Development of a platform for automated, two-photon targeted quad patch-clamping of neurons

Abstract

Whole-cell patch-clamp allows for the study of ion channel biophysics, membrane properties, and synaptic responses in intact brains. Multi-patching promises to elucidate the functional and connectivity properties of neuronal microcircuits, but the high skill and labour required (and low throughput) hinders its widespread dissemination. Automated multi-patching may solve this problem, but current ‘blind’ autopatchers are impractical for targeting non-primary cell types or perform connectivity studies. We have developed an automated two-photon targeted quad-channel patch-clamping technology platform for ex vivo electrophysiology, extending the system of Annecchino et al. (Neuron 2017, 95:1048-55), as a promising step towards an in vivo targeted, quad-patching setup. The platform comprises a two-photon microscope, four micromanipulators (Sensapex/Scientifica) fitted with mechanical stability clamps (to improve pipette resistance against vibration, tissue deformation and pressure changes), custom-developed electronically-controlled pressure regulator, patch-clamp amplifier and DAQs. Control is via a custom-developed LabView program which acquires frames directly from the microscope. After each trial, pipettes are automatically cleaned and returned to their previous positions, allowing for their reuse. A ‘follow’ function is available to allow the user to move a single pipette and have the rest follow, both in the z and x/y coordinates, to facilitate patching in new, undisturbed brain regions. Temperature control for the perfusion system is also possible within the software. The output and cell information of each channel is automatically recorded for each trial. Basic injection and connectivity protocols are also available to run within the program, making the software a complete set for the basic needs of any experimenter, regardless of their skill set. Our targeted quad patch-clamp system allows scalable and reproducible electrophysiology studies to be conducted across a variety of laboratory settings, offering for the first time robotically automated recording of subthreshold signals from multiple genetically and optically targeted cells simultaneously.