title: Production and isolation of axons from sensory neurons for biochemical analysis using porous filters.
abstract: Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Unsain
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University.
authors.forename: Kristen N
authors.initials: KN
authors.lastname: Heard
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Higgins
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University; phil.barker@mcgill.ca.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Barker
mesh_terms: D000818:Animals; D001369:Axons / Q000502:physiology*; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D005260:Female; D005374:Filtration / Q000295:instrumentation / Q000379:methods; D005727:Ganglia, Spinal / Q000166:cytology; D051379:Mice; D011247:Pregnancy; D011984:Sensory Receptor Cells / Q000166:cytology*