Establishing a Whole-Cell Patch Clamp for Electrophysiological Recording from Vomeronasal Sensory Neurons

Abstract

Source: Ackels, T., et al. In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ. J. Vis. Exp. (2016)

The protocol video describes establishing a whole-cell patch clamp for electrophysiological recording from the sensory neurons of the vomeronasal organ (VNO) in mice. A coronal VNO slice with fluorescently labeled sensory neurons is placed in a recording setup. A labeled neuron is patched with a recording pipette in a whole-cell configuration for electrophysiological recording in response to chemical stimuli.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Solution Preparation

1. Prepare extracellular solution S₁: 4-(2-Hydroxy-ethyl)piperazine-1-ethanesulfonic acid (HEPES) buffered extracellular solution containing (in mM) 145 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES; pH = 7.3 (adjusted with NaOH); osmolarity = 300 mOsm (adjusted with glucose).
2. Prepare extracellular solution S₂: Carbogen-oxygenated (95% O₂, 5% CO₂) extracellular solution containing (in mM) 125 NaCl, 25 NaHCO

Results

Figure 1: Dissection of the VNO. (A) Side view on the mouse head to illustrate the position and angle at which the incisors are cut. (B) Ventral view depicting the best point to grab and peel back the upper palate (UP). (

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals
Agarose (low-gelling temperature)	PeqLab	35-2030
ATP (Mg-ATP)	Sigma-Aldrich	A9187
Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES)	Sigma-Aldrich	B9879
Calcium chloride	Sigma-Aldrich	C1016
Ethylene glycol tetraacetic acid (EGTA)	Sigma-Aldrich	E3889
Glucose	Sigma-Aldrich	G8270
GTP (Na-GTP)	Sigma-Aldrich	51120
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Sigma-Aldrich	H3375
Magnesium chloride	Sigma-Aldrich	M8266
Potassium chloride	Sigma-Aldrich	P9333
Potassium hydroxide	Sigma-Aldrich	3564
Sodium chloride	Sigma-Aldrich	S7653
Sodium hydrogen carbonate	Sigma-Aldrich	S5761
Sodium hydroxide	Sigma-Aldrich	S8045
Surgical tools and consumables
Large petri dish, 90 mm	VWR		decapitation, dissection of VNO capsule
Small petri dish, 35 mm	VWR		lid for VNO dissection, dish for embedding in agarose
Sharp large surgical scissor	Fine Science Tools		decapitation, removal of lower jaw
Strong bone scissors	Fine Science Tools		cutting incisors
Medium forceps, Dumont tweezers #2	Fine Science Tools		removing skin and palate
Micro spring scissors, 8.5 cm, curved, 7 mm blades	Fine Science Tools		cutting out VNO
Two pairs of fine forceps, Dumont tweezers #5	Fine Science Tools		dissecting VNO out of cartilaginous capsule
Small stainless steel spatula	Fine Science Tools		handling agarose block and tissue slices
Surgical scalpel			cutting agarose block into pyramidal shape
Equipment
Amplifier	HEKA Elektronik	EPC-10
Borosilicate glass capillaries (1.50 mm OD/0.86 mm ID)	Science Products
CCD-camera	Leica Microsystems	DFC360FX
Filter cube, excitation: BP 450-490, suppression: LP 515	Leica Microsystems	I3
Fluorescence lamp	Leica Microsystems	EL6000
Hot plate magnetic stirrer	Snijders	34532
Microforge	Narishige	MF-830
Micromanipulator Device	Luigs & Neumann	SM-5
Micropipette puller, vertical two-step	Narishige	PC-10
Microscope	Leica Microsystems	CSM DM 6000 SP5
Noise eliminator 50/60 Hz (HumBug)	Quest Scientific
Objective	Leica Microsystems	HCX APO L20x/1.00 W
Oscilloscope	Tektronik	TDS 1001B
Osmometer	Gonotec	Osmomat 030
Perfusion system 8-in-1	AutoMate Scientific
pH Meter five easy	Mettler Toledo
Pipette storage jar	World Precision Instruments	e212
Recording chamber	Luigs & Neumann	Slice mini chamber
Razor blades	Wilkinson Sword GmbH	Wilkinson Sword Classic
Oxygenating slice storage chamber; alternative commercial chambers are e.g. BSK1 Brain Slice Keeper (Digitimer) or the Pre-chamber (BSC-PC; Warner Instruments)	custom-made
Stereo microscope	Leica Microsystems	S4E
Trigger interface	HEKA Elektronik	TIB-14 S
Vibratome	Leica Microsystems	VT 1000 S
Water bath	Memmert	WNB 45