Automated and parallelized spike collision tests to identify spike signal projections

The spike collision test is a highly reliable technique to identify the axonal projection of a neuron recorded electrophysiologically for investigating functional spike information among brain areas. It is potentially applicable to more neuronal projections by combining multi-channel recording with optogenetic stimulation. Yet, it remains inefficient and laborious because an experimenter must visually select spikes in every channel and manually repeat spike collision tests for each neuron serially. Here, we automated spike collision tests for all channels in parallel (Multi-Linc analysis) in a multi-channel real-time processing system. The rat cortical neurons identified with this technique displayed physiological spike features consistent with excitatory projection neurons. Their antidromic spikes were similar in shape but slightly larger in amplitude compared with spontaneous spikes. In addition, we demonstrated simultaneous identification of reciprocal or bifurcating projections among cortical areas. Thus, our Multi-Linc analysis will be a powerful approach to elucidate interareal spike communication.