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 (Figure 2B and Video 1), whereas TMRE fluorescence was lost from axonal mitochondria (Figure 2C and Video 1). The video was captured using a fully integrated digital widefield microscope (see Table of Materials).
Figure 1: Microfluidic devices allow the fluidic isolation of axons. (A) Schematic of the microfluidic device used in this study. The silicone disk (diameter 21 mm) fits easily into a six-well plate. a) Well on soma side, b) Entrance of channel on soma side, c) Well on axon side, d) Channel on soma side, e) Microgrooves, f) Channel on axon side, g) Exit of the channel on soma side, h) Well on soma side, i) Well on axon side. (B) Schematic detailing how the different fluid levels create a fluidic pressure gradient across the microchannels. (C) Demonstration of the fluidic isolation by addition of Trypan blue to one side of the chamber. Note that due to the fluidic pressure gradient, the blue dye does not equilibrate across the microchannels. Please click here to view a larger version of this figure.
Figure 2: Selective treatment of axons depolarizes axonal but not cell body mitochondria. (A) Representative micrograph presenting an overview of the effectivity of TMRE staining in microfluidic devices. Scale bar = 100 µm. (B–C) The representative micrograph shows the same area in the cell body and the axonal compartment before and after adding 20 µM Antimycin A (AA) to the axonal compartment. Scale bar = 20 µm. Please click here to view a larger version of this figure.
Video 1: Effect of Antimycin A treatment on mitochondria in axons and cell bodies. Live cell imaging of the loss of TMRE fluorescence upon adding Antimycin A. Please click here to download this Video.
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 readouts9. Other neuronal cell types can also be grown in microfluidic chambers, such as primary cortical neurons10 or induced pluripotent stem cell (ipSC)-derived motorneurons11, making this platform a versatile tool to study mitochondrial function in neurodegeneration in the neuronal cell type of interest. The assembly of microfluidic devices is crucial to achieving an efficient seal and is most easily explained by watching an experienced researcher perform the assembly. The downstream labeling and treatment procedure described here are meant to be exemplary and may be adjusted to fit the protocols currently established in the respective labs while maintaining the fluidic pressure gradient.
The seeding technique described here differs from published protocols and the manufacturer’s description, as we skip the suggested washes of the assembled device and instead directly seed the dissociated neurons into the dry chamber (section 2). It has been observed that this reduces the number of neurons needed, as it increases the density of neurons within the channel (e) and limits the spread of neurons into the wells far away from the microchannels. The dry seeding is aided by tapping the bottom of the plate (step 2.5), which may or may not be necessary depending on the force applied previously when assembling the device.
However, certain limitations exist in this procedure. There is some variability in the tightness of the seal due to differences in the force applied during assembly that may lead to restricted growth through the microgrooves. Also, remaining moisture can disturb the seal formation and allow axonal growth or even cell migration underneath the device. Both problems can easily be spotted prior to staining, leading to the exclusion of faulty chambers from the experiment.
The authors have nothing to disclose.
This study was supported by the German Research Foundation (HA 7728/2-1 and EXC2145 Project ID 390857198) and the Max Planck Society.
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 |