Summary Synapses, the connections between neurons, play a crucial role in determining circuit connectivity and function. Visualizing their structure and dynamics is challenging due to the vast number of synapses and the complex interactions between them. Prior studies have explored some aspects of synaptic plasticity, primarily focusing on postsynaptic neurons and utilizing imaging techniques to observe dendritic spines, which are widely recognized as key synaptic structures. Nevertheless, there remains a significant gap in our understanding of how these changes are linked to their specific input sources, leaving the identity of these synapses largely uncharted. To overcome this limitation, researchers have developed dual-component synapse detectors, like the neurexin-neuroligin complex and split fluorescent proteins. However, split fluorescent proteins have limitations, such as slow maturation and irreversible associations, hindering real-time synaptic observation. To address these challenges, we developed a reversible fluorescent sensor technology using dimerization-dependent fluorescent proteins (ddFPs) named SynapShot. These ddFPs emit fluorescence only when they heterodimerize, which happens quickly and reversibly. With this technique, we successfully monitored synaptic dynamics in real time, including synaptic formation and elimination, as indicated by changes in dendritic spine size. We also distinguished connections from one postsynaptic neuron to multiple presynaptic neurons. In this proposal, we will further explore the uncharted territory of synaptic dynamics in both local and long-range circuits in in vivo setups. In the first aim, we will investigate the functional implications of synaptic labeling detected by the SynapShot. Synaptic plasticity will be tested in single dendritic spines to demonstrate the ability of tracking structural changes in real-time. In the second aim, we will explore inhibitory synaptic connections, especially those involving genetically-defined interneuron cell types, using the SynapShot method. In the third aim, we will employ synapShot to observe synaptic changes during the acquisition of a new learning task, study dendritic integration dynamics, and examine the rules governing connections among engram neurons. In summary, SynapShot provides a means to observe bidirectional changes in synaptic contacts both in vitro and in vivo and can differentiate distinct populations of synapses when used in dual-colored configurations. This platform contributes to understanding the relationship between time-dependent synaptic structure changes and various brain functions and diseases. The successful completion of this project promises to be a valuable asset in advancing the field of neural circuit research.