Isolation of Functional Presynaptic Complexes from CNS Neurons: A Cell-Free Preparation for the Study of Presynaptic Compartments In Vitro
journal contributionposted on 18.08.2010, 00:00 by Anna Lisa Lucido, Gopakumar Gopalakrishnan, Patricia T. Yam, David R. Colman, R. Bruce Lennox
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.