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

David G. Keener; Amy Cheung; Kensuke Futai

doi:10.3791/61662

JoVE Journal > Neuroscience

Neurosciences

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

Published: October 06, 2020

doi:

10.3791/61662

David G. Keener*^1,2, Amy Cheung*^1,3, Kensuke Futai¹

¹Brudnick Neuropsychiatric Research Institute, Department of Neurobiology,University of Massachusetts Medical School, ²Interdisciplinary Graduate Program,University of Massachusetts Medical School, ³UMMS MD/PhD Program,University of Massachusetts Medical School

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

Prepare mouse organotypic hippocampal slice cultures as previously described¹¹, using postnatal 6- to 7-day old mice of either sex.
1. 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.
2. 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 µg/mL), and 0.06 mM ascorbic acid, pH adjusted to 7.3. Adjust the osmolarity to 310-330 osmol using an osmometer.
3. Dissect hippocampi out from the whole brain by using two spatulas, and slice (400 µm) 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 µL) underneath the inserts.
Store organotypic slice cultures in a tissue culture incubator (35°C, 5% CO₂) and change the slice culture media every two days.

2. Plasmid preparation

Prepare the plasmid for the gene of interest.
1. Subclone enhanced green fluorescent protein (EGFP) gene into a pCAG vector.
2. 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 µg/µL).

3. Glass pipette preparation

Pull borosilicate glass pipettes (4.5 – 8 MΩ) on a micropipette puller (Figure 1A).
NOTE: The glass pipettes used for whole-cell patch clamp recordings are ideal for electroporation.
Bake glass pipettes overnight at 200°C to sterilize.
Check the size of the pipette tip under a dissection microscope to approximate the electrical resistance.
1. 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 MΩ. Glass pipettes with high pipette resistance (Figure 1B) often clog at the tip during repeated electroporation.

4. Electroporation rig setup

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.
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.

5. Electroporation preparation

Transfer slice culture inserts from 6 well plates to 3 cm Petri dishes loaded with 900 µL of culture media and store in a tabletop CO₂ incubator until ready to perform electroporation.
1. 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.
Clean and prepare the rig for electroporation.
1. Perfuse the lines with 10% bleach for 5 min to sterilize the tubing and chamber prior to beginning the experiment for the day.
2. Perfuse the lines with deionized autoclaved water for at least 30 min to rinse completely.
3. 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.
Set the electroporator’s pulse parameters: amplitude of –5 V, square pulse, train of 500 ms, frequency of 50 Hz, and a pulse width of 500 µs.
Fill glass pipette with 5 µL of plasmid-containing internal solution.
1. Remove any trapped air bubbles from the pipette tip by flicking and gently tapping the tip multiple times.
2. 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.
Securely attach the pipette tip to the electrode and turn the speakers on. Record the readout (the pipette’s resistance) of the electroporator when the tip has made contact with the aCSF medium.
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.
1. 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.

6. Electroporate cells of interest

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.
Use the micromanipulator’s 3-dimensional knob controls to maneuver the pipette tip near the surface of the slice culture.
Choose a target cell and approach it, keeping the positive pressure applied until a dimple forms on the cell surface, visible on the microscope.
Perform pressure cycles.
1. 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.
2. 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.
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.
Gently retract the pipette approximately 100 µm from the cell without applying pressure.
Re-apply positive pressure, verifying that the resistance is similar to the recorded readout in step 5.5, then approach the next cell.
1. Remove potential clogs, indicated visually or by a significantly increased (>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.
After electroporation, transfer the slice culture onto a fresh culture insert, and incubate at 35°C in the incubator for up to 3 days.

7. Fixation, staining and imaging of organotypic hippocampal slice cultures

Fix electroporated organotypic slice cultures 2 – 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.
Remove fixative and incubate slices in 30% sucrose in 0.1 M phosphate buffer (1x PB) for 2 h.
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.
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.
Wash slices with 1x PBS three times at room temperature for 10 min each wash.
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 µg/mL) in 1x PBS for 10 min at room temperature.
Wash slices with 1x PBS three times at room temperature for 3 min each wash.
Mount slices on glass slides using mounting medium and perform fluorescence imaging.

Representative Results

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 Pv^cre (JAX #008069) or VGT3^cre (JAX #018147) lines with TdTomato (a variant of red fluorescent protein) reporter line (Jax #007905), respectively named Pv/TdTomato and VGT3/TdTomato lines. Organotypic slice cultures were prepared from C57BL/6J, Pv/TdTomato, and VGT3/TdTomato mice.

First, electroporation was performed in CA1 pyramidal neurons (Py) at either 7, 14, or 21 days in vitro (DIV). EGFP was transfected into 5-20 pyramidal neurons in the hippocampal CA1 area across these slice culture ages (Figure 2B-D). CA1 pyramidal neurons were identified using differential interference contrast (DIC). To demonstrate the anatomical distribution and morphological differences between pyramidal neurons and inhibitory interneurons in slice culture, CA1 pyramidal neurons were electroporated with EGFP in a DIV7 Pv/TdTomato mouse and nuclear counterstaining was performed to display the distinct location of EGFP-positive neurons in the CA1 pyramidal cell layer (Figure 2A).

Next, this protocol was also applied to TdTomato-positive Pv and VGT3 interneurons. EGFP electroporation was carried out in 1-10 fluorescently labeled interneurons. TdTomato-positive Pv (Figure 3) and VGT3 (Figure 4) neurons were successfully electroporated in the hippocampal CA1 area. Interestingly, transfection of the EGFP gene of interest in all of these inhibitory neuronal types was not significantly affected by DIV and did not differ with the transfection efficiency (~80%) observed in CA1 pyramidal neurons (Figure 5).

Figure 1: Two representative glass pipette images.
(A) Display lower resistance (6.5 MΩ) pipettes, used in this protocol, and (B) higher resistance (10.4 MΩ) pipettes typical for electroporation protocols. Please click here to view a larger version of this figure.

Figure 2: Organotypic hippocampal slice cultures were electroporated with EGFP (green) at three different time points.
(A) Representative organotypic hippocampal slice culture in a DIV7 Pv/TdTomato mouse. CA1 pyramidal neurons were electroporated with EGFP (green, white arrowheads) and showed no overlap with TdTomato (TdT)-positive Pv interneurons (red, yellow arrowheads). DAPI nuclear counterstaining (blue) was performed. DG: dentate gyrus. (B-D) CA1 pyramidal neurons were electroporated with EGFP at three different time points: (B) DIV7, (C) DIV14 and (D) DIV21. Organotypic slice cultures were fixed with 4% sucrose, 4% paraformaldehyde/ 1x PBS and imaged without further sectioning. Top left, low magnification images of the hippocampal CA1 area. Arrowheads represent individual CA1 pyramidal neurons targeted for electroporation. Transfected neurons with yellow arrowheads are zoomed in the bottom panels. White arrowheads signify additional electroporated neurons outside of the high magnification view. Top right, low magnification images of superimposed (Sup) fluorescent and Nomarski images. Scale bars: 500, 50, 100, 20 μm respectively. Please click here to view a larger version of this figure.

Figure 3: Pv/TdTomato organotypic hippocampal slice cultures were electroporated with EGFP (green) at three different time points.
(A) DIV7, (B) DIV14 and (C) DIV21. Overlap with Pv-labeled TdTomato (TdT)-positive cells (red) was observed in the hippocampal CA1 pyramidal cell layer and oriens. In the low magnification insets (top row), yellow arrowheads represent individual Pv interneurons targeted for electroporation. White arrowheads signify additional TdTomato-positive Pv interneurons electroporated outside of the high magnification view. Scale bars: 50, 20 μm. Please click here to view a larger version of this figure.

Figure 4: VGT3/TdTomato organotypic hippocampal slice cultures were electroporated with EGFP (green) at three different time points.
(A) DIV7, (B) DIV14 and (C) DIV21. Overlap with VGT3-labeled TdTomato (TdT)-positive cells (red) was observed in the hippocampal CA1 pyramidal cell layer and oriens. In the low magnification insets (top row), yellow arrowheads represent individual VGT3 interneurons targeted for electroporation. White arrowheads signify additional TdTomato-positive VGT3 interneurons electroporated outside of the high magnification view. Scale bars: 50, 20 μm. Please click here to view a larger version of this figure.

Figure 5: Comparable levels of transfection efficiency in CA1 pyramidal neurons, Pv/TdTomato, and VGT3/TdTomato interneurons at three different in vitro slice culture ages.
Summary bar graphs of three different slice culture ages: DIV7 (left), DIV14 (middle) and DIV21 (right). Each symbol represents the transfection efficiency obtained from one organotypic slice culture CA1 Py: DIV7 (12 slice cultures from 2 mice), DIV14 (8/2), DIV21 (8/2); Pv: DIV7 (5/2), DIV14 (6/2), DIV21 (6/2); VGT3: DIV7 (6/2), DIV14 (7/2), DIV21 (6/2). One-way ANOVA; n.s. (not significant). Data shown are mean ± SEM. Please click here to view a larger version of this figure.

Discussion

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 clogging. In addition, it is possible that the lower resistance pipettes allow for the use of milder electrical pulse parameters compared with previous methods while still achieving the desired result³. Next, repeated pressure cycling before electroporation markedly reduced cell death¹⁰. We often observed that with the application of a single pulse of negative pressure before electroporating, plasma membrane stuck to the inside of the pipette tip, damaging the cell. The pressure cycles helped far less plasma membrane stick to the pipette, which improved cell survival and recovery during the procedure¹⁰. Finally, the addition of TTX into aCSF greatly improved the success of electroporation in inhibitory neurons¹⁰. We consider that electroporation can cause lethal cell overexcitation which can be prevented by TTX. Above all, these critical improvements offer remarkably high success rates in electroporation to both excitatory and inhibitory neurons (Figure 5). Although we found no electrophysiological abnormalities in our neurons after transfection with this method¹⁰ in the targeted cells, it has been reported that local pH changes during electroporation reduce cell viability and the optimization of electroporation parameters can be relatively difficult compared with other gene transfection methods¹³^,¹⁴. Therefore, it is important to consider unforeseen side effects of electroporation and to re-optimize the electrical parameters as needed to achieve a high transfection efficiency for any specific application.

Microinjection technology has also been used as a powerful approach to deliver transgenes to cells²^,¹⁵^,¹⁶. However, this approach generally produces a lower yield of transfection and requires a high level of skill. In contrast, our method can be used with relative ease and at minimal cost to laboratories that routinely perform whole-cell electrophysiology studies.

Recently, we showed that multiple genes can be transfected into both excitatory and somatostatin expressing inhibitory neurons with no side effects on electrophysiological properties¹⁰. In this study, we demonstrate that this electroporation method is highly efficient in CA1 pyramidal neurons (Figure 2) and Pv (Figure 3) and VGT3 (Figure 4) inhibitory interneurons. Moreover, this electroporation technique allows us to transfect genes of interest with an ~80% success rate regardless of cell type or number of days in vitro (Figure 5). Although the technique has only been tested in vitro, organotypic hippocampal slice cultures at the same DIV time points we tested have been shown to follow age-matched in vivo synaptic morphology and activity, as seen in acutely prepared hippocampal slices¹⁷. Moreover, as we have only tested this method in hippocampal neurons, it is possible that there could be unanticipated challenges in applying this technique to neurons in other brain regions. The method invites exploration of genes during organotypic slice culture development in which synaptic transmission and density, among other properties, change over time.

This protocol is an improvement over previously established ones in that it is simultaneously low-cost, less technically challenging, and more efficient in generating single-neuron gene transfection⁵^,⁶^,⁷^,⁸^,⁹. It also appears to be an improvement over previous methods in terms of lower rates of cell damage or death, as we observed that ~80% of cells were successfully transfected and healthy after the procedure. This method, therefore, provides a new opportunity to examine the roles of genes in multiple neuronal cell types using fluorescent mouse models. Future studies using this method can focus on protein-protein interactions between cells to examine specific molecular or physiological functions, including trans-synaptic protein interactions.

Divulgations

The authors have nothing to disclose.

Acknowledgements

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.

Materials

Plasmid preparation
Plasmid Purification Kit	Qiagen	12362

Organotypic slice culture preparation
6 Well Plates	GREINER BIO-ONE	657160
Dumont #5/45 Forceps	FST	#5/45	Angled dissection forceps for organotypic slice culture preparation
Flask Filter Unit	Millipore	SCHVU02RE	Filtration and storage of culture media
Incubator	Binder	BD C150-UL
McIlwain Tissue Chopper	TED PELLA, INC.	10180	Tissue chopper for organotypic slice culture preparation
Millicell Cell Culture Insert, 30 mm	Millipore	PIHP03050	Organotypic slice culture inserts
Osmometer	Precision Systems	OSMETTE II
PTFE coated spatulas	Cole-Parmer	SK-06369-11
Scissors	FST	14958-09
Stereo Microscope	Olympus	SZ61
Sterile Vacuum Filtration System	Millipore	SCGPT01RE	Filtration and storage of aCSF

Electrode preparation
Capillary Glasses	Warner Instruments	640796
Micropipetter Puller	Sutter Instrument	P-1000	Puller
Oven	Binder	BD (E2)
Puller Filament	Sutter Instrument	FB330B	Puller

Single-cell electroporation and fluorescence imaging #1
3.5 mm Falcon Petri Dishes	BD Falcon	353001
Airtable	TMC	63-7512E
CCD camera	Q Imaging	Retiga-2000DC	Camera
Electroporation System	Molecular Devices	Axoporator 800A	Electroporator
Fluorescence Illumination System	Prior	Lumen 200
Manipulator	Sutter Instrument	MPC-385	Manipulator
Metamorph software	Molecular Devices		Image acquisition
Peristaltic Pump	Rainin	Dynamax, RP-2	Perfusion pump
Shifting Table	Luigs & Neuman	240 XY
Speaker	Unknown		Speakers connected to the electroporator
Stereo Microscope	Olympus	SZ30
Table Top Incubator	Thermo Scientific	MIDI 40
Upright Microscope	Olympus	BX61WI

Fluorescence imaging #2
All-in-One Fluorescence Microscope	Keyence	BZ-X710

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Keener, D. G., Cheung, A., Futai, K. Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons. J. Vis. Exp. (164), e61662, doi:10.3791/61662 (2020).