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J E Dowling

Publications and source records attributed to J E Dowling.

At least 37 records · Page 2Linked to original sources

Retinal patterning in the zebrafish mutant cyclops.

Determination of cell fate in the vertebrate retina has been shown to be largely independent of lineage. After cell fates are determined, retinal neurons become organized in a precise laminar pattern. The mechanisms for this patterning could involve morphogens distributed in gradients or, alternatively, direct cell-cell interactions. In the zebrafish mutant cyclops (Cyc(b16)), most embryos have two partial retinas joined in the ventral midline. This presents developing retinal cells near the midline with abnormal cellular environments, whereas laterally the pattern of developing cells is normal. We examined the consequences of this for patterning in the mutant's retina. We found that the retinas are joined in the midline at the apical surfaces of the photoreceptor layers. A laminar pattern emerges in the midline that preserves normal positional relationships between retinal cell types locally but is abnormal with respect to patterning over the entire retina. Lateral to the midline, retinal patterning appears normal. Metabolic labeling experiments showed that late rounds of DNA synthesis precede the emergence of the novel pattern in this midline region. We conclude that these observations in the cyclops mutant are compatible with mechanisms of pattern formation in the retina involving local cell interactions.

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Mutations affecting eye morphology in the developing zebrafish (Danio rerio).

The zebrafish (Danio rerio) has received considerable attention as a mainstream model for the molecular and genetic study of vertebrate development. In our laboratory, we have conducted a third-generation screen of chemically mutagenized zebrafish for recessive mutations affecting the visual system. This report describes the visible phenotypes and number of morphological mutants so far observed and presents a more detailed histological analysis of six of these mutations. Through analysis of mutant larvae, it was determined that several of the subtle morphological mutations resulted in degeneration of specific cellular layers of the retina. Other mutations resulted in some degeneration distributed diffusely across the entire retina or concentrated at the retinal margin. A single mutation affecting invagination of the optic cup and lens vesicle formation resulted in a failure to develop an anterior chamber. These results demonstrate the utility of a small-scale, highly focused screen for uncovering novel loci involved in retinal and eye development.

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Retinoic acid alters photoreceptor development in vivo.

Application of exogenous retinoic acid (RA) to zebrafish during the initial stages of photoreceptor differentiation results in a precocious development of rod photoreceptors and an inhibition of cone photoreceptor maturation. The acceleration of rod differentiation is observed initially within the ventral retina 3 days after fertilization, following 24 hr of RA application, and within the dorsal retina 4 days after fertilization, following 48 hr of RA application. The differentiation of rods was impeded significantly when the synthesis of endogenous retinoic acid was inhibited by citral prior to the initial stage of rod differentiation. RA-treated embryos labeled for bromodeoxyuridine (BrdU) uptake revealed that RA exerts its effect on a postmitotic cell population within the developing retina. During normal development in zebrafish, rod differentiation is most robust within the ventral retina, a region previously shown to be rich in RA. Our data suggest that the RA signaling pathway is involved in the differentiation and maturation of both the rod and cone photoreceptors within the developing zebrafish retina.

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Comparison of topographical patterns of ganglion and photoreceptor cell differentiation in the retina of the zebrafish, Danio rerio.

Earlier studies suggested retinal differentiation in the zebrafish commences ventrally rather than centrally as is the case in other vertebrates. Here we describe the topographical spread of cell differentiation for ganglion cells, double cones and rods in the zebrafish retina between 36 and 72 hours postfertilization (hpf), by using immunohistochemical markers in retinal wholemounts. Staining for all three cell types commenced within the ventral retina on the nasal side of the optic nerve and choroid fissure, at 38 hpf for ganglion cells and 50 hpf for double cones and rods. Within 3 to 4 hours, the staining of ganglion cells and double cones spread in a continuous wave-like fashion into the nasal region of the ventral retina. After this time, the staining patterns for ganglion cells and double cones progressed dorsally into the central and temporal retina. Finally, stained somata of ganglion cells were observed within the temporal-ventral region by approximately 48 hpf, more than 8 hours later than the first ganglion cells within the nasal retina. The topographical spread of double cone staining was slightly less orderly. After staining had extended into the nasal retina between 50 and 54 hpf, a small group of stained double cones often appeared at the temporal edge of the choroid fissure by 56 hpf, simultaneously with initial staining observed dorsal and temporal to the optic nerve. The topographical spread of rod staining in the ventral retina was more symmetrical. After rod staining appeared near the nasal edge of the choroid fissure at 50 hpf, rods accumulated within a localized patch nasal to the fissure. Approximately 5 hours after initial rod staining, scattered rod staining appeared on the temporal side of the choroid fissure (approximately 55-57 hpf). Rods increased rapidly within the ventral retina, and a dense symmetrical patch extended out from the choroid fissure into the nasal and temporal regions of the ventral retina by 70 hpf. A scattered pattern of rod staining also occurred within the dorsal retina at this time.

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On bipolar cell responses in the teleost retina are generated by two distinct mechanisms.

1. ON Bipolar cells were recorded in slices obtained from hybrid bass retinas. Cells were identified as bipolar cells by position in the slice, by characteristic voltage- and ligand-gated currents, and by filling with the fluorescent dye Lucifer yellow. Cells were recorded with the use of either whole cell or perforated-patch techniques. Standard electrophysiological protocols were used. Drugs were applied by puffing and by local superfusion. 2. Application of exogenous glutamate to ON bipolar cells generated two characteristic responses. One effect of glutamate was to open a conductance with a reversal potential close to the chloride equilibrium potential. The other effect of glutamate was to close a conductance with a reversal potential near 0 mV. These two effects of glutamate on ON bipolar cells match the effects of light described previously with the use of intracellular recordings. Thus the effects of glutamate that we report here appear to underlie the rod and cone inputs to these cells. 3. Many of the ON bipolar cells recorded demonstrated both classes of responses to glutamate. To isolate the two responses, 500 microM glutamate was first applied, and then glutamate in the presence of 5 microM 2-amino-4-phosphonobutyric acid (APB). APB specifically blocks the effects of glutamate on the putative roddriven glutamate receptor (the glutamate-elicited conductance decrease), allowing us to study in isolation the effects of glutamate on the cone component, the glutamate-activated chloride current (IGlu). 4. By isolating IGlu as described above, and taking advantage of the fact that amphotericin-perforated-patch recordings limit the diffusion of chloride ions between the patch pipette and the cell body, we found the physiological reversal potential of IGlu to be -58.9 +/- 7.7 (SD) mV. 5. Both the putative rod- and cone-mediated glutamatergic inputs to these bipolar cells could be activated by driving the photoreceptors with puffs of potassium. The currents recorded with this technique were very similar to those seen with direct application of glutamate.

Aminobutyrates↗

Retinoic acid establishes ventral retinal characteristics.

The developing eye is known to be rich in retinoic acid (RA), and perturbations in RA levels during formation of the optic primordia, as well as RA receptor mutations, cause retinal malformations, especially in ventral eye regions. To test the hypothesis that RA plays a role in the establishment of ventral retinal characteristics, we examined several dorsal and ventral ocular markers in RA-treated zebrafish. The optic stalk represents the ventral-most region of the early eye field. During normal development, the optic stalks constrict, decreasing in width and are gradually replaced by the optic nerve. Systemic high RA levels cause an expansion in the optic stalk with an increased cell content and a patent lumen. In addition, the stalks do not constrict and persist into later stages of development indicating an enhancement of early ventral eye characteristics. Expression of the transcription factor pax[b], normally confined to the ventral retina, expands into the dorsal retina following RA treatment, whereas msh[c], normally expressed in the dorsal retinal pole, disappears. Activity of an aldehyde dehydrogenase that normally occupies the dorsal third of the retina is reduced or abolished following high systemic RA. When a localized RA source, an RA-soaked bead, is placed next to the developing eye, a fissure resembling the choroid fissure appears in the eye facing the bead. Taken together, these observations suggest that RA is involved in the determination of the ventral retina.

Aldehyde Oxidoreductases↗

A behavioral screen for isolating zebrafish mutants with visual system defects.

Optokinetic and phototactic behaviors of zebrafish larvae were examined for their usefulness in screening for recessive defects in the visual system. The optokinetic response can be reliably and rapidly detected in 5-day larvae, whereas the phototactic response of larvae is variable and not robust enough to be useful for screening. We therefore measured optokinetic responses of mutagenized larvae as a genetic screen for visual system defects. Third-generation larvae, representing 266 mutagenized genomes, were examined for abnormal optokinetic responses. Eighteen optokinetic-defective mutants were identified and two mutants that did not show obvious morphological defects, no optokinetic response a (noa) and partial optokinetic response a (poa), were studied further. We recorded the electroretinogram (ERG) to determine whether these two mutations affect the retina. The b-wave of noa larvae was grossly abnormal, being delayed in onset and significantly reduced in amplitude. In contrast, the ERG waveform of poa larvae was normal, although the b-wave was reduced in amplitude in bright light. Histologically, the retinas of noa and poa larvae appeared normal. We conclude that noa larvae have a functional defect in the outer retina, whereas the outer retina of poa larvae is likely to be normal.

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GABAA and GABAC receptors on hybrid bass retinal bipolar cells.

1. gamma-Aminobutyric acid (GABA) responses from solitory hybrid bass retinal bipolar cells were studied with the use of conventional and perforated whole cell patch-clamp recording. 2. GABA elicited a chloride current in bipolar cells that had both transient and sustained components. The transient component was sensitive to bicuculline and resembled GABAA-mediated currents, whereas the more sustained component was resistant to bicuculline and resembled the responses mediated by GABAC receptors. 3. The bicuculline-resistant GABA responses recorded from the bipolar cells could not be modulated by either diazepam or pentobarbital sodium, and they were unaffected by phaclofen and 2-hydroxysaclofen, GABAB receptor antagonists. On the other hand, the bicuculline-resistant GABA responses could be blocked substantially by imidazole-4-acetic acid (I4AA), a competitive antagonist of GABAC receptors. 4. Noise analysis of the GABA-elicited currents suggested a different single channel conductance for GABAA (10.1 pS) and GABAC receptors (3.6 pS). 5. Zinc, a putative modulator of synaptic transmission, strongly inhibited the GABAC responses on bipolar cells, whereas the GABAA responses were not significantly affected by zinc. 6. The proportion of the GABAC to GABAA responses varied widely between bipolar cells. Local application of GABA onto dendrites or axon terminals showed that both types of GABA receptors are present on both regions of the cell. 7. The distinct properties of these two GABA receptor types suggest that they play different roles in retinal function.

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A glutamate-activated chloride current in cone-driven ON bipolar cells of the white perch retina.

Cone-driven ON-type bipolar cells were patch clamped in white perch retinal slices. Application of glutamate activated a current (IGlu) that was mediated by a conductance increase. The reversal potential for IGlu followed ECl closely when the intracellular chloride concentration was varied. IGlu was not blocked by 100 microM picrotoxin or 1 microM strychnine, indicating that it was not caused by inhibitory input. IGlu is not mediated by a typical ionotropic glutamate receptor since it was not activated by kainate, AMPA, or NMDA, or blocked by kynurenic acid, CNQX, DNQX, or AP-V. Further, IGlu is not mediated by a known metabotropic glutamate receptor since it was not activated by quisqualic acid, AP-4, ACPD, or ibotenate. IGlu required the presence of extracellular sodium and could be partially inhibited by the glutamate uptake inhibitors THA and tPDC. This is suggestive of sodium-dependent glutamate transport. However, when intracellular sodium was greatly increased, neither the magnitude nor reversal potential of IGlu was substantively affected. Thus, IGlu appears to involve a chloride channel activated by a glutamate receptor with transporter-like pharmacology. IGlu is localized to the dendrites of the bipolar cell, where bipolar cells receive an endogenous glutamatergic input from photoreceptors. Further, the reversal potential of the light response in these cells is the same as that of IGlu. Thus, it seems likely that IGlu is the current responsible for the cone component of the ON bipolar cell light response in the teleost retina.

2-Amino-5-phosphonovalerate↗

Threshold and chromatic sensitivity changes in fish cone horizontal cells following prolonged darkness.

The light-evoked responses of L-type cone horizontal cells in the teleost retina were studied following a prolonged period of complete darkness. Intact, isolated white perch retinas were superfused in complete darkness for more than 90 min, following which horizontal cells were impaled without the aid of any light flashes. Following this prolonged darkness, L-type cone horizontal cell light responses to dim and bright full-field stimuli were slow and small in amplitude and response duration to bright stimuli was considerably longer than stimulus duration. In addition, absolute threshold was 2 log units lower than typical for cone horizontal cells and spectral sensitivity to shorter wavelengths was increased. Following bright light stimulation, light responses became more transient and increased in amplitude, reaching 40-50 mV to bright flashes. Moreover, absolute threshold increased and responses to spectral stimuli were similar to those observed typically for L-type cone horizontal cells after light-sensitization. These results suggest that following prolonged darkness, cone input to cone horizontal cells is reduced and rod input is present.

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Protein content and cAMP-dependent phosphorylation of fractionated white perch retina.

In the retinas of teleost fish dopamine, released from interplexiform cells, modulates synaptic transmission at both the chemical and electrical synapses of retinal horizontal cells. This modulation is due to activation of adenylate cyclase and phosphorylation by protein kinase A, perhaps of the synaptic ion channel proteins themselves. In this study we have fractionated the white perch retina by Percoll density gradient centrifugation in order to identify proteins which coenrich with horizontal cells. In addition we have tested retinal fractions for phosphorylation by native cAMP-dependent kinase. Our findings indicate that there are at least 3 proteins of molecular weights 28, 43/44 and 50 kDa which coenrich with horizontal cells and 3 proteins of 30/31 kDa, 35 kDa (putative rhodopsin) and 48 kDa (putative arrestin) which coenrich with photoreceptor fractions. The 43/44 kDa phosphoprotein is a target for cAMP-dependent protein phosphorylation and thus is apparently an element of the dopaminergic modulatory pathway in perch horizontal cells.

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Retinoic acid is necessary for development of the ventral retina in zebrafish.

In the embryonic zebrafish retina, as in other vertebrates, retinoic acid is synthesized from retinaldehyde by two different dehydrogenases, one localized dorsally, the other primarily ventrally. Early in eye development only the ventral enzyme is present. Citral competitively inhibits the ventral enzyme in vitro and decreases the production of retinoic acid in the ventral retina in vivo. Treatment of neurula-stage zebrafish embryos with citral during the formation of the eye primordia results in eyes lacking a ventral retina. This defect can be partially rescued by retinoic acid. The results demonstrate that synthesis of retinoic acid can be selectively inhibited in vivo and suggest that retinoic acid is necessary for the proper development of the ventral retina.

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Early eye morphogenesis in the zebrafish, Brachydanio rerio.

Early eye morphogenesis in the zebrafish between 12 and 36 hours postfertilization was studied by light- and scanning electron microscopy. Overall, early eye morphogenesis in the zebrafish is similar to that of other vertebrates even though the optic primordia evaginate from the forebrain as solid masses of cells. After initial evagination (6-7 somite stage [SS]), the optic primordia take on a wing-like shape (8-9 SS). Subsequently, they bend ventrally and rotate slightly in an anterior direction (10-12 SS). These changes serve to bring the primordia from a horizontal to a more vertical orientation in relation to the embryonic neural axis. Invagination commences from the center of each primordium (14 SS) and progresses symmetrically out towards the periphery (14-20 SS). The choroid fissure forms by an involution along the anterior region of the eyecup (18-20 SS). By 24 hours postfertilization (pf), the eyecups are well formed. Between 24 and 36 hours pf, the eyes rotate further in relation to the axis of the embryo, and this repositions the choroid fissue to a typical ventral location by 36 hours pf. Because of the two rotations of the eye during early morphogenesis, particularly the later one, the anterior-posterior orientation of the emerging optic primordium ultimately becomes the ventral-dorsal axis of the completed eyecup.

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Pharmacology of novel GABA receptors found on rod horizontal cells of the white perch retina.

A novel type of GABA receptor is present on rod-driven (H4) horizontal cells of the white perch retina (Qian and Dowling, 1993a). These receptors have been tentatively termed GA-BAC receptors. In this study, the pharmacological properties of these receptors were further investigated by applying several conformationally restricted GABAA receptor agonists, GABAA antagonists, and a GABAB agonist to the H4 horizontal cells. GABA analogs locked in a partially folded conformation had a variety of effects. Isonipecotic acid had no effect on these receptors, whereas isoguvacine activated them but with low potency (EC50 = 137 microM). THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) acted as a competitive antagonist on these receptors with an inhibition constant of 82.5 microM. P4S (piperidine-4-sulfonic acid) activated the receptors at high concentrations (> 1 mM), but at lower concentrations it was a competitive antagonist with an inhibition constant of 80.9 microM. 14AA (imidazole-4-acetic acid), a GABA analog with an extended conformation, potently inhibited the GABA responses on H4 horizontal cells with an inhibition constant of 1.67 microM. Muscimol, which can assume both partially folded and extended conformations, acted as a mixed agonist-antagonist. The GABA responses on H4 horizontal cells were resistant to several competitive GABAA receptor antagonists including bicuculline, hydrastine, and SR-95531, but they were very sensitive to picrotoxin (IC50 = 237 nM). The inhibition by picrotoxin was both competitive and noncompetitive in nature. On the other hand, TBPS (tert-butyl-bicyclophosphorothionate), another GABAA receptor channel blocker, had minimal effects on these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

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