Neuroscience Letters, 129 (1991) 51-54 ADONIS 030439409100400E
51
NSL 07911
Caffeine-induced epileptic discharges in CA3 neurons of hippocampal slices of the guinea pig I. M o r a i d i s l, D. B i n g m a n n 1, A. Lehrnenkiib_ler2 a n d E.-J. S p e c k m a n n 2 Ilnstitut f~r Physiologic, Essen ( F.R.G. ) and ~Institut J~r Physiologic, M ~nster ( F.R.G. )
(Received 15 March 1991;Revisedversionreceived24 April 1991;Accepted26 April 1991) Key words: Caffeine;CA3-neuron;Paroxysmaldepolarization;Organiccalciumantagonist;Tetrodotoxin
In order to analyzethe elementarymechanismsunderlyingcaffeine-inducedepileptiformdischarges,hippocampalslicesof guineapigs were exposed to this drug. When the bath concentrationof caffeineexceeded0.2 raM, periodicallyoccurringparoxysmaldepolarizations(PD) in CA3 neurons appeared. They were accompaniedby declinesof extracellularfree calciumconcentrationand were suppressed by the organiccalciumantagonists verapamiland flunarizine.PD-likefluctuationsof the membranepotentialcould be evokedalso in CA3 neuronsfunctionallyisolatedby tetrodotoxin (TTX). The observationsindicate that caffeine-inducedPD are generated endogenouslyand that transmembranouscalciumcurrents contribute to these mechanisms.
Overdosage of caffeine resulting in plasma concentrations of more than 100/zM was found to lead to seizures in man which may be attributed either to general rise of excitability or to modulation of synaptic transmission [4-9, 15]. However, the epileptogenic mechanism is still unclear. To contribute to a clarification, the present experiments were carried out on hippocampal slices. Transverse slices (400-450/lm thick) were prepared from guinea pigs (300--400 g) under ether anesthesia. Slices were preincubated for 2 h in 28°C warm saline, equilibrated with 5% CO2 in 02. The saline contained (in mM): NaC1 124, KC1 5 or 3, CaCI2 0.75, MgSO41.3, KH2PO4 1.25, NaHCO3 26 and glucose 10. The experiments were performed in a perspex recording chamber (vol. 2 ml) which was mounted on an inverted microscope. The chamber was continuously perfused at the rate of 2 ml/min by 32°C warm saline. The saline was of the same composition as mentioned above except for the calcium concentration which was elevated to 1.75 mM. Caffeine (Sigma) and tetrodotoxin (TTX; Sigma) as well as the organic calcium antagonists verapamil (Knoll) and flunarizine (solved in cyclodextrine; Janssen) were added to the saline without affecting the pH of 7.4 . Intracellular recordings from neurons were obtained with glass micropipettes filled with potassium methylsulfate (2 M; Atlanta). The resistance of the electrodes ranged from 80 to 200 MI2. Current was Correspondence: I. Moraidis,Institut fiir Physiologic,Hufelandstr.55, 4300 Essen 1, F.R.G.
injected intracellularly through the recording microelectrode using a bridge circuit. Calcium selective microelectrodes were constructed from thick septum 0-capillary borosilicate glass with a tip diameter of 2-3 gm. After silanization of the later ion-selective barrel, both channels were backfilled with appropriate electrolyte solutions. The Ca2+-selective cocktail (ETH 1001; Fluka No. 21048) was sucked into the silanized channel containing 100 m M CaC12. The reference channel contained 150 mM NaCI. Typical ion-selective electrodes had a slope of 29 mV per decade when tested in a set of solutions containing Ca 2+ concentrations of 0.1-10 mM at a fixed background of 5 m M KCI and 150 mM NaCI. The signals of the ion-selective electrodes were amplified as described elsewhere [12, 13]. Under control conditions, intracellular recordings from CA3 neurons most often exhibited spontaneous aperiodic activity when the concentration of KCI in the superfusate was 5 (n = 7) or 3 mM (n = 34). After adding 0.2-5 mM caffeine to the superfusate containing 5 m M KC1, periodic epileptic paroxysmal discharges (PD) developed in 71 out of 74 tested CA3 neurons. These PD consisted of a steep depolarization, a plateau-like decrease of membrane potential, a rapid repolarization and a prolonged afterhyperpolarization. The epileptic activity strictly resembled that elicited in CA neurons by pentylenetetrazol [1-3]. A typical example of caffeine-induced, periodically occurring PD is shown in Fig. 1A. The latency of the onset of PD
52
t rnV -45 -55 L..urr~llt~
B
C FP
[Ca*"]° ~
FP
~
rco÷"Jo
[7.z5,..
l
i I
5s
1, 7 0
30s
L1,7O
Fig. 1. Caffeine-induced paroxysmal depolarizations (PD) in CA3 neurons of guinea pig in vitro. The tracings of the membrane potentials 1 and 2 (note different time scales) are related to each other by straight lines. A: aperiodic discharges (la) were replaced by stereotype and periodically generated PD (lb-f). B and C: epileptiform activity of CA3 neurons was accompanied by periodic field potentials (FP) and corresponding declines of extracellular free calcium concentration ([Ca2+]o). Stratum pyramidale. Caffeine-induced discharges. Different horizontal and vertical amplifications.
sequencies ranged from 2 to 5/min when the bath concentration was 1 mM. Lowering of the caffeine concentration in the bath tended to prolong this latency. The epileptic activity often developed at constant resting membrane potential. Sometimes, however, a slight depolarization up to 5 mV was observed. The frequency of occurrence of PD was in the range of 15-60/min. The amplitude of PD was about 30 mV and the duration varied from 80 to 500 ms. A dependency of these PD parameters on the caffeine concentration could not be found. The same PD sequencies were observed at lowered potassium concentration (n = 34 ), which indicates that this PD generation is not due to a critical elevation of potassium [20]. During caffeine-induced epileptic activity, periodic field potentials (FP) which had the same frequencies of occurrence like the PD described, were recorded in CA3 area (Fig. 1B). In the stratum pyramidale FP consisted of an initial positive deflection (amplitude, 0.5-1 mV; duration, 200 ms) followed by a smaller negative one (amplitude: 0.1 mV; duration: 600 ms). FP reversed in polarity in stratum radiatum (not shown). The periodic occurrence of stereotype FP of large amplitudes in the CA3 area and the transient declines in extracellular free
A
rr~ -50 -60
~ra~arn~/ 750~un'm~
B
o 2O mV
I j
rnV -50 . -60
Fig. 2. Effect of the organic calcium antagonist verapamil on caffeine induced spontaneous depolarization shifts. A: suppression of caffeine induced PD (lb-d). PD were reduced in amplitude and duration until epileptic activity failed. B: recovery of PD after removal of verapamil from the bath saline (la~l).
53
calcium concentration ([Ca2+]o) also described for other epilepsy models [10, 1l, 17] reflects the synchronization of epileptic activity of the neurons. In the present model the decreases of [Ca2+]o amounted to 30-50 #M. Minimum calcium values were observed after 300 ms (Fig. 1C). [Ca2+]o recovered within 6 s. When the organic calcium antagonists flunarizine (40 gM added to the bath solution; n=4) and verapamil (80-150 gM; n = 14) were given during caffeine exposure, PD were reduced in amplitude and duration until the generation of PD failed (Fig. 2). Both changes occurred either simultaneously or successively and were accompanied by a reduction of frequency of occurrence of PD. Epileptic activity was completely blocked within 20-60 min, while the resting membrane potential was unaffected in most cases. The suppressive effects of verapamil and flunarizine were reversible after 30-90 min. Addition of these organic calcium antagonists to control saline did
~
not affect resting membrane potentials, action potentials and postsynaptic potentials of CA3 neurons [2, 3]. In order to analyze the contribution of synaptic transmission to the generation of PD in the present case, TTX was added when PD occurred in caffeine-containing saline (Fig. 3). During TTX exposure (0.2 gM) the amplitude of the sodium spikes were reduced and spontaneous, periodic PD generation stopped. Injection of depolarizing currents, however, still generated fluctuations of the membrane potential resembling those underlying typical PD (Fig. 3A-lc,d). Sometimes, such membrane potential fluctuations even appeared spontaneously (Fig. 3A-le). The PD-like membrane potential fluctuations were reduced as well in amplitude and duration during additional exposure to verapamil (Fig. 3B-la,b). After withdrawal of verapamil from the caffeine- and TTX-containing bath solution the amplitude of PD-like membrane potential fluctuations recovered
~,
2
/L ........ -it ~''~ fr~. -If-
,x
c
Jl /~.,,,.~/~
d
e
- ~:.-:l:,~';"t..... "_.." ~'- =--t_u~--r c
/ /
t_50 -
X rnV
-40
...... . . . . . . . . . . .
T TX ,Verapamd
// I
-- . . . . . . .
"-'
') ~ ' J ' - ~
TTX , 5rnm , /i/
f
-50
-60
Fig. 3. Effects of TTX (2 x 10-7 M) and verapamil (80/zM) on caffeine-induced spontaneous PD. A: suppression of PD during TTX exposure and persistence of electrically induced PD-like fluctuations of the membrane potential (lc~i). The membrane potential fluctuation (le) occurred spontaneously. B: reversible suppression of TTX resistant PD-like signals in amplitude and duration by verapamil (la-c). Membrane potential recordings 1 and 2 (different time scales) are related to each other by straight lines.
54 (Fig. 3B-ld,e). Similar findings have been o b s e r v e d in pentylenetetrazol-treated CA3 neurons during TTX e x p o s u r e o r after w i t h d r a w a l o f s o d i u m f r o m the superfusate [2]. It is k n o w n t h a t caffeine acts as a c o m p e t i t i v e a n t a g o nist at a d e n o s i n e r e c e p t o r sites b l o c k i n g the i n h i b i t o r y effect o f a d e n o s i n e o n a d e n y l a t e cyclase. This disinhibition increases the i n t r a c e l l u l a r c A M P - l e v e l [4-9, 16]. There are hints t h a t caffeine-induced epileptic discharges are also m e d i a t e d b y such a n a d e n o s i n e r e c e p t o r b l o c k a d e [14] elevating the i n t r a c e l l u l a r c A M P level to a b n o r m a l l y high c o n c e n t r a t i o n s which eventually m a y lead to excessive t r a n s m e m b r a n o u s c a l c i u m currents a n d consequently to decreases in [Ca2+]o. F u r t h e r m o r e , it s h o u l d be c o n s i d e r e d t h a t the used doses o f caffeine m a y also lead to a release o f C a 2+ f r o m stores at the i n t r a c e l l u l a r site which c o u l d facilitate the d e v e l o p m e n t o f non-specific i n w a r d currents [19]. T h e p r e s e n t results indicate: (1) t r a n s m e m b r a n o u s calc i u m currents essentially c o n t r i b u t e to caffeine-induced p a r o x y s m a l d e p o l a r i z a t i o n shifts. D u e to a b l o c k a d e o f these excessive calcium currents P D were suppressed b y o r g a n i c calcium a n t a g o n i s t s like P D elicited by pentylenetetrazol a n d bicuculline in C A 3 n e u r o n s [1, 2, 18]; (2) caffeine-evoked P D - l i k e m e m b r a n e p o t e n t i a l fluctuations are m e d i a t e d by e n d o g e n o u s m e c h a n i s m s which persist d u r i n g T T X exposure. S y n a p t i c processes are s u p p o s e d to trigger these m e c h a n i s m s a n d synchronize the caffeine-induced epileptic activity o f C A 3 neurons. I Bingmann, D. and Speckmann, E.-J., Actions of pentylenetetrazol (PTZ) on CA3 neurons in hippocampal slices of guinea pigs, Exp. Brain Res., 64 (1986) 94-104. 2 Bingmann, D. and Speckmann, E.-J., Depression of pentylenetetrazol-induced epileptiform discharges in CA3 neurons of hippocampal slices by flunarizine and verapamil. In E.-J. Speckmann, H. Schulze and J. Walden (Eds.), Epilepsy and Calcium, Urban and Schwarzenberg, MiJnchen, 1986, pp. 301-317. 3 Bingmann, D. and Speckmann, E.-J., Specific suppression of pentylenetetrazol-induced epileptiform discharges in CA3 neurons (hippocampal slice, guinea pig) by the organic calcium antagonists flunarizine and verapamil, Exp. Brain Res., 74 (1989) 239-248. 4 Daly, J.W., Bruns, R.F. and Snyder, S.H., Adenosine receptors in the central nervous system: relationship to the central actions of methylxanthines, Life Sci., 28 (1981) 2083-2097. 5 Daly, J.W., Buns-Lamb, P. and Padgett, W., Subclasses of adenosine receptors in the central nervous system: interaction with caf-
feine and related methylxanthines, Cell. Mol. Neurobiol., 3 (1983) 69-80.
6 Fredholm, B.B., On the mechanism of theophylline and caffeine, Acta Med. Scand., 217 (1985) 149-153. 7 Gerber, U., Greene, R.W., Haas, H.L. and Stevens, D.R., Characterization of inhibition mediated by adenosine in the hippocampus of the rat in vitro, J. Physiol., 417 (1989) 567-578. 8 Greene, R.W., Haas, H.L. and Hermann, A., Effects of caffeine on hippocampal pyramidal cells in vitro, Br. J. Pharmacol., 85 (1985) 163-169. 9 Haas, H.L. and Greene, R.W., Endogenous adenosine inhibits hippocampal CA 1 neurones: further evidence from extra- and intraeellular recording, Naunyn-Schmiedeberg's Arch. Pharmacol., 337 (1988) 561-565. I0 Heinemann, U., Lux, H.D. and Gutnick, M.J., Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat, Exp. Brain Res., 27 (1977) 237-243. 11 Heinemann, U., Konnerth, A., Pumain, R. and Wadman, W., Extracellular calcium and potassium concentration changes in chronic epileptic tissue, Adv. Neurol., 44 (1986) 641-661. 12 Lehmenkiihler, A., Caspers, H. and Kersting U., Relations between DC potentials, extracellular ion activities and extracellular volume fraction in the cerebral cortex with changes in pCO2. In M. Kessler, D.K. Harrison and J. Hrper (Eds.), Ion Measurements in Physiology and Medicine, Springer, Berlin, 1985, pp. 199-205. 13 Liicke, A., Lehmenkiihler, A. and Speckmann, E.-J., The influence of the convulsant pentylenetetrazol on Ca2*-selective microelectrodes (neutral carrier ETH 1001), J. Neurosci. Methods, 29 (1989) 139-142. 14 Moraidis, I., Lehmenkiihler, A., Straub, H. and Bingmann, D., Decline of extracellular free calcium concentration during caffeine induced paroxysmal discharges of CA3 neurons in hippocampal slices (guinea pig), Pfliiger's Arch., S 415 (1990) R 84. 15 Rail, Th.W., Central nervous system stimulants. In A. Goodman Gilman, L.S. Goodman, Th.W. Rail and F, Murad (Eds.), The Pharmacological basis of Therapeutics, McMillan, New York, 1985, pp. 589-603. 16 Snyder, S.H., Katims, J.J., Annau, Z., Bruns, R.F. and Daly, J.W., Adenosine receptors and the behavioural actions of methylxanthines, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 3260. 17 Speckmann, E.-J., Walden, J. and Bingmann, D., Contribution of calcium ions to epileptogenesis, J. Basic Clin. Physiol., 1 (1990) 95105. 18 Straub, H., Speckmann, E.-J., Bingmann, D. and Walden, J., Paroxysmal depolarization shifts induced by bicuculline in CA3 neurons of hippocampal slices: suppression by the organic calcium antagonist verapamil, Neurosci. Lett., 111 (1990) 99-101. 19 Swandulla, D. and Lux, H.D., Activation of a non-specific cation conductance by intracellular Ca2+ activation in bursting pacemaker neurons, J. Neurophysiol., 54 (1985) 1430-1443. 20 Yaari, Y., Konnerth, A. and Heinemann, U., Non-synaptic epileptogenesis in the mammalian hippocampus in vitro. II. Role of extracellular potassium, J. Neurophysiol., 56 (1986) 424-438.
51
NSL 07911
Caffeine-induced epileptic discharges in CA3 neurons of hippocampal slices of the guinea pig I. M o r a i d i s l, D. B i n g m a n n 1, A. Lehrnenkiib_ler2 a n d E.-J. S p e c k m a n n 2 Ilnstitut f~r Physiologic, Essen ( F.R.G. ) and ~Institut J~r Physiologic, M ~nster ( F.R.G. )
(Received 15 March 1991;Revisedversionreceived24 April 1991;Accepted26 April 1991) Key words: Caffeine;CA3-neuron;Paroxysmaldepolarization;Organiccalciumantagonist;Tetrodotoxin
In order to analyzethe elementarymechanismsunderlyingcaffeine-inducedepileptiformdischarges,hippocampalslicesof guineapigs were exposed to this drug. When the bath concentrationof caffeineexceeded0.2 raM, periodicallyoccurringparoxysmaldepolarizations(PD) in CA3 neurons appeared. They were accompaniedby declinesof extracellularfree calciumconcentrationand were suppressed by the organiccalciumantagonists verapamiland flunarizine.PD-likefluctuationsof the membranepotentialcould be evokedalso in CA3 neuronsfunctionallyisolatedby tetrodotoxin (TTX). The observationsindicate that caffeine-inducedPD are generated endogenouslyand that transmembranouscalciumcurrents contribute to these mechanisms.
Overdosage of caffeine resulting in plasma concentrations of more than 100/zM was found to lead to seizures in man which may be attributed either to general rise of excitability or to modulation of synaptic transmission [4-9, 15]. However, the epileptogenic mechanism is still unclear. To contribute to a clarification, the present experiments were carried out on hippocampal slices. Transverse slices (400-450/lm thick) were prepared from guinea pigs (300--400 g) under ether anesthesia. Slices were preincubated for 2 h in 28°C warm saline, equilibrated with 5% CO2 in 02. The saline contained (in mM): NaC1 124, KC1 5 or 3, CaCI2 0.75, MgSO41.3, KH2PO4 1.25, NaHCO3 26 and glucose 10. The experiments were performed in a perspex recording chamber (vol. 2 ml) which was mounted on an inverted microscope. The chamber was continuously perfused at the rate of 2 ml/min by 32°C warm saline. The saline was of the same composition as mentioned above except for the calcium concentration which was elevated to 1.75 mM. Caffeine (Sigma) and tetrodotoxin (TTX; Sigma) as well as the organic calcium antagonists verapamil (Knoll) and flunarizine (solved in cyclodextrine; Janssen) were added to the saline without affecting the pH of 7.4 . Intracellular recordings from neurons were obtained with glass micropipettes filled with potassium methylsulfate (2 M; Atlanta). The resistance of the electrodes ranged from 80 to 200 MI2. Current was Correspondence: I. Moraidis,Institut fiir Physiologic,Hufelandstr.55, 4300 Essen 1, F.R.G.
injected intracellularly through the recording microelectrode using a bridge circuit. Calcium selective microelectrodes were constructed from thick septum 0-capillary borosilicate glass with a tip diameter of 2-3 gm. After silanization of the later ion-selective barrel, both channels were backfilled with appropriate electrolyte solutions. The Ca2+-selective cocktail (ETH 1001; Fluka No. 21048) was sucked into the silanized channel containing 100 m M CaC12. The reference channel contained 150 mM NaCI. Typical ion-selective electrodes had a slope of 29 mV per decade when tested in a set of solutions containing Ca 2+ concentrations of 0.1-10 mM at a fixed background of 5 m M KCI and 150 mM NaCI. The signals of the ion-selective electrodes were amplified as described elsewhere [12, 13]. Under control conditions, intracellular recordings from CA3 neurons most often exhibited spontaneous aperiodic activity when the concentration of KCI in the superfusate was 5 (n = 7) or 3 mM (n = 34). After adding 0.2-5 mM caffeine to the superfusate containing 5 m M KC1, periodic epileptic paroxysmal discharges (PD) developed in 71 out of 74 tested CA3 neurons. These PD consisted of a steep depolarization, a plateau-like decrease of membrane potential, a rapid repolarization and a prolonged afterhyperpolarization. The epileptic activity strictly resembled that elicited in CA neurons by pentylenetetrazol [1-3]. A typical example of caffeine-induced, periodically occurring PD is shown in Fig. 1A. The latency of the onset of PD
52
t rnV -45 -55 L..urr~llt~
B
C FP
[Ca*"]° ~
FP
~
rco÷"Jo
[7.z5,..
l
i I
5s
1, 7 0
30s
L1,7O
Fig. 1. Caffeine-induced paroxysmal depolarizations (PD) in CA3 neurons of guinea pig in vitro. The tracings of the membrane potentials 1 and 2 (note different time scales) are related to each other by straight lines. A: aperiodic discharges (la) were replaced by stereotype and periodically generated PD (lb-f). B and C: epileptiform activity of CA3 neurons was accompanied by periodic field potentials (FP) and corresponding declines of extracellular free calcium concentration ([Ca2+]o). Stratum pyramidale. Caffeine-induced discharges. Different horizontal and vertical amplifications.
sequencies ranged from 2 to 5/min when the bath concentration was 1 mM. Lowering of the caffeine concentration in the bath tended to prolong this latency. The epileptic activity often developed at constant resting membrane potential. Sometimes, however, a slight depolarization up to 5 mV was observed. The frequency of occurrence of PD was in the range of 15-60/min. The amplitude of PD was about 30 mV and the duration varied from 80 to 500 ms. A dependency of these PD parameters on the caffeine concentration could not be found. The same PD sequencies were observed at lowered potassium concentration (n = 34 ), which indicates that this PD generation is not due to a critical elevation of potassium [20]. During caffeine-induced epileptic activity, periodic field potentials (FP) which had the same frequencies of occurrence like the PD described, were recorded in CA3 area (Fig. 1B). In the stratum pyramidale FP consisted of an initial positive deflection (amplitude, 0.5-1 mV; duration, 200 ms) followed by a smaller negative one (amplitude: 0.1 mV; duration: 600 ms). FP reversed in polarity in stratum radiatum (not shown). The periodic occurrence of stereotype FP of large amplitudes in the CA3 area and the transient declines in extracellular free
A
rr~ -50 -60
~ra~arn~/ 750~un'm~
B
o 2O mV
I j
rnV -50 . -60
Fig. 2. Effect of the organic calcium antagonist verapamil on caffeine induced spontaneous depolarization shifts. A: suppression of caffeine induced PD (lb-d). PD were reduced in amplitude and duration until epileptic activity failed. B: recovery of PD after removal of verapamil from the bath saline (la~l).
53
calcium concentration ([Ca2+]o) also described for other epilepsy models [10, 1l, 17] reflects the synchronization of epileptic activity of the neurons. In the present model the decreases of [Ca2+]o amounted to 30-50 #M. Minimum calcium values were observed after 300 ms (Fig. 1C). [Ca2+]o recovered within 6 s. When the organic calcium antagonists flunarizine (40 gM added to the bath solution; n=4) and verapamil (80-150 gM; n = 14) were given during caffeine exposure, PD were reduced in amplitude and duration until the generation of PD failed (Fig. 2). Both changes occurred either simultaneously or successively and were accompanied by a reduction of frequency of occurrence of PD. Epileptic activity was completely blocked within 20-60 min, while the resting membrane potential was unaffected in most cases. The suppressive effects of verapamil and flunarizine were reversible after 30-90 min. Addition of these organic calcium antagonists to control saline did
~
not affect resting membrane potentials, action potentials and postsynaptic potentials of CA3 neurons [2, 3]. In order to analyze the contribution of synaptic transmission to the generation of PD in the present case, TTX was added when PD occurred in caffeine-containing saline (Fig. 3). During TTX exposure (0.2 gM) the amplitude of the sodium spikes were reduced and spontaneous, periodic PD generation stopped. Injection of depolarizing currents, however, still generated fluctuations of the membrane potential resembling those underlying typical PD (Fig. 3A-lc,d). Sometimes, such membrane potential fluctuations even appeared spontaneously (Fig. 3A-le). The PD-like membrane potential fluctuations were reduced as well in amplitude and duration during additional exposure to verapamil (Fig. 3B-la,b). After withdrawal of verapamil from the caffeine- and TTX-containing bath solution the amplitude of PD-like membrane potential fluctuations recovered
~,
2
/L ........ -it ~''~ fr~. -If-
,x
c
Jl /~.,,,.~/~
d
e
- ~:.-:l:,~';"t..... "_.." ~'- =--t_u~--r c
/ /
t_50 -
X rnV
-40
...... . . . . . . . . . . .
T TX ,Verapamd
// I
-- . . . . . . .
"-'
') ~ ' J ' - ~
TTX , 5rnm , /i/
f
-50
-60
Fig. 3. Effects of TTX (2 x 10-7 M) and verapamil (80/zM) on caffeine-induced spontaneous PD. A: suppression of PD during TTX exposure and persistence of electrically induced PD-like fluctuations of the membrane potential (lc~i). The membrane potential fluctuation (le) occurred spontaneously. B: reversible suppression of TTX resistant PD-like signals in amplitude and duration by verapamil (la-c). Membrane potential recordings 1 and 2 (different time scales) are related to each other by straight lines.
54 (Fig. 3B-ld,e). Similar findings have been o b s e r v e d in pentylenetetrazol-treated CA3 neurons during TTX e x p o s u r e o r after w i t h d r a w a l o f s o d i u m f r o m the superfusate [2]. It is k n o w n t h a t caffeine acts as a c o m p e t i t i v e a n t a g o nist at a d e n o s i n e r e c e p t o r sites b l o c k i n g the i n h i b i t o r y effect o f a d e n o s i n e o n a d e n y l a t e cyclase. This disinhibition increases the i n t r a c e l l u l a r c A M P - l e v e l [4-9, 16]. There are hints t h a t caffeine-induced epileptic discharges are also m e d i a t e d b y such a n a d e n o s i n e r e c e p t o r b l o c k a d e [14] elevating the i n t r a c e l l u l a r c A M P level to a b n o r m a l l y high c o n c e n t r a t i o n s which eventually m a y lead to excessive t r a n s m e m b r a n o u s c a l c i u m currents a n d consequently to decreases in [Ca2+]o. F u r t h e r m o r e , it s h o u l d be c o n s i d e r e d t h a t the used doses o f caffeine m a y also lead to a release o f C a 2+ f r o m stores at the i n t r a c e l l u l a r site which c o u l d facilitate the d e v e l o p m e n t o f non-specific i n w a r d currents [19]. T h e p r e s e n t results indicate: (1) t r a n s m e m b r a n o u s calc i u m currents essentially c o n t r i b u t e to caffeine-induced p a r o x y s m a l d e p o l a r i z a t i o n shifts. D u e to a b l o c k a d e o f these excessive calcium currents P D were suppressed b y o r g a n i c calcium a n t a g o n i s t s like P D elicited by pentylenetetrazol a n d bicuculline in C A 3 n e u r o n s [1, 2, 18]; (2) caffeine-evoked P D - l i k e m e m b r a n e p o t e n t i a l fluctuations are m e d i a t e d by e n d o g e n o u s m e c h a n i s m s which persist d u r i n g T T X exposure. S y n a p t i c processes are s u p p o s e d to trigger these m e c h a n i s m s a n d synchronize the caffeine-induced epileptic activity o f C A 3 neurons. I Bingmann, D. and Speckmann, E.-J., Actions of pentylenetetrazol (PTZ) on CA3 neurons in hippocampal slices of guinea pigs, Exp. Brain Res., 64 (1986) 94-104. 2 Bingmann, D. and Speckmann, E.-J., Depression of pentylenetetrazol-induced epileptiform discharges in CA3 neurons of hippocampal slices by flunarizine and verapamil. In E.-J. Speckmann, H. Schulze and J. Walden (Eds.), Epilepsy and Calcium, Urban and Schwarzenberg, MiJnchen, 1986, pp. 301-317. 3 Bingmann, D. and Speckmann, E.-J., Specific suppression of pentylenetetrazol-induced epileptiform discharges in CA3 neurons (hippocampal slice, guinea pig) by the organic calcium antagonists flunarizine and verapamil, Exp. Brain Res., 74 (1989) 239-248. 4 Daly, J.W., Bruns, R.F. and Snyder, S.H., Adenosine receptors in the central nervous system: relationship to the central actions of methylxanthines, Life Sci., 28 (1981) 2083-2097. 5 Daly, J.W., Buns-Lamb, P. and Padgett, W., Subclasses of adenosine receptors in the central nervous system: interaction with caf-
feine and related methylxanthines, Cell. Mol. Neurobiol., 3 (1983) 69-80.
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