1 Department of Applied Physics and Chemistry, Division of Bio-Informatics, 2 Department of Information Network Science, The University of Electro-Communications, Chofu, Tokyo 182 - 8585, Japan; and 3 Department of Physiology, Saar University, D-66421 Homburg, Germany
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ABSTRACT |
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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.
Ca2+-gated Cl channel; reversal
potential; imaging; N-(ethoxycarbonylmethyl)-6-methoxyquinolinium
bromide
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INTRODUCTION |
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WHEN VERTEBRATE
OLFACTORY 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 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.
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MATERIALS AND METHODS |
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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.
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 µm) 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 µm) 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 µl) 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 µl 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 charge-coupled 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 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 ![]() |
RESULTS |
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In parallel to the imaging experiment, we tested the viability of
the isolated rat cells by recording odor-induced 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.
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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. 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.
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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
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(1) |
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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 µm) 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 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.
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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.
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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)
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(2) |
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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 and soma to be ~0.65, which suggests a
comparably high correlation between the concentrations at the two sites.
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DISCUSSION |
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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
µm (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 µm in length and 0.1 µm 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.
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
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(3) |
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 530/B2.
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: tad{at}pc.uec.ac.jp).
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.
Received 6 October 2000; accepted in final form 22 January 2001.
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