Excitatory (glutamatergic) synapses in the mammalian brain are usually situated on dendritic spines, a postsynaptic microcompartment that also harbors organelles involved in protein synthesis, membrane trafficking, and calcium metabolism. The postsynaptic membrane contains a high concentration of glutamate receptors, associated signaling proteins, and cytoskeletal elements, all assembled by a variety of scaffold proteins into an organized structure called the postsynaptic density (PSD). A complex machine made of hundreds of distinct proteins, the PSD dynamically changes its structure and composition during development and in response to synaptic activity. The molecular size of the PSD and the stoichiometry of many major constituents have been recently measured. The structures of some intact PSD proteins, as well as the spatial arrangement of several proteins within the PSD, have been determined at low resolution by electron microscopy. On the basis of such studies, a more quantitative and geometrically realistic view of PSD architecture is emerging.

The structure and function of presynaptic and postsynaptic components of the synapse are highly coordinated. How such coordination is achieved and the molecules involved in this process have not been clarified. Several lines of evidence suggest that presynaptic functionalities are regulated by retrograde mechanisms from the postsynaptic side. We therefore sought postsynaptic mechanisms responsible for trans-synaptic regulation of presynaptic function at excitatory synapses in rat hippocampal CA1 pyramidal neurons. We show here that the postsynaptic complex of scaffolding protein PSD-95 and neuroligin can modulate the release probability of transmitter vesicles at synapse in a retrograde way, resulting in altered presynaptic short-term plasticity. Presynaptic beta-neurexin serves as a likely presynaptic mediator of this effect. Our results indicate that trans-synaptic protein-protein interactions can link postsynaptic and presynaptic function.

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. We report here that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with mu2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2mu and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre- and postsynaptic membrane proteins.

Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons–a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.

Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.

Neural activity regulates dendrite and synapse development, but the underlying molecular mechanisms are unclear. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an important sensor of synaptic activity, and the scaffold protein liprinalpha1 is involved in pre- and postsynaptic maturation. Here we show that synaptic activity can suppress liprinalpha1 protein level by two pathways: CaMKII-mediated degradation and the ubiquitin-proteasome system. In hippocampal neurons, liprinalpha1 mutants that are immune to CaMKII degradation impair dendrite arborization, reduce spine and synapse number, and inhibit dendritic targeting of receptor tyrosine phosphatase LAR, which is important for dendrite development. Thus, regulated degradation of liprinalpha1 is important for proper LAR receptor distribution, and could provide a mechanism for localized control of dendrite and synapse morphogenesis by activity and CaMKII.

The scaffold protein PSD-95 promotes the maturation and strengthening of excitatory synapses, functions that require proper localization of PSD-95 in the postsynaptic density (PSD). Here we report that phosphorylation of ser-295 enhances the synaptic accumulation of PSD-95 and the ability of PSD-95 to recruit surface AMPA receptors and potentiate excitatory postsynaptic currents. We present evidence that a Rac1-JNK1 signaling pathway mediates ser-295 phosphorylation and regulates synaptic content of PSD-95. Ser-295 phosphorylation is suppressed by chronic elevation, and increased by chronic silencing, of synaptic activity. Rapid dephosphorylation of ser-295 occurs in response to NMDA treatment that causes chemical long-term depression (LTD). Overexpression of a phosphomimicking mutant (S295D) of PSD-95 inhibited NMDA-induced AMPA receptor internalization and blocked the induction of LTD. The data suggest that synaptic strength can be regulated by phosphorylation-dephosphorylation of ser-295 of PSD-95 and that synaptic depression requires the dephosphorylation of ser-295.

In neuroscience, rats have several advantages over mice as a model organism. For instance, behavioral experiments are more advanced and the larger size of the brain is better suited for surgical manipulation and biochemistry. Furthermore, the vascular physiology of rats is considered closer to human, providing clinical relevance. Because transgenesis rates achieved by conventional pronuclear injection are extremely low (0.2-3.5%), the availability of transgenic rats in neuroscience is limited. Lentivirus infection is an efficient way to integrate exogenous genes into the genome of a one-cell embryo to generate transgenic animals. We report here the generation of synapsin I promoter driven GRIP1-transgenic rats using lentiviral transgenesis. GRIP1 was chosen as a transgene because it interacts with AMPA receptors and is involved in glutamate receptor signaling. From a single infection experiment, 45% of the offspring carried the transgene and 40% achieved germ-line transmission. The expression of GRIP1 was observed at low levels in brain, spinal cord and testis. Interestingly, one transgenic copy lacked a 147 bp fragment in the GRIP1 coding region most likely caused by alternative splicing of genomic lentiviral RNA. Co-immunoprecipitation from rat brains showed that transgenic GRIP1 is in complex with the endogenous GluR2 subunit of AMPA receptors. These results indicate that functional transgenic GRIP1 protein is expressed in rat brain using lentiviral vectors containing a human synapsin I promoter. Tissue specific lentiviral transgenic rats will be a powerful tool for various applications in modern neuroscience.

The compound NTA-DCF consists of two components, a dichlorofluorescein (DCF) reporter and a nitrilotriacetic acid (NTA) functionality. The latter binds polyhistidine sequences selectively through a bridging metal ion. The NTA-DCF conjugate has photophysical properties similar to those of the parent DCF fluorophore both by itself and as its nickel(II) complex. The insensitivity of the emission to paramagnetic ions allows the probe to label His6-tagged proteins fluorescently on the extracellular surfaces of HEK 293-T and HeLa cells.

Excitatory synapses are formed on dendritic spines, postsynaptic structures that change during development and in response to synaptic activity. Once mature, however, spines can remain stable for many months. The molecular mechanisms that control the formation and elimination, motility and stability, and size and shape of dendritic spines are being revealed. Multiple signaling pathways, particularly those involving Rho and Ras family small GTPases, converge on the actin cytoskeleton to regulate spine morphology and dynamics bidirectionally. Numerous cell surface receptors, scaffold proteins and actin binding proteins are concentrated in spines and engaged in spine morphogenesis.