Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices

Abstract

Source: Wu, Q., et al. Application of automated image-guided patch clamp for the study of neurons in brain slices. J. Vis. Exp. (2017).

In this video, brain slices are immobilized in a recording chamber, and cells of interest are located and focused on using a computer-controlled microscope. The automated image processing system then precisely positions a patch pipette to seal the cell membrane and monitor electrical signals.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

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(Light-emitting diodes), Metal-Oxide Semiconductor field-effect transistors (MOSFETs), capacitors, and connectors. Solder solenoid valves onto the PCB (Pri....

Results

Table 1. Printed Circuit Board (PCB) to secondary data acquisition (DAQ) board connection configuration. Use this table to connect PCB outputs (first column from left) to ports on the DAQ board (second column from left). The port name and number on the secondary DAQ refer to single-ended mode.

.......

Outlet on the PCB

Port name on the DAQ board

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	488nm 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 (2-Pin)	Sparkfun	PRT - 08084	Screw Terminals 3.5mm Pitch (2-Pin)
N-Channel MOSFET 60V 30A	Sparkfun	COM - 10213
DIP Sockets Solder Tail - 8-Pin	Sparkfun	PRT-07937
LED - Basic Red 5mm	Sparkfun	COM-09590
LED - Basic Green 5mm	Sparkfun	COM-09592
DC Barrel Power Jack/Connector (SMD)	Sparkfun	PRT-12748
Wall Adapter Power Supply - 12VDC 600mA	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