Activation of silent and weak synapses by cAMP-dependent protein kinase in cultured cerebellar granule neurons.

Presynaptic long term potentiation of synaptic transmission activates silent synapses and potentiates existing active synapses. We sought to visualise these two processes by studying the cAMP-dependent protein kinase (PKA) potentiation of presynaptic vesicle cycling in cultured cerebellar granule neurons.Using FM dyes to label the pool of recycling synaptic vesicles,we found that trains of electrical stimulation which do not potentiate already active synapses are sufficient to rapidly activate a discrete population comprising silent and very low activity synapses. Silent synapse activation required PKA activity and conversely, active synapses could be silenced by PKA inhibition. Surprisingly, the recycling pool of synaptic vesicles in recently activated synapses was larger than in already active synapses and equivalent to synapses treated with forskolin. Imaging of synaptic vesicle cycling and cytosolic Ca(2+) in individual nerve terminals confirmed that silent synapses have evoked Ca(2+) transients comparable to those of active synapses. Furthermore, across populations of active synapses, changes in Ca(2+) influx did not correlate with changes in the size of the pool of recycling synaptic vesicles. Finally, we found that stimulation of synapsin phosphorylation, but not RIM1α, by PKA was frequency dependent and long lasting. These data are consistent with the idea that PKA regulates synaptic vesicle recycling downstream of Ca(2+) influx and that this pathway is highly active in recently activated synapses.