Caffeine and adenosine A 2a receptor antagonists prevent β-amyloid (25–35)-induced cognitive deficits in mice

Caffeine and adenosine A 2a receptor antagonists prevent β-amyloid (25–35)-induced cognitive deficits in mice

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  This article was srcinally published in a journal published byElsevier, and the attached copy is provided by Elsevier for theauthor’s benefit and for the benefit of the author’s institution, fornon-commercial research and educational use including withoutlimitation use in instruction at your institution, sending it to specificcolleagues that you know, and providing a copy to your institution’sadministrator.All other uses, reproduction and distribution, including withoutlimitation commercial reprints, selling or licensing copies or access,or posting on open internet sites, your personal or institution’swebsite or repository, are prohibited. For exceptions, permissionmay be sought for such use through Elsevier’s permissions site at:     A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y Caffeine and adenosine A 2a   receptor antagonists prevent   β -amyloid(25 – 35)-induced cognitive deficits in mice Oscar P. Dall'Igna  a  , Paulo Fett   a  , Marcio W. Gomes  a  , Diogo O. Souza  a  ,Rodrigo A. Cunha  b, ⁎ , Diogo R. Lara  c a   Departamento de Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil   b Centro de Neurociências de Coimbra, Instituto de Bioquímica, Faculdade de Medicina, Universidade de Coimbra, Portugal  c  Departamento de Ciências Fisiológicas, Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil  Received 5 May 2006; revised 27 July 2006; accepted 10 August 2006Available online 27 September 2006 Abstract Consumption of caffeine, an adenosine receptor antagonist, was found to be inversely associated with the incidence of Alzheimer's disease.Moreover, caffeine protects cultured neurons against   β -amyloid-induced toxicity, an effect mimicked by adenosine A 2A  but not A 1  receptor antagonists. We now tested if caffeine administration would prevent  β -amyloid-induced cognitive impairment in mice and if this was mimicked byA 2A  receptor blockade. One week after icv administration of the 25 – 35 fragment of   β -amyloid (A β , 3 nmol), mice displayed impaired performance in both inhibitory avoidance and spontaneous alternation tests. Prolonged treatment with caffeine (1 mg/ml) had no effect alone but  prevented the A β -induced cognitive impairment in both tasks when associated with acute caffeine (30 mg/kg) 30 min treatment before A β administration. The same protective effect was observed after subchronic (4 days) treatment with daily injections of either caffeine (30 mg/kg) or the selective adenosine A 2A  receptor antagonist SCH58261 (0.5 mg/kg). This provides the first direct   in vivo  evidence that caffeine and A 2A receptor antagonists afford a protection against A β -induced amnesia, which prompts their interest for managing Alzheimer's disease.© 2006 Elsevier Inc. All rights reserved.  Keywords:  Adenosine; Caffeine; A 2A  receptor;  β -amyloid; Alzheimer's disease; Memory; Neuroprotection Introduction Caffeine, the most widely consumed psychoactive drug,acts as an adenosine receptor antagonist at non-toxic doses(Fredholm et al., 1999). Some of the most evident effects of caffeine, such as its psychomotor effects (e.g. Svenningssonet al., 1997) or its memory enhancing effects (e.g. Prediger et al., 2005), are now recognized to be due to the ability of caffeine to antagonize adenosine A 2A  receptors. Interestingly,the blockade of A 2A  receptors has consistently been foundto afford neuroprotection against different brain insults, aneffect mimicked by caffeine (reviewed in Cunha, 2005). In  particular, caffeine consumption has been found to beinversely correlated with the incidence of Alzheimer'sdisease (AD) (Maia and de Mendonça, 2002). This is in agreement with our findings showing that caffeine andselective A 2A  receptor antagonists afford a robust protectionagainst   β -amyloid peptide (A β ) toxicity in cerebellar neuroncultures (Dall'Igna et al., 2003). In fact, soluble forms of A β  are considered the most likely culprit for the earlydevelopment of AD (Hardy and Selkoe, 2002). Accordingly, cerebral microinjection of A β  causes amnesia and isconsidered a suitable animal model to test new protectivestrategies eventually relevant to manage the early phases of AD (Harkany et al., 1999). Thus, we now used an  in vivo model of centrally administered A β  in mice to test if acuteand/or more prolonged treatment with caffeine or an A 2A receptor antagonist could prevent the A β -induced cognitivedeficit in inhibitory avoidance and spontaneous alternationtasks. Experimental Neurology 203 (2007) 241 –  ⁎ Corresponding author. Center for Neuroscience of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra,Portugal.  E-mail address:  (R.A. Cunha). URL:  (R.A. Cunha).0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.expneurol.2006.08.008     A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y Materials and methods  Animals Experiments were performed with male adult mice (CF1strain) maintained in our own animal facilities under controlledenvironment (23±2°C, 12 h-light/dark cycle, free access tofood and water) until 3 – 4 months old (35 – 45 g). All behavioralexperiments were conducted between 10:00 a.m. and 2:00 p.m.  Drugs and administration procedures The  β -amyloid (25 – 35) peptide fragment (A β ) wasdissolved in bidistilled water at a concentration of 2 mg/mland stored at  − 20°C until use. A β (1 mg/ml) was then incubatedfor 4 days at 37°C to allow the formation of birefringent fibril-like structures (Maurice et al., 1996) before intracerebroven-tricular (icv) administration. Mice were anesthetized with60 mg/kg of thiopental and placed in a stereotaxic frame. A28-gauge 5 mm-long stainless-steel needle was insertedunilaterally 1 mm lateral to bregma, 1 mm posterior and2.5 mm deep from the pial surface (Paxinos and Franklin,2001). A β  or vehicle (3  μ l) was delivered gradually withinapproximately 10 s. Correct icv administration was confirmedin preliminary experiments by injecting methylene blue intomice lateral ventricles. Control animals were injected icv withthe same amount of distilled water (3  μ l).Caffeine treatment consisted of either acute, subchronic,more prolonged or combined prolonged and acute treatments.Acute treatment was a single intraperitoneal (ip) administrationofcaffeineatthedosesof30or80mg/kg(10ml/kg,dissolvedinsaline), 30minbefore A β administration. Thedoseof30mg/kg,correspondingtotheequivalentof4 – 6cupsofcoffeeinhumans,causes the maximal behavior effects in rodents (Fredholm et al.,1999), whereas the dose of 80 mg/kg is still below toxic dosageand is associated with higher antagonism of adenosine A 2A receptors (Quarta et al., 2004; Solinas et al., 2002). Prolonged caffeine treatment was achieved as previously described(Ciruela et al., 2006; Quarta et al., 2004) by supplying caffeine (1 mg/ml) through the drinking solution for 12 days, with A β administration on day 7. The combined prolonged and acutetreatments consisted of 12 days of free access to the caffeine(1 mg/ml) drinking solution for 12 days and administration of caffeine(30mg/kg)30minbeforeA β administration.Thistakesadvantage of the ability of prolonged caffeine treatment todesensitize A 1  receptor-mediated responses while increasing theability of caffeine to block A 2A  receptors (Karcz-Kubicha et al.,2003; Quarta et al., 2004). Subchronic caffeine treatment  consisted in a daily administration of caffeine (30 mg/kg, ip)from 2 days before until 1 day after A β administration (4 days).The A 2A  receptor antagonist SCH58261 (0.5 mg/kg) wasadministered subchronically as described for caffeine, i.e. it wasadministeredipdailyfrom2daysbeforeuntil1dayafterA β administration (4 days). The dose of SCH58261 used is withinthe range of doses peripherally administered that have beenshowntoaffordneuroprotection againstdifferent brain insults  invivo  (consult  Cunha, 2005).In all experiments, we included four groups of animals: (1)control (sham-operated and water/saline treated); (2) A β -treated (icv injection of A β  and water/saline treated); (3) A β -treated together with the different treatments with caffeine or SCH58261; (4) sham-operated and treated with caffeine or SCH58261. As the combination of prolonged and acutecaffeine treatment failed to affect cognitive performance incontrol mice, we did not test the individual effects of only prolonged or only acute caffeine treatments in control mice.Furthermore, using the same protocol of A β  injection, wehave shown that locomotion was not altered (Dall'Igna et al.,2004), so we have not shown these control data again.Locomotion is also not expected to change due to caffeine or SCH58261 treatment as they were administered only aroundthe time of A β  injection, but not when behavioral tests were performed (i.e. at least 3 days after the last administration of adenosine receptor antagonists).  Inhibitory avoidance The inhibitory avoidance is a classical test; mice areshocked when leaving a platform in a training session makingthem more prone to remain in the platform during asubsequent test session. We have previously used this test to monitor aversive memory in rodents (de Oliveira et al.,2005; Kazlauckas et al., 2005). Mice performance in the step-down inhibitory avoidance was examined 9 days after A β administration. The training apparatus is a 50×25×25 cm plastic box with a 2-cm-high, 4×6-cm-wide platform at the box center. The floor of the apparatus was made of parallel0.1-cm caliber stainless steel bars spaced 1.0 cm apart. In thetraining session, the mouse was placed on the platform andlatency to step-down the four paws on the grid was measured;upon stepping down, the mouse received a 2 s intermittent foot shock (three 0.5 s shocks, 0.2 mA, with a 0.25 s interval between them). Mice step-down latency was measured in atest session 24 h after training (equal to training sessionexcept for the absence of shock), keeping a ceiling time of 180 s. Spontaneous alternation The Y-maze is an ancillary behavioral test that allowsevaluating cognitive searching behavior, although it obviouslydoes not allow isolating memory performance (reviewed inHughes, 2004). This test evaluates the searching behavior of animals rated as their spontaneous alternation in a Y-mazecomposed of 3 arms (each with 30 cm long, 20 cm height and6 cm wide) converging to an equal angle. Eight days after distilled water or A β icv administration, each mouse was placedat the end of one arm and allowed to freely move through themaze during 8 min. The series of arm entries was recordedvisually. An alternation was defined as entries in all three armson consecutive occasions. The percentage of alternation wascalculated as total of alternations/(total arm entries − 2),according to Maurice et al. (1996). Evaluation was performedunder blind conditions to different treatments. 242  O.P. Dall'Igna et al. / Experimental Neurology 203 (2007) 241  –  245     A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y Statistical analysis Step-down latency is expressed as medians±interquartileranges since, despite being a continuous variable, valuesdistribute in a non-normal pattern due to a fixed limit or cut-off time (ceiling time of 180 s). To determine differences amongthe tested groups, step-down latency values were comparedusing the Kruskal – Wallis non-parametric test followed byMann – Whitney test and Bonferroni correction for   α  valueadjustment. Spontaneous alternation values are presented asmean±SEM and were analyzed with one-way ANOVAfollowed by Duncan's post hoc test. A value of   p <0.05 wasconsidered statistically significant. Results As previously reported (Maurice et al., 1996), central administration of   β -amyloid (25 – 35) peptide (A β ) caused animpairment of mice performance in both inhibitory avoidance(Fig. 1a) and spontaneous alternation tasks (Fig. 1 b). Acute administration (ip, 30 min before A β administration) of caffeineat the dose of 80 mg/kg completely prevented the A β -inducedamnesic effects in both tasks, whereas the dose of 30 mg/kg wasdevoid of effects (Fig. 1). Since acute administration of caffeine(up to 50 mg/kg) mostly acts by antagonism of A 1  receptorswhereas higher, but still non-toxic doses (<100 mg/kg, seeFredholm et al., 1999), of caffeine applied acutely favor A 2A receptor antagonism (Karcz-Kubicha et al., 2003; Quarta et al.,2004; Solinas et al., 2002), these results suggest a preferential Fig. 1. Caffeine prevents  β -amyloid effects on (A) inhibitory avoidance and(B) spontaneous alternation tasks. Mice were treated (3 nmol, icv) with  β -amyloid (25 – 35) fragment (+) or vehicle ( − ). Caffeine acute treatment consisted of ip administration of doses of 0, 30 or 80 mg/kg, 30 min before  β -amyloid icv administration. Prolonged treatment (chronic) consisted of caffeineadministration in drinking water at a dose of 1 mg/ml for 6 days before until6 days after   β -amyloid administration. (A) Step-down latency is shown asmedian±interquartile range and analyzed by the Kruskal – Wallis non- parametric test, followed by Mann – Whitney test. No significant modificationof performance in the training trial was observed between any of the groups of mice (not shown). (B) Spontaneous alternation is expressed as mean±SEMand analyzed by one-way ANOVA followed by Duncan post hoc test. Data are n =25 – 26 for control and amyloid-injected groups and  n =9 – 14 for other groups. *  p <0.05 vs. control (sham operated). #  p <0.05 vs.  β -amyloid.Fig. 2. Subchronic treatments with caffeine or with the selective A 2A  receptor antagonist SCH58261 prevent   β -amyloid-induced impairment of spatialmemory performance. Mice were treated (3 nmol, icv) with  β -amyloid (25 – 35) fragment (+) or vehicle ( − ). Subchronic treatment with caffeine (30 mg/kg)or SCH58261 (0.5 mg/kg) consisted of four daily ip injections from 2 days before until 1 day after   β -amyloid administration. (A) Step-down latency isshown as median±interquartile range and analyzed by the Kruskal – Wallis non- parametric test, followed by Mann – Whitney test. No significant modification of  performance inthe trainingtrial wasobservedbetweenanyof the groupsofmice(not shown). (B) Spontaneous alternation is expressed as mean±SEM andanalyzed by one-way ANOVA followed by Duncan post hoc test. Data are n =25 – 26 for control and amyloid-injected groups and  n =9 – 14 for other groups. *  p <0.05 vs. control (sham operated). #  p <0.05 vs.  β -amyloid.243 O.P. Dall'Igna et al. / Experimental Neurology 203 (2007) 241  –  245     A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y role of A 2A  rather than A 1  receptors in the prevention of A β -induced cognitive dysfunction by caffeine.Previous studies have established that the prolongedtreatment with caffeine (1 mg/ml – mean dose of 22 mg/kg/ day) leads to the development of tolerance to the behavioraleffects of A 1 , but not A 2A , receptor blockade while increasingthe impact of A 2A  receptor blockade by caffeine (Karcz-Kubicha et al., 2003; Solinas et al., 2002). We found that the prolonged treatment with caffeine (1 mg/ml) was not able to prevent A β -induced amnesic effects, but when it wasassociated with the acute administration of caffeine (30 mg/ kg ip 30 min before A β  administration), the A β -inducedamnesic effects were now abrogated (Fig. 1). This combina-tion of prolonged (1 mg/ml) and acute (30 mg/kg, ip) caffeinetreatments had no effect on the performance of control micein both tasks (Fig. 1).Again in an attempt to maximize the effects of caffeine onA 2A  rather than A 1  receptors, we decided to test a subchronictreatment with a dose of caffeine of 30 mg/kg for 4 days. Thissubchronic administration of caffeine abolished the A β -inducedamnesic effects (Fig. 2). To confirm the involvement of A 2A receptors in this protective effect of caffeine, we tested the effect of a similar subchronic administration of a selective A 2A receptor antagonist, SCH58261. We found that the subchronictreatment with SCH58261 (0.5 mg/kg for 4 days) completely prevented the A β -induced amnesic effects (Fig. 2). Subchronictreatment with SCH58261 had no effect on inhibitory avoidanceor spontaneous alternation performance in control mice. Discussion The present results demonstrate that the blockade of adenosine A 2A  receptors prevents  β -amyloid-induced impair-ment of cognitive performance in the inhibitory avoidance andspontaneous alternation tasks, an effect mimicked by asubchronic administration of the non-selective adenosinereceptor antagonist caffeine.Current evidence favors the idea that soluble A β species playa prominent role in the pathogenesis of Alzheimer's disease(AD), a pathology that disrupts memory performance (reviewedin Hardy and Selkoe, 2002; Klein et al., 2004). One avenue of  research hopefully allowing to interfere with AD is to seek for strategies to counteract A β -induced neuronal failure (Colemanet al., 2004; Hardy and Selkoe, 2002). This A β -inducedcognitive disruption may primarily result from a synapticdysfunction which then spreads to include a pattern of neuronaldeath (reviewed in Coleman et al., 2004), and thereforesynaptically located modulatory systems may be particularlyeffective to counteract the initial A β -induced cognitiveimpairment. Accordingly, we now report that the blockade of adenosine A 2A  receptors, which have a synaptic localization inthe hippocampus (Rebola et al., 2005), prevents  β -amyloid-induced amnesia, extending our previous observation that A 2A receptor antagonists prevent   β -amyloid-induced toxicity incultured neurons (Dall'Igna et al., 2003). This is in accordance with the general ability of A 2A  receptor antagonists to afford protection against different brain insults, in particular in animalmodels of other neurodegenerative conditions, such as Parkin-son's disease (reviewed in Cunha, 2005). Although themechanisms by which A 2A  receptors control the disruption of  brain function in these conditions are still unresolved (reviewedin Cunha, 2005), it has been proposed that the A 2A  receptor-mediated control of neuroinflammation could play a role (Sauraet al., 2005) in accordance with the detection of A 2A  receptors inmicroglia-like profiles in patients with AD (Angulo et al.,2003).We also found that the administration of the non-selectiveadenosine receptor antagonist caffeine (see Fredholm et al.,1999) also prevented  β -amyloid-induced impairment in spatialmemory performance. This was observed upon acute adminis-tration of a dose of caffeine of 80 mg/kg, which enhances thecontribution of A 2A  receptor blockade (Quarta et al., 2004;Solinas et al., 2002) but probably lacks the selectivity for adenosine receptors since it begins affecting the activity of  phosphodiesterases (see Fredholm et al., 1999). However, the neuroprotective effect was also observed by pairing prolongedcaffeine treatment with an acute administration of caffeine,which causes a tolerance to the effects of A 1  receptor blockadewhile enhancing the behavioral effects resulting from A 2A receptor blockade (see Karcz-Kubicha et al., 2003; Quarta et al., 2004). Finally, the parallel protective effects recorded uponsubchronic treatment with 30 mg/kg caffeine (corresponding to4 – 6 cups of coffee in humans), which selectively antagonizesadenosine receptors (Fredholm et al., 1999), and withSCH58261 further strengthen the role of A 2A  receptors in thecontrol of A β -induced cognitive dysfunction. This is inagreement with our previous findings that caffeine protectionagainst   β -amyloid-induced neurotoxicity was mimicked byselective A 2A  but not A 1  receptor antagonists (Dall'Igna et al.,2003). Interestingly, this protective effect seems not to undergotolerance as the combination of chronic and acute caffeinetreatments was more effective than each treatment alone, inaccordance with the lack of desensitization over time of theneuroprotective effects of A 2A  receptor antagonists in animalmodels of Parkinson's disease (Xu et al., 2002). Finally, the present observation that caffeine prevented  β -amyloid-inducedcognitive deficits corroborates the proposal that caffeineconsumption is a protective factor in AD (Maia and deMendonça, 2002) and bolsters the interest of A 2A  receptor antagonists as candidate drugs to manage AD. Furthermore, both caffeine (e.g. Angelucci et al., 1999; Johnson-Kozlow et al., 2002) and selective A 2A  receptor antagonists (Kopf et al.,1999; Prediger et al., 2005) improve mnemonic functions, aneffectmost evident in aged subjects (e.g. Johnson-Kozlow et al.,2002) or animals (Prediger et al., 2005) in accordance with the age-related increase in the density of A 2A  receptors in corticalregions (e.g. Rebola et al., 2003). In conclusion, this reported ability of caffeine and A 2A receptor antagonists to prevent   β -amyloid-induced cognitivedeficit provides the rationale to evaluate putative therapeuticactions of caffeine and of A 2A  receptor antagonists in AD patients, with the potential to provide not only symptomaticimprovement, but also a delay on cognitive decline andneurodegenerative changes. 244  O.P. Dall'Igna et al. / Experimental Neurology 203 (2007) 241  –  245
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