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Non-synaptic modulation of repetitive firing by adenosine is antagonized by 4-aminopyridine in a rat hippocampal slice
Neuroscience Letters, 67 (1986) 334-338 Elsevier Scientific Publishers Ireland L t d
334
NSL 04000
NON-SYNAPTIC MODULATION OF REPETITIVE FIRING BY ADENOSINE IS ANTAGONIZED BY 4-AMINOPYRIDINE IN A RAT HIPPOCAMPAL SLICE
PETER SCHUBERT* and KEVIN S. LEE
Max Planck Institute for Psychiatry, Department of Neuromorphology, am Klopferspitz 18a. D-8033 Martinsried (F. R.G. ) (Received January 10th, 1986; Revised version received and accepted March 24th, 1986)
Key words: adenosine - modulation - 4-aminopyridine - potassium conductance - A-current - epileptic -
hippocampal slice - rat
In rat hippocampal slices which were superfused with low calcium (0.2 mM) medium, stimulation of the alvear fibers elicited an extracellularly recorded antidromic population spike in CAI pyramidal neurons which was followed by 2-4 afterpotentials. Adenosine (20-40 laM) and the Al-adenosine agonist lphenylisopropyladenosine (L-PIA) blocked these afterpotentials without affecting the first spike. Addition of up to 5 m M tetraethylammonium to the superfused medium did not interfere with this adenosine action. But the addition of only 50 jaM 4-aminopyridine (4-AP) antagonized almost completely the adenosineor L-PIA-induced depression of antidromically evoked repetitive firing. It is concluded that functioning of 4-AP-sensitive potassium channels is a prerequisite for this 'antiepileptic' adenosine action. Since a similar pharmacological characteristic has been described for the A-current, it is likely that adenosine acts by turning on this particular potassium current.
Neuromodulation by adenosine is achieved by an ensemble of different effects including a depression of synaptic transmission which can be distinguished from a non-synaptic influence on nerve cell firing (see refs. 5 and 6). The finding of an adenosine-induced membrane hyperpolarization seen in conjunction with a reduction of the input resistance, provided an early indication that adenosine increases membrane potassium conductances [9, 11]. But there are several potassium currents which can be distinguished on the basis of their kinetics and pharmacological characteristics, and the possibility that adenosine exerts its multiple effects by a selective modulation of individual ion channels should be considered. In the present paper we have attempted to obtain some information about the ionic mechanism of the non-synaptic action of adenosine by which the pattern of evoked firing is modulated and thus the tendency to generate burst discharges is reduced. There is recent evidence that tetraethylammonium (TEA) and 4-aminopyridine (4AP) can be used as pharmacological tools to differentiate between individual potassium currents [3, 10]. These antagonists are apparently highly selective with respect *Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
335 to their action on the individual potassium channels. Whereas TEA antagonizes the delayed rectifier current and some calcium-dependent potassium currents [1], it is rather ineffective in antagonizing the transient and calcium-independent potassium current, the so-called A-current. But the latter is rather selectively blocked by 4-AP [3, 10]. Taking advantage of such a pharmacological differentiation of potassium fluxes, we tested the efficacy and the relative potency of TEA vs 4-AP in antagonizing the depressive effect of adenosine on the generation of burst discharges, Experiments were performed on 400 lam rat hippocampal slices which were prepared by means of a tissue chopper after decapitation and rapid removal of the hippocampus. After transfer into a chamber, the slices were kept on a nylon net and constantly superfused at 32'C with oxygenated medium containing (in mM): CaCI~ 0.24, MgSO4 4.0, KCI 3.3, NaC1 124, NaHCO3 25.7, KHzPO4 1.25, glucose 10. Within one hour of superfusion a steady state concentration of extracellular free Ca 2' ions of 0.2 mM was reached and synaptic transmission was apparently blocked. Orthodromic stimulation of the Schaffer collateral/commissural fibers elicited a presynaptic fiber volley only and no evoked postsynaptic potentials were extracellularly recorded. Under these conditions hippocampal neurons tend to generate spontaneous burst discharges, a phenomenon which has been used as an experimental model for the non-synaptic generation of epileptiform activity [5, 13, 15]. In order to overcome such spontaneous bursting, the concentration of Mg 2+ ions in the superfused medium was raised to a level at which CAl-neurons did not fire spontaneously, but still responded to antidromic stimulation with repetitive discharges. A single stimulation pulse applied to the alvear fibers elicited an antidromic action potential which was usually followed by 2-4 afterpotentials (Fig. 1). Addition of 0.2-20 ~tM adenosine reduced the amplitude of afterpotentials in a concentration-dependent manner [6]. The degree of sensitivity to adenosine varied to some extent between the different hippocampal preparations. A relatively high adenosine concentration (40 ~tM) was therefore used in most of the present experiments in order to ensure that a strong and consistent depression of afterpotentials was obtained for the subsequent testing of K +-channel antagonists. In each experiment, sensitivity to adenosine was determined followed by a washout period of ca. 20 min. Then 4-AP was added and the effect of adenosine was tested again in the presence of this potassium channel blocker (14 experiments). In addition, the remaining inhibitory action of the adenosine analogue, L-phenylisopropyladenosine (L-PIA) was tested at a concentration of 0.03 ~tM (9 experiments) and 0.3 laM (5 experiments) utilizing the same paradigm as used with adenosine. Antagonism of the adenosine effect by TEA was determined in 6 experiments, preceding the test of 4-AP. The results were quantified by measuring the amplitudes of the first antidromic potential and the first afterpotential by means of an on-line computer. Responses generated at a constant stimulus strength were recorded and measured during the entire timecourse of the experiment. In addition input-output curves were generated by testing a variety of stimulation intensities at certain time points (see Fig. l). tion intensities at certain time points (see Fig. 1). Adenosine led to a marked depression of the afterpotentials without affecting the
336
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Fig. 1. Differential effect of TEA and 4-AP on the adenosine-induced depression of antidromically elicited afterpotentials. A: the amplitudes of antidromic action potentials (upper row of dots) and of the first subsequent afterpotentials (lower row of dots) were continuously measured on-line by a computer during the course of a typical experiment. Each dot represents one measurement at the time point indicated. During the time marked by arrows, 40 laM adenosine was added to the superfused medium. Adenosine had no effect on the antidromic spike, but it depressed the first afterpotential in low Ca medium and also in the presence of 5 mM TEA. The depression by adenosine was eliminated in the presence of 50 IxM 4-AP. B: antidromically evoked responses in the absence (left) and in the presence of 40 BM adenosine (right). Upper traces, in low Ca medium; middle traces, in 5 mM TEA; lower traces, in 0.05 mM 4-AP. Calibration: 1 mV, 2 ms. C: input-output curves (same experiment) of the antidromically elicited first afterpotential. The abscissa gives the voltage of the stimulus pulse, the ordinate gives the amplitude of the evoked response. Squares, in 5 mM TEA; dots, in 0.05 mM 4-AP; open symbols, in the absence of adenosine: filled symbols, in the presence of adenosine.
height o f the initial a n t i d r o m i c spike (Fig. 1). A t the i n d i c a t e d c o n c e n t r a t i o n s , the first a f t e r p o t e n t i a l was r e d u c e d by 70-80% on a v e r a g e a n d the consecutive a f t e r p o tentials were n e a r l y a b o l i s h e d (Table I). L-PIA, the p o t e n t A l - r e c e p t o r a g o n i s t usually d e p r e s s e d the g e n e r a t i o n o f a f t e r p o t e n t i a l s b y m o r e t h a n 80% at a c o n c e n t r a tion o f 0.01-0.03 ~tM. The d e p r e s s i o n o f a f t e r p o t e n t i a l s by a d e n o s i n e a n d L - P I A was n o t a t t e n u a t e d in the presence o f u p to 5 m M T E A (Fig. 1). But in the presence o f o n l y 50 ~tM 4 - A P the depressive effect o f 40 ~tM a d e n o s i n e a n d 0.03 ~tM L - P I A was a l m o s t e l i m i n a t e d (Table I). This a n t a g o n i s m was seen o v e r the w h o l e r a n g e o f the g e n e r a t e d i n p u t o u t p u t curves (Fig. 1). Even when L - P I A was increased to 0.3 ~tM - this is 10 times
,
337 TABLE 1 A M P L I T U D E OF THE FIRST A F T E R P O T E N T I A L 4-AP was tested at a concentration of 50 I~M; its addition to the superfused medium increased the amplitude of the first afterpotential by 51 +34% (S.D.). TEA was tested at concentrations of 2 5 mM (n =6). The presence of TEA did in no case affect the adenosine-induced depression of afterpotentials. Since L-PIA is difficult to wash out, it was tested only in the presence of 4-AP. From other experiments L-PIA is known to block the afterpotentials at a concentration of 0.03 p.M by more than 80% [6]. Following addition of
Adenosine (20 /aM) Adenosine (40 /aM) L-PIA (0.03 laM) L-PIA (0.3 laM)
In low Ca 2+ medium
In low Ca 2+ medium + 4-AP
100%
100%
Depression by (%)
Depression by (%)
70+_12 76+14
10_+8 (S.D.) 2 + 3 (S.D.)
(S.D.) (S.D.)
{0 5-+2 /0 / 6_+4
(S.D.) (S.D.)
(n=7) (n=7) (n =4) (n=5) (n=2) (n=3)
the concentration needed to depress the generation of afterdischarges m the absence of 4-AP only a minimal effect was achieved (Table I). The antagonistic effect of 4-AP on the depression of afterpotentials by adenosine indicates that functioning 4-AP-sensitive potassium channels are a prerequisite for this particular action. Some sensitivity to 4-AP has also been reported for the reduction of spontaneous firing as observed in cortical neurons in the presence of adenosine [8]. On the other hand, there are certainly effects, for example the adenosineinduced changes of the resting membrane properties [9, 11], which are not antagonized by 4-AP [2, 9]. Whereas these effects, i.e. the membrane hyperpolarization and decrease of resistance, have been reported to be voltage-insensitive [2], direct measurements with the voltage-clamp technique revealed that adenosine also affects potassium currents which show a marked voltage-dependency [14]. These data suggest that adenosine acts via different ionic mechanisms which also seems to be the case for the 'antiepileptic' effects. The reported enhanced accommodation of cell firing was seen at the end of a long-lasting (500 ms) depolarizing pulse [4]; it is a slowly developing event and has been explained by a change in the regulation of intracellular calcium [2]. In contrast, the investigated depressive effect of adenosine on the generation of afterpotentials is seen already within 2-30 ms after initiation of the antidromic spike and must be mediated by a mechanism with a rapid onset. The observed pharmacological characteristics of this adenosine effect, i.e. strong antagonism by 4-AP and inefficiency of TEA, resemble those of the A-current. This current is characterized as a transient potassium current with a rapid onset; it is turned on by a slight membrane depolarization, reaches a peak within 15 20 ms and is again rapidly inactivated [3, 10]. Thus, the A-current reduces the probability of consecutive cell firing as soon as a first action potential has been generated. Turn-
338 ing o n the A - c u r r e n t by adenosine, as suggested by the data, m a y therefore provide a powerful m e c h a n i s m to prevent a priori the i n i t i a t i o n o f burst discharges a n d the d e v e l o p m e n t o f epileptic activity. There is recent evidence that v o l t a g e - d e p e n d e n t calcium currents are also m o d u lated by adenosine. Specifically, a depression o f c a d m i u m - s e n s i t i v e currents has been d e m o n s t r a t e d which went a l o n g with a decrease o f m e m b r a n e c o n d u c t a n c e a n d hence is a p p a r e n t l y n o t reflecting a m o d u l a t i o n , i.e. a n increase o f p o t a s s i u m currents [7]. This depression o f calcium fluxes seems to be rather selective c o n c e r n i n g the site of action: the spike-induced presynaptic influx o f calcium ions into the a x o n terminal was f o u n d to be particularly sensitive a n d the power o f this a d e n o s i n e effect, which m a y largely c o n t r o l t r a n s m i t t e r release, was even e n h a n c e d in the presence of 4-AP [12]. In view o f these findings, a d e n o s i n e should be regarded as a rather u n i q u e neur o m o d u l a t o r which is capable o f m o d u l a t i n g different ion currents a n d which influences n e u r o n a l p o t a s s i u m as well as calcium fluxes. The study was performed in c o o p e r a t i o n with R e g i n a K o l b p r o v i d i n g skilful technical help. 1 Brown, D.A. and Griffith, W.H., Calcium-activated outward current in voltage-clamped hippocampal neurons of the guinea pig, J. Physiol. (London), 237 (1983) 287-301. 2 Greene, R.W. and Haas, H.L., Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices, J, Physiol. (London), 366 (1985) 119-127. 3 Gustafsson, B., Galvan, M., Grafe, P. and Wigstroem, H., A transient outward current in a mammalian central neurone blocked by 4-aminopyridine, Nature (London), 199 (1982) 252-254. 4 Haas, H.L. and Greene, R.W., Adenosine enhances afterhyperpolarization and accommodation in hippocampal pyramidal cells, Pfluegers Arch., 402 (1984) 244-247. 5 Haas, H.L., Jefferys, J.G.R., Slater, N.T. and Carpenter, D.O., Modulation of low calcium induced field bursts in the hippocampus by monoamines and cholinomimetics, Pfliigers Arch., 400 (1984) 2833. 6 Lee, K.S., Schubert, P. and Heinemann, U., The anticonvulsive action of adenosine: a postsynaptic dendritic action by a possible endogenous anticonvulsant, Brain Res., 321 (1984) 160-164. 7 MacDonald, R.L., Skerritt, J.H. and Werz, M,A., Adenosine agonists reduce voltage-dependent calcium conductance of mouse sensory neurones in cell culture, J. Physiol. (London), 370 (1986) 75-90. 8 Perkins, M.N. and Stone, T.W., 4-aminopyridine blockade of neuronal depressant responses to adenosine triphosphate, Br. J. Pharmacol., 70 (1980) 425-428. 9 Segal, M., Intracellular analysis of a postsynaptic action of adenosine in the rat hippocampus, Eur. J. Pharmacol., 79 (1982) 193-199. 10 Segal, M., Rogawski, M.A. and Barker, J.L., A transient potassium conductance regulates the excitability of cultured hippocampal and spinal neurons, J. Neurosci., 4 (1984) 604-609. 11 Siggins,G.R. and Schubert, P., Adenosine depression of hippocampal neurons in vitro: an intracellular study of dose-dependent actions on synaptic and membrane potentials. Neurosci. Lett.. 23 (1981) 55-60. 12 Schubert, P., Heinemann, U. and Kolb, R., Differential effect of adenosine on pre- and postsynaptic calcium fluxes, Brain Res., in press. 13 Taylor, C.P. and Dudek, F.E., Synchronous neural afterdischarges in rat hippocampal slices without active chemical synapses, Science,218 (1982) 810--812. 14 Trusset, L.O. and Meyer, B.J., Adenosine-activated potassium conductance in cultured striatal neurons, Proc. Natl. Acad. Sci. USA, 82 (1985) 4857-4861. 15 Yaari, Y., Konnerth, A. and Heinemann, U., Spontaneous epileptiform activity of CA1 hippocampal neurons in tow extracellular calcium solutions, Exp. Brain Res., 51 (1983) 151-156.
334
NSL 04000
NON-SYNAPTIC MODULATION OF REPETITIVE FIRING BY ADENOSINE IS ANTAGONIZED BY 4-AMINOPYRIDINE IN A RAT HIPPOCAMPAL SLICE
PETER SCHUBERT* and KEVIN S. LEE
Max Planck Institute for Psychiatry, Department of Neuromorphology, am Klopferspitz 18a. D-8033 Martinsried (F. R.G. ) (Received January 10th, 1986; Revised version received and accepted March 24th, 1986)
Key words: adenosine - modulation - 4-aminopyridine - potassium conductance - A-current - epileptic -
hippocampal slice - rat
In rat hippocampal slices which were superfused with low calcium (0.2 mM) medium, stimulation of the alvear fibers elicited an extracellularly recorded antidromic population spike in CAI pyramidal neurons which was followed by 2-4 afterpotentials. Adenosine (20-40 laM) and the Al-adenosine agonist lphenylisopropyladenosine (L-PIA) blocked these afterpotentials without affecting the first spike. Addition of up to 5 m M tetraethylammonium to the superfused medium did not interfere with this adenosine action. But the addition of only 50 jaM 4-aminopyridine (4-AP) antagonized almost completely the adenosineor L-PIA-induced depression of antidromically evoked repetitive firing. It is concluded that functioning of 4-AP-sensitive potassium channels is a prerequisite for this 'antiepileptic' adenosine action. Since a similar pharmacological characteristic has been described for the A-current, it is likely that adenosine acts by turning on this particular potassium current.
Neuromodulation by adenosine is achieved by an ensemble of different effects including a depression of synaptic transmission which can be distinguished from a non-synaptic influence on nerve cell firing (see refs. 5 and 6). The finding of an adenosine-induced membrane hyperpolarization seen in conjunction with a reduction of the input resistance, provided an early indication that adenosine increases membrane potassium conductances [9, 11]. But there are several potassium currents which can be distinguished on the basis of their kinetics and pharmacological characteristics, and the possibility that adenosine exerts its multiple effects by a selective modulation of individual ion channels should be considered. In the present paper we have attempted to obtain some information about the ionic mechanism of the non-synaptic action of adenosine by which the pattern of evoked firing is modulated and thus the tendency to generate burst discharges is reduced. There is recent evidence that tetraethylammonium (TEA) and 4-aminopyridine (4AP) can be used as pharmacological tools to differentiate between individual potassium currents [3, 10]. These antagonists are apparently highly selective with respect *Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
335 to their action on the individual potassium channels. Whereas TEA antagonizes the delayed rectifier current and some calcium-dependent potassium currents [1], it is rather ineffective in antagonizing the transient and calcium-independent potassium current, the so-called A-current. But the latter is rather selectively blocked by 4-AP [3, 10]. Taking advantage of such a pharmacological differentiation of potassium fluxes, we tested the efficacy and the relative potency of TEA vs 4-AP in antagonizing the depressive effect of adenosine on the generation of burst discharges, Experiments were performed on 400 lam rat hippocampal slices which were prepared by means of a tissue chopper after decapitation and rapid removal of the hippocampus. After transfer into a chamber, the slices were kept on a nylon net and constantly superfused at 32'C with oxygenated medium containing (in mM): CaCI~ 0.24, MgSO4 4.0, KCI 3.3, NaC1 124, NaHCO3 25.7, KHzPO4 1.25, glucose 10. Within one hour of superfusion a steady state concentration of extracellular free Ca 2' ions of 0.2 mM was reached and synaptic transmission was apparently blocked. Orthodromic stimulation of the Schaffer collateral/commissural fibers elicited a presynaptic fiber volley only and no evoked postsynaptic potentials were extracellularly recorded. Under these conditions hippocampal neurons tend to generate spontaneous burst discharges, a phenomenon which has been used as an experimental model for the non-synaptic generation of epileptiform activity [5, 13, 15]. In order to overcome such spontaneous bursting, the concentration of Mg 2+ ions in the superfused medium was raised to a level at which CAl-neurons did not fire spontaneously, but still responded to antidromic stimulation with repetitive discharges. A single stimulation pulse applied to the alvear fibers elicited an antidromic action potential which was usually followed by 2-4 afterpotentials (Fig. 1). Addition of 0.2-20 ~tM adenosine reduced the amplitude of afterpotentials in a concentration-dependent manner [6]. The degree of sensitivity to adenosine varied to some extent between the different hippocampal preparations. A relatively high adenosine concentration (40 ~tM) was therefore used in most of the present experiments in order to ensure that a strong and consistent depression of afterpotentials was obtained for the subsequent testing of K +-channel antagonists. In each experiment, sensitivity to adenosine was determined followed by a washout period of ca. 20 min. Then 4-AP was added and the effect of adenosine was tested again in the presence of this potassium channel blocker (14 experiments). In addition, the remaining inhibitory action of the adenosine analogue, L-phenylisopropyladenosine (L-PIA) was tested at a concentration of 0.03 ~tM (9 experiments) and 0.3 laM (5 experiments) utilizing the same paradigm as used with adenosine. Antagonism of the adenosine effect by TEA was determined in 6 experiments, preceding the test of 4-AP. The results were quantified by measuring the amplitudes of the first antidromic potential and the first afterpotential by means of an on-line computer. Responses generated at a constant stimulus strength were recorded and measured during the entire timecourse of the experiment. In addition input-output curves were generated by testing a variety of stimulation intensities at certain time points (see Fig. l). tion intensities at certain time points (see Fig. 1). Adenosine led to a marked depression of the afterpotentials without affecting the
336
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! m
.... •'7'-...
4-AP
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......
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Fig. 1. Differential effect of TEA and 4-AP on the adenosine-induced depression of antidromically elicited afterpotentials. A: the amplitudes of antidromic action potentials (upper row of dots) and of the first subsequent afterpotentials (lower row of dots) were continuously measured on-line by a computer during the course of a typical experiment. Each dot represents one measurement at the time point indicated. During the time marked by arrows, 40 laM adenosine was added to the superfused medium. Adenosine had no effect on the antidromic spike, but it depressed the first afterpotential in low Ca medium and also in the presence of 5 mM TEA. The depression by adenosine was eliminated in the presence of 50 IxM 4-AP. B: antidromically evoked responses in the absence (left) and in the presence of 40 BM adenosine (right). Upper traces, in low Ca medium; middle traces, in 5 mM TEA; lower traces, in 0.05 mM 4-AP. Calibration: 1 mV, 2 ms. C: input-output curves (same experiment) of the antidromically elicited first afterpotential. The abscissa gives the voltage of the stimulus pulse, the ordinate gives the amplitude of the evoked response. Squares, in 5 mM TEA; dots, in 0.05 mM 4-AP; open symbols, in the absence of adenosine: filled symbols, in the presence of adenosine.
height o f the initial a n t i d r o m i c spike (Fig. 1). A t the i n d i c a t e d c o n c e n t r a t i o n s , the first a f t e r p o t e n t i a l was r e d u c e d by 70-80% on a v e r a g e a n d the consecutive a f t e r p o tentials were n e a r l y a b o l i s h e d (Table I). L-PIA, the p o t e n t A l - r e c e p t o r a g o n i s t usually d e p r e s s e d the g e n e r a t i o n o f a f t e r p o t e n t i a l s b y m o r e t h a n 80% at a c o n c e n t r a tion o f 0.01-0.03 ~tM. The d e p r e s s i o n o f a f t e r p o t e n t i a l s by a d e n o s i n e a n d L - P I A was n o t a t t e n u a t e d in the presence o f u p to 5 m M T E A (Fig. 1). But in the presence o f o n l y 50 ~tM 4 - A P the depressive effect o f 40 ~tM a d e n o s i n e a n d 0.03 ~tM L - P I A was a l m o s t e l i m i n a t e d (Table I). This a n t a g o n i s m was seen o v e r the w h o l e r a n g e o f the g e n e r a t e d i n p u t o u t p u t curves (Fig. 1). Even when L - P I A was increased to 0.3 ~tM - this is 10 times
,
337 TABLE 1 A M P L I T U D E OF THE FIRST A F T E R P O T E N T I A L 4-AP was tested at a concentration of 50 I~M; its addition to the superfused medium increased the amplitude of the first afterpotential by 51 +34% (S.D.). TEA was tested at concentrations of 2 5 mM (n =6). The presence of TEA did in no case affect the adenosine-induced depression of afterpotentials. Since L-PIA is difficult to wash out, it was tested only in the presence of 4-AP. From other experiments L-PIA is known to block the afterpotentials at a concentration of 0.03 p.M by more than 80% [6]. Following addition of
Adenosine (20 /aM) Adenosine (40 /aM) L-PIA (0.03 laM) L-PIA (0.3 laM)
In low Ca 2+ medium
In low Ca 2+ medium + 4-AP
100%
100%
Depression by (%)
Depression by (%)
70+_12 76+14
10_+8 (S.D.) 2 + 3 (S.D.)
(S.D.) (S.D.)
{0 5-+2 /0 / 6_+4
(S.D.) (S.D.)
(n=7) (n=7) (n =4) (n=5) (n=2) (n=3)
the concentration needed to depress the generation of afterdischarges m the absence of 4-AP only a minimal effect was achieved (Table I). The antagonistic effect of 4-AP on the depression of afterpotentials by adenosine indicates that functioning 4-AP-sensitive potassium channels are a prerequisite for this particular action. Some sensitivity to 4-AP has also been reported for the reduction of spontaneous firing as observed in cortical neurons in the presence of adenosine [8]. On the other hand, there are certainly effects, for example the adenosineinduced changes of the resting membrane properties [9, 11], which are not antagonized by 4-AP [2, 9]. Whereas these effects, i.e. the membrane hyperpolarization and decrease of resistance, have been reported to be voltage-insensitive [2], direct measurements with the voltage-clamp technique revealed that adenosine also affects potassium currents which show a marked voltage-dependency [14]. These data suggest that adenosine acts via different ionic mechanisms which also seems to be the case for the 'antiepileptic' effects. The reported enhanced accommodation of cell firing was seen at the end of a long-lasting (500 ms) depolarizing pulse [4]; it is a slowly developing event and has been explained by a change in the regulation of intracellular calcium [2]. In contrast, the investigated depressive effect of adenosine on the generation of afterpotentials is seen already within 2-30 ms after initiation of the antidromic spike and must be mediated by a mechanism with a rapid onset. The observed pharmacological characteristics of this adenosine effect, i.e. strong antagonism by 4-AP and inefficiency of TEA, resemble those of the A-current. This current is characterized as a transient potassium current with a rapid onset; it is turned on by a slight membrane depolarization, reaches a peak within 15 20 ms and is again rapidly inactivated [3, 10]. Thus, the A-current reduces the probability of consecutive cell firing as soon as a first action potential has been generated. Turn-
338 ing o n the A - c u r r e n t by adenosine, as suggested by the data, m a y therefore provide a powerful m e c h a n i s m to prevent a priori the i n i t i a t i o n o f burst discharges a n d the d e v e l o p m e n t o f epileptic activity. There is recent evidence that v o l t a g e - d e p e n d e n t calcium currents are also m o d u lated by adenosine. Specifically, a depression o f c a d m i u m - s e n s i t i v e currents has been d e m o n s t r a t e d which went a l o n g with a decrease o f m e m b r a n e c o n d u c t a n c e a n d hence is a p p a r e n t l y n o t reflecting a m o d u l a t i o n , i.e. a n increase o f p o t a s s i u m currents [7]. This depression o f calcium fluxes seems to be rather selective c o n c e r n i n g the site of action: the spike-induced presynaptic influx o f calcium ions into the a x o n terminal was f o u n d to be particularly sensitive a n d the power o f this a d e n o s i n e effect, which m a y largely c o n t r o l t r a n s m i t t e r release, was even e n h a n c e d in the presence of 4-AP [12]. In view o f these findings, a d e n o s i n e should be regarded as a rather u n i q u e neur o m o d u l a t o r which is capable o f m o d u l a t i n g different ion currents a n d which influences n e u r o n a l p o t a s s i u m as well as calcium fluxes. The study was performed in c o o p e r a t i o n with R e g i n a K o l b p r o v i d i n g skilful technical help. 1 Brown, D.A. and Griffith, W.H., Calcium-activated outward current in voltage-clamped hippocampal neurons of the guinea pig, J. Physiol. (London), 237 (1983) 287-301. 2 Greene, R.W. and Haas, H.L., Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices, J, Physiol. (London), 366 (1985) 119-127. 3 Gustafsson, B., Galvan, M., Grafe, P. and Wigstroem, H., A transient outward current in a mammalian central neurone blocked by 4-aminopyridine, Nature (London), 199 (1982) 252-254. 4 Haas, H.L. and Greene, R.W., Adenosine enhances afterhyperpolarization and accommodation in hippocampal pyramidal cells, Pfluegers Arch., 402 (1984) 244-247. 5 Haas, H.L., Jefferys, J.G.R., Slater, N.T. and Carpenter, D.O., Modulation of low calcium induced field bursts in the hippocampus by monoamines and cholinomimetics, Pfliigers Arch., 400 (1984) 2833. 6 Lee, K.S., Schubert, P. and Heinemann, U., The anticonvulsive action of adenosine: a postsynaptic dendritic action by a possible endogenous anticonvulsant, Brain Res., 321 (1984) 160-164. 7 MacDonald, R.L., Skerritt, J.H. and Werz, M,A., Adenosine agonists reduce voltage-dependent calcium conductance of mouse sensory neurones in cell culture, J. Physiol. (London), 370 (1986) 75-90. 8 Perkins, M.N. and Stone, T.W., 4-aminopyridine blockade of neuronal depressant responses to adenosine triphosphate, Br. J. Pharmacol., 70 (1980) 425-428. 9 Segal, M., Intracellular analysis of a postsynaptic action of adenosine in the rat hippocampus, Eur. J. Pharmacol., 79 (1982) 193-199. 10 Segal, M., Rogawski, M.A. and Barker, J.L., A transient potassium conductance regulates the excitability of cultured hippocampal and spinal neurons, J. Neurosci., 4 (1984) 604-609. 11 Siggins,G.R. and Schubert, P., Adenosine depression of hippocampal neurons in vitro: an intracellular study of dose-dependent actions on synaptic and membrane potentials. Neurosci. Lett.. 23 (1981) 55-60. 12 Schubert, P., Heinemann, U. and Kolb, R., Differential effect of adenosine on pre- and postsynaptic calcium fluxes, Brain Res., in press. 13 Taylor, C.P. and Dudek, F.E., Synchronous neural afterdischarges in rat hippocampal slices without active chemical synapses, Science,218 (1982) 810--812. 14 Trusset, L.O. and Meyer, B.J., Adenosine-activated potassium conductance in cultured striatal neurons, Proc. Natl. Acad. Sci. USA, 82 (1985) 4857-4861. 15 Yaari, Y., Konnerth, A. and Heinemann, U., Spontaneous epileptiform activity of CA1 hippocampal neurons in tow extracellular calcium solutions, Exp. Brain Res., 51 (1983) 151-156.