Am J Physiol Cell Physiol
280: C1387–C1393, 2001.
Noninvasive measurement of chloride concentration in rat
olfactory receptor cells with use of a fluorescent dye
HIROSHI KANEKO,1 TADASHI NAKAMURA,1,2 AND BERND LINDEMANN3
1
Department of Applied Physics and Chemistry, Division of Bio-Informatics, 2Department of
Information Network Science, The University of Electro-Communications, Chofu, Tokyo 182–8585,
Japan; and 3Department of Physiology, Saar University, D-66421 Homburg, Germany
Received 6 October 2000; accepted in final form 22 January 2001
100–120 mM (17, 30). On the other hand, we (23) found
that [Cl⫺]i of the isolated newt olfactory cell perfused in
normal Ringer solution ([Cl⫺]: 100 mM) was about 40
mM when measured with the Cl⫺-sensitive fluorescent
dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium
bromide (MQAE) (8, 29). Other groups have attempted
measurements of [Cl⫺]i by patch-clamp experiments (2)
and energy-dispersive X-ray (EDX) microanalysis (28).
They reported a low value of 23 mM in the mud puppy (2),
but 69 mM in the rat (28). Furthermore, a chloride inward current induced by the odorants has been demonstrated electrophysiologically in rat olfactory cells (17).
Here, using the fluorescent Cl⫺ probe MQAE with in
situ calibration, we reexamined the distribution of Cl⫺ in
the rat olfactory neuron. The method allowed us to estimate [Cl⫺] in the soma and olfactory knob of ORNs, but
not in the cilia, the site of odorant transduction. Nevertheless, the measured values are of interest because the
ciliary chloride content is expected to equilibrate with
that of the knob in the resting state. Thus the [Cl⫺] of the
knob provides a starting point for the decrease in ciliary
[Cl⫺] that is predicted to occur during transduction.
MATERIALS AND METHODS
receptor neurons (ORN) receive odorants at their ciliary receptor membrane, intracellular cAMP increases and causes opening of cyclic
nucleotide gated channels (1, 20, 22), allowing influx of
sodium (Na⫹) and calcium (Ca2⫹) into the cells (3, 13, 15,
25). The increased Ca2⫹ in the cell activates Cl⫺ channels
in the ciliary membrane (10, 24). It has been generally
accepted that Cl⫺ ions flow out through these channels to
enlarge the inward receptor current (6, 9, 14, 17, 30).
Intracellular Cl⫺ concentration ([Cl⫺]i) needs to be high
to allow outward movement of Cl⫺. The value of [Cl⫺]i,
however, is controversial. Based on reversal potentials of
the receptor current, the [Cl⫺]i was found in the range
Isolation of olfactory cells. Wistar rats aged 2–7 wk
(Charles River, Nuremberg, Germany or Nippon SLC,
Hamamatsu, Japan) were narcotized with carbon dioxide,
then decapitated. Their heads were cut into two halves at the
central lines to expose the nasal cavities. Using forceps, we
quickly removed turbinates from the nasal cavity and immersed them in Tyrode’s solution containing (in mM) 140
NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES,
pH 7.4, on ice. One or two turbinates were used to prepare 1
ml cell suspension. The olfactory epithelium was removed
from the turbinates and, with a “microknife” (a splinter
broken from a razor blade), cut into fragments of ⬃0.5 ⫻ 0.5
mm in phosphate-buffered saline (PBS) containing (in mM)
140 NaCl, 2 KCl, 1.9 NaH2PO4, and 8.1 Na2HPO4, pH 7.4.
The fragments were incubated with PBS containing 0.1%
trypsin for 10 min at room temperature in a plastic tube, then
trypsin was removed by dilution with Tyrode’s solution. The
tissue fragments were suspended in 2 ml Tyrode’s solution
containing 0.1% albumin and dissociated by shaking in the
plastic tube several times by hand.
Address for reprint requests and other correspondence: T. Nakamura, Div. of Bio-Informatics, Dept. of Applied Physics and Chemistry, Univ. of Electro-Communications, Chofugaoka 1–5-1, Chofu,
Tokyo 182–8585, Japan (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
2⫹
⫺
Ca -gated Cl channel; reversal potential; imaging; N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide
WHEN VERTEBRATE OLFACTORY
http://www.ajpcell.org
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society
C1387
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Kaneko, Hiroshi, Tadashi Nakamura, and Bernd Lindemann. Noninvasive measurement of chloride concentration
in rat olfactory receptor cells with use of a fluorescent dye. Am
J Physiol Cell Physiol 280: C1387–C1393, 2001.—Inwardly
directed Ca2⫹-dependent chloride currents are thought to prolong and boost the odorant-induced transient receptor currents
in olfactory cilia. Cl⫺ inward current, of course, requires a
sufficiently high intracellular Cl⫺ concentration ([Cl⫺]i). In
previous measurements using a fluorescent Cl⫺ probe,
N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide
(MQAE), [Cl⫺]i of newt olfactory cells was estimated to be only
40 mM. This low value led us to reexamine the [Cl⫺]i by an
improved procedure. When isolated rat olfactory neurons were
bathed in Tyrode’s solution (150 mM Cl⫺) at room temperature,
the [Cl⫺] was 81.5 ⫾ 13.5 mM (mean ⫾ SE) in the tip of the
dendrite (olfactory knob) and 81.8 ⫾ 10.2 mM (mean ⫾ SE) in
the soma. The corresponding Cl⫺ equilibrium potentials were
⫺15.4 and ⫺15.3 mV, respectively. Therefore, at resting potentials in the range of ⫺90 to ⫺50 mV, Cl⫺ currents are predicted
to be inward and capable of contributing to the depolarization
induced by odorants. Yet, if the cell was depolarized beyond
⫺15 mV, somal Cl⫺ currents would be outward and facilitate
repolarization during excitation. The measured [Cl⫺] in soma
and knob are of interest, because in the cilia the chloride
content may be expected to equilibrate with that of the knob in
the resting state. They provide a starting point for the decrease
in ciliary [Cl⫺] predicted to occur during transduction.
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CHLORIDE CONCENTRATION IN RAT OLFACTORY CELLS
olfactory knob, and fluorescence intensities in these regions
were plotted as a function of time.
Calibration. MQAE is less sensitive to chloride when the
dye is intracellular rather than dissolved in saline. According
to Koncz and Daugirdas (11), this is due to a splitting of the
intramolecular ester bond, generating at a low rate a dye of
lower chloride affinity. This chemical change makes in situ
calibration essential. We used the “double ionophore” calibration procedure that was developed by Krapf et al. (12) for a
related dye, 6-methoxy-N-(3-sulfopropyl) quinolinium, and
used by Koncz and Daugirdas (11) for MQAE.
To shift the [Cl⫺]i for the in situ calibration, we replaced
the solution in the chamber with one containing different
[Cl⫺] and two ionophores, the Cl⫺/OH⫺ antiporter tributyltin
(5 ␮M) and the K⫹/H⫹ antiporter nigericin (3.5 ␮M). Tributyltin (20 mM, Sigma, Deisenhofen, Germany) and nigericin
(15 mM, Sigma) in ethanol were stocked at ⫺20°C. The drugs
were diluted into standard Cl⫺ solutions immediately before
use. The [Cl⫺] standards were prepared by substitution of
NO3⫺ for Cl⫺ in the K⫹-rich solution containing (in mM) 150
KCl, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4.
RESULTS
In parallel to the imaging experiment, we tested the
viability of the isolated rat cells by recording odorinduced receptor currents by a suction electrode technique. We recorded the odor-induced receptor currents
as shown in Fig. 1A. This record shows that the isolation procedure did not cause severe damage to the cells.
It was possible that MQAE at 5 mM, the concentration required to load this dye into cells, stimulates the
receptor cells as an odorant does. We used EOG recording to examine effects of MQAE on the cell activity
Fig. 1. Test of viability of the preparations used. A: inward receptor
currents were recorded from an isolated olfactory cell by suction
electrode method in response to a pulse (50 ms) of odorant mixture.
B: electroolfactograms (EOGs) were recorded in response to pulses
of Tyrode’s solution (1), odorant mixture (2, 4), and 5 mM N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) (3).
MQAE was loaded into the epithelium between records 3 and 4.
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Electrophysiology. Possible deterioration of the receptor
cells during preparation was tested by recording odor-induced response with a suction electrode technique (18, 27) or
by electroolfactogram (EOG) (19). Briefly, an isolated olfactory cell was sucked into a glass pipette electrode so that the
olfactory cilia protruded from the electrode tip (opening diameter, ⬃5 ␮m) into the bath solution. In a stream of Tyrode’s solution, pulses (50 ms) of odorant mixture containing
(in mM) 0.28 2-heptanone, 0.32 pinacolone, 0.23 geraniol,
0.23 citral, 0.25 l-carvone, and 5.4 n-amyl acetate were
ejected toward the cilia from a micropipette (opening diameter: ⬃1 ␮m) connected to an electromagnetic valve-controlled air pressure (⬃6 kPa). Inward current at the cilia
leaked out from another part of the cell and was collected by
the suction electrode and recorded through a patch-clamping
amplifier (List EPC-7, List Electronic, Darmstadt, Germany).
On the other hand, EOGs were recorded from dissected
olfactory epithelia set in a superfusion chamber. Pulses (⬃30
␮l) of the odorant mixture containing (in mM) 1.34 n-amyl
acetate, 1.19 cineole, 1.23 d-limonene were given to the
receptor side by a switching valve in the solution line that
continuously superfused the receptor side of the epithelium
(perfusion rate, 4 ml/min). Voltage across the epithelium was
measured between two Ag/AgCl electrodes connected by salt
bridges to each side of the tissue. DC-amplifier was made in
our laboratory. Both type of records were low-pass filtered at
20 Hz and digitized at 100–300 Hz to be stored in computers.
Measuring chamber. A rectangular filter paper having an
elliptical hole (⬃5 ⫻ 20 mm) was soaked in paraffin at 60°C
and attached to a glass coverslip (24 ⫻ 60 mm), such that the
paraffin formed the rim of a flat chamber. The glass bottom of
the chamber was coated with Cell-Tak (Collaborative Biochemical Products, Labor Schubert, Munich, Germany). On
the stage of the microscope, the chamber was mechanically
stabilized with a metal plate to minimize bending of the glass
while solutions were changed.
Imaging. Dye loading to the cells was achieved by incubating the dissociated cells with 5 mM MQAE (Molecular
Probes, Eugene, OR) in Tyrode’s solution for 30 min at room
temperature. Fifty microliters of the cell suspension was
added to 50 ␮l Tyrode’s solution in the measuring chamber.
In 20 min, the cells attached to the bottom of the chamber. To
wash away the extracellular dye, we added 0.5–0.6 ml Tyrode’s solution to the chamber with a Pasteur pipette while
removing overflow through a syringe needle (24 gauge) set on
the chamber. The solution in the chamber was completely
replaced within 1 s. Ten to twenty minutes after washing the
dye, we began the measurement.
The measuring chamber was placed onto the stage of a
conventional inverted fluorescence microscope (IMT2-F, Olympus, Hamburg, Germany). The objective was a Nikon Fluor ⫻40
of 0.85 NA. Excitation light of 380 nm was provided by a
monochromatic light source (Polychrome II, T.I.L.L. Photonics,
Planegg, Germany) that was coupled to the microscope with a
light guide. A dichroic mirror having a separation wavelength of
400 nm was used. Light color was under computer control
(T.I.L.L. Vision 3.02). The slow-scan camera was a Sensicam
(PCO, Kelheim, Germany), containing an integrating chargecoupled device chip of 12-bit pixel depth. Integration times
(exposure times) were typically 500 ms. The excitation light was
applied every 15 s to minimize bleaching of the dye. In a typical
experiment, 61 images (as in Fig. 2B) were recorded in 15 min
and stored on a personal computer. In the intervals of 15 s, cell
morphology was monitored with transmission images (as in Fig.
2A) recorded under green light. On the recorded fluorescence
images, regions of interest were identified on the soma and the
CHLORIDE CONCENTRATION IN RAT OLFACTORY CELLS
(Fig. 1B). First, we recorded EOGs induced by a pulse
of odorant mixture (trace 2) then by a pulse of 5-mM
MQAE solution (trace 3). These waveforms were basically the same. Thus 5 mM MQAE stimulated olfactory
cells as an odorant would. Then, we tested the effect of
the loaded dye on the receptor cell activity. For about
30 min, we incubated the same epithelium in the same
recording chamber with 5 mM MQAE to load it into the
cells. After flushing free MQAE with Tyrode’s solution
for about 15 min, we recorded EOG again with the
same odorant mixture (trace 4). Loading of the dye into
the cells in the epithelium was confirmed with the
fluorescence microscope after the EOG recordings.
EOGs before and after the dye loading had basically
the same waveform. Therefore, although 5 mM MQAE
works like an odorant, MQAE loaded in the cells does
not interfere largely with the cell responsibility.
Conventional transmission and fluorescence images
of an isolated rat olfactory cell loaded with MQAE are
shown in Fig. 2, A and B. The strongest fluorescence
was seen in the soma, mainly due to the thickness of
this structure. Dendrite and its tip, the olfactory knob,
which are much thinner, had less fluorescence. Images
such as those in Fig. 2B were captured repetitively at
intervals of 15 s.
The fluorescence of MQAE in the cell showed an
exponential decrease with time (Fig. 3A). Such decreases have been attributed mainly to the leak of the
dye from the cell to the bathing medium (8, 29). However, when we changed the rate of imaging, the decrease in fluorescence changed accordingly (Fig. 3A),
suggesting that bleaching of the dye caused by the
excitation light was significant. The decline of fluorescence could be fitted with a single exponential function
F ⫽ Fb ⫹ Fae ⫺ t/␶
(1)
where F is the fluorescence intensity at time t, ␶ is the
time constant of the decline, Fb is the fluorescence
intensity of the background, and Fa is the fluorescence
intensity in excess of Fb at time zero. By fitting Eq. 1 to
each record, we obtained ␶ for various rates of imaging.
Figure 3B shows that the rate of decay (␶⫺1) was
proportional to the inverse interval between images
and that a regression line fitted to these data crossed
the ordinate at a point not significantly different from
zero. Therefore, the decay of fluorescence was completely described by a first-order kinetic process driven
by light exposure, i.e., it was probably due to bleaching,
dye leakage being negligible in the olfactory cells. Subsequently, we used single exponential functions to correct the fluorescence decrease during the in situ calibration, as described next.
After recording 10 fluorescence images in intervals of
15 s, the in situ calibration was begun. In the intervals,
calibration solutions containing ionophores were applied. When the solution containing the ionophores was
first added to the chamber, small protrusions (diameter, 2–3 ␮m) appeared on some of the cells tested. The
protrusions seemed to be unstable and to burst. In such
cases, a rapid and large decrease of the fluorescence
intensity was observed, most likely due to a loss of
membrane integrity. When this happened, recording
had to be stopped. Only with those cells that were not
Fig. 3. Effect of light exposure on the decay of MQAE fluorescence in
the olfactory cell. A: exposures for 500 ms at 380 nm and intervals of
60, 30, 15, or 10 s. Exponentials (Eq. 1) were fitted to these data. B:
the rate constant (inverse time constant) of the fluorescence decay
was plotted against the rate of irradiation (inverse time interval of
exposures). Each point represents an average of 4–8 measurements,
except at 1/60 s⫺1 (n ⫽ 1). The vertical bars are SE. The straight line
was obtained by linear regression. The dashed lines delimit the 95%
confidence interval.
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Fig. 2. An isolated olfactory cell loaded with MQAE: transmission
image (A) under green light (⬎500 nm) and fluorescent image (B)
under excitation light at 380 nm, showing knob (K), dendrite (D), and
soma (S). In A, olfactory cilia (C) are scarcely visible due to their
small diameters. Scale bar: 10␮m.
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CHLORIDE CONCENTRATION IN RAT OLFACTORY CELLS
Fig. 4. Time course of fluorescence intensity at the olfactory knob
(solid line) and soma (broken line) at various intracellular Cl⫺
concentrations ([Cl⫺]i). After recording in normal Tyrode’s solution
for 90 s, ionophores were applied, and [Cl⫺]i was shifted successively
to 15, 30, 45, and 60 mM. At 60 mM [Cl⫺]i, recording was continued
for 10 min to determine the time constant of dye leakage from the
cell.
noticeably damaged by the ionophores was the measuring protocol carried out.
Figure 4 shows a representative time course of
MQAE fluorescence intensities at knob and soma obtained in one experiment. Each time course had three
sections, 1) a period before application of ionophores;
the fluorescence recorded here was used to estimate
the “original” [Cl⫺]i of the resting ORN; 2) in situ
calibration by equilibration of the cells with the standard solutions of 15, 30, 45, and 60 mM chloride in the
presence of ionophores; and 3) a period of 10 min
without solution changes that served to obtain the time
constant of the fluorescence decrease.
The overall decrease in MQAE fluorescence intensity
is obvious from the last section of the time courses in
Fig. 4. Equation 1 was fitted to the fluorescence time
course observed at 60 mM [Cl⫺]i ,yielding ␶ and Fb. The
exponential time course was retropolated to time zero
(Fig. 5, dashed curve). Intensity scaling of Fa was then
used to obtain the dotted curves of Fig. 5 that show the
time course predicted for various [Cl⫺]i. We thus obtained Fa as a function of [Cl⫺]i for each cell.
In Fig. 6, the mean Fa values obtained at each [Cl⫺]i
were plotted against [Cl⫺]i. These points were well
fitted with the Stern-Volmer equation (identical with
the law of mass action for a second-order reaction
between dye and chloride ions)
Fa ⫽ F0 /共1 ⫹ Kq关Cl⫺兴i兲
(2)
where Fa is the fluorescence intensity obtained from
Eq. 1 for a given [Cl⫺], F0 is the fluorescence intensity
Fig. 5. Comparison of decreasing fluorescence at various [Cl⫺]i for
knob (A) and soma (B). The time constant (␶) of the decrease in
fluorescence towards the background fluorescence intensity (Fb) was
obtained by fitting the traces at 60 mM [Cl⫺]i with a single exponential (Eq. 1). The exponential was retropolated to time zero (dashed
curve). Intensity scaling of Fa was used to obtain the dotted curves,
which show the predicted time course at various [Cl⫺]i. The zero time
values, Fa, of the dotted curves were used for calibration in Fig. 6.
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at 0 [Cl⫺], and Kq is the quenching constant (the
inverse dissociation constant). Kq was 3.7 ⫻ 10⫺2
mM⫺1 at the knob and 7.9 ⫻ 10⫺3 mM⫺1 at the soma.
These differences, which are probably due to the binding of dye to subcellular structures, serve to highlight
the importance of an in situ calibration that is performed separately for different parts of the cells. Similar differences in quenching constants between in
vitro and in situ calibrations have also been noted by
others (5, 29).
For calibration, Eq. 2 was used after normalizing the
Fa values with respect to those at 15 mM [Cl⫺]i . The
original [Cl⫺]i in each cell was estimated by inserting
the fluorescence intensity recorded before adding solutions containing ionophores into Eq. 2. Alternatively, a
graphical procedure was used (Fig. 6). The mean [Cl⫺]i
from the 11 cells were thus calculated to be 81.5 ⫾ 13.5
and 81.8 ⫾ 10.2 mM (mean ⫾ SE) in the knob and the
soma, respectively. By examining data obtained at the
two sites in the same cells (n ⫽ 8), we found the
correlation factor between the concentrations in knob
CHLORIDE CONCENTRATION IN RAT OLFACTORY CELLS
and soma to be ⬃0.65, which suggests a comparably
high correlation between the concentrations at the two
sites.
DISCUSSION
The chloride-sensitive dye MQAE is a methoxyquinolin
compound. The amphophilic properties of this class of
compounds suggest that MQAE will bind to subcellular
structures, a feature that makes in situ calibration of the
[Cl⫺] dependence of the fluorescence essential. On the
other hand, as MQAE is membrane permeant, it may be
expected that constant loss of dye to the bathing medium
will occur and affect the measurements. However, we
observed that leakage of MQAE was negligible in the
ORNs, as reported previously (23). In the present study,
the decay of the fluorescence intensity had a single exponential time course, apparently due to bleaching (Fig. 3,
A and B). MQAE can be hydrolyzed by esterases, forming
N-(6-methoxyquinolyl) acetic acid (11). This compound is
somewhat less sensitive to Cl⫺ (8, 29) and probably less
membrane permeant.
We found that, under resting conditions, the [Cl⫺]i of
isolated ORNs of the rat to be near 80 mM in both knob
and soma. This value is twice as large as the [Cl⫺]
obtained for isolated ORNs of the newt (23), but not
much larger than the 69 mM obtained for the rat by
EDX microanalysis (28). Because the fluorescence intensity was low at the knob compared with that of
soma, the obtained concentrations at the knob might
include certain measurement errors. It was possible
that a subset of the receptor cells were stimulated by
MQAE to change their [Cl⫺]i during the loading procedure. In fact, we observed that 5 mM MQAE induced
the EOG (Fig. 1B). However, as normal EOG was
induced by stimulation with conventional odorants
from an olfactory epithelium loaded with MQAE (Fig.
1B), the cells loaded with this dye seemed to maintain
the original ionic compositions.
Due to the comparatively large volume of knob and
soma, the high [Cl⫺]i of 80 mM may be expected to
change little when the cell responds to odorants. The
Cl⫺ equilibrium potential (ECl) measured by electrophysiology on rat olfactory cells is not yet certain in
literature. However, the ECl at the somal membrane
would be ⫺15 mV, if a [Cl⫺] of 150 mM may be
assumed for the interstitial space of the olfactory
epithelium.
It does not seem possible that the high [Cl⫺] found is
due to a chloride leak which may have developed during cell isolation. Given well-polarized cells (see above
for responsiveness to odorants), a passive distribution
of chloride ions would place ECl near the resting potential (⫺70 mV) and not at the value found (⫺15 mV). At
the same time, the cytosolic [Cl⫺] would be near 9 mM
and not near the value found, which is about ninefold
larger.
In neurons, the major chloride-accumulating transporters are Na⫹-driven cation-chloride cotransporters,
especially the furosemide-sensitive Na⫹-K⫹-2Cl⫺ cotransporter. Its occurrence in olfactory neurons has not
yet been described. In future experiments, furosemide
could be used to test for the role of cotransporters in
ORNs, as done with central neurons by Ehrlich et al. (4).
The conductance change induced in ORNs by odorants occurs predominantly in the plasma membrane of
the olfactory cilia (22). Therefore, the [Cl⫺] in the cilia
is of primary interest. Unfortunately, the cilia’s small
diameters of 0.1–0.2 ␮m (16, 26) made it impossible for
us to image the MQAE within the intraciliary space. It
is instructive, however, to make a rough calculation of
the changes in [Cl⫺] expected to occur in a cilium
during olfactory stimulation.
A model cilium of 20 ␮m in length and 0.1 ␮m in
diameter will be considered. Its volume is 0.16 fl. Due
to microtubules contained in the cilium, only a fraction
of this volume is available for free diffusion. Setting
this fraction to 50%, we obtain a diffusional space of
W ⫽ 0.08 fl. During stimulation with odorants, channels open and an inward current flows through the
ciliary membrane. We choose a value of ⫺2 pA for this
current. According to Lowe and Gold (17), this is a very
moderate value. Of this current, a large fraction, say
50%, is carried by chloride ions.
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Fig. 6. Estimation of original [Cl⫺]i in the knob (A) and soma (B) of
olfactory receptor neurons. F, Mean relative fluorescence intensities,
Fa, found by in situ calibration for each [Cl⫺]i (n ⫽ 11). The Fa value
for 15 mM [Cl⫺]i was normalized to 1. The vertical bars are SE. The
solid curves represent the Stern-Volmer equation (Eq. 2). By inserting normalized fluorescence intensities obtained in the original
states into the diagram (E, left), we could read out original [Cl⫺]i on
the abscissa. Mean original [Cl⫺]i were 81.5 ⫾ 13.5 and 81.8 ⫾ 10.2
mM (mean ⫾ SE) at the knob and soma, respectively.
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CHLORIDE CONCENTRATION IN RAT OLFACTORY CELLS
The initial [Cl⫺] in the ciliary compartment is similar to that in the knob, i.e., 80 mM. Therefore, the
initial ECl at the ciliary membrane is positive, near 10
mV, when the [Cl⫺] in the mucus surrounding the cilia
is 55 mM (28). The rate of change in the ciliary [Cl⫺] is
d关Cl⫺]/dt ⫽ ⫺ 共Im ⫺ Ia兲/共zFW兲
(3)
During this work, T. Nakamura and H. Kaneko were at Saar
University as an Overseas Research Scholar of the Ministry of
Education, Science, Sports and Culture of Japan and a Special Guest
Student of the Graduiertenkolleg der Medizinischen Fakultät der
Universität des Saarlandes, respectively.
This work was supported by Deutsche Forschungsgemeinschaft
Grant SFB 530/B2.
REFERENCES
1. Dhallan RS, Yau KW, Schrader KA, and Reed RR. Primary
structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature 347: 184–187,
1990.
2. Dubin AE and Dionne VE. Action potentials and chemosensitive conductances in the dendrites of olfactory neurons suggest
new features for odor transduction. J Gen Physiol 103: 181–201,
1994.
3. Dzeja C, Hagen V, Kaupp UB, and Frings S. Ca2⫹ permeation in cyclic nucleotide-gated channels. EMBO J 18: 131–144,
1999.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
where z and F are valence (⫺1) and Faraday’s constant
(97 C/mmol), respectively, Im is the partial chloride
current at the membrane (inward current negative),
and Ia is the axial chloride current at the base of the
cilium. Based on our estimate of the [Cl⫺] in the knob,
we allow for Ia only 30% of the total current, the
remainder being carried by cations and nonchloride
anions.
We thus obtain for the initial change in [Cl⫺] in the
cilium ⫺60 mmol/s. Therefore, starting from a resting
value of 80 mM, the supply of chloride ions driving Im
tends to be exhausted within seconds. Thereby the
ciliary reversal potential for the chloride current shifts
to more negative values and Im decreases. At the same
time, Ia increases by the development of a [Cl⫺] gradient along the cilium. The limiting stationary value is
reached when Im ⫽ Ia. Complete reloading of the ciliary
chloride occurs in the intervals between stimuli by
diffusion from the knob compartment. Furthermore, a
strong depolarization, as it occurs at the cilia by retrograde conduction of action potentials (7), may resupply
chloride ions from the mucus compartment.
The above considerations suggest a significant depletion in ciliary chloride content during transduction. A more precise estimate requires measurements of the [Cl⫺] in the mucus compartment
surrounding the cilia. EDX microanalysis yielded 55
mM (28). This, however, is a mean value that does
not account for the compartmentalization of the mucus (21). A complementary measurement of local
[Cl⫺] in the mucus, by means of fluorescent dyes,
would seem necessary. Of foremost interest, however, is the future measurement of ciliary [Cl⫺], both
in the resting state and during stimulation.
4. Ehrlich I, Lohrke S, and Friauf E. Shift from depolarizing to
hyperpolarizing glycine action in rat auditory neurones is due to
age-dependent Cl⫺ regulation. J Physiol (Lond) 520: 121–137,
1999.
5. Engblom AC and Akerman KE. Determination of the intracellular free chloride concentration in rat brain synaptoneurosomes using a chloride-sensitive fluorescent indicator. Biochim
Biophys Acta 1153: 262–266, 1993.
6. Firestein S and Shepherd GM. Interaction of anionic and
cationic currents leads to a voltage dependence in the odor
response of olfactory receptor neurons. J Neurophysiol 73: 562–
567, 1995.
7. Frings S and Lindemann B. Single unit recording from olfactory cilia. Biophys J 57: 1091–1094, 1990.
8. Inoue M, Hara M, Zeng XT, Hirose T, Ohnishi S, Yasukura
T, Uriu T, Omori K, Minato A, and Inagaki C. An ATP-driven
Cl⫺ pump regulates Cl⫺ concentrations in rat hippocampal neurons. Neurosci Lett 134: 75–78, 1991.
9. Kleene SJ. Origin of the chloride current in olfactory transduction. Neuron 11: 123–132, 1993.
10. Kleene SJ and Gesteland RC. Calcium-activated chloride
conductance in frog olfactory cilia. J Neurosci 11: 3624–3629,
1991.
11. Koncz C and Daugirdas JT. Use of MQAE for measurement of
intracellular [Cl⫺] in cultured aortic smooth muscle cells. Am J
Physiol Heart Circ Physiol 267: H2114–H2123, 1994.
12. Krapf R, Berry CA, and Verkman AS. Estimation of intracellular chloride activity in isolated perfused rabbit proximal convoluted tubules using a fluorescent indicator. Biophys J 53:
955–962, 1988.
13. Kurahashi T and Shibuya T. Ca2⫹-dependent adaptive properties in the solitary olfactory receptor cell of the newt. Brain Res
515: 261–268, 1990.
14. Kurahashi T and Yau KW. Co-existence of cationic and chloride components in odorant-induced current of vertebrate olfactory receptor cells. Nature 363: 71–74, 1993.
15. Leinders-Zufall T, Rand MN, Shepherd GM, Greer CA, and
Zufall F. Calcium entry through cyclic nucleotide-gated channels in individual cilia of olfactory receptor cells: spatiotemporal
dynamics. J Neurosci 17: 4136–4148, 1997.
16. Lidow MS and Menco BP. Observations on axonemes and
membranes of olfactory and respiratory cilia in frogs and rats
using tannic acid-supplemented fixation and photographic rotation. J Ultrastruct Res 86: 18–30, 1984.
17. Lowe G and Gold GH. Nonlinear amplification by calciumdependent chloride channels in olfactory receptor cells. Nature
366: 283–286, 1993.
18. Lowe G and Gold GH. The spatial distributions of odorant
sensitivity and odorant-induced currents in salamander olfactory receptor cells. J Physiol (Lond) 442: 147–168, 1991.
19. Lowe G, Nakamura T, and Gold GH. Adenylate cyclase mediates olfactory transduction for a wide variety of odorants. Proc
Natl Acad Sci USA 86: 5641–5645, 1989.
20. Ludwig J, Margalit T, Eismann E, Lancet D, and Kaupp
UB. Primary structure of cAMP-gated channel from bovine olfactory epithelium. FEBS Lett 270: 24–29, 1990.
21. Menco BP and Farbman AI. Ultrastructural evidence for
multiple mucous domains in frog olfactory epithelium. Cell Tissue Res 270: 47–56, 1992.
22. Nakamura T and Gold GH. A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature 325: 442–444, 1987.
23. Nakamura T, Kaneko H, and Nishida N. Direct measurement of the chloride concentration in newt olfactory receptors
with the fluorescent probe. Neurosci Lett 237: 5–8, 1997.
24. Nakamura T, Lee HH, Kobayashi H, and Satoh TO. Gated
conductances in native and reconstituted membranes from frog
olfactory cilia. Biophys J 70: 813–817, 1996.
25. Nakamura T, Tsuru K, and Miyamoto S. Regulation of Ca2⫹
concentration by second messengers in newt olfactory receptor
cell. Neurosci Lett 171: 197–200, 1994.
26. Pongracz F, Firestein S, and Shepherd GM. Electrotonic
structure of olfactory sensory neurons analyzed by intracellular
and whole cell patch techniques. J Neurophysiol 65: 747–758,
1991.
CHLORIDE CONCENTRATION IN RAT OLFACTORY CELLS
27. Reisert J and Matthews HR. Na⫹-dependent Ca2⫹ extrusion
governs response recovery in frog olfactory receptor cells. J Gen
Physiol 112: 529–535, 1998.
28. Reuter D, Zierold K, Schroder WH, and Frings S. A depolarizing chloride current contributes to chemoelectrical transduction in olfactory sensory neurons in situ. J Neurosci 18:
6623–6630, 1998.
C1393
29. Verkman AS, Sellers MC, Chao AC, Leung T, and Ketcham
R. Synthesis and characterization of improved chloride-sensitive
fluorescent indicators for biological applications. Anal Biochem
178: 355–361, 1989.
30. Zhainazarov AB and Ache BW. Odor-induced currents in
Xenopus olfactory receptor cells measured with perforated-patch
recording. J Neurophysiol 74: 479–83, 1995.
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