Ketamine selectively suppresses synchronized afterdischarges in immature hippocampus

Neuroscience Letters, 69 (1986) 143-149

143

Elsevier Scientific Publishers Ireland Ltd. NSL 04117

KETAMINE SELECTIVELY S U P P R E S S E S S Y N C H R O N I Z E D A F T E R D | S C H A R G E S IN I M M A T U R E H I P P O C A M P U S

ROBERT J. BRADY* and JOHN W. SWANN ll'adsworth ('enter/or Laboratories and Research, New York Stale Department qf Health. Alhany. ~\' Y 12201 ( ['.S.A. ;

(Received January 23rd. 1986: Revised version received and accepted May 29th, 1986)

Key wor&v

cpilepsy afterdischarge N-methyl-D-aspartate noic acid hippocampus rat

ketamine

2-amino-7-phosphonohepta-

The role of excitatory amino acid neurotransmission in epileptogenesis was investigated in the developmg hippocampus, Bath application of ketamine blocked penicillin-induced, synchronized al'tcrdischarges in immature rat CA, hippocampal neurons. Ketamine also decreased the duration of the preceding inlracellularly recorded depolarization shift but had no measurable effect on the resting membrane potential or input impedance of pyramidal ceils. Concentrations of ketamine that blocked afterdischarge generation dramatically depressed intracellular depolarizations produced by iontophoretic application of N-mcth~lD-aspartate (NMDA) but not quisqualate. The effects of the NMDA antagonist 2-amino-7-phosphonohcptanoic acid on epileptiform discharges were identical to those of ketamine. These results suggest that an endogenous excitatory amino acid acting on an NMDA receptor plays a key role in the pronounced capacity of immature hippocampus for seizures.

At least 3 distinct receptors for e x c i t a t o r y a m i n o acids occur on n e u r o n s o f m a m m a l i a n b r a i n [24]. The dissociative anesthetic k e t a m i n e m a y act p r i m a r i l y by suppressing responses m e d i a t e d by one o f these r e c e p t o r types, the N - m e t h y l - D - a s p a r t a t c ( N M D A ) - p r e f e r r i n g r e c e p t o r [1, 8, 12, 21]. In n e o c o r t i c a l slices a p p a r e n t N M D A r e c e p t o r - m e d i a t e d e x c i t a t o r y s y n a p t i c p o t e n t i a l s have been suppressed by k e t a m i n e [21]. O t h e r areas o f the b r a i n have yet to be studied in this regard. R e c u r r e n t e x c i t a t o r y s y n a p t i c p a t h w a y s a p p e a r to p l a y a key role in the g e n e r a t i o n o f c p i l e p t i f o r m discharges in the h i p p o c a m p u s [6, 16, 17, 22, 23]. These synapses are p r e s u m e d to utilize an e x c i t a t o r y a m i n o acid as their n e u r o t r a n s m i t t e r [19]. Indeed, a p p l i c a t i o n o f e x c i t a t o r y a m i n o acid a n t a g o n i s t s can alter e p i l e p t i f o r m discharges in m a t u r e h i p p o c a m p a l n e u r o n s [13, 14, 16]. W e have p r e v i o u s l y shown that the CA3 subfield o f the i m m a t u r e rat h i p p o c a m p u s has a p r o n o u n c e d c a p a c i t y for p r o l o n g e d s y n c h r o n i z e d a f t e r d i s c h a r g e s when e x p o s e d to y - a m i n o b u t y r i c acid a n t a g o n i s t s [20]. In the e x p e r i m e n t s r e p o r t e d here we have

*Author for correspondence. 0304-3940'86,'$ 03.50 © 1986 Elsevier Scientilic Publishers Ireland Ltd.

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used ketamine to examine the role of N M D A receptor-mediated synaptic events in the generation of this epileptiform activity. The methods employed have been described previously [20]. Transverse slices of ventral hippocampus, 400/am thick, were obtained from immature rats 10-16 days of age. After sectioning, the slices were immediately transferred to the surface of a nylon net in an experimental chamber. There they rested at the interface of circulating artificial cerebrospinal fluid (ACSF) and humidified 95% 02/5% CO2. The chamber was continuously perfused with ACSF, which had the following composition (in raM): NaCI 122.75, K('I 5.0, CaCI2 2.0, MgSO4 2.0, NaHPO4 1.25, NaHCO~ 26.0, glucose 10.0. A mixture of 95% 02/5% CO2 was bubbled through the solution to maintain a pH of 7.4. The sodium salt of penicillin (1.7 mM) or ketamine-HC1 or 2-amino-7-phosphonoheptanoic acid (AP7) was dissolved in this medium for bath application. During iontophoretic experiments in which synaptic transmission was abolished, tetrodotoxin (TTX, 10 6 g/ml) was added to the bathing medium; the Ca :~ content was decreased to 0.2 raM: and MgC12 was added to a final concentration of 5 raM. All experiments were performed at 3 2 - 3 3 C . Iontophoretic application of N M D A and quisqualate (each 50 mM in 100 mM NaCI, pH 7.4) was performed with triplebarrel, fiber-filled microelectrodes. The third barrel was filled with 2 M NaC1 for iontophoretic current balancing. Hippocampal slices were taken from 36 different rat pups. Intracellutar recordings were made from 27 CA3 pyramidal cells, each from a separate animal. The resting membrane potentials ranged from - 5 5 to - 7 2 mV and averaged -68_+ 5.5 mV (S.E.M.). The microelectrodes were filled with 4 M potassium acetate and had resistances of 100 180 M~. For extracellular field recordings the microelectrodes contained 2 M NaCI and had resistances of 5- 15 M~. Monopolar, electrolytically sharpened tungsten electrodes were used Jbr orthodromic stimulation of CA3 neurons via fiber tracts in the stratum radiatum. Data were stored on FM magnetic tape (DC~5 kHz) for further analysis. Selected portions were digitized (10 kHz) and drawn with a HP 7470A plotter. To display the activity at a slower time base, data were played back from tape on a pen recorder at 25% of the original tape speed. The effects of ketamine on evoked penicillin-induced epileptiform activity are illustrated in Fig. 1. The control panel shows a response to a single orthodromic stimulus. The depolarization shift and coincident extracellular epileptiform burst, which are seen clearly in the lower traces, were followed by a prolonged afterdischarge. Bath application of 250 /aM ketamine eliminated the afterdischarges but had no effect on the resting membrane potential, resistance or stimulus threshold of the evoked response. Stimulation of the stratum radiatum still produced a depolarization shift and epileptiform burst extracellularly. However, the average duration of the depolarization shift at half-maximal amplitude was reduced from 141 ±5.2 ms (mean _+S.E.M., n = 6) during the control period to 91 ± 3.7 ms (n = 6) in the presence of ketamine. This is an average decrease of 36%. When ketamine was washed out, the afterdischarges reappeared. The lowest effective dose of ketamine for complete suppression of afterdischarges

145

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Fig. I. Effects of ketamine on penicillin-induced epileptiform discharges. Simultaneous intracellular {A) and exlracellular field (B) recordings were obtained from the CA~ cell body layer of a rat hippocampal slice taken on postnatal day 9. A pulse of hyperpolarizing current was passed intracellularly just berate the response was evoked by orthodromic stimuli applied to the stratum radiatum. Responses were elicited by identical stimuli belBre (left panels), during (center panels), and after (right panels) bath application of ketamine (250 I,tM). The lower sets of traces are the initial segments of those shown above but at a faster time base. The resting membrane potential of the cell was - 62 mV.

was 100 g M , but c o m p l e t e suppression was then seen in only 5 o f 8 slices tested. At 250 ~M k e t a m i n e there was c o m p l e t e s u p p r e s s i o n in all slices tested (5 o f 5). W e next investigated the effects o f k e t a m i n e on the response o f i m m a t u r e h i p p o c a m p a l CA3 p y r a m i d a l cells to two e x c i t a t o r y a m i n o acid agonists, N M D A and quisqualate. To ensure that the responses were p o s t s y n a p t i c a l l y m e d i a t e d , the slices were b a t h e d in a s o l u t i o n c o n t a i n i n g T T X , high M g 2+ a n d low Ca 2+. T h e i o n t o p h o r e t i c m i c r o p i p e t t e was placed in the p r o x i m a l p o r t i o n o f the basilar dendritic layer. W h e n N M D A a n d q u i s q u a l a t e were i o n t o p h o r e s e d , while h y p e r p o l a r i z i n g cond u c t a n c e pulses were passed intracellularly, the responses o f a p y r a m i d a l cell (Fig. 2, panel 1, row A) were very similar to those r e c o r d e d in the m a t u r e h i p p o c a m p u s [7, 10, 11]. N M D A caused the cell to d e p o l a r i z e , first with an a p p a r e n t associated increase and then with a decrease in m e m b r a n e resistance. Q u i s q u a l a t e , on the o t h e r h a n d , p r o d u c e d a d e p o l a r i z a t i o n a s s o c i a t e d solely with a decrease in m e m b r a n e resistance. D e p o l a r i z a t i o n s elicited by N M D A a n d q u i s q u a l a t e are shown in the absence o f h y p e r p o l a r i z i n g pulses (row B). K e t a m i n e d r a m a t i c a l l y decreased the N M D A e v o k e d d e p o l a r i z a t i o n but h a d no effect on the q u i s q u a l a t e response (row C). This high degree o f selectivity was o b s e r v e d in all 7 cells studied in this m a n n e r . In the presence o f 250 btM k e t a m i n e a m u c h greater N M D A i o n t o p h o r e t i c c u r r e n t was needed to p r o d u c e a d e p o l a r i z a t i o n (Fig. 2, panel 2). K e t a m i n e d r a m a t i c a l l y shifted the N M D A d o s e - r e s p o n s e curve, a n d b o t h the slope o f the curve a n d the m a x i m a l d e p o l a r i z a t i o n were decreased. These latter effects are consistent with the view that k e t a m i n e is n o t a c o m p e t i t i v e N M D A a n t a g o n i s t .

146

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Fig. 2. Effects of ketamine on N M D A - and quisqualate-evoked depolarizations. Intracellular records were obtained from CA 3 pyramidal cells on postnatal day 13 (panel 1) and 14 (panel 2). The iontophoretic electrodes were placed in the infrapyramidal zone of the stratum oriens, 50 p,m from the intracellular recording sites in the cell body layer. Panel 1 shows the response of a cell to iontophoretic pulses of N M D A and quisqualate. In row A changes in membrane resistance are monitored by hyperpolarizing current pulses (0.2 nA/100 ms). Row B shows the control responses without current pulses. Row C shows the responses after 20 min of bath application of 250 laM ketamine. The resting membrane potential of this cell was - 7 2 mV. Panel 2 shows the alteration of the N M D A dose-response relationship by ketamine (250/aM). It is a semilog plot of the peak amplitude of the depolarization induced by iontophoretic currents of varying amplitude. The resting potential of the cell was 58 mV. For the studies in both panels the slices were bathed in medium containing TTX (10 6 g/ml), high M g ~ ~ (5 m M ) and low Ca 2+ (0.2 mM).

Because ketamine had effects solely on N M D A responses, its ability to suppress afterdischarge generation appeared to be mediated by its actions at this excitatory amino acid receptor subtype. To test this inference we applied the N M D A antagonist AP7 to penicillin-treated slices from immature rats. Its effects on epileptiform discharges (Fig. 3, panel 1), were identical to those of ketamine, although an approximately 20-fold higher concentration of AP7 was required. AP7 eliminated the afterdischarges and decreased the duration of the epileptiform burst. These effects were reversed by washing AP7 from the chamber, Suppression of afterdischarges was seen in all of 3 slices tested when 5 mM AP7 was bath-applied. However, bath applica tion of 1 mM AP7 failed to completely block afterdischarges in both of two experiments. Iontophoretic experiments (Fig. 3, panel 2) show that even at a concentration of 5 raM, AP7 selectively suppresses the response of CA3 pyramidal cells to NMDA. Responses to quisqualate are relatively unaffected. The results of the microiontophoretic experiments show that CA3 hippocampal pyramidal cells from 10- to 20-day-old rats respond to excitatory amino acid agonists much like their mature counterparts. Moreover, these results indicate that ketamine is a selective antagonist of N M D A receptors in the immature hippocampus. Ketamine had no measurable effect on the resting membrane potential or input impedance of pyramidal cells. These findings are in agreement with recent reports [1, 8, 12, 21]

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Fig. 3. Effects of AP7 on penicillin-induced epileptiform activity (panel l) and on N M D A - and quisquahUe-evoked depolarizations (panel 2). Panel 1 shows extracellular recordings obtained from the CA~ cell body layer of a hippocampal slice taken on postnatal day 16. These responses were evoked by orthodromic stimuli applied in the stratum radiatum. The responses were obtained before (panel 1A), during (panel 1B) and aflcr (panel IC) bath application of AP7 (5 mM). Panel 2 shows intracellular recordings obtained from a CA~ pyramidal cell on postnatal day 12. Recordings were obtained as in Fig. 2. Control responses 1o the iontophoretic pulses (4 nA/s) are shown in the presence of hyperpolarizing current pulses (panel 2A) and without them (panel 2B). Panel 2C shows the responses after 15 min of bath application of 5 mM AP7. The resting membrane potential of this cell was - 7 1 mV.

that ketamine may act exclusively by suppressing the action of an endogenous excitatory amino acid on N M D A receptors. Thus our results show that N M D A receptormediated neurophysiologic events play a key role in the genesis of synchronized afterdischarges recorded during bath application of penicillin. N M D A antagonists, especially AP7, have attenuated or blocked seizure generation in several experimental model systems [2, 5, 15, 18]. Our results show that although AP7 can selectively suppress afterdischarge generation in the CA3 subfield of the immature hippocampus, its effective concentration is high - at least 20-fold higher than that of ketamine. This relative inefficiency of AP7 in the developing hippocampus may stem from its mode of interaction with the N M D A receptor. AP7 is thought to be a competitive N M D A antagonist [9, 12]. During penicillin-induced epileptogenesis the synaptic activation of immature pyramidal cells by an endogenous excitatory amino acid neurotransmitter may be so intense, and such large quantities of receptor agonist may be released, that unusually high concentrations of AP7 are required to compete effectively for the receptor and to suppress afterdischarges. The same suppression is effected by a much lower concentration of ketamine, which apparently does not compete directly for the N M D A binding site (Fig. 2, panel 2; see also ref. 12). The physiologic mechanisms responsible for afterdischarge generation and for the transition from interictal to ictal-like events in penicillin foci have been sought for more than 20 years. One recent study suggests that chemical synaptic transmission may be important in these processes [23]. Our results extend these observations by

148

showing that N M D A receptor-mediated synaptic potentials play a key role in the genesis of seizure-like events. Under normal physiologic conditions the responses of hippocampal neurons to orthodromic stimulation appear not to be mediated by an N M D A receptor [4]. However, under conditions of increased excitability (e.g. refs. 3 and 4) N M D A receptors appear to mediate at least a portion of the excitatory synaptic activation. For instance, the duration of the depolarization shift produced in mature CA j neurons in the presence of bicuculline is reduced by the N MDA receptor antagonist 2-amino-5-phosphonovaleric acid [14]. Our results are clearly in agreement with this observation (Figs. I and 3). However, we show here that subsequent synchronized afterdischarges are particularly sensitive to N M D A antagonists. Drugs such as ketamine will be important pharmacologic tools for further investigations of the neurophysiologic origins of seizures.

This research was supported by Grants NS-23071 (to R.J.B.) and NS-18309 (to J.W.S.) from the National Institute of Neurological and Communicative Disorders and Stroke - National Institutes of Health, and a National Research Service Award postdoctoral fellowship (to R.J.B.) NS-07395. Ketamine was provided by the Warner-Lambert Co. We thank Dr. David Carpenter for his critical reading of the manuscript, Ms. Karen L. Smith for expert technical assistance, Ms. Carolyn S. Wieland and Ms. Elizabeth Larkins for skilled preparation of the manuscript. 1 Annis, N.A., Berry, S.C., Burton, N.R. and Lodge, D., The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N-methyl-aspartate, Br. J. Pharmacol., 79 (1983) 565-573. 2 Bourn, W.M., Yang, D3. and Davisson, J.N., Effect of ketamine on sound-induced convulsions in epilepsy prone rats, Pharmacol. Res. Commun., 15 (9) (1983) 815-824. 3 Coan, E.J. and Collingridge, G.L., Magnesium ions block an N-methyl-D-aspartate receptor-mediated component of synaptic transmission in rat hippocampus, Neurosci. Lett., 53 (1985) 21-26. 4 Collingridge, G.L., Kehl, S.J. and McLennon, H., Excitatory amino acids in synaptic transmission in the Schaeffer collateral-commisural pathway of the rat hippocampus, J. Physiol. (London), 334 (1983) 33-46. 5 Coucher, M.J., Collins, J.F. and Meldrum, B.S., Anticonvulsant actions of excitatory amino acid antagonists, Science, 216 (1982) 899-90 I. 6 Dichter, M. and Spencer, W.A., Penicillin-induced interictal discharges from cat hippocampus. II Mechanisms underlying origin and restriction, J. Neurophysiol., 32 (1969) 663-687. 7 Dingledine, R., N-methyl-aspartate activates voltage-dependent calcium conductance in rat hippocampal pyramidal cells, J. Physiol. (London), 343 (1983) 385~405. 8 Duchen, M.R., Barton, N.R. and Biscoe, T.J., An intracellular study of the interactions of N-methylDe-aspartate with ketamine in the mouse hippoeampal slice, Brain Res., 342 (1985) 149-153. 9 Evans, R.H., Francis, A.A., James, A.W, Smith, D.A.S. and Watkins, J.C., The effects of a series of c0-phosphonic a-carboxylic amino acids on electrically evoked and excitant amino acid-induced responses in isolated spinal cord preparations, Br. J. Pharmacol,, 75 (1982) 65-76. l0 Hablitz, J.J.. Conductance changes induced by DL-homocysteic acid and N-methyl-De-aspartate in hippocampal neurons, Brain Res., 247 (1982) 149-152. l 1 Hablitz, J.J., Action of excitatory amino acids and their antagonists on hippocampal neurons, Cell. Mol. Neurobiol., 5(4) (1985) 389~05. 12 Harrison, N.L. and Simmonds, M.A., Quantitative studies on some antagonists of N-methyI-D-aspartare in slices of rat cerebral cortex, Br. J. Pharmacol., 84 (1985) 381-391.

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13 l lerron, C.E., Williamson, R. and Collingridge, G.L., A selective N-methyl-D-aspartate antagonist dcpresses cpileptiform activity in rat hippocampal slices, Ncurosci. Lett., 61 (1985) 255- 260. 14 ttynes, M.A. and Dingledine, R., Attenuation of epileptiform burst firing in the rat hippocampal slice by antagonists of N-methyI-D-aspartate receptors, Soc. Neurosci. Abstr., 10 (1984) 229. 15 Meldruln, B.S., Amino acid neurotransmitters and new approaches to anticonvulsant drug action, Epilepsia, 24, Suppl. 2 (1984) s140-s149. 16 Miles+ R., Wong, R.K.S. and Traub, R.D., Synchronized afterdischarges in the hippocampus: contribution of local interactions, Neuroscience, 12(4) (1984) 1179 1189. 17 Miles, R. and Wong, R.K.S., Single neurons can initiate synchronized population discharge in the ('A3 region of guinea pig hippocampus, Nature (London) 306 (1983) 371 373. 18 Ryan, G.P., Hackman, J.C. and Davidoff, R.A., Spinal seizures and excitatory amino acid-mediated synaptic transmission, Neurosci. Lett., 44 (1984) 161 166. 19 Storm-Mathisen, J., Glutamate in hippocampal pathways. In G. Dichara and G.L. Gessa (Eds.}, Glutamate as a Neurotransmitter, Raven Press, New York+ 1981, pp. 43 55. 2[) Swann, .I.W. and Brady, R.J., Penicillin-induced epileptogenesis in immature hippocampal pyramidal cells, Dev. Brain Res., 12 (1984) 243 254. 21 Thomson, A.M., West, D.C. and Lodge+ D., An N-methyl-aspartate receptor-mediated synapse in rat cerebral cortex: a site of action of ketamine, Nature (London) 313 (I 985) 479481. 22 Traub. R.D. and Wong+ R.K.S., Cellular mechanism of neuronal synchronization in epilepsy, Science, 216 (1982) 745 747. 23 Traub, R.D., Knowles, W.D., Miles, R. and Wong, R.K.S., Synchronized afterdischarges in the hippocampus: stimulation studies of the cellular mechanism, Neuroscience, 12(4) (1984) 1191 1200. 24 Watkins, J.C. and Evans, R.H,, Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 1652[)4.