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</su...

<ol>
	<li>Washbourne, P., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+gene+transfer+into+neurons.">Techniques for gene transfer into neurons.</a> Current Opinion in Neurobiology. 12 (5), 566-573 (2002).</li><li>Capecchi, M. R. <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+transformation+by+direct+microinjection+of+DNA+into+cultured+mammalian+cells.">High efficiency transformation by direct microinjection of DNA into cultured mammalian cells.</a> Cell. 22 (2), 479-488 (1980).</li><li>Rae, J. L., Levis, R. 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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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+single-cell+manipulation+in+brain+tissue.">High-throughput single-cell manipulation in brain tissue.</a> PLoS One. 7 (4), 35603 (2012).</li><li>Keener, D. G., Cheung, A., Futai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+method+for+single-cell+electroporation+in+mouse+organotypic+hippocampal+slice+culture.">A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture.</a> Journal of Neuroscience Methods. 337, 108632 (2020).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Ibata, K., Sun, Q., Turrigiano, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+synaptic+scaling+induced+by+changes+in+postsynaptic+firing.">Rapid synaptic scaling induced by changes in postsynaptic firing.</a> Neuron. 57 (6), 819-826 (2008).</li><li>Li, Y., 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=Electroporation+on+microchips:+the+harmful+effects+of+pH+changes+and+scaling+down.">Electroporation on microchips: the harmful effects of pH changes and scaling down.</a> Science Reports. 5, 17817 (2015).</li><li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> Journal of Neuroscience. 30 (18), 6171-6177 (2010).</li><li>Taverna, E., Haffner, C., Pepperkok, R., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+manipulate+the+fate+of+single+neural+stem+cells+in+tissue.">A new approach to manipulate the fate of single neural stem cells in tissue.</a> Nature Neurosciences. 15 (2), 329-337 (2011).</li><li>Zhang, Y., Yu, L. C. <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+microinjection+technology+in+cell+biology.">Single-cell microinjection technology in cell biology.</a> Bioessays. 30 (6), 606-610 (2008).</li><li>De Simoni, A., Griesinger, C. B., Edwards, F. 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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 ...

Gene transfection is a vital approach for neurobiology studies. Our electroporation technique enables us to transfect gene of interest interneurons in organotypic hippocampus slice culture with no detectable side effects. This protocol has a much higher transfection efficiency compared to other single cell protocols, but it is still relatively inexpensive and simple to perform.This technique will be useful for researchers interested in understanding specific molecular and physiological functions of neurons, including cell autonomous mechanisms and transsynaptic protein interactions. To prepare slice cultures for electroporation, transfer the culture inserts of interest into individual three centimeter petri dishes loaded with 900 microliters of culture medium, and place the cultures in a tabletop carbon dioxide incubator. Next, pre-incubate fresh culture inserts with one milliliter of slice culture medium per insert in a 3.5 centimeter petri dish for at least 30 minutes, and sterilize the lines of the electroporation rig with 10%bleach for five minutes.At the end of the perfusion, rinse the lines with the ionized autoclaved water for at least 30 minutes before perfusing with filter sterilized ACSF containing 0.001 millimolar tetrodotoxin. Set the electroporator pulse to an amplitude of minus five volts, a square pulse, a train of 500 milliseconds, a frequency of 50 hertz, and a pulse width of 500 microseconds. Fill a glass pipette with five microliters of plasmid-containing internal solution, and gently flick and tap the tip multiple times to remove any trapped bubbles.Use a dissection microscope to confirm that the tip is not damaged, and securely attach the pipette tip to the electrode. When the tip makes contact with the ACSF, record the pipette resistance readout of the electroporator. To isolate a slice culture, cut the culture insert membrane with a sharp blade, and use sharp angled forceps to carefully transfer the slice culture to the electroporation chamber.Then fix the culture position with a slice anchor. For electroporation of the cells of interest, apply positive pressure to the pipette by mouth, and use the three-dimensional knob controls to maneuver the pipette tip to near the surface of the slice culture. Viewing the culture with the microscope, approach the target cell with the tip, keeping the positive pressure applied until a dimple forms on the cell surface.Upon visualization of the dimple, quickly apply mild negative pressure by mouth so that a loose seal forms between the pipette tip and the plasma membrane. The membrane will slightly enter the pipette. An approximately 2.5x increase in the initial resistance of the pipette should be observed, as indicated by an increase in tone from the speakers.Quickly reapply positive pressure so that the dimple reforms. Then immediately complete at least two more pressure cycles as just demonstrated without pausing. After the last pressure pulse, hold the negative pressure for one second.When tone from the speakers reaches a stable apex in pitch, use the foot pedal to quickly pulse the electroporator. After the electroporation, gently retract the pipette approximately 100 microns from the cell without applying pressure, and reapply positive pressure, verifying that the resistance is similar to the baseline readout before approaching the next cell. When all of the cells of interest have been electroporated, transfer the slice culture into one of the prepared fresh culture inserts, and place the insert at 35 degrees Celsius for up to three days.In this representative experiment, EGFP was transfected into five to 20 pyramidal neurons in the hippocampal CA1 area across seven, 14, and 21 days in vitro slice culture ages. Parvalbumin and vesicular glutamate type 3 interneurons can also be successfully electroporated within the hippocampal CA1 area. Interestingly, transfection is not significantly affected by the day in vitro slice culture age, and does not differ with the transfection efficiency observed in CA1 pyramidal neurons.Neurons are very fragile. You must be extremely cautious and gentle when approaching and pressure cycling the cells and retracting the pipette tip to causing serious damage. After electroporation, the cells can be subjected to various experimental analysis, including electrophysiology and imaging.

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Research

JoVE Journal

Neuroscience

3.5K visualizações.  University of Massachusetts Medical School. Gene transfection is a vital approach for neurobiology studies. Our electroporation technique enables us to transfect gene of interest interneurons in organotypic hippocampus slice culture with no detectable side effects. This protocol has a much higher transfection efficiency compared to other single cell protocols, but it is still relatively inexpensive and simple to perform.This technique will be useful for researchers interested in understanding specific molecular and physiological...