Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Gap Junctions among Taste Bud Cells in Mouse Fungiform Papillae
Graduate school of Life Science and Systems Engineering, Kyushu Institute of Technology, Wakamatsu-Ku, Kitakyushu 808-0196, Japan
Correspondence to be sent to: Kiyonori Yoshii, e-mail: yoshii{at}brain.kyutech.ac.jp
Key words: cell-networks, dye-coupling, in situ patch clamp
 |
Introduction |
|---|
 Top Introduction Materials and methodsResults and discussionReferences |
|---|
Mouse taste buds in fungiform papillae consist of
50 cells (TBCs; unpublished
data), but only a few of them have synaptic contacts with taste nerves (Kinnamon et al., 1993
;
Seta and Toyoshima, 1995). Neither
chemical nor electrical synapses/gap junctions have been confirmed in mammalian taste
buds, though a subpopulation of TBCs expressed a variety of neurotransmitter receptors
(Kumazawa et al., 2001;
Hayato et al., 2002).
Non-innervated TBCs thus have been assumed to be supportive. However, we considered their
roles in taste transduction.
We tested this hypothesis under in situ whole-cell patch clamp and optical
recording conditions (Ohtubo et al.,
2001). Patch clamp studies showed that a part of TBCs generated depolarizing
or hyperpolarizing receptor potentials in response to taste substances. Optical
recordings with a voltage-sensitive dye showed that such chemosensitive TBCs tended to
form colonies by the polarity of their receptor potentials, suggesting the existence of
any interactions among TBCs.
In the present study, we show the diffusion of probe dyes from injected TBCs to their neighbors (dye-couplings), which evidences the existence of intercellular corridor between TBCs. These results suggest that there are TBC networks within mammalian taste buds, and that taste buds are miniature processing units rather than the aggregation of chemosensitive cells.
|
Materials and methods |
|---|
|
TopIntroductionMaterials and methodsResults and discussionReferences |
|---|
Peeled lingual epithelia
We prepared peeled lingual epithelia as described previously (Furue and Yoshii, 1997, 1998) in accordance with Guiding
Principles for the Care and Use of Animals in the Field of Physiological Sciences
approved by the Council of the Physiological Society of Japan. In brief, we obtained
tongues from mice etherized and then decapitated, subcutaneously injected a collagenase
or elastase solution into tongues, peeled the epithelia with forceps 5 min after the
injection, and mounted on a recording platform (Figure
1). Under the conditions, the
preserved epithelium and taste bud structures protected the basolateral membranes of TBCs
as an effective barrier against deionized water and taste solutions such as high
concentration of HCl or NaCl for >60 min (Furue
and Yoshii, 1997, 1998).
|
Detection of dye-couplings
We injected Lucifer Yellow CH (LY, 2 mg/ml) or biocytin (BC, 2 mg/ml) into TBCs through patch pipettes placed on the basolateral membranes of the TBC under in-situ voltage-clamp conditions (Figure 1). The peeled epithelia that contained LY-injected TBCs were examined either in fresh or after fixation. Those that contained BC-injected TBCs were fixed and then stained with streptavidin rhodamine red-X conjugate. The nuclei of TBCs in the fixed preparations were stained with acridine orange, a membrane permeable dye. The identification of nuclei within biocytin-coupled TBCs evidenced that stained structures were dye-coupled TBCs but not single complex TBCs, because TBCs are mononuclear.
We epi-illuminated the fresh preparations through a dichroic mirror unit, which
reflected wavelengths between 400 and 440 nm, excited LY (
max =
428 nm) through the 60x water-immersion objective and transmitted fluorescence
through a high pass filter (>475 nm) to a cooled CCD camera. The fixed preparation was
examined with a confocal laser microscope with similar filters.
|
Results and discussion |
|---|
|
TopIntroductionMaterials and methodsResults and discussionReferences |
|---|
Dye couplings
Many cells absorb biocytin leaked from recording patch electrodes and yielded false biocytin-couplings. Although no TBCs absorbed biocytin when the electrode was placed 30 µm apart for 60 s, they absorbed it when the electrodes were placed 1 µm apart from them for >30 s. We eliminated such artifacts by forming giga-ohm seal in 10 s when recording electrodes left the 30 µm apart position. No TBCs absorbed LY.
The diffusion of biocytin from injected TBCs to another (biocytin-coupling) occurred in 10 TBCs of 56 examined and that of LY in five TBCs of 29 examined. There was no difference between biocytin-couplings and LY couplings in the occurrence ratio of dye-couplings. Single biocytin-couplings involved 2–5 TBCs with mean ± SD of 3.1 ± 1.2 (n = 9). TBCs were different in morphology; rod, branched, flat and round types. Also, a group of TBCs extended their apical portions to the taste pore of respective taste buds and others did not. It is likely that dye-couplings occur irrespective of such morphological differences.
TBCs elicited various voltage-gated currents, such as TTX-sensitive
Na+ currents, TEA-sensitive outwardly rectifying K+
currents, inwardly rectifying K+ currents, Cl– currents,
high-voltage and low-voltage activated Ca2+ currents (Furue and Yoshii, 1997;
Noguchi et al., 2003). There
are no differences between coupled and non-coupled TBCs in the magnitude of their pooled
currents. Also, there were no differences in the pooled magnitude of membrane conductance
and membrane capacitance.
Junctional conductance
These results suggest that the junctional conductance of gap junctions is as low as
membrane conductance. However, the occurrence of LY-couplings suggests that the
junctional conductance is more than 2 nS (Dermietzel and Spray, 1993). On the other hand, the membrane
conductance of non-coupled cells was 1 nS or less. Under whole-cell clamp
conditions, the simplified equivalent electric circuit for coupled TBCs is a 2 nS
conductor (junctional conductance) in series with a 1 nS conductor (membrane conductance
of a coupled TBC). The total membrane conductance and capacitance must be higher than
those of non-coupled TBCs.
The membrane conductance and membrane capacitance of TBCs were different from cell to cell. Averaging procedures seem to conceal the differences between coupled and non-coupled TBCs in the magnitude of these membrane properties. If it was possible to regulate the gating of gap junctions, statistic tests such as paired t-test would discriminate coupled and uncoupled TBCs.
TBC networks
Dye-couplings (Sata et al.,
1992) and both dye-couplings and electrical couplings (Bigiani and Roper, 1993) showed gap junctions in amphibian
TBCs. However, extensive electron microscopic studies have failed to confirm the
occurrence of gap junctions in mammalian taste buds, though they had been reported 36
years ago in rat vallate papillae (Akisaka and
Oda, 1978). Dye-couplings appear to be more sensitive in detecting gap
junctions than electron microscopy. Gap junctions are thus found in both mammalian and
amphibian taste buds. They may be fundamental devices in taste transduction.
In mouse fungiform papillae, only type III cells have synaptic contacts with taste nerve. It appears that type II cells transmit taste information to type III cells through gap junctions. When type III cells have neurotransmitter receptors, paracrine systems in addition to gap junctions can transmit taste information. Thus gap junctions together with paracrine systems contribute in forming TBC networks which process taste information (Figure 2).
|
|
References |
|---|
|
TopIntroductionMaterials and methodsResults and discussionReferences |
|---|
Akisaka, T. and Oda, M. (1978) Taste buds in the vallate papillae of the rat studied with freeze-fracture preparation. Arch. Histol. Jpn, 41, 87–98.[Medline]
Bigiani, A. and Roper, S.D. (1993) Identification of electrophysiologically distinct cell subpopulations in Necturus taste buds. J. Gen. Physiol., 102, 143–170.
Dermietzel, R. and Spray, D.C. (1993) Gap junctions in the brain: where, what type, how many and why? Trends Neurosci., 16, 186–192.[CrossRef][Web of Science][Medline]
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]
Furue, H. and Yoshii, K. (1998) A method for in-situ tight-seal recordings from single taste bud cells of mice. J. Neurosci. Methods, 84, 109–114.
Hayato, R., Kitabori, T., Katayama, Y., Takeuchi, K., Kumazawa, T., Endo, S. and Yoshii, K. (2002) Oscillating currents and [Ca2+] in increase in response to 5-HT applied to the basolateral membranes of mouse taste bud cells. Soc. Neurosci. Abstr., 355, 12.
Kinnamon, J.C., Henzler, D.M. and Royer, S.M. (1993) HVEM ultrastructural analysis of mouse fungiform taste buds, cell types, and associated synapses. Microsc. Res. Technol., 26, 142–156.[CrossRef][Web of Science][Medline]
Kumazawa, T., Shiobara, S., Matsumoto, T., Ohtubo, Y., Nakatani, K. and Yoshii, K. (2001) In situ imaging of cytosolic calcium in mouse taste bud cells. Soc. Neurosci. Abstr., 287, 7.
Noguchi, T., Ikeda, Y., Miyajima, M. and Yoshii, K. (2003) Voltage-gated channels involved in taste responses and characterizing taste bud cells in mouse soft palates. Brain Res., 982, 241–259.[CrossRef][Web of Science][Medline]
Ohtubo, Y., Suemitsu, T., Shiobara, S., Matsumoto, T., Kumazawa, T. and Yoshii, K.Y. (2001) Optical recordings of taste responses from fungiform papillae of mouse in situ. J. Physiol., 530, 287–293.
Sata, O., Okada, Y., Miyamoto, T. and Sato, T. (1992) Dye-coupling among frog (Rana catesbeiana) taste disk cells. Comp. Biochem. Physiol., 103, 99–103.[CrossRef]
Seta, Y. and Toyoshima, K. (1995) Three-dimensional structure of the gustatory cell in the mouse fungiform taste buds: a computer-assisted reconstruction from serial ultrathin sections. Anat. Embryol. (Berl.), 191, 83–88.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. M. Breza, K. S. Curtis, and R. J. Contreras Monosodium Glutamate but not Linoleic Acid Differentially Activates Gustatory Neurons in the Rat Geniculate Ganglion Chem Senses, November 1, 2007; 32(9): 833 - 846. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||