title: Isolation of functional presynaptic complexes from CNS neurons: a cell-free preparation for the study of presynaptic compartments In vitro.
abstract: The difficulty in developing successful treatments to facilitate nerve regeneration has prompted a number of new in vitro experimental methods. We have recently shown that functional presynaptic boutons can be formed when neuronal cells are cocultured with surface-modified artificial substrates including poly(d-lysine)-coated beads and supported lipid bilayer-coated beads (Lucido(2009) J. Neurosci.29, 12449-12466; Gopalakrishnan(2010) ACS Chem. Neurosci.1, 86-94). We demonstrate here, using confocal microscopy combined with immunocytochemistry, that it is possible to isolate such in vitro presynaptic endings in an exclusive fashion onto glass substrates through a simple "sandwich/lift-off" technique (Perez(2006) Adv. Funct. Mater.16, 306-312). Isolated presynaptic complexes are capable of releasing and recycling neurotransmitter in response to an external chemical trigger. These bead-presynaptic complexes are facile to prepare and are readily dispersible in solution. They are thus compatible with many experimental methods whose focus is the study of the neuronal presynaptic compartment.
authors: Anna Lisa
authors: AL
authors: Lucido
authors: Gopakumar
authors: G
authors: Gopalakrishnan
authors: Patricia T
authors: PT
authors: Yam
authors: David R
authors: DR
authors: Colman
authors: R Bruce
authors: RB
authors: Lennox
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D002474:Cell-Free System; D002478:Cells, Cultured; D002490:Central Nervous System / Q000166:cytology*; D020099:Coated Materials, Biocompatible; D005089:Exocytosis; D005456:Fluorescent Dyes / Q000032:analysis; D006624:Hippocampus / Q000166:cytology / Q000196:embryology; D018613:Microscopy, Confocal; D008863:Microspheres; D009419:Nerve Tissue Proteins / Q000032:analysis; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011107:Polylysine; D011137:Polystyrenes; D017729:Presynaptic Terminals / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D061251:Primary Cell Culture / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020099:Coated Materials, Biocompatible; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; D011137:Polystyrenes; D011107:Polylysine; C003321:polystyrene sulfonic acid