Brain Research, 106 (1976) 321-331 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

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METABOLIC REGULATIONS OF THE R H Y T H M I C ACTIVITY IN PACEM A K E R NEURONS. III. N E U T R A L I Z A T I O N OF THE PENTYLENETETRAZOL EFFECT IN R E G U L A R L Y BEATING A P L Y S 1 A NEURONS BY METABOLIC MODIFIERS

GONTER KR~MER* AND RONALD A. CHAPLAIN Department of Physiology, University of Mainz, D-65 Mainz (G.F.R.)

(Accepted September 1lth, 1975)

SUMMARY

In regular beating pacemaker neurons isolated from the sea hare Aplysia californica, 30-50 m M pentylenetetrazol (PTZ) induces spike doublets and triplets, multi-spike bursts, and paroxysmal depolarizing shifts. The development of the characteristic PTZ-induced changes in impulse pattern is preceded by a transient increase in discharge frequency and a reduction in after-hyperpolarization. According to earlier findings, the rhythmic spike activity of pacemaker neurons is governed by a phosphofructokinase-fructose-l,6-diphosphatase-mediated substrate cycle, the activation of which leads to the appearance of spike bursts. Inhibitory modifiers of this neuronal substrate cycle, such as citrate, ATP, and 3-phosphoglycerate, are able to neutralize the changes in the impulse characteristics occurring as the result of PTZ action. Since the reinitiated single-spike trains reverted again to the PTZ pattern following the further addition of activating modifiers, such as fructose-l,6-diphosphate or fructose-6-phosphate, it appears likely that PTZ exerts an influence on the metabolic reactions driving the rhythmic spike activity. In contrast to the reversible effects of the metabolic modifiers the conventional anti-convulsants, phenobarbital and pentobarbital, merely block all spike activity.

INTRODUCTION Considerable attention has been focused over the last two decades on the mechanisms leading to the initiation of epileptic seizures in the hope of localizing the neuronal reactions which terminate these high-frequency synchronous discharges in a * Submitted in partial fulfillmentof the requirements for an M. D. Thesisat the Faculty of Theoretical Medicine.

322 group of cortical neurons. At present the most commonly accepted hypothesis is that the impulse bursts in epileptic neurons are the result of increased excitatory synapnc inputs or blockade of inhibitory synaptic events, without any changes in the properties of the neuron20,21,23,31. However, the postulates that neurons in epileptic focl exhibit a characteristically high autorhythmic activityaz and that there is a change m the properties of the membranes of individual 'epileptic' neurons 1,7,9 can certamly not be ruled out by any of the results obtained so far. In support of a concept which envisages changes in basic neuronal parameters, Speckmann and Caspers 26 have been able to induce 'paroxysmal depolarizing shifts' in isolated Helix pacemaker neurons, using the convulsant drug pentylenetetrazol (synonymous with Metrazol, trade name: Cardiazol). This kind of neuronal activity closely resembles the interictal spike pattern of mammalian cortical neurons13, is. Pentylenetetrazol-mduced spike bursts recorded from neurons in intact ganglia of the molluscs Aplysia, Helix and Tritonia3,9-11 have been shown to be associated with an increased inactivation of the delayed outward current and a pronounced anomalous rectification of the neuronal membraneT,XL As pentylenetetrazol (PTZ) induces epileptogenic activity in mammalian cortical neurons 27,2s this model system has been studied in more detail using completely isolated Aplysia pacemaker neurons. With such regularly beating pacemaker neurons isolated from Aplyaia californica, it has been recently demonstrated that the rhythmic changes in spike activity are controlled by two membrane-bound enzymes which determine the rate and direction of the carbon flux through glycolysis and gluconeogenesis 4. These neuronal enzymes - - phosphofructokinase and fructose-l,6diphosphatase - - catalyze a recycling of fructose-6-phosphate in which ATP is hydrolyzed and H + ions are produced 4. The activity of the enzyme couple is controlled by allosteric activators and inhibitors which differ in their intracellular concentrations quite markedly among pacemaker and silent neurons 4. As the neuronal membrane is readily permeable to these metabolites a tool has become available for manipulating the rhythmic activity of pacemaker neurons4. In the search for the possible mechanism underlying the high-frequency spike discharges in epileptic neurons it appeared therefore promising to investigate the interaction between the PTZ-induced effects and the enzyme-mediated substrate cycle driving the autorhythmic neuronal activity. For comparison, conventional anti-convulsants, phenobarbital and pentobarbital, have been included in this study. MATERIAL AND METHODS

Regularly beating pacemaker neurons of the types Ra-R14 (for terminology see ref. 12) have been isolated from the abdominal ganglion of the sea hare Aplysia californica as described in earlier publications4-6. The spike activity was recorded with the aid of a microelectrode impaling the soma 4-6. The basic medium was artificial sea water consisting of (in mM): 425 NaC1, 10 KCI, 10 CaClz, 22 MgCI2, 26 MgSOa, 2.5 NaHCOs, supplemented with 10 mM triethanolamine buffer, pH 7.8. The free ion concentration in the presence of pentylenetetrazol (Sigma Chemical Co.) or a given

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Fig. 1. Action of pentylenetetrazol on isolated Aplysia pacemaker neurons. A: regular spike train of a Re neuron. B: following the addition of 50 mM PTZ to the medium characteristic changes in the shape of the impulse develop with a given time course; a: 5 min, b: 10 min, c: 20 min, d: 25 min, e: 27 rain, and f: 29 min after PTZ administration. C and D: conversion of the spike train from single to double spikes at two different expansions of the time scale, both recordings were taken at 30 and 31 min after PTZ addition. E: reductions in spike amplitude and interspike distance recorded after 45 rain. F: appearance of triple spikes after 56 min. G: development of depolarizing shifts with multi-spike bursts on the crest of the wave. Calibrations: vertical line 100 mV; horizontal line in A, and D-G 6 sec, in C 1.2 sec, in B and the inserts in D-G the time calibration corresponds to 240 msec. Only for the display of the spike pattern in G has the potential change been amplified by a factor of two, thus the vertical calibration corresponds to 50 mV. metabolite has been calculated f r o m the k n o w n association constants o f the organometal complexes and the total ion concentration adjusted accordingly (compare

ref. 4). The recordings have not been retouched. All illustrations are characteristic for at least 10 similar recordings. RESULTS

Pentylenetetrazol-induced changes in impulse pattern of isolated pacemaker neurons In an attempt to extend the studies o f Speckmann and Caspers z8 the effect o f P T Z has been investigated on regularly beating pacemaker neurons. F o r this purpose Rz-R14 cells have been isolated free o f any synaptic, ephaptic or h u m o r a l inputs. In the majority o f all neurons investigated (nearly 60 % o f the cells) concentrations o f 50 m M P T Z induced the appearance o f double spikes. This pattern develops in a characteristic time course as illustrated in Fig. 1. Starting f r o m a n o r m a l regular spike train (the frequency o f which transiently increases, see Fig. 2) there is an initial broadening o f the spike as the result o f an extension o f the late-potential phase (Fig. 1B). The ' h u m p ' in the late phase o f the spike becomes more and more p r o n o u n c e d until

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about 20-30 min after PTZ addition to the medium a second spike is triggered (Fig. 1C and D). Towards longer recording times the amplitude of the second spike decreases (Fig. 1E). After 45-60 min the latter pattern transforms into triple spikes (Fig. 1F). For spike triplets to appear it was always necessary for the amplitude of the second spike to be reduced by virtually half compared with the first spike. All these patterns can be maintained for prolonged periods at any desired stage provided the PTZ concentration is stringently controlled. For example doublets can be induced in the presence of 50 m M PTZ; this pattern persists if the PTZ concentration in the medium is lowered to 30 mM. If a high PTZ concentration is present for as long as 90 min, m some neurons depolarizing shifts become apparent with spike bursts superimposed on the crest of the wave (Fig. 1G). Generally, at the longer time periods in the presence of PTZ, the amplitudes of the spikes merely decrease continously until they become as small as 30 mV. On washing the PTZ-treated Ra-Rla cells with artificial sea water the PTZ effect could be completely relieved in virtually all preparations. In yet another neuronal sample (mainly R8 and R18 cells) PTZ induced characteristic paroxysmal depolarizations (Fig. 3), which have been reported to be the predominant discharge pattern in PTZ-treated. isolated snail neurons 26. As shown for a Rs neuron, paroxysmal depolarizing shifts (PDS) developed within 30 min after PTZ additions (Fig. 2B). After 40 min a prolonged phase of depolarizations became apparent and as the interval covered by the initial paroxysmal depolarizing shift progressively shortened the actual depolariziation was maintained for several minutes (Fig. 2E). In a fraction of 15-18 ~ of the pacemaker neurons all attempts to induce double

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Fig. 3. Reversible neutralization of the PTZ effectswith metabolic modifiers of the autorhythmic spike activity. After administration of 50 mM PTZ had produced a maintained train of double spikes subsequent addition of 5 mM citrate as recorded 5 mln afterwards (A) produced changes in the shape of the double spike (inserts: a recorded 5 mm, and b 10 min, after citrate addition). B: conversion of the doublets into single spikes after immersion of the PTZ-treated neuron for 12-13 min in the presence of citrate. C: reappearance of the characteristic PTZ pattern following the further addition of 5 mM F-1,6-P2 (indicated by the arrow). D: return of single spikes in the discharge pattern of a PTZ-treated R6 neuron as the result of the addition of 10 mM citrate (see arrow). E: subsequent inclusion of 5 mM F-6-P after 6 min led to occurrence of multi-spike bursts. F: after a period of 11 mm the bursts alternated with single spikes. Time calibrations: 12 sec (in the inserts: 240msec). or multiple spikes or PDS with 30-70 m M P T Z only resulted in marked increases in spike frequency, followed by a progressive reduction in spike amplitude (Fig. 2F). In this way all spike activity ceased within 15-25 min, but reappeared on washing out the P T Z from the medium.

The effect of metabolic modifiers on PTZ-induced double spikes Earlier investigations on the biochemical mechanisms governing the rhythmic spike activity indicate a close coupling with two allosteric enzymes: the phosphofructokinase (PFK) and fructose-l,6-diphosphatase (FDPase)4, 5. Both the enzyme activity and the neuronal discharge frequency are increased by the allosteric effectors fructose-l,6-diphosphate (F-1,6-Ps), adenosine monophosphate (AMP), and fructose6-phosphate (F-6-P), while the substrate cycle and the rhythmic spike activity are inhibited by citrate, A T P and 3-phosphoglycerate (3-PGA). As the appearance of doublets, triplets and spike bursts in normally regular pacemaker neurons can be induced by the activating modifiers A M P and F-6-P (see ref. 5), the hypothesis was tested that P T Z may have a similar metabolic site of action. I f both allosteric activators of the PFK-FDPase-mediated substrate cycle and P T Z increase the neuronal pacemaker activity in a similar manner, it should be possible to revert the PTZ-induced double spikes to single spike discharges. F o r this purpose 5 m M citrate was added to a medium containing 50 m M PTZ, after an isolated

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F~g. 4. Effect o f 3-phosphoglycerate on the PTZ-induced changes in neuronal discharge pattern. The results shown have been obtained on an isolated R14 neuron. A: time course o f the PTZ-induced changes in spike activity (addition o f 50 m M PTZ denoted by the arrow). B: train o f spike doublets after 12 mm. C: further a d d i t m n of 5 m M 3-PGA led to single spikes after 7 mm, the relative fractions o f which increased with time, as recorded at intervals after 19 rain (D), 24 min (E) and 62 min (F). W h e n a R14 neuron had been in contact with 50 m M P T Z for 60 min somewhat distorted spike triplets appeared (G) which transformed into PDS after 75 min (H). Administration o f 10 m M 3-PGA (see arrow in H) restored regular single-spike trains after 20 min (I). Time cahbration: in A and F the horizontal line corresponds to 1 min, and in B - D and G - I to 12 sec.

neuron (illustrated for a R 6 cell in Fig. 3) had developed a regular train of doublets. About 5 min after citrate addition the second spike became flattened and its duration was prolonged (Fig. 3A, right-hand side). The interval between the two spikes in the doublet increased until after 12 min only single spikes were generated again. If the PFK-FDPase-mediated substrate cycle was indeed involved it should be possible to neutralize the citrate effect by an activator and in this way reestablish the PTZinduced spike pattern. In support of this postulate it could be demonstrated that 5 m M F-1,6-P2 did in fact overcome the citrate-promoted inhibition (Fig. 3C). In other experiments in which 10 m M citrate was added to PTZ-treated neurons, citrate at first lengthened the interval between the doublets and soon only single spikes could be recorded (Fig. 3D). Adding a further 5 m M F-6-P, an activator of the neuronal PFK, to these neurons resulted in the appearance of short bursts (Fig. 3E), rather similar to the twin-doublets in Fig. 1D; 10-15 min later the length of these

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Fig. 5. Action of the anti-convulsants phenobarbital and pentobarbital on the PTZ-induced double spike discharges. A: the anti-convulsant phenobarbital (20 raM) was added to an isolated R6 neuron which discharges a maintained train of double spikes obtained in the presence of 30 raM PTZ. The barbiturate-mediated changes have been recorded after 1.5 rain (B) and 3.5 rain (C). D: after 5 min the phenobarbital concentration was increased to 30 mM (D), the changes in the spike pattern due to the second addition of phenobarbital are illustrated for the time intervals covering the first 5 rain (D-F). On the expanded time scales in F, alterations in the shape of the spike are shown for the recording periods 1 min (a), 3 min (b) and 5 min (c) after increasing the phenobarbital level in the medium to 30 raM. G: effect of 25 mM pentobarbital applications (see arrow) on the PTZinduced doublets. Note that the interburst interval gradually increases, as recorded after 5 rain (H) and 10 rain (I) until the spike discharges are completely suppressed after 12 ram. Time calibration: the horizontal line corresponds to a time scale in A-C and G-I equal to 6 sec, in D and E (as well as inserts in A and B) equal to 1.2 see, while in F it marks an interval of 1.2 see for a, and of 240 msec for b and c. b u r s t s was extended, with the bursts always i n t e r s p a c e d by single spikes (Fig. 3F). T o verify the h y p o t h e s i s o f a possible P T Z a c t i o n o n the i n t e r m e d i a r y m e t a b o l i s m two o t h e r inhibitors o f the P F K - F D P a s e - m e d i a t e d s u b s t r a t e cycle - - 3-phosp h o g l y c e r a t e a n d A T P - - have been a d d e d to P T Z - t r e a t e d neurons. Once d o u b l e spikes h a d b e c o m e firmly e s t a b l i s h e d in an R14 n e u r o n (note the r e d u c t i o n in spike a m p l i t u d e a n d the t r a n s i e n t increase in spike frequency i n d u c e d b y 50 m M PTZ), 10 m M 3 - P G A was a d d e d . A f t e r an interval o f 12-15 min the first single spikes a p p e a r e d , which after 25 min h a d b e c o m e very frequent (Fig. 4C a n d D). A c o m p l e t e r e t u r n to the r e g u l a r discharge p a t t e r n characteristic f o r the R14 n e u r o n (Fig. 4A) c o u l d be o b s e r v e d s o m e 60 m i n after 3 - P G A a d d i t i o n (Fig. 4F). Even u n d e r c o n d i t i o n s where p a r o x y s m a l d e p o l a r i z i n g shifts h a d d e v e l o p e d after 70 min e x p o s u r e in presence o f 50 m M P T Z , (Fig. 4H), a d d i t i o n o f 5 m M 3 - P G A r e t u r n e d a n o r m a l r e g u l a r spike

328 discharge (Fig. 41) within a short hme period. However. 3-PGA was unable to restore the reduced spike amplitude to the full height. Thus the long-term effects of PTZ may well impair membrane ion transport. Simdar results have also been observed w~th other neurons of the regular beating type Ra-R~a, except for the R10 cell which is not inh~bited by 3-PGA (see ref. 4). The effect of 3-PGA could be duplicated by adding 2-4 mM ATP to the PTZ-treated neurons.

The actton of antwonvulsants on the pentylenetetrazol-in&wed changes & spike activity The classical representatwe of antiepdept~c drugs is the barbiturate phenobarbital (5-ethyl-5-phenyl-malonylurea) whmh is known to block all spontaneous neuronal activityz4. When R3 - R14 pacemaker neurons are treated with 50 mM PTZ for 25 rain the subsequent addition of 20-30 mM sodium phenobarbital within 1 min started to affect the PTZ-induced spike doublets (Fig. 5B). About 1.5 min after administration of the barbiturate, triple spikes developed which after 3.5 min started to degenerate again (Fig. 5D and E). Thus phenobarbital, at least transiently, seems to promote the PTZ effect. The exact changes m the shape of mplets are illustrated in Fig. 5F. Between 4 and 5 rain after phenobarbital administration in place of the second and third spike a very broad shoulder appears. After 10 rain all spike activity has been depressed by the anticonvulsant. Suppression of spike activity was also noted with another barbiturate, pentobarbital, which at first lengthened the interval between the PTZ-induced double spikes (Fig. 5G and H), untd the impulse discharges ceased after 10-12 min (Fig. 5I). DISCUSSION

The changes in impulse pattern which can be induced by pentylenetetrazol in isolated pacemaker neurons of the sea hare Aplysia californica are generally the same as those reported for other invertebrate and mammalian systemsa,l°-l~,~7,2s. The initial increase in the discharge frequency paralleled by a reduction in after-hyperpoiarization (Fig. l) has been observed previously in a variety of neuronal systems s,11,14, as. The decrease in spike amplitude is equally consistent with similar results on other molluscan neurons11,14, ~6 and the changes in the impulse characteristics immediately preceding the ictal period in electrically induced seizures of the vertebrate cortex 2s,29. The appearance of doublet and triplet bursts of spikes has been noted in virtually all drug-induced seizuresg-llA ~-27, while the finding of recurring depolarizing shifts with impulse bursts superimposed (Fig. 1G), although previously noted in Tritonia neurons 11, may be a precursor state to the paroxysmal depolarizing shifts (PDS). The latter, which developed under PTZ-treatment only in some of the regular beating Aplysia pacemakers (Fig. 2), appear to be a more common pattern of neuronal activity in Helixa,lo, 26 and in the mammalian brain 18,27. The use of completely isolated pacemaker neurons rules out that the effects of PTZ on spike activity are mediated in any way by synaptic, ephaptic or humoral inputs. Further, the finding that inhibitory modifiers of the PFK-FDPase-mediated substrate cycle, such as ATP, citrate or 3-PGA, are able to revert the PTZ-induced doublets,

329 triplets and PDS to single spikes suggests a common site of action. This hypothesis receives additional support from the observation that the impulse pattern characteristic for the PTZ-treated neurons can be restored simply on neutralizing the effects induced by the allosteric inhibitors through such metabolites which normally activate the neuronal substrate cycle (Fig. 3). As selective activators of the neuronal PFK such as F-6-P or AMP are themselves able to induce the spike doublets and triplets 5 shown in Fig. 1, there exists a real possibility that PTZ affects in some way the metabolic control mechanisms. Thus epileptic seizure may in fact be nothing else but the loss of the reciprocal control of the neuronal PFK and FDPase in pacemaker neurons described in an earlier communication4. A number of findings reported in the literature appear to be in good agreement with the hypothesis that 'epileptic' neurons are functionally impaired in the regulatory mechanisms normally slowing the PFK-FDPase-mediated substrate cycle. First, one of the most effective inhibitory modifiers of the substrate cycle, ATP, is known to decrease considerably during experimental seizures 22. Second, free fatty acids (which stimulate the substrate cycle in perfused tissues by activating the FDPase 2) and fluoracetate (which increases the PFK activity by lowering the inhibitory citrate levels a3) are known to promote convulsive activity16,30. Third, the concentration of glucoplastic amino acids decreases during drugreduced seizures25,a°. In this respect it is of interest that in Aplysia pacemaker gluconeogenesis provides the substrate for the PFK-FDPase couple 4, with the addition of gluconeogenic substrate pyruvate leading to spike burstsL Since in brain systems NH4 ÷ ions constitute an activator of the PFK ~7, the deamination in the conversion of amino acids to glycolytic intermediates will provide a positive feedback for the operation of the substrate cycle. The outlined metabolic regulatory effects equally provide an explanation of the finding of an insulin-induced convulsive activity16,ao, an effect which is paralleled by increased cerebral ammonia levelsZS,a°. Insulin-induced hypoglycemia actually promotes gluconeogenesis from amino acids via pyruvate. The postulate of an 'epileptogenic' neuron which exhibits convulsive activity as the result of an error in metabolic regulations is by no means irreconcilable with the diversity of neuronal responses observed in epileptogenic foci. The differences between the actwe and passive neurons of Matsumoto and Ajmone Marsan la and the different states of involvement reported by Prince and Futamachi z4 may in fact reflect the finding that the PFK-FDPase-mediated substrate cycle only operates in pacemaker cells but not in silent neurons4. Naturally, the synaptic inputs from the pacemaker neurons will themselves influence other elements of neuronal circuits, giving rise to the more complex patterns in seizures16,2°,21,2a,al. Provided one is able to perfuse inhibitory modifiers of the PFK-FDPase cycle into an epileptic focus, this could perhaps offer a new approach to terminate seizure discharges. In contrast to the action of the anti-convulsants phenobarbital and pentobarbital which ultimately block all spike activity in the 'epileptic' neurons, with metabolites it will be possible to restore the normal autorhythmic impulse discharges.

330 ACKNOWLEDGEMENTS This work was supported by the Deutsche Forschungsgemeinschaft, Research G r a n t Ch 25/1. The authors wish to t h a n k Prof. R. y o n B a u m g a r t e n for his advice a n d contin u e d interest d u r i n g the course o f this study.

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