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Updated in 6/21/2018 8:54:33 AM      Viewed: 1014 times      (Journal Article)
Journal of visualized experiments : JoVE (19) (2008)

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Maria F Pazyra-Murphy , Rosalind A Segal
ABSTRACT
FROM JOVE.COM:

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.

DOI: yle="">10.3791/951      ISSN: 1940-087X     
Notes
Preparation of reagents Collagen coating: collagen coat p35 tissue culture plates and place in an oven at 37°C for 2 days before greasing the dividers. The final concentration of collagen should be at .71 mg/ml diluted in .001N HCl.  Then, add 1 ml of mixture per plate. Grease loaders: In order to fill the grease loader, a 60mL syringe must first be filled with Corning vacuum grease. Use the syringe to fill the grease loader, wrap it in foil and then autoclave for 45 minutes. Teflon dividers: the dividers can be re-used after each experiment but must first be properly cleaned. Remove the divider from the plate, wipe off all of the remaining grease and place in sulfuric acid for 2 days. After removing from the acid, rinse with water 3X, boil for 20 minutes, allow to dry, place in a glass p100 petri dish and autoclave for 20 minutes. N2-methylcellulose: Weigh out 1.5g of methylcellulose and place it in a 500mL bottle. Add a stir bar and autoclave it for 20 minutes on dry (from this poin Read more...