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 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.
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.
The care and treatment of animals in this protocol were performed under the supervision and approval of the Tel Aviv University Committee for Animal Ethics.
1. MFC preparation
2. Neuronal culture plating
3. Axonal transport (Figure 4A)
4. Image analysis (Figures 4-5)
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 were imaged, and the movies were analyzed as follows: First, we compared the general movement distribution using kymograph analysis (Figures 4B,D). This analysis revealed a bias in the retrograde direction only in acidic compartments (nonmoving = 77.1 ± 9.5%; retrograde = 16.9 ± 8.3%, anterograde = 6 ± 5%; Figure 4E) but not in mitochondrial transport (nonmoving = 83.4 ± 6.8%; retrograde = 10.5 ± 6.9%; anterograde = 6.8 ± 5.1%; Figure 4F). Kymograph analysis was used to quantify the particle density, revealing a higher number of mitochondrial particles compared to acidic compartments in HB9::GFP spinal cord explant axons (Mitochondria = 0.46 ± 0.13; Acidic compartments = 0.3 ± 0.07 particles/µm axon, Figure 4G).
Next, single particle transport analysis was conducted using semiautomated software followed by in-house code (Figure 5A-B). This analysis revealed that despite having similar particle velocity in general (Figure 5C), when observing the distribution of velocities (Figure 5D) only acidic compartments but not mitochondria displayed a bias towards retrograde movement.
Figure 1: Silicone mold preparation. Schematic drawing describing the procedure of chlorotrimethylsilane wafer cleansing. (A) First, 50 mL of liquid nitrogen were added to an appropriate container. Working in a chemical hood, a syringe and needle were used to draw 8 mL of liquid nitrogen. The entire content of the syringe was injected into the chlorotrimethylsilane bottle. The bottle was turned with the cap facing down and 8 mL of chlorotrimethylsilane were drawn back. (B) Chlorotrimethylsilane spread in the container (not directly on the wafer). The container needs to be closed, followed by 5 min incubation for each mold. (C) Liquid PDMS was poured into each wafer up to the desired height. (D) All plates were placed together inside a vacuum desiccator for 2 h, followed by 3 h-overnight in a 70 °C oven. Please click here to view a larger version of this figure.
Figure 2: MFC specialized design. (A) Polymerized PDMS template taken out of the mold using a metal scalpel. (B) Depending on the experimental setup either 6 mm, 7 mm, or 1 mm punchers were used for punching the PDMS templates. (C) For explant culture in the MFC, 7 mm and 1 mm punchers were used, and a 20 G syringe was utilized for making "caves" for easy explant insertion. (D) For dissociated MN culture MFC, a 6 mm puncher was used to create four wells at the channel edges. (E-F) Illustrations of the final MFC shapes described in C and D, respectively. Please click here to view a larger version of this figure.
Figure 3: Neuronal culture. (A) E12.5 mouse embryo was placed in position after the head, tail, and skin were removed in order to expose the neural tube. (B) Dissection of the whole spinal cord. (C) Using gentle forceps, the meninges was peeled away from the spinal cord. (D) Left panel: Removal of the spinal cord lateral segments from the ventral spinal cord to yield better MN purification. Right Panel: Representative image of dissociated MN culture in the MFC. HB9::GFP axons crossed to the distal compartment (green). Hoechst staining indicates neuronal nuclei (blue). (E) Spinal cord explants generated by dissecting 1 mm thick transverse sections of the ventral spinal cord. Representative image of HB9::GFP (green) spinal cord explant axons in an MFC. Please click here to view a larger version of this figure.
Figure 4: Axonal transport of mitochondria and acidic compartment in MNs. (A) Illustration of the axonal transport essay. Lysotracker Red and Mitotracker Deep Red were added to both the proximal and distal compartments of the MFC, containing HB9::GFP ventral spinal cord explant. (B) Kymograph analysis. Moving particles were defined as moving anterograde or retrograde following displacement of more than 10 µm in that direction. Rotating or immobile particles were counted as nonmoving. Scale bar = 10 µm. (C) First frame of a time-lapse movie displaying primary HB9::GFP mouse spinal cord explant axons dyed with Lysotracker red to tag acidic compartments and Mitotracker Deep Red to tag mitochondria. Scale bar = 10 µm. (D) Representative kymographs displaying a typical axonal movement of acidic compartments and mitochondria. Scale bar = 10 µm. (E) Kymograph analysis of mitochondrial axonal transport, ****p < 0.0001, Anova with Holm-Sidak correction (n = 77 axons). Scale bar = 10 µm (F) Kymograph analysis of acidic compartment axonal transport, ** p < 0.01, ****p < 0.0001, Anova with Holm-Sidak correction (n = 77 axons). (G) Axonal particle density analysis of mitochondria and acidic compartments, ****p < 0.0001, Student's t-test (n = 77 axons). Error bars represent values with SD. Please click here to view a larger version of this figure.
Figure 5: Semi-automated single particle analysis to measure organelle velocity. (A) Schematic workflow for semiautomated single particle transport analysis. (B) Ventral spinal cord explant axons were analyzed for single particle tracking. The analysis software is capable of tracking single axonal particles in time-lapse movies, as indicated for mitochondria (yellow dots, upper panel) and acidic compartments (green dots, lower panel). (C) The average velocity did not change between mitochondria and acidic compartments, Mann Whitney test (n = 417 mitochondria, n = 371 acidic compartments). Error bars represent values with SD. (D) Distribution of mitochondrial and acidic compartments retrograde and anterograde velocities. Please click here to view a larger version of this figure.
Complete Neurobasal Medium – for 50mL | ||
Ingredient | Volume | Concentration |
Neurobasal | 47mL | |
B27 | 1 mL | 2% |
Horse serum | 1 mL | 2% |
P/S | 0.5 mL | 1% |
L-Glutamine (Glutamax) | 0.5 mL | 1% |
Beta-Mercaptoethanol (50mM) | 25 µL | 25µM |
BDNF (10ug/mL) | 5 µL | 1ng/mL |
GDNF (10ug/mL) | 5 µL | 1ng/mL |
CNTF (10ug/mL) | 2.5 µL | 0.5ng/mL |
Table 1: Recipe for preparation of complete neurobasal (CNB) solution.
Optiprep Solution – for 10mL | ||
Ingredient | Volume | Concentration |
DDW | 5.27 mL | |
Density Gradient Medium (Optiprep) 60% | 1.73 mL | 10.4% (w/v) |
Tricine 100mM | 1 mL | 2% |
Glucose 20% (w/v) | 2 mL | 2% |
Table 2: Recipe for preparation of density gradient medium solution.
Spinal Cord Explant Medium (SCX) – for 20mL | ||
Ingredient | Volume | Concentration |
Neurobasal | 19.5 mL | |
B27 | 200 µL | 2% |
P/S | 100 µL | 1% |
L-Glutamine (Glutamax) | 100 µL | 1% |
BDNF | 50 µL | 25ng/mL |
Table 3: Recipe for preparation of spinal cord explant (SCEX) solution.
MN Culture | Spinal Cord Explants |
Longer Procedure prior to plating | Short procedure – Dissect & Plate |
Extra caution needed for plating in MFC | Easier to plate in the MFC |
High concentration of motor neurons with no glial cells – more accurate | Presence of glial cells and other neuronal types – more physiological |
Unlimited manipulation possibilities on both Soma and axons | Limited manipulation possibilities on cell bodies |
Easy immunostaining for both cell bodies and axons | Low efficiency during immunostaining of cell bodies within the explant. |
High efficiency of viral infection | Very low efficiency of viral infection |
Table 4: Comparison between spinal cord explants and dissociated MN culture based on perimeters of speed, feasibility, glial presence, manipulation possibilities, immunostaining, and viral infection.
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 disease10,16. Moreover, this system can also be applied for studying transport of trophic factors9,16, microRNA10, mRNA8, and labeled proteins in healthy and diseased MNs or in other neurons, such as sensory9 or sympathetic axons14. A similar method can also be applied to study organelle transport in a coculture system, such as MNs cultured with skeletal muscle cells12 or sympathetic neurons cocultured with cardiomyocytes14. The MFC system can be utilized to generate active NMJs17,18 and study the effect of synapse formation on axonal transport16.
This protocol has several advantages compared to other protocols for culturing of neurons in MFC and using live imaging to analyze axonal transport: 1) MFCs are commercially available by several manufacturers. However, self-manufacturing of the MFCs is extremely cost effective compared to the commercial alternative. A single PDMS wafer yields four large MFCs or nine small ones at the minimal expense of several US dollars for the PDMS resin itself. 2) The self-manufactured MFCs can be modified to answer specific experimental needs (e.g., changing the size and the location of the wells, increasing the thickness of the MFC PDMS). 3) The MFCs can be irreversibly attached to a plate (via plasma bonding) but can also be recycled multiple times to reduce the possibility of contamination. 4) The PDMS MFCs are transparent, making them ideal for live imaging by having reduced background, which is critical for axonal transport assays where signal-to-noise distinction could be a limiting factor. 5) Spinal cord explant culture is very efficient and fast. One embryonic spinal cord can yield up to 30 explants, enough for 10 MFCs. This can help to save time and materials, and ensures that even a pregnant mouse with few embryos can produce a successful experiment. See a detailed comparison of spinal cord explant and dissociated MN culture in Table 4.
This protocol is relatively simple and inexpensive. However, it requires expertise in several technical matters. The manufacturing and handling of the MFC needs to be accurate and gentle, to avoid structural defects and creation of air bubbles in the obligatory vacuum step (1.1.10), for example. During the optional vacuum step (1.6.2) it is important to clear all the air, or it will block axonal crossing, but also keep the vacuum short to avoid detachment of the MFC from the glass. Pay close attention to the dissection protocol steps, as it is important to properly remove the meninges and dorsal horns, as well as to try and cut the pieces to the right size in order to insert them to the MFC's cave without using physical force. Any excessive physical force applied to the MFC can easily detach the grooves or the entire MFC, thus the procedure should be done gently. Culturing of MNs and plating them in the MFC needs to be relatively swift, as MNs tend to aggregate and lose viability quickly under stress conditions such as the low medium volume of the plating step. The plating in the MFC should be done in low volume to allow proper attachment of the neurons in the MFC channel, and after no more than 30 min warmed medium should be added very gently to prevent detachment of the MNs.
After several days in culture, and once axons have extended to the distal compartment, the cultures are ready to undergo organelle staining followed by acquisition of axonal transport movies and finally a specific procedure for image analysis. The analysis can be performed either by single particle tracking over time, or by using an automated or semiautomated tracking algorithm. In our experience, automated methods are less time-consuming, but have several disadvantages. Mainly, automated tracking reliability is decreased, with crowded axons that cross paths. Furthermore, automated algorithms have a decreased ability to distinguish between overlapping particles. Consequently, performing manual or semiautomated tracking is recommended. In general, semiautomated tracking is preferable as it is less time-consuming, but manual tracking can be a good option when there is no available paid software. A thorough comparison between manual and automatic analysis can be found in Gluska et al.9.
In conclusion, adopting this simple protocol can yield important data for the study of axonal transport of various cellular components. It can be adapted to fit a diversity of imaging setups and neuronal subtypes, as well as to cocultures of neurons with other cells. It also allows distinct spatial pharmacological manipulation of either cell bodies or axons in order to understand basic mechanisms of neuronal health and to improve drug development for diseases with altered axonal transport.
The authors have nothing to disclose.
This work was supported by grants from the Israel Science foundation (ISF, 561/11) and the European Research Council (ERC, 309377).
35mm Fluodish – glass bottom dish | World Precision Instruments WPI | FD35-100 | |
50mm Fluodish – glass bottom dish | World Precision Instruments WPI | FD5040-100 | |
Andor iXon DU-897 EMCCD camera | Andor | ||
ARA-C (Cytosine β-D-arabinofuranoside) | Sigma-Aldrich | C1768 | stock of 2mM in filtered DDW |
B-27 Supplement (50X) | Thermo Fisher | 17504044 | |
BDNF | Alomone Labs | B-250 | Dilute to 10 µg/mL in filtered ddw with 0.01% BSA) |
Biopsy punch 1.25mm | World Precision Instruments WPI | 504530 | For preperation of large MFC |
Biopsy punch 6mm | World Precision Instruments WPI | 504533 | For preperation of small MFC |
Biopsy punch 7mm | World Precision Instruments WPI | 504534 | For preperation of large MFC |
Bitplane Imaris software – version 8.4.1 | Imaris | ||
Bovine Serum Albumine (BSA) | Sigma-Aldrich | #A3311-100G | 5% w/v in ddw |
Chlorotrimetylsilane | Sigma-Aldrich | #386529-100ML | |
CNTF | Alomone Labs | C-240 | Dilute to 10 µg/mL in filtered ddw with 0.01% BSA) |
Density Gradient Medium – Optiprep | Sigma-Aldrich | D1556 | |
Deoxyribonuclease I (DNAse) from bovine pancreas | Sigma-Aldrich | DN-25 | stock 10mg/mL in neurobasal |
Dow Corning High-vacuum silicone grease | Sigma-Aldrich | Z273554-1EA | For epoxy mold preperation |
DPBS 10X | Thermo Fisher | #14200-067 | dilute 1:10 in ddw |
Dumont fine forceps #55 0.05 × 0.02 mm | F.S.T | 1125520 | |
Epoxy Hardener | Trias Chem S.R.L | IPE 743 | For epoxy mold preperation |
Epoxy Resin | Trias Chem S.R.L | RP 026UV | For epoxy mold preperation |
FIJI software | ImageJ | ||
GDNF | Alomone Labs | G-240 | Dilute to 10 µg/mL in filtered ddw with 0.01% BSA) |
Glutamax 100X | Thermo Fisher | #35050-038 | |
HB9:GFP mice strain | Jackson Laboratories | 005029 | |
HBSS 10X | Thermo Fisher | #14185-045 | Dilute 1:10 in ddw with addition of 1% P/S and filter |
iQ software | Andor | ||
Iris scissors, curved, 10 cm | AS Medizintechnik | 11-441-10 | |
Iris scissors, straight, 9 cm | AS Medizintechnik | 11-440-09 | |
Laminin | Sigma-Aldrich | #L-2020 | |
Leibovitz's L-15 Medium | Thermo Fisher | 11415064 | |
LysoTracker Red | Thermo Fisher | L7528 | |
Mitotracker Deep-Red FM | Thermo Fisher | M22426 | |
Neurobasal medium | Thermo Fisher | 21103049 | |
Nikon Eclipse Ti micorscope | Nikon | ||
Penicillin-Streptomycin (P/S) Solution | Biological Industries | 03-031-1 | |
Poly-L-Ornithin (PLO) | Sigma-Aldrich | #P8638 | Dilute 1:1000 in flitered 1X PBS |
Sylgard 184 silicone elastomer kit | DOW Corning Corporation | #3097358-1004 | |
Trypsin from bovine pancreas | Sigma-Aldrich | T1426 | stock 25 mg/mL in 1XPBS |
Vannas spring microdissection scissors, 3 mm blade | F.S.T | 15000-00 | |
Yokogawa CSU X-1 | Yokogawa |