Abstract
Neuronal chloride concentration ([Cl−]i) is known to be dynamically modulated and alterations in Cl− homeostasis may occur in the brain at physiological and pathological conditions, being also likely involved in glioma-related seizures. However, the mechanism leading to changes in neuronal [Cl−]i during glioma invasion are still unclear. To characterize the potential effect of glioma released soluble factors on neuronal [Cl−]i, we used genetically encoded CFP/YFP-based ratiometric Cl-(apical) Sensor transiently expressed in cultured hippocampal neurons. Exposition of neurons to glioma conditioned medium (GCM) caused rapid and transient elevation of [Cl−]i, resulting in the increase of fluorescence ratio, which was strongly reduced by blockers of ionotropic glutamate receptors APV and NBQX. Furthermore, in HEK cells expressing GluR1-AMPA receptors, GCM activated ionic currents with efficacy similar to those caused by glutamate, supporting the notion that GCM contains glutamate or glutamatergic agonists, which cause neuronal depolarization, activation of NMDA and AMPA/KA receptors leading to elevation of [Cl−]i. Chromatographic analysis of the GCM showed that it contained several aminoacids, including glutamate, whose release from glioma cells did not occur via the most common glial mechanisms of transport, or in response to hypoosmotic stress. GCM also contained glycine, whose action contrasted the glutamate effect. Indeed, strychnine application significantly increased GCM-induced depolarization and [Cl−]i rise. GCM-evoked [Cl−]i elevation was not inhibited by antagonists of Cl− transporters and significantly reduced in the presence of anion channels blocker NPPB, suggesting that Cl− selective channels are a major route for GCM-induced Cl− influx. Altogether, these data show that glioma released aminoacids may dynamically alter Cl− equilibrium in surrounding neurons, deeply interfering with their inhibitory balance, likely leading to physiological and pathological consequences.
Highlights
In order to assess whether substances released from glioma cells may alter neuronal chloride homeostasis, we used a CFP/YFPbased ratiometric Cl-Sensor expressed in primary hippocampal neuronal cultures (Figure 1A)
Since glioma cells may release glutamate in the extracellular space (Ye and Sontheimer, 1999; Buckingham et al, 2011) we wondered whether glioma conditioned medium (GCM)-induced neuronal [chloride concentration (Cl−]i) rise could be triggered by the activation of ionotropic glutamate receptors on neurons
We characterized the effect of neuroactive aminoacids released by glioma cells on neuronal [Cl−]i, taking advantage of a genetically encoded CFP/YFP-based ratiometric Cl-Sensor (Markova et al, 2008; Bregestovski et al, 2009; Waseem et al, 2010), which was transiently expressed in cultured hippocampal neurons
Summary
In the central nervous system (CNS), a tight regulation of intracellular chloride concentration ([Cl−]i) is important for a number of cellular functions, including the stabilization of resting membrane potential, the regulation of both intracellular pH and Abbreviations: ACM, Astrocyte conditioned medium; APV, D-2-amino-5phosphonopentanoic; [Cl−]i, chloride concentration; CNS, central nervous system; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, Dimethyl sulfoxide; EAATs, Excitatory aminoacid transporters; FBS, Fetal Bovine Serum; FFA, flufenamic acid; GABA, γ-aminobutyric acid; GCM, glioma conditioned medium; Glu, glutamate; GluR1-HEK cells, HEK 293 stably expressing the rat flip variant of wild-type glutamate receptor 1; HBSS, Hank’s balanced salt solution; HPLC, High Performance Liquid Chromatography; HyperGCM, hyperosmotic glioma conditioned medium; NBQX, 2,3-dihydroxy-6-nitro-7sulfamoyl-benzo[f]quinoxaline-2,3-dione; NES, normal external solution; NFA, niflumic acid; NPPB, 5-Nitro-2-(3-phenylpropylamino)benzoic acid; OPA, ophthaldialdehyde; SAS, Sulfasalazine; TBOA, DL-threo-β-Benzyloxyaspartic acid; TTX, Tetrodotoxin.cell-volume (Pasantes-Morales et al, 2006; Suzuki et al, 2006)and the strength and polarity of γ-aminobutyric acid (GABA) and glycine-mediated neurotransmission (Payne et al, 2003). Several different pathways allow Cl− movements across neuronal membranes, determining Cl− equilibrium. Among these are the ligand-gated anion channels (GABAA and glycine receptors), the cation-chloride cotransporters (KCC2 and NKCC1) and a variety of Cl− channels, including Ca2+-, volume-, and voltageactivated Cl− channels (Payne et al, 2003; Suzuki et al, 2006; Jentsch, 2008; Deisz et al, 2011). Even modest increases in extracellular glutamate concentration can alter synaptic transmission (Araque et al, 1999) and reduced activity of glial glutamate transporters has been suggested to contribute to and exacerbate a number of neurological conditions, including stroke, epilepsy, cerebral ischaemia, amyotrophic lateral sclerosis, and others (O’Shea, 2002)
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