Adenosine A_2A Receptors are Up‐regulated in Pick’s Disease Frontal Cortex

Abstract

Adenosine A_2A receptors (A_2AR) are highly expressed in striatum. However, they are also present in extrastriatal structures. A_2AR were studied in post‐mortem human frontal cortex from Pick’s disease (PiD) and age‐matched non‐demented controls by radioligand binding assays, Western‐blotting, real‐time PCR and adenylyl cyclase activity determination. Saturation binding assay using [³H]ZM 241385, a selective A_2A antagonist, as radioligand revealed a significant increase in total adenosine A_2AR numbers (B_max) in frontal cortex from PiD samples (191% of control B_max), suggesting up‐regulation of this receptor. A significant increase in the level of A_2AR was also detected by Western‐blotting. Furthermore, expression of mRNA coding A_2AR determined by quantitative real‐time PCR was enhanced. In agreement, stimulation of adenylyl cyclase by CGS 21680, a selective A_2A receptor agonist, was significantly strengthened. Up‐regulation of A_2B receptors and their corresponding mRNA was also observed. These results show that A_2A adenosine receptor/adenylyl cyclase transduction pathway is up‐regulated and sensitized in frontal cortex brain from PiD.

INTRODUCTION

Pick’s disease (PiD) is a fronto‐temporal dementia characterized by severe atrophy of the frontal and temporal lobes that spares the pre‐central gyrus and the posterior two‐thirds of the superior temporal gyrus. This is accompanied by marked neuron loss, mainly in the upper cortical layers, and the appearance of typical phospho‐tau‐immunoreactive intraneuronal inclusions named Pick bodies, principally in the dentate gyrus of the hippocampus, CA1 region of the hippocampus, amygdala, septal nuclei, and upper layers of the entorhinal cortex and isocortex, together with phospho‐tau‐immunoreactive thorn‐shaped and ramified astrocytes and tau‐positive bodies in oligodendroglia (4, 5, 32). Immunoelectrophoresis and Western blotting of fractions enriched with abnormal filaments have shown two main bands of 55 and 64 kDa, mostly consisting of three‐repeat tau but also of four‐repeat tau in significant amounts (5, 6, 13, 55).

Adenosine is a neuroprotective metabolite in central nervous system which modulates the release of neurotransmitters, mainly glutamate (11, 15, 41). The actions of adenosine are mediated through G‐protein‐coupled receptors, which have been classified into four types: A₁, A_2A, A_2B and A₃. A₁ and A₃ receptors inhibit adenylyl cyclase through Gi/o proteins, while A_2A and A_2B receptors stimulate adenylyl cyclase through Gs proteins. In addition, A_2B receptors are coupled to other signaling pathways, including phospholipase C activation. A₂ receptors have different distribution and affinity for adenosine. A_2A receptors have higher affinity for adenosine than A_2B receptors. A_2A receptors appear to be mainly expressed in striatum, olfactory tubercle and nucleus accumbens. A_2B receptors are more diffusely distributed in the brain, although the number of receptors is small and relatively high concentrations of adenosine are generally needed to evoke a response (18, 40).

Most of the neuroprotective effects of adenosine in the adult brain are thought to be mediated by neuronal A₁ receptors (19). Accordingly, the acute administration of A₁ receptor agonists affords brain neuroprotection and, conversely, A₁ receptor antagonists exacerbate brain damage in adult animals (34). Despite the low levels of A_2A in brain regions other than the striatum, blocking A_2A receptor function using antagonists or deleting the A_2A receptor gene results in a decrease in the extent of neuronal damage in adult animals. However, the mechanisms of this neuroprotection remain unknown (8). There exist a number of findings suggesting that A_2A receptor antagonists may have a role to play in the treatment of Parkinson’s disease (PD) (38).

Neurodegenerative diseases have been associated with a neurotoxicity elicited by glutamate. Adenosine inhibits glutamate release, thereby acting as a neuroprotector (15). It has been shown that adenosine and drugs that affect adenosine receptors may help to inhibit the progressive neurodegenerative process in dementia (43). Alterations in adenosine A₁ receptors have been described in patients with dementia, mainly those with Alzheimer disease (12, 22, 51). However, there are no data in the literature about adenosine receptors in PiD. We have analyzed adenosine A₂ receptors and their stimulatory coupling to adenylyl cyclase activity in frontal cortex from patients with PiD. Results show increased levels of A_2A receptor protein and mRNA, and enhanced function, in PiD. Moreover, A_2B receptors are also increased in PiD cases as compared with age‐matched controls.

MATERIALS AND METHODS

Materials.  [³H]adenosine 3′,5′‐cyclic phosphate ([³H]cAMP 27.4 Ci/mmol) was from PerkinElmer (Madrid, Spain) and [2‐³H](4‐(2‐[7‐amino‐2(2‐furyl)[1,2,4] triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino] ethyl]phenol) ([³H]ZM 241385 27.4 Ci/mmol) from Tocris (Bristol, UK). Anti‐A_2A and Anti‐A_2B antibodies were from Santa Cruz (Madrid, Spain) and Abcam (Cambridge, UK), respectively. Guanosin triphosphate was purchased from Roche (Barcelona, Spain). Calf intestine adenosine deaminase (ADA), 2‐[4‐[(2‐ carboxyethyl)phenyl]ethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) and theophylline were from Sigma (Madrid, Spain). All other reagents were of analytical grade and obtained from commercial sources.

Tissue samples.  Brain samples were obtained from the brain banks of the Institute of Neuropathology and the University of Barcelona‐Clinic Hospital following the guidelines of the local ethics committees. The brains of four patients with PiD and five age‐matched controls were obtained from 1 to 6 h after death, and were immediately prepared for morphological and biochemical studies. The cases with PiD were two men and two women aged 65, 68, 71 and 66 years old, with sporadic fronto‐temporal dementia and severe fronto‐temporal lobar atrophy on neuroimaging studies (CT and MRI). The fresh brain weights were 950, 1000, 850 and 900 g. At autopsy, half of each brain was fixed in formalin, while the other half was cut in coronal sections 1 cm thick, frozen on dry ice and stored at −80°C until use. For diagnostic morphological studies, the brains were fixed by immersion in 10% buffered formalin for 2 or 3 weeks. The neuropathological study was carried out on formalin‐fixed, paraffin‐embedded samples of the frontal (area 8), primary motor, primary sensory, parietal, temporal superior, temporal inferior, anterior cingulate, anterior insular, and primary and associative visual cortices; entorhinal cortex and hippocampus; caudate, putamen and pallidum; medial and posterior thalamus; subthalamus; Meynert nucleus; amygdala; midbrain (two levels), pons and medulla oblongata; and cerebellar cortex and dentate nucleus. De‐waxed sections, 5 µm thick, were stained with hematoxylin and eosin, and Klüver Barrera, or processed for immunohistochemistry following the streptavidin LSAB method (Dako, Dakopats, Barcelona, Spain). After incubation with methanol and normal serum, the sections were incubated with one of the primary antibodies at 4°C overnight. Antibodies to phosphorylated neurofilaments of 170 kDa or 200 kDa (clones BF10 and RT97, Boehringer‐Mannheim, Barcelona, Spain) were used at dilutions of 1:100 and 1:50, respectively. Antibodies to glial fibrillary acidic protein (GFAP, Dako, Dakopats, Barcelona, Spain), βA4‐amyloid (Boehringer‐Mannheim, Barcelona, Spain) and ubiquitin (Dako, Dakopats, Barcelona, Spain) were used at dilutions of 1:250, 1:50, and 1:200, respectively. Antibodies to α‐synuclein (Dako, Dakopats, Barcelona, Spain) were used at a dilution of 1:100. Antibodies to pan‐tau (Sigma, Madrid, Spain) were used at a dilution of 1:100. In addition, the following phospho‐specific tau rabbit polyclonal antibodies were used: Thr181, Ser199, Ser202, Ser214, Ser231, Ser262, Ser396 and Ser422 (all of them from Calbiochem, VWR, Barcelona, Spain). These antibodies were used at a dilution of 1:100, excepting anti‐phospho‐tauThr181, which was used at a dilution of 1:250. Following incubation with the primary antibody, the sections were incubated with LSAB for 1 h at room temperature. The peroxidase reaction was visualized with 0.05% diaminobenzidine and 0.01% hydrogen peroxide. Neuropathological findings were characteristic of PiD (14, 32). Neurofibrillary tangles, amyloid plaques and α‐synuclein inclusions were absent in all cases.

Control cases were three men and two women (70, 62, 69, 71 and 68 years old) with no neurological disease. The neuropathological examination was carried out in similar corresponding sections using the same immunohistochemical methods. The nine cases (controls and PiD cases) were processed for all the biochemical studies and morphological studies.

Plasma membrane isolation.  Plasma membranes from frontal cortex samples were isolated as described previously (9). Samples were homogenized in 20 volumes of isolation buffer (50 mM Tris‐HCl, pH 7.4 containing 10 mM MgCl₂ and protease inhibitors) in Dounce homogenizer (10 × A, 10 × B). After homogenization, brain preparations were centrifuged for 5 minutes at 1000 g in a Beckman JA 21 centrifuge. Supernatant was centrifuged for 20 minutes at 27 000 g and the pellet was finally resuspended in isolation buffer. Protein concentration was measured by the method of Lowry, using bovine serum albumin as a standard.

[ ³H]ZM 241385 binding assays to plasma membranes.  Binding assays were performed as follows. Plasma membranes were incubated with 5 U/mg ADA in 50 mM Tris, 2 mM MgCl₂, 100 mM NaCl, pH 7.4, for 30 minutes at 25°C, in order to eliminate endogenous adenosine from membrane preparations. Then, plasma membranes (100 µg of protein) were incubated with [³H]ZM 241385 for 2 h at 25°C. Saturation assays were carried out at different [³H]ZM 241385 concentrations (0.5–50 nM) using 5 mM theophylline to obtain nonspecific binding. Binding assays were stopped by rapid filtration through Whatman GF/B filters, and then immediately washed and transferred to vials to count the radioactivity.

Determination of adenylyl cyclase activity.  Adenylyl cyclase activity was determined in brain plasma membranes as previously described (31), with several modifications. Assay was performed with 15–20 µg of protein in a final volume of 0.25 mL of 50 mM Tris‐HCl pH 7.4, 5 mM MgCl₂, 1 mM DTT, 1 mg/mL BSA, 1 mg/mL creatine kinase, 10 mM creatine phosphate and 0.1 mM Ro 20‐1724 (specific phosphodiesterase inhibitor). Plasma membranes previously incubated with ADA (5 U/mg protein) in order to remove endogenous adenosine were preincubated for 15 minutes at 37°C in the absence and in the presence of 1 mM CGS 21680. Reaction was started by the addition of 200 µM ATP and incubation at 37°C for 10 minutes. Reaction was stopped by boiling samples and centrifuging at 12 000 g for 4 minutes. Twenty microliters of supernatant was used to determine cAMP accumulation. Samples were incubated with 0.25 pmol of [³H]cAMP and 6.25 µg PKA in a final volume of 200 µL of buffer assay (Tris‐HCl 50 mM pH 7.4, EDTA 4 mM) for 2–8 h at 4°C. Standard samples were prepared in the same buffer in the range of concentrations of from 0 to 16 pmol. Reaction was stopped by rapid filtration through Whatman GF/B filters, followed by washing with ice‐cold buffer. Filters were then counted in a Microbeta Trilux (Perkin Elmer, Madrid, Spain) liquid scintillation counter.

Western‐blotting assays.  Plasma membranes from frontal cortex were subjected to electrophoresis on 10% polyacrylamide gels in the presence of 10% SDS, electrophoretically transferred to nitrocellulose and blocked for 1 h with 5% (w/v) non‐fat skimmed milk in PBS (pH 7.2). Blots were probed with the commercial antisera anti‐A_2A (1:500), anti‐A_2B (1:500) and anti‐β‐actin (1:2000). After washing with 0.3% Tween 20 in PBS, a second goat anti‐rabbit antiserum coupled to horseradish peroxidase (BioRad, Madrid, Spain) was added (1:2000 in PBS). After washing, specific bands corresponding to A_2A and A_2B receptors were visualized using the ECL chemiluminescent reagent kit from Amersham (Madrid, Spain) and quantified by densitometry, using a GS‐690 densitometer from BioRad (Madrid, Spain).

Preparation of Total RNA and cDNA.  Total RNA was extracted using an ABI 6100 Nucleic Acid PrepStation according to the manufacturer’s protocol. All chemicals for the ABI 6100 were purchased from Applied Biosystems (Madrid, Spain). Total RNA from frontal cortex was isolated and stored at −80°C individually. The purity of RNA was assessed by absorption at 260 and 280 nm (values of the ratio of A₂₆₀/A₂₈₀ of 1.9–2.1 were considered acceptable). RNA concentrations were determined from the A₂₆₀. One microgram of total RNA was reverse‐transcribed using Applied Biosystems High‐Capacity cDNA Archive Kit.

Quantitative real‐time RT‐PCR Analysis.  Quantitative real‐time RT‐PCR analysis (21) was performed with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan^® universal PCR master mix according to the manufacturer’s specifications (Applied Biosystems Inc., Foster City, CA, USA) for A_2A, A_2B and β‐actin genes, for which validated TaqMan Gene Expression Assays are available. The TaqMan probes and primers for A_2A (Hs00169123_m1), A_2B (Hs00386497_m1) and β‐actin (Hs99999903_m1) were assay‐on‐demand gene expression products (Applied Biosystems, Madrid, Spain). The TaqMan^® primer and probe sequences are packaged together in a 20× solution. The sequences are proprietary, so they are not available. Human β‐actin gene was used as endogenous control. The gene‐specific probes were labeled by using reporter dye FAM. A nonfluorescent quencher and the minor groove binder were linked at the 3′ end of the probe as quenchers. The thermal cycler conditions were as follows: hold for 20 s at 95°C, followed by two‐step PCR for 40 cycles of 95°C for 3 s, followed by 60°C for 30 s. Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems, Madrid, Spain) according to the 2^−ΔΔCt method. Briefly, expression results of a gene were normalized to internal control β‐actin and relative to a calibrator, consisting of the mean expression level of the corresponding gene in control samples as follows: 2^−ΔΔCt= 2^{−[(Ct receptor gene − Ct actin) patient −(Ct receptor gene − Ct actin) calibrator]}. The results from three independent repeat assays, performed in different plates each using different cDNAs from the patients analyzed, were averaged to produce a single mean quantity value for each mRNA for each patient.

Statistical and data analysis.  Data statistical analysis was performed using Student t‐test. Differences between mean values were considered statistically significant at P < 0.05. The binding data were analyzed with the GraphPad Prism 3.03 program (GraphPad Software, San Diego, CA, USA).

RESULTS

General neuropathological findings.  The four cases of PiD showed the same neuropathological findings. Marked neuronal loss accompanied by severe astrocytic gliosis was found in the frontal and temporal cortices. Reduced myelin and myelin pallor, as seen with Klüver‐Barrera, was found in the white matter of the frontal and temporal lobes. Changes were more marked in cases with severe atrophy (cases with brain weights of 850 and 900 g), whereas neuron loss and gliosis was less pronounced in the other cases. Pick bodies, as visualized with immunohistochemistry to phosphorylated neurofilament epitopes, pan‐tau and ubiquitin antibodies, were observed in the dentate gyrus, CA1 area of the hippocampus, subiculum, entorhinal cortex, and upper and inner layers of the frontal and temporal cortex (9). No abnormalities were seen in the occipital cortex (primary visual cortex and neighboring association areas). Phospho‐tau inclusions were better visualized with specific anti‐phospho tau antibodies including Pick bodies and diffuse deposits in the cytoplasm of neurons, neuropil threads, astrocytic inclusions, and coiled bodies in oligodendrocytes. Special attention was paid to rule out the presence of neurofibrillary tangles and β‐amyloid deposits in these cases. Lewy bodies, as revealed with anti‐α‐synuclein antibodies, were absent. This may explain the limited number of cases in this study as no cases with combined pathology were included in the present series.

Age‐matched control cases showed no morphological abnormalities. Hyper‐phosphorylated tau inclusions, α‐synuclein deposits and β‐amyloid plaques were absent in every control case.

Adenosine A_2A receptors in PiD by radioligand binding assays.  Adenosine A_2A receptors were studied in human brain frontal cortex from control and PiD cases by radioligand binding assay using the selective A_2A antagonist [³H]ZM 241385 as radioligand. Non‐linear regression analysis of binding data indicated that a single binding model fit better than a double binding model, with a total receptor number of 259.4 ± 18.6 (5) fmol/mg protein and a K_d value of 24.2 ± 1.3 (5) nM in control samples. Adenosine A_2A receptors were significantly increased (191% of controls) in samples from PiD brain, suggesting the up‐regulation of these receptors. K_d value was also increased in PiD (194% of controls, suggesting a decrease in the receptor affinity (Figure 1).

Figure 1.

Adenosine A_2A receptor detection by radioligand binding assay. Saturation curves of [³H]ZM 241385 binding to plasma membranes were performed by incubation of 100 µg of protein from control and Pick brains with increasing concentrations of the radioligand, as described in Materials and methods, after pre‐incubation with adenosine deaminase in order to remove endogenous adenosine. Total receptor number (B_max) and receptor affinity (K_d) determined by Scatchard and nonlinear regression analysis are shown in the inset. Data are mean ± SEM of control and Pick’s disease cases performed in triplicate using plasma membrane isolations from different samples. ***P < 0.001 significantly different from control values.

Adenosine A_2A and A_2B receptors in PiD by Western‐blotting.  Adenosine A_2A and A_2B receptor levels in control and PiD frontal cortex samples were also analyzed by Western‐blot using specific antibodies. As can be observed in Figure 2, adenosine A_2A receptors were significantly increased in membranes from PiD in agreement with the increase detected by binding assays. Moreover, a narrow band corresponding to A_2B receptor was observed in control samples which were enhanced in PiD samples.

Figure 2.

Western‐blotting of adenosine A_2A and A_2B receptors. One hundred micrograms of protein was subjected to SDS/PAGE, transferred electrophoretically to nitrocellulose and probed with antisera anti‐A_2A (panel A) or anti‐A_2B (panel B), as described in Materials and methods. For the control of protein loading membranes were incubated with anti‐β‐actin. Data are means ± SEM of four Pick’s disease (PiD) and five control cases. Inset shows bands corresponding to a representative experiment. **P < 0.01 and ***P < 0.001 significantly different from control value. Mw, molecular weight marker.

Adenosine A_2A receptor/adenylyl cyclase coupling in PiD.  To assess whether variations observed in adenosine A_2A receptors affect their functionality we determined the stimulatory effect exhibited by CGS 21680, a selective adenosine A_2A receptor agonist, on adenylyl cyclase activity, by quantification of cAMP levels. As Figure 3 shows, a significant increase in the CGS 21680‐stimulated adenylyl cyclase was observed in PiD cases (P < 0.05) suggesting a sensitization of the A_2A/AC system.

Figure 3.

Adenylyl cyclase activity in brain plasma membranes from control and Pick’s disease cases. Fifteen to twenty micrograms of plasma membranes, previously treated with adenosine deaminase, was used to determine basal cAMP level and stimulation after incubation with 1 mM CGS 21680 (selective A_2A receptor agonist). Basal cAMP level was 123.6 ± 13.3 and 92.0 ± 7.5 pmol/mg protein in control and PiD samples, respectively. Data are means ± SEM of experiments performed in triplicate using membrane preparations from four Pick’s disease and five control cases. *P < 0.05 significantly different from control value.

Expression of gene coding adenosine A_2A and A_2B receptors in PiD.  To determine whether increased adenosine A_2A and A_2B receptor levels was due, at least in part, to enhanced gene transcription, we performed quantitative real‐time PCR assays with total RNA isolated from controls and PiDs. Results showed a significant increase in mRNA expression of both A_2A and A_2B receptors (Figure 4), suggesting increased rate of gene transcription as responsible mechanism in the variation on A_2A and A_2B levels detected in PiD cases.

Figure 4.

Real‐time analysis of A_2A and A_2B mRNA expression. Expression of adenosine A₂ receptors mRNA in human cortex brain (Brodmann area 8) in control and Pick’s disease (PiD) cases as determined by real‐time TaqMan® PCR using subtype‐specific primers and probes. Values were normalized for β‐actin relatively to a calibrator, consisting of the mean expression level of the corresponding gene in control samples. Data are represented as means ± SEM of four PiD and five control cases, after three independent experiments, each using different cDNA preparations. *P < 0.05 significantly different from control value.

DISCUSSION

Little is known about modulation of receptors in PiD. The number of muscarinic acetylcholine receptors is either preserved or reduced to some extent, although ChAT activity, which indicates presynaptic cholinergic innervation, is preserved (20, 54). In contrast, m₂ muscarinic receptors have been shown to be higher in atypical PiD than in controls (35). Metabotropic glutamate receptors are also impaired in the cerebral cortex in PiD (10). In the present work, we provide evidence that adenosine A_2A and A_2B receptors are up‐regulated, and the stimulatory pathway of adenosine mediated by A_2A receptors is sensitized in cerebral cortex from PiD cases.

Only a few studies have detected, characterized and examined the distribution of adenosine A_2A receptors in human brain. Using autoradiographic studies it has been shown that adenosine A_2A receptors are present only in the putamen, nucleus accumbens, olfactory tubercle and lateral part of the globus pallidus (33). However, studies using membrane preparations have demonstrated the presence of adenosine A_2A receptors in other structures such as cerebral cortex, thalamus and hippocampus (23, 25, 53). The presence of A_2A receptors in extrastriatal structures has also been reported by quantitative autoradiography, demonstrating that distribution of adenosine A_2A receptors in human brain is more widespread than previously thought (50). Here we have confirmed the presence of A_2A receptors in frontal cortex from human brain by Western‐blotting and real‐time PCR assays. Moreover, we have quantified these receptors in membrane from human brain frontal cortex by radioligand binding assay using [³H]ZM 241385. This selective A_2A receptor antagonist (37, 39) is considered a highly suitable ligand for A_2A receptor binding analysis and characterization in membrane preparations (2, 52). Specific [³H]ZM 241385 binding sites found in cortex are completely lost in adenosine A_2A receptor knockout mice (28). However, [³H]ZM 241385 has shown moderate affinity for A_2B receptors when used to label recombinant human A_2B receptors in cells which do not express A_2A receptors, such as HEK‐293 and CHO cells (24, 36).

Binding parameters (K_d and B_max) obtained here using [³H]ZM 241385 show similarity to those obtained previously in human striatal membranes using [³H]CGS 21680, a selective A_2A agonist (25). These authors also found that A_2A receptors are present in the human cortex, albeit at a much lower density than in the striatum. [³H]CGS 21680 was also used by Wan and co‐workers to determine, by saturation studies in human basal ganglia, a single class of high‐affinity binding sites with values for K_d of 22 ± 0.5 nM and B_max of 444 ± 63 fmol/mg protein in the presence of exogenously added ADA (53). Adenosine is known to be abundant in brain and it is expected that it remains bound to the A_2A receptor in high‐affinity state during the membrane preparation as well (52).

Adenosine is particularly important in excitable tissues such as brain, in which it reduces cellular activity by coupling energy consumption and energy supply (17). The neuroprotective effects of adenosine are mediated through A₁ receptor activation which results in the inhibition of glutamate release and reduction of its excitatory effects at a postsynaptic level (1, 42, 49). An increase in extracellular adenosine level can result from both physiological and pathological conditions. In many diseases, extracellular adenosine concentrations can rise through the regulation of enzymes involved in the intracellular metabolism of adenine nucleotides. Thus, disorders in which the energy requirements of the brain surpass its ability to synthesize ATP increase adenosine release. This can lead to adenosine concentrations 100 times higher than normal in ischemia, hypoxia and oxidative stress (29, 44). Higher adenosine concentration is responsible for the down‐regulation of adenosine receptors by overexposure to the agonist, as has been reported (30, 45, 46). Conversely, low agonist levels can elicit up‐regulation of specific receptors. The adenosine level seems to be altered in other neurodegenerative diseases as well. A significant decrease in the plasma concentration of adenosine in patients with AD has been described (48). Moreover, adenosine and pharmaceutical agents that raise the extracellular adenosine concentration have been suggested as reinforcements to the conventional treatment with acetylcholinesterase inhibitors in AD (16, 47). Therefore, the up‐regulation of adenosine A₂ receptors could be the response to decreased endogenous adenosine levels in PiD.

Alterations in adenosine A₂ receptors have been previously described in human brain with other neurodegenerative diseases. A_2A receptors became detectable in glial cells in the hippocampus and cerebral cortex of AD patients but not in controls (3). While in PD the density of A_2A binding sites was comparable to that seen in control cases, density values of A₂ sites were dramatically decreased in the basal ganglia of patients with Huntington’s chorea (33). In contrast, A_2A receptor mRNA level and [³H]SCH 58261 specific binding to adenosine A_2A receptors were increased in the external globus pallidus of PD patients compared with controls regardless of the dyskinesigenic response to Levodopa (7). Accordingly, adenosine A_2A receptor antagonists have been suggested as potent therapeutic agents for the treatment of PD (27, 38). Moreover, adenosine receptor antagonists have been proposed as cognitive enhancers, based on the facilitatory effects of these compounds on LTP as well as on behavioral models of learning and memory, and the known effects of caffeine in humans (8, 26). Similarly, these antagonists could be also useful in treating PiD.