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 meget nyttige diskussioner. Forfatterne takker Ilan Breskin og Jordi Soriano for at udvikle tidlige versioner af teknologien. Forfatterne takker Tsvi Tlusty og Jean-Pierre Eckmann om hjælp med de teoretiske begreber. Denne forskning blev støttet af Minerva Foundation, Ministeriet for Videnskab og Teknologi, Israel, og af Israel Science Foundation tilskud 1320-1309 og Bi-National Science Foundation tilskud 2.008.331.

Etik erklæring: Procedurer, der involverer håndtering af dyr blev udført i overensstemmelse med retningslinjerne fra Institutional Animal Care og brug Udvalg (IACUC) fra Weizmann Institute of Science, og det passende Israelsk lov. Den Weizmann Institute er akkrediteret af Association for Vurdering og akkreditering af Laboratory Animal Care International (AAALAC). Den Weizmann Institutional Animal Care og brug Udvalg godkendte denne undersøgelse, udført med hippocampusneuroner. 1. Fremstil...

<ol>
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1D mønstring er et vigtigt redskab, der kan anvendes til en lang række applikationer. For eksempel har vi anvendt 1D mønsterdannelse til at skabe logiske porte fra neuronale kulturer 29 og mere for nylig at måle Chronaxie og Rheobase af rotte hippocampale neuroner 5, og opbremsningen i signaludbredelseshastighed af affyringsaktivitet i Down syndrom hippocampale neuroner sammenlignet med den vildtype (WT) hippocampusneuroner 27. Den foreslåede p...

Samspillet mellem neuroner og eksterne elektriske felter har grundlæggende konsekvenser samt praktiske. Medens det er kendt siden de tider af Volta at et eksternt påført elektrisk felt kan excite væv, er de mekanismer, der er ansvarlige for produktionen af en resulterende virkningspotentiale i neuroner først for nylig begyndt at blive bragt i orden 1, 2, 3, 4. Dette omfatter at finde svar på spørgsmål vedrørende den mekanisme, der forårsager depolarisering af membranpotentiale, rolle membranegenskaber og ionkanaler, og selv region i neuron, der imødekommer det elektriske felt 2, 5. Terapeutisk neurostimulation 6, 7, 8, 9, <supclass = &quot;xref&quot;&gt; 10 metoder er særligt afhængig af disse oplysninger, som kan være afgørende for målretning de ramte områder og for forståelse af resultatet af terapien. En sådan forståelse kan også hjælpe med at udvikle behandlingsprotokoller og nye tilgange til stimulering af forskellige områder i hjernen. Måling af interaktionen inden in vivo hjernen tilføjer en vigtig komponent til denne forståelse, men hæmmes af unøjagtighed og ringe styrbarhed af målinger inden kraniet. I modsætning hertil kan målinger i kulturer let udføres i høj volumen med høj præcision, fremragende signal til støj ydeevne og en høj grad af reproducerbarhed og kontrol. Anvendelse af kulturer en lang række af neuronale egenskaber af kollektive netværk adfærd kan belyses 11, 12, 13, 14, </sup&gt; 15, 16. Ligeledes forventes denne velkontrolleret system højeffektive til at belyse den mekanisme, hvorved andre stimuleringsmetoder arbejde, for eksempel hvordan kanalåbning under optisk stimulering i optogenetically aktive neuroner 17, 18, 19 er ansvarlig for at skabe aktionspotentiale. Her er fokus på at beskrive den udvikling og forståelse for værktøjer, der effektivt kan ophidse neuron via et eksternt elektrisk felt. I denne artikel beskriver vi fremstilling af todimensionale og endimensional mønstrede hippocampuskulturer, stimulering ved hjælp af forskellige konfigurationer og orientering af en direkte påtrykt elektrisk felt ved bad elektroder og endelig stimulering af todimensional og mønstrede endimensionale kulturer ved en tidsvarierende magnetfelt, som inducerer et 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 neuron vil fyre et aktionspotentiale når dens membranpotentialet overstiger en vis tærskel. I typiske aktivitet i hjernen, dette sker som et resultat af kemiske input til sine synapser. Dog kan neuroner også ophidset af en pålagt elektrisk felt. Især de seneste kliniske anvendelser aktiverer neuroner ved at skabe et elektrisk felt eksternt. Det er derfor af interesse at undersøge, hvordan den neuron reagerer på det eksterne område og hvad der forårsager aktionspotentialet. Heldigvis, præcis og kontrolleret påføring af et ydre elektrisk felt er muligt for embryonale neuronale celler, som er udskåret, dissocieret og dyrkes i kulturer. Dette giver mulighed for undersøgelse af disse spørgsmål i en meget reproducerbar system. I dette papir nogle af de teknikker, der anvendes til styret påføring af ydre elektrisk felt på neuronale kulturer gennemgås. Netværkene kan være enten en dimensionel, dvs. mønstrede i linear former eller lov til at vokse på hele substratets plan, og således todimensionale. Desuden kan excitationen skabes ved direkte anvendelse af elektrisk felt via elektroder nedsænket i fluidet (bad elektroder) eller ved at inducere det elektriske felt ved hjælp af fjernbetjeningen skabelse af magnetiske impulser.

<html> Protokollen præsenteret tillader let mønsterdannelse af neuronale kulturer. Når den er kombineret med flere metoder vi udviklet til stimulering, det gør det muligt at foretage målinger af nogle iboende neuron egenskaber såsom Chronaxie og Rheobase 5, for at sammenligne egenskaberne af raske og syge neuroner 27, at finde optimale måder at stimulere kulturer som en funktion af deres struktur og mange flere nye tilgange. Nogle eks...

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

Neuronale kulturer er en god model til at studere nye brain stimulation teknikker via deres effekt på enkelte neuroner eller en population af neuroner. Her præsenteres forskellige metoder til stimulering af mønstrede neuronale kulturer ved et elektrisk felt, der frembringes direkte af bad elektroder eller fremkaldt af en tidsvarierende magnetfelt.

Ekstern ekspression af neuroner ved hjælp af elektriske og magnetiske felter i en- og todimensionelle 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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