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Olfactory Epithelium (Neurogenesis/Cell Culture) D Proc. Nail. Acad. Sci. USA Vol. 82, pp. 7782-7786, November 1985 Neurobiology Ontogeny of electrically excitable cells in cultured olfactory epithelium (neurogenesis/cell culture) D. SCHUBERT,* W. STALLCUP, M. LACORBIERE, Y. KIDOKORO, AND L. ORGEL The Salk Institute, P.O. Box 85800, San Diego, CA 92138 Communicated by W. Maxwell Cowan, July 2S, 1985 ABSTRACT A primary system has been developed in Hepes-buffered Vogt-Dulbecco modified Eagle's medium. which it is possible to study the production of electrically The dissociated cell suspension was washed twice with excitable neuron-like cells from a precursor population of modified Eagle's medium containing 10% cadet calf serum olfactory epithelial cells. Rat nasal epithelium was dissociated (Biocell Laboratories), plated on 60-mm plastic tissue culture and placed in culture. The initial surviving cells are flat and dishes (Falcon) at about 106 cells per dish, and cultured at ciliated and contain glial fibrillary acidic protein (GFAP). After 370C. A variety of other dissociation procedures, sera, 3-5 days electrically excitable cells appear that contain neuron- substrata, and culture media was tested; none was effective specific enolase but not GFAP. These round cells originate by in producing the cell populations described below. Macro- means of the differentiation of the GFAP-positive flat cell to a phage cultures were prepared by the method of Wahl et al. round cell, followed by the division of the round cell. There- (5). B65 is a rat neuroblastoma, B15 is a rat glioma, and C1 fore, neuron-like cells can be derived from cells that synthesize is a mouse lymphoid cell line (6, 7). GFAP. Immunohistochemistry. Rabbit anti-glial fibrillary acidic protein (GFAP) was obtained from A. Bignami, mouse The olfactory epithelium ofmost animals contains three types monoclonal anti-rat T200 protein was obtained from R. of cells-receptor, sustentacular, and basal. The receptor Hyman, and monoclonal anti-S100 and anti-neuron-specific cells contain sensory receptors on their epithelial surface and enolase was from B. Boss. For staining with anti-T200, extend their axons directly to the brain. These neurons cultures were incubated with the primary antibody for 30 min occupy about 70% of the total epithelial volume. The at room temperature. The cultures were then washed and sustentacular cells, whose function is unknown, but which exposed for an additional 30 min to a rhodamine-labeled goat are thought to resemble glial or Schwann cells in the nervous antibody against mouse immunoglobulin (Tago, Burlingame, system, are interdigitated between the receptor cells. Under- CA). Finally, the cultures were washed, air dried, and fixed lying the receptor and supporting cells are the basal cells (see, with 95% ethanol for 10 min at -20°C. For labeling with for example, refs. 1 and 2). When the olfactory axons in mice anti-GFAP, cultures were first fixed with 95% ethanol for 10 are lesioned, the receptor cells degenerate and are replaced min at -20°C, dried, and then incubated for 30 min with the through the mitosis of the basal cells and the subsequent primary antiserum. After washing, the cultures were exposed differentiation of the daughter cells into receptor cells (3, 4). to a fluorescein-labeled goat antibody against rabbit immu- The question ofwhether or not the basal cells in normal adult noglobulin for an additional 30 min, followed by more olfactory epithelium are able to divide and give rise to washing and a final air drying. For double-labeling the cells sensory neurons was answered by autoradiographic tech- were first stained with anti-T200, fixed, and then stained with niques following pulse labeling of animals with [3H]thymi- anti-GFAP. Staining with anti-S100 and anti-nerve-specific dine. The results in mice showed that most of the isotopic enolase was done according to published procedures (8). labeling of nuclei occurred in the basal cell compartment (1). Time-Lapse Photography. Time-lapse photography was The labeled nuclei later appeared in receptor cells and, to a done on a Zeiss Universal microscope equipped with a Cohu-4400 video camera and a Panasonic time-lapse video lesser extent, in sustentacular cells. These results, and in similar data in frogs (2), strongly suggest that receptor and recorder. The cells were cultured on glass coverslips the division standard medium and maintained at 37°C and at the proper sustentacular cells are continuously renewed by pH with a Bachofer culture chamber. of basal cells. Since the olfactory epithelium is continually Thynudine Labeling. Cells were labeled in complete growth producing neurons from a less-differentiated cell type, it medium for 4 hr with 2 ,Ci (1 Ci = 37 GBq) of [3H]thymidine would be an excellent system in which to study neurogenesis per ml and fixed with ethanol, and the presence of isotopi- if it were possible to reproduce this event in culture. Ideally, cally labeled nuclei was determined by autoradiography. At one would like to have primary cultures in which nerve cells least 500 cells were examined under each growth condition. are produced from their precursors as well as a method for Carnosine Synthesis. To measure the rate of carnosine establishing permanent cell lines of the precursor and prog- synthesis, cells were labeled for 6 hr with [14C]histidine (2 eny populations. The experiments outlined here describe a ,uCi/ml) and [3H]leucine (10 ,Ci/ml) in Vogt-Dulbecco primary culture system in which the formation of electrically modified Eagle's medium containing 1% of the normal excitable cells can be examined. amount of histidine. The cells were then washed once with the culture medium and the amount of [3H]leucine incorpo- MATERIAL AND METHODS rated into protein was determined by trichloroacetic acid Cell Cultures. Olfactory epithelia were dissected from precipitation of an aliquot of cells. The remaining cells were 2-month-old Sprague-Dawley rats. The tissue was dissoci- extracted with ethanol and the amount of [14C]histidine ated with 20% pancreatin (GIBCO) for 45 min at 37°C in incorporated into carnosine was determined by high-voltage The publication costs of this article were defrayed in part by page charge Abbreviations: GFAP, glial fibrillary acidic protein; Bt2cAMP, payment. This article must therefore be hereby marked "advertisement" N6,02'-dibutyryladenosine 3',5'-cyclic monophosphate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7782 Downloaded by guest on September 26, 2021 Neurobiology: Schubert et al. Proc. Natl. Acad. Sci. USA 82 (1985) 7783 electrophoresis (9). The data are expressed as the ratio of cells' cytoskeleton was initiated within 10 min (Fig. 2A) and isotope incorporated into carnosine to that of total protein was complete within 2 hr (Fig. 2B). A similar cAMP-induced synthesis. cytoplasmic "shrinkage" has been reported in cultured glial Electrophysiology. Intracellular recording ofthe membrane cells (11, 12). potential was performed with conventional procedures (10). Also at 5 days a population of loosely attached phase-bright The microelectrodes were filled with 4 M potassium acetate round cells appeared in the cultures. These increased in number and had a resistance of about 80 MC. The bath saline had the for several days (Fig. 1C). By gentle trituration it was possible following salt composition (in mM): NaCl, 142.6; KCI, 5.6; to remove the majority ofthe round cells from the attached flat CaCl2, 10; Hepes/NaOH, 5 (pH 7.4). The experiments were cells, thus isolating a morphologically homogeneous population carried out at room temperature (20-220C). Successful re- of round cells (Fig. ID). Several weeks after they were pro- cordings were made from 17 round cells; all gave similar duced, some of the round cells attached to the substratum and results. grew short neurite-like processes (Fig. iD Inset). Electrophysiology and Neurotrans itter Synthesis. Two RESULTS properties of olfactory neurons are electrical excitability and Culture System. Primary cultures of olfactory epithelia the ability to synthesize the f-alanyl-L-histidine dipeptide were made fromjuvenile Sprague-Dawley rats. The epithelia carnosine, which is thought to be the primary neurotrans- were dissociated and plated into tissue culture dishes con- mitter in this cell type (13). When the round cells were taining modified Eagle's medium and 10% newborn calf examined by electrophysiological techniques, they were serum. After 3 days in culture the majority of the plated cells found to be able to generate action potentials following the were dead, but small patches of cells were attached to the passage of a depolarizing current pulse (Fig. 3). It has been culture dish substratum (Fig. 1A). The majority ofthese cells impossible to obtain stable, reproducible recordings from the had actively beating cilia on their upper surface. After 5 days flat cell population. To assay the rate of carnosine synthesis some of the cells within the patches divided and migrated out relative to total protein synthesis in the small amounts of from the initial clumps, frequently forming a semiconfluent available cells, cultures were labeled with both [14C]histidine monolayer of very large (100-150 Am in diameter) flat cells and [3H]leucine, and the amount of each incorporated into (Fig. 1B). If N6,02'-dibutyryladenosine 3',5'-cyclic mono- carnosine and total protein, respectively, was determined. phosphate (Bt2cAMP) was added to this cell population a Table 1 shows that the round cell population synthesizes retraction ofthe plasma membrane and cytoplasm around the significantly more carnosine than the flat cells. The rat neuroblastoma, glioma, and mouse lymphoid cells synthe- sized relatively little of the dipeptide. Immunological Properties of Cells. The ability of the round cell population to produce action potentials suggests that they are neuronal. However, since lymphoid cells such as mac- rophages are also able to generate action potentials (14), cells were stained by fluorescent antibody techniques for a variety of nervous system-specific antigens.
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