Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Summary

The primary goal of this article is to provide details of how to record membrane potential (V_m) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

Abstract

Membrane potential (V_m) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate V_m in disease conditions could potentially alter V_m, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record V_m in healthy and diseased states are essential. This method will allow modulating V_m using different pharmacological agents to restore V_m. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record V_m from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The V_m signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the V_m of a vessel wall without damaging the cells and without changing the membrane resistance.

Introduction

The membrane potential (V_m) of a cell refers to the relative difference of ionic charge across the plasma membrane and the relative permeability of the membrane to these ions. The V_m is generated by the differential distribution of ions and is maintained by ion channels and pumps. Ion channels such as K⁺, Na⁺, and Cl⁻ contribute substantially to the resting V_m. Vascular smooth muscle cells (VSMCs) express more than four different types of K⁺ channels¹, two types of voltage-gated Ca²⁺ channels (VGCC)², more than two types of Cl

Protocol

The male rats were housed in the Animal Care Facility at UMMC, which is approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals had free access to food and water throughout the study. Animals were maintained in a controlled environment with temperature at 24 ± 2 °C, humidity levels of 60–80% and 12 h light/dark cycles. All protocols were approved by the Animal Care and Use Committee of UMMC.

1. Preparation of Equipment

1. Place a dual channel differential electrometer amplifier (see the Table of Materials) close to the vessel chamber and at the desire....

Results

The presented method can be reliably used to record V_m in cannulated vessels. A brief procedure describing how to isolate MCA from the brain is presented in Figure 1A. After separating the brain from the skull, the MCA was dissected out and placed in a Petri dish containing low calcium PSS. Part of the connective tissue that was attached was also dissected along with MCA using spring scissors and forceps to prevent damage to MCA during the isolatio.......

Discussion

This article provides the necessary steps on how to use a sharp microelectrode impalement method to record V_m from a cannulated vessel preparation. This method is widely used, and offers high-quality, consistent recordings of V_m that answer a wide range of experimental questions.

Some critical considerations and troubleshooting steps are described here to ensure success of the method. The quality of the microelectrode (its sharpness and resistance) and the cellular proces.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dissection instruments
Aneshetic Vaporiser	Parkland scientific	V3000PK
Dissection microscope	Nikon Instruments Inc., NY	Eclipse Ti-S
Kleine Guillotine Type 7575	Harvard Apparatus, MA	73-198
Littauer Bone Cutter	Fine science tools	16152-15
Moria MC40 Ultra Fine Forceps	Fine science tools	11370-40
Surgical scissors Sharp-Blunt	Fine science tools	14008-14
Suture	Harvard Apparatus	72-3287
Vannas Spring Scissors	Fine science tools	15018-10
Electrophysiology Instruments
Charge-coupled device camera	Qimaging, , BC	Retiga 2000R
Differential electrometer amplifier	WPI	FD223A
In-line pressure transducer	Harvard Apparatus, MA	MA1 72-4496
Micromanipulator	Thor labs	PCS-5400
Microelectrodes	Warner Instruments LLC, CT	G200-6,
Micro Fil (Microfiber syringe)	WPI	MF28G67-5
Microelectrode holder	WPI	MEH1SF
Myograph	Living Systems Instrumentation, VT	CH-1-SH
Puller	Sutter Instrument, San Rafael, CA	P-97
Vibration-free table	TMC	3435-14
Softwares
Clampex 10	Molecular devices
p Clamp 10	Molecular devices
Imaging software	Nikon, NY	NIS-elements
Chemicals
NaCl	Sigma	S7653
KCl	Sigma	P4504
MgSO4	Sigma	M7506
CaCl2	Sigma	C3881
HEPES	Sigma	H7006
Glucose	Sigma	G7021
NaH2PO4	Sigma	S0751
NaHCO3	Sigma	S5761