Pyramidal neuron output is shaped by thousands of excitatory synapses distributed across the dendritic arbor. Activation of single excitatory synapses causes calcium accumulations in individual dendritic spines, mediated by NMDA-Rs32,33, which can be imaged to measure the tuning of single synapses in vivo10,33. We used GCaMP6s to image synaptic calcium signals within dendritic spines over chronic timescales. In V1 with sparsely labeled L2/3 pyramidal neurons (Methods) small dendritic branches were imaged at high magnification during visual stimulation (Fig. 4a). We first focused on neurons that did not fire visually evoked action potentials (∼40% of neurons) to avoid calcium changes caused by action potentials back-propagating into dendrites7. Individual spines showed large fluorescence transients, often independent of their parent dendrites (Fig. 4b,d and Supplementary Video 2). Spine responses were orientation-tuned (Fig. 4b-e), as expected from the fact that most input to V1 neurons originates from (presumably orientation-tuned) V1 neurons34. Neighboring spines were often tuned with different preferred orientations. The correlation between orientation tuning and distance between spines was weak (R=0.08; p>0.05). Overall, 27% of spines (62/228, 15 dendrites, 4 mice) were visually
