- Email: [email protected]
Rij Inbred Strain
Brain ResearchBulletin,Vol. 33, No. 6, pp. 715-718, 1994 Copyright8 1994ElsevierScience Ltd P&tedin the USA. All rights reserved 0361-9230/94$6.00 + .@I
0361-9230(93)EOO32-H
Interactions Between NMDA and NonNMDA Receptors in Nonconvulsive Epilepsy in the WAG/Rij Inbred Strain B. W. M. M. PEETE?RS,*’ G. M. J. RAMAKERS,t B. A. ELLENBROEK,$ J. M. H. VOSSENg AND A. M. L. COENENS *Department of ~earopharmac~la~, Organon International B.V., P.U. Box 20, 5340BH Oss, The Netherlands ~R~o~~agn~ institute forP~rmaco~o~, U~iversi~ of Utrecht, Utrecht, The Netherian~ #Psychop~rmacuiog~cal Research Unit, department of Pha~acoZo~, University of Nijmegen, Nijmegen, l’he Net~er~a~s $Department of Comparative and Physiological Psychology, University of Nijmegen, Nijmegen, The Netherlands Received 17 March 1993; Accepted 17 November 1993 PEETERS, B. W. M. M., G. M. J. RAMAKERS, B. A. ELLENBROEK, J. M. H. VOSSEN AND A. M. L. COENEN, Znteractiom Between NMDA and NonNMDA Receptors in Nonconvulsive Epilepsy in the WAGIRij Inbred Strain. BRAIN RES BULL 33(6) 715-718, 199ATLe interaction between NMDA and nonNMDA receptors was studied in nonconvulsive epilepsy in WAG/Rij rats. Compounds acting on NMDA (NMDA, APH) and nonNMDA (AMPA, GDEE, kainic acid, kynurenic acid) receptors were coinjected intracerebroventricularly. The WAG/Rij rat strain may be an animal model for human nonconvulsive absence epilepsy. The effects on the epilepsy, l%G and behaviour were measured. It appeared that the epilepsy increase, induced by the nonNMDA receptor agonist AMPA, and in a fess obvious way, kainic acid, was blocked by the NMDA receptor antagonist APH. The effects of NMDA were completely blocked by the nonNMDA receptor antagonists GDEE and kymuenic acid. These results suggest that there is an interaction between NMDA and nonNMDA receptors. It might be that nonNMDAergic compounds act via activation or inactivation of NMDA receptors and that this latter receptor subtype is the trigger for an epileptic seizure. NMDA receptor NonNMDA receptors nonN~Aergic drugs
Absence epilepsy
IN previous studies we showed that all three ionotropic glutamate
WAGjRij inbred strain
NMDAergic drugs
In the present study we investigated whether there is also an interaction between NMDA and nonNMDA receptors in nonconvulsive epilepsy and whether the NMDA receptor is indeed the trigger for an epileptic seizure. We tried to study this by coinjection expe~men~ in which compounds, acting on different receptor subtypes, were administered simult~~usly. The compounds used were NMDA ~-me~yl-D-~pa~te, a NMDA receptor agonist (12)), APH (2-amino-Tphosphonoheptanoic acid, a NMDA receptor antagonist (12)), AMPA ((RS)-alpha-amino-3-hydroxy-5methyl-Q-isoxazole propionic acid, an AMPA receptor agonist (5,8)), GDEE (glutamic acid diethyl ester, an AMPA receptor antagonist (4)), kainic acid (I&in, a kainate receptor agonist (1)) and kynurenic acid (Kyn, a broad spectrum antagonist (7)). The drugs were administered intracerebroventricularly in WAG/Rij rats and their effects on epilepsy, EEG and behaviour were studied. In our opinion, if the NMDA receptor is indeed the epileptic trigger, then all manipulations on nonNMDA receptors should be blocked by the NMDA receptor antagonist API% Furthermore, it should be
receptor subtypes are involved in nonconvulsive epilepsy as observed in the WAG/Rij rat inbred strain (9,10,11). Activation of NMDA, AMPA and in a less obvious way, kainate receptors, increased the epilepsy while blockage of the receptor subtypes caused a decrease. Recently, it was suggested that the three glutamate receptor subtypes do not act independently. It is possible that some excitatory amino acid synapses have more than one receptor subtype at their disposal: nonNMDA (AMPA and kainate) receptors for “normal” transmi~ion and NMDA receptors for special circumstances. Under “normal” conditions, excitatory transmission depends on nonNMDA receptors. But when these receptors are strongly stimulated, the NMDA receptor is “‘switched on.” In this case glutamate also acts on this latter receptor subtype and a strong response is evoked. It may be that activation of the NMDA receptor and stimulation of this site by glutamate results in an epileptic seizure (2,3).
’ To whom requests for reprints should be addressed,
715
716
I’EETEKS
5 pmol/B j.d
25.0
1
5 j.4moll5 ~1 50 pmol/5 ~1
IT 12.5
II
5 /_tmol/5~1 5 nmol/5 wl
hT Al
2.2 mmol/l; Cl 162 mmol/l) (CSF). Kyn was dissolved in CSP to which a minimum of NaOH was added (end-pH =: 9). All compounds were administered intracerebroventricularly. The rats were habituated to the experimental conditions for 18 h, and a baseline EEG was then recorded for 1 h. Subscquently, the animals were taken out of the observation cage and the cannula was opened. A needle via a tube connected to a five microliter syringe was then inserted (the needle extended 0.5 mm below the cannula), thus permitting free movement during injection and registration. The rats were returned to their cages, and after about 5 min they received the first injection (5 $). About 30 min later this procedure was repeated and the second injection administered. The EEG was then recorded for 5 h. During all recording hours the rats were closely observed and videotaped. The records were analyzed to detect behavioural changes induced by the drugs. The group of 18 rats was divided into six groups of three. All animals were used several times as shown in Table 1. Because of this repeated injection schedule, CSF injections were included to check for long lasting effects of previous injections. These effects were never observed.
100
#
0
1
0.0 B
1
2
3
4
5
FIG. 1. Effects of GDEE (5 /Lmol/5 ~1; 5 pmol/5 ~1 coinjected with NMDA (50 pmol/5 ~1); 5 pmol/S ~1 coinjected with NMDA (5 nmol/5 ~1)) on the number (#) of the spike-wave discharges. B = baseline hour; 1, 2, 3, 4, 5 = post-injection hour 1, 2, 3, 4, 5. Each bar represents the mean scores and SEMs for 6 rats. Statistical analyses were performed by means of Wilcoxon matched-pairs signed ranks test (GDEE versus CSF; 0 p < 0.05) and by means of Mann-Whitney U-test (coinjections versus monoinjections; 0 p < 0.05).
10 pmol/S ~1 5 nmous CL1
possible to modify the effect of a given NMDA dosage by manipulations on nonNMDA receptors. METHODS
Eighteen WAG/Rij rats (male and female) weighing between 200 and 390 g were used. They were maintained on a 12-12 h light-dark regime with lights on at 19.00 h. Each rat was anaesthetized with Ketamine/Xylazine and implanted with EEG electrodes and a ventricle cannula. EEG electrodes (Plastic Products Company, Roanoke, VA, MS 333/2A) were placed on the surface of the cortex; one in the frontal region (coordinates with skull surface flat and bregma zero-zero: A (anterior) 2.0 L (lateral) 3.5) and a second one in the parietal region (A -6.0 L 4.0). The ground electrode was placed in the cerebellum (6). A polyethylene cannula (inner diameter 0.4 mm/outer diameter 0.8 mm) was positioned in the left lateral ventricle (A 0.7 L -1.3) and had a depth of 3.5 mm from the skull surface. Following surgery, rats were allowed to recover S-7 days before drug administration. AMPA, GDEE, Kain, NMDA, and APH were dissolved in artificial cerebrospinal fluid (Na+ 154 mmol/l; K+ 4 mmol/l; Ca*’
10 pmol/S ~1 50 nmol/s /d
0 B
1
2
3
4
5
FIG. 2. Effects of AMPA (10 pmoll5 ~1; 10 pmol/S ~1 coinjected with APH (5 nmol/S ~1); 10 pmol/5 ,ul coinjected with APH (50 nmol/5 ~1)) on the number (#) of the spike-wave discharges. B = baseline hour; 1, 2,3,4,.5 = post-injection hour 1,2,3,4,5. Each bar represents the mean scores and SEMs for 6 rats. Statistical analyses were performed by means of Wilcoxon matched-pairs signed ranks test (AMPA versus CSF; 0 p < 0.05) and by means of Mann-Whitney U-test (coinjections versus monoinjections; 0 p < 0.05).
NMDA
AND NON-NMDA
RECEPTOR
717
I~~~ONS
5 nmol/5 ~1 and 50 nmol/5 ~1 produced scores which were significantly lower than those obtained after AMPA injection alone
I
600 nmol/s pl 60 pmol/S PI
T-T,
600 nmol/6 pl 6 nmoU6 /Al
(during post-injection hour 1 and post-injection hour 1 and 2, respectively) (Fig. 2). Kyn (500 nmol/S ~1) caused a decrease in the number of spike-wave discharges (data from (9)). A similar decrease was observed after coinjection of Kyn with NMDA (50 pmoh’5 ~1 and 5 nmoll5 ~1). The scores obtained after coinjection with NMDA 50 pmoll5 ~1 might suggest that the Kyn induced decrease is shortened (during ~st-injection hour 3, the coinjection scores were significantly higher than the Kyn scores). This, however, was not confirmed after coinjection of Kyn with the higher NMDA dosage (5 nmol/S ~1) (Fig. 3). Injection of Kain (0.01 nmolf5 ~1) had no consistent effects (data from (9)). First there was an increase (during post-injection hour 1, 2 and 3; not significant) but then during post-injection hour 5 there was a decrease (significant). Coinjection of Kain with APH (50 nmol/S ~1) revealed scores that were not significantly different from the Kain scores. However, visual inspection of both graphs might suggest a decrease after coinjection (Fig. 4). None of the herein mentioned combinations of compounds induced consistent changes in the mean duration of the discharges. Also the background EEG and overall behaviour of the animals were not changed.
12.5
DISCUSSION
0.0 B
1
2
3
4
5
FIG. 3. Effects of Kyn (500 nmoV5 ~1; 500 nmoV5 ~1 coinjected with NMDA (50 pmoli5 ~1); 500 nmoV5 ~1 coinjected with NMDA (5 nmot/ 5 ~1)) on the number (#) of the spike-wave discharges. B = baseline hour; 1,2,3,4,5 = ~st-~je~ion hour 1,2,3,4,X Bach bar represents the mean scores and SEMs for 6 rats. Statistical analyses were performed by means of Wilcoxon matched-pairs signed ranka test (Kyn versus CSF; 0 p < 0.05) and by means of Mann-Whitney U-test (coinjections versus monoinjections; 0 p < 0.05).
For each rat, the number and mean duration of discharges were measured (6) during the baseline hour and the five successive post-injection hours. Statistical analyses were performed by means of the Mann-Whitney U-test which compared coinjections and monoinjections. On completion of the studies, the brains of the implanted rats were fixed and sectioned to verify the correct position of the injection site. Histological verification showed that all injections
In the introduction it was stated that it may be possible that some excitatory amino acid systems have more than one receptor subtype at their disposal. NonNMDA receptors for the ‘Lnormal” transmission and NMDA receptors for special circumstances. Activation of the NMDA receptor and stimulation of this site
“:1 T / -r
25.0
were given into the left lateral ventricle. 12.5
RESULTS
Intracerebroventricular injection of GDEE (5 pmol/S ~1) caused a decrease in the number of spike-wave discharges (data from (9)). This decrease was even more pronounced after coinjection with NMDA (50 pmol/5 ~1) and NMDA (5 nmol/5 ~1). Coinjection of GDEE with both NMDA dosages caused a significant reduction, during post-injection hour 1 and 2, compared to GDEE injection alone (Fig. 1). AMPA (10 pmoli5 ~1) caused an increase in the number of discharges (data from (9)). After coinjection of AMPA with APH, however, this increase disappeared. Coinjection with APH
0.01nmol/s pl
1
0.01 lImoI/ 60 MlolRi
T
a
1
2
3
4
pl &al
5
FIG. 4. Effects of Kain (0.01 nmol/5 ~1; 0.01 nmoV5 ~1 coinjected with APW (50 nmoW ~1)) on the number (#) of the spike-wave discharges. B = baseline hour; 1, 2, 3, 4, 5 = post-injection hour 1, 2, 3, 4, 5. Each bar represents the mean scores and SEMs for 6 rats. Statistical analyses were performed by means of Wilwxon matched-pairs signed ranks test (Kain versus CSF; l p < 0.05) and by means of Mann-~i~ey U-test (coinjections versus monoinjections; 0 p < 0.05).
71x
PEETERS
TABLE INJECTION
F’l‘ AI
I
SCHEDULE
Group Inj. Time Day
(min)
1
t=O t=o t = 30 t=o t=o t = 30 t=o t=o t = 30 t=o
2 3 4 5 6 7
1
CSF GDEE 5 pmol NMDA 50 pmol CSF APH 5 nmol AMPA 10 pmol CSF Kyn 500 nmol NMDA 5 nmol CSF
2
CSF Kyn 500 nmol NMDA 5 nmol CSF APH 5 nmol AMPA 10 pmol CSF GDEE 5 pmol NMDA 50 pmol CSF
3
CSF APH 50 nmol AMPA 10 pmol CSF GDEE 5 pmol NMDA 5 nmol CSF
CSF GDEE 5 pmol NMDA 5 nmol CSF APH 50 nmol AMPA 10 pmol CSF
CSF Kyn 500 nmol NMDA 50 pmol CSF APH 50 nmol Kdin 0.01 nmol (‘SF
(‘SF APH 50 nmol Kain 0.01 nmol CSF Kyn 500 nmol NMDA SO pmol <‘SF
-
may result in an epileptic seizure (2,3). In the case of WAG/Rij rats, spike-wave discharges spontaneously occur, and this suggests that some NMDA receptors are already activated. This is supported by the observation that NMDA antagonists are able to block the epileptic events (10). But also nonNMDAergic agents are able to influence the discharges (9,ll). Whether these nonNMDAergic agents act via activation or inactivation of NMDA receptors is, however, unknown. If this is the case, then an increase in the amount of epilepsy, caused by activation of nonNMDA receptors, should be blocked by a NMDA receptor antagonist. Furthermore, nonNMDA receptor antagonists should be able to inactivate NMDA receptors that cause NMDA agonists to have smaller effects.
Our results show that the epilepsy increase, induced by the nonNMDA receptor agonist AMPA and in a less obvious way, Kain, was blocked by the NMDA receptor antagonist APH. The effects of NMDA were completely blocked by the nonNMDA receptor antagonists GDEE and Kyn. Keeping in mind that not all the used compounds are equally selective, these results still suggest that induced changes in the amount of epilepsy nonNMDAergic agents are mediated by NMDA receptors. In conclusion, it appears that in nonconvulsive epilepsy
by as
observed in WAG/Rij rats, all three glutamate receptor subtypes are involved. Their actions are, however, not independent but are strongly linked. NonNMDA receptors appear to act via NMDA receptors which might suggest that the NMDA receptor is the trigger for an epileptic seizure.
REFERENCES 1. Cotman, C. W.; Iversen, L. L. Excitatory amino acids in the brainfocus on NMDA receptors. Trends Neurosci. 10:263-264; 1987. 2. Cotman, C. W.; Monaghan, D. T.; Ganong, A. H. Excitatory amino acid neurotransmission: NMDA receptors and Hebb-type synaptic plasticity. Arm. Rev. Neurosci. 11:61-80; 1988. 3. Dingledine, R. NMDA receptors: What do they do? TINS 91:4749; 1986. 4. Haldeman, S.; McLennan, H. The antagonistic action of GDEE towards amino acid induced and synaptic excitation of central neurons. Brain Res. 45:393-400; 1972. 5. Krogsgaard-Larsen, P.; Honore, T.; Hansen, J. J.; Curtis, D. R.; Lodge, D. New class of glutamate agonists structurally related to ibotenic acid. Nature 28464-66; 1980. 6. Luijtelaar van, E. L. J. M.; Coenen, A. M. L. Two types of electrocortical paroxysms in an inbred strain of rats. Neurosci. Lett. 70:393-397; 1986. 7. Meldrum, B. S.; Chapman, A. G. Excitatory amino acid antagonists as anticonvulsant agents: Receptor subtype involvement in different
8.
9.
10.
Il.
12.
seizure models. In: Nistico, G., ed. Neurotransmitters, seizures and epilepsy III. New York: Raven Press; 1986:223-233. Monaghan, D. T.; Yao, D.; Cotman, C. W. Distribution of “H-AMPA binding sites in rat brain as determined by quantitative autoradiography. Brain Res. 324:160-164; 1984. Peeters, B. W. M. M.; Ramakers, G. M. J.; Vossen, J. M. H.; Coenen, A. M. L. The WAG/Rij rat model for nonconvulsive absence epilepsy: Involvement of nonNMDA receptors. Submitted for publication. Peeters, B. W. M. M.; Rijn van, C. M.; Vossen, J. M. H.; Coenen, A. M. L. Involvement of NMDA receptors in nonconvulsive epilepsy in WAG/Rij rats. Life Sci. 47:523-529; 1990. Ramakers, G. M. J.; Peeters, B. W. M. M.; Vossen, J. M. H.; Coenen, A. M. L. CNQX, a new nonNMDA receptor antagonist, reduces spike-wave discharges in the WAG/Rij rat model of absence epilepsy. Epilepsy Res. 9:127-131; 1991. Watkins, J. C.; Olverman, H. J. Agonists and antagonists for excitatory amino acid receptors. Trends Neurosci. 10:265-272: 1987.
0361-9230(93)EOO32-H
Interactions Between NMDA and NonNMDA Receptors in Nonconvulsive Epilepsy in the WAG/Rij Inbred Strain B. W. M. M. PEETE?RS,*’ G. M. J. RAMAKERS,t B. A. ELLENBROEK,$ J. M. H. VOSSENg AND A. M. L. COENENS *Department of ~earopharmac~la~, Organon International B.V., P.U. Box 20, 5340BH Oss, The Netherlands ~R~o~~agn~ institute forP~rmaco~o~, U~iversi~ of Utrecht, Utrecht, The Netherian~ #Psychop~rmacuiog~cal Research Unit, department of Pha~acoZo~, University of Nijmegen, Nijmegen, l’he Net~er~a~s $Department of Comparative and Physiological Psychology, University of Nijmegen, Nijmegen, The Netherlands Received 17 March 1993; Accepted 17 November 1993 PEETERS, B. W. M. M., G. M. J. RAMAKERS, B. A. ELLENBROEK, J. M. H. VOSSEN AND A. M. L. COENEN, Znteractiom Between NMDA and NonNMDA Receptors in Nonconvulsive Epilepsy in the WAGIRij Inbred Strain. BRAIN RES BULL 33(6) 715-718, 199ATLe interaction between NMDA and nonNMDA receptors was studied in nonconvulsive epilepsy in WAG/Rij rats. Compounds acting on NMDA (NMDA, APH) and nonNMDA (AMPA, GDEE, kainic acid, kynurenic acid) receptors were coinjected intracerebroventricularly. The WAG/Rij rat strain may be an animal model for human nonconvulsive absence epilepsy. The effects on the epilepsy, l%G and behaviour were measured. It appeared that the epilepsy increase, induced by the nonNMDA receptor agonist AMPA, and in a fess obvious way, kainic acid, was blocked by the NMDA receptor antagonist APH. The effects of NMDA were completely blocked by the nonNMDA receptor antagonists GDEE and kymuenic acid. These results suggest that there is an interaction between NMDA and nonNMDA receptors. It might be that nonNMDAergic compounds act via activation or inactivation of NMDA receptors and that this latter receptor subtype is the trigger for an epileptic seizure. NMDA receptor NonNMDA receptors nonN~Aergic drugs
Absence epilepsy
IN previous studies we showed that all three ionotropic glutamate
WAGjRij inbred strain
NMDAergic drugs
In the present study we investigated whether there is also an interaction between NMDA and nonNMDA receptors in nonconvulsive epilepsy and whether the NMDA receptor is indeed the trigger for an epileptic seizure. We tried to study this by coinjection expe~men~ in which compounds, acting on different receptor subtypes, were administered simult~~usly. The compounds used were NMDA ~-me~yl-D-~pa~te, a NMDA receptor agonist (12)), APH (2-amino-Tphosphonoheptanoic acid, a NMDA receptor antagonist (12)), AMPA ((RS)-alpha-amino-3-hydroxy-5methyl-Q-isoxazole propionic acid, an AMPA receptor agonist (5,8)), GDEE (glutamic acid diethyl ester, an AMPA receptor antagonist (4)), kainic acid (I&in, a kainate receptor agonist (1)) and kynurenic acid (Kyn, a broad spectrum antagonist (7)). The drugs were administered intracerebroventricularly in WAG/Rij rats and their effects on epilepsy, EEG and behaviour were studied. In our opinion, if the NMDA receptor is indeed the epileptic trigger, then all manipulations on nonNMDA receptors should be blocked by the NMDA receptor antagonist API% Furthermore, it should be
receptor subtypes are involved in nonconvulsive epilepsy as observed in the WAG/Rij rat inbred strain (9,10,11). Activation of NMDA, AMPA and in a less obvious way, kainate receptors, increased the epilepsy while blockage of the receptor subtypes caused a decrease. Recently, it was suggested that the three glutamate receptor subtypes do not act independently. It is possible that some excitatory amino acid synapses have more than one receptor subtype at their disposal: nonNMDA (AMPA and kainate) receptors for “normal” transmi~ion and NMDA receptors for special circumstances. Under “normal” conditions, excitatory transmission depends on nonNMDA receptors. But when these receptors are strongly stimulated, the NMDA receptor is “‘switched on.” In this case glutamate also acts on this latter receptor subtype and a strong response is evoked. It may be that activation of the NMDA receptor and stimulation of this site by glutamate results in an epileptic seizure (2,3).
’ To whom requests for reprints should be addressed,
715
716
I’EETEKS
5 pmol/B j.d
25.0
1
5 j.4moll5 ~1 50 pmol/5 ~1
IT 12.5
II
5 /_tmol/5~1 5 nmol/5 wl
hT Al
2.2 mmol/l; Cl 162 mmol/l) (CSF). Kyn was dissolved in CSP to which a minimum of NaOH was added (end-pH =: 9). All compounds were administered intracerebroventricularly. The rats were habituated to the experimental conditions for 18 h, and a baseline EEG was then recorded for 1 h. Subscquently, the animals were taken out of the observation cage and the cannula was opened. A needle via a tube connected to a five microliter syringe was then inserted (the needle extended 0.5 mm below the cannula), thus permitting free movement during injection and registration. The rats were returned to their cages, and after about 5 min they received the first injection (5 $). About 30 min later this procedure was repeated and the second injection administered. The EEG was then recorded for 5 h. During all recording hours the rats were closely observed and videotaped. The records were analyzed to detect behavioural changes induced by the drugs. The group of 18 rats was divided into six groups of three. All animals were used several times as shown in Table 1. Because of this repeated injection schedule, CSF injections were included to check for long lasting effects of previous injections. These effects were never observed.
100
#
0
1
0.0 B
1
2
3
4
5
FIG. 1. Effects of GDEE (5 /Lmol/5 ~1; 5 pmol/5 ~1 coinjected with NMDA (50 pmol/5 ~1); 5 pmol/S ~1 coinjected with NMDA (5 nmol/5 ~1)) on the number (#) of the spike-wave discharges. B = baseline hour; 1, 2, 3, 4, 5 = post-injection hour 1, 2, 3, 4, 5. Each bar represents the mean scores and SEMs for 6 rats. Statistical analyses were performed by means of Wilcoxon matched-pairs signed ranks test (GDEE versus CSF; 0 p < 0.05) and by means of Mann-Whitney U-test (coinjections versus monoinjections; 0 p < 0.05).
10 pmol/S ~1 5 nmous CL1
possible to modify the effect of a given NMDA dosage by manipulations on nonNMDA receptors. METHODS
Eighteen WAG/Rij rats (male and female) weighing between 200 and 390 g were used. They were maintained on a 12-12 h light-dark regime with lights on at 19.00 h. Each rat was anaesthetized with Ketamine/Xylazine and implanted with EEG electrodes and a ventricle cannula. EEG electrodes (Plastic Products Company, Roanoke, VA, MS 333/2A) were placed on the surface of the cortex; one in the frontal region (coordinates with skull surface flat and bregma zero-zero: A (anterior) 2.0 L (lateral) 3.5) and a second one in the parietal region (A -6.0 L 4.0). The ground electrode was placed in the cerebellum (6). A polyethylene cannula (inner diameter 0.4 mm/outer diameter 0.8 mm) was positioned in the left lateral ventricle (A 0.7 L -1.3) and had a depth of 3.5 mm from the skull surface. Following surgery, rats were allowed to recover S-7 days before drug administration. AMPA, GDEE, Kain, NMDA, and APH were dissolved in artificial cerebrospinal fluid (Na+ 154 mmol/l; K+ 4 mmol/l; Ca*’
10 pmol/S ~1 50 nmol/s /d
0 B
1
2
3
4
5
FIG. 2. Effects of AMPA (10 pmoll5 ~1; 10 pmol/S ~1 coinjected with APH (5 nmol/S ~1); 10 pmol/5 ,ul coinjected with APH (50 nmol/5 ~1)) on the number (#) of the spike-wave discharges. B = baseline hour; 1, 2,3,4,.5 = post-injection hour 1,2,3,4,5. Each bar represents the mean scores and SEMs for 6 rats. Statistical analyses were performed by means of Wilcoxon matched-pairs signed ranks test (AMPA versus CSF; 0 p < 0.05) and by means of Mann-Whitney U-test (coinjections versus monoinjections; 0 p < 0.05).
NMDA
AND NON-NMDA
RECEPTOR
717
I~~~ONS
5 nmol/5 ~1 and 50 nmol/5 ~1 produced scores which were significantly lower than those obtained after AMPA injection alone
I
600 nmol/s pl 60 pmol/S PI
T-T,
600 nmol/6 pl 6 nmoU6 /Al
(during post-injection hour 1 and post-injection hour 1 and 2, respectively) (Fig. 2). Kyn (500 nmol/S ~1) caused a decrease in the number of spike-wave discharges (data from (9)). A similar decrease was observed after coinjection of Kyn with NMDA (50 pmoh’5 ~1 and 5 nmoll5 ~1). The scores obtained after coinjection with NMDA 50 pmoll5 ~1 might suggest that the Kyn induced decrease is shortened (during ~st-injection hour 3, the coinjection scores were significantly higher than the Kyn scores). This, however, was not confirmed after coinjection of Kyn with the higher NMDA dosage (5 nmol/S ~1) (Fig. 3). Injection of Kain (0.01 nmolf5 ~1) had no consistent effects (data from (9)). First there was an increase (during post-injection hour 1, 2 and 3; not significant) but then during post-injection hour 5 there was a decrease (significant). Coinjection of Kain with APH (50 nmol/S ~1) revealed scores that were not significantly different from the Kain scores. However, visual inspection of both graphs might suggest a decrease after coinjection (Fig. 4). None of the herein mentioned combinations of compounds induced consistent changes in the mean duration of the discharges. Also the background EEG and overall behaviour of the animals were not changed.
12.5
DISCUSSION
0.0 B
1
2
3
4
5
FIG. 3. Effects of Kyn (500 nmoV5 ~1; 500 nmoV5 ~1 coinjected with NMDA (50 pmoli5 ~1); 500 nmoV5 ~1 coinjected with NMDA (5 nmot/ 5 ~1)) on the number (#) of the spike-wave discharges. B = baseline hour; 1,2,3,4,5 = ~st-~je~ion hour 1,2,3,4,X Bach bar represents the mean scores and SEMs for 6 rats. Statistical analyses were performed by means of Wilcoxon matched-pairs signed ranka test (Kyn versus CSF; 0 p < 0.05) and by means of Mann-Whitney U-test (coinjections versus monoinjections; 0 p < 0.05).
For each rat, the number and mean duration of discharges were measured (6) during the baseline hour and the five successive post-injection hours. Statistical analyses were performed by means of the Mann-Whitney U-test which compared coinjections and monoinjections. On completion of the studies, the brains of the implanted rats were fixed and sectioned to verify the correct position of the injection site. Histological verification showed that all injections
In the introduction it was stated that it may be possible that some excitatory amino acid systems have more than one receptor subtype at their disposal. NonNMDA receptors for the ‘Lnormal” transmission and NMDA receptors for special circumstances. Activation of the NMDA receptor and stimulation of this site
“:1 T / -r
25.0
were given into the left lateral ventricle. 12.5
RESULTS
Intracerebroventricular injection of GDEE (5 pmol/S ~1) caused a decrease in the number of spike-wave discharges (data from (9)). This decrease was even more pronounced after coinjection with NMDA (50 pmol/5 ~1) and NMDA (5 nmol/5 ~1). Coinjection of GDEE with both NMDA dosages caused a significant reduction, during post-injection hour 1 and 2, compared to GDEE injection alone (Fig. 1). AMPA (10 pmoli5 ~1) caused an increase in the number of discharges (data from (9)). After coinjection of AMPA with APH, however, this increase disappeared. Coinjection with APH
0.01nmol/s pl
1
0.01 lImoI/ 60 MlolRi
T
a
1
2
3
4
pl &al
5
FIG. 4. Effects of Kain (0.01 nmol/5 ~1; 0.01 nmoV5 ~1 coinjected with APW (50 nmoW ~1)) on the number (#) of the spike-wave discharges. B = baseline hour; 1, 2, 3, 4, 5 = post-injection hour 1, 2, 3, 4, 5. Each bar represents the mean scores and SEMs for 6 rats. Statistical analyses were performed by means of Wilwxon matched-pairs signed ranks test (Kain versus CSF; l p < 0.05) and by means of Mann-~i~ey U-test (coinjections versus monoinjections; 0 p < 0.05).
71x
PEETERS
TABLE INJECTION
F’l‘ AI
I
SCHEDULE
Group Inj. Time Day
(min)
1
t=O t=o t = 30 t=o t=o t = 30 t=o t=o t = 30 t=o
2 3 4 5 6 7
1
CSF GDEE 5 pmol NMDA 50 pmol CSF APH 5 nmol AMPA 10 pmol CSF Kyn 500 nmol NMDA 5 nmol CSF
2
CSF Kyn 500 nmol NMDA 5 nmol CSF APH 5 nmol AMPA 10 pmol CSF GDEE 5 pmol NMDA 50 pmol CSF
3
CSF APH 50 nmol AMPA 10 pmol CSF GDEE 5 pmol NMDA 5 nmol CSF
CSF GDEE 5 pmol NMDA 5 nmol CSF APH 50 nmol AMPA 10 pmol CSF
CSF Kyn 500 nmol NMDA 50 pmol CSF APH 50 nmol Kdin 0.01 nmol (‘SF
(‘SF APH 50 nmol Kain 0.01 nmol CSF Kyn 500 nmol NMDA SO pmol <‘SF
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may result in an epileptic seizure (2,3). In the case of WAG/Rij rats, spike-wave discharges spontaneously occur, and this suggests that some NMDA receptors are already activated. This is supported by the observation that NMDA antagonists are able to block the epileptic events (10). But also nonNMDAergic agents are able to influence the discharges (9,ll). Whether these nonNMDAergic agents act via activation or inactivation of NMDA receptors is, however, unknown. If this is the case, then an increase in the amount of epilepsy, caused by activation of nonNMDA receptors, should be blocked by a NMDA receptor antagonist. Furthermore, nonNMDA receptor antagonists should be able to inactivate NMDA receptors that cause NMDA agonists to have smaller effects.
Our results show that the epilepsy increase, induced by the nonNMDA receptor agonist AMPA and in a less obvious way, Kain, was blocked by the NMDA receptor antagonist APH. The effects of NMDA were completely blocked by the nonNMDA receptor antagonists GDEE and Kyn. Keeping in mind that not all the used compounds are equally selective, these results still suggest that induced changes in the amount of epilepsy nonNMDAergic agents are mediated by NMDA receptors. In conclusion, it appears that in nonconvulsive epilepsy
by as
observed in WAG/Rij rats, all three glutamate receptor subtypes are involved. Their actions are, however, not independent but are strongly linked. NonNMDA receptors appear to act via NMDA receptors which might suggest that the NMDA receptor is the trigger for an epileptic seizure.
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