Transfection of rat or mouse neurons by biolistics or electroporation

Abstract

Properties of ion channels are affected by the background of the cells in which they are expressed. Thus, it is important for investigators interested in neuronal function to study these proteins in post-mitotic neurons. However, post-mitotic neurons, and many cell lines, are difficult to transfect by standard methods. Here we provide detailed protocols for two different procedures, biolistic and electroporation, which have been used to transfect peripheral sensory neurons from mice or rats with expression constructs of voltage-gated sodium channels. Neurons can be prepared, transfected and currents recorded within 48 h. Using these methods, primary sensory neurons can be transfected with an efficiency of 5-20%, which has permitted studying biophysical properties of sodium channels and their naturally occurring mutants in a native neuronal cell background. Although we have used sodium channels for the examples that we show here, these methods can also be used to study other types of molecules.

Voltage-clamp and current-clamp recordings from mammalian DRG neurons

“…Transfection by electroporation has proved to be the most successful method and the best compromise. Details of how to transfect by electroporation and a more extensive discussion of the technique can be found in Dib-Hajj et al 10 .…”

“…As noted in the protocol on transfection of neurons 10 , most neurons co-express multiple isoforms of sodium channels, making it difficult to discriminate, in isolation, the current produced by the channel isoform of interest. For most types of neurons, this problem can be obviated by transfecting with a tetrodotoxinresistant (TTX-R) version of the channel of interest and then exposing the cells to 250-300 nM TTX during voltage-clamp recording, so as to block the other endogenous TTX-sensitive (TTX-S) sodium channels, leaving the current from the channel of interest in isolation 14 .…”

“…An example is presented by the erythromelalgia Na v 1.7-L858H mutation, which was investigated by our laboratory 9 , as discussed next. Na v 1.7 is normally expressed in both sensory DRG neurons and sympathetic SCG neurons (Table 1 in Dib-Hajj et al 10 ) and it is important to examine the effects of the mutated channel on firing behavior of both types of neuron. Voltage-clamp experiments indicate that the mutation shifts the voltage dependence of activation in a hyperpolarized direction and enhances the current in response to small, slow depolarization (ramp current) 35 .…”

“…Here we describe protocols that can be used to study sodium channels in mammalian DRG neurons, including those transfected by biolistics or electroporation, as described in our accompanying protocol 10 . We describe how to perform whole-cell voltage-clamp recordings from DRG neurons to obtain accurate measurements of the voltage-dependent and kinetic properties of ion channels in their native environment.…”

A Gain-of-Function Mutation in Nav1.6 in a Case of Trigeminal Neuralgia

“…TRG from adult male Sprague-Dawley albino rats (4-6 wks old, Harlan) were isolated and cultured as described previously (24). WT and Met136Val Na V 1.6 R channels were transiently transfected into TRG neurons, along with EGFP, by electroporation with AmaxaTM Basic Neuron SCN Nucleofector Kit (Lonza) using program SCN-BNP6 on Nucleofector IIS (Lonza), as described previously (26). The ratio of sodium channel to GFP constructs was 10:1 (2 μg of WT or Met136Val plus 0.2 μg of GFP).…”