The vertebrate retina receives, processes and transforms visual information. Light absorbed in photoreceptors is transduced into electrical signals which evoke the light modulated release of glutamate onto horizontal and bipolar cells (HCs and BCs). HCs provide lateral feedback regulating transmitter release from photoreceptors according to conditions of ambient illumination, while BCs transfer light signals forward to ganglion cells and amacrine cells. Ganglion cells transform light signals into trains of action potentials which propagate to brain visual centers; amacrine cells are retinal interneurons with several signal processing roles. As images pass through retinal circuitry, they are decomposed into component parts, so that special sets of ganglion cells extract different components of the image. Some signal highlights, others shadows, movement, direction or color. Signal processing within the retina is achieved by specialized circuits, patterns of connections among neurons, and specialized receptors for neurotransmitters. The research program studies the relationships between receptor expression on retinal neurons, the neural circuitry of the retina, and retinal information processing tasks. Zebrafish provides a model of human genetic disease. Through studies of this model, genetic and other perturbations of human vision may be better understood. We examine zebrafish using acutely dissociated retinal neurons, light responsive in vitro eyecup preparations, and zebrafish retinal slices. Cell structure, a key element in discerning retinal circuits, is beautifully delineated in the zebrafish model. Acutely dissociated BC, HC and photoreceptor cells are readily identified morphologically. Retinal slices can be impregnated with DiI coated microcarriers using a gene gun. Diffusion of the lipophilic dye along membranes reveals the shapes of individual neurons. Retinal ganglion cells are rapidly stained through back labeling of the optic nerve. In retinal slices, cell structure may also be seen as dyes introduced through the microelectrode diffuse through cytoplasm, outling the cell. The neurotransmitter responses of cell types are key to understanding information processing in retinal circuits. Physiological responses to neurotransmitters can be assayed with voltage-sensitive probes in isolated cells, and by whole-cell patch recording in retinal slices. Circuitry models of retina must be consistent with whole-system responses. In zebrafish eyecup preparations electroretinographic responses can be recorded, and selective agonists and antagonists used to discover neurotransmitter systems at work in the component elements of this field potential. Whole-cell patch recording and puff pipette techniques have identified glutamate receptor mechanisms on BC dendrites in the zebrafish retinal slice. Lucifer Yellow stains of these BCs reveal axons with terminal varicosities, as well as one or two varicosities along the axon. These are the sites of synaptic ouput in the inner plexiform layer (IPL). Axon varicosities divide the IPL into 2 major zones: a thick sublamina a, with three bands of varicosities, and a thin sublamina b, with two bands. BCs occurred with varicosities restricted to sublamina a (Group a), sublamina b (Group b) or both sublaminae (Group a/b). BCs hyperpolarized by light (HBCs) are depolarized by glutamate. HBCs belonged to Group a or Group a/b. These cells responded to glutamate or kainate with a CNQX-sensitive conductance increase. Reversal potential (Erev) ranged from -0.6 to +18 mV. Bipolar cells stimulated sequentially with both kainate and glutamate revealed a population of glutamate-insensitive, kainate-sensitive cells in addition to cells sensitive to both agonists. In culture, cells depolarized only by kainate were not immunoreactive (IR) for kainate receptors. BCs depolarized by light (DBCs)are hyperpolarized by glutamate. Intense Kainate receptor-like IR was seen in DBCs. By axon stratification, DBCs belonged to Group b or Group a/b and responded to glutamate via one of three mechanisms: (a) a conductance decrease with Erev ~ 0 mV, mimicked by L-(+)-2-amino- 4-phosphonobutyric acid (APB), (b) a glutamate-gated chloride conductance increase (Iglu-like), characterized by Erev near ECl (where ECl is the chloride equilibrium potential), and partially blocked by extracellular Li+/Na+ substitution, or (c) the activation of both APB and chloride mechanisms simultaneously to produce a response with outward currents at all holding potentials. APB-like responses were found only among DBCs in Group b, with a single terminal varicosity ramifying deep within sublamina b; whereas, cells expressing Iglu-like currents had one or more axonal varicosities, and occurred within Groups b or a/b. Multistratified cells (Group a/b) were common and occurred with either HBC or DBC physiology. Multistratified DBCs expressed the IGlu- like mechanism only. Visual processing in the zebrafish retina involves at least 13 BC types. Using a voltage probe (oxonol, DiBaC4(5)) to study dissociated zebrafish HCs and HBCs, we found that glutamate causes a significant post excitatory, long-term hyperpolarization of membrane potential or after-hyperpolarization (AHP). AHP was blocked by CNQX. It was evoked by kainate, AMPA, and the AMPA-selective agonist (S)-5-Fluorowillardiine, but not by NMDA, D-aspartate, or the kainate selective agonist SYM 2081. Dissociated HCs and HBCs were commonly found in a tonically depolarized state. Resting potentials were restored by nifedipine, suggesting a tonic, depolarizing action of L-type Ca2+ channels. AHP was not blocked by nifedipine. AHP was insensitive to [Cl-]o. AHP was blocked by [Li+]o substitution for [Na+]o and by ouabain. AHP was never seen in depolarizing (ON-type) bipolar cells (DBC's). A mechanism is proposed in which Na+ entering through ionotropic AMPA channels stimulates Na+, K+ -ATPase, which, by electrogenic action, restores membrane potential, generating the AHP response. Patterns of ATPase IR support localization in cone pedicles, HC's, and BC's. Labeling was weaker in the IPL than in nuclear layers. In the IPL 2 bands of IR bipolar terminals were observed, one in sublamina a and the other in sublamina b. Persistent stimulation of distal retina by photoreceptor glutamate may induce increased expression and activity of Na+, K+ -ATPase, with a consequent impact on distal signal processing.
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