Laser microsurgery for presynaptic interrogation.

Synaptic connections among neurons are critical for information processing and memory storage in the brain, making them hotspots for neuropathologies. Understanding the physiology of synapses, therefore, may facilitate the development of therapeutic approaches. However, synapses are micrometer-sized functional structures involved in many neuronal processes, where the challenge is deciphering differential signaling in presynaptic and postsynaptic compartments of relatively intact microcircuits. Here we developed a method combining two-photon laser microsurgery with compartment-specific electrophysiological activation and readout to improve the specificity with which neuronal signaling is detected. After finding a connection, femtosecond laser pulses are used to sever the presynaptic axon from the cell body with micrometer precision. This microdissection method is effective to a depth of at least 100 µm. The initial segment of the isolated axon is extracellularly stimulated and activated to release neurotransmitters, as detected via a recipient whole-cell neuron, which is being recorded. This methodology is an alternative to axonal patch-clamp recordings, which are short-lasting and difficult. Together with pharmacology and genetic manipulation, our approach allows the interrogation of compartmentalized signaling in intact synapses. The total time of laser exposure is a few seconds and the microsurgery takes 5-10 min, which enables the interrogation of multiple synapses within an experiment. Our protocol provides a tool to investigate compartment-specific signaling in relatively intact brain tissue, enabling a more comprehensive understanding of neuronal synapses.