Pflttgers Arch (1985) 403 : 141-145 PflLigers Archiv European Journal of Physiology

�9 Springer-Verlag 1985

Adenosine-induced depression of synaptic transmission in the isolated olfactory cortex: receptor identification J. McCabe and C. N. Scholfield

Physiology Department, Queen's University, Medical Biology Centre, 97, Lisburn Road, Belfast, UK

Abstract. 1. We have investigated the type of purine receptor in the guinea-pig olfactory cortex, using pial surface slices maintained in vitro.

2. Adenosine (0.1 to 100 lamol/1) bath applied in the presence of the uptake inhibitor nitrobenzylthioinosine, depressed the evoked potentials in a dose related fashion.

3. Synthetic and uptake resistant adenosine analogues had the same effect as adenosine and the order of potency of these was: 5'-N-ethylcarboxamide adenosine > L-N 6- phenylisopropyl adenosine (L-PIA) = N6-cyclohexyl - adenosine = 2-chtoroadenosine > adenosine > D-N6-phe - nylisopropyladenosine (D-PIA).

4. The D-stereoisomer of PIA was 45 times less potent than L-PIA.

5. The methylxanthine compounds 8-phenyltheophylline (3 gmol/1) and 3-isobutyl-l-methylxanthine (50gmol/1) antagonised the depression produced by L-PIA.

6. Rolipram, a phosphodiesterase inhibitor, in concen- trations up to 100 gmol/1 had no effect on the evoked potentials or on adenosine action.

7. Forskolin, a cAMP stimulant, slightly increased the amplitude of the evoked potential, and partly reversed the depressant effect of adenosine. Noradrenaline had no effect either alone or in the presence of adenosine.

8. The results of these experiments indicate the existence of A1 subtype adenosine receptors in the guinea pig olfac- tory cortex probably linked to a depression of intracellular cAMP.

Key words: Adenosine - Adenosine analogues - Forskolin - Theophylline - Rolipram - Adenosine AI receptors

Introduction

Two distinct subclasses of adenosine receptors are at present described, A1 and A2. The AI receptors have a high (nanomolar) affinity for adenosine and inhibit adenylate cyclase activity. The A2 receptors have a lower (micromolar) affinity for adenosine and stimulate adenylate cyclase activ- ity (Van Calker et al. 1979; Londos et al. 1980; Snyder 1981).

The central actions of adenosine itself already have been studied (Scholfield 1978; Kuroda t983; Stone 1981). Re- cently various synthetic and enzyme stable adenosine analogues have been developed which exhibit differing

Address for correspondence: C. N. Scholfield at the above address

potencies at the A1 and A2 receptor in cell free preparations (Williams and Risley 1980; Bruns et al. 1980; Daly 1983). Comparison of the relative potencies of these analogues at any particular site can therefore be used to give an indication of the receptor population of the area. In particular, the L- and D-stereoisomers of phenylisopropyladenosine (PIA) may be used to differentiate between the two types of recep- tor: at the A1 receptor the L-isomer is up to 100 times more potent than the D-isomer, while the A2 receptors exhibit rather less, or perhaps no stereoselectivity, in the guinea-pig (Smellie et al. 1979; Fredholm et al. 1982). On the other hand, A2 receptors are much more sensitive to N-ethyl- carboxamide adenosine (NECA) than cyclohexyladenosine. The availability of these synthetic analogues has therefore made it possible to determine the class of receptors present in the guinea pig olfactory cortex, by examining their relative potencies in depressing the evoked potentials of the brain slice preparation.

The aim of the present study was to see at which type of receptor adenosine acts by using two approaches: (a) by assessing the sensitivity of the olfactory cortex to adenosine analogues, and (b) to manipulate the intracellular levels of cAMP perhaps thereby mimicking or opposing adenosine action. We also wanted to observe the action of 8- phenyltheophylline, a selective adenosine antagonist (Bruns et al. 1980; Snyder et al. 1981) since previously (Scholfield 1978) it was unclear whether theophylline was acting as an adenosine antagonist or a phosphodiesterase inhibitor.

Methods

Guinea-pigs of either sex, weighing 200-400 g were de- capitated, and their brains removed and a pial surface slice of olfactory cortex cut to a thickness of 0.45 - 0 . 6 ram, using a bow cutter and guide (Scholfietd 1980).

The slice was placed for at least 2 h in a holder suspended in Krebs solution at room temperature (23 - 25 ~ C), through which a 95% 02/5% CO2 gas mixture was bubbled. The slice was then placed in the recording bath described previously (Scholfield 1980). Its cut surface rested on the nylon mesh in the bath, and its uppermost (pial) surface was covered with a piece of nylon mesh, held in place with a wire clip (Scholfield 1980). Krebs solution equilibrated with 95% 02/ 5% COz flowed through the bath at about 10 ml/min.

The lateral olfactory tract (LOT) was stimulated using a pair of silver wires contained in glass tubes (1 mm diameter), placed across its severed end, with single supramaximal stim- uli (about 10 V, duration 0.4 ms, I00 f2 source impedence)

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continuously at frequencies of 0 .01-1 Hz. Potentials (the pial surface negative wave, see results) were recorded from the pyriform cortex using a saline filled glass electrode with a tip diameter of 0.2 ram, positioned on the surface of the slice with a binocular microscope. The recording electrode was connected via a pre-amplifier to a Gould Advance digital storage oscilloscope. Traces were recorded using an X-Y plotter linked to this oscilloscope. All potential mea- surements from the tissue were taken with respect to an indifferent silver electrode in the recording bath.

Recordings were taken from the slice every 5 min, until the potentials reached a constant level. The adenosine analogues were made up in Krebs solution and fed through the bath for 5 rain, and a further recording taken. Adenosine compounds were applied sequentially with a 15 min period of washing to allow full recovery after the first two doses before proceding with higher concentrations. Drug effects were determined by expressing the amplitude of the record as a percentage of that before adding and after recovery from the drug.

We noticed that the sensitivity and the maximum depres- sion produced by adenosine and its analogues varied over a 2 year period. To minimise this effect, we made comparisons within a short period (2 months) and wherever possible, we compared effects on the same slice or pairs of slices from the same animal.

The composition of the Krebs solution used was (mmol/1): Na + 144, K + 3.0, Ca 2+ 2.5, Mg 2+ 1.2, C1- 129, HCO~- 25, SO 2- 1.2, HzPO2 1.1, and D-glucose 11.

The drugs used were: Adenosine (Sigma Chemical Co, Poole, GB, Lot A9251); 2-chloroadenosine (Sigma Chemical Co, Poole, GB, Lot C5134); N6-(S)-l-phenyl-2-pro - pyladenosine (Boehringer Mannheim, FRG, Lot 1382101) (D-phenylisopropyladenosine, D-PIA); N6-(R)-l-phenyl-2- propyladenosine (Boehringer Mannheim, FRG, Lot 1332201) (L-phenylisopropyladenosine, L-PIA); N6-cyclo - hexyladenosine (Calbiochem-Behring Corp., CA, USA, Lot 110101); 5'-N-ethylcarboxamide adenosine (gift from Dr. Schick of Byk-Gulden, Konstanz, FRG, Lot K 8/82/3); Rolipram (a gift from Schering Aktiengesellschaft, Berlin, Lot 01220/81); Forskolin (Calbiochem-Behring Corp., CA, USA, Lot 202828); 8-phenyltheophylline (Calbiochem-Be- hring Corp., CA, USA, Lot 110087); 3-isobutyl-l-methyl- xanthine (Sigma Chemical Co, Poole, GB, Lot 23F-0144).

Results

Upon stimulation of the lateral olfactory tract (LOT, the main afferent input to the olfactory cortex), potentials with a surface-negative/surface-positive sequence were recorded from the pial surface of the pyriform cortex. All potentials were recorded with respect to the cut surface, therefore superficial depolarizations were manifested as surface nega- tive waves, and deep depolarization as surface positive waves (Fig. 1).

The large early part of the negative wave was used since this arises as the summed monosynaptic epsps in the apical dendrites of superficial pyramidal cells (Yamamoto and Mcllwain 1966; Richards and Sercombe 1968; Biedenbach and Stevens 1969; Halliwell 1976; Gilbey and Wooster 1979) - see Fig. 1. This wave is generated in structures near to the pial surface and so the actions of drugs subject to up- take will be less attenuated than those in deeper layers. This

A Monosy~ B

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Fig. 1 A--D. Voltage recordings from the pial surface of the guinea- pig olfactory cortex. A A single supramaximal stimulus was applied to the LOT at the time coinciding with the arrows and the stimulus artefacts. The upper traces were from two different slices, in normal solution, and adenosine (10-4mol/1) added to slice A or cyclohexyladenosine (10 -s tool/l) to B. B Time-courses for the ac- tion of adenosine C or chloroadenosine D at sequentially increasing concentrations in two other slices. The record was obtained from a sample-and-hold amplifier taking alternate samples of the slice surface potential before the stimulus and at the peak of the mono- synaptic epsp. They appear respectively as the baseline and the upper extremity shown in this record

component could be recorded over all areas of the slice, but exhibited variations in amplitude between different areas. Preliminary experiments were performed to find the optimum frequency of stimulation for adenosine action and the regional sensitivity to adenosine over the slice surface. The LOT was stimulated at various rates between 0.01 and 1.0 Hz, and the response to adenosine plotted against frequency. Adenosine action was found to be potentiated at higher frequencies of stimulation. A maximum adenosine induced depression was observed at 0.2 Hz: it was 1.5 x greater than that at 0.01 Hz. Since stimulation at higher frequencies tended to lead to deterioration of the responses over a 2 h period, we comprimised with a 0.1 Hz stimulus rate. Adenosine became increasingly effective the more post- eriorly the recording electrode was placed up to a distance of 3 mm from the pyriform end of the LOT. Beyond this, the depression decreased again. Therefore the recording electrode was placed 3 mm from the LOT.

Effect of adenosine on the evoked potentials

Adenosine is subject to cellular uptake and its potency on bath application is reduced whereas the adenosine analogues

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Adenosine concn.u.moL/L Fig. 2. Dose-response curves for the action of adenosine and various analogues on the epsp. Ordinate: maximum height of the monosynaptic epsp as a ratio of that before adding the adenosine analogue. Abscissa: concentration of the appropriate adenosine analogue denoted by the key

used in these experiments are thought to be resistant to uptake (Collis 1983). Therefore, to make its effects more comparable with the analogues, we used adenosine in the presence of the uptake inhibitor, nitrobenzylthioinosine (Marangos et al. 1982).

Adenosine in the presence of nitrobenzylthioinosine (5 pmol/1) depressed the evoked potentials (Fig. 1). The manner of the depression is more fully described in Schol- field (1978). There was a 10 s delay in the onset of adenosine action (see Scholfield 1978 and Fig. 1C). This delay is the time taken for the solution to arrive at the slice. After the onset, adenosine action increased rapidly during the first minute of application and then levelled out (Fig. 1C). Complete recovery required up to 20 rain. Adenosine was effective over the dose range of 0 .1 -100 gmol/1 (Fig. 1C). Dose response curves were constructed for adenosine (Fig. 2) and a smooth curve drawn through the points by "eye". Application of concentrations higher than 100 gmol/1 did not produce any further depression of the epsp under these conditions. The concentrations required to reduce the amplitude of the monosynaptic epsp to 50% of that before adding the adenosine analogue (IDso) was read off the graphs. This value for adenosine was 2.90 lamol/1 (and 7.6 pmol/l in the absence of uptake inhibitor).

Relative potencies of adenosine analogues

Cyclohexyladenosine (Fig. 1), 2-chloroadenosine, 5'-N- ethylcarboxamide adenosine (NECA) and the L-stereo- isomer of phenylisopropyladenosine (L-PIA) at 0.01-- 10 gmol/1 each depressed the monosynaptic epsp in a manner very similar to adenosine (Fig. 2). In contrast, the D-stereoisomer of PIA was moderately weak in its depressant effect. High doses of this adenosine analogue were untested

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because of its poor solubility in water. The IDso for these compounds are listed below in order of potency:

Agonist IDs0 SEM n

5'-N-Ethylcarboxamide adenosine 0.17 4- 0.06 5 Cyclohexyladenosine 0.58 + 0.12 20 L-PIA 0.78 +_ 0.42 6 Chloro-adenosine 0.96 4- 0.22 7 Adenosine (+ nitrobenzylthioinosine) 2.9 4- 0.4 6 D-PIA 35 4- 20** 7

** This value was obtained by extrapolation

Analysis of variance revealed no significant difference be- tween IDso vslues for chloroadenosine, L-PIA and cyclohexyladenosine. The difference in potency between these analogues and adenosine was, however, highly signifi- cant (unpaired t-test, P < 0.0001) and NECA significantly different from cyclohexyladenosine, chloroadenosine and L- PIA (P < 0.002). D-PIA was the least potent of all the analogues tested: the D isomer of PIA was 45 times less potent than the L-form. These analogues, like adenosine (Scholfield 1978) had no effect on the presynaptic compound action potential. However, the recovery of the preparations from these substances was prolonged compared to adeno- sine (about 5 h for full recovery). Although the onset in their action was just as rapid as for adenosine, the potent adenosine analogues required 5 min or more to produce their full effect (compared D and C in Fig. 1).

The effect of other agents influencing intracellular cAMP levels

1. Forskolin

Forskolin stimulates intracellular cAMP independent of any membrane drug receptor (Seaman and Daly 1981). On its own, forskolin increased the amplitude of the monosynaptic epsp by 14.7 4- 3.9%. Forskolin in four experiments at 50 pmol/1 and in one at 100 gmol/1, antagonised the effect of adenosine: for 50 pmol/1 forskolin, it shifted the IDs0 from 2.50 +_ 0.25 to 6.15 + 0.87 gmol/1 ( P < 0.002 for the difference) (Fig. 3 a). The maximum depression produced by adenosine was also less in the presence of forskolin (Fig. 3 a). A limited amount of the substance and its poor solubility prevented us testing higher forskolin concentrations.

2. Noradrenaline

At concentrations of 10 and 100 pmol/1 noradrenaline had no effect on the epsp alone or on the effectiveness of adeno- sine in three experiments. Isoprenaline (1 and I00 pmol/1) had no influence of the effect of cyclohexyladenosine.

3. "Phosphodiesterase inhibitors"

The effect of Rolipram, a potent phosphodiesterase inhibitor (ZK 62711 - see Puurunen et al. 1978), was investigated. At concentrations of 0.1 to 100 pmol/1 it had no effect on the evoked potentials when applied alone nor any effect on the action of cyclohexyladenosine. We also examined the effect of Rolipram in Krebs solution containing only 1.25 mmol/1 Ca instead of 2.5 retool/1. The lowered Ca re- duced the amplitude of the evoked potential by about 50%. The principle behind the idea was that in the higher Ca concentration, synaptic transmission might be already

144

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A ADENOSINE CONCENTRATION.moI/I a L-PIA CONCENTRATION,moI/I Fig. 3. (A) Dose-response curve for adenosine alone or in the presence of 50 gmol/1 forskolin. Various concentrations of adenosine (0.1 to 100 I~mol/1) were applied to the preparation. The preparation was allowed to recover and forskolin applied for 15 rain before retesting adenosine in the presence of forskolin. All tests were performed in the presence of 0.2% ethanol. This was because forskolin had to be dissolved in a small quantity of ethanol before adding to the Krebs solution. (B) The effect of 3 gmol/1 8-phenyltheophylline on the depression of the epsp produced by L-HA. Each of the two slices of olfactory cortex from 6 animals received doses (0.01-10/.unol/1) of L-HA either alone or with 8-phenyltheophylline

maximal and not further increased by any action of Rolipram via increased cAMP. However, Rolipram was still without any effect by itself or on the action of cyclo- hexyladenosine.

We also performed two experiments using 3-isobutyl-l- methylxanthine (IBMX) as a phosphodiesterase inhibitor (Wells and Kramer 1981). IBMX (30 t~mol/1) appeared to produce similar shift in the L-HA dose-response curve as did 8-phenyltheophylline (see below). IBMX (30 gmol/l) by itself consistantly increased the amplitude of the evoked potential by 24 _+ 4% (P < 0.0005) in eight preparations.

The antagonists

The antagonists action of the methylxanthine compound 8- phenyltheophylline was investigated, using the metabolically stable adenosine analogue E-HA, in order to ascertain that the drugs used exerted their depressant effects via the methylxanthine sensitive P1 receptors described earlier.

Dose response curves were constructed for E-HA alone, and in the presence of 3 gmol/1 phenyltheophylline (Fig. 3 b). 8-Phenyltheophylline antagonised the depression produced by L-HA, increasing its IDs0 from I to 15 gmol/1. This concentration of 8-phenyltheophylline slightly increased the amplitude of the evoked potential (by 4.6 __ 2.0%).

Discussion

The aim of the present study was to decide whether adeno- sine depresses synaptic transmission in this preparation by either causing a decrease (via the Al-receptor) or an increase (A2-receptor) in the level of intracellular cAMP. According to other studies on a wide variety of tissues the adenosine analogues we used should clearly differentiate between A~ and A2 receptors. I t is evident that D-HA was much less potent than the L-form, an observation which itself indicates

the presence of the Al-receptor. Most of the adenosine analogues we tested had effects down at the 10 nmol/1 level of concentrations, which is similar to the binding constants of around 3 - 8 nmol/l for these compounds in guinea-pig brain homogenates (Bruns et al. 1980). This high sensitivity to the adenosine analogues also suggests Al-receptor activa- tion. However, it is difficult to make firm comparisons be- tween binding constants obtained in celt free and cell culture systems and in these experiments with intact synapses. In the latter, there are several stages between the binding of adenosine to the external receptor and the end result on synaptic transmission. The A2 type of receptor is 2 0 - 1 0 0 times more sensitive to NECA than L-PIA cyclohexyladenosine (Londos et al. 1980). In the present experiments it was 3.4 times more sensitive than cyclohexyladenosine, a similar potency ratio as the guinea- pig atrium (Collis 1983). This small ratio in the olfactory cortex tends to contradict A2 receptor mediation in this depression.

I f the depressant effect of adenosine was mediated through AI receptors, procedures which increased the level of intracellular cAMP should reverse this depression. Forskolin did partly achieve this. On the other hand noradrenaline was ineffective in this way but this might have been a consequence of the appropriate receptors being absent. We also attempted to increase the intracellular con- centration of cAMP by the application of phosphodiesterase inhibitors. Rolipram had no effect. However, the prepara- tion may contain a phosphodiesterase isozyme resistant to this agent, perhaps a Ca-dependent one (Wells and Miller 1983). We also tested IBMX as a phosphodiesterase inhibi- tor. It was not very clear in the present experiments whether IBMX was acting as a phosphodiesterase inhibitor, an adenosine receptor antagonist or both. 30 ~mol/1 IBMX antagonised L-PIA in a manner similar to 3 gmol/1 8-phe- nyltheophylline. When either compound was applied by it-

145

self, there was an increase in the ampl i tude of the monosynapt ic epsp. However, the effect of 8-phenyl- theophyll ine was much smaller than that of I B M X and of forskolin. N o w 8-phenyltheophyll ine is regarded as a specific adenosine receptor blocker, so the difference in ac- t ion between this and I B M X might be a t t r ibutable to I B M X having an addi t ional phosphodies terase inhibi tory action.

Direct measurements of c A M P in the guinea-pig olfac- tory cortex have shown that adenosine (at 10 gmol/1) in- creases c A M P (Kuroda 1983; Mot ley and Collins 1983). However, c A M P st imulat ion occurred at adenosine concen- trat ions higher than those we used and only after a delay: at times after the epsp is depressed (see K u r o d a 1983). These very large c A M P rises had been measured in whole tissues: A raised c A M P in one cellular element would mask any decrease in other structures. Thus a l though A2 receptors are apparent ly present in this tissue, they are unlikely to be responsible for the adenosine-induced depression of the epsp. This idea is suppor ted by the enhanced transmission observed with compounds such a 8 -b romo-cAMP and dibutyryl c A M P which mimick an increased c A M P (Kuroda 1983). Many substances which influence intracellular c A M P have limited usefulness in the present type of analysis be- cause they often have mult iple actions on the adenosine receptor, on the adenosine uptake site or on phosphodi - esterases (Schwabe et al. 1976).

So these experiments s trongly indicate the presence of the A t type of receptor p robab ly decreasing intracellular c A M P and it is the act ion of adenosine on these receptors which is responsible for the depression in exci tatory transmission at the LOT synapse.

Acknowledgements. We thank Dr. N. Sprzagala (of Schering) for supplying the Rolipram and Dr. Schick of Byk-Gulden for the NECA.

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Received April 20/Accepted October 24, 1984