Neurons extract information from light. In the eye, retinal neural circuits process images. One of the fundamental challenges for eyes 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 in turn is served by dedicated sets of interneurons forming intra-retinal rod and cone networks. Mammals such as cats, rabbits, rats and mice provide good models for rod neural circuits. In primates cone types and cone circuits have evolved for color vision. Common laboratory mammals have poorly developed color vision and a low density of cones. On the other hand, zebrafish, like primates, have evolved color vision, using 4 specialized cone types sensitive to different spectral regions, and possess retinal neural circuits that process spectral information. Zebrafish is advantageous for ease of genetic manipulations and for extensive libraries of mutants and transgenics. It is for these reasons that, over the past decade, this lab and others have worked to develop zebrafish as a model for electrophysiological and neuroanatomical studies of visual system circuitry. Membrane receptors for neurotransmitters drive neural circuits. In retina receptor expression pattern often provides a unique signature for neural types. Receptor patterns dictate functional properties of circuits and provide molecular handles for experimental 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 receptors, AMPA-kainate receptors, and transporter-associated chloride channels. GABA, a retinal inhibitory neurotransmitter, evoked responses from transporters, and a variety of ionotropic GABA receptors. In parallel with studies of neurotransmitter receptors, a morphological library of horizontal, bipolar, and amacrine cell types was developed, both through patch microelectrode staining, and through 'diolistic' staining, both in retinal slice. Neural light responses are the circuitry product. A flattened, perfused eyecup preparation provides microelectrode access to retinal interneurons within 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 revealed 6 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 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 wavelength. Reconstruction of from image stacks of microelectrode injected cells revealed unique stratification patterns associated with physiological wavefoms. 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 ganglion cells (sublamina b). The responses of ON-OFF 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. 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. Altogether zebrafish lives up to the expectation of rich processing networks for wavelength discrimination. Transgenic insertions of reporter genes selectively label retinal neurons. Our physiology laboratory collaborates with molecular laboratories to use transgenic marking in studies of neural circuits. GE4a was developed in the Fumihito Ono Lab, NIAAA. 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 appears to involve the Musashi1 promoter on chromosome 8. The mutation is also associated with a 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 receptor (trb2) is required for the differentiation of long wavelength cones. The Rachel Wong lab developed transgenic lines crx:mYFP-2A-trb2 and gnat2:mYFP-2A-trb2 both of which overexpress trb2 receptor, the first early in development, before any cone types are born, the second late in development, within differentiated cones of all types. The crx line causes early overproduction of red cones, at the expense green, blue, and UV cones. The gnat2 line causes mixed opsin expression in green, blue and UV cones, and may eventually trans-differentiate mixed cones into red cones. In crx physiology, spectral sensitivity shifts towards long wavelengths by day 5 for both cone PIII and ON-bipolar (b2) ERG components. The effect persists from larvae to adults. A long-wavelength spectral shift in cone PIII begins by day 6 in the gnat line, and b-wave development is delayed. In both lines both cone and bipolar cell physiology are altered, suggesting far reaching changes in retinal processing are controlled by the trb2 nuclear receptor.
