This study was supported by the German Research Foundation (HA 7728/2-1 and EXC2145 Project ID 390857198) and the Max Planck Society.

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
<ol>
	<li>Coat one six-well glass-bottom tissue culture plate with a final concentration of 20 &#181;g/mL of Poly-D-Lysine and 3.4 &#181;g/mL of Lam...

<ol>
	<li>Murali Mahadevan, H., Hashemiaghdam, A., Ashrafi, G., Harbauer, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+neuronal+health:+from+energy+metabolism+to+Parkinson's+disease.">Mitochondria in neuronal health: from energy metabolism to Parkinson's disease.</a> Advanced Biology. 5 (9), 2100663 (2021).</li><li>Dauer, W., Przedborski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+mechanisms+and+models.">Parkinson's disease: mechanisms and models.</a> Neuron. 39 (6), 889-909 (2003).</li><li>Moratalla, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+vulnerability+of+primate+caudate-putamen+and+striosome-matrix+dopamine+systems+to+the+neurotoxic+effects+of+1-methyl-4-phenyl-1,2,3,6-+tetrahydropyridine.">Differential vulnerability of primate caudate-putamen and striosome-matrix dopamine systems to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine.</a> Proceedings of the National Academy of Sciences. 89 (9), 3859-3863 (1992).</li><li>Cheng, H. -. C., Ulane, C. M., Burke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+progression+in+Parkinson+disease+and+the+neurobiology+of+axons.">Clinical progression in Parkinson disease and the neurobiology of axons.</a> Annals of Neurology. 67 (6), 715-725 (2010).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2 (8), 599-605 (2005).</li><li>Harbauer, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+mitochondria+transport+Pink1+mRNA+via+synaptojanin+2+to+support+local+mitophagy.">Neuronal mitochondria transport Pink1 mRNA via synaptojanin 2 to support local mitophagy.</a> Neuron. 110 (9), 1516-1531 (2022).</li><li>Ashrafi, G., Schlehe, J. S., LaVoie, M. J., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+of+damaged+mitochondria+occurs+locally+in+distal+neuronal+axons+and+requires+PINK1+and+Parkin.">Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin.</a> Journal of Cell Biology. 206 (5), 655-670 (2014).</li><li>Shipman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic+acid+(HEPES)+as+a+tissue+culture+buffer.">Evaluation of 4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic acid (HEPES) as a tissue culture buffer.</a> Proceedings of the Society for Experimental Biology and Medicine. 130 (1), 305-310 (1969).</li><li>Harbauer, A. B., Schneider, A., Wohlleber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondria+by+single-organelle+resolution.">Analysis of mitochondria by single-organelle resolution.</a> Annual Review of Analytical Chemistry. 15, 1-16 (2022).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+mRNA+in+uninjured+and+regenerating+cortical+mammalian+axons.">Axonal mRNA in uninjured and regenerating cortical mammalian axons.</a> The Journal of Neuroscience. 29 (15), 4697-4707 (2009).</li><li>Altman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+TDP-43+condensates+drive+neuromuscular+junction+disruption+through+inhibition+of+local+synthesis+of+nuclear+encoded+mitochondrial+proteins.">Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins.</a> Nature Communications. 12 (1), 1-17 (2021).</li></ol>

The authors declare no competing interests.

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<sup class='xref'...

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&#39;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 &#181;m 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well Glass bottom plate</td>
			<td>Cellvis</td>
			<td>P06.1.5H-N</td>
			<td>Silicone device</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS M5000 widefield microscope</td>
			<td>Thermofischer Scientific</td>
			<td>EVOS M5000</td>
			<td>fully integrated digital widefield microscope</td>
		</tr>
		<tr>
			<td>Hibernate E</td>
			<td>BrainBits</td>
			<td>HE500</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted spinning disk confocal</td>
			<td>Nikon</td>
			<td>TI2-E + CSU-W1</td>
			<td>With incubator chamber</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Invitrogen</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic devices</td>
			<td>XONA microfluidics</td>
			<td>RD450</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>TMRE</td>
			<td>Sigma</td>
			<td>87917</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidics-assisted selective depolarization of axonal mitochondria

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.

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...

Watch this Scientific Journal Video about Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria at JoVE.com

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

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 ...

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.

microfluidics-assisted-selective-depolarization-of-axonal-mitochondria

Research

JoVE Journal

Neuroscience

1.6K Görüntüleme.  Technical University of Munich. Mitokondri nöronal sağlık için gereklidir. Birçok nörodejeneratif hastalıkta, aksonal mitokondri ilk acı çekenlerdir, ancak aksonal mitokondrileri bu kadar özel kılan şeyin ne olduğu açık değildir. Bu protokolde kullanılan odalardaki akışkan basınç gradyanı, mitokondri aksonlarının seçici tedavisine izin verir.Bu, hücre gövdesi süreçlerini rahatsız etmeden bırakır ve aksonal biyolojiye odaklanmamızı sağlar. Burada membran potansiyeline duyarlı bir bo...