FM1-43 imaging reveals cGMP-dependent long-term depression of presynaptic transmitter release

A persistent question concerning mechanisms underlying longterm, activity-d ependent synaptic plasticity is whether the sites of alterations are presyn aptic, postsynaptic, or both. Recently, we discovered a chemical method of inducing long-term depression (LTD) of synaptic strength at Schaffer collat eral-CA1 synapses by simultaneously elevating [cGMP] and inhibiting cAMP-de pendent protein kinase (PKA). Chemical LTD (CLTD) is activity-independent, occluded by stimulus-evoked LTD, and requires access of pharmacologic agent s to presynaptic terminals. In the present study, we used fluorescence and two-photon Imaging of presynaptic terminals with the fluorescent dye N-(3-t riethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1 -43) to determine directly if inducing CLTD is associated with a long-term reduction in transmitter release. In presynaptic Schaffer collateral-CA1 te rminals of control hippocampal slices loaded with FM1-43, electrical stimul ation (10 Hz/2 min) elicited a frequency-dependent destaining that peaked a t 20% reduction in fluorescence. In contrast, when we first induced CLTD by a 30 min treatment of slices with the type V phosphodiesterase inhibitor z aprinast (20 muM) plus the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]- 5-isoquinolinesulfonamide (H-89; 10 muM), then washed for 60 min, the desta ining of FM1-43 fluorescence evoked by the same stimulation was reduced to 4%. Treatment and washout of slices with either drug singly had a significa ntly smaller effect on stimulus-evoked FM1-43 destaining. Only CLTD was ass ociated with virtually complete suppression of stimulus-evoked FM1-43 relea se, the first direct evidence for at least one form of LTD being mediated b y persistent reduction of presynaptic transmitter release.