Localization of Rod Bipolar Cells in the Mammalian Retina Using an Antibody Against the α1c L-type Ca(2+) Channel

Abstract

Bipolar cells transmit stimuli via graded changes in membrane potential and neurotransmitter release is modulated by Ca(2+) influx through L-type Ca(2+) channels. The purpose of this study was to determine whether the α1c subunit of L-type voltage-gated Ca(2+) channel (α1c L-type Ca(2+) channel) colocalizes with protein kinase C alpha (PKC-α), which labels rod bipolar cells. Retinal whole mounts and vertical sections from mouse, hamster, rabbit, and dog were immunolabeled with antibodies against PKC-α and α1c L-type Ca(2+) channel, using fluorescein isothiocyanate (FITC) and Cy5 as visualizing agents. PKC-α-immunoreactive cells were morphologically identical to rod bipolar cells as previously reported. Their cell bodies were located within the inner nuclear layer, dendritic processes branched into the outer plexiform layer, and axons extended into the inner plexiform layer. Immunostaining showed that α1c L-type Ca(2+) channel colocalized with PKC-α in rod bipolar cells. The identical expression of PKC-α and α1c L-type Ca(2+) channel indicates that the α1c L-type Ca(2+) channel has a specific role in rod bipolar cells, and the antibody against the α1c L-type Ca(2+) channel may be a useful marker for studying the distribution of rod bipolar cells in mouse, hamster, rabbit, and dog retinas.

Keywords: L-type Ca2+ channel; immunocytochemistry; mammalian retina; protein kinase C; rod bipolar cell.

Fig. 1.

Photomicrographs of the distribution and control of α₁c L-type Ca²⁺ channel-immunoreactive cells in the retina. (A, D, G, J) Anti-α₁c L-type Ca²⁺ channel-immunoreactive cells. (B, E, H, K) Sections before preabsorption. (C, F, I, L) The α₁c L-type Ca²⁺ channel antibody was preabsorbed with antigen prior to tissue incubation. Preabsorption control test of antibody against the α₁c L-type Ca²⁺ channel showed no immunoreactive cells in four different mammalian retinas. INL, inner nuclear layer; IPL, inner plexiform layer. Bar=20 μm.

Fig. 2.

Confocal micrographs of vertical sections from the mouse, hamster, rabbit, and dog retinas. (A, E, I, M) DIC images were used to reveal the layers of retina. (B, F, J, N) Retinal sections were immunostained for PKC-α. (C, G, K, O) Retinal sections were immunostained for α₁c L-type Ca²⁺ channel. In both labelings, the immunoreactive cells displayed the morphology of rod bipolar cells as their cell bodies in the INL extended long axonal process throughout the IPL. (D, H, L, P) Superimposed images of PKC-α and α₁c L-type Ca²⁺ channel immunolabeling revealed an almost complete of colocalization. DIC, differential interference contrast; INL, inner nuclear layer; IPL, inner plexiform layer. Bar=20 μm.

Fig. 3.

Confocal micrographs of whole mounts from the mouse, hamster, rabbit, and dog retinas. (A, D, G, J) Whole mounts were immunostained for PKC-α. (B, E, H, K) Whole mounts were immunostained for α₁c L-type Ca²⁺ channel. Immunolabeling was visible within the cytoplasm of rod bipolar cells. The immunoreactive cells in the mouse and hamster formed a tight mosaic compared to those in the rabbit and dog. (C, F, I, L) Superimposed images of PKC-α and α₁c L-type Ca²⁺ channel immunostaining revealed that these proteins were colocalized. Bar=20 μm.