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

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; fu....

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

1. 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 ....

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 li.......

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 protocols^3,5,6. This change enabled us to electroporate many neurons without pipette clogg.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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