Research PaperNBQX, a highly selective competitive antagonist of AMPA and KA ionotropic glutamate receptors, increases seizures and mortality following picornavirus infection
Introduction
Seizures occur due to an imbalance between excitation and inhibition with the balance tipping towards excitation (Nadler, 2012). Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) of mammals with fully 60–70% of all synapses being glutamate synapses (Nadler, 2012). Therefore upregulation of expression and/or function of glutamate receptors can contribute to seizures (Dingledine, 2012; Nadler, 2012). There are three subfamilies of ionotropic glutamate receptors, named based on their activation by selective agonists: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate), NMDA (N-methyl-d-aspartate) and KA (kainate) receptors (Dingledine, 2012). AMPA, NMDA and KA receptors are expressed on microglia, astrocytes, oligodendrocytes, and neurons (Gottlieb and Matute, 1997; Hagino et al., 2004; Lees, 2000; Matute, 2006; Murugan et al., 2011; Nadler, 2012; Noda et al., 2000). The region of the brain with the highest density of NMDA and AMPA receptors is the hippocampus, specifically CA1 for AMPA receptors (Rainbow et al., 1984; Wong et al., 1986). KA receptors are found to be most abundant in brain regions where NMDA receptors are least abundant (Nadler, 2012). Nevertheless, hippocampal neurons have been shown to express KA receptors (Paternain et al., 1995). The kinetic properties of KA receptors are intermediate between AMPA and NMDA receptors and there is considerable overlap in agonist action between KA and AMPA receptors (Nadler, 2012). Antagonists to the various ionotropic glutamate receptors, used in this study, include: NBQX [2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline], a highly selective competitive antagonist of AMPA and KA receptors (Sheardown et al., 1990); GYKI-52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine], a non-competitive antagonist, or negative allosteric modulator, of AMPA receptors (Lees, 2000; Wilding and Huettner, 1995); and MK 801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], also known as Dizocilpine, a potent non-competitive antagonist of NMDA receptors (Rod and Auer, 1989; Wong et al., 1986). NBQX, GYKI-52466 and MK 801 all readily penetrate into the CNS when administered peripherally (Sheardown et al., 1990; Smith et al., 1991; Vizi et al., 1996; Wong et al., 1986).
Previous studies have shown that NBQX, GYKI-52466 and MK 801 block seizures in various animal seizure models (Barton et al., 2003, Chapman et al., 1991, Loscher and Honack, 1994, Twele et al., 2015). We determined the effects of these antagonists on acute seizure development in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model, an infection-driven animal model for epilepsy [reviewed in (Libbey and Fujinami, 2011)]. In this model, approximately 50% of C57BL/6J mice infected with TMEV experience acute behavioral seizures between days 3 and 10 post-infection (p.i.) (Libbey et al., 2008; Stewart et al., 2010b). The infected mice clear the virus by about day 14 p.i. (Kirkman et al., 2010; Libbey et al., 2011b, Libbey et al., 2010, and then approximately 50% of the mice that had acute seizures develop spontaneous seizures (epilepsy) following a latent period (Stewart et al., 2010a). Hippocampal neurons, particularly within the CA1 region, are infected and undergo cell death (Kirkman et al., 2010; Libbey et al., 2008; Stewart et al., 2010b).
We found that, of the three antagonists of ionotropic glutamate receptors tested, only NBQX had an effect on acute seizure development in the TMEV-induced seizure model. Treatment with NBQX resulted in a significantly higher number of mice experiencing seizures, an increase in the number of seizures per mouse, a greater cumulative seizure score per mouse and a significantly higher mortality rate among the mice. Therefore, although NBQX has previously been shown to be a potent anticonvulsant, it has the opposite effect in the TMEV-induced seizure model. It should be noted that animal seizure models in which the seizures are induced by electrical stimulation, drug administration or as a result of genetic predisposition may differ greatly in terms of mechanism of seizure development from our virus-induced seizure model.
Section snippets
Animal experiments
Four week old, male C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All animal experiments were reviewed and approved by the University of Utah Institutional Animal Care and Use Committee (Protocol #12-09006) and conducted in accordance with the guidelines prepared by the Committee on Care and Use of Laboratory Animals, Institute of Laboratory Animals Resources, National Research Council. All animal studies complied with the ARRIVE guidelines. All efforts were made to
Results
The effect of treatment, with antagonists of ionotropic glutamate receptors, on the development of seizures was examined in the TMEV-induced seizure model. TMEV-infected C57BL/6J mice treated with MK 801, GYKI-52466, NBQX or PBS as control (treatment: day 2.5–10.5 p.i.) were monitored for seizures through day 21 p.i. Mice were observed for 2 h each day immediately following the first injection of antagonist of the day. The time course of seizure occurrence was not altered by antagonist
Discussion
NBQX competitively binds to the glutamate-binding site on both AMPA and KA receptors (Lees, 1996, Lees, 2000). In terms of selectivity, NBQX has been demonstrated to be more potent against AMPA receptors compared to KA receptors (Lees, 2000, Sheardown et al., 1990; Wilding and Huettner, 1996). NBQX treatment was found to protect hippocampal CA1 neurons against ischemic injury (Sheardown et al., 1990), but not against damage caused by status epilepticus (SE) in an adult mouse model (KA injection
Acknowledgements
We would like to thank Jordan T. Sim, BA, Mitchell A. Wilson, Kelley M. Ingram and Samantha P. Duzy, for excellent technical assistance, Ana Bea DePaula-Silva, PhD, for many helpful discussions, and Daniel J. Harper for the outstanding preparation of the manuscript
This work was supported by NIH 5R01NS065714. The funding source had no involvement in the study design, in collection, analysis and interpretation of data, in writing the article or in the decision to submit the article for
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