In the eye, neural circuits process images. One of the fundamental challenges for retinal neural circuits is to maintain image constancy over illuminations ranging from starlight to the brightest noontime sun. To span the brightness range, separate classes of photoreceptors have evolved, rods for nocturnal vision, and cones for diurnal vision. Each is served by dedicated sets of interneurons for processing rod and signals. Mammals such as cats, rabbits, rats and mice provide reasonable models for human rod circuitry. In primates cone types and cone circuits evolved for color vision, but common laboratory mammals cone density is low, and color sense is weak. Zebrafish, like primates, evolved color vision. Zebrafish employ 4 specialized cone types sensitive to different spectral wavebands. Zebrafish cone neural circuits process this spectral information. The ease of genetic manipulation is advantageous in zebrafish and there are extensive libraries of mutants and transgenics. For these reasons, over the past decade, this lab and others have worked to develop zebrafish as a model for electrophysiological and neuroanatomical studies of visual system development, circuitry and function. Membrane receptor expression at neural synapses dictates functional properties of circuits and provides molecular handles for experimental and therapeutic manipulation. The receptors on individual zebrafish retinal neurons, either dissociated, or in retinal slice, were investigated for neurotransmitter-induced changes in membrane potential (using a fluorescent voltage probe) or for neurotransmitter-induced changes in membrane currents (using patch electrodes). Cone bipolar cells (retinal interneurons) responded to glutamate (the cone neurotransmitter) through metabotropic glutamate receptors, AMPA-kainate receptors, and transporter-associated chloride channels. GABA, a retinal inhibitory neurotransmitter, evoked responses from GABA transporters, and a variety of ionotropic GABA receptors. In retinal slice and in retinal wholemounts., a library of horizontal, bipolar, and amacrine cell morphology was developed, both through patch and sharp microelectrode staining, and through gene-gun 'diolistic' staining. Neural responses to light are the product of retinal circuitry. A flattened, perfused eyecup preparation provides microelectrode access to retinal interneurons functioning deep within the circuitry of the light-responsive tissue. Cell bodies, dendrites and axons of horizontal cells were revealed in wet epifluorescence microscopy following microelectrode injection of alexafluor 594. Light-response physiology multiple spectral types, including trichromatic UV color opponent cells, with dominant UV cone signals being opposed by blue and green cone signals, but reinforced by red cone signals. The axons of UV trichromatic cells are longer and the dendritic fields wider than other spectral types, resembling the anatomical H3 types. A UV color-opponent physiology has not been reported for horizontal cells of other species. Tetrachromatic responses were depolarized by UV, hyperpolarized by far blue, depolarized by blue-green, and hyperpolarized by yellow or red. As the spectral responses of adult horizontal cells contain so many different cone signals, the 570nm-peaking red cones, the 480nm-peaking green cones, the 410nm-peaking blue cones, and the 362nm-peaking UV cones, a model was devised to infer the signal composition. This consists of the sum of four saturable Hill functions, one for each cone spectral type. It is a three-dimensional response-wavelength-irradiance function. The model quantifies the stimulus color calculations that horizontal cells, or other zebrafish retinal neurons perform. Amacrine cells revealed four temporal patterns: 1) Depolarizing transients at ON and at OFF. 2) Sustained depolarization. 3) A hyperpolarizing or biphasic ON response followed by a transient OFF depolarization. 4) Color opponent responses with response sign determined by stimulus wavelength. Reconstruction of image stacks from microelectrode injected cells revealed unique stratification patterns associated with amacrine light-response waveforms. ON-OFF cells are almost exclusively bistratified within the retinal inner plexiform layer (IPL), though with several patterns of bistratification; ON cells are monostratified near the middle, or just below the middle of the IPL; OFF cells are monstratified in the distal IPL, near amacrine cell bodies (sublamina a); color opponent cells are monostratified in the proximal IPL, near the ganglion-cell layer (sublamina b). The responses of ON-OFF amacrines are dominated by red cones, both at ON and at OFF, and serve red-cone function. Both ON and OFF amacrine types mix red with green or blue cone signals. Color opponent cells sample all cone types in various patterns. Ganglion cells are the output neurons of retina, sending axons to the brain. As seen in loose-patch recordings, the impulse discharges of larval ganglion cells are color coded. The most common type is UV triphasic, excited by red and UV stimuli, but inhibited in the mid-spectral range, similar to triphasic horizontal cells. Altogether zebrafish lives up to the expectation of rich processing networks for spectral waveband discrimination. Transgenic insertions of reporter genes selectively label retinal neurons. Our physiology laboratory collaborates with molecular laboratories for transgenic marking in studies of neural circuits. GE4a was developed in the Fumihito Ono Lab, Osaka Medical College. In addition to populations of amacrine and ganglion cells, there is label in a select horizontal cell type, perhaps the H2 anatomical type. The gene insertion is in a non-coding region of chromosome 14. The y245 line, a Gal4:UAS line developed in the Harry Burgess lab (NICHD), labels red and green cones brightly, in addition to a Muller-cell population. More faintly marked are amacrine and ganglion cells. The transgene insertion occurs in the musashi1 promoter on chromosome 8, and affects cone development. There is slow retinal degeneration of UV cones, reduced sensitivity, and aberrant spectral pattern, seen in the isolated cone PIII responses of both larvae and adults. The thyroxin beta 2 nuclear receptor (trb2) is natively induces the differentiation and development of long wavelength cones. The Rachel Wong lab made transgenic lines crx:mYFP-2A-trb2 and gnat2:mYFP-2A-trb2 to study red cone development. Both lines miss-express trb2. In crx:mYFP-2A-trb2, early expression in uncommitted retinal progenitors, results in all cone types, and some bipolar types expressing this gene. Red cones are overproduced, at the expense of green, blue and UV cones. In gnat2:mYFP-2A-trb2 expression is restricted to cones, but of all types rather than just red cones. The gnat2 line induces mixed opsin expression in green, blue and UV cones. In crx:mYFP-2A-trb2 physiology, spectral sensitivity shifts towards long wavelengths by larval day 5 for both cone PIII signals and ON-bipolar (b2) ERG signals. The effect persists in adults, which are transformed into red cone monochromats. A long-wavelength spectral shift in cone PIII begins by day 6 in the gnat2:mYFP-2A-trb2 line, and b-wave development is delayed. Adult cone PIII signals are dominated by red cones, with severe loss of other cone signals. In trb2 miss-expression lines both cone and bipolar cell physiology are altered, suggesting changes in retinal processing are influenced by the trb2 nuclear receptor. In loose-patch recordings of larval ganglion cells, more red-cone OFF responses are seen in crx:mYFP-2A-trb2, and larval cone morphology is altered.
