Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed. INTRODUCTIONEvery gene transfer method aims to achieve high transfection efficiency, low toxicity and long-lasting expression. These goals are especially difficult to achieve for postmitotic primary neurons due to their high sensitivity to any microenvironmental change 1-5 . Many currently available transfection methods do not yield sufficiently good gene expression results to allow functional gene analysis in differentiated adherent neurons in vitro. A recent report provides a useful overview of seven different gene delivery methods, including two often-used transfection techniques (calcium-phosphate and lipofection), but excluding magnetofection 6 . Magnetofection is a technique that can be used to reliably and efficiently introduce DNA into a variety of cell types 7 . Therefore, we have invested in designing a reliable, magnetofection-based protocol to express DNA or shRNA constructs in rat hippocampal neurons (embryonic day (E)18/19) cultured for several hours to 21 d in vitro (DIV). After transfection, neurons expressing exogenous proteins can develop normally in culture for 5-10 d. We have determined the optimum parameters for transfection efficiency such as age of cells at transfection time point, expression levels during the time after transfection and parameters for doubletransfection. This protocol shows low toxicity, uses a combination of a transfection reagent, CombiMag, and magnetic force and shows a sufficiently high efficiency in neurons (ca. 5%) to allow single-cell analysis.We present a core protocol and illustrate the critical steps, modifications and applications carried out by our three laboratories (Medina, Fuhrer and Fritschy) [8][9][10] . This protocol allows single and double transfections of DNA vectors at different time points after plating. First, we present a detailed characterization of magnetofection parameters such as age of neurons, time course of protein expression after magnetofection, and transfection efficiency in pyramidal neurons and GABAergic interneurons. Three specific