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Department of Molecular Physiology

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olfaction
Fluorescence - Lifetime imaging

Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of cells loaded with the Cl-sensitive dye MQAE makes it possible to measure the intracellular Cl concentration in living olfactory sensory neurons. With a spatial resolution of roughly 1 µm3, this technique reveals the local Cl concentrations within the somata, the dendrites and the dendritic knobs of the sensory neurons. Cilia, however, remain invisible because they do not accommodate a sufficient amount of dye to produce a detectable fluorescence signal. In collaboration with the Institute for Biological Signal Processing at the Jülich Research Center we have examined the mechanism of Cl accumulation in olfactory sensory neurons and found that the Na-K-Cl-cotransporter NKCC1 contributes to Cl uptake. Moreover, the neurons suppress the expression of the Cl-exporter KCC2 and thus allow intracellular Cl to accumulate to a steady-state level of 50 mM.



The figure shown here is a single shot from a 3D-reconstruction of a section from the uppermost part of the olfactory epithelium. The figuer was constructed from a stack of 15 individual 2P-FLIM images, taken at intervals of 2 µm. The colours indicate intracellular Cl concentrations, with warmer colours indicating higher Cl levels inside the cells. Dendrites of olfactory sensory neurons (indicated by the dashed lines) reach the apical surface between supporting cells. The dendrites are able to support an axial Cl gradient, with highest Cl levels at the apical knobs and lower levels in the more proximal parts of the dendrites. This suggests that the site of Cl uptake is the apical knobs and/or the cilia. It thus appears that Cl uptake mechanisms are located in or near the cilia, where Cl is needed as charge carrier of the receptor current.

For a better look at the 3D reconstruction, please download the low-resolution animation (1.4 MByte) or  the high-resolution version (4.7 MByte).

low-resolution (new window)

high-resolution (new window)






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