Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Summary

The present protocol describes the seeding and staining of neuronal mitochondria in microfluidic chambers. The fluidic pressure gradient in these chambers allows for the selective treatment of mitochondria in axons to analyze their properties in response to pharmacological challenges without affecting the cell body compartment.

Abstract

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many neurodegenerative diseases. Given some axons' elaborate architecture and extreme length, it is not surprising that mitochondria in axons can experience different environments compared to their cell body counterparts. Interestingly, dysfunction of axonal mitochondria often precedes effects on the cell body. To model axonal mitochondrial dysfunction in vitro, microfluidic devices allow treatment of axonal mitochondria without affecting the somal mitochondria. The fluidic pressure gradient in these chambers prevents diffusion of molecules against the gradient, thus allowing for analysis of mitochondrial properties in response to local pharmacological challenges within axons. The current protocol describes the seeding of dissociated hippocampal neurons in microfluidic devices, staining with a membrane-potential sensitive dye, treatment with a mitochondrial toxin, and the subsequent microscopic analysis. This versatile method to study axonal biology can be applied to many pharmacological perturbations and imaging readouts, and is suitable for several neuronal subtypes.

Introduction

Mitochondria are the main suppliers of ATP (adenosine triphosphate) in neurons. As neuronal health is intimately linked to mitochondrial function, it is not surprising that dysfunctional regulation of these organelles has been associated with the onset of various neurodegenerative diseases, including Parkinson's disease¹. Furthermore, mitochondrial intoxication has successfully been used to model Parkinsonian symptoms in animals². In both animal models and human disease, the demise of neurons starts at the distal parts^3,4, hinting that a....

Protocol

All animal experiments were performed following the relevant guidelines and regulations of the Government of Upper Bavaria. The primary neurons were prepared from E16.5 C57BL/6 wild-type mouse embryos of both sexes following standard methods as previously described⁶.

1. Assembly of the microfluidic device

1. Coat one six-well glass-bottom tissue culture plate with a final concentration of 20 µg/mL of Poly-D-Lysine and 3.4 µg/mL of Lam.......

Discussion

The present protocol describes a method to seed and culture dissociated hippocampal neurons in a microfluidic device to treat axonal mitochondria separately. The utility of this approach with the membrane-sensitive dye TMRE and the complex III inhibitor Antimycin A (as previously demonstrated⁷) is demonstrated here, but this method can be easily adapted to other mitochondrial dyes or genetically encoded sensors of mitochondrial functions that allow local, microscopy-based readouts

Materials

Name	Company	Catalog Number	Comments
6-well Glass bottom plate	Cellvis	P06.1.5H-N	Silicone device
Antimycin A	Sigma	A8674
B27	Gibco	17504044
EVOS M5000 widefield microscope	Thermofischer Scientific	EVOS M5000	fully integrated digital widefield microscope
Hibernate E	BrainBits	HE500
Inverted spinning disk confocal	Nikon	TI2-E + CSU-W1	With incubator chamber
Laminin	Invitrogen	L2020
Microfluidic devices	XONA microfluidics	RD450
Neurobasal medium	Gibco	21103049
Poly-D-Lysine	Sigma	P2636
TMRE	Sigma	87917