This work was supported by National Institutes of Health Grants (R01NS085215 to K.F., T32 GM107000 and F30MH122146 to A.C.). The authors thank Ms. Naoe Watanabe for skillful technical assistance.

All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Massachusetts Medical School. Slice culture preparation, plasmid preparation, and electroporation are also detailed in our previously published methods and can be referred to for additional information10.
1. Slice culture preparation
<ol>
	<li>Prepare mouse organotypic hippocampal slice cultures as previously described11, using postnatal 6- to 7-day old mice of either sex.
	<ol>
		<li>Prepare dissection media for organotypic slice culture consisting of (in mM): 238 sucrose, 2.5 KCl, 1 CaCl2, 4 MgCl2, 26 NaHCO3, 1 NaH2PO4, and 11 glucose in deionized water, then gas with 5% CO2/95% O2 to a pH of 7.4.</li>
		<li>Prepare organotypic slice culture media consisting of: 78.8% (v/v) Minimum Essential Medium Eagle, 20% (v/v) horse serum, 17.9 mM NaHCO3, 26.6 mM glucose, 2 M CaCl2, 2 M MgSO4, 30 mM HEPES, insulin (1 &#181;g/mL), and 0.06 mM ascorbic acid, pH adjusted to 7.3. Adjust the osmolarity to 310-330 osmol using an osmometer.</li>
		<li>Dissect hippocampi out from the whole brain by using two spatulas, and slice (400 &#181;m) using a tissue chopper. Separate slices by using two forceps and transfer to 30 mm cell culture inserts in a 6 well plate filled with culture media (950 &#181;L) underneath the inserts.</li>
	</ol>
	</li>
	<li>Store organotypic slice cultures in a tissue culture incubator (35&#176;C, 5% CO2) and change the slice culture media every two days.</li>
</ol>
2. Plasmid preparation
<ol>
	<li>Prepare the plasmid for the gene of interest.
	<ol>
		<li>Subclone enhanced green fluorescent protein (EGFP) gene into a pCAG vector.</li>
		<li>Purify pCAG-EGFP plasmid with an endotoxin-free purification kit and dissolve in an internal solution that consists of diethyl pyrocarbonate-treated water containing 140 mM K-methanesulfonate, 0.2 mM EGTA, 2 mM MgCl2, and 10 mM HEPES, adjusted to pH 7.3 with KOH (plasmid concentration: 0.1 &#181;g/&#181;L).</li>
	</ol>
	</li>
</ol>
3. Glass pipette preparation
<ol>
	<li>Pull borosilicate glass pipettes (4.5 &#8211; 8 M&#937;) on a micropipette puller (Figure 1A). 
	NOTE: The glass pipettes used for whole-cell patch clamp recordings are ideal for electroporation.</li>
	<li>Bake glass pipettes overnight at 200&#176;C to sterilize.</li>
	<li>Check the size of the pipette tip under a dissection microscope to approximate the electrical resistance.
	<ol>
		<li>Optional:Verify pipette resistance by attaching the pipette to the electroporation electrode and use the micromanipulator to maneuver the pipette tip into filter-sterilized artificial cerebrospinal fluid (aCSF) containing (in mM): 119 NaCl, 2.5 KCl, 0.5 CaCl2, 5 MgCl2, 26 NaHCO3, 1 NaH2PO4 and 11 glucose in deionized water, gassed with 5% CO2/95% O2 to a pH of 7.4. Confirm the actual resistance using the readout on the electroporator. 
		NOTE: The sharper the pipette tip, the larger the electrical resistance. The pipette resistance should be below 10 M&#937;. Glass pipettes with high pipette resistance (Figure 1B) often clog at the tip during repeated electroporation.</li>
	</ol>
	</li>
</ol>
4. Electroporation rig setup
<ol>
	<li>Install the electroporator to a standard whole-cell electrophysiology rig, equipped with an upright microscope mounted on a shifting table with a micromanipulator and peristaltic pump.</li>
	<li>Install the headstage of the electroporator onto a micromanipulator and connect a pair of speakers to the electroporator. Connect the electroporator to a foot pedal which can be used to send a pulse when ready. 
	NOTE: The speakers emit a tone when turned on, which is an indicator of the electrical resistance at the electrode. This makes it possible to determine relative changes in resistance without pulling attention away from the procedure.</li>
</ol>
5. Electroporation preparation
<ol>
	<li>Transfer slice culture inserts from 6 well plates to 3 cm Petri dishes loaded with 900 &#181;L of culture media and store in a tabletop CO2 incubator until ready to perform electroporation.
	<ol>
		<li>Preincubate fresh culture inserts with slice culture media (1 mL) for at least 30 min in a 3.5 cm Petri dish to culture slices after electroporation.</li>
	</ol>
	</li>
	<li>Clean and prepare the rig for electroporation.
	<ol>
		<li>Perfuse the lines with 10% bleach for 5 min to sterilize the tubing and chamber prior to beginning the experiment for the day.</li>
		<li>Perfuse the lines with deionized autoclaved water for at least 30 min to rinse completely.</li>
		<li>Perfuse the lines with filter-sterilized aCSF containing 0.001 mM tetrodotoxin (TTX). 
		NOTE: TTX minimizes cellular toxicity and death due to overexcitation of interneurons10.</li>
	</ol>
	</li>
	<li>Set the electroporator&#8217;s pulse parameters: amplitude of &#8211;5 V, square pulse, train of 500 ms, frequency of 50 Hz, and a pulse width of 500 &#181;s.</li>
	<li>Fill glass pipette with 5 &#181;L of plasmid-containing internal solution.
	<ol>
		<li>Remove any trapped air bubbles from the pipette tip by flicking and gently tapping the tip multiple times.</li>
		<li>Check the tip for damage by visualizing it under a dissection microscope or by repeating step 3.3.1 to check the pipette resistance. 
		NOTE: If the tip is damaged, the glass pipette must be discarded, and this step must be repeated with a new glass pipette previously prepared in step 3.</li>
	</ol>
	</li>
	<li>Securely attach the pipette tip to the electrode and turn the speakers on. Record the readout (the pipette&#8217;s resistance) of the electroporator when the tip has made contact with&#160;the aCSF medium.</li>
	<li>Cut the culture insert membrane using a sharp blade and isolate one slice culture. Carefully transfer the slice culture to the electroporation chamber by using sharp angled forceps and fix its position with a slice anchor.
	<ol>
		<li>Do not keep the slice culture outside of the incubator for more than 30 min at a time to prevent side effects such as changes in neuronal health or function12.</li>
	</ol>
	</li>
</ol>
6. Electroporate cells of interest
<ol>
	<li>Apply positive pressure to the pipette with mouth or by using a 1 mL syringe (0.2 - 0.5 mL pressure) attached to the tubing.</li>
	<li>Use the micromanipulator&#8217;s 3-dimensional knob controls to maneuver the pipette tip near the surface of the slice culture.</li>
	<li>Choose a target cell and approach it, keeping the positive pressure applied until a dimple forms on the cell surface, visible on the microscope.</li>
	<li>Perform pressure cycles.
	<ol>
		<li>Quickly apply mild negative pressure by mouth so that a loose seal forms between the pipette tip and the plasma membrane, indicated visually by the membrane going up into the pipette tip somewhat. Observe an increase (~2.5x the initial resistance) in pipette resistance by listening for an increase in tone coming from the speakers. Quickly re-apply positive pressure so that the dimple re-forms.</li>
		<li>Immediately complete at least two more pressure cycles without pausing, then hold negative pressure for 1 s. 
		NOTE: Pausing between cycles, applying too much pressure, or holding the negative pressure for too long can cause significant cell damage and possibly cause the cell to die during electroporation.</li>
	</ol>
	</li>
	<li>Quickly pulse the electroporator once using the foot pedal when the tone from the speakers reaches a stable apex in pitch, indicating peak electrical resistance. Do not wait at the peak resistance for more than 1 s before sending the pulse. 
	NOTE: We have observed no off-target electroporation when using this protocol10. Only the cells in contact with the glass pipette during pressure cycles were transfected. Positioning the pipette near other neurons does not result in gene transfection.</li>
	<li>Gently retract the pipette approximately 100 &#181;m from the cell without applying pressure.</li>
	<li>Re-apply positive pressure, verifying that the resistance is similar to the recorded readout in step 5.5, then approach the next cell.
	<ol>
		<li>Remove potential clogs, indicated visually or by a significantly increased (&#62;15% higher) pipette resistance after electroporation, by applying positive pressure. 
		NOTE: If there is no visible clog and the resistance is still significantly higher, discard the pipette and use a new one. On an average, a pipette can be used for up to 20 electroporation events if the user is careful10.</li>
	</ol>
	</li>
	<li>After electroporation, transfer the slice culture onto a fresh culture insert, and incubate at 35&#176;C in the incubator for up to 3 days.</li>
</ol>
7. Fixation, staining and imaging of organotypic hippocampal slice cultures
<ol>
	<li>Fix electroporated organotypic slice cultures 2 &#8211; 3 days after transfection with 4% paraformaldehyde and 4% sucrose in 0.01 M phosphate buffered saline (1x PBS) for 1.5 h at room temperature.</li>
	<li>Remove fixative and incubate slices in 30% sucrose in 0.1 M phosphate buffer (1x PB) for 2 h.</li>
	<li>Place slices on a slide glass and freeze them by putting the slide glass on top of crushed dry ice. Thaw the slices at room temperature and transfer them to a 6 well plate filled with 1x PBS.</li>
	<li>Stain the slices with mouse anti-GFP and rabbit anti-RFP antibodies in GDB buffer (0.1% gelatin, 0.3% Triton X-100, 450 mM NaCl, and 32% 1x PB, pH 7.4) for 2 h at room temperature.</li>
	<li>Wash slices with 1x PBS three times at room temperature for 10 min each wash.</li>
	<li>Incubate slices with anti-mouse Alexa 488-conjugated secondary antibody and anti-rabbit Alexa 594-conjugated secondary antibody in GDB buffer for 1 h at room temperature. Incubate slices with DAPI (4 &#181;g/mL) in 1x PBS for 10 min at room temperature.</li>
	<li>Wash slices with 1x PBS three times at room temperature for 3 min each wash.</li>
	<li>Mount slices on glass slides using mounting medium and perform fluorescence imaging.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation.">Single-cell electroporation.</a> Pflugers Archives: European Journal of Physiology. 443 (4), 664-670 (2002).</li><li>Teruel, M. N., Blanpied, T. A., Shen, K., Augustine, G. J., Meyer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+microporation+technique+for+the+transfection+of+cultured+CNS+neurons.">A versatile microporation technique for the transfection of cultured CNS neurons.</a> Journal of Neuroscience Methods. 93 (1), 37-48 (1999).</li><li>Rathenberg, J., Nevian, T., Witzemann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transfection+of+individual+neurons+using+modified+electrophysiology+techniques.">High-efficiency transfection of individual neurons using modified electrophysiology techniques.</a> Journal of Neuroscience Methods. 126 (1), 91-98 (2003).</li><li>Tanaka, M., Yanagawa, Y., Hirashima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+small+interfering+RNA+by+single-cell+electroporation+in+cerebellar+cell+cultures.">Transfer of small interfering RNA by single-cell electroporation in cerebellar cell cultures.</a> Journal of Neuroscience Methods. 178 (1), 80-86 (2009).</li><li>Wiegert, J. S., Gee, C. E., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Electroporation+of+Neurons.">Single-Cell Electroporation of Neurons.</a> Cold Spring Harbor Protocols. 2017 (2), 094904 (2017).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robot+for+high+yield+electrophysiology+and+morphology+of+single+neurons+in+vivo.">A robot for high yield electrophysiology and morphology of single neurons in vivo.</a> Nature Communication. 8, 15604 (2017).</li><li>Steinmeyer, J. D., Yanik, M. 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The authors declare that they have no conflicts of interest.

We describe here an electroporation method that transfects both excitatory and inhibitory neurons with high efficiency and precision. Our optimized electroporation protocol has three innovative breakthroughs to achieve highly efficient gene transfection. Our first modification was to increase pipette size compared with previously published protocols3,5,6. This change enabled us to electroporate many neurons without pipette clogg...

In molecular biology, one of the most important considerations to an investigator is how to deliver a gene of interest into a cell or population of cells to elucidate its function. The different methods of delivery can be categorized as either biological (e.g., a viral vector), chemical (e.g., calcium phosphate or lipid), or physical (e.g., electroporation, microinjection, or biolistics)1,2. Biological methods are highly efficient and can be cell type-specific but are limited by the development of specific genetic tools. Chemical approaches are very powerful in vitro, but transfections are generally random; further, these approaches are mostly reserved for primary cells only. Of the physical approaches, biolistics is the simplest and easiest from a technical point of view, but again produces random transfection results at a relatively low efficiency. For applications which require transfer into specific cells without the need for developing genetic tools, we look toward single-cell electroporation3,4.
Whereas electroporation used to refer only to field electroporation, over the past twenty years, multiple in vitro and in vivo single-cell electroporation protocols have been developed to improve specificity and efficiency5,6,7, demonstrating that electroporation can be used to transfer genes to individual cells and can, therefore, be extremely precise. However, the procedures are technically demanding, time-consuming, and relatively inefficient. Indeed, more recent papers have investigated the feasibility of mechanized electroporation rigs8,9, which can help to eliminate several of these barriers for investigators interested in installing such robotics. But for those looking for simpler means, the problems with electroporation, namely cell death, transfection failure, and pipette clogging, remain a concern.
We recently developed an electroporation method that uses larger-tipped glass pipettes, milder electrical pulse parameters, and a unique pressure cycling step, which generated a much higher transfection efficiency in excitatory neurons than previous methods, and enabled us for the first time to transfect genes in inhibitory interneurons, including somatostatin-expressing inhibitory interneurons in the hippocampal CA1 region of mouse organotypic slice culture10. However, the reliability of this electroporation method in different inhibitory interneuron types and neuronal developmental stages has not been addressed. Here, we demonstrated that this electroporation technique is capable of transfecting genes into both excitatory neurons and different classes of interneurons. Importantly, transfection efficiency was high regardless of days in vitro (DIV) slice culture age tested. This established and user-friendly technique is highly recommended to any investigator interested in using single-cell electroporation for different cell types in the context of in vitro mouse brain tissue.

<table>
	<tbody>
		<tr>
			<td>Plasmid preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Purification Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Organotypic slice culture preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Plates</td>
			<td>GREINER BIO-ONE</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>FST</td>
			<td>#5/45</td>
			<td>Angled dissection forceps for organotypic slice culture preparation</td>
		</tr>
		<tr>
			<td>Flask Filter Unit</td>
			<td>Millipore</td>
			<td>SCHVU02RE</td>
			<td>Filtration and storage of culture media</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>BD C150-UL</td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>TED PELLA, INC.</td>
			<td>10180</td>
			<td>Tissue chopper for organotypic slice culture preparation</td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert, 30 mm</td>
			<td>Millipore</td>
			<td>PIHP03050</td>
			<td>Organotypic slice culture inserts</td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>Precision Systems</td>
			<td>OSMETTE II</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE coated spatulas</td>
			<td>Cole-Parmer</td>
			<td>SK-06369-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14958-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Vacuum Filtration System</td>
			<td>Millipore</td>
			<td>SCGPT01RE</td>
			<td>Filtration and storage of aCSF</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glasses</td>
			<td>Warner Instruments</td>
			<td>640796</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipetter Puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Binder</td>
			<td>BD (E2)</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller Filament</td>
			<td>Sutter Instrument</td>
			<td>FB330B</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single-cell electroporation and fluorescence imaging #1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 mm Falcon Petri Dishes</td>
			<td>BD Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Airtable</td>
			<td>TMC</td>
			<td>63-7512E</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Q Imaging</td>
			<td>Retiga-2000DC</td>
			<td>Camera</td>
		</tr>
		<tr>
			<td>Electroporation System</td>
			<td>Molecular Devices</td>
			<td>Axoporator 800A</td>
			<td>Electroporator</td>
		</tr>
		<tr>
			<td>Fluorescence Illumination System</td>
			<td>Prior</td>
			<td>Lumen 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulator</td>
			<td>Sutter Instrument</td>
			<td>MPC-385</td>
			<td>Manipulator</td>
		</tr>
		<tr>
			<td>Metamorph software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Rainin</td>
			<td>Dynamax, RP-2</td>
			<td>Perfusion pump</td>
		</tr>
		<tr>
			<td>Shifting Table</td>
			<td>Luigs &#38; Neuman</td>
			<td>240 XY</td>
			<td></td>
		</tr>
		<tr>
			<td>Speaker</td>
			<td>Unknown</td>
			<td></td>
			<td>Speakers connected to the electroporator</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ30</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Incubator</td>
			<td>Thermo Scientific</td>
			<td>MIDI 40</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Microscope</td>
			<td>Olympus</td>
			<td>BX61WI</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence imaging #2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell electroporation across different organotypic slice culture of mouse hippocampal excitatory and class-specific inhibitory neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Our single-cell electroporation is capable of precisely delivering genes into visually identified excitatory and inhibitory neurons. We electroporated three different neuronal cell types at three different time points. Parvalbumin (Pv) or vesicular glutamate type 3 (VGT3) expressing neurons were visualized by crossing Pvcre (JAX #008069) or VGT3cre (JAX #018147) lines with TdTomato (a variant of red fluorescent protein) reporter line (Jax #007905), respectively named Pv/TdTomato and VGT3/TdTomato li...

Watch this Scientific Journal Video about Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons at JoVE.com

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

single-cell-electroporation-across-different-organotypic-slice

Research

JoVE Journal

Neuroscience

3.6K Views.  University of Massachusetts Medical School. Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Video: Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...