Long-term potentiation (LTP) at glutamatergic synapses is considered to underlie learning and memory and is associated with the enlargement of dendritic spines. Because the consolidation of memory and LTP require protein synthesis, it is important to clarify how protein synthesis affects spine enlargement. In rat brain slices, the repetitive pairing of postsynaptic spikes and two-photon uncaging of glutamate at single spines (a spike-timing protocol) produced both immediate and gradual phases of spine enlargement in CA1 pyramidal neurons. The gradual enlargement was strongly dependent on protein synthesis and brain-derived neurotrophic factor (BDNF) action, often associated with spine twitching, and was induced specifically at the spines that were immediately enlarged by the synaptic stimulation. Thus, this spike-timing protocol is an efficient trigger for BDNF secretion and induces protein synthesis-dependent long-term enlargement at the level of single spines.The consolidation of memory and long-term potentiation (LTP) require protein synthesis (1, 2). Therefore, it is important to clarify whether protein synthesis can regulate synaptic plasticity at the level of a single synapse and how it affects synaptic structure. The spine enlargement associated with LTP can be immediately induced by intensive stimulation of postsynaptic N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors (the conventional protocol) in CA1 pyramidal neurons (3)(4)(5). This spine enlargement can be induced even in the absence of postsynaptic spikes (3), although if synaptic stimulation is closely followed in time by postsynaptic spikes (a spike-timing protocol), a more robust form of LTP is induced that plays an important role in the development and learning of neuronal networks (6). In rat brain slices, we examined the structural plasticity of dendritic spines induced by the stimulation of single spines, using two-photon uncaging of glutamate (7) in the absence or presence of postsynaptic spikes in CA1 pyramidal neurons (uncaging is photorelease from a biologically inert precursor).CA1 pyramidal neurons in slice culture were subjected to whole-cell perfusion with a solution containing the fluorescent dye Alexa594 (50 μM) and β-actin (5 μM) (8). The latter protein was included because we found that it delayed the washout of plasticity (3) ( fig. S1 and supporting online text). We detected marked (>50%) increases in spine-head volume (ΔV H ) in most (37 of 41) small spines stimulated by repetitive (80 times at 1 Hz) uncaging of 4-methoxy-7-nitroindolinyl (MNI)-glutamate paired with post-synaptic spikes within 20 ms (spike-timing protocol or uncaging plus spikes) (Fig. 1, A to C). Spine enlargement was not induced by repetitive glutamate uncaging (1.6 ± 6.6%, n =9 spines, in the presence of Mg 2+ ) or spike application alone (-3.0 ± 2.6%, n = 54). It was also not induced when spikes were triggered >50 ms after uncaging (3.9 ± 3.4%, n = 10). Spine enlargement was restricted to stimulated spines; it did not spread to neighboring ...