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Gas ChromatographyMass Fragmentography of Amino Acids

The coupling of a gas chromatograph (GC) to a mass spectrometer (MS) results in the marriage of two powerful technologies. The tremendous separative power of the GC is coupled to the direct identification and quantitation capabilities of the MS. The MS produces ions by electron bombardment of el ...

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Double-Isotope Dansyl Microassay for Cerebral Amino Acids

1-Dimethylammo-naphthalene-5-sulfonyl chloride (dansyl chloride, Fig. 1) was originally used by Weber in 1952 for the formation of fluorescent conjugates of albumin. Dansyl chloride reacts with primary and secondary amino groups and with phenols. Fig 1Structure of dansyl chlori ...

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Liquid Chromatographic Determination of Amino Acids After Precolumn Fluorescence Derivatization

Certain amino acids are now well established as the transmitters employed by the majority of neurons in the central nervous system (Curtis and Johnston, 1974; Watkins and Evans, 1981; Fagg and Foster, 1983). Investigation of the physiology, pharmacology, and biochemistry of these amino aci ...

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Radioreceptor Assays for Amino Acids and Related Compounds

The recognition of the significance of amino acids as mediators of chemical neurotransmission in the mammalian central nervous system has grown extensively in recent years, and no fewer than 16 of these simple compounds have been proposed to function as excitatory or inhibitory neurotr ...

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Immunocytochemical Techniques

One of the most powerful tools in the identification of various neuronal types and then connectivities is immunocytochemical localization of specific neuronal markers, e g., synthetic enzymes for neurotransmitters at precise cellular and subcellular levels. Several methods ex ...

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Determination of Transmitter Amino Acid Turnover

Five amino acids have received considerable attention as putative neurotransmitters in mammalian brain; namely, γ-ammobutyric acid (GABA), glycine, glutamate, aspartate, and taurine Fonnum, 1978).

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In vitro Autoradiographic Localization of Amino Acid Receptors and Uptake Sites

Some amino acids may function as neurotransmitters and are presumably released at nerve terminals to interact with specific receptors postsynaptically. Biochemical studies of high-affinity uptake, a potential marker of nerve terminals and of receptor binding, have been employ ...

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Use of Cre-Lox Technology to Analyze Integrin Functions in Astrocytes

Astrocytes communicate with the vascular endothelium via direct cell–cell contacts as well as a variety of secreted growth factors and extracellular matrix (ECM) proteins. Integrins are heterodimeric cell surface receptors for ECM protein ligands, and many integrin subunits are e ...

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In Vivo Imaging of Ca2+ Signaling in Astrocytes Using Two-Photon Laser Scanning Fluorescent Microscopy

Astrocytes are the predominant nonneuronal cell type in the central nervous system. Although they are electrically nonexcitable, they have been found to play an active role in modulation of neuronal function and plasticity through Ca2+ excitability. Thus, Ca2+ signaling in astrocytes ...

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Transgenic Techniques for Cell Ablation or Molecular Deletion to Investigate Functions of Astrocytes and Other GFAP-Expressing Cell Types

Genetic tools are enabling the molecular dissection of the functions and mechanisms of many biological processes. Transgenic manipulations provide powerful tools with which to test hypotheses regarding functions of specific cell types and molecules in vivo in combination with d ...

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Imaging In Vivo Astrocyte/Endothelial Cell Interactions at the BloodBrain Barrier

The goal of this chapter is to highlight methods used to demonstrate in vivo changes in astrocyte expression at the blood–brain barrier (BBB). Loss of BBB integrity is seen in many acute and chronic disease conditions. However, despite the importance of the BBB to homeostasis and correct functio ...

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Reprogramming of Postnatal Astroglia of the Mouse Neocortex into Functional, Synapse-Forming Neurons

Direct conversion of glia into neurons by cellular reprogramming represents a novel approach toward a cell-based therapy of neurodegenerative processes. Here we describe a protocol that allows for the direct and efficient in vitro reprogramming of mouse astroglia from the early post ...

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An In Vitro Model of Ischemic Stroke

Brain stroke is a devastating cerebrovascular disease and ranks as the third most common cause of death and disability in the US. Altered blood–brain barrier (BBB) signaling and permeability characteristics during stroke can increase the risk for life-threatening hemorrhagic tran ...

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In Vitro Models of the BloodBrain Barrier

The chapter provides an introduction and brief overview of currently available in vitro blood–brain barrier models, pointing out the major advantages and disadvantages of the respective models and potential applications. Bovine brain microvessel endothelial cell isolation, ...

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An Improved In Vitro BloodBrain Barrier Model: Rat Brain Endothelial Cells Co-cultured with Astrocytes

In vitro blood–brain barrier (BBB) models using primary cultured brain endothelial cells are important for establishing cellular and molecular mechanisms of BBB function. Co-culturing with BBB-associated cells especially astrocytes to mimic more closely the in vivo condition ...

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Investigation of Astrocyte Oligodendrocyte Interactions in Human Cultures

Multiple sclerosis (MS) is characterized by CNS demyelination and oligodendrocyte depletion, axonal loss, and reactive astrogliosis. Myelin loss causes conduction block, while remyelination is associated with recovery of conduction and return of function. Reactive astroc ...

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An In Vitro Assay to Examine Oligodendrocyte Precursor Cell Migration on Astrocytes

Oligodendrocyte migration is required for the myelination of axons during development and also following demyelinating lesions of the central nervous system. Oligodendrocytes arise from oligodendrocyte precursor cells (OPCs) which are present within the brain and spinal co ...

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AstrocyteSchwann-Cell Coculture Systems

Schwann cells are one of the cellular candidates used in repair strategies following trauma and demyelination of the spinal cord. One of the major obstacles in the use of Schwann cells is their limited migratory ability within the astrocytic environment of the CNS and boundary formation betw ...

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Use of Astrocyte-Microglial Cocultures to Examine the Regulatory Influence of Astrocytes on Microglial Activation

Microglia are the principal immune effector cells of the central nervous system (CNS). Under normal conditions, they occupy a quiescent surveillance phenotype, but following stimulation by microorganisms or inflammatory cytokines, microglia transform into highly activat ...

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Cocultures of Neurons and Astrocytes as a Model for Examining Hypoxia-Induced Neuronal Death

Astrocytes perform critical functions necessary for neuronal survival. Thus, examining the influence of astrocyte function on neuronal cell death during disease, including hypoxia/ischemia, has become an important avenue of investigation. In this chapter we detail the method ...

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