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Odor discrimination in single turtle olfactory receptor neuron
Neuroscience Letters 170 (1994) 233-236
ELSEVIER
NEUROSCIENCE LETTERS
Odor discrimination in single turtle olfactory receptor neuron Makoto Kashiwayanagi*, Kenzo Kurihara Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan
Received 5 January 1994; Revised version received 3 February 1994; Accepted 3 February 1994
Al~traet To explore the ability of odor discrimination of olfactory receptor neurons, current responses to odorant cocktails were recorded from an isolated olfactory neuron of the turtle. Twenty-five percent of the neurons tested responded to both cAMP-dependent and the IP3-dependent odorant cocktails. Application of the cAMP-dependent (or the IP3-dependent) odorant cocktail to the neuron after an inward current induced by the IP3-dependent (or the cAMP-dependent) odorant cocktail was adapted induced a large inward current in the neuron. The results suggest that at least two different receptors exist in a single olfactory neuron. Key words: Isolated olfactory neuron; Whole-cell patch-clamp; Odorant-activated current; Odor discrimination; Transduction; Second messenger
It is now generally believed that the G-protein-linked cAMP pathway contributes to the generation of olfactory responses [1,5,9,10,16,17]. It is also pointed out that the IP3-dependent pathway plays an important role in olfactory transduction [2,6,7,13,14,18]. Recently, we showed a new pathway independent of neither cAMP nor IP 3 also contributes to the turtle olfactory response [8]. These results suggest that there are multiple transduction pathways in the olfactory transduction systems. The first aim in the present study is to explore whether a single neuron of the turtle has only a single second messenger pathway. In an earlier study, Sicard examined the odor responses of frog single olfactory neurons to 20 odorants having different molecular structures and odor qualities [15]. In 75% of them, more than 2 odorants induced odor responses. Firestein et al. also recorded that 47% of isolated salamander olfactory neurons responded to different odorants [4]. These results suggest that multiple receptors exist in the single olfactory neurons of the frog and salamander. On the other hand, Raming et al. demonstrated that in OR5 (G-protein-coupled receptor)-expressing St9 cells, IP3 was produced by various odorants
*Corresponding author. Fax: (81) 11-717-3267. 0304-3940/94/$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved SSDI 0304-3940(94)00139-2
having different molecular structures and odor qualities [11]. These broad tunings of odor response suggest that the responsiveness of a single neuron to different odorants does not always mean existence of multiple receptors in the single olfactory neurons. In addition, on the basis of results of gene expression of putative olfactory receptors in the rat olfactory epithelium, Ressler et al. discussed that a single neuron may select one gene for expression [12]. In the present study, we examine whether a single olfactory neuron isolated from the turtle olfactory epithelium has different receptors by using the cross-adaptation method. Results suggest that at least two different receptors exist in a single olfactory neuron and a single neuron has an ability of discriminating odorants. Turtles, Geoclemys reevesii, weighing 150-300 g, were obtained from commercial suppliers and maintained at 22°C. Animals were fed porcine and bovine liver ad libitum. Animals were cooled to 0°C and decapitated. The nasal cavities were opened, and the olfactory epithelia were quickly removed. The epithelia were cut into slices of about 300 p m thickness in normal Ringer solution at 0°C and stored at 4°C. Slices were incubated for 30 min to 2 h at 37°C in Ca-free Ringer solution for the isolation of olfactory neurons. Immediately prior to recording, one slice of the epithelium was placed in 500 ¢tl normal Ringer solution in the recording chamber and
234
M. Kashiu'ayanagi, K. Kurihara / Neuroscience Letters 170 (1994) 233 236
shaken. No enzymes or proteases were added. Turtle olfactory neurons were easily distinguished from other types of cells such as respiratory and basal cells based on their characteristic morphology. Recordings were made with an Axopatch 1C amplifier (Axon Instruments, Inc., Foster City, CA) using patch electrodes of borosilicate glass. Electrodes with resistance of 5-10 Mr2 were manufactured on a Narishige PP83 puller (Narishige Co., Tokyo, Japan) using a double stage pull. Gigaohm seals were obtained by applying negative pressure (-30 to -100 cmH20). The whole-cell configuration was attained by application of additional negative pressure. The current signal was digitized and stored on video tape. Gravity was used to deliver a constant stream of Ringer solution from the stimulating tube. Three electrically actuated valves were used to switch adapting Ringer solution and odorant cocktail solutions. The stimulating tube with a lumen 160 200/~m in diameter was placed under visual control within about 500/~m of the cell. Normal Ringer solution contained (mM): 116 NaC1, 4 KC1, 2 CaCI2, 2 MgC12, 15 glucose, 5 sodium pyruvate, 10 HEPES-NaOH (pH 7.4). For the incubation of slices, 2 mM CaCI2 was removed from normal Ringer solution. Patch pipettes were filled with an internal solution (mM): 115 KCI, 5 NaCI, 2 MgC12, 5 ATP, 10 HEPES-KOH (pH 7.4). The cAMP-dependent odorant cocktail consisted of 200/JM each of citralva, hedione, eugenol,/-carvone and cineole, which were reported to increase cAMP concentration in the rat and bullfrog cilia preparations but unchanged IP3 concentration [2,16], in normal Ringer solution. The IP3-dependent odorant cocktail contained ~M): 40 lilial, 40 lyral, 20 ethyl vanillin and 100 limonene, which are reported to increase IP3 concentration in the rat cilia preparation but unchange cAMP concentration in the rat and the bullfrog [2,16]. Although we have no evidence, it is highly probable that the same species of odorants increase the same second messenger in the turtle olfactory neuron because the same results were obtained from quite different animal such as the bullfrog and the rat. Therefore, we used above odorants as cAMP-dependent or IP3-dependent odorants. All odorants were kindly supplied from Takasago International (Tokyo, Japan). Whole-cell recordings were obtained from a total of 44 olfactory neurons. Only neurons with motile cilia were used for the recording. Fig. la,b show inward currents induced by application of the odorant cocktails in an isolated olfactory neuron. Application of the cAMP-dependent odorant cocktail (Fig. la) and the IP3-dependent odorant cocktail (Fig. lb) induced inward currents in the same olfactory neuron. Fig. lc shows number of neurons which responded to both cocktails, the cAMP-dependent odorant cocktail, or the IP3-dependent odorant cocktail. Eleven olfactory neurons (25% of neurons recorded in
a
b
cAMP-dependent Odorants g,"/,/,/,///////////////,d
IP3-dependent Odorants I I
12s]10 pA
C
BothCocktails
cAMP-dependent Odorants
IP3-dependent Odorants
No ReSponse~
~
[
I
, i
0
10
20
Number of Neurons
Fig. 1. Inward current in response to a cAMP-dependent odorant cocktail (a) and to an 1P3-dependent odorant cocktail (b). Both responses were recorded from the same neuron. Bars above traces indicate period of each odor stimulation. Numbers of preparations which responded to both cocktails, the cAMP-dependent odorant cocktail, and the IP3-dependent odorant cocktail (c). Holding potential was -50 mV.
the study) responded to both odorant cocktails, suggesting that both the cAMP-dependent pathway and the IP3-dependent pathway exist in a single olfactory neuron. These data are consistent with a recent immunohistochemical data reported by Cunningham et al.; Golfand IP 3 receptor were expressed in essentially all olfatory neuron of the rat [3]. Cross-adaptation experiments were undertaken to examine whether different receptors exist in a single olfactory neuron (Fig. 2). Application of the cAMP-dependent odorant cocktail induced an inward current. After the current was adapted to the basal level, the IP3-dependent odorant cocktail induced an inward current (Fig. 2a). Similar results were obtained from 6 different olfactory neurons. Mean magnitude of inward current induced by the IP3-dependent odorant cocktail after the cAMP-dependent one was 89 + 43 pA (S,E.M., n = 6). Fig. 2b shows the results of the cross-adaptation experi-
M. Kashiwayanagi, K. Kuriharal Neuroscience Letters 170 (1994) 233-236 cAMP-dependent IP3-clependent IP3-dapendent cAMP-dependent Odorants Odorants Odorants Odorants ~"///////,~ P'//////A I
a
cAMP-dependent IP3-dependent Odorants Odorants ~///I//1111/,,41 I
IP3-dependent cAMP-dependent Odorants Odorants I
,
v////4
d 50 pA Fig. 2. Current response to an IP3-dependent odorant cocktail after the response to a cAMP-dependent odorant cocktail were adapted (a,c) and those to the cAMP-dependent odorant cocktail after the IP3-dependent odorant cocktail (b,d). An isolated olfactory neuron was first stimulated by application of the cAMP-dependent (a,c) and IPa-dependent odorant cocktails, respectively. After the response to the cocktail was adapted, the solution was changed to a different cocktail. Bars above traces indicate period of each odor stimulation. Traces a and b, c and d were recorded from the same olfactory neuron, respectively. Holding potential was -50 mV.
ments when the order of application of the cocktail was reversed. Traces of a and b were recorded from the same neuron. As shown in Fig. 2b, the cAMP-dependent odorants also induced an inward current after the IPsdependent odorants. Similar results were obtained from 3 different neurons. Mean magnitude of cAMP-induced inward current after the IP3-dependent odorant cocktail was 25 +__9 pA (S.E.M., n = 3). These results indicate that single olfactory neurons discriminate odorants in the cAMP-dependent and the IP3-dependent odorant cocktails and that at least two different receptors exist in a single olfactory neuron. Fig. 2c,d also show current traces recorded from another neuron. In this neuron, both odorant cocktails failed to induced an inward current after the different odorant cocktail. These results suggest that odor specificities of receptors to the cAMP-dependent odorants and the IP3-dependent odorants in this neuron are quite similar to each other. It is likely that both the cAMPdependent and IP3-dependent odorants bind to similar receptors in this type of neuron. Turtle olfactory systems have remarkable ability of
235
odor discrimination. For example, optical isomers such as d-carvone and /-carvone were discriminated at the olfactory bulb level [19]. In a separate study, we examined whether a single olfactory neuron discriminates between d-carvone and/-carvone by measuring action potentials from the olfactory cilia. Application of 0.1 mM d-carvone to the turtle olfactory epithelium increased impulse frequency recorded from single olfactory cilium. After the response to d-carvone was adapted, application of 0.1 mM l-carvone increased impulse frequency (unpublished data). The fact that the response to/-carvone appeared after adaptation of d-carvone suggests that the receptors for d-carvone and l-carvone are different from each other. Hence the above result suggests that a single olfactory neuron has both receptors for d-carvone and l-carvone. These results together with the present results suggest that multiple receptors exist in a single olfactory neuron. [1] Boekhoff, I., Tarelius, E., Strotmann, J. and Breer, H., Rapid activation of alternative second messenger pathways in olfactory cilia from rats by different odorants, EMBO J., 9 (1990) 24532458. [2] Breer, H. and Boekhoff, I., Odorants of the same odor class activate different second messenger pathways, Chem. Senses, 16 (1991) 19 29. [3] Cunningham, A.M., Ryugo, D.K., Sharp, A.H., Reed, R.R., Snyder, S.H. and Ronnett, G.V., Neuronal inositol 1,4,5-trisphosphate receptor localized to the plasma membrane of olfactory cilia, Neuroscience, 57 (1993) 339-352. [4] Firestein, S.F., Picco, C. and Menini, A., The relation between stimulus and response in olfactory receptor cells of the tiger salamander, J. Physiol., 468 (1993) 1-10. [5] Frings, S. and Lindemann, B., Current recording from sensory cilia of olfactory receptor cells in situ. I. The neuronal response to cyclic nucleotides, J. Gen. Physiol., 97 (1990) 1-16. [6] Kahn, A.A., Steiner, J.P. and Snyder, S.H., Plasma membrane inositol 1,4,5-triphosphate receptor of lymphocytes: selective enrichment in sialic acid and unique binding specificity, Proc. Natl. Acad. Sci. USA., 89 (1992) 2849 2853. [7] Kalinoski, D.L., Aldinger, S.B., Boyle, A.G., Huque, T., Marecek, J.F., Prestwich, G.D. and Restrepo, D., Characterization of a novel inositol 1,4,5-triphosphate receptor inisolated olfactory cilia, Biochem. J., 281 (1992) 449456. [8] Kashiwayanagi, M., Kawahara, H., Hanada, T. and Kurihara, K., A large contribution of cyclic AMP independent pathway to the turtle olfactory transduction, J. Gen. Physiol., in press. [9] Nakamura, T. and Gold, G.H., A cyclic nucleotide-gated conductance in olfactory receptor cilia, Nature, 325 (1987) 442~t44. [10] Pace, U., Hasnski, E., Salomon, ¥. and Lancet, D., Odorantsensitive adenylate cyclase may mediate olfactory reception, Nature, 316 (1985) 255 258. [11] Raining, K., Krieger, J., Strotmann, J., Boekhoff, I., Kubick, S., Baumstarkm, C. and Breer, H., Cloning and expression of odorant receptors, Nature, 361 (1993) 353-356. [12] Ressler, K.J., Sullivan, S.L. and Buck, L.B., A zonal organization of odorant receptor gene expression in the olfactory epithelium, Cell, 73 (1993) 597~09. [13] Restrepo, D., Miyamoto, T., Bryant, B.P. and Teeter, J.H., Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish, Science, 249 (1990) 1166-1168. [14] Restrepo, D., Teeter, J.H., Honda, E., Boyle, A.G., Marecek, J.F., Prestwich, G.D. and Kalinoski, D.L., Evidence for an InsP3-gated
236
M. Kashiwayanagi, K. Kurihara/Neuroscience Letters 170 (1994) 233 236
channel protein in isolated rat olfactory cilia, Am. J. Physiol., 63 (1992) C667-C673. [15] Sicard, G.,Olfactorydiscriminationofstructure related molecules: receptor cell responses to camphoraceous odorants, Brain Res., 326 (1985) 203-212. [16] Sklar, P.B., Anholt, R.H. and Snyder, S.H., The odorant-sensitive adenylate cyclase of olfactory receptor cells: differential stimulation by distinct classes of odorants, J. Biol. Chem., 261 (1986) 15538 15543. [17] Suzuki, N., Voltage- and cyclic nucleotide-gated currents in iso-
lated olfactory receptor cells. In J.G. Brand, J.H. Teeter, R.H. Cagan and M.R. Kare (Eds.), Chemical Senses, Vol. 1: Receptor Events and Transduction in Taste and Olfaction, Marcel Dekker, New York, 1989, pp. 469-494. [18] Suzuki, N., IP3-activated ion channel activity in frog olfactory cell, Chem. Senses, 17 (1992) 87 (abstr.). [19] Taniguchi, M., Kashiwayanagi, M. and Kurihara, K., Quantitative analysis on odor intensity and quality of optical isomers in turtle olfactory system, Am. J. Physiol., 62 (1992) R99-R104.
ELSEVIER
NEUROSCIENCE LETTERS
Odor discrimination in single turtle olfactory receptor neuron Makoto Kashiwayanagi*, Kenzo Kurihara Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan
Received 5 January 1994; Revised version received 3 February 1994; Accepted 3 February 1994
Al~traet To explore the ability of odor discrimination of olfactory receptor neurons, current responses to odorant cocktails were recorded from an isolated olfactory neuron of the turtle. Twenty-five percent of the neurons tested responded to both cAMP-dependent and the IP3-dependent odorant cocktails. Application of the cAMP-dependent (or the IP3-dependent) odorant cocktail to the neuron after an inward current induced by the IP3-dependent (or the cAMP-dependent) odorant cocktail was adapted induced a large inward current in the neuron. The results suggest that at least two different receptors exist in a single olfactory neuron. Key words: Isolated olfactory neuron; Whole-cell patch-clamp; Odorant-activated current; Odor discrimination; Transduction; Second messenger
It is now generally believed that the G-protein-linked cAMP pathway contributes to the generation of olfactory responses [1,5,9,10,16,17]. It is also pointed out that the IP3-dependent pathway plays an important role in olfactory transduction [2,6,7,13,14,18]. Recently, we showed a new pathway independent of neither cAMP nor IP 3 also contributes to the turtle olfactory response [8]. These results suggest that there are multiple transduction pathways in the olfactory transduction systems. The first aim in the present study is to explore whether a single neuron of the turtle has only a single second messenger pathway. In an earlier study, Sicard examined the odor responses of frog single olfactory neurons to 20 odorants having different molecular structures and odor qualities [15]. In 75% of them, more than 2 odorants induced odor responses. Firestein et al. also recorded that 47% of isolated salamander olfactory neurons responded to different odorants [4]. These results suggest that multiple receptors exist in the single olfactory neurons of the frog and salamander. On the other hand, Raming et al. demonstrated that in OR5 (G-protein-coupled receptor)-expressing St9 cells, IP3 was produced by various odorants
*Corresponding author. Fax: (81) 11-717-3267. 0304-3940/94/$7.00© 1994 Elsevier ScienceIreland Ltd. All rights reserved SSDI 0304-3940(94)00139-2
having different molecular structures and odor qualities [11]. These broad tunings of odor response suggest that the responsiveness of a single neuron to different odorants does not always mean existence of multiple receptors in the single olfactory neurons. In addition, on the basis of results of gene expression of putative olfactory receptors in the rat olfactory epithelium, Ressler et al. discussed that a single neuron may select one gene for expression [12]. In the present study, we examine whether a single olfactory neuron isolated from the turtle olfactory epithelium has different receptors by using the cross-adaptation method. Results suggest that at least two different receptors exist in a single olfactory neuron and a single neuron has an ability of discriminating odorants. Turtles, Geoclemys reevesii, weighing 150-300 g, were obtained from commercial suppliers and maintained at 22°C. Animals were fed porcine and bovine liver ad libitum. Animals were cooled to 0°C and decapitated. The nasal cavities were opened, and the olfactory epithelia were quickly removed. The epithelia were cut into slices of about 300 p m thickness in normal Ringer solution at 0°C and stored at 4°C. Slices were incubated for 30 min to 2 h at 37°C in Ca-free Ringer solution for the isolation of olfactory neurons. Immediately prior to recording, one slice of the epithelium was placed in 500 ¢tl normal Ringer solution in the recording chamber and
234
M. Kashiu'ayanagi, K. Kurihara / Neuroscience Letters 170 (1994) 233 236
shaken. No enzymes or proteases were added. Turtle olfactory neurons were easily distinguished from other types of cells such as respiratory and basal cells based on their characteristic morphology. Recordings were made with an Axopatch 1C amplifier (Axon Instruments, Inc., Foster City, CA) using patch electrodes of borosilicate glass. Electrodes with resistance of 5-10 Mr2 were manufactured on a Narishige PP83 puller (Narishige Co., Tokyo, Japan) using a double stage pull. Gigaohm seals were obtained by applying negative pressure (-30 to -100 cmH20). The whole-cell configuration was attained by application of additional negative pressure. The current signal was digitized and stored on video tape. Gravity was used to deliver a constant stream of Ringer solution from the stimulating tube. Three electrically actuated valves were used to switch adapting Ringer solution and odorant cocktail solutions. The stimulating tube with a lumen 160 200/~m in diameter was placed under visual control within about 500/~m of the cell. Normal Ringer solution contained (mM): 116 NaC1, 4 KC1, 2 CaCI2, 2 MgC12, 15 glucose, 5 sodium pyruvate, 10 HEPES-NaOH (pH 7.4). For the incubation of slices, 2 mM CaCI2 was removed from normal Ringer solution. Patch pipettes were filled with an internal solution (mM): 115 KCI, 5 NaCI, 2 MgC12, 5 ATP, 10 HEPES-KOH (pH 7.4). The cAMP-dependent odorant cocktail consisted of 200/JM each of citralva, hedione, eugenol,/-carvone and cineole, which were reported to increase cAMP concentration in the rat and bullfrog cilia preparations but unchanged IP3 concentration [2,16], in normal Ringer solution. The IP3-dependent odorant cocktail contained ~M): 40 lilial, 40 lyral, 20 ethyl vanillin and 100 limonene, which are reported to increase IP3 concentration in the rat cilia preparation but unchange cAMP concentration in the rat and the bullfrog [2,16]. Although we have no evidence, it is highly probable that the same species of odorants increase the same second messenger in the turtle olfactory neuron because the same results were obtained from quite different animal such as the bullfrog and the rat. Therefore, we used above odorants as cAMP-dependent or IP3-dependent odorants. All odorants were kindly supplied from Takasago International (Tokyo, Japan). Whole-cell recordings were obtained from a total of 44 olfactory neurons. Only neurons with motile cilia were used for the recording. Fig. la,b show inward currents induced by application of the odorant cocktails in an isolated olfactory neuron. Application of the cAMP-dependent odorant cocktail (Fig. la) and the IP3-dependent odorant cocktail (Fig. lb) induced inward currents in the same olfactory neuron. Fig. lc shows number of neurons which responded to both cocktails, the cAMP-dependent odorant cocktail, or the IP3-dependent odorant cocktail. Eleven olfactory neurons (25% of neurons recorded in
a
b
cAMP-dependent Odorants g,"/,/,/,///////////////,d
IP3-dependent Odorants I I
12s]10 pA
C
BothCocktails
cAMP-dependent Odorants
IP3-dependent Odorants
No ReSponse~
~
[
I
, i
0
10
20
Number of Neurons
Fig. 1. Inward current in response to a cAMP-dependent odorant cocktail (a) and to an 1P3-dependent odorant cocktail (b). Both responses were recorded from the same neuron. Bars above traces indicate period of each odor stimulation. Numbers of preparations which responded to both cocktails, the cAMP-dependent odorant cocktail, and the IP3-dependent odorant cocktail (c). Holding potential was -50 mV.
the study) responded to both odorant cocktails, suggesting that both the cAMP-dependent pathway and the IP3-dependent pathway exist in a single olfactory neuron. These data are consistent with a recent immunohistochemical data reported by Cunningham et al.; Golfand IP 3 receptor were expressed in essentially all olfatory neuron of the rat [3]. Cross-adaptation experiments were undertaken to examine whether different receptors exist in a single olfactory neuron (Fig. 2). Application of the cAMP-dependent odorant cocktail induced an inward current. After the current was adapted to the basal level, the IP3-dependent odorant cocktail induced an inward current (Fig. 2a). Similar results were obtained from 6 different olfactory neurons. Mean magnitude of inward current induced by the IP3-dependent odorant cocktail after the cAMP-dependent one was 89 + 43 pA (S,E.M., n = 6). Fig. 2b shows the results of the cross-adaptation experi-
M. Kashiwayanagi, K. Kuriharal Neuroscience Letters 170 (1994) 233-236 cAMP-dependent IP3-clependent IP3-dapendent cAMP-dependent Odorants Odorants Odorants Odorants ~"///////,~ P'//////A I
a
cAMP-dependent IP3-dependent Odorants Odorants ~///I//1111/,,41 I
IP3-dependent cAMP-dependent Odorants Odorants I
,
v////4
d 50 pA Fig. 2. Current response to an IP3-dependent odorant cocktail after the response to a cAMP-dependent odorant cocktail were adapted (a,c) and those to the cAMP-dependent odorant cocktail after the IP3-dependent odorant cocktail (b,d). An isolated olfactory neuron was first stimulated by application of the cAMP-dependent (a,c) and IPa-dependent odorant cocktails, respectively. After the response to the cocktail was adapted, the solution was changed to a different cocktail. Bars above traces indicate period of each odor stimulation. Traces a and b, c and d were recorded from the same olfactory neuron, respectively. Holding potential was -50 mV.
ments when the order of application of the cocktail was reversed. Traces of a and b were recorded from the same neuron. As shown in Fig. 2b, the cAMP-dependent odorants also induced an inward current after the IPsdependent odorants. Similar results were obtained from 3 different neurons. Mean magnitude of cAMP-induced inward current after the IP3-dependent odorant cocktail was 25 +__9 pA (S.E.M., n = 3). These results indicate that single olfactory neurons discriminate odorants in the cAMP-dependent and the IP3-dependent odorant cocktails and that at least two different receptors exist in a single olfactory neuron. Fig. 2c,d also show current traces recorded from another neuron. In this neuron, both odorant cocktails failed to induced an inward current after the different odorant cocktail. These results suggest that odor specificities of receptors to the cAMP-dependent odorants and the IP3-dependent odorants in this neuron are quite similar to each other. It is likely that both the cAMPdependent and IP3-dependent odorants bind to similar receptors in this type of neuron. Turtle olfactory systems have remarkable ability of
235
odor discrimination. For example, optical isomers such as d-carvone and /-carvone were discriminated at the olfactory bulb level [19]. In a separate study, we examined whether a single olfactory neuron discriminates between d-carvone and/-carvone by measuring action potentials from the olfactory cilia. Application of 0.1 mM d-carvone to the turtle olfactory epithelium increased impulse frequency recorded from single olfactory cilium. After the response to d-carvone was adapted, application of 0.1 mM l-carvone increased impulse frequency (unpublished data). The fact that the response to/-carvone appeared after adaptation of d-carvone suggests that the receptors for d-carvone and l-carvone are different from each other. Hence the above result suggests that a single olfactory neuron has both receptors for d-carvone and l-carvone. These results together with the present results suggest that multiple receptors exist in a single olfactory neuron. [1] Boekhoff, I., Tarelius, E., Strotmann, J. and Breer, H., Rapid activation of alternative second messenger pathways in olfactory cilia from rats by different odorants, EMBO J., 9 (1990) 24532458. [2] Breer, H. and Boekhoff, I., Odorants of the same odor class activate different second messenger pathways, Chem. Senses, 16 (1991) 19 29. [3] Cunningham, A.M., Ryugo, D.K., Sharp, A.H., Reed, R.R., Snyder, S.H. and Ronnett, G.V., Neuronal inositol 1,4,5-trisphosphate receptor localized to the plasma membrane of olfactory cilia, Neuroscience, 57 (1993) 339-352. [4] Firestein, S.F., Picco, C. and Menini, A., The relation between stimulus and response in olfactory receptor cells of the tiger salamander, J. Physiol., 468 (1993) 1-10. [5] Frings, S. and Lindemann, B., Current recording from sensory cilia of olfactory receptor cells in situ. I. The neuronal response to cyclic nucleotides, J. Gen. Physiol., 97 (1990) 1-16. [6] Kahn, A.A., Steiner, J.P. and Snyder, S.H., Plasma membrane inositol 1,4,5-triphosphate receptor of lymphocytes: selective enrichment in sialic acid and unique binding specificity, Proc. Natl. Acad. Sci. USA., 89 (1992) 2849 2853. [7] Kalinoski, D.L., Aldinger, S.B., Boyle, A.G., Huque, T., Marecek, J.F., Prestwich, G.D. and Restrepo, D., Characterization of a novel inositol 1,4,5-triphosphate receptor inisolated olfactory cilia, Biochem. J., 281 (1992) 449456. [8] Kashiwayanagi, M., Kawahara, H., Hanada, T. and Kurihara, K., A large contribution of cyclic AMP independent pathway to the turtle olfactory transduction, J. Gen. Physiol., in press. [9] Nakamura, T. and Gold, G.H., A cyclic nucleotide-gated conductance in olfactory receptor cilia, Nature, 325 (1987) 442~t44. [10] Pace, U., Hasnski, E., Salomon, ¥. and Lancet, D., Odorantsensitive adenylate cyclase may mediate olfactory reception, Nature, 316 (1985) 255 258. [11] Raining, K., Krieger, J., Strotmann, J., Boekhoff, I., Kubick, S., Baumstarkm, C. and Breer, H., Cloning and expression of odorant receptors, Nature, 361 (1993) 353-356. [12] Ressler, K.J., Sullivan, S.L. and Buck, L.B., A zonal organization of odorant receptor gene expression in the olfactory epithelium, Cell, 73 (1993) 597~09. [13] Restrepo, D., Miyamoto, T., Bryant, B.P. and Teeter, J.H., Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish, Science, 249 (1990) 1166-1168. [14] Restrepo, D., Teeter, J.H., Honda, E., Boyle, A.G., Marecek, J.F., Prestwich, G.D. and Kalinoski, D.L., Evidence for an InsP3-gated
236
M. Kashiwayanagi, K. Kurihara/Neuroscience Letters 170 (1994) 233 236
channel protein in isolated rat olfactory cilia, Am. J. Physiol., 63 (1992) C667-C673. [15] Sicard, G.,Olfactorydiscriminationofstructure related molecules: receptor cell responses to camphoraceous odorants, Brain Res., 326 (1985) 203-212. [16] Sklar, P.B., Anholt, R.H. and Snyder, S.H., The odorant-sensitive adenylate cyclase of olfactory receptor cells: differential stimulation by distinct classes of odorants, J. Biol. Chem., 261 (1986) 15538 15543. [17] Suzuki, N., Voltage- and cyclic nucleotide-gated currents in iso-
lated olfactory receptor cells. In J.G. Brand, J.H. Teeter, R.H. Cagan and M.R. Kare (Eds.), Chemical Senses, Vol. 1: Receptor Events and Transduction in Taste and Olfaction, Marcel Dekker, New York, 1989, pp. 469-494. [18] Suzuki, N., IP3-activated ion channel activity in frog olfactory cell, Chem. Senses, 17 (1992) 87 (abstr.). [19] Taniguchi, M., Kashiwayanagi, M. and Kurihara, K., Quantitative analysis on odor intensity and quality of optical isomers in turtle olfactory system, Am. J. Physiol., 62 (1992) R99-R104.