Intracellular chloride concentration influences the GABAA receptor subunit composition

GABAA receptors (GABAARs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABAAR are largely unknown. The intracellular concentration of chloride ([Cl−]i), the main ion permeating through GABAARs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl−]i could modulate the sequential expression of specific GABAARs subtypes in primary cerebellar neurons. We show that [Cl−]i regulates the expression of α3-1 and δ-containing GABAA receptors, responsible for phasic and tonic inhibition, respectively. Our findings highlight the role of [Cl−]i in tuning the strength of GABAergic responses by acting as an intracellular messenger

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

Efficient and long-lasting transfection of primary neurons is an essential tool to address 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 (shRNA) vectors, using magnetofection, into rat hippocampal neurons (E18/19) cultured for several hours to 21 days in vitro. This protocol even allows for 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. The 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 as done by our three laboratories are detailed

Molecular architecture of potassium chloride co-transporter KCC2

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