Noninvasive measurement of chloride concentration in rat olfactory receptor cells with use of a fluorescent dye

Hiroshi Kaneko1, Tadashi Nakamura1,2, and Bernd Lindemann3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-]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 µM) and the K+/H+ antiporter nigericin (3.5 µM). 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 NO<UP><SUB>3</SUB><SUP>−</SUP></UP> for Cl- in the K+-rich solution containing (in mM) 150 KCl, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (15K):
[in this window]
[in a new window]
 
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.

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.


View larger version (121K):
[in this window]
[in a new window]
 
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: 10µm.

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<IT>=</IT>F<SUB>b</SUB><IT>+</IT>F<SUB>a</SUB><IT>e<SUP>−t/&tgr;</SUP></IT> (1)
where F is the fluorescence intensity at time t, tau  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 tau  for various rates of imaging. Figure 3B shows that the rate of decay (tau -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.


View larger version (17K):
[in this window]
[in a new window]
 
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.

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.


View larger version (15K):
[in this window]
[in a new window]
 
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.

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 tau  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.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Comparison of decreasing fluorescence at various [Cl-]i for knob (A) and soma (B). The time constant (tau ) 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.

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)
F<SUB>a</SUB><IT>=</IT>F<SUB><IT>0</IT></SUB><IT>/</IT>(<IT>1+K<SUB>q</SUB></IT>[Cl<SUP>−</SUP>]<SUB>i</SUB>) (2)
where Fa is the fluorescence intensity obtained from Eq. 1 for a given [Cl-], F0 is the fluorescence intensity 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).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Estimation of original [Cl-]i in the knob (A) and soma (B) of olfactory receptor neurons. , 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 (open circle , 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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
d[Cl<SUP>−</SUP>]/d<IT>t=−</IT>(<IT>I</IT><SUB>m</SUB><IT>−I</IT><SUB>a</SUB>)<IT>/</IT>(<IT>zFW</IT>) (3)
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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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[ISI][Medline].

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[Abstract].

3.   Dzeja, C, Hagen V, Kaupp UB, and Frings S. Ca2+ permeation in cyclic nucleotide-gated channels. EMBO J 18: 131-144, 1999[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[ISI][Medline].

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[Abstract/Free Full Text].

7.   Frings, S, and Lindemann B. Single unit recording from olfactory cilia. Biophys J 57: 1091-1094, 1990[Abstract].

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[ISI][Medline].

9.   Kleene, SJ. Origin of the chloride current in olfactory transduction. Neuron 11: 123-132, 1993[ISI][Medline].

10.   Kleene, SJ, and Gesteland RC. Calcium-activated chloride conductance in frog olfactory cilia. J Neurosci 11: 3624-3629, 1991[Abstract].

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[Abstract/Free Full Text].

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[Abstract].

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[ISI][Medline].

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[ISI][Medline].

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[Abstract/Free Full Text].

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[ISI][Medline].

17.   Lowe, G, and Gold GH. Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells. Nature 366: 283-286, 1993[ISI][Medline].

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[Abstract].

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[Abstract].

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[ISI][Medline].

21.   Menco, BP, and Farbman AI. Ultrastructural evidence for multiple mucous domains in frog olfactory epithelium. Cell Tissue Res 270: 47-56, 1992[ISI][Medline].

22.   Nakamura, T, and Gold GH. A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature 325: 442-444, 1987[ISI][Medline].

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[ISI][Medline].

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[Abstract].

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[ISI][Medline].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[ISI][Medline].

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[Abstract/Free Full Text].


Am J Physiol Cell Physiol 280(6):C1387-C1393
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society