Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP)

In the “GFP reconstitution across synaptic partners” (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, Caenorhabditis elegans and Drosophila melanogaster, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same interneurons remain unlabeled. We also generated antisera that label reconstituted GFP but neither fragment in order to amplify the GRASP signal and thereby increase the sensitivity of the method

A synaptic nidogen: developmental regulation and role of nidogen-2 at the neuromuscular junction

Background: The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.Results: In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen- 2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.Conclusion: All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction

Spotted substrates for focal presentation of proteins to cells

Neurites and other cell processes encounter localized deposits of signaling factors as they grow. The difficulty in generating patterned artificial substrates has hindered the analysis of these instructive factors in vitro. Here we report a simple method for presenting cultured cells with small spots of protein on an otherwise uniform substrate. We use a biolistic device called a gene gun to deposit 0.1-5 microm fluorescent dots of pure protein on or beneath a growth-promoting substrate. Using this technique, we demonstrate local presynaptic differentiation of motoneurons in response to dots of a polycation. We also show that biotin-avidin and antibody-antigen interactions can be used to prepare spots from more dilute, more labile, or less abundant proteins. This method should prove useful for analyzing extracellular signaling molecules that act focally on neurons or other cell types

Kinase- and rapsyn-independent activities of the muscle-specific kinase (MuSK).

The muscle-specific receptor tyrosine kinase (MuSK) is co-localized with nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane of the skeletal neuromuscular junction, and is required for all known aspects of postsynaptic differentiation. Studies in vitro have shown that Z(+)-agrin, a nerve-derived proteoglycan, activates MuSK's kinase activity to promote clustering of AChRs and MuSK itself with a cytoplasmic, receptor-associated protein, rapsyn. These studies, however, have used soluble forms of agrin, whereas agrin is cell- or matrix-attached in vivo. We show here that immobilized (particle- or cell-attached) agrin but not soluble agrin is able to aggregate MuSK in the absence of rapsyn and that this aggregation does not require MuSK's kinase activity but does require MuSK's cytoplasmic domain. Moreover, immobilized agrin can promote clustering of AChRs by a mechanism that requires MuSK and rapsyn but does not require MuSK's kinase activity. These results imply that rapsyn and signaling components activated by MuSK kinase may be dispensable for some early aspects of postsynaptic differentiation.Peer reviewe

Welcome to Neural Development

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract[There is no abstract for this article

Viewpoints: contrasting opinions in Neural Development.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Laminar Restriction of Retinal Ganglion Cell Dendrites and Axons: Subtype-Specific Developmental Patterns Revealed with Transgenic Markers

Retinal ganglion cells (RGCs), which transfer information from the eye to the brain, are heterogeneous in structure and function, but developmental studies have generally treated them as a single group. Here, we investigate the development of RGC axonal and dendritic arbors using four mouse transgenic lines in which nonoverlapping subsets of RGCs are indelibly labeled with a fluorescent protein. Each subset has a distinct functional signature, size, and morphology. Dendrites of each subset are restricted to specific sublaminae within the inner plexiform layer in adulthood, but acquire their restriction in different ways: one subset has lamina-restricted dendrites from an early postnatal stage, a second remodels an initially diffuse pattern, and two others develop stepwise. Axons of each subset arborize in discrete laminar zones within the lateral geniculate nucleus or superior colliculus, demonstrating previously unrecognized subdivisions of retinorecipient layers. As is the case for dendrites, lamina-restricted axonal projections of RGC subsets develop in different ways. For example, while axons of two RGC subsets arborize in definite zones of the superior colliculus from an early postnatal stage, axons of another subset initially occupy a deep layer, then translocate to a narrow subpial zone. Together, these results show that RGC subsets use a variety of strategies to construct lamina-restricted dendritic and axonal arbors. Taking account of these subtype-specific features will facilitate identification of the molecules and cells that regulate arbor formation.Molecular and Cellular Biolog

Age-Related Alterations in Neurons of the Mouse Retina

The behavioral consequences of age-related alterations in neural function are well documented, but less is known about their cellular bases. To characterize such changes, we analyzed 14 molecularly identified subsets of mouse retinal projection neurons (retinal ganglion cells or RGCs) and interneurons (amacrine, bipolar, and horizontal cells). The retina thinned but expanded with age, maintaining its volume. There was minimal decline in the number of RGCs, interneurons, or photoreceptors, but the diameter of RGC dendritic arbors decreased with age. Together, the increased retinal area and the decreased dendritic area may lead to gaps in RGC coverage of the visual field. Axonal arbors of RGCs in the superior colliculus also atrophied with age, suggesting that the relay of visual information to central targets may decline over time. On the other hand, the laminar restriction of RGC dendrites and the interneuronal processes that synapse on them were not detectably disturbed, and RGC subtypes exhibited distinct electrophysiological responses to complex visual stimuli. Other neuronal types aged in different ways: amacrine cell arbors did not remodel detectably, whereas horizontal cell processes sprouted into the photoreceptor layer. Bipolar cells showed arbor-specific alterations: their dendrites sprouted but their axons remained stable. In summary, retinal neurons exhibited numerous age-related quantitative alterations (decreased areas of dendritic and axonal arbors and decreased density of cells and synapses), whereas their qualitative features (molecular identity, laminar specificity, and feature detection) were largely preserved. Together, these data reveal selective age-related alterations in neural circuitry, some of which could underlie declines in visual acuity