Neuroscience Letters, 119 (1990) 53-55

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Elsevier Scientific Publishers Ireland Ltd. NSL 07250

NMDA receptor-independent epileptiform activity induced by magnesium-free solution in rat amygdala neurons is blocked by CNQX Po-Wu Gean Department of Pharmacology, Collegeof Medicine, National Cheng-Kung University, Tainan City, Taiwan (Republic of China) (Received 23 May 1990; Revised version received 9 July 1990; Accepted 10 July 1990)

Key words: NMDA receptor; Magnesium; CNQX; D-APV; Amygdala; Brain slice The effect of 6-~yano-7-nitroquinoxaline-2,3-dione(CNQX), a specific non-N-methyl-o-aspartate (non-NMDA) receptor antagonist, on NMDAindependent epileptiform activity induced by Mge+-free medium was studied in rat basolateral amygdala (BLA) neurons using intracellular recording techniques. Twenty to 30 min after switching to Mg2+-frce medium, spontaneous and evoked epileptiform activity were observed in 16 out of 18 amygdala slices. Superfusion of o-2-amino-5-phosphonovalerate (D-APV), a selective NMDA receptor antagonist, reduced the duration of epilcptiform activity by an average of 83%. However, there was a residual depolarizing component which remained in the presence of D-APV. This o-APVresistant component could be completely blocked by CNQX suggesting that it is mediated by non-NMDA receptors.

Recent evidence has indicated that the channels activated by N-methyl-o-aspartate (NMDA) are gated in a voltage-dependent manner by Mg 2+ [10, 14]. When Mg 2+ is omitted from the perfusate, the NMDA receptor system can be activated at potentials close to resting membrane potentials and trigger bursting discharges. Indeed, spontaneous and evoked epileptiform activity have been observed in various brain regions including hippocampus [11], neocortex [17] and amygdala [5]. We have previously shown that the epileptiform activity induced by MgE+-free medium in basolateral amygdala (BLA) neurons could be blocked by D-2-amino-5-phosphonovalerate (D-APV) [5], a specific NMDA receptor antagonist. However, there was still a D-APV-resistant component remained in Mg2+-free solution. Since BLA neurons receive excitatory input from the pyriform cortex and this input is likely mediated by excitatory amino acids [15], I therefore examined the effect of 6-cyano-7nitroquinoxaline-2,3-dione (CNQX), a specific nonNMDA antagonist [1, 8], on NMDA-independent component in Mg2+-free medium. The preparation of the amygdala slices was similar to that reported previously [6]. In brief, male SpragueDawley rats weighing 125-200 g were decapitated and transverse amygdala slices of 500/~m thickness were prepared using a Vibroslice (Campden Inst.). Slices were submerged in the recording chamber and maintained at Correspondence: P.-W. Gean, Department of Pharmacology, College of Medicine, National Cheng-Kung University, Taiwan City, Taiwan 70101, Republic of China. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

32 _+1°C. The artificial cerebral spinal fluid (ACSF) solution superfusing the preparation had the following composition (in raM): NaCI 117, KCI 4.7, CaCI2 2.5, MgC12 1.2, NaHCO3 25, NaH2PO4 1.2 and glucose 11. Intracellular recordings were obtained from neurons of the basolateral amygdala nucleus using conventional intracellular recording techniques. Microelectrodes were pulled from microfiber-fillexi 1.0 mm capillary tubing on a Brown-Flaming electrode puller (Sutter Instruments). Electrodes were filled with 4 M potassium acetate with resistance ranging from 90-150 Mr2. Electrical signals were amplified using an Axoclamp 2A amplifier and recorded on a Gould 3200 chart recorder. A bipolar stimulating electrode was placed in the ventral endopyriform nucleus [4] and stimulated with monophasic constant voltage pulses from a Grass $88 stimulator. Stimulus-intensities varied between 5 and 50 V with a pulse duration ranging from 50 to 100 /zs. All data were expressed as mean_+ S.E.M. Statistic analysis was performed using the paired Student's t-test. Ten to 15 min after switching to magnesium-free solution, synaptic noises and frequency of spontaneous excitatory postsynaptic potential (EPSP)-Iike activity with or without action potential initiation increased progressively. Later, the spontaneous epileptiform activity consisting of an initial burst followed by a number of afterdischarges (Fig. 1A). were observed in 16 out of 18 slices studied. The spontaneous epileptiform activity had an average duration of 4.98 _+0.45 s (n= 16) with a frequency of 0.6 to 3.1 bursts/min, or could be triggered by stimulation of the ventral endopiriform nucleus (Fig. 1A).

54 A

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Mg~- free + CNQX

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M9~ _ free

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Mg - f r e e + C N Q X + O - A P V

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Fig. 1. Effects of D-APV and CNQX on the epileptiform activity induced in Mg2+-free solution. A: intracellular recordings of spontaneous (right) and triggered (left) bursts in a BLA neuron 40 rain after the superfusion with Mg2+-freesolution. B: application of D-APV(50 /~M) reduced the duration of epileptiform activity. C: the D-APV-resistant component could be blocked completelyby CNQX (8 pM). D: the effectof CNQX was reversibleupon 50 min of washing. All records were taken from the same neuron. Filled triangles represent point of stimulus. Resting membrane potential of this neuron was -66 inV. Superfusion of D-APV reduced the duration of epileptiform activity induced in Mg2+-free medium. Fig. 1A,B show intracellular recordings of spontaneous and single stimulus-evoked epileptiform bursts before and after application of D-APV. At a concentration of 50 pM, DAPV shortened the duration of spontaneous epileptiform activity by an average of 83% (4.98 + 0.45 s before and 0.85 +0.12 s after the superfusion of 50/~M D-APV, n = 16). Often, the plateau amplitude of the initial depolarizing wave was also reduced with a concomitant decrease in the number of spikes atop it. Thus, the major component of epileptiform activity induced by Mg 2+free solution is likely mediated by N M D A receptors as reflected by the largely shortened burst duration in the presence of N M D A antagonist. The D-APV-resistant depolarizing component could then be blocked completely by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Fig. 1C) indicating that n o n - N M D A receptors may also contribute to the epileptiform activity induced by Mg 2+free solution in BLA neurons.

Fig. 2. Effectof CNQX on the epileptiform activity induced in Mg2÷free medium. A: stimulation of ventral endopyriform nucleus evoked an EPSP which reached threshold and initiated an action potential. B: 30 min after switching to Mg2+-freemedium, same stimulus intensity now evoked a burst response. C: superfusion of CNQX (10 gM) depressed the fast component of the depolarizing wave. D: same as C with different time scale. E,F: the CNQX-resistant depolarizing wave could be reversiblyblocked by D-APV(50 gM). All records were taken from the same neuron. Filled triangles represent point of stimulus. Resting membrane potential of this neuron was ~a4 inV. To further test this hypothesis, the effect of C N Q X on the epileptiform activity induced in Mg2+-free medium was examined. Fig. 2A shows that orthodromic stimulation of ventral endopyriform nucleus evoked an EPSP. With an increase in stimulus intensity or duration, EPSP amplitude could be increased sufficiently to trigger an action potential. Thirty minutes after exposure to Mg 2+free solution, the same stimulus intensity could now evoke an epileptiform burst with spikes superimposed on a large depolarizing wave up to 30 mV (Fig. 2B). Superfusion of C N Q X (10 p M ) depressed the amplitude of depolarizing wave induced in Mg2+-free solution. Note that the initial fast component of the depolarizing shift was blocked by C N Q X resulting in a slower rising time course (Fig. 2C) which is a characteristic of N M D A receptor-mediated synaptic response [3]. The CNQX-resistant depolarizing shift could be reversibly blocked by D-APV ( 5 0 / t M ) (Fig. 2E) further confirming that it is mediated by the activation of N M D A receptors. In this study, I have shown that N M D A receptor activation does not appear to be the only factor that contributes to the epileptiform activity induced by Mg2+-free solution in BLA neurons. After exposure to D-APV at

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a concentration of 50 pM, which has been reported to be a near-maximal concentration of D-APV necessary to block the N M D A responses [2], a residual depolarizing component was still present. Previous study in hippocampal CA1 neurons showed that this N M D A receptorindependent enhancement of synaptic potentials in the absence of extracellular Mg 2+ ions is due to a relief of the antagonistic action of Mg 2+ on presynaptic Ca 2+ entry which would lead to enhanced transmitter release [7]. Furthermore, Stone et al. proposed that low magnesium enhancement of synaptic potentials could be attributed to the reduced presynaptic inhibitory efficacy of endogenous adenosine since superfusion of 8-phenyltheophylline or adenosine deaminase precludes the enhancement [16]. I tested the possible involvement of increased transmitter release by applying CNQX because the neurotransmitter for pyriform cortex-amygdala pathway is likely to be glutamate [15]. Superfusion of CNQX completely blocked the NMDA-independent depolarizing component indicating that it is mediated by activation of the non-NMDA receptors. The present result suggests that both N M D A and non-NMDA receptors are involved in spontaneous and evoked epileptiform activity induced by Mg2+-free solution in BLA neurons. It is likely that removal of extracellular Mg 2+ may increase Ca 2+ channel activity or reduce the inhibitory action of adenosine at presynaptic terminals which results in enhanced transmitter release. The rapid depolarization produced by the activation of non-NMDA receptors would bring the membrane potential above the threshold at which the blocking action of Mg 2+ on N M D A channel is reduced. The subsequent recruitment of N M D A receptors would then add and prolong the epileptiform activity. Finally, this study as well as those of others [9] suggest that CNQX is not effective in blocking bursts induced by N M D A agonists or in Mg2+-free solution. On the other hand, epileptiform activity induced by kainate or 4-aminopyridine could be blocked by CNQX [4, 12] while NMDA receptor antagonists were without effect [13]. It appears that the ability of CNQX or o-APV to suppress epileptiform bursts depends on which types of excitatory amino acid receptors are involved. In MgE+-free model of epilepsy, N M D A receptor activation accounts for approximately 83% of the epileptiform activity, therefore, D-APV is more effective to suppress these bursts. In contrast, non-NMDA receptors play a major role in kainate or 4-aminopyridine induced epileptiform discharges, thus, CNQX is more effective in blocking this type of bursts. I thank Dr. J.D. Huang for reading the manuscript. This work was supported by the National Science Council of Taiwan, R.O.C. (NSC 79-0412-B006-43).

1 Andreasen, M., Lambert, J.D.C. and Jensen, M.S., Effects of new non-N-methyl-o-aspartate antagonists on synaptic transmission in the in vitro rat hippocampus, J. Physiol., 414 (1989) 317-336. 2 Collingridge, G.L. and Lester, R.A., Excitatory amino acid receptors in the vertebrate central nervous system, Pharmacological Reviews, 40 (1989) 143-210. 3 Gallagher, J.P. and Hasuo, H., Excitatory amino acid-receptormediated EPSPs in rat dorsolateral septal nucleus neurons in vitro, J. Physiol., 418 (1989) 353-365. 4 Gean, P.W., Chou, S.M. and Chang, F.C., Epileptiform activity induced by 4-aminopyridine in rat amygdala neurons: the involvement of N-methyl-D-aspartate receptors, Eur. J. Pharmacol., in press. 5 Gean, P.W. and Shinnick-Gallagher, P., Epileptiform activity induced by magnesium-free solution in slices of rat amygdala: antagonism by N-methyl-v-aspartate receptor antagonists, Neuropharmacology, 27 (1988) 557-562. 6 Gean, P.W. and Shinnick-Gallagber, P., The transient potassium current, the A-current, is involved in spike frequency adaptation in rat amygdala neurons, Brain Res., 480 (1989) 160-169. 7 Hamon, B., Stanton, P.K. and Heinemann, U., An N-methyl-Daspartate receptor-independent excitatory action of partial reduction of extracellular [Mg2+] in CAI region of rat hippocampal slices, Neurosei. Lett., 75 (1987) 240-245. 8 Honore, T., Davies, S.N., Drejer, J., Fletcher, E.J., Jacobsen, P., Lodge, D. and Nielsen, F.E., Quinoxalinediones: potent competitive non-N-methyl-D-aspartateglutamate receptor antagonists, Science, 241 (1988) 701-703. 9 Jensen, L.H. and Sheardown, M., Lack of potent antiepileptic effect of specific non-NMDA EAA receptor antagonist. In E.A. Cavalheiro, J. Lehmann and L. Turski (Eds.), Frontiers in Excitatory Amino Acid Research, Alan R. Liss, New York, 1988, pp. 219-226. 10 Mayer, M.L., Westbrook, G.L. and Guthrie, P.B., Voltage-dependent block by Mg 2+ of NMDA responses in spinal cord neurons, Nature, 309 (1984) 261-263. 11 Mody, I., Lambert, J.D.C. and Heinemann, U., Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices, J. NeurophysioL, 57 (1987) 869-888. 12 Neuman, R.S., Ben-Aft, Y. and Cherubini, E., Antagonism of spontaneous and evoked bursts by 6-cyano-7-nitroquinoxaline-2,3dione (CNQX) in the CA3 region of the in vitro hippocampus, Brain Res., 474 (1988) 201-203. 13 Neuman, R., Cherubini, E. and Ben-Aft, Y., Epileptiform bursts elicited in CA3 hippocampal neurons by a variety of convulsants are not blocked by N-methyl-v-aspartate antagonists, Brain Res., 459 (1988) 265-274. 14 Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A., Magnesium gates giutamate-activated channels in mouse central neurons, Nature, 307 (1984) 462--465. 15 Ottersen, O.P., Fischer, B.O., Rinvik, E. and Storm-Mathisen, J., Putative amino acid transmitters in the amygdala. In R. Schwarcz and Y. Ben-Ari (Eds.), Excitatory Amino Acids and Epilepsy, Plenum, New York, 1986 pp. 53~6. 16 Stone, T.W., Connick, .I.H. and Bartrup, J.T., NMDA-receptor-independent effects of low magnesium: involvement of adenosine, Brain Res., 508 (1990) 333-336. 17 Thomson, A.M. and West, D.C., N-methylaspartate receptors mediate epileptiform activity evoked in some, but not all conditions in rat neocortical slices, Neuroscience, 19 (1986) 1161-1177.

NMDA receptor-independent epileptiform activity induced by magnesium-free solution in rat amygdala neurons is blocked by CNQX.

The effect of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a specific non-N-methyl-D-aspartate (non-NMDA) receptor antagonist, on NMDA-independent epi...
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