On the epileptogenic effects of kainic acid and dihydrokainic acid in the dentate gyrus of the rat

NeuropharmacologyVol. 21, No. 4, pp. 375-381, 1988 Printed in Great Britain

0028-3908/88$3.00+ 0.00 Pergamon Press plc

ON THE EPILEPTOGENIC EFFECTS OF KAINIC ACID AND DIHYDROKAINIC ACID IN THE DENTATE GYRUS OF THE RAT S. P. BUTCHER,*? I. JACOBSONand A. HAMBERGER Institute of Neurobiology, University of Goteborg, Goteborg, Sweden (Accepted 1 October 1987)

Summary-The in uivo effects of the acidic amino receptor agonist, kainic acid and the inhibitors of the uptake of glutamate, dihydrokainic acid and threo-3_hydroxyaspartate, on spontaneous activity and perforant path evoked field potentials were examined in the dentate gyrus of the rat. The effect of these compounds on extracellular levels of endogenous amino acids in the hippocampus was assessed simultaneously using in viuo microdialysis. Kainic acid (10-100 PM) and dihydrokainic acid (l-10mM) both evoked epileptiform activity and an apparent loss of recurrent inhibition (as assessed using the paired-pulse technique). Extracellular increases in taurine, alanine and phosphoethanolamine were noted following administration of kainate (100 PM) and dihydrokainate (l-10 mM). An increase in extracellular glutamate and aspartate was also noted in rats treated with dihydrokainate (100 PM-10 mM). In contrast, threo-3-hydroxyaspartate did not induce epileptiform activity, suggesting that the epileptogenic effects of dihydrokainate and kainate are not mediated by inhibition of uptake. The effect of the N-methylD-aspartate receptor antagonist, n-2-amino-5phosphonovalerate on these responses was studied. This compound attenuated the epileptiform activity and reversed the apparent loss of recurrent inhibition in response to both kainic acid and dihydrokainic acid. These data suggest that activation of N-methylo-aspartate receptors underlies the epileptogenic effects of both compounds, and the possible mechanisms which might be involved in this response are discussed. Key words: field potential, perforant path, epilepsy, acidic amino acids, 2-amino-5phosphonovalerate, brain dialysis, hippocampus.

Recent studies in the brain suggest that there may be a link between epilepsy and putative excitatory amino acid neurotransmitters, such as glutamate and aspartate. Thus, an increased release of these substances has been demonstrated in several animal models of epilepsy (Koyama, 1972; Dodd and Bradford, 1976; Dodd, Bradford, Abdul-Ghani, Cox and CoutinhoNetto, 1980; de Boer, Stoof and van Duijn, 1982; Skerritt and Johnston, 1983; Peterson, Collins and Bradford, 1983) and acidic amino acid receptor particularly antagonists, N-methyl-D-aspartate (NMDA) receptor selective blockers, are reported to be effective inhibitors of seizure activity (Croucher, Collins and Meldrum, 1982; Czuczwar and Meldrum, 1982; Meldrum, Croucher, Badman and Collins, 1983). Although the precise mechanism(s) underlying the initiation and propagation of epilepsy are uncertain, electrophysiological experiments in the hippocampus have demonstrated that a loss of recurrent inhibition may be intimately involved in these events (Wong and Prince, 1979; Ben Ari, Krnjevic and Reinhardt,

*To whom all correspondence and reprint requests should be sent. tPresent address: Department of Pharmacology, University of Edinburgh Medical School, 1 George Square, Edinburgh, U.K.

1979; Schwarzkroin and Prince, 1980; Dingledine and Gjerstad, 1980). Recurrent inhibition has been demonstrated previously in the dentate gyrus using a paired pulse stimulation of the afferent perforant path fibres (McNaughton and Barnes, 1977). This response is believed to be mediated through a negative feedback loop involving inhibitory interneurones (Anderson, Holmqvist and Voorhoeve, 1966). The finding that application of the rigid glutamate receptor agonist, kainic acid, induces epileptiform activity and an apparent loss of recurrent inhibition in the dentate gyrus was therefore of great interest (Sloviter and Damiano, 1981a). Furthermore, recent studies have demonstrated that chronic stimulation of the afferent perforant pathway exerts similar effects (Sloviter and Damiano, 198 1b). This finding is particularly relevant since these fibres are believed to utilise glutamate and/or aspartate as their transmitter (White, Nadler, Hamberger, Cotman and Cummins, 1977; Nadler, White, Vaca, Perry and Cotman, 1978). The aim of the present study was to investigate the mechanism(s) underlying the epileptogenic actions of kainic acid and endogenous glutamate/aspartate in the dentate gyrus of the rat. The method involved simultaneous analysis of the electroencephalogram (EEG), evoked field potentials and concentrations of extracellular amino acids, using an implanted dialysis probe with combined electrode (Sandberg, Butcher 375

S. P. BUTCHER etal.

376

and Hagberg, 1986). Epileptiform activity was induced by application of either kainic acid or inhibitors of the uptake of glutamate, such as dihydrokainate (DHK; Johnston, Kennedy and Twitchen, 1979) and threo-3-hydroxyaspartate (THA; Balcar, Johnston and Twitchen, 1977), through the dialysis probe. The effects of D-2-amino-S-phosphonovalerate (D-APV), an acidic alnino acid receptor antagonist which is sensitive to NMDA, on acidic amino acidinduced epileptiform activity have been assessed. METHODS Surgical procedures

Sprague-Dawley rats (250-300 g; either sex) were anaesthetised initially with methohexital (75 mg/kg; Brietal@; Lilly and Co.), tracheotomised and placed in a Kopf stereotaxic frame. Anaesthesia was then maintained using halothane ( < 1.5%). The skull was exposed and burr holes (2 mm dia) were drilled directly above the dentate gyrus (AP - 3.8; ML f 2; Paxinos and Watson, 1982) and the angular bundle (AP - 8.0; ML & 4.5; Paxinos and Watson, 1982). A cannulla design dialysis probe (1 mm active dialysis length; 0.3 mm dia; see Sandberg et al. 1986; Jacobson, Butcher and Hamberger, 1986) was then positioned in the dentate gyrus. This device incorporates a tungsten electrode adjacent to the dialysis probe which is used for electrical recordings (EEG and evoked potentials). Stimulation

of the perforant path

A concentric bipolar electrode (Clark Electromedical; U.K.) was positioned in the angular bundle and the perforant path was stimulated at 0.7-1.0 mA for 100 psec at 1 set intervals. These stimulus parameters were just above threshold with regard to the firing of population spikes. The dialysis probe was then repositioned until the maximum evoked response, corresponding to an electrode position in the hilus of the dentate gyrus, was observed. The position of the dialysis probe within the dentate gyrus was later confirmed histologically. Evoked potentials and EEG activity were amplified using a Digitimer “Neurolog” system and field potentials were stored and averaged using an ABC microcomputer (Jacobson et al., 1986). The EEG activity was recorded continuously using a Grass recorder. Recurrent inhibition was studied using a paired pulse technique (two identical stimuli given 20msec apart). The amplitude of the population spike 1 (PSI; the synchronous discharge of granule cells following the first stimulus) and of the population spike 2 (PS2; the synchronous discharge of granule cells following the second stimulus) was determined as shown in Fig. 2. Experiment

protocol

The dialysis probe was perfused continuously at 1.25 pl/min with oxygenated Krebs-bicarbonate buffer (composition in mM; NaCl 122; KC1 3.0;

MgSO, 1.12; KH, PO, 0.04; NaHCO, 25; CaCl, 1.2; pH 7.4). After a 60min equilibrium washout, samples of dialysate (20 min; 25 ~1) were collected throughout the experiment and analysed for content of amino acids. Control EEG and field potential responses were also recorded before the compounds to be tested (kainic acid; DHK; THA) were introduced into the perfusion buffer for periods of 60 min. The concentration of each agent was increased sequentially until epileptiform EEG activity (fast spike deflections greater than 250 PA amplitude) was noted and the field potential responses were then recorded. After perfusion of the drug for 60 min and after a period of epileptiform EEG activity lasting at least 20 min, D-APV (1 mM) was introduced into the perfusing buffer together with the compound to be tested. The records of EEG and field potentials were then recorded 10 min later. Analysis

of amino acids

The concentration of amino acids was determined by reverse phase liquid chromatography and fluorescent detection following precolumn derivatisation with o-phthaldialdehyde (Lindroth, Hamberger and Sandberg, 1986). Derivatised amino acids were separated by reverse phase liquid chromatography on a Nucleosil 5 pm column (200 x 4.6 mm), using gradient elution with methanol (CrlOO%) in sodium phosphate buffer (50 mM; pH 5.4). Quantification was performed by peak height analysis with regard to an amino acid standard. Materials

Kainic acid, DHK and NMDA were all supplied by Sigma. Threo-3-hydroxyaspartate (THA) was supplied by Calbiochem and APV by Tocris Chemicals, U.K.

RESULTS EEG activity

Perfusion of 1 PM KA did not affect the pattern of EEG activity. Increasing the concentration of kainic acid to 10 PM led to the appearance of epileptiform activity after 30-40 min in 3/6 animals. A further increase in concentration to 100pM evoked epileptiform activity almost immediately in the remaining 3 animals ( < 10 min; Fig. 1). Addition of 100 PM DHK to the perfusion buffer did not affect the EEG activity. At 1 mM, 2/6 animals exhibited epileptiform activity after 30-40 min. Increasing the concentration of DHK to 1OmM induced epileptiform activity almost immediately in the remaining 4 animals (< 10 min; Fig. 1). Threo-3-hydroxyaspartate was also perfused over a wide concentration range (lOpM-10 mM). No alterations in the pattern of EEG activity were noted (Fig. 1).

Epileptogenic effects of kainic acid and dihydrokainic acid KA

DHK

317

THA

Fig. 1. Effects of acidic amino acid derivatives on EEG activity in the dentate gyrus of the rat. Results show typical records obtained from each experimental groups of rats. Voltage calibration 500 pV; time scale 500 msec. The upper records show control activity obtained immediately before perfusion with drug. Kainic acid (KA, 100 PM) and DHK (10 mM) (middle records) are taken from animals which exhibited epileptiform activity for at least 20 min. The record of THA was taken after perfusion with THA (10 mM) for 60 min. The lower records show the effects of D-APV (1 mM; coperfused with either KA or DHK) in the same animal 10min later.

Inclusion of D-APV (1 mM) in the perfusing buffer together with epileptogenic doses of either kainic acid or DHK attenuated the epileptiform activity within 10 min (Fig. 1). A decrease in both the amplitude and frequency of epileptiform activity was noted.

(Al

Perforant path evokedfield potentials

Perforant path evoked field potentials were recorded in the hilus of the dentate gyrus (Fig. 2). The amplitude of the population spikes (which represent a synchronous discharge of granule cells) was determined after twin stimuli given 20 msec apart. No interactions between epileptiform activity and evoked potentials were noted. Thus, stimulation of the perforant pathway did not noticeably affect the pattern of randomly firing epileptic spikes. Kainic acid (1 PM) and DHK (100 PM) did not affect the amplitude of either population spike (data not shown). Threo-3-hydroxyaspartate (10 mM) also did not affect the amplitude of either PSI or 2 (PSl; control, 0.2mV; THA, OmV; PS2; control 0.2mV; THA OmV). In those rats perfused with either 10 PM kainic acid (n = 3) or 1 nM DHK (n = 2), which exhibited epileptiform activity, there was an increase in the amplitude of both population spikes 1 and 2. these alterations were only noted However, 30-40min after administration of kainic acid and DHK, i.e. concomitant with the appearance of epileptiform activity. In rats perfused with either 10 PM kainic acid (n = 3) or 1 nM DHK (n = 4) which did not exhibit epileptiform activity, no such alterations were noted. An increase in the amplitude of both population spike 1 and 2 was noted in all animals when the concentration of kainic acid was raised to 100 g M, and that of DHK to 1OmM. This again occurred simultaneously with the onset of epileptiform activity (< 1Omin after administration). Data from animals displaying epileptiform activity are pooled in Table 1. When expressed as a ratio of PS2: PSI, a control value of 0.10-0.17 was noted (Table 1). In animals

-“IL (El

PSI PS2

%

(Cl

L

Fig. 2. Effects of analogues of acidic amino acids on evoked potentials in the dentate gyrus of the rat. Results show typical records obtained in response to stimulation of the perforant path (twin pulses at 1 Hz; 0.7-1.0 mA; 100 pse~ duration; 20 msec inter-stimulus interval). Voltage calibration, I mV; time scale, 10 msec. Control responses (trace A) were obtained immediately before perfusion of the drugs of interest. The middle record (trace B) demonstrates the effects of kainic acid (1OOpM; 20min following administration of drug) on this response. The method used to calculate the amplitude of the population spike is shown in this record. The lower record (trace C) shows the effect of D-APV (1 mM; perfused together with kainic acid) in the same animal 10 min later.

378 Table I.

Effects

of D-APV on acidic amino acid-induced alterations in the amplitude of evoked field potentials in the perforant path Population spike 1 amplitude (PSI) (mV)

Population spike 2 amplitude (PS2) (mV)

PS2:PSI ratio

Control DHK DHK + APV

0.70 + 0.16 1.65 + 0.34’. I .61 f 0.62

0.12 & 0.05 I .40 + 0.25* 0.39 k0.16t

0.17 0.85 0.24

Control KA KA + APV

1.1 + 0.30 1.41 f 0.22” 1.30+0.15

0.10 f 0.07 I .48 f 0.47’ 0.32 + 0.1 It

0.13 I .05 0.25

Recurrent inhibition was examined by stimulating the perforant path at 1Hz with paired electrical pulses (0.7-I mA intensity; 100 bsec duration; 20 msec stimulus interval). The mean response of 4-S stimuli was analysed and the amplitude of the two population spikes was determined. Results are the meansf SEM of at least 6 independent experiments. Statistical differences between groups were assessed using an analysis of variance (ANOVA) test combined with Bonferroni analysis [control (PSI) vs KA (PSl)/DHK (PSI); l * = P c 0.01;KA vs KA + APV and DHK + APV Call PSI) = non sienificant: control (PS2) vs KA (PSZ)/DHK (PSZ)/DHK (PS2); * = P < 0.02: Kl (PS2)‘vs KA +‘AP+ (PS2), t = P < 0.02; DHK (PS2) vs DHK _( APV (PS2); t = P < 0.021.

which displayed epileptiform activity, this value increased to approximately 1. Application of D-APV (1 mM) through the dialysis probe reversed the increase in the amplitude of population spike 2 evoked by both kainic acid and DHK in animals which displayed epileptiform activity (Table 1). However, the amplitude of PSI was not affected by D-APV (Table 1). Accordingly, the ratio of PS2: PSI fell to close to control levels after administration of D-APV (approximately 0.25); D-APV did not affect the amplitude of either population spike under control conditions (data not shown).

The effects of THA on the content of amino acids in the dialysate could not be assessed because this compound interferes with the detection of amino

ASP

GLU

200

200

100

100

Content of amino acid, in dialysate

The basal concentration of amino acids in dialysates was: aspartate (asp), 0.2 PM; asparagine (asn), 0.2 PM; glutamate (glu), 0.8 PM; glutamine (gln), 8 PM; phosphoethanolamine (PEA), 0.9 PM; taurine (tau), 1.7 PM; alanine (ala), 0.9 PM; methionine (met), 0.4pM; valine (val), 1.3 PM. The concentration of asparagine, methionine and valine in the dialysate was not altered under any conditions. Application of kainic acid (1 and 10 PM) did not significantly affect the concentration of any of the other amino acids in the dialysate (Fig. 3). No significant differences were noted between those animals perfused with 10 PM, which displayed epileptiform activity and those which did not. However, at 100 PM, increases in the dialysate concentration of taurine, alanine and phosphoethanolamine in the dialysate were noted (Fig. 3). Dihydrokainate (100 PM) induced an increase in the concentration of glutamate and aspartate in the dialysate (Fig. 4). At 1 mM, the responses to glutamate and aspartate were larger, and there was also an increase in the concentration of taurine, phosphoethanolamine and alanine in the dialysate. However, this occurred irrespective of whether the animals displayed epileptiform activity and occurred within 40min of administration of the drug. A larger response of each of these amino acids was noted after perfusion of 10mM DHK (Fig. 4).

w

w

01 1

ii

2

3

4

5

L----J 1

GLN 300

I-

I

1

2

3

4

5

PEA

I

I

I

1

2

3

4

5

r

Of--%--z ALA

TAU ml-200

100

FRACTION

NUMBER

Fig. 3. Effects of kainate on the content of endogenous amino acids in the dialysate. Each fraction represents a

20 min collection period. Kainic acid was added after fraction 2. Results are the meansf SEM of at least 4 independent experiments. Statistical differences between control and drug fractions were assessed using analysis of variance (*; P ~0.05). Key; 0, 1 PM; n , IOlM; A, 1OOnM. asp = aspartate; glu = glutamate; gln = glutamine; PEA = phosphoethanolamine; tau = taurine; ala = alanine.

Epileptogenic effects of kainic acid and dihydrokainic acid GLU

379

amplitude of both PSI and PS2 and increased the PS2: PSI ratio from 0.14 to approximately 1. The l l l inhibition of the amplitude of PS2 compared with l 300 l PSI under control conditions is believed to be mediated by inhibitory interneurones (Anderson et al., 150 1966; Sloviter and Damiano, 1981a) and the effects of + t l kainic acid and DHK noted in the present study may 0 Ez therefore represent a loss of recurrent inhibition as 1 2 3 4 5 suggested previously (Sloviter and Damiano, 1981b). PEA GLN However, although increases in the amplitude of both 300 150 r r PSI and 2 occurred simultaneously with the appearance of epileptiform activity, only the increase the amplitude of PS2 was sensitive to D-APV. Since this compound also attenuated epileptiform activity a connection between these two events is therefore suggested. Thus, activation of NMDA receptors appears to mediate both the epileptiform activity and ALA TAU the increase in the amplitude of PS2 evoked by kainic XI0 300 acid and DHK. Similar inhibitory effects of APV l l against seizures evoked by kainic acid have been 200 t * l reported previously in the deep prepyriform cortex (Piredda and Gale, 1986) and after injection of kainic loo acid and APV together into the ventricles (Turski, Meldrum and Collins, 1985). This finding may be 1 2 3 4 5 3 4 5 explained in terms of the activation of a voltage-gated membrane channel sensitive to NMDA (Nowak, FRACTION NUMBER Bregestovski, Ascher, Herbert and Prochiantz, 1985; Fig. 4. Effects of dihydrokainate on the content of endoMayer, Westbrook and Guthrie, 1985). Thus, an genous amino acids in the dialysate. Each fraction represents a 20min collection period. DHK was added after alteration in the state of depolarisation of the memperiod 2. Results are the means f SEM of at least 4 indepen- brane induced by either kainic acid or DHK may dent experiments. Statistical differencesbetween control and allow endogenous glutamate/aspartate to activate drug were assessed using analysis of variance (*; P < 0.05). this membrane channel. Such an alteration could be Key; 0, 100pM; n , 1 mM; A, 1OmM. Abbreviations as caused by a variety of mechanisms. A depolarisation in Fig. 4. of granule cells in the dentate gyrus induced by kainic acid has been reported previously (Crunelli, Forda, Collingridge and Kelly, 1983; Westbrook and Lothman, 1984). However, in this case an NMDA DISCUSSION receptor-mediated facilitation of the amplitude of the The present results demonstrate that locally ap- PSI might be anticipated. Moreover, in pyramidal cells in the hippocampus kainic acid applied acutely plied kainic acid and DHK evoked epileptiform activity in the dentate gyrus of the rat. This finding produced epileptiform activity at doses which did not directly influence membane depolarisation (Westis in agreement with data concerning systemicallyapplied kainic acid (Sloviter and Damiano, 1981a). brook and Lothman, 1984; Fisher and Alger, 1984). The threshold concentration required to produce Since kainic acid blocked inhibition mediated by this effect was l&100 PM for kainic acid and y-aminobutyric acid (GABA) at these small doses I--1OmM in the case of DHK. However, the more (Fisher and Alger, 1984), a disinhibitory process may in fact underlie the appearance of the component of potent inhibitor of uptake, threo-3-hydroxyaspartate the evoked field potential and the induction of epi(Balcar et al., 1977) was devoid of epileptiform activity even at 10mM. This suggests that the epi- leptiform activity mediated by an NMDA receptor. recent intracellular electroleptogenic action of both kainic acid and DHK is In this respect, likely to be mediated through a direct activation of physiological studies have demonstrated that D-APV inhibited epileptiform activity evoked in the presence acidic amino acid receptors, rather than by an inof drugs which reduce synaptic inhibition, such hibition of uptake. In this respect, the potent neuroas bicuculline and pentylenetetrazole (Herron, excitatory effects of kainic acid and the weaker Williamson and Collingridge, 1985; Dingledine, actions of DHK have been demonstrated (Johnson, Hynes and King, 1986). Curtis, Davis and McCulloch, 1974). The facilitation of the amplitude of PSI (insensitive The effects of DHK, THA and kainic acid on to D-APV) might be explained by the kainic acidevoked field potentials in the perforant path were also mediated potentiation of extracellular synaptic reexamined. Concentrations of kainic acid and DHK sponses noted by several workers (Sloviter and which induced epileptiform activity increased the ASP

450

* w

O!---eFz

&-----

Of--F--

380

S. P. BUTCHER et al.

Damiano, 1981a; Coilingridge, Kehl, Loo and McLennan, 1983). A prolongation of synapti~~ly evoked, intracellular excitatory postsynaptic potentials induced by kainic acid has also been reported previously and this has also been suggested to be mediated by a disinhibitory action of kainic acid (Westbrook and Lothman, 1984; Fisher and Alger, 1984). This would increase the likelihood of a synchronous discharge of granule cells after stimulation of the perforant path, thereby increasing the amplitude of PSI. Furthermore, if the second stimulus is delivered during the prolonged excitatory postsynaptic potentials (i.e. when the membrane is depolarised to some extent), an activation of NMDA receptors might be expected. The present data are also relevant to the effects of kainic acid and DHK on levels of extracellular amino acids. Although their significance is uncertain, in uivo alterations in taurine, phosphoethanolamine and alanine have been reported previously in several animal models of epilepsy (Lehmann, Hagberg, Jacobson and Hamberger, 198.5; Vezzani, Ungerstedt, French and Schwartz, 1985). Kainic acid and DHK induced alterations in these compounds and a DHK-mediated increase in dialysate glutamate and aspartate, were also noted in the present study and in previous reports (Lehmann, Jsacsson and Hamberger, 1983; Lehmann and Hamberger, 1983; Butcher, Lazarewicz and Hamberger, 1988; Munoz, Herranz, Solis, Martin de1 Rio and Let-ma, 1987). However, these alterations occurred irrespective of whether epileptiform activity or alterations in the amplitude of the population spike occurred. This response may therefore reflect the degree of activation of excitatory amino acid receptors rather more closely than the appearance of epileptiform activity. A similar dissociation between alterations in neurochemical markers and epileptifo~ activity has been reported previously when using quinolinic acid (Vezzani et al., 1986). Moreover, lesion studies in the striatum suggest that the effects of kainic acid on the efilux of amino acids are mediated predominantly at nonneuronal sites (Butcher et al., 1988) and the releasing

action of kainic acid may therefore be distinguished from its direct effect on neuronal excitability. The lack of effect of kainic acid on the efflux of glutamatejaspartate certainly suggests that a presynaptic action of this drug is not involved in the induction of epilepsy. In the case of DHK, the increase in glutamate and aspartate in the dialysate suggests that an inhibition of uptake was indeed occurring. This is unlikely to be related to the epileptogenic activity because the more potent uptake inhibitor, THA does not exhibit epileptogenic activity even at 10 mM, whereas a concentration of 20 PM affects glutamate and aspartate in the dialysate in the striatum (Young and Bradford, 1987). Acknowledgement-S.P.B.

is supported by a Royal Society European Exchange Fellowship.

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