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

Abstract

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 (beta-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

“…In conclusion, the results of this study extend our previous findings [Josephson et al, 2004] to show that OE-OB cocultures older than 21 DIV, even as old as 121 DIV, can produce stable EOG responses to air-borne odorants. The results also suggest a functional difference between younger ( !…”

“…We achieved both objectives by collecting and analyzing EOG responses from 29 cocultures ranging in age from 1 to 121 DIV. The EOG response kinetics recorded from mouse OE grown in OE-OB coculture were similar in several respects to those recorded from in vivo OE, acutely dis- sected OE, and dissociated ORN [Gesteland, 1964;Ezeh et al, 1995;Ma et al, 1999;Reisert and Matthews, 2001;Josephson et al, 2004;Reisert et al, 2007;Waggener and Coppola, 2007;Pinato et al, 2008]. The typical EOG response of both cultured and dissected OE have rise and decay phases of about a few hundred milliseconds with the rise 2-3 times faster than the decay.…”

“…Our previous work [Josephson et al, 2004] described some of the anatomical and physiological characteristics of a long-lived OE-OB coculture, including responsiveness to air-borne odorants, as measured by EOG. Our objective in conducting this study was to better characterize the EOG responses at different in vitro ages of mouse OE grown in coculture with olfactory bulb.…”

“…These include resilience to environmental exposure, regenerative capacity, and odorant responsiveness. Our previous work [Josephson et al, 2004] established some of the characteristics of a long-lived OE-olfactory bulb (OB) coculture system, including longevity to 68 days in vitro (DIV), odorant responsiveness to 21 DIV, and ORN maturity as demonstrated by OMP expression.…”

Cell surface expression of 27C7 by neonatal rat olfactory ensheathing cells in situ and in vitro is independent of axonal contact

“…BrieXy, the isolated olfactory bulb from 1-day-old rats was placed on a plate of low-adhesive plastic (Vulcolan ® ) and sections were cut (350 m) using a tissue chopper (Mc Illwain, Mickle Lab, Engineering Surrey, UK). Slices were transferred to organotypic inserts (Millicell Cell Culture Inserts, Millipore, Schwalbach, Germany) placed in 6-well plates (Nunclon, Nunc) and maintained in DME medium (Invitrogen Inc.) containing 25% Hank's balanced salt solution (HBSS, PAA, Marburg, Germany) and 25% normal horse serum (NHS, PAA, Josephson et al 2004). After 2 and 8 days, slices were cut out of the membrane using a scalpel and embedded in cryomatrix (Wewetzer et al 2005).…”