The expression of tissue plasminogen activator (tPA) is increased during activity-dependent forms of synaptic plasticity. We have found that inhibitors of tPA inhibit the late phase of long-term potentiation (L-LTP) induced by either forskolin or tetanic stimulation in the hippocampal mossy fiber and Schaffer collateral pathways. Moreover, application of tPA enhances L-LTP induced by a single tetanus. Exposure of granule cells in culture to forskolin results in secretion of tPA, elongation of mossy fiber axons, and formation of new, active presynaptic varicosities contiguous to dendritic clusters of the glutamate receptor R1. These structural changes are blocked by tPA inhibitors and induced by application of tPA. Thus, tPA may be critically involved in the production of L-LTP and specifically in synaptic growth.

Neuronal signaling requires that synaptic proteins be appropriately localized within the cell and regulated there. In mammalian neurons, polyribosomes are found not just in the cell body, but also in dendrites where they are concentrated within or beneath the dendritic spine. The ␣ subunit of Ca 2؉ -calmodulin-dependent protein kinase II (CaMKII␣) is one of only five mRNAs known to be present within the dendrites, as well as in the soma of neurons. This targeted subcellular localization of the mRNA for CaMKII␣ provides a possible cell biological mechanism both for controlling the distribution of the cognate protein and for regulating independently the level of protein expression in individual dendritic spines. To characterize the cis-acting elements involved in the localization of dendritic mRNA we have produced two lines of transgenic mice in which the CaMKII␣ promoter is used to drive the expression of a lacZ transcript, which either contains or lacks the 3-untranslated region of the CaMKII␣ gene. Although both lines of mice show expression in forebrain neurons that parallels the expression of the endogenous CaMKII␣ gene, only the lacZ transcripts bearing the 3-untranslated region are localized to dendrites. The ␤-galactosidase protein shows a variable level of expression along the dendritic shaft and within dendritic spines, which suggests that neurons can control the local biochemistry of the dendrite either through differential localization of the mRNA or variations in the translational efficiency at different sites along the dendrite.

We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrincoated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is colocalized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.The Eps15 homology (EH) 1 domain is an important proteinprotein interaction module functioning in endocytosis. The core of the EH domain-binding motif is composed of the amino acids asparagine-proline-phenylalanine (NPF) (1-3). This sequence is often found at the C terminus of EH domain-binding proteins where the free carboxylate can contribute to binding (3). The EH domain was originally identified in the epidermal growth factor receptor phosphorylation substrate Eps15 (4). Through its EH domains, Eps15 binds to epsin, a recently identified protein implicated in endocytosis (5). Eps15 is localized to the rim of clathrin-coated pits (6), likely through its interactions with AP2 (7-10) and/or with epsin (5). EH domains are also found in the yeast proteins Pan1p and End3p, which are required for endocytosis and normal organization of the actin cytoskeleton (11-15).The Src homology 3 (SH3) domain, a 50 -70-amino acid motif that binds to proline-rich ligands (16, 17) has also been implicated in endocytosis (18). For example, amphiphysins I and II are nerve terminal-enriched proteins that demonstrate SH3 domain-dependent binding to proline-rich sequences in dynamin and synaptojanin (19 -23), enzymes which function in the endocytosis of clathrin-coated vesicles (20,24,25). In fact, overexpression of the SH3 domains of amphiphysins I and II leads to a functional block in endocytosis in a number of different systems (26 -29).A link between EH and SH3 domain-mediated protein-protein interactions has been revealed with the identification and cloning of Xenopus laevis intersectin, a protein containing two N-terminal EH domains, a central helix f...

We present a novel approach for patterning cultured neural networks in which a particular geometry is achieved via anchoring of cell clusters (tens of cells/each) at specific positions. In addition, compact connections among pairs of clusters occur spontaneously through a single non-adherent straight bundle composed of axons and dendrites. The anchors that stabilize the cell clusters are either poly-D-lysine, a strong adhesive substrate, or carbon nanotubes. Square, triangular and circular structures of connectivity were successfully realized. Monitoring the dynamics of the forming networks in real time revealed that the self-assembly process is mainly driven by the ability of the neuronal cell clusters to move away from each other while continuously stretching a neurite bundle in between. Using the presented technique, we achieved networks with wiring regions which are made exclusively of neuronal processes unbound to the surface. The resulted network patterns are very stable and can be maintained for as long as 11 weeks. The approach can be used to build advanced neuro-chips for bio-sensing applications (e.g. drug and toxin detection) where the structure, stability and reproducibility of the networks are of great relevance.