Repetitive activation of protein kinase A induces slow and persistent potentiation associated with synaptogenesis in cultured hippocampus

Mammalian brain memory is hypothesized to be established through two phases; short-term plasticity, as exemplified by long-term potentiation (LTP) where pre-existing synapses change transmission efficiency, and long-lasting plasticity where new synapses are formed. This hypothesis, however, has not been verified experimentally. Using cultured hippocampal slices, we show that the repeated induction of late-phase LTP by brief applications of forskolin (FK) led to a slowly-developing long-lasting synaptogenesis, as judged from electrophysiological, cytological and ultrastructural indices. These indices include (1) field postsynaptic potential standardized by field action potential, which should represent the number of synapses per neuron; (2) the amounts of synaptic marker proteins; (3) the number of synaptophysin-immunopositive puncta; (4) the number of dendritic spines per length; (5) the density of synaptic ultrastructures; (6) ultrastructures similar to synapse perforation. Increment in these indices occurred approximately 10 days after FK-application and outlasted the following weeks. The increment depended on the times and intervals of FK-application. A biologically inert FK analogue failed to produce the similar effect. An inhibitor for cyclic AMP-dependent protein kinase (PKA) blocked the synaptogenesis. The cultured brain slice repeatedly exposed to FK should serve as a good model system for the analysis of persistent synaptogenesis possibly related to long-term memory in mammalian CNS.