by 10 microM D-tubocurarine, but was insensitive to both 1 microM tetrodotoxin and 1 microM alpha-bungarotoxin. Concanavalin A (50 micrograms/ml) inhibited agonist-induced ion flux by 80% at 4 degrees C. Succinyl-concanavalin A was ineffective at concentrations up to 250 micrograms/ml, and could not protect against the concanavalin A inhibition. However, inhibition by concanavalin A was eliminated by prior incubation of the lectin with 0.2 M alpha-methyl-D-mannoside and subsequent co-incubation with the sugar. Wheat germ agglutinin, lentil lectin, cholera toxin and tetanus toxin were without effect at either 4 degrees C or 37 degrees C. These results suggest a specific interaction between concanavalin A and neuronal nicotinic acetylcholine receptors.     

Autoradiographic localization of high affinity GABA, benzodiazepine, dopaminergic, adrenergic and muscarinic cholinergic receptors in the rat, monkey and human retina

High affinity gamma-aminobutyric acid, benzodiazepine, strychnine (glycine), dopamine, spirodecanone, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic and muscarinic cholinergic binding sites were localized by semiquantitative autoradiography in rat and, in some instances, in monkey and human retinae using [3H]muscimol, [3H]flunitrazepam, [3H]strychnine, [3H]spiperone, [3H]prazosin, [3H]para-aminoclonidine, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate, respectively. In nearly every case, the inner plexiform layer (IP) contained a high receptor density. The distribution of alpha 1 sites was unusual in that binding was concentrated in the outer plexiform layer (OP). Dopaminergic and, to a lesser extent, beta-adrenergic binding was diffusely distributed in the outer nuclear layer, the OP, the inner nuclear layer and the IP. The ganglion cell layer displayed significant benzodiazepine binding. The intraretinal distribution of pre- and postsynaptic markers of these neurotransmitters is discussed.     

Immunohistochemical localization of cathechol-O-methyltransferase in circumventricular organs of the rat: Potential variations in the blood-brain barrier to native catechols

Catechol-O-methyltransferase (COMT) was localized in cells of the pia-arachnoid, and in epithelial cells of the choroid plexus, using an indirect immunofluorescence technique. The specific activity of COMT derived from these tissues was determined by radioenzymatic assay, and in the case of the choroid plexus was found to be 9-fold greater than that measured in whole rat brain. The level of COMT specific activity in pia-arachnoid was twice as high as that in whole brain.Indirect immunofluorescence studies also revealed an intensity of COMT immunofluorescence in the ciliary epithelium at the blood-aqueous barrier in the rat eye, similar to that visualized in the epithelium of the choroid plexus at the blood-cerebrospinal fluid barrier.The localization of COMT in the leptomeninges, choroid plexus, and ciliary epithelium is consistent with a role for this enzyme in the separation of catechol compounds synthesized in the central nervous system, from those of peripheral origin. Thus, catecholamines derived from the peripheral sympathetic system may be prevented from entering the brain parenchyma, which is innervated by the functionally distinct central catecholaminergic systems.     

Heterogeneity of muscarinic cholinergic receptors in the developing chick embryo retina

Heterogeneity of muscarinic cholinergic receptors was investigated in chick embryo retina throughout development and in chicks immediately after hatching. The presence of a homogeneous receptor population was evidenced by antagonist binding. The affinity of antagonists increased up to day 14 of incubation, when synaptogenesis occurs. After this stage, it remained substantially unchanged. The number of receptors increased in embryos until hatching. On the contrary, agonists, such as acetylcholine and carbachol, bound to two (high- and low-affinity) binding sites. Through development, the affinity of both significantly increased until day 14, further substantiating the hypothesis of a maturation of the receptor pattern which precedes synapse formation. Muscarinic cholinergic binding seems to identify 3 critical steps in retinal neuronal development. The first is between 7 and 9 days of incubation, the second when synaptogenesis occurs and the third after initiation of function.     

Neuroprotective role of erythropoietin by antiapoptosis in the retina

Erythropoietin (EPO) stimulates red blood cell production, in part by inhibiting apoptosis of the red blood cell precursors. The erythropoietic effects of EPO are circadian stage dependent. Retinal injury due to light occurs through oxidative mechanisms and is manifest by retinal and retinal pigment epithelium (RPE) cells apoptosis. The visual cycle might be circadian coordinated as a means of effectively protecting the retina from the detrimental effects of light‐induced, oxygen‐dependent, free radical–mediated damage, especially at the times of day when light is more intense. We show that the retinal expression of EPO and its receptor (EPOR), as well as subsequent Janus kinase 2 (Jak2) phosphorylations, are each tightly linked to a specific time after oxidative stress and in anticipation of daily light onset. This is consistent with physiological protection against daily light‐induced, oxidatively mediated retinal apoptosis. In vitro, we verify that EPO protects RPE cells from light, hyperoxia, and hydrogen peroxide–induced retinal cell apoptosis, and that these stimuli increase EPO and EPOR expression in cultured RPE cells. Together, these data support the premise that EPO and its EPOR interactions represent an important retinal shield from physiologic and pathologic light‐induced oxidative injury. © 2009 Wiley‐Liss, Inc.     

Rapid activation of presynaptic nicotinic acetylcholine receptors by nerve-released transmitter

Nicotine's ability to enhance neurotransmitter release has implicated presynaptic nicotinic acetylcholine receptors (nAChRs) in synaptic modulation, but there aze few examples where presynaptic nAChRs are known to be activated by nerve-released transmitter. We searched for endogenous activation of presynaptic nAChRs in the calyceal nerve terminals of the chick ciliary ganglion by imaging presynaptic calcium transients using dextran-coupled indicator dyes. The amplitude of Ca(+)signals recorded in individual nerve terminals was frequency dependent over 2-50 Hz. Calcium transients evoked by stimulation of the preganglionic nerve were significantly reduced (approximately 10-15%) by the nonspecific nAChR antagonist d-tubocurarine (d-TC; 100 microM) and the alpha7-specific antagonist methyllycaconitine (20-50 nM) but were not affected by 10 microM dihydro-beta-erythroidine, which should inhibit several non-alpha7 nAChRs. Feedback was rapid and did not require a stimulation-dependent build-up of transmitter, as d-TC and MLA reduced the amplitude of the first calcium transient in a 2-Hz train. Choline is an agonist at alpha7 nAChRs but is not the sole agonist in this system, as inhibition of acetylcholinesterase by echothiophate failed to reduce calcium transients. These results show that nerve-released acetylcholine (ACh) feeds back onto presynaptic alpha7 nAChRs to enhance calcium signals within the terminal. This feedback may help maintain the high rate of transmission at this cholinergic synapse.     

Retinal pigment epithelial correlates of avian retinal degeneration: electron microscopic analysis

The delayed amelanotic (DAM) strain of domestic chicken is characterized by an early, developmental onset of choroidal inflammation and destruction of both feather and choroidal melanocytes. Secondarily, retinal pigment epithelial (RPE) cells in the peripapillary region develop abnormalities, and a series of progressive histopathological changes ensues which includes reduction and ultimate loss of RPE-melanin granules and RPE-cell atrophy. The earliest sign of RPE-cell abnormality is a dramatic alteration in the distribution of intracellular melanin granules. Apical processes also show a lessening of contact with photoreceptor outer segments, leading in more advanced stages to their retraction and development of retinal detachments. Other progressive alterations in RPE cells include disorganization and loss of basal infoldings; size reductions and density increases in both mitochondria and myeloid bodies from early to advanced stages; appearance of large macrophages in the subretinal space; Loss of intercellular junctional complexes; and progressive reduction in the density of melanin granules. These abnormalities appear to spread in a cell-by-cell, radial pattern, until widespread areas of the retina become severely pathologic and atrophic. The DAM chorioretinal disorder appears to show many of the histopathologic features which characterize experimentally induced uveitis and other ocular diseases which may result from hypersensitivity to, or autoimmune reaction against, pigments of the uveal tract.     

Distribution of muscarinic acetylcholine receptors on processes of isolated retinal cells

Binding of propylbenzilylcholine mustard, a muscarinic acetylcholine receptor antagonist, to isolated retinal cells was examined with light microscopic autoradiography. Dissociation of the adult tiger salamander retina yielded identifiable rod, cone, horizontal, bipolar, amacrine/ganglion, and Müller cells. Preservation of fine structure was assessed with conventional electron microscopy. For all cell types, the plasmalemma was intact and free of adhering debris; in addition, presynaptic ribbon complexes were present in photoreceptor and bipolar axon terminals indicating that synaptic structures were retained. Specific binding to cell bodies and processes was analyzed separately by using morphometric and statistical techniques. The highest grain densities occurred on processes of amacrine/ganglion cells and axons and 2 degrees and 3 degrees dendrites of bipolar neurons. Bipolar cells, however, seemed to be a heterogeneous population because there was great variation in the density of binding sites on both their axons and distal dendrites. Intermediate levels of binding were found on bipolar 1 degree dendrites and horizontal cells. No specific binding was detected on Müller cells and most parts of photoreceptors. Comparisons between cells showed that grain densities were similar for bipolar axons and amacrine/ganglion cell processes but bipolar dendrites were richer in binding sites than horizontal cell dendrites. Thus, muscarinic receptors in the salamander retina are located on amacrine/ganglion, bipolar, and horizontal cells and primarily confined to the processes which compose the two synaptic layers. In the inner plexiform layer, muscarinic receptors reside on processes from all three inner retinal neurons: in the outer synaptic layer, receptors are only on second-order cells and are more numerous along bipolar than horizontal cell dendrites.     

Synaptogenesis in the rat retina: subcellular localization of glycine receptors,GABAA receptors,and the anchoring protein gephyrin

The mechanisms by which neurotransmitter receptors are clustered at postsynaptic sites of neurons are largely unknown. The 93-kDa peripheral membrane protein gephyrin has been shown to be essential for the formation of postsynaptic glycine receptor clusters, and there is now evidence that gephyrin can also be found at gamma-aminobutyric acid (GABA)ergic synapses. In this study, we have analyzed the synaptic localization of glycine receptors, GABA_A receptors, and the anchoring protein gephyrin in the inner plexiform layer of the developing rat retina, by using immunofluorescence with subunit specific antibodies. At early postnatal stages, the antibodies produced a diffuse staining, suggesting that early retinal neurons can express glycine and GABA_A receptors. A clustered distribution of the subunits in “hot spots” was also observed. The number of “hot spots” increased during development and reached adult levels in about 2 weeks. Electron microscopy showed that synapses of the conventional type are present in the inner plexiform layer of the postnatal retina and that the hot spots correspond to an aggregation of receptors at postsynaptic sites. Gephyrin was also localized to “hot spots,” and double immunofluorescence revealed a colocalization of gephyrin with the α2 subunit of the GABA_A receptor. These results indicate that clustering of receptor subunits occurs in parallel with the formation of morphologically identifiable synaptic specializations and suggest that gephyrin may be involved in clustering of GABA_A receptors at postsynaptic sites.J.Comp. Neurol. 381:158-174, 1997. © 1997 Wiley-Liss, Inc.     

The nutrient supply of the chick visual system: Responses to form and light deprivation

Blood flow and uptake of two nutrient analogues have been studied in the neural retina, pigment epithelium and optic lobes of 5-7-day-old chicks after systemic administration of isotopically labeled compounds. The pigment epithelium accumulated the inert amino acid, alpha-aminoisobutyric acid, 6 times as effectively on a wet weight basis than did the neural retina or optic lobes. However, the neural retina was over 3 times as active in transport of deoxyglucose as was either the pigment epithelium or optic lobe. These differences could not be attributed to variations in blood flow since the penetrance of the freely diffusible antipyrine was low in the neural retina and roughly equal in the vascularized epithelium and optic lobes. Two days of unilateral eyelid suture significantly depressed blood flow and deoxyglucose uptake in the optic lobe innervated by the occluded eye. The neural retina of the eye receiving reduced light input also had a reduced level of deoxyglucose accumulation but the capacity to concentrate alpha-aminoisobutyrate was increased. The corresponding dark-adapted pigment epithelium had a major (48%) increase in the uptake of this inert amino acid. The data show that regional glucose utilization may not be directly proportional to blood flow or glucose diffusion rates. The non-vascularized neural retina possesses an unusually powerful glucose transport mechanism while the pigment epithelium has a high capacity for amino acid accumulation.     

Extra-synaptic localization of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

Surface [¹²⁵I]α-bungarotoxin receptors were localized on dissociated cultures of embryonic chick ciliary ganglion neurons by electron microscopic autoradiography. Grain density was 4–5-fold higher on cell body membrane versus neuritic process membrane, and there was no apparent concentration of grains at morphologically identifieble inter-neuronal synapses.     

Afferent specific localization of muscarinic acetylcholine receptors in cingulate cortex

The laminar distribution of acetylcholine receptors in rat cingulate cortex and their localization to axons of neurons in the anterior thalamic nuclei (ATN) were evaluated with the muscarinic antagonist [3H]propylbenzilylcholine mustard (PrBCM) in vitro. Specific binding of PrBCM in granular area 29 was heterogeneous, with a 57% variation from the highest binding in layer Ia to the lowest in layer II-III. In contrast, binding in area 24 was homogeneous, with only a 14% variation. The heterogeneity of PrBCM binding almost exactly duplicated the distribution of termination of ATN afferents in layers I to IV of area 29c. Four experiments indicated that 50% of the excess binding in layers Ia and IV was due to axonal receptor sites. First, ATN lesions abolished 41% and 27% of total specific binding in layers Ia and IV, respectively. Second, an undercut procedure that totally deafferented layers I to Va showed changes similar to those following ATN lesions, suggesting that other afferents to these layers may not have muscarinic receptors associated with them. Third, the sequence of losses in receptor binding and acetylcholinesterase (AChE) activity was evaluated 2, 3, 5, 9, and 14 days following ATN lesions. Since AChE was present in ATN axons, as evidenced by early postlesion losses, the correlation of both losses as well as previous analyses of axon degeneration in this cortex confirmed that these receptors were in axons. Fourth, binding peaks in layers Ia and IV remained in area 29c following destruction of virtually all neurons with the neurotoxin ibotenic acid. This is the first evidence that the activity of a major neocortical thalamic afferent may be regulated by axonal acetylcholine receptors.     

GABA-like immunoreactive cells containing nicotinic acetylcholine receptors in the chick retina.

The possibility that GABA-like immunoreactive cells of the chick retina also contain neuronal nicotinic acetylcholine receptors was investigated by means of immunohistochemical techniques. Double-labeled cell bodies containing GABA-like immunoreactivity and nicotinic receptor-like immunoreactivity were seen in the inner third of the inner nuclear layer and were presumably amacrine cells. Approximately 29-36% of the GABA-positive cells in the inner nuclear layer contained nicotinic receptor immunoreactivity. Their soma sizes ranged from 5-12 microns. Some double-labeled cells ranging from 7-21 microns were observed in the ganglion cell layer as well. Between 9-37% of the GABA-positive cells in this layer contained nicotinic receptor-like immunoreactivity. Following injection of a retrograde tracer into the optic tectum, some of the retrogradely labeled cells were also double labeled with antibodies against GABA and nicotinic receptors. This indicates that at least some of the GABA-positive cells containing nicotinic acetylcholine receptors in the ganglion cell layer are indeed ganglion cells. The present data appear to represent the first demonstration of the presence of acetylcholine receptors in GABA-containing cells in the retina, thus providing a basis for a possible influence of acetylcholine upon those presumptive GABAergic cells.