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神经生物学

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Generation of Primary Astrocyte Cultures Devoid of Contaminating Microglia

Microglia, resident phagocytic cells of the central nervous system, are frequent contaminants of astrocyte cultures. Unfortunately and not always fully appreciated, contamination by microglia can confound results of studies designed to elucidate the molecular mechanisms u ...

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Preparation of Mixed Glial Cultures from Postnatal Rat Brain

We describe a method to prepare postnatal rat brain primary cell cultures composed of astrocytes, oligodendrocytes, and microglia. After 1 week in vitro, the mixed glial cell cultures are free of neurons, meningeal cells and fibroblasts. We developed a simple procedure to selectively harv ...

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Cell Culturing of Caenorhabditis elegans Glial Cells for the Assessment of Cytosolic Ca2+ Dynamics

Cell culture has emerged as an important research method for studying various types of primary cells, including neurons and glial cells. This method has been especially instrumental in assessing intracellular Ca2+ dynamics of neural cells. The invertebrate model organism Caenorha ...

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Isolation and Culture of Human Astrocytes

Although rodent models have been essential to unveil the emerging functions of astrocytes, the existence of interspecies differences calls for caution in extrapolating data from rodent to human astrocytes. We have developed highly enriched primary astrocyte cultures from human f ...

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Isolation and Cultivation of Porcine Astrocytes

A procedure for the isolation and cultivation of astrocytes from swine is described. More specifically, the donor animals are adolescent minipigs about 3 months in age and 10 kg in weight. About 20 g of cerebral tissue can be isolated from the piglet, yielding enough astrocytes of homogeneous gene ...

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Heterogeneity of Astrocytic Form and Function

Astrocytes participate in all essential CNS functions, including blood flow regulation, energy metabolism, ion and water homeostasis, immune defence, neurotransmission, and adult neurogenesis. It is thus not surprising that astrocytic morphology and function differ betwe ...

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Neurogenic Astrocytes and Their Glycoconjugates: Not Just Glue Anymore

Cells with certain attributes of very immature astroglial cells and their radial precursors can act as stem and/or progenitor cells during developmental and persistent neurogenesis. Neural stem/progenitor cells both express and are affected by a variety of developmentally regu ...

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Astrocytes: Multitalented Stars of the Central Nervous System

Astrocytes contribute to virtually every aspect of brain function, including ionic homeostasis, energy metabolism, and synaptic signaling. The varied and important roles of astrocytes have evolved to allow increasingly complex nervous systems to operate efficiently and with h ...

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The Use of Small Noncoding RNAs to Silence Transcription in Human Cells

In human cells, small noncoding RNAs have been shown to possess an ability to exert regulatory control of gene expression when they are generated to target a gene promoter specifically. Mechanistically, small RNA-directed transcriptional gene suppression functions by targeting of e ...

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Rapid Cloning and Validation of MicroRNA Shuttle Vectors: A Practical Guide

MicroRNAs (miRNAs) have emerged as important modulators of eukaryotic gene expression through a process called RNA interference (RNAi). Over the last several years, a large amount of work has focused on understanding how miRNAs are expressed and processed to a biologically functional f ...

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Designing Hairpin-Based RNAi Shuttles

The intrinsic potency of a hairpin-based silencing trigger is one of the most critical determinants for applications such as the creation of stable knockdown cell lines or transgenic knockdown animals. To facilitate the creation of potent silencing constructs, we systematically in ...

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Application of RIP-Chip for the Identification of miRNA Targets

MicroRNAs (miRNAs) are a class of noncoding small RNAs that can regulate gene expression at the posttranscriptional level. To understand how miRNAs function, it is crucial to determine the mRNA targets that are regulated by specific miRNAs. Based on known miRNA:mRNA interactions, miRNA ta ...

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Delivery Strategies for RNAi to the Nervous System

Drug and gene delivery to the central nervous system poses significant challenges to basic researchers and clinicians. The blood–brain barrier prevents most substances from reaching the desired target cells. Strategies for RNA interference (RNAi) have been proposed for certain ne ...

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Cloning Small RNAs

Small, noncoding RNAs have proven to be powerful and ubiquitous agents of gene regulation in eukaryotic genomes. Since their initial discovery, several methods have been developed for directly cloning and sequencing these tiny RNA species. These cloning methods are presented along wi ...

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Profiling the miRNome: Detecting Global miRNA Expression Levels with DNA Microarrays

The ability to profile the miRNome (global microRNA/miRNA expression levels) accurately has become essential for multiple aspects of biological research. However, this process is complicated by the short length of the mature miRNA, the existence of multiple intermediate miRNA for ...

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High-Throughput Profiling of Mature MicroRNA by Real-Time PCR

Real-time quantitative PCR has become a staple technique of most molecular biology laboratories. Configuration of quantitative PCR instruments into 384-well plates has allowed the technology to function as a low-density gene expression array. In this chapter, we present protocols ...

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In Situ Detection of MicroRNAs in Paraffin-Embedded, Formalin-Fixed Tissues: Different Methodologies and Co-localization with Possible Targets

The in situ detection of microRNAs (miRNAs) has been greatly facilitated by several recent technologic advances. These include the locked nucleic acid (LNA)-modified nucleotide, in situ extension of the mature miRNA, and RT in situ PCR amplification of the pre- and pri-microRNA. Furtherm ...

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Tracing of siRNAs Inside Cells by FRET Imaging

The structural integrity of siRNA carrying fluorescent dyes attached to its different strands can be �monitored through the efficiency of fluorescence resonance energy transfer between the dyes. In this chapter, the experimental details of the construction of dye-labeled siRNA a ...

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Fluorescence Imaging with Single-Molecule Sensitivity and Fluorescence Correlation Spectroscopy of Cell-Penetrating Neuropeptides

Neuropeptide–plasma membrane interactions in the absence of a corresponding specific receptor may result in neuropeptide translocation into the cell. Translocation across the plasma membrane may represent a previously unknown mechanism by which neuropeptides can signal i ...

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Visualization of Peptide Secretory Vesicles in Living Nerve Cells

Analysis of real-time movements of peptidergic vesicles in live neurons provides insight into molecular mechanism(s) supporting the activity-dependent secretion of neurotrophins and neuropeptides. We examined the effect of overexpression of exogenous peptides compri ...

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