Unconventional But Potential 3-mercaptopyruvate sulfurtransferase Methods

Given the stoichiometry of ion coupling to glutamate uptake, the theoretical lower limit of extracellular glutamate in brain is approximately 2?nM (Zerangue and Kavanaugh, 1996?and?Levy et al., 1998). Many studies using intracerebral microdialysis have reported levels of ambient glutamate???2?��M, three orders of magnitude higher than the theoretical lower limit (Benveniste et al., 1984?and?Lerma et al., 1986; for reviews see Cavelier et al., 2005?and?Nyitrai et al., 2006). By contrast, reports of ambient glutamate concentration estimated from electrophysiological measurement of tonic NMDA receptor activity in hippocampal slice range from 87 to 89?nM (Cavelier and Attwell, 2005?and?Le Meur et al., 2007) to as low as 25?nM (Herman and Jahr, 2007). Accurate 3-mercaptopyruvate sulfurtransferase knowledge of the ambient glutamate concentration in different brain regions is important Selleck XAV-939 for evaluating its effects on synaptic transmission. Several ionotropic and metabotropic glutamate receptor subtypes are activated by low micromolar concentrations of glutamate, and tonic exposure in this range profoundly inhibits synaptic circuitry in vitro ( Zorumski et al., 1996). Glutamate transporters play a dominant role in limiting ambient glutamate, as pharmacological inhibition of transport has been shown to lead to a rapid increase in ambient glutamate causing increased tonic NMDA receptor signaling ( Jabaudon et al., 1999, Cavelier and Attwell, 2005, Le Meur et al., 2007?and?Herman and Jahr, 2007). In this work we attempt to integrate data in the literature with new in vitro measurements and in vivo modeling of diffusion gradients formed by glutamate transporters. Proceeding from the assumption that in steady-state conditions, the volume-averaged rates of release and uptake of glutamate are equal, we show the influence of glutamate transporter membrane density on steady-state diffusion gradients in a density range relevant to in vivo brain expression. We suggest that metabolic impairment of glutamate transport in a shallow boundary region of a microdialysis probe can account for the discrepancies between estimates of ambient glutamate from dialysis and electrophysiological approaches. Approximately 50?ng of human EAAT3 cRNA was microinjected into stage V�CVI Xenopus oocytes and check details recordings were made 1�C6?d later. Recording solution contained 96?mM NaCl, 2?mM KCl, 1?mM MgCl2, 1.8?mM CaCl2, and 5?mM Hepes (pH 7.5). Microelectrodes were pulled to resistances between 1 and 3?M�� and filled with 3?M KCl. Data were recorded with Molecular Devices amplifiers and analog�Cdigital converters interfaced to Macintosh computers. Data were analyzed offline with Axograph X (v.1.0.8) and KaleidaGraph (v 3.6; Synergy) software. For stopped flow measurements, oocytes were voltage clamped at ?60?mV in a perspex recording chamber in which glutamate depletion in the absence of perfusion was