This TechNote describes fluorescent retrograde labeling of neurons via pseudorabies viral infection of axons isolated within the axonal compartment of XonaChips®. Results using this method are illustrated using E18 rat hippocampal neurons and similar results are found using human stem cell derived neurons.
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Introduction

This TechNote describes a procedure for retrograde labeling of neurons within XonaChips® using G-deleted rabies virus. Retrograde tracers such as G-deleted rabies virus expressing fluorescent proteins (incompetent for trans-synaptic transfer) are used to identify neurons that project their axons into the axonal compartment. Pseudorabies virus also retrogradely labels neurons in vivo to trace neuronal circuitry1. Using this virus we labeled both human and murine derived neurons in compartmentalized microfluidic devices2-7. Importantly, fluorescence labeling of neurons with this virus helps to visualize the entire dendritic tree, including fine structures such as filopodia and spines.

In addition, several other viruses have been used in Xona Microfluidic’ devices including Alpha-Herpesvirus, Herpes Simplex Virus 1, Enterovirus, and, Human Coronavirus to study their axonal transport and spread within neurons 8-12. Hence, these microfluidic devices provide a novel method for understanding the route of viral infections and how they modulate neuronal function in CNS.

For more information on cyclic olefin copolymer XonaChips® 4,6, see the Introducing XonaChips® technote.

Methods

Neurons should be first cultured within XonaChips® for at least 1 week or until sufficient axonal growth is present in the axonal compartment before proceeding with labeling steps below. Refer to the appropriate XonaChip® protocol for details.

NOTE: G-deleted rabies virus was purchased from GT3 Core Facility of the Salk Institute by University of North Carolina at Chapel Hill4,6 in collaboration with Xona Microfludics, Inc. Handle potentially infectious materials according to the local organization’s guidelines. Additional training may be required.

  1. Warm up fresh neuronal culture media to 37 °C. Use approximately 400 µl to 500 µl of media per chip.
  2. Remove 50 μl of media from either well of the axonal compartment and store in a 1.5 ml centrifuge tube.
  3. Add 100,000 viral units of the selected modified rabies virus to the 1.5 ml centrifuge tube from step-2.
    NOTE: Tips and tubes in contact with the virus need to be disposed according to your organization-approved protocol.
  4. Gently remove the remaining media from the wells of the axonal compartment into a centrifuge tube and store it at 37 °C.
  5. Add 150 µL of fresh warm media with the diluted virus to the axonal compartment. Place the XonaChip® in the incubator for 2 h at 37° C.
  6. After incubation, gently remove and properly dispose of the virus-containing media from the axonal compartment.
    NOTE: Air bubbles may become trapped in the chip if fluid is vigorously removed or aspirated from the main channels.
  7. Gently add 75 µL of fresh media to one axon well and allow it to flow through to the connecting axonal well.
  8. Remove and dispose properly the flow-through from the second axonal well.
  9. Repeat steps 7 and 8 once. Add reserved media from step 4 to the axonal compartments. Add approximately 50 µL fresh media, if necessary, to maintain adequate volume and return the cells to the incubator.

Results

E18 rat hippocampal neurons were cultured within two compartment XonaChips® (XC450) coated with XC Pre-Coat and XC PDL as described in the XonaChip® protocol. We retrogradely labeled neurons that extended axons into the axonal compartment by applying a G-deleted mCherry rabies virus (incompetent for trans-synaptic transfer) to the axonal compartment at 18 days in-vitro. Neurons were imaged 3 days post-infection (21 days in-vitro). Montages of fluorescently labeled live neurons were captured within the chip to visualize the entire neuron including dendrites in the somato-dendritic compartment and axons that extend through the microgrooves into the axonal compartment (Fig. 1). Images were acquired with a laser scanning Confocal microscope using a 30×/1.05 N.A. silicone oil (ne = 1.406) objective lens.

Conclusion

In summary, neurons can be easily retrogradely labeled via viral infection within both Xona’s Silicone Devices and XonaChips®.

About Xona Microfluidics Inc

Xona Microfluidics, Inc is a life sciences company based in Research Triangle Park, North Carolina. More information can be found at xonamicrofluidics.com.

If you are interested in testing XonaChips® contact us at info@xona.us

References

  1. Wickersham, I.R., et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron. 2007 Mar 1; 53(5):639-47.
  2. Bigler, R. L., Kamande, J. W., Dumitru, R., Niedringhaus, M. & Taylor, A. M. Messenger RNAs localized to distal projections of human stem cell derived neurons. Sci Rep. 2017; 7 (1), 611, doi:10.1038/s41598-017-00676-w, (2017).
  3. Nagendran, T., Larsen, R.S., Bigler, R.L., Frost, S.B., Philpot, B.D., Nudo, R.J., and Taylor, A.M. Distal axotomy enhances presynaptic excitability onto injured pyramidal neurons via trans-synaptic signaling. Nat Commun. 2017 Sep 20; 8(1):625. doi:10.1038/s41467-017-00652-y
  4. Nagendran, T., Poole, V., Harris, J. & Taylor, A. M. Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons. Vis. Exp. 2018; (141), e58421, doi:10.3791/58421.
  5. Kamande J.W., Nagendran, T., Harris, J., and Taylor, A.M. Multi-compartment microfluidic device geometry and covalently bound poly-D-lysine influence neuronal maturation. Front. Bioeng. Biotechnol. 2019; 7:84.doi: 10.3389/fbioe.2019.00084.
  6. Paranjape, S.R., Nagendran, T., Poole, V., Harris, J., and Taylor, A.M. Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips. J. Vis. Exp. 2019; (147), e59250, doi:10.3791/59250.
  7. Nagendran, T and Taylor, A.M. Unique axon-to-soma signaling pathways mediate dendritic spine loss and hyper-excitability post-axotomy. Cell. Neurosci. 2019; Sep 24;13:431. doi: 10.3389/fncel.2019.00431.
  8. Liu, W.W., Goodhouse, J., Jeon, N.L., Enquist, L.W., 2008. A microfluidic chamber for analysis of neuron-to-cell spread and axonal transport of an alpha-herpesvirus. PLoS One 3, e2382-e2382.
  9. Cabrera, J.R., Charron, A.J., Leib, D.A., 2018. Neuronal Subtype Determines Herpes Simplex Virus 1 Latency-Associated-Transcript Promoter Activity during Latency. J Virol 92, e00430-00418.
  10. Hixon, A.M., Clarke, P., Tyler, K.L., 2019. Contemporary Circulating Enterovirus D68 Strains Infect and Undergo Retrograde Axonal Transport in Spinal Motor Neurons Independent of Sialic Acid. J Virol 93, e00578-00519.
  11. Sundaramoorthy, V., Green, D., Locke, K., O’Brien, C.M., Dearnley, M., Bingham, J., 2020. Novel role of SARM1 mediated axonal degeneration in the pathogenesis of rabies. PLOS Pathogens 16, e1008343.
  12. Desforges, M., Le Coupanec, A., Dubeau, P., Bourgouin, A., Lajoie, L., Dubé, M., Talbot, P.J., 2019. Human Coronaviruses and Other Respiratory Viruses: Underestimated Opportunistic Pathogens of the Central Nervous System? Viruses 12, 14.
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