Abstract
Purpose:
Ribbon synapses of sensory neurons tonically release glutamate to signal graded changes in stimulus intensity over a large operating range. The ribbon structure is found at photoreceptor, hair cell, and bipolar cell synapses where it tethers synaptic vesicles in close proximity to the presynaptic active zone. The zebrafish hi1482 mutant was isolated in a screen for abnormal visual system development and results from a retroviral insertion in the gene encoding tryptophan rich basic protein (Wrb). The goal of this study was to gain insight into the role of this novel protein in ribbon synapse structure and function.
Methods:
Electroretinography (ERG), optokinetic response measurements (OKR), Immunohistochemistry (IHC) and electron microscopy (EM) analyses were employed to assess ribbon synapse function, protein expression, and ultrastructure. Real-time q RT-PCR was used for relative quantification of wrb expression. As homozygous mutants do not survive to adulthood, all fish were analyzed at 5 days post fertilization, when rods are not active.
Results:
The hi1482 retroviral insertion represents a hypomorphic mutation, with expression of wrb reduced to less than 1% of WT. Mutant OKR was minimal, with saccade frequency reduced to 15% of WT and gain of OKR slow phase negligible at the highest intensity and contrast. Mutants have severely diminished b-wave responses, with maximal b-wave amplitudes only ~20% of WT. ERG a-waves had comparable amplitudes in mutant vs. WT. Since photoreceptor number appears normal in mutant fish, ERG and OKR results point to a specific defect in cone photoreceptor synaptic transmission. Mutant cone photoreceptor synapses had a greater number of misaligned, floating ribbons than WT and a partial disruption in SV2 and ribeye localization. More severe disruptions of ribbon architecture were encountered in mechanosensory hair cells of mutants, where ribeye-positive presynaptic ribbons were scarce. Despite disrupted signaling, the post-synaptic contacts of bipolar cells at cone synaptic terminals were structurally normal in wrb mutants.
Conclusions:
The attenuated synaptic transmission at ribbon synapses and the mislocalization of key presynaptic components in the wrb mutant suggest that wrb is important for the assembly or maintenance of ribbon synapses.
Keywords: 648 photoreceptors •
728 synapse •
689 retina: distal (photoreceptors, horizontal cells, bipolar cells)