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INNOVATIVE METHODOLOGY

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

¹Division of Biophysics, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, and Center for NanoBio Integration, The University of Tokyo, Tokyo; ²Precursory Research Organization for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan; and ³Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania

Submitted 12 October 2007; accepted in final form 16 January 2008

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 x 600 x 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.