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Differential effects of cortical spreading depression on epileptic foci induced by various convulsants
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Electroencephalography and Clinical Neurophysiology, 1977, 4 3 : 6 6 6 - - 6 7 4 © Elsevier/North-Holland Scientific Publishers, L t d
D I F F E R E N T I A L E F F E C T S OF CORTICAL SPREADING DEPRESSION ON EPILEPTIC FOCI INDUCED BY VARIOUS CONVULSANTS M. UEDA * and J. BURE~
Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Accepted for publication: March 21, 1977)
A close relationship between spreading depression of EEG activity (SD -- Le~o 1944) and convulsions was recognized even in the firsts reports on SD (for review see Marshall 1959; Ochs 1962; Bure~ et al. 1974). Electrical stimulation of cerebral cortex can induce either after-discharge or SD (Le~o 1944). SD is often accompanied b y paroxysmal activity (Van Harreveld and Stamm 1953) and epileptic discharge can be terminated by SD (Van Harreveld and Stamm 1954). When SD and epileptic discharge were independently evoked in the same hemisphere, SD appeared to be prepotent. It was used to block strychnine or penicillin spikes elicited by local application of the drug (Sloan and Jasper 1950; Atsev 1966), to interfere with the electrically evoked cortical or hippocampal after-discharge (Aqufno-Cfas and Buret 1966) and to decrease the audiogenic seizure susceptibility (Bureg and Bure~ova 1956; Chocholov~ 1962; Kesner et al. 1965}. More recent evidence indicated that this relationship can sometimes be reversed; Aqulno-Cias et al. (1971) reported that SD waves generated in intact cortex do not suppress the activity of an epileptic focus induced by topical application of picrotoxin. Bure~ et al. (1975} confirmed this finding and demonstrated that the effect is related to the increased concentration of extracellular potassium [K+]e in the focus. Epileptic foci induced by penicillin and strychnine (Aqufno* Visiting scientist from Keio University, School of Medicine, Tokyo, Japan.
Cfas et al. 1971; Aqul~no-Cfas, Thesis, Havana, 1975), Metrazol (Buret et al. 1975) or cobalt (Holubar and Fischer 1967) did not block SD propagation. The purpose of the present paper is to examine systematically the possibility of blocking SD by topically applied convulsants and to analyse the mechanism of this effect.
Method Experiments were performed on 45 male hooded rats (Druckrey strain), aged 3 months. Under Nembutal anaesthesia (40 mg/kg) five trephine holes were made over the frontal, parietal and occipital cortex of one hemisphere and over the frontal area of the contralateral hemicortex, according to the scheme in Fig. 1. The rostral opening (1) was 4 mm in diameter and served for the topical application of drugs or for insertion of intracortical electrodes. The caudal opening (2 mm in diameter) was employed for intracortical injection (1/~l} of 25% KC1 solution. The remaining three trephine holes (2 mm in diameter) were used for ECoG and slow potential recording. Calomel cell electrodes with wick or agar-saline bridges were placed in the trephine openings. A similar electrode contacting the exposed nasal bone served as reference. Electrodes 1 to 3 (Fig. 1) were connected to the inputs of a conventional EEG apparatus and to high impedance input operational amplifiers, the outputs of which were fed through a slow mechanical chopper (3/sec) to
SPREADING DEPRESSION AND EPILEPSY
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Fig. 1. Blockade of SD propagation into a picrotoxin focus of epileptic activity. Brain diagram indicates the position of the trephine openings and electrodes (0--4), as well as the location of the focus (drug) and of the potassium chloride microinjection (KCI). Monopolar DC (above) and ECoG records (below) from the electrodes shown. Time of KCI injection is denoted by the arrow. Calibration: 10 mV for the slow potentials (negativity of active electrode downward), 100 pV for the EEG (negativity of active electrode upward). Time calibration: 1 min.
other EEG channels. Only EEG activity was recorded from opening 4. The experiments started by eliciting SD under control conditions. KC1 was injected into the occipital cortex and the change in ECoG and slow potentials was observed. Thirty minutes later a piece o f filter paper (3 X 3 mm) soaked in convulsant solution was applied on the exposed frontal cortex. The drugs used were 5% picrotoxin (Fluka), 10% sodium 5-methyl-3-phenyl-4-isoxazolyl penicillin hydrate (Spofa), 10% Metrazol, 10% strychnine nitrate (Spofa), 4 % Aldactone (K-3-[3-oxo17-beta-hydroxy:4,6-androstadien-17alpha-yl] propionate). The focal epileptic discharge was recorded for 2--3 h. In a seriesof experiments activity of a fully developed picrotoxin focus was suppressed by graded application of tetrodotoxin (TTX, Sankyo, 10 -4 M ) o n the focal region SD waves were evoked from the occipital cortex at various stages of the devel-
o p m e n t o f the focus or at regular 30 min intervals. In a separate series of experiments extracellular potassium concentration in the focus was measured with potassium-selective microelectrodes (Walker 1971; Vysko~il et al. 1972). The potassium electrode was a glass capillary filled with 0.5 M KC1 contacting the liquid ion exchanger (Coming 4 7 7 3 1 7 ) i n the siliconized tip (1--2/~m in diameter). A similar capillary filled with physiological saline was m o u n t e d parallel to the K ÷ electrode so that the tips were n o t more than 50 #m apart. Chlorided silver wires contacting the solutions in the electrode vessels were connected to high impedance input operational amplifiers. The K ÷ electrode resistance was 200--300 M ~ . The electrode system was calibrated in solutions of KC1 in 150 mM NaC1. The response was a linear function o f log[K÷]e above 4 mM and reached 40--50 mV for a tenfold
668
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increase of potassium concentration. The calibrated electrode system was inserted through intact dura 1 mm below the cortical surface exposed by trephine opening 1. The difference between the K ÷ electrode potential and the capillary electrode potential corresponding to E K w a s recorded in one channel of a polygraph or of a recording millivoltmeter, whereas other channels were used for ECoG and slow potential recording. Application of a filter paper (2 × 2 mm) soaked with 3% KC1 solution to the exposed occipital cortex was used for eliciting SD in this case.
the concentrations employed. The first interictal spikes were observed after 5 min. The discharge rate and amplitude reached maxim u m after 20 min and were relatively stable for the subsequent 2--3 h. Alternating periods of ictal and interictal activity were observed at this stage in the cases of picrotoxin and penicillin foci. Typical records of the fully developed epileptic discharge are shown in Fig. 1 and 2. The spikes propagated from the focus into the symmetrical cortical area and into the remote regions of the same hemisphere, their amplitude decreasing with distance from the focus {Fig. 1).
Results
Effects of the focus on SD propagation (a) Picrotoxin. During the initial phase of
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Fig. 2. Failure of an Aldactone focus of epileptic activity to block S D propagation. The SD wave is the last in a train o f 4 elicited b y a single KCI injection into the occipital cortex. F o r description see Fig. 1. N o t e sudden cessation o f t h e epileptic discharge when SD enters the focus.
SPREADING DEPRESSION AND EPILEPSY pital cortex were n o t significantly modified. When SD invaded the focus, epileptic discharge was disrupted for 0.5--3 min. With increasing rate o f interictal activity, amplitude of the slow potential decreased in the focus by 70% and the overall conduction time between trephine openings 3 and 1 increased by 44%. At the same time the duration of the SD-induced spike discharge blockade dropped to 50 sec. After the spike rate reached 40-50/min, SD penetration into the focus w a s completely prevented. The amplitude o f the negative slow potential was reduced also in lead 0--2 (Fig. 1), whereas the positive comp o n e n t o f the slow potential was enhanced. The epileptic discharges o f the focus continued while SD invaded the surrounding cortex. The projected spikes recorded from the parieto-occipital cortex were, however, diminished. The blockade of SD lasted for several hours and subsided only after the focus started to decline. (b) Penicillin. The effect on SD was essentially similar to that of picrotoxin. When the focal activity reached 20--30/min, the amplitude o f the slow potential change decreased b y 88% and the conduction time increased b y 26%. Full blockade of SD was seen only in 20% of cases, however. In 60% of the experiments the spike discharge was slowed d o w n or interrupted for more than 60 sec when SD approached the focus. (c) Strychnine. Spike discharge had little effect on SD propagation. The amplitude of the slow potential decreased only by 23% and complete blockade of SD was never observed. On the other hand, SD regularly suppressed the focal discharge, for 158 sec on average. (d) Metrazol. In spite o f high discharge rates ( 5 0 - ~ 0 / m i n ) , the slow potential decrem e n t was only 32%. The spreading rate was decreased b y 17%, b u t SD always reached the focus and blocked the spikes for 180 sec. (e) Aldactone. Aldactone usually evoked an SD wave soon after application. The initial depression was gradually replaced b y epileptic activity which was essentially similar to t h a t evoked by other convulsants. SD was n o t
669 blocked b u t the amplitude o f the slow potential penetrating into the focus was increased by 22% and the spreading rate by 9%. Invasion of the Aldactone focus b y SD was accompanied b y a prolonged blockade of spiking which lasted usually more than 5 min. A remarkable effect of Aldactone was the increased efficiency o f the SD-evoking stimulus. Whereas under control conditions or with foci due to other drugs microinjection of 1/~1 of 25% KC1 into the occipital cortex usually elicited single SD waves, the presence o f the Aldactone focus in the same hemisphere increased the number o f SD waves generated b y the same KCI stimulus to 5 on the average. There was brief recovery of spike discharge between the individual SD waves generated, with an average interval o f 8.9 min. Such spontaneous SD waves (Fig. 2) were often characterized b y apparently faster propagation of the slow potential between electrodes 3--2 (interelectrode distance 3 m m ) than between electrodes 2--1 (interelectrode distance 4.5 mm). :An alternative explanation is that the repeated SD waves were n o t generated at the injection site, b u t at some distance from it (in Fig. 2 from an area a b o u t 3 mm lateral and 2 mm rostral to the KC1 injection). The results of all experiments are summarized in Fig. 3, which shows the relationship b e t w e e n the average discharge rate o f the
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focus and the decrement of the slow potential waves penetrating into the focus. For any given drug SD blockade was proportional to discharge rate but with the same discharge rate SD blockade depended on the type of the drug. The SD change reached rapidly a constant level which remained stable for hours. Fig. 4 illustrates the reverse relationship, that is, the effect of SD on the activity of the focus. Duration of the complete blockade of spike discharge was established for each SD wave elicited after application of the drug. Evaluation of each drug was based on 20 SD waves. The spike blockade was classified as short-lasting (< 1 min), intermediate (1--5 min) or long-lasting ( > 5 min). In the early phase, spike discharge was suppressed for 2--3 min on average. The duration of this
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Fig. 5. Changes of cortical [K+]e accompanying S D waves penetrating an Aldactone (above) or penicillin (below) focus. The upper curve is the slow potential record, the lower curve the [K÷]e record. Aid or Pnc --application of Aldactone or penicillin to the exposed frontal cortex. Calibration: 5 rain, 10 m V for the slow potential change, logarithmic scale for the [K÷]e record. The dashed horizontal line indicates 4 m M [K ÷] e level. Negativity of the slow potential record upwards.
SPREADING DEPRESSION AND EPILEPSY effect decreased in the case of picrotoxin foci (62% SD waves blocked the spike discharge for less than I min) and increased in the case o f Aldactone foci (71% SD waves blocked the spike discharge for more than 5 min). The duration o f the spike blockade was short in foci elicited by penicillin but was almost unaffected by Metrazol and strychnine.
Removal of the SD blockade In 4 rats TTX (10 -4 M) was applied to the picrotoxin focus after full SD block had been produced. The discharge rate rapidly decreased to 50% of the m a x i m u m and SD evoked from the occipital cortex started to penetrate into the focus again. A similar effect could be achieved also by washing the picrotoxin focus with saline but the discharge rate decreased more slowly in this case. Application of TTX to intact cortex did not cause any appreciable change in the penetration o f the slow potential wave into the treated cortical area.
Changes of [K+]~ in the focus The effects of two drugs yielding intermediate and minimum blockade of SD were analysed with the potassium electrodes. In four experiments [K +] ~ was measured in the penicillin focus. With the development o f epileptic discharge [K+]e increased from the resting level o f 3.5 mM up to 12 mM. SD waves evoked when the focal [K÷]e did not exceed 5 mM (Fig. 5) penetrated into the focus, but the amplitude of the [K+]e change rapidly decreased with repeated SDs. SD waves induced when [K+]e in the focus reached 8-9 mM did not penetrate the focus. In 5 experiments with Aldactone foci the resting [K+]e level of 3.5 mM increased after 2--3 h of Aldactone treatment to 4.2 mM on average. SD penetration into the focus was not blocked and the [K÷]~ increase accompanying SD was not diminished. A typical experiment is shown in Fig. 5. The potassium electrodes were also used to estimate potassium activity in the Aldactone solution applied to the cerebral cortex. It contained about 20
671 mM potassium, that is, a concentration subthreshold for eliciting SD.
Discussion The results of the present study show that SD can be used to differentiate between various convulsants the topical application of which produces apparently similar epileptic foci. Although the spike amplitudes and discharge rates elicited by picrotoxin and Aldactone are almost the same, the two drugs affect SD propagation in opposite ways. Other drugs assume intermediate positions between these two extremes. The correlation between SD blockade and [K+]e in the focus, described in a previous paper (Bure~ et al. 1975), was confirmed in the present study. SD did not penetrate into penicillin foci in which [K+]e exceeded 8 mM but was not stopped by foci with lower [K÷]~. Similar results were obtained with Metrazol (Buret et al. 1975), which did not block SD propagation and raised the [K+]e level only to 4.6 mM on average. Also, the slight [K+]~ increase induced by Aldactone is compatible with the facilitatory effect of the drug on SD propagation. The mechanism of the convulsant action o f the drugs used is not fully understood (see Ajmone Marsan 1969; Prince 1972 for reviews). They may depress inhibitory processes by interfering with the presynaptic release o f transmitter or by blocking the postsynaptic receptor sites. They m a y potentiate excitatory processes by enhancing the release o f transmitter (e.g. by blocking presynaptic i n h i b i t i o n - - D a v i d o f f 1972) or by potentiating the recurrent excitatory circuits. They may directly influence the membrane properties o f the postsynaptic neurones, e.g., by increasing the membrane resistance or by selectively reducing membrane conductance for one species of ions. The above mechanisms are not mutually exclusive and their combination probably accounts for the effect on the complex neural circuitry o f the cere-
672 bral cortex. Picrotoxin antagonizes the inhibitory effect of GABA (Galindo 1969; Kawaguchi and Ono 1973) and penicillin acts in a similar way (Curtis et al. 1972). Strychnine blocks the inhibition mediated by glycine (Obata and Highstein 1970) but its effects at the cortical level may also be due to changes of membrane conductance or of the IPSP equilibrium potential (Van Duijn et al. 1973). Metrazol seems to affect membrane properties more than synaptic transmission (David et al. 1974). According to Straschill and Schick (1975) the epileptogenic effect of Aldactone is due to inhibition of Na ÷, K ÷ ATPase and to impaired reabsorbtion o f potassium released during excitation. This action, analogous to that of ouabaine (Aqu~no-Cias et al. 1967) may trigger the initial SD waves, which cannot be evoked by the low potassium content of the Aldactone solution (about 20 mM K ÷ as compared with the 80 mM t h r e s h o l d Bure~ et al. 1974). Low levels of Aldactone outside the focus may perhaps account for the enhanced effect o f potassium ions injected into a distant cortical area. Schmiedek et al. (1974) suggested that Aldactone not only interferes with the sodium pump by antagonizing the aldosterone effect on electrolyte transport (Woodburry 1972), but that blocking the effector site o f aldosterone increases, by a feedback mechanism, the endogenous production of the hormone to overcome the block. Although this mechanism may explain why the beneficial effects of systemic Aldactone treatment on brain oedema (Baethmann et al. 1970) can be reproduced by aldosterone administration {Schmiedek et al. 1974), it cannot account for the action of topically applied Aldactone, the explanation of which must be sought in the spatial organization of the focus. The repolarization phase of SD is faster and [K+]~ level lower in the deep layers (1 mm) of Aldactone foci than in penicillin foci. This indicates that the Aldactone effect is either limited to the surface layers of the cerebral cortex or perhaps only to a fraction of the
M. UEDA, J. BURE~ neuronal population of the focus. Similar factors may also contribute to the differential effect of other epileptic foci on SD propagation. Although the foci employed in the present study were approximately equated for average frequency and amplitude of surface discharge, no attempt was made to establish their transcortical profiles. It is conceivable that complete SD blockade occurs o n l y . when the focus activates all cortical layers. This is indicated by high [K÷]e 1 m m below the cortical surface whereas low [ K÷] e at this depth suggests that the deep layers axe relatively unaffected. Since SD can pass through deep cortical layers after the superficial ones have been eliminated (Grafstein 1956) SD can enter such foci from below and block epileptic discharges in the surface layers by potassium released from the deep layers. This assumption is supported by the observation (Bure~ et al. 1975) that the picrotoxin focus can be suppressed by topical application of 2% KC1 and that elimination of the epileptic discharge restores penetration of SD waves in the potassium-treated area. The TTX effect described in the present study can be interpreted in a similar way. As shown by Sugaya et al. (1975) TTX prevents elicitation of SD by electrical stimulation of cerebral cortex, but not by local application of KCI. Blockade of the sodium conductance of the neural membrane interferes with the mechanism of the epileptic discharge. Both picrotoxin and TTX are applied on the cortical surface and form similar concentration gradients. This accounts for the marked interference with the generation of epileptic spikes and for minimal interference with SD propagation, since TTX concentration in the deep cortical layers is far below the blocking level.
Sun,mary Interaction between epileptic loci and spreading depression (SD) was studied in the cerebral cortex of rats anesthetized with Nembutal. At comparable discharge rates, picro-
SPREADING DEPRESSION AND EPILEPSY
toxin and penicillin caused complete and partial SD blockade respectively, strychnine and Metrazol were ineffective and Aldactone facilitated SD. Conversely, the duration of SD-induced blockade o f epileptic activity was maximal for Aldactone and minimal for picrotoxin. Treatment of the picrotoxin focus with tetrodotoxin (10 -4 M) reduced the discharge rate and reinstated SD propagation into the focus. [K÷]e measured with ion-selective K ÷ electrodes 1 mm below the cortical surface increased to 8 mM in penicillin foci blocking SD and remained below 5 mM in Aldactone foci. It is concluded that the differential effect o f various convulsants on SD propagation depends on the potassium concentration in the depth of the focus rather than on the discharge rate or on the mechanism of the epileptogenic effect.
R~sum~ Diffdrences d'effets de la depression lYropagde corticale, sur des foyers dpileptiques suscitds par divers convulsivants L'~tude concerne l'interaction entre foyers ~pileptiques et d~pression propag~e (DP) dans le cortex c~r~bral du rat anesth~si~ au Nembutal. Pour des taux de d~charges comparables, la picrotoxine et la p~nicilline ont provoqu~ un bloc respectivement complet et patiel de la DP. La strychnine et le M~trazol ont ~t~ sans effet, et l'Aldactone a facilit~ la DP. Inversement, la dur~e de suppression des activit~s ~pileptiques par la DP fut maximale pour l'Aldactone et minimale pour la picrotoxine. Le traitement du foyer ~ la picrotoxine par la tdtrodotoxine (10 -4 M), a r~duit le taux de d~charge et r~tabli la propagation de la ddpression dans le f o y e r [K÷]e, ~valude par ~lectrodes s~lectives ~ 1 mm au-dessous de la surface corticale, d~passait 8 mM dans les foyers ~ la p~nicilline, bloquants de la DP, tandis qu'elle restait au-dessous de 5 raM dans les foyers ~ l'Aldactone. On conclut que cette difference d'effets de divers convulsivants sur
673
la propagation de la d~pression d~pend de la concentration de K ÷ en profondeur du foyer, plutSt que du taux de d~charge, ou du m~canisme propre de l'action ~pileptog~ne.
The authors would like to thank Dr L. Ha~kovec from the Psychiatric Clinic, Medical Faculty, Charles University, Prague for drawing their attention to the epileptogenic effects of Aldactone and for stimulating discussion of experimental results.
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M. UEDA, J. BURE~ Ochs, S. The nature of spreading depression in neural networks. Int. Rev. Neurobiol., 1962, 4: 1--69. Prince, D.A. Topical convulsant drugs and metabolic antagonists. In D.P. Purpura, J.K. Penry, D.B. Tower, D.M. Woodburry and R.D. Walter (Eds.), Experimental Models of Epilepsy, Raven Press, N e w York, 1972: 51--83. Schmiedek, P., Ottinger, W., Baethmann, A., Enzenbach, R. and Marguth, F. Aldosterone. A new therapeutic principle for the treatment of brain oedema in man. Acta Neurochirurgica, 1974, 30: 59-68. Sloan, N. and Jasper, J. The identity of spreading depression and "suppression". Electroenceph. clin. Neurophysiol., 1950, 2: 59--78. Straschill, M. and Schick, F. The effects of a spirolactone derivative on EEG and cortical single unit activity in the cat Electroenceph. clin. Neurophysiol., 1975, 39: 473---477. Sugaya, E., Takato, M. and Noda, Y. Neuronal and glial activity during spreading depression in cerebral cortex of cat. J. Neurophysiol.~ 1975, 38: 822--841. Van Duijn, H., Schwartzkroin, P.A. and Prince, D.A. A c t i o n of penicillin on inhibitory processes in the cat's cortex. Brain Research, 1973, 53: 470--476. Van Harreveld, A. and Stature, J.S. Spreading cortical convulsions and depressions. J. Neurophysiol., 1953, 16: 352--366. Van Harreveld, A. and Stature, J.S. Consequences of cortical convulsive activity in rabbit. J. Neurophysiol., 1954, 17: 505--520. Vysko~il, F. K~i~, N. and Buret, J. Potassium-selective microeleetrodes used for measuring the extracellular brain potassium during spreading depression and anoxic depolarisation in rats. Brain Res., 1972, 3 9 : 2 5 5 --259. Walker, J.L., Jr. Ion specific liquid ion exchange microelectrodes. Analyt. Chem., 1971, 43: 89A-92A. Woodburry, D.M. Biochemical effects of adrenocortical steroids on the central nervous system. In: A. Laitha (Ed.). Handbook of Neurochemistry, Vol. 7, Plenum Press, 1972:255-287.
Electroencephalography and Clinical Neurophysiology, 1977, 4 3 : 6 6 6 - - 6 7 4 © Elsevier/North-Holland Scientific Publishers, L t d
D I F F E R E N T I A L E F F E C T S OF CORTICAL SPREADING DEPRESSION ON EPILEPTIC FOCI INDUCED BY VARIOUS CONVULSANTS M. UEDA * and J. BURE~
Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Accepted for publication: March 21, 1977)
A close relationship between spreading depression of EEG activity (SD -- Le~o 1944) and convulsions was recognized even in the firsts reports on SD (for review see Marshall 1959; Ochs 1962; Bure~ et al. 1974). Electrical stimulation of cerebral cortex can induce either after-discharge or SD (Le~o 1944). SD is often accompanied b y paroxysmal activity (Van Harreveld and Stamm 1953) and epileptic discharge can be terminated by SD (Van Harreveld and Stamm 1954). When SD and epileptic discharge were independently evoked in the same hemisphere, SD appeared to be prepotent. It was used to block strychnine or penicillin spikes elicited by local application of the drug (Sloan and Jasper 1950; Atsev 1966), to interfere with the electrically evoked cortical or hippocampal after-discharge (Aqufno-Cfas and Buret 1966) and to decrease the audiogenic seizure susceptibility (Bureg and Bure~ova 1956; Chocholov~ 1962; Kesner et al. 1965}. More recent evidence indicated that this relationship can sometimes be reversed; Aqulno-Cias et al. (1971) reported that SD waves generated in intact cortex do not suppress the activity of an epileptic focus induced by topical application of picrotoxin. Bure~ et al. (1975} confirmed this finding and demonstrated that the effect is related to the increased concentration of extracellular potassium [K+]e in the focus. Epileptic foci induced by penicillin and strychnine (Aqufno* Visiting scientist from Keio University, School of Medicine, Tokyo, Japan.
Cfas et al. 1971; Aqul~no-Cfas, Thesis, Havana, 1975), Metrazol (Buret et al. 1975) or cobalt (Holubar and Fischer 1967) did not block SD propagation. The purpose of the present paper is to examine systematically the possibility of blocking SD by topically applied convulsants and to analyse the mechanism of this effect.
Method Experiments were performed on 45 male hooded rats (Druckrey strain), aged 3 months. Under Nembutal anaesthesia (40 mg/kg) five trephine holes were made over the frontal, parietal and occipital cortex of one hemisphere and over the frontal area of the contralateral hemicortex, according to the scheme in Fig. 1. The rostral opening (1) was 4 mm in diameter and served for the topical application of drugs or for insertion of intracortical electrodes. The caudal opening (2 mm in diameter) was employed for intracortical injection (1/~l} of 25% KC1 solution. The remaining three trephine holes (2 mm in diameter) were used for ECoG and slow potential recording. Calomel cell electrodes with wick or agar-saline bridges were placed in the trephine openings. A similar electrode contacting the exposed nasal bone served as reference. Electrodes 1 to 3 (Fig. 1) were connected to the inputs of a conventional EEG apparatus and to high impedance input operational amplifiers, the outputs of which were fed through a slow mechanical chopper (3/sec) to
SPREADING DEPRESSION AND EPILEPSY
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Fig. 1. Blockade of SD propagation into a picrotoxin focus of epileptic activity. Brain diagram indicates the position of the trephine openings and electrodes (0--4), as well as the location of the focus (drug) and of the potassium chloride microinjection (KCI). Monopolar DC (above) and ECoG records (below) from the electrodes shown. Time of KCI injection is denoted by the arrow. Calibration: 10 mV for the slow potentials (negativity of active electrode downward), 100 pV for the EEG (negativity of active electrode upward). Time calibration: 1 min.
other EEG channels. Only EEG activity was recorded from opening 4. The experiments started by eliciting SD under control conditions. KC1 was injected into the occipital cortex and the change in ECoG and slow potentials was observed. Thirty minutes later a piece o f filter paper (3 X 3 mm) soaked in convulsant solution was applied on the exposed frontal cortex. The drugs used were 5% picrotoxin (Fluka), 10% sodium 5-methyl-3-phenyl-4-isoxazolyl penicillin hydrate (Spofa), 10% Metrazol, 10% strychnine nitrate (Spofa), 4 % Aldactone (K-3-[3-oxo17-beta-hydroxy:4,6-androstadien-17alpha-yl] propionate). The focal epileptic discharge was recorded for 2--3 h. In a seriesof experiments activity of a fully developed picrotoxin focus was suppressed by graded application of tetrodotoxin (TTX, Sankyo, 10 -4 M ) o n the focal region SD waves were evoked from the occipital cortex at various stages of the devel-
o p m e n t o f the focus or at regular 30 min intervals. In a separate series of experiments extracellular potassium concentration in the focus was measured with potassium-selective microelectrodes (Walker 1971; Vysko~il et al. 1972). The potassium electrode was a glass capillary filled with 0.5 M KC1 contacting the liquid ion exchanger (Coming 4 7 7 3 1 7 ) i n the siliconized tip (1--2/~m in diameter). A similar capillary filled with physiological saline was m o u n t e d parallel to the K ÷ electrode so that the tips were n o t more than 50 #m apart. Chlorided silver wires contacting the solutions in the electrode vessels were connected to high impedance input operational amplifiers. The K ÷ electrode resistance was 200--300 M ~ . The electrode system was calibrated in solutions of KC1 in 150 mM NaC1. The response was a linear function o f log[K÷]e above 4 mM and reached 40--50 mV for a tenfold
668
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increase of potassium concentration. The calibrated electrode system was inserted through intact dura 1 mm below the cortical surface exposed by trephine opening 1. The difference between the K ÷ electrode potential and the capillary electrode potential corresponding to E K w a s recorded in one channel of a polygraph or of a recording millivoltmeter, whereas other channels were used for ECoG and slow potential recording. Application of a filter paper (2 × 2 mm) soaked with 3% KC1 solution to the exposed occipital cortex was used for eliciting SD in this case.
the concentrations employed. The first interictal spikes were observed after 5 min. The discharge rate and amplitude reached maxim u m after 20 min and were relatively stable for the subsequent 2--3 h. Alternating periods of ictal and interictal activity were observed at this stage in the cases of picrotoxin and penicillin foci. Typical records of the fully developed epileptic discharge are shown in Fig. 1 and 2. The spikes propagated from the focus into the symmetrical cortical area and into the remote regions of the same hemisphere, their amplitude decreasing with distance from the focus {Fig. 1).
Results
Effects of the focus on SD propagation (a) Picrotoxin. During the initial phase of
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Fig. 2. Failure of an Aldactone focus of epileptic activity to block S D propagation. The SD wave is the last in a train o f 4 elicited b y a single KCI injection into the occipital cortex. F o r description see Fig. 1. N o t e sudden cessation o f t h e epileptic discharge when SD enters the focus.
SPREADING DEPRESSION AND EPILEPSY pital cortex were n o t significantly modified. When SD invaded the focus, epileptic discharge was disrupted for 0.5--3 min. With increasing rate o f interictal activity, amplitude of the slow potential decreased in the focus by 70% and the overall conduction time between trephine openings 3 and 1 increased by 44%. At the same time the duration of the SD-induced spike discharge blockade dropped to 50 sec. After the spike rate reached 40-50/min, SD penetration into the focus w a s completely prevented. The amplitude o f the negative slow potential was reduced also in lead 0--2 (Fig. 1), whereas the positive comp o n e n t o f the slow potential was enhanced. The epileptic discharges o f the focus continued while SD invaded the surrounding cortex. The projected spikes recorded from the parieto-occipital cortex were, however, diminished. The blockade of SD lasted for several hours and subsided only after the focus started to decline. (b) Penicillin. The effect on SD was essentially similar to that of picrotoxin. When the focal activity reached 20--30/min, the amplitude o f the slow potential change decreased b y 88% and the conduction time increased b y 26%. Full blockade of SD was seen only in 20% of cases, however. In 60% of the experiments the spike discharge was slowed d o w n or interrupted for more than 60 sec when SD approached the focus. (c) Strychnine. Spike discharge had little effect on SD propagation. The amplitude of the slow potential decreased only by 23% and complete blockade of SD was never observed. On the other hand, SD regularly suppressed the focal discharge, for 158 sec on average. (d) Metrazol. In spite o f high discharge rates ( 5 0 - ~ 0 / m i n ) , the slow potential decrem e n t was only 32%. The spreading rate was decreased b y 17%, b u t SD always reached the focus and blocked the spikes for 180 sec. (e) Aldactone. Aldactone usually evoked an SD wave soon after application. The initial depression was gradually replaced b y epileptic activity which was essentially similar to t h a t evoked by other convulsants. SD was n o t
669 blocked b u t the amplitude o f the slow potential penetrating into the focus was increased by 22% and the spreading rate by 9%. Invasion of the Aldactone focus b y SD was accompanied b y a prolonged blockade of spiking which lasted usually more than 5 min. A remarkable effect of Aldactone was the increased efficiency o f the SD-evoking stimulus. Whereas under control conditions or with foci due to other drugs microinjection of 1/~1 of 25% KC1 into the occipital cortex usually elicited single SD waves, the presence o f the Aldactone focus in the same hemisphere increased the number o f SD waves generated b y the same KCI stimulus to 5 on the average. There was brief recovery of spike discharge between the individual SD waves generated, with an average interval o f 8.9 min. Such spontaneous SD waves (Fig. 2) were often characterized b y apparently faster propagation of the slow potential between electrodes 3--2 (interelectrode distance 3 m m ) than between electrodes 2--1 (interelectrode distance 4.5 mm). :An alternative explanation is that the repeated SD waves were n o t generated at the injection site, b u t at some distance from it (in Fig. 2 from an area a b o u t 3 mm lateral and 2 mm rostral to the KC1 injection). The results of all experiments are summarized in Fig. 3, which shows the relationship b e t w e e n the average discharge rate o f the
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focus and the decrement of the slow potential waves penetrating into the focus. For any given drug SD blockade was proportional to discharge rate but with the same discharge rate SD blockade depended on the type of the drug. The SD change reached rapidly a constant level which remained stable for hours. Fig. 4 illustrates the reverse relationship, that is, the effect of SD on the activity of the focus. Duration of the complete blockade of spike discharge was established for each SD wave elicited after application of the drug. Evaluation of each drug was based on 20 SD waves. The spike blockade was classified as short-lasting (< 1 min), intermediate (1--5 min) or long-lasting ( > 5 min). In the early phase, spike discharge was suppressed for 2--3 min on average. The duration of this
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Fig. 5. Changes of cortical [K+]e accompanying S D waves penetrating an Aldactone (above) or penicillin (below) focus. The upper curve is the slow potential record, the lower curve the [K÷]e record. Aid or Pnc --application of Aldactone or penicillin to the exposed frontal cortex. Calibration: 5 rain, 10 m V for the slow potential change, logarithmic scale for the [K÷]e record. The dashed horizontal line indicates 4 m M [K ÷] e level. Negativity of the slow potential record upwards.
SPREADING DEPRESSION AND EPILEPSY effect decreased in the case of picrotoxin foci (62% SD waves blocked the spike discharge for less than I min) and increased in the case o f Aldactone foci (71% SD waves blocked the spike discharge for more than 5 min). The duration o f the spike blockade was short in foci elicited by penicillin but was almost unaffected by Metrazol and strychnine.
Removal of the SD blockade In 4 rats TTX (10 -4 M) was applied to the picrotoxin focus after full SD block had been produced. The discharge rate rapidly decreased to 50% of the m a x i m u m and SD evoked from the occipital cortex started to penetrate into the focus again. A similar effect could be achieved also by washing the picrotoxin focus with saline but the discharge rate decreased more slowly in this case. Application of TTX to intact cortex did not cause any appreciable change in the penetration o f the slow potential wave into the treated cortical area.
Changes of [K+]~ in the focus The effects of two drugs yielding intermediate and minimum blockade of SD were analysed with the potassium electrodes. In four experiments [K +] ~ was measured in the penicillin focus. With the development o f epileptic discharge [K+]e increased from the resting level o f 3.5 mM up to 12 mM. SD waves evoked when the focal [K÷]e did not exceed 5 mM (Fig. 5) penetrated into the focus, but the amplitude of the [K+]e change rapidly decreased with repeated SDs. SD waves induced when [K+]e in the focus reached 8-9 mM did not penetrate the focus. In 5 experiments with Aldactone foci the resting [K+]e level of 3.5 mM increased after 2--3 h of Aldactone treatment to 4.2 mM on average. SD penetration into the focus was not blocked and the [K÷]~ increase accompanying SD was not diminished. A typical experiment is shown in Fig. 5. The potassium electrodes were also used to estimate potassium activity in the Aldactone solution applied to the cerebral cortex. It contained about 20
671 mM potassium, that is, a concentration subthreshold for eliciting SD.
Discussion The results of the present study show that SD can be used to differentiate between various convulsants the topical application of which produces apparently similar epileptic foci. Although the spike amplitudes and discharge rates elicited by picrotoxin and Aldactone are almost the same, the two drugs affect SD propagation in opposite ways. Other drugs assume intermediate positions between these two extremes. The correlation between SD blockade and [K+]e in the focus, described in a previous paper (Bure~ et al. 1975), was confirmed in the present study. SD did not penetrate into penicillin foci in which [K+]e exceeded 8 mM but was not stopped by foci with lower [K÷]~. Similar results were obtained with Metrazol (Buret et al. 1975), which did not block SD propagation and raised the [K+]e level only to 4.6 mM on average. Also, the slight [K+]~ increase induced by Aldactone is compatible with the facilitatory effect of the drug on SD propagation. The mechanism of the convulsant action o f the drugs used is not fully understood (see Ajmone Marsan 1969; Prince 1972 for reviews). They may depress inhibitory processes by interfering with the presynaptic release o f transmitter or by blocking the postsynaptic receptor sites. They m a y potentiate excitatory processes by enhancing the release o f transmitter (e.g. by blocking presynaptic i n h i b i t i o n - - D a v i d o f f 1972) or by potentiating the recurrent excitatory circuits. They may directly influence the membrane properties o f the postsynaptic neurones, e.g., by increasing the membrane resistance or by selectively reducing membrane conductance for one species of ions. The above mechanisms are not mutually exclusive and their combination probably accounts for the effect on the complex neural circuitry o f the cere-
672 bral cortex. Picrotoxin antagonizes the inhibitory effect of GABA (Galindo 1969; Kawaguchi and Ono 1973) and penicillin acts in a similar way (Curtis et al. 1972). Strychnine blocks the inhibition mediated by glycine (Obata and Highstein 1970) but its effects at the cortical level may also be due to changes of membrane conductance or of the IPSP equilibrium potential (Van Duijn et al. 1973). Metrazol seems to affect membrane properties more than synaptic transmission (David et al. 1974). According to Straschill and Schick (1975) the epileptogenic effect of Aldactone is due to inhibition of Na ÷, K ÷ ATPase and to impaired reabsorbtion o f potassium released during excitation. This action, analogous to that of ouabaine (Aqu~no-Cias et al. 1967) may trigger the initial SD waves, which cannot be evoked by the low potassium content of the Aldactone solution (about 20 mM K ÷ as compared with the 80 mM t h r e s h o l d Bure~ et al. 1974). Low levels of Aldactone outside the focus may perhaps account for the enhanced effect o f potassium ions injected into a distant cortical area. Schmiedek et al. (1974) suggested that Aldactone not only interferes with the sodium pump by antagonizing the aldosterone effect on electrolyte transport (Woodburry 1972), but that blocking the effector site o f aldosterone increases, by a feedback mechanism, the endogenous production of the hormone to overcome the block. Although this mechanism may explain why the beneficial effects of systemic Aldactone treatment on brain oedema (Baethmann et al. 1970) can be reproduced by aldosterone administration {Schmiedek et al. 1974), it cannot account for the action of topically applied Aldactone, the explanation of which must be sought in the spatial organization of the focus. The repolarization phase of SD is faster and [K+]~ level lower in the deep layers (1 mm) of Aldactone foci than in penicillin foci. This indicates that the Aldactone effect is either limited to the surface layers of the cerebral cortex or perhaps only to a fraction of the
M. UEDA, J. BURE~ neuronal population of the focus. Similar factors may also contribute to the differential effect of other epileptic foci on SD propagation. Although the foci employed in the present study were approximately equated for average frequency and amplitude of surface discharge, no attempt was made to establish their transcortical profiles. It is conceivable that complete SD blockade occurs o n l y . when the focus activates all cortical layers. This is indicated by high [K÷]e 1 m m below the cortical surface whereas low [ K÷] e at this depth suggests that the deep layers axe relatively unaffected. Since SD can pass through deep cortical layers after the superficial ones have been eliminated (Grafstein 1956) SD can enter such foci from below and block epileptic discharges in the surface layers by potassium released from the deep layers. This assumption is supported by the observation (Bure~ et al. 1975) that the picrotoxin focus can be suppressed by topical application of 2% KC1 and that elimination of the epileptic discharge restores penetration of SD waves in the potassium-treated area. The TTX effect described in the present study can be interpreted in a similar way. As shown by Sugaya et al. (1975) TTX prevents elicitation of SD by electrical stimulation of cerebral cortex, but not by local application of KCI. Blockade of the sodium conductance of the neural membrane interferes with the mechanism of the epileptic discharge. Both picrotoxin and TTX are applied on the cortical surface and form similar concentration gradients. This accounts for the marked interference with the generation of epileptic spikes and for minimal interference with SD propagation, since TTX concentration in the deep cortical layers is far below the blocking level.
Sun,mary Interaction between epileptic loci and spreading depression (SD) was studied in the cerebral cortex of rats anesthetized with Nembutal. At comparable discharge rates, picro-
SPREADING DEPRESSION AND EPILEPSY
toxin and penicillin caused complete and partial SD blockade respectively, strychnine and Metrazol were ineffective and Aldactone facilitated SD. Conversely, the duration of SD-induced blockade o f epileptic activity was maximal for Aldactone and minimal for picrotoxin. Treatment of the picrotoxin focus with tetrodotoxin (10 -4 M) reduced the discharge rate and reinstated SD propagation into the focus. [K÷]e measured with ion-selective K ÷ electrodes 1 mm below the cortical surface increased to 8 mM in penicillin foci blocking SD and remained below 5 mM in Aldactone foci. It is concluded that the differential effect o f various convulsants on SD propagation depends on the potassium concentration in the depth of the focus rather than on the discharge rate or on the mechanism of the epileptogenic effect.
R~sum~ Diffdrences d'effets de la depression lYropagde corticale, sur des foyers dpileptiques suscitds par divers convulsivants L'~tude concerne l'interaction entre foyers ~pileptiques et d~pression propag~e (DP) dans le cortex c~r~bral du rat anesth~si~ au Nembutal. Pour des taux de d~charges comparables, la picrotoxine et la p~nicilline ont provoqu~ un bloc respectivement complet et patiel de la DP. La strychnine et le M~trazol ont ~t~ sans effet, et l'Aldactone a facilit~ la DP. Inversement, la dur~e de suppression des activit~s ~pileptiques par la DP fut maximale pour l'Aldactone et minimale pour la picrotoxine. Le traitement du foyer ~ la picrotoxine par la tdtrodotoxine (10 -4 M), a r~duit le taux de d~charge et r~tabli la propagation de la ddpression dans le f o y e r [K÷]e, ~valude par ~lectrodes s~lectives ~ 1 mm au-dessous de la surface corticale, d~passait 8 mM dans les foyers ~ la p~nicilline, bloquants de la DP, tandis qu'elle restait au-dessous de 5 raM dans les foyers ~ l'Aldactone. On conclut que cette difference d'effets de divers convulsivants sur
673
la propagation de la d~pression d~pend de la concentration de K ÷ en profondeur du foyer, plutSt que du taux de d~charge, ou du m~canisme propre de l'action ~pileptog~ne.
The authors would like to thank Dr L. Ha~kovec from the Psychiatric Clinic, Medical Faculty, Charles University, Prague for drawing their attention to the epileptogenic effects of Aldactone and for stimulating discussion of experimental results.
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