Functions of the two glutamate transporters GLAST and GLT-1 in the retina

In the retina, the glutamate transporter GLAST is expressed in Müller cells, whereas the glutamate transporter GLT-1 is found only in cones and various types of bipolar cells. To investigate the functional role of this differential distribution of glutamate transporters, we have analyzed GLAST and GLT-1 mutant mice. In GLAST-deficient mice, the electroretinogram b-wave and oscillatory potentials are reduced and retinal damage after ischemia is exacerbated, whereas GLT-1-deficient mice show almost normal electroretinograms and mild increased retinal damage after ischemia. These results demonstrate that GLAST is required for normal signal transmission between photoreceptors and bipolar cells and that both GLAST and GLT-1 play a neuroprotective role during ischemia in the retina.

Retinoic acid has light-adaptive effects on horizontal cells in the retina

Ambient light conditions affect the morphology of synaptic elements within the cone pedicle and modulate the spatial properties of the horizontal cell receptive field. We describe here that the effects of retinoic acid on these properties are similar to those of light adaptation. Intraorbital injection of retinoic acid into eyes of dark-adapted carp that subsequently were kept in complete darkness results in the formation of numerous spinules at the terminal dendrites of horizontal cells, a typical feature of light-adapted retinae. The formation of these spinules during light adaptation is impaired in the presence of citral, a competitive inhibitor of the dehydrogenase responsible for the generation of retinoic acid in vivo. Intracellularly recorded responses of horizontal cells from dark-adapted eyecup preparations superfused with retinoic acid reveal typical light-adapted spatial properties. Retinoic acid thus appears to act as a light-signaling modulator. Its activity appears not to be at the transcriptional level because its action was not blocked by actinomycin.

Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector

The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

An alternative pathway for rod signals in the rodent retina: Rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors

In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.

Cannabinoid CB1 receptors and ligands in vertebrate retina: Localization and function of an endogenous signaling system

CB1, a cannabinoid receptor enriched in neuronal tissue, was found in high concentration in retinas of rhesus monkey, mouse, rat, chick, goldfish, and tiger salamander by using a subtype-specific polyclonal antibody. Immunolabeling was detected in the two synaptic layers of the retina, the inner and outer plexiform layers, of all six species examined. In the outer plexiform layer, CB1 was located in and/or on cone pedicles and rod spherules. Labeling was detected in some amacrine cells of all species and in the ganglion cells and ganglion cell axons of all species except fish. In addition, sparse labeling was found in the inner and/or outer segments of the photoreceptors of monkey, mouse, rat, and chick. Using GC/MS to detect possible endogenous cannabinoids, we found 3 nmol of 2-arachidonylglycerol per g of tissue, but no anandamide was detectable. Cannabinoid receptor agonists induced a dramatic reduction in the amplitude of voltage-gated L-type calcium channel currents in identified retinal bipolar cells. The presence and distribution of the CB1 receptor, the large amounts of 2-arachidonylglycerol found, and the effects of cannabinoids on calcium channel activity in bipolar cells suggest a substantive role for an endogenous cannabinoid signaling system in retinal physiology, and perhaps vision in general.

Horizontal cells reveal cone type-specific adaptation in primate retina

The human cone visual system maintains contrast sensitivity over a wide range of ambient illumination, a property known as light adaptation. The first stage in light adaptation is believed to take place at the first neural step in vision, within the long, middle, and short wavelength sensitive cone photoreceptors. To determine the properties of adaptation in primate outer retina, we measured cone signals in second-order interneurons, the horizontal cells, of the macaque monkey. Horizontal cells provide a unique site for studying early adaptational mechanisms; they are but one synapse away from the photoreceptors, and each horizontal cell receives excitatory inputs from many cones. Light adaptation occurred over the entire range of light levels evaluated, a luminance range of 15–1,850 trolands. Adaptation was demonstrated to be independent in each cone type and to be spatially restricted. Thus, in primates, a major source of sensitivity regulation occurs before summation of cone signals in the horizontal cell.

Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by α-toxin

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho → Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal α-toxin, including an α-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [γ-32P]ATP to α-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.

X-ngnr-1 and Xath3 promote ectopic expression of sensory neuron markers in the neurula ectoderm and have distinct inducing properties in the retina

Xath3 encodes a Xenopus neuronal-specific basic helix–loop–helix transcription factor related to the Drosophila proneural factor atonal. We show here that Xath3 acts downstream of X-ngnr-1 during neuronal differentiation in the neural plate and retina and that its expression and activity are modulated by Notch signaling. X-ngnr-1 activates Xath3 and NeuroD by different mechanisms, and the latter two genes crossactivate each other. In the ectoderm, X-ngnr-1 and Xath3 have similar activities, inducing ectopic sensory neurons. Among the sensory-specific markers tested, only those that label cranial neurons were found to be ectopically activated. By contrast, in the retina, X-ngnr-1 and Xath3 overexpression promote the development of overlapping but distinct subtypes of retinal neurons. Together, these data suggest that X-ngnr-1 and Xath3 regulate successive stages of early neuronal differentiation and that, in addition to their general proneural properties, they may contribute, in a context-dependent manner, to some aspect of neuronal identity.

Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814–4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription–PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of ≈7.5 kb, ≈3.0 kb, and ≈2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

The number of unidentified amacrine cells in the mammalian retina

The three largest known populations of amacrine cells in the rabbit retina were stained with fluorescent probes in whole mounts and counted at a series of retinal eccentricities. The retinas were counterstained using a fluorescent DNA-binding molecule and the total number of nuclei in the inner nuclear layer were counted in confocal sections. From the total number of inner nuclear layer cells and the known fraction of them occupied by amacrine cells, the fraction of amacrine cells made up by the stained populations could be calculated. Starburst cells made up 3%, indoleamine-accumulating cells made up 4%, and AII cells made up 11% of all amacrine cells. By referring four smaller populations of amacrine cells to the number of indoleamine-accumulating cells, they were estimated to make up 4% of all amacrine cells. Thus, 78% of all amacrine cells in the rabbit’s retina are known only from isolated examples, if at all. This proportion is similar in the retinas of the mouse, cat, and monkey. It is likely that a substantial fraction of the local circuit neurons present in other regions of the central nervous system are also invisible as populations to current techniques.

The Optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.

Suppression of trkB expression by antisense oligonucleotides alters a neuronal phenotype in the rod pathway of the developing rat retina.

trkB is the high-affinity receptor for brain-derived neurotrophic factor (BDNF), a trophic molecule with demonstrated effects on the survival and differentiation of a wide variety of neuronal populations. In the mammalian retina, trkB is localized to both ganglion cells and numerous cells in the inner nuclear layer. Much information on the role of BDNF in neuronal development has been derived from the study of trkB- and BDNF-deficient mutant mice. This includes an attenuation of the numbers of cortical neurons immunopositive for the calcium-binding proteins, parvalbumin, and calbindin. Unfortunately, these mutant animals typically fail to survive for > 24-48 hr after birth. Since most retinal neuronal differentiation occurs postnatally, we have devised an alternative scheme to suppress the expression of trkB in the retina to examine the role of BDNF on the postnatal development of neurons of the inner retina. Neonatal rats were treated with intraocular injection of an antisense oligonucleotide (1-2 microliters of 10-100 microM solution) targeted to the trkB mRNA. Immunohistochemistry with a polyclonal antibody to trkB showed that the expression of trkB in retinal neurons was suppressed 48-72 hr following a single injection. Northern blot analysis demonstrated that antisense treatment had no effect on the level of trkB mRNA, even after multiple injections. This suggests an effect of trkB antisense treatment on protein translation, but not on RNA transcription. No alterations were observed in the thickness of retinal cellular or plexiform layers, suggesting that BDNF is not the sole survival factor for these neurons. There were, however, alterations in the patterns of immunostaining for parvalbumin, a marker for the narrow-field, bistratified AII amacrine cell-a central element of the rod (scotopic) pathway. This was evidenced by a decrease in both the number of immunostained somata (> 50%) and in the intensity of immunolabeling. However, the immunostaining pattern of calbindin was not affected. These studies suggest that the ligands for trkB have specific effects on the neurochemical phenotypic expression of inner retinal neurons and in the development of a well-defined retinal circuit.

Low-calcium-induced enhancement of chemical synaptic transmission from photoreceptors to horizontal cells in the vertebrate retina.

According to the classical calcium hypothesis of synaptic transmission, the release of neurotransmitter from presynaptic terminals occurs through an exocytotic process triggered by depolarization-induced presynaptic calcium influx. However, evidence has been accumulating in the last two decades indicating that, in many preparations, synaptic transmitter release can persist or even increase when calcium is omitted from the perfusing saline, leading to the notion of a "calcium-independent release" mechanism. Our study shows that the enhancement of synaptic transmission between photoreceptors and horizontal cells of the vertebrate retina induced by low-calcium media is caused by an increase of calcium influx into presynaptic terminals. This paradoxical effect is accounted for by modifications of surface potential on the photoreceptor membrane. Since lowering extracellular calcium concentration may likewise enhance calcium influx into other nerve cells, other experimental observations of "calcium-independent" release may be reaccommodated within the framework of the classical calcium hypothesis without invoking unconventional processes.