A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
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.
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.
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 parts3,4, hinting that axonal mitochondria might be more susceptible to insults. However, the biology of mitochondria in axons is not well understood due to the difficulties associated with targeted treatment and analysis of axonal mitochondria without simultaneous disturbance of cell body processes.
Recent advances in culturing techniques of dissociated neurons in vitro now allow the fluidic separation of axons and cell bodies through microfluidic devices5. As depicted in Figure 1A, these devices feature four access wells (a/h and c/i), with two channels connecting each pair (d and f). The large channels are connected with each other by a series of 450 µm long microchannels (e). Intentional differences in the fill levels between the two chambers create a fluid pressure gradient (Figure 1B) that prevents the diffusion of small molecules from the channel with a lower fluid level to the other side (Figure 1C, illustrated with Trypan blue dye).
We recently used microfluidic devices to study local translation requirements in axonal mitophagy, the selective removal of damaged mitochondria6. In the present protocol, different steps are presented to induce local mitochondrial damage through selective treatment of axons using the mitochondrial complex III inhibitor Antimycin A6,7.
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
2. Seeding and maintaining of neurons
3. Staining with mitochondrial membrane potential sensitive dye
4. Live-cell imaging
NOTE: Still images shown were acquired on a spinning disk confocal microscope, using a 40x NA 1.25 immersion objective (see Table of Materials). 200 ms exposure time and 10% laser power for the red channel and 500 ms exposure time for brightfield were chosen. However, regular confocal or widefield inverted microscopes can also be used to study TMRE intensity.
Primary hippocampal neurons were grown in microfluidic devices for 7-8 days before mitochondria were stained with the membrane-sensitive dye (TMRE) for 25 min in both the channels. As shown in Figure 2A, this yielded homogenous staining of mitochondria on both sides of the microgrooves, yet it was insufficient to equilibrate the staining into the middle of the microgrooves. Upon addition of Antimycin A to the axonal side, somal mitochondria retained the TMRE signal (Figu...
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
The authors declare no competing interests.
This study was supported by the German Research Foundation (HA 7728/2-1 and EXC2145 Project ID 390857198) and the Max Planck Society.
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 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved