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

We are grateful to R. Guti&#233;rrez and A. Morales for their excellent technical assistance and to L. A. Weiss for editing. C.G.B. is funded by the Spanish Ministerio de Ciencia e Innovaci&#243;n (MICINN), FPI-BES-2012-056011. This work was funded by a grant from BBVA Foundation and SAF2014-58598-JIN (MINECO) to M. Navarrete and by a grant from the Ram&#243;n Areces Foundation and grants SAF2014-52119-R and BFU2014-55738-REDT (from MINECO) to M. Nieto.

All animal procedures were approved by the Community of Madrid Animal Care and Use Committee, in compliance with national and European legislation (PROEX 118/14; PROEX 331/15). Maintain sterile conditions during the procedure.
1. In Utero Electroporation
NOTE: This protocol for IUE is adapted from others that have been previously published5,6,7. This manuscript descr...

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This protocol describes in detail how to label neurons of the somatosensory cortex of C75BL/6 mice in order to analyze their connectivity and their excitability. With respect to existing methods, it visualizes discriminating aspects of connectivity, such as the number of axonal branches per neuron, their precise topography, and their anatomical location. By altering the position of the electrodes, it is possible to target other neuron populations, such as the cingulate cortex (keep the same angle between the electrodes a...

The development of dendritic and axonal structures is an important facet of circuit regulation in the nervous system, including in the cerebral cortex. It plays a critical role during the selective wiring of the diverse neuronal subpopulations. A number of recent reports have shown that, in addition to connectivity, the molecular diversity of neurons is reflected by the acquisition of highly specific modes of firing. However, the mechanisms determining the excitability and connectivity of the distinct neuronal subtypes during development, as well as their degree of coordination, are still poorly understood1,2.
In vivo loss- and gain-of-function analyses allow for the study of the relationship between the expression level of specific genes and their influence in the development of the circuit. In utero electroporation (IUE) is a technique widely used to study the function of a gene of interest in specific neuronal populations and to study the overall patterns of their connectivity. However, to determine the morphological characteristics of axons and dendrites in cortical layers in living mice, it is essential to label neurons sparsely. A Cre recombination system combined with IUE can be used to mark a sparse population of neurons at a sufficiently low density to resolve the projections emitted by individual cells of the identified cortical laminas. This method labels a sufficient number of neurons per cortex to obtain quantitative data after the analysis of reasonable numbers of electroporated brains (Figure 1). This manuscript presents a method for such fine analysis of connectivity. It also presents a similar strategy to analyze, in separate experiments, the electrical properties of neurons by performing current-clamp recordings on green fluorescence protein (GFP)-electroporated cells from acute cortical slices. These protocols are versatile and can be applied to the study of the excitability and connectivity of neurons of WT and transgenic animals, and also of neurons in which losses and gains of function are introduced by additional plasmids during IUE.
Although this protocol describes the electroporation of mice at embryonic day (E)15.5, this technique can be performed at any age between E9.53 and postnatal day (P)24. While electroporation at early stages targets neurons and precursors of the thalamus and deep layers of the cortex, later-stage electroporation marks more superficial layers (e.g., E15.5 IUE targets layer II-III neurons). In summary, the combination of IUE with single-cell morphological analysis and electrophysiology is a useful tool to elucidate the molecular mechanisms underlying the enormous structural and functional diversity of neurons in the nervous system.

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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>Carprofen (Rimadyl)</td>
			<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></td>
			<td></td>
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			<td>Sigma -Aldrich</td>
			<td>S0389</td>
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			<td>Paraformaldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>158127</td>
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		<tr>
			<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>
		</tr>
		<tr>
			<td>GFP Tag Polyclonal Antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11122</td>
		</tr>
		<tr>
			<td>Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11008</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270106&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-500ML</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Cryostat - CM 1950</td>
			<td>Leica</td>
			<td>-</td>
		</tr>
		<tr>
			<td>P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
		</tr>
		<tr>
			<td>Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)</td>
			<td>Molecular Devices</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Acquisition software (MultiClamp 700B Amplifier)</td>
			<td>Molecular Devices</td>
			<td>DD1440A</td>
		</tr>
		<tr>
			<td>Motorized Micromanipulator + Rotating Base&#160;</td>
			<td>Sutter Instrument</td>
			<td>MP-225</td>
		</tr>
		<tr>
			<td>Air Table</td>
			<td>Newport</td>
			<td>-</td>
		</tr>
		<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

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

To characterize the morphological changes of neurons in detail and throughout development, it is essential to label neurons sparsely. A Cre-recombinase diluted system allows for the expression of a gene of interest in a sparse population of neurons, so that only those neurons that incorporate this enzyme express GFP (Figure 1A). Using this strategy, layer II-III is targeted and labeled by IUE at E15.5. CAG-DsRed2 at 1 &#181;g/&#181;L, is co-electroporated as a control and...

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

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

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

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

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

 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

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

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

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

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

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