Endogenous adenosine modulates long-term potentiation in the hippocampus

hhroscienee Vol. 62, No. 2, pp. 385-390, 1994 Elscvicr Science Ltd

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ENDOGENOUS ADENOSINE MODULATES LONG-TERM POTENTIATION IN THE HIPPOCAMPUS A. DE MENDON~A*~ and J. A. RIBEIRO# SLaboratory of Pharmacology, Gulbenkian Institute of Science, 2781 Oeiras, Portugal tCentro de Estudos Egas Moniz, Hospital de Santa Maria, Lisboa, Portugal Absfraet-The effect of endogenous adenosine on fr~uency-indeed long-term potentiation of the responses evoked by stimulation of the SchaiTerfibres and recorded in the CA1 area was studied in hippocampal slices of the rat. Long-term potentiation was facilitated in the presence of the selective A, adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (10-20 nM), and was reduced in the presence of the adenosine uptake blocker, nitro~n~l~ioinos~e (SpM), suggesting that endo~no~ adenosine exerted a tonic inhibitory role on long-term potentiation, which was mediated through adenosine A, receptors. We also found that long-term potentiation was increased in the presence of the selective A, receptor agonist, CGS 21680(30 nM), suggesting that the activation of adenosine A, receptors may have excitatory effects on long-term potentiation. We suggest that, endogenous adenosine is able to modulate mechanisms of synaptic plasticity, such as long-term potentiation, in the hippocampus.

Adenosine is considered to have an important role in the modulation of central synaptic transmission and neuronai excitability (see e.g. Ribeiro and SebastiI0). I4 We have shown previously that the stabie adenosine analogue 2-chloroadenosine (CADO) decreases f~quency-endued long-term potentiation (LTP) of the responses evoked by stimulation of the Schaffer fibres and recorded in the CA1 area, in hippocampal slices of the rats7This effect is mediated through the activation of a xanthine-sensitive adenosine receptor’ and may involve modulation of the N-methyl+aspartate receptor-mediated responses.’ The present work was undertaken to test the hypothesis that endogenous adenosine may modulate LTP in the same experimental model, by comparing the magnitudes of LTP induced in the absence and in the presence of the selective A, adenosine receptor antagonist, 1,3-dipropyl-8-cyclo~ntylxanthin~ (DPCPX), the adenosine uptake blocker, nitroben~lthio~nosine (NBI), the enzyme that degrades extracellular adenosine, adenosine deaminase (ADA), the selective A, receptor agonist, 2-p(2-carboxy-ethyl)phenethylamino-S-N-ethylcarboxamidoadenosine (CGS 21680), and the selective A, receptor antagonist, 8-(3-chloros~y~l~affeine (CSC). *To whom wrr~ponden~

should be addressed.

Abbreviations: ADA, adenosine deaminase; CADO, 2-

chloroadenosine; CGS 21680, 2-p(Zcarboxy-ethyl) phenethylamino-5’-N-ethylcarboxamidoadenosine; CSC, 8-(3~hlorosty~l~ffeine; DMSO, dimethylsulphoxide~ DPCPX, 1,3_dipropyI-8fycloptylxanthine; IZPSP, field excitatory postsynaptic potential; LTP, long-term potentiation; NBI, nitrobenzylthioinosine; PS, population spike; T, high-frequency stimulation train.

EXPERiMENTAL PROCEDURES The experiments were performed on hipp~mpal slice preparations taken from Wistar rats weighing 13&15Og. The animals were decapitated and the hippocampi dissected free within an ice-cold (Krebs) solution, previously gassed with a 95% 0, + 5% CO2mixture, of the follo~ng composition (mM): NaCl 124,KC13, NaH,P04 1.25, NaHCO, 26, MgSO, 1, CaCl? 2, glucose 10. Slices 400 orn thick were cut perpendicular to thk long axis of the hippocampus with a Mcllwain tissue chopper, and kept in a chamber within the same gassed medium it room temperature (22-25°C). After 1 h, individual slices were eventualy transferred to the recording submerged chamber of 1ml capacity, where they were continuously superfused at a rate of 3 ml/min with the same gassed solution. Drugswere added to this superfusing soiution. In the experiments performed in the presence of DPCPX, relatively low concentrations (l&20 nM) of DPCPX were used and the CA3 area was usually removed from the slices, in order to minimize epileptiform activity.’ Monopolar stimulation was delivered with the aid of a wire electrode placed in the stratum radiatum @chaffer collateral~~ommissura~ fib@ under ~sillumination of the slice. Rectangular pulses of 0.1 ms duration were applied once every 15s. Evoked responses were extracellularly recorded from CA1 area stratum pyramidale-population soikes (PSs), or stratum radiatu&&eld excit&oiy postsynaptic potentials (fEPSPs), using micropioettes filled with 4M NaCl and of 2-4 Gfi n&tame, &J displayed on a Tektronix digitizing oscilloscope. The averages of eight or 16 consecutive responses were obtained and recorded with a Tektmate computer on magnetic disks, from which thev could be later iransferred to-a pen-recorder. The ampiitud; of the PS was measure@ as the difference between the spike peak negativity and the following positivity of the potential. The fEPSPs were quantified as the slooe of the initial x nhase of the potential. . LTP was induced by applying a high-fauns stimulation train of 100Hz during 100ms (10 pulses),using the sameintensity as that used to obtain the control responses. After the high-frequency stimulation, the pre-train conditions were resumed and the responses recorded for at least 32 min. LTP was quantified as the percentage increase in the

385

386

A. de Mendonca

average amplitude of the PS or in the average initial slope of the fEPSP, taken between the 20th and the 28th minutes after the train, in relation to the average pm-train value. determined during the 8 mm that preceded the application of the train. In preliminary experiments, we observed that variation of LTP between the slices might prevent the achievement of statistical significance when studying the effects of an adenosine receptor antagonist on LTP.’ As LTP may progressively develop following successive trains,’ we chose an experimental protocol in which one high-frequency stimulation train (Tl) was first applied to the slice, eventually ehciting LTP (LTPI). After that, a second highfrequency stimulation train (T2) was applied, agam eventually eliciting LTP (LTPZ). The ratio LTPZ/LTPl was calculated for each slice. We investigated whether the average ratios LTPZ/LTPI were different in the control slices. in which both high-frequency stimulation trains were

and J. A. Riheiro applied in the control bathing solution, and m the test slices. in which either Tl or T2 was applied in the presence of a drug, assuming that eventual differences should rcvcal the effects of the tested drug. For one test slice the corresponding control slice was usually morphologically stmilar and taken from the same hippocampus. The intervals between the trams were adjusted in control slices to match the intervals in the test slices, determined by the times of application or washout of the drugs. The intensmes of the stimuli were adjusted so that the magnitudes of the potentials before the high-frequency stimulation trains were similar in the control slice and in the corresponding test slice. The order of assessing the test slice and the control slice was changed in different expertments. Values are given as the means + S. E. M The significance of the differences between the means was calculated with the Student’s r-test. P values less than 0.05 were considered statistically significant.

B

A

4.5 -

4.5

-DpCPX

Tl

t

4.0 3.5 ( >^

3.0,

g cl

2.5

9

2.0,

4.0 * 3.5 .

b . ..a...

g 1.5.

1.0,

. .

.

9..

C

a

d

- 3.0 . > B 2.5 ’

..*

2.0.

b

C.

.

.,a.

1.0.

0.5 <

d

.

l=.

n.=

. ..=

..

a

C

1.5 *

l.=.=

(10 nM) -

T’ i

.Q

.

.”

..

0.5 *

O_, a

0-p 0 I2

44

Time (min)

b

C

110

140

Time (min)

d

a

b

C

d

I mV

10ms Fig. I. Effect of the selective A, adenosine receptor antagonist, DPCPX (IO nM), on LTP of the PSs. (A) Time course of a typical experiment in a control slice, showing the amplitudes of the averages of 16 consecutive PSs; in this slice, two high-frequency stimulation trains (Tl, T2) were applied in the control bathing solution; in the lower part, the recordings corresponding to the letters in the time course of the experiment are represented; LTP induced by Tl (LTPl) was larger than that induced by T2 (LTPZ). (B) Time course of a typical experiment in a test slice, morphologically similar and taken from the same hippocampus as the control slice. showing the amplitudes of the averages of 16 consecutive PSs; in this slice, Tl was applied in the control bathing solutton; after decreasing the intensity of the stimulus. DPCPX (IO nM) was added to the superfusing control solution, exerting an excitatory effect on the amplitude of the PS; after readjusting the intensity of the stimulus, T2 was applied in the presence of DPCPX (10 nM); in the lower part, the recordings corresponding to the Letters in the time course of the experiment are represented; in this slice, LTP2 was of the same order of magnitude as LTPI.

Endogenous

adenosine modulates

The drugs used were: DPCPX (Research ~i~~~~~~ls Inc.), made up as a 5 mM stock solution in dimethylsulphoxide (DMSO; 99% v/v containing &Of M NaOH); NM (Sigma), made up as a 20 mM stock solution in DMSO; ADA (type VI, Sigma); CGS 21680 (Research Biochemicals Inc.), made up as a IO mM stock solution in DMSO CSC, made up as a 20 mM stock solution in DMSO. The solvents of the drugs were added to the control solutions so that the solvent concentrations were kept constant throughout the experiments. RESULTS

387

LTP

{E = 4) was obtained for the control slices and a value of O,95& 0.19 (8 = 5) for the test slices. This difference is statistically significant (P < 0.05), and represents an excitatory effect of DPCPX (10nM) on LTP2 of the PS in the test slices. Next, we investigated whether the excitatory effects of DPCPX were also observed when studying LTP of the fEPSP, An increase of about 13.5 +6,7% in the slope of the initial phase of the fEPSP obtained under conditions of low-frequency stimulation was observed in the presence of DPCPX @OnM), A value of LTP2,/LTPI of 0.78 kO.18 (n = 5) was obtained for the controf slices and a value of 1.46 & 0.25 (n = 5) for the test slices, in which T2 was applied in the presence of DPCPX (20 nM). This difference is statistically significant (U < 0.05), and represents an excitatory effect of DPCPX (20 nM) on LTP2 of the fEPSP in the test slices.

The time course and recordings of a typical experiment showing the effect of DPCPX (IO nM) on LTP of the PS is depicted in Fig. 1. In the control slice (Fig. lA), after the responses were stable, Tl was applied, eliciting a relatively long-lasting increase in of nitrobenzylthioinosine on long-ttwn the magnitude of the responses, which corresponds to Efict LTPI. After decreasing the intensity of the stimulus potentiutinn The effect of the adenasine uptake blocker, NE1 in order to equalize the pre-train values, T2 was applied, again eliciting a long-lasting increase in the (5 FM), on LTP of the PS is shown in Table 2. A magnitude of the. responses (LTP2). In this slice, decrease of 39.4 +5.4% in the amphtude of the E’S LTPI was larger than L’I’PZ.In the test slice (Fig. obtained under conditions of how-~equency stimuI3)% rno~holo~~l~y similar and taken from the lation was observed in the presence of NBI (5 FM). same hippo~mpus, a similar expe~men~l protocol In control slices (Table 2), LTPI was larger than was carried out, except that TZ was applied in the LTPZ, whereas in the test slices (Table 2) in which Tl presence of DPCPX (IO nM). In this slice, LTP2 was was applied in the presence of NBI (5 PM), LTPl was of the same order of magnitude as LTPl. smaller than LTP2. Calculating the ratio The results obtained in several slices were LTP2/LTPl, a value of 0.81 f 0.21 (n = 5) was found quantified (Table 1). An increase of about 72.4 for the control slices and a value of 1.42 It 0.52 f 17.2% in the amplitude of the PS obtained under (n = 5) for the test slices. This difference is statisticonditions of low-frequency stimulation was ob- cally significant (P < 0.05), and represents an inhibiserved in the presence of DPCPX (IO nM). Calculat- tory effect of NBI (5yM) on LTPl of the PS in the ing the ratio LTPZ/LTPI, I value of 0.65 +O. 19 test slices. Table I. Effect of the selective A, adenosine receptor antagonist, 1,3dipropyl-8-cyclo~n~~ixanthine (IO nM), on long-term ~t~nt~t~on of the population spikes &e-T I (mV) DPCPX (10 nMf Control

1.06+ 0.06 I .osIO.02

Post-T 1 (mV1

Pre-T2 (mv)

Post-T2 (mV)

2.37 f 0.19 2.54 IO.31

I .09+ 0.07

2.35 + 0.19 1.98$.12

1.09 7 0,w

LTP2/LTPl 0.95 + 0. LB* 0.65iO.19

Values are given as the means f S. E. M., and were obtained from five test slices and four control slices. The average amplitudeof the PSsdeterminedduring the 8 min that preceded the application of the train @e-T), and the average amplitude of the PSs taken between the 20th and the 28th minutes after the train (Post-T), are shown for the two high-frequency stimulation trains fT1, T2). LTP was quantified for each slice, and for both trains, as the percentage increase in the Post-T amplitude, in relation to the Pre-T amplitude. Individual LTPZjLTPl ratios were also determined for each slice, and the average LTP2JLTPI ratio is shown. In the test slices, T2 was applied in the presence of DPCPX (IO &I).* Significant difference (P K 0.05) from control. Table 2. Effect

of the adenosine uptake Wcker, potentiation

NBI (5~iM) Control

ni~o~n~lth~o~nos~ne of the population spikes

(5 pM), on bng-term

R&T I (mV)

Post-T1 (mV)

Pre-T2 (mV)

Post-T2 (mV)

I .05 F 0.08 0.99 ;t a.05

1.92* 0.20

I .09f 0.09

2.43 + 0.49

1.01 E 0.05

2.21 ‘ro.15 2.22 T 0.50

LTP2/LTPI 1.42 + 0.52* 0.8t $21

Values are given as the means & S. E. M., and were obtained from five test slices and five control slices. In the test slices, Tl was applied in the presence of NBI (5 PM). Abbreviations and notation are as in Table 1 and in the text.

3X8

A. de Mendonca

and J. A. Rlberro

Table 3. Effect of the enzyme that degrades extracellular adenosine, on long-term potentiation of the population Pre-Tl (mV) ADA (2 U/ml)

Control

0.99 + 0.05 0.99 & 0.04

Post-T (mV)

I

2.75 +0.51 2.69 + 0.44

adenosine spikes

deaminase

Pre-T2 (mV)

Post-T2 (mV)

0.94 * 0.03 0.98 + 0.04

2.00 i 0.26 2.43 & 0.44

(2 U/ml),

LTPZ/LTPl

0.64 * 0.20 0.87 + 0.40

Values are given as the means & S. E. M., and were obtained from five test slices and four control slices. In the test slices, T2 was applied in the presence of ADA (2 U/ml). Abbr~iations are as in Table 1 and in the text.

Table 4. E&t of the selective A, receptor agonist, CGS 21680 (30 nM), on long-term potentiation population Pre-T 1 (mV) CGS 21680 (30 nM) Control

0.97 + 0.03 1.01 f 0.03

Post-T

1

of the

spikes Pre-T2

(mV)

(mV)

Post-T2 (mV)

2.04 + 0.13 2.05 +0.15

1.01 * 0.04 1.03 * 0.04

2.25 * 0.1 I l&&O.14

LTP2/LTPI 1.13 + 0.10* 0.69 * 0.39

given as the means + S. E. M., and were obtained from five test slices and five control slices. in the test slices. T2 was aImlied in the nreSence of CGS 21680 (30 nM). Abbreviations and notation are as in Table t and in the text. L

Values are

1.

The effect of the enzyme that degrades extracellular adenosine, ADA (2 U/ml), on LTP of the PS is shown in Table 3. An increase of about 98.8 k 36.2% in the amplitude of the PS obtained under conditions of low-frequency stimulation was observed in the presence of ADA (ZU/ml). Both in the control slices (Table 3) and in the test slices (Table 3) in which T2 was applied in the presence of ADA (2 U/ml), LTP2 was smaller than LTPl. Calculating the ratio LTP2,/LTPl, a value of 0.87 f0.40 (n = 4) was found for the control slices and a value of 0.64 +0.20 (n = 5) for the test slices, which represent a non-significant difference. In a few experiments, the effect of ADA (2 U/ml) on LTP of the fEPSP was studied; again, no significant differences were observed between the test slices and the control slices. Effect of 2-p (2-carboxy -ethyl) ethylcarboxamidoadenosine

phenethylamino-5’-N-

on long-term

potentiation

The effect of the selective A, receptor agonist, CGS 21680 (30 nM), on LTP of the PS is shown in Table 4. An increase ofabout 44.0 +9.1% in the amplitude of the PS obtained under conditions of low-frequency stimulation was observed in five slices in the presence of CGS 21680 (30nM). In two slices, CGS 21680 (30 nM) appeared to have no excitatory effects on the amplitude of the PS obtained under conditions of low-frequency stimulation, and these slices were not considered further. In the control slices (Table 4),

LTP2 was smaller than LTPl, whereas in the test slices (Table 4) in which T2 was applied in the presence of CGS 21680 (30 nM), LTP2 was larger than LTPl. Calculating the ratio LTP2/LTPI, a value of 0.69 +0.39 (n = 5) was obtained for the control slices and a value of 1.13 k 0.10 (n = 5) for the test slices. This difference is statistically significant (P < 0.05), and represents an excitatory effect of CGS 21680 (30 nM) on LTP2 of the PS in the test slices. Effect of 8-(3-chlorosryryl] cajkine on long-term potentiation A new selective A, receptor antagonist, CSC, has recently been developed.” The effect of CSC (2.5 PM) on LTP of the PS is shown in Table 5. A concentration (2.5 p M) of antagonist was chosen that is about 50 times the K, for the A2 receptors and about one-tenth the K, for the A, receptors, as determined for the inhibition of agonist binding in the rat brain membranes.” We observed that CSC (2.5 PM) could prevent the excitatory effects of CGS 21680 (30 nM) on the amplitude of the PS obtained under conditions of low-frequency stimulation. CSC (2.5 yM) itself had no consistent effects on the amplitude of the PS obtained under these conditions of stimulation. Both in the control siices (Table 5) and in the test slices (Table 5) in which Tl was applied in the presence of CSC (2.5 FM), LTP2 was smaller than LTPI. Caiculating the ratio LTPZ/LTPl, values of 0.65 $0.08 were found both for the control slices (n = 4) and the test slices (n = 4).

Table 5. Effect of the selective A, receptor antagonist, 8-(3-chlorostyryl) caffeine (2.5pM), on long-term potentiation of the population spikes

CSC (2.5 PM) Control

Pre-Tl (mV)

Post-T1 (mV)

Pre-T2 (mV)

Post-T2 (mv)

LTP2/LTPl

1.10f0.06 1.03 + 0.04

3.19 + 0.28 3.02 +_0.29

1.20 + 0.02 1.03 k 0.04

2.64 IO.16 2.34 1: 0.25

0.65 f 0.08 0.65 k 0.08

Values are given as the means & S. E. M., and were obtained from four test slices and four control slices. In the test slices, TI was applied in the presence of CSC (2.5 FM). Abb~viations are as in Table 1 and in the text.

Endogenous adenosine modulates LTP DISCUSSION

389

tori’), refine previous results showing that the moderately selective A, receptor antagonist, CP 66713 (which has a selectivity of 1Zfold for the A2 receptor15), does not modify LTP of the PS, although this compound was reported to prevent the induction of LTP of the fEPSP.“’ The present results showing that the enzyme that degrades extracellular adenosine, ADA, did not significantly modify LTP might be explained by the hypothesis that ADA, by degrading endogenous adenosine, would remove not only the inhibitory effects of adenosine on LTP mediated by A, receptors, but also presumably excitatory effects mediated by AZ receptors. However, as mentioned above, in the present experimental conditions no consistent effect of endogenous adenosine mediated by the A2 receptor on LTP could be detected. Another possible explanation would be that ADA at the concentration we used (2 U/ml) might have non-specific effects in this preparation. However, we do not think this is the case, because the excitatory effects of this concentration of ADA on the amplitude of the PS can be virtually totally reversed by the specific ADA inhibitor, eryrhro-9-(2-hydroxy-3-nonyl)adenine (Cunha R., personal communication). Another hypothesis to explain why ADA did not modify LTP involves a possible interaction between the two main types (A, and AJ of adenosine receptors. It is known that part of the augmentation in the PS amplitude that corresponds to LTP can be mediated through an increase in intracellular cyclic adenosine monophosphate.” The adenosine A, receptor is negatively coupled and the adenosine A, receptor is positively coupled to the adenylate cyclase/cAMP system.*’ It could be that some degree of activation of the A, receptor would be required for the operation of this transduction system by different substances, as recently shown in the peripheral nervous system.(’ In other words, some degree of activation of the A2 receptor would be necessary for the relief of the inhibitory effects of A, receptor on this transduction system to become apparent as an increase in LTP. Further work on the interaction between the different types of adenosine receptors may clarify this issue. One further point deserves comment: the hypothesis was recently advanced that the effects of adenosine on LTP were merely due to the inhibitory actions of this purine on synaptic transmission.12 The observation that the removal of endogenous adenosine by ADA markedly increases the amplitude of the responses evoked by low-frequency stimulation, and yet does not facilitate LTP, suggests that the modulatory role of adenosine on LTP may not be a non-specific consequence of the inhibitory effects of this purine on synaptic transmission,

In the present study, the role of endogenous adenosine in the modulation of LTP was investigated. We found that endogenous adenosine exerted a tonic inhibitory effect on LTP, which was mediated by adenosine A, receptors. Under certain experimental conditions, the activation of adenosine A2 receptors may facilitate LTP. It is well known that exogenously administered adenosine has inhibitory effects on evoked synaptic responses in hippocampal CAI neurons.‘6,‘9 These inhibitory effects are mediated through adenosine A, receptors.*J* More recently, it was found that activation of adenosine A, receptors has excitatory effects on evoked synaptic responses in the same hippocampal area. I7 In relation to endogenous adenosine, several authors described that this nucleoside exerts an inhibitory tone on evoked synaptic responses and neuronal excitability in hippocampal slices.9~‘o The actions of adenosine and adenosine analogues on phenomena of synaptic plasticity were also investigated in the hippocampus. Both exogenously administered adenosine and the adenosine analogue, CADO, decrease LTP of the responses evoked by stimulation of the Schaffer fibres and recorded in the CA1 area.4,7 In a recent report, it was described that DPCPX, used at a very high concentration (100 nM), increases LTP of the fEPSP induced by theta pattern stimulatioms In the present work, we found that DPCPX facilitates LTP of both the PS and the fEPSP, induced by high-frequency stimulation, at a rather lower concentration (10-20 nM), at which it is expected to selectively antagonize the A, adenosine receptor.‘* Consistent with these findings, we observed that the adenosine uptake blocker, NBI (5pM), decreased LTP, confirming the existence of a tonic inhibitory role of endogenous adenosine on LTP. This tonic inhibitory effect is presumably accentuated in consequence of the increase in extracellular adenosine levels caused by adenosine uptake blockade. Another point of interest in the present work is that activation of adenosine AZ receptors, by using the selective AZ receptor agonist, CGS 21680 (30 nM), facilitates LTP. These results further extend the description of excitatory effects of adenosine mediated through the A, receptor in the hippocampus, which could be observed both on the PS evoked by low-frequency stimulation” and on the modifications of synaptic plasticity associated with highfrequency stimulation, as shown in the present work. The observation that the selective A, receptor antagonist, CSC (2.5 PM), did not modify LTP does not support the hypothesis that endogenous adenosine may modulate LTP through activation of CONCLUSION adenosine A, receptors. These findings using the selective A, receptor antagonist, CSC (2SpM) We conclude that endogenous adenosine is able to (which has a selectivity of 520-fold for the A, recep- modulate mechanisms of synaptic plasticity, such as

.4. de Mendonp

LTP, in the hippocampus. As a corollary, drugs that modify purinergic neuromodulation might hopefully prove to be efficient in the treatment of cognitive deficits like those observed in dementia disorders.

and J. .A. Rib&o

Acknowleriqements-We would like to thank Prof. I(. Jacobson for the generous gift of CSC. Dr A. M. Sebrstiao and Dr R. Cunha for helpful comments and suggestions. and Dr M. J. de Mendon@ for assistance in the preparation of the manuscript. A. de MendonCa thanks Prof. A. Castro Caldas for encouragement.

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20. Sekino Y., Ito K.-I., Miyakawa H., Kato H. and Kuroda Y. (1991) Adenosine (A,) antago~st inhibits induction of long-term potentiation of evoked synaptic potentials but not of the population spike in hippocampal CA1 neurons. Biochem. biophys. Rex. Commun. 181, 1010-1014. 21. van Calker D., Miller M. and Hamprecht B. (1979) Adenosine regulates via two different types of receptors the accumulation of cyclic AMP in cultured brain cells. J. Neurochem. 33, 999-1005. (Accepted 26 April 1994)