Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Taste Receptor Cells Responding with Action Potentials to Taste Stimuli and their Molecular Expression of Taste Related Genes
Section of Oral Neuroscience, Graduate School of Dental Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Correspondence to be sent to: Yuzo Ninomiya, e-mail: nino{at}dent.kyushu-u.ac.jp
Key words: apical taste stimulation, gustatory sensitivity, loose patch recording, mouse fungiform papillae, single cell RT–PCR, taste receptor, taste transduction
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Introduction |
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 Top Introduction Materials and methodsResultsDiscussionAcknowledgementsReferences |
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Recent molecular biological studies have revealed many molecular aspects of taste transduction in receptor cells for each taste categories which are referred to as salty, sweet, sour, bitter and umami in human respectively. (Lindemann, 2001
;
Gilbertson and Boughter, 2003). For
example, ion channels such as ENaC, ASIC and HCN mediate salty and sour taste and
G-protein-coupled receptors such as T1Rs and T2Rs play a key role in sweet, bitter and
umami taste. However, there is little evidence for involvements of these molecules in
taste reception and transduction at the cellular level. It is necessary to examine both
molecular and physiological properties in single taste receptor cell. In this study, by
use of loose patch recording technique combined with single cell multiplex RT–PCR,
we characterized single taste cells by their responses to four basic taste stimuli and
expression of taste-related molecules. |
Materials and methods |
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TopIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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All experimental procedures were approved by the committee for Laboratory Animal Care and Use at Kyushu University, Japan. Subjects were adult C57BL/6N or C57BL/KsJ mice at >2 months of age. Animals were anesthetized and killed by cervical dislocation. The anterior part of tongue was removed and subjected to enzyme treatment. The lingual epithelium was peeled from underlying tissue. Individual taste buds of fungiform papilla were excised from the epithelial sheet and drawn into the orifice of the stimulating pipette (40–50 µm). A gentle suction on the pipette was maintained to perfuse taste solutions and to hold the taste bud in place. The bath solution [Tyrode solution containing (in mM): NaCl, 140; KCl, 5; CaCl2, 1; MgCl2, 1; HEPES, 10; glucose, 10; sodium pyruvate, 10; pH = 7.40) was continuously perfused. The electrical responses of taste receptor cells in intact taste buds were recorded with a loose patch configuration from basolateral side in voltage or current clamp mode.
For data analysis, the number of spikes in unit time was counted throughout the recording. The mean spontaneous impulse discharge for each taste cells was calculated by averaging the number of spikes over the 10 s period that distilled water was applied to the taste pore. Numbers of spikes larger than the mean plus one standard deviation of the spontaneous impulse discharges was taken as the final criterion for the occurrence of a response.
For the RT–PCR experiment, a single taste cell was collected from a taste bud and transferred to a PCR tube after recording of responses. Several target sequences were simultaneously amplified by multiplex PCR with nested PCR primers and two rounds of amplification. Each pair of primers spanned at least one intron sequence to distinguish between products amplified from cDNA and genomic DNA. PCR product size for each genes was 900–1000 base pairs for RT and first round PCR and 300–400 base pairs for second round PCR.
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Results |
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TopIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Using our experimental setup, spontaneous spike activities were recorded in some taste cells under loose patch configuration. Each spike was recorded as negative current followed by positive current in V-clamp mode and as positive potential followed by negative potential in I-clamp mode. We examined the effect of voltage gated sodium channel blocker, tetrodotoxin (TTX) on these spike activities. Bath application of 1 µM TTX completely inhibited spike activities and they recovered after wash out of TTX (Figure 1A), indicating that spike activities result from action potentials (currents) in taste receptor cells. In our experiments, action currents of taste receptor cells were also affected by potassium channel blocker, tetraethylammonium (TEA). The peak size of positive current was decreased and duration between negative and positive peak was prolonged by bath application of 20 mM TEA and recovered after wash out of TEA (Figure 1A,B). Because bath application of these blockers affected action currents of taste cells, these channels exist on the basolateral membrane of taste cells.
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Next, we examined response of taste receptor cells to apical taste stimuli. NaCl, saccharin, HCl and quinine were used as taste stimulants. We recorded responses to taste stimuli as increase in firing frequency of taste receptor cells. Many taste cells responded to one of four taste stimuli and some clearly responded to multiple taste stimuli. After recording of taste response, taste receptor cells were collected from taste buds to examine their molecular expression by multiplex RT–PCR. Collection of single taste receptor cell was visually confirmed. In RT–PCR experiments, we investigated expression of sweet receptor component, T1r3 and G-protein
-subunit,
gustducin in taste receptor cells responding to saccharin. Our preliminary results of
these experiments were summarized in Table
1. This suggests a possibility of the
existence of multiple receptor and transduction systems for sweet taste. Thus, this
technique might be useful to investigate molecular aspects of taste receptor cells
responding to taste stimuli.
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Discussion |
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TopIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We recorded impulse discharges of taste cells under the loose patch configuration. Action potentials in taste receptor cells were described first in amphibian species (Kashiwayanagi et al., 1983
;
Roper, 1983) and have been reported
in mammals (Avenet and Lindemann,
1991;
Gilbertson et al., 1992;
Cummings et al., 1993). The
underlying current properties of action potentials in mammalian taste cells have been
well documented (Herness and Sun,
1995;
Chen et al., 1996). In our
experiments, bath application of TTX and TEA inhibited or modulated action currents of
taste cells, indicating voltage gated sodium and potassium channels contribute to
generation of action potentials in taste receptor cells. It has been reported that sweet
(Cummings et al., 1993),
salty (Avenet and Lindemann, 1991),
sour (Gilbertson et al.,
1992) and bitter (Furue and Yoshii,
1997) stimuli elicited action potentials in taste receptor cells. In our
study, responses of taste receptor cells to apical taste stimuli were recorded as
increase in firing frequency. These results suggest that voltage gated
Na+ and K+ channels might be necessary for taste
transduction in taste receptor cells.
There is little evidence for molecular expression in taste receptor cells responding
to taste stimuli. Recently, expression of G-protein subunit -gustducin and
Gi was examined in taste cells responding to bitter stimuli by using
Ca2+ imaging and immunohistochemical method (Caicedo et al., 2003). In the present study,
molecular expression of taste receptor cells responding to taste stimuli was examined by
using loose patch recording and multiplex RT–PCR. Both techniques may be useful for
investigating physiological and molecular properties of taste receptor cells; however,
there are some problems in detection of molecular expression. In immunohistchemical
experiment, the number of genes of which expression can be simultaneously detected is
limited. In multiplex RT–PCR experiment, we cannot distinguish between PCR products
amplified from cDNA or genomic DNA if mRNA is transcribed from single exon sequence such
as T2Rs. In order to investigate molecular expression in taste receptor cells more in
detail, further improvements are needed.
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Acknowledgements |
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TopIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We thank Dr Stephen D. Roper for valuable comments on the manuscript and Drs Bernd Lindemann, Satoru Ishizuka and Akihiko Okabe for technical advice. This work was supported by Grants-in-Aid 15209061 (Y.N.) and 16791127 (N.S.) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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References |
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TopIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Avenet, P. and Lindemann, B. (1991) Non-invasive recording of receptor cell action potentials and sustained currents from single taste buds maintained in the tongue: the response to mucosal NaCl and amiloride. J. Membr. Biol., 124, 33–41.[CrossRef][Web of Science][Medline]
Caicedo, A., Pereira, E., Margolskee, R.F. and Roper, S.D. (2003) Role of the G-protein subunit -gustducin in taste cell responses to bitter stimuli. J. Neurosci., 23, 9947–9952.
Chen, Y., Sun, X.D. and Herness, S. (1996) Characteristics of action potentials and their underlying outward currents in rat taste receptor cells. J. Neurophysiol., 75, 820–831.
Cummings, T.A., Powell, J. and Kinnamon, S.C. (1993) Sweet taste transduction in hamster taste cells: evidence for the role of cyclic nucleotides. J. Neurophysiol., 70, 2326–2336.
Furue, H. and Yoshii, K. (1997) In situ tight seal recordings of taste substance elicited action currents and voltage-gated Ba currents from single taste bud cells in the peeled epithelium of mouse tongue. Brain Res., 776, 133–139.[CrossRef][Web of Science][Medline]
Gilbertson, T.A. and Boughter, J.D., Jr (2003) Taste transduction: appetizing times in gustation. Neuroreport, 14, 905–911.[CrossRef][Web of Science][Medline]
Gilbertson, T.A., Avenet, P., Kinnamon, S.C. and Roper, S.D. (1992) Proton currents through amiloride-sensitive Na+ channels in hamster taste cells. Role in acid transduction. J. Gen. Physiol., 100, 803–824.
Herness, M.S. and Sun, X.D. (1995) Voltage-dependent sodium currents recorded from dissociated rat taste cells. J. Membr. Biol., 146, 73–84.[Web of Science][Medline]
Kashiwayanagi, M., Miyake, M. and Kurihara, K. (1983) Voltage-dependent Ca2+ channel and Na+ channel in frog taste cells. Am. J. Physiol., 244, C82–C88.
Lindemann, B. (2001) Receptors and transduction in taste. Nature, 413, 219–225.[CrossRef][Medline]
Roper, S.D. (1983) Regenerative impulses in taste cells. Science, 220, 1311–1312.
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