The Cerebellum

, Volume 12, Issue 1, pp 16–26 | Cite as

Polarised Localisation of the Voltage-Gated Sodium Channel Nav1.2 in Cerebellar Granule Cells

  • José Martínez-Hernández
  • Carmen Ballesteros-Merino
  • Laura Fernández-Alacid
  • Joel C. Nicolau
  • Carolina Aguado
  • Rafael Luján
Original Paper

Abstract

Voltage-gated sodium channels are responsible for action potential initiation and propagation in electrically excitable cells. In this study, we used biochemical, immunohistochemical and quantitative immunoelectron microscopy techniques to reveal the temporal and spatial expression of the Nav1.2 channel subunit in granule cells of cerebellum. Using histoblot, we detected Nav1.2 widely distributed in the adult brain, but prominently expressed in the cerebellum. During postnatal development, Nav1.2 mRNA and protein were detected low during the first and second postnatal week, increased to P15 and then continue to decrease until adult levels. At the light microscopic level, Nav1.2 immunoreactivity concentrated in the molecular layer of the cerebellar cortex. Using immunofluorescence, Nav1.2 colocalised with VGluT1, but not with VGluT2, demonstrating that the subunit was preferentially present in parallel fibre axons and axon terminals. At the electron microscopic level, Nav1.2 immunoparticles were exclusively detected at presynaptic sites in granule cell axons and axon terminals of granule cells, with occasional clustering in their axon initial segment. This was demonstrated using quantitative immunogold analysis. In the axon terminals, the distribution of Nav1.2 was relatively uniform along the extrasynaptic plasma membrane and never detected in the active zone. We could not find detectable levels of Nav1.2 at postsynaptic elements of granule cells or other cerebellar cell types. The present findings show a polarised distribution of Nav1.2 along the neuronal surface of granule cells and suggest its primary involvement in the transmission of information from granule cells to Purkinje cells.

Keywords

Sodium channel Nav1.2 Cerebellum Electron microscopy Development Immunohistochemistry 

Supplementary material

12311_2012_387_Fig6_ESM.jpg (53 kb)
Supplementary Figure 1

Immunoreactivity for Nav1.2 in the cerebellar cortex in the mouse during postnatal development using a pre-embedding immunoperoxidase method. (AE). Parasagittal photomicrographies of the cerebellum. Strong immunolabelling for Nav1.2 was found in the molecular layer (ml) and very weak in the granule cell layer (gc) at all developmental ages studied. During postnatal development, a progressive increase in the intensity of immunoreactivity for Nav1.2 was observed in the molecular layer and in processes ascending from the granular cell layer to the molecular layer, likely corresponding to bundles of granule cell axons. Immunoreactivity for Nav1.2 was not detected in the granule cell somata or dendrites in the external (egl) or internal granular layer (igl) or in Purkinje cell bodies (pc) at any postnatal age. Scale bars: AE, 50 μm (JPEG 52 kb)

12311_2012_387_MOESM1_ESM.tif (7.5 mb)
High resolution image file (TIFF 7702 kb)

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • José Martínez-Hernández
    • 1
  • Carmen Ballesteros-Merino
    • 1
  • Laura Fernández-Alacid
    • 1
  • Joel C. Nicolau
    • 1
  • Carolina Aguado
    • 1
  • Rafael Luján
    • 1
  1. 1.Department of Ciencias Médicas, Instituto de Investigación en Discapacidades Neurológicas (IDINE), Facultad de MedicinaUniversidad Castilla-La ManchaAlbaceteSpain

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