Breunig E , Kludt E , Czesnik D , Schild D .

	FIGURE 1. FM1-43-stained ORNs in the OE and their responses to odorants. A, OE of a tadpole after a 7-min exposure to FM1-43. B, z-projection (three-dimensional projection) of a number of zoomed cells shown in A illustrating the morphology of FM1-43-labeled ORNs. C, control OE. D, the spectrally different fluorescence intensities of ORNs labeled with FM1-43 (red) and backfilled with Alexa Fluor 488 (green) were unmixed (upper panel, combined fluorescence; center panel, backtraced ORNs in green; lower panel, FM1-43-stained ORNs in red). E–I, [Ca2+]i transients of an FM1-43-labeled ORN evoked by the odorant mixture but recorded at different times (E and I), amino acids (F), and alcohols (G and H) shown in chronological order. Scale bars = 50 μm (A and C), 10 μm (B), 150 μm (D). E–I, black lines under the traces indicate odorant applications. Time scale (s) and change of fluorescence intensity ΔF/F (%) are indicated by the bars in the lower right corner. The boundaries of the OEs are shown by white dotted lines.
	FIGURE 2. Evidence for FM1-43 passing CNG channels. Incubation of the tadpoles in FM1-43 solution with 2 mM CaCl2 (A) or 1 mM MgCl2 (B) almost completely blocked FM1-43 uptake. The unspecific CNG channel blockers LY-83583 (D) and amiloride (E) also blocked FM1-43 uptake. C and D, two control OEs, each with a large number of ORNs labeled by FM1-43 (2 μM). G, fluorescence emission of FM1-43 alone (red traces) and mixed with 200 μM LY-83583 or 1 mM amiloride. The emission maximum of FM1-43 decreases to 60% in the presence of LY-83583 and slightly increases with amiloride. H, forskolin-evoked [Ca2+]i transients. I, [Ca2+]i transients induced by uncaging of cAMP in individual FM1-43-stained ORNs. Similar results were obtained in 10 of 13 cells (five slices, the non-responding cells came all from the same slice) for stimulation with forskolin and five of five cells (three slices) for stimulation with uncaging of caged cAMP. Scale bars = 200 μm (A–F). OE boundaries are illustrated by white dotted lines. H and I, the black line under the trace indicates the application of forskolin, and the black dot the time point of uncaging. Time scale (s) and change of fluorescence intensity ΔF/F (%) are indicated by the bars in the upper right corners.
	FIGURE 3. CNG currents are inhibited by FM1-43. A–C, current traces of patch-clamped cells in bath solution. The upper traces show action potential-associated capacitative currents in the voltage clamp, each current pulse indicating one action potential. Forskolin increased the spiking rate in cAMP-dependent (A and B, upper black traces) but not in cAMP-independent ORNs (C, upper black trace). A, lower trace, inward current after breakthrough into the whole-cell mode and after changing to the Ca2+- and Mg2+-free bath solution (0 Ca/0 Mg). B, lower trace, markedly reduced inward current with FM1-43 being present in the 0 Ca/0 Mg bath solution. C, lower trace, no inward current was observed in ORNs that were not responsive to forskolin. The same current and time scales apply to A, B, and C. D, bar graph of inward current amplitudes with (left panel) and without (right panel) FM1-43 in the bath solution. ***, p < 0.001.
	FIGURE 4. Extracellular FM1-43 blocks odorant transduction. A, overview over the OE and the principal cavity (PC). The black rectangle indicates the position of the area of the OE measured in B using two-photon excitation microscopy. Forskolin-induced fluorescence changes of fluo-4 are illustrated at the response peak. C, forskolin-evoked [Ca2+] transients of the ORN encircled ORN in B (black trace) were reduced upon FM1-43 in the bath (10 μM, red trace). Also shown is recovery after washout of the dye (gray trace). D, average amplitudes and their S.E. for 10 μM FM1-43 and the washout (n = 61, four animals) are quantified in the bar graph. E, overview over the olfactory bulb (OB), the black rectangle indicating the position of the area of the olfactory bulb shown in F, i.e. the medial cluster of the olfactory bulb explant preparation shown. Forskolin-induced fluorescence changes of fluo-4 are illustrated at the response peak. G, forskolin-evoked [Ca2+] transients of glomeruli in this cluster (black trace, corresponding to the glomerulus marked in F) were reduced upon FM1-43 in the bath (10 μM, red trace). Recovery of the response amplitude after washout of the dye (gray trace). The black line indicates the application of forskolin. H, reduction of response amplitudes (and S.E.) for FM1-43 bath concentrations of either 10 μM (n = 11, three animals) or 20 μM (n = 17, three animals). **, p < 0.01; ***, p < 0.001. Scale bars = 250 μm (A), 20 μm (B), 250 μm (E), 30 μm (F).

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