Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Summary

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Abstract

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

Introduction

The patch clamp technique was first developed by Neher and Sakmann in the 1970s to study the ionic channels of excitable membranes¹. Since then, patch clamping has been applied to the study of many different subjects at the cellular, synaptic, and circuit level—both in vitro and in vivo—in many different cell types, including neurons, cardiomyocytes, Xenopus oocytes, and artificial liposomes². This process involves the correct identification and targeting of a cell of interest, intricate micromanipulator control to move the patch pipette in close proximity to the cell, the application of pos....

Protocol

1. System Setup

1. Construct the pressure control unit.
   1. Assemble the pressure control unit according to the circuit map (Figure 1). Solder the necessary parts onto the Printed Circuit Board (PCB) manufactured according to the electrical circuit schematics (Figure 1b). Use standard resistors, LEDs, Metal-Oxide Semiconductor Field-Eeffect Transistors (MOSFETs), capacitors, and connectors (see the Table of Materials). Solder solenoid valves onto the PCB. Connect the air pump and air pressure sensor to the PCB with electrical wire.
      NOTE: It should take about 2 h to co....

Results

Our system has been tested on its ability to patch cells in acute brain slices, mouse induced Pluripotent Stem Cells (iPSCs) differentiated into neurons, and HEK 293 cells artificially expressing channels of interest. Figure 3 shows an experiment using Thy1-ChR2-YFP transgenic mice (B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J) targeting fluorescently labeled layer 5 pyramidal neurons in the visual cortex. The target cell was one of the automatically identified green flu.......

Discussion

Here, we describe a method for automatic image-guided patch clamp recordings in vitro. The key steps in this process are summarized as follows. First, computer vision is used to automatically recognize the pipette tip using a series of images acquired via a microscope. This information is then used to calculate the coordinate transformation function between the microscope and the manipulator coordinate systems. Computer vision is used to automatically detect fluorescently labeled cells and to identify their coor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CCD Camera	QImaging	Rolera Bolt
Electrophysiology rig	Scientifica	SliceScope Pro 2000	Include microscope and manipulators. The manufacturer provided manipulator control software demonstrated in this manuscript is “Linlab2”.
Amplifier	Molecular Devices	MultiClamp 700B	computer-controlled microelectrode amplifier
Digitizer	Molecular Devices	Axon Digidata 1550
LED light source	Cool LED	pE-100	488 nm wavelength
Data acquisition board	Measurement Computing	USB1208-FS	Secondary DAQ. See manual at : http://www.mccdaq.com/pdfs/manuals/USB-1208FS.pdf
Solenoid valves	The Lee Co.	LHDA0531115H
Air pump	Virtual industry	VMP1625MX-12-90-CH
Air pressure sensor	Freescale semiconductor	MPXV7025G
Slice hold-down	Warner instruments	64-1415 (SHD-40/2)	Slice Anchor Kit, Flat for RC-40 Chamber, 2.0 mm, 19.7 mm
Python	Anaconda	version 2.7 (32-bit for windows)	https://www.continuum.io/downloads
Screw Terminals	Sparkfun	PRT - 08084	Screw Terminals 3.5 mm Pitch (2-Pin)
(2-Pin)
N-Channel MOSFET 60 V 30 A	Sparkfun	COM - 10213
DIP Sockets Solder Tail - 8-Pin	Sparkfun	PRT-07937
LED - Basic Red 5 mm	Sparkfun	COM-09590
LED - Basic Green 5mm	Sparkfun	COM-09592
DC Barrel Power Jack/Connector (SMD)	Sparkfun	PRT-12748
Wall Adapter Power Supply - 12 V DC 600 mA	Sparkfun	TOL-09442
Hook-Up Wire - Assortment (Solid Core, 22 AWG)	Sparkfun	PRT-11367
Locking Male x Female x Female Stopcock	ARK-PLAS	RCX10-GP0
Fisherbrand Tygon S3 E-3603 Flexible Tubings	Fisher scientific	14-171-129	Outer Diameter: 1/8 in. Inner Diameter: 1/16 in.
BNC male to BNC male coaxial cable	Belkin Components	F3K101-06-E
560 Ohm Resistor (5% tolerance)	Radioshack	2711116
Picospritzer	General Valve	Picospritzer II