This work was supported in part by grants from the Intramural support research program (IRSP) from UMMC, AHA Scientist Development Grant (13SDG14000006) awarded to Mallikarjuna R. Pabbidi.

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 &#177; 2 &#176;C, humidity levels of 60&#8211;80% and 12 h light/dark cycles. All protocols were approved by the Animal Care and Use Committee of UMMC.
1. Preparation of Equipment
<ol>
	<l...

<ol>
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The authors have nothing to disclose.

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

The membrane potential (Vm) 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 Vm is generated by the differential distribution of ions and is maintained by ion channels and pumps. Ion channels such as K+, Na+, and Cl&#8722; contribute substantially to the resting Vm. 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&#8722; channels3,4,5, store-operated Ca2+ channels6, stretch-activated cation channels7,8, and electrogenic sodium-potassium ATPase pumps9 in their plasma membranes, all of which may be involved in the regulation of Vm.
The Vm of VSMCs depends on lumen pressure. In non-pressurized vessels, Vm varies from -50 to -65 mV, however, in pressurized arterial segments, Vm ranges from -37 to -47 mV10. Elevation of intravascular pressure causes VSMCs to depolarize11, decreases the threshold for VGCC opening, and increases calcium influx contributing to the development of myogenic tone12. On the contrary, in passive or non-pressurized vessels, membrane hyperpolarization, due to high K+ channel activity, will prevent VGCC from opening, resulting in limited calcium entry and a decrease in intracellular calcium, contributing to less vascular tone13. Thus, Vm due to changes in lumen pressure appears to play an essential role in vascular tone development, and both VGCC and K+ channels play a crucial role in the regulation of Vm.
Vm varies between vessel type and species. Vm is -54 &#177; 1.3 mV in guinea pig superior mesenteric arterial strips14, -45 &#177; 1 mV in the rat middle cerebral arteries at 60 mmHg lumen pressure12, and -35 &#177; 1 mV in rat parenchymal arteries at 40 mmHg lumen pressure15. The resting Vm recorded in unstretched rat lymphatic muscle is -48 &#177; 2 mV16. Vm of cerebral VSMCs is more negative than in peripheral arteries. In comparison, feline middle cerebral arteries were reported to have a Vm of approximately -70 mV, while mesenteric and coronary arteries were reported to have -49 and -58 mV, respectively17,18. Differences in the Vm across vascular beds may reflect the differences in the expression and function of ion channels and electrogenic sodium-potassium pumps.
Increases and decreases in Vm are referred to as membrane depolarization and hyperpolarization, respectively. These alterations in Vm play a central role in many physiological processes, including ion-channel gating, cell signaling, muscle contraction, and action potential propagation. At a fixed pressure, many endogenous and synthetic vasodilator compounds that activate K+ channels cause membrane hyperpolarization, resulting in vasodilation1,13. Conversely, sustained membrane depolarization is vital in agonist-induced or receptor-mediated vasoconstriction19. Vm is a critical variable that not only regulates Ca2+ influx through VGCC13 but also influences the release of Ca2+ from internal stores20,21 and Ca2+-sensitivity of the contractile apparatus22.
While there are several methods to record Vm from different cell types, data collected from the microelectrode impalement method of cannulated vessels appears to be more physiological than data obtained from isolated VSMCs. When recorded from isolated VSMC using current clamp methods, Vm is seen as spontaneous transient hyperpolarizations in VSMCs24. Isolated VSMCs are not in the syncytium, and the changes in the series resistance may contribute to the oscillatory behavior of Vm. On the other hand, oscillatory behavior is not observed when Vm is recorded from intact vessels, probably due to cell-cell contact between VSMCs that are in syncytium in the artery and are summated throughout the vessel leading to a stable Vm24. Thus, measurement of Vm from pressurized vessels using standard microelectrode impalement technique is relatively close to the physiological conditions.
Recording Vm from cannulated vessels could provide vital information, since Vm of VSMCs that are in syncytium is one of the major determinants of vascular tone and blood flow, and modulation of the Vm could provide a way to dilate or constrict blood vessels. Thus, it is essential to understand the methodology involved in recording Vm. This article describes intracellular recording of Vm from cannulated middle cerebral arteries (MCAs) using a microelectrode impalement method. This protocol will describe how to prepare MCAs, microelectrodes, set up the electrometer and perform the impalement method to record Vm. Also, representative data, common issues that were encountered when using this method and potential issues are discussed.

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

microelectrode impalement method to record membrane potential from a cannulated middle cerebral artery

Membrane potential (Vm) 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 Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm 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 Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The presented method can be reliably used to record Vm 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...

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

The primary goal of this article is to provide details of how to record membrane potential (Vm) 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.

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

This protocol can be used to measure the membrane potential of normal and diseased blood vessels and to evaluate pharmacological agents that modulate membrane potential, vascular tone, and blood flow. The membrane potentials generated from this technique are close to the physiological range since vascular smooth muscles within a vessel are in a syncytium. Before beginning the procedure, place a dual channel differential electrometer amplifier close to the vessel chamber at the desired location.Use a BNC-BNC cable to connect the output of the amplifier channel A or B to the channel input of the digitizer. Mount the probe in the micromanipulator, and turn the micromanipulator towards the microscope and the myograph on a vibration-free table. Place the knobs and switches on the front of the amplifier in the appropriate positions for the experiment, as described in the manual.Then connect the bath ground to the circuit ground of the amplifier with an appropriate electrode, and ensure that the cage is grounded to the chassis of the amplifier. To prepare the borosilicate glass microelectrodes, use a standard puller to achieve a short, gradual, eight-to 10-millimeter taper with a less-than-one-micrometer diameter, looping twice for higher resistances and smaller tips. Use a microfiber syringe to fill the microelectrode with three-molar potassium chloride, slowly retracting the plunger during the injection to allow space for the fluid to fill and to prevent the formation of bubbles inside the microelectrode.Place the fully loaded microelectrode onto the microelectrode holder, and carefully but firmly push the electrode shank into the holder through the bored hole. Remove any excess fluid with a lab tissue, and connect the electrode holder assembly to the amplifier probe. Conduct an electrode test to measure the electrode resistance.Then open the recording software, assign a name to the file, and save the file for future analysis. Next, rinse the myograph chamber with distilled water several times before loading the chamber with five milliliters of normal physiological salt solution, or PSS. Using a five-or 10-milliliter syringe, fill both glass cannulas and the attached tubing with filtered normal PSS without introducing any air bubbles, and use blunt forceps to make a half-knot in two 10-0 monofilament nylon sutures.Then, using dissection forceps under a dissection microscope, place the partially closed suture knots on both cannulas slightly away from the tips. For middle cerebral artery isolation, use spring scissors and forceps to identify and dissect out an unbranched segment of the middle cerebral artery, with an inner diameter of 100 to 200 micrometers, of the rat brain. Use fine forceps to mount the middle cerebral artery onto the glass cannulas, and tighten the sutures to secure the artery to the cannula.Close off the distal cannula so that there will be no flow within the artery, and connect the inflow pipette to a reservoir of PSS. Visualize the cannulated middle cerebral artery using a charge-coupled device camera mounted on an inverted microscope and imaging software. Set the axial length of the MCA to an approximate length such that it appears neither rigid nor flaccid.Equilibrate the bath solution with 95%oxygen and 5%carbon dioxide at 37 degrees Celsius. Immerse the ground of the amplifier in the PSS of the myograph. Illuminate the vessel chamber, and visualize the tip of the microelectrode in the bath solution through the microscope.Using the micromanipulator, move the tip of the microelectrode close to the outer wall of the blood vessel, and begin the recording. Slowly move the tip of the microelectrode toward the vessel, aiming for the center of the vessel. When the tip comes in close proximity to the vessel, advance the electrode forward in one rapid motion to impale the membrane of the muscle.Once the membrane has been penetrated, changes in the membrane potential may be observed. Do not touch the micromanipulator once the membrane has been impaled. When the membrane potential changes have been recorded, use the manipulator to remove the microelectrode in one rapid movement, and stop the recording before saving the data files.Impalement is considered successful when there is a rapid deflection to negative values, the membrane potential is stable for a minimum of 30 seconds, and the voltage returns abruptly to zero millivolts upon removal of the electrode. After a successful impalement and membrane potential stabilization, drugs of interest can be perfused into the bath and changes in the membrane potential can be recorded. In this representative experiment, perfusion with potassium chloride depolarized the membrane by approximately six millivolts, while perfusion with a calcium-dependent activator of large conductance calcium-activated potassium channels hyperpolarized the membrane by nearly four millivolts.The most important thing to remember is to pull highly resistant microelectrodes that have a short, gradual taper with a less-than-one-micrometer diameter.

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Research

JoVE Journal

Medicine

6.6K Görüntüleme.  University of Mississippi Medical Center. Bu protokol, normal ve hastalıklı kan damarlarının membran potansiyelini ölçmek ve membran potansiyelini, vasküler tonuve kan akışını modüle eden farmakolojik ajanları değerlendirmek için kullanılabilir. Bu teknikten üretilen membran potansiyelleri, damar içindeki vasküler düz kaslar bir senitoyumda olduğundan fizyolojik menzile yakındır. İşleme başlamadan önce, çift kanallı diferansiyel elektrometre amplifikatörü istediğiniz yere yakın bir damar odasına yerl...