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.

Current needs of central nervous system therapy urge for the identification of scaffolds supporting the generation and long-term maintenance of healthy and functional neuronal tissue. We compared for the first time the viability of hippocampal neurons and astrocytes grown on conventional 2-dimensional (2D) conditions with that of cells grown on an aragonite bioactive 3-dimensional (3D) scaffold prepared from coralline exoskeleton. Cultures in 3D showed significantly lower mortality rate and higher neurons/astrocytes ratio than 2D cultures. Moreover, whereas cell survival in 2D was arrested in the absence of the supporting substrates poly-D-lysine and laminin, these substrates had negligible effect on the 3D cultures. Furthermore, aragonite matrices supported cell survival and growth under conditions of calcium and nutrients deprivation, whereas in 2D such treatments led to death of all neurons and of almost all astrocytes. To show that the aragonite matrices are permissive for neural cells also in vivo, aragonite matrices having no substrate coating grafted into postnatal rat cortex were invaded by neurons growing on the surface and in multilayer structures resembling those seen in the 3D culture in vitro. Hence, culture of neurons and astrocytes on 3D aragonite coralline matrices is a novel mean for production of stable neuronal tissue, with significant implication to the field of neuronal tissue restoration.

Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures. However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses. To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granuleto-pyramidal cells. As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization). The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methly-D-aspartate-independent long-term potentiation. Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system. Several features of the hippocampal mossy fiber (MF) pathway are interesting for studying synaptic plasticity (1). First, repeated activation of MFs results in long-term potentiation (LTP) in the CA3 subfield (2) that does not require activation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. Second, MFs are capable of extensive sprouting following epilepsy (3) and contribute to the susceptibility of CA3 neurons to epileptic seizures (4, 5). Finally, the enpassant connections established by MF with dendritic spines of CA3 pyramidal cells have unique morphological characteristics. The presynaptic endings of the MF axons are among the largest in the brain, and the postsynaptic target of the MFs often take the form of giant, multi-invaginating spines termed thorny excresences, making these connections readily identifiable (1, 6, 7).Study of the MF pathway in hippocampal slices has been limited by the complex circuitry of the CA3 region and the extremely low incidence of MF synapses (1, 2). Here we describe a dissociated cell culture system where mossy fiber plasticity can be characterized at the level of individual neurons and elementary synaptic connections. In culture, the MF connections retain the major morphological and immunocytochemical characteristics of the MF pathway in vivo. Thus, this reduced preparation provides an experimentally accessible, in vitro model of the MF pathway that allows the study of its cellular and molecular properties and plastic capabilities at the level of individual, identified synapses. In a companion study, Lopez-Garcia et al. (8) have further demonstrated that the in vitro granule-to-pyramidal connection exhibits NMDAindependent LTP similar to that evident in vivo. MATERIALS AND METHODSCell Culture. Hippocampi of P1-P5 rat pups were split longitudinally along the midportion of the CA3 region (see Fig. 1A). Tissue was...

Tissue-like structures of hippocampal neurons were established in a crystalline three-dimensional (3D) aragonite biomatrix obtained from the exoskeleton of the coral Porites lutea. Cultures were maintained in vitro for up to 5 weeks. Cell viability and regeneration of neuronal properties were studied by immunocytochemical methods, light microscopy image analysis techniques, and scanning electron microscopy. Some portions of the cell population acquired the morphological characteristics of hippocampal pyramidal or granule neurons with axons and dendrites extending in a 3D manner along the surfaces of the crystalline biomatrix. The neurons usually grew on a sheet of glial cells. Within the pore void areas, multiple layers of neurons were formed, many of the neurons growing with no attachment to the crystalline surfaces. The neurons developed mature synaptic connections, with presynaptic sites expressing the synaptic vesicle protein 2 and postsynaptic sites having the shape of dendritic spines and expressing type 1 glutamate receptors, as these cells do under conventional culture conditions. The findings of the present study suggest that neuronal networks growing in a strong 3D aragonite support may find application as tissue replacement material for the central nervous system.

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