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.

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Research

JoVE Journal

Neuroscience

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 neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

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

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

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

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

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

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

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

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

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

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 this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

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

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

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

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

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

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 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 modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

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 the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

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

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

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

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

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

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

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

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

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 dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

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