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 är tacksamma för R. Gutiérrez och A. Morales för deras utmärkta tekniskt bistånd och till LA Weiss för redigering. CGB finansieras av den spanska Ministerio de Ciencia e Innovación (MICINN), FPI-BES-2012-056011. Detta arbete har finansierats genom ett bidrag från BBVA Foundation och SAF2014-58598-JIN (Mineco) M. Navarrete och genom ett bidrag från Ramón Areces Foundation och bidrag SAF2014-52119-R och BFU2014-55738-REDT (från Mineco) till M. Nieto.

Alla djurförsök har godkänts av regionen Madrid Animal Care och användning kommittén, i enlighet med nationell och europeisk lagstiftning (PROEX 118/14, PROEX 331/15). Bibehålla sterila betingelser under förfarandet. 1. I livmodern elektroporering OBS: Detta protokoll för IUE är anpassad från andra som har tidigare publicerats 5, 6, 7. Detta manuskript b...

<ol>
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Detta protokoll beskriver i detalj hur man märka neuroner i somatosensoriska cortex av C75BL / 6-möss för att analysera deras anslutning och deras retbarhet. När det gäller befintliga metoder, visualiserar det diskriminerande aspekter av anslutningsmöjligheter, till exempel antalet axonal förgreningar per neuron, deras exakta topografi, och deras anatomiska läge. Genom att ändra placeringen av elektroderna, är det möjligt att rikta andra neuron populationer, såsom cingulatecortexen (behålla samma vinkel mel...

Utvecklingen av dendritiska och axonal strukturer är en viktig aspekt av kretsreglering i nervsystemet, bland annat i hjärnbarken. Den spelar en avgörande roll under den selektiva ledningar av de olika neuronala subpopulationer. Ett antal nya rapporter har visat att förutom anslutning, är den molekylära mångfalden av nervceller som reflekteras av förvärvet av mycket specifika typer av bränning. Men de mekanismer som bestämmer retbarhet och anslutning av de distinkta neuronala subtyper under utveckling, liksom deras grad av samordning, fortfarande dåligt kända 1, 2. In vivo förlust- och få-av-funktion analyser möjliggör studier av förhållandet mellan uttrycksnivån av specifika gener och deras inflytande i utvecklingen av kretsen. I livmodern elektroporering (IUE) är en teknik som används i stor utsträckning för att studerafunktionen av en gen av intresse i specifika neuronala populationer och att studera de övergripande mönster av deras anslutning. Men för att bestämma morfologiska egenskaperna hos axoner och dendriter i kortikala skikt i levande möss, är det viktigt att märka nervceller glest. En Cre rekombination kombinerat med IUE kan användas för att markera en gles befolkning av nervceller vid en tillräckligt låg densitet för att lösa de prognoser som avges av enskilda celler i de identifierade kortikala lamellerna. Denna metod etiketter ett tillräckligt antal neuroner per cortex att få kvantitativa data efter analys av rimliga mängder electroporated hjärnor (Figur 1). Detta manuskript presenterar en metod för sådan fin analys av anslutning. Den presenterar också en liknande strategi för att analysera, i separata experiment, de elektriska egenskaperna hos nervceller genom att utföra strömkläm inspelningar på grönt fluorescerande protein (GFP) -electroporated celler från akut kortikala skivor. dessa protokoler är mångsidiga och kan användas för att studera i retbarhet och anslutning av neuroner i WT och transgena djur, och även neuroner där vinster och förluster på funktion införs genom ytterligare plasmider under IUE. Även om detta protokoll beskriver elektroporering av möss vid embryonal dag (E) 15,5, kan denna teknik utföras vid alla åldrar mellan E9.5 3 och postnatal dag (P) 2 4. Medan elektroporering i tidiga skeden riktar neuroner och förstadier i thalamus och djupa lagren av hjärnbarken, senare skede elektroporation märken mer ytliga skikt (t.ex. E15.5 IUE mål skikt II-III neuroner). Sammanfattningsvis är kombinationen av IUE med encelliga morfologisk analys och elektro ett användbart verktyg för att klarlägga de molekylära mekanismerna bakom den enorma strukturella och funktionella mångfald av nervceller i nervsystemet.

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			<td>pCAG-Cre</td>
			<td>Addgene</td>
			<td>13775</td>
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			<td>Addgene</td>
			<td>13770</td>
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			<td>Addgene</td>
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			<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>QIAGEN</td>
			<td>12362</td>
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			<td>Pfizer GmbH</td>
			<td>1615 ESP</td>
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			<td>Abbott (Esteve)</td>
			<td>1385 ESP</td>
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			<td>Merial</td>
			<td>2528-ESP</td>
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			<td>Calier</td>
			<td>0682-ESP</td>
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			<td>Meda</td>
			<td>694109.6</td>
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			<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>Sigma -Aldrich</td>
			<td>P4333</td>
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			<td>Scalpel Handle #3 - 12cm</td>
			<td>Fine Science Tools</td>
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			<td>Fine Science Tools</td>
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			<td>Fine Science Tools</td>
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			<td>Fine Science Tools</td>
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			<td>Scissors Mezenbaum-Nelson Curved L=14,5cm</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>Braintree scientific, Inc</td>
			<td>ACS&#160;APL</td>
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			<td>MikRon Precision, Inc.</td>
			<td>205016</td>
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			<td>Sutures - Polysorb 6-0</td>
			<td>Covidien</td>
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			<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>
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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>Becton, Dickinson and Company</td>
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			<td>Sigma -Aldrich</td>
			<td>158127</td>
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			<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>Sigma-Aldrich</td>
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			<td>Electroporator ECM 830&#160;</td>
			<td>BTX Harvard Apparatus</td>
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			<td>Nepagene</td>
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			<td>Microscope&#160;for Fluorescent Imaging - MZ10F</td>
			<td>Leica</td>
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			<td>Matrx</td>
			<td>-</td>
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			<td>Leica</td>
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			<td>Axiovert 200 Microscope</td>
			<td>Zeiss</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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			<td>Air Table</td>
			<td>Newport</td>
			<td>-</td>
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			<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

Nervsystemet består av ett enormt utbud av olika neuronala typer. Dessa neuronala subpopulationer kännetecknas av, bland andra funktioner, deras distinkta dendritiska morfologier, deras specifika mönster av axonal anslutning, och deras selektiva skjut svar. De molekylära och cellulära mekanismer som är ansvariga för dessa aspekter av differentiering under utveckling fortfarande dåligt kända. Här beskriver vi kombinerade protokoll för märkning och karakterisera den strukturella anslutningsmöjligheter och retbarhet kortikala neuroner. Ändring av protokollet i livmodern elektroporering (IUE) kan märkningen av en gles befolkning av nervceller. Detta i sin tur gör det möjligt att identifiering och spårning av dendriter och axoner av enskilda nervceller, exakt karakterisering av den laminära lokaliseringen av axonal prognoser och morfometrisk analys. IUE kan också användas för att undersöka förändringar i retbarhet avvild-typ (WT) eller genetiskt modifierade nervceller genom att kombinera den med helcells-inspelning från akut skivor electroporated hjärnor. Dessa två tekniker bidra till en bättre förståelse för kopplingen av strukturell och funktionell uppkoppling och av de molekylära mekanismer som styr neuronal mångfald under utveckling. Dessa utvecklingsprocesser har viktiga konsekvenser för axonal ledningar, den funktionella mångfalden av nervceller, och biologi kognitiva störningar.

För att karakterisera de morfologiska förändringar av nervceller i detalj och hela utveckling, är det viktigt att märka nervceller glest. En Cre-rekombinas utspätt system möjliggör uttryck av en gen av intresse i en gles befolkning av nervceller, så att endast de nervceller som innehåller detta enzym uttrycka GFP (Figur 1A). Med hjälp av denna strategi är skiktet II-III riktade och märkt av IUE vid E15.5. CAG-DsRed2 vid 1 mikrogram / mikroliter, är co-elekt...

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

Detta manuskript ger protokoll som använder i livmodern elektroporering (IUE) för att beskriva den strukturella anslutning av nervceller vid encelliga nivå och retbarhet av fluorescerande nervceller. Histologi används för att karakterisera dendritiska och axonal prognoser. Hela-cell inspelning i akuta skivor används för att undersöka retbarhet.

I livmodern elektroporering Approaches att studera retbarhet av neuronala Subpopulationer och encelliga Connectivity

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

The overall goal of this in utero electroporation approach is to characterize the structural connectivity and excitability of cortical neurons under normal and experimental conditions. This method can help answer key questions in the developmental neurobiology, such as dissecting the structure and functional connectivity of the brain and the biology of cognitive disorders. The main advantage of this technique is that it allows the marking of a sparse population of neurons and allows you to dress the dendrites and access the individual neurons which leads to more efficient, more fimetic quantitative analysis.Before the injection, prepare ten microliters of DNA mixture per surgery for either single cell labeling or standard patch clamp study. Then, manipulate the embryos gently with fingers to locate the telencephalon. Next, place a prepared borosilicate capillary in a mouth pipette.Pass the tip of the needle through the uterus, avoiding blood vessels, until it reaches the lateral ventricle. After that, slowly inject approximately one microliter of Fast Green colored DNA solution until a large blue spot is observed. A critical step in this procedure is the injection of the DNA.This should be done as gently as possible. Now, place the seven millimeter platinum electrodes laterally on the head of an embryo and apply voltage via the platinum electrodes. In this procedure, cryoprotect the fixed brains in ten milliliters of 30%sucrose in PBS at four degrees Celsius for one to two days until they sink.Then prepare one cubed centimeter aluminum foil cubes and fill the cubes 2/3 or the way with OCT. Afterward, put the brains in the cubes and freeze them on dry ice. Subsequently store the frozen brains at 80 degrees Celsius.To section the brains in the cryostat, place a drop of OCT on the surface of the specimen disc. Peel the aluminum foil from the histology block and position the block at the desired orientation on top of the liquid OCT. Apply firm pressure until the histological block is fixed.Then insert the specimen disc into the specimen head of the cryostat and orient the specimen for sectioning. Then slice 50-100 micrometer thick sections and transfer the floating cryosections to PBS using a fine brush. Subsequently, block the sections for one hour at room temperature with 5%fetal bovine serum in PBS, containing 0.5%Triton X-100.Next, incubate them overnight at four degrees Celsius with 1:500 primary antibody diluted in blocking solution. The next day, wash the sections three times in PBS. Add 1:500 secondary antibody diluted in blocking solution and incubate the sections for one hour at room temperature.Then wash the sections three times in PBS. Afterward, counterstain the sections with dapE in PBS containing 0.5%Triton X-100 for ten minutes. Rinse the sections with PBS and mount them with mounting medium.To reconstruct the neurons, acquire images of the brain sections with high magnification and high resolution. Next, select Tile Scan in the acquisition software to cover the area of interest, spanning all dendrites and axonal processes. Acquire a sufficient number of stacks on the Z axis to avoid information loss.After that, open the image and select the segmented line option from the menu. Draw a line following the structure of the neuron. Subsequently, go to Analyze, Tools, followed by ROI manager, then Add to save the line.Repeat this process for every axon or dendrite of the analyzed neuron. In the ROI Manager menu, press Measure to get the length. Export the measurements to a text file or spreadsheet for analysis.To perform a recording of GFP expressed neuron from a GFP electroporated mouse, place the brain in a chilled culture dish. Cut off the cerebellum with small scissors. Then pick up the brain with a spatula and blot it dry on a paper towel.Glue the ventral caudal plane of the brain on a Vibratome holder. Place the holder on a Vibratome filled with ice cold ACSF and ensure the Vibratome chamber has continuous carbogenation. Following this, obtain acute slices by cutting 300 micrometer coronal sections within 15 minutes.Next, incubate the acute slices at 25 degrees Celsius for at least 60 minutes in ACSF while bubbling with carbogen. After 60 minutes, transfer a slice to the recording chamber using a Pasteur pipette or a small brush. Hold down the slice with a harp and profuse it with ACSF at a rate of two milliliters per minute.To patch a GFP positive neuron, locate the area of interest through the microscope at 10x. Then find a GFP positive cell using the 60x objective. Next, fill the recording electrode with intracellular solution.Subsequently, place a glass pipette in the pipette holder. Afterward, place the pipette tip in the bath and focus on the tip. Once the pipette is in the bath, apply positive pressure through the back pressure control system.Approach the cell of interest under visual guidance while maintaining back pressure in the pipette. Upon the appearance of a small dimple on the cell's surface, release the pressure. At this point, a tight seal with a resistance larger than one giga-ohm may be formed.Otherwise, apply a light negative pressure to facilitate it. While the seal is being formed, bring the holding voltage clamp to 60 millivolts. Once the giga-ohm seal is formed, apply a pulse of suction to rupture the cell membrane and break into the whole cell mode.Once it is in whole cell mode, switch from voltage clamp to current clamp mode and start recording. These images show the delivery of vectors to layer 2/3 neurons by in utero electroporation at embryonic day 15.5, and the coronal sections were prepared at post-natal day 16. The CAG DsRed2 vector was cotransfected as a control.GFP is expressed only in those neurons that also incorporated cre, allowing the recombination of the LoxP sites in the CALNL GFP vector. Here is a high magnification confocal image of the dendritic arbors of another sparsely labeled GFP neuron. This is a pyramidal neuron electroporated with GFP which was observed under bright fields and green fluorescence conditions.Here is a typical, regular spiking response of a CAG-GFP electroporated control neuron at layer 2/3. Once mastered, this technique can be done in 30 minutes for in utero electroporations and half a day for the patch clamp recording if it's performed properly. While attempting this procedure, it's important to remember to do the surgery as fast as possible to reduce the stress of the mother and increase the chances of survival of the pups.Following this procedure, other methods like expression can be performed in order to answer additional questions, like the relationship between expression of specific genes and their influence in the development of the surrogate. After its development, this technique paved the way for researchers in the field of developmental neuroscience to explore connectivity and morphology of neurons in mice. After watching this video, you should have a good understanding of how to use in utero operation to describe the structure and connectivity of neurons at the single cell level and the excitability of fluorescently labeled neurons.

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

I livmodern elektroporation följt av primär Neuronala Kultur för att studera geners funktion i Delmängd av kortikal Neuroner

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

Mus I livmodern Elektroporation: Kontrollerad Spatiotemporal Gene Transfektion

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 för studier av Neuronal Morfogenes:<em&gt; Ex vivo</em&gt; RNAi Elektroporation i embryonal Murine 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 och Genetic Manipulation av dendriter och ryggar i mus hjärnbarken och hippocampus med användning av<em&gt; I utero</em&gt; Elektroporering

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 av Lokalt Transgena råttor genom<em&gt; I livmodern</em&gt; Elektroporering och<em&gt; In vivo</em&gt; Bioluminescence 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

Genmanipulation av Mouse Utveckla Hypothalamus genom<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

Snabb Micro-iontophoresis av glutamat och GABA: Ett användbart verktyg för att undersöka 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

Genetic Manipulation av Cerebellar granulneuroner<em&gt; In Vitro</em&gt; Och<em&gt; In Vivo</em&gt; Att studera neuronal morfologi och migration

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

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 av en Isoelectric hjärntillstånd för att undersöka inverkan av endogen Synaptic aktivitet på neuronala retbarhet<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 ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Tidsberoende ökning av nätverkssvaret till stimulering av neuronala cellkulturer på mikroelektroder

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...