Detailed confocal fluorescent visualization of Ca 2+ and membrane potential changes in pulmonary neuroepithelial bodies (NEBs) in a murine lung slice model

Abstract

Conclusive physiological data on potential stimuli for pulmonary NEBs are very scarce because of the lack of a reliable in vitro model. We recently developed a method for selective staining of NEBs by 4-Di-2-ASP in vibratome slices of live mouse lungs. Aim of the study was optimizing the model for live cell imaging, allowing detailed confocal visualization of changes in intracellular free calcium ([Ca2+]i) and membrane potential, using the fluorescent indicators Fluo-4 and DiBac4 (3), respectively. In all NEBs, experimental short-term elevation of extracellular potassium ([K+]o) was seen to evoke a reproducible rise in cytoplasmic fluorescence for both Fluo-4, corresponding to an increase in [Ca2+]i, and DiBac4 (3), indicative for membrane depolarization. So-called Clara-like cells, known to almost completely shield NEBs from the airway lumen, appeared to exhibit a slightly delayed increase in Fluo-4 fluorescence after high [K+]o, while DiBac4 (3) did not reveal changes. The Ca2+ response in NEBs and Clara-like cells was abolished in Ca2+ free solution. This study provides the first evidence that physiological activity of pulmonary NEBs, and surrounding tissues, can be visualized in live lung slices using fluorescent indicators. The delayed increase in [Ca2+]i in Clara-like cells might be explained by stimulation of Clara-like cells via depolarization-induced neurotransmitter release from NEB cells. Support: FWO grant G.0085.04 (D.A.); UA grants NOI-BOF 2003 (D.A.) and KP-BOF 2006 (I.B.)