Synaptic plasticity involves a series of coordinate changes occurring both pre- and postsynaptically, of which alpha-synuclein is an integral part. We have investigated on mouse primary hippocampal neurons in culture whether redistribution of alpha-synuclein during plasticity involves retrograde signaling activation through nitric oxide (NO), cGMP, cGMP-dependent protein kinase (cGK) and calmodulin-dependent protein kinase II. We have found that deletion of the alpha-synuclein gene blocks both the long-lasting enhancement of evoked and miniature transmitter release and the increase in the number of functional presynaptic boutons evoked through the NO donor, DEA/NO, and the cGMP analog, 8-Br-cGMP. In agreement with these findings both DEA/NO and 8-Br-cGMP were capable of producing a long-lasting increase in number of clusters for alpha-synuclein through activation of soluble guanylyl cyclase, cGK and calcium/calmodulin-dependent protein kinase IIalpha. Thus, our results suggest that NO, cGMP, GMP-dependent protein kinase and calmodulin-dependent protein kinase II play a key role in the redistribution of alpha-synuclein during plasticity.