Two specific tongue regions receive bilateral hypoglossal innervation: A study in neonatal rat pups

https://doi.org/10.1016/j.archoralbio.2014.12.002Get rights and content

Highlights

  • Two specific tongue regions receive bilateral XII innervation in neonatal rats.

  • Contralaterally projecting XII nerve components innervate tongue protrudor muscles.

  • Bilateral XII projections relate to suckling capability in unilateral nerve injury.

  • Contralateral XII nerve components decrease gradually during development.

Abstract

Objective

The purpose of this study was to investigate the functional role of bilateral hypoglossal (XII) nerve innervation of the tongue.

Materials and methods

The possibility of bilateral innervation of XII nerve in the tongue was examined using postmortem fibre tracing in normal neonatal rats. In addition, immunohistochemical testing for synaptophysin or vesicular acetylcholine transporter (VAChT) was carried out in unilaterally XII nerve-resected neonatal rats.

Results

Postmortem fibre tracing revealed constant distribution of the nerve fibres extending across the midline and existence of bilateral innervated area in the rostroventral and caudodorsal regions of the tongue. Synaptophysin-positive and VAChT-positive nerve terminals were also distributed continuously from the XII nerve-intact to the nerve-resected side across the midline of the tongue. The contralaterally projecting VAChT-positive nerve terminals were more numerous in suckling P2 rats (6.6 ± 0.5/section) than those in non-suckling P2 rats (4.9 ± 0.3/section) 24 h after nerve resection. Furthermore, the contralaterally projecting VAChT-positive nerve terminals were more numerous in P7 rats with nerve resection on P1 (6.3 ± 0.2/section) than those in P7 rats with nerve resection on P6 (3.1 ± 0.8/section).

Conclusion

We concluded that neonatal rats have two specific tongue regions receiving bilateral XII innervation, which allowed suckling in unilaterally XII nerve-resected neonatal rats.

Introduction

We have previously reported axonal sprouting across the midline of the tongue from the intact side to the hypoglossal (XII) nerve-resected side in neonatal rats.1 Although the speed of axonal sprouting to the contralateral tongue remains uncertain, early onset of axonal sprouting within 24 h after a nerve insult still raises the possibility of residual nerve components of bilateral innervation of the XII nerve. Although the problem of postmortem fibre tracing comes from difficulties in constant visualization of finely arborized neural networks as judged from the relatively large number of unsuccessful cases, we re-examined XII innervation in neonatal rat tongues using a large number of normal newborn pups and many tongue preparations cut serially with shorter intervals. In addition, synaptophysin and vesicular acetylcholine transporter (VAChT), presynaptic terminal markers for the neuromuscular junction,2, 3, 4, 5 were immunohistochemically detected in the unilaterally XII nerve-resected tongues, because tongue movement is essential for suckling in developing rats.6, 7, 8 We performed quantitative analyses of the nerve terminals across the midline of the tongue in neonatal rats with suckling disturbance induced by unilateral resection of the XII nerve. In this study, we will discuss the functional role of bilateral XII innervation of the tongue.

Section snippets

Animals

Newborn Wistar rats (Japan SLC Inc., Shizuoka, Japan) of both sexes were used in this study. Postnatal day (P) 0 refers to the first 24 h after birth. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and protocols were approved by our Institutional Animal Care and Use Committee. Every effort was made to minimize animal suffering and pain.

Postmortem neuronal tracing

Normal pups were euthanatized with sodium pentobarbital (100 mg/kg,

DiI-labelled XII nerve components across the midline of the tongue

The characteristics of the DiI-labelled XII nerve components in the neonatal rats’ tongues are summarized in Table 1. DiI application to the XII nerve stumps produced various stages of neural fibre labelling in neonatal rats, from unsuccessful cases without fine-labelled fibres to successful cases with finely arborized labelled fibres distributed throughout the left side of the tongue. In the unsuccessful cases, DiI labelling occurred only in thick fibre bundles in the posterior one-third of

Discussion

Postmortem unilateral DiI labelling of the XII nerve in the tongue of the normal neonatal rats and synaptophysin/VAChT immunohistochemistry of the tongue of the unilaterally XII nerve-resected neonatal rats demonstrated the presence of two specific tongue regions where nerve fibres extended to the contralateral side across the midline at the early neonatal stage just after birth. Furthermore, by postmortem bilateral labelling of the left (DiI) and right (4-Di-10-ASP) XII nerves, it became

Conclusions

We concluded that neonatal rats have two specific tongue regions receiving bilateral XII innervation, which allowed suckling in unilaterally XII nerve-resected neonatal rats.

Funding

None.

Conflict of interest

None declared.

Ethical approval

The protocol was approved by the Institutional Animal Care and Use Committee of Shinshu University (#190016).

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