High-efficiency transfection of individual neurons using modified electrophysiology techniques

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Abstract

Transfection of cells by electroporation is a widely used and efficient method. Recently, it has been shown that single neurons in brain slice cultures can be transfected using micropipettes loaded with plasmid DNA expression constructs. However, the transfection efficiencies were very low. Routine employment of single-cell electroporation (SCE) for transfection of neurons requires high and reliable efficiency together with good cell survival. Here, we describe the modification of electrophysiology techniques for SCE leading to very simple and efficient (up to 80%) transfection of neurons in organotypic rat hippocampus and mouse cortex slice cultures. Electroporation-mediated transfection was visualized in real-time by two-photon microscopy at the cellular level using fluorescently labeled oligonucleotides and plasmid DNA. Small oligonucleotides enter the cell immediately during pulse application while large plasmids remain localized for more than 10 min at the cell membrane before they enter the cell by an, as yet, unknown process. SCE does not affect the electrophysiology of transgene-expressing cells. Expression of several neuronal green fluorescent protein-tagged proteins demonstrates that the method can be employed to analyze subcellular trafficking and targeting in single living neurons.

Introduction

Electroporation is a method to insert DNA, RNA, dyes, peptides and proteins into cells (Neumann et al., 1999). Compared to other methods, such as viral gene transfer and ‘gene gun’ biolistics potential toxic or mechanical damage is avoided. Furthermore, using these methods it is nearly impossible to transfect selectively a single cell within a brain slice culture.

Electroporation occurs when the voltage applied across the membrane exceeds the dielectric breakdown voltage of the membrane (∼0.2–1.5 V), leading to the formation of minute pores (20–120 nm diameter) through which molecules can enter the cell. The opening of the pores occurs in the microsecond range whereas their closure requires tens to hundreds of milliseconds (Teruel et al., 1997). Traditionally short-duration dielectric field pulses are applied between two relatively large plate electrodes (Teruel et al., 1999). However, this approach does not allow the transfection of single cells. For single-cell electroporation (SCE) a micropipette is filled with a DNA solution and connected to the cathode of a voltage pulse generator through a thin silver wire, which is in contact with the DNA solution. The ground electrode connected to the anode is in contact with the medium/buffer reservoir. Under visual guidance the pipette can be positioned with a micromanipulator into neural tissue. This method can be utilized to transfect neurons in organotypic hippocampal slice cultures, and even in living animals (Haas et al., 2001). However, there were low transfection rates. High transfection rates (up to ∼84%) could be achieved with optimized electroporation parameters and high-power visualization of the cell and the pipette using the lens epithelial cell line α-NT4 (Rae and Levis, 2002).

Direct visualization of the electroporation process is a prerequisite to improve the transfection efficiency for electroporated neurons. Fluoresceine-conjugated oligonucleotides and fluorescent dyes attached to DNA are tools to directly observe the electroporation process before, during and after pulse application. The green fluorescent dye TOTO-1 was employed to investigate electrically mediated gene delivery into cultured cells (Golzio et al., 2002). We analyzed factors which significantly enhance the transfection efficiency of individual neurons using SCE by visualization of the transfer processes by real-time imaging at high magnification. Our results show that electroporation does not affect the electrophysiological properties of SCE-transfected cells. Several green fluorescent protein (GFP)-tagged neuronal proteins were successfully expressed by this method. In addition to the transfection of hippocampal neurons we also show very efficient transfection of cortical neurons.

Section snippets

Organotypic brain slice cultures

Organotypic brain slice cultures were prepared as previously described (Stoppini et al., 1991). Hippocampi were isolated from 7-day-old Wistar rats, cortex was isolated from 7-day-old C57Bl/6 mice. Three hundred and fifty micrometer slices were cut using a McIlwain Tissue Chopper. The slices were cultured on Millicell CM culture plate inserts with 5-mm low wall (Millipore, Billerica, USA). The culture medium consisted of 50% MEM (Gibco Invitrogen, Karlsruhe, Germany), 25% EBSS (Gibco), 25%

Real-time SCE visualization of a fluoresceine-conjugated oligonucleotide

To increase the efficiency of SCE it is essential to understand what happens during the pulse application, and whether pulse application is correlated with oligonucleotide or plasmid DNA transfer through the cell membrane. Electroporation was therefore visualized using an electrophysiology setup equipped with a two-photon laser-scanning microscope. The pipette was filled with a fluoresceine-conjugated oligonucleotide and electrical pulses were applied as described in Section 2. A series of

Discussion

SCE, i.e. electroporation with a DNA filled micropipette, has been used for the transfection of individual neurons of organotypic brain slice cultures (Haas et al., 2001). However, the reported transfection efficiencies were too low for routine experiments. To improve SCE, we visualized in real-time the electroporation process to define optimal electroporation conditions.

The most critical and important factor for successful molecule transfer into the cell is the direct contact between the

Acknowledgements

We thank Dr R. Sprengel for the GluRB-GFP expression construct and Dr T. Kuner for the synaptophysin-GFP expression construct. Furthermore, we thank Dr Aron Jaffe for critically reading the manuscript.

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These authors contributed equally to this paper.

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