Correspondence to: Makoto Kashiwayanagi, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan., yanagi{at}hucc.hokudai.ac.jp (E-mail), Fax: +81-11-706-4991; (fax)
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents, and inward currents in response to water-soluble and volatile odorants from receptor neurons in the lateral diverticulum (water nose) of the olfactory sensory epithelium of Xenopus laevis. The resting membrane potential was -46.5 ± 1.2 mV (mean ± SEM, n = 68), and a current injection of 13 pA induced overshooting action potentials. Under voltage-clamp conditions, a voltage-dependent Na+ inward current, a sustained outward K+ current, and a Ca2+-activated K+ current were identified. Application of an amino acid cocktail induced inward currents in 32 of 238 olfactory neurons in the lateral diverticulum under voltage-clamp conditions. Application of volatile odorant cocktails also induced current responses in 23 of 238 olfactory neurons. These results suggest that the olfactory neurons respond to both water-soluble and volatile odorants. The application of alanine or arginine induced inward currents in a dose-dependent manner. More than 50% of the single olfactory neurons responded to multiple types of amino acids, including acidic, neutral, and basic amino acids applied at 100 µM or 1 mM. These results suggest that olfactory neurons in the lateral diverticulum have receptors for amino acids and volatile odorants.
Key Words: olfactory receptor neuron, odor response, voltage-clamp, action potential, amino acid
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Xenopus laevis, the olfactory organ is compartmentalized into two independent subregions, the medial and lateral diverticulums. Anatomical observation has indicated that the olfactory sensory epithelium in the medial diverticulum comes into contact with air, while the epithelium in the lateral diverticulum is in contact with water (
Xenopus laevis possesses a gene repertoire encoding two distinct classes of olfactory G-proteincoupled receptors; one class is related to the olfactory G-proteincoupled receptors of fish, and the other is related to the olfactory G-proteincoupled receptors of mammals (
In the present study, we recorded the responses to water-soluble odorants as well as to volatile odorants from Xenopus laevis lateral olfactory receptor neurons under whole-cell voltage-clamp conditions. The results obtained indicated that the lateral olfactory neurons responded to both water-soluble and volatile odorants. Many single olfactory neurons responded to a variety of amino acids, including acidic, basic, and neutral amino acids.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Slice Preparation of Xenopus laevis Olfactory Sensory Epithelium
The method of preparation was essentially the same as that described in a previous paper (
Data Recording and Analysis
The conventional whole-cell patch clamp method was used to measure transmembrane currents ( were made from borosilicate glass capillaries with an inner filament (GD-1.5; Narishige Co.) using a two-stage electrode puller (PP853; Narishige Co.), and then heat-polished. Gigaohm seals were obtained by applying negative pressure (-30 to -100 cm H2O). The whole-cell configuration was attained through the application of additional negative pressure. Membrane currents (holding potential, -70 mV) and voltages were recorded in the whole-cell configuration. Data were recorded continuously using an EPC-7 patch clamp amplifier (List Electronic) and stored on video cassette via a digital audio processor (PCM-501; Sony Corp.). All recordings were carried out at room temperature. Membrane currents were low-pass filtered with two-poles, Bessel filter of EPC-7. The filter frequency was 10 kHz. We estimated the magnitude of inward currents from just before the response to the peak. Analysis was carried out on a personal computer using pCLAMP software (Axon Instruments). All values are given as a mean ± SEM. A liquid junction potential was measured as described by
Lucifer Yellow Dialysis
Lucifer yellow CH was dialyzed intracellularly by using patch pipettes filled with 1% Lucifer yellow solution as described previously (
Stimulation
Olfactory neurons were stimulated with extracellular solutions by bath application from the outlet of the stimulating tube. Ringer solution, delivered by gravity, was alternated with odorant solutions by means of eight electrically actuated valves. The volume of the solutions in the chamber was ~90350 µl. A stimulating tube with a lumen 160200 µm in diameter was placed under visual control ~3 mm from the cell to eliminate the mechanical effects of alternating among solutions. The flow rate, which was adjusted with needle valves, was 12.323.3 µl/s. The delay, due to dead space and to exchange solution in the chamber, was 325 s, depending on the flow rate. Our attempt to eliminate deviations in the delay was unsuccessful because the precision of the needle valves was not sufficient to regulate the flow of small amounts of the solutions. To examine the extent of the dilution during stimulation, 50 mM 1-anilinonaphtalene-8-sulfonate (ANS) was applied from the stimulating tube to the recording chamber, and the fluorescence intensity of ANS at the point where the epithelium was usually placed was measured with an inverted microscope (Axiovert 135; Carl Zeiss, Inc.). The fluorescence intensity reached a saturated level within 418 s. This saturated fluorescence intensity was similar to that of 50 mM ANS.
Solutions
The extracellular Ringer solution consisted of (mM): 116 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES-NaOH, ph 7.4. Patch pipettes were usually filled with a normal internal solution containing (mM): 115 KCl, 2 MgCl2, 2 EGTA, 10 HEPES-KOH, pH 7.4. Further details on the solutions are shown in the individual figure legends. Lucifer yellow was dissolved in the internal solution at a concentration of 1%. The odorant cocktails were vigorously stirred with the magnetic stirrer for more than 30 min at room temperature. We confirmed that the solutions of odorant cocktails were completely clear. The final concentrations of each odorant in volatile odorant cocktail I, which induced inositol 1,4,5-trisphosphate accumulation in rat olfactory cilia, were 40 µM lilial, 40 µM lyral, and 20 µM ethyl vanillin (
Chemicals
Tetrodotoxin (TTX)1 and Lucifer yellow CH were obtained from Sankyo and the Aldrich Chemical Co., respectively. Amino acids (l-alanine, l-arginine, l-glutamic acid, and l-methionine) and N-methyl-D-glucamine were obtained from Wako. All volatile odorants were kindly supplied by Takasago International. All chemicals used were of the highest grade available.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Morphology
Figure 1A and Figure B, show micrographs of the olfactory epithelia of the lateral diverticulum and medial diverticulum of Xenopus laevis bathed in water containing 0.1% methylene blue. The surface of the lateral diverticulum was clearly stained with methylene blue, while that of the medial diverticulum was not stained at all. These results support a previous finding that the lateral and medial olfactory epithelia demonstrate differentiated contact with water-soluble odorants and volatile odorants, respectively (
|
Current-Clamp Recordings
Initial studies of the whole-cell clamp technique were made using the current-clamp mode. With K+ and Na+ as the main internal and external membrane-permeable cations, respectively, the mean resting membrane potential for these neurons was -46.5 ± 1.2 mV (n = 68); a value similar to the value measured in the Xenopus olfactory receptor neuron (
|
Voltage Response to Injected Current
Voltage-clamp recordings confirm that the current responsible for the rising phase of the current-induced action potential is carried by Na+. Figure 3 A shows the two major currents elicited by the depolarizing steps of voltage from a holding potential of -70 mV. A transient inward current appeared at greater than -40 mV, and an outward current was observed after the rapid inward current. The currentvoltage curves taken at the peak of the inward and outward currents are shown in Figure 3 B.
|
To examine the transient inward currents, internal KCl was substituted with CsCl. Replacing Na+ with choline in the external solution reversibly and completely abolished the transient inward current in all five neurons (Figure 3 C). The addition of 1 µM TTX to the external solution also completely inhibited this current in all four neurons (Figure 3 D). These results suggest that the current is probably carried by Na+.
To study the outward K+ currents, 25 mM tetraethylammonium (TEA) was substituted with an equivalent amount of NaCl, and 1 µM TTX was added to the external medium. The outward current was partially blocked by the addition of 25 mM TEA to the external solution (Figure 3 E) in all six neurons. The magnitude of the outward currents was reduced to 32.3 ± 4.4% (n = 6) by 25 mM TEA. The presence of a Ca2+-dependent K+ current in the neuron was studied by measuring the outward current in an external solution with and without Ca2+, respectively. The outward current was partially attenuated by the elimination of Ca2+ into the external solution (Figure 3 F), suggesting the presence of a Ca2+-activated K+ channel. Recently, K+ currents mediated with RELK1 channels insensitive to 100 mM TEA have been shown to be blocked by Ba2+ (
Current Responses to Water-soluble and Volatile Odorants
To evaluate the odor selectivity of the olfactory neuron of the lateral diverticulum, an amino acid cocktail and two volatile odorant cocktails were separately applied to neurons. The application of these three odorant cocktails induced inward currents in 46 of 238 neurons (Figure 4). In 7 of 32 neurons responding to the amino acid cocktail, the magnitude of the inward current was reduced rapidly but not completely during continuous stimulation (Figure 4 A). The remaining responses to the amino acid cocktail did not show sharp peaks (Figure 4 B). The peak amplitude of inward currents in response to the amino acid cocktail ranged, typically, from 0 to 126 pA. The mean amplitude of inward currents in response to odorant cocktails was calculated using neurons that responded to any one of the three odorant cocktails.
|
The volatile odorant cocktails I and II induced inward current responses in 17 (37%) and 14 (30%) neurons, respectively (Figure 4C and Figure D). The peak amplitudes of inward currents induced by the odorant cocktails I and II ranged, typically, from 0 to 100 and 0 to 164 pA, respectively. There are single neurons that responded to both cocktails. Some single neurons responded to all three cocktails (i.e., the amino acid cocktail as well as the two volatile odorant cocktails). One example of plural responses is shown in Figure 4 E. In this neuron, the response to the amino acid cocktail is larger than that to the volatile odorant cocktail I or the volatile odorant cocktail II.
Figure 5 shows the response profiles of individual olfactory neurons to the amino acid cocktail, the volatile odorant cocktail I, and the volatile odorant cocktail II. The population of neurons responding to the amino acid cocktail was larger than that responding to either of the two volatile odorant cocktails. 5 of 46 single olfactory neurons responded to all three odorant cocktails. Seven neurons responded to two odorant cocktails. Thus, ~26% of the single olfactory neurons responded to plural odorant cocktails. More than 19% of the neurons responded to both the amino acid cocktail and the volatile odorant cocktail.
|
Alanine of varying concentrations was applied to the receptor neurons (Figure 6 A). The magnitude of the inward current induced by alanine increased with increases in alanine concentrations. Figure 6 B shows the magnitudes of the responses to alanine of various concentrations. The currents appeared at 100 µM and increased with increases in concentration. The application of arginine also induced inward current responses in a dose-dependent manner (Figure 6 C).
|
To explore the selectivity of single olfactory neurons with neutral, basic, and acidic amino acids, alanine, arginine, and glutamic acid were applied to the olfactory neuron of the lateral diverticulum (Figure 7). The application of 100 µM of individual amino acids induced inward current responses in the single sensory neuron, as shown in Figure 7 A. The magnitude of the response to arginine was larger than that to alanine or glutamic acid. The neurons shown in Figure 7 B responded to either 1 mM alanine or 1 mM glutamic acid, but not to 1 mM arginine. Thus, although single olfactory neurons responded to plural amino acids, the response selectivity was differentiated among neurons. Figure 7 C shows the response profiles of individual olfactory neurons to 100 µM or 1 mM alanine, arginine, and glutamic acid, respectively. These amino acids induced responses in 13 of 152 neurons. More than 50% of the sensory neurons responded to plural amino acids. In particular, four neurons (31% of neurons stimulated by three odorants) responded to all three amino acids.
|
Lilial and citralva at different concentrations were applied to single olfactory neurons. The neuron shown in Figure 8 A responded to 1 and 10 µM, and to 1 mM alanine. Six of nine cells responded to alanine, and to lilial, and/or citralva. Figure 8B and Figure C, shows the magnitudes of response to lilial and citralva of varying concentrations. Distinct responses appeared at 1 µM for the odorants, indicating that the olfactory receptor neuron of the water nose responds sensitively to volatile odorants.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we examined the electrophysiological properties of receptor neurons in the lateral diverticulum of the olfactory sensory epithelium of Xenopus laevis using the whole-cell voltage-clamp technique, and showed that olfactory receptor neurons in the water nose respond to both amino acids and volatile odorants.
Odorants are considered to be received by olfactory G-proteincoupled receptors, which are linked to adenylyl cyclase or phospholipase C via the G-proteins (
In the present study, we showed that >50% of single olfactory neurons of Xenopus laevis respond to a variety of amino acids, including acidic, neutral, and basic amino acids. Single olfactory neurons in catfish also responded to plural amino acids (
An in situ hybridization study of the olfactory G-proteincoupled receptors of Xenopus laevis has shown that G-proteincoupled receptors similar to those in fish exist not in the epithelium of the medial diverticulum, but in the epithelium of the lateral diverticulum, while G-proteincoupled receptors similar to those in mammals exist in the epithelium of the medial diverticulum, but not in the epithelium of the lateral diverticulum (
It should be noted, however, that many olfactory neurons in the lateral diverticulum respond not only to amino acids, but also to volatile odorants. The sensitivity of the neurons to volatile odorants is similar to the olfactory neurons of salamanders (
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We gratefully thank Prof. Kenzo Kurihara for his support and for his critical review of the manuscript.
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.
Submitted: March 29, 1999; Revised: May 18, 1999; Accepted: May 19, 1999.
1used in this paper: TEA, tetraethylammonium; TTX, tetrodotoxin
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altner, H. (1962) Untersuchungen uber Leistungen und bau der Nase des Sudafrikanischen Krallenfrosches Xenopus laevis (Daudin, 1803). Z. Vgl. Physiol. 45:272-306.
Axel, R. (1995) The molecular logic of smell. Sci. Am. 273:154-159[Medline].
Breer, H., Boekhoff, I. (1991) Odorants of the same odor class activate different second messenger pathways. Chem. Senses. 16:19-29.
Breer, H., Raming, K., Krieger, J. (1994) Signal recognition and transduction in olfactory neurons. Biochim. Biophys. Acta. 1224:277-287[Medline].
Buck, L., Axel, R. (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell. 65:175-187[Medline].
Engeland, B., Neu, A., Ludwig, J., Roeper, J., Pongs, O. (1998) Cloning and functional expression of rat ether-á-go-go-like K+ channel genes. J. Physiol. 513:647-654
Firestein, S., Werblin, F. (1989) Odor-induced membrane currents in vertebrate-olfactory receptor neurons. Nature. 244:79-82.
Freitag, J., Krieger, J., Strotmann, J., Breer, H. (1995) Two classes of olfactory receptors in Xenopus laevis. Neuron. 15:1383-1392[Medline].
Gesteland, R.C., Yancey, R.A., Farbman, A.I. (1982) Development of olfactory receptor neuron selectivity in the rat fetus. Neuroscience. 7:3127-3136[Medline].
Hamill, O.P., Marty, A., Neher, E., Sackmann, B., Sigworth, E.J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391:85-100[Medline].
Hofmann, M.H., Meyer, D.L. (1991) Functional subdivisions of the olfactory system correlate with lectin-binding properties in Xenopus. Brain Res. 564:344-347[Medline].
Ivanova, T.T., Caprio, J. (1993) Odorant receptors activated by amino acids in sensory neurons of the channel catfish Ictalurus punctatus. J. Gen. Physiol. 102:1085-1105[Abstract].
Kang, J., Caprio, J. (1995) In vivo responses of single olfactory receptor neurons in the channel catfish, Ictalurus punctatus. J. Neurophysiol. 73:172-177
Kashiwayanagi, M., Kurihara, K. (1994) Odor discrimination in single turtle olfactory receptor neuron. Neurosci. Lett. 170:233-236[Medline].
Kashiwayanagi, M., Shimano, K., Kurihara, K. (1996) Existence of multiple receptors in single neurons: responses of single bullfrog olfactory neurons to many cAMP-dependent and -independent odorants. Brain Res. 738:222-228[Medline].
Kishimoto, J., Cox, H., Keverne, E.B., Emson, P.C. (1994) Cellular localization of putative odorant receptor mRNAs in olfactory and chemosensory neurons: a non-radioactive in situ hybridization study. Mol. Brain Res. 23:33-39[Medline].
Lischka, F.W., Schild, D. (1993) Effects of nitric oxide upon olfactory receptor neurons in Xenopus laevis. Neuroreport. 4:582-584[Medline].
Neher, E. (1992) Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 208:123-131.
Ngai, J., Chess, A., Dowling, M.M., Necles, N., Macagno, E.R., Axel, R. (1993) Coding of olfactory information: topography of odorant receptor expression in the catfish olfactory epithelium. Cell. 72:667-680[Medline].
Raming, K., Krieger, J., Strotmann, J., Boekhoff, I., Kubick, S., Baumstark, C., Breer, H. (1993) Cloning and expression of odorant receptors. Nature. 361:353-356[Medline].
Ressler, K.J., Sullivan, S.L., Buck, L.B. (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell. 73:597-609[Medline].
Ressler, K.J., Sullivan, S.L., Buck, L.B. (1994) A molecular dissection of spatial patterning in the olfactory system. Curr. Opin. Neurobiol. 4:588-596[Medline].
Schild, D. (1989) Whole-cell currents in olfactory receptor cells of Xenopus laevis. Exp. Brain Res. 78:223-232[Medline].
Schild, D., Lischka, F.W. (1994) Amiloride-insensitive cation conductance in Xenopus laevis olfactory neurons: a combined patch clamp and calcium imaging analysis. Biophys. J. 66:299-304[Abstract].
Sicard, G., Holley, A. (1984) Receptor cell responses to odorants: similarities and differences among odorants. Brain Res. 292:283-296[Medline].
Sklar, P.B., Anholt, R.R., Snyder, S.H. (1986) The odorant-sensitive adenylate cyclase of olfactory receptor cells: differential stimulation by distinct classes of odorants. J. Biol. Chem. 261:15538-15543
Sullivan, S.L., Ressler, K.J., Buck, L.B. (1995) Spatial patterning and information coding in the olfactory system. Curr. Opin. Genet. Dev. 5:516-523[Medline].
Taniguchi, M., Kashiwayanagi, M., Kurihara, K. (1996) Intracellular dialysis of cyclic nucleotides induces inward currents in turtle vomeronasal receptor neurons. J. Neurosci. 16:1239-1246[Abstract].
Vassar, R., Ngai, J., Axel, R. (1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell. 74:309-318[Medline].