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. New method: 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 100 min 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. Comparison with existing method: 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.
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