Chem. Senses 24: 211-216,
1999
© Oxford University Press
Short Communication |
A Method for Maintaining Odor-responsive Adult Rat Olfactory Receptor Neurons in Short-term Culture
Correspondence to be addressed to: Gricelly Vargas, Department of Physiology, University of Utah, 410 Chipeta Way, Rm 155, Salt Lake City, UT 84108, USA. e-mail: gricelly.vargas{at}m.cc.utah.edu
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
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We report a culture system requiring the addition of freshly made ascorbic acid to the medium, that supports the short-term survival of adult rat olfactory receptor neurons. The cultured neurons exhibit typical voltage-gated currents and are responsive to application of odorants.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Olfactory receptor neurons (ORNs), the sensory components of the olfactory neuroepithelium, are unique in their ability to undergo cellular turnover and replacement throughout life. Due to this unique characteristic, several culture systems have been developed to maintain and study these cells in vitro. Differentiation and survival of dissociated rat ORNs have been documented for olfactory cell cultures derived from embryonic (Chuah and Farbman, 1983
;
Calof and Chikaraishi, 1989;
Chuah et al., 1991)
or newborn rats
(Noble et al., 1984;
Pixley and Pun, 1990;
Ronnett et al., 1991;
Trombley and Westbrook, 1991;
Pixley, 1992).
However, the differentiation and survival of ORNs in those culture systems required plating onto
a feeder layer of CNS astrocytes (coculture), addition of growth factors or contact with the
olfactory bulb. Survival of dissociated cells from adult mammalian olfactory mucosa has also
proven to be difficult. Primary cultures of ORNs from adult mice required induction of chemical
injury to the olfactory epithelium by ZnSO4 to activate progenitor mitogenic activity in
situ prior to culture
(Sosnowski et al., 1995;
Liu et al., 1998),
while primary cultures from adult rats required coculture with a supportive bed layer of cortical
glia
(Grill and Pixley, 1997).
Also, addition of brain-derived neurotrophic factor to the culture medium was necessary to
increase the number of bipolar cells (ORNs) in culture
(Liu et al., 1998).
We have developed and report here a culture system that supports the short-term survival of
dissociated adult rat ORNs without requiring coculture or addition of growth factors to the
culture medium. The neurons in our culture system are immunoreactive for the general neuronal
marker neuron-specific tubulin (NST) and olfactory marker protein (OMP), show typical
voltage-gated currents and respond to odorants. While our system is not designed to study the
process of neurogenesis of ORNs, it provides an efficient and convenient technique to study the
physiology of adult rat ORNs, which has been done primarily on acutely dissociated ORNs. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Cell Preparation and Culture
Rat ORNs were dissociated with a modification of previously described procedures
(Lynch and Barry, 1991a;
Ronnett et al., 1991;
Vargas and Lucero, 1999).
Briefly, adult male Simonsen albino rats (~200 g) were deeply anesthetized with 150 mg/kg
ketamine + 15 mg/kg
rompum (Mallinckrodt Veterinary, Inc., Mundelein, IL) and sacrificed by decapitation. The
olfactory epithelium from the nasal septum and turbinates of one rat was dissected under 100%
oxygen vapor saturated with rat Ringer's [6 ml of rat Ringer's nebulized with
100% oxygen in a Respigard 2 Nebulizer System (Marquest Medical, Englewood, CO)]. The
tissue was placed in 5 ml of divalent-cation-free rat ringers (in mM: 145 NaCl, 5.6 KCl, 10
HEPES, 10 glucose, 4 EGTA, pH 7.4, 300 mOsm) containing 10 mg/ml bovine serum albumin, 1
mg/ml collagenase (Gibco BRL, Grand Island, NY), 50 µg/ml deoxyribonuclease II and
44 U/ml dispase (Gibco BRL), and incubated with gentle shaking (80 r.p.m.) at 37°C for 45
min. Following incubation, the tissue was transferred to 5 ml of fresh divalent-cation-free rat
Ringer's and incubated with gentle shaking at 37°C for 5 min. The tissue was then
transferred to 2 ml of fresh divalent-cation-free rat Ringer's and triturated using a
fire-polished Pasteur pipette. The resulting cell suspension was filtered using a 53 micron
monofilament cloth (Small Parts Inc., Miami Lakes, FL). Cells (200 µl) were plated onto
Concanavalin A (10 mg/ml; Sigma type IV)-coated glass coverslips placed in 35 mm Petri
dishes. Following a 20 min settling time, 2 ml of culture medium was added to each dish. The
dishes were placed at 37°C in a CO2 incubator until used (up to 4 days). The
culture medium was replaced daily and consisted of Dulbecco's modified Eagle's
medium (Gibco BRL) supplemented with 100 µM ascorbic acid, 1x
insulin–transferrin–selenium-X (Gibco BRL), 2 mM glutamine, 100 U/ml
penicillin G and 100 mg/ml streptomycin (Irvine Scientific, Santa Ana, CA). All chemicals were
obtained from Sigma Chemical Company (St Louis, MO) unless stated otherwise.
Immunocytochemistry
Primary cultures of rat olfactory receptor neurons were characterized by immunostaining for
NST and OMP according to a previously described procedure
(Pixley, 1992).
Briefly, rat ORN
cultures were prepared as described above and fixed at 0, 1, 2, 3 and 4 days in culture with 4%
paraformaldehyde in 0.1 M phosphate buffer (PB) for 15 min at room temperature. Fixed cells
were incubated at room temperature in blocking buffer [0.1 M phosphate buffered saline (PBS:
0.1 M phosphate, 0.15 M NaCl) with 10% horse serum, 0.2% Triton X-100 and 0.02% sodium
azide] for 1 h and overnight with the primary antibodies [monoclonal anti-ß tubulin isotype
III (mouse ascites fluid) 1:1500 and goat anti-OMP 1:5000, a gift from Dr F. Margolis]. The
cultures were then washed with PBS and incubated for 2 h with the secondary antibodies
(biotinylated horse anti-mouse IgG 1:500 and rabbit anti-goat 1:500; Vector Laboratories, Inc.,
Burlingame, CA). The staining was performed using the Vectastain Elite ABC kit (Vector
Laboratories, Inc.) and was visualized using 3,3'-diaminobenzidine (0.5 mg/ml in 0.1 M
PB; 20 s). After immunostaining, coverslips were mounted on microscope slides using Gelvatol
(Harlow and Lane, 1988).
Electrophysiological recordings
Standard whole-cell voltage-clamp recording techniques
(Hamill et al., 1981)
were
performed. Electrodes (10–12 M
resistance) were pulled on a Flaming/Brown
P87 puller from thick walled (0.64 mm) borosilicate filament glass (Sutter Instrument Co., San
Rafael, CA). Coverslips with adherent cells were placed into the recording chamber and perfused
with external bath solution at a rate of 1–2 ml/min. The external bath solution was
maintained at 35°C and was grounded with a 3 M KCl agar bridge to a AgCl wire.
Voltage-clamp recordings were performed and digitized with a Digidata 1200 interface, a 200A patch clamp amplifier, PClamp software (Axon Instruments, Foster City, CA) and a 486-33 IBM clone computer. Na+ and K+ currents were evoked by depolarizing voltage pulses from a holding potential of -80 mV. The hyperpolarization-activated current, Ih, was evoked by hyperpolarizing voltage pulses from a holding potential of -50 mV. Data were sampled at 1–5 kHz and filtered off-line at 500–5000 Hz.
Intracellular calcium measurements
Ca+2 imaging experiments were carried out on rat ORNs dissociated and plated
as described above. Cells were loaded with 5 µM Fura-2/AM
(Grynkiewicz et al., 1985)
plus pluronic acid F127 (80 µg/ml) in filtered rat Ringer's at 16°C in
the dark for 20–30 min (followed by four washes in rat Ringer's). Intracellular Ca2+ ([Ca2+]i) was approximated from the background
corrected ratio of fluorescence at 340 nm/380 nm using a two-point calibration scheme and the
equation:
 |
where R is the fluorescence ratio (F340/F380), Rmin and Rmax are the fluorescence ratios of 5 µM Fura standards at the limiting low (0 µM Ca2+) and high (10 mM Ca2+) concentrations, Kd is the calcium dissociation constant, and (Fo/Fs) is the ratio of fluorescence intensities when excited at 380 nm at the limiting low (Fo) and high (Fs) Ca+2 concentrations. A Zeiss-Attofluor imaging system and software (Atto Instruments Inc., Rockville MD) was used to make the calibration curves, acquire and analyze the data. Data points were sampled at 1 Hz. Basal intracellular Ca+2 levels estimated using the two-point calibration method described above averaged 65 ± 4 nM (mean ± SEM, n = 50 cells). Odorants and IBMX were applied to the cells using a loop injector (100 µl).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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We characterized our culture system by immunostaining for a general neuronal marker antibody (NST) and for an olfactory specific marker (OMP). NST staining identifies immature as well as mature neurons while OMP staining identifies only mature ORNs (Pixley, 1992
).
The
immunostaining was performed on cultures from day 0 (day of cell dissociation) to day 4 in
culture. Figure 1 shows examples of typical immunostaining at days 0 (Figure 1A1–3) and
2 (Figure 1B1–3) in culture. At day 0, the cultures typically
exhibited a large number of
small round cells (suggesting that some ORNs had lost their processes during the dissociation
procedure) and cells with a more characteristic ORN morphology (soma extending a dendrite that
terminates in a dendritic knob). Staining for both NST (Figure 1A2) and
OMP (Figure 1A3) was
abundant, and observed on cells with characteristic ORN morphology as well as small round
cells (Figure 1A2 and 1A3). By day 1 in culture, only small changes in
the cultures were
observed: some cells had started to extend short processes. NST and OMP staining was observed
in small round cells, cells with ORN morphology and cells starting to extend processes (data not
shown). At day 2 in culture, a large number of cells had extended processes. Some cells extended
only one process (unipolar) while others extended two processes (bipolar). The processes were
typically long, usually extending to nearby neurons. NST (Figure 1B–2)
and OMP (Figure
1B–3) staining was still abundant: round cells, cells with ORN morphology,
unipolar and
bipolar cells were immunostained. These positively stained cells were present at day 3 and 4 in
culture; however, the total cell density was reduced (data not shown). Larger round cells and cells
with atypical morphology (flat large cells) were not immunostained (indicated by arrows).
Controls in which the primary antibodies were not included showed no staining for NST nor
OMP on either day 0 (Figure 1A1) or day 2 in culture (Figure
1B1).
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To evaluate the yield of neurons from our dissociation procedure and the survival of those neurons under our culture conditions, we quantified the total number of NST and OMP-immunoreactive neurons present from day 0 to day 4 in culture. The number of immunoreactive neurons was obtained for six random areas (2.4 mm2) at x10 magnification for two different coverslips from each day in culture (day 0 to day 4). At day 0, 814 ± 89 neurons/coverslip (mean ± SEM, n = 12 areas) and 1036 ± 80 neurons/coverslip (mean ± SEM, n = 12 areas) were immunoreactive for NST and OMP respectively (Figure 2). No significant difference was observed between the number of NST and OMP-immunoreactive cells (Student's t-test, P> 0.05). Significant decreases in the number of both NST and OMP-immunostained neurons were observed up to day 3 and day 2 in culture respectively. After these time points, the number of cells remained stable and no further decreases were observed (Figure 2). No significant differences were observed between the total number of immunostained neurons for NST and OMP at each day in culture (Student's t-test, P > 0.05). This suggests that the majority of the cells present in our cultures are mature or well differentiated ORNs, since the OMP protein is a specific marker of mature ORNs and does not appear in the basal or stem cells. Therefore, our culture conditions are able to support the short-term survival of well differentiated or mature ORNs. In the search for these optimal culture conditions, we found that daily addition of freshly made ascorbic acid to the culture medium was critical not only for the survival of cells but also for process extension. At 24 h after the dissociation procedure, a significant decrease (~50%) in the number of cells that normally survived in culture was observed when ascorbic acid was omitted from the culture medium. The cells that initially survived in the absence of ascorbic acid failed to extend processes and died within 48–72 h in culture.
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Voltage-gated currents, including inward Na+ currents, outward K+ currents and the hyperpolarization-activated current, Ih, are present in acutely dissociated rat ORNs (Lynch and Barry, 1991a
,
b;
Rajendra et al., 1992b).
We
found, using whole-cell voltage-clamp recordings, that these conductances were present in every
cultured ORN tested (>200 cells) regardless of the day in culture. Figure 3A shows an example
of a typical family of currents activated by depolarizing voltage steps up to 40 mV from a
holding potential of -80 mV. Fast transient inward Na+ currents followed by
slowly inactivating K+ currents were activated. The outward K+
currents were sensitive to internal appliction of 5 mM tetraethylammonium (TEA, insert in
Figure 3A). Ih was also present in the cultured rat
ORNs (Figure 3B). It was
activated by hyperpolarizing voltage steps to -150 mV from a holding potential of
-50 mV and was reversibly blocked by external application of 5 mM CsCl (data not
shown). The patterns of these voltage-gated currents are similar to the ones described previously
in the acutely dissociated adult rat ORNs
(Lynch and Barry, 1991a,
b;
Rajendra et al., 1992b);
therefore, our system is suitable for the study of the electrophysiological properties of
ORNs. We did not observe any changes in the properties of the voltage-gated currents studied
over the 1–4 days in culture; however, we found that our success rate in obtaining stable
recordings was greater with cultures on days 2 and 3.
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Since ORNs are the sensory transducers of odorant stimuli, we used Ca2+-imaging techniques to study whether cultured rat ORNs respond to odors. Cultured ORNs were loaded with the Ca2+ indicator dye, Fura-2, and an odor cocktail (50 µM r-carvone, 50 µM s-carvone and 50 µM amyl acetate in rat Ringer's) was applied. Figure 3C shows the results of a representative experiment. Application of the odor cocktail produced an increase in [Ca2+]i, as expected from the activation of odorant receptors. The percent of cultured ORNs responsive to the application of odors was consistent throughout all the days in culture. On days 0, 1, 2, 3 and 4 in culture, 10/38 (26%), 48/180 (27%), 22/82 (27%), 14/45 (31%) and 8/34 (24%) cells tested responded to odors respectively. We also tested the effect of 100 µM isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Application of 100 µM IMBX to the cultured ORNs allowed basal levels of cAMP to rise, resulting in the activation of cyclic nucleotide gated (CNG) channels and subsequent increase in [Ca2+]i, as shown in Figure 3D. When an odor cocktail was applied to the same cell, an odorantelicited Ca2+ influx was also observed (Figure 3D). These experiments indicate that all of the elements necessary for the odorant transduction cascade are conserved in the cultured ORNs, making our culture system suitable for the study of such events.
We have reported a culture system that supports the short term survival of adult rat ORNs
without the addition of growth factors or coculture. While our system does not support
neurogenesis like previously reported culture systems
(Grill and Pixley, 1997;
Liu et al., 1998),
it provides a simple and convenient alternative to the use of acutely dissociated ORNs for
the study of every aspect of olfactory signal transduction, including odor sensitivity,
desensitization, adaptation, neurotransmitter modulation and membrane excitability. Compared
with the acute isolation of cells, short-term cultures reduce the number of animals sacrificed and
time spent on everyday isolation. Both, the number of cultured ORNs present after the cultures
have stabilized and the overall health of the cultures support the realization of physiological
experiments.
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Acknowledgments |
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We thank Dr S. Pixley for her help during the immunocytochemical studies, Dr F. Margolis for providing anti-OMP, Dr M. Michel for his help during the analysis of the immunocytochemical studies and D. Piper for technical assistance during the Ca2+ imaging experiments. This work was supported by a Ford Foundation Predoctoral Fellowship to G. Vargas and an NIH NIDCD R01 # DC02994-02 to M.T. Lucero.
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Accepted December 4, 1998
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