Abstract
Purpose: :
There is growing evidence that mammalian horizontal cells (HCs) possess the molecular machinery for vesicular exocytosis. As conventional transmitter release is triggered by voltage-gated calcium channels, we investigated the subcellular localization of N- and L-type calcium channels in mouse horizontal cells.
Methods: :
Mouse retinas were fixed for 15-30 min with 4% paraformaldehyde. Cryostat vertical sections were processed for indirect immunofluorescence immunohistochemistry and confocal microscopy. Double label experiments were carried out with rabbit polyclonal antibodies (Ab) to N- (Cav2.2) and L-type (Cav1.2) voltage-gated calcium channels (Alomone, Israel) and mouse monoclonal Ab to cell type-specific markers for HCs (calbindin), for rod bipolar cells (PKC) and to synaptic ribbons (CtBP2).
Results: :
Immunolabeling for Cav2.2 calcium channels was most prominent in ribbon-like structures at the very tips of HC axons. Cav2.2 labeling also occurred in clusters at HC dendrites apposed to the base of cone pedicles and less intensely in HC bodies. To ascertain whether the Cav2.2 was at synaptic ribbons of presynaptic photoreceptor terminals, double label experiments were done with CtBP2 Ab. The labeling patterns were not identical, indicating that the calcium channel staining was not recognizing synaptic ribbons, but that the Cav2.2 labeling followed the shape of the ribbon. The invaginating dendrites of rod bipolar cells (RBCs), labeled for PKC, were capped by the crescent-shaped Cav2.2 immunolabeling, indicating a lack of localization to RBC dendrites. Double labelings for Cav1.2 and calbindin produced immunoreactivity at HC tips as well as the stalk of the endings, and at HC processes adjacent to the base of cone pedicles.
Conclusions: :
Mouse HCs express both N- and L-type calcium channels. N-type (Cav2.2) channels localized to the distal tips of HC endings in close relation to the presynaptic synaptic ribbon. While L-type (Cav1.2) channels were also expressed in horizontal cell tips, they were also found along the stalks and processes of HCs. Regulated transmitter release likely occurs in HC tips within the triad synapse and possibly along HC processes in a somatodendritic manner.
Keywords: horizontal cells • calcium • synapse