characterizing extracellular space in a brain slice using ion-selective and iontophoresis microelectrodes

Characterizing Extracellular Space in a Brain Slice Using Ion-Selective and Iontophoresis Microelectrodes

Place a 400-micrometer thick brain slice in the recording chamber, ensuring that it is fully submerged in the flowing ACSF. Next, move both the iontophoresis microelectrode and the ISM above the field of interest on the brain slice. Submerge both electrodes in the flowing ACSF but above the slice. Then, offset the voltage for both the reference and the ion-sensing channels to 0 millivolts. Wait for the voltage in both channels to stabilize.
On the chart recorder, Mark the voltage measured on the ion-sensing channel of the ISM. Next, place the ISM and iontophoresis microelectrodes 200 micrometers deep in the slice and 120 micrometers away from each other. Calculate the voltage difference between the TMA signals measured in the flowing ACSF and in the brain and input this value into the baseline volt millivolt field in the measuring electrode box of the Wanda GUI.
On the left side of the GUI, ensure that all the experimental parameters are correctly entered. Start the recording by clicking Acquire, and allow it to take a full recording. After moving both microelectrodes out of the slice, use the chart recorder to determine any change between the voltage measured now, and its measurement from before.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;&#160;&#160;&#160;
1. Real-time Iontophoresis in Brain Slices
<ol>
	<li>Place a 400 &#181;m-thick brain slice in the recording chamber, ensuring that it is fully submerged in the flowing artificial cerebrospinal fluid (ACSF). Position the slice using a watercolor paintbrush and gently secure it with a grid.</li>
	<li>Move both the iontophoresis microelectrode and the ISM above the field of interest on the brain slice. Submerge both in the flowing ACSF but above the slice.</li>
	<li>Offset the voltage for both the reference and the ion-sensing channels to &#34;0&#34; mV. Wait for the voltage in both channels to stabilize. On the chart recorder, mark the voltage measured on the ion sensing channel of the ISM. Use this to calculate the baseline V parameter in Wanda.</li>
	<li>Place the ISM and iontophoresis microelectrode 200 &#181;m deep in the slice and 120 &#181;m away from each other. Wait for the stabilization of the signal after moving the microelectrode into the brain slice. 
	NOTE: The bias current applied to the iontophoresis microelectrode causes a small accumulation of tetramethylammonium (TMA). It is a common mistake to take a recording too soon and underestimate the signal buildup.</li>
	<li>On the chart recorder, mark the stabilized voltage measured in the brain slice on the ion-sensing channel of the ISM. Calculate the voltage difference between the TMA signal measured in step 8.3 and step 8.4 and input this value into the &#34;Baseline V (mV)&#34; field in the Measuring Electrode box of the Wanda GUI.</li>
	<li>On the left side of the GUI, ensure that all experimental parameters are correctly recorded/entered. Set &#34;Medium&#34; to &#34;Brain,&#34; &#34;Transport number&#34; to the average value calculated for the iontophoresis microelectrode in step 7.4, and &#34;Temperature&#34; to the temperature of the bath containing the slice. 
	NOTE: V must be recorded for each set of measurements. The baseline V will be converted by Wanda into the baseline C (mM) parameter (i.e., the concentration of TMA in the brain tissue).</li>
	<li>Start the recording by clicking &#34;Acquire&#34; and allow it to take a full recording. Wait until the TMA signal returns to baseline before acquiring a new recording.</li>
	<li>Take two to three successive recordings before removing the microelectrodes from the chosen brain location. Input the temperature measured into the Wanda software immediately before each recording.</li>
	<li>Move both microelectrodes diagonally back to the surface of the slice. Raise both to at least 50 &#181;m above the slice. Using the chart recorder, determine any change between the V measured now and its measurement from step 8.3.</li>
	<li>Center the tips of the ISM and the iontophoretic microelectrodes relative to each other in the x-, y-, and z-axes. Obtain spacing changes, if any, from the display of the micromanipulator control box.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>amplifier for ISM</td>
			<td>Dagan</td>
			<td>Model IX2-700 Dual Intracellular Preamplifier</td>
			<td>ion and reference voltage amplifier with N=0.1 (for reference barrel) and N=0.001 (for ion barrel) headstages</td>
		</tr>
		<tr>
			<td>biological compound miscroscope (with 4x and 10x objective)</td>
			<td></td>
			<td></td>
			<td>for chipping the microelectrode tips and inspecting microelectrodes; various suppliers, e.g. AmScope</td>
		</tr>
		<tr>
			<td>borosilicate theta capillary glass tubing</td>
			<td>Harvard Apparatus</td>
			<td>Warner Instruments model TG200-4; order #64-0811</td>
			<td>double-barreled glass tubing for ion-selective microelectrodes and iontophoretic microelectrodes; O.D. 2.0 mm, I.D. 1.4 mm, septum 0.2 mm, length 10 cm</td>
		</tr>
		<tr>
			<td>chart recorder</td>
			<td></td>
			<td></td>
			<td>to record continuously voltages on ion-selective microelectrode during calibration in tetramethylammonium standards and during RTI experiment; e.g. Kipp &#38; Zonen type BD112 dual-cannel chart recorded, available refurbished</td>
		</tr>
		<tr>
			<td>Commercial Software</td>
			<td>The MathWorks</td>
			<td>MATLAB, Data acquisition toolbox</td>
			<td>for data acquisition and analysis using Wanda and Walter programs. Note that an academic license is available.</td>
		</tr>
		<tr>
			<td>eye protective goggles</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fixed-stage compound microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>can use other compound microscopes with fixed stages</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fine Science Tools</td>
			<td>#11251-10</td>
			<td>to chip glass capillary; Dumond #5, preferably used and no longer needed for fine work</td>
		</tr>
		<tr>
			<td>iontophoretic unit</td>
			<td>Dagan</td>
			<td>ION-100 and PS-100</td>
			<td>ION-100 is a single channel iontophoresis unit +/- 130 V compliance; PS-100 is an external power supply; alternatives: e.g. Axoprobe-1A made by Axon Instruments (now Molecular Devices), out of production, check for availability of refurbished units (eBay and other sites)</td>
		</tr>
		<tr>
			<td>microelectrode holder</td>
			<td>WPI</td>
			<td>M3301EH</td>
			<td>to hold ion-selective microeletrode prefabricate for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; WPI sells two versions of this holder, clear M3301EH and black M3301EH. In our experience, the clear M3301EH appears to be sturdier then the black M3301EH.</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>MM-3</td>
			<td>to position ion-selective microelectrode prefabricate during silanization and filling the tip of ion-selective barrel with liquid ion exchanger; can be substituted with any three-axis micromanipulator in good working condition</td>
		</tr>
		<tr>
			<td>objective 5x dry</td>
			<td>Olympus</td>
			<td>MPlan N</td>
			<td></td>
		</tr>
		<tr>
			<td>objective 10x water immersion</td>
			<td>Olympus</td>
			<td>UMPlan FL N</td>
			<td>10x objective is water immersion, numerical aperture is 0.3, working distance is 3.3 mm</td>
		</tr>
		<tr>
			<td>platform and x-y translation stage for fixed-stage microscope</td>
			<td>EXFO</td>
			<td>Gibraltar Burleigh</td>
			<td>platform holds slice chamber, micromanipulators and accesorries, x-y translational stage moves microscope without compromising recording stability</td>
		</tr>
		<tr>
			<td>robotic micromanipulator with precise x,y,z positioning</td>
			<td>Sutter Instruments</td>
			<td>MP-285</td>
			<td>two mircomanipulators are needed to hold separately ion-selective microelectrode and iontophoretic microelectrode. Also possible to glue micropipettes in a spaced array (see text).</td>
		</tr>
		<tr>
			<td>signal conditioning unit with low-pass filter</td>
			<td>Axon Instruments</td>
			<td>CyberAmp 320 or 380</td>
			<td>no longer available from the manufacturer but may be available from E-Bay; alternatives: e.g. FLA-01 Filter/Amplifier from Cygnus Technology. This is a single channel instrument with a minimum cutoff at 10 Hz using a multipole Bessel filter but the company may be willing to modify it for a lower cutoff frequency (2 Hz) if needed.</td>
		</tr>
		<tr>
			<td>silver wire</td>
			<td>A-M Systems</td>
			<td>#7830</td>
			<td>diameter 0.015&#34;, bare (no coating)</td>
		</tr>
		<tr>
			<td>slice chamber</td>
			<td>Harvard Apparatus</td>
			<td>Warner Model RC-27L</td>
			<td>this is submersion slice chamber; do not use interface slice chamber</td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td></td>
			<td></td>
			<td>for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; horizontally mounted; various suppliers</td>
		</tr>
		<tr>
			<td>tetramethyammonium (TMA) chloride</td>
			<td>Sigma-Aldrich</td>
			<td>T-3411</td>
			<td>5 M solution; CAUTION: acute toxicity (oral, dermal, inhalation), carcinogenicity, hazardous to the aquatic environment, see Sigma-Aldrich Safety Information for full description</td>
		</tr>
	</tbody>
</table>

Source:&#160; Odackal, J., et al. <a href="https://app.jove.com/v/55755">Real-time iontophoresis with tetramethylammonium to quantify volume fraction and tortuosity of brain extracellular space.</a> J. Vis. Exp. (2017)
This video demonstrates how to characterize the extracellular space in a brain slice by using ion-selective and iontophoresis microelectrodes to measure and compare electrical signals. This process helps determine the space&#39;s volume fraction and degree of twisting.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;&#160;&#160;&#160;
1. ...

Begin with a recording chamber containing a secured thick brain slice immersed in artificial cerebrospinal fluid or aCSF.
This brain slice includes interstitial fluid and extracellular matrix, forming the interconnected extracellular space that surrounds brain cells.
Submerge the dual-channel ion-selective microelectrode and the iontophoresis microelectrode into the aCSF.
The dual-channel ion-selective microelectrode features a sensing channel for tetramethylammonium or TMA ions and a reference channel, while the iontophoresis microelectrode contains a TMA solution.
The iontophoresis electrode releases TMA ions, which diffuse toward the ion-selective microelectrode.
The sensing channel detects these TMA ions and generates an electrical signal. Record this as a baseline.
Insert both electrodes into the brain slice, keeping them slightly apart.
As the TMA ions diffuse through the extracellular space, they reach the ion-selective microelectrode.
Rerecord the signal and compare it with the baseline to analyze extracellular space properties, such as the volume fraction and the degree of twisting.

15 조회수. Ion-Selective and Iontophoresis microelectrodes를 사용한 뇌 절편의 세포외 공간 특성화

비디오: Ion-Selective and Iontophoresis microelectrodes를 사용한 뇌 절편의 세포외 공간 특성화 - 실험

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

통합 된 마이크로 전극을 가진 바이오 영감 소프트 로봇

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

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JoVE Journal

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Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

이온 선택적 자체 참조 미세 전극 기술을 이용하여 세포 외 이온 플럭스 측정

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

생물학

Performing Laser-Induced Brain Injury in a Rat Model

To perform a laser-induced brain injury, first, assign twenty 300- to 350-gram Sprague Dawley rats into the laser group. After confirming a lack of response to withdrawal reflex, place one rat on a rectal temperature-regulated heating pad, and use a shaver to remove enough hair from the injury site, with an approximately two-centimeter hair-free margin around the incision. Disinfect the exposed skin with 70% ethanol, and place the rat in the prone position in a stereotaxic head holder.
Make a three-centimeter incision to allow lateral reflection of the scalp to expose the area between bregma and lambda. Holding an Nd:YAG laser with peak wavelength of 1,064 nanometers at a two-millimeter distance from the skull, administer 50 joules times 10 points, with a one-second pulse duration, to the exposed area of the skull above the right hemisphere. After the laser injury has been delivered, remove the rat from the device, and close the scalp with 3-0 silk surgical sutures. Then, place the rat in its cage with monitoring until full recumbency.

Characterizing Extracellular Space in a Brain Slice Using Ion-Selective and Iontophoresis Microelectrodes

내레이션 대본

Characterizing Extracellular Space in a Brain Slice Using Ion-Selective and Iontophoresis Microelectrodes