157 research outputs found
Glia-neuron signaling
This dissertation deals with release of neurotransmitter from glia. It has been demonstrated that bradykinin causes a receptor-mediated release of excitatory amino acids (EAAs), glutamate and aspartate, from glial cultures obtained from dorsal root ganglia (DRG) together with an increase in the cytoplasmic level of glial free calcium. Perturbations which inhibited bradykinin-induced calcium mobilization prevented the release of EAAs from glia. The addition of ionomycin caused a calcium-dependent release of EAAs. Taken together, these data demonstrate that calcium is both necessary and sufficient for stimulating the release of EAAs from DRG glia;Bradykinin was applied to mixed neuron-glial cultures derived from rat cerebral cortex while monitoring calcium levels. Bradykinin elevated calcium levels in neurons only when neurons contacted glia. The general glutamate receptor antagonist, D-glutamylglycine (DGG), prevented bradykinin-induced neuronal calcium elevation. These data indicate that bradykinin elevates neuronal calcium levels through the action of glutamate that is released from glia. While addition of bradykinin to mixed neuron-glial culture is the simplest experimental method of testing the hypothesis that glia can signal to neurons, a second technique allowed a more direct test of this hypothesis. Direct photo-stimulation of glia was used to increase glial calcium levels. A portion of glia was exposed to focal application of UV light while monitoring the calcium response. Photo-stimulation reliably raised the level of calcium in the glial cell. Since elevated calcium is sufficient to stimulate the release of glutamate from glia, this perturbation induced the calcium-dependent release of glutamate. By monitoring calcium levels from adjacent neurons, it was possible to determine that photo-stimulation of glia caused an elevation in calcium levels of adjacent unstimulated neurons. This effect was greatly reduced by DGG. Thus, glia can regulate neuronal calcium levels through glutamate-mediated actions
Astrocyte glutamine synthetase : pivotal in health and disease
The multifunctional properties of astrocytes signify their importance in brain physiology and neurological function. In addition to defining the brain architecture, astrocytes are primary elements of brain ion, pH and neurotransmitter homoeostasis. GS (glutamine synthetase), which catalyses the ATP-dependent condensation of ammonia and glutamate to form glutamine, is an enzyme particularly found in astrocytes. GS plays a pivotal role in glutamate and glutamine homoeostasis, orchestrating astrocyte glutamate uptake/release and the glutamate-glutamine cycle. Furthermore, astrocytes bear the brunt of clearing ammonia in the brain, preventing neurotoxicity. The present review depicts the central function of astrocytes, concentrating on the importance of GS in glutamate/glutamine metabolism and ammonia detoxification in health and disease
Micropatterned Substrates for Studying Astrocytes in Culture
Recent studies of the physiological roles of astrocytes have ignited renewed interest in the functional significance of these glial cells in the central nervous system. Many of the newly discovered astrocytic functions were initially demonstrated and characterized in cell culture systems. We discuss the use of microculture techniques and micropatterning of cell-adhesive substrates in studies of astrocytic Ca2+ excitability and bidirectional neuron-astrocyte signaling. This culturing approach aims to reduce the level of complexity of the system by limiting the interacting partners and by controlling the localization of cells. It provides tight control over experimental conditions allowing detailed characterization of cellular functions and intercellular communication. Although such a reductionist approach yields some difference in observations between astrocytic properties in culture and in situ, general phenomena discovered in cell culture systems, however, have also been found in vivo
SNAREs: Could They be the Answer to an Energy Landscape Riddle in Exocytosis?
During exocytosis, chemical transmitters stored in secretory vesicles can be released upon fusion of these intracellular organelles to the plasma membrane. In this process, SNARE proteins that form a ternary core complex play a central role. This complex could provide the means for generation/storage of energy necessary for driving the fusion of vesicular and plasma membranes. Recently, the amount of energy for (dis)assembly of the ternary complex has been measured using various experimental approaches, including atomic force microscopy, the surface force apparatus, and isothermal titration calorimetry. The obtained measurements are in good agreement with the calculated energy required for membrane fusion achieved by theoretical modeling approaches. Whether the energy expenditure to form the ternary SNARE complex can be utilized towards membrane fusion and/or docking/tethering of vesicles to the plasma membrane still remains one of the key contemporary issues in biophysics and neuroscience
Single Molecule Probing of Exocytotic Protein Interactions Using Force Spectroscopy
Relatively recently, the Atomic Force Microscope (AFM) emerged as a powerful tool for single molecule nanomechanical investigations. Parameters that can be measured by force spectroscopy using AFM, such as the force and total mechanical extension required to break bonds between various proteins can yield valuable insights into the nature of the bond (zippering vs. highly localized binding site), the sequence of its interactions and the energy landscape along the length of the interaction. In this review we discuss the use of AFM in force spectroscopy mode to study intermolecular interactions between the exocytotic proteins of the core SNARE complex. Information gathered by force spectroscopy of protein-protein interactions of this complex supplement previous results acquired with other techniques, and allows a deeper understanding of SNARE protein interactions and their role in exocytosis
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