Subcellular electrical stimulation of neurons enhances the myelination of axons by oligodendrocytes

<div><p>Myelin formation has been identified as a modulator of neural plasticity. New tools are required to investigate the mechanisms by which environmental inputs and neural activity regulate myelination patterns. In this study, we demonstrate a microfluidic compartmentalized culture system with integrated electrical stimulation capabilities that can induce neural activity by whole cell and focal stimulation. A set of electric field simulations was performed to confirm spatial restriction of the electrical input in the compartmentalized culture system. We further demonstrate that electrode localization is a key consideration for generating uniform the stimulation of neuron and oligodendrocytes within the compartments. Using three configurations of the electrodes we tested the effects of subcellular activation of neural activity on distal axon myelination with oligodendrocytes. We further investigated if oligodendrocytes have to be exposed to the electrical field to induce axon myelination. An isolated stimulation of cell bodies and proximal axons had the same effect as an isolated stimulation of distal axons co-cultured with oligodendrocytes, and the two modes had a non-different result than whole cell stimulation. Our platform enabled the demonstration that electrical stimulation enhances oligodendrocyte maturation and myelin formation independent of the input localization and oligodendrocyte exposure to the electrical field.</p></div

CONFORMAL PHASED SURFACES FOR WIRELESS POWERING OF BIOELECTRONIC MICRODEVICES

10.1038/s41551-017-0043NATURE BIOMEDICAL ENGINEERING1

Whole cell and subcellular ESTIM enhances oligodendrocyte precursor cells (OPCs) differentiation into oligodendrocytes (OLs) and supports axon myelination.

<p>(A-D) Representative images of axons in the axonal compartment stained against neurofilaments (NF, red) and OLs stained against a set of differentiation markers: (A) O4 signal (green) expressed by premature OPCs (white arrows) after 3 days of stimulation; (B) CNPase signal (green) expressed by mature OLs (white arrows) after 7 days of stimulation; (C) MBP signal (green) expressed by mature OLs (white arrows) after 7 days of stimulation; (D) MBP signal (green) expressed by mature OLs after 14 day of stimulation demonstrating formed myelin fragments (white arrows). Scale bar (A—D): 50μm. The images for all experimental conditions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179642#pone.0179642.s006" target="_blank">S6 Fig</a>. (E) The percentage of O4-positive OPCs decreased slightly after 3 days in the control group outlining the baseline level of the differentiation. ESTIM further decreased the percentage of O4-positive OPCs. (F-G) 7 days of ESTIM supported OLs maturation as indicated by the increase in the percentage of (F) MBP-positive and (G) CNPase-positive cells. (H) 14 days of ESTIM increased the number of formed myelin fragments. The data are presented as mean ± S.E.M. For individual data points see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179642#pone.0179642.s007" target="_blank">S7 Fig</a>. The numbers on the graphs stand for the numbers of experimental replicates. The groups were compared with one-way ANOVA at a significance level of α = 0.05; *** p<0.001.</p

Integrated compartmentalized and electrical system for subcellular stimulation of neurons.

<p>(A) Dorsal root ganglion neurons and oligodendrocyte precursor cells were cultured in microfluidics and electrically stimulated in three different modes of electrode configuration: whole cell stimulation (WholeSTIM), somatic compartment stimulation (SomaSTIM) and axonal compartment stimulation (AxonSTIM). (B) Schematic of stimulation protocol.</p

Electric field strength within the long compartments.

<p>(A) Schematic of the simulation model with larger compartment length (15 mm). (B) Cross-section of model along indicated plane. (C-E) Contour maps of electric field magnitudes within the compartments for different electrode configurations.</p

Electrode configuration affects the effectiveness of neuron stimulation.

<p>(A) The same central regions of a microfluidic neuron culture (broken line boxes) were first imaged under electrode configuration D than electrode configuration C. (B) DIC images of the representative region of the imaged areas. (C-D) Calcium response of cell bodies (<i>C</i>) and axons (<i>D</i>) to ESTIM. E Average calcium changes (ΔF/F_B) pooled from the same cell bodies in two different electrode configurations. The inset shows frequency of the firing events (f_ΔF/FB) in 3 min following ESTIM onset. The results are expressed as mean ± SEM (n = 10 cells). Statistical analysis was performed with paired student-t test ** p<0.005.</p

Electric field amplitude within the compartments.

<p>(A) Schematic of the simulation model. (B) Cross-section of model along indicated plane. (C-H) Contour map of electric field amplitude within compartments for different stimulation electrode configurations. (I-K) Profile of electric field amplitude along dashed lines in (C-H) spanning two wells. (L) Measured (gray; relative electrical potential difference) and simulated (black; relative electric magnetic field) at 6 locations, as numbered in (<i>C</i>) and (<i>E</i>) and outlined (white x and dotted lines) in all electrodes configurations (<i>C-H</i>). The data for G and H come from the same set of simulations and measurements. Measurement data are mean ± S.D. For individual data points see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179642#pone.0179642.s002" target="_blank">S2 Fig</a>.</p