Vesicular ATP pools in zebrafish horizontal cells

Abstract

Purpose : The discovery of a vesicular nucleotide transporter (VNUT) has established that ATP can be concentrated and stored in neuronal vesicles. VNUT expression has been localized to horizontal cells in the outer retina. Therefore, we hypothesize that an ATP pool is present in horizontal cells and contributes to ATP release which can serve as a precursor to adenosine during nocturnal conditions.

Methods : Experiments were performed on both intact retinas and isolated horizontal cells from zebrafish retina. Confocal live cell imaging of quinacrine or MANT-ATP were used to monitor ATP levels and release. Immunohistochemical analysis using confocal microscopy of zebrafish retina was performed on vertical sections and whole mounts. Vesicle cycling was monitored using a VNUT antibody targeted to the luminal face of the protein conjugated to CF488 in order to characterize vesicle turnover in horizontal cells.

Results : Vesicles containing ATP labeled stores co-localized with VNUT in horizontal cells. Antibodies targeted to the luminal face of VNUT transporters labelled cycling vesicles within horizontal cell processes. Uptake was stimulation and Ca²⁺-dependent arguing in favour of a vesicular mechanism, and reduced in the absence of Ca²⁺ and Ca²⁺ channel blockers. Synaptic vesicle recycling and retrieval was demonstrated with sequential Ca²⁺ labelling of vesicles with fluorescently labelled VNUT antibody followed by a secondary antibody targeted to the VNUT antibody.

Conclusions : These findings support the hypothesis that horizontal cells contain ATP in VNUT expressing vesicles and release ATP from horizontal cells in the outer retina. Thus, during nocturnal conditons it is possible that outer retinal adenosine is derived form ATP.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.