In the eye, neural circuits process images. Retinal circuits maintain image constancy over illuminations ranging from starlight to the brightest noontime sun. To span the brightness range, separate photoreceptor types evolved, rods for nocturnal vision, and cones for diurnal vision. Dedicated sets of interneurons separately process rod and cone signals. Mammals such as cats, rabbits, and rodents, are reasonable models for human rod circuitry. In primates further cone types and cone circuits evolved for color vision, but in 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 in zebrafish is advantageous, and there are extensive libraries of visual-system mutants and transgenics. For these reasons, this lab and others have worked to develop zebrafish as a model for electrophysiological and neuroanatomical studies of visual system development, circuitry, and function. Neurotransmitter receptors at neural synapses dictate functional properties of circuits and provide molecular handles for experimental and therapeutic manipulation. 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 morphologies was developed, through patch and sharp microelectrode staining, and through gene-gun 'diolistic' staining. A flattened, perfused eyecup provides microelectrode access to retinal interneurons functioning deep within the retinal circuitry. Cell bodies, dendrites and axons of individual horizontal cells (HCs) and amacrine cells (ACs) are revealed in wet epifluorescence microscopy following microelectrode injection of alexafluor 594. Light-response physiology revealed multiple color-texture types, including multichromatic UV color opponent HCs and ACs, with UV cone signals being opposed or reinforced by signals of other cone types. The axons of UV trichromatic HCs are longer and the dendritic fields wider than other types and resemble the anatomical H3 type. Tetrachromatic HC 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 HCs contain so many different cone signals, red cones, green cones, blue cones, and 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 HCs, ACs or ganglion cells (GCs) perform. In cone PIII spectral recordings, a refined version of the model directly determines the multiple underlying opsin peaks within ERG waveforms and provides physiological confirmation of shifts in opsin expression during zebrafish development as seen by in-situ hybridization. These include particularly a shift from the shorter wavelength red-cone LWS2 opsin (556 nm) in larvae to the longer wavelength LWS1 opsin (575 nm) in adults. ACs revealed four temporal-chromatic 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 wavelength. Reconstruction of stain-injected cells revealed unique stratification patterns associated with AC temporal-chromatic patterns. ON-OFF cells were almost exclusively bistratified within the retinal inner plexiform layer (IPL), though with several bistratification patterns; ON cells are monostratified in mid-IPL; OFF cells are monstratified in the distal IPL, near AC cell bodies (sublamina a); color opponent cells are monostratified in the proximal IPL, near GCs (sublamina b). The responses of ON-OFF ACs are dominated by red cones, both at ON and at OFF. Both ON and OFF AC types mix red with green or blue cone signals. Color opponent cells sample all cone types in various patterns of excitation and inhibition. GCs, the output neurons of retina, send axons to the brain. As seen in loose-patch recordings, the impulse discharges of larval GCs are color coded. The overall GC spectral pattern involves primary excitation in the ultraviolet, secondary excitation in the red, and mid-spectral inhibition. In individual GCs, signals from as many as 5 of the 7 opsins expressed in larvae can be identified, though more commonly, 3 or 4 opsin signals are intermixed. Altogether zebrafish lives up to the expectation of rich processing networks for spectral waveband discrimination. To investigate neural circuits, we collaborate with molecular laboratories whose transgenic lines selectively mark neurons. The GE4a line, developed in the Fumihito Ono Lab, Osaka Medical College marks select populations of HCs, ACs and GCs; the HC label may be selective for H2. GE4a transgene 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 ACs and GCs. 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) induces differentiation and development of red cones. Transgenic lines from the Rachel Wong lab (crx:mYFP-2A-trb2 and gnat2:mYFP-2A-trb2) are gain-of-function lines, mis-expressing trb2. In crx:trb2, early expression in uncommitted retinal progenitors, results in all cone types, and many BC types, expressing trb2. Red cones are overproduced, at the expense of green, blue and UV cones. In gnat2:trb2, transgene expression is restricted to differentiated cones of all types. The gnat2:trb2 line mixes red opsin expression into differentiated green, blue and UV opsin-expressing cones. In crx:trb2, ERG spectral sensitivity shifts towards long wavelengths by larval day 5, for both cone PIII signals and ON-bipolar (b2) signals. More red-cone OFF responses are seen in loose-patch recordings of larval GCs. Larval cone morphology is altered. Adults are transformed into red cone monochromats. A slight long-wavelength shift in cone PIII begins by day 6 in gnat2:trb2, and b-wave development is delayed. By adulthood, cone PIII signals are red-cone dominated, with severe loss of other cone signals. In trb2 gain-of-function lines, alteration of both cone, BC, and GC physiology suggest trb2 changes retinal circuits. A trb2 -/- mutant line with no larval or adult red-cone signals, and enhanced UV-cone signals has been established by Crispr corruption of the first-exon trb2 reading frame. This line lacks larval optomotor or optokinetic responses for both red/black and green/black color contrasts. The arrestin 3a antigen, (zpr-1 antibody), normally expressed in red-green double cones, appears to be lost.
