Fungiform taste bud degeneration in C57BL/6J mice following chorda-lingual nerve transection

Abstract

Taste buds are dependent on innervation for normal morphology and function. Fungiform taste bud degeneration after chorda tympani nerve injury has been well documented in rats, hamsters, and gerbils. The current study examines fungiform taste bud distribution and structure in adult C57BL/6J mice from both intact taste systems and after unilateral chorda-lingual nerve transection. Fungiform taste buds were visualized and measured with the aid of cytokeratin 8. In control mice, taste buds were smaller and more abundant on the anterior tip (<1 mm) of the tongue. By 5 days after nerve transection taste buds were smaller and fewer on the side of the tongue ipsilateral to the transection and continued to decrease in both size and number until 15 days posttransection. Degenerating fungiform taste buds were smaller due to a loss of taste bud cells rather than changes in taste bud morphology. While almost all taste buds disappeared in more posterior fungiform papillae by 15 days posttransection, the anterior tip of the tongue retained nearly half of its taste buds compared to intact mice. Surviving taste buds could not be explained by an apparent innervation from the remaining intact nerves. Contralateral effects of nerve transection were also observed; taste buds were larger due to an increase in the number of taste bud cells. These data are the first to characterize adult mouse fungiform taste buds and subsequent degeneration after unilateral nerve transection. They provide the basis for more mechanistic studies in which genetically engineered mice can be used.

Fig. 1

Cytokeratin 8-positive taste buds. Taste bud cells immunopositive for cytokeratin 8 appear as white. A–E: Serial sections of a taste bud from an intact (control) mouse. F–I: A taste bud from a mouse 10 days after an ipsilateral chorda-lingual transection. Scale bar = 20 µm.

Fig. 2

Diagram from the tongue of an intact mouse to illustrate the divisions used for analyses. The mouse tongue was divided into anterior (anteriormost 1 mm) and intermediate (next 4 mm) divisions for anterior–posterior analyses and medial (midline to 350 µm lateral from midline), middle (next 550 µm), and lateral divisions for medial–lateral analyses. CV, circumvallate papilla; FP, foliate papillae; IE, intermolar eminence. Scale bar = 1 mm.

Fig. 3

A: Percent of taste buds remaining after nerve transection: anterior vs. intermediate tongue. The intermediate tongue was particularly susceptible to taste bud loss. The anterior tongue lost as much as half its taste bud population after chorda-lingual transection. The intermediate tongue lost 100% and 97% of taste buds at 15 and 20 days, respectively. Asterisks (*) denote significant differences between anterior and intermediate regions at each time period posttransection. B: Total volume of taste buds (±SEM) on the anterior and intermediate tongue after transection. The intermediate tongue had significantly fewer taste buds, and in turn much less total taste tissue than the anterior tongue (P’s < 0.01). Asterisks (*) indicate significant differences from control volumes on the anterior tongue. The number symbol (#) represents significant differences between taste bud volumes on the intermediate tongue from control volumes.

Fig. 4

Frequency distributions (percent of the total number/group) of taste bud volumes from the side of the tongue ipsilateral to the nerve section (open bars; curve fit by dotted line), the intact control tongue (solid bars; curve fit by solid line), and the side contralateral to the nerve transection (gray bars; curve fit by dashed line).

Fig. 5

Two taste buds immunopositive for cytokeratin 8 (magenta) and the nuclear stain Sytox (green). A taste bud from a control mouse (A) and from the transected side of the tongue in a mouse sacrificed 10 days after nerve transection (B). Scale bar = 20 µm.

Fig. 6

Mean number of Sytox/cytokeratin 8-positive cells per taste bud in unoperated control mice (intact) and in the transected (ipsilateral cut) and intact (contralateral intact) sides of the tongue of mice 10 days after receiving chorda-lingual nerve transection. Taste buds on the transected side of the tongue had significantly fewer cells than those in control mice and on the intact side of the tongue at 10 days post-nerve transection (*P’s < 0.001). Also, the side of the tongue contralateral to the nerve transection had more mean taste buds compared to intact control mice (*P = 0.04).

Fig. 7

Innervation after chorda-lingual nerve transection. A–C: Profiles of taste buds from a mouse 10 days after unilateral chorda-lingual nerve transection. Fibers positive for neurofilament and PGP 9.5 are in green, cytokeratin 8-positive cells are in magenta. Taste buds on the contralateral, intact side of the tongue were richly innervated with neurofilament 150-positive fibers (A). On the transected side of the tongue, no fibers were observed in empty papilla (B) or papilla with remnant fungiform taste buds (C). The arrow indicates hypoglossal fibers innervating underlying muscle tissue. D–F: Profiles of taste buds 20 days after chorda-lingual nerve transection. These include an intact taste bud contralateral to nerve transection (D), neural fibers in an empty fungiform papilla (E), and fibers approaching a small, remnant fungiform taste bud (F). To confirm the lack of chorda-lingual fibers 10 days after nerve transection, G–I show PGP 9.5 immuno-like staining. Taste buds on the intact side of the tongue show prolific staining for PGP 9.5 (G), while the cut side lacks PGP 9.5 staining in empty fungiform papilla (H) and in the presence of a fungiform taste bud (I). Scale bars = 20 µm.

Fig. 8

A fungiform papilla containing a taste bud (A,B) and another papilla containing a remnant or atrophic taste bud (C,D). Panels A and C were stained with hematoxylin and eosin and panels B and D were stained with the cytokeratin 8 antibody. The solid outline in A denotes the position of the taste bud as estimated from hematoxylin and eosin stained tissue. A similar structure of the same taste bud is also shown in B. Note that the papilla in C does not contain slender cells and nuclei in a position characteristic of taste buds, and that there are no immunopositive cells labeled in D. The label shown in D was the only immunopositive label in the entire papilla (magenta) and may represent remnant processes of the original taste bud; this papillae would be scored as not containing a taste bud. Scale bar = 20 µm.