dizocilpine-maleate and 5-nitro-2-(3-phenylpropylamino)benzoic-acid

Excitotoxicity is associated with stroke, brain trauma, and a number of neurodegenerative disorders. In the brain, during excitotoxic insults, neurons undergo rapid swelling in both the soma and dendrites. Focal swellings along the dendrites called varicosities are considered to be a hallmark of acute excitotoxic neuronal injury. However, it is not clear what pathway is involved in the neuronal anion flux that leads to the formation and resolution of excitotoxic varicosities. Here, we assessed the roles of the volume-sensitive outwardly rectifying (VSOR) Cl- channel in excitotoxic responses in mouse cortical neurons. Whole-cell patch-clamp recordings revealed that the VSOR Cl- channel in cultured neurons was activated by NMDA exposure. Moreover, robust expression of this channel on varicosities was confirmed by on-cell and nystatin-perforated vesicle patch techniques. VSOR channel blockers, but not blockers of GABA(A) receptors and Cl- transporters, abolished not only varicosity resolution after sublethal excitotoxic stimulation but also necrotic death after sustained varicosity formation induced by prolonged NMDA exposure in cortical neurons. The present slice-patch experiments demonstrated, for the first time, expression of the VSOR Cl- channels in somatosensory pyramidal neurons. NMDA-induced necrotic neuronal death in slice preparations was largely suppressed by a blocker of the VSOR Cl- channel but not of the GABA(A) receptor. These results indicate that VSOR Cl- channels exert dual, reciprocal actions on neuronal excitotoxicity by serving as major anionic pathways both for varicosity recovery after washout of an excitotoxic stimulant and for persistent varicosity formation under prolonged excitotoxic insults leading to necrosis in cortical neurons.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; Animals; Apoptosis; Benzothiadiazines; Bicuculline; Bumetanide; Cell Size; Cells, Cultured; Cerebral Cortex; Chlorides; Dendrites; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; GABA-A Receptor Antagonists; Glycolates; Ion Channels; Mice; Mice, Inbred C57BL; N-Methylaspartate; Necrosis; Neurons; Neurotoxins; Nitrobenzoates; Patch-Clamp Techniques; Phloretin; Picrotoxin; Potassium Channel Blockers; Potassium Channels; Quinine; Receptors, N-Methyl-D-Aspartate; Sodium Chloride Symporter Inhibitors; Sodium Chloride Symporters; Somatosensory Cortex; Tetrodotoxin

Volume-sensitive organic anion channels (VSOACs) in astrocytes are activated by cell swelling and are permeable to organic anions, such as glutamate and taurine. We have examined the release of glutamate through VSOACs during the propagation of spreading depression (SD). SD was induced by bath application of ouabain in hippocampal brain slices and was monitored by imaging intrinsic optical signals, a technique that provides a measure of cellular swelling. The onset of SD was associated with increased light transmittance, confirming previous studies that cellular swelling occurs during SD. NMDA receptor antagonists, either noncompetitive (MK-801, 10-50 microM) or competitive (CGS-17355, 100 microM), reduced the rate of propagation of SD, indicating that glutamate release contributes to SD onset. SD still occurred in zero Ca(2+)-EGTA (0-Ca(2+)-EGTA) solution, a manipulation that depresses synaptic transmission. HPLC measurements indicated that, even in this solution, there was significant glutamate release. Two lines of experiments indicated that glutamate was released through VSOACs during SD. First, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a blocker of VSOACs, depressed the rate of propagation of SD in a manner similar to NMDA antagonists. Second, NPPB inhibited the release of glutamate during SD in 0-Ca(2+)-EGTA external solution. These results indicate that cellular swelling during SD causes the activation of VSOACs and the release of glutamate by permeation through this channel. Cellular swelling is a result of neuronal activity and is observed during excitotoxicity. Therefore, glutamate release from VSOAC activation could occur under conditions of cell swelling and contribute to excitotoxic damage.

Topics: Animals; Anion Transport Proteins; Carrier Proteins; Chelating Agents; Cortical Spreading Depression; Dizocilpine Maleate; Egtazic Acid; Excitatory Amino Acid Antagonists; Furocoumarins; Glutamic Acid; In Vitro Techniques; Nitrobenzoates; Pipecolic Acids; Rats; Rats, Sprague-Dawley