Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variet
y of cellular processes, including synaptic vesicle recycling. However, lit
tle is known about the spatial distribution of this phospholipid in neurons
and its dynamics. In this study, we have focused on these questions by tra
nsiently expressing the phospholipase C (PLC)-delta1 pleckstrin homology (P
H) domain fused to green fluorescent protein (GFP) in cultured hippocampal
neurons. This PH domain binds specifically and with high affinity to PIP2.
Live confocal imaging revealed that in resting cells, PH-GFP is localized p
redominantly on the plasma membrane. Interestingly, no association of PH-GF
P with synaptic vesicles in quiescent neurons was observed, indicating the
absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulat
ion of hippocampal neurons resulted in a decrease of the PH-GFP signal at t
he plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2.
This was accompanied in the majority of presynaptic terminals by a marked
increase in the cytoplasmic PH-GFP signal, localized most probably on fresh
ly endocytosed membranes. Further investigation revealed that the increase
in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate re
ceptors and the consequent production of nitric oxide (NO). Thus, PIP2 in t
he presynaptic terminal appears to be regulated by postsynaptic activity vi
a a retrograde action of NO.