Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) alpha7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates alpha7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells, Xenopus oocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of alpha7 nAChRs. Tyrosine kinase inhibition by genistein decreased alpha7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of alpha7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased alpha7 nAChR-mediated responses, whereas expression of active Src reduced alpha7 nAChR activity. Mutant alpha7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface alpha7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of alpha7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin‐treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR‐associated MuSK, as demonstrated by MuSK phosphorylation. In agrin‐treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the β subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin‐induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.

At the neuromuscular junction, aggregates of acetylcholine receptors (AChRs) are anchored in the muscle membrane by association with rapsyn and other postsynaptic proteins. We have investigated the interactions between the AChR and these proteins in cultured C2 myotubes before and after treatment with agrin, a nerve-derived protein that induces AChRs to cluster. When AChRs were isolated from detergent extracts of untreated C2 myotubes, they were associated with rapsyn and, to a lesser degree, with utrophin, ␤-dystroglycan, MuSK, and src-related kinases, but not with syntrophin. Treatment with agrin increased the association of AChRs with MuSK, a receptor tyrosine kinase that forms part of the agrin receptor complex, without affecting other interactions. Analysis of rapsyndeficient myotubes, which do not form protein clusters in response to agrin, revealed that rapsyn is required for association of the AChR with utrophin and ␤-dystroglycan, and for the agrin-induced increase in association with MuSK, but not for constitutive interactions with MuSK and src-related kinases. In rapsyn Ϫ/Ϫ myotubes, agrin caused normal tyrosine phosphorylation of AChR-associated and total MuSK, whereas phosphorylation of the AChR ␤ subunit, both constitutive and agrininduced, was strongly reduced. These results show first that aneural myotubes contain preassembled AChR protein complexes that may function in the assembly of the postsynaptic apparatus, and second that rapsyn, in addition to its role in AChR phosphorylation, mediates selected protein interactions with the AChR and serves as a link between the AChR and the dystrophin/utrophin glycoprotein complex.

Mice deficient in src and fyn or src and yes move and breathe poorly and die perinatally, consistent with defects in neuromuscular function. Src and Fyn are associated with acetylcholine receptors (AChRs) in muscle cells, and Src and Yes can act downstream of ErbB2, suggesting roles for Src family kinases in signaling pathways regulating neuromuscular synapse formation. We studied neuromuscular synapses in src Ϫ/Ϫ ; fynand src Ϫ/Ϫ ; yes Ϫ/Ϫ mutant mice and found that muscle development, motor axon pathfinding, clustering of postsynaptic proteins, and synapse-specific transcription are normal in these double mutants, showing that these pairs of kinases are not required for early steps in synapse formation. We generated muscle cell lines lacking src and fyn and found that neural agrin and laminin-1 induced normal clustering of AChRs and that agrin induced normal tyrosine phosphorylation of the AChR ␤ subunit in the absence of Src and Fyn. Another Src family member, most likely Yes, was associated with AChRs and phosphorylated by agrin in myotubes lacking Src and Fyn, indicating that Yes may compensate for the loss of Src and Fyn. Nevertheless, PP1 and PP2, inhibitors of Src-class kinases, did not inhibit agrin signaling, suggesting that Src class kinase activity is dispensable for agrin-induced clustering and tyrosine phosphorylation of AChRs. AChR clusters, however, were less stable in myotubes lacking Src and Fyn but not in PP1-or PP2-treated wild-type cells. These data show that the stabilization of agrin-induced AChR clusters requires Src and Fyn and suggest that the adaptor activities, rather than the kinase activities, of these kinases are essential for this stabilization.

The acetylcholine receptor (AChR)-associated protein rapsyn is essential for neuromuscular synapse formation and clustering of AChRs, but its mode of action remains unclear. We have investigated whether agrin, a key nerve-derived synaptogenic factor, influences rapsyn-AChR interactions and how this affects clustering and cytoskeletal linkage of AChRs. By precipitating AChRs and probing for associated rapsyn, we found that in denervated diaphragm rapsyn associates with synaptic as well as with extrasynaptic AChRs showing that rapsyn interacts with unclustered AChRs in vivo. Interestingly, synaptic AChRs are associated with more rapsyn suggesting that clustering of AChRs may require increased interaction with rapsyn. In similar experiments in cultured myotubes, rapsyn interacted with intracellular AChRs and with unclustered AChRs at the cell surface, although surface interactions are much more prominent. Remarkably, agrin induces recruitment of additional rapsyn to surface AChRs and clustering of AChRs independently of the secretory pathway. This agrin-induced increase in rapsyn-AChR interaction strongly correlates with clustering, because staurosporine and herbimycin blocked both the increase and clustering. Conversely, laminin and calcium induced both increased rapsynAChR interaction and AChR clustering. Finally, time course experiments revealed that the agrin-induced increase occurs with AChRs that become cytoskeletally linked, and that this precedes receptor clustering. Thus, we propose that neural agrin controls postsynaptic aggregation of the AChR by enhancing rapsyn interaction with surface AChRs and inducing cytoskeletal anchoring and that this is an important precursor step for AChR clustering.

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

Tyrosine phosphorylation of the ␤ subunit of the acetylcholine receptor (AChR) has been postulated to play a role in AChR clustering during development of the neuromuscular junction. We have investigated the mechanism of this phosphorylation in mammalian C2 myotubes and report that the tyrosine kinase Src binds and phosphorylates glutathione S-transferase fusion proteins containing the N-terminal half of the cytoplasmic loop of the ␤ subunit. No binding occurs to the related kinases Fyn or Yes or to the corresponding regions from the ␥ and ␦ subunits. Furthermore, AChRs affinity-isolated from C2 myotubes using ␣-bungarotoxin-Sepharose were specifically associated with Src and Fyn and had tyrosine-phosphorylated ␤ subunits. We suggest that AChRs are initially phosphorylated by Src and subsequently bind Fyn in a phosphotyrosine-dependent manner. These interactions are likely to play an important role in construction of the specialized postsynaptic membrane during synaptogenesis.Protein-tyrosine phosphorylation is a widely used mechanism for regulating cellular functions, particularly those involving growth or differentiation factors. Several protein-tyrosine kinases are highly expressed in brain (1-3) and are associated with synaptic structures (4), suggesting that they play a general role in synaptic function. At the neuromuscular junction and at its homologous synapse in the electric organ of Torpedo, tyrosine phosphorylation appears to be important for regulating both the function and the distribution of the nicotinic acetylcholine receptor (AChR) 1 during development (5, 6). The AChR is a ligand-gated ion channel with a pseudosymmetric pentameric structure consisting of four homologous subunits in the ratio ␣ 2 ␤␥␦. Each subunit traverses the membrane four times, with a long, cytoplasmic loop between transmembrane domains 3 and 4 (7, 8). In the Torpedo AChR, a single conserved tyrosine residue in the cytoplasmic loop of each of the ␤, ␥, and ␦ subunits is phosphorylated by a kinase activity in the postsynaptic membrane (9). In this tissue, two members of the Src family of tyrosine kinases, Fyn and Fyk, account for a substantial fraction of the total tyrosine kinase activity and have been shown in immunoprecipitation experiments to be associated with tyrosine-phosphorylated AChRs (10, 11). Phosphorylation of the AChR subunits is accompanied by an increase in the rate of rapid desensitization of the receptor by cholinergic ligands, a change that is also produced by phosphorylation of the receptor on serine residues (12, 13).Tyrosine phosphorylation of the AChR appears to play an important role in synaptogenesis. At the mammalian neuromuscular junction, tyrosine phosphorylation in the postsynaptic membrane, possibly of the AChR, increases during the late, post-natal stage of synaptic maturation (14). Tyrosine phosphorylation of the AChR may also be related to one of the earliest steps in synapse formation, the clustering of AChRs in the postsynaptic membrane underlying the nerve terminal (5). Studies on the devel...

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol-rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve-muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout-induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src(-/-);fyn(-/-) myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src-family kinases were strongly enriched in lipid microdomains prepared from wild-type myotubes. Microdomain disruption by cholesterol-sequestering methyl-beta-cyclodextrin disassembled AChR clusters and decreased AChR-rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src(-/-);fyn(-/-) myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol-rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain-association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components.