Changes in the axon terminals of primary afferents from a single vibrissa in the rat trigeminal nuclei after active touch deprivation or exposure to an enriched environment

Abstract

Lasting modifications of sensory input induce structural and functional changes in the brain, but the involvement of primary sensory neurons in this plasticity has been practically ignored. Here, we examine qualitatively and quantitatively the central axonal terminations of a population of trigeminal ganglion neurons, whose peripheral axons innervate a single mystacial vibrissa. Vibrissa follicles are heavily innervated by myelinated and unmyelinated fibers that exit the follicle mainly through a single deep vibrissal nerve. We made intraneural injections of a mixture of cholera-toxin B (CTB) and isolectin B4, tracers for myelinated and unmyelinated fibers, respectively, in three groups of young adult rats: controls, animals subjected to chronic haptic touch deprivation by unilateral whisker trimming, and rats exposed for 2 months to environmental enrichment. The regional and laminar pattern of terminal arborizations in the trigeminal nuclei of the brain stem did not show gross changes after sensory input modification. However, there were significant and widespread increases in the number and size of CTB-labeled varicosities in the enriched condition, and a prominent expansion in both parameters in laminae III–IV of the caudal division of the spinal nucleus in the whisker trimming condition. No obvious changes were detected in IB4-labeled terminals in laminae I–II. These results show that a prolonged exposure to changes in sensory input without any neural damage is capable of inducing structural changes in terminals of primary afferents in mature animals, and highlight the importance of peripheral structures as the presumed earliest players in sensory experience-dependent plasticity.

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Abbreviations

ABC:

Avidin-biotynilated peroxidase complex

Cb:

Cerebellum

CE:

Coefficient of error

CTB:

Cholera toxin, fraction B

DAB:

3,3′-Diaminobenzidine

DVN:

Deep vibrissal nerve

DRG:

Dorsal root ganglion

HRP:

Horseradish peroxidase

IB4:

Isolectin B4 from Griffoniasimplicifolia

IoN:

Infraorbital nerve

PB:

Phosphate buffer

Pr5:

Principal trigeminal nucleus

Sp5c:

Spinal trigeminal nucleus, pars caudalis

Sp5ip:

Spinal trigeminal nucleus, pars interpolaris

Sp5o:

Spinal trigeminal nucleus, pars oralis

t5:

Spinal trigeminal tract

TG:

Trigeminal ganglion

WGA:

Wheat germ agglutinin

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Acknowledgements

The authors gratefully acknowledge Ms. Begoña Rodriguez for her skilled technical help in the preparation of the histological material, and the Servicios de Microscopía Confocal and Microscopía Electrónica de Transmisión of the SIDI-UAM for their help in the confocal microscopy analyses. We also thank Dr. A. Krzyzanowska for reading a final draft of the manuscript and making useful style suggestions. This study was supported by Grants BFU2012-39960 and BFU2015-66941R from Spain’s Ministerio de Economía Industria y competitividad/Fondo Europeo de Desarrollo Regional (MINECO/FEDER).

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429_2017_1472_MOESM1_ESM.tif

Fig. 1:Injection of CTB and IB4 in a single deep vibrissal nerve (DVN). (a) Exposure of the deep nerve from vibrissal follicle C1. Star, follicle; arrows, stretch of the nerve attaching to the follicle wall. (b) Intraneural deposit of a mixture of tracers with Light Green to help visualization of the same nerve; the picture was taken immediately after finishing the injection. (c) Confocal immunofluorescence image of a cross section of C1 DVN, 2 mm proximal to the injection. Most, if not all, medium-sized and large axons are labeled by CTB (red). Asterisk marks a blood vessel artifact. (d) Semithin section of C1 DVN injected with both tracers, subjected to a preembedding immunohistochemistry and counterstained with toluidine blue. The axoplasm of most myelinated axons, normally unstained by toluidine blue, exhibits now a dark color due to the immunoreaction. (e) Low-power electron micrograph of a cross section of C1 DVN in a naïve case without injection of tracers. (f, g) High power electron micrographs of the same C1 DVN as in (d), showing details of several labeled myelinated and unmyelinated axons. The axoplasm of myelinated axons seems uniformly filled with a diffuse precipitate which masks the axonal cytoskeleton. Unmyelinated axons are irregularly filled with dark deposits (arrows). Scale bars 200 µm (a, b), 20 µm (c), 50 µm (d, e), and 2 µm (f, g) (TIFF 42358 kb)

429_2017_1472_MOESM2_ESM.tif

Fig. 2: Panels showing examples of neuron somata in the TG of a control case that are single-labeled by either CTB (a, b, c: red, left boxes of each panel) or IB4 (a, b, c: green, middle boxes). Merged images in the boxes on the right show no colocalization of the tracers. In (c), however, a few small neurons in the same case show colocalization (arrowheads). Scale bars: 40 µm (TIFF 19353 kb)

429_2017_1472_MOESM3_ESM.tif

Fig. 3: Confocal merged images with orthogonal projections showing three examples of varicosities double-labeled for CTB (red) and IB4 (green) in lamina II of Sp5c in a control case. (a) Example of a fairly large double-labeled varicosity (arrow) located very close to another varicosity that is only labeled for CTB. (b, c) Crosshairs marking two small double-labeled profiles, probably corresponding to small varicosities. Scale bars 50 µm (a), 10 µm (b, c) (TIFF 20286 kb)

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Fernández-Montoya, J., Martin, Y.B., Negredo, P. et al. Changes in the axon terminals of primary afferents from a single vibrissa in the rat trigeminal nuclei after active touch deprivation or exposure to an enriched environment. Brain Struct Funct 223, 47–61 (2018). https://doi.org/10.1007/s00429-017-1472-5

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Keywords

  • Isolectin B4
  • Primary afferent
  • Input-dependent plasticity
  • Trigeminal ganglion
  • Transganglionic