Name: external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures
Uploaded: 2017-05-07T14:00:00
Description: Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

Forfatterne takker Ofer Feinerman, Fred Wolf, Menahem Segal, Andreas Neef og Eitan Reuveny for svært nyttige diskusjoner. Forfatterne takker Ilan Breskin og Jordi Soriano for utvikling av tidlige versjoner av teknologien. Forfatterne takker Tsvi Tlusty og Jean-Pierre Eckmann for hjelp med de teoretiske begreper. Denne forskningen ble støttet av Minerva Foundation, Ministry of Science and Technology, Israel, og Israel Science Foundation bevilgning 1320-1309 og Bi-National Science Foundation bevilgning 2.008.331.

Etikk Uttalelse: Prosedyrer som involverer dyr behandling ble gjort i samsvar med retningslinjene i Institutional Animal Care og bruk Committee (IACUC) av Weizmann Institute of Science, og den aktuelle israelsk lov. Weizmann Institute er akkreditert av Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Weizmann Institutional Animal Care og bruk komité godkjente denne studien, utført med hippocampalneuroner. 1. Fremstilling av to-dimensjonale (2D) o...

<ol>
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1D mønster er et viktig verktøy som kan brukes for en rekke anvendelser. For eksempel har vi brukt 1D mønstring for å lage logiske porter fra neuronale kulturer 29 og mer nylig for å måle Chronaxie og Rheobase av rotte-hippokampale neuroner 5, og nedbremsing av signalforplantningshastigheten til avfyring aktivitet i Down syndrom hippocampalneuroner forhold til villtype (WT) hippocampalneuroner 27. Den foreslåtte protokoll for 1D mønster er r...

Samspillet mellom nevroner og eksterne elektriske felt har fundamentale konsekvenser samt praktiske seg. Mens det er kjent siden tider av Volta som en ytre påført elektrisk felt kan eksitere vev, blir de mekanismer som er ansvarlig for produksjonen av en resulterende virkningspotensial i neuroner bare nylig begynt å bli avslørt 1, 2, 3, 4. Dette inkluderer å finne svar på spørsmål angående den mekanisme som forårsaker depolarisering av membranpotensialet, rollen av membranegenskaper og ione-kanaler, og til og med regionen i nervecelle som reagerer på det elektriske felt 2, 5. Terapeutisk nerve 6, 7, 8, 9, <supclass = &quot;ekstern referanse&quot;&gt; 10 metoder er spesielt avhengig av denne informasjonen, noe som kan være avgjørende for å målrette de berørte områdene, og for å forstå utfallet av terapien. En slik forståelse kan også bidra i utviklingen av behandlingsprotokoller og nye tilnærminger for stimulering av ulike områder i hjernen. Måling av vekselvirkning inne i hjernen in vivo gir en viktig komponent til denne forståelse, men er hemmet av den unøyaktighet og lav kontrollerbarhet av målinger i kraniet. I motsetning til dette, kan målinger i kulturer enkelt bli utført i høyt volum med høy presisjon, utmerket signal til støyforhold og en høy grad av reproduserbarhet og av kontroll. Ved bruk av kulturer et stort utvalg av nevronale egenskaper for kollektive forbindelsene oppførsel kan bli belyst 11, 12, 13, 14, </sup&gt; 15, 16. Tilsvarende er det godt kontrollert system forventes være svært effektiv i å belyse mekanismen ved hvilken andre stimuleringsmetoder arbeide, for eksempel hvor kanalen åpner seg under optisk stimulering i optogenetically aktive neuroner 17, 18, 19 er ansvarlig for å opprette aksjonspotensial. Her fokuseres det på å beskrive utvikling og forståelse av verktøy som effektivt kan opphisse nervecellen via en ekstern elektrisk felt. I denne beskrivelsen beskriver vi fremstilling av todimensjonale og endimensjonal mønstrede hippocampal kulturer, stimulering ved hjelp av forskjellige konfigurasjoner og orientering av en direkte påtrykt elektrisk felt av badelektroder, og endelig stimulering av to-dimensjonale og mønstrede endimensjonale kulturer ved en tidsvarierende magnetisk felt som induserer en elektrisk felt5, 20, 21.

<table>
	<tbody>
		<tr>
			<td>APV</td>
			<td>Sigma-Aldrich</td>
			<td>A8054</td>
			<td>Disconnect the network. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>B27 supp</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Plating medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Sigma-Aldrich</td>
			<td>14343</td>
			<td>Disconnect the network&#160;. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>Borax (sodium tetraborate decahydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>S9640</td>
			<td>Borate buffer. Mentioned in Section 1.1.2</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Frutarom LTD</td>
			<td>5550710</td>
			<td>Borate buffer. Mentioned in Section 1.1.2</td>
		</tr>
		<tr>
			<td>CaCl2 , 1M</td>
			<td>Fluka&#160;</td>
			<td>21098</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma-Aldrich</td>
			<td>C239</td>
			<td>Disconnect the network&#160;. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>COMSOL</td>
			<td>COMSOL Inc</td>
			<td>Multiphysics 3.5</td>
			<td>Numerical simulation. Mentioned in Section 3.5.2</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>65146</td>
			<td>Plating medium, Extracellular recording solution&#160;. Mentioned in Section 1.1.1&#160;&#160;&#160; 1.5.2</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Cell Cultures. Mentioned in Section 1.2.4&#160;&#160;&#160; 1.2.6</td>
		</tr>
		<tr>
			<td>FCS(FBS)</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td>Plating medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td>Bio Coating. Mentioned in Section 1.2.6</td>
		</tr>
		<tr>
			<td>Fluo4, AM</td>
			<td>Life technologies</td>
			<td>F14201</td>
			<td>Imaging of spontaneous or evoked activity&#160;. Mentioned in Section 1.5.1&#160;&#160;&#160; 1.5.3&#160;&#160;&#160; 1.5.5</td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td>Changing medium. Mentioned in Section 1.4.1</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>GlutaMAX 100X</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>Hepes, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>HI HS&#160;</td>
			<td>BI</td>
			<td>04-124-1A</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1&#160;&#160;&#160; 1.4.1&#160;&#160;&#160; 1.4.2</td>
		</tr>
		<tr>
			<td>KCl,&#160; 3M</td>
			<td>Merck</td>
			<td>1049361000</td>
			<td>Extracellular recording solution. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Laminin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Bio Coating. Mentioned in Section 1.2.6</td>
		</tr>
		<tr>
			<td>MEM x 1</td>
			<td>Gibco</td>
			<td>21090-022</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1&#160;&#160;&#160; 1.4.2</td>
		</tr>
		<tr>
			<td>MgCl2 , 1M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Extracellular recording solution. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>NaCl, 4M</td>
			<td>Bio-Lab</td>
			<td>19030591</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Octadecanethiol</td>
			<td>Sigma-Aldrich</td>
			<td>01858</td>
			<td>Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3</td>
		</tr>
		<tr>
			<td>Pluracare F108 NF Prill</td>
			<td>BASF Corparation&#160;</td>
			<td>50475278</td>
			<td>Bio-Rejection Coating, Bio Coating. Mentioned in Section 1.2.4&#160;&#160;&#160; 1.2.6</td>
		</tr>
		<tr>
			<td>Poly-L-lysine 0.01% solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;P47075</td>
			<td>Promote cell division. Mentioned in Section 1.1.4</td>
		</tr>
		<tr>
			<td>Sucrose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>S1888</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Thiol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>1858</td>
			<td>Bio-Rejection Coating. Mentioned in Section 1.2.3</td>
		</tr>
		<tr>
			<td>URIDINE</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td>Changing medium. Mentioned in Section 1.4.1</td>
		</tr>
		<tr>
			<td>Sputtering machine</td>
			<td>AJA International, Inc</td>
			<td>ATC Orion-5Series&#160;</td>
			<td>coating glass with thin layers of metal. Mentioned in Section 1.2.2</td>
		</tr>
		<tr>
			<td>Pen plotter&#160;</td>
			<td>Hewlett Packard&#160;</td>
			<td>HP 7475A</td>
			<td>Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5</td>
		</tr>
		<tr>
			<td>Electrodes wires&#160;</td>
			<td>A-M Systems, Carlsborg WA</td>
			<td>767000</td>
			<td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1&#160;&#160;&#160; 2.2&#160;&#160;&#160; 2.3&#160;&#160; 2.4.5</td>
		</tr>
		<tr>
			<td>Signal generator</td>
			<td>BKPrecision</td>
			<td>4079</td>
			<td>Shaping of the electric signal. Mentioned in Section 2.3</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Homemade</td>
			<td></td>
			<td>Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Matrix&#160;</td>
			<td>MPS-3005 LK-3&#160;</td>
			<td>Power supply to the sputtering machine. Mentioned in Section 1.2.2.3</td>
		</tr>
		<tr>
			<td>Transcranial magnetic stimulation</td>
			<td>Magstim, Spring Gardens, UK</td>
			<td>Rapid 2</td>
			<td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.1&#160;&#160; 3.3&#160;&#160; 3.4</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Cognis</td>
			<td>Versamid 140</td>
			<td>Casting of homemade coils. Mentioned in Section 3.4</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Shell</td>
			<td>EPON 815&#160;</td>
			<td>Casting of homemade coils. Mentioned in Section 3.4</td>
		</tr>
		<tr>
			<td>Platinum wires 0.005&#39;&#39; thick; A-M Systems,&#160;</td>
			<td>&#160;Carlsborg WA</td>
			<td>&#160;767000</td>
			<td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1</td>
		</tr>
		<tr>
			<td>Circular magnetic coil</td>
			<td>Homemade</td>
			<td></td>
			<td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.3</td>
		</tr>
		<tr>
			<td>WaveXpress SW</td>
			<td>B&#38;K Precision&#160;</td>
			<td></td>
			<td>Waveform editing software. Mentioned in Section 2.1.32</td>
		</tr>
		<tr>
			<td>Xion Ultra 897</td>
			<td>Andor</td>
			<td></td>
			<td>Sensitive EMCCD camera. Mentioned in Section 2.4.4</td>
		</tr>
	</tbody>
</table>

external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures

En nevron vil avfyre ​​et aksjonspotensial når dens membranpotensialet overstiger en viss terskel. I typisk aktiviteten i hjernen, skjer dette som et resultat av kjemiske innganger til sine synapser. Imidlertid kan neuroner også bli eksitert av et pålagt elektrisk felt. Spesielt de siste kliniske applikasjoner aktivere nerveceller ved å skape et elektrisk felt eksternt. Det er derfor av interesse å undersøke hvordan nervecellen reagerer på ytre felt og hva som forårsaker handlingen potensial. Heldigvis er nøyaktig og kontrollert påføring av et eksternt elektrisk felt mulig for embryonale neuronale celler som er skåret ut, dissosierte og dyrket i kulturer. Dette gjør etterforskningen av disse spørsmålene i en svært reproduserbar system. I dette papiret noen av de teknikker som brukes for styrt påføring av ytre elektrisk felt på neuronale kulturer blir gjennomgått. Nettverkene kan enten være endimensjonal, det vil si mønstret i linear former eller tillatt å vokse på hele planet til substratet, og således todimensjonale. Videre kan eksitasjon skapes ved direkte anvendelse av elektrisk felt via elektroder nedsenket i væsken (badelektroder), eller ved å indusere det elektriske feltet ved hjelp av fjern opprettelse av magnetiske pulser.

<html> Protokollen present gir mulighet for enkel mønstring av neuronale kulturer. Når den kombineres med flere metoder vi utviklet for stimulering, gjør det mulig å foreta målinger av noen iboende neuron egenskaper som Chronaxie og Rheobase 5, for å sammenligne egenskapene av friske og syke neuroner 27, for å finne optimale måter å stimulere kulturer som en funksjon av sin struktur og mange flere nye tilnærminger. Noen eksempler e...

Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

Nevrale kulturer er en god modell for å studere nye hjernestimuleringsteknikker via deres effekt på enkeltnerveceller eller en populasjon av nerveceller. Presenteres her er forskjellige metoder for stimulering av mønstrede neuronale kulturer av et elektrisk felt frembragt direkte ved badelektroder eller indusert av et tidsvarierende magnetfelt.

Ekstern Eksitasjon av nerveceller ved hjelp av elektriske og magnetiske felt i en-og to-dimensjonale kulturer

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

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