A technique for the primary dissociation of neurons from restricted regions of the vertebrate CNS.
Acute isolation of vertebrate neurons has been used extensively to characterize membrane properties in the absence of circuit connections or extensive dendritic arborizations. We describe a technique that allows cells to be dissociated from anatomically defined regions of a tissue slice at a resolution beyond that attainable by micro-dissection. Dissociation is performed by using a fire-polished electrode with a tip diameter of 40-100 microns connected by tubing to a micrometer syringe that allows graded levels of positive or negative pressure to be applied at the electrode tip. The electrode tip is placed under microscopic observation upon a cell group within an enzymatically treated slice and negative pressure is applied to dissociate cells into the electrode shaft. Positive pressure is used to eject the cells onto the surface of poly-L-lysine-coated glass coverslips. We have used this technique to dissociate and culture cells from specific laminae of separate sensory maps in a medullary nucleus of adult weakly electric fish. Isolated cells were viable, could be identified by morphological criteria, and exhibited process extension within 2 h of plating. This technique greatly increases the probability of isolating morphologically identifiable vertebrate neurons for electrophysiological analysis or for the reconstruction of neural circuits in vitro.
Turner
R W
RW
Department of Medical Physiology, University of Calgary, Alberta, Canada.
Borg
L L
LL
Syed
N I
NI
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002490:Central Nervous System / Q000166:cytology*; D004555:Electric Fish; D007150:Immunohistochemistry; D009474:Neurons; D009488:Neurosciences / Q000379:methods*; D014714:Vertebrates