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Potential use of D P C P X as probe for in vivo localization of brain A 1 adenosine receptors J.C. Bisserbe a, O. Pascal b, j. Deckert c,, and B. Mazi~re b " lnstitut National de la Santd et de la Recherche Mddicale (INSERM), Senice Hospitalier Frdddric Joliot (SHFJ), Orsay (France), t, Sercice Hospitalier Fr~ddric Joliot (SHFJ), Orsay (France) and " Unit on Neurochemistry, National Institute of Mental Health, Bethesda MD 2089 (USA)
(Accepted 21 July 1992)
Key words: DPCPX; Adenosine A l receptor; Ex vivo autoradiography; Rat
The suitability of (3H)DPCPX (8-cyclopentyl-l,3-dipropylxanthine), a xanthine derivative, as an vivo probe for labelling adenosine A j receptors was studied in rats. [3H]DPCPX (nM) penetrated largely into the brain (0.8% of the injected dose per gram of brain tissue 5 min after injection). Brain concentrations stayed at a plateau level from 5 to 15 rain after the injection. The distribution in the different brain regions was heterogeneous with the highest amount of [3HIDPCPX in cerebellum and hippocampus and the lowest concentrations in hypothalamus and brain stem. Displacement (45-70% of total radioactivity) was obtained by the injection of 250 nM of cold DPCPX or cyclopentylxanthine, an analog of DPCPX. The ex vivo autoradiographic distribution of [3H]DPCPX was similar to the in vitro autoradiographic distribution of tritiated A x adenosine receptor ligand as [3H]CHA. These results suggest the potential use of DPCPX for further in vivo investigation of A 1 adenosine receptors with techniques such as positron emission tomography.
INTRODUCTION A d e n o s i n e , in a d d i t i o n to its f u n c t i o n in i n t e r m e d i ary m e t a b o l i s m , is, b e s i d e s G A B A , t h e m a j o r n e u r o m o d u l a t o r in m a m m a l i a n b r a i n k n o w n t o d a y t9'35. Its C N S actions a r e m a i n l y e x e r t e d via two r e c e p t o r s , A~ and A236. R e u p t a k e is b e l i e v e d to b e the r a t e - l i m i t i n g step for its inactivation 2'3. T h e actions of m e t h y i x a n thines such as caffeine a n d t h e o p h y l l i n e a r e m e d i a t e d by a n t a g o n i s m o f e n d o g e n o u s a d e n o s i n e at its r e c e p tors 13. R a d i o l a b e l l e d a d e n o s i n e a n d x a n t h i n e derivatives have b e e n utilized to visualize a n d q u a n t i f y A l r e c e p tors in r o d e n t 16JT"28m, cat 1, d o g 15 a n d also in h u m a n b r a i n s ~8. W i d e l y d i s t r i b u t e d A l r e c e p t o r s a p p e a r to m e d i a t e t h e a d e n o s i n e ' s d e p r e s s i o n o f synaptic transmission jg. A e r e c e p t o r s have also r e c e n t l y b e e n m a p p e d with t h e specific l i g a n d [ 3 H ] C G S 21680 25 a n d m o r e r e c e n t l y in h u m a n b r a i n by in situ h y b r i d i z a t i o n with the A 2 r e c e p t o r m R N A 34. T h e l i m i t e d localization o f
the A 2 r e c e p t o r to c a u d a t e , p u t a m e n a n d a c c u m b e n s a n d t h e effect o f local injection o f N E C A in c a u d a t e 2° suggest a specific role o f this r e c e p t o r in the physiology o f t h e b a s a l g a n g l i a 24. T h e a n a t o m y o f the ' a d e n o s i n e system' has b e e n c o m p l e t e d with the m a p p i n g o f the a d e n o s i n e t r a n s p o r t e r 4"5, of t h e e n z y m e s a d e n o s i n e d e a m i n a s e 3° a n d a d e n o s i n e 5 ' - n u c l e o t i d a s e 17, a n d also of a d e n o s i n e - c o n t a i n i n g n e u r o n s ~. B a s e d on a n i m a l w o r k a n d h u m a n p o s t m o r t e m studies, a d e n o s i n e has b e e n i m p l i c a t e d in the p a t h o p h y s i o l ogy a n d b i o c h e m i s t r y o f c o n d i t i o n s as diverse as anxiety a n d s l e e p d i s o r d e r s , psychosis, n e u r o d e g e n e r a t i v e p a t h o l o g y , c e r e b r a l i s c h e m i a a n d convulsions a n d also in the m e c h a n i s m o f a c t i o n o f p s y c h o t r o p i c drugs 14"33. U p r e g u l a t i o n o f a d e n o s i n e A I r e c e p t o r s has b e e n f o u n d in a n i m a l m o d e l s of anxiety 6'27'29"37. In h u m a n s caffeine has shown specific a n x i o g e n i c p r o p e r t i e s in p a n i c disord e r p a t i e n t s 7'12. A r e d u c t i o n o f a d e n o s i n e A~ r e c e p t o r s has b e e n f o u n d in several p o s t m o r t e m h u m a n b r a i n studies o f A l z h e i m e r p a t i e n t s 22'23"2~'. F u r t h e r explo-
Correspondence: J.C. Bisserbe. Present address: Inserm U 302, Pavilion Cl~rambault, H6pital La Salp~tri6re, 47 boulevard de l'H6pital, 75014
Paris, France, * Present address: Universi6its-Nervenklinik, Fiichsleinstrasse 15, 87 Wiirzburg, Germany.
ration of the role of adenosine in neuropsychiatric diseases will require direct in vivo exploration of the adenosine receptors. We therefore decided to investigate the suitability of available adenosine ligands as tools for in vivo labelling of A t adenosine brain receptors and for their potential use in adenosine receptor exploration with positron emission tomography in humans. We present here the results obtained in rats with the highly A 1 specific adenosine ligand DPCPX.
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cerebellum hippocampus striatum cortex
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hypot.halamus brain stem plasma 0,6
0,4
MATERIALS
&
/
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IIII
AND METHODS
Determination of the ligand tissue kinetics Male Sprague-Dawley rats (200-250 g) with food and water ad libitum were used for all the experiments. Rats were injected in a tail vein with 5 - 8 /zCi of [3H]DPCPX (95 C i / m m o l A m e r s h a m ) in 0.2-0.3 ml of a 50% ethanol:saline solution. Animals were killed by decapitation at sequential times after the injection of [3H]DPCPX (2.5 min, 5 min, 10 min, 15 rain, 20 min and 30 rain). The brains were rapidly removed and dissected on ice to obtain samples of 7 anatomical regions: cerebellum, hippocampus, hypothalamus, cortex, striaturn, thalamus and brain stem. Blood was collected on ice, aliquoted, then centrifuged for 1 min, the plasma was aliquoted, then precipitated with T C A and the deproteinated plasma was also aliquoted. tleart, kidney, liver, muscle and lung tissues samples were also removed. The samples were weighed and dissolved overnight in a scintillation vial containing 1 ml of Beckman Tissue Solubilizer (BTS 450). After neutralization with acetic acid and addition of scintillation liquid (Ready-Solv Beckman) the samples were counted for 5 rain in a Packard Liquid Scintillation Counter. The amount of radioactive tracer present in each tissue at the different times was expressed as a percentage of the injected dose per gram of tissue (% i.d./g).
0,2
i
1
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20
30
rain
Fig. 1. Time course of [3H]DPCPX concentration in different brain areas: brainstem, cerebellum, hypothalamus, hippocampus, striatum, cortex and in deproteinated plasma. Rats were injected in a tail vein with 5 - 8 p, Ci of [3H]DPCPX (95 C i / m m o l A m e r s h a m ) in 0.2-0.3 ml of a 50% ethanol saline solution and sacrificed at 2.5 min, 5 rain, 10 min, 20 rain and 30 rain. The brains were dissected on ice to obtain samples of the different anatomical regions the samples were weighed in scintillation vials and 1 ml of Beckman Tissue So[ubilizer (BTS 450) was added for overnight dissolution. Radioactivity was counted for 5 min in a Packard Liquid Scintillation Counter after neutralization with acetic acid and addition of scintillation liquid (Ready-Solv Beckman). The concentrations of radioactive tracer present in each tissue at the different times were expressed as a percentage of the injected dose per gram of tissue (% i d / g ) and are the mean value of 3 - 6 experiments.
Saturation and di,splacement experiments For the determination of non-displaceable radioactivity, a bolus of 250 nM of either DPCPX in a 10% ethanol isotonic saline solution or cyclopentyl theophylline (CPT) in isotonic saline with 1.5% D M S O was injected in the tail vein at different times before (saturation experiment) or after (displacement experiment) the injection of 8-10 #Ci of [~H]DPCPX (0.08-0.1 nM). For total radioactivity determination, rats were injected under identical condition with [3H]DPCPX and with the vehicle of the cold compound. The animals were killed at different times after the two injections. The time of sacrifice was selected based on [~H]DPCPX kinetic data. Samples of the different tissues were then obtained and treated as described in the above section.
In Hco autoradiography with [¢H]DPCPX Rats were sacrificed 10 rain after the injection of 10 /~Ci of [3H]DPCPX. After rapid removal, the brain was frozen in isopentane at -40°C, fixed on a microtome chuck, then cut in 20 p,m sections. The sections were fixed on microscope cover slips and apposed to a tritium-sensitive film (Ultrofilm LKB, Sweden) for 8 - 1 2 weeks. The films were developed with Kodak D-19.
Stability of/~tt/DPCPX Stability of the injection solution of [3H]DPCPX were tested using thin layer chromatography. The T L C plates were read with a static radiochromatograph reader (Chromelec) with an efficiency of 9% for tritium. Biological stability of [3H]DPCPX was also tested in plasma after chloroform extraction with 80% recovery.
RESULTS
[~H]DPCPX tissue kinetics The time course of the brain concentration of the ligand showed a rapid penetration in the brain followed by a plateau from 5 to 15 min after the injection. The ligand very largely crossed the blood brain barrier; 0.85% i.d./g (S.D. = 0.22%) was present in the cerebellum 5 min after injection. Distribution in the different brain regions was heterogeneous with the highest amount of [3H]DPCPX in the cerebellum and hippocampus and lowest concentrations in hypothalamus and brain stem (Fig. 1). These differences were observed at each time point from 5 to 30 rain. In peripheral tissues, the highest concentrations were observed in the liver with respectively 1.5% and 1% i.d./g, 5 rain and 15 min after [~H]DPCP X injection. There was no apparent accumulation of the radioactivity in any of the peripheral organs examined (Fig. 2). The b l o o d / b r a i n concentration ratio was close to 1 and the ligand concentrations in deproteinated plasma, repre-
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Fig. 2. Time course of [3H]DPCPX concentrations in peripheral organs. All tissues samples were obtained during the experiment described in Fig. 1. Blood was collected on ice, plasma was separated by a 1 min centrifugation, deproteinated plasma was obtained after a protein TCA precipitation. Heart tissue samples were obtained from the ventricular part; kidney samples were cut on the cortical part of the organ; muscle samples were removed from the abdominal and thigh muscles; liver samples were obtained from two different lobes; lung tissue was removed from right and left upper and lower lobes.; blood was wiped off all tissue samples with filter paper. The samples were treated as described under Fig. 1. Concentrations are expressed as percentage of the injected dose. and are the mean value of 3-6 experiments.
senting the free [3H]DPCPX plasma concentrations, were below the brain concentration.
Saturation and displacement experiments In the saturation experiments with cold DPCPX described in Fig. 3, values for displaceable radioactivity varied largely with the different brain regions. Highest concentrations were found in the cerebellum and hippocampus with respective values of 0.41% i.d./g (S.D. = 0.02%) and 0.35% i.d./g (S.D. = 0.04%). Low-
hippocampus
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striatum
hypotharamus
brain stem
Fig. 3. Regional distribution of displaccablc and non-displaceahle radioactivity in selected rat brain regions in a saturation experiment. Rats were injected in the tail vein with 8 # C of [3tt]DPCPX and with 250 ~zM of cold DPCPX or an equivalent amount of vehicle (10% ethanol in isotonic saline) and were killed 10 min after the injection. The brains were then removed, dissected, tissue samples obtained from different anatomical regions were treated and radioactivity counted as described in the Methods section. The local [3H]DPCPX concentrations expressed as percentage of the injected dose (% id/g) are the mean value and standard errors obtained from two rats. Displaceable radioactivity varied from 0.42% i d / g (0.035 nM [3H]DPCPX) in the cerebellar cortex to 0.14% (0.01I nM [3H]DPCPX) in the brain stem. Deproteinated plasma concentrations at the sacrifice time in the two conditions were respectively 0.3% and 0.303% of the injected dose. The relative percentage of displaceable over non-displaceable varying from 71 to 43% arc indicated in each bar graph.
est concentrations were found in the brainstem and hypothalamus with values of 0.14% i.d./g (S.D. = 0.04%) and 0.14% i.d./g (S.D. = 0.01%). As expected, non-displaceable radioactivity concentrations were quite homogeneous among the different anatomical regions ranging from 0.15% i.d./g (S.D. = 0.(18%) in cortex to 0.19% i.d./g (S.D. = 0.08%) in brainstem. In structures with high [3H]DPCPX concentrations such as cerebellum, hippocampus, cortex or striatum displaceable radioactivity represented between 63% and 71% of the total radioactivity. Radioactivity concentra-
TABLE 1
Regiona! distribution of total and non-displaceable radioactivity in [JH]DPCPX displacement experiments with cold DPCPX or cych)pentyhheophylline Rats were injected with 250 nM of DPCPX/cyclopentyltheophylline or with the vehicles 12 min after 8-10 mCi of [3H]DPCPX and sacrificed 5 min after the cold ligand injection. Tissue samples were obtained and treated as described in Materials and Methods. Concentrations expressed in percentage of the injected dose per gram of tissue are mean and standard error values obtained with 3 rats per group in DPCPX displacement and two rats per group in cyclopentyltheophylline displacement.
Region
Radioactiuity concentration (% of injected dose / g of tissue) Displacement with DPCPX
Cerebellum Hippocampus Striatum Diencephalon Cortex Hypothalamus Brain stem
Displacement with CPT
Total R.A.
Non-disptaceable R.A.
Total R.A.
Non-displaceable R.A.
0.521 0.464 0.436 0.358 0.452 0.27 0.259
0.283 0.292 0.283 0.23 0.192 0.217 0.2
0.684 0.624 0.590 0.469 0.550 0.362 0.364
0.367 0.353 0.327 0.259 [].32 0.207 0.217
(0.027) (0.025) (0.027) (0.017) (0.021) (0.009) (0.007)
(0.045) (0.03) (0.024) (0.026) (0.028) (0.069) (0.03)
(0.008) (0.045) (0.013) (0.013) (0.016) (0.027) (0)
(0.091) (0.059) (0.054) (0.045) (0.013) (0.023) (0.034)
tions in the deproteinated plasma were identical both in presence (0.30% i.d./g) or in absence (0.303% i.d./ g) of cold DPCPX. Two comparable displacement experiments, using as cold ligand DPCPX or CPT are reported in Table I. Displaceable radioactivity ranged from 0.23% i.d./g in cerebellum to 0.6% i.d./g in brain stem when cold DPCPX was used and from 0.31% i.d./g in cerebellum to 0.14% i.d./g in brain stem when CPT was used as
cold ligand. The relative regional distribution of total and displaceable radioactivity was quite similar in the two experiments. In two other displacement experiments, CPT was injected 6 min or 30 rain after [~H]DPCPX injection, and the animals sacrificed 5 min after. In these two conditions non-displaceable radioactivity concentrations were quite identical but, as expected, values of displaceable radioactivity varied with time from 0.26%
GL
A
ML
B
(3 Fig. 4. Ex vivo autoradiographic distribution of [3H]DPCPX in representative antero-posterior sections of rat brain. Rats were injected with 10 /xCi of [3H]DPCPX alone (section A&B) or with 250 nM cold D P C P X (section C) and sacrificed after 10 min. The brains were removed, frozen and sectioned as described in the text. The identification of the anatomical structures was made with reference to the rat brain atlas of Paxinos and Watson 32. Darker areas represent regions with higher concentrations of the tritiated ligand. Abbreviations are: I, cortical layer 1; IV, cortical layer ~; C, caudate nucleus; LS, lateral septum; a, thalamic anterior nucleus; P, thalamic posterior nucleus; G, lateral geniculate body; DG, gyrus dentatus; cal, hippocampus cal area; SO, stratum oriens of the hippocampus; GL, granular layer of the cerebellum; ML, molecular layer of the cerebellum; WM, white matter; sc, superficial layer of the superior colliculus.
i.d./g in the brain stem and 0.47% i.d./g in the cerebellum at 6 rain to 0.04% i.d./g in the brain stem and 0.17% i.d./g in the cerebellum at 30 rain.
Autoradiography Ex vivo autoradiograms of [3H]DPCPX confirmed the heterogeneity of the anatomical distribution found in pharmacokinetic, saturation and displacement experiments and demonstrated discrete anatomical localization patterns in the different brain structures (Fig. 4). The typical pattern of adenosine A~ receptor distribution was observed in the cerebellum with highest densities in the molecular layer, intermediate density in the granular layer and background levels in the white matter. A heterogeneous distribution was observed in the hippocampus with higher densities in the stratum oriens and the stratum radiatum. In the cerebral cortex, layer I and IV showed higher densities of activity. The thalamic region also showed a heterogeneous distribution with higher density in the anteroventral nucleus. The lateral geniculate body and the superficial gray layer of the colliculi were also high density structures. This distribution was identical to the characteristic in vitro distribution of A 1 receptors obtained with ligands such as [3H]CHA2s. DISCUSSION In a first set of experiments we have described the tissue kinetics of [3H]DPCPX. The ligand penetrated brain tissues very extensively, since the injection of 0.1 nM of [3H]DPCPX (0.1 /zg/kg) led to a brain concentration around 1 nmol from 5 to 15 min after the injection. Brain concentrations of the tracer were above free tracer plasmatic concentrations (deproteinated plasma concentration). Assuming a linear relationship between increasing injected doses and brain concentration, a dose of 1 mg/ kg as used in behavioural experiments 2~ could lead to micromolar brain tissue concentrations. In comparison using the same experimental conditions only 0.03% (i.e. 0.05 nM brain concentration) of an injected dose of the specific adenosine A~ receptor antagonist XAC would be present in the brain 15 rain after injection. Similar values would be obtained with A1 agonists such as CHA or PIA (paper in preparation). Thus such compounds should require about a 50-fold higher dose in order to obtain a significant presence in brain tissue. A second set of experiments supported the existence of a specific in vivo binding of [3H]DPCPX to adenosine A~ receptors. First we have found [3H]DPCPX accumulated preferentially in areas known - - from in vitro binding studies - - to contain high levels of the
TABLE I1
Regional distribution oJ" ~pecific [ ¢tt]DP(TPA binding m ra~ /,rum ~:~pressed in q~ of values/or .~triatunt Ex vivo values were obtained from lhe displacement experimem described in Fig. 3. in vitro results were taken horn Bruns cl al."
Region Cerebellum Hippocampus Cortex Striatum Hypothalamus Brain stem
Specific binding (f~; value qf striatum) In vivo
In t'itro
120 109 101 100 45 45
102 1Z0 11 I I~!0 57 53
adenosine A~ receptor (e.g. cerebellum and hippocampus) and to a lower degree in areas with low densities of adenosine receptors (e.g. brain stem, hypothalamus). Further we have shown that [3H]DPCPX could be displaced by the administration of cold DPCPX and also by another potent A i adenosine antagonist: CPT "~. As expected the absolute values of non-disptaceable radioactivity (expressed in % of i.d./g) corresponding to the sum of the non-specifically bound ligand, plus the free ligand in brain tissue and the ligand present in the blood trapped into the brain tissue were quite identical in all brain regions in saturation and displacement experiments. By contrast the absolute amount of disptaceable [3H]DPCPX (specific binding) varied among brain regions, ranging from values of 0.4% of the injected dose for structures known to contain a high density of adenosine A ~ receptors such as cerebellum and hippocampus to values about 0.18% of the injected dose for structures with a low densities adenosine of A l receptors such as brainstem and hypothalamus (Fig. 3). The comparison of ex vivo regional distribution of [3H]DPCPX with the relative distribution of [3H]DPCPX from in vitro binding, reported previously by Bruns 9, presented in Table II shows similar distribution patterns. Nonetheless the differences observed between in vivo and in vitro experiments could be due to our sampling methods which required small tissue samples to facilitate dissolution and radioactive counting. Indeed for large stuctures such as cerebellum we collected only a small sample of cortical tissue rich in adenosine receptors, thus excluding cerebellar whitc matter which was devoid of adenosine receptors. Compared to in vivo binding methods, where the whole brain region is processed, our method could result in a relative adenosine receptor enrichment. In different displacement and saturation experiments, including results not presented, on the ratio of displaceable/nondisplaceable radioactivity, we observed significant variations in the values of total binding according to the
11
selected time interval between the injection of hot and cold ligand and also with the time interval between cold injection and sacrifice of the animals. The best ratios were obtained when the cold ligand was injected from 0 to 5 - 7 min after [3H]DPCPX and when the animals were sacrificed about 10 min after the injection of the cold ligand. The best ratio of specific/nonspecific radioactivity was obtained in a saturation experiment where the animals were sacrificed after the coinjection of both hot and cold DPCPX. In these conditions, displaceable radioactivity could reach 71% of total radioactivity in the cerebellum (Fig. 3). Finally, the A 1 adenosine receptor distribution revealed by [3H]DPCPX was confirmed at the histological level by ex vivo autoradiography. Indeed autoradiography results showed the pattern of distribution typical of in vitro binding studies with A l ligands 4"15'>. Particularly noticeable was the characteristic laminar distribution of [3H]DPCPX in the cerebellum with highest densities in the molecular layer, intermediate density in the granular layer and background levels in the white matter, as well as the distribution pattern observed in the hippocampus with higher densities in the stratum oriens and the stratum radiatum. In summary, [~H]DPCPX pharmacokinetics and A~ specificity, as demonstrated by in vitro studies m and our present results, indicate that DPCPX should be a suitable pharmacological tool for in vivo brain A ! r e c e p t o r blockade. The acceptable solubility of DPCPX and its very high affinity demonstrated in vitro, with a K d of 0.46 nM would further support this proposition I i. In addition we have shown that [3H]DPCPX is an appropriate ligand for ex vivo labelling of adenosine A L receptors in rodents. An important point remaining to be clarified is the metabolic stability of [3H]DPCPX. Preliminary results on thin layer chromatography analysis of plasmatic extract obtained after large [3H]DPCPX injections suggest no major metabolic degradation in the first 20 min after administration(data not presented). When considering the development of an in vivo ligand for A, adenosine receptor brain imaging methodology as positron emission tomography, the very favourable characteristics of DPCPX should be considered. These include a large penetration in the brain to obtain a good signal with a limited dose of radioactive tracer; a sizeable accumulation with a plateau of brain concentration to allow the detection of the receptorligand interaction; a low ratio blood/brain ligand concentration to avoid interference from the radioactive blood present in the brain; a specificity for the adenosine A l receptor with a good ratio specific/non-specific binding. It is thus expected that DPCPX, when appro-
priately labelled with positron emission isotope could be a good candidate ligand for in vivo exploration of adenosine A~ receptors in primates. Acknowledgements. J.C.B. held a Fellowship of the Fondation pour I'Etude du Syst~me Nerveux (Geneva, Switzerland). Thanks are due to M. Ottaviani and O. Stulzaft for technical assistance.
REFERENCES 1 Aohi, C., Development of the A i adenosine receptor in the visual cortex of cats, dark-reared and normally reared, Det'. Brain Res., 22 (1985) 125-133. 2 Barberis, C., Minn, A., Gayet, J., Adenosine transport in guinea pig synaptosomes, J. Neurochem., 362 ( 1981 ) 347-354. 3 Bender, A.S., Wu, P.H. and Phillis, J.W., The characterization of [3H]adenosine uptake into rat cerebral cortex synaptosomes, J. Neurochem., 35 (1980) 629-640. 4 Bisserbe, J.C., Deckert, J. and Marangos, P.J., Autoradiographic localization of adenosine uptake sites in guinea pig brain using [3 H] dipyridamole, Neurosci. Lett., 66 (1986) 341-345. 5 Bisserbe, J.C., Patel, J. and Marangos, P.J., Autoradiographic localization of adenosine uptake sites in the guinea pig brain using [3H]nitrobenzylthioinosine, J. Neurosci., 5 (1985) 544-55(I. 6 Boulenger, J.P., Patel, J., Post, R.M., Parma, A.M. and Marangos, P.J., Chronic caffeine consumption increases the number of brain adenosine receptors, Life Sci., 32 (1983) 1135-1142. 7 Boulenger, J.P., Uhde, T.W., Wolff, E.A. anti Post, R.M., Increased sensitivity to caffeine in patients with panic disorder: preliminary evidence, Arch. Gen. P~yehiatry. 41 (1984) 1067-1071. 8 Braas K.M., Newby A.C., Wilson, V.S. and Snyder. S.tt., Adenosine-containing neurones in the brain localized by immunocytochemistry, J. Neurosci., 6 (1986) 1952-1961. 9 Bruns, R.F., Fergus, J.H., Badger, E.W., Bristol, J.A., Santay, L.A. and Hays, S.J., PD 115,119: an antagonist ligand for adenosine A~ receptors, Nuuynyn-Sehmiedeherg's Arch. Pharrnacol., 335 (1987) 64 69. 10 Bruns, R.F., Fergus, J.H., Badger, E.W., Bristol, J.A., Santay, L.A., Hartman, J.D., Hays, S.J. and Huang, C.C., Binding of the Ai-selective adenosine antagonist 8-cyclopentyl-l,3 dipropylxanthine to rat brain membranes, Nauynyn-S~tlnliedeberg~ Arch. Pharmacol., 335 (1987) 59-63. 11 Bruns, R.F. and Fergus, J.H., Solubilities of adenosine antagonists determined by radioreceptor assay, J. Pharm. Pharmacol., 41 (1989) 590-594. 12 Charney, D.S., [teninger, G.R. and Jaflow, P.I., Increased anxiogenic effects of caffeine in panic disorders, Arch. Gen. Psyehiato'. 42 (1985) 233-243. 13 Daly, J.W. and Snyder, S.H., Adenosine receptors in the central nervous system: relationship to central action of methylxanthines, L~fe Sci., 28 (1981) 2083-2(/87. 14 Daval, J.L., Nehlig, A. and Nicolas, F., Physiological and pharmacological properties of adenosine: therapeutic implications, l,ife Sci., in press. 15 Deckert, J., Bisserbe, J.C., Klein, E. and Marangos, P.J., Adenosine uptake sites in brain: regional distribution of putative subtypes in relationship to adenosine At-receptors, .l.Neurosci., 8 (1988) 2338-2349. 16 Deckert, J., Morgan, P.J., Bisserbe, J.C., Jacobson, K.A., Kirk, K.L., Daly, J.W. and Marangos, P.J., Autoradiographic localization of mouse brain adenosine receptors with a [3H]xanthine amine congener of 1,3-dipropyl-8-phenylxanthine, J.Neurochem., 48 (1987) Suppl.1, 92. 17 Fastbom, J., Pazos, A. and Palacios, J.M., The distribution of adenosine A t receptors and 5'-nucleotidase in the brain of some commonly used experimental animals, Neuroscience, 22 (1987) 813 826. 18 Fastbom, J., Pazos, A., Probst, A. and Patacios, J.M., Adenosine A~ receptors in the human brain: a quantitative autoradiographic study, Neuroscience, 22 (1987) 827-839.
12 19 Fredholm, B.B. and Dunwiddie, T.V., How does adenosine inhibit transmitter release, Trends Pharmacol. Sci., 11988) 131/-134. 20 Green, R.D., Proudfit, H.K. and Yeung, S.M.H., Modulation of striatal dopaminergic function by local injection of 5'-N-ethylcarboxamide adenosine, Science, 218 (1982) 58-61. 21 Griebel, G., Saffroy-Spittler, M.. Misslin, R., Remmy, D. and Vogel, E., Comparison of an adenosine A 1 / A 2-receptor antagonist, COS 15943, and an Al-selective antagonist, DPCPX. P,9'chopharmacolog% 103 ( 1991 ) 54 1-544. 22 Jaarsma, D., Sehens, J.B. and Korf, J., Reduction of adenosine A~ receptors in the perforant pathway terminals in Alzheimer hippocampus, Neurosci. Lett., 121 (1991) 111 114. 23 Jansen, K.L.R., Faull, R.L.M., Dragunow, M. and Synek, B.L., Alzheimer's disease changes in hippocampal N-methyl-D-aspartate, quisqualate, neurotensin, adenosine, benzodiazepine, serotonin and opioid receptors - - an autoradiographic study, Neuroscience, 39 (1990) 613-627. 24 Jarvis, M.F. and Williams, M., Adenosine and dopamine function in the CNS, Trends Pharmacol. Sci., 8 (1987) 330-331. 25 Jarvis, M.F. and Williams, M., Direct autoradiographic localization of adenosine receptors in rat brain using the A z selective agonist (3H)-CGS 21680, Eur. J. PharmacoL, 168 (1989) 243-246. 26 Kalaria, R.N., Sromek S., Wilcox, B.J. and Unnerstal, J.R., Hippocampal adenosine A 1 receptors are decreased in Alzheimer's disease, Neurosci. Lett., 118 (1990) 257-260. 27 Klein, E., Marangos, P.J., Montgomery, P., Bacher, J. and Uhde, T.W., Adenosine receptors alterations in nervous pointer dogs: a preliminary report, Clin. NeuropharmacoL, 10 (1987) 462-469. 28 Lewis, M.E., Patel, J., Moon Edley, S.M. and Marangos, P.J.,
29
30
31
32 33
34
35 36
37
Autoradiographic visualization of A i adenosine receptol:s using N~'-cyclohexyl(3H)adenosine, Ear. Z Pharmacol., 73 {19~1) 10t)I10. Marangos, P.J., lnsel, T.R., Montgomery, f'. and Tamborska, liH Brain adenosine receptors in Maudsley reactive and non-reactive: rats, Brain Res., 421 (1987) 69-74. Nagy, J.I., La Belta, N., Buss, M. and Daddona, P.E.. hnmunohistochemistry of adenosine deaminase: implications for adenosine neurotransmission, Science, 224 (1984) 166-168. Onodera, H. and Kogure, K., Autoradiographic visualizalion of A l adenosine receptors in the gerbil hippocampus: changes in the receptor densities after transient ischemia, Brain Res., 345 (1985) 406-408. Paxinos, O. and Watson, C, The Rat Brain m Stereotaxic Coordinates, Academic Press, New York, 1982. Phillis J.W., O'Reagan, M.H., The role of adenosine in the central action of benzodiazepines, Prog. NeuropsychopharmacoL Biol. Psychiato', 12 (1988) 389-404. Schiffmann, S.N., Liebert, F., Vassart, G. and Vanderhaeghen, J.J., Distribution of adenosine A z receptor mRNA in the human brain, Neurosci. Lett.,130 (1991) 177-181. Snyder, S.H., Adenosine as a neuromodulator, Annu. Ret,. Neurosci., 8 (1985) 103-124. Van Calker, D., Muller, D.M. and Hamprecht, B., Adenosine regulates via two different types of receptors: the accumulation of cyclic AMP in cultured brain cells, J. Neurochem.. 33 11979) 999-1005. Yanik, G.M. and Radulovacki, M., REM sleep deprivation upregutates adenosine A~ receptors, Brain Res., 402 (1987) 362-364.
Brain Rc,,'ar~/J. :,~m ( l ~;92) i~ [
¢ 19t;2 Elsevier Science Publishers B.V. All lights rcsc]~'cd 11l~l}6-S~m3 t)2 5!!5il~i BRES 18352
Potential use of D P C P X as probe for in vivo localization of brain A 1 adenosine receptors J.C. Bisserbe a, O. Pascal b, j. Deckert c,, and B. Mazi~re b " lnstitut National de la Santd et de la Recherche Mddicale (INSERM), Senice Hospitalier Frdddric Joliot (SHFJ), Orsay (France), t, Sercice Hospitalier Fr~ddric Joliot (SHFJ), Orsay (France) and " Unit on Neurochemistry, National Institute of Mental Health, Bethesda MD 2089 (USA)
(Accepted 21 July 1992)
Key words: DPCPX; Adenosine A l receptor; Ex vivo autoradiography; Rat
The suitability of (3H)DPCPX (8-cyclopentyl-l,3-dipropylxanthine), a xanthine derivative, as an vivo probe for labelling adenosine A j receptors was studied in rats. [3H]DPCPX (nM) penetrated largely into the brain (0.8% of the injected dose per gram of brain tissue 5 min after injection). Brain concentrations stayed at a plateau level from 5 to 15 rain after the injection. The distribution in the different brain regions was heterogeneous with the highest amount of [3HIDPCPX in cerebellum and hippocampus and the lowest concentrations in hypothalamus and brain stem. Displacement (45-70% of total radioactivity) was obtained by the injection of 250 nM of cold DPCPX or cyclopentylxanthine, an analog of DPCPX. The ex vivo autoradiographic distribution of [3H]DPCPX was similar to the in vitro autoradiographic distribution of tritiated A x adenosine receptor ligand as [3H]CHA. These results suggest the potential use of DPCPX for further in vivo investigation of A 1 adenosine receptors with techniques such as positron emission tomography.
INTRODUCTION A d e n o s i n e , in a d d i t i o n to its f u n c t i o n in i n t e r m e d i ary m e t a b o l i s m , is, b e s i d e s G A B A , t h e m a j o r n e u r o m o d u l a t o r in m a m m a l i a n b r a i n k n o w n t o d a y t9'35. Its C N S actions a r e m a i n l y e x e r t e d via two r e c e p t o r s , A~ and A236. R e u p t a k e is b e l i e v e d to b e the r a t e - l i m i t i n g step for its inactivation 2'3. T h e actions of m e t h y i x a n thines such as caffeine a n d t h e o p h y l l i n e a r e m e d i a t e d by a n t a g o n i s m o f e n d o g e n o u s a d e n o s i n e at its r e c e p tors 13. R a d i o l a b e l l e d a d e n o s i n e a n d x a n t h i n e derivatives have b e e n utilized to visualize a n d q u a n t i f y A l r e c e p tors in r o d e n t 16JT"28m, cat 1, d o g 15 a n d also in h u m a n b r a i n s ~8. W i d e l y d i s t r i b u t e d A l r e c e p t o r s a p p e a r to m e d i a t e t h e a d e n o s i n e ' s d e p r e s s i o n o f synaptic transmission jg. A e r e c e p t o r s have also r e c e n t l y b e e n m a p p e d with t h e specific l i g a n d [ 3 H ] C G S 21680 25 a n d m o r e r e c e n t l y in h u m a n b r a i n by in situ h y b r i d i z a t i o n with the A 2 r e c e p t o r m R N A 34. T h e l i m i t e d localization o f
the A 2 r e c e p t o r to c a u d a t e , p u t a m e n a n d a c c u m b e n s a n d t h e effect o f local injection o f N E C A in c a u d a t e 2° suggest a specific role o f this r e c e p t o r in the physiology o f t h e b a s a l g a n g l i a 24. T h e a n a t o m y o f the ' a d e n o s i n e system' has b e e n c o m p l e t e d with the m a p p i n g o f the a d e n o s i n e t r a n s p o r t e r 4"5, of t h e e n z y m e s a d e n o s i n e d e a m i n a s e 3° a n d a d e n o s i n e 5 ' - n u c l e o t i d a s e 17, a n d also of a d e n o s i n e - c o n t a i n i n g n e u r o n s ~. B a s e d on a n i m a l w o r k a n d h u m a n p o s t m o r t e m studies, a d e n o s i n e has b e e n i m p l i c a t e d in the p a t h o p h y s i o l ogy a n d b i o c h e m i s t r y o f c o n d i t i o n s as diverse as anxiety a n d s l e e p d i s o r d e r s , psychosis, n e u r o d e g e n e r a t i v e p a t h o l o g y , c e r e b r a l i s c h e m i a a n d convulsions a n d also in the m e c h a n i s m o f a c t i o n o f p s y c h o t r o p i c drugs 14"33. U p r e g u l a t i o n o f a d e n o s i n e A I r e c e p t o r s has b e e n f o u n d in a n i m a l m o d e l s of anxiety 6'27'29"37. In h u m a n s caffeine has shown specific a n x i o g e n i c p r o p e r t i e s in p a n i c disord e r p a t i e n t s 7'12. A r e d u c t i o n o f a d e n o s i n e A~ r e c e p t o r s has b e e n f o u n d in several p o s t m o r t e m h u m a n b r a i n studies o f A l z h e i m e r p a t i e n t s 22'23"2~'. F u r t h e r explo-
Correspondence: J.C. Bisserbe. Present address: Inserm U 302, Pavilion Cl~rambault, H6pital La Salp~tri6re, 47 boulevard de l'H6pital, 75014
Paris, France, * Present address: Universi6its-Nervenklinik, Fiichsleinstrasse 15, 87 Wiirzburg, Germany.
ration of the role of adenosine in neuropsychiatric diseases will require direct in vivo exploration of the adenosine receptors. We therefore decided to investigate the suitability of available adenosine ligands as tools for in vivo labelling of A t adenosine brain receptors and for their potential use in adenosine receptor exploration with positron emission tomography in humans. We present here the results obtained in rats with the highly A 1 specific adenosine ligand DPCPX.
% id/g
cerebellum hippocampus striatum cortex
0,8
hypot.halamus brain stem plasma 0,6
0,4
MATERIALS
&
/
/s
IIII
AND METHODS
Determination of the ligand tissue kinetics Male Sprague-Dawley rats (200-250 g) with food and water ad libitum were used for all the experiments. Rats were injected in a tail vein with 5 - 8 /zCi of [3H]DPCPX (95 C i / m m o l A m e r s h a m ) in 0.2-0.3 ml of a 50% ethanol:saline solution. Animals were killed by decapitation at sequential times after the injection of [3H]DPCPX (2.5 min, 5 min, 10 min, 15 rain, 20 min and 30 rain). The brains were rapidly removed and dissected on ice to obtain samples of 7 anatomical regions: cerebellum, hippocampus, hypothalamus, cortex, striaturn, thalamus and brain stem. Blood was collected on ice, aliquoted, then centrifuged for 1 min, the plasma was aliquoted, then precipitated with T C A and the deproteinated plasma was also aliquoted. tleart, kidney, liver, muscle and lung tissues samples were also removed. The samples were weighed and dissolved overnight in a scintillation vial containing 1 ml of Beckman Tissue Solubilizer (BTS 450). After neutralization with acetic acid and addition of scintillation liquid (Ready-Solv Beckman) the samples were counted for 5 rain in a Packard Liquid Scintillation Counter. The amount of radioactive tracer present in each tissue at the different times was expressed as a percentage of the injected dose per gram of tissue (% i.d./g).
0,2
i
1
i
10
20
30
rain
Fig. 1. Time course of [3H]DPCPX concentration in different brain areas: brainstem, cerebellum, hypothalamus, hippocampus, striatum, cortex and in deproteinated plasma. Rats were injected in a tail vein with 5 - 8 p, Ci of [3H]DPCPX (95 C i / m m o l A m e r s h a m ) in 0.2-0.3 ml of a 50% ethanol saline solution and sacrificed at 2.5 min, 5 rain, 10 min, 20 rain and 30 rain. The brains were dissected on ice to obtain samples of the different anatomical regions the samples were weighed in scintillation vials and 1 ml of Beckman Tissue So[ubilizer (BTS 450) was added for overnight dissolution. Radioactivity was counted for 5 min in a Packard Liquid Scintillation Counter after neutralization with acetic acid and addition of scintillation liquid (Ready-Solv Beckman). The concentrations of radioactive tracer present in each tissue at the different times were expressed as a percentage of the injected dose per gram of tissue (% i d / g ) and are the mean value of 3 - 6 experiments.
Saturation and di,splacement experiments For the determination of non-displaceable radioactivity, a bolus of 250 nM of either DPCPX in a 10% ethanol isotonic saline solution or cyclopentyl theophylline (CPT) in isotonic saline with 1.5% D M S O was injected in the tail vein at different times before (saturation experiment) or after (displacement experiment) the injection of 8-10 #Ci of [~H]DPCPX (0.08-0.1 nM). For total radioactivity determination, rats were injected under identical condition with [3H]DPCPX and with the vehicle of the cold compound. The animals were killed at different times after the two injections. The time of sacrifice was selected based on [~H]DPCPX kinetic data. Samples of the different tissues were then obtained and treated as described in the above section.
In Hco autoradiography with [¢H]DPCPX Rats were sacrificed 10 rain after the injection of 10 /~Ci of [3H]DPCPX. After rapid removal, the brain was frozen in isopentane at -40°C, fixed on a microtome chuck, then cut in 20 p,m sections. The sections were fixed on microscope cover slips and apposed to a tritium-sensitive film (Ultrofilm LKB, Sweden) for 8 - 1 2 weeks. The films were developed with Kodak D-19.
Stability of/~tt/DPCPX Stability of the injection solution of [3H]DPCPX were tested using thin layer chromatography. The T L C plates were read with a static radiochromatograph reader (Chromelec) with an efficiency of 9% for tritium. Biological stability of [3H]DPCPX was also tested in plasma after chloroform extraction with 80% recovery.
RESULTS
[~H]DPCPX tissue kinetics The time course of the brain concentration of the ligand showed a rapid penetration in the brain followed by a plateau from 5 to 15 min after the injection. The ligand very largely crossed the blood brain barrier; 0.85% i.d./g (S.D. = 0.22%) was present in the cerebellum 5 min after injection. Distribution in the different brain regions was heterogeneous with the highest amount of [3H]DPCPX in the cerebellum and hippocampus and lowest concentrations in hypothalamus and brain stem (Fig. 1). These differences were observed at each time point from 5 to 30 rain. In peripheral tissues, the highest concentrations were observed in the liver with respectively 1.5% and 1% i.d./g, 5 rain and 15 min after [~H]DPCP X injection. There was no apparent accumulation of the radioactivity in any of the peripheral organs examined (Fig. 2). The b l o o d / b r a i n concentration ratio was close to 1 and the ligand concentrations in deproteinated plasma, repre-
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0,5
-
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-
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i
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i
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20
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Fig. 2. Time course of [3H]DPCPX concentrations in peripheral organs. All tissues samples were obtained during the experiment described in Fig. 1. Blood was collected on ice, plasma was separated by a 1 min centrifugation, deproteinated plasma was obtained after a protein TCA precipitation. Heart tissue samples were obtained from the ventricular part; kidney samples were cut on the cortical part of the organ; muscle samples were removed from the abdominal and thigh muscles; liver samples were obtained from two different lobes; lung tissue was removed from right and left upper and lower lobes.; blood was wiped off all tissue samples with filter paper. The samples were treated as described under Fig. 1. Concentrations are expressed as percentage of the injected dose. and are the mean value of 3-6 experiments.
senting the free [3H]DPCPX plasma concentrations, were below the brain concentration.
Saturation and displacement experiments In the saturation experiments with cold DPCPX described in Fig. 3, values for displaceable radioactivity varied largely with the different brain regions. Highest concentrations were found in the cerebellum and hippocampus with respective values of 0.41% i.d./g (S.D. = 0.02%) and 0.35% i.d./g (S.D. = 0.04%). Low-
hippocampus
codex
striatum
hypotharamus
brain stem
Fig. 3. Regional distribution of displaccablc and non-displaceahle radioactivity in selected rat brain regions in a saturation experiment. Rats were injected in the tail vein with 8 # C of [3tt]DPCPX and with 250 ~zM of cold DPCPX or an equivalent amount of vehicle (10% ethanol in isotonic saline) and were killed 10 min after the injection. The brains were then removed, dissected, tissue samples obtained from different anatomical regions were treated and radioactivity counted as described in the Methods section. The local [3H]DPCPX concentrations expressed as percentage of the injected dose (% id/g) are the mean value and standard errors obtained from two rats. Displaceable radioactivity varied from 0.42% i d / g (0.035 nM [3H]DPCPX) in the cerebellar cortex to 0.14% (0.01I nM [3H]DPCPX) in the brain stem. Deproteinated plasma concentrations at the sacrifice time in the two conditions were respectively 0.3% and 0.303% of the injected dose. The relative percentage of displaceable over non-displaceable varying from 71 to 43% arc indicated in each bar graph.
est concentrations were found in the brainstem and hypothalamus with values of 0.14% i.d./g (S.D. = 0.04%) and 0.14% i.d./g (S.D. = 0.01%). As expected, non-displaceable radioactivity concentrations were quite homogeneous among the different anatomical regions ranging from 0.15% i.d./g (S.D. = 0.(18%) in cortex to 0.19% i.d./g (S.D. = 0.08%) in brainstem. In structures with high [3H]DPCPX concentrations such as cerebellum, hippocampus, cortex or striatum displaceable radioactivity represented between 63% and 71% of the total radioactivity. Radioactivity concentra-
TABLE 1
Regiona! distribution of total and non-displaceable radioactivity in [JH]DPCPX displacement experiments with cold DPCPX or cych)pentyhheophylline Rats were injected with 250 nM of DPCPX/cyclopentyltheophylline or with the vehicles 12 min after 8-10 mCi of [3H]DPCPX and sacrificed 5 min after the cold ligand injection. Tissue samples were obtained and treated as described in Materials and Methods. Concentrations expressed in percentage of the injected dose per gram of tissue are mean and standard error values obtained with 3 rats per group in DPCPX displacement and two rats per group in cyclopentyltheophylline displacement.
Region
Radioactiuity concentration (% of injected dose / g of tissue) Displacement with DPCPX
Cerebellum Hippocampus Striatum Diencephalon Cortex Hypothalamus Brain stem
Displacement with CPT
Total R.A.
Non-disptaceable R.A.
Total R.A.
Non-displaceable R.A.
0.521 0.464 0.436 0.358 0.452 0.27 0.259
0.283 0.292 0.283 0.23 0.192 0.217 0.2
0.684 0.624 0.590 0.469 0.550 0.362 0.364
0.367 0.353 0.327 0.259 [].32 0.207 0.217
(0.027) (0.025) (0.027) (0.017) (0.021) (0.009) (0.007)
(0.045) (0.03) (0.024) (0.026) (0.028) (0.069) (0.03)
(0.008) (0.045) (0.013) (0.013) (0.016) (0.027) (0)
(0.091) (0.059) (0.054) (0.045) (0.013) (0.023) (0.034)
tions in the deproteinated plasma were identical both in presence (0.30% i.d./g) or in absence (0.303% i.d./ g) of cold DPCPX. Two comparable displacement experiments, using as cold ligand DPCPX or CPT are reported in Table I. Displaceable radioactivity ranged from 0.23% i.d./g in cerebellum to 0.6% i.d./g in brain stem when cold DPCPX was used and from 0.31% i.d./g in cerebellum to 0.14% i.d./g in brain stem when CPT was used as
cold ligand. The relative regional distribution of total and displaceable radioactivity was quite similar in the two experiments. In two other displacement experiments, CPT was injected 6 min or 30 rain after [~H]DPCPX injection, and the animals sacrificed 5 min after. In these two conditions non-displaceable radioactivity concentrations were quite identical but, as expected, values of displaceable radioactivity varied with time from 0.26%
GL
A
ML
B
(3 Fig. 4. Ex vivo autoradiographic distribution of [3H]DPCPX in representative antero-posterior sections of rat brain. Rats were injected with 10 /xCi of [3H]DPCPX alone (section A&B) or with 250 nM cold D P C P X (section C) and sacrificed after 10 min. The brains were removed, frozen and sectioned as described in the text. The identification of the anatomical structures was made with reference to the rat brain atlas of Paxinos and Watson 32. Darker areas represent regions with higher concentrations of the tritiated ligand. Abbreviations are: I, cortical layer 1; IV, cortical layer ~; C, caudate nucleus; LS, lateral septum; a, thalamic anterior nucleus; P, thalamic posterior nucleus; G, lateral geniculate body; DG, gyrus dentatus; cal, hippocampus cal area; SO, stratum oriens of the hippocampus; GL, granular layer of the cerebellum; ML, molecular layer of the cerebellum; WM, white matter; sc, superficial layer of the superior colliculus.
i.d./g in the brain stem and 0.47% i.d./g in the cerebellum at 6 rain to 0.04% i.d./g in the brain stem and 0.17% i.d./g in the cerebellum at 30 rain.
Autoradiography Ex vivo autoradiograms of [3H]DPCPX confirmed the heterogeneity of the anatomical distribution found in pharmacokinetic, saturation and displacement experiments and demonstrated discrete anatomical localization patterns in the different brain structures (Fig. 4). The typical pattern of adenosine A~ receptor distribution was observed in the cerebellum with highest densities in the molecular layer, intermediate density in the granular layer and background levels in the white matter. A heterogeneous distribution was observed in the hippocampus with higher densities in the stratum oriens and the stratum radiatum. In the cerebral cortex, layer I and IV showed higher densities of activity. The thalamic region also showed a heterogeneous distribution with higher density in the anteroventral nucleus. The lateral geniculate body and the superficial gray layer of the colliculi were also high density structures. This distribution was identical to the characteristic in vitro distribution of A 1 receptors obtained with ligands such as [3H]CHA2s. DISCUSSION In a first set of experiments we have described the tissue kinetics of [3H]DPCPX. The ligand penetrated brain tissues very extensively, since the injection of 0.1 nM of [3H]DPCPX (0.1 /zg/kg) led to a brain concentration around 1 nmol from 5 to 15 min after the injection. Brain concentrations of the tracer were above free tracer plasmatic concentrations (deproteinated plasma concentration). Assuming a linear relationship between increasing injected doses and brain concentration, a dose of 1 mg/ kg as used in behavioural experiments 2~ could lead to micromolar brain tissue concentrations. In comparison using the same experimental conditions only 0.03% (i.e. 0.05 nM brain concentration) of an injected dose of the specific adenosine A~ receptor antagonist XAC would be present in the brain 15 rain after injection. Similar values would be obtained with A1 agonists such as CHA or PIA (paper in preparation). Thus such compounds should require about a 50-fold higher dose in order to obtain a significant presence in brain tissue. A second set of experiments supported the existence of a specific in vivo binding of [3H]DPCPX to adenosine A~ receptors. First we have found [3H]DPCPX accumulated preferentially in areas known - - from in vitro binding studies - - to contain high levels of the
TABLE I1
Regional distribution oJ" ~pecific [ ¢tt]DP(TPA binding m ra~ /,rum ~:~pressed in q~ of values/or .~triatunt Ex vivo values were obtained from lhe displacement experimem described in Fig. 3. in vitro results were taken horn Bruns cl al."
Region Cerebellum Hippocampus Cortex Striatum Hypothalamus Brain stem
Specific binding (f~; value qf striatum) In vivo
In t'itro
120 109 101 100 45 45
102 1Z0 11 I I~!0 57 53
adenosine A~ receptor (e.g. cerebellum and hippocampus) and to a lower degree in areas with low densities of adenosine receptors (e.g. brain stem, hypothalamus). Further we have shown that [3H]DPCPX could be displaced by the administration of cold DPCPX and also by another potent A i adenosine antagonist: CPT "~. As expected the absolute values of non-disptaceable radioactivity (expressed in % of i.d./g) corresponding to the sum of the non-specifically bound ligand, plus the free ligand in brain tissue and the ligand present in the blood trapped into the brain tissue were quite identical in all brain regions in saturation and displacement experiments. By contrast the absolute amount of disptaceable [3H]DPCPX (specific binding) varied among brain regions, ranging from values of 0.4% of the injected dose for structures known to contain a high density of adenosine A ~ receptors such as cerebellum and hippocampus to values about 0.18% of the injected dose for structures with a low densities adenosine of A l receptors such as brainstem and hypothalamus (Fig. 3). The comparison of ex vivo regional distribution of [3H]DPCPX with the relative distribution of [3H]DPCPX from in vitro binding, reported previously by Bruns 9, presented in Table II shows similar distribution patterns. Nonetheless the differences observed between in vivo and in vitro experiments could be due to our sampling methods which required small tissue samples to facilitate dissolution and radioactive counting. Indeed for large stuctures such as cerebellum we collected only a small sample of cortical tissue rich in adenosine receptors, thus excluding cerebellar whitc matter which was devoid of adenosine receptors. Compared to in vivo binding methods, where the whole brain region is processed, our method could result in a relative adenosine receptor enrichment. In different displacement and saturation experiments, including results not presented, on the ratio of displaceable/nondisplaceable radioactivity, we observed significant variations in the values of total binding according to the
11
selected time interval between the injection of hot and cold ligand and also with the time interval between cold injection and sacrifice of the animals. The best ratios were obtained when the cold ligand was injected from 0 to 5 - 7 min after [3H]DPCPX and when the animals were sacrificed about 10 min after the injection of the cold ligand. The best ratio of specific/nonspecific radioactivity was obtained in a saturation experiment where the animals were sacrificed after the coinjection of both hot and cold DPCPX. In these conditions, displaceable radioactivity could reach 71% of total radioactivity in the cerebellum (Fig. 3). Finally, the A 1 adenosine receptor distribution revealed by [3H]DPCPX was confirmed at the histological level by ex vivo autoradiography. Indeed autoradiography results showed the pattern of distribution typical of in vitro binding studies with A l ligands 4"15'>. Particularly noticeable was the characteristic laminar distribution of [3H]DPCPX in the cerebellum with highest densities in the molecular layer, intermediate density in the granular layer and background levels in the white matter, as well as the distribution pattern observed in the hippocampus with higher densities in the stratum oriens and the stratum radiatum. In summary, [~H]DPCPX pharmacokinetics and A~ specificity, as demonstrated by in vitro studies m and our present results, indicate that DPCPX should be a suitable pharmacological tool for in vivo brain A ! r e c e p t o r blockade. The acceptable solubility of DPCPX and its very high affinity demonstrated in vitro, with a K d of 0.46 nM would further support this proposition I i. In addition we have shown that [3H]DPCPX is an appropriate ligand for ex vivo labelling of adenosine A L receptors in rodents. An important point remaining to be clarified is the metabolic stability of [3H]DPCPX. Preliminary results on thin layer chromatography analysis of plasmatic extract obtained after large [3H]DPCPX injections suggest no major metabolic degradation in the first 20 min after administration(data not presented). When considering the development of an in vivo ligand for A, adenosine receptor brain imaging methodology as positron emission tomography, the very favourable characteristics of DPCPX should be considered. These include a large penetration in the brain to obtain a good signal with a limited dose of radioactive tracer; a sizeable accumulation with a plateau of brain concentration to allow the detection of the receptorligand interaction; a low ratio blood/brain ligand concentration to avoid interference from the radioactive blood present in the brain; a specificity for the adenosine A l receptor with a good ratio specific/non-specific binding. It is thus expected that DPCPX, when appro-
priately labelled with positron emission isotope could be a good candidate ligand for in vivo exploration of adenosine A~ receptors in primates. Acknowledgements. J.C.B. held a Fellowship of the Fondation pour I'Etude du Syst~me Nerveux (Geneva, Switzerland). Thanks are due to M. Ottaviani and O. Stulzaft for technical assistance.
REFERENCES 1 Aohi, C., Development of the A i adenosine receptor in the visual cortex of cats, dark-reared and normally reared, Det'. Brain Res., 22 (1985) 125-133. 2 Barberis, C., Minn, A., Gayet, J., Adenosine transport in guinea pig synaptosomes, J. Neurochem., 362 ( 1981 ) 347-354. 3 Bender, A.S., Wu, P.H. and Phillis, J.W., The characterization of [3H]adenosine uptake into rat cerebral cortex synaptosomes, J. Neurochem., 35 (1980) 629-640. 4 Bisserbe, J.C., Deckert, J. and Marangos, P.J., Autoradiographic localization of adenosine uptake sites in guinea pig brain using [3 H] dipyridamole, Neurosci. Lett., 66 (1986) 341-345. 5 Bisserbe, J.C., Patel, J. and Marangos, P.J., Autoradiographic localization of adenosine uptake sites in the guinea pig brain using [3H]nitrobenzylthioinosine, J. Neurosci., 5 (1985) 544-55(I. 6 Boulenger, J.P., Patel, J., Post, R.M., Parma, A.M. and Marangos, P.J., Chronic caffeine consumption increases the number of brain adenosine receptors, Life Sci., 32 (1983) 1135-1142. 7 Boulenger, J.P., Uhde, T.W., Wolff, E.A. anti Post, R.M., Increased sensitivity to caffeine in patients with panic disorder: preliminary evidence, Arch. Gen. P~yehiatry. 41 (1984) 1067-1071. 8 Braas K.M., Newby A.C., Wilson, V.S. and Snyder. S.tt., Adenosine-containing neurones in the brain localized by immunocytochemistry, J. Neurosci., 6 (1986) 1952-1961. 9 Bruns, R.F., Fergus, J.H., Badger, E.W., Bristol, J.A., Santay, L.A. and Hays, S.J., PD 115,119: an antagonist ligand for adenosine A~ receptors, Nuuynyn-Sehmiedeherg's Arch. Pharrnacol., 335 (1987) 64 69. 10 Bruns, R.F., Fergus, J.H., Badger, E.W., Bristol, J.A., Santay, L.A., Hartman, J.D., Hays, S.J. and Huang, C.C., Binding of the Ai-selective adenosine antagonist 8-cyclopentyl-l,3 dipropylxanthine to rat brain membranes, Nauynyn-S~tlnliedeberg~ Arch. Pharmacol., 335 (1987) 59-63. 11 Bruns, R.F. and Fergus, J.H., Solubilities of adenosine antagonists determined by radioreceptor assay, J. Pharm. Pharmacol., 41 (1989) 590-594. 12 Charney, D.S., [teninger, G.R. and Jaflow, P.I., Increased anxiogenic effects of caffeine in panic disorders, Arch. Gen. Psyehiato'. 42 (1985) 233-243. 13 Daly, J.W. and Snyder, S.H., Adenosine receptors in the central nervous system: relationship to central action of methylxanthines, L~fe Sci., 28 (1981) 2083-2(/87. 14 Daval, J.L., Nehlig, A. and Nicolas, F., Physiological and pharmacological properties of adenosine: therapeutic implications, l,ife Sci., in press. 15 Deckert, J., Bisserbe, J.C., Klein, E. and Marangos, P.J., Adenosine uptake sites in brain: regional distribution of putative subtypes in relationship to adenosine At-receptors, .l.Neurosci., 8 (1988) 2338-2349. 16 Deckert, J., Morgan, P.J., Bisserbe, J.C., Jacobson, K.A., Kirk, K.L., Daly, J.W. and Marangos, P.J., Autoradiographic localization of mouse brain adenosine receptors with a [3H]xanthine amine congener of 1,3-dipropyl-8-phenylxanthine, J.Neurochem., 48 (1987) Suppl.1, 92. 17 Fastbom, J., Pazos, A. and Palacios, J.M., The distribution of adenosine A t receptors and 5'-nucleotidase in the brain of some commonly used experimental animals, Neuroscience, 22 (1987) 813 826. 18 Fastbom, J., Pazos, A., Probst, A. and Patacios, J.M., Adenosine A~ receptors in the human brain: a quantitative autoradiographic study, Neuroscience, 22 (1987) 827-839.
12 19 Fredholm, B.B. and Dunwiddie, T.V., How does adenosine inhibit transmitter release, Trends Pharmacol. Sci., 11988) 131/-134. 20 Green, R.D., Proudfit, H.K. and Yeung, S.M.H., Modulation of striatal dopaminergic function by local injection of 5'-N-ethylcarboxamide adenosine, Science, 218 (1982) 58-61. 21 Griebel, G., Saffroy-Spittler, M.. Misslin, R., Remmy, D. and Vogel, E., Comparison of an adenosine A 1 / A 2-receptor antagonist, COS 15943, and an Al-selective antagonist, DPCPX. P,9'chopharmacolog% 103 ( 1991 ) 54 1-544. 22 Jaarsma, D., Sehens, J.B. and Korf, J., Reduction of adenosine A~ receptors in the perforant pathway terminals in Alzheimer hippocampus, Neurosci. Lett., 121 (1991) 111 114. 23 Jansen, K.L.R., Faull, R.L.M., Dragunow, M. and Synek, B.L., Alzheimer's disease changes in hippocampal N-methyl-D-aspartate, quisqualate, neurotensin, adenosine, benzodiazepine, serotonin and opioid receptors - - an autoradiographic study, Neuroscience, 39 (1990) 613-627. 24 Jarvis, M.F. and Williams, M., Adenosine and dopamine function in the CNS, Trends Pharmacol. Sci., 8 (1987) 330-331. 25 Jarvis, M.F. and Williams, M., Direct autoradiographic localization of adenosine receptors in rat brain using the A z selective agonist (3H)-CGS 21680, Eur. J. PharmacoL, 168 (1989) 243-246. 26 Kalaria, R.N., Sromek S., Wilcox, B.J. and Unnerstal, J.R., Hippocampal adenosine A 1 receptors are decreased in Alzheimer's disease, Neurosci. Lett., 118 (1990) 257-260. 27 Klein, E., Marangos, P.J., Montgomery, P., Bacher, J. and Uhde, T.W., Adenosine receptors alterations in nervous pointer dogs: a preliminary report, Clin. NeuropharmacoL, 10 (1987) 462-469. 28 Lewis, M.E., Patel, J., Moon Edley, S.M. and Marangos, P.J.,
29
30
31
32 33
34
35 36
37
Autoradiographic visualization of A i adenosine receptol:s using N~'-cyclohexyl(3H)adenosine, Ear. Z Pharmacol., 73 {19~1) 10t)I10. Marangos, P.J., lnsel, T.R., Montgomery, f'. and Tamborska, liH Brain adenosine receptors in Maudsley reactive and non-reactive: rats, Brain Res., 421 (1987) 69-74. Nagy, J.I., La Belta, N., Buss, M. and Daddona, P.E.. hnmunohistochemistry of adenosine deaminase: implications for adenosine neurotransmission, Science, 224 (1984) 166-168. Onodera, H. and Kogure, K., Autoradiographic visualizalion of A l adenosine receptors in the gerbil hippocampus: changes in the receptor densities after transient ischemia, Brain Res., 345 (1985) 406-408. Paxinos, O. and Watson, C, The Rat Brain m Stereotaxic Coordinates, Academic Press, New York, 1982. Phillis J.W., O'Reagan, M.H., The role of adenosine in the central action of benzodiazepines, Prog. NeuropsychopharmacoL Biol. Psychiato', 12 (1988) 389-404. Schiffmann, S.N., Liebert, F., Vassart, G. and Vanderhaeghen, J.J., Distribution of adenosine A z receptor mRNA in the human brain, Neurosci. Lett.,130 (1991) 177-181. Snyder, S.H., Adenosine as a neuromodulator, Annu. Ret,. Neurosci., 8 (1985) 103-124. Van Calker, D., Muller, D.M. and Hamprecht, B., Adenosine regulates via two different types of receptors: the accumulation of cyclic AMP in cultured brain cells, J. Neurochem.. 33 11979) 999-1005. Yanik, G.M. and Radulovacki, M., REM sleep deprivation upregutates adenosine A~ receptors, Brain Res., 402 (1987) 362-364.
