Three-Dimensional Mapping of Unitary Synaptic Connections by Two-Photon Macro Photolysis of Caged Glutamate

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Abstract
To understand the precise microarchitecture of the cortical circuitry, it is crucial to know the distribution of synaptic connections and their synaptic strengths at the level of a single cell, rather than a group of cells. Here, we describe a new application of two-photon photolysis of caged glutamate that enabled us to induce an action potential in only a small number (about five) of pyramidal neurons by increasing the volume of two-photon excitation by reducing the effective numerical aperture of the objective. We performed whole cell patch-clamp recordings from layer 2/3 pyramidal neurons in the rat visual cortex and stimulated many neurons in a large three-dimensional space (∼600 × 600 × 100 μm) including neurons in layers 2/3 and 4 using this new technique. We mapped the density and amplitude of unitary excitatory postsynaptic currents and found that the basic microarchitecture of excitatory synaptic connections consists of two regions: a columnar, dense core region with a radius of 150 μm and an outer, sparse region. The dense core region includes the majority of strong synaptic connections in layer 2/3. Our results reveal the columnar organization of synaptic connectivity in the rat visual cortex, where functional columns have not been clearly demonstrated. Thus this technique will be a uniquely powerful tool for quantifying synaptic connectivity and manipulating neural activity at the single-cell level.

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