Name: microelectrode impalement method to record membrane potential from a cannulated middle cerebral artery
Uploaded: 2019-07-02T14:00:00
Description: Watch this Scientific Journal Video about Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery at JoVE.com

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
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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...

Watch this Scientific Journal Video about Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery at JoVE.com

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 ...

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Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...