Key Event Overview
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AOPs Including This Key Event
Level of Biological Organization
How this Key Event works
Biological state: L-glutamate (Glu) is a neurotransmitter with important role in the regulation of brain development and maturation processes. Two major classes of Glu receptors, ionotropic and metabotropic, have been identified. Due to its physiological and pharmacological properties, Glu activates three classes of ionotropic receptors named: α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA receptors), 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (kainate receptors) and N-methyl-D-aspartate (NMDA receptors, NMDARs), which transduce the postsynaptic signal. Ionotropic glutamate receptors are integral membrane proteins formed by four large subunits that compose a central ion channel pore. In case of NMDA receptors, two NR1 subunits are combined with either two NR2 (NR2A, NR2B, NR2C, NR2D) subunits and less commonly are assembled together with a combination of NR2 and NR3 (A, B) subunits (reviewed in Traynelis et al., 2010). To be activated NMDA receptors require simultaneous binding of both glutamate to NR2 subunits and of glycine to either NR1 or NR3 subunits that provide the specific binding sites named extracellular ligand-binding domains (LBDs). Apart from LBDs, NMDA receptor subunits contain three more domains that are considered semiautonomous: 1) the extracellular amino-terminal domain that plays important role in assembly and trafficking of these receptors; 2) the transmembrane domain that is linked with LBD and contributes to the formation of the core of the ion channel and 3) the intracellular carboxyl-terminal domain that influences membrane targeting, stabilization, degradation and post-translation modifications.
Biological compartments: The genes of the NMDAR subunits are expressed in various tissues and are not only restricted to the nervous system. The level of expression of these receptors in neuronal and non-neuronal cells depends on: transcription, chromatin remodelling, mRNA levels, translation, stabilization of the protein, receptor assembly and trafficking, energy metabolism and numerous environmental stimuli (reviewed in Traynelis et al., 2010).
In hippocampus region of the brain, NR2A and NR2B are the most abundant NR2 family subunits. NR2A-containing NMDARs are mostly expressed synaptically, while NR2B-containing NMDARs are found both synaptically and extrasynaptically (Tovar and Westbrook, 1999).
General role in biology: NMDA receptors, when compared to the other Glu receptors, are characterized by higher affinity for Glu, slower activation and desensitisation kinetics, higher permeability for calcium (Ca2+) and susceptibility to potential-dependent blockage by magnesium ions (Mg2+). NMDA receptors are involved in fast excitatory synaptic transmission and neuronal plasticity in the central nervous system (CNS). Functions of NMDA receptors:
1. They are involved in cell signalling events converting environmental stimuli to genetic changes by regulating gene transcription and epigenetic modifications in neuronal cells (Cohen and Greenberg, 2008).
2. In NMDA receptors, the ion channel is blocked by extracellular Mg2+ and Zn2+ ions, allowing the flow of Na+ and Ca2+ ions into the cell and K+ out of the cell which is voltage-dependent. Ca2+ flux through the NMDA receptor is considered to play a critical role in pre- and post-synaptic plasticity, a cellular mechanism important for learning and memory (Barria and Malinow, 2002).
3. The NMDA receptors have been shown to play an essential role in the strengthening of synapses and neuronal differentiation, through long-term potentiation (LTP), and the weakening of synapses, through long-term depression (LTD). All these processes are implicated in the memory and learning function (Barria and Malinow, 2002).
How it is Measured or Detected
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?
No OECD methods are available to measure the activation state of NMDA receptors.
The measurement of the activation or the inhibition of NMDA receptors is done indirectly by recording the individual ion channels that are selective to Na+, K+ and Ca+2 by the patch clamp technique. This method relies on lack of measurable ion flux when NMDA ion channel is closed, whereas constant channel specific conductance is recorded at the open state of the receptor (Blanke and VanDongen, 2009). Furthermore, this method is based on the prediction that activation or inhibition of an ion channel results from an increase in the probability of being in the open or close state, respectively.
The whole-cell patch clamp recording techniques have also been used to study synaptically-evoked NMDA receptor-mediated excitatory or inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) in brain slices and neuronal cells, allowing the evaluation of the activated or inhibited state of the receptor (Ogdon and Stanfield, 2009; Zhao et al., 2009).
Microelectrode array (MEA) recordings are used to measure electrical activity in cultured neurons in response to NMDA receptor activation or inactivation (Keefer et al., 2001, Gramowski et al., 2000 and Gopal, 2003; Johnstone et al., 2010). MEAs can also be applied in higher throughput platforms to facilitate screening of numerous chemical compounds based on electrical activity measurements (McConnell et al., 2012).
Evidence Supporting Taxonomic Applicability
The cellular expression of the NMDAR subunits has been studied in both adult human cortex and hippocampus (Scherzer et al., 1998) as well as during the development of the human hippocampal formation (Law et al., 2003). The whole-cell patch clamp recording techniques have been used in NMDA receptors expressed in human TsA cells (derivative of the human embryonic kidney cell line HEK-293) (Ludolph et al., 2010). Cell-attached single-channel recordings of NMDA channels has been carried out in human dentate gyrus granule cells acutely dissociated from slices prepared from hippocampi surgically removed from human patients (Lieberman and Mody, 1999).
It is important to note that in invertebrates the glutamatergic synaptic transmission has inhibitory and not excitatory role like in vertebrates. This type of neurotransmission is mediated by glutamate-gated chloride channels that are members of the ‘cys-loop’ ligand-gated anion channel superfamily found only in invertebrates. The subunits of glutamate-activated chloride channel have been isolated from C. elegans and from Drosophila (Blanke and VanDongen, 2009).
Barria A, Malinow R. (2002) Subunit-specific NMDA receptor trafficking to synapses. Neuron 35: 345-353.
Blanke ML, VanDongen AMJ. (2009) Activation Mechanisms of the NMDA Receptor. In: Van Dongen AM, editor. Biology of the NMDA Receptor. Boca Raton (FL): CRC Press; Chapter 13. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5274/
Cohen S, Greenberg ME. (2008) Communication between the synapse and the nucleus in neuronal development, plasticity, and disease. Ann Rev Cell Dev Biol 24: 183-209.
Gopal K. (2003) Neurotoxic effects of mercury on auditory cortex networks growing on microelectrode arrays: a preliminary analysis. Neurotoxicol Teratol. 25: 69-76.
Gramowski A, Schiffmann D, Gross GW. (2000) Quantification of acute neurotoxic effects of trimethyltin using neuronal networks cultures on microelectrode arrays. Neurotoxicology 21: 331-342.
Johnstone AFM, Gross GW, Weiss D, Schroeder O, Shafer TJ. (2010). Use of microelectrode arrays for neurotoxicity testing in the 21st century Neurotoxicology 31: 331-350.
Keefer E, Norton S, Boyle N, Talesa V, Gross G. (2001) Acute toxicity screening of novel AChE inhibitors using neuronal networks on microelectrode arrays. Neurotoxicology 22: 3-12.
Law AJ, Weickert CS, Webster MJ, Herman MM, Kleinman JE, Harrison PJ. (2003) Expression of NMDA receptor NR1, NR2A and NR2B subunit mRNAs during development of the human hippocampal formation. Eur J Neurosci. 18: 1197-1205.
Lieberman DN, Mody I. (1999) Properties of single NMDA receptor channels in human dentate gyrus granule cells. J Physiol. 518: 55-70.
Ludolph AG, Udvardi PT, Schaz U, Henes C, Adolph O, Weigt HU, Fegert JM, Boeckers TM, Föhr KJ. (2010) Atomoxetine acts as an NMDA receptor blocker in clinically relevant concentrations. Br J Pharmacol. 160: 283-291.
McConnell ER, McClain MA, Ross J, LeFew WR, Shafer TJ (2012). Evaluation of multi-well microelectrode arrays for neurotoxicity screening using a chemical training set Neurotoxicology 33: 1048-1057.
Ogdon D, Stanfield P. (2009) Patch clamp techniques for single channel and whole-cell recording. Chapter 4, pages 53-78. http://www.utdallas.edu/~tres/microelectrode/microelectrodes_ch04.pdf
Scherzer CR, Landwehrmeyer GB, Kerner JA, Counihan TJ, Kosinski CM, Standaert DG, Daggett LP, Veliçelebi G, Penney JB, Young AB. (1998) Expression of N-methyl-D-aspartate receptor subunit mRNAs in the human brain: hippocampus and cortex. J Comp Neurol. 390: 75-90.
Tovar KR, Westbrook GL. (1999) The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci. 19: 4180–4188.
Traynelis S, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R. (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 62: 405-496.
Zhao Y, Inayat S, Dikin DA, Singer JH, Ruoff RS, Troy JB. (2009) Patch clamp technique: review of the current state of the art and potential contributions from Nanoengineering. Proc. IMechE 222, Part N: J. Nanoengineering and Nanosystems 149. DOI: 10.1243/17403499JNN149