A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu(5) receptor in cerebellar granule cells

We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a r ate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transf ast to other transfection reagents, we found this compound to be the most e fficient. GFP-transfected mouse cerebellar granule cells displayed normal w hole-cell voltage-sensitive and unitary big K+ channel currents. We also us ed this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding f or the metabotropic glutamate receptor type 5 (mGlu(5) receptor). Ninety pe rcent of the cells expressing GFP also expressed mGlu(5) receptor. Though n eurones were best transfected one day after plating, they still expressed b oth GFP and mGlu(5) receptor proteins 2 weeks after plating, i.e. after ful l differentiation. A functional test of the expressed mGlu(5) receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu(5) receptor induced single big K+ channel activity, as it was the case for the native mGlu(1) receptor. This indicated that the transfected mGlu(5) receptor plas mid was functionally expressed and that both mGlu(1) and mGlu(5) receptors may share common coupling mechanisms to big K+ channels in neurones. (C) 19 99 Elsevier Science Ltd. All rights reserved.