Simultaneous isolation of glial and neuronal fractions from rat brain homogenates: comparison of high-affinity L-glutamate transport properties.
An improved three-step Percoll density gradient centrifugation technique is described for simultaneous isolation of glial plasmalemmal vesicles (GPV) and synaptosomal vesicles (SYN) from a rat brain homogenate. While electron microscopy revealed that fractions contained intact vesicles with markedly distinct morphological features, measures of high-affinity [3H]choline uptake, glutamine synthetase and carbonic anhydrase activities, as well as Western blot analyses for glial fibrillary acidic protein and neuron specific enolase, served to confirm the low level of neuronal contamination in GPV fractions as well as the low level of glial contamination in SYN fractions. In addition, GPV and SYN fractions were used to characterize the kinetic and pharmacological properties of sodium-dependent [3H]L-glutamate transport. In conclusion, these results demonstrate the usefulness of this method for obtaining highly-enriched, functionally viable populations of glial and neuronal elements which are suitable for studies of their respective cell functions in vitro.
Daniels
K K
KK
Department of Neuroscience, University of Florida, Gainesville 32610, USA.
Vickroy
T W
TW
D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000648:ultrastructure*; D002256:Carbonic Anhydrases / Q000032:analysis; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D002794:Choline / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000032:analysis; D018698:Glutamic Acid / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D009457:Neuroglia / Q000648:ultrastructure*; D009474:Neurons / Q000648:ultrastructure*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure
D018698:Glutamic Acid; D012964:Sodium; D002256:Carbonic Anhydrases; D005974:Glutamate-Ammonia Ligase; D002794:Choline