title: Three-dimensional mapping of unitary synaptic connections by two-photon macro photolysis of caged glutamate.
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 (approximately 600 x 600 x 100 microm) 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 microm 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.
authors.affiliation: Division of Biophysics, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. mzakim@m.u-tokyo.ac.jp
authors.forename: Masanori
authors.initials: M
authors.lastname: Matsuzaki
authors.forename: Graham C R
authors.initials: GC
authors.lastname: Ellis-Davies
authors.forename: Haruo
authors.initials: H
authors.lastname: Kasai
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D000818:Animals; D000831:Animals, Newborn; D001931:Brain Mapping; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D005971:Glutamates / Q000494:pharmacology*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D066298:In Vitro Techniques; D009474:Neurons / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D010782:Photolysis; D017785:Photons; D051381:Rats; D017207:Rats, Sprague-Dawley; D013569:Synapses / Q000187:drug effects* / Q000502:physiology*; D014793:Visual Cortex / Q000166:cytology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D005971:Glutamates; C082764:alpha-(4,5-dimethoxy-2-nitrobenzyl) glutamate; D005680:gamma-Aminobutyric Acid