Role of the NR2A/2B subunits of the N-methyl- d -aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro

• Published in: Neuroscience Vol. 171; no. 4; pp. 1075 - 1090
• Main Authors: Monnerie, H, Hsu, F.-C, Coulter, D.A, Le Roux, P.D
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 29.12.2010; Elsevier
• Subjects: Analysis of Variance; Animals; Bicuculline - pharmacology; Biological and medical sciences; calpain; Calpain - pharmacology; Cell Survival - drug effects; Cells, Cultured; cerebral cortex; Cerebral Cortex - cytology; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors - pharmacology; Excitatory Amino Acid Agents - pharmacology; excitatory amino acids; excitotoxicity; Fundamental and applied biological sciences. Psychology; GABA Agents - pharmacology; GABAergic neurons; gamma-Aminobutyric Acid - metabolism; Gene Expression Regulation - drug effects; Glutamate Decarboxylase - metabolism; Glutamic Acid - pharmacology; Glycine Agents - pharmacology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Mice; Mice, Inbred BALB C; Neurology; Neurons - drug effects; NMDA receptors; Patch-Clamp Techniques - methods; Receptors, N-Methyl-D-Aspartate - physiology; Strychnine - pharmacology; Vertebrates: nervous system and sense organs; FDA; N-methyl- d-aspartate; N-[N-[(Phenylmethoxy)carbonyl]-l-valyl]-phenylalaninal; 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide; fluorescein diacetate; (RS)-α-methyl-4-sulfonophenylglycine; spontaneous excitatory postsynaptic currents; MDL 28170; Ro 25-6981; calpain; MK-801; PEAQX; propidium iodide; NBM; NMDA; TBI; excitotoxicity; AP5; excitatory amino acids; TBOA; (RS)-1-aminoindan-1,5-dicarboxylic acid; traumatic brain injury; (5R,10S)-(-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo hepten-5,10-imine hydrogen maleate; (aR,bS)-a-(4-hydroxyphenyl)-b-methyl-4-(phenylmethyl)-1-piperidinepropanol maleate; sEPSCs; GAD; [[[(1S)-1-(4-Bromophenyl)ethyl]amino](1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl] phosphonic acid; NMDA receptors; N-methyl- d-aspartate receptor; dl-threo-β-benzyloxyaspartic acid; glutamic acid decarboxylase; neurobasal medium; DIV; cerebral cortex; 2-amino-5-phosphonovaleric acid; MSPG; NMDAR; PI; GABAergic neurons; days in vitro; AIDA; NBQX; Cerebral cortex; Enzyme; Toxicity; Cysteine endopeptidases; Central nervous system; Calpain; Lyases; Glutamate receptor; Glutamate decarboxylase; In vitro; Encephalon; Peptidases; Carbon-carbon lyases; Carboxy-lyases; Excitotoxicity; Neurotransmitter; Excitatory aminoacid; Hydrolases; NMDA receptor; Gabaergic neuron
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2010.09.050

Abstract The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [ Monnerie and Le Roux, (2007) Exp Neurol 205:367–382, (2008) Exp Neurol 213:145–153]. An N-methyl- d -aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.