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First published online July 14, 2008
Journal of Experimental Biology 211, 2417-2422 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018796

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The elusive crypt olfactory receptor neuron: evidence for its stimulation by amino acids and cAMP pathway agonists

Alex Vielma, Alvaro Ardiles, Luz Delgado and Oliver Schmachtenberg^*

Centro de Neurociencia de Valparaíso, Universidad de Valparaíso, Avda. Gran Bretaña 1111, Casilla 5029, Correo 4 2360102 Valparaíso, Chile

View larger version (49K): [in this window] [in a new window]   Fig. 1. Cell-attached recordings of isolated crypt olfactory receptor neurons (ORNs) and their respective spike-frequency plots (f, s–1). (A) Amino acids, 8Br-cAMP and IBMX plus forskolin caused a transient increase of the spike frequency in crypt ORNs. Individual cells responded to all three stimuli. In experiments with amino acids or 8Br-cAMP, the excitatory effect reverted to baseline levels within seconds following termination of the stimulus, whereas the combination of IBMX and forskolin induced stronger and longer lasting effects. (B) Temporal response characteristics of silent and tonically active crypt ORNs stimulated with IBMX alone. Higher stimulus concentrations, expressed as the pressure applied to the stimulus pipette by the picospritzer, frequently caused spike suppression during the stimulus followed by prolonged excitation during the post-pulse period. Burst-like activity was observed in a significant proportion of cells (cell 3).

View larger version (49K): [in this window] [in a new window]   Fig. 2. Calcium imaging of isolated crypt olfactory receptor neurons (ORNs). In each panel the upper row of images shows total fluorescence; the lower row shows a bright-field image, maximum relative fluorescence increase (F/F) and an overlay of both. The corresponding plots display the time course of the normalized fluorescence average of the whole crypt ORN and its supporting cell. Amino acids (A), IBMX plus forskolin (B) and 8Br-cAMP (C) induced a transient rise of intracellular calcium in the crypt ORN. Some supporting cells, which are coupled to the crypt ORN by gap junctions (Schmachtenberg, 2006), were included in the calcium signal (B,C) whereas others were apparently excluded (D). The effects were similar in amplitude and duration to those of stimulation with KCl (50 mmol l–1 in the stimulus pipette). Bile salts (E) and the combination of PGF2 and 17,20βP (F) did not cause significant and reproducible effects. bf, bright-field image; co, crypt ORN; sc, supporting cell.

View larger version (39K): [in this window] [in a new window]   Fig. 3. Immunocytochemistry and western blot of adenylate cyclase III (AC III). (A,B) Bright-field images and immunocytochemical labeling of AC III (red) in isolated crypt olfactory receptor neurons (ORNs) counterstained with DAPI (blue). (C) Near-absence of labeling after preadsorption of the antiserum with excess antigen. Scale bar: 10 mm. (D) Immunoblot of olfactory epithelium from fish and rat. The AC III antiserum marked a band of about 130 kDa in both species. 95 µg of fish protein were loaded as opposed to 15 µg of rat protein.

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