Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Recovery of Salt Taste Responses and PGP 9.5 Immunoreactive Taste Bud Cells during Regeneration of the Mouse Chorda Tympani Nerve
Section of Oral Neuroscience, Graduate School of Dental Sciences, Kyushu University, Fukuoka 812-8582, Japan
Correspondence to be sent to: Yuzo Ninomiya, e-mail: nino{at}dent.kyushu-u.ac.jp
Key words: amiloride, fungiform papillae, PGP9.5, regeneration, sodium taste, taste nerve
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Introduction |
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Taste receptor cells are replaced with an average life span of ~10 days in mammals. This turnover is accompanied by continuing synaptic reconnection between newly formed taste cells and gustatory fibers. However, little is known of how sensory information of the fibers is maintained during the synaptic reconnection. Our previous study examined taste responses of regenerated mouse chorda tympani (CT) fibers and revealed that each fiber type classified based on sensitivity to amiloride maintains its characteristics after synapse reformation between regenerated taste axons and receptor cells. That is, we found that there are approximately equal numbers of two types of NaCl-responsive neurons; one type showed strong suppression by amiloride [amiloride-sensitive (AS), N-type fiber], and the other type showed only weak or no suppression by amiloride [amiloride-insensitive (AI), E-type fiber] in intact, regenerated and cross-regenerated taste nerve (Ninomiya, 1998
). Our subsequent study investigated the processes of reformation of AI
and AS neural systems during the CT regeneration. We postulated and verified whether
incoming regenerated CT axons would (i) induce AI and AS properties after synapse
formation with identical progenitors; (ii) innervate taste cells randomly followed by
elimination of mismatched branches; or (iii) selectively innervate AS or AI taste
progenitor cells (Yasumatsu et al.,
2003). The results revealed that NaCl responses of the CT recovered from 3
weeks after the nerve crush and most NaCl responsive fibers showed AI (E-type). The
number of fibers responding to NaCl after amiloride formed a bimodal distribution from 4
weeks and there were no clusters of fibers with intermediate sensitivity to amiloride
(Yasumatsu et al., 2003,
figure 6). Moreover, N-type and E-type were clearly different in Kd
value and response selectivity (KCl/NaCl response ratios) right from the beginning of
their appearance. Thus, these findings from our electrophysiological studies are
consistent with the view that regenerating taste axons selectively innervate their
corresponding classes of taste progenitor cells. In the present study, to test the
possibilities further, we examined PGP9.5, a maker of neurons (Thompson et al., 1983) and sensory paraneurons
(Iwanaga et al., 1992),
immunoreactive (IR) taste bud cells and the number of taste buds after crushing the mouse
CT nerve. |
Materials and methods |
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Experimental manipulation
Subjects were adult male and female C57BL/6N Crj mice (Charles River Japan, Tokyo,
Japan), 8–20 weeks of age, ranging in weight from 20 to 32 g. At 8–10 weeks
of age, mice were divided into five groups, one intact control group and four nerve-crush
groups, respectively 2, 3, 4 and 
5 weeks after bilateral CT nerves were crushed.
Animals were anesthetized with pentobarbital sodium (40–50 mg/kg i.p.), and the
bilateral CT nerves were exposed at ~5 mm rostrally apart from their entry to the bulla
and repeatedly crushed at a single point with number 5 forceps.
Tissue preparation
The animals from the five groups were anesthetized with pentobarbital and perfused through the left ventricle with a physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Tongues were excised and immersed in the same fixative for 3 h at 4°C and then rinsed with 0.1 M PB containing 20% sucrose for 12 h. The tongues were divided longitudinally at the median sulcus into two pieces with equal width, one piece for immunohistochemistry of PGP 9.5 and the other for hematoxylin and eosin staining to measure the number of taste buds. Cryostat sections were cut and thawed onto gelatine-coated slides.
Immunohistochemistry
Sections, 10 µm thick, were treated with 10% normal donkey serum in PBS for 60 min, having first been immunoreacted with rabbit anti-PGP9.5 polyclonal antibody (Biogenesis) at a dilution of 1:600 in PBS for 12 h at 4°C. The sections were then reacted with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Jackson Labs) at a dilution of 1:600 for 2 h at room temperature. Slides were rinsed with PBS and coverslipped. PGP9.5-IR cells were counted in complete sets of serial sections of randomly selected taste buds. The number of taste buds with taste pores was counted in the remaining halves of the tongues. Complete serial sections with 20 µm thickness were stained with hematoxylin and eosin. All images were collected with a CCD camera using DP controller (Olympus) software.
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Results and discussion |
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Fibers and bundles in the containing PGP 9.5 were frequently seen in the whole papilla, including the epithelium and the core of the papilla containing connective tissue. Fibers were seen entering epithelium and taste buds when taste buds were present (Figure 1A,C–E). Some taste bud cells were found to exhibit PGP 9.5-IR. The immunoreactive cells are slender and contain round or invaginated nucleus (Figure 1A). At 2 weeks after crushing the CT, dermal indentations into the epithelium, papillae without taste buds, could be seen (Figure 1B). There was no significant difference in the number of papillae among all groups (data not shown). Nerve fibers often penetrate into epithelium of the papillae (Figure 1B), and some fungiform papillae contain one taste bud primordium or a taste bud containing a taste pore. The existence of a taste pore is often used as a criterion of functional maturation of regenerating and developing taste buds (Segerstad and Hellekant, 1989
;
Harada et al., 2000). The
mean number of PGP-IR cells in a bud was 47.6% of the control PGP-IR cells (Table
1). The CT nerve of this time
responded to tactile but not to taste stimuli.
Cheal et al. (1977) reported
that it required about three days for axons to re-establish a taste bud with functional
receptors in gerbils. It may be plausible that some molecules involved in taste
transduction or synapses did not exist. At 3 weeks, the number of taste buds containing a
pore increased to 50% of control. The number of taste buds with a pore in a
papilla was 0.43, and PGP-IR cells in a bud were 76.2% of the control (Figure
1C). In 2–3 weeks, although no
AS taste responses were observed in the CT, there were taste bud cells expressing
, ß, 
subunits of ENaC (Shigemura et al., unpublished
observation), suggesting differentiation of taste cells is independent of synapse
formation. These results weaken possibility (i); regenerated CT axons would induce AI and
AS properties after synapse formation with identical progenitors. At 4 weeks, the
proportion of buds with a pore and the number of PGP-IR cells became larger than that at
3 weeks after nerve crush (Figure
1D). At > 5 weeks, the
number of taste buds with a pore in a papilla and PGP-IR cells increased, suggesting
functional recovery of these papillae (Figure
1E). If the number of PGP-IR cells
decreased after increment, possibility (ii) might be supported. Although in fact, the
number of PGP-IR cells and taste buds kept increasing during the course of regeneration.
This observation is consistent with the finding that impulse discharges of both E-type
and N-type fibers kept increasing during regeneration (Yasumatsu et al., 2003). Taken together, in the
present study, results suggest that (iii) individual axons in each type may increase
branches to their matched type of taste progenitor cells with maintaining the
selectivity.
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This work was supported by Grant-in-Aids 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 methodsResults and discussionAcknowledgementsReferences |
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Cheal, M., Dickey, W.P., Jones, L.B. and Oakley, B. (1977) Taste fiber responses during reinnervation of fungiform papillae. J. Comp. Neurol., 172, 627–646.[CrossRef][ISI][Medline]
Harada, S., Yamaguchi, K., Kanemaru, N. and Kasahara, Y. (2000) Maturation of taste buds on the soft palate of the postnatal rat. Physiol. Behav., 68, 333–339.[CrossRef][Medline]
Segerstad, H.C. and Hellekant, G. (1989) Changes in number and morphology of fungiform taste buds in rat after transection of the chorda tympani or chorda-lingual nerve. Chem. Senses, 14, 335–348.
Iwanaga, T., Han, H., Kanazawa, H. and Fujita, T. (1992) Immunohistochemical localization of protein gene product 9.5 (PGP9.5) in sensory paraneurons of the rat. Biomed. Res., 13, 225–230.
Ninomiya, Y. (1998) Reinnervation of cross-regenerated gustatory nerve fibers into amiloride-sensitive and amiloride-insensitive taste receptor cells. Proc. Natl Acad. Sci. USA, 95, 5347–5350.
Thompson, R.J., Doran, J.F., Jackson, P., Dhillon, A.P. and Rode, J. (1983) PGP 9. 5–a new marker for vertebrate neurons and neuroendocrine cells. Brain Res., 278, 224–228.[CrossRef][ISI][Medline]
Yasumatsu, K., Katsukawa, H., Sasamoto, K. and Ninomiya, Y. (2003) Recovery of amiloride-sensitive neural coding during regeneration of the gustatory nerve: behavioral-neural correlation of salt taste discrimination. J. Neurosci., 23, 4362–4368.
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