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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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⁻ channels^3,4,5, store-operated Ca²⁺ channels⁶, stretch-activated cation channels^7,8, and electrogenic sodium-potassium ATPase pumps⁹ in their plasma membranes, all of which may be involved in the regulation of V_m.

The V_m of VSMCs depends on lumen pressure. In non-pressurized vessels, V_m varies from -50 to -65 mV, however, in pressurized arterial segments, V_m 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, V_m 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 V_m.

V_m varies between vessel type and species. V_m is -54 ± 1.3 mV in guinea pig superior mesenteric arterial strips¹⁴, -45 ± 1 mV in the rat middle cerebral arteries at 60 mmHg lumen pressure¹², and -35 ± 1 mV in rat parenchymal arteries at 40 mmHg lumen pressure¹⁵. The resting V_m recorded in unstretched rat lymphatic muscle is -48 ± 2 mV¹⁶. V_m of cerebral VSMCs is more negative than in peripheral arteries. In comparison, feline middle cerebral arteries were reported to have a V_m of approximately -70 mV, while mesenteric and coronary arteries were reported to have -49 and -58 mV, respectively^17,18. Differences in the V_m across vascular beds may reflect the differences in the expression and function of ion channels and electrogenic sodium-potassium pumps.

Increases and decreases in V_m are referred to as membrane depolarization and hyperpolarization, respectively. These alterations in V_m 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 vasodilation^1,13. Conversely, sustained membrane depolarization is vital in agonist-induced or receptor-mediated vasoconstriction¹⁹. V_m is a critical variable that not only regulates Ca²⁺ influx through VGCC¹³ but also influences the release of Ca²⁺ from internal stores^20,21 and Ca²⁺-sensitivity of the contractile apparatus²².

While there are several methods to record V_m 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, V_m 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 V_m. On the other hand, oscillatory behavior is not observed when V_m 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 V_m²⁴. Thus, measurement of V_m from pressurized vessels using standard microelectrode impalement technique is relatively close to the physiological conditions.

Recording V_m from cannulated vessels could provide vital information, since V_m of VSMCs that are in syncytium is one of the major determinants of vascular tone and blood flow, and modulation of the V_m could provide a way to dilate or constrict blood vessels. Thus, it is essential to understand the methodology involved in recording V_m. This article describes intracellular recording of V_m 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 V_m. Also, representative data, common issues that were encountered when using this method and potential issues are discussed.

Protokół

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 desired location.
2. Connect the output of the amplifier channel A or B to the channel input of the digitizer with a BNC-BNC cable.
3. Mount the probe in the micromanipulator and place it near the microscope and the myograph. The recording setup must be installed on a vibration-free table.
4. Place the knobs and switches on the front of the amplifier in positions that configure it for this experiment as described in the manual.
5. Connect the bath ground to the circuit ground of the amplifier via with an appropriate electrode. Similarly, ensure that the cage is grounded to the chassis of the amplifier.

2. Preparation of Microelectrodes and Assembly

1. Use borosilicate glass microelectrodes (see the Table of Materials) and pull the glass tip to have a 8–10 mm taper, diameter of <1 µm and resistance of 80–120 MΩ when filled with 3 M KCl.
   1. Use a standard puller to achieve a short gradual taper using the following settings: heat = 650; velocity = 20; pull = 25; time = 250 and loop twice for higher resistances and smaller tips. See the Table of Materials as the settings are instrument-specific.
      NOTE: Tip diameters <1 µm will cause minimal damage to the cell when impaled.
2. Fill the microelectrode with 3 M KCl using a microfiber syringe (see the Table of Materials).
   1. Slowly pull the plunger of the microfiber syringe up while injecting the 3 M KCl into the microelectrode to allow space for the fluid to fill and to prevent the formation of air bubbles inside the microelectrode.
   2. Fill the microelectrode until full and ensure that there are no air bubbles before placing it in the microelectrode holder. If bubbles are present, gently tap the microelectrode with a finger to remove the bubbles.
3. Exercising care, firmly push the electrode shank into the holder through the bored hole. If excess fluid is present, remove it with a tissue.
4. Connect the electrode holder assembly to the amplifier probe. Conduct an electrode test, adjust the input offset, verify zero setting and check the probe input leakage as per the amplifier manual.
5. Measure electrode resistance using an electrode test as shown in Table 1.
6. Note that a working electrode displays a positive DC voltage shift of 1 mV/MΩ at the channel output. On the other hand, if a large voltage appears at the channel output and on the meter, this indicates a blocked or broken electrode.
7. Open the recording software, assign a name to the file and save it for future analysis in a storing software.

3. Isolation and Cannulation of the Middle Cerebral Artery

1. Preparation the reagents.
   1. Prepare normal and low calcium physiological salt solution (PSS) as described in Table 2.
2. Prepare the myograph.
   1. Rinse the myograph chamber (see the Table of Materials) with distilled water multiple times to keep it free of debris. Load the chamber with 5 mL of normal PSS.
   2. Fill both glass cannulas with filtered normal PSS using a 5–10 mL syringe. Carefully fill the entire cannula and the attached tubing without introducing any air bubbles.
   3. Prepare two monofilament nylon sutures (10-0, 0.02 mm) with a half-knot each using blunt forceps.
   4. Place the partially closed suture knots on both cannulas slightly away from the tip using dissection forceps under a dissection microscope. Later these knots will be slid off and tied carefully onto the cannulated arterial ends to secure the vessel.
3. Isolate and cannulate the middle cerebral artery.
   1. Induce deep anesthesia in a Sprague Dawley rat by using 2–4% inhaled isoflurane.
   2. Decapitate the rat using guillotine under deep anesthesia.
   3. Carefully remove the skull using a bone cutter and a scissor.
   4. Remove the brain from the skull and place it in 5 mL of low calcium PSS on ice.
   5. Identify and dissect out an unbranched segment of rat middle cerebral artery (MCA) with an inner diameter of 100–200 μm from the brain using spring scissors and forceps.
   6. Mount the MCA onto the glass cannulas using fine forceps and secure by tightening the sutures in the myograph containing normal PSS.
   7. Close off the distal cannula so that there will be no flow within the MCAs.
   8. Connect the inflow pipette to a reservoir holding PSS to allow for control of intraluminal pressure which will be monitored with an in-line pressure transducer.
   9. Visualize the cannulated MCAs using a charge-coupled device camera (see the Table of Materials) mounted on an inverted microscope and an imaging software.
   10. Set the axial length of the MCA to an approximate length where it should appear neither rigid nor flaccid.
   11. Equilibrate the bath solution with O₂ (95%) and CO₂ (5%) at 37 °C to provide adequate oxygenation, temperature and to maintain pH at 7.4.
4. Impale (penetrate the cell plasma membrane) the vascular smooth muscle cells.
   1. Connect the ground electrode and keep it immersed in the PSS of the myograph.
   2. Illuminate the vessel chamber and look through the microscope to visualize the tip of the microelectrode in the bath solution.
      NOTE: Alternatively, one can visualize the MCA and microelectrode on a computer having an imaging software.
   3. Use the controls of the micromanipulator to move the tip of the microelectrode close to the outer wall of the blood vessel. The micromanipulator and the tip of the microelectrode must be in a stable position in relation to the tissue.
      NOTE: Before beginning experiments, confirm that the membrane voltage has stabilized. If the measured voltage is unstable, the connection between the electrode and the cell is not sealed, indicating a leak.
   4. Begin the recording.
   5. Slowly move the tip of the microelectrode towards the vessel, aiming for the center of the vessel using course or fine control of the micromanipulator.
      NOTE: Occasionally, a small deflection in the recording may be observed when the microelectrode tip contacts a muscle fiber membrane.
   6. When the tip comes in close proximity to the vessel, advance the electrode forward in one rapid motion using the micromanipulator to impale the membrane of the muscle.
   7. At this point, one can begin observing the changes in V_m being recorded. Do not touch the micromanipulator when the microelectrode impales the membrane of the cell.
      NOTE: The difference in voltage between the recording and reference electrode decreases from 0 mV to between -40 mV and -75 mV depending on the level of intravascular pressure or other excitatory or inhibitory stimuli. These readings characterize the transmembrane potential difference of the current cell.
   8. Perform multiple impalements on a single vessel in different areas of the vessel without damaging VSMCs in order to get accurate measurements.
   9. After recording, use the manipulator to remove the microelectrode in one rapid movement.
   10. Stop the recording and save data files for further analysis.

Wyniki

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...

Dyskusje

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...

Materiały

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