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

Gephyrin is a multifunctional protein contributing to molybdenum cofactor (Moco) synthesis and postsynaptic clustering of glycine and GABAA receptors. It contains three major functional domains (G-C-E) and forms cytosolic aggregates and postsynaptic clusters by unknown mechanisms. Here, structural determinants of gephyrin aggregation and clustering were investigated by neuronal transfection of EGFP-tagged deletion and mutant gephyrin constructs. EGFP-gephyrin formed postsynaptic clusters containing endogenous gephyrin and GABAA-receptors. Isolated GC- or E-domains failed to aggregate and exerted dominant-negative effects on endogenous gephyrin clustering. A construct interfering with intermolecular E-domain dimerization readily auto-aggregated but showed impaired postsynaptic clustering. Finally, two mutant constructs with substitution of vertebrate-specific E-domain sequences with homologue bacterial MoeA sequences uncovered a region crucial for gephyrin clustering. One construct failed to aggregate, but retained Moco biosynthesis capacity, demonstrating the independence of gephyrin enzymatic activity and aggregation. Reinserting two vertebrate-specific residues restored gephyrin aggregation and increased formation of postsynaptic clusters containing GABAA receptors at the expense of PSD-95 clusters – a marker of glutamatergic synapses. These results underscore the key role of specific E-domain regions distinct from the known dimerization interface for controlling gephyrin aggregation and postsynaptic clustering and suggest that formation of gephyrin clusters influences the homeostatic balance between inhibitory and excitatory synapses.

Type 2 glycine transporter (GlyT2) mediates intracellular glycine transport and is expressed selectively in glycinergic neurons. Expression of GlyT2 gene promoter-driven enhanced green fluorescent protein (eGFP) in BAC transgenic mice allows selective visualization of glycinergic neurons by fluorescence microscopy. Here, we show that cerebellar interneuron precursors identified by the transcription factor Pax2, including gamma-aminobutyric acid (GABA)ergic interneurons of the molecular layer (ML; basket and stellate cells), transiently express GlyT2-eGFP during development. In contrast, expression of endogenous GlyT2 is restricted to glycinergic Golgi cells. Comparison with knock-in mice expressing eGFP in GABAergic neurons [glutamic acid decarboxylase (GAD)67-eGFP] revealed that GlyT2-eGFP expression often precedes GAD67-eGFP and is therefore a marker of immature GABAergic interneurons. In the internal granule cell layer, GABAergic Golgi cells differentiated shortly after birth, prior to glycinergic Golgi cells. In the ML, GlyT2-eGFP-positive precursor cells migrated until the boundary with the external granule cell layer, forming an inside-out maturation gradient that determined the final position of interneurons in the ML. After migration, GlyT2-eGFP gradually disappeared, while interneurons differentiated morphologically and became immunoreactive for parvalbumin, the GABA(A) receptor alpha1 subunit, and the K(+)Cl(-) exchanger KCC2 (K(+)Cl(-) cotransporter type 2). Numerous presumptive GABAergic synaptic terminals were seen on immature ML interneurons as early as P4, preceding the expression of these neurochemical markers. These results suggest that GABAergic synaptogenesis marks the onset of differentiation of basket and stellate cells in the mouse cerebellum, and that GABAergic synaptic function might contribute to the differentiation of interneurons in the cerebellar cortex.

Formation of chemical synapses in the central nervous system is a highly regulated, multistep process that requires bidirectional communication across the synaptic cleft. Neurotransmitter receptors, scaffolding proteins, and signaling molecules need to be concentrated in the postsynaptic density, a specialized membrane microdomain apposed to the active zone of presynaptic terminals, where transmitter release occurs. This precise, synapse-specific matching implicates that sorting and targeting mechanisms exist for the molecular constituents of different types of synapses to ensure correct formation of neuronal circuits in the brain. There is considerable evidence from in vitro and in vivo studies that neurotransmitter signaling is not required for proper sorting during synapse formation, whereas active neurotransmission is essential for long-term synapse maintenance. Here, the authors review recent studies on the role of cell adhesion molecules in synaptogenesis and on possible mechanisms ensuring correct matching of pre- and postsynaptic sites. They discuss the role of neurotransmitter receptors and scaffolding proteins in these processes, focusing on fundamental differences between synapse formation during development and synapse maintenance and plasticity in adulthood.

Aims/hypothesis: Some cyclooxygenase-2 (COX2, also known as prostaglandin-endoperoxide synthase 2 [PTGS2]) inhibitors have been shown to increase insulin sensitivity in man or induce hypoglycaemic episodes when overconsumed or taken in combination with oral hypoglycaemic drugs. These side-effects and their impact on patients are not always recognised in routine clinical practice. We investigated whether these side-effects of COX2 (PTGS2) inhibitors result from stimulation of the glucose transport system in skeletal muscle cells. Materials and methods: L6 myotube cultures were used to study effects of COX2 (PTGS2) inhibitors on the glucose transport system and their relationship to PTGS2 expression, insulin action and AMP-activated protein kinase α (AMPKα) activity. Results: The inhibitors niflumic acid, nimesulide and rofecoxib increased the rate of hexose uptake in L6 myotubes in the absence of insulin and in a dose-and time-dependent manner. They did this by increasing the total cell content of member 4 of the solute carrier family 2 (SCLC2A4, previously known as glucose transporter 4 [GLUT4]) (but not SCLC2A1 [previously known as GLUT1]) mRNA and protein and the amount of it in the plasma membrane. AMPKα was not involved in the latter effect since the inhibitors did not activate it. In addition, none of the inhibitors modulated the rate of hexose transport in vascular endothelial and smooth muscle cells expressing PTGS2 and SCLC2A1. Prostaglandin-endoperoxide synthase 1 (also known as cyclooxygenase 1) inhibitors (acetylsalicylic acid and indomethacin) did not alter the rate of hexose uptake and SCLC2A4 subcellular distribution in L6 myotubes. Conclusions/interpretation: This study suggests that certain COX2 (PTGS2) inhibitors can alter glucose homeostasis in vivo by stimulating glucose uptake in skeletal muscles that express PTGS2.