Three-dimensional Mapping of Unitary Synaptic Connections by Two-photon Macro Photolysis of Caged Glutamate Slice Preparation

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.

[1]  Yun Wang,et al.  Synaptic connections and small circuits involving excitatory and inhibitory neurons in layers 2-5 of adult rat and cat neocortex: triple intracellular recordings and biocytin labelling in vitro. , 2002, Cerebral cortex.

[2]  T. Harkany,et al.  Pyramidal cell communication within local networks in layer 2/3 of rat neocortex , 2003, The Journal of physiology.

[3]  R. Kötter,et al.  Cell Type-Specific Circuits of Cortical Layer IV Spiny Neurons , 2003, The Journal of Neuroscience.

[4]  Benjamin R. Arenkiel,et al.  In Vivo Light-Induced Activation of Neural Circuitry in Transgenic Mice Expressing Channelrhodopsin-2 , 2007, Neuron.

[5]  R. Silver,et al.  Synaptic connections between layer 4 spiny neurone‐ layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: physiology and anatomy of interlaminar signalling within a cortical column , 2002, The Journal of physiology.

[6]  R. Pascher,et al.  Heterogeneity in the columnar number of neurons in different neocortical areas in the rat , 1996, Neuroscience Letters.

[7]  W. C. Hall,et al.  High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice , 2007, Proceedings of the National Academy of Sciences.

[8]  H. Markram,et al.  The neocortical microcircuit as a tabula rasa. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Stewart,et al.  Distribution of neurons and glia in the visual cortex (area 17) of the adult albino rat: A quantitative description , 1987, Neuroscience.

[10]  Sooyoung Chung,et al.  Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex , 2005, Nature.

[11]  Haruo Kasai,et al.  4-Carboxymethoxy-5,7-Dinitroindolinyl-Glu: An Improved Caged Glutamate for Expeditious Ultraviolet and Two-Photon Photolysis in Brain Slices , 2007, The Journal of Neuroscience.

[12]  A. Burkhalter,et al.  Intrinsic connections of rat primary visual cortex: Laminar organization of axonal projections , 1989, The Journal of comparative neurology.

[13]  R. Douglas,et al.  Neuronal circuits of the neocortex. , 2004, Annual review of neuroscience.

[14]  E. Callaway,et al.  Excitatory cortical neurons form fine-scale functional networks , 2005, Nature.

[15]  Shy Shoham,et al.  Rapid neurotransmitter uncaging in spatially defined patterns , 2005, Nature Methods.

[16]  Yasushi Miyashita,et al.  Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons , 2001, Nature Neuroscience.

[17]  Lawrence C. Katz,et al.  Scanning laser photostimulation: a new approach for analyzing brain circuits , 1994, Journal of Neuroscience Methods.

[18]  A. Peters,et al.  The neuronal composition of area 17 of rat visual cortex. III. Numerical considerations , 1985, The Journal of comparative neurology.

[19]  Y. Fukui,et al.  Estimation of the numerical densities of neurons and synapses in cerebral cortex. , 1997, Brain research. Brain research protocols.

[20]  Karel Svoboda,et al.  Circuit Analysis of Experience-Dependent Plasticity in the Developing Rat Barrel Cortex , 2003, Neuron.

[21]  H. Kasai,et al.  Number and Density of AMPA Receptors in Single Synapses in Immature Cerebellum , 2005, The Journal of Neuroscience.

[22]  G. Ellis‐Davies,et al.  Structural basis of long-term potentiation in single dendritic spines , 2004, Nature.

[23]  Ian R. Wickersham,et al.  Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons , 2007, Neuron.

[24]  L. Maffei,et al.  Functional postnatal development of the rat primary visual cortex and the role of visual experience: Dark rearing and monocular deprivation , 1994, Vision Research.

[25]  Karel Svoboda,et al.  NMDA Receptor Subunit-Dependent [Ca2+] Signaling in Individual Hippocampal Dendritic Spines , 2005, The Journal of Neuroscience.

[26]  B. Sabatini,et al.  State-Dependent Calcium Signaling in Dendritic Spines of Striatal Medium Spiny Neurons , 2004, Neuron.

[27]  Yuji Ikegaya,et al.  Synfire Chains and Cortical Songs: Temporal Modules of Cortical Activity , 2004, Science.

[28]  John E. W. Mayhew,et al.  Retinotopy within rat primary visual cortex using optical imaging , 2005, NeuroImage.

[29]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[30]  D. Hubel,et al.  Receptive fields, binocular interaction and functional architecture in the cat's visual cortex , 1962, The Journal of physiology.

[31]  I. Ohzawa,et al.  Functional Micro-Organization of Primary Visual Cortex: Receptive Field Analysis of Nearby Neurons , 1999, The Journal of Neuroscience.

[32]  E. Callaway,et al.  Laminar sources of synaptic input to cortical inhibitory interneurons and pyramidal neurons , 2000, Nature Neuroscience.

[33]  E. Callaway,et al.  Photostimulation using caged glutamate reveals functional circuitry in living brain slices. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[34]  V. Mountcastle.  Modality and topographic properties of single neurons of cat's somatic sensory cortex. , 1957, Journal of neurophysiology.

[35]  R. Lund,et al.  Receptive field properties of single neurons in rat primary visual cortex. , 1999, Journal of neurophysiology.

[36]  R. Huganir,et al.  Synapse-specific regulation of AMPA receptor function by PSD-95 , 2006, Proceedings of the National Academy of Sciences.

[37]  J. B. Levitt,et al.  Comparison of intrinsic connectivity in different areas of macaque monkey cerebral cortex. , 1993, Cerebral cortex.

[38]  G. Shepherd,et al.  Geometric and functional organization of cortical circuits , 2005, Nature Neuroscience.

[39]  Rafael Yuste,et al.  Multiphoton stimulation of neurons. , 2002, Journal of neurobiology.

[40]  Rafael Yuste,et al.  Two-photon photostimulation and imaging of neural circuits , 2007, Nature Methods.

[41]  A. Peters,et al.  The neuronal composition of area 17 of rat visual cortex. I. The pyramidal cells , 1985, The Journal of comparative neurology.

[42]  J. Magee,et al.  Mechanism of the distance‐dependent scaling of Schaffer collateral synapses in rat CA1 pyramidal neurons , 2003, The Journal of physiology.

[43]  Roberto Araya,et al.  The spine neck filters membrane potentials , 2006, Proceedings of the National Academy of Sciences.

[44]  Mark Hübener,et al.  Mouse visual cortex , 2003, Current Opinion in Neurobiology.