Establishing a functional brain circuitry: the development of the retino-tectal projection

Topographic matching between pre- and postsynaptic ganglion cell sheets appears to be a typical way in which different regions of the vertebrate brain are interconnected. The visual system provides a well-suited model to study the processes underlying the generation of 'topographic projections' during development. In this review, we will discuss several mechanisms which are possibly involved in the sorting of retinal axon terminals within their target field, the optic tectum. These mechanisms include target recognition by 'cytochemical matching' between axons and target cells, fiber-fiber interactions, and synapse stabilization and elimination based on impulse activity

Protein analysis on two-dimensional polyacrylamide gels in the femtogram range: use of a new sulfur-labeling reagent

An ultrasensitive staining procedure for two-dimensional polyacrylamide gels of protein homogenates has been developed. It combines the use of ultrathin gels and the labeling of proteins by a 35S-labeling reagent

Affinity purification of monospecific antibodies from polyclonal sera as a means for the identification of differentially expressed genes and proteins

The identification and characterization of genes and proteins that are either cell-type specific or exhibit an otherwise spatially or temporally restricted expression pattern is a crucial step toward understanding the complex interactions between the different cell types in an organism. We demonstrate here that monospecific polyclonal antibodies purified from rabbit antisera by a simple Western blotting and reelution technique can be successfully used for the detection of such proteins, as well as the corresponding genes by screening cDNA expression libraries. This strategy is discussed as a supplement to other approaches, such as the generation of tissue-specific monoclonal antibodies and the "subtractive hybridization technique.

Axonal Growth on Solubilized and Reconstituted Matrix from the Embryonic Chicken Retina Inner Limiting Membrane

Basal laminae, thin sheets of extracellular matrix covering the basal side of all neuroepithelia, are strongly supportive for neurite outgrowth in vitro and may provide a permissive environment for growing neurites in vivo. To gain information about the biological activity and composition of in situ-derived basal laminae the inner limiting membranes from embryonic day (E) 7 to E11 chick and quail retinae were isolated. The basal laminae were solubilized with high-molar guanidine hydrochloride or urea, and the solubilized proteins reconstituted by dialysis. The matrix proteins were spotted or dried onto nitrocellulose or polylysine-coated dishes. When explants from retina or from dorsal root ganglia were incubated on the protein spots, neurite extension was very robust, at a level as high as on authentic basal lamina. Extracts from the pigment epithelial basement membrane did not support neurite extension. Western blot analysis showed that the explant from the retinal inner limiting membrane contained predominantly basal lamina-type proteins, such as laminin, collagen type IV and heparan sulphate proteoglycan, whereas the matrix extract from the pigment epithelium contained predominantly mesenchymal-type proteins, like collagen type I and tenascin. JG22, a beta1 integrin antibody that inhibited neurite extension on EHS tumour laminin substrate, had no effect on neurite outgrowth on retinal basal lamina matrix, indicating that embryonic basal laminae contain other or additional growth promoting substrate molecules

Repair strategies for traumatic spinal cord injury, with special emphasis on novel biomaterial-based approaches

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Use of the biotin-avidin system for labelling, isolation and characterization of neural cell-surface proteins

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated

Guidance and topographic stabilization of nasal chick retinal axons on target-derived components in vitro

We studied mechanisms underlying the generation of topographic order within the developing chick retinotectal connection by combining the recently introduced stripe assay with a novel membrane protein fractionation technique. Our experiments show a preference of temporal and nasal retinal fibers for growing on cell membranes prepared from their proper target area. In addition, membrane preparations from posterior tectum were found to prolong substantially the survival of nasal neurites in vitro. We conclude that tropic as well as trophic interactions contribute to the generation of topographic maps during embryogenesis, in our case to the homing of nasal axons within the posterior tectum

Local embryonic matrices determine region-specific phenotypes in neural crest cells

Membrane microcarriers were used to determine the ability of regional extracellular matrices to direct neural crest cell differentiation in culture. Neural crest cells from the axolotl embryo responded to extracellular matrix material explanted from the subepidermal migratory pathway by dispersing and by differentiating into pigment cells. In contrast, matrix material from the presumptive site of dorsal root ganglia stimulated pronounced cell-cell association and neurotypic expression. Cell line segregation during ontogeny of the neural crest that leads to diversification into pigment cells of the skin or into elements of the peripheral nervous system appears to be controlled in part by local cell-matrix interactions