This work was supported by grants from the Israel Science foundation (ISF, 561/11) and the European Research Council (ERC, 309377).

<ol><li>Misgeld, T., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitostasis+in+Neurons%3A+Maintaining+Mitochondria+in+an+Extended+Cellular+Architecture%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Mitostasis in Neurons: Maintaining Mitochondria in an Extended Cellular Architecture.</a> Neuron. 96, 651-666 (2017).</li>
<li>Devine, M. J., Kittler, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+at+the+neuronal+presynapse+in+health+and+disease%5BTitle%5D)+AND+%22Nature+Reviews+Neuroscience%22%5BJournal%5D)">Mitochondria at the neuronal presynapse in health and disease.</a> Nature Reviews Neuroscience. 19, 63-80 (2018).</li>
<li>Gibbs, K. L., Kalmar, B., Sleigh, J. N., Greensmith, L., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+axonal+transport+in+murine+motor+and+sensory+neurons%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">In vivo imaging of axonal transport in murine motor and sensory neurons.</a> Journal of Neuroscience Methods. 257, 26-33 (2016).</li>
<li>Mandal, A., Drerup, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Axonal+Transport+and+Mitochondrial+Function+in+Neurons%5BTitle%5D)+AND+%22Frontiers+in+Cellular+Neuroscience%22%5BJournal%5D)">Axonal Transport and Mitochondrial Function in Neurons.</a> Frontiers in Cellular Neuroscience. 13, 373(2019).</li>
<li>Magrané, J., Cortez, C., Gan, W. B., Manfredi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abnormal+mitochondrial+transport+and+morphology+are+common+pathological+denominators+in+SOD1+and+TDP43+ALS+mouse+models%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models.</a> Human Molecular Genetics. 23, 1413-1424 (2014).</li>
<li>Anderson, R. G. W., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+view+of+acidic+intracellular+compartments%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">A view of acidic intracellular compartments.</a> Journal of Cell Biology. 106, 539-543 (1988).</li>
<li>Kiral, F. R., Kohrs, F. E., Jin, E. J., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rab+GTPases+and+Membrane+Trafficking+in+Neurodegeneration%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Rab GTPases and Membrane Trafficking in Neurodegeneration.</a> Current Biology. 28, R471-R486 (2018).</li>
<li>Ya-Cheng Liao, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+Granules+Hitchhike+on+Lysosomes+for+Long-Distance+Transport%2C+Using+Annexin+A11+as+a+Molecular+Tether%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether.</a> Cell. 179, 147-164 (2019).</li>
<li>Gluska, S., Chein, M., Rotem, N., Ionescu, A., Perlson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tracking+Quantum-Dot+labeled+neurotropic+factors+transport+along+primary+neuronal+axons+in+compartmental+microfluidic+chambers%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Tracking Quantum-Dot labeled neurotropic factors transport along primary neuronal axons in compartmental microfluidic chambers.</a> Methods in Cell Biology. 131, 365-387 (2016).</li>
<li>Gershoni-Emek, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+RNAi+Machinery+to+Axonal+Branch+Points+and+Growth+Cones+Is+Facilitated+by+Mitochondria+and+Is+Disrupted+in+ALS%5BTitle%5D)+AND+%22Frontiers+in+Molecular+Neuroscience%22%5BJournal%5D)">Localization of RNAi Machinery to Axonal Branch Points and Growth Cones Is Facilitated by Mitochondria and Is Disrupted in ALS.</a> Frontiers in Molecular Neuroscience. 11, 311(2018).</li>
<li>Neto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compartmentalized+Microfluidic+Platforms%3A+The+Unrivaled+Breakthrough+of+In+Vitro+Tools+for+Neurobiological+Research%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of In Vitro Tools for Neurobiological Research.</a> Journal of Neuroscience. 36 (46), 11573-11584 (2016).</li>
<li>Ionescu, A., Zahavi, E. E., Gradus, T., Ben-Yaakov, K., Perlson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compartmental+microfluidic+system+for+studying+muscle-neuron+communication+and+neuromuscular+junction+maintenance%5BTitle%5D)+AND+%22European+Journal+of+Cell+Biology%22%5BJournal%5D)">Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance.</a> European Journal of Cell Biology. 95, 69-88 (2016).</li>
<li>Zahavi, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+compartmentalized+microfluidic+neuromuscular+co-culture+system+reveals+spatial+aspects+of+GDNF+functions%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions.</a> Journal of Cell Science. 128, 1241-1252 (2015).</li>
<li>Altman, T., Geller, D., Kleeblatt, E., Gradus-Perry, T., Perlson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vitro+compartmental+system+underlines+the+contribution+of+mitochondrial+immobility+to+the+ATP+supply+in+the+NMJ%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">An in vitro compartmental system underlines the contribution of mitochondrial immobility to the ATP supply in the NMJ.</a> Journal of Cell Science. 132 (23), (2019).</li>
<li>Schaller, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+combinatorial+screening+identifies+neurotrophic+factors+for+selective+classes+of+motor+neurons%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+USA%22%5BJournal%5D)">Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons.</a> Proceedings of the National Academy of Sciences USA. 114, E2486-E2493 (2017).</li>
<li>Ionescu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+Sigma-1+Receptor+via+Pridopidine+Ameliorates+Central+Features+of+ALS+Pathology+in+a+SOD1G93A+Model%5BTitle%5D)+AND+%22Cell+Death+%26+Disease%22%5BJournal%5D)">Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1G93A Model.</a> Cell Death & Disease. 10 (3), 210(2019).</li>
<li>Zahavi, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+compartmentalized+microfluidic+neuromuscular+coculture+system+reveals+spatial+aspects+of+GDNF+functions%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">A compartmentalized microfluidic neuromuscular coculture system reveals spatial aspects of GDNF functions.</a> Journal of Cell Science. 128, 1241-1252 (2015).</li>
<li>Maimon, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mir126-5p+downregulation+facilitates+axon+degeneration+and+nmj+disruption+via+a+non-cell-autonomous+mechanism+in+ALS%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Mir126-5p downregulation facilitates axon degeneration and nmj disruption via a non-cell-autonomous mechanism in ALS.</a> Journal of Neuroscience. 38, 5478-5494 (2018).</li>
</ol>

The authors declare no conflict of interest.

In this protocol, we describe a system to track axonal transport of mitochondria and acidic compartments in motor neurons. This simplified in vitro platform allows precise control, monitoring, and manipulation of subcellular neuronal compartments, enabling experimental analysis of motor neuron local functions. This protocol can be useful for studying MN diseases such as ALS, to focus on understanding the underlying mechanism of axonal transport dysfunction in the disease10,<sup class="x...

MNs are highly polarized cells with long axons, reaching up to one meter long in adult humans. This phenomenon creates a critical challenge for the maintenance of MN connectivity and function. Consequently, MNs depend on proper transport of information, organelles, and materials along the axons from their cell body to the synapse and back. Various cellular components, such as proteins, RNA, and organelles are shuttled regularly through the axons. Mitochondria are important organelles that are routinely transported in MNs. Mitochondria are essential for proper activity and function of MNs, responsible for ATP provision, calcium buffering, and signaling processes1,2. The axonal transport of mitochondria is a well-studied process3,4. Interestingly, defects in mitochondrial transport were reported to be involved in several neurodegenerative diseases and specifically in MN diseases5. Acidic compartments serve as another example for intrinsic organelles that move along MN axons. Acidic compartments include lysosomes, endosomes, trans-Golgi apparatus, and certain secretory vesicles6. Defects in the axonal transport of acidic compartments were found in several neurodegenerative diseases as well7, and recent papers highlight their importance in MN diseases8.
To efficiently study axonal transport, microfluidic chambers that separate somatic and axonal compartments are frequently used9,10. The two significant advantages of the microfluidic system, and the compartmentalization and the isolation of axons, render it ideal for the study of subcellular processes11. The spatial separation between the neuronal cell bodies and axons can be used to manipulate the extracellular environments of different neuronal compartments (e.g., axons vs. soma). Biochemical, neuronal growth/degeneration, and immunofluorescence assays all benefit from this platform. MFCs can also assist in studying cell-to-cell communication by coculturing neurons with other cell types, such as skeletal muscles12,13,14.
Here, we describe a simple yet precise protocol for monitoring mitochondria and acidic compartment transport in motor neurons. We further show the use of this method by comparing the relative percentage of retrograde and anterograde moving organelles, as well as the distribution of transport velocity.

<table><tbody><tr><td>35mm Fluodish &amp;ndash; glass bottom dish</td><td>World Precision Instruments WPI</td><td>FD35-100</td><td></td></tr><tr><td>50mm Fluodish &amp;ndash; glass bottom dish</td><td>World Precision Instruments WPI</td><td>FD5040-100</td><td></td></tr><tr><td>Andor iXon DU-897 EMCCD camera</td><td>Andor</td><td></td><td></td></tr><tr><td>ARA-C (Cytosine &amp;beta;-D-arabinofuranoside)</td><td>Sigma-Aldrich</td><td>C1768</td><td>stock of 2mM in filtered DDW</td></tr><tr><td>B-27 Supplement (50X)</td><td>Thermo Fisher</td><td>17504044</td><td></td></tr><tr><td>BDNF</td><td>Alomone Labs</td><td>B-250</td><td>Dilute to 10 &amp;micro;g/mL in filtered ddw with 0.01% BSA)</td></tr><tr><td>Biopsy punch 1.25mm</td><td>World Precision Instruments WPI</td><td>504530</td><td>For preperation of large MFC</td></tr><tr><td>Biopsy punch 6mm</td><td>World Precision Instruments WPI</td><td>504533</td><td>For preperation of small MFC</td></tr><tr><td>Biopsy punch 7mm</td><td>World Precision Instruments WPI</td><td>504534</td><td>For preperation of large MFC</td></tr><tr><td>Bitplane Imaris software - version 8.4.1</td><td>Imaris</td><td></td><td></td></tr><tr><td>Bovine Serum Albumine (BSA)</td><td>Sigma-Aldrich</td><td>#A3311-100G</td><td>5% w/v in ddw</td></tr><tr><td>Chlorotrimetylsilane</td><td>Sigma-Aldrich</td><td>#386529-100ML</td><td></td></tr><tr><td>CNTF</td><td>Alomone Labs</td><td>C-240</td><td>Dilute to 10 &amp;micro;g/mL in filtered ddw with 0.01% BSA)</td></tr><tr><td>Density Gradient Medium - Optiprep</td><td>Sigma-Aldrich</td><td>D1556</td><td></td></tr><tr><td>Deoxyribonuclease I (DNAse) from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN-25</td><td>stock 10mg/mL in neurobasal</td></tr><tr><td>Dow Corning High-vacuum silicone grease</td><td>Sigma-Aldrich</td><td>Z273554-1EA</td><td>For epoxy mold preperation</td></tr><tr><td>DPBS 10X</td><td>Thermo Fisher</td><td>#14200-067</td><td>dilute 1:10 in ddw</td></tr><tr><td>Dumont fine forceps #55 0.05 &amp;times; 0.02 mm</td><td>F.S.T</td><td>1125520</td><td></td></tr><tr><td>Epoxy Hardener</td><td>Trias Chem S.R.L</td><td>IPE 743</td><td>For epoxy mold preperation</td></tr><tr><td>Epoxy Resin</td><td>Trias Chem S.R.L</td><td>RP 026UV</td><td>For epoxy mold preperation</td></tr><tr><td>FIJI software</td><td>ImageJ</td><td></td><td></td></tr><tr><td>GDNF</td><td>Alomone Labs</td><td>G-240</td><td>Dilute to 10 &amp;micro;g/mL in filtered ddw with 0.01% BSA)</td></tr><tr><td>Glutamax 100X</td><td>Thermo Fisher</td><td>#35050-038</td><td></td></tr><tr><td>HB9:GFP mice strain</td><td>Jackson Laboratories</td><td>005029</td><td></td></tr><tr><td>HBSS 10X</td><td>Thermo Fisher</td><td>#14185-045</td><td>Dilute 1:10 in ddw with addition of 1% P/S and filter</td></tr><tr><td>iQ software</td><td>Andor</td><td></td><td></td></tr><tr><td>Iris scissors, curved, 10 cm</td><td>AS Medizintechnik</td><td>11-441-10</td><td></td></tr><tr><td>Iris scissors, straight, 9 cm</td><td>AS Medizintechnik</td><td>11-440-09</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>#L-2020</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Thermo Fisher</td><td>11415064</td><td></td></tr><tr><td>LysoTracker Red</td><td>Thermo Fisher</td><td>L7528</td><td></td></tr><tr><td>Mitotracker Deep-Red FM</td><td>Thermo Fisher</td><td>M22426</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermo Fisher</td><td>21103049</td><td></td></tr><tr><td>Nikon Eclipse Ti micorscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin (P/S) Solution</td><td>Biological Industries</td><td>03-031-1</td><td></td></tr><tr><td>Poly-L-Ornithin (PLO)</td><td>Sigma-Aldrich</td><td>#P8638</td><td>Dilute 1:1000 in flitered 1X PBS</td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>DOW Corning Corporation</td><td>#3097358-1004</td><td></td></tr><tr><td>Trypsin from bovine pancreas</td><td>Sigma-Aldrich</td><td>T1426</td><td>stock 25 mg/mL in 1XPBS</td></tr><tr><td>Vannas spring microdissection scissors, 3 mm blade</td><td>F.S.T</td><td>15000-00</td><td></td></tr><tr><td>Yokogawa CSU X-1</td><td>Yokogawa</td><td></td><td></td></tr></tbody></table>

axonal transport of organelles in motor neuron cultures using microfluidic chambers system

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Following the described protocol, mouse embryonic HB9::GFP spinal cord explants were cultured in MFC (Figure 4A). Explants were grown for 7 days, when axons fully crossed into the distal compartment. Mitotracker Deep Red and Lysotracker Red dyes were added to the distal and proximal compartments in order to label the mitochondria and acidic compartments (Figure 4C). Axons in the distal grooves we...

Watch this Scientific Journal Video about Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System at JoVE.com

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

axonal-transport-organelles-motor-neuron-cultures-using-microfluidic

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JoVE Journal

Neuroscience

10.0K Views.  Tel Aviv University. Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Video: Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biology

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

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.

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 ...

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

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Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

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