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Innovative Methodology

Reverse Optical Trawling for Synaptic Connections In Situ

¹Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo; and ²Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Tokyo, Japan

Submitted 6 January 2009; accepted in final form 19 April 2009

We introduce a new method to unveil the network connectivity among dozens of neurons in brain slice preparations. While synaptic inputs were whole cell recorded from given postsynaptic neurons, the spatiotemporal firing patterns of presynaptic neuron candidates were monitored en masse with functional multineuron calcium imaging, an optical technique that records action potential–evoked somatic calcium transients with single-cell resolution. By statistically screening the neurons that exhibited calcium transients immediately before the postsynaptic inputs, we identified the presynaptic cells that made synaptic connections onto the patch-clamped neurons. To enhance the detection power, we devised the following points: 1) [K⁺]_e was lowered and [Ca²⁺]_e and [Mg²⁺]_e were elevated, to reduce background synaptic activity and minimize the failure rate of synaptic transmission; and 2) a small fraction of presynaptic neurons was specifically activated by glutamate applied iontophoretically through a glass pipette that was moved to survey the presynaptic network of interest ("trawling"). Then we could theoretically detect 96% of presynaptic neurons activated in the imaged regions with a 1% false-positive error rate. This on-line probing technique would be a promising tool in the study of the wiring topography of neuronal circuits.