Confocal Microscopy of Skate Electroreceptors: Fluorescent Antibodies Used to Localize Cav1.3 and BK Channels

Abstract

Skate electroreceptors are exquisitely sensitive to small voltage changes in the environment. This sensitivity is due to co-localization of voltage-sensitive calcium channels (Cav1.3) and large calcium-activated K+ channels (BK) in the apical membranes of the receptor cells. Regenerative responses depolarize the basal membranes, which form synapses with afferent nerve fibers and have presynaptic calcium channels. We characterized this localization using fluorescent antibodies against conserved Cav1.3(α) and BK(α) sequences. Single ampullae were dissected and fixed with paraformaldehyde and treated with detergent. Images were initially obtained with a stereomicroscope. Both antibodies caused bright staining of the receptor epithelium. For confocal microscopy, both primary antibodies were applied together. Stacks of 1μm merged and single color images were obtained at 10x and 40x. One alveolus filled the field at 40x. Staining of Cav1.3 produced green images, while staining of BK produced red images. Receptor cells were most clearly identified as a monolayer of reddish disks, presumably nuclei, which harbor BK (Li et al 2014). Some cell images do not show the nucleus but appear as a black void with a fluorescent rim. There is a rim of bright green fluorescence around the outer surface of the alveolus in some views. There is also localization of green fluorescence at the basal surface of some receptor cells (the presynaptic region). With optimal imaging, the membrane near the receptor cell apex is visible and appears yellow, which suggests staining for both BK and Cav1.3. Refinement of these methods should lead to additional insights concerning electroreception.