Brain Revearch Bullerm, Printed in the USA.
Vol. 28,
pp. 417-42 I, 1992 Copyright
0363.9230192 $5.00 i .OO ((.I 1992 Pergamnn Press Ltd.
Ethanol Inhibits Epileptiform Activity and NMDA Receptor-Mediated Synaptic Transmission in Rat Amygdaloid Slices PO-WU
Department
GEAN
ofPharmacology, College qf‘,illedicinc, Nutional C’heng-Kmg C’nivcrsir~~. Taimn City, Taiwan 70101, R.U.C. Received
24 May
1991
GEAN, P-W. Ethanol inhibits epilept(/bn actid), und NMD.4 rt~~i~~?tor-mpdiatEd .s~xqtic transmission in rut awqyluloid sliw BRAIN RES BULL 28(3) 4 17-42 I. 1992.-The effect of ethanol on the epileptiform activity induced by Mg++-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg’ ‘-free medium. Superfusion with ethanol (20-100 mM) reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentrationdependent manner. Synaptic response mediated by N-mcthyI-D-aspa~ate (NMDA) receptor activation was isolated hy application of a solution containing the non-~MDA receptor antagonist ~~yano-?-nitr~uinoxaiine-2.3-dione (CNQX) and either in Mg’ ‘+ free solution or in the presence of 50 PM bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation. In addition, the observed effect ofethanol on NMDA receptor-mediated synaptic response could be relevant to the cognitive and behavioral manifestations seen in some alcoholics. Ethanol
NMDA receptors
Epileptiform activity
Amygdala
-THE mechanisms underIying the effects of ethanol in the central nervous system (CNS) are not fully understood, although many studies implicate GABA,ergic action of ethanol (3,19). However. more evidence has been accumulated to suggest the involvement of excitatory amino acid system(s) in the action of ethanol (8). For example, ethanol has been reported to inhibit N-methyl-Daspartate (NMDA)- and glutamate-evoked [‘Hlnoradrenaline release in the rat brain cortex (9,lO) and block NMDA-induced ion current in cultured hippo~ampal neurons (1516). Ethanol has also been shown to block tetanic and calcium-induced longterm potentiation (LTP) (2,27), a phenomenon associated with NMDA receptor activation (4,22). Furthermore, in vivo experiments showed that ethanol reduced the severity and duration of convulsion and provided protection against mortality following convulsions induced by picrotoxin and NMDA. The anticonvulsant effect of ethanol could be partly attributed to its ability to antagonize NMDA-mediated excitatory response (14). The ion currents activated by the NMDA class of glutamate receptors are gated in a voItage-de~ndent manner by Mgif (l&23). When Mg’+ is removed from the perfusate. the NMDA receptor system can be activated to trigger bursting discharges; this is currently used as an in vitro model of epilepsy (1,6,20). This study examined the effect of ethanol on the epileptiform
activity induced in Mg’+-free medium in rat basolateral amygdata (BLA) neurons. I found that ethanol possesses anticonvulsant activity through its blocking action on the NMDA receptormediated synaptic transmission. METHOD
In this study. intracellular recordings were made from brain slices ofthe amygdala complex. The preparation of the amygdaiar slices was similar to that reported previously (5). In brief. male Sprague-Dawley rats weighing 125-200 g were decapitated using a guillotine. and the brains were removed rapidly from the skull. Transverse slices of 500-wrn thickness were cut and the appropriate slices were placed in a beaker of artificial cerebrospinal fluid (ACSF) gassed with 95% OZ. 5% COZ at room temperature for at least I h before recording. A single slice was then transferred to the recording chamber, where it was held submerged between two nylon nets. A bipolar stimulating electrode, which was insulated except at the tip, was placed in the ventral endopy~form nucleus and stimulated with monophasic constant voltage pulses from a Grass S88 stimulator. Stimulus intensities varied between 5 and 50 V with a pulse duration ranging from 50 to 100 ps. The slice was maintained at 32 + 1“C and superfused with ox-
Requests for reprints should be addressed to PO-Wu Gean, PhD. Department of Pharmacology. College of Medicine. National Cheng-Kung University. Tainan City, Taiwan 70 101. R.O.C.
417
418
ygenated ACSF having the following composition (in mM): NaCl 117, KCI 4.7, CaCl, 2.5, MgC& 1.2. NaHCOs 25, NaH2P04 1.2, and glucose Il. Intracellular recordings were obtained from neurons of the basolateral amygdala nucleus using conventional intracellular recording techniques. Microelectrodes were pulled from microfiber-filled l.O-mm capillary tubing on a Brown-Flaming electrode puller (Sutter Instruments). Electrodes were filled with 4 M potassium acetate with resistance ranging from 90- I50 M!& Electrical signals were amplified using an Axoclamp 2A amplifier and recorded on a Gould 3200 chart recorder. Very fast transient potentials, which could not be adequately resolved by chart recorder, were digitized using a Data-6100 digital oscilloscope (Data Precision Co.) and were reproduced on a Hewlett-Packard ColorPro graphics plotter. All data were expressed as mean + SE. Statistic analysis was performed using the paired Student’s t test, and a p value of less than 0.05 was considered to be statistically significant. DL-2-amino-5-phosphonovaleate (DL-APV, Sigma), bicuculline methiodide (BMI, Sigma), ethanol (Merck), and 6-cyano7-nitroquinoxaline-2,3-dione (CNQX, Cambridge Research Biochemicals) of known concentrations were delieved to the slices by switching a three-way stopcock to an alternate reservoir.
,n=*,
(“CO,
(i,.F,
FIG. 2. Graphic analysis of the effect of ethanol on the epileptiform activity induced in Mg++-free solution. Bar graphs represent the mean + SE of the duration as a percentage of control responses. *p < 0.05 vs. control. **p < 0.02 vs. control. ***,LI< 0.005 vs. control. Duration of epileptiform activity was measured as the time from the initial depolarization to 90% of its decay phase.
The control and test solutions were exchanged completely in the recording chamber within a few minutes after switching the taps. The Mg++-free solution was made by eliminating MgCl* without replacement.
Mg*-Fr-Free
A RESULTS
Ethanol 1OOmM
B
C
0
I
20 mV
20s
Wash
Mg*Free
FIG. 1. Effect of ethanol on the epileptiform activity induced in Mg++free medium (A). Chart records of spontaneous bursts in a BLA neuron 40 min after superfusion with Mg++-freesolution. Action potential amplitude was attenuated by chart recorder frequency response (B). Application of ethanol ( 100 mM) reduced the duration of epileptiform activity (C and D) are continuous records showing that the duration of the epileptiform activity gradually returned to control level when ethanol was washed out of the recording chamber. The resting membrane potential (RMP) of this neuron was -65 mV.
Stable intracellular recordings were obtained in 38 neurons with resting membrane potential and neuronal input resistance of -65 + I mV (n = 38) and 43 + 2 Ma (n = 28). respectively. Twenty to thirty minutes after switching to Mg++-free medium, spontaneous and evoked epileptiform bursts were observed in 34 of 38 neurons tested. Superfusion of ethanol (100 mM) reversibly reduced the duration of epileptifarm activity. Fig. 1 shows intracellular recordings of spontaneous epileptiform bursts before, during, and after application of ethanol. At a concentration of 100 mM, ethanol shortened the duration of epiieptiform activity (measured as the time from initial depolarization to the 90% of its decay phase) by an average of 48.1% ( 12 10 +- 1 IO ms before and 628 + 76 ms after the superfusion of 100 mM ethanol, p < 0.005, n = 9). At 50 mM, ethanol produced a 40.6% (1083 + 164 ms before and 643 + 114 ms after the application of ethanol, p < 0.02, n = 8) reduction of the duration. The inhibition observed with 20 mM ethanol was 20.7% (1100 + 146 ms before and 872 + 142 ms after the superfusion of ethanol, p < 0.05, n = 5) (Fig. 2). The effect of ethanol was fully reversible, as illustrated in Figs. 1C and D. The duration of the epileptiform activity gradually returned to control level when ethanol was washed out of the recording chamber. The duration of epileptiform burst triggered by a single stimulus was also reduced in the presence of ethanol (Fig. 3). In 12 neurons, the duration of single-stimulus-evoked epileptiform activity in Mg++-free solution was 12 15 + 177 ms. Superfusion of ethanol at a concentration of 100 mM reduced the duration by an average of 52.7% (575 + 90 ms after the superfusion of ethanol, p < 0.001, n = 12). I have shown that in the Mg++-free model of epilepsy, NMDA receptor activation accounts for approximately 83% of the duration of epileptiform activity (7). Thus, the suppression of epileptiform activity by ethanol may be due to ethanol’s blocking
ETHANOL
A
INHIBITS
EPILEPTIFORM
419
ACTIVITY
Mg”Free
Ethanol 1OOmM
1
20mV
Wash
polarizing potential could be reversibly abolished by DL-APV (50 FM), indicating that it is mediated by NMDA receptors (Fig. 5C). Fig. 5E showed that superfusion of ethanol suppressed the NMDA receptor-mediated synaptic responses. On average, 100 mM ethanol produced a 52.9 ? 5.3% (n = 7, p < 0.02) reduction in the amplitude of NMDA receptor-mediated synaptic component. The degree of inhibition is similar to ethanol-induced inhibition of NMDA receptor-mediated svnaptic response in CNQX and Mg+ ‘-free solution. This indicates that the inhibition of the NMDA receptor-mediated synaptic response by ethanol in the experiments carried out in Mg’ +-free solution was not dependent on the low Mg’ ’ concentration. The present result also shows that ethanol was effective in suppressing the amplitude of the NMDA receptormediated synaptic response in the presence of the GABA* receptor antagonist bicuculline. In agreement with the observation in hippocampal neurons, superfusion of ethanol (SO-100 mM) produces no consistent effect on the resting membrane potentials (26). In I6 of 34 BLA neurons tested. resting membrane potentials were unchanged, whereas IO neurons were weakly depolarized (range = 2-8 mV, mean = 3.9 mV). Hyperpolarization. usually in a small ampli-
*I!
Ma”- Free +CNQX
B
FIG. 3. Ethanol shortened the duration of evoked epileptiform bursts. (A) In the absence of magnesium. orthodromic stimulation evoked an epileptiform burst. (B and C) Application of ethanol (100 mM) reversibly suppressed the epileptiform burst. Filled triangles represent point of stimulus. RMP = ~62 mV.
Mg”-Free+CNClX+D-APV
C Mg--F,ee + CNQX
action on the NMDA receptors. To test this possibility, I applied CNQX to the Mg++-free medium to isolate the NMDA receptormediated synaptic component. As depicted in Fig. 4B. the depolarizing wave that remained in the presence of CNQX was abolished by DL-APV, indicating that it is an NMDA receptormediated synaptic response. Application of ethanol (100 mM) then reversibly suppressed the NMDA receptor-mediated synaptic response. Figs. 4C, D, and E show intracellular recordings of NMDA receptor-mediated synaptic responses before, during, and after the superfusion of ethanol. At a concentration of 100 mM, ethanol shortened the duration of NMDA receptor-mediated synaptic response by an average of 50.9% (489 * 56 ms before and 240 ? 33 ms after the superfusion of 100 mM ethanol, n = IO, p < 0.001). Often, the plateau amplitude of the depolarizing wave was also depressed. To determine if an inhibition of the NMDA receptor-mediated synaptic response does not result from a secondary effect of ethanol that enhances Gamma-aminobutyric acid (GABA)-mediated inhibition. I examined the action of ethanol on synaptic response recorded in 8 PM CNQX. I .2 As illustrated in Fig. 5A, mM Mg+‘. and 50 FM bicuculline. bicuculline induced epileptiform activity in normal Mg++ solution. Superfusion of CNQX (8 PM) suppressed the epileptiform activity; however, a depolarizing potential persisted in the presence of CNQX (Fig. 5B). This CNQX-resistant de-
D Ethanol ,OOmM
E Wash
II
FIG. 4. Reversible inhibition of NMDA receptor-mediated synaptic excitation by ethanol (A). A triggered response in the presence of CNQX and Mg”-free solution (B and C). The CNQX-resistant depolarizing wave was reversibly blocked by D-APV (50 PM) (D and E). Superfusion of ethanol ( 100 mM) reversibly suppressed the NMDA receptor-mediated synaptic excitation. All records were taken from the same cell. RMP =
-64 mV.
B BYI+ CNQX
I I
C
0 BMI*CNQX+APV
E
Eth.“ol
BYI+CNQX
F
W.lh -I 50ms
zomv
G
_I
IOrnV
SOIllS
FIG. 5. Ethanol inhibition of NMDA-mediated synaptic response in the presence ofCNQX, 1.2 mM Mg++,and bicuculline. (A) A burst response evoked by single stimulus pulse in the presence of bicuculline methiodide (BMI, 5d PM). (B) A depolarizing potential remained after application of CNQX (8 PM) (C and D). The CNQX-resistant depolarizing potential was reversibly abolished by DLAPV (50 NM). (E and F) Superfusion of ethanol (100 mM) reversibly suppressed NMDA receptor-mediated synaptic response. (G) Superimposed traces of B, C, D. E, and F. All records
were taken from the same cell. RMP = -70 mV.
tude (2-6 mV, mean = 3.3 mV), was seen in 8 neurons. This could be explained by ethanol’s dual effects of blocking the Mcurrent (21) while inhibiting NMDA-activated currents (25), which depolarized and hyperpolarized the membrane potentials, respectively. No profound changes in apparent input resistance were noted. DISCUSSION
The present study demonstrates that ethanol reversibly shortened the duration of evoked epileptiform activity induced in Mg++-free medium in neurons of rat basolateral amygdaloid nucleus. The duration of spontaneous bursts was also reduced and the effect was concentration dependent. The epileptiform activity induced by Mg++-free solution has two components: one is sensitive to D-APV and the other is independent of NMDA receptor activation (7,ll). In this study, the inhibition of epileptiform activity by ethanol correlated well with its blockade of the NMDA receptor-mediated synaptic excitation. Thus, it appears likely that the depression of epileptiform activity by ethanol is due primarily to ethanol’s blocking action on the NMDA receptors. However, the mechanism by which ethanol produces its effect on the NMDA response remains unknown. An allosteric site which is reg-
ulated by glycine has been proposed to be mvolved in this action of ethanol (24,28). Further study is necessary to determine if glycine can reverse ethanol’s inhibition of NMDA receptor-mediated synaptic response. The introduction of CNQX that selectively blocks nonNMDA excitatory amino acid receptors (12) allows us to directly test the effect of ethanol on the NMDA receptor-mediated synaptic response. Two types of experiments were performed. First, I examined the effect of ethanol on the synaptic response recorded in the presence of CNQX and Mg’ ‘-free solution. 1 found that 100 mM ethanol inhibited the synaptic response under this condition by 50.9%. Note, however, that this experiment could not exclude the possibility that the inhibition of NMDA receptor-mediated synaptic transmission by ethanol might be mediated through a GABAergic mechanism because it has been reported that ethanol can potentiate the openings of GABA, receptor-gated chloride channels (3,19). Accordingly. I carried out a second study in which the NMDA receptormediated synaptic response was isolated by superfusing the amygdaloid slices with CNQX and bicuculline. Application of ethanol again reversibly suppressed the amplitude of NMDA receptor-mediated synaptic response by 52.94. The degree of inhibition was similar to that produced by ethanol in the absence of bicuculline, suggesting that the depression of NMDA receptor-mediated synaptic response by ethanol does not depend on GABAergic transmission. Ethanol has been shown to selectively block ion currents activated by NMDA in hippocampal cells ( 15,I6) and it is suggested that NMDA receptors may be a plausible neurochemical target for mediating certain pharmacological effects of ethanol (8). Indeed, more evidence has been accumulated so NMDA-stimulated norepinephrine release from rat cortical slices was inhibited by ethanol (9,lO). Furthermore, Lovinger et al. showed that ethanol suppressed the NMDA receptor-mediated population EPSPs in rat hippocampal slices (17). Using the intracellular recording technique, the present result extends this observation by showing that the NMDA receptor-mediated synaptic excitation is depressed in the presence of ethanol in neurons of rat basolateral amygdaloid nucleus. A blood ethanol level of 100 mg/dl, corresponding to tissue concentration of 22 mM, is considered being under the influence of intoxicating beverages. Ataxia, impaired mental and motor skills, and impaired short-term memory can be detected at blood concentrations of 100-150 mg/dl (22-33 mM). At higher concentrations of 250-500 mg/dl (54- 108 mM), ethanol produces coma and has a lethal effect ( 13). In the present study, the observation that high concentrations of ethanol (20-100 mM) reduce the NMDA receptor-mediated synaptic response suggests that inhibition of NMDA receptor activation might contribute to ethanol’s intoxicating effects. In conclusion, my results suggest that intoxicating doses of ethanol possess anticonvulsant activity through ethanol’s blocking action on the NMDA receptor-mediated synaptic excitation. Because of the large body of data implicating the role of the NMDA receptor in long-term potentiation (4,22), the observed inhibitory effect of ethanol on the NMDA receptor-mediated response could contribute to the cognitive impairments associated with toxication. ACKNOWLEDGEMENT
This work was supported by the National Science Council of Taiwan, R.O.C. (NSC 80-0412~BOO6-41).
ETHANOL
INHIBITS
EPILEPTIFORM
421
ACTIVITY REFERENCES
1. Anderson. W. W.: Lewis, D. V.; Swartzwelder, H. S.: Wilson. W. A. Magnesium-free medium activates seizure-like events in the rat hippocampal slice. Brain Res. 398:2 15-2 19: 1986. 2. Blitzer. R. D.: Gil, 0.; Landau. E. M. Long-term potentiation in rat hippocampus is inhibited by low concentrations of ethanol. Bram Res. 537:203-20X; 1990. 3. Celantano, J. J.: Gibbs. T. T.: Farb. D. H. Ethanol potentiates GABAand glycine-induced chloride currents in chick spinal cord neurons. Brain Res. 455:377-380: 1988. 4. Collingridge. Ci. L.: Bliss. 7‘. V. P. NMDA receptors-their role in long-term potentiation. Trends Neurosci. 10:288-293; 1987. 5. Gean. P. W.: Chou. S. M.; Chang, F. C. Epileptiform activity induced by 4-aminopyridine in rat amygdala neurons: the involvement of Nmethyl-D-aspartate receptors. Eur. J. Pharmacol. 184:2 13-222: 1990. 6. Clean. P. W.; Shinnick-Gallagher. P. Epileptiform activity induced by magnesium-free solution in slices of rat amygdala: antagonism by N-methyl-D-aspartate receptor antagonists. Neuropharmacology 271557-562; lY88. 7. Gean. P. W.. NMDA receptor-independent epileptiform activity induced by magnesium-free solution in rat amygdala neurons is blocked by CNQX. Neurosci. Lett. 119:53-55: 1990. 8. Gonzales, R. A. NMDA receptors excite alcohol research. Trends Pharmacol. Sci. I I : 137- 139: 19YO. 9. Gonzales. R. A.: Woodward. J. J. Ethanol inhibits N-methyl-Daspartate-stimulated [‘Hlnorepinephrine release from rat cortical slices. J. Pharmacol. Exp. Ther. 253: 1138- 1 144: 1990. 10. Ciothert. M.: Fink. K. Inhibition of N-methyl-D-aspartate (NMDA)and glutamate-induced norepinephrine and acetylcholine release in the rat brain by ethanol. N-S Arch. Pharmacol. 340:5 16-52 1: 1989. I I. Hamon. B.: Stanton, P. K.: Heinemann. U. An N-methyl-D-aspartate receptor-independent excitatory action of partial reduction of extracellular [Mg’ ‘] in CAlregion of rat hippocampal slices. Neurosci. Lett. 75:240-235; 1987. 12. Honorc. T.: Davis. S. N.; Drejer. J.: Fletcher. E. J.: Jacobsen, P.: Lodge. D.: Nielsen. F. E. Quinoxalinediones: potent competitive non-N-methyl-D-aspartate glutamate receptors antagonists. Science 241:701-703: 1988. I?. Hunt. W. A. Ethanol and other aliphatic alcohols. In: Craig. L. R.; Stitzel, R. E., eds. Modern pharmacology. Boston: Little. Brown and Company: lYYO:533-541. 14. Kulkarni. S. K.: Mehta. A. K.: Ticku. M. K. Comparison of anticonvulsant cfIect of ethanol against NMDA-, kainic acid- and picrotoxin-mduccd convulsions in rats. Life Sciences 46:48 l-487: 1990.
M. T. R.; Albuquerque, E. X. Ethanol potentiates 15. Lima-Landman. and blocks NMDA-activated single-channel currents in rat hippocampal pyramidal cells. FEBS Lett. 2476 l-67: 1989. 16. Lovinger. D. M.: White, G.: Weight, F. F. Ethanol inhibits NMDAactivated ion current in hippocampal neurons, Science 243: 172 l1724: 1989. 17. Lovinger. D. M.: White, C.: Weight. F. F. NMDA receptor-mediated synaptic excitation selectively inhibited by ethanol in hippocampal slice from adult rat. J. Neurosci. 10: 1372- 1379: 1990. 18. Mayer. M. L.; Westbrook, G. L.; Guthrie. P. B. Voltage-dependent block by Mg’ ’ of NMDA responses in spinal cord neurons. Nature 309:261-263: 1984. of GABAergic 19. Mehta. A. K.: Ticku. M. K. Ethanol potentiation transmission in cultured spinal cord neurons involves gamma-aminobutyric acid,-gated chloride channels. J. Pharmacol. Exp. Ther. 246:558-564: 198X. 20. Mody. I.; Lambert. J. D. C.; Heinemann. U. Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices. J. Neurophysiol. 57:X69-888: 1987. 21. Moore. S. D.: Madamba. S. Cl.: Siggins. G. R. Ethanol diminished a voltage-dependent K’ current. the M-current. in CA 1 hippocampal pyramidal neurons in vitro. Brain Res. 5 16222-228: 1990. 22. Nicoll. R. A.; Kauer, J. .4.; Malenka. R. C. The current excitement in long-term potentiation. Neuron I :97- 103: 1988. 23. Nowak, L.; Bregestovski. P.: Ascher. P.; Herbet. A.: Prochiantr. A. Magnesium gates glutamate-activated channels in mouse central neurons. Nature 307:462-465; 1984. 24. Rabe. C. S.: Tabakoff. B. Glycine site-directed agonists reverse the actions of ethanol at the N-methyl-D-aspartate receptor. Mol. Pharmacol. 38:753-757: 1990. 25. Sah. P.: Hestrin. S.: Nicoll. R. A. Tonic activation of NMDA receptors by ambient glutamate enhances cxcitahility of neurons. Science 246:815-817; 198’). 26. S&ins. Cl. R.: Pittman, Q. J.: French. E. D. Effects of ethanol on CA 1 and CA3 pyramidal cells in the hippocampal slice preparation: an intracellular study. Brain Res. 414:22-34: 19X7. 27. Sinclair, J. G.: Lo, G. F. Ethanol blocks tetanic and calcium-induced long-term potentiation in the hippocampal slice. Gen. Pharmacol. 17231-233: 1986. 28. Woodward. J. J.: Gonzales. R. A. Ethanol inhibition of N-methylD-aspartate-stimulated endogenous dopamine release from rat striatal slices: reversal by glycine. J. Neurochem. 54:7 12-7 15: 1990.
Vol. 28,
pp. 417-42 I, 1992 Copyright
0363.9230192 $5.00 i .OO ((.I 1992 Pergamnn Press Ltd.
Ethanol Inhibits Epileptiform Activity and NMDA Receptor-Mediated Synaptic Transmission in Rat Amygdaloid Slices PO-WU
Department
GEAN
ofPharmacology, College qf‘,illedicinc, Nutional C’heng-Kmg C’nivcrsir~~. Taimn City, Taiwan 70101, R.U.C. Received
24 May
1991
GEAN, P-W. Ethanol inhibits epilept(/bn actid), und NMD.4 rt~~i~~?tor-mpdiatEd .s~xqtic transmission in rut awqyluloid sliw BRAIN RES BULL 28(3) 4 17-42 I. 1992.-The effect of ethanol on the epileptiform activity induced by Mg++-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg’ ‘-free medium. Superfusion with ethanol (20-100 mM) reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentrationdependent manner. Synaptic response mediated by N-mcthyI-D-aspa~ate (NMDA) receptor activation was isolated hy application of a solution containing the non-~MDA receptor antagonist ~~yano-?-nitr~uinoxaiine-2.3-dione (CNQX) and either in Mg’ ‘+ free solution or in the presence of 50 PM bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation. In addition, the observed effect ofethanol on NMDA receptor-mediated synaptic response could be relevant to the cognitive and behavioral manifestations seen in some alcoholics. Ethanol
NMDA receptors
Epileptiform activity
Amygdala
-THE mechanisms underIying the effects of ethanol in the central nervous system (CNS) are not fully understood, although many studies implicate GABA,ergic action of ethanol (3,19). However. more evidence has been accumulated to suggest the involvement of excitatory amino acid system(s) in the action of ethanol (8). For example, ethanol has been reported to inhibit N-methyl-Daspartate (NMDA)- and glutamate-evoked [‘Hlnoradrenaline release in the rat brain cortex (9,lO) and block NMDA-induced ion current in cultured hippo~ampal neurons (1516). Ethanol has also been shown to block tetanic and calcium-induced longterm potentiation (LTP) (2,27), a phenomenon associated with NMDA receptor activation (4,22). Furthermore, in vivo experiments showed that ethanol reduced the severity and duration of convulsion and provided protection against mortality following convulsions induced by picrotoxin and NMDA. The anticonvulsant effect of ethanol could be partly attributed to its ability to antagonize NMDA-mediated excitatory response (14). The ion currents activated by the NMDA class of glutamate receptors are gated in a voItage-de~ndent manner by Mgif (l&23). When Mg’+ is removed from the perfusate. the NMDA receptor system can be activated to trigger bursting discharges; this is currently used as an in vitro model of epilepsy (1,6,20). This study examined the effect of ethanol on the epileptiform
activity induced in Mg’+-free medium in rat basolateral amygdata (BLA) neurons. I found that ethanol possesses anticonvulsant activity through its blocking action on the NMDA receptormediated synaptic transmission. METHOD
In this study. intracellular recordings were made from brain slices ofthe amygdala complex. The preparation of the amygdaiar slices was similar to that reported previously (5). In brief. male Sprague-Dawley rats weighing 125-200 g were decapitated using a guillotine. and the brains were removed rapidly from the skull. Transverse slices of 500-wrn thickness were cut and the appropriate slices were placed in a beaker of artificial cerebrospinal fluid (ACSF) gassed with 95% OZ. 5% COZ at room temperature for at least I h before recording. A single slice was then transferred to the recording chamber, where it was held submerged between two nylon nets. A bipolar stimulating electrode, which was insulated except at the tip, was placed in the ventral endopy~form nucleus and stimulated with monophasic constant voltage pulses from a Grass S88 stimulator. Stimulus intensities varied between 5 and 50 V with a pulse duration ranging from 50 to 100 ps. The slice was maintained at 32 + 1“C and superfused with ox-
Requests for reprints should be addressed to PO-Wu Gean, PhD. Department of Pharmacology. College of Medicine. National Cheng-Kung University. Tainan City, Taiwan 70 101. R.O.C.
417
418
ygenated ACSF having the following composition (in mM): NaCl 117, KCI 4.7, CaCl, 2.5, MgC& 1.2. NaHCOs 25, NaH2P04 1.2, and glucose Il. Intracellular recordings were obtained from neurons of the basolateral amygdala nucleus using conventional intracellular recording techniques. Microelectrodes were pulled from microfiber-filled l.O-mm capillary tubing on a Brown-Flaming electrode puller (Sutter Instruments). Electrodes were filled with 4 M potassium acetate with resistance ranging from 90- I50 M!& Electrical signals were amplified using an Axoclamp 2A amplifier and recorded on a Gould 3200 chart recorder. Very fast transient potentials, which could not be adequately resolved by chart recorder, were digitized using a Data-6100 digital oscilloscope (Data Precision Co.) and were reproduced on a Hewlett-Packard ColorPro graphics plotter. All data were expressed as mean + SE. Statistic analysis was performed using the paired Student’s t test, and a p value of less than 0.05 was considered to be statistically significant. DL-2-amino-5-phosphonovaleate (DL-APV, Sigma), bicuculline methiodide (BMI, Sigma), ethanol (Merck), and 6-cyano7-nitroquinoxaline-2,3-dione (CNQX, Cambridge Research Biochemicals) of known concentrations were delieved to the slices by switching a three-way stopcock to an alternate reservoir.
,n=*,
(“CO,
(i,.F,
FIG. 2. Graphic analysis of the effect of ethanol on the epileptiform activity induced in Mg++-free solution. Bar graphs represent the mean + SE of the duration as a percentage of control responses. *p < 0.05 vs. control. **p < 0.02 vs. control. ***,LI< 0.005 vs. control. Duration of epileptiform activity was measured as the time from the initial depolarization to 90% of its decay phase.
The control and test solutions were exchanged completely in the recording chamber within a few minutes after switching the taps. The Mg++-free solution was made by eliminating MgCl* without replacement.
Mg*-Fr-Free
A RESULTS
Ethanol 1OOmM
B
C
0
I
20 mV
20s
Wash
Mg*Free
FIG. 1. Effect of ethanol on the epileptiform activity induced in Mg++free medium (A). Chart records of spontaneous bursts in a BLA neuron 40 min after superfusion with Mg++-freesolution. Action potential amplitude was attenuated by chart recorder frequency response (B). Application of ethanol ( 100 mM) reduced the duration of epileptiform activity (C and D) are continuous records showing that the duration of the epileptiform activity gradually returned to control level when ethanol was washed out of the recording chamber. The resting membrane potential (RMP) of this neuron was -65 mV.
Stable intracellular recordings were obtained in 38 neurons with resting membrane potential and neuronal input resistance of -65 + I mV (n = 38) and 43 + 2 Ma (n = 28). respectively. Twenty to thirty minutes after switching to Mg++-free medium, spontaneous and evoked epileptiform bursts were observed in 34 of 38 neurons tested. Superfusion of ethanol (100 mM) reversibly reduced the duration of epileptifarm activity. Fig. 1 shows intracellular recordings of spontaneous epileptiform bursts before, during, and after application of ethanol. At a concentration of 100 mM, ethanol shortened the duration of epiieptiform activity (measured as the time from initial depolarization to the 90% of its decay phase) by an average of 48.1% ( 12 10 +- 1 IO ms before and 628 + 76 ms after the superfusion of 100 mM ethanol, p < 0.005, n = 9). At 50 mM, ethanol produced a 40.6% (1083 + 164 ms before and 643 + 114 ms after the application of ethanol, p < 0.02, n = 8) reduction of the duration. The inhibition observed with 20 mM ethanol was 20.7% (1100 + 146 ms before and 872 + 142 ms after the superfusion of ethanol, p < 0.05, n = 5) (Fig. 2). The effect of ethanol was fully reversible, as illustrated in Figs. 1C and D. The duration of the epileptiform activity gradually returned to control level when ethanol was washed out of the recording chamber. The duration of epileptiform burst triggered by a single stimulus was also reduced in the presence of ethanol (Fig. 3). In 12 neurons, the duration of single-stimulus-evoked epileptiform activity in Mg++-free solution was 12 15 + 177 ms. Superfusion of ethanol at a concentration of 100 mM reduced the duration by an average of 52.7% (575 + 90 ms after the superfusion of ethanol, p < 0.001, n = 12). I have shown that in the Mg++-free model of epilepsy, NMDA receptor activation accounts for approximately 83% of the duration of epileptiform activity (7). Thus, the suppression of epileptiform activity by ethanol may be due to ethanol’s blocking
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polarizing potential could be reversibly abolished by DL-APV (50 FM), indicating that it is mediated by NMDA receptors (Fig. 5C). Fig. 5E showed that superfusion of ethanol suppressed the NMDA receptor-mediated synaptic responses. On average, 100 mM ethanol produced a 52.9 ? 5.3% (n = 7, p < 0.02) reduction in the amplitude of NMDA receptor-mediated synaptic component. The degree of inhibition is similar to ethanol-induced inhibition of NMDA receptor-mediated svnaptic response in CNQX and Mg+ ‘-free solution. This indicates that the inhibition of the NMDA receptor-mediated synaptic response by ethanol in the experiments carried out in Mg’ +-free solution was not dependent on the low Mg’ ’ concentration. The present result also shows that ethanol was effective in suppressing the amplitude of the NMDA receptormediated synaptic response in the presence of the GABA* receptor antagonist bicuculline. In agreement with the observation in hippocampal neurons, superfusion of ethanol (SO-100 mM) produces no consistent effect on the resting membrane potentials (26). In I6 of 34 BLA neurons tested. resting membrane potentials were unchanged, whereas IO neurons were weakly depolarized (range = 2-8 mV, mean = 3.9 mV). Hyperpolarization. usually in a small ampli-
*I!
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B
FIG. 3. Ethanol shortened the duration of evoked epileptiform bursts. (A) In the absence of magnesium. orthodromic stimulation evoked an epileptiform burst. (B and C) Application of ethanol (100 mM) reversibly suppressed the epileptiform burst. Filled triangles represent point of stimulus. RMP = ~62 mV.
Mg”-Free+CNClX+D-APV
C Mg--F,ee + CNQX
action on the NMDA receptors. To test this possibility, I applied CNQX to the Mg++-free medium to isolate the NMDA receptormediated synaptic component. As depicted in Fig. 4B. the depolarizing wave that remained in the presence of CNQX was abolished by DL-APV, indicating that it is an NMDA receptormediated synaptic response. Application of ethanol (100 mM) then reversibly suppressed the NMDA receptor-mediated synaptic response. Figs. 4C, D, and E show intracellular recordings of NMDA receptor-mediated synaptic responses before, during, and after the superfusion of ethanol. At a concentration of 100 mM, ethanol shortened the duration of NMDA receptor-mediated synaptic response by an average of 50.9% (489 * 56 ms before and 240 ? 33 ms after the superfusion of 100 mM ethanol, n = IO, p < 0.001). Often, the plateau amplitude of the depolarizing wave was also depressed. To determine if an inhibition of the NMDA receptor-mediated synaptic response does not result from a secondary effect of ethanol that enhances Gamma-aminobutyric acid (GABA)-mediated inhibition. I examined the action of ethanol on synaptic response recorded in 8 PM CNQX. I .2 As illustrated in Fig. 5A, mM Mg+‘. and 50 FM bicuculline. bicuculline induced epileptiform activity in normal Mg++ solution. Superfusion of CNQX (8 PM) suppressed the epileptiform activity; however, a depolarizing potential persisted in the presence of CNQX (Fig. 5B). This CNQX-resistant de-
D Ethanol ,OOmM
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FIG. 4. Reversible inhibition of NMDA receptor-mediated synaptic excitation by ethanol (A). A triggered response in the presence of CNQX and Mg”-free solution (B and C). The CNQX-resistant depolarizing wave was reversibly blocked by D-APV (50 PM) (D and E). Superfusion of ethanol ( 100 mM) reversibly suppressed the NMDA receptor-mediated synaptic excitation. All records were taken from the same cell. RMP =
-64 mV.
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FIG. 5. Ethanol inhibition of NMDA-mediated synaptic response in the presence ofCNQX, 1.2 mM Mg++,and bicuculline. (A) A burst response evoked by single stimulus pulse in the presence of bicuculline methiodide (BMI, 5d PM). (B) A depolarizing potential remained after application of CNQX (8 PM) (C and D). The CNQX-resistant depolarizing potential was reversibly abolished by DLAPV (50 NM). (E and F) Superfusion of ethanol (100 mM) reversibly suppressed NMDA receptor-mediated synaptic response. (G) Superimposed traces of B, C, D. E, and F. All records
were taken from the same cell. RMP = -70 mV.
tude (2-6 mV, mean = 3.3 mV), was seen in 8 neurons. This could be explained by ethanol’s dual effects of blocking the Mcurrent (21) while inhibiting NMDA-activated currents (25), which depolarized and hyperpolarized the membrane potentials, respectively. No profound changes in apparent input resistance were noted. DISCUSSION
The present study demonstrates that ethanol reversibly shortened the duration of evoked epileptiform activity induced in Mg++-free medium in neurons of rat basolateral amygdaloid nucleus. The duration of spontaneous bursts was also reduced and the effect was concentration dependent. The epileptiform activity induced by Mg++-free solution has two components: one is sensitive to D-APV and the other is independent of NMDA receptor activation (7,ll). In this study, the inhibition of epileptiform activity by ethanol correlated well with its blockade of the NMDA receptor-mediated synaptic excitation. Thus, it appears likely that the depression of epileptiform activity by ethanol is due primarily to ethanol’s blocking action on the NMDA receptors. However, the mechanism by which ethanol produces its effect on the NMDA response remains unknown. An allosteric site which is reg-
ulated by glycine has been proposed to be mvolved in this action of ethanol (24,28). Further study is necessary to determine if glycine can reverse ethanol’s inhibition of NMDA receptor-mediated synaptic response. The introduction of CNQX that selectively blocks nonNMDA excitatory amino acid receptors (12) allows us to directly test the effect of ethanol on the NMDA receptor-mediated synaptic response. Two types of experiments were performed. First, I examined the effect of ethanol on the synaptic response recorded in the presence of CNQX and Mg’ ‘-free solution. 1 found that 100 mM ethanol inhibited the synaptic response under this condition by 50.9%. Note, however, that this experiment could not exclude the possibility that the inhibition of NMDA receptor-mediated synaptic transmission by ethanol might be mediated through a GABAergic mechanism because it has been reported that ethanol can potentiate the openings of GABA, receptor-gated chloride channels (3,19). Accordingly. I carried out a second study in which the NMDA receptormediated synaptic response was isolated by superfusing the amygdaloid slices with CNQX and bicuculline. Application of ethanol again reversibly suppressed the amplitude of NMDA receptor-mediated synaptic response by 52.94. The degree of inhibition was similar to that produced by ethanol in the absence of bicuculline, suggesting that the depression of NMDA receptor-mediated synaptic response by ethanol does not depend on GABAergic transmission. Ethanol has been shown to selectively block ion currents activated by NMDA in hippocampal cells ( 15,I6) and it is suggested that NMDA receptors may be a plausible neurochemical target for mediating certain pharmacological effects of ethanol (8). Indeed, more evidence has been accumulated so NMDA-stimulated norepinephrine release from rat cortical slices was inhibited by ethanol (9,lO). Furthermore, Lovinger et al. showed that ethanol suppressed the NMDA receptor-mediated population EPSPs in rat hippocampal slices (17). Using the intracellular recording technique, the present result extends this observation by showing that the NMDA receptor-mediated synaptic excitation is depressed in the presence of ethanol in neurons of rat basolateral amygdaloid nucleus. A blood ethanol level of 100 mg/dl, corresponding to tissue concentration of 22 mM, is considered being under the influence of intoxicating beverages. Ataxia, impaired mental and motor skills, and impaired short-term memory can be detected at blood concentrations of 100-150 mg/dl (22-33 mM). At higher concentrations of 250-500 mg/dl (54- 108 mM), ethanol produces coma and has a lethal effect ( 13). In the present study, the observation that high concentrations of ethanol (20-100 mM) reduce the NMDA receptor-mediated synaptic response suggests that inhibition of NMDA receptor activation might contribute to ethanol’s intoxicating effects. In conclusion, my results suggest that intoxicating doses of ethanol possess anticonvulsant activity through ethanol’s blocking action on the NMDA receptor-mediated synaptic excitation. Because of the large body of data implicating the role of the NMDA receptor in long-term potentiation (4,22), the observed inhibitory effect of ethanol on the NMDA receptor-mediated response could contribute to the cognitive impairments associated with toxication. ACKNOWLEDGEMENT
This work was supported by the National Science Council of Taiwan, R.O.C. (NSC 80-0412~BOO6-41).
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