The formation and elimination of spine synapses can be visualized both in vitro as well as in vivo using advanced imaging techniques. It is known that the number of dendritic spines changes both during development as well as during the acquisition of new memories. However, up to now these observations are entirely correlative. It is unclear if the presynaptic neuron forming a functional contact on a newly formed spine indeed was the one that showed correlated activity with the postsynaptic cell as would be required for a learning mechanism following classical Hebbian plasticity. In fact, since it has never been shown what information is transmitted via a newly formed spine synapse in vivo, it has yet to be demonstrated that new long-term stable spines indeed could carry a memory trace related to a specific previous experience.Here, I propose to functionally identify the presynaptic partners of newly formed spine synapses. I will assess if the information carried by a new spine can be predicted from readily observable global changes in neuronal activation strength in a well-defined sensory deprivation paradigm in vivo. Furthermore, I will study whether indeed neurons that ‘fire together’ also ‘wire together’ by forming new stable spine synapses in vitro.I will develop chronic single spine Ca2+ imaging techniques and novel optophysiological tools like a dual-color genetically encoded Ca2+ indicator (GCaMP3-based) and an activity-dependent structural tag (TetTag-based) of active axons. I aim to functionally define the active presynaptic population in during visual and electrical stimulation in vivo and in vitro. Furthermore, I will assess if deletion of proteins involved in the functional maintenance of synaptic strengthening (LTP-maintenance) prevents the formation and/or stabilization of new synapses between defined coactive partners. This work will take our understanding of memory formation forward while providing a range of broadly applicable new techniques.