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Excitatory and inhibitory effects of adenosine on the neurotransmission in the hippocampal slices of guinea pig
Brain Research, 525 (1990) 165-169 Elsevier
165
BRES 24226
Excitatory and inhibitory effects of adenosine on the neurotransmission in the hippocampal slices of guinea pig Shunji Nishimura, Mitsuhiro Mohri, Yasuhiro Okada and Masahiro Mori Department of Physiology, School of Medicine, Kobe University, Kobe (Japan)
(Accepted 1 May 1990) Key words: Hippocampalslice; Neurotransmission;Postsynapticpotential; Adenosine; Adenosine triphosphate; Excitation; Inhibition
Postsynaptic potentials (PSPs) were recorded from the granular cell layer of guinea pig hippocampal slices. Application of adenosine at a concentration of more than 10/~M in the incubation medium depressed the amplitude of the PSP, but adenosine at a concentration of 10 nM to 1/~M enhanced the amplitude of the PSP. The dose-response curve of the effect of adenosine showed an excitatory and inhibitory biphasic pattern. Adenosine analogues such as adenosine triphosphate (ATP), adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD) showed similar biphasic effects, howeverfl-y-methyleneadenosinetriphosphate(fl-y-ATP),5"-N-ethylcarboxamidoadenosine(NECA), N~-cyclohexyladenosine(CHA) and N~-R-phenylisopropyladenosine(RPIA) revealed only inhibitory effects. Adenosine has been considered as an inhibitory neurotransmitter in the gut I and neuromodulator in the CNS on synaptic transmission 14. Using tissue slices incubated in a bath, Okada et al. observed distinct inhibitory effects with adenosine and its derivatives on the neurotransmission of hippocampal and olfactory cortex slices 1~-13. Phillis et al. reported that microiontophoretic application of adenosine showed potent inhibitory action on the transmission in the cortical neurons ~5. The inhibitory action of adenosine has been attributed to be mediated through the inhibition of transmitter release at the presynaptic site or through changes of the potassium conductance in the postsynaptic cell4'6. On the other hand, the excitatory effect of adenosine triphosphate (ATP), an adenine nucleotide, has been reported in cuneate neurons in vivo and in spinal ganglion cells in vitro 5'9. In this paper, we are reporting concentrationdependent excitatory and inhibitory biphasic effects of adenosine on neurotransmission in hippocampal slices. Guinea pigs weighing 250-300 g were used. Hippocampal slices were prepared and preincubated for at least 20 rain in standard medium (in mM): NaCI 125, NaHCO 3 26, KC1 5, CaCI2 2, KH2PO 4 1.2, MgSO 4 1.3, glucose 10. The details of the techniques used to prepare slices have been reported elsewhere 13. The slices were placed in the observation chamber and fully submerged in the standard medium continuously perfused (5 ml/min). The temperature was kept at 36 °C throughout the experiment. Under a stereomicroscope, the perforant path was stimulated electrically by means of a pair of silver wire
electrodes, and the postsynaptic field potential (PSP; population spike) was recorded in the granular cell layer of the dentate gyrus with a glass microelectrode. The stimulation was given every 2 s (square pulse 0.1 ms in duration) and the strength of electrical stimulation was adjusted to obtain the PSP at half of the maximum amplitude elicited by supramaximal stimulation. A stable PSP was recorded at least 30 min before the addition of adenosine and its analogues to the medium. Drugs were applied to the perfusion medium in the circulatory unit, and removed by washing the slices. Only one slice was used for one trial to test the effect of adenosine at single concentration applied. Adenosine, ATP, adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NAD), fl-y-methyleneadenosinetriphosphate (fl-y-ATP) and N6-R-phenylisopropyladenosine (RPIA) were purchased from Sigma. 5"-N-Ethylcarboxamidoadenosine (NECA) was purchased from Research Biochemicals Inc. N~-Cyclohexyladenosine (CHA) was purchased from Kohjin Co. Other chemicals were obtained from Nakarai, Kyoto. A typical PSP recorded from the granular cell layer is shown in Fig. 1A. After obtaining the supramaximal response of the PSP, the amplitude of the PSP was adjusted to half of the maximum amplitude (Fig. 1A-l). The amplitude of PSP increased gradually after the application of adenosine at a concentration of 0.5/~M (Fig. 1A-2), and reached a plateau after 15 min (Fig. 1A-3). After washing the slices, the PSP returned to near the original level within 15 min (Fig. 1A-4). In addition to the
Correspondence: Y. Okada, Department of Physiology,School of Medicine, Kobe University, 7-5-1 Kusunoki-cho,Chuoku, Kobe, Japan.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166
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Adenosine
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.
.
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Fig. 1. Excitatory and inhibitory actions of adenosine on the neurotransmission in the granular cell layer of hippocampal slice. In the inset figures in A, A-1 shows the population spike (PSP) before application of adenosine; A-2 shows the PSP 5 min after application of adenosine at a concentration of 0.5/~M; A-3 shows the PSP 15 min after application of adenosine; A-4 shows the PSP 15 min after the removal of adenosine. In the trace of the PSP, upper deflection indicates positivity. Calibrations are indicated in the fight bottom. Bottom figure shows the time course of excitatory actions of adenosine. The amplitude of the PSP was measured from the peak of the negativity to that of positivity. A stable amplitude of the PSP observed at least for 30 min and the amplitude just before the application of drugs was taken as 100% and the effect of drugs was indicated as the percentages of this original amplitude. In the inset figures in B, B-1 shows the PSP before the application of adenosine; B-2 shows the PSP 5 min after application of adenosine at a concentration of 100/~M; B-3 shows that 15 rain after the removal of adenosine. Bottom figure shows the time course of the inhibitory action of adenosine. At the point of the arrow, adenosine was applied to the medium or removed from the medium.
increase in the amplitude, the p e a k latency of the PSP was slightly s h o r t e n e d after application of adenosine. The a m p l i t u d e o f the PSP could be m a i n t a i n e d at the plateau level if the slice was not washed. In 4 of 102 cases the a m p l i t u d e was increased gradually for 2 h even after the r e m o v a l of adenosine. A n increase in amplitude of the PSP by a d e n o s i n e was not always observed, especially
when the slices had been preincubated for a long time or the amplitude of the PSP was small. However, application of adenosine at a concentration of 0.5/~M usually increased the amplitude of the PSP by an average of 25%. A p p l i c a t i o n of a d e n o s i n e at a concentration g r e a t e r than 10 p M , d e p r e s s e d the a m p l i t u d e as has b e e n previously r e p o r t e d 11-]3. Fig. 1B shows the time course
167
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100
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0 0 -s
,
, -r 10
'
,
,
, -6 10
,
,
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,
~-
**
7 -4 10
Adenosine(M) Fig. 2. The dose-response curve of the excitatory and inhibitory biphasic actions of adenosine. The ordinate shows the change of amplitude. The amplitude of PSP before application of adenosine was taken as 100% and the changes of amplitude of PSP after application of adenosine are indicated by percentage. The abscissa shows the concentrations of adenosine on a logarithmic scale. Each plot shows the mean of the results obtained from 7-16 trials (slices). Only one slice was used for one trial to test the effect of adenosine at one single concentration applied. The vertical bars indicate the S.E. of the mean. Asteriks indicate that the differences between the original amplitude and the amplitude after addition of adenosine are statistically significant (two-tailed t-test, *P < 0.01; * * P < 0.05).
of inhibitory effect of adenosine on the PSP at a concentration of 100/~M. The dose-response curve of adenosine on neurotransmission is shown in Fig. 2. When the PSP was evoked by supramaximal stimulation to obtain the maximum amplitude, the excitatory effect of adenosine was not observed, and only an inhibitory effect was observed. The adenine nucleotides and adenosine
TABLE I
Excitatory and inhibitory effects of adenosine and its analogues on PSP in hippocampal slicesof guineapig Table also shows the range of concentrations of the compounds necessary to produce excitation or inhibition. + shows the presence of excitation or inhibition. To test the effect and dose-response of adenosine on neurotransmission, 102 slices were used as shown in Fig. 2. In other drugs, 7-10 slices were used, respectively, to determine the effect of the drug.
Compound Excitation
Adenosine ATP ADP NAD fl-r-ATP CHA RPIA NECA
Inhibition
Presence Effective concentration range
Presence Effective concentration range
+ + + + -
+ +
lOnMto 1/~M
+ + + +
>IO/~M
O.lnMtolOOnM
+
>O.1/~M
+
derivatives such as ATP, ADP and N A D showed both excitatory and inhibitory biphasic effects in the doseresponse curve as indicated in Fig. 2. Adenosine analogues such as NECA, RPIA and CHA, A 1 and A 2 receptor agonists at concentrations of more than 100 nM showed only inhibitory effects and at lower doses showed no excitatory effects, fl-y-ATP which cannot be hydrolysed as ATP had no excitatory effect at the concentration of 0.1-1 MM and showed only inhibitory effect at the concentration of 10-100 MM. These results are summarized in Table I. The inhibitory actions of adenosine in the CNS have been described in many reports 11-15. In our previous reports, adenosine at concentrations over 100 MM inhibited the PSP but at lower concentrations it did not show any excitatory actions n-13. This discrepancy can be explained by the difference in stimulation strength used to evoke PSP. In the previous preparations, the stimulation strength used to evoke PSP was fixed to obtain maximum amplitude before application of adenosine. The dose-response curves for the inhibitory action agreed well with the results obtained in the present experiments. Adenosine at lower concentrations showed no excitatory effects because the amplitude of the PSP was already maximally saturated. In the present experiments, however, the strength of the stimulation was adjusted to obtain half of the maximum amplitude in each slice. This allowed excitatory actions of adenosine to
168 be observed. In the iontophoretic application of adenosine and adenosine derivatives, Phillis et al. reported only inhibitory action in the cerebral cortical neurons. However, in the case of iontophoretic application the concentrations cannot be determined accurately 14'~5. In this experiment (Fig. 1B), the inhibitory action appeared immediately after the application of adenosine as has been observed in the olfactory cortex, hippocampus and cerebral cortex 11-13. The mechanism of the inhibitory action of adenosine has been attributed to the inhibition of transmitter release by way of depression of calcium influx ~2 or partly by increasing the potassium conductance in the postsynaptic cell 4. Concerning the dose-response curve, the concentration range for excitation and inhibition is similar to that for the effect of adenosine on adenylate cyclase activity by which adenosine receptors are classified into A~ and A 2 receptors ~6. Neither C H A and RPIA, A 1 receptorselective agonists, nor NECA, considered an A 2 receptor agonist 3, showed excitatory effects in this experiment. Application of adenosine at a concentration of 100 p M actually increased the level of cyclic AMP in the tissue slices, but the inhibitory effects of neurotransmission were not well correlated with the increase of cAMP ~1. Thus, the excitatory and inhibitory effects may not be mediated through A~ nor A 2 receptor which was identified by the effect of adenosine on the adenylate cyclase activity. However, Dunwiddie et al. reported that the inhibition by adenosine is mediated by A1 receptor 4. The excitatory effect of adenine nucleotides such as ATP have been demonstrated in the spinal cord 7, in cuneate neurons 5 and in dorsal horn neurons 9. However, the mechanisms of excitation have been unclear, a chelating effect of ATP or transmitter action of ATP has been proposed 5. It is interesting to note that the mode of excitatory action of adenosine on neurotransmission reported here is different from that reported above 5'9, in
which excitation appeared immediately after the addition of ATP, suggesting that the action may be receptor dependent. However, in the present experiment the effect of excitation by adenosine, ATP, A D P and N A D appeared gradually and slowly as shown in Fig. 1. Adenosine derivatives such as N E C A , RPIA, and C H A which cannot be metabolized showed only inhibitory effects on neurotransmission. Furthermore, fl-7-ATP which cannot be hydrolyzed and metabolized to adenosine showed no excitatory effect. These findings suggest that adenosine and adenine nucleotides and adenosine analogues may act on the same receptor to produce the inhibition. On the other hand, adenosine and the adenine nucleotides which can be hydrolyzed to adenosine are taken up into the cell to produce the excitation whose mechanism is not clarified yet. The slow time course of increase in the amplitude by adenosine resembles that during formation of long-term potentiation (LTP), which is elicited after tetanic stimulation 2. Increase in the amplitude during LTP is considered to be mediated through a metabolic process involving the protein kinase C (PKC) system 1°. It is not known whether adenosine can activate phosphoinositol diphosphate (PIP2) hydrolysis whose product may stimulate PKC as ATP does 8'17. However, the similarity in the time course of the increase of the PSP indicates that the excitatory action may be mediated by a metabolic process within the cell. We have preliminarily tried intracellular recording of CA3 pyramidal cells during the application of adenosine and observed that adenosine in low doses markedly increased the amplitude of the excitatory postsynaptic potential (EPSP) without altering the membrane potential. This may suggest that adenosine enhances the release of transmitters, although further study should be performed to clarify the mechanism of excitatory action of adenosine and adenine nucleotide on neurotransmission.
1 Burnstock, G., Purinergic nerves, Pharmacol. Rev., 24 (1972) 509-581. 2 Collingridge, G.L. and Bliss, T.V.P., NMDA receptors - - their role in long-term potentiation, Trends Neurosci., 10 (1987) 288-293. 3 Daly, J.W., Kuroda, Y., Phillis, J.W., Shimizu, H. and Ui, M., Physiology and Pharmacology of Adenosine Derivatives, Raven Press, New York, 1983. 4 Dunwiddie, T.V., The physiological role of adenosine in the central nervous system, Int. Rev. Neurobiol., 27 (1985) 63-139. 5 Galindo, A., Krnjevic, K. and Schwartz, S., Microiontophoretic studies on neurones in the cuneate nucleus, J. Physiol., 192 (1967) 359-377. 6 Gerber, U., Greene, W.R., 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. 7 Holton, F.A. and Holton, P., The capillary dilator substances in dry powders of spinal roots, a possible role of adenosine triphosphate in chemical transmission from nerve endings, J.
Physiol., 126 (1954) 124-140. 8 Huang, H.M. and Sun, G.Y., Effects of ATP on phosphatidylinositol-phospholipase C and inositol 1-phosphate accumulation in rat brain synaptosomes, J. Neurochem., 50 (1988) 366-374. 9 Jahr, C.E. and Jessel, T.M., ATP excites a subpopulation of rat dorsal horn nucleus, Nature, 304 (1983) 730-733. 10 Lovinger, D.M., Wong, Ka L., Murakami, K. and Routtenberg, A., Protein kinase C inhibitors eliminate hippocampal long-term potentiation, Brain Research, 436 (1987) 177-183. 11 Okada, Y. and Saito, M., Inhibitory action of adenosine, 5-HT (serotonine) and GABA (7-aminobutyric acid) on the postsynaptic potential (PSP) of slices from olfactory cortex and superior colliculus in correlation to the level of cyclic AMP, Brain Research, 160 (1979) 368-371. 12 Okada, Y. and Kuroda, Y., Inhibitory action of adenosine and adenosine analogs on neurotransmission in the olfactory cortex slice of guinea pig, structure-activity relationships, Eur. J. Pharmacol., 61 (1980) 137-146. 13 Okada, Y. and Ozawa, S., Inhibitory action of adenosine on
169 synaptic transmission in the hippocampus of the guinea pig in vitro, Eur. J. Pharmacol., 68 (1980) 483-492. 14 Phillis, J.W. and Kostpoulos, G.K., Adenosine as a putative transmitter in the cerebral cortex. Studies with potentiators and antagonists, Life Sci., 17 (1975) 1085-1094. 15 Phillis, J.W., Edstrom, J.P., Kostpoulos, G.K. and Kirkpatrick, J.R., Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex, Can. J. Physiol. Pharmacol.,
57 (1979) 1289-1312. 16 Van Calker, D., MOiler, M. and Hamprecht, B., Adenosine regulates via two different types of receptors, the accumulation of cyclic AMP in cultured brain cells, J. Neurochem., 33 (1979) 999-1005. 17 Weraszko, A. and Seyfried, T.N., ATP-induced synaptic potentiation in hippocampal slices, Brain Research, 491 (1989) 356-359.
165
BRES 24226
Excitatory and inhibitory effects of adenosine on the neurotransmission in the hippocampal slices of guinea pig Shunji Nishimura, Mitsuhiro Mohri, Yasuhiro Okada and Masahiro Mori Department of Physiology, School of Medicine, Kobe University, Kobe (Japan)
(Accepted 1 May 1990) Key words: Hippocampalslice; Neurotransmission;Postsynapticpotential; Adenosine; Adenosine triphosphate; Excitation; Inhibition
Postsynaptic potentials (PSPs) were recorded from the granular cell layer of guinea pig hippocampal slices. Application of adenosine at a concentration of more than 10/~M in the incubation medium depressed the amplitude of the PSP, but adenosine at a concentration of 10 nM to 1/~M enhanced the amplitude of the PSP. The dose-response curve of the effect of adenosine showed an excitatory and inhibitory biphasic pattern. Adenosine analogues such as adenosine triphosphate (ATP), adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD) showed similar biphasic effects, howeverfl-y-methyleneadenosinetriphosphate(fl-y-ATP),5"-N-ethylcarboxamidoadenosine(NECA), N~-cyclohexyladenosine(CHA) and N~-R-phenylisopropyladenosine(RPIA) revealed only inhibitory effects. Adenosine has been considered as an inhibitory neurotransmitter in the gut I and neuromodulator in the CNS on synaptic transmission 14. Using tissue slices incubated in a bath, Okada et al. observed distinct inhibitory effects with adenosine and its derivatives on the neurotransmission of hippocampal and olfactory cortex slices 1~-13. Phillis et al. reported that microiontophoretic application of adenosine showed potent inhibitory action on the transmission in the cortical neurons ~5. The inhibitory action of adenosine has been attributed to be mediated through the inhibition of transmitter release at the presynaptic site or through changes of the potassium conductance in the postsynaptic cell4'6. On the other hand, the excitatory effect of adenosine triphosphate (ATP), an adenine nucleotide, has been reported in cuneate neurons in vivo and in spinal ganglion cells in vitro 5'9. In this paper, we are reporting concentrationdependent excitatory and inhibitory biphasic effects of adenosine on neurotransmission in hippocampal slices. Guinea pigs weighing 250-300 g were used. Hippocampal slices were prepared and preincubated for at least 20 rain in standard medium (in mM): NaCI 125, NaHCO 3 26, KC1 5, CaCI2 2, KH2PO 4 1.2, MgSO 4 1.3, glucose 10. The details of the techniques used to prepare slices have been reported elsewhere 13. The slices were placed in the observation chamber and fully submerged in the standard medium continuously perfused (5 ml/min). The temperature was kept at 36 °C throughout the experiment. Under a stereomicroscope, the perforant path was stimulated electrically by means of a pair of silver wire
electrodes, and the postsynaptic field potential (PSP; population spike) was recorded in the granular cell layer of the dentate gyrus with a glass microelectrode. The stimulation was given every 2 s (square pulse 0.1 ms in duration) and the strength of electrical stimulation was adjusted to obtain the PSP at half of the maximum amplitude elicited by supramaximal stimulation. A stable PSP was recorded at least 30 min before the addition of adenosine and its analogues to the medium. Drugs were applied to the perfusion medium in the circulatory unit, and removed by washing the slices. Only one slice was used for one trial to test the effect of adenosine at single concentration applied. Adenosine, ATP, adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NAD), fl-y-methyleneadenosinetriphosphate (fl-y-ATP) and N6-R-phenylisopropyladenosine (RPIA) were purchased from Sigma. 5"-N-Ethylcarboxamidoadenosine (NECA) was purchased from Research Biochemicals Inc. N~-Cyclohexyladenosine (CHA) was purchased from Kohjin Co. Other chemicals were obtained from Nakarai, Kyoto. A typical PSP recorded from the granular cell layer is shown in Fig. 1A. After obtaining the supramaximal response of the PSP, the amplitude of the PSP was adjusted to half of the maximum amplitude (Fig. 1A-l). The amplitude of PSP increased gradually after the application of adenosine at a concentration of 0.5/~M (Fig. 1A-2), and reached a plateau after 15 min (Fig. 1A-3). After washing the slices, the PSP returned to near the original level within 15 min (Fig. 1A-4). In addition to the
Correspondence: Y. Okada, Department of Physiology,School of Medicine, Kobe University, 7-5-1 Kusunoki-cho,Chuoku, Kobe, Japan.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166
A
..•5m=ecImV 200.
Adenosine
%
Wash
co o.. 100" E ,<
-1o
1'o
-'5
,'s
2'0
2's
3'0
Time (min)
5mlll¢ 200%
Adenosines,
¢.0 ¢L
ish
IO0E
--15 . . --10 . .
.
.
.
0
10 "
- - '
20 3'0 Time (rain)
;o
0
6'0
Fig. 1. Excitatory and inhibitory actions of adenosine on the neurotransmission in the granular cell layer of hippocampal slice. In the inset figures in A, A-1 shows the population spike (PSP) before application of adenosine; A-2 shows the PSP 5 min after application of adenosine at a concentration of 0.5/~M; A-3 shows the PSP 15 min after application of adenosine; A-4 shows the PSP 15 min after the removal of adenosine. In the trace of the PSP, upper deflection indicates positivity. Calibrations are indicated in the fight bottom. Bottom figure shows the time course of excitatory actions of adenosine. The amplitude of the PSP was measured from the peak of the negativity to that of positivity. A stable amplitude of the PSP observed at least for 30 min and the amplitude just before the application of drugs was taken as 100% and the effect of drugs was indicated as the percentages of this original amplitude. In the inset figures in B, B-1 shows the PSP before the application of adenosine; B-2 shows the PSP 5 min after application of adenosine at a concentration of 100/~M; B-3 shows that 15 rain after the removal of adenosine. Bottom figure shows the time course of the inhibitory action of adenosine. At the point of the arrow, adenosine was applied to the medium or removed from the medium.
increase in the amplitude, the p e a k latency of the PSP was slightly s h o r t e n e d after application of adenosine. The a m p l i t u d e o f the PSP could be m a i n t a i n e d at the plateau level if the slice was not washed. In 4 of 102 cases the a m p l i t u d e was increased gradually for 2 h even after the r e m o v a l of adenosine. A n increase in amplitude of the PSP by a d e n o s i n e was not always observed, especially
when the slices had been preincubated for a long time or the amplitude of the PSP was small. However, application of adenosine at a concentration of 0.5/~M usually increased the amplitude of the PSP by an average of 25%. A p p l i c a t i o n of a d e n o s i n e at a concentration g r e a t e r than 10 p M , d e p r e s s e d the a m p l i t u d e as has b e e n previously r e p o r t e d 11-]3. Fig. 1B shows the time course
167
150"
100
*
,
r~
E <
50-
.:#
0 0 -s
,
, -r 10
'
,
,
, -6 10
,
,
i
1 0-s
,
~-
**
7 -4 10
Adenosine(M) Fig. 2. The dose-response curve of the excitatory and inhibitory biphasic actions of adenosine. The ordinate shows the change of amplitude. The amplitude of PSP before application of adenosine was taken as 100% and the changes of amplitude of PSP after application of adenosine are indicated by percentage. The abscissa shows the concentrations of adenosine on a logarithmic scale. Each plot shows the mean of the results obtained from 7-16 trials (slices). Only one slice was used for one trial to test the effect of adenosine at one single concentration applied. The vertical bars indicate the S.E. of the mean. Asteriks indicate that the differences between the original amplitude and the amplitude after addition of adenosine are statistically significant (two-tailed t-test, *P < 0.01; * * P < 0.05).
of inhibitory effect of adenosine on the PSP at a concentration of 100/~M. The dose-response curve of adenosine on neurotransmission is shown in Fig. 2. When the PSP was evoked by supramaximal stimulation to obtain the maximum amplitude, the excitatory effect of adenosine was not observed, and only an inhibitory effect was observed. The adenine nucleotides and adenosine
TABLE I
Excitatory and inhibitory effects of adenosine and its analogues on PSP in hippocampal slicesof guineapig Table also shows the range of concentrations of the compounds necessary to produce excitation or inhibition. + shows the presence of excitation or inhibition. To test the effect and dose-response of adenosine on neurotransmission, 102 slices were used as shown in Fig. 2. In other drugs, 7-10 slices were used, respectively, to determine the effect of the drug.
Compound Excitation
Adenosine ATP ADP NAD fl-r-ATP CHA RPIA NECA
Inhibition
Presence Effective concentration range
Presence Effective concentration range
+ + + + -
+ +
lOnMto 1/~M
+ + + +
>IO/~M
O.lnMtolOOnM
+
>O.1/~M
+
derivatives such as ATP, ADP and N A D showed both excitatory and inhibitory biphasic effects in the doseresponse curve as indicated in Fig. 2. Adenosine analogues such as NECA, RPIA and CHA, A 1 and A 2 receptor agonists at concentrations of more than 100 nM showed only inhibitory effects and at lower doses showed no excitatory effects, fl-y-ATP which cannot be hydrolysed as ATP had no excitatory effect at the concentration of 0.1-1 MM and showed only inhibitory effect at the concentration of 10-100 MM. These results are summarized in Table I. The inhibitory actions of adenosine in the CNS have been described in many reports 11-15. In our previous reports, adenosine at concentrations over 100 MM inhibited the PSP but at lower concentrations it did not show any excitatory actions n-13. This discrepancy can be explained by the difference in stimulation strength used to evoke PSP. In the previous preparations, the stimulation strength used to evoke PSP was fixed to obtain maximum amplitude before application of adenosine. The dose-response curves for the inhibitory action agreed well with the results obtained in the present experiments. Adenosine at lower concentrations showed no excitatory effects because the amplitude of the PSP was already maximally saturated. In the present experiments, however, the strength of the stimulation was adjusted to obtain half of the maximum amplitude in each slice. This allowed excitatory actions of adenosine to
168 be observed. In the iontophoretic application of adenosine and adenosine derivatives, Phillis et al. reported only inhibitory action in the cerebral cortical neurons. However, in the case of iontophoretic application the concentrations cannot be determined accurately 14'~5. In this experiment (Fig. 1B), the inhibitory action appeared immediately after the application of adenosine as has been observed in the olfactory cortex, hippocampus and cerebral cortex 11-13. The mechanism of the inhibitory action of adenosine has been attributed to the inhibition of transmitter release by way of depression of calcium influx ~2 or partly by increasing the potassium conductance in the postsynaptic cell 4. Concerning the dose-response curve, the concentration range for excitation and inhibition is similar to that for the effect of adenosine on adenylate cyclase activity by which adenosine receptors are classified into A~ and A 2 receptors ~6. Neither C H A and RPIA, A 1 receptorselective agonists, nor NECA, considered an A 2 receptor agonist 3, showed excitatory effects in this experiment. Application of adenosine at a concentration of 100 p M actually increased the level of cyclic AMP in the tissue slices, but the inhibitory effects of neurotransmission were not well correlated with the increase of cAMP ~1. Thus, the excitatory and inhibitory effects may not be mediated through A~ nor A 2 receptor which was identified by the effect of adenosine on the adenylate cyclase activity. However, Dunwiddie et al. reported that the inhibition by adenosine is mediated by A1 receptor 4. The excitatory effect of adenine nucleotides such as ATP have been demonstrated in the spinal cord 7, in cuneate neurons 5 and in dorsal horn neurons 9. However, the mechanisms of excitation have been unclear, a chelating effect of ATP or transmitter action of ATP has been proposed 5. It is interesting to note that the mode of excitatory action of adenosine on neurotransmission reported here is different from that reported above 5'9, in
which excitation appeared immediately after the addition of ATP, suggesting that the action may be receptor dependent. However, in the present experiment the effect of excitation by adenosine, ATP, A D P and N A D appeared gradually and slowly as shown in Fig. 1. Adenosine derivatives such as N E C A , RPIA, and C H A which cannot be metabolized showed only inhibitory effects on neurotransmission. Furthermore, fl-7-ATP which cannot be hydrolyzed and metabolized to adenosine showed no excitatory effect. These findings suggest that adenosine and adenine nucleotides and adenosine analogues may act on the same receptor to produce the inhibition. On the other hand, adenosine and the adenine nucleotides which can be hydrolyzed to adenosine are taken up into the cell to produce the excitation whose mechanism is not clarified yet. The slow time course of increase in the amplitude by adenosine resembles that during formation of long-term potentiation (LTP), which is elicited after tetanic stimulation 2. Increase in the amplitude during LTP is considered to be mediated through a metabolic process involving the protein kinase C (PKC) system 1°. It is not known whether adenosine can activate phosphoinositol diphosphate (PIP2) hydrolysis whose product may stimulate PKC as ATP does 8'17. However, the similarity in the time course of the increase of the PSP indicates that the excitatory action may be mediated by a metabolic process within the cell. We have preliminarily tried intracellular recording of CA3 pyramidal cells during the application of adenosine and observed that adenosine in low doses markedly increased the amplitude of the excitatory postsynaptic potential (EPSP) without altering the membrane potential. This may suggest that adenosine enhances the release of transmitters, although further study should be performed to clarify the mechanism of excitatory action of adenosine and adenine nucleotide on neurotransmission.
1 Burnstock, G., Purinergic nerves, Pharmacol. Rev., 24 (1972) 509-581. 2 Collingridge, G.L. and Bliss, T.V.P., NMDA receptors - - their role in long-term potentiation, Trends Neurosci., 10 (1987) 288-293. 3 Daly, J.W., Kuroda, Y., Phillis, J.W., Shimizu, H. and Ui, M., Physiology and Pharmacology of Adenosine Derivatives, Raven Press, New York, 1983. 4 Dunwiddie, T.V., The physiological role of adenosine in the central nervous system, Int. Rev. Neurobiol., 27 (1985) 63-139. 5 Galindo, A., Krnjevic, K. and Schwartz, S., Microiontophoretic studies on neurones in the cuneate nucleus, J. Physiol., 192 (1967) 359-377. 6 Gerber, U., Greene, W.R., 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. 7 Holton, F.A. and Holton, P., The capillary dilator substances in dry powders of spinal roots, a possible role of adenosine triphosphate in chemical transmission from nerve endings, J.
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