Solomon Yilma

RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein α-subunits αi and αq, lack such activity for αs (refs 3,4,5,6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the αs family member αolf links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on αolf but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.

Structure and function of long-lived olfactory organotypic cultures from postnatal mice

The first synapse in the olfactory pathway mediates a significant transfer of information given the restricted association of specific olfactory receptor neurons with specific glomeruli in the olfactory bulb. To understand better how this connection is made and what the functional capacities of the participating cells are, we created a long‐lived culture system composed of olfactory epithelium and olfactory bulb tissues. Using the roller tube method of culturing, we grew epithelium‐bulb cocultures, explanted from 1–4‐day‐old Swiss Webster mice, on Aclar for periods ranging from 18 hr to 68 days. The explants flattened so that in some areas the culture was only a few cells thick, making individual cells distinguishable. From 107 cultures studied, we identified the following cell types by expression of specific markers (oldest culture expressing marker, days in vitro, DIV): olfactory receptor neurons (neural cell adhesion molecule, 42 DIV); mature receptor neurons (olfactory marker protein, 28 DIV); postmitotic olfactory receptor neurons and olfactory bulb neurons (β‐tubulin, 68 DIV); astrocytes (glial fibrillary acidic protein, glutamate/aspartate transporter, 68 DIV); olfactory horizontal basal cells (cytokeratin, 22 DIV). Neuronal processes formed glomeruli in 2–4‐week‐old cultures. We also recorded electro‐olfactography responses to puffs of vapor collected over an odorant mixture containing ethyl butyrate, eugenol, (+) carvone, and (−) carvone from cultures as old as 21 DIV. These features of our olfactory culture system make this model useful for studying properties of immature and mature olfactory receptor neurons, pathfinding strategies of receptor axons, and mechanisms of information transfer in the olfactory glomerulus.

Odorant Response Kinetics from Cultured Mouse Olfactory Epithelium at Different Ages in vitro

Mammalian olfactory epithelium can withstand the external environment, undergo life-long regeneration, and respond to thousands of odorant stimuli, making it an attractive system for a variety of studies. Previously, we described a long-lived olfactory coculture of olfactory epithelium and bulb tissues and we present here the kinetic properties of that culture system. Neonatal mouse epithelial-bulbar explants were grown for periods as long as 121 days in vitro (DIV), nearly doubling the survival time of our previously longest lived cultures. Cultures at all ages responded to air-borne odorants. The youngest cultures (1–15 DIV) showed shorter half-rise and half-decay times than older cultures (21–121 DIV), and were more variable in their half-decay times. Zinc nanoparticles enhanced electro-olfactogram responses of both younger and older cultures and both groups were immunopositive for olfactory marker protein. The results show that our olfactory culture model can support mature, odorant-responsive olfactory receptor neurons that possess many of the response features of in situ olfactory receptor neurons.

Contents Vol. 192, 2010

Stem Cells and Tissue Engineering S.F. Badylak, Pittsburgh, Pa. E-Mail: badylaks@msx.upmc.edu A. Müller, Würzburg E-Mail: albrecht.müller@mail.uni-wuerzburg.de L.E. Niklason, New Haven, Conn. E-Mail: laura.niklason@yale.edu A. Ratcliffe, San Diego, Calif. E-Mail: anthonyratcliffe@synthasome.com A.M. Wobus, Gatersleben E-Mail: wobusam@ipk-gatersleben.de Tumor Cell Plasticity E. Thompson, Melbourne E-Mail: rik@medstv.unimelb.edu.au