Congenital stationary night blindness in mice - a tale of two Cacna1f mutants.

BACKGROUND: Mutations in CACNA1F, which encodes the Ca(v)1.4 subunit of a voltage-gated L-type calcium channel, cause X-linked incomplete congenital stationary night blindness (CSNB2), a condition of defective retinal neurotransmission which results in night blindness, reduced visual acuity, and diminished ERG b-wave. We have characterized two putative murine CSNB2 models: an engineered null-mutant, with a stop codon (G305X); and a spontaneous mutant with an ETn insertion in intron 2 of Cacna1f (nob2). METHODS: Cacna1f ( G305X ): Adults were characterized by visual function (photopic optokinetic response, OKR); gene expression (microarray) and by cell death (TUNEL) and synaptic development (TEM). Cacna1f ( nob2 ): Adults were characterized by properties of Cacna1f mRNA (cloning and sequencing) and expressed protein (immunoblotting, electrophysiology, filamin [cytoskeletal protein] binding), and OKR. RESULTS: The null mutation in Cacna1f ( G305X ) mice caused loss of cone cell ribbons, failure of OPL synaptogenesis, ERG b-wave and absence of OKR. In Cacna1f ( nob2 ) mice alternative ETn splicing produced ~90% Cacna1f mRNA having a stop codon, but ~10% mRNA encoding a complete polypeptide. Cacna1f ( nob2 ) mice had normal OKR, and alternatively-spliced complete protein had WT channel properties, but alternative ETn splicing abolished N-terminal protein binding to filamin. CONCLUSIONS: Ca(v)1.4 plays a key role in photoreceptor synaptogenesis and synaptic function in mouse retina. Cacna1f ( G305X ) is a true knockout model for human CSNB2, with prominent defects in cone and rod function. Cacna1f ( nob2 ) is an incomplete knockout model for CSNB2, because alternative splicing in an ETn element leads to some full-length Ca(v)1.4 protein, and some cones surviving to drive photopic visual responses.

Conductance changes associated with receptor potentials in Limulus photoreceptors.

The receptor potential in Limulus photoreceptors appears to be a consequence not of permeability changes in the cell membrane but of alterations in a light-sensitive constant-current generator.

Structural basis for on-and off-center responses in retinal bipolar cells.

Electron microscopy of Golgi preparations of goldfish retina shows that dendrites of type a (hyperpolarizing, off-center) bipolar cells make wide cleft junctions unassociated with synaptic ribbons, while those of type b (depolarizing, on-center) bioplar cells make narrow cleft junctions and synaptic ribbon contacts, with rods and cones. This suggests that wide cleft junctions are the site of sign-conserving, and narrow cleft junctions or ribbon contacts (or both) are the site of sign-inverting synaptic transmission from photoreceptors to bipolars.

Goldfish retina: functional polarization of cone horizontal cell dendrites and synapses.

In serial electron micrographs we observed that dendrites of goldfish cone horizontal cells are either central or lateral in ribbon synaptic triads, depending on cone and horizontal cell type. The chromatic properties of cone horizontal cell responses may be explained if the cone horizontal cells act as interneurons, receiving from cones through their central processes but acting on cones through their lateral processes.

A role for the sodium pump in photoreception in Limulus.

The membranes of photoreceptor cells in Limulus have an electrogenic sodium pump which contributes directly to membrane potential and whose activity is changed by light. These light-induced changes in pump activity underlie the receptor potential.

Light- and focus-dependent expression of the transcription factor ZENK in the chick retina.

Ocular growth and refraction are regulated by visual processing in the retina. We identified candidate regulatory neurons by immunocytochemistry for immediate-early gene products, ZENK (zif268, Egr-1) and Fos, after appropriate visual stimulation. ZENK synthesis was enhanced by conditions that suppress ocular elongation (plus defocus, termination of form deprivation) and suppressed by conditions that enhance ocular elongation (minus defocus, form deprivation), particularly in glucagon-containing amacrine cells. Fos synthesis was enhanced by termination of visual deprivation, but not by defocus and not in glucagon-containing amacrine cells. We conclude that glucagon-containing amacrine cells respond differentially to the sign of defocus and may mediate lens-induced changes in ocular growth and refraction.

Channeling Vision: CaV1.4-A Critical Link in Retinal Signal Transmission.

Voltage-gated calcium channels (VGCC) are key to many biological functions. Entry of Ca2+ into cells is essential for initiating or modulating important processes such as secretion, cell motility, and gene transcription. In the retina and other neural tissues, one of the major roles of Ca2+-entry is to stimulate or regulate exocytosis of synaptic vesicles, without which synaptic transmission is impaired. This review will address the special properties of one L-type VGCC, CaV1.4, with particular emphasis on its role in transmission of visual signals from rod and cone photoreceptors (hereafter called "photoreceptors," to the exclusion of intrinsically photoreceptive retinal ganglion cells) to the second-order retinal neurons, and the pathological effects of mutations in the CACNA1F gene which codes for the pore-forming α1F subunit of CaV1.4.

Cone dystrophy and ectopic synaptogenesis in a Cacna1f loss of function model of congenital stationary night blindness (CSNB2A).

Congenital stationary night blindness 2A (CSNB2A) is an X-linked retinal disorder, characterized by phenotypically variable signs and symptoms of impaired vision. CSNB2A is due to mutations in CACNA1F, which codes for the pore-forming α1F subunit of a L-type voltage-gated calcium channel, Cav1.4. Mouse models of CSNB2A, used for characterizing the effects of various Cacna1f mutations, have revealed greater severity of defects than in human CSNB2A. Specifically, Cacna1f-knockout mice show an apparent lack of visual function, gradual retinal degeneration, and disruption of photoreceptor synaptic terminals. Several reports have also noted cone-specific disruptions, including axonal abnormalities, dystrophy, and cell death. We have explored further the involvement of cones in our 'G305X' mouse model of CSNB2A, which has a premature truncation, loss-of-function mutation in Cacna1f. We show that the expression of genes for several phototransduction-related cone markers is down-regulated, while that of several cellular stress- and damage-related markers is up-regulated; and that cone photoreceptor structure and photopic visual function - measured by immunohistochemistry, optokinetic response and electroretinography - deteriorate progressively with age. We also find that dystrophic cone axons establish synapse-like contacts with rod bipolar cell dendrites, which they normally do not contact in wild-type retinas - ectopically, among rod cell bodies in the outer nuclear layer. These data support a role for Cav1.4 in cone synaptic development, cell viability, and synaptic transmission of cone-dependent visual signals. Although our novel finding of cone-to-rod-bipolar cell contacts in this mouse model of a retinal channelopathy may challenge current views of the role of Cav1.4 in photopic vision, it also suggests a potential new target for restorative therapy.

Horizontal cell axons and axon terminals in goldfish retina.

Retinas of ordinary and black moor varieties of goldfish (Carassius auratus) were prepared by the Golgi method, mounted flat or sectioned vertically, and studied in the light microscope. Three types of horizontal cells whose dendrites contact only cones, and one type whose dendrites contact only rods, were observed. The cone horizontal cells (Cajal's "external horizontal cells") all have slender axons which descend gradually to the inner nuclear layer and terminate there in long, fusiform expansions (Cajal's "internal horizontal cells"). The thin and thick portions of the axons, as well as the perikarya of the horizontal cells, bear small numbers of straight, horizontally-directed, knobby filamentous appendages which may be sites of synaptic contact. The cone horizontal cell axons in goldfish, unlike those in higher vertebrates, do not terminate in contact with synaptic endings of photoreceptor cells, but in proximity to cells and processes deep in the inner nuclear layer. Axons have not yet been demonstrated on rod horizontal cells in goldfish.

Choline acetyltransferase immunoreactivity suggests that ganglion cells in the goldfish retina are not cholinergic.

Published evidence that ganglion cells in the retinae of nonmammalian species are cholinergic is strong but indirect. In this paper we report results of attempts to demonstrate choline acetyltransferase immunoreactivity in ganglion cells of goldfish retina using two different antisera against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme. We obtained ChAT-immunoreactive staining of amacrine and displaced amacrine cells in the retina and type XIV cells in the tectum, but we obtained no direct immunocytochemical evidence that ganglion cells in the goldfish retina are cholinergic. Thus, ganglion cells identified by retrograde transport of propidium iodide were never ChAT-immunoreactive. Intraocular injections of colchicine did not result in the appearance of a population of ChAT-immunoreactive neurons in the ganglion cell layer. ChAT-immunoreactive axons were not observed in intact, ligated, or transected optic nerves. And finally, the ChAT immunoreactivity of cells and fibers in the optic tectum was unaffected by deafferentation. These experiments provide no positive evidence that any ganglion cells in goldfish retina contain the acetylcholine-synthesizing enzyme, ChAT. While it is possible that our method is too insensitive to detect the enzyme in ganglion cell somata or too specific to recognize the form of ChAT present in these cells, the fact that we can stain putatively cholinergic retinal amacrine cells and tectal neurons makes these alternative explanations improbable. We conclude that it is unlikely that any of the ganglion cells in the retina are cholinergic and that alternative explanations should be sought for previously published results that suggest that they are.

Color-specific interconnections of cones and horizontal cells in the retina of the goldfish.

In Golgi preparations of goldfish retina we have observed three types of horizontal cell which receive exclusively from cones and one which receives exclusively from rods. The cone horizontal cells were designated H1, H2 and H3, in order of increasing dendritic spread, increasing separation from the outer synaptic layer, decreasing size of perikaryon, and decreasing density of cone contacts. Slender appendages with knobby terminal enlargements project horizontal cells by alalyzing serial 1 mum sections with the light microscope. The probable inputs, in terms of visual pigments in the cones which contact them, are: H1, red+green+blue; H2, green+blue; H3, blue. Analysis of previously published work suggests (1) that H1 cells generate monophasic or L-type responses, H2 cells generate biphasic or C1-type responses, and H3 cells generate triphasic or C2-type responses; (2) that H1 cells receive direct functional input at least from red-sensitive cones, H2 cells from green-sensitive cones, and H3 cells from blue-sensitive cones, and (3) that H1 constitute pathways from cones to H2 cells, and H2 cells, and H2 cells constitute pathways from cones and H1 cells to H3 cells. The precise location and route of the transfers, from H1 to H2 and from H2 to H3, are not yet known.

Nitric oxide synthase-containing cells in the retina, pigmented epithelium, choroid, and sclera of the chick eye.

Nitric oxide is a nonconventional neurotransmitter that is produced as needed by the enzyme nitric oxide synthase (NOS). NOS has been detected in numerous neural structures, including distinct populations of retinal neurons in a variety of vertebrate species. The purpose of this study was to identify NOS-containing cells in the retina and extraretinal ocular tissues of hatched chicks. NOS was detected in frozen sections by using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and antisera to neuronal NOS. In the retina, NADPH-diaphorase and NOS immunolabelling were present in four subtypes of amacrine cells, some ganglion cells, efferent fibers, efferent target cells, and neuronal processes in both plexiform layers, whereas diaphorase alone was detected in photoreceptor ellipsoids and Müller cells. In addition, NADPH-diaphorase and immunoreactive NOS were detected in axon bundles and innervation to vascular smooth muscle in the choroid, whereas stromal and endothelial cells in the choroid, scleral chondrocytes, and the retinal pigmented epithelium contained only NADPH-diaphorase. The excitotoxin quisqualate destroyed all but one subtype of NOS-immunoreactive amacrine cell and caused increased NADPH-diaphorase activity in Müller cells. We conclude that nitric oxide is produced by many different cells in the chick eye, including retinal amacrine and ganglion cells, Müller cells, retinal pigmented epithelium, and cells in the choroid, and likely has a broad range of visual and regulatory functions.

Immunocytochemical and morphological evidence for a retinopetal projection in anuran amphibians.

The centrifugal projection to the retina in anuran amphibians (Rana catesbeiana and Xenopus laevis) has been investigated by immunocytochemistry, HRP transport, and optic nerve lesionings. FMRFamide- and N-terminal substance P-immunoreactive (Fa-ir and SP(3-7)-ir) fibers were abundant in the normal retina and optic nerve but almost absent distal to an optic nerve section or crush after 7-14 days survival. Fa-ir and SP(3-7)-ir fibers were traced to the optic nerve from the lamina terminalis (or the septopreoptic junctional area), where there are many Fa-ir and SP(3-7)-ir perikarya. After application of HRP to the optic nerve and survival for 9-10 days, retrogradely labeled neurons were observed in the lamina terminalis. Conversely, following HRP injection into the septal and preoptic area, labeled fibers were observed in the optic nerve. These results suggest that Fa-ir and SP(3-7)-ir efferent fibers project from the lamina terminalis to the retina. But in anurans, unlike teleosts, these fibers are not gonadotropin-releasing-hormone (GnRH)-ir. The morphological relations of this retinopetal pathway with the GnRH-ir nervus terminalis are discussed.

Electron microscopic study of immunocytochemically labeled centrifugal fibers in the goldfish retina.

The centrifugal fibers innervating the goldfish retina were studied quantitatively by light and electron microscopy. These fibers originating from cell bodies in the olfactory bulb were labeled by antiserum to the tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide). The number of FMRFamide-immunoreactive (ir) centrifugal fibers in each eye of the adult goldfish (body length: 12-15 cm) was 65 +/- 14 (mean +/- S.D., n = 7). All of these fibers in the optic nerve and the retina were unmyelinated. Each FMRFamide-ir centrifugal fiber runs along the optic fiber layer and gives several terminal arborizations in the outermost layer (layer 1) of the inner plexiform layer. Layer 1 is, therefore, densely covered by a plexus of terminal arborizations. Along these terminal arborizations, we found output synapses characterized by a cluster of small clear vesicles (40 nm in diameter) at the presynaptic site and a thickened membrane in the apposed retinal cell processes. In a sample area of 2,000 microns 2, such synapses occurred at a density of one per 105 microns 2, or about 13,000 per centrifugal fiber. Thus, the FMRFamide-ir centrifugal fibers are likely to modulate retinal cell activity through an estimated total of 840,000 output synapses per retina.

Synaptic contacts of somatostatin-immunoreactive amacrine cells in goldfish retina.

Retinal amacrine cells containing somatostatinlike immunoreactivity (SLI) were labeled by the peroxidase-antiperoxidase technique and their connections were analyzed by light and electron microscopy. The labeled processes were found in two distinct plexuses--one near the most proximal border of the inner plexiform layer and the other near the most distal border. They received most (89%) of their input from amacrine cells and the remainder from bipolar cells. A majority (56%) of their output synapses go to processes of amacrine cells, a substantial proportion (38%) go to ganglion cell dendrites, and the remainder go to bipolar cell axon terminals. The relative frequencies of each of the types of contacts were nearly identical in the distal and proximal plexuses. The cells containing SLI are different in their morphology and synaptic connections from any goldfish amacrine cells containing conventional neurotransmitters, but one type of amacrine cell containing SLI resembles certain other peptidergic amacrine cells in the goldfish retina.

Rod and cone inputs to bipolar cells in goldfish retina.

Five morphological types of bipolar cells which make synaptic contact with rods and cones are distinguished in the retina of adult goldfish (Carassius auratus) by characteristics readily observable in the light microscope. Cells were designated type a or type b according to whether their axons terminate in the distal part (sublamina a) or proximal part (sublamina b) of the inner plexoform layer, respectively. Analysis of serial semi-thin sections of Golgi-impregnated cells demonstrates that each subtype of bipolar contacts rods and a characteristic set of chromatic subtypes of cones: types a1 and b1 cells contact rods and red-sensitive cones, while types a2, b2 and b3 contact rods and red- and green-sensitive cones. Comparison with published descriptions of cells stained with Procion Yellow after intracellular recordings had been made suggests that type a cells should be off-center types and type b on-center. Furthermore, it is suggested that the receptive fields of cell types a1 and b1 should be non-color-coded, and those of a2, b2, and b3 color-coded.

GABA-ergic pathways in the goldfish retina.

A high-affinity uptake mechanism for [3H]-gamma-aminobutyric acid (GABA) has been localized to type H1 cone horizontal cells and type Ab pyriform amacrine cells in the retina of the goldfish by light and electron microscopy autoradiography. By stimulating isolated retinas with colored lights during incubation we have been able to use [3H]-GABA uptake as a probe of light-evoked changes in membrane potential. All colors of lights increase and darkness decreases [3H]-GABA uptake by H1 cone horizontal cells. Our model of voltage dependence of GABA uptake predicts that all colors of light should hyperpolarize H1 cone horizontal cells and other investigators have shown by intracellular recording and dye-marking that type H1 cone horizontal cells hyperpolarize to all wavelengths of light. We have also obtained evidence that dark-induced depolarization of cone horizontal cells leads to release of GABA. Type Ab pyriform amacrine cells show maximal [3H]-GABA uptake in darkness and when exposed to green or blue lights, but red lights dramatically suppress uptake. We predict these neurons to be red-depolarizing, and recent intracellular recordings and dye-marking by Famiglietti et al. ('77) support our conclusions. Synaptic relations of apparently GABA-ergic neurons were investigated in the electron microscope. We propose type H1 cone horizontal cells to be both pre- and post-synaptic to red-sensitive cones and type Ab pyriform amacrine cells to be both pre- and post-synaptic to red-sensitive center-depolarizing bipolar cells.

Immunocytochemical characterization of quisqualic acid- and N-methyl-D-aspartate-induced excitotoxicity in the retina of chicks.

A single, large dose of N-methyl-D-aspartate (NMDA) or quisqualic acid (QA) injected into the chick eye has been shown previously to destroy many retinal amacrine cells and to induce excessive ocular growth accompanied by myopia. The purpose of this study was to identify distinct populations of retinal cells, particularly those believed to be involved in regulating ocular growth, that are sensitive to NMDA or QA. Two pmol of NMDA or 0.2 micromol of QA were injected unilaterally into eyes of 7-day-old chicks, and retinas were prepared for observation 1, 3, or 7 days later. Retinal neurons were identified by using immunocytochemistry, and cells containing fragmented DNA were identified by 3'-nick-end labelling in frozen sections. NMDA and QA destroyed many amacrine cells, including those immunoreactive for vasoactive intestinal polypeptide, Met-enkephalin, and choline acetyltransferase, but they had little effect upon tyrosine hydroxylase-immunoreactive cells. Other cells affected by both QA and NMDA included those immunoreactive for glutamic acid decarboxylase, gamma-aminobutyric acid, parvalbumin, serotonin, and aminohydroxy methylisoxazole propionic acid (AMPA) receptor subunits GluR1 and GluR2/3. Cells largely unaffected by QA or NMDA included bipolar cells immunoreactive for protein kinase C (alpha and beta isoforms) and amacrine cells immunoreactive for glucagon. DNA fragmentation was detected maximally in many amacrine cells and in some bipolar cells 1 day after exposure to QA or NMDA. We propose that excitotoxicity caused by QA and NMDA induces apoptosis in specific populations of amacrine cells and that these actions are responsible for the ocular growth-specific effects of QA and NMDA reported elsewhere.

Localization of D2 dopamine receptors in vertebrate retinae with anti-peptide antibodies.

Dopamine plays an important role in modulating various aspects of retinal signal processing. The morphology of dopaminergic neurons and its physiological effects are well characterized. Two classes of receptor molecules (D1 and D2) were shown pharmacologically to mediate specific actions, with differences between individual groups of vertebrates. In an attempt to better understand dopaminergic mechanisms at the cellular level, we used antisera against D2 receptors and investigated the localization of the dopamine D2 receptor in the retinae of rat, rabbit, cow, chick, turtle, frog, and two fish species with immunofluorescence techniques. Antisera were raised in rabbits to two oligopeptides predicted from rat D2 receptor cDNA; one specific for the splice-variant insertion in the third cytoplasmic loop and the other directed towards the extracellular amino terminal region shared by both short and long isoforms. Preadsorption with the synthetic peptide resulted in a significant reduction of label, indicating the presence of specific binding in all species except turtle and goldfish. The pattern of labelling produced by the two antisera was essentially identical; however, the staining obtained with antiserum to the extracellular motif was always more intense. Specific staining was present in photoreceptor inner and outer segments, and in the outer and inner plexiform layers of all species. In mammals and chick, strongly fluorescent perikarya were observed in the ganglion cell layer and at the proximal margin of the inner nuclear layer. Label may be present in the pigment epithelium but could not be established beyond doubt. This pattern of labelling is in accordance with previous observations on D2 receptor localization by means of radioactive ligand binding and in situ hybridization techniques. It suggests that retinal dopamine acts as a neuromodulator as well as a transmitter. In the distal retina, it may reach its targets via diffusion over considerable distances, even crossing the outer limiting membrane; in the inner and outer plexiform layers, conventional synaptic transmission seems to coexist with paracrine addressing of more distant targets, and D2 receptors are expressed by both amacrine and ganglion cells.

Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick.

The purpose of this study was to characterize the distribution of muscarinic acetylcholine receptors (mAChRs) in the ocular tissues of hatched chicks. In the chick, different isoforms of these receptors have been detected in the brain, heart, and retina, and mAChRs in ocular tissues have been implicated in the pathogenesis of form-deprivation myopia. However, the precise anatomical distribution of mAChRs within the retina, retinal pigment epithelium, choroid, ciliary body, and ciliary ganglion remains unknown. We used affinity-purified, type-specific antibodies directed to three different chick mAChR subtypes (cm2, cm3, and cm4) to detect receptor immunoreactivity in sections and extracts of these ocular tissues. We found cm2, cm3, and cm4 in the retina, retinal pigment epithelium, choroid, and ciliary body. Within the retina, cm2 was expressed in numerous amacrine and ganglion cells; cm3 was expressed in many bipolar cells and small subsets of amacrine cells; and cm4 was found in most, if not all, amacrine and ganglion cells. Each mAChR was localized to distinct strata within the inner plexiform layer that cumulatively form three broad bands that closely match previously described localizations of subtype-nonspecific muscarinic ligand binding. Only cm3 was detected in the outer plexiform layer, and only cm4 was detected in the ciliary ganglion. We propose that each mAChR subtype has unique functions in each ocular tissue.