Abstract
Purpose: :
Sensory neurons in the retina utilize ribbon synapses to release neurotransmitter in response to graded changes in membrane potential. The active zone of these specialized synapses utilizes a "ribbon" structure, which is surrounded by and physically contacting numerous synaptic vesicles. We have identified a zebrafish mutant, which affects the wrb gene, with visual and mechanosensory defects resulting from ribbon synapse dysfunction. The wrb gene encodes a novel 170 amino acid protein with no predicted functional or enzymatic domains. Mutants lacking the Wrb protein show anatomical defects in ribbon synapse structure and function. In order to gain a better understanding of the mechanisms underlying ribbon synapse function, we have investigated the cellular and physiological phenotypes of the wrb mutant. The wrb gene thus represents a novel candidate for human disorders characterized by loss of vision and hearing.
Methods: :
Zebrafish wrb mutants have been examined by immunohistochemistry to examine various presynaptic and post-synaptic proteins on zebrafish retinas at 5 days post fertilization. Apical endocytosis of presynaptic membranes was monitored by the uptake of the vital dye FM1-43. Electron microscopy was used to analyze ribbon synapse architecture. Optokinetic response (OKR) behaviors and electroretinography have been analyzed to examine visual function.
Results: :
The presynaptic proteins SV2 and Ribeye were strongly mislocalized in wrb mutant photoreceptors at 5 dpf and in the synaptic terminals of lateral line hair cells, indicating a common defect at ribbon synapses. FM1-43 uptake was severely reduced in wrb mutants, suggesting that mechanotransduction is blocked. By electron microscopy, secondary neurons appeared to invaginate normally in wrb mutants but fewer ribbons were observed and several ribbons were "floating" within the cytoplasm, suggesting a defect in ribbon assembly. ERG waveforms were also different. The wrb mutants had much smaller b- and d-waves and the a-wave was more noticeable.
Conclusions: :
Zebrafish mutants in the wrb gene exhibit defects in ribbon synapse structure and function. The wrb gene encodes a novel protein with unknown function. Mutations in wrb result in mislocalization of components of the ribbon synapse and block neurotransmission to postsynaptic cells. As FM1-43 staining does not occur, we hypothesize that Wrb participates in either assembly or maintenance of the synaptic ribbons but not in vesicle recycling. Thus, wrb represents a novel functional components of the in ribbon synapse represents a novel candidate for hereditary deafness/blindness disorders.
Keywords: synapse • photoreceptors • transgenics/knock-outs