Neuroprotocols最新文献

For a given neuron, the development of its axonal and dendritic arborizations depends on many external factors, such as matrix molecules, growth factors, depolarization, electric fields, and adhesion molecules. In this paper, we summarize and comment on several protocols that can be used to modulate axonal or dendritic elongation and/or modify the shape of the neurites. A first series of protocols is based on the modulation of neuron-substratum adhesion by the addition of extra cellular matrix molecules. Indeed, axons initiate and elongate under low adhesion conditions, whereas dendrites grow only on highly adhesive substrata. A second series of protocols involves the use of drugs affecting the organization of the cytoskeleton. They suggest that the different behaviors of the axonal and dendritic compartments, in particular under low adhesion conditions, are due partly to the organization of the microtubule and actin networks. Third, we describe a protocol based on the internalization of Antennapedia homeodomain that translocates through the cell membrane and is conveyed to neuronal nuclei. Using this technique, we demonstrated that homeoproteins are involved in the morphological differentiation of postmitotic neurons and, in the case of the motoneurons, in axonal elongation. Furthermore, fusion polypeptides up to 109 amino acids and encompassing the 60-amino-acid translocating homeodomain are also transported through the membrane, thus offering a way to introduce exogenous biologically active peptides into live neurons.

During the development of the nervous system, axons grow over considerable distances to reach their appropriate targets. Information derived from a range of experimental systems suggests that a multiplicity of guidance cues govern growth cone navigation. Among these may be physical features of the environment, pathways of extracellular matrix molecules such as laminin, and distributed positional information cues on the surfaces of neuroepithelial cells. Yet some of these guidance mechanisms may act only over a short range, and it is uncertain whether any of them can specify direction. A possibility that is theoretically attractive is that axons might be directed by diffusible signals emanating from their targets. Although this idea was first proposed by Ramón y Cajal at the beginning of the century, accumulating evidence that chemotropism plays a role in neural development has only recently become compelling. Some in vivo experiments have hinted strongly at chemotropism, as when axons navigate to their target along ectopic routes. But there is only one way of unequivocally demonstrating a chemotropic response of growing neurites. This involves placing an explant containing the neurons of interest at some distance from their target tissue In a three-dimensional collagen matrix devoid of other landmarks. Within such gels it has been demonstrated that gradients of diffusible molecules can be established [T. Ebendal (1977) Cell Tissue Res.175, 439-458]. During the culture period, axons may then display direct or arcuate trajectories toward the target across the neutral matrix. If this phenomenon is observed in the presence of the target but not in the presence of control tissues, this suggests that chemotropism participates in axonal pathfinding during normal development.

There is increasing evidence that molecules that inhibit growth cone motility are involved in the guidance of axons to their appropriate targets during neural development and contribute to the suppression of axon regeneration in the mammalian CNS. Two tissue culture phenomena have been used to detect and monitor these molecules: inhibition of neurite outgrowth and growth cone collapse. In neurite outgrowth assays the inhibitory material is used as a culture substratum. It can be presented to neurons either as a continuous layer or in a form that growing axons will encounter, such as an explant or a stripe. Tissue explants or sections, monolayer cultures of cells, membrane fractions, and purified or partially purified material have all been used. In the growth cone collapse assay, the growth cones of axons extending on a permissive substratum are treated with liposomes incorporating the putative inhibitory material. This method is particularly useful for testing the inhibitory effects of membrane-derived molecules. The relevance of results obtained with these in vitro assays to axon growth phenomena in vivo must always be established. Their principal value lies in the provision of a means of monitoring biochemical purification procedures aimed at identifying and characterizing molecules that inhibit nerve growth.

In vitro methods for studying interactions between axons and their target cells are presented. The methods maximize the number of cultures that can be produced by limiting the volume and area of the cultures. Small cultures promote cell-cell Interactions and permit rapid conditioning of medium. In addition, valuable reagents added to these microcultures are conserved. The methods include: (a) the manufacture of 40-μl well-volume, coverslip-bottomed culture dishes with plating area of less than 24 mm² the dishes allow the small working distances of high-resolution light microscopy; (b) a micromethod to test for the Involvement of secreted factors in cell-cell interactions; cells on different surfaces are cocultured in shared medium; (c) a method to plate explant sources of neurites at a controlled distance from target cells to facilitate neurite identification and to control the timing of growth cone-target cell contacts; and (d) nonisotopic in situ hybridization for chamber-slide cultures combined with immunolabeling of cells in the hybridized culture. These methods can be used in culture assays to identify cell types or molecules involved in a variety of neuronal or, more generally, cell-cell interactions.

Proteoglycans are a structurally diverse class of molecules that interact with many ECM and cell surface components, thereby contributing significantly to a multitude of processes. One function for these macromolecules is the regulation of neurite outgrowth. Proteoglycans are present in axon-free regions of the developing nervous system, where the temporal pattern of their expression suggests a possible role as barrier molecules. In other regions, they are expressed where axons grow and may exist at these sites in combination with growth-promoting molecules, such that their influence is not inhibitory, but rather modulatory. In vitro, when presented in high concentrations in combination with laminin, chondroitin sulfate proteoglycan (CSPG) is inhibitory to growth cone advance for each of three neuronal types tested. Enzymatic degradation of the carbohydrate portion of this molecule (glycosaminoglycan) indicates that it is responsible for the inhibition. However, growth cones can grow on CSPG (bound to laminin) when presented in a stepwise, graded distribution, with the response to the CSPG step gradient being different for each of three neuronal populations. Although the behavior of each cell type is unique, a common behavior of each cell type on the CSPG step gradient is a decrease in the rate of neurite outgrowth with increasing CSPG concentration. These data suggest that different patterns of neurite outgrowth may result from the regulation of the ratio of growth-promoting to growth-inhibiting molecules in the growth cones immediate environment.

Important information about the mechanisms of axon guidance in the developing and regenerating brain has been obtained from in vitro experiments. One particular system, the retinotectal projection of vertebrates, has been analyzed by several in vitro assays, three of which are described here in detail: (i) the stripe choice assay, (ii) the collapse assay, and (iii) the gradient assay. Each of these has revealed position-specific behavior of retinal axons in response to cell membranes derived from different regions of the optic tectum. The stripe choice assay tests the ability of growing axons to discriminate between two membrane substrates offered as alternating stripes. The gradient assay assesses whether growth cones can detect (and be guided by) smooth transitions from one substrate type to another. The collapse assay reveals instantaneous reactions of growth cones to inhibitory or repellent factors present in their environment. The protocols describe the preparation of retinal explants and tectal membranes, as well as the assays proper. Particular emphasis is placed on the gradient assay, which has not yet been described in detail. All of the approaches discussed here have in common that they are applicable to axon guiding components bound to cell membranes. By a few modifications, however, it should be possible to extend this type of investigation to a wider range of related questions, including cell migration and guidance. We do not consider the important aspect of chemotropic guidance of axons in response to diffusible factors.