IP3 receptors are present at ribbon synapses of both rod and cone bipolar cell terminals in mammalian retina

Abstract

Abstract: : Ribbon synapses are important sites of L–glutamate release from bipolar cells in the retina. Transmitter release is regulated by Ca²⁺ entry into the synaptic terminal of these cells. Purpose: Release from IP₃ receptor (IP₃R)–mediated Ca²⁺ stores has been shown to play a critical role in regulating excitatory transmitter release and plasticity at central neurons, but their role in the retina has not been elucidated. We therefore tested whether IP₃Rs are present at cone and rod bipolar cell synapses and determined their impact on Ca²⁺ dynamics at bipolar cell terminals. Methods: [Ca²⁺]_i changes were assessed in bipolar cells using the dyes, fluo–4 or calcium orange. To test for immunolocalization of IP₃Rs, rat or mouse retinas were fixed for 1 or 2 hrs with 4% paraformaldehyde and processed for indirect immunofluorescence immunohistochemistry.Results: Using a pan–IP₃R antibody that recognizes all types of IP₃Rs, immunoreactivity was present on both cone and rod bipolar cells with the most intense labeling at the synaptic terminals. Double label experiments with antibodies to VGLUT1, PSD–95, and CtBP2 revealed significant overlap with IP₃R immunoreactivity, suggesting that IP₃Rs are present at rod and cone bipolar synaptic terminals. Double labeling experiments with bipolar cell markers, PKCα, VGLUT1 and CtBP2, revealed that glutamatergic terminals of bipolar cells possess IP3Rs. Imaging [Ca²⁺]_i revealed that IP₃–mediated stores can be activated at terminals of rod and cone bipolar cells. Conclusions: These findings suggest that IP₃Rs are localized to synaptic terminals of bipolar cells, and that stimulation of IP₃R–mediated pathways can alter calcium signaling at ribbon synapses, which in turn can modulate transmitter release.

Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • bipolar cells • second messengers