1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School
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F. Pazyra-Murphy, M., A. Segal, R. Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures. J. Vis. Exp. (20), e951, doi:10.3791/951 (2008).
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.
Preparation of reagents
Setting up the compartmented chambers (start this process 1-2 days before the dissection)
Maintaining DRG neurons in compartmented cultures
In this video, we have demonstrated how to prepare and maintain compartmented chambers for use in culturing DRG neurons. Done properly, this system allows separation of the cell body from the axon in order to study mechanisms by which neurotrophins signal across long axons. Since there is fluidic isolation between the compartments, it allows for selective stimulation or treatment of one compartment without the other compartments being affected. Compartmented chamber cultures can support other cell types including sympathetic neurons from the superior cervical ganglia, retinal ganglion neurons, and cortical neurons. Spatial understanding of neurotrophin signal transduction may provide novel insights into treatments of neurodegenerative disorders. Several neurodegenerative disorders, including Alzheimer's disease, Huntington's disease and motor neuron disease, are associated with defects in axonal transport. Recent studies have used microfluidic chambers instead of these compartmented chambers. The microfluidic chambers4,5 have several advantages for imaging analysis.
Prior studies have tested the ability of these cultures to prevent diffusion between the axon and the cell body compartment1,3,6. This can easily be tested by adding low concentrations of a dye such as trypan blue to one compartment only, and look for diffusion of the dye. There should be little or no diffusion visible within 24 hours.
We would like to thank Katharina Cosker and Stephanie Courchesne for helpful discussions.
|N2-methylcellulose 400CPS||Reagent||Sel-Win Chemicals|
|Teflon divider||Other||Tyler Research||CAMP10||many other types of dividers are available|
|Pin rake||Tool||Tyler Research||Camp-PR|
|Grease loader||Tool||Tyler Research||Camp-GLSS|
|High vacuum grease||Reagent||Fisher Scientific||14-635-5D|
|23 gauge luer stub adapter||Tool||Fisher Scientific||427565|
|90° angle hemostats||Tool||Roboz Surgical Instruments Co.||RS-7035|
1. Campenot, RB. Independent Control of the Local Environment of Somas and Neurites. Methods in Enzymology 58 302-7, (1979).
2. Watson, FL., et al. Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nature Neuroscience 4 981-88, (2001).
3. Heerssen, HM., et al. Dynein motors transport activated Trks to promote survival of target-dependent neurons. Nature Neuroscience 7 596-603, (2004).
4. Taylor, AM., et al. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods 2 599-605, (2005).
5. Park JW., et al. Microfluidic culture platform for neuroscience research. Nat Protoc. 4 2128-36, (2006).
6. Ure DR., et al. Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures. J Neuroscience 4 1282-90, (1997)