Synaptic plasticity, the ability for synapses to change their connection strength, is thought to underlie learning and memory. Cascades of biochemical reactions in dendritic spines, tiny (~0.1 femtoliter) postsynaptic compartments emanating from dendritic surface, trigger diverse forms of synaptic plasticity. These reactions are mediated by signaling networks consist of hundreds of species of signaling proteins. Our focus is to elucidate some of the operation principles of such signaling networks in dendritic spines using various optical techniques. First, we have been developing techniques to image activity of various proteins in single dendritic spines using 2-photon fluorescence lifetime imaging microscopy (2pFLIM) in combination with new biosensors extensively optimized for 2pFLIM. Using this technique, we have succeeded in imaging signaling proteins, CaMKII, HRas, RhoA and Cdc42, in single dendritic spines undergoing synaptic potentiation. Our results indicate that signaling activity is subjected to a complicated spatiotemporal regulation: some signals are compartmentalized in single synapses, while others spread to affect a short stretch (micrometers) of dendritic segments including multiple synapses. This rich spatiotemporal regulation plays an essential role in coordinating cellular events in different dendritic micro-compartments. Further, Ca2+ elevation in a synapse is relayed in several different stages to induce long-term plasticity: First, Ca2+ signals in the synapse are integrated by a signaling protein CaMKII, of which activity decays over ~10 s. Then, Ras, Cdc42 and RhoA are activated by CaMKII, and relay this transient signal into signals lasting 10-30 min.
We have been developing many more sensors to reveal the precise temporal sequences of signaling events in different microcompartments. In addition, we have been developing tools to activate and inactivate signaling proteins in spines optically. Recently we have developed a photo-inducible CaMKII inhibitor, and demonstrated that 60 s of CaMKII activation is required to activate downstream signaling. By monitoring and manipulating activity of signaling proteins with high spatiotemporal resolution, we hopefully disentangle the complicated signaling networks and understand the signaling mechanisms underlying synaptic plasticity and ultimately learning and memory.