Anaesthetized C57Bl/6 mice between postnatal day 22 and 26 were injected with the calcium-sensitive dye Oregon Green Bapta-1 AM into monocular V1 as described previously11 and in vivo two-photon calcium imaging14 ,15 was used to record responses of layer 2/3 neurons to 8 different drifting square-wave gratings (0.035 cycles/degree, 2 cycles/s, 100% contrast) and natural movie sequences. Spike trains were inferred from calcium signals using a non-negative deconvolution method. Preferred orientation and direction, as well as orientation selectivity index (OSI) and direction selectivity index (DSI) were calculated using Fourier-interpolated tuning curves. Pearson’s correlation coefficient was used to obtain pair-wise response correlations, either from average responses to the stimulus (signal correlation) or from mean-subtracted responses (noise correlation). Small volumes of fluorescent microspheres were injected into the imaged region to facilitate identification of the region in the sliced brain. Coronal slices were cut after dissection of the brain, and whole-cell recordings from up to four cells simultaneously were carried out in the vicinity of the microsphere tract (identified by two-photon microscopy). The presence of synaptic connections was tested by evoking five spikes at 30-Hz in each cell, repeated for 30-90 times. Connection probability is the number of detected connections over the total number of potential connections assayed. Probability of finding uni- or bidirectionally connected pairs was calculated as the number of uni- or bidirectionally connected pairs over the total number of pairs. To register in vivo and in vitro image stacks and to match the same neurons imaged in vivo and recorded from in vitro, three□dimensional image registration by affine transformation using custom-written MATLAB software was performed subsequent to the experiment. To relate connectivity to functional properties, the asymptotic Cochran-Armitage test for trend was used to test for significance.