Springer protocols feed by Neuroscience 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Morpholino Studies in Xenopus Brain Development
Antisense morpholino oligonucleotides (MOs) have become a valuable method to knock down protein levels, to block mRNA splicing, and to interfere with miRNA function. MOs are widely used to alter gene expression during development of Xenopus and zebra fish, where they are typically injected into the fertilized egg or blastomeres. Here, we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells in the brain with cu...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Microinjection Manipulations in the Elucidation of Xenopus Brain Development
Microinjection has a long and distinguished history in Xenopus and has been used to introduce a surprisingly diverse array of agents into embryos by both intra- and intercellular means. In addition to nuclei, investigators have variously injected peptides, antibodies, biologically active chemicals, lineage markers, mRNA, DNA, morpholinos, and enzymes. While enumerating many of the different microinjection approaches that can be taken, we will focus upon the mechanical operations and options available to introduce mRNA, DNA, and morpholinos intracellularly into early stage embryos for the study of neurogenesis. (Source: Spr...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Methods in Brain Development of Molluscs
Representatives of the phylum Mollusca have long been important models in neurobiological research. Recently, the routine application of immunocytochemistry in combination with confocal laser scanning microscopy has allowed fast generation of highly detailed reconstructions of neural structures of even the smallest multicellular animals, including early developmental stages. As a consequence, large-scale comparative analyses of neurogenesis—an important prerequisite for inferences concerning the evolution of animal nervous systems—are now possible in a reasonable amount of time. Herein, we describe immunocytoch...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Dye Coupling and Immunostaining of Astrocyte-Like Glia Following Intracellular Injection of Fluorochromes in Brain Slices of the Grasshopper, Schistocerca gregaria
Injection of fluorochromes such as Alexa Fluor® 568 into single cells in brain slices reveals a network of dye-coupled cells to be associated with the central complex. Subsequent immunolabeling shows these cells to be repo positive/glutamine synthetase positive/horseradish peroxidase negative, thus identifying them as astrocyte-like glia. Dye coupling fails in the presence of n-heptanol indicating that dye spreads from cell to cell via gap junctions. A cellular network of dye-coupled, astrocyte-like, glia surrounds and infiltrates developing central complex neuropils. Intracellular dye injection techniques complement c...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Using MARCM to Study Drosophila Brain Development
Mosaic analysis with a repressible cell marker (MARCM) generates positively labeled, wild-type or mutant mitotic clones by unequally distributing a repressor of a cell lineage marker, originally tubP-driven GAL80 repressing the GAL4/UAS system. Variations of the technique include labeling of both sister clones (twin spot MARCM), the simultaneous use of two different drivers within the same clone (dual MARCM), as well as the use of different repressible transcription systems (Q-MARCM). MARCM can be combined with any UAS-based construct, such as localized GFP fusions to visualize subcellular compartments, genes for rescue an...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Immunofluorescent Labeling of Neural Stem Cells in the Drosophila Optic Lobe
The Drosophila visual system is an excellent model system to study the switch from proliferating to differentiating neural stem cells. In the developing larval optic lobe, symmetrically dividing neuroepithelial cells transform to asymmetrically dividing neuroblasts in a highly ordered and sequential manner. This chapter presents a protocol to visualize neural stem cell types in the Drosophila optic lobe by fluorescence confocal microscopy. A main focus is given on how to dissect, fix, immunolabel, and mount brains to reveal cellular morphology during early larval brain development. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Flybow to Dissect Circuit Assembly in the Drosophila Brain
Visualization of single neurons within their complex environment is a pivotal step towards uncovering the mechanisms that control neural circuit development and function. This chapter provides detailed technical information on how to use Drosophila variants of the mouse Brainbow-2 system, called Flybow, for stochastic labeling of cells with different fluorescent proteins in one sample. We first describe the genetic strategies and the heat shock regime required for induction of recombination events. This is followed by a detailed protocol as to how to prepare samples for imaging. Finally, we provide specifications to facili...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Analysis of Complete Neuroblast Cell Lineages in the Drosophila Embryonic Brain via DiI Labeling
Proper functioning of the brain relies on an enormous diversity of neural cells generated by neural stem cell-like neuroblasts (NBs). Each of the about 100 NBs in each side of brain generates a nearly invariant and unique cell lineage, consisting of specific neural cell types that develop in defined time periods. In this chapter we describe a method that labels entire NB lineages in the embryonic brain. Clonal DiI labeling allows us to follow the development of a NB lineage starting from the neuroectodermal precursor cell up to the fully developed cell clone in the first larval instar brain. We also show how to ablate indi...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Non-fluorescent RNA In Situ Hybridization Combined with Antibody Staining to Visualize Multiple Gene Expression Patterns in the Embryonic Brain of Drosophila
In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for non-fluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to an...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Immunostaining of the Developing Embryonic and Larval Drosophila Brain
Immunostaining is used to visualize the spatiotemporal expression pattern of developmental control genes that regulate the genesis and specification of the embryonic and larval brain of Drosophila. Immunostaining uses specific antibodies to mark expressed proteins and allows their localization to be traced throughout development. This method reveals insights into gene regulation, cell-type specification, neuron and glial differentiation, and posttranslational protein modifications underlying the patterning and specification of the maturing brain. Depending on the targeted protein, it is possible to visualize a multitude of...
Source: Springer protocols feed by Neuroscience - September 24, 2013 Category: Neuroscience Source Type: news
Monitoring Synaptic Plasticity by Imaging AMPA Receptor Content and Dynamics on Dendritic Spines
Time-lapse imaging techniques are widely used to monitor dendritic spine dynamics, a measurement of synaptic plasticity. However, it is challenging to follow the dynamics of spines over an extended period in vivo during development or in deep brain structures that are beyond the reach of traditional microscopes. Here, we describe an AMPA receptor-based optical approach to monitor recent history of synaptic plasticity. This method allows the identification of spines that have recently acquired synaptic AMPA receptors in a single imaging session, so that synaptic plasticity that occurs in vivo in a variety of conditions can ...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Semiautomated Analysis of Dendrite Morphology in Cell Culture
Quantifying dendrite morphology is a method for determining the effect of biochemical pathways and extracellular agents on neuronal development and differentiation. Quantification can be performed using Sholl analysis, dendrite counting, and length quantification. These procedures can be performed on dendrite-forming cell lines or primary neurons grown in culture. In this protocol, we describe the use of a set of computer programs to assist in quantifying many aspects of dendrite morphology, including changes in total and localized arbor complexity. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Microcontact Printing of Substrate-Bound Protein Patterns for Cell and Tissue Culture
Patterned distributions of signalling molecules play fundamental roles during embryonic development. Several attempts have been made to reproduce these patterns in vitro. In order to study substrate-bound or membrane proteins, microcontact printing (μCP) is a suitable method for tethering molecules on various surfaces. Here, we describe three μCP variants to produce patterns down to feature sizes of about 300 nm, which are highly variable with respect to shape, protein spacing, and density. Briefly, the desired pattern is etched into a silicon master, which is then used as a master for the printing process. Each vari...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
The Stripe Assay: Studying Growth Preference and Axon Guidance on Binary Choice Substrates In Vitro
Stripe assays are frequently used for studying binary growth decisions of cells and axons towards surface-bound molecules in vitro. In particular in the fields of neurodevelopment and axon guidance, stripe assays have become a routine tool. Several variants of the stripe assay have been developed since its introduction by Bonhoeffer and colleagues in 1987 (Development 101:685–696, 1987). In all variants, however, the principle is the generation of a structured binary growth substrate, consisting of two sets of cues, arranged in alternating stripes. There are two major classes of stripe assays, mainly distinguished by...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Growth Cone Collapse Assay
Growth cone collapse is an easy and efficient test for detecting and characterizing axon guidance activities secreted or expressed by cells. It can also be used to dissect signaling pathways by axon growth inhibitors and to isolate therapeutic compounds that promote axon regeneration. Here, we describe a growth cone collapse assay protocol used to study signal transduction mechanisms of the repulsive axon guidance molecule ephrin-A5 in hippocampal neurons. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
