Purpose: The molecular identity of the calcium channels that mediate glutamate release from photoreceptors is unknown. Mutations in the recently identified, retina-specific alpha(1F) calcium channel subunit cause incomplete X-linked congenital stationary night blindness (CSNB2), the phenotype of which is consistent with a defect in neurotransmission within the retina. The purpose of this study was to determine the cellular distribution of the alpha(1F) subunit in the retina.
Methods: Antibodies were raised against a unique peptide from the human alpha(1F) sequence. Rat retina sections were labeled with affinity-purified alpha(1F) antibodies and the immunofluorescence analyzed by confocal microscopy. The alpha(1F) staining was compared with that obtained with a pan-alpha(1) antibody, used to reveal the distribution of known voltage-gated calcium channels in the retina. Some sections were double labeled for alpha(1F) and the photoreceptor synaptic ribbon marker, bassoon.
Results: Staining of retina sections with anti-alpha(1F) resulted in strong punctate labeling in the outer plexiform layer (OPL) and weak punctate labeling in the inner plexiform layer (IPL), consistent with a synaptic localization. Staining was also observed in the outer nuclear layer. Within the OPL, alpha(1F) immunoreactivity was clustered in discrete, horseshoe-shaped patches, the shape and dimensions of which are characteristic of rod active zones. Similar structures were labeled with the pan-alpha(1) antibody. Localization of alpha(1F) immunoreactivity to rod active zones was confirmed by double labeling for bassoon, a component of photoreceptor synaptic ribbons.
Conclusions: The distribution of alpha(1F) immunoreactivity in the OPL suggests that calcium influx through alpha(1F) or alpha(1F)-like channels mediates glutamate release from rod photoreceptors.