Tracking Quantum Dot–Tagged Calcium Channels at Vertebrate Photoreceptor Synapses: Retinal Slices and Dissociated Cells

Abstract

At synapses in the central nervous system, precisely localized assemblies of presynaptic proteins, neurotransmitter-filled vesicles, and postsynaptic receptors are required to communicate messages between neurons. Our understanding of synaptic function has been significantly advanced using electrophysiological methods, but the dynamic spatial behavior and real-time organization of synapses remains poorly understood. In this unit, we describe a method for labeling individual presynaptic calcium channels with photo-stable quantum dots for single-particle tracking analysis. We have used this technique to examine the mobility of L-type calcium channels in the presynaptic membrane of rod and cone photoreceptors in the retina. These channels control release of glutamate-filled synaptic vesicles at the ribbon synapses in photoreceptor terminals. This technique offers the advantage of providing a real-time biophysical readout of ion channel mobility and can be manipulated by pharmacological or electrophysiological methods. For example, the combination of electrophysiological and single-particle tracking experiments has revealed that fusion of nearby vesicles influences calcium channel mobility and changes in channel mobility can influence release. These approaches can also be readily adapted to examine membrane proteins in other systems.

Expression and cellular localization of the voltage‐gated calcium channel α2δ3 in the rodent retina

“…The presence of a 2 d 1 (Huang et al, 2013) and a 2 d 4 (Ruether et al, 2000;Wycisk et al, 2006a,b;Mercer et al, 2011;P erez de Sevilla M€ uller et al, 2013;Mercer and Thoreson, 2013) Ca channel subunits in the ret-ina has been reported. This study extends these earlier findings and reports the expression of the a 2 d 3 subunit by multiple retinal cell types in the mouse and rat retina.…”

Endocytosis sustains release at photoreceptor ribbon synapses by restoring fusion competence

“…Methods for QD attachment to individual Ca 2+ channels are described in detail elsewhere ( Mercer and Thoreson, 2013 ). Isolated cells or retinal slices were incubated with a primary anti-α 2 δ 4 subunit antibody (1:1,000; Qin et al, 2002 ) in amphibian saline solution for 3 h at 4°C.…”