Neurons act in concert to perform remarkable computational feats. Among 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 in turn 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. The zebrafish model provides a cone dominated retina, rich in 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. Membrane receptors for neurotransmitters are a vital component of neural circuits. The pattern of receptor expression on different neural types suggests potential circuitry pathways. The first research phase focused on neurotransmitter responses of isolated 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. The distribution of GABA transporters, and a variety of ionotropic GABA receptors on neural types was later uncovered. 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 neurotransmission. 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 even serve as a GABA sensor mechanism. During phase one, 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, each technique in retinal slice. During the second phase of the research program, the 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, though still only about 40 microns diameter. This UV color type has not previously been reported in horizontal cells of other species. Tetrachromatic responses were also seen, being depolarized in the UV, hyperpolarized in the far blue, depolarized in the 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 compositiion. 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, as well as amacrine cells, or ERG components perform. Studies of amacrine types reveal four temporal patterns: 1. a depolarizing transient at ON and at OFF, 2. sustained depolarization, 3. a hyperpolarizing or biphasic ON response followed by a transient OFF depolarization 4. various color opponent responses with response pattern determined by wavelength. The dendritic stratification pattern of each physiological type has been reconstructed from image stacks of microelectrode injected cells using Neurolucida (MBF Bioscience). Each physiological type reveals a unique branching pattern: Type 1 cells are almost exclusively bistratified, though with several patterns of bistratification;type 2 cells are monostratified near the middle, or just below the middle of the inner plexiform layer;type 3 cells are monstratified in the distal IPL, near amacrine cells (sublamina a);type 4 cells are monostratified in the proximal inner plexiform layer, near ganglion cells (sublamina b). Amacrine cell dendritic fields are much larger than those of horizontal cells, and range from 150 to 300 microns. Some are redially symetric, others branch profusely only to one side of the cell body. Still others have one or more axon-like processes extending away from a central dendritic arbor. The responses of types 1,2 and 3 are dominated by red cones. The color opponent patterns in type 4 are variuous, but include UV tetraphasic types. This present research stage revisits 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 (Molecular Probes) and modern long focal length water objectives allow visualization of live, stained neurons deep within the tissue. Depending on experimental goals, dyes with different fluorescence spectra, or different gap-junction permeance can be selected. UV compliant microscope optics provide stimulation of zebrafish UV cone circuitry, all 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 color circuitry in this animal model from the perspective of neural responses to visual stimuli. The proposal is to continue to elaborate this new research tool to gain further insight into population of neurons serving retinal cone system function, and in the future to study the disruption of these circuits in neural mutants.
