Name: in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity
Uploaded: 2017-02-15T13:00:00
Description: Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

Vi er taknemmelige for R. Gutiérrez og A. Morales for deres fremragende teknisk bistand og til LA Weiss til redigering. CGB er finansieret af den spanske Ministerio de Ciencia e Innovación (MICINN), FPI-BES-2012-056011. Dette arbejde blev finansieret af en bevilling fra BBVA Foundation og SAF2014-58598-JIN (MINECO) til M. Navarrete og ved en bevilling fra Ramón Areces Foundation og tilskud SAF2014-52119-R og BFU2014-55738-REDT (fra MINECO) til M. Nieto.

Alle dyreforsøg blev godkendt af Madrid Animal Care og brug Udvalg i overensstemmelse med national og europæisk lovgivning (PROEX 118/14; PROEX 331/15). Oprethold sterile forhold under proceduren. 1. I Utero Elektroporation BEMÆRK: Denne protokol for IUE er tilpasset fra andre, der tidligere er blevet offentliggjort 5, 6, 7. Dette håndskrift beskriver en proto...

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Denne protokol beskriver i detaljer, hvordan at mærke neuroner i somatosensoriske cortex af C75BL / 6-mus for at analysere deres tilslutning og deres ophidselse. Med hensyn til de eksisterende metoder, det visualiserer diskriminerende aspekter af tilslutningsmuligheder, såsom antallet af axonale grene pr neuron, deres præcise topografi, og deres anatomiske placering. Ved at ændre positionen af elektroderne, er det muligt at målrette andre neuron populationer, såsom cingulate cortex (holde den samme vinkel mellem e...

Udviklingen af ​​dendritiske og axonal strukturer er en vigtig facet af kredsløb regulering i nervesystemet, herunder i hjernebarken. Det spiller en afgørende rolle under den selektive ledningsføring af de forskellige neuronale delpopulationer. En række nylige rapporter har vist, at der ud over forbindelsen, er den molekylære mangfoldighed af neuroner afspejles af købet af yderst specifikke former for affyring. Imidlertid er de mekanismer, der bestemmer ophidselse og tilslutning af de distinkte neuronale undertyper under udvikling, samt deres grad af koordinering, stadig dårligt forstået 1, 2. In vivo tabs- og få af funktion analyser muliggør studiet af forholdet mellem ekspressionsniveauet af specifikke gener og deres indflydelse på udviklingen af kredsløbet. In utero elektroporation (IUE) er en teknik meget anvendt til at studerefunktionen af ​​et gen af ​​interesse i specifikke neuronale populationer og for at undersøge den overordnede mønstre i deres forbindelse. Men for at bestemme de morfologiske karakteristika af axoner og dendritter i kortikale lag i levende mus, er det vigtigt at mærke neuroner tyndt. En Cre rekombination system kombineret med IUE kan anvendes til at markere en sparsom population af neuroner ved en tilstrækkeligt lav densitet til at løse fremspringene udsendes af individuelle celler i de identificerede kortikale lamellerne. Denne metode etiketter et tilstrækkeligt antal neuroner pr cortex at få kvantitative data efter analyse af rimelige antal elektroporerede hjerner (figur 1). Dette håndskrift præsenterer en metode til en sådan fin analyse af tilslutningsmuligheder. Den indeholder også en lignende strategi til at analysere, i separate eksperimenter, de elektriske egenskaber af neuroner ved at udføre strøm-clamp optagelser på grønt fluorescensprotein (GFP) -electroporated celler fra akut kortikale skiver. Disse proprotokoller er alsidige og kan anvendes til studiet af ophidselse og tilslutning af neuroner i WT og transgene dyr, og også af neuroner, hvor tab og gevinster på funktion er indført ved yderligere plasmider under IUE. Selvom denne protokol beskriver elektroporering af mus ved embryonisk dag (E) 15,5, kan denne teknik udføres på alle alderstrin mellem E9.5 3 og postnatal dag (P) 2 4. Mens elektroporering ved tidlige stadier målrettet neuroner og forstadier for thalamus og dybe lag af cortex, senere stadier elektroporation varemærker mere overfladiske lag (f.eks E15.5 IUE mål lag II-III neuroner). Sammenfattende kombinationen af ​​IUE med enkelt-celle morfologisk analyse og elektrofysiologi er et nyttigt værktøj til at belyse de molekylære mekanismer bag den enorme strukturelle og funktionelle alsidighed neuroner i nervesystemet.

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			<td>pCAG-Cre</td>
			<td>Addgene</td>
			<td>13775</td>
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			<td>pCALNL-GFP</td>
			<td>Addgene</td>
			<td>13770</td>
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			<td>pCAG-DsRed2</td>
			<td>Addgene</td>
			<td>15777</td>
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			<td>pCAG-GFP</td>
			<td>Addgene</td>
			<td>11150</td>
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			<td>Fast Green</td>
			<td>Carl Roth</td>
			<td>301.1</td>
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			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
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			<td>Pfizer GmbH</td>
			<td>1615 ESP</td>
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			<td>Isoflurane (IsoFlo)</td>
			<td>Abbott (Esteve)</td>
			<td>1385 ESP</td>
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			<td>Ketamine (Imalgene)</td>
			<td>Merial</td>
			<td>2528-ESP</td>
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			<td>Xylazine (Xilagesic)</td>
			<td>Calier</td>
			<td>0682-ESP</td>
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			<td>Povidone Iodine</td>
			<td>Meda</td>
			<td>694109.6</td>
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			<td>Eye Ointment (Lipolac)</td>
			<td>Angelini</td>
			<td>65.277</td>
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			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Gibco by Life Technologies</td>
			<td>24020-091</td>
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			<td>Penicillin-Streptomycin</td>
			<td>Sigma -Aldrich</td>
			<td>P4333</td>
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			<td>Scalpel Handle #3 - 12cm</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
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			<td>Scalpel Blades #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
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			<td>Adson Forceps-Serrated - Straight 12 cm</td>
			<td>Fine Science Tools</td>
			<td>1106-12</td>
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			<td>Hardened Fine Scissors - Straight &#160; 11 cm</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
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			<td>Scissors Mezenbaum-Nelson Curved L=14,5cm</td>
			<td>Teleflex</td>
			<td>PO143281</td>
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			<td>Thin curved tips - Style 7 Dumoxel</td>
			<td>Dumont</td>
			<td>0303-7-PO</td>
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			<td>Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
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			<td>Mathieu Needle Holder - Serrated</td>
			<td>Fine Science Tools</td>
			<td>12010-14</td>
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			<td>AutoClip Applier</td>
			<td>Braintree scientific, Inc</td>
			<td>ACS&#160;APL</td>
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			<td>9mm AutoClips</td>
			<td>MikRon Precision, Inc.</td>
			<td>205016</td>
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			<td>Sutures - Polysorb 6-0</td>
			<td>Covidien</td>
			<td>UL-101</td>
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			<td>Electric Razor&#160;</td>
			<td>Panasonic</td>
			<td>ER 240</td>
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			<td>Borosilicate glass capillaries (100mm, 1.0/0.58 Outer/Inner diameter)</td>
			<td>Wold Precision Instrument Inc.</td>
			<td>1B100F-4</td>
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			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma -Aldrich</td>
			<td>A5177-5EA</td>
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			<td>Gauze (Aposan)</td>
			<td>Laboratorios Indas, S.A.U.</td>
			<td>C.N. 482232.8</td>
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			<td>Cotton Swabs (Star Cott)</td>
			<td>Albasa</td>
			<td>-</td>
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			<td>Needle 25G (BD Microlance 3)</td>
			<td>Becton, Dickinson and Company</td>
			<td>300600</td>
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			<td>Sigma -Aldrich</td>
			<td>S0389</td>
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			<td>Sigma -Aldrich</td>
			<td>158127</td>
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			<td>OCT Compound</td>
			<td>Sakura</td>
			<td>4583</td>
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			<td>Tissue Culture Dish 100 x 20 mm</td>
			<td>Falcon</td>
			<td>353003</td>
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			<td>GFP Tag Polyclonal Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11122</td>
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			<td>Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11008</td>
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			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542&#160;</td>
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			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270106&#160;</td>
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			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
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			<td>Electroporator ECM 830&#160;</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0002</td>
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			<td>Platinum electrodes 650P 7 mm</td>
			<td>Nepagene</td>
			<td>CUY650P7</td>
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			<td>Microscope&#160;for Fluorescent Imaging - MZ10F</td>
			<td>Leica</td>
			<td>-</td>
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			<td>VIP 3000 Isofluorane Vaporizer</td>
			<td>Matrx</td>
			<td>-</td>
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			<td>TCS-SP5 Laser Scanning System</td>
			<td>Leica</td>
			<td>-</td>
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			<td>Axiovert 200 Microscope</td>
			<td>Zeiss</td>
			<td>-</td>
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			<td>Cryostat - CM 1950</td>
			<td>Leica</td>
			<td>-</td>
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			<td>P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
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			<td>Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)</td>
			<td>Molecular Devices</td>
			<td>-</td>
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			<td>Acquisition software (MultiClamp 700B Amplifier)</td>
			<td>Molecular Devices</td>
			<td>DD1440A</td>
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			<td>Motorized Micromanipulator + Rotating Base&#160;</td>
			<td>Sutter Instrument</td>
			<td>MP-225</td>
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		<tr>
			<td>Air Table</td>
			<td>Newport</td>
			<td>-</td>
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		<tr>
			<td>Miniature Peristaltic Pumps</td>
			<td>WPI</td>
			<td>-</td>
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in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity

Nervesystemet består af et enormt udvalg af forskellige neuronale typer. Disse neuronale delpopulationer er karakteriseret ved, blandt andre funktioner, deres distinkte dendritiske morfologier, deres specifikke mønstre af axonal tilslutningsmuligheder, og deres selektive fyring svar. De molekylære og cellulære mekanismer, der er ansvarlige for disse aspekter af differentiering under udviklingen stadig dårligt forstået. Her beskriver vi kombinerede protokoller for mærkning og karakterisere strukturelle tilslutningsmuligheder og ophidselse af kortikale neuroner. Ændring af i livmoderen elektroporation (IUE) protokol tillader mærkning af en sparsom befolkning af neuroner. Dette på sin side muliggør identifikation og sporing af dendritter og axoner individuelle neuroner, den præcise karakterisering af det laminare placering af axonale projektioner og morfometrisk analyse. IUE kan også anvendes til at undersøge ændringer i ophidselse afvildtype (WT) eller genetisk modificerede neuroner ved at kombinere det med hel-celle optagelse fra akutte skiver af elektroporerede hjerner. Disse to teknikker bidrage til en bedre forståelse af koblingen af ​​strukturelle og funktionelle forbindelse og af de molekylære mekanismer, der styrer neuronal mangfoldighed under udviklingen. Disse udviklingsprocesser har vigtige konsekvenser for axonal ledninger, den funktionelle mangfoldighed af neuroner, og biologi kognitive forstyrrelser.

At karakterisere de morfologiske ændringer af neuroner i detaljer og hele udvikling, er det vigtigt at mærke neuroner tyndt. En Cre-rekombinase fortyndet system giver mulighed for ekspression af et gen af interesse i en sparsom population af neuroner, således at kun de neuroner, der indeholder dette enzym udtrykke GFP (figur 1A). Under anvendelse af denne strategi, er lag II-III målrettet og mærket ved IUE ved E15.5. CAG-DsRed2 ved 1 ng / nl, er coelektroporeret som...

Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

Dette håndskrift indeholder protokoller, der bruger i livmoderen elektroporation (IUE) til at beskrive den strukturelle konnektivitet af neuroner på encellede niveau og ophidselse af fluorescens-mærkede neuroner. Histologi anvendes til at karakterisere dendritiske og axonale projektioner. Helcelle-optagelse i akutte skiver anvendes til at undersøge ophidselse.

I Utero Elektroporation Approaches to Undersøgelse ophidselse af neuronale subpopulationer og Single-celle Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Research

JoVE Journal

Neuroscience

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Metoder til Undersøgelse af Neuronal morfogenese:<em&gt; Ex vivo</em&gt; RNAi Elektroporation i embryoniske murin Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualisering og genetisk manipulering af dendritter og udløberne i mus cerebrale cortex og hippocampus under anvendelse<em&gt; I utero</em&gt; Elektroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generering af Topisk Transgene rotter ved<em&gt; In Utero</em&gt; Elektroporation og<em&gt; In vivo</em&gt; Bioluminescens Screening

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetisk Manipulation af musen Udvikling Hypothalamus gennem<em&gt; In Utero</em&gt; Elektroporering

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetisk manipulation af Cerebellær granuleneuroner<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; At studere Neuronal morfologi og Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Udarbejdelse af neuronale Co-kulturer med Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

Induktion af en Isoelectric Brain State til at undersøge betydningen af ​​endogene Synaptic aktivitet på neuronexcitabilitet<em&gt; In vivo</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...