Isolation of hippocampal synaptosomes on Percoll gradients: cholinergic markers and ligand binding sites.
The S1 Percoll procedure, devised empirically for cortical tissue, provides highly purified, functionally viable synaptosomes on a four-step Percoll gradient. Here, for the first time, the procedure has been applied to rat hippocampus, and the gradient fractions have been analysed with respect to cholinergic markers and the synaptosomal index, lactate dehydrogenase. The presynaptic cholinergic markers choline acetyltransferase and [3H]choline uptake were most enriched in fraction 4. In contrast, acetylcholinesterase activity was broadly distributed across the gradient, consistent with the separation of synaptic plasma membranes (in fractions 1 and 2) from synaptosomes (in fractions 3 and 4). This is supported by the recovery of muscarinic binding sites labelled with [3H]quinuclidinylbenzilate in fractions 1 and 2. (-)-[3H]-Nicotine binding sites, however, were most enriched in fraction 4, consistent with their predominantly presynaptic localisation in the CNS. These results demonstrate the applicability of the S1 Percoll method to discrete brain regions for the recovery of homogeneous and viable synaptosome fractions. The separation of presynaptic terminals from post-synaptic membranes is a further advantage of this technique.
Thorne
B
B
Department of Biochemistry, University of Bath, England.
Wonnacott
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S
Dunkley
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PR
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002794:Choline / Q000378:metabolism*; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D006624:Hippocampus / Q000648:ultrastructure*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D009538:Nicotine / Q000378:metabolism; D011205:Povidone; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism; D012822:Silicon Dioxide; D013574:Synaptosomes / Q000378:metabolism / Q000648:ultrastructure*
D011976:Receptors, Muscarinic; D011978:Receptors, Nicotinic; C016039:Percoll; D011813:Quinuclidinyl Benzilate; D009538:Nicotine; D012822:Silicon Dioxide; D007770:L-Lactate Dehydrogenase; D002795:Choline O-Acetyltransferase; D000110:Acetylcholinesterase; D011205:Povidone; D002794:Choline