Quantitative analysis of axonal transport by using compartmentalized and surface micropatterned culture of neurons

Abstract

Mitochondria, synaptic vesicles, and other cytoplasmic constituents have to travel long distance along the axons from cell bodies to nerve terminals. Interruption of this axonal transport may contribute to many neurodegenerative diseases including Alzheimer's disease (AD). It has been recently shown that exposure of cultured neurons to β-amyloid (Aβ) resulted in severe impairment of mitochondrial transport. This Letter describes an integrated microfluidic platform that establishes surface patterned and compartmentalized culture of neurons for studying the effect of Aβ on mitochondria trafficking in full length of axons. We have successfully quantified the trafficking of fluorescently labeled mitochondria in distal and proximal axons using image processing. Selective treatment of Aβ in the somal or axonal compartments resulted in considerable decrease in mitochondria movement in a location dependent manner such that mitochondria trafficking slowed down more significantly proximal to the location of Aβ exposure. Furthermore, this result suggests a promising application of microfluidic technology for investigating the dysfunction of axonal transport related to neurodegenerative diseases.

Figure 1

Schematic diagram of experimental setup. (A) Procedure for micropatterning of PLL strip on a substrate and its integration with a compartmentalized microfluidic neuron culture device. A PDMS stamp with embossed line pattern (30 × 50 μm) was placed on a PLL precoated glass coverslip. Then reactive oxygen plasma selectively removed PLL in regions that were not covered by PDMS. The substrate patterned with the PLL strip was aligned and bonded to PDMS neuron culture device. (B) Micrograph showing primary neurons cultured in the integrated microfluidic multicompartment chamber with PLL strip. Surface patterned PLL was used to control growth of axons. (C) Fluidic isolation was demonstrated by adding 10 kDa of FITC-dextran to the axonal chamber. No fluorescence was observed in the somal chamber for 24 h. (D) Neurons transfected with GFP on the left side of the device (green), and neurons transfected on the right side of the device with RFP (red). Compartmentalization and fluidic isolation allow for the transfection of two different proteins simultaneously.

Figure 2

Demonstration of image processing. (A) mito-GFP signal along an axon (green) and extracted axon centerline (red). (B) Space–time diagram showing fluorescence along the axon centerline. Automatically decomposed into static component and dynamic component. Static and rapidly moving mitochondria are represented as vertical lines and diagonals, respectively. E-18 rat cortical neurons were cultured on nonpatterned substrate for demonstrating similar complexity in culture but not in the analysis using an automated trafficking method.

Figure 3

Trafficking of mitochondria in proximal and distal axons under selective treatment of Aβ_1–42. Kymographs were generated using time-lapse images that were taken before and after 1 μM of Aβ_1–42 was applied to the axonal compartment. (A,B) Mitochondria trafficking of proximal and distal axons before treatment. (C,D) Mitochondria trafficking after 1 μM of Aβ_1–42 treatment on axonal compartment.

Figure 4

Quantification of mitochondrial transport at various distances away from Aβ_1–42 exposure. The average velocity of mitochondria was determined in each Kymograph for five different regions of neurons in a microfluidic device (i.e., soma side, proximal axon, middle axon, distal axon, axonal side). (A) Velocity of mitochondria following Aβ treatment on soma compartment (*p = 0.0172). (B) Velocity of mitochondria following Aβ treatment on axonal compartment (*p = 0.0013, **p < 0.0001). Asterisks indicate a statistical significance when comparing with the control (CTRL) at the corresponding region. Error bar indicates SEM.