Name: real-time iontophoresis with tetramethylammonium to quantify volume fraction and tortuosity of brain extracellular space
Uploaded: 2017-07-24T13:00:00
Description: Watch this Scientific Journal Video about Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space at JoVE.com

Arbejdet blev støttet af NIH NINDS tilskud R01 NS047557.

Alle dyreprocedurer, der bruges til at opnå vævsprøver, blev godkendt af dyreetikudvalget ved SUNY Downstate Medical Center. 1. Fremstilling af løsninger og udstyr <ol><li> Forbered en 150 mM NaCl-påfyldningsopløsning til ISM&#39;s referencebeholder. Opbevares i en 10 ml sprøjte, der er fastgjort til et 0,22 μm filter (for at fjerne bakterier eller partikler). </li><li> Forbered en 150 mM TMA chlorid (TMA-Cl) -fyldningsopløsning for mikroelektroderne. Opbevares i en 10 ml sprøjte...

<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="Figur 10" class="xfigimg" src="/files/ftp_upload/55755/55755fig10.jpg" /> Figur 10: Ikke-ideelle data, der viser almindelige tekniske problemer. ( A ) Diagrammer af fælles tekniske problemer med iontofores mikroelektroder: Sammenligning af den normale frigivelse af TMA fra en fungerende iontofores mikroelektrode med tre kilder, der viser tekniske problemer. [Høj forstørrelse, a1] Strømmen i en ideel iontoforetisk kilde bæres lige ved TMA frigivelse og chloridoptagels...

<html> Det ekstracellulære rum (ECS) er netværket af indbyrdes forbundne kanaler udvendigt til alle hjerneceller og indeholder både interstitiell væske og ekstracellulær matrix ( figur 1a og figur 1b ). Fordelingen af ​​mange stoffer, der kræves til hjernecellens funktion, herunder næringsstoffer, hormoner og neurotransmittere, forekommer ved diffusion gennem ECS. Ændringer i de fysiske parametre i dette rum, herunder volumen, geometri og ekstracellulær matrix, kan drastisk påvirke diffusion gennem ECS og de lokale ionkoncentrationer, badinghjerneceller, som har en dybtgående indvirkning på hjernecellens funktion 1 , 2 . Real-time iontoforese (RTI) bruges til at bestemme to strukturelle egenskaber i en hjerneområde: volumenfraktion og tortuosity 3 , 4 ,&quot;Xref&quot;&gt; 5. Volumenfraktion ( α ) er andelen af ​​vævsvolumen optaget af ECS ( V ECS ) i forhold til det totale vævsvolumen ( V væv ) i et repræsentativt elementært volumen; <img alt="ligning" src="/files/ftp_upload/55755/55755eq1.jpg" /> Tortuosity ( λ ) er den relative hindring, som et stof møder, når det diffunderer gennem en hjernegruppe sammenlignet med et medium uden forhindringer; <img alt="ligning" src="/files/ftp_upload/55755/55755eq2.jpg" /> Hvor D * (cm 2 s -1 ) er den effektive diffusionskoefficient af stoffet i hjerne og D (cm 2 s -1 ) er stoffets fri diffusionskoefficient i et frit medium, såsom fortyndet agarosegel. I dag er det mest anvendte probesubstans for RTI-metoden er det lille kationtetramethylammonium (TMA). TMA har en molekylvægt på 74 g / mol, dissocieres fuldstændigt i opløsning og har en positiv ladning. RTI undersøgelser med denne ion har vist at α <img alt="ligning" src="/files/ftp_upload/55755/55755eq3.jpg" /> 0,2 og λ <img alt="ligning" src="/files/ftp_upload/55755/55755eq3.jpg" /> 1,6 1 , 2 . Det betyder, at ECS er ca. 20% af det samlede hjernevolumen, og at diffusionen af ​​et lille inert molekyle forekommer ca. 2,5 gange langsommere i ECS end i et medium uden forhindringer 3 . Imidlertid varierer både a og λ med hjernens alder, region og tilstand og i patologiske forhold 1 . Ændringer af disse parametre har været forbundet med hjernens udvikling, aldring, søvn, epilepsi og mange andre grundlæggende processer og hjernens sygdomme 1, 6 . Mens andre teknikker måler α og λ , kan RTI måle både i lokaliserede områder af levende væv i realtid. Af denne grund er RTI blevet et uundværligt redskab til at undersøge ændringer i α og λ under akutte og reversible udfordringer. Teorien til støtte for RTI blev oprindeligt valideret af Nicholson og Phillips, og teknikken er blevet brugt udbredt siden dengang 4 , 7 . Eksperimenter, der anvender RTI, begynder med frigivelsen af ​​en puls af TMA fra en kilde-mikroelektrode ved iontoforese i en fortyndet agarosegel. Når de er udkastet, diffunderer ionerne frit fra punktkilden og vælger fra et potentielt uendeligt antal tilfældige stier ( figur 1d ). Den ændrede koncentration af ionen måles over tid ved anvendelse af en ion-selektiv mikroelektrode (ISM) placeret groft100 μm væk ( figur 1c ). Ændringerne i TMA-koncentrationen er graferet og monteret på en kurve, der gør det muligt at beregne både D og transportnummeret for iontofores mikroelektroden (parametre diskuteret i protokollen). Med disse værdier gentages proceduren i en hjerneområde af interesse for at opnå D * og at beregne både α og λ . Kontrol af iontofores mikroelektroden, dataindsamling, grafing og montering af TMA-koncentrationskurven og beregning af eksperimentelle parametre udføres typisk af programmerne Wanda og Walter, som er specielt designet til dette formål (softwaren og deres manualer er Frit tilgængelig fra forfatterne efter anmodning). Protokollafsnittet i denne gennemgang beskriver de grundlæggende procedurer, der er nødvendige for at designe og udføre RTI i hjerneskiver af gnavere. Teknikken er også blevet brugt i ikke-stangEnt modeller, herunder humane hjerne skiver og in vivo hjernen præparater 1 , 4 , 6 , 8 , 9 . Afsnittet Repræsentative resultater giver både ideelle og ikke-ideelle resultater for at fremhæve nuancer i datatolkning. Endelig dækker diskussionsafsnittet kortfattet fejlfindingsteknikker, begrænsninger af RTI, alternative teknikker, der bruges til at studere ECS, og fremtidige anvendelser af RTI. <img alt="figur 1" class="xfigimg" src="/files/ftp_upload/55755/55755fig1.jpg" /> Figur 1: Diagrammer af diffusion gennem ECS. ( A ) Diagram over ECS: Viser størrelsen og placeringen af ​​ECS i en typisk hjerneafdeling. Gul markerer ECS mellem de grå hjernecelleprocesser. Volumenet af ECS er ca. 20% af den samlede vævsvolumen ( dvs. volumenfraktion = 0.2) under fysiologiske forhold. ( B ) Forstørret diagram af ECS: Fremhæver fysiske parametre, der bidrager til tortuositet, herunder hjernecellegeometri (grå) og ekstracellulær matrix (skitseret som et maske af flerfarvede glycosaminoglycaner og proteoglycaner). ( C ) 3D-diagram over diffusion fra en punktkilde: demonstrerer netbevægelsen af ​​inerte molekyler fra en iontoforetisk kilde til en ISM. Udelukkende diffusionsbarrierer og cellulær optagelse diffunde molekyler udad i alle retninger og producerer en sfærisk koncentrationsfront. ISM kvantificerer den lokale koncentration af de inerte molekyler frigivet fra den iontoforetiske kilde. ( D ) Computer simulering af diffusion i hjerne ECS: [langt til venstre] Setup for Monte Carlo simulering; Grønne kugler repræsenterer hjernecelleprocesser, og det røde kors repræsenterer en punktkilde. Denne opsætning modellerer hjernevævet diagrammet i figur 1a . [Mellembilleder] 3 og6 molekyler udfører tilfældige bevægelser, da de diffunderer gennem hjernens ekstracellulære rum, vist i 2 dimensioner. [Langt til højre] Tilfældige vandringer af mange molekyler frigivet fra punktkilden. Netto bevægelsen af ​​alle molekyler fra punktkilden er udad som vist i figur 1c . De kumulative tilfældige gåture skitserer mellemrummene mellem cellerne ( dvs. ECS, se reference 5 for yderligere forklaring). <a href="http://ecsource.jove.com/files/ftp_upload/55755/55755fig1large.jpg" target="_blank">Klik her for at se en større version af denne figur.</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

Denne gennemgang beskriver de grundlæggende begreber og protokollen til at udføre realtids-iontoforese-metoden (GTI), guldstandarden til at udforske og kvantificere det ekstracellulære rum (ECS) i den levende hjerne. ECS omgiver alle hjerneceller og indeholder både interstitiel væske og ekstracellulær matrix. Transporten af ​​mange stoffer, der kræves til hjernens aktivitet, herunder neurotransmittere, hormoner og næringsstoffer, sker ved diffusion gennem ECS. Ændringer i volumen og geometri i dette rum forekommer under normale hjerneprocesser, som søvn og patologiske tilstande, som iskæmi. Men strukturen og reguleringen af ​​hjerne ECS, især i syge stater, forbliver stort set uudforsket. RTI-metoden måler to fysiske parametre af levende hjerne: volumenfraktion og tortuositet. Volumenfraktion er andelen af ​​vævsvolumen optaget af ECS. Tortuosity er et mål for den relative hindring, et stof møder, når det diffunderer gennem en hjerne reGion i forhold til et medium uden forhindringer. I RTI pulses et inert molekyle fra en kilde-mikroelektrode til hjernens ECS. Da molekyler diffunderer væk fra denne kilde, måles den ændrede koncentration af ionet over tid ved anvendelse af en ion-selektiv mikroelektrode placeret omtrent 100 μm væk. Fra den resulterende diffusionskurve kan både volumenfraktion og tortuositet beregnes. Denne teknik er blevet brugt i hjerneskiver fra flere arter (herunder mennesker) og in vivo for at studere akutte og kroniske ændringer i ECS. I modsætning til andre metoder kan RTI bruges til at undersøge både reversible og irreversible ændringer i hjernens ECS i realtid.

Anvendelsen af ​​RTI-teknikken er demonstreret i et eksperiment designet til at måle ændringerne i a og under en hypoosmolær udfordring ( figur 8 og figur 9 ). Det har tidligere vist sig, at reduktion af osmolariteten af ​​ECS ved vask på hypotonisk ACSF vil medføre et fald i α og en stigning i λ 13 . I...

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

Denne protokol beskriver realtids-iontoforese, en metode, som måler fysiske parametre for hjernens ekstracellulære rum (ECS). Diffusionen af ​​et inert molekyle, der frigives i ECS, anvendes til at beregne ECS-volumenfraktionen og tortuositeten. Det er ideelt til at studere akut reversible ændringer i hjernens ECS.

Real-time iontophorese med tetramethylammonium til kvantificering af volumen fraktion og tortuosity af hjernens ekstracellulære rum

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

Research

JoVE Journal

Neuroscience

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Hurtig Mikro-iontophoresis af glutamat og GABA: et nyttigt redskab til Undersøge Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Real-time Imaging af axonal Transport af Quantum Dot-mærket BDNF i Primary neuroner

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

En<em&gt; Ex vivo</em&gt; Laser-induceret Rygmarvsskader model til opgørelse Mekanismer axon Degeneration i realtid

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Real-time Elektrofysiologi: Brug lukkede kredsløb protokoller til Probe Neuronal Dynamics og Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

En protokol til Administration af Real-Time fMRI Neurofeedback træning

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Analyse til måling af nukleocytoplasmisk transport i realtid inden for motor neuronlignende NSC-34 celler

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

Live-celle måling af lugtstof Receptor aktivering ved hjælp af en Real-time lejr Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Optagelse synaptisk plasticitet i akut hippocampus skiver vedligeholdes i en lille-volumen genanvendelse - Perfusion- og neddykning-type kammer System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Udforskning af Deep Space - afdække anatomi af Periventricular strukturer til at afsløre de laterale ventrikler af den menneskelige hjerne

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In Vivo Mikrodalyse metode til at indsamle store ekstracellulære proteiner fra hjernen interstitiel væske med høj Molekylær vægt Cut-off sonder

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...