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quisqualate receptor antagonist, CNQX, blocks the fast component of spontaneous epileptiform activity in organotypic cultures of rat hippocampus
Neuroscience Letters, 93 (1988) 341 345
341
Elsevier Scientific Publishers Ireland Ltd. NSL 05636
The kainate/quisqualate receptor antagonist, CNQX, blocks the fast component of spontaneous epileptiform activity in organotypic cultures of rat hippocampus C.J. McBain, P. Boden and R.G. Hill* Parke°Davis Research Unit, Cambridge ( U.K.)
(Received 17 May 1988; Revised version received 29 June 1988; Accepted 2 July 1988) Key words." Organotypic culture; Hippocampus; Epileptiform activity; 6-Cyano-2,3-dihydroxy-7-nitro-
quinoxaline; D-2-Amino-5-phosphonovalerate Intracellular recordings were made from CA 1 pyramidal neurones of hippocampus maintained in organotypic culture. Both spontaneous interictal and ictal epileptiform activity was observed. CNQX, an antagonist at kainate/quisqualate but not at N-methyl-D-aspartate (NMDA)-sensitive excitatory amino acid receptors depressed but did not abolish spontaneous epileptiform activity. Addition of the specific NMDA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) abolished the remaining activity. Similar effects were observed on electrically evoked excitatory post synaptic potentials (EPSPs). This suggests a role for endogenous excitatory amino acids acting at both kainate/quisqualate and NMDA sensitive excitatory amino acid receptors in the generation and maintainance of epileptiform activity within these organotypic cultures.
T h e e x c i t a t o r y a m i n o acids ( E A A ) L - g l u t a m a t e a n d L - a s p a r t a t e are t h o u g h t to be the m o s t likely t r a n s m i t t e r c a n d i d a t e s at the synapses between s t r a t u m p y r a m i d a l e o f C A 3 a n d C A l in the rat h i p p o c a m p u s . These a m i n o acids are k n o w n to act on three m a i n E A A receptors, defined in terms o f their sensitivity to the u n n a t u r a l a m i n o acids kainate, q u i s q u a l a t e a n d N - m e t h y l - o - a s p a r t a t e ( N M D A ) [16]. M o s t e l e c t r o p h y s i o l o g i c a l studies to d a t e have c o n c e n t r a t e d on the N M D A r e c e p t o r as selective r e c e p t o r a n t a g o n i s t s for this site are readily available [17]. N M D A r e c e p t o r s h o w e v e r are voltage-sensitive a n d m a y n o t be a c t i v a t e d u n d e r c o n d i t i o n s o f ' n o r m a l ' s y n a p t i c t r a n s m i s s i o n [6, 1 l]. A role for N M D A receptors has been d e m o n s t r a t e d in long term p o t e n t i a t i o n [6] a n d in convulsive c o n d i t i o n s such as e x p e r i m e n t a l epilepsy [7]. N M D A r e c e p t o r i n v o l v e m e n t in e p i l e p t i f o r m activity has been d e m o n -
*Present address: Smith, Kline and French Research Ltd., The Frythe, Welwyn, Herts AL6 9AR, U.K. Correspondence." C.J. McBain, Parke-Davis Research Unit, Cambridge CB2 2QB, U.K. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
342 strated to be restricted to the latter portion of the recorded event [I, 7, 9], leaving the initial fast depolarising shift and first action potentials observed intact. Investigations into the involvement of that component of epileptiform activity insensitive to N M D A receptor antagonists have been severely hampered by the lack of suitable specific antagonists for kainate and quisqualate receptors. CNQX (6-cyano-2,3dihydroxy-7-nitro-quinoxaline) has recently been shown to be an antagonist which blocks the actions of kainate and quisqualate but not those of N M D A [10]. CNQX has also been demonstrated to reduce the amplitude of excitatory postsynaptic potentials (EPSPs) at CAI neurones in the hippocampal slice preparation [2]. We have studied the effects of CNQX on spontaneous interictal and ictal activity recorded from hippocampus maintained in organotypic culture [3]. Hippocampal organotypic cultures were prepared using the technique first described by Gahwiler [8]. Briefly, both hippocampi were aseptically dissected free from 6 day neonate Wistar rats killed by cervical dislocation prior to decapitation. Transverse slices (400/~m) were cut on a McIlwain tissue sectioner and incubated for 30 rain in Geys' balanced salt solution (BSS) enriched with glucose (36 raM) and KC1 (30 mM) at 4°C. Individual slices were then placed on a clean glass coverslip and held in place by a chicken plasma clot. Coverslips were transferred to a tissue culture tube containing 1 ml of medium (50% Basal medium eagles, 25% Hanks BSS, 25% heat-inactivated horse serum and glucose (36 mM). Tubes were placed in a roller drum within an incubator at 36°C in dry air. Cultures were fed once weekly. Conventional intracellular recording techniques were used throughout these experiments. Intracellular electrodes were filled with 3 M potassium acetate and had DC resistances of 80-200 Mg2 when measured in physiological saline. Recordings were made from CA1 neurones of stratum pyramidale using an Axoclamp amplifier in bridge mode in cultures that had been maintained for periods 3-10 weeks in vitro. Only results from neurones impaled for periods greater than 45 min are included in the study. Cultures were perfused with Hanks' BSS flowing through the recording chamber (volume 0.8 ml) at 2 ml/min at 37°C. The composition of this medium was (mM), NaC1 136.9, KCI 5.4, MgSO4 0.4, Na2HPO4 0.26, CaC12 1.25, KH2PO4 0.44, MgCI2 0.49 NaHCO3 4.1, glucose 5.5. Drugs were added in known concentrations by changing the superfusion solution by means of 3-way taps. Synaptic potentials were evoked by stimulation of the Schaffer collateral pathway at 0.03 Hz. CNQX was obtained from Tocris Neuramin and D-2-amino-5-phosphonovalerate (D-APV) from Cambridge Research Biochemicals. Epileptiform discharges were recorded from cultures following approximately 3 weeks in culture [3]. Both spontaneous interictal and ictal events were observed, identified as such using criteria derived from motor seizures described by Matsumoto and Ajmone-Marsan [12, 13]. Neurones had an input resistance of 6 8 + 6 Mg2 (n= 10) (mean _+S.E.M., the number of cells in parentheses) and resting membrane potential of 54.6+ 1.0 mV (n= 10). CNQX (10/tM) was applied to 9 neurones. In 8 neurones CNQX attenuated both the amplitude and duration of the paroxysmal depolarising shift (PDS). The amplitude was reduced by 61 _+5% ( n = 8 ) while the duration was 42_+ 9% (n = 8) that of control epileptiform activity (Fig. 1). CNQX had no effect on
343
CONTROL
CNQX (10~M)
I ILli CNQX (101~M), D-APV (30[~M)
WASH
' illllllillllillllllllllllllllll 20mY 20sec Fig. 1. Intracellular recordings, made using 3 M potassium acetate-filled microelectrodes of spontaneous epileptiform events recorded from a hippocampal organotypic culture after 63 days in vitro. The paroxysmal activity typically commenced with a large depolarising shift (PDS) of up to 40 mV in amplitude. Superimposed on this shift was a high frequency (often upwards of 100 Hz) burst of action potentials. Paroxysmal events had durations of up to 3 min. Upon termination of these events the membrane hyperpolarised to potentials below control levels and this period constituted a period of relative inexcitability. Addition of CNQX to the perfusate resulted in cessation of the high frequency action potential firing and this was associated with a decrease in both the amplitude and duration of the underlying depolarising shift. Addition of D-APV in the continued presence of CNQX abolished all spontaneous synaptic activity. These drugs, separately or in combination, had no effect on the resting membrane parameters of the neurone. Upon return to drug-free perfusate only the D-APV-sensitive component returned following one hour of wash. This neurone maintained its resting membrane potential for the duration of the experiment.
the m e a n i n p u t resistance ( 9 8 _ 2%, n = 8 o f c o n t r o l ) o r resting m e m b r a n e p o t e n t i a l ( 1 0 4 _ 2%, n = 8). C N Q X d i d n o t affect the d i s c h a r g e frequency o b s e r v e d in 7 cultures (0.08-1.0 Hz), n o r d i d it effect the p o s t - d e p o l a r i s i n g s h i f t - a f t e r h y p e r p o l a r i s a t i o n . In two n e u r o n e s however, C N Q X r e d u c e d the frequency whilst a u g m e n t i n g the d u r a t i o n o f the r e m a i n i n g P D S activity. F o l l o w i n g C N Q X a d d i t i o n the r e m a i n i n g d e p o l a r i s i n g shift was never o f sufficient a m p l i t u d e to p r e c i p i t a t e a c t i o n p o t e n t i a l firing. Single a c t i o n p o t e n t i a l s c o u l d still be e v o k e d h o w e v e r b y direct s t i m u l a t i o n o f the n e u r o n e by the p a s s a g e o f d e p o l a r i s i n g c u r r e n t t h r o u g h the r e c o r d i n g electrode, d e m o n s t r a t i n g t h a t C N Q X h a d n o t merely affected the spike g e n e r a t i n g m e c h a n i s m o f the cell. W h e n the N M D A r e c e p t o r a n t a g o n i s t D - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e ( o - A P V ) was a p p l i e d in the c o n t i n u e d presence o f C N Q X all r e m a i n i n g s p o n t a n e o u s s y n a p t i c activity was a b o l i s h e d . This c o m b i n a t i o n o f a n t a g o n i s t s was also w i t h o u t
344
5mY lOOms Fig. 2. This figure illustrates that following the onset of epileptiform activity subsequent low frequency (0.03 Hz) electrical stimulation of the Schaffer collateral pathway would evoke EPSPs which typically displayed a two component decay phase (record a). This decay can be described by two time constants zfa~,, 30.4 ms and r~bow,121.0 ms (see text for details). Addition of a mixture of CNQX (10/IM) and D-APV (30 /~M) to the superfusate bathing the culture depressed but did not totally abolish the EPSP (record b). The EPSP amplitude in this experiment was depressed by 87%. Following 1 h of wash with drug-free perfusate the EPSP amplitude recovered to 54% that of the pre-drug control value (record c). This partial recovery was probably attributable to the prolonged action of CNQX in this preparation (see text). The resting membrane potential for this neurone was - 5 7 mV. Each trace is the average of 10 consecutive synaptic potentials.
effect on the resting membrane parameters of the neurone. We have previously demonstrated that epileptiform events are of synaptic origin within these cultures [4]. All activity was blocked by addition of solutions containing high magnesium ion concentration or tetrodotoxin. Epileptiform events are reversed at membrane potentials approaching the synaptic equilibrium potential. These experiments suggest that both CNQX and D-APV are blocking components of synaptic transmission. In order to examine this further, we observed the effects of CNQX and D-APV on electrically evoked EPSPs. Following the onset of epileptiform activity evoked EPSPs have a decay phase that can be fitted by a double exponential. We have previously demonstrated that only the slow component of the decay phase is due to N M D A receptor activation and this component is only observed following the onset of epileptiform activity [4]. In these experiments addition of CNQX (10/tM) together with D-APV (30/tM) depressed the amplitude of the EPSP by up to 89-+-3% (n = 5). CNQX and D-APV never totally abolished this evoked EPSP. The effects of CNQX were only partially reversed following 1 h of wash and the EPSP returned to 58_+ 3% (n = 5) of the control value after this period. Evoked synaptic potentials recorded from organotypic cultures prior to the onset of epileptiform activity display sensitivity to CNQX but not D-APV. This suggests that only kainate/quisqualate receptors are involved in synaptic transmission under 'normal' conditions. This has recently been confirmed in conventional in vitro hippocampal slices [5]. These results suggest that following the onset of epileptiform activity, both NMDA and non-NMDA EAA receptor activation are involved in both spontaneous and evoked excitatory synaptic events. Activation of kainate/quisqualate receptors would appear to be responsible for the initiation of the event. The rapid depolarisation caused by this activation presumably would bring the membrane potential into the range for relief of the voltage-dependent block (by Mg 2+) of the channel asso-
345
ciated with the NMDA receptor [14, 15]. This subsequent recruitment of the NMDA receptor-operated component would contribute to the maintenance of the epileptiform activity. These results also demonstrate that these NMDA receptors can be activated independent of the activation of other receptors and these data are in agreement with Verdoorn and Dingledine's observations on NMDA receptors expressed following mRNA injection into Xenopusoocytes (personal communication). Thus drugs which act specifically as antagonists at kainate/quisqualate receptors may prove to be of use as anticonvulsant agents and should be particularly effective when used in conjunction with specific NMDA receptor antagonists. 1 Ashwood, T.J. and Wheal, H.V., The expression of N-methyl-D-aspartate-receptor-mediated component during epileptiform synaptic activity in the hippocampus. Br. J. Pharmacol., 91 (1987) 815-822. 2 Blake, J.F., Brown, M.W. and Collingridge, G.L., Selective blockade of non-NMDA receptor mediated depolarizations and synaptic components by CNQX. Neurosci. Len., Suppl. 32 (1988) $4. 3 Boden, P., Hill, R.G. and McBain, C.J., Long term organotypic cultures of rat hippocampal neurones display changes in neuronal excitability, J. Physiol. (Lond.), 396 (1987) 62P. 4 Boden, P., Hill, R.G. and McBain, C.J., Rat hippocampal slices in vitro display spontaneous epileptiform activity following long term organotypic culture, J. Neurosci. Methods, in press. 5 Collingridge, G.L. and Davies, S.N., Synaptic transmission following blockade of non-NMDA-type excitatory amino acid receptors in rat hippocampus in vitro, J. Physiol. (Lond.), in press. 6 Collingridge, G.L., Kehl, S.J. and Mclennan, H., Excitatory amino acids in synaptic transmission in the Schaffer collateral-commisural pathway of the rat hippocampus, J. Physiol. (Lond.), 334 (1983) 33-46. 7 Dingledine, R., Hynes, M.A. and King, G.L., Involvement of N-methyl-o-aspartate receptors in epileptiform bursting in the rat hippocampal slice, J. Physiol. (Lond.), 380 (1986) 175 189. 8 Gahwiler, B.H., Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods, 4 (1981) 329 342. 9 Herron, C.E., Williamson, R. and Collingridge, G.L., A selective N-methyl-o-aspartate antagonist depresses epileptiform activity in rat hippocampal slices. Neurosci. Lett., 61 (1985) 225-260. 10 Honore, T., Davies, S.N., Drejer, J., Fletcher, E.J., Jacobsen, P., Lodge, D. and Nielsen, F.E., Potent and competitive antagonism at non-NMDA receptors by FG 9041 and FG 9065, Soc. Neurosci. Abstr., 13 (1987) 109.7. 11 Koerner, J.F. and Cotman, C.W., Response of Schaffer collateral CAI pyramidal cell synapses of the hippocampus to analogues of acidic amino acids, Brain Res., 251 (1982) 105-I 15. 12 Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: interictal manifestations, Exp. Neurol., 9 (1964) 28f~304. 13 Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: ictal manifestations, Exp. Neurol., 9 (1964) 305-326. 14 Mayer, M.L., Westbrook, G.L. and Guthrie, P.B., Voltage-dependent block by Mg ÷ ÷ of NMDA responses in spinal cord neurones, Nature (Lond.), 309 (1984) 261-263. 15 Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A., Magnesium gates glutamateactivated channels in mouse central neurones. Nature (Lond.), 307 (1984) 462-465. 16 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 17 Watkins, J.C. and Olverman, H.J., Agonists and antagonists for excitatory amino acid receptors, Trends Neurosci. 10 (1987) 265 272.
341
Elsevier Scientific Publishers Ireland Ltd. NSL 05636
The kainate/quisqualate receptor antagonist, CNQX, blocks the fast component of spontaneous epileptiform activity in organotypic cultures of rat hippocampus C.J. McBain, P. Boden and R.G. Hill* Parke°Davis Research Unit, Cambridge ( U.K.)
(Received 17 May 1988; Revised version received 29 June 1988; Accepted 2 July 1988) Key words." Organotypic culture; Hippocampus; Epileptiform activity; 6-Cyano-2,3-dihydroxy-7-nitro-
quinoxaline; D-2-Amino-5-phosphonovalerate Intracellular recordings were made from CA 1 pyramidal neurones of hippocampus maintained in organotypic culture. Both spontaneous interictal and ictal epileptiform activity was observed. CNQX, an antagonist at kainate/quisqualate but not at N-methyl-D-aspartate (NMDA)-sensitive excitatory amino acid receptors depressed but did not abolish spontaneous epileptiform activity. Addition of the specific NMDA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) abolished the remaining activity. Similar effects were observed on electrically evoked excitatory post synaptic potentials (EPSPs). This suggests a role for endogenous excitatory amino acids acting at both kainate/quisqualate and NMDA sensitive excitatory amino acid receptors in the generation and maintainance of epileptiform activity within these organotypic cultures.
T h e e x c i t a t o r y a m i n o acids ( E A A ) L - g l u t a m a t e a n d L - a s p a r t a t e are t h o u g h t to be the m o s t likely t r a n s m i t t e r c a n d i d a t e s at the synapses between s t r a t u m p y r a m i d a l e o f C A 3 a n d C A l in the rat h i p p o c a m p u s . These a m i n o acids are k n o w n to act on three m a i n E A A receptors, defined in terms o f their sensitivity to the u n n a t u r a l a m i n o acids kainate, q u i s q u a l a t e a n d N - m e t h y l - o - a s p a r t a t e ( N M D A ) [16]. M o s t e l e c t r o p h y s i o l o g i c a l studies to d a t e have c o n c e n t r a t e d on the N M D A r e c e p t o r as selective r e c e p t o r a n t a g o n i s t s for this site are readily available [17]. N M D A r e c e p t o r s h o w e v e r are voltage-sensitive a n d m a y n o t be a c t i v a t e d u n d e r c o n d i t i o n s o f ' n o r m a l ' s y n a p t i c t r a n s m i s s i o n [6, 1 l]. A role for N M D A receptors has been d e m o n s t r a t e d in long term p o t e n t i a t i o n [6] a n d in convulsive c o n d i t i o n s such as e x p e r i m e n t a l epilepsy [7]. N M D A r e c e p t o r i n v o l v e m e n t in e p i l e p t i f o r m activity has been d e m o n -
*Present address: Smith, Kline and French Research Ltd., The Frythe, Welwyn, Herts AL6 9AR, U.K. Correspondence." C.J. McBain, Parke-Davis Research Unit, Cambridge CB2 2QB, U.K. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
342 strated to be restricted to the latter portion of the recorded event [I, 7, 9], leaving the initial fast depolarising shift and first action potentials observed intact. Investigations into the involvement of that component of epileptiform activity insensitive to N M D A receptor antagonists have been severely hampered by the lack of suitable specific antagonists for kainate and quisqualate receptors. CNQX (6-cyano-2,3dihydroxy-7-nitro-quinoxaline) has recently been shown to be an antagonist which blocks the actions of kainate and quisqualate but not those of N M D A [10]. CNQX has also been demonstrated to reduce the amplitude of excitatory postsynaptic potentials (EPSPs) at CAI neurones in the hippocampal slice preparation [2]. We have studied the effects of CNQX on spontaneous interictal and ictal activity recorded from hippocampus maintained in organotypic culture [3]. Hippocampal organotypic cultures were prepared using the technique first described by Gahwiler [8]. Briefly, both hippocampi were aseptically dissected free from 6 day neonate Wistar rats killed by cervical dislocation prior to decapitation. Transverse slices (400/~m) were cut on a McIlwain tissue sectioner and incubated for 30 rain in Geys' balanced salt solution (BSS) enriched with glucose (36 raM) and KC1 (30 mM) at 4°C. Individual slices were then placed on a clean glass coverslip and held in place by a chicken plasma clot. Coverslips were transferred to a tissue culture tube containing 1 ml of medium (50% Basal medium eagles, 25% Hanks BSS, 25% heat-inactivated horse serum and glucose (36 mM). Tubes were placed in a roller drum within an incubator at 36°C in dry air. Cultures were fed once weekly. Conventional intracellular recording techniques were used throughout these experiments. Intracellular electrodes were filled with 3 M potassium acetate and had DC resistances of 80-200 Mg2 when measured in physiological saline. Recordings were made from CA1 neurones of stratum pyramidale using an Axoclamp amplifier in bridge mode in cultures that had been maintained for periods 3-10 weeks in vitro. Only results from neurones impaled for periods greater than 45 min are included in the study. Cultures were perfused with Hanks' BSS flowing through the recording chamber (volume 0.8 ml) at 2 ml/min at 37°C. The composition of this medium was (mM), NaC1 136.9, KCI 5.4, MgSO4 0.4, Na2HPO4 0.26, CaC12 1.25, KH2PO4 0.44, MgCI2 0.49 NaHCO3 4.1, glucose 5.5. Drugs were added in known concentrations by changing the superfusion solution by means of 3-way taps. Synaptic potentials were evoked by stimulation of the Schaffer collateral pathway at 0.03 Hz. CNQX was obtained from Tocris Neuramin and D-2-amino-5-phosphonovalerate (D-APV) from Cambridge Research Biochemicals. Epileptiform discharges were recorded from cultures following approximately 3 weeks in culture [3]. Both spontaneous interictal and ictal events were observed, identified as such using criteria derived from motor seizures described by Matsumoto and Ajmone-Marsan [12, 13]. Neurones had an input resistance of 6 8 + 6 Mg2 (n= 10) (mean _+S.E.M., the number of cells in parentheses) and resting membrane potential of 54.6+ 1.0 mV (n= 10). CNQX (10/tM) was applied to 9 neurones. In 8 neurones CNQX attenuated both the amplitude and duration of the paroxysmal depolarising shift (PDS). The amplitude was reduced by 61 _+5% ( n = 8 ) while the duration was 42_+ 9% (n = 8) that of control epileptiform activity (Fig. 1). CNQX had no effect on
343
CONTROL
CNQX (10~M)
I ILli CNQX (101~M), D-APV (30[~M)
WASH
' illllllillllillllllllllllllllll 20mY 20sec Fig. 1. Intracellular recordings, made using 3 M potassium acetate-filled microelectrodes of spontaneous epileptiform events recorded from a hippocampal organotypic culture after 63 days in vitro. The paroxysmal activity typically commenced with a large depolarising shift (PDS) of up to 40 mV in amplitude. Superimposed on this shift was a high frequency (often upwards of 100 Hz) burst of action potentials. Paroxysmal events had durations of up to 3 min. Upon termination of these events the membrane hyperpolarised to potentials below control levels and this period constituted a period of relative inexcitability. Addition of CNQX to the perfusate resulted in cessation of the high frequency action potential firing and this was associated with a decrease in both the amplitude and duration of the underlying depolarising shift. Addition of D-APV in the continued presence of CNQX abolished all spontaneous synaptic activity. These drugs, separately or in combination, had no effect on the resting membrane parameters of the neurone. Upon return to drug-free perfusate only the D-APV-sensitive component returned following one hour of wash. This neurone maintained its resting membrane potential for the duration of the experiment.
the m e a n i n p u t resistance ( 9 8 _ 2%, n = 8 o f c o n t r o l ) o r resting m e m b r a n e p o t e n t i a l ( 1 0 4 _ 2%, n = 8). C N Q X d i d n o t affect the d i s c h a r g e frequency o b s e r v e d in 7 cultures (0.08-1.0 Hz), n o r d i d it effect the p o s t - d e p o l a r i s i n g s h i f t - a f t e r h y p e r p o l a r i s a t i o n . In two n e u r o n e s however, C N Q X r e d u c e d the frequency whilst a u g m e n t i n g the d u r a t i o n o f the r e m a i n i n g P D S activity. F o l l o w i n g C N Q X a d d i t i o n the r e m a i n i n g d e p o l a r i s i n g shift was never o f sufficient a m p l i t u d e to p r e c i p i t a t e a c t i o n p o t e n t i a l firing. Single a c t i o n p o t e n t i a l s c o u l d still be e v o k e d h o w e v e r b y direct s t i m u l a t i o n o f the n e u r o n e by the p a s s a g e o f d e p o l a r i s i n g c u r r e n t t h r o u g h the r e c o r d i n g electrode, d e m o n s t r a t i n g t h a t C N Q X h a d n o t merely affected the spike g e n e r a t i n g m e c h a n i s m o f the cell. W h e n the N M D A r e c e p t o r a n t a g o n i s t D - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e ( o - A P V ) was a p p l i e d in the c o n t i n u e d presence o f C N Q X all r e m a i n i n g s p o n t a n e o u s s y n a p t i c activity was a b o l i s h e d . This c o m b i n a t i o n o f a n t a g o n i s t s was also w i t h o u t
344
5mY lOOms Fig. 2. This figure illustrates that following the onset of epileptiform activity subsequent low frequency (0.03 Hz) electrical stimulation of the Schaffer collateral pathway would evoke EPSPs which typically displayed a two component decay phase (record a). This decay can be described by two time constants zfa~,, 30.4 ms and r~bow,121.0 ms (see text for details). Addition of a mixture of CNQX (10/IM) and D-APV (30 /~M) to the superfusate bathing the culture depressed but did not totally abolish the EPSP (record b). The EPSP amplitude in this experiment was depressed by 87%. Following 1 h of wash with drug-free perfusate the EPSP amplitude recovered to 54% that of the pre-drug control value (record c). This partial recovery was probably attributable to the prolonged action of CNQX in this preparation (see text). The resting membrane potential for this neurone was - 5 7 mV. Each trace is the average of 10 consecutive synaptic potentials.
effect on the resting membrane parameters of the neurone. We have previously demonstrated that epileptiform events are of synaptic origin within these cultures [4]. All activity was blocked by addition of solutions containing high magnesium ion concentration or tetrodotoxin. Epileptiform events are reversed at membrane potentials approaching the synaptic equilibrium potential. These experiments suggest that both CNQX and D-APV are blocking components of synaptic transmission. In order to examine this further, we observed the effects of CNQX and D-APV on electrically evoked EPSPs. Following the onset of epileptiform activity evoked EPSPs have a decay phase that can be fitted by a double exponential. We have previously demonstrated that only the slow component of the decay phase is due to N M D A receptor activation and this component is only observed following the onset of epileptiform activity [4]. In these experiments addition of CNQX (10/tM) together with D-APV (30/tM) depressed the amplitude of the EPSP by up to 89-+-3% (n = 5). CNQX and D-APV never totally abolished this evoked EPSP. The effects of CNQX were only partially reversed following 1 h of wash and the EPSP returned to 58_+ 3% (n = 5) of the control value after this period. Evoked synaptic potentials recorded from organotypic cultures prior to the onset of epileptiform activity display sensitivity to CNQX but not D-APV. This suggests that only kainate/quisqualate receptors are involved in synaptic transmission under 'normal' conditions. This has recently been confirmed in conventional in vitro hippocampal slices [5]. These results suggest that following the onset of epileptiform activity, both NMDA and non-NMDA EAA receptor activation are involved in both spontaneous and evoked excitatory synaptic events. Activation of kainate/quisqualate receptors would appear to be responsible for the initiation of the event. The rapid depolarisation caused by this activation presumably would bring the membrane potential into the range for relief of the voltage-dependent block (by Mg 2+) of the channel asso-
345
ciated with the NMDA receptor [14, 15]. This subsequent recruitment of the NMDA receptor-operated component would contribute to the maintenance of the epileptiform activity. These results also demonstrate that these NMDA receptors can be activated independent of the activation of other receptors and these data are in agreement with Verdoorn and Dingledine's observations on NMDA receptors expressed following mRNA injection into Xenopusoocytes (personal communication). Thus drugs which act specifically as antagonists at kainate/quisqualate receptors may prove to be of use as anticonvulsant agents and should be particularly effective when used in conjunction with specific NMDA receptor antagonists. 1 Ashwood, T.J. and Wheal, H.V., The expression of N-methyl-D-aspartate-receptor-mediated component during epileptiform synaptic activity in the hippocampus. Br. J. Pharmacol., 91 (1987) 815-822. 2 Blake, J.F., Brown, M.W. and Collingridge, G.L., Selective blockade of non-NMDA receptor mediated depolarizations and synaptic components by CNQX. Neurosci. Len., Suppl. 32 (1988) $4. 3 Boden, P., Hill, R.G. and McBain, C.J., Long term organotypic cultures of rat hippocampal neurones display changes in neuronal excitability, J. Physiol. (Lond.), 396 (1987) 62P. 4 Boden, P., Hill, R.G. and McBain, C.J., Rat hippocampal slices in vitro display spontaneous epileptiform activity following long term organotypic culture, J. Neurosci. Methods, in press. 5 Collingridge, G.L. and Davies, S.N., Synaptic transmission following blockade of non-NMDA-type excitatory amino acid receptors in rat hippocampus in vitro, J. Physiol. (Lond.), in press. 6 Collingridge, G.L., Kehl, S.J. and Mclennan, H., Excitatory amino acids in synaptic transmission in the Schaffer collateral-commisural pathway of the rat hippocampus, J. Physiol. (Lond.), 334 (1983) 33-46. 7 Dingledine, R., Hynes, M.A. and King, G.L., Involvement of N-methyl-o-aspartate receptors in epileptiform bursting in the rat hippocampal slice, J. Physiol. (Lond.), 380 (1986) 175 189. 8 Gahwiler, B.H., Organotypic monolayer cultures of nervous tissue, J. Neurosci. Methods, 4 (1981) 329 342. 9 Herron, C.E., Williamson, R. and Collingridge, G.L., A selective N-methyl-o-aspartate antagonist depresses epileptiform activity in rat hippocampal slices. Neurosci. Lett., 61 (1985) 225-260. 10 Honore, T., Davies, S.N., Drejer, J., Fletcher, E.J., Jacobsen, P., Lodge, D. and Nielsen, F.E., Potent and competitive antagonism at non-NMDA receptors by FG 9041 and FG 9065, Soc. Neurosci. Abstr., 13 (1987) 109.7. 11 Koerner, J.F. and Cotman, C.W., Response of Schaffer collateral CAI pyramidal cell synapses of the hippocampus to analogues of acidic amino acids, Brain Res., 251 (1982) 105-I 15. 12 Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: interictal manifestations, Exp. Neurol., 9 (1964) 28f~304. 13 Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: ictal manifestations, Exp. Neurol., 9 (1964) 305-326. 14 Mayer, M.L., Westbrook, G.L. and Guthrie, P.B., Voltage-dependent block by Mg ÷ ÷ of NMDA responses in spinal cord neurones, Nature (Lond.), 309 (1984) 261-263. 15 Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A., Magnesium gates glutamateactivated channels in mouse central neurones. Nature (Lond.), 307 (1984) 462-465. 16 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 17 Watkins, J.C. and Olverman, H.J., Agonists and antagonists for excitatory amino acid receptors, Trends Neurosci. 10 (1987) 265 272.