In the cerebral cortex, local circuits consist of tens of thousands of neurons, each of which makes thousands of synaptic connections. Perhaps the biggest impediment to understanding these networks is that we have no wiring diagrams of their interconnections. Even if we had a partial or complete wiring diagram, however, understanding the network would also require information about each neuron's function. Here we show that the relationship between structure and function can be studied in the cortex with a combination of in vivo physiology and network anatomy. We used two-photon calcium imaging to characterize a functional property-the preferred stimulus orientation-of a group of neurons in the mouse primary visual cortex. Large-scale electron microscopy of serial thin sections was then used to trace a portion of these neurons' local network. Consistent with a prediction from recent physiological experiments, inhibitory interneurons received convergent anatomical input from nearby excitatory neurons with a broad range of preferred orientations, although weak biases could not be rejected. Past studies of the synaptic connections in the cortex have focused on a few cells at a time, so that today we have only partial information about the structure of highly interconnected cortical networks. Until recently, anatomists relied on the sparse labelling of individual neurons to trace the extensive axonal and dendritic arbors of cortical neurons. Synaptic connectivity was originally inferred from the overlap of axonal and dendritic arbors 1 , an approach that remains fruitful today 2,3 . Combined physiological and anatomical studies of cortical slices in vitro have extended this analysis to include rules of pair-wise connectivity between different cell types and cortical layers 4-6 . It has been argued that the cortical network might be random beyond these simple statistical rules 7 , but recent studies suggest that there could be higher-order patterns of connections in cortical networks, such as mutually interconnected triplets of cells 8 , and subnetworks of neurons that are highly connected within a group, but not between groups 9,10 . Although these hypothesized patterns are based on in vitro physiological data, they have never been demonstrated anatomically, nor have they been related to information processing as measured from neural response properties in vivo. Electron microscopy (EM) is an ideal tool for characterizing the highly interconnected structure of cortical networks. From visualizing quantal release by synaptic vesicles 11 to generating a complete wiring diagram of a model organism 12 , it has provided definitive data for examining the relationship between structure and function in the nervous system. Electrophysiology combined with light microscopy and serial-section EM has allowed the inspection of structure-function relationships of single cells within neural circuits, such as the hippocampus 13 , retina 14,15 , thalamus 16 and cortex 17-20 . A crucial difference from light microscopy is that serial-section EM can be used to follow the three-dimensional contours of neuronal membranes, so that any given axon or dendrite can be traced over hundreds of micrometres without selective, sparse staining of individual neurons. This feature of serial-section EM has been used to examine small volumes of cortical tissue, typically numbering in the thousands of cubic micrometres, in which portions of multiple dendrites and axons were reconstructed to examine synaptic relationships amongst them (refs 21, 22, compare ref. 23). Here we exploited recent improvements in computer speed and storage capacity to perform serial-section EM of a volume that encompasses millions of cubic micrometres, sufficient to contain large portions of the dendritic and axonal arbors of more than 1,000 cells. With this data set, we could attempt a sampling-targeted to a subset of functionally imaged cells-of the dense interconnections found in a cortical network. In particular, we tested a somewhat controversial prediction from recent physiological work (refs 24-27, compare refs 28, 29): that inhibitory interneurons in the mouse primary visual cortex receive dense, convergent input from nearby excitatory (pyramidal) neurons with widely varying preferred stimulus orientations (Fig. 1a) . We used in vivo two-photon calcium imaging to determine the preferences for stimulus orientation of a cohort of cells in layer 2/3 of mouse primary visual cortex 27, 30 . After loading cells with the fluorescent calcium indicator Oregon Green BAPTA-1 AM (OGB) 31 , we recorded neuronal activity as reflected by increases in intracellular calcium from a single plane of cells 186 mm beneath the brain surface. Black and white bars of varying orientations and directions were presented to the anaesthetized animal, and the cellular responses were used to generate a map of orientation preference ( Fig. 1b and Supplementary Fig. 1; Methods) . We concluded the in vivo experiment by collecting a stack of images from a volume surrounding the calciumimaged plane ( Fig. 1c ; red, vessels; green, neurons). We then immediately perfused the animal and prepared the corresponding volume of visual cortex for serial-section transmission EM (TEM; Methods). After finding the calcium-imaged region of cortex (Methods), we cut a series of 1,215 thin sections (40-45 nm), oriented radially, at right angles to the functionally imaged plane (Fig. 1b, c) . Each section was wide enough (450 mm) to encompass the imaged plane, and tall enough (350 mm) to include cortical layers 1, 2/3 and upper 4 ( Fig. 2a, b, Supplementary Movie 1) . Although cortical axons can
Serial section imaging is the only technique to record data volumes of millions of cubic micrometers REF .
Network anatomy and in vivo physiology of visual cortical neurons
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