title: Optical detection of neuron connectivity by random access two-photon microscopy.
abstract: BACKGROUND
  Knowledge about the distribution, strength, and direction of synaptic connections within neuronal networks are crucial for understanding brain function. Electrophysiology using multiple electrodes provides a very high temporal resolution, but does not yield sufficient spatial information for resolving neuronal connection topology. Optical recording techniques using single-cell resolution have provided promise for providing spatial information. Although calcium imaging from hundreds of neurons has provided a novel view of the neural connections within the network, the kinetics of calcium responses are not fast enough to resolve each action potential event with high fidelity. Therefore, it is not possible to detect the direction of neuronal connections.
  
  
  METHODS
  We took advantage of the fast kinetics and large dynamic range of the DiO/DPA combination of voltage sensitive dye and the fast scan speed of a custom-made random-access two-photon microscope to resolve each action potential event from multiple neurons in culture.
  
  
  RESULTS
  Long-duration recording up to 100min from cultured hippocampal neurons yielded sufficient numbers of spike events for analyzing synaptic connections. Cross-correlation analysis of neuron pairs clearly distinguished synaptically connected neuron pairs with the connection direction.
  
  
  CONCLUSIONS
  The long duration recording of action potentials with voltage-sensitive dye utilized in the present study is much longer than in previous studies. Simultaneous optical voltage and calcium measurements revealed that voltage-sensitive dye is able to detect firing events more reliably than calcium indicators.
  
  
  CONCLUSIONS
  This novel method reveals a new view of the functional structure of neuronal networks.
authors: Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan.
authors: Nasrin
authors: N
authors: Shafeghat
authors: Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan.
authors: Morteza
authors: M
authors: Heidarinejad
authors: Department of Electrical Engineering and Bioscience, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan.
authors: Noboru
authors: N
authors: Murata
authors: Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan.
authors: Hideki
authors: H
authors: Nakamura
authors: Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan. Electronic address: inoue.t@waseda.jp.
authors: Takafumi
authors: T
authors: Inoue
mesh_terms: D015761:4-Aminopyridine / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003198:Computer Simulation; D004558:Electric Stimulation; D018756:GABA Antagonists / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology*; D008959:Models, Neurological; D009415:Nerve Net / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D061848:Optical Imaging; D018408:Patch-Clamp Techniques; D010852:Picrotoxin / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology; D017208:Rats, Wistar; D013569:Synapses / Q000502:physiology; D056969:Voltage-Sensitive Dye Imaging
chemical_list: D015761:4-Aminopyridine; D002118:Calcium; D018756:GABA Antagonists; D010852:Picrotoxin; D026902:Potassium Channel Blockers
keywords: Neural connectivity; Population spike recording; Random access two-photon microscopy; Voltage sensitive dye