Structure And Function In Retinal Neurons

National Institute Of Neurological Disorders And Stroke

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Abstract

In the eye, neural circuits process images. These circuits maintain image constancy over illuminations ranging from starlight to noontime sun. Rods evolved for nocturnal vision, and cones for diurnal vision. Separate interneurons process these rod or 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, employing four cone types specialized for different spectral wavebands, and developing complementary cone neural circuits. 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 on retinal interneurons shape circuit function and provide molecular handles for experimental and therapeutic manipulation. Previously these were investigated through neurotransmitter-induced influence on membrane potential using techniques such as fluorescent voltage probe in dissociated cells or patch electrodes in retinal slice. Cone bipolar cells (BCs, retinal interneurons post-synaptic to cones) responded to glutamate (the cone neurotransmitter) through metabotropic glutamate receptors, AMPA-kainate receptors, and glutamate 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 (HC), bipolar, and amacrine cell (AC) morphologies was developed, through patch and sharp microelectrode staining, and through gene-gun 'diolistic' staining. The library of dendritic morphologies helps to identify connectivity patterns and neural circuits. A flattened, perfused eyecup allowed microelectrode access to retinal interneurons functioning deep within the retinal circuitry. Cell bodies, dendrites and axons of individual HCs and ACs were revealed in live 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 were 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, analytic models were devised to infer the signal composition. These models consisted of sums of multiple, single cone type, saturable Hill functions. They are three-dimensional response-wavelength-irradiance functions replicating the stimulus color calculations that HCs, ACs or ganglion cells (GCs) perform. In cone PIII electroretinogram (ERG) 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 previously 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 different bistratification arrangements; 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. Color opponent cells sample all cone types combining 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 cone 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 identify gene action in neural circuits we collaborate with molecular laboratories for electrophysiology studies in mutant and transgenic lines. The thyroxin beta 2 nuclear receptor (trb2) has been a recent focus. This gene is required for 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 causes all later developing cone types, and many BC types, to express trb2. Red cones are overproduced, at the expense of green, blue and UV cones. In crx:trb2, ERG spectral sensitivity shifts towards long wavelengths by larval day 5, for both cone PIII ERG signals and ON-bipolar (b2 ERG) signals. More red-cone OFF responses are seen in loose-patch recordings of larval GCs. Larval cone morphology is altered. Adults become red cone monochromats. In gnat2:trb2, transgene expression only occurs in differentiated cones. The gnat2:trb2 line mixes red opsin expression into green, blue and UV opsin-expressing cones. A slight long-wavelength shift in cone PIII begins by day 6, 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 downstream retinal circuits. A trb2 -/- mutant line with no larval or adult red-cone signals, and enhanced UV-cone signals was established by Crispr corruption of the first-exon trb2 reading frame. We find this line lacks larval optomotor or optokinetic responses for both red/black and green/black color contrasts, and lacks ERG responses to red stimuli. The arrestin 3a antigen, (zpr-1 antibody), normally expressed in red-green double cones, appears to be lost.