Mitochondria are essential for neuronal health. In many neurodegenerative diseases, axonal mitochondria are the first ones to suffer, but it is not clear what makes axonal mitochondria so special. The fluidic pressure gradient in the chambers used in this protocol allows for the selective treatment of mitochondrion axons.
This leaves cell body processes undisturbed, and allows us to focus on axonal biology. We show here the depolarization of mitochondria with a membrane potential sensitive dye, but this method can be applied to many different neuronal cell types, and fluorescent readouts. To begin, place the coded six well plates in a sterile hood, and wash them twice with sterile double distilled water.
Allow the plates to dry in a tilted position for three to five minutes. Remove excess water by vacuum suction or pipetting. Then soak the microfluidic chamber in 80%ethanol.
Again, dry the microfluidic chamber for three to five minutes in a tilted position, and remove excess ethanol with a pipette tip. When completely dry, place the microfluidic chamber in the center of the well and the plate. Gently tap the microfluidic chamber at its borders and microgroove section in the middle for proper attachment to the glass plate.
First, collect the desired number of dissociated hippocampal neurons in a 1.5 milliliter reaction tube. Centrifuge the tube at 1000 times G for four minutes at room temperature. Discard the supernatant, and resuspend the pellet in eight microliters of B-27 neuro basal media.
Then dispense the cell solution into the channel entrance of the microfluidic chamber. Tap on the back of the plate to assist the flow through the channel. Using a pipette, aspirate any remaining cell suspension at the exit of the channel.
Incubate the microfluidic chamber with cells for 15 to 20 minutes at 37 degrees Celsius and 5%carbon dioxide. Next, fill the top axonal well with 50 microliters of B-27 neurobasal media, and tap on the back of the plate to assist the flow. Fill each well on the soma side with 150 microliters of B-27 neurobasal media, creating a tension bubble on top.
Also fill both wells of the axonal side with 100 microliters of B-27 neurobasal media each. After seven to eight days of in-vitro culture, ensure that the axons have grown through the microgrooves, and extended into the axonal compartment. Wash the device by removing the B-27 neurobasal medium, and adding 100 microliters of pre-warmed imaging medium to the top wells of both the axonal and soma channels.
Let the medium flow through to the lower wells. Remove the medium from the lower wells, and any leftover medium from the top wells, and repeat with fresh medium, leaving the wells empty at the end of the wash. Then add 100 microliters of five nanomolar TMRE dilution to both the somatic and the axonal top wells.
Let the medium flow through both compartments, until there is an equal volume in all wells. Fill up with the TMRE containing medium. Select a region of interest in the somatic compartment, and follow it through the microgrooves into the axonal compartment.
Ensure that the micro grooves imaged in the somatic and axonal compartments are matched to each other. After changing the file name, acquire red fluorescence images using 561 nanometers excitation and 625 nanometers emission wavelengths. Ensure a volume difference between the soma and axonal chambers by checking the presence of a tension bubble on the somatic side.
Then remove the imaging medium from both wells of the axonal side, except for the medium inside the channel. To induce mitochondrial depolarization, add 160 microliters of 20 micromolar antimycin A diluted in the imaging medium to the top axonal well. Let it flow through the channel until there is an equal volume in both axonal wells.
Repeat the imaging, ensuring that the same positions are imaged as during the baseline acquisition, by identifying the microgrooves. Using this protocol, primary hippocampal neurons were grown in microfluidic devices, followed by mitochondrial staining with a membrane sensitive dye, TMRE. Homogenous staining of mitochondria was observed on both sides of the microgrooves, but not in the middle.
Upon addition of antimycin A to the axonal side, somal mitochondria retained the TMRE signal, whereas the fluorescence was lost from the axonal Mitochondria Ensure that no moisture is left in the well or on the device before you assemble the device. Otherwise, the chambers will not be sealed. Using this method, we can study the effects of loss of mitochondrial function in the axon and local functions, as well as its influence on retrograde signaling and global cell survival.
Mitochondrial biogenesis was long been thought to happen exclusively in the cell body. This method allowed us to reveal that axonal mitochondria damage required local translation of the short of protein PINK1.
