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The Journal of Neuroscience, October 1, 1999, 19(19):8260-8270
An Olfactory Sensory Neuron Line, Odora, Properly
Targets Olfactory Proteins and Responds to Odorants
Julie R.
Murrell1 and
Dale D.
Hunter1, 2
1 Program in Cell, Molecular, and Developmental Biology
and Departments of Neuroscience, Anatomy and Cell Biology, and
2 Ophthalmology, Tufts University School of Medicine,
Boston, Massachusetts 02111
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ABSTRACT |
The site for interactions between the nervous system and much of
the chemical world is in the olfactory sensory neuron (OSN). Odorant
receptor proteins (ORPs) are postulated to mediate these interactions.
However, the function of most ORPs has not been demonstrated in
vivo or in vitro. For this and other reasons, we
created a conditionally immortalized cell line derived from the OSN
lineage, which we term odora. Odora cells, under control conditions, are phenotypically similar to the OSN progenitor, the
globose basal cell. After differentiation, odora
cells more closely resemble OSNs. Differentiated odora
cells express neuronal and olfactory markers, including components of
the olfactory signal transduction pathway. Unlike other cell lines,
they also efficiently target exogenous ORPs to their surface.
Strikingly, differentiated odora cells expressing ORPs
respond to odorants, as measured by an influx of calcium. In
particular, cells expressing one ORP demonstrate a specific response to
only one type of tested odorant. Odora cells, therefore,
are ideal models to examine the genesis and function of olfactory
sensory neurons.
Key words:
olfactory epithelium; globose basal cell; olfactory
sensory neuron; olfactory receptor protein; odorant signal
transduction; conditional immortalization
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INTRODUCTION |
The mature sensory neurons of the
mammalian olfactory epithelium (OE) are the primary transducers of
odorant signals from the external world to the CNS. Olfactory
sensory neurons (OSNs) receive and transmit their stimuli directly:
their dendritic termini lie in the nasal cavity, directly apposed to
the environment; their axonal termini, unlike those of other sensory
neurons, synapse directly onto second-order neurons within the
forebrain (Graziadei and Metcalf, 1971
; Moulton, 1974; Costanzo and
Morrison, 1989). At least partly because of their constant exposure to
environmental insults, OSNs die and are replenished throughout life
(Farbman, 1990; Crews and Hunter, 1994); this property makes them
unlike most other neurons, which, under normal conditions, show limited regeneration in mature mammals (Brustle and McKay, 1996; Weiss et al.,
1996; Kuhn et al., 1997; Fawcett and Geller, 1998).
There are three dividing cell types within the mature olfactory
epithelium, only one of which gives rise to OSNs (Fig.
1; Caggiano et al., 1994). These are: (1)
the horizontal basal cell (HBC), whose nucleus resides in the
horizontal cell zone (HCZ); (2) the sustentacular cell (SC), whose
nucleus resides in the sustentacular cell zone (SCZ); and (3) the
globose basal cell (GBC), whose nucleus resides in the globose cell
zone (GCZ). Of these, the GBC is the most prolific in vivo
(Graziadei and Monti Graziadei, 1979; Schwartz Levey et al., 1991;
Caggiano et al., 1994); for example, retroviral lineage tracing
suggests that at least 50% of all cell division occurring just after
birth in the rat OE is within the GCZ (Caggiano et al., 1994).
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Figure 1.
Major cellular elements of the olfactory
epithelium. The olfactory epithelium contains the primary cell in the
olfactory pathway, the OSN, which expresses general neuronal markers,
including neurotubulin (left panel). In addition,
there are several other cell types within the epithelium; the nuclei of
all types are present in cell type-specific zones (right
panel; cell sizes are exaggerated). Included are those of the
three dividing progenitors (asterisks) that give rise to
the cells of the mature olfactory epithelium. Two of these, the
horizontal basal cell within the horizontal cell zone
(HCZ) and the sustentacular cell within the
sustentacular cell zone (SCZ), give rise only to
themselves. The third, the globose basal cell within the globose cell
zone (GCZ), gives rise to itself, as well as to immature
OSNs within the middle neuron zone (MNZ;
arrow), which in turn give rise to mature OSNs in the
upper neuron zone (UNZ; arrow). Together,
these zones span the width of the epithelium from the basal lamina
(BL), which separates the epithelium from the underlying
lamina propria in which OSN axons extend in bundles (small
arrowheads), to the nasal cavity, in which odorants are
presented to OSN dendrites located at the lumenal surface
(LS). Adapted from Caggiano et al. (1994). Scale bar, 20 µm.
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It has been difficult to recapitulate GBC turnover and differentiation
in vitro, both in primary cultures (Calof and Chikaraishi, 1989; Pixley, 1992; Mahanthappa and Schwarting, 1993) and in cell lines
(Goldstein et al., 1997; Coronas et al., 1997a). In addition, primary
culture of OSNs is frequently inefficient and cumbersome (Bozza and
Kauer, 1998). Because of these technical obstacles, many questions
about the turnover and function of OSNs have remained unanswered.
Perhaps the most vexing has been the role of the family of genes
encoding putative odorant receptor proteins (ORPs; Buck and Axel,
1991); the lack of robust in vitro models has particularly hindered any direct functional investigations into the coupling by
specific members of this family to a physiological odorant response.
Although ORPs can be expressed intracellularly via baculoviral infection of insect cells (Raming et al., 1993; Nekrasova et al., 1996), chimeric ORPs can be expressed in heterologous cells (Krautwurst et al., 1998), and at least two receptors have been successfully introduced to OSNs in vivo (Zhao et al., 1998; Touhara et
al., 1999); functional surface expression of ORPs in intact OSNs has not been achieved in vitro. We, therefore, have created a
cell line that we propose will be useful in defining: (1) the role of
ORPs in odorant detection; (2) the transduction apparatus within OSNs;
(3) signals that influence trafficking of ORPs and other olfactory
proteins within OSNs; and (4) factors that influence GBC turnover,
differentiation, and maturation.
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MATERIALS AND METHODS |
Creation of the odora line. A
temperature-sensitive mutant (tsA58) (Jat et al., 1986) of the SV40
large T antigen (Southern and Berg, 1982) in a retroviral backbone
(Cepko et al., 1984) was used to create cell lines from the olfactory
epithelium. Postnatal day 3 rats were killed according to
protocols approved by Tufts University, the Society for Neuroscience,
and the National Institutes of Health. Olfactory epithelia from eight
pups were dissociated using a method (Hunter et al., 1992) we had
developed for dissociation of retinae, and were incubated at 33°C (at
which tsA58 should be active) in normal medium (DMEM;
Bio-Whittaker, Walkersville, MD) containing 10% fetal bovine serum
(FBS; Hyclone, Logan, UT), 100 U/ml penicillin (Irvine Scientific,
Santa Ana, CA) and 100 µg/ml streptomycin (Irvine Scientific). During
plating, 30 µl of a tsA58-Tag viral concentrate (Jat and Sharp, 1989)
(~106 pfu/ml, prepared as in Caggiano et
al., 1994) was added to each dish; after 2.5 hr, an additional 30 µl
was added. After 2 d, the medium was supplemented with 400 µg/ml
G418 (active drug concentration; Life Technologies, Gaithersburg, MD).
After 5 weeks, eighteen colonies had formed. All were isolated in
cloning rings, then replated in G418-containing medium. After reaching
confluency, all lines were frozen and stored in liquid nitrogen. After
thawing, only five remained viable; all have similar characteristics.
None was able to survive our attempts at single-cell cloning in 96-well plates; we presume that this reflects a requirement for autocrine factors. Interestingly, all adhere tenaciously to both plastic and
glass, suggesting that these cells produce strong adhesion molecules.
The cells cannot be removed easily by standard trypsinization (0.05%
in DMEM), even when incubated at 37°C for 1 hr; however, cells
readily detach when divalent cations are chelated, suggesting that they
adhere via a calcium- (perhaps cadherin-) mediated mechanism. One of
these lines we now call odora.
Odora culture. Odora cells were grown at
33°C to ~30% confluency, then shifted to 39°C (at which
tsA58-Tag should be degraded; see Fig. 3) and incubated for 5-7 d in
normal medium (see above) supplemented with 1 µg/ml insulin, which is
important in maintaining an independent olfactory cell line (Goldstein
et al., 1997), and 20 µM dopamine and 100 µM ascorbic acid (both from Sigma, St. Louis,
MO), which can elicit differentiation in yet another cell line (Coronas
et al., 1997a). Together, these changes resulted in differentiation of
odora cells. Control cells remained in unsupplemented normal
medium and at 33°C. For assessment of proliferation, odora cells were cultured with a combination of fluoro- and
bromo-deoxyuridine (cell proliferation labeling reagent; Amersham,
Arlington Heights, IL) during the last 18 hr of culture. Cells were
then rinsed, fixed in absolute methanol at 
20°C, and processed for
BrdU immunohistochemistry. The average number of cells from 14 random
fields taken from three individual coverslips was determined, and the
percent that had incorporated BrdU was calculated. The total number of
labeled cells present at 33°C was 4.8-fold higher than that at 6 d after the shift to 39°C, reflecting the more rapid proliferation
seen when tsA58-T antigen is active.
Immunohistochemistry and immunofluorescence.
Immunohistochemistry was performed as previously described (Libby et
al., 1996). For sections, the rostral portion of the entire head of a
postnatal day 1 to 3 rat, frozen in liquid nitrogen-cooled isopentane,
was cut at 10-12 µm and placed onto ethanol-cleaned glass slides. After drying, the sections were either left unfixed or fixed with 4%
paraformaldehyde (Polysciences, Warrington, PA) in PBS (in mM: 137 NaCl, 2.7 KCl, 10 Na2HPO4, and 1.8 KH2PO4, pH 7.2-7.3) for 5 min at ambient temperature. Odora cells on 12 mm round glass coverslips were washed with PBS, then fixed in 4% paraformaldehyde in
PBS for 5 min at ambient temperature. The primary antibodies used are
given in Table 1. Most have been
shown by other laboratories to react specifically with their stated
antigen in tissue homogenates; we, and our collaborators (B. Talamo,
unpublished observations), have additionally demonstrated that the
antibodies recognizing the signal transduction components
(G
olf, ACIII, and oCNG
) react with
appropriately sized species on protein transfer (Western) blots of
extracts of odora cells (see Fig. 6). Secondary antibodies coupled to fluorescein, rhodamine, and R-phycoerythrin were
obtained from Sigma and Molecular Probes (Eugene, OR). All antibodies
were diluted in a potassium Ringer's buffer (KRB; in
mM: 140 NaCl, 5 KCl, 5 NaHCO3, 2 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES, pH 7.4)
containing 2% bovine serum albumin (Sigma), and 0.01% Triton X-100
(Sigma); the wash solution was KRB. For an assessment of percentage of
cells expressing a given marker, the total numbers of cells in random
fields (assessed by contrast or with a nuclear stain) were counted, and
the numbers of cells within those fields expressing the marker were
calculated. Data are expressed as average percent of cells (± SD) expressing detectable levels of the marker in the given
conditions.
Protein transfer blot. Control and differentiated
odora cells, grown as described above, were solubilized in
10 mM Tris-HCl, pH 7.5, containing 1%
SDS, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 5 µg/ml aprotinin, 5 µg/ml chymostatin, and 100 µg/ml pefabloc. After collection and sonication, two volumes of
sample buffer (192 mM Tris-HCl, pH 6.8, containing 9% SDS, 15% glycerol, and 2% 2-mercaptoethanol) were
added. Lysates were boiled and separated by electrophoresis on a
denaturing 10% polyacrylamide gel, then transferred to Immobilon-P (Millipore, Bedford, MA). The membrane was blocked in 5% (w/v) nonfat
milk in 200 mM NaCl, 0.1% Tween 20, 50 mM Tris, pH 7.4 for 1 hr, incubated in primary
antibody for 2 hr and peroxidase-conjugated secondary antibody (New
England Biolabs, Beverly, MA) for 1 hr, all at ambient temperature.
After washing, the membrane was incubated in a chemiluminescent
substrate (New England Nuclear, Boston, MA) for 1 min and exposed to
x-ray film. Samples were normalized by nucleic acid content in order to
load extracts from approximately equal numbers of cells per lane.
RT-PCR. RNA was isolated from dissected adult rat
olfactory epithelium and retina using RNAzol B (Biotecx, Houston, TX)
and from odora cells using Ultraspec (Biotecx) following the
manufacturer's recommendations. For oCNG
, oCNG, and rCNG-t, 4 µg of RNA was reverse-transcribed using SuperScript II (Life
Technologies) primed with random hexamers following the manufacturer's
recommendations. In addition, for oCNG, 100 ng of RNA was similarly
reverse-transcribed using a specific oligonucleotide primer
(5'-TACATCTCTCGGCCAATGTC-3'). The resultant cDNAs (5% of the randomly
primed, or all of the specifically primed) were amplified in PCRs using
Platinum Taq High Fidelity (Life Technologies) in 2 mM MgSO4. Oligonucleotides used were: oCNG, forward, 5'-GTCATCATCCACTGGAATGCTTG-3' and reverse, 5'-ATCAGCTACCACTGCCAACTTGCCC-3' (Kingston et al., 1996); oCNG, forward, 5'-ACCATGCGCTGGTAAAGAAG-3' and reverse,
5'-TACATCTCTCGGCCAATGTC-3' (sequences obtained from the Mombaerts
laboratory); and rCNG-t, forward, 5'-TCCATGCTGTGCCAATCACA-3' and
reverse, 5'-CTGGTCCACATCAGCCTGCA-3' (Sautter et al., 1998). Cycling
protocol was: 94°C, 2 min; 35-40 cycles of 94°C, 45 sec; 52°C,
45 sec; 72°C, 2 min; and 10 min at 72°C. Fifty percent of each
reaction was analyzed on a 1% agarose/1% NuSieve (FMC
Bioproducts, Rockland, ME) gel. The primers used for oCNG and
rCNG-t have been shown previously not to amplify sequences from
related CNG cDNAs. In addition, the primers used for oCNG and
oCNG span introns in genomic DNA; both sets would, therefore, detect
contaminating genomic DNA in our RNA samples. We did not detect these
larger products derived from genomic DNA in our samples, demonstrating
a lack of contamination.
Calcium imaging. Differentiated cells were loaded at 39°C
with the ratiometric calcium imaging dye, fura-2 AM (4 µM; also contains 0.01% Pluronic F-127; both from
Molecular Probes) on 31 mm round glass coverslips in odorant Ringer's
solution (ORS; in mM: 140 NaCl, 5 KCl, 10 glucose, 1 Na
pyruvate, 1 CaCl2, 1 MgCl2,
and 10 HEPES, pH 7.2) as previously described for primary cultures of
OSNs (Bozza and Kauer, 1998). After 1 hr, cells were rinsed and placed
into a perfusion chamber through which odorants (Table
2; final concentrations of 50 µM each in ORS that had been stored in a polypropylene
tube) were presented for 16-24 sec. Two controls were used:
"Ringer's", which was a pulse of Ringer's stored in polystyrene,
and "no odorant", which was a pulse of Ringer's stored in
polypropylene, which has an intrinsically different odor than that of
polystyrene. No differences were ever seen between the two controls.
Unless otherwise noted, data were collected at 340 and 380 nm every 4 sec, stored, and analyzed and plotted using Quattro Pro versions 5 and
7 (Corel, Ottawa, Canada). For simplicity, the signals are shown as the
ratios of the fluorescence intensity at 340 nm divided by that at 380 nm; this roughly reflects the concentration of intracellular calcium (Bozza and Kauer, 1998, their references). Note that the baseline ratio
varies with each experiment, presumably reflecting such parameters as
length and efficiency of dye loading, as well as the ambient
temperature during the experiment (which varied from 26 to 33°C).
Transfection of odora cells. Odora
cells, grown on 31 mm round glass coverslips in individual 35 mm tissue
culture dishes, were differentiated as described above. Five days after
differentiation was begun, odora cells were transfected for
2 hr with 0.625 µg of a plasmid containing the
2-adrenergic receptor or an ORP construct (U131 or OR5) and a hemagglutinin tag (McClintock et al., 1997), using
3.75 µl of the SuperFect reagent (Qiagen, Chatsworth, CA) per dish,
then were washed and returned to the appropriate medium for 2 d.
SuperFect was by far the most efficient transfection reagent tested,
resulting in 50-80% transfection efficiency. To assess surface
expression, cells were removed to 0°C to prevent receptor and
antibody internalization and incubated for 2 hr with a monoclonal mouse
anti-HA antibody (BAbCo, Berkeley, CA), then fixed in 4%
paraformaldehyde in PBS. Cells were washed and incubated in a secondary
antibody, coupled to R-phycoerythrin (Molecular Probes) for
45-60 min at ambient temperature and coverslipped in ProLong
(Molecular Probes). Intracellular calcium responses were assayed (see
above) in the presence of the 2-adrenergic receptor agonist, isoproterenol (25 mM; Sigma) in
cells transfected with the 2-adrenergic
receptor; cells transfected with ORP constructs were assayed for
responses to our odorant mixtures (see above).
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RESULTS |
Creation of an olfactory sensory neuron line
We chose to immortalize cells derived from the OE of perinatal
rats; at this age, the OE has achieved a mature morphology, but is
still undergoing massive proliferation (Caggiano et al., 1994). Because
of the high degree of division occurring in the basal cell populations
at this age, a large number of dividing progenitors (HBC, GBC, and SC;
Fig. 1) are present. We have previously shown that ~50% of the
mitotically active cells present just after birth are HBCs, and ~50%
are GBCs or their immediate progeny (Caggiano et al., 1994); an
immortalizing oncogene could, therefore, be transmitted to these
dividing populations via infection with a replication-incompetent
retrovirus. After infection of a cell, the oncogene will be
incorporated into the DNA of one of its daughter cells and subsequently
passed onto that daughter's progeny. We chose a temperature-sensitive
oncogene in the hope that cells that were proliferating at the
permissive temperature of the oncogene would cease proliferation
and differentiate when the cells were removed to a nonpermissive temperature.
Cells from freshly dissociated perinatal rat OE were infected with a
retrovirus carrying a neomycin resistance gene and a temperature-sensitive mutant of an oncogene, large T antigen
(tsA58-Tag), driven by the murine moloney leukemia virus long terminal
repeat (MMLV-LTR) (Southern and Berg, 1982; Cepko et al., 1984; Jat et al., 1986; Jat and Sharp, 1989). After selection at the permissive temperature (33°C), 18 colonies were isolated; of these, five remained viable after freezing and replating. Of these five, we have
chosen one for further study; we now call these odora cells, for olfactory-derived, odorant
receptor activatable cells. Surprisingly, the
other four lines are qualitatively similar in growth properties, gross
morphology, and antigen expression (data not shown), suggesting that
they were derived from similar progenitor cells. Thus, one population
of progenitors appears to be more easily immortalized.
Characterization of odora cells
Odora cells grow rapidly, attach readily to uncoated
glass, and are easily dissociated when grown at 33°C in normal medium (see Materials and Methods). This line, which was originally created in
1995, has now been in continuous culture for 2 years; over that time,
the gross characteristics of the cells have not changed. Briefly, the
cells achieve an epithelial morphology quickly, only rounding up
occasionally (Fig. 2A).
After differentiation [shift to the nonpermissive temperature (39°C)
and addition of insulin and dopamine; see Materials and Methods],
odora cells become somewhat flatter and larger (Fig.
2B); in addition, they begin to attain a bipolar
morphology, frequently extending long processes across many tens of
micrometers (Fig. 2C, D). Their division rate
also decreases dramatically: after an 18 hr pulse of the thymidine analog bromodeoxyuridine (BrdU), >90% of the nuclei of the control cells have incorporated BrdU, whereas only 20% of the nuclei of the
differentiated cells have (see Materials and Methods).
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Figure 2.
Morphology of odora cells.
Odora cells, immortalized by infection with a
temperature-sensitive variant of large T antigen (tsA58-Tag), exhibit
an epithelial morphology when grown at 33°C in normal medium
(A). Dividing cells, which normally round up from
the epithelial sheet, are frequent. In contrast, after differentiation,
cells remain largely flattened (B); when sparse,
their bipolar morphology is apparent. Differentiated cells occasionally
extend long, sometimes branched, processes (arrowheads)
over many tens of micrometers (C, D).
These processes occasionally contact those from other cells
(asterisk) or terminate in a filopodium
(arrow). Scale bar, 40 µm.
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Immunohistochemically, control odora cells express nuclear
T-antigen (Fig. 3), and do not express
markers of glia (GFAP; Fig. 3), of horizontal basal cells (keratins;
Fig. 3), or of sustentacular cells (SUS-4; data not shown). However,
control odora cells do express a marker of the globose basal
cell (GBC-1; Fig. 3), as well as the neural cell adhesion molecule,
NCAM (as do GBCs; Caggiano et al., 1994; Fig.
4). Together, these data suggest that
odora cells were derived from infection of a dividing
globose basal cell. Odora cells do not express appreciable
quantities of several neuronal markers, including the
microtubule-associated protein MAP5 and the growth-associated protein
GAP-43; they also do not appear to assemble neurotubulin into
microtubules (Fig. 4). Thus, control odora cells share some
characteristics with the undifferentiated OSN progenitor, the GBC. As
noted above, our other lines appear qualitatively similar; this
suggests that cells within the GBC lineage are more easily
immortalized, or more easily cultured, than those within the HBC
lineage.
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Figure 3.
Odora cells express markers of the
olfactory sensory neuron lineage, but not of the other two lineages
within the mature olfactory epithelium. T-antigen, the immortalizing
oncogene, is expressed in the nuclei of odora cells at
the permissive temperature (control), but only
infrequently after a shift to the nonpermissive temperature
(treated) in nuclei (arrow), and
occasionally in perinuclear regions (arrowheads).
Markers of glia (GFAP), horizontal basal cells
(keratins), and sustentacular cells (SUS-4; data not
shown) are not expressed in either condition. Importantly,
odora cells express a marker of globose basal cells,
GBC-1, under control conditions; this expression
decreases dramatically with differentiation, as it does in OSNs. Scale
bar, 20 µm.
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Figure 4.
Odora cells express neuronal
markers. Under control conditions, odora cells, like
globose basal cells, express NCAM, but little MAP5, and no GAP-43.
After differentiation, all three markers are expressed. Neurotubulin,
although expressed in control conditions, is not assembled into
discernible microtubules until the cells are differentiated, where it
is present both in long processes (arrow) and in bundles
within the cytoplasm (arrowheads). Scale bar, 20 µm.
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Differentiated odora cells largely do not express nuclear
T-antigen (Fig. 3), consistent with their slowing in division (cf. Jat
and Sharp, 1989). They also have an antigenic profile similar to that
for OSNs: they largely cease expression of GBC-1 (Fig. 3), maintain
expression of NCAM (Fig. 4), and begin to express MAP5 and GAP-43 (Fig.
4). In addition, they appear to assemble neurotubulin into discernible
microtubules within their somata and processes (Fig. 4). Thus, in many
respects, differentiated odora cells are similar to
olfactory sensory neurons.
Odora cells also express markers that are more selective for
OSNs. These include components of the olfactory signal transduction machinery: the olfactory G-protein (Golf),
adenylyl cyclase type III (ACIII), and the subunit of the olfactory
cyclic nucleotide-gated channel (oCNG), as well as olfactory marker
protein (OMP). In tissue sections of the olfactory epithelium, the
components of the transduction machinery are located within the
dendrites at the lumenal surface, whereas OMP is throughout the somata
of OSNs (Fig. 5). In odora
cells, all of these markers are expressed to some degree
before differentiation (Fig. 5); in this regard, control odora cells appear to be more differentiated than GBCs.
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Figure 5.
Odora cells express markers that
are expressed by olfactory sensory neurons in vivo,
including components of the olfactory signal transduction machinery.
Olfactory cells, both in situ and in
vitro, express the olfactory G-protein
(Golf), adenylyl cyclase type
III (ACIII), the subunit of the olfactory cyclic
nucleotide-gated channel (oCNG), and olfactory marker
protein (OMP). In situ, mature sensory
neurons express all four (OE; oCNG section was fixed
in 4% paraformaldehyde; others were unfixed). The basal lamina
(BL) and lumenal surface (LS) are noted
for orientation (compare Fig. 1); for ACIII, two sections are shown,
because the expression within the somata is variable. Note that in
control odora cells, oCNG appears trapped in
perinuclear compartments, some of which are strikingly bar- or
crescent-shaped (asterisk), whereas others are more
diffuse (arrowhead); after differentiation, the channel
largely moves away from these sites, including to the distal ends of
cellular processes (arrow), although some remains
diffusely perinuclear (arrowhead). OMP also appears to
be distributed to additional, peripheral, locations after
differentiation (arrow). Scale bar, 40 µm for tissue
sections; 20 µm for odora cells.
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However, the expression of Golf and OMP
increases after differentiation. For Golf, the
protein level of each expressing cell increases: although the percent
of cells that produce immunohistochemically detectable levels of
Golf does not change with
differentiation (Figs. 5, 6), the
overall amount of Golf does increase after differentiation, as assayed by protein transfer (Western) blot (Fig.
6). In contrast, for OMP, this change appears to be in the number of
cells that express immunohistochemically detectable amounts of OMP:
although some cells produce detectable levels before differentiation,
nearly all cells produce detectable OMP after differentiation (Figs. 5,
6). This increase in OMP expression coincides with the dramatic
decrease in numbers of cells producing detectable levels of GBC-1 after
differentiation (Figs. 3, 6).
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Figure 6.
Golf, OMP, and GBC-1 levels change
after differentiation of odora cells. Protein transfer
blots (left) of control (C) and
treated (T) odora cell extracts
reveal an increase in overall Golf expression after
differentiation. However, the percentage of cells
(right, average ± SD) expressing
immunohistochemically detectable levels of Golf does not
increase after treatment, suggesting an increase in Golf
levels in each expressing cell. In contrast, the percent of cells
expressing detectable levels of OMP increases, and the percent
expressing GBC-1 decreases after differentiation.
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In addition, the distributions of oCNG and OMP change after
differentiation (Fig. 5). Both shifts are consistent with the differentiation of odora cells to a phenotype more like that
of an OSN. Strikingly, the altered distribution of oCNG after
differentiation suggests a novel regulation of subcellular distribution
within OE-derived cells that may be partly responsible for localizing olfactory-specific components of OSNs (including, potentially, ORPs;
see below) to the plasma membrane.
We have shown that odora cells produce the subunit of
the olfactory cyclic nucleotide-gated channel (Fig. 5). However, the cyclic nucleotide-gated channel in OSNs is thought to consist of at
least two subunits, oCNG (Dhallan et al., 1990) (also known as
oCNC1, CNG2, or CNC3) and oCNG (Bradley et al., 1994; Liman and
Buck, 1994) (also known as oCNC2, CNG5, or CNC4). In addition, the
olfactory epithelium specifically produces a further potential subunit,
rCNG-t (also known as CNG4.3 or CNC1b), which is produced from an
alternative transcript of the retinal rod subunit (Sautter et al.,
1998; Bönigk et al., 1999). The exact combination of subunits
that is used in native OSNs is not known, but coexpression of all three
cDNAs results in a channel similar to that found in OSNs (Sautter et
al., 1998; Bönigk et al., 1999), and all three polypeptides are
expressed in the OE (Bönigk et al., 1999), suggesting that the
native channel is a heterotrimer.
We used RT-PCR to demonstrate the presence of RNAs encoding all three
of these potential subunits. Using primers for oCNG that have been
shown to amplify that subunit specifically (Kingston et al., 1996), we
find that odora cells, like OE (but not retina), produce the
oCNG RNA, and that differentiation of odora cells appears
to increase the amount of oCNG RNA (Fig.
7). Using primers for rCNG-t that have
similarly been shown to be specific (Sautter et al., 1998), we also
find that odora cells, like OE (but not retina), produce the
rCNG-t RNA (Fig. 6). Finally, using primers for oCNG, we find
that odora cells, like OE (but not retina) produce the
oCNG RNA; however, this RNA appears to be in low abundance in
odora cells relative to OE, because it is difficult to
amplify from odora-derived cDNA that has been randomly
primed (requiring additional cycles of amplification), or requires
specific priming in order to produce substantial amplification (Fig.
7). The products we amplified were not derived from genomic DNA, as at
least two of the primer sets (those for oCNG and oCNG) span introns, and would have resulted in larger products than those we
amplified in these reactions. Together, these RT-PCR data suggest that
odora cells, like OSNs, produce three potential cyclic
nucleotide-gated channel subunits, and are, therefore, likely to
assemble an OSN-like channel. Although we have shown
immunohistochemically that the oCNG protein is present in
odora cells and in OSNs (Fig. 5), the precise combination of
proteins that is produced and used in odora cells and in
OSNs awaits further characterization of the native channels.
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Figure 7.
Odora cells express RNAs encoding
three potential subunits of a cyclic nucleotide-gated channel. RNAs
were reverse-transcribed and amplified in PCRs as described in
Materials and Methods. Products can be amplified in 35 cycles for
oCNG (674 nt) and rCNG-t (293 nt) using randomly primed cDNAs,
and for oCNG (313 nt) using specifically primed cDNAs, from
olfactory epithelium (OE), control odora
cells (C), and treated odora cells
(T). Products were not derived from genomic DNA,
because two of the primer sets (those for oCNG and oCNG) span
introns and would have resulted in larger products than those we
amplified in these reactions. No products were amplified when cDNAs
from retina (R) were used in parallel
reactions.
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Responses to stimuli in odora cells
As differentiated odora cells contain many of the
components of the putative signal transduction machinery, we next asked whether these cells can respond to odorants. These cells, unlike many
neurons, frequently did not respond (as assayed by changes in
intracellular calcium levels; see Materials and Methods) to a
potentially depolarizing concentration of potassium (100 mM; Fig. 8). The
reasons that they may not always respond to a high concentration of
potassium are many; for example, they may not express voltage-activated
calcium channels, or they may have an unusually high resting potential.
An accurate description of this phenomenon awaits a full
characterization of the electrophysiological properties of
odora cells.
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Figure 8.
Odora cells can target functional
exogenous proteins to their plasma membrane. Top,
Untransfected odora cells do not respond to pulses
(16-24 sec, displayed as bars below the response traces) of our
odorant mixtures (data from one cell shown; compare Table 2), or to a
pulse of Ringer's (Ring) or odorant diluent
(NoOd). Odora cells frequently do not
respond to 100 mM potassium (K). Bottom,
Odora cells, when transfected with a plasmid containing the
2-adrenergic receptor and an HA tag, properly target the
exogenous protein their surface, as detected by immunoreactivity for
the HA tag in live (non-permeabilized) cells (inset).
Transfected odora cells respond to isoproterenol
(iso) but not to odorant mixtures
(A). Response for one cell shown.
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We have not yet determined whether odora cells express an
endogenous ORP; however, because there are hundreds of members of the
odorant receptor gene family (Buck and Axel, 1991), the likelihood of
testing the cells with an odorant that happened to stimulate their
endogenous receptors seems slight. We have used six mixtures of
odorants (containing a total of 30 odorants; Table 2), which, because
of the broad odorant responsivity in individual cells (Bozza and Kauer,
1998; Malnic et al., 1999), is likely to stimulate, at least partially,
many more than 30 different ORPs. Differentiated odora cells
never responded to these mixes (Fig. 8, top; Table 3); although this is consistent with a
lack of endogenous ORP expression, it could just as easily reflect our
relatively small odorant profile.
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Table 3.
Responses to odorant mix B and selected components (Comp),
including those cells that responded to: EA (total; includes 34 that
responded only to EA); PA (only); and both EA and
PA
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Others have shown that the 2-adrenergic
receptor can activate the olfactory G-protein
(Golf) (Jones et al., 1990), resulting in a
transduction cascade similar to that after odorant exposure. Because we
had never elicited a response to odorants in differentiated odora cells (see above), and because we knew that
odora cells could produce, and distribute, many components
of the transduction machinery (see above), we transfected cells with a
plasmid containing the 2-adrenergic receptor
(see Materials and Methods). In this plasmid, the
2-adrenergic receptor is coupled to a
hemagglutinin (HA) tag (McClintock et al., 1997), which can
subsequently be detected in living cells with an antibody directed
against HA. Unlike many neuronally-derived cells, odora
cells are readily transfected: we typically achieve 50-80%
transfection efficiency (as assayed by expression of the
2-adrenergic receptor in live cells; Fig. 8,
inset) in transient transfections.
Odora cells transfected with the
2-adrenergic receptor are able to respond with
an increase in intracellular calcium to a saturating concentration of
an appropriate agonist (isoproterenol, 6 of 12 cells; Fig. 8).
Importantly, when presented with our odorant mixtures before
isoproterenol, these transfected cells did not respond (0 of 12 on two
coverslips; Fig. 8), demonstrating that expression of a non-ORP
receptor is not sufficient to confer an odorant response. Although
odora cells, like OSNs, appear to produce some
Gs (data not shown), these data, nevertheless,
suggest that odora cells can couple ligand activation of a
receptor (in this case, the 2-adrenergic
receptor) to an increase in intracellular calcium, presumably via a
stimulation of adenylyl cyclase by Golf (or
Gs). The resultant increase in cAMP is likely
to result in opening of cyclic nucleotide-gated channels within the
plasma membrane, as is thought to occur in OSNs.
Expression and function of ORPs in odora cells
In marked contrast to the inability of other cell lines to direct
ORPs to the plasma membrane (McClintock et al., 1997), odora cells properly and efficiently target exogenous ORPs to their surface
in a punctate pattern (Fig. 9,
inset). This pattern was seen with both ORP constructs
tested (U131, Fig. 9; OR5, data not shown), and was similar to that
seen after expression of the 2-adrenergic
receptor in odora cells (compare Fig. 8). Odora cells are, therefore, unique in their ability to direct ORPs to their
surface. As these ORP-transfected odora cells express (1) an
ORP, (2) Golf, (3) ACIII, and (4) subunits of
the cyclic nucleotide-gated channel, we reasoned that these cells
should respond to stimuli for the exogenous ORP. We have assayed
responses by detecting changes in intracellular calcium by ratiometric
imaging with fura-2 AM (see Materials and Methods), focusing on those cells that were transfected with the rat ORP, U131. This ORP was isolated in 1997 (McClintock et al., 1997), but its function has not
been previously characterized.
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Figure 9.
Odora cells can express functional
exogenous odorant receptor proteins. Top, Odora cells,
when transfected with a plasmid containing an ORP (U131) and an HA tag,
properly target the exogenous ORP to their surface
(inset). The transfected odora cells
respond to a mix of odorants (B), but not to
others (A, CD, EF);
therefore, expression of U131 confers odorant responsiveness. Cells
occasionally respond to 100 mM potassium (K). The response
to mix B desensitizes: the second response is markedly smaller than the
first, and the third pulse elicited no response. The response to a
mixture of A and B (AB) is smaller than that for B
alone; this may, in part, reflect the dilution inherent in mixing A and
B, but could also be caused by more complicated interactions among the
various components of A and B. The data shown are the average of the
responses from four cells; the break in the line reflects a sampling
error at 380 nm. Bottom, Cells that responded to mix B
were divided into two categories: those that responded to
n-enanthic acid (EA; solid
line; average of five cells), and those that responded both to
EA and n-pelargonic acid (PA;
dotted line; average of five cells); responses to PA are
never seen in cells that do not respond to EA. Importantly, expression
of U131 does not confer response to compounds with structures that are
similar to EA, including n-valeric acid
(VA), benzyl alcohol (BA), and heptanol
(Table 2). The responses desensitize, both to mix B (presented three
times in 20 min) and to the individual components (each presented twice
in 10 min). Together, these data suggest that U131 acts as a receptor
for seven- and nine-carbon saturated fatty acids, and that
odora cells can appropriately couple this receptor to a
physiological response. We did not detect a response to 100 mM potassium (K) in the
differentiated odora cells, even when data were
collected every 880 msec during and after the K pulse (data collection
returned to every 4 sec after 92 sec).
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We have used, as noted above, six mixtures of odorants (Table 2) to
test for potential odorant responses. As also noted above, the
relatively broad responsivity displayed by OSNs (Bozza and Kauer, 1998;
Malnic et al., 1999) suggests that our mixes might contain a ligand
that would interact with the transfected ORP, particularly since
expression of transfected constructs can be quite high.
Differentiated odora cells that had been transiently
transfected with an ORP, U131, respond to one of our six mixes of
odorants (mix B), but not to the five others (mixes A, C-F; Fig. 9).
This increase in intracellular calcium is completely blocked when
extracellular calcium is chelated (G. Liu and B. Talamo, personal
communication), suggesting that the increase results from
calcium influx rather than from release of calcium from intracellular
stores. On 18 coverslips, 32% of the cells responded to odorant mix B
(Table 3), suggesting that a large fraction (~50%) of the
transfected cells (themselves 50-80% of the total number of cells)
displayed an odorant response. Those cells that were transfected but
did not respond to mix B may express lower levels of transfected ORP, may have been incapable of responding because of changes in other components of the transduction pathway, or may have had inadequate access to the odorant. Nevertheless, after transient transfection with
a specific ORP, a relatively large number of cells produced responses
to one specific odorant mixture.
Intriguingly, odora cells transfected with U131 display
odorant desensitization, a phenomenon that has been described in OSNs (Bozza and Kauer, 1998, their references). We routinely observe a
diminished response to successive challenges of odorant (Fig. 9), even
when those presentations are minutes apart. Remarkably, within a given
experiment, there is little difference among individual cells, both in
initial amplitude of response and subsequent desensitization. Odora cells present a rich, relatively uniform, system in
which to study this phenomenon in detail.
Which of the components of the stimulating mix can elicit a response?
When tested individually, only two, the seven-carbon saturated fatty
acid (enanthic acid; EA) and nine-carbon satu- rated fatty acid
(pelargonic acid; PA), ever elicited a response in odora
cells (Fig. 9; Table 3). Strikingly, the five-carbon saturated fatty
acid (valeric acid; VA) and the seven-carbon saturated alcohol
(heptanol) gave no measurable response (0 of 225 cells). Of those cells
that responded to odorant mix B, 73% responded to EA (Table 3); of
those that responded to EA, 29% also responded to PA. The only cells
that responded to EA or PA were among those that had responded to
odorant mix B; similarly, no cells ever responded to PA that did not
respond to EA. Remarkably, as noted earlier with odorant mixtures, the
amplitude and duration of responses among cells within each of the
groups were consistent within a given experiment, even in the degree of
desensitization. A more exhaustive characterization of the response
profile awaits additional experiments; nevertheless, it is clear that
U131 provides odora cells with the capacity to respond to
seven- and nine-carbon saturated fatty acids.
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DISCUSSION |
The nature of the odora progenitor
The postnatal rodent olfactory epithelium contains three major
dividing cell types: sustentacular cell, horizontal basal cell, and
globose basal cell. We have previously shown, under the conditions we
have studied, that these three cell types appear to be in independent lineages, and that olfactory sensory neurons appear to be exclusively derived from GBCs (Caggiano et al., 1994). Similar results have been
reported by others (Schwob et al., 1994). However, these lineal
relationships are not absolute: under some experimental conditions,
such as exposure to methyl bromide gas, a single, HBC-like progenitor
is competent to give rise to all cell types within the olfactory
epithelium (Huard et al., 1998). Thus, there is some plasticity within
the progenitors which, under most conditions, is dampened.
It is possible that factors present in the resting olfactory epithelium
act to repress transdifferentiation among the three lineages. Indeed,
within the OSN lineage itself, it is thought that some aspects of the
mature OSN phenotype are transcriptionally repressed in the progenitor
cells (Tsai and Reed, 1997; Wang et al., 1997). It is conceivable that
some of these controls are lost after transfer of cells ex
vivo. The loss of these controls would, at least in part, explain
the difficulty that many laboratories have had in recapitulating OSN
generation in vitro, and may also partly account for the
partially differentiated phenotype that we observe in control
odora cells.
Within the usual OSN lineage, i.e., from GBC to OSN, cells move through
several stages of development (for review, see Calof et al., 1998).
During the initial stages, the cell is still mitotically active; as it
progresses through the subsequent stages, mitosis ceases. We could have
infected any of these dividing cells within the early stages of OSN
production with our retrovirus containing the immortalizing oncogene,
thereby obtaining cell lines with a phenotype between GBC and OSN. Our
immunohistochemical results with the odora line are
consistent with our immortalization of a slightly differentiated OSN progenitor.
Dopamine as a differentiation agent
Olfactory sensory neurons express D2 dopamine receptor RNA and
protein within the olfactory epithelium (Coronas et al., 1997b), as
well as at their target sites in the glomeruli of the olfactory bulb
(Nickell et al., 1991; Coronas et al., 1997b). One synaptic target of
OSNs is the dopaminergic periglomerular cell, which may, therefore,
provide the neurotransmitter that acts on D2 receptors expressed by
OSNs. It is conceivable that one form of communication between an OSN
and its target is the detection of dopamine by presynaptic receptors on
OSN terminals. Thus, dopamine is ideally situated as a potential
modulator of OSN function. Indeed, dopamine has been shown to modulate
adenylyl cyclase levels in olfactory epithelium (Mania-Farnell et al.,
1993) and to modulate an inwardly rectifying
hyperpolarization-activated current in OSNs (Vargas and Lucero,
1999).
Dopamine may also modulate OSN maturation. Others have shown that
activation of a D2 receptor in an olfactory epithelium-derived cell
line can lead to differentiation of those cells (Coronas et al.,
1997a). Similarly, we have shown here that dopamine elicits differentiation in odora cells; we have also found that
odora cells express D2 receptors (J. R. Murrell and
D. D. Hunter, unpublished observations). Thus, dopamine, derived
from targets within the olfactory bulb, and acting on D2 receptors in
OSNs, may act as a differentiation signal for some of the final aspects
of OSN maturation.
The cyclic nucleotide-gated channel
One component of the odorant signal transduction system that is
thought to be vital is the cyclic nucleotide-gated channel. Animals in
which one subunit of the channel has been disrupted by homologous
recombination display anosmia to all tested odorants (Brunet et al.,
1996), suggesting that all of these compounds use activation of this
channel as part of the signal transduction process. However, the exact
nature of the channel as it exists in OSNs is unclear. The olfactory
channel consists of at least two subunits, oCNG (Dhallan et al.,
1990; also known as oCNC1, CNG2, or CNC3) and oCNG (Bradley et
al., 1994; Liman and Buck, 1994; also known as oCNC2, CNG5, or
CNC4), and perhaps a third, rCNG-t (Sautter et al., 1998;
Bönigk et al., 1999; also known as CNG4.3 or CNC1b). It seems
plausible that the native channel is a heterotrimer of all three
subunits (Sautter et al., 1998; Bönigk et al., 1999). We have
shown that odora cells produce RNAs encoding all three
potential subunits of the olfactory cyclic nucleotide-gated channel.
Therefore, odora cells are likely to serve as a good model
for studying this channel.
Activation of the olfactory cyclic nucleotide-gated channel can lead to
an influx of calcium through the OSN membrane, which could subsequently
activate a variety of channels within the plasma membrane (for review,
see Schild and Restrepo, 1998). Whether a similar cascade exists within
odora cells is unclear; however, the influx in calcium we
measure in response to odorants suggests that at least some portion of
this pathway is operating as it does in OSNs.
Odorant receptor proteins and odor codes
We have, for the first time, demonstrated the expression of
full-length exogenous odorant receptor proteins in a cell line. The
ready availability of large numbers of cells expressing the same
odorant receptor will allow for a careful analysis of the odorant
response profiles conferred by a given odorant receptor protein. Here,
we have shown a moderate degree of flexibility, but also a fair amount
of selectivity, in the chemical nature of the odorant that stimulates a
given odorant receptor protein. Similar results have been obtained in
OSNs that were infected with a single odorant receptor (I7, Zhao et
al., 1998; MOR23, Touhara et al., 1999), and are consistent with the
many reports that the primary reception event is somewhat broadly tuned
(Bozza and Kauer, 1998; Malnic et al., 1999; Duchamp-Viret et al.,
1999).
Where, then, is odorant selectivity achieved? The bulk of the data
generated within the olfactory system in the last several decades has
led to the concept that much of the selectivity is achieved via
interactions among the neurons of the network within the olfactory bulb
(Schild, 1988; Kauer, 1991). An understanding of the relationships
among individual odorants and their receptors will, therefore, only lay
a partial groundwork for an understanding of odor perception.
Nevertheless, it will be important to show carefully whether
reproducible response profiles contribute to the combinatorial events
that occur within the olfactory bulb and result in odorant
discrimination. Such profiles can be more easily generated in the
defined, consistent system provided by odora cells.
Summary
Our creation and characterization of odora cells is a
major step forward: although other lines have been created (Goldstein et al., 1997; Coronas et al., 1997a), none has been as extensively characterized as this line, none appears to follow the course of
differentiation (similar to that of GBCs) displayed by this line, and,
in particular, none shows the capacity to respond to odorants when
expressing a known ORP that we have shown here. Together, these
properties of odora cells should allow for an extensive
characterization of the generation and function of odorant-responsive cells, a task that has been limited in the past by the necessity to
study OSNs in vivo or in relatively inefficient primary
culture systems.
The presence of a family of G-protein-coupled odorant receptors was
originally proposed in 1991 (Buck and Axel, 1991), based on the initial
identification of a multitude of RNAs that could potentially encode
these proteins. A major problem in previous experiments has been a lack
of correct targeting of heterologously expressed ORPs; the shift in
targeting that occurs when odora cells are differentiated
provides us with an ideal model system for studying trafficking of
membrane proteins, including ORPs. In addition, we are now in a
position that allows for a thorough analysis of the issues surrounding
the contribution that individual ORPs may make to odorant sensitivity
and selectivity. Similarly, the ready isolation of large quantities of
the signal transduction components from a cell line represents a unique
opportunity for biochemical elucidation of many aspects of this cascade
which have, for now, remained a mystery, including such issues as
odorant desensitization. In summary, as models of the OSN lineage,
odora cells will now allow us to study the genesis,
maturation, function, death, and replenishment of OSNs in a controlled
environment, enabling us to ask fundamental questions concerning the
olfactory system that could previously not be asked.
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FOOTNOTES |
Received April 30, 1999; revised July 7, 1999; accepted July 19, 1999.
This work was supported by funds provided by Tufts University. We
particularly thank Tom Bozza for many helpful discussions during the
course of this work, Tim McClintock for providing advice and receptor
constructs, and members of the laboratory of Barbara Talamo for
comments and sharing unpublished data, as well as Atsuko Polzin for
help in performing some of the original infections, Joel White and
members of the laboratory of Jim Wang for comments, and Dona
Chikaraishi for encouragement during the beginning of this project. We
also thank William Brunken for many hours of support and advice.
Correspondence should be addressed to Dr. Dale D. Hunter, Department of
Neuroscience SC-6, Tufts University School of Medicine, 136 Harrison
Avenue, Boston, MA 02111.
| |
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TOP
ABSTRACT
INTRODUCTION
