Long-term potentiation (LTP) at hippocampal CA3-CA1 synapses, can be subdivided into two mechanistically different forms of NMDAR-dependent synaptic potentiation based on their independence of (LTP1) or dependence on (LTP2) de novo protein synthesis (PS). Previous experiments reported the requirement of CP-AMPAR activation and intracellular calcium stores for LTP2 but not LTP11,2. Here the requirement for CP-AMPARs and intracellular calcium stores in structural LTP (sLTP) were investigated. Simultaneous field potential recordings and two-photon imaging were carried out in acute hippocampal slices of adult mice (P70-100) expressing Thy1-EGFP. Additionally, FM 4-64 was used to label presynaptic boutons of activated synapses3. Two TBS protocols (3xTBS) with different inter-episode intervals (IEI) were used – a compressed TBS (cTBS) protocol (IEI 10 s) to induce LTP1 and a spaced TBS (sTBS) protocol (IEI 10 min) to induce LTP2 and LTP1. Custom written scripts in MATLAB and python were used to automate the analysis of sLTP. Throughout the 3h recording session, both spine fluorescence and synaptic transmission remained stable. Delivery of sTBS but not cTBS, led to a substantial and sustained increase in spine volume at FM+ synapses. Further analysis will look at differences in spine clustering under these conditions. This persistent increase in spine volume induced by sTBS, was prevented by treatment with D-AP5 (50 μM), anisomycin (30 μM), ryanodine (30 μM), IEM- 1460 (30 μM), suggesting that activation of NMDARs, de novo PS, CP-AMPARs and intracellular calcium signalling are vital for sLTP.