Neurons act in concert to perform remarkable computational feats. One of the more amazing of these is extracting information from light. In the eye, retinal neural circuits process images. One of the fundamental challenges for eyes is to maintain constancy of neural product over ambient illuminations ranging from starlight to the brightest noontime sun. To cover this range separate classes of photoreceptors have evolved, rods for nocturnal vision, and cones for diurnal vision. Each is served by dedicated sets of interneurons forming intraretinal rod and cone pathways. Mammals such as cats, rabbits,rats and mice provide excellent models for rod system circuitry. Cone system circuitry is relatively less well studied. Color vision is a characteristic of cones and their associated interneurons. The common mammalian models have a poorly developed color vision and a low density of cones. Zebrafish provides a lower vertebrate model with a cone dominated retina, rich color vision, and with the opportunity to study cone circuitry. The zebrafish is also well known for ease of genetic manipulations and an extensive library of mutants and transgenics. It is for these reasons that, over the past decade my lab and others have worked to develop zebrafish as a model for electrophysiological study of the visual system. Channels on neural membranes lead to circuitry properties. The earlier phases of this research program focused on isolated membrane properties of zebrafish retinal neurons, either dissociated or in retinal slice. Glutamatergic actions on cone bipolar cells revealed a variety of mechanisms, including metabotropic receptors, AMPA-kainate receptors, and transporter-associated chloride channels. In parallel with these studies, a morphological library of horizontal, bipolar, and amacrine cell types was developed, both through patch microelectrode staining, and through 'diolistic'staining, each technique in retinal slice. Recently the GABAergic properties of horizontal and bipolar cells have been added to the inventory of membrane properties on identified zebrafish retinal interneurons. There appear to be at least three GABA receptor types on bipolar cells, a GABA-A receptor and both picrotoxin sensitive and insensitive GABA-C receptors. Some bipolar cells express a GABA transporter, a signature for GABAergic neurons. Horizontal cells lack GABA receptors, but a subset transport GABA and are also GABAergic. As seen in voltage-probe techniques, GABA transporter signals are very large, and may serve as a GABA sensor mechanism. During the past review period, the physiological focus shifted to neural light responses. A flattened, perfused eyecup preparation was developed that provided access to horizontal cell light responses through sharp microelectrodes. Cell bodies, dendrites and axons of these horizontal cells were revealed in wet epifluorescence microscopy following injection of alexafluor 594. Spectral studies of horizontal cell light responses revealed 6 chromatic 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 morphology of these UV trichromatic cells is characterized by long axons and comparatively wide dendritic fields. This color type has not previously been reported in horizontal cells of any other species. Several horizontal cell types receive inputs from all four cone types in zebrafish. These are 570nm-peaking red cones, 480nm-peaking green cones, 410nm-peaking blue cones, and 362nm-peaking UV cones. A linear model composed of the sum of four Hill functions, one for each cone signal, has been devised to measure all these color inputs. We call this the unified response-spectral-irradiance model. The model quantifies the stimulus color calculations that horizontal cells perform. The current research program has revisited the sharp electrode technique, but in modernized version. Amplifiers can now handle gigohm dye-filled electrodes, and micropositioners are vastly improved. In particular, the Alexa dyes and modern long focal length wet objectives allow visualization of live, stained neurons deep within the tissue. UV compliant objectives allow stimulation of zebrafish UV cone circuitry on the microscope stage. Computer driven optical benches provide time-efficient stimulus delivery. It is exciting to find that this combination of new and old techniques is well suited to the tiny neurons of zebrafish retina, and that it promises to open up knowledge of retinal circuitry in this animal model. The proposal is to continue to elaborate this new research tool to gain further insight into retinal cone system function.
