Isolation of synaptosomes
Synaptosomes were isolated as previously described by
(51). Briefly, mice at postnatal days
90-95 were treated as described above. Brain hippocampi from the two
animal groups were collected, rinsed and homogenized in solution A,
consisting of 0.32 M Sucrose, 1 mM NaHCO3, 1 mM
MgCl2, 0.5 mM CaCl2•H2O, 10 mM sodium
pyrophosphate and protease inhibitors using a dounce homogenizer. After
centrifugation at 1,400 x g for 10 min at 4 oC, the
resulting supernatants were kept and the pellets were diluted 10 % w/v
in solution A and spun at 710xg for 10 min. The supernatants were
collected and centrifuged at 13,800 x g for 10 min at 4oC. The pellets were resuspended in 0.32 M sucrose and
1 mM NaHCO3 using a dounce homogenizer and layered on a
discontinuous sucrose gradient (10ml-layers of 1.2 M, 1.0 M and 0.85 M
sucrose). After centrifugation at 82,500 x g for 2 h, the synaptosomes
from U50,488- or saline-injected mice were isolated from the 1.2-1 M
sucrose layer.