The work was supported by NIH NINDS grant R01 NS047557.

All animal procedures, used to obtain tissue samples, were approval by the animal ethics committee at SUNY Downstate Medical Center.
1. Preparation of Solutions and Equipment
<ol>
	<li>Prepare a 150 mM NaCl backfill solution for the reference barrel of the ISM. Store it in a 10 mL syringe attached to a 0.22 &#181;m filter (to remove bacteria or particles).</li>
	<li>Prepare a 150 mM TMA chloride (TMA-Cl) backfill solution for the microelectrodes. Store it in a 10 mL syringe attached to a 0.2...

<ol>
	<li>Sykova, E., Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+in+brain+extracellular+space.">Diffusion in brain extracellular space.</a> Physiol Rev. 88 (4), 1277-1340 (2008).</li><li>Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+and+related+transport+mechanisms+in+brain+tissue.">Diffusion and related transport mechanisms in brain tissue.</a> Rep Prog Phys. 64 (7), 815-884 (2001).</li><li>Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-selective+microelectrodes+and+diffusion+measurements+as+tools+to+explore+the+brain+cell+microenvironment.">Ion-selective microelectrodes and diffusion measurements as tools to explore the brain cell microenvironment.</a> J Neurosci Methods. 48 (3), 199-213 (1993).</li><li>Nicholson, C., Phillips, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+diffusion+modified+by+tortuosity+and+volume+fraction+in+the+extracellular+microenvironment+of+the+rat+cerebellum.">Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum.</a> J Physiol. 321, 225-257 (1981).</li><li>Nicholson, C., Sykova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+space+structure+revealed+by+diffusion+analysis.">Extracellular space structure revealed by diffusion analysis.</a> Trends Neurosci. 21 (5), 207-215 (1998).</li><li>Xie, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+drives+metabolite+clearance+from+the+adult+brain.">Sleep drives metabolite clearance from the adult brain.</a> Science. 342 (6156), 373-377 (2013).</li><li>Hrabetova, S., Nicholson, C., Michael, A. C., Borland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+of+brain+extracellular+space+explored+with+ion-selective+microelectrodes,+integrative+optical+imaging+and+related+techniques.">Biophysical properties of brain extracellular space explored with ion-selective microelectrodes, integrative optical imaging and related techniques.</a> Electrochemical Methods for Neuroscience Neuroscience. , 167-204 (2007).</li><li>Rice, M. E., Okada, Y. C., Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+and+heterogeneous+diffusion+in+the+turtle+cerebellum:+implications+for+volume+transmission.">Anisotropic and heterogeneous diffusion in the turtle cerebellum: implications for volume transmission.</a> J Neurophysiol. 70 (5), 2035-2044 (1993).</li><li>Vargova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+parameters+of+the+extracellular+space+in+human+gliomas.">Diffusion parameters of the extracellular space in human gliomas.</a> Glia. 42 (1), 77-88 (2003).</li><li>Haack, N., Durry, S., Kafitz, K. W., Chesler, M., Rose, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-barreled+and+concentric+microelectrodes+for+measurement+of+extracellular+ion+signals+in+brain+tissue.">Double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue.</a> J Vis Exp. (103), (2015).</li><li>Xiao, F., Hrabetova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+extracellular+space+of+aquaporin-4-deficient+mice+does+not+enhance+diffusion+of+Alexa+Fluor+488+or+dextran+polymers.">Enlarged extracellular space of aquaporin-4-deficient mice does not enhance diffusion of Alexa Fluor 488 or dextran polymers.</a> Neuroscience. 161 (1), 39-45 (2009).</li><li>Sherpa, A. D., Pvan de Nes, ., Xiao, F., Weedon, J., Hrabetova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gliotoxin-induced+swelling+of+astrocytes+hinders+diffusion+in+brain+extracellular+space+via+formation+of+dead-space+microdomains.">Gliotoxin-induced swelling of astrocytes hinders diffusion in brain extracellular space via formation of dead-space microdomains.</a> Glia. 62 (7), 1053-1065 (2014).</li><li>Kume-Kick, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independence+of+extracellular+tortuosity+and+volume+fraction+during+osmotic+challenge+in+rat+neocortex.">Independence of extracellular tortuosity and volume fraction during osmotic challenge in rat neocortex.</a> J Physiol. 542 (Pt 2), 515-527 (2002).</li><li>Saghyan, A., Lewis, D. P., Hrabe, J., Hrabetova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+diffusion+in+laminar+brain+structures+exemplified+by+hippocampus.">Extracellular diffusion in laminar brain structures exemplified by hippocampus.</a> J Neurosci Methods. 205 (1), 110-118 (2012).</li><li>Fedirko, N., Svichar, N., Chesler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+use+of+high-speed,+concentric+H+-+and+Ca2+-selective+microelectrodes+suitable+for+in+vitro+extracellular+recording.">Fabrication and use of high-speed, concentric H+- and Ca2+-selective microelectrodes suitable for in vitro extracellular recording.</a> J Neurophys. 96 (2), 919-924 (2006).</li><li>Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+from+an+injected+volume+of+a+substance+in+brain+tissue+with+arbitrary+volume+fraction+and+tortuosity.">Diffusion from an injected volume of a substance in brain tissue with arbitrary volume fraction and tortuosity.</a> Brain Res. 333 (2), 325-329 (1985).</li><li>Nicholson, C., Tao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindered+diffusion+of+high+molecular+weight+compounds+in+brain+extracellular+microenvironment+measured+with+integrative+optical+imaging.">Hindered diffusion of high molecular weight compounds in brain extracellular microenvironment measured with integrative optical imaging.</a> Biophys J. 65 (6), 2277-2290 (1993).</li><li>Thorne, R. G., Nicholson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+diffusion+analysis+with+quantum+dots+and+dextrans+predicts+the+width+of+brain+extracellular+space.">In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space.</a> Proc Natl Acad Sci U S A. 103 (14), 5567-5572 (2006).</li><li>Wolak, D. J., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+macromolecules+in+the+brain:+implications+for+drug+delivery.">Diffusion of macromolecules in the brain: implications for drug delivery.</a> Mol Pharm. 10 (5), 1492-1504 (2013).</li></ol>

<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/55755/55755fig10.jpg" /> 
Figure 10: Non-ideal Data Demonstrating Common Technical Issues. (a) Diagrams of common technical issues with iontophoresis microelectrodes: Comparison of the normal release of TMA from a functioning iontophoresis microelectrode with three sources demonstrating technical issues. [High magnification, a1] The current in an ideal iontophoretic source is carried equally by TMA release and chloride upta...

The extracellular space (ECS) is the network of interconnected channels exterior to all brain cells and contains both interstitial fluid and extracellular matrix (Figure 1a and Figure 1b). The distribution of many substances required for brain cell function, including nutrients, hormones, and neurotransmitters, occurs by diffusion through the ECS. Changes in the physical parameters of this space, including volume, geometry, and extracellular matrix, can drastically affect diffusion through the ECS and the local ion concentrations bathing brain cells, which have a profound impact on brain cell function1,2.
Real-time iontophoresis (RTI) is used to determine two structural characteristics of a brain region: volume fraction and tortuosity3,4,5. Volume fraction (&#945;) is the proportion of tissue volume occupied by the ECS (VECS) relative to the total tissue volume (Vtissue) in a representative elementary volume;
<img alt="Equation" src="/files/ftp_upload/55755/55755eq1.jpg" />
Tortuosity (&#955;) is the relative hindrance that a substance encounters when diffusing through a brain region as compared to a medium with no obstructions;
<img alt="Equation" src="/files/ftp_upload/55755/55755eq2.jpg" />
where D* (cm2 s-1) is the effective diffusion coefficient of the substance in brain and D (cm2s-1) is the free diffusion coefficient of the substance in a free medium, such as dilute agarose gel.
Today, the most commonly used probe substance for the RTI method is the small cation tetramethylammonium (TMA). TMA has a molecular weight of 74 g/mol, completely dissociates in solution, and has one positive charge. RTI studies with this ion have demonstrated that &#945; <img alt="Equation" src="/files/ftp_upload/55755/55755eq3.jpg" /> 0.2 and &#955; <img alt="Equation" src="/files/ftp_upload/55755/55755eq3.jpg" /> 1.61,2. This means that the ECS is roughly 20% of the total brain volume and that the diffusion of a small, inert molecule occurs roughly 2.5 times slower in the ECS than in a medium with no obstructions3. However, both &#945; and &#955; vary with brain age, region, and state and in pathological conditions1. Alterations of these parameters have been linked to brain development, aging, sleep, epilepsy, and many other fundamental processes and diseases of the brain1,6. While other techniques measure &#945; and &#955;, RTI can measure both in localized regions of living tissue in real time. For this reason, RTI has become an indispensable tool for investigating changes in &#945; and &#955; during acute and reversible challenges.
The theory supporting RTI was originally validated by Nicholson and Phillips, and the technique has been used extensively since that time4,7. Experiments employing RTI begin with the release of a pulse of TMA from a source microelectrode by iontophoresis into a dilute agarose gel. Once ejected, the ions freely diffuse away from the point source, choosing from a potentially infinite number of random paths (Figure 1d). The changing concentration of the ion is measured over time using an ion-selective microelectrode (ISM) positioned roughly 100 &#181;m away (Figure 1c). The changes in TMA concentration are graphed and fitted to a curve that allows for the calculation of both D and the transport number of the iontophoresis microelectrode (parameters discussed in the Protocol). With these values, the procedure is repeated in a brain region of interest to obtain D* and to calculate both &#945; and &#955;. Control of the iontophoresis microelectrode, data collection, graphing and fitting of the TMA concentration curve, and calculation of the experimental parameters are all typically done by the programs Wanda and Walter, which have been specifically designed for this purpose (the software and their manuals are freely available from the authors upon request).
The Protocol section of this review describes the basic procedures needed to design and perform RTI in rodent brain slices. The technique has also been used in non-rodent models, including human brain slices and in vivo brain preparations1,4,6,8,9. The Representative Results section provides both ideal and non-ideal results to highlight nuances in data interpretation. Finally, the Discussion section briefly covers troubleshooting techniques, limitations of RTI, alternative techniques used to study the ECS, and future applications of RTI.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55755/55755fig1.jpg" /> 
Figure 1: Diagrams of Diffusion through ECS. (a) Diagram of ECS: Demonstrates the size and location of the ECS in a typical brain section. Yellow marks the ECS between the gray brain cell processes. The volume of the ECS is roughly 20% of the total tissue volume (i.e., volume fraction = 0.2) under physiological conditions. (b) Magnified diagram of the ECS: Highlights physical parameters contributing to tortuosity, including brain cell geometry (gray) and extracellular matrix (diagramed as a mesh of multicolored glycosaminoglycans and proteoglycans). (c) 3D diagram of diffusion from a point source: Demonstrates the net movement of inert molecules from an iontophoretic source to an ISM. Excluding diffusion barriers and cellular uptake, molecules diffuse outwards in all directions, producing a spherical concentration front. The ISM quantifies the local concentration of the inert molecules released from the iontophoretic source. (d) Computer simulation of diffusion in ECS of brain: [Far left] Setup for Monte Carlo simulation; green spheres represent brain cell processes and the red cross represents a point source. This setup models the brain tissue diagrammed in Figure 1a. [Middle images] 3 and 6 molecules performing random movements as they diffuse through the extracellular space of the brain, shown in 2 dimensions. [Far right] Random walks of many molecules released from the point source. The net movement of all molecules from the point source is outwards as depicted in Figure 1c. The cumulative random walks outline the spaces between the cells (i.e., the ECS; see reference5 for further explanation). <a href="http://cloudflare.jove.com/files/ftp_upload/55755/55755fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A/D and D/A converter</td>
			<td>National Instruments Corporation</td>
			<td>NI USB-6221 DAQ</td>
			<td>The NI USB-6221 is still sold as a &#39;Legacy&#39; device by NI. They recommend using NI USB-6341 X Series DAQs for new installations, however we have not tested the newer units. We describe the use of the NI USB-6221 with MATLAB and Windows 7 (32-bit). Alternatives: the much older PCI-MIO-16E-4 A/D converter (Used under Windows XP or older OS only) with BNC-2090 BNC connector panel and SH68-68-EP cable. As noted in the Wanda Manual, an experimental MATLAB program to use Axon Binary Files is available.</td>
		</tr>
		<tr>
			<td>agarose</td>
			<td>Lonza</td>
			<td>NuSieve GTG Agarose #50081</td>
			<td>to prepare dilute agarose gel for RTI measurements</td>
		</tr>
		<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>brush</td>
			<td>Winsor &#38; Newton</td>
			<td>University Series 233, size 0</td>
			<td>round shoft handle brush, available from Amazon</td>
		</tr>
		<tr>
			<td>bunsen burner</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera for visualizing micropipettes</td>
			<td>Olympus</td>
			<td>OLY-150</td>
			<td>requires monitor, IR filter on substage illuminator is optional</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>chlorotrimethylsilane, puriss., &#62; 99%</td>
			<td>Sigma-Aldrich</td>
			<td>catalog # 92360</td>
			<td>for silanization; CAUTION: flammable, acute toxicity (oral, dermal, inhalation), skin corrosion, eye damage, reacts violently with water, see Sigma-Aldrich Safety Information for full description</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>fume hood</td>
			<td></td>
			<td></td>
			<td>for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; various supliers, e.g. Captair with approriate filter sold by Erlab</td>
		</tr>
		<tr>
			<td>glass microscope slide</td>
			<td>Fisher</td>
			<td>#12-550A</td>
			<td>to chip microelectrode tips</td>
		</tr>
		<tr>
			<td>heater/stirrer</td>
			<td>Fisher</td>
			<td>Corning PC-420D</td>
			<td>to prepare dilute agarose gel and stir solutions</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>liquid ion exchanger (LIX) for tetramethylammonium</td>
			<td>World Precision Instruments</td>
			<td>IE190 Potassium Ion Exchanger</td>
			<td>Note: this is equivalent to the original Corning potassium exchanger 477317 based on tetraphenlyborate - do not confuse with neutral carrier potassium exchanger originating from the laboartory of Dr. Simon, ETH, Zurich, which does not sense tetramethylammonium, and is sold by Fluka. You can also make liquid ion exchanger for tetramethylammonium yourself: 3% by weight potassium tetrakis = (p-chlorophenyl) borate dissolved in 2,3-dimethylnitrobenzene. Buy chemicals from Fluka (now part of Sigma). See Oehme and Simon (1976) Anal. Chim. Acta 86: 21-25; CAUTION: The toxicological properties of this liquid ion exchanger have not been fully determined. Ingestion or contact with the human body may be harmful. Exercise due care! Liquid ion exchangers should be stored in a cool place out of direct sunlight.</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>micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-97</td>
			<td>to pull double-barreled glass tubing; other pullers can be used as long as they can accommodate large diameter double-barreled glass tubing</td>
		</tr>
		<tr>
			<td>microprobe thermometer</td>
			<td>Physiotemp</td>
			<td>Model BAT-12R</td>
			<td>fine probe of this thermometer is placed close to recording site</td>
		</tr>
		<tr>
			<td>needle</td>
			<td>BD</td>
			<td>Syringes and Needles # 305122 (25 gauge)</td>
			<td>for silanization; BD PrecisionGlide needles 25 G x 5/8 in (0.5mm x 16mm)</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>plastic containers (with lids)</td>
			<td>Fisher</td>
			<td>#14-375-148</td>
			<td>to store tetramethylammonium standard solutions and microelectrodes</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>porous minicup</td>
			<td></td>
			<td></td>
			<td>for RTI measurements in a dilute agarose gel; homemade</td>
		</tr>
		<tr>
			<td>reusable adhesive</td>
			<td>Bostik</td>
			<td>Blu-Tack</td>
			<td>for securing microelectrodes to holding vessel and other uses; various suppliers, available from Amazon</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>syringe, 10 mL</td>
			<td>BD</td>
			<td>Syringes and Needles #309604</td>
			<td>to backfill microelectrodes and for silanization; BD Luer-Lok tip</td>
		</tr>
		<tr>
			<td>syringe filter 0.22&#181;m pore</td>
			<td>Whatman</td>
			<td>#6780-1302</td>
			<td>to filter backfill solutions; available from Fisher</td>
		</tr>
		<tr>
			<td>syringe needle, 28 gauge, 97mm</td>
			<td>World Precision Instruments</td>
			<td>MicroFil MF28G-5</td>
			<td>to backfill microelectrodes</td>
		</tr>
		<tr>
			<td>Teflon (=PTFE) tubing</td>
			<td>Component Supply</td>
			<td>STT-28 PTFE tube light wall (28 gauge)</td>
			<td>for silanization of ion-selective barrel; fits on BD PrecisionGlide needles 25 G x 5/8 in. Note: Teflon is essential, PVC tubing would melt by hot wax.</td>
		</tr>
		<tr>
			<td>temperature control system</td>
			<td>Harvard Apparatus</td>
			<td>Warner Models TC-344B and SH-27A</td>
			<td>TC-344B is a dual automatic temperature controller, SH-27A is an in-line heater; controller and heater work with Warner slice chambers</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>
		<tr>
			<td>vibrating blade microtome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td>to cut brain slices</td>
		</tr>
		<tr>
			<td>xylenes</td>
			<td>Fisher</td>
			<td>X5-1</td>
			<td>for silanization; CAUTION: flammable, acute toxicity (oral, dermal, inhalation), skin corrosion, eye damage, carcinogenicity, see Fisher Safety Information for full description</td>
		</tr>
	</tbody>
</table>

real-time iontophoresis with tetramethylammonium to quantify volume fraction and tortuosity of brain extracellular space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

The utility of the RTI technique is demonstrated in an experiment designed to measure the changes in &#945; and during a hypoosmolar challenge (Figure 8 and Figure 9). It has previously been shown that reducing the osmolarity of the ECS by washing on hypotonic ACSF will produce a decrease in &#945; and an increase in &#955;13.
<p class="jove_content" fo:keep-together.within-p...

Watch this Scientific Journal Video about Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space at JoVE.com

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

The overall goal of this procedure is to quantify the volume fraction and tortuosity of the extracellular space in brain tissue. This method answers key questions regarding the relationship between the extracellular space and the physiology and disease. The main advantage of this technique is that it allows the real-time characterization of two important parameters of the extracellular space.Generally, individuals new to this method will struggle because the iontophoretic microelectrode exhibits a tendency to change its properties during experiments and frequently requires troubleshooting during experiments. Visual demonstration of this method is critical as the precise manipulation of an ion-selective microelectrode, an iontophoretic electrode over multiple steps is required to obtain a successful recording. To begin this procedure, run ACSF containing tetramethylammonium, or TMA, through the submersion chamber at two milliliters per minute.Set the temperature controller to a desired temperature and bubble the ACSF with 95%oxygen, 5%carbon dioxide for the duration of the experiment. Next, mount the ground electrode on a holder and submerge its tip into the ACSF running through the submersion chamber. Then, fill the porous cup with 0.3%agarose containing TMA prepared previously and place it in the submersion chamber.Now, secure an iontophoresis microelectrode to the pipette holder of one micromanipulator. Connect the iontophoresis microelectrode wire to the head stage of the iontophoresis unit. Next, secure a calibrated ISM to the second micromanipulator and connect the ISM to its head stages.Afterwards, turn on the computer containing the Wanda and Walter programs. In the Wanda GUI, click Calibrate. Then, in the Calibration box, fill in the voltages measured during the ISM calibration and click Fit Data.Following that, click Accept in the Calibrate box to automatically transfer the slope and interference generated to the main GUI. On the left side of the GUI, ensure that all the experimental parameters are set in the corresponding entries. Next, place a temperature probe in the agar cup.Record the measured temperature in the Temperature entry in the Measuring Electrode box of the GUI. Then, lower the microelectrodes at least 1, 000 micrometers deep into the agarose and center them in the cup. Visualize them under the microscope using a 10X objective.On the two-channel amplifier, manually move the ISM channel connector to the voltage subtraction output to set the subtraction on between the reference and ISM channels. Center the tips on each other in all three directional axes. Next, zero the relative positions of both microelectrodes on the micromanipulator control boxes and ensure that the microelectrodes are centered accurately and precisely.Then, move the ISM 120 micrometers away from the iontophoresis microelectrode in one axis. Subsequently, start a recording by clicking on Acquire in the GUI and allow the program to run for a full recording. In this procedure, open the Walter program on the computer.In the Zero Records From menu, click the Wanda Voltoro button to read the records generated by Wanda. In the next popup window, select one or more records to be read and click Open. In the One Write Excel menu, click Sheet 1.3 and follow prompts until the records are automatically graphed.To begin the fitting procedure, in the Two Options menu, click on the select rec button. Then, in the figure two popup window, use the mouse to move the cross hairs over the first record to be processed and press either mouse button to choose the record. After that, click on fit curve in the menu and use at least 20 iterations of fitting to obtain an accurate fit of the data.Next, select all to fit all data points and select continue. The program will fit the displayed curve. Then, select the option to write the result to the appropriate spreadsheet program by clicking Excel in the seven results menu.Name the Excel file. Note and record the data generated which will be used to determine the functionality of the iontophoresis microelectrode. If the iontophoresis microelectrode is deemed suitable for the experiment, record the average transport number from all trials in the TransportNum end field in the Wanda GUI.In this procedure, 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 zero 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. To analyze the recordings from the brain, repeat the procedures for agarose data analysis using the Walter program.Write the data onto the spreadsheet program by clicking Excel in the Walter menu. Then, record the volume fraction of the brain ECS, tortuosity of the brain ECS and the non-specific clearance. To check the transport number, take new recordings in agarose and repeat the procedures in the Walter program to obtain the transport number.Inspect the spreadsheet and check if the transport number has changed by more than 10%from the transport number obtained prior to the brain measurements. If so, the data obtained with the iontophoretic microelectrode are not reliable. The transport number measured in this step must closely match that obtained prior to brain measurements, otherwise the volume fraction cannot be considered accurate.Use newly obtained ISM calibration data as the input in the Wanda Calibrate box and check that the slope value differs by less than 10%from the previous calibration. Shown here is the representative data from a single trial obtained in agar demonstrating the concentration curve of TMA. And this is a fitted curve obtained from data processing in Walter.The overlap of the data in the fitted curve demonstrates that the curve fitting done by Walter accurately models diffusion in this trial. Here is the table of agar measurements before experimentation in the brain. Here is a summary of measurements taken in a mouse brain slice under the control and experimental conditions.Alpha decreased during a hypoosmolar challenge while tortuosity increased as expected. Alpha overshot the control values in the recovery conditions. And here is the table of agra measurements after experimentation in the brain.The average transport numbers before and after the experiment were within 10%of each other which indicate that the values recorded in the brain were reliable. Once mastered, this procedure can be performed in 14 hours over two days. On day one, ISMs are fabricated and calibrated.On day two, ISMs are re-calibrated and real-time iontophoresis is performed. While attempting this procedure, it's important to first be precise in the spacing of your electrodes. Secondly, it's important to only accept an experiment if the transport number of the iontophoretic microelectrode remains stable throughout the course of the experiment.Following this procedure, which characterizes the diffusion of small cations in the extracellular space, you can go onto to perform other techniques like the integrative optical imaging method that allows you to answer the question of how large micromolecules move in the extracellular space. After watching this video, you should have a good understanding of how to perform the real-time iontophoresis method to quantify brain extracellular space volume and tortuosity in living brain in health and disease. Thank you for watching and good luck with your experiments.

real-time-iontophoresis-with-tetramethylammonium-to-quantify-volume

Research

JoVE Journal

Neuroscience

11.7K Views.  University of Virginia. The overall goal of this procedure is to quantify the volume fraction and tortuosity of the extracellular space in brain tissue. This method answers key questions regarding the relationship between the extracellular space and the physiology and disease. The main advantage of this technique is that it allows the real-time characterization of two important parameters of the extracellular space.Generally, individuals new to this method will struggle because the iontophoretic microelectrod...