Node of Ranvier formation on motoneurons in vitro

Abstract

One of the most significant interactions between Schwann cells and neurons is myelin sheath formation. Myelination is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems provide a unique modality to study both factors influencing myelination and diseases associated with myelination. Currently, no in vitro system for motoneuron myelination by Schwann cells has been demonstrated. This work details the myelination of motoneuron axons by Schwann cells, with complete Node of Ranvier formation, in a defined in vitro culture system. This defined system utilizes a novel serum-free medium in combination with the non-biological substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA). The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This defined system provides a highly controlled, reproducible model for studying Schwann cell interactions with motoneurons as well as the myelination process and its effect on neuronal plasticity. Furthermore, an in vitro system that would allow studies of motoneuron myelination would be beneficial for understanding peripheral demyelinating neuropathies such as diabetes induced peripheral neuropathy and could lead to a better understanding of CNS demyelinating diseases like multiple sclerosis, as well as neuromuscular junction maturation and maintenance.

Figure 1

XPS and contact angle analysis of DETA monolayer on glass coverslips. (A) XPS survey spectra analysis of DETA coverslip, (B) XPS high resolution spectrum of N_1s peak on DETA coverslip, (C) XPS high resolution spectrum of Si_2p peak on DETA coverslip, (D) contact angle image of water on DETA coverslip.

Figure 2

Phase contrast images of motoneuron + Schwann cell co-cultures. (A) EMN culture image at day seven, (B) pure neonatal Schwann cell culture at day 14 (C) EMN+SC co-culture at day 7 (arrow indicating MN). Scale bars = 60µm.

Figure 3

Immunocytochemical evaluation of the myelination of motoneurons by Schwann cells. (A–D) embryonic MN+SC co-culture images at day 29, (A) phase contrast image of the MN+SC co-culture, (B) NF-H antibody staining neuronal processes throughout the culture, (C) MBP antibody staining showing a segment of compact myelin and the outline of the Schwann cell, (D) merge image showing the colocalization of the NF-H and MBP antibody staining, (E–H) embryonic MN+SC culture images at day 27, (E) phase contrast image of the MN+SC co-culture, (F) NF-H antibody staining showing neuronal process, (G) MBP antibody staining revealing a segment of compact myelin in the culture, (H) merge image showing co-localization of the NF-H and MBP antibody staining. Scale bars = 50µm.

Figure 4

Immunocytochemical characterization of node of Ranvier formation on motoneurons. (A) phase contrast image of day 29 MN+SC co-culture showing an axonal segment and multiple Schwann cell bodies, (B) MBP and vgsc staining indicating node formation, (C) CASPR staining indicating paranode formation, (D) vgpc staining indicating juxtaparanode formation. Scale bar = 50µm.