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Preparation of Synaptosomal Plasma Membranes by Subcellular Fractionation

Brain tissue is extremely heterogeneous—being comprised of neurons (which are often myelinated) and a number of glial cell types—and, therefore, requires some purification in order to study neuronal and glial functioning in isolation at the molecular level. The introduction of the ult ...

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Immunocytochemical Detection of Amino Acid Neurotransmitters in Paraformaldehyde-Fixed Tissues

It is generally thought that up to 90% of all synapses in the mammalian brain use amino acids as neurotransmitters (1). Neurotransmitter amino acids may either be those that are also used directly as protein constituents (such as glutamate) or modified amino acids, such as γ-amino butyric acid (GAB ...

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cGMP-Immunocytochemistry

Immunocytochemistry (ICC) of small, water-soluble molecules requires some special precautions compared to ICC of large protein molecules. In general, protein antigens are relatively insensitive to the type of fixative used. Apparently, the epitopes are not altered in a decisive way ...

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Investigation of the Intracellular Regulators and Components of the Exocytotic Pathway

Exocytotic fusion of secretory vesicles with the plasma membrane occurs in cells in either a constitutive or regulated fashion (Burgess and Kelly, 1987). Constitutive exocytosis is the mechanism by which membrane components and certain soluble proteins are released. In the regulated ...

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Intracellular Messengers in Vertebrate Photoreceptors: Electrophysiological Techniques

Rod and cone photoreceptors transduce the absorption of a photon of light into an electrical response. Within the last decade, our understanding of photoreceptors has increased enormously, and this growth in understanding has resulted in large part from the application of new electrop ...

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Intracellular Messengers in Invertebrate Photoreceptors Studied in Mutant Flies

Invertebrate phototransduction is a central subject in neuroscience that includes several key topics with implications to a wide variety of cells and tissues. The phototransduction cascade begins with isomerization of the retinoid chromophore by photons that activate the phot ...

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Inositol Triphosphate Receptors and Intracellular Calcium: Experimental Approaches

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) was first shown to stimulate mobilization of intracellular Ca2+ from permeabilized pancreatic acinar cells (Streb et al., 1983) and has since become recognized as an intracellular messenger formed after receptor activation and then res ...

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Methods for the Analysis of Phosphoinositides and Inositol Phosphates

There is now compelling evidence that stimulation of inosito1 phospholipid metabolism via phosphoinositidase C (MC) is the signal transduction pathway for a wide variety of receptors in the mammalian nervous system. A by no means comprehensive list is shown in Table 1. Receptors are thoug ...

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Identification and Analysis of Function of Heterotrimeric Guanine Nucleotide-Binding Proteins Expressed in Neural Tissue

Most neurotransmitters, hormones, and growth factors that cause alterations in the rate of synthesis of intracellular messengers interact with cell surface receptors that are members of a family of single polypeptide proteins. Secondary and tertiary structure predictions for s ...

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Measurement of Intracellular Calcium with Fluorescent Calcium Indicators

Changes in cytosolic free calcium ion concentration (i) accompany many cellular transitions and stimulation of cell surface receptors. Both release of Ca2+ from internal stores and Ca2+ entry through plasma membrane ion channels can contribute to cellular Ca2+ transients. In the past, d ...

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Analysis of Protein Kinase C Function

Protein kinase C is a key element in signal transduction, cell regulation, and tumor promotion, influencing cellular responses to many stimuli at a variety of levels, including receptors, transduction systems, ion channels, and membrane pumps. Although it was once thought of as a single enti ...

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Single-Cell Imaging Technology

This chapter will describe and assess the technology by which optical indicators are used to produce images that show the distribution of a measured quantity, such as free ion concentration or electric potential, within individual cells. Common ions whose free concentrations, or activi ...

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Synthetic Analogs of Intracellular Messengers

Signaling pathways that convert extracellular stimuli into intracellular responses regulate many diverse physiological and biochemical processes, and share many characteristics. An external signal binds to a cell surface receptor and stimulates a transducer to activate an ...

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Methods in Cyclic Nucleotide Research

Cyclic nucleotides were discovered about 30 years ago, becoming the prototype intracellular messengers (Robison et al., 1971). Intracellular messenger signaling systems may be split into the following divisions:

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Caged Intracellular Messengers and the Inositol Phosphate Signaling Pathway

The development of caged intracellular messenger compounds offers an elegant new approach to quantitative, time-resolved studies of intracellular signaling pathways. Several reviews of caged compounds have recently appeared (Kaplan and Somlyo, 1989; McCray and Trentham, 19 ...

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Field Potentials in the Central Nervous System: Recording, Analysis, and Modeling

In contrast to unit action potentials recorded from discrete neurons, field potentials, as the name implies, are not restricted to the spatial domain of a neuron or of the brain. As a result of the volume-conducting properties of the brain, field potentials reflect activity of a population of neuro ...

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Human Evoked Potentials

After the current section, the history (1.2.) of evoked potentials (EP—for abbreviations, see the end of this chapter) is briefly presented, followed by a discussion of how EPs can be broken down into components (1.3.). General methodology (2.) is then presented, covering recording techniques (2 ...

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Techniques to Study Spinal-Cord, Peripheral Nerve, and Muscle Activity in Freely Moving Animals

In the past dozen years, newly developed chronic recording techniques have made possible the direct study of peripheral nerve and spinal cord function in conscious, freely moving animals. Two complementary approaches were introduced in the mid-1970s: floating microelectrodes to r ...

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Regulation of Receptor Expression: Analysis of Receptor mRNA and Gene Transcription

In addition to desensitization and downregulation of receptors at the cell membrane, the number of receptors expressed is modulated by regulation of steady-state levels of mRNA and the rate of gene transcription (see refs. 1 and 2 for reviews). Regulation of receptor mRNA and gene transcript ...

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Radioligand Binding Measurement of Receptor Sequestration in Intact and Permeabilized Cells

A frequently observed adaptive response to chronic agonist occupancy of cell surface receptors is a process in which receptors become internalized or sequestered into a cell compartment to which hydrophilic ligands have only a limited access (1–3). Although receptor sequestration ...

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