Little is known about the influence of substrate-bound gradients on neuronal development, since it has been difficult to fabricate gradients over the distances typically required for biological studies (a few hundred micrometers). This article demonstrates a generally applicable technique for the fabrication of substratebound gradients of proteins with complex shapes, using laminar flows in microchannels. Gradients that range from pure laminin to pure BSA were formed in solution by using a network of microchannels, and these proteins were allowed to adsorb onto a homogeneous layer of poly-L-lysine. Rat hippocampal neurons were cultivated on these substrate-bound gradients. Analysis of optical images of these neurons showed that axon specification is oriented in the direction of increasing surface density of laminin. Linear gradients in laminin adsorbed from a gradient in solution having a slope of ١[laminin] > about 0.06 g (ml⅐m) ؊1 (defined by dividing the change of concentration of laminin in solution over the distance of the gradient) orient axon specification, whereas those with ١[laminin] < about 0.06 g (ml⅐m) ؊1 have no effect. microfluidics ͉ neuronal polarity ͉ hippocampal G radients of chemoattractant and chemorepellent substances play central roles in controlling the development of the brain (1). Although the influence of gradients of soluble substances on neuronal behavior has been studied extensively and has been used to unravel the molecular and cellular mechanisms of axon guidance (2), much less is known about gradients of substrate-bound substances (3). Generating consistent substrate-bound gradients over the distances required for biological studies (a few hundred micrometers) has been difficult, and this difficulty hindered the investigation of the role of immobilized gradients in neuronal development (e.g., establishment of cellular polarity and axon guidance) and cell migration. This article demonstrates a generally applicable technique for the fabrication of substrate-bound gradients that uses laminar flow of fluids in microchannels; we have described the microfluidic systems used to form these gradients (4). By generating gradients in the concentration of proteins in solution and allowing them to adsorb on a surface, we fabricated substratebound gradients that range from pure laminin to pure BSA immobilized on a uniform layer of poly-L-lysine (PLL) over distances of a few hundred micrometers. We demonstrate that axon specification of rat hippocampal neurons cultivated on these gradients is oriented in the direction of increasing concentration of laminin. We demonstrate that gradients of extracellular molecules adsorbed on the surface are capable of orienting the polarity of cultured hippocampal neurons.Hippocampal neurons in culture develop their characteristic structural and functional polarity in a stereotyped sequence of developmental events (5). Cells first attach to the substrate and then develop processes. When new processes start to form, they cannot be distinguished as either axons o...

The XR-1 Chinese hamster ovary cell line is impaired in DNA double-strand break repair (DSBR) and in ability to support V(D)J recombination of transiently introduced substrates. We now show that XR-1 cells support recombination-activating gene 1- and 2-mediated initiation of V(D)J recombination within a chromosomally integrated substrate, but are highly impaired in ability to complete the process by forming coding and recognition sequence joins. On this basis, we isolated a human cDNA sequence, termed XRCC4, whose expression confers normal V(D)J recombination ability and significant restoration of DSBR activity to XR-1, clearly demonstrating that this gene product is involved in both processes. The XRCC4 gene maps to the previously identified locus on human chromosome 5, is deleted in XR-1 cells, and encodes a ubiquitously expressed product unrelated to any described protein.

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Synaptic vesicles are recycled locally within presynaptic specializations. We examined how vesicles are reused after endocytosis, using transgenic mice expressing the genetically encoded fluorescent indicator synaptopHluorin in subsets of neurons. At both excitatory and inhibitory synapses in cultured hippocampal neurons, newly endocytosed vesicles did not preferentially enter the releasable pool of vesicles. Rather, they entered the reserve pool first and subsequently the readily releasable pool over a period of several minutes. All vesicles in the recycling pool could be accessed by spaced stimuli, arguing against preferential local reuse of the readily releasable vesicles. Interestingly, nearly half the vesicles at excitatory synapses, and a third at inhibitory synapses, could not be recruited for release even by sustained stimuli. We conclude that, at presynaptic terminals in the hippocampus, most vesicles vacate release sites after exocytosis and are replaced by existing vesicles from the reserve pool, placing constraints on kiss-and-run recycling.endocytosis ͉ exocytosis ͉ hippocampus ͉ kiss and run S ynaptic vesicles are recycled locally within the presynaptic terminal for reuse (1, 2). Several mechanisms of recycling have been proposed, including a conventional pathway involving clathrin coat formation (1, 2) and a clathrin-independent pathway involving kiss-and-run exocytosis (3-9) that might explain rapid endocytosis observed at some synapses (5, 10-12). Although recent attention has centered on modes of endocytosis, several key questions can also be raised regarding postendocytic traffic of synaptic vesicles. Where do vesicles go after endocytosis? Are recently recaptured vesicles preferentially reused? Rapid reuse of recently recaptured vesicles (13, 14) might be advantageous to synapses if it allows vesicles to become release-competent more rapidly than would be possible by recruitment from the reserve pool.Studies in hippocampal and cortical cultures generally have focused on glutamatergic synapses or assumed that the synapses under investigation are glutamatergic synapses because they are more abundant (15). It is unclear whether inhibitory presynaptic terminals have the same mechanisms of recycling as excitatory synapses. Some differences might be anticipated based on previous studies establishing differences in the molecular composition (16, 17) and short-term synaptic dynamics (16,18,19). Additionally, the loss of specific proteins, for example, synapsin I (20), appears to have different consequences in glutamatergic and GABAergic synapses. Therefore, it is important to understand the properties of and mechanisms in vesicle recycling at inhibitory synapses.Genetically encoded probes such as synaptopHluorin (spH), which is a fusion protein of the vesicle protein VAMP2 and a pH-sensitive EGFP (21), offer a method to target tracers to selected populations of neurons. We have generated lines of transgenic mice that express spH in subsets of neurons in the brain. We first show that exocytosis and en...