Vasco C Sousa

Joaquim Natália Assaife-Lopes

Alexandre Ribeiro

Judith A Pratt

Ros R Brett

Ana Maria Sebastião

Natalia Assaife-Lopes

Joaquim A Ribeiro

Affiliations

Regulation of hippocampal cannabinoid CB receptor actions by adenosine A receptors and chronic caffeine administration: implications for the effects of ∆-tetrahydrocannabinol on spatial memory

Keywords: adenosine, cannabinoid, hippocampus, caffeine, THC, memory

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Introduction

The fine-tuning of neuronal activity to suit specific cognitive functions is a major task of endogenous neuromodulators of which adenosine and the endocannabinoids are two important examples. Both modulators are released by neurons and activate G proteincoupled receptors (GPCRs) that represent some of the most widely and densely expressed GPCRs in the brain [START_REF] Dunwiddie | The role and regulation of adenosine in the central nervous system[END_REF][START_REF] Herkenham | Cannabinoid receptor localization in brain[END_REF]).

In the hippocampus, the predominant adenosine and cannabinoid receptors are the A 1 and CB 1 receptors, respectively. Several forms of learning, memory and other cognitive functions require the integrity of the hippocampal circuitry, where A 1 and CB 1 receptors were shown to have important roles due to their presynaptic regulation of neurotransmitter release (e.g. [START_REF] Ohno | Working memory failure by stimulation of hippocampal adenosine A1 receptors in rats[END_REF][START_REF] Wise | Hippocampal CB(1) receptors mediate the memory impairing effects of Delta(9)-tetrahydrocannabinol[END_REF]. Moreover, in the hippocampus these receptors are the main targets for the cognitive effects of two of the most heavily consumed psychoactive substances worldwide: caffeine and Δ 9tetrahydrocannabinol (THC) [START_REF] Barone | Caffeine consumption[END_REF][START_REF] Leggett | A review of the world cannabis situation[END_REF].

Caffeine is present in various dietary sources such as coffee, tea and soft drinks, and at moderate doses is an adenosine receptor antagonist with cognitive enhancing properties [START_REF] Fredholm | Actions of caffeine in the brain with special reference to factors that contribute to its widespread use[END_REF][START_REF] Ribeiro | Caffeine and adenosine[END_REF]. As customary in most coffee consumers, long-term intake of caffeine leads to the development of tolerance to some of its acute effects by mechanisms not yet fully understood, although most studies found an increased number of A 1 , but not A 2A receptors, in several brain areas [START_REF] Jacobson | Adenosine receptor ligands: differences with acute versus chronic treatment[END_REF]. Chronic caffeine intake has also been associated with increased behavioral effects of some drugs of abuse, e.g. amphetamine and cocaine [START_REF] Gasior | Changes in the ambulatory activity and discriminative stimulus effects of psychostimulant drugs in rats chronically exposed to caffeine: effect of caffeine dose[END_REF][START_REF] Justinova | Effects of chronic caffeine exposure on adenosinergic modulation of the discriminative-stimulus effects of nicotine, methamphetamine, and cocaine in rats[END_REF]. THC is the main psychoactive constituent of the cannabis plant, which is consumed recreationally or used for medicinal purposes; it mainly activates cannabinoid CB 1 receptors in the central nervous system (CNS) to produce motor and cognitive disrupting effects (see [START_REF] Pertwee | The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta9-tetrahydrocannabinol, cannabidiol and delta9-tetrahydrocannabivarin[END_REF].

Hippocampal CB 1 receptors are primarily found in presynaptic terminals of cholecystokinin (CCK)-expressing GABAergic interneurons from the CA1 and CA3 subfields [START_REF] Hájos | Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations[END_REF][START_REF] Katona | Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons[END_REF]. CCK-expressing GABAergic interneurons regulate the temporal coordination in the activity of principal cell assemblies, which is critical for the integrity of hippocampal-dependent memory [START_REF] Freund | Perisomatic inhibition[END_REF]. Accordingly, it was recently demonstrated that presynaptic CB 1 receptors at GABAergic, but not glutamatergic, neurons are required for THC-induced amnesia [START_REF] Puighermanal | Cannabinoid modulation of hippocampal long-term memory is mediated by mTOR signaling[END_REF]. In contrast, A 1 receptors mostly affect excitatory synaptic transmission [START_REF] Dunwiddie | Adenosine A1 receptors inhibit adenylate cyclase activity and neurotransmitter release and hyperpolarize pyramidal neurons in rat hippocampus[END_REF][START_REF] Sebastião | The inhibitory adenosine receptor at the neuromuscular junction and hippocampus of the rat: antagonism by 1,3,8-substituted xanthines[END_REF], having no direct influence upon GABAergic transmission in mature hippocampal neurons [START_REF] Jeong | Adenosine A1 receptor-mediated presynaptic inhibition of GABAergic transmission in immature rat hippocampal CA1 neurons[END_REF][START_REF] Lambert | Adenosine depresses excitatory but not fast inhibitory synaptic transmission in area CA1 of the rat hippocampus[END_REF][START_REF] Li | Adenosine action on interneurons and synaptic transmission onto interneurons in rat hippocampus in vitro[END_REF][START_REF] Yoon | Adenosine inhibits excitatory but not inhibitory synaptic transmission in the hippocampus[END_REF] or on GABA release from isolated nerve terminals [START_REF] Cunha | Purinergic modulation of [(3)H]GABA release from rat hippocampal nerve terminals[END_REF].

However, A 1 receptors are present in hippocampal GABAergic interneurons [START_REF] Ochiishi | Cellular localization of adenosine A1 receptors in rat forebrain: immunohistochemical analysis using adenosine A1 receptor-specific monoclonal antibody[END_REF], where they control the actions of vasoactive intestinal peptide [START_REF] Cunha-Reis | A1 and A2A receptor activation by endogenous adenosine is required for VIP enhancement of K+ -evoked [3H]-GABA release from rat hippocampal nerve terminals[END_REF].

Both A 1 and CB 1 receptors regulate synaptic transmission through activation of Gprotein α i/o subunits [START_REF] Straiker | G-protein alpha subunit isoforms couple differentially to receptors that mediate presynaptic inhibition at rat hippocampal synapses[END_REF], which inhibit adenylyl cyclase, block voltagegated calcium channels (VGCCs) and activate inwardly rectifying potassium channels [START_REF] Dunwiddie | The role and regulation of adenosine in the central nervous system[END_REF][START_REF] Howlett | Cannabinoid receptor signaling[END_REF]. In the cerebellum, A 1 receptors modulate the motor incoordination effects induced by acute administration of THC or CB 1 receptor agonist CP55,940 [START_REF] Dar | Cerebellar CB(1) receptor mediation of Delta(9)-THC-induced motor incoordination and its potentiation by ethanol and modulation by the cerebellar adenosinergic A(1) receptor in the mouse[END_REF][START_REF] Dar | Acute ethanol/cannabinoid-induced ataxia and its antagonism by oral/systemic/intracerebellar A1 adenosine receptor antisense in mice[END_REF][START_REF] Desanty | Involvement of the cerebellar adenosine A(1) receptor in cannabinoid-induced motor incoordination in the acute and tolerant state in mice[END_REF]. Furthermore, prolonged intracerebellar administration of a CB 1 or A 1 agonist induces cross-tolerance (DeSanty and [START_REF] Desanty | Involvement of the cerebellar adenosine A(1) receptor in cannabinoid-induced motor incoordination in the acute and tolerant state in mice[END_REF], and similar observations were obtained in two subsequent studies [START_REF] Kouznetsova | Desensitization of cannabinoid-mediated presynaptic inhibition of neurotransmission between rat hippocampal neurons in culture[END_REF][START_REF] Selley | Long-term administration of Delta9tetrahydrocannabinol desensitizes CB1-, adenosine A1-, and GABAB-mediated inhibition of adenylyl cyclase in mouse cerebellum[END_REF]. A more recent study observed that CB 1 -mediated inhibition of excitatory synaptic transmission in the hippocampus is modulated by endogenous adenosine, through A 1 receptor activation [START_REF] Hoffman | Control of cannabinoid CB1 receptor function on glutamate axon terminals by endogenous adenosine acting at A1 receptors[END_REF]; but see [START_REF] Serpa | Cannabinoid CB(1) and adenosine A(1) receptors independently inhibit hippocampal synaptic transmission[END_REF]. These previous findings raised the hypothesis that a functional interaction between A 1 and CB 1 receptors in the hippocampus may have cognitive and pathophysiological implications, particularly for the effects of cannabis and caffeine consumption in humans.

The present study initially focused upon the possibility that an A 1 -CB 1 interaction influences GABA and glutamate release. We found that A 1 receptor activation attenuated the CB 1 -mediated inhibition of GABA and glutamate release and that this interaction is manifested at the level of G-protein activation. We then evaluated the functional consequences of chronic caffeine administration on the memory deficits induced by acute THC administration. Caffeine (i.p., 3mg/kg/day, for 15 days, >12h before trials) increased A 1 receptor levels, and did not by itself cause measurable effects on spatial memory, but led to an A 1 -mediated exacerbation of the CB 1dependent acute effects of THC in a spatial memory task.

Methods

Animals

Adult male 6-8 weeks old Wistar rats (Harlan Interfauna Iberica, Spain), and 12-16 weeks old C57Bl/6J mice (Harlan-Olac, UK) were used. Animals were housed in a temperature and humidity regulated room with a 12h dark/light cycle, and free access to food and water. Experiments were performed during the light phase. All experimentation followed the UK Animals (Scientific Procedures) Act, 1986, Portuguese and European Union law concerning animal care. C57Bl/6J mice were used in all experiments involving chronic caffeine administration for logistic advantages and because mice have been extensively used in behavioral studies where the systemic effects of cannabinoids on motor and cognitive function have been assessed [START_REF] Lichtman | Endocannabinoids in cognition and dependence[END_REF].

Drugs 4-amino-[2,3-3 H]butyric acid ([ 3 H]GABA), L-[G-3 H]glutamic acid ([ 3 H]glutamate), 1,3- [ 3 H]-dipropyl-8-cyclopentylxanthine ([ 3 H]-DPCPX),

Synaptosomal preparation

For each experiment, hippocampal synaptosomes were prepared from 2 rats, or corticohippocampal synaptosomes from 1 mouse. Animals were decapitated under halothane anesthesia and synaptosomes were prepared as previously described (Assaife-Lopes solution containing 1 mM EDTA and 10 mM HEPES, pH 7.4. The homogenate was then centrifuged at 3000 g for 10 min, and the supernatant obtained was centrifuged at 14000 g for 12 min to obtain a stratified pellet, containing synaptosomes [START_REF] Mcmahon | Tetanus toxin and botulinum toxins type A and B inhibit glutamate, gamma-aminobutyric acid, aspartate, and met-enkephalin release from synaptosomes. Clues to the locus of action[END_REF][START_REF] Phelan | Isolation of synaptosomes, growth cones, and their subcellular components[END_REF].

[ 3 H]-neurotransmitter release assays

For [ 3 H]GABA release assays, the pellet was resuspended and synaptosomes were incubated for 20 min, at 37ºC, with [ 3 H]GABA (1.5 µCi/ml, 1.85 nM), and 0.625 µM of unlabelled GABA to decrease specific activity of [ 3 H]GABA to 2.3 µCi/nmol. Incubation and superfusion solutions consisted of oxygenated aCSF containing the GABA transaminase inhibitor AOAA. For [ 3 H]glutamate experiments, the synaptosomal pellet was resuspended in aCSF which did not contain AOAA, and synaptosomes were incubated for 5 min, at 37ºC with 10 μCi/ml [ 3 H]glutamate. Synaptosomes were then layered over GF/C filters (Milipore, MA, USA) on an 8-chamber superfusion (0.8 ml/min) apparatus [START_REF] Raiteri | A simple apparatus for studying the release of neurotransmitters from synaptosomes[END_REF]. This constant and rapid flow rate washes out endogenously released substances thus ensuring drug effect specificity (see [START_REF] Raiteri | Synaptosomes still viable after 25 years of superfusion[END_REF]. After a 30 min washout period, samples were continuously collected for 36 min, in 2 min fractions. Synaptosomes were stimulated during 2 min with 15 mM K + (isomolar substitution of Na + with K + ) at the 6 th (S 1 ) and 24 th (S 2 ) minutes of collection time. CB 1 agonists were added to the superfusion medium from the 18 th minute onward, to measure effects on S 2 . The CB 1 antagonist, AM251, or A 1 and GABA B receptor ligands were added from the 15 th minute of the washout period onward, i.e. present during S 1 and S 2 in order to assess their ability to modify the effect of WIN55,212-2 (applied before S 2 ). Each condition was tested in duplicate, as commonly accepted in this paradigm (e.g. [START_REF] Cunha | Purinergic modulation of [(3)H]GABA release from rat hippocampal nerve terminals[END_REF]. Under similar conditions, the percentage of GABA and glutamate in the K + -evoked outflow is >90% of the total tritium in the sample [START_REF] Cunha | Inhibition of [3H] gamma-aminobutyric acid release by kainate receptor activation in rat hippocampal synaptosomes[END_REF][START_REF] Lopes | Adenosine A(2A) receptor facilitation of hippocampal synaptic transmission is dependent on tonic A(1) receptor inhibition[END_REF]. Fractional [ 3 H]neurotransmitter release was expressed as the percentage of total radioactivity present in the synaptosomes at each time point (fractional release). The amount of tritium released after each pulse of K + (S 1 or S 2 ) was calculated by integration of the peak area. Effects were calculated by normalizing the S 2 /S 1 values of corresponding controls from the same batch of synaptosomes to 0% effect. For example, the effect of WIN55,212-2 (added before S 2 ), in the presence of CPA (during S 1 and S 2 ), was calculated using the S 2 /S 1 of CPA alone (during S 1 and S 2 ) as a control, which was obtained from the same experiment and batch of synaptosomes.

Binding assays

Rat hippocampal, or mouse cortico-hippocampal membranes were prepared as previously described (e.g. [START_REF] Cunha | G protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors[END_REF], with modifications. Tissue was homogenized in ice-cold 0.32 M sucrose solution containing 2 mM EGTA, 1 mM DTT and 50 mM Tris, pH 7.6. The homogenate was centrifuged at 1000 g for 10 min, and the supernatant obtained was centrifuged at 14000 g for 12 min. The pellets were resuspended in assay buffer and incubated with 4 U/ml ADA for 30 min at 37º C, followed by centrifugation at 14000 g for 12 min and resuspension in assay buffer.

Assay buffer composition, in mM, for radioligand binding assays was: Tris 50, MgCl 2 2, pH 7.4. For [ 35 S]-GTPγS binding assays: Tris 50, MgCl 2 5, NaCl 100, EGTA 0.2, pH 7.4.

Protein content was determined by the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. For [START_REF] Breivogel | Cannabinoid receptor agonist efficacy for stimulating [35S]GTPgammaS binding to rat cerebellar membranes correlates with agonistinduced decreases in GDP affinity[END_REF]. Specific binding was calculated by subtracting nonspecific binding obtained by incubation with 10 µM GTPγS. The effect of co-application of CPA or baclofen with CB 1 agonists was calculated by subtracting the increase in [ 35 S]-GTPγS induced by CPA or baclofen alone. The reactions were stopped by vacuum filtration through GF/C filters, followed by washing with ice-cold buffer.

In vivo drug administration

Mice were randomly assigned to various groups and habituated to the handling during 5 days before testing began. For chronic treatment with caffeine, animals received caffeine (3 mg/kg/day), or vehicle (saline: 0.9% NaCl), >12 h before trials, for at least 15 days prior to experimental days, and throughout the course of behavioral testing, in order to avoid withdrawal effects. Total caffeine exposure was for 22-24 days, and euthanization occurred 24 h after last injection. The half-life of caffeine for doses lower than 10 mg/kg ranges from 0.7 to 1.2 h in the rat and mouse [START_REF] Fredholm | Actions of caffeine in the brain with special reference to factors that contribute to its widespread use[END_REF], therefore the estimated concentrations of caffeine present in plasma or brain during behavioral testing were negligible. For acute administration, animals received a single dose of vehicle (8% Tween 80 in saline), THC (5 mg/kg), AM251 (3 mg/kg), DPCPX (1 mg/kg), or WIN55,212-2 (1 mg/kg). THC was prepared in Tween 80 as previously described [START_REF] Pertwee | Inhibitory effects of certain enantiomeric cannabinoids in the mouse vas deferens and the myenteric plexus preparation of guinea-pig small intestine[END_REF]; AM251, DPCPX, and WIN55,212-2 were suspended in the vehicle and carefully sonicated. All drugs were given by intraperitoneal injection (i.p.) in a volume of 2 ml/kg weight. The concentration of Tween 80 used was previously

shown not to affect motor activity in mice [START_REF] Castro | Behavioral effects of vehicles: DMSO, ethanol, Tween-20, Tween-80, and emulphor-620[END_REF].

Water maze experiments (trials to criterion task)

We performed 2 separate sets of water maze experiments in which mice were randomly assigned to 4 experimental groups of 7 to 8 subjects (total of 57 animals). The protocol is a version of the Morris water maze test that is sensitive to hippocampal-dependent short-term spatial learning [START_REF] Chen | A learning deficit related to age and beta-amyloid plaques in a mouse model of Alzheimer's disease[END_REF][START_REF] Daumas | Transient activation of the CA3 Kappa opioid system in the dorsal hippocampus modulates complex memory processing in mice[END_REF]. To form a stable representation of the environment, mice were first trained to quickly find a hidden platform at a fixed platform location for 5 consecutive days. Subjects then performed several tasks, each consisting of a new platform position. Each animal was given a maximum of 8 trials per day, to perform the task until reaching a performance criterion of ≤7 s average latency on three consecutive trials. A 15 min inter-trial interval was applied, during which animals were allowed to dry under a ceramic heat lamp. Once the criterion was reached, trials stopped and a new task began on the following testing day.

Animals first performed 4-5 training tasks in order to learn to optimize their search strategies, then the effects of acute drug administration were tested in separate tasks, as described in the results section.

A black infrared-translucent Perspex™ tank (1 m in diameter) of water (temperature, 22±1ºC) was placed over an infrared lightbox (Tracksys, Nottingham, UK) in a room with various visible external cues. A transparent platform was ~0.5 cm below the water surface and its position varied between several possible locations, on two concentric circles, according to the original protocol. An infrared-sensitive automated tracking system (Noldus Ethovision 7.0, Noldus Information Technology, Netherlands) monitored all performances .

Statistical analysis

Statistical significance was tested using paired Student's t-test, one-way or two-way analysis of variance (ANOVA), followed by Dunnett's or Tukey's post hoc tests, as indicated. The two-way ANOVA and post hoc tests were performed using the Predictive Analytics Software 18.0 (SPSS Inc., an IBM Company, Chicago, IL). GraphPad Prism 5.0 (GraphPad Prism Software Inc., San Diego, CA) was used for all other statistical tests and nonlinear regression curve fitting. Differences in parameters between binding curves were tested using extra sum-of-squares F test.

Results

A 1 receptor activation attenuates the CB 1 receptor-dependent inhibition of (20 μM) inhibited its release by 34±1.6% (n=8, p<0.001, data not shown).

To induce a CB 1 receptor-dependent effect on [ 3 H]GABA release, we used the potent cannabinoid receptor agonist WIN55,212-2, which has been previously shown to inhibit evoked [ 3 H]GABA release from hippocampal synaptosomes through a CB 1 specific mechanism, having a maximum CB 1 selective effect at 1 μM [START_REF] Köfalvi | Anandamide and NADA bi-directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus[END_REF]. Application of 1 μM WIN55,212-2 six minutes before S 2 , caused a decrease of basal [ 3 H]GABA outflow and inhibited evoked GABA release (Figure 1a) with an average S 2 /S 1 of 0.78±0.01 (n=31) that represents an inhibition of 16.7±1.4% (n=31), when compared to control S 2 /S 1 within each experiment. Blockade of VGCCs by Cd 2+

(CdCl 2 , 200 μM) completely abolished the effect of WIN55,212-2 at its maximum CB 1specific concentration (n=5, p<0.01, Figure 1b). Conversely, blockade of GABA transporters with SKF89976a (20 μM) did not alter the effect of WIN55,212-2 (n=5, Figure 1b), which suggests that the effect of 1 μM WIN55,212-2 upon [ 3 H]GABA release is exerted through the inhibition of Ca 2+ -dependent exocytotic release. The effect of WIN55,212-2 (0.01-10 μM) on K + -evoked [ 3 H]GABA release was concentrationdependent (Figure 1c). Since WIN55,212-2 is known to directly block N-type VGCCs at concentrations above 1 μM [START_REF] Németh | CB1 receptor-dependent and -independent inhibition of excitatory postsynaptic currents in the hippocampus by WIN 55,212-2[END_REF][START_REF] Shen | The cannabinoid agonist Win55,212-2 inhibits calcium channels by receptor-mediated and direct pathways in cultured rat hippocampal neurons[END_REF], we tested the specificity of its effect in our preparation using the CB 1 antagonist AM251, as well as WIN55,212-3, an enantiomer of WIN55,212-2 that does not activate the CB 1 receptor but maintains the Ca 2+ channel blocking properties [START_REF] Shen | The cannabinoid agonist Win55,212-2 inhibits calcium channels by receptor-mediated and direct pathways in cultured rat hippocampal neurons[END_REF]. AM251 (1 μM) fully blocked the effect of 1 μM but not of 10 μM of WIN55,212-2 (Figure 1c). Higher concentrations of AM251 were not used to avoid loss of selectivity (see [START_REF] Köfalvi | Cannabinoids and the Brain[END_REF][START_REF] Köfalvi | Anandamide and NADA bi-directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus[END_REF]. The enantiomer had no significant effect applied at 1 μM but it inhibited evoked [ 3 H]GABA release by 22.4±1.4% at 10 μM (n=5, p<0.05. Figure 1c), which indicates that the effect of 1 μM WIN55,212-2 upon [ 3 H]GABA release is CB 1 receptor-dependent. It is noteworthy that the effect of 10 μM WIN55,212-2 was larger than the effect of 10 μM WIN55,212-3, which indicates that the effect of 10 μM WIN55,212-2 still encompasses a CB 1 receptor-dependent component. The partial CB 1 agonist Δ 9 -Tetrahydrocannabinol (THC, 1 μM) inhibited K + -evoked [ 3 H]GABA release by 8.9±0.9% (n=8, p<0.05, Figure 1c).

Consistent with previous observations [START_REF] Cunha | Purinergic modulation of [(3)H]GABA release from rat hippocampal nerve terminals[END_REF], adenosine A 1 receptor activation with the selective agonist CPA (100 nM) before S 2 , did not affect K +evoked [ 3 H]GABA release (2.7±3% of control S 2 /S 1 , n=3, p>0.05, data not shown). To evaluate the influence of A 1 receptors on the CB 1 -mediated inhibition of GABA release, we tested the effect of WIN55,212-2 (applied before S 2 ) in the presence of CPA (100 nM, applied throughout the experiment, S 1 +S 2 ). Under these conditions, the effect of WIN55,212-2 at 1 μM (n=10) and at 10 μM (n=7) was significantly attenuated (p<0.01 and p<0.05, respectively; Figure 2a). The average effect of 1 μM WIN55,212-2 was 18±3% (n=10) and CPA attenuated this effect to 12±2% (p<0.01, n=10), which represents a 33±13% decrease in the average effect of WIN55,212-2 alone. To test if the attenuation of the effect of 10 μM WIN55,212-2 caused by CPA occurred through a Ca 2+ channel-dependent mechanism and not involving CB 1 receptors, we performed the experiments using WIN55,212-3. Application of WIN55,212-3 (10 μM, S 2 ) by itself inhibited [ 3 H]GABA release by 22±2%, while in presence of CPA (100 nM) its effect was unaltered (22±2%, n=3, Figure 2a). This indicates that the CPA-induced attenuation of the effect of WIN55,212-2 was exerted at the CB 1 receptor-dependent component, but not upon the Ca 2+ channel-dependent mechanisms, affected by high micromolar concentrations of WIN55,212-2. The blockade of A 1 receptors with the antagonist DPCPX (50 nM) did not, on its own, alter the effects of 1 μM and 10 μM WIN55,212-2, but it fully prevented the CPA-induced attenuation (p>0.05, n=7, paired Student's t-test, Figure 2b).

The signaling pathways of CB 1 , A 1 and GABA B are known to converge when coexpressed in cerebellar neurons [START_REF] Selley | Long-term administration of Delta9tetrahydrocannabinol desensitizes CB1-, adenosine A1-, and GABAB-mediated inhibition of adenylyl cyclase in mouse cerebellum[END_REF]. Furthermore, both CB 1 and GABA B receptors are present in inhibitory interneurons [START_REF] Katona | Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons[END_REF][START_REF] Sloviter | Localization of GABA(B) (R1) receptors in the rat hippocampus by immunocytochemistry and high resolution autoradiography, with specific reference to its localization in identified hippocampal interneuron subpopulations[END_REF], couple to the same Gα i/o subunits [START_REF] Straiker | G-protein alpha subunit isoforms couple differentially to receptors that mediate presynaptic inhibition at rat hippocampal synapses[END_REF] and exhibit reciprocal inhibition [START_REF] Cinar | Reciprocal inhibition of G-protein signaling is induced by CB(1) cannabinoid and GABA(B) receptor interactions in rat hippocampal membranes[END_REF] in hippocampal neurons. We therefore evaluated whether the A 1 receptor-dependent attenuation of the effect of WIN55,212-2 was mimicked by activation of GABA B receptors. As shown in Figure 2c, the effect of WIN55,212-2 (1 μM)

was unchanged (p>0.05, n=5, paired Student's t-test) by the presence of the GABA B receptor agonist baclofen (10 μM). Altogether, these results indicate that the cannabinoid CB 1 receptors in GABAergic nerve terminals are under the modulatory influence of adenosine A 1 receptors, but not GABA B receptors.

Despite the predominant influence of CB 1 receptors in hippocampal circuitry being exerted through the inhibition of GABA release, CB 1 receptors at glutamatergic presynaptic nerve terminals [START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF]) also inhibit the K + -evoked release of glutamate (e.g. [START_REF] Cannizzaro | Presynaptic effects of anandamide and WIN55,212-2 on glutamatergic nerve endings isolated from rat hippocampus[END_REF][START_REF] D'amico | Inhibition by anandamide and synthetic cannabimimetics of the release of [3H]D-aspartate and [3H]GABA from synaptosomes isolated from the rat hippocampus[END_REF][START_REF] Köfalvi | Anandamide and NADA bi-directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus[END_REF]. Importantly, the regulation of excitatory synaptic transmission is the most relevant role of the hippocampal A 1 receptors [START_REF] Dunwiddie | Adenosine A1 receptors inhibit adenylate cyclase activity and neurotransmitter release and hyperpolarize pyramidal neurons in rat hippocampus[END_REF][START_REF] Sebastião | The inhibitory adenosine receptor at the neuromuscular junction and hippocampus of the rat: antagonism by 1,3,8-substituted xanthines[END_REF]. To investigate whether A 1 receptors also regulate the CB 1dependent effects upon glutamate release, the influence of A 1 receptor activation on the CB 1 -mediated inhibition of K + -evoked [ 3 H]glutamate release was also tested. The absence of a tonic activation of A 1 receptors by endogenous adenosine was first assessed by using A 1 receptor blocker DPCPX. As Figure 3a shows, WIN55,212-2 (1 μM) inhibited the release of [ 3 H]glutamate by 16±2% and blockade of A 1 receptors with DPCPX (50 nM) did not modify the effect of WIN55,212-2 (15±1%, p>0.05, n=4). This indicates that endogenous adenosine was effectively washed out by the continuous vertical flow of superfusion medium (see Methods).

As shown in Figure 3b, the inhibitory effect of 1 μM WIN55,212-2 upon glutamate release (19±2%) was significantly attenuated to 7±2% (n=5, p<0.01, Figure 3b) in the presence of CPA. We then evaluated whether the A 1 receptor-mediated inhibition of glutamate release is also under the modulatory control of CB 1 receptors, by comparing the effect of CPA (before S 2 ) in the absence and in the presence of 1 μM WIN55,212-2.

CPA (100 nM) inhibited [ 3 H]glutamate release by 12±4% and this effect was not modified in the presence of 1 μM WIN55,212-2 (13±2%, n=4, p>0.05, Figure 3b). These findings further indicate that A 1 receptors negatively modulate the CB 1 -mediated effects in the hippocampus and support recent evidence that CB 1 -mediated inhibition of excitatory synaptic transmission in the hippocampus is modulated by A 1 receptor activation [START_REF] Hoffman | Control of cannabinoid CB1 receptor function on glutamate axon terminals by endogenous adenosine acting at A1 receptors[END_REF]. 1). This indicates a functional interaction between co-localized CB 1 and A 1 receptors in hippocampal membranes, which impacts on the ability of CB 1 receptors to activate Gα i/o -proteins. Similarly, the co-application of THC (0.1 nM-1 μM) with 100 nM CPA (Figure 4b) significantly decreased the E max of THC from 163±8% (when applied alone) to 129±13% (

Chronic caffeine administration increases acute THC-induced spatial memory deficits in mice

The evidence that A 1 receptor activation attenuates CB 1 receptor signaling raised the hypothesis that this A 1 -CB 1 interplay has a functional impact upon hippocampaldependent memory. Chronic caffeine consumption is known to induce an increase in adenosine A 1 , but not A 2A receptors (reviewed by [START_REF] Jacobson | Adenosine receptor ligands: differences with acute versus chronic treatment[END_REF]. Acute systemic THC administration induces CB 1 -dependent deficits in working memory [START_REF] Wise | Hippocampal CB(1) receptors mediate the memory impairing effects of Delta(9)-tetrahydrocannabinol[END_REF]. Therefore, we used a hippocampal-dependent, short-term spatial memory testing experiments, there were no differences between groups and no 'group task' interaction in any parameter during the training period (p>0.05 for all parameters). Thus, chronic caffeine administration by itself did not affect memory performance or motor activity.

For the first set of experiments, the effect of THC (5 mg/kg), or vehicle, given at task 6 (30 min before first trial), as well as the modification of this effect by AM251 (3 mg/kg),

given at task 7 (15 min before THC, or vehicle), are displayed in Figure 5a-d When mice received AM251 pre-treatment, there were no significant effects of any treatment group on all parameters, indicating that the effects of THC were dependent on the activation of CB 1 receptors. These results show that chronic caffeine exacerbates the CB 1 -dependent actions of THC in a short-term spatial memory task.

For the second set of experiments, the effect of THC (5 mg/kg, i.p.), or vehicle, given at task 5 (30 min before first trial), the modification of this effect by DPCPX (1 mg/kg, i.p.), given at task 7 (15 min before THC, or vehicle), as well as the effect of WIN55,212- vehicle) did not induce significant effects in the control (vehicle treated) group at any parameter, the effect of THC was exacerbated in the chronic caffeine group, on 'latency' (p=0.02, Tukey post hoc, Figure 6b) and 'pathlength' (p=0.01, Tukey post hoc, Figure 6c). There were also significant differences between THC on the chronic caffeine group vs. THC in the vehicle group on 'pathlength' (p=0.04); and between THC on the chronic caffeine group vs. the vehicle control group on 'trials to criterion' (p=0.05), 'latency' (p=0.01), and 'pathlength' (p=0.006).

When mice were tested 48 h after the last THC injection, there were no significant effects of any treatment group on all parameters, indicating that the effects of THC were not prevailing after this period. Importantly, when mice received a pre-treatment of DPCPX, there were also no significant effects of any treatment group on all parameters, indicating that the effects of THC were reversed by the blockade of A 1 receptors.

Finally, there was a significant effect of WIN55,212-2 on 'trials to criterion' 6b) and 'pathlength' (p=0.03, Tukey post hoc, Figure 6c). There were also significant differences between WIN55,212-2 on the chronic caffeine group vs. the vehicle control group on 'latency' (p=0.03), and 'pathlength' (p=0.03). The lower effect of WIN55,212-2, compared to that of THC, could be due to pharmacokinetic differences, since the penetration of WIN55,212-2 in the brain following i.p. injection is much lower than that of THC [START_REF] Petitet | Cannabinoid penetration into mouse brain as determined by ex vivo binding[END_REF]. Higher doses of WIN55,212-2 were not used to avoid non-specific effects [START_REF] Varvel | Evaluation of CB1 receptor knockout mice in the Morris water maze[END_REF]. These findings show a significant chronic caffeineinduced, and A 1 receptor-mediated, exacerbation of the CB 1 -dependent effects on short-term spatial memory.

Chronic caffeine and A 1 receptor number

To quantify the influence of chronic caffeine administration upon A 1 receptor number and affinity in cortico-hippocampal membranes, [ 3 H]DPCPX (0.1-10 nM) binding assays were performed (Figure 7). In vehicle-treated mice, the total number of specific binding sites obtained by nonlinear regression analysis (B max ) was 848±44 fmol/mg of protein, while the equilibrium dissociation constant (K D ) was 1.20±0.21 nM. In the chronic caffeine group, the B max was increased to 980±50 fmol/mg of protein (p<0.05, n=6, vs vehicle group) but K D (1.31±0.23 nM) was not significantly (p>0.05) affected. Thus, animals under chronic caffeine had ~16% higher density of A 1 receptor without changes in affinity.

Chronic caffeine and CB 1 receptor signaling in mouse cortico-hippocampal tissue

We first tested the consistency of our in vitro results between rats and mice by testing the effect of CPA (100 nM) on the WIN55,212-2-mediated inhibition of K +evoked [ 3 H]GABA release from cortico-hippocampal synaptosomes prepared from untreated mice (Figure 8). Consistent to previous observations in rats, the effect of 1 μM WIN55,212-2 alone was 19±1%, and it was significantly attenuated to 9±1% in the presence of CPA (p<0.01, n=4, paired Student's t-test, Figure 8). We then analyzed the influence of chronic caffeine administration upon the CB 1 receptor-mediated inhibition of K + -evoked [ 3 H]GABA release (Figure 8). In control (vehicle-treated) mice, 1 μM WIN55,212-2 inhibited [ 3 H]GABA release by 17±1%, while in the chronic caffeine group, the effect of WIN55,212-2 was significantly reduced to 11±2% (p<0.05, n=4, paired Student's t-test, Figure 8).

As Figure 9 and Table 2 show, in cortico-hippocampal membranes of vehicle-treated mice, WIN55,212-2 stimulated [ 35 S]-GTPγS binding (% of basal) with an EC 50 ≈989 nM and E max =321±11% (n=5, Figure 9a), while THC had an EC 50 ≈41 nM and E max =167±11% (n=4, Figure 9c). Chronic caffeine administration did not affect the EC 50 of WIN55,212-2 or THC, but it significantly reduced the E max of WIN55,212-2 to 269±8%, and of THC to 135±8% (p<0.05, n=4-5, extra sum-of-squares F test, Figure 9a,c). As observed in rats (Figure 4a), the co-application of 100 nM CPA in control mice significantly decreased the E max of WIN55,212-2 (254±13%, p<0.05, n=5, extra sum-ofsquares F test, Figure 9b, Table 2), but not the EC 50 . This reduction in WIN55,212-2stimulated [ 35 S]-GTPγS binding, caused by CPA in membranes from control animals, was of similar magnitude as the decrease observed in the chronic caffeine-treated group in the absence of CPA (Table 2). In the chronic caffeine group, CPA did not induce a further decrease in the efficacy of WIN55,212-2 to stimulate G-protein activation (E max =252±8%, Figure 9a,b, Table 2), which may suggest that chronic caffeine treatment and A 1 receptor activation do not have additive effects upon the modification of CB 1 receptor signaling.

The receptors to activate G-proteins is unaltered in chronic caffeine treated mice.

Accordingly, there were no statistically significant differences in the bottom of the nonlinear regression binding curves between chronic caffeine and vehicle treated animals, in the presence of CPA (p>0.05, n=5, extra sum-of-squares F test, Figure 9b).

Chronic caffeine and CB 1 receptor number

The effects of chronic caffeine administration upon CB 1 signaling in vitro suggested that CB 1 receptor number and/or affinity are decreased in these mice. We then directly analyzed the effect of chronic caffeine administration in CB 1 receptor number and affinity by performing [ 3 H]SR141716A (0.1-4nM) saturation binding assays in tissue collected from mice used in the behavioral experiments. The non-linear regression analysis of [ 3 H]SR141716A binding to cortical membranes of vehicle treated mice showed a B max = 1425±123 fmol/mg of protein, and a K D =1.4±0.3 nM (n=10, Figure 10a).

In hippocampal membranes, the B max of [ 3 H]SR141716A was 1322±97 fmol/mg of protein, and the K D =1.0±0.2 nM (n=5, Figure 10b). In the chronic caffeine group, the B max of [ 3 H]SR141716A binding was lower (p<0.05, compared to vehicle group) and this reduction was observed both in cortical membranes (B max =1151±65 fmol/mg of protein, n=10) and hippocampal membranes (B max =1089±57 fmol/mg of protein, n=5). There were no significant differences in affinity, as in the chronic caffeine group the K D values for [ 3 H]SR141716A binding were 1.0±0.2nM in cortical, and 0.9±0.1nM in hippocampal membranes.

Discussion

The present study demonstrates that adenosine A 1 receptors located in GABAergic and glutamatergic nerve terminals of the hippocampus exert a negative modulatory effect on the cannabinoid CB 1 receptor-mediated inhibition of GABA and glutamate release. CB 1 -mediated G-protein activation is also impaired by A 1 receptor activation. In addition, chronic administration of caffeine leads to an A 1 receptor-mediated enhancement of the CB 1 -dependent effects of THC upon short-term spatial memory, despite a reduction in CB 1 receptor number and signaling. This provides first evidence for chronic caffeine-induced alterations in cannabinoid actions in the cortex and hippocampus.

The CB 1 -A 1 receptor cross-talk might occur at the G-protein level since A 1 receptor activation with CPA reduced the efficacy of CB 1 receptor agonists to stimulate [ 35 S]-GTPγS binding in the hippocampus. This is in accordance with a previous observation that simultaneous application of CB 1 and A 1 agonists produces less than additive stimulation of [ 35 S]-GTPγS binding in cerebellar membranes [START_REF] Selley | Long-term administration of Delta9tetrahydrocannabinol desensitizes CB1-, adenosine A1-, and GABAB-mediated inhibition of adenylyl cyclase in mouse cerebellum[END_REF].

Similarly to the A 1 receptors, GABA B receptors couple to Gα i/o proteins and are expressed in the same interneuron populations as CB 1 receptors [START_REF] Neu | Postsynaptic origin of CB1-dependent tonic inhibition of GABA release at cholecystokinin-positive basket cell to pyramidal cell synapses in the CA1 region of the rat hippocampus[END_REF][START_REF] Sloviter | Localization of GABA(B) (R1) receptors in the rat hippocampus by immunocytochemistry and high resolution autoradiography, with specific reference to its localization in identified hippocampal interneuron subpopulations[END_REF]. However, CB 1 receptor-mediated signaling, assessed either as inhibition of [ 3 H]GABA release or stimulation of [ 35 S]-GTPγS binding, was unaffected by GABA B receptor activation, which indicates that the modulation of CB 1 receptor signaling by A 1 receptors is not shared by all Gα i/o -coupled receptors.

The CB 1 receptor agonist WIN55,212-2 inhibited calcium-dependent [ 3 H]GABA release with a maximum specific effect at 1 μM, in agreement with previous studies [START_REF] Katona | GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus[END_REF][START_REF] Köfalvi | Anandamide and NADA bi-directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus[END_REF]. There are clear differences in the magnitude of the reported effects of WIN55,212-2 in studies using different methodologies. For example, we and others [START_REF] Köfalvi | Anandamide and NADA bi-directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus[END_REF] observed that 1 μM WIN55,212-2 induces 15-20% inhibition of K + -evoked [ 3 H]GABA release from rat hippocampal synaptosomes, while several reports show that the same concentration of WIN55,212-2, by activating presynaptic CB 1 receptors, inhibits GABAergic inhibitory postsynaptic currents (IPSCs) in rat hippocampal slices by ~50% (e.g. [START_REF] Hájos | Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations[END_REF][START_REF] Hoffman | Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus[END_REF][START_REF] Wilson | Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses[END_REF]). These differences are likely due to a combination of factors.

The main reason possibly lies in the fact that [ 3 H]GABA release assays provide a quantitative measurement of the amount of GABA released from the whole population of GABAergic nerve terminals at the hippocampus, while patch clamp techniques provide a quantification of endogenous GABA release by measuring the post-synaptic responses of a single hippocampal pyramidal neuron. In addition, differences are likely due to the type of stimulus used (electrical vs. high K + ), the time and length of WIN55,212-2 application, and to an amplifying effect of multiple afferents upon IPSC measurements. The effect of 1 μM WIN55,212-2 on the release of [ 3 H]GABA from rat hippocampal slices [START_REF] Katona | Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons[END_REF] is also larger than in synaptosomes. Again, a longer exposure time (6 min vs. 18 min) to WIN55,212-2 and/or the amplification by intrinsic circuits in the slices is a likely explanation for these differences.

The CB 1 receptor-mediated modulation of GABA release from hippocampal CCKpositive interneurons, which express large quantities of CB 1 receptors, is a critical mechanism for spatial and episodic memory, as these interneurons regulate the temporal coordination of principal cell assemblies [START_REF] Hájos | Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations[END_REF][START_REF] Robbe | Alteration of theta timescale dynamics of hippocampal place cells by a cannabinoid is associated with memory impairment[END_REF][START_REF] Robbe | Cannabinoids reveal importance of spike timing coordination in hippocampal function[END_REF]. However, the CCK-expressing interneuron populations mostly receive input from glutamatergic neurons (see [START_REF] Freund | Interneurons of the hippocampus[END_REF][START_REF] Freund | Perisomatic inhibition[END_REF], which also express CB 1 [START_REF] Katona | Molecular composition of the endocannabinoid system at glutamatergic synapses[END_REF][START_REF] Kawamura | The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum[END_REF] and A 1 [START_REF] Ochiishi | Cellular localization of adenosine A1 receptors in rat forebrain: immunohistochemical analysis using adenosine A1 receptor-specific monoclonal antibody[END_REF] receptors. We found that A 1 receptor activation also attenuates CB 1 receptor-mediated inhibition of glutamate release from hippocampal synaptosomes. It was recently reported that endogenous adenosine, by activating A 1 receptors, regulates CB 1 -mediated inhibition of glutamatergic synaptic transmission [START_REF] Hoffman | Control of cannabinoid CB1 receptor function on glutamate axon terminals by endogenous adenosine acting at A1 receptors[END_REF]; but see [START_REF] Serpa | Cannabinoid CB(1) and adenosine A(1) receptors independently inhibit hippocampal synaptic transmission[END_REF].

Thus, A 1 and CB 1 receptors also interact at glutamatergic neurons, which indicates that the inhibitory effect of A 1 receptor activation upon the CB 1 -dependent stimulation of [ 35 S]-GTPγS binding might be derived from an A 1 -CB 1 receptor interaction at both GABAergic and glutamatergic neurons. Interestingly, WIN55,212-2 did not attenuate the inhibitory action of CPA upon glutamate release, suggesting that the modulatory action of A 1 receptors upon CB 1 receptors is not reciprocal.

The relevance of the GABAergic circuitry for the CB 1 receptor-mediated influences upon memory function became firmly established after the demonstration that intraperitoneal THC administration disrupts hippocampal-dependent memory through the activation of CB 1 receptors [START_REF] Wise | Hippocampal CB(1) receptors mediate the memory impairing effects of Delta(9)-tetrahydrocannabinol[END_REF] in GABAergic, but not glutamatergic, neurons [START_REF] Puighermanal | Cannabinoid modulation of hippocampal long-term memory is mediated by mTOR signaling[END_REF]. We now show that chronic administration of a moderate dose of caffeine leads to increased levels of A 1 receptors in the corticohippocampal membranes, and to an A 1 receptor-mediated increase of the disruptive effects of acute THC in a hippocampal-dependent short-term spatial memory task. This finding points towards a significant functional relevance of the cross-talk between A 1 and CB 1 receptors in the hippocampus. Interestingly, the motor impairments induced by THC are enhanced by acute activation of A 1 receptors [START_REF] Dar | Cerebellar CB(1) receptor mediation of Delta(9)-THC-induced motor incoordination and its potentiation by ethanol and modulation by the cerebellar adenosinergic A(1) receptor in the mouse[END_REF]. In contrast, acute administration of caffeine antagonizes THC-induced changes in cortico-hippocampal EEG wave recordings [START_REF] Consroe | EEG and behavioral effects of delta9-tetrahydrocannabinol in combination with stimulant drugs in rabbits[END_REF]. Several studies show that chronic exposure to adenosine receptor antagonists causes similar actions to acute agonist exposure (see [START_REF] Jacobson | Adenosine receptor ligands: differences with acute versus chronic treatment[END_REF][START_REF] Lubitz | Effects of chronic administration of adenosine A1 receptor agonist and antagonist on spatial learning and memory[END_REF], while acute administration of caffeine is expected to have opposite effects to acute agonist exposure. The timing of caffeine administration and the presence of caffeine in the blood during testing must also be taken into account when comparing data from different studies. The behavioral tests now reported were performed in the absence of relevant plasma concentrations of caffeine (>12 h after caffeine injection), which was given 2 h after the last behavioral trial, to prevent effects on memory consolidation [START_REF] Angelucci | Effects of caffeine on learning and memory in rats tested in the Morris water maze[END_REF]. It is therefore not surprising that acute caffeine administration prevents THC-induced effects [START_REF] Consroe | EEG and behavioral effects of delta9-tetrahydrocannabinol in combination with stimulant drugs in rabbits[END_REF] whereas chronic caffeine exposure exacerbates the memory disruption induced by CB 1 receptor agonists (present work). In a recent study, chronic administration of a high dose of caffeine (210 mg/kg/day) in rats was shown to potentiate CB 1 -dependent effects at striatal GABAergic, but not glutamatergic, synapses [START_REF] Rossi | Caffeine drinking potentiates cannabinoid transmission in the striatum: Interaction with stress effects[END_REF]. However, it is difficult to draw a comparison with the present study, given the differences in the experimental approach, namely the dose of caffeine used, which is not adenosine receptor-selective and is more than about 70 times higher than the equivalent daily human intake. Exposure to high doses of caffeine (~100 mg/kg/day) leads to altered brain levels of several receptors [START_REF] Shi | Chronic caffeine alters the density of adenosine, adrenergic, cholinergic, GABA, and serotonin receptors and calcium channels in mouse brain[END_REF][START_REF] Shi | Effects of chronic caffeine on adenosine, dopamine and acetylcholine systems in mice[END_REF], inhibit

phosphodiesterases and may even block GABA A receptors, among others (see [START_REF] Daly | Caffeine--an atypical drug of dependence[END_REF].

The conclusion that A 1 receptors are involved in the chronic caffeine-induced exacerbation of the effects of THC presently reported is reinforced by the finding that A 1 receptor blockade with DPCPX fully prevented the effects of THC in the chronic caffeine group. DPCPX was administered at a dose that occupies A 1 receptors [START_REF] Baumgold | Penetration of adenosine antagonists into mouse brain as determined by ex vivo binding[END_REF][START_REF] Hooper | Effects of purine analogues on spontaneous alternation in mice[END_REF] while not affecting motor activity (present data and (Von [START_REF] Lubitz | Effects of chronic administration of adenosine A1 receptor agonist and antagonist on spatial learning and memory[END_REF]. In addition, DPCPX by itself had no effects in the absence of THC, which suggests that A 1 receptors do not directly influence short-term spatial memory.

Furthermore, acute application of DPCPX did not influence the action of THC in vehicletreated animals, which further supports previous evidence (see above) that chronic and acute blockade of A 1 receptors have different functional consequences. The effects of DPCPX also exclude the involvement of A 2A receptors, which are known to modulate the actions of CB 1 receptors in the striatum [START_REF] Carriba | Striatal adenosine A2A and cannabinoid CB1 receptors form functional heteromeric complexes that mediate the motor effects of cannabinoids[END_REF][START_REF] Tebano | Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum[END_REF]. A 1 and A 2A receptors have similar affinities for caffeine [START_REF] Fredholm | Nomenclature and classification of purinoceptors[END_REF]), yet the expression of A 2A receptors in the hippocampus and cortex is much lower than that of A 1 receptors (reviewed by [START_REF] Ribeiro | Adenosine receptors in the nervous system: pathophysiological implications[END_REF]. Furthermore, chronic caffeine exposure does not alter the expression of A 2A receptors [START_REF] Jacobson | Adenosine receptor ligands: differences with acute versus chronic treatment[END_REF].

The increase in A 1 receptor expression caused by moderate doses of chronically administered caffeine results from prevention of tonic adenosine-mediated receptor down-regulation (see [START_REF] Fredholm | Actions of caffeine in the brain with special reference to factors that contribute to its widespread use[END_REF]. The dose of caffeine we have administered to mice is equivalent to the estimated US average human daily caffeine consumption [START_REF] Barone | Caffeine consumption[END_REF] and, in addition to the expected increase in A 1 receptor levels, it also caused a decrease of cortical and hippocampal CB 1 receptors.

Accordingly, in chronic caffeine-treated mice there was a reduction in the CB 1 receptormediated inhibition of GABA release and stimulation of G-protein activation. Since tonic activation of A 1 receptors was prevented through elimination of endogenous adenosine by vertical superfusion in the [ 3 H]GABA release assays, and by ADA in the [ 35 S]-GTPγS binding assays, it is unlikely that chronic caffeine-induced A 1 receptor up-regulation could be responsible for reduction of CB 1 dependent actions in the in vitro assays. Most probably, chronic caffeine intake, by inducing an imbalance in adenosinergic signaling, disturbs the A 1 -CB 1 cross-talk, which reflects in CB 1 receptor down-regulation.

Independently of the exact mechanisms involved, it is clear that CB 1 receptors are affected after chronic caffeine exposure.

Given that chronic caffeine decreases CB 1 and increases A 1 receptor levels, and that activation of A 1 receptors inhibited the CB 1 -mediated actions in the in vitro assays, it was somewhat surprising that the memory impairment caused by the CB 1 receptor agonists was exacerbated by chronic caffeine intake. It is therefore evident that changes observed in vitro do not necessarily reflect, in a linear way, the effects upon the integrated hippocampal circuitry in vivo. An imbalance in GABAergic transmission resulting from the chronic caffeine-induced alterations of A 1 and CB 1 levels may have occurred, leading to some adaptive changes in the pyramidal cells and/or in the parvalbumin-expressing (PV) GABAergic neurons, which do not express CB 1 receptors [START_REF] Katona | Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons[END_REF]. Interestingly, the blockade of GABAergic transmission was shown to reverse the cognitive effects of acute THC in vivo [START_REF] Varvel | Delta(9)-THC-induced cognitive deficits in mice are reversed by the GABA(A) antagonist bicuculline[END_REF]. A critical imbalance in the temporal coordination of pyramidal cell firing could have become evident when THC was administered, if there was an enhanced sensitivity to the fast spiking activity of PV cells, hence leading to increased inhibition of pyramidal cell firing.

In summary, the present work highlights two relevant factors influencing cannabinoid CB 1 signaling in the hippocampus: the activity of A 1 receptors, and the chronic consumption of caffeine. This A 1 -CB 1 receptor interaction therefore points towards the possibility that the pathophysiological or therapeutically relevant actions operated by CB 1 receptors can be significantly affected by interference with A 1 receptor activity, as is the case of chronic caffeine intake.

Tables:

  and [ 3 H]SR141716A were obtained from GE Healthcare Life Sciences (Buckinghamshire, UK). Guanosine 5'-(γ-[ 35 S]-thio) Triphosphate, ([ 35 S]-GTPγS) was from Perkin Elmer NEN Radiochemicals (Boston, MA, USA). Adenosine deaminase (ADA, EC 3.5.4.4) was from Roche Diagnostics (Indianapolis, IN. USA). ∆ 9 -Tetrahydrocannabinol (THC, >98% purity) was from THC Pharm (Frankfurt, Germany), or Tocris Bioscience (Bristol, UK). CdCl 2 , caffeine (anhydrous base), GABA, aminooxyacetic acid (AOAA), guanosine diphosphate (GDP), guanosine 5'-O-[gamma-thio]triphosphate (GTPγS) were from Sigma (St Louis, MO, morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone monomethanesulfonate (WIN55,212-3), and N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), were from Tocris Bioscience (Bristol, UK). ((RS)-4-Amino-3-(4-chlorophenyl)butanoic acid (baclofen) was from Ascent Scientific (Bristol, UK). All other reagents were from Sigma. For in vitro experiments, non-water soluble drugs were dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at -20º C. The amount of DMSO in solutions was normalized for all conditions in any given experiment, and always <0.02%.

  et al, 2010), with modifications. Briefly, tissue was dissected in a continuously oxygenated (95% O 2 , 5% CO 2 ) ice-cold artificial cerebrospinal fluid (aCSF) of the following composition (mM): NaCl 125, KCl 3, NaH 2 PO 4 1, NaHCO 3 25, CaCl 2 1.5, MgSO 4 1.2, glucose 10, pH 7.4. Samples were homogenized in ice-cold 0.32 M sucrose

[

[ 3 H

 3 ]GABA and [ 3 H]glutamate release from rat hippocampal nerve terminalsIn control conditions, pooling data from all experiments performed, the average basal release of [ 3 H]GABA from rat hippocampal synaptosomes was 0.76±0.02% (n=31, average of first 6 min of collection, Figure1a) of the total tritium retained by synaptosomes at the same time points. Depolarization of the hippocampal synaptosomes with K + (15 mM) for 2 min induced a 3-fold increase in the [ 3 H]GABA release with an average peak of 2.5±0.1% during S 1 , and 2.3±0.1% (n=31, Figure1a) during S 2 , giving an average S 2 /S 1 of 0.94±0.01. Depolarization by K + mainly induced a calcium-dependent release of [ 3 H]GABA since blockade of voltage-gated Ca 2+ channels (VGCCs) by Cd 2+ (CdCl 2 , 200 μM) inhibited its release by 70±3.0% (n=8, p<0.001, data not shown). The GABA transporters account for the remaining percentage of [ 3 H]GABA released upon K + depolarization, since blockade of GABA transporters with SKF89976a

A 1

 1 receptor activation attenuates CB 1 receptor-induced stimulation of G-proteins in rat hippocampal membranesTo test whether the adenosine-cannabinoid interaction occurs at the level of Gprotein activation, we measured agonist-stimulated [ 35 S]-GTPγS binding in hippocampal membranes with the full agonist of CB 1 receptors, WIN55,212-2, or with the partial agonist THC, in absence and in the presence of A 1 receptor agonist CPA. The basal [ 35 S]GTPγS binding in the absence of WIN55,212-2 (Figure4a) or THC (Figure4b) is represented as 100% in the ordinates, which corresponds to (fmol/mg protein): Figure4a: 149 ± 16 in control ( , n=7), 311 ± 50 in presence of CPA ( , n=4), and 331 ± 74 in presence of baclofen ( , n=4); Figure4b: 98 ± 4 in control ( , n=4), 255 ± 25 in presence of CPA ( , n=4), and 161 ± 15 in presence of baclofen( , n=4

  protocol(Chen et al, 2000, see methods), to study the effects of chronic caffeine administration on the memory deficits induced by an acute systemic THC injection in mice. Two separate sets of experiments were performed, in which caffeine (3 mg/kg/day), or vehicle, was administered >12 h before trials, and for at least 15 days before the first test with THC.During the training phase, all subjects learned to perform efficiently at all test parameters, in both sets of experiments (see Figure5a-c, 6a-c). The number of trials, the total latency and total pathlength needed to reach the criterion, decreased progressively from task to task reaching a plateau in the last training task. A repeated measures 2-way analysis of variance (ANOVA) on these parameters revealed a significant overall learning effect. For 'pathlength' (representative parameter of memory performance), significance values were F(4,100)=10.2, p<0.0001 (Figure5c); and F(3,72)=13.5, p<0.0001 (Figure6c). The average swim speed (control parameter for motor activity) was constant throughout the training tasks [Figure5d: F(4,100)=0.4, p=0.8; Figure 6d: F(3,72)=1, p=0.4] with no significant differences between groups[Figure 5d: F(3,25)=1.8, p=0.2; Figure 6d: F(3,24)=0.2, p=0.9]. In both sets of

  . After completion of task 6, each subject rested for one day to allow for the metabolic clearance of THC.There was a significant effect of THC on 'trials to criterion' [F(1,25)=4.24, p=0.05],'latency' [F(1,25)=6.98, p=0.01], and 'pathlength' [F(1,25)=7.18, p=0.01], but no significant effect on 'average speed' [F(1,25)<0.001, p=0.98]. There were no effects of chronic caffeine treatment on all parameters, but a marginally significant 'chronic caffeine THC' interaction on 'pathlength' [F(1,25)=3.11, p=0.09]. Whilst acute THC injection (vs. vehicle) did not induce significant effects in the control (vehicle treated) group at any parameter, the effect of THC was exacerbated in the chronic caffeine group, on 'latency' (p=0.03, Tukey post hoc, Figure5b) and 'pathlength' (p=0.02, Tukey post hoc, Figure5c).

  basal [ 35 S]GTPγS binding in the absence of WIN55,212-2 (Figure 9a,b) or THC (Figure 9c) is represented as 100% in the ordinates, which corresponds to (fmol/mg protein): Figure 9a,b: 143 ± 10 ( , n=5), 154 ± 15 ( , n=5), 324 ± 13 ( , n=5), and 327 ± 25 ( , n=5); Figure 9c: 53 ± 9 ( , n=4), 67 ± 10 ( , n=4). CPA (100 nM), by itself, enhanced [ 35 S]-GTPγS binding by 122±9% (over 2 fold net increase from basal binding) in membranes prepared from vehicle-treated subjects, and by 111±7% in the chronic caffeine group (p>0.05, n=5, Student's t-test, data not shown), hence the ability of A 1

Figure 2 .

 2 Figure 2. A 1 receptor activation significantly attenuates the CB 1 -mediated inhibition of K + -evoked [ 3 H]GABA release from rat hippocampal synaptosomes. (a) Effects of

Figure 3 .

 3 Figure 3. A 1 receptors modulate the CB 1 -mediated inhibition of [ 3 H]glutamate. (a)

Figure 4 .

 4 Figure 4. Influence of A 1 or GABA B receptor activation on CB 1 -induced stimulation of Gproteins, as assayed by WIN55,212-2 or THC-induced [ 35 S]GTPγS binding. Rat

Figure 7 .

 7 Figure 7. Saturation analysis of specific [ 3 H]DPCPX binding (0.1 -10 nM) to cortico-

Figure 8 .

 8 Figure 8. Influence of the adenosine A 1 receptor agonist, CPA (100 nM), and of chronic caffeine administration on the CB 1 -mediated inhibition of K + -evoked [ 3 H]GABA release

Figure 9 .

 9 Figure 9. Influence of chronic caffeine administration on CB 1 -induced stimulation of Gproteins, as assayed by WIN55,212-2 or THC-induced [ 35 S]GTPγS binding. Cortico-

Figure 10 .

 10 Figure 10. Saturation analysis of specific [ 3 H]SR141716A binding (0.1 -4 nM) to (a)

Table 1

 1 To examine if other Gα i/o coupled receptors are also capable of interfering with the G-protein coupling of CB 1 receptors, we tested whether combined activation of CB 1 and GABA B receptors in the hippocampus would also affect the efficacy of WIN55,212-2 in [ 35 S]-GTPγS binding. To activate GABA B receptors we used 100 μM baclofen, which by itself induced a 118±11% net increase from basal [ 35 S]-GTPγS binding (n=4), that was not significantly different from the effect of 100 nM CPA. As shown in Figure4a,band Table1, 100 μM baclofen did not affect the WIN55,212-2-induced (E max=257±12%, n=4) or THC-induced (E max =157±9%, n=4) stimulation of [ 35 S]-GTPγS binding. The reduced efficacy in stimulation of [ 35 S]-GTPγS binding by CB 1 with A 1 but not GABA B suggests that the adenosine A 1 receptors play a specific role in modulating CB 1 signaling in hippocampal presynaptic terminals.

	, n=4, p<0.05), but not the

Table 1 .

 1 E max and log EC 50 values of agonist-stimulated [ 35 S]GTPγS binding in rat hippocampal membranes.

		EC 50	E max
		-log, M	% of stimulation
	WIN	-6.589 ± 0.11	251 ± 7
	WIN + CPA	-6.752 ± 0.23	204 ± 10 *
	WIN + baclofen	-6.554 ± 0.20	257 ± 12
	THC	-6.837 ± 0.20	163 ± 8
	THC + CPA	-6.870 ± 0.72	129 ± 13 *
	THC + baclofen	-6.765 ± 0.26
	Note that the A 1 receptor agonist CPA (100 nM) significantly decreased the E max of
	WIN55,212-2 (WIN) and THC-stimulated [ 35 S]GTPγS binding, but not the EC 50 ; The
	GABA B receptor agonist baclofen (100 μM) had no significant influence. Data represent
	mean values ± s.e.m (n=4) obtained from nonlinear regression analyses of the data
	shown in Figure 3. *p<0.05, compared to appropriate control, calculated using the extra
	sum-of-squares F test.

Table 2 .

 2 E max and log EC 50 values of WIN55,212-2 and THC-stimulated [ 35 S]GTPγS binding in mouse cortico-hippocampal membranes. GABA release induced by WIN (1 μM) in the absence or in the presence of the calcium channel blocker, CdCl 2 (Cd 2+ , 200 μM), or the GABA transporter inhibitor, SKF89976A (SKF, 20 μM), as indicated below each bar. Note that Cd 2+ fully blocked the μM WIN, and 1 μM WIN-3. The S 2 /S 1 values from corresponding controls were taken as 0% within each experiment. P-values were obtained by a one-way ANOVA test with Dunnett post hoc, compared to control (0%). SKF, Cd 2+ and AM251 were applied 15

			EC 50	E max
			-log, M	% of stimulation
	Vehicle	WIN WIN + CPA -5.654 ± 0.16 254 ± 13 * -6.005 ± 0.09 321 ± 11 THC -7.389 ± 0.37 167 ± 11
	Chronic	caffeine	WIN WIN + CPA -6.035 ± 0.10 -6.006 ± 0.08 THC -7.660 ± 0.56	269 ± 8 * 252 ± 8 * 135 ± 8 *
	Note that the E max , but not the EC 50 , of WIN55,212-2 (WIN) and THC-stimulated
	[ 35 S]GTPγS binding was significantly decreased in the chronic caffeine group (3
	mg/kg/day, for 22 days), compared to control (vehicle treated) mice. The A 1 receptor
	agonist, CPA (100nM) reduced the E max of WIN55,212-2 in control mice, but did not
	further decrease the E max of WIN55,212-2 in chronic caffeine treated mice. The EC 50 of
	WIN in either vehicle or chronic caffeine groups was not significantly affected by CPA
	(p>0.05, extra sum-of-squares F test). Data represent mean values ± s.e.m (n=4-5)
	obtained from nonlinear regression analyses of the data shown in Figure 8. *p<0.05,
	versus corresponding control, calculated using the extra sum-of-squares F test.

effect of WIN (n=5, **p<0.01, paired Student's t-test vs. effect of WIN alone within the same batch of synaptosomes), whereas SKF did not alter the WIN-induced inhibition (n=5, p>0.05). Data represent mean ± s.e.m. from 5 experiments, performed in duplicate. (c) Concentration-dependent inhibition of K + -evoked release of [ receptor-selective at 1 μM, but not at 10 μM. Each point represents the mean ± s.e.m. of 4 to 10 independent experiments performed in duplicate, except (n=2) for 0.01 μM and 3

Acknowledgments:

We thank Dr. Stephanie Daumas and Dr. Bruno da Silva (University of Edinburgh) for helpful assistance in the experimental design of the behavioural tests. We acknowledge the Institute of Physiology at the Faculty of Medicine (University of Lisbon) for the animal house facility, Prof. Alexandre de Mendonça (University of Lisbon) and Dr. Attila Kofalvi (University of Coimbra) for useful scientific discussions, and Dr. João Maroco (Instituto Superior de Psicologia Aplicada, ISPA) for valuable advice with statistical analysis. This work, was supported by grants from Fundação para a Ciência e Tecnologia (FCT, V.C.S.: SFRH/BD/21359/2005 and N.A.L.: SFRH/BD/21374/2005), and the European Union [European Cooperation in Science and Technology (COST) COST B30 concerted action, Neural Regeneration and Plasticity (NEREPLAS)].

Disclosure/Conflict of Interest

The authors declare that, except for income received from primary employers, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal holdings that could be perceived as constituting a potential conflict of interest. (3 mg/kg). Subjects rested for one day off drug after task 6, to allow full metabolization of THC. For clarity of comparison between groups, symbols were nudged at tasks 6 and 7. Note that chronic caffeine exacerbated the spatial memory deficits induced by acute THC, and this effect of THC was fully prevented by previous administration of AM251.

All data represent mean ± s.e.m. of n=7-8. *p<0.05, 2-way analysis of variance, followed by Tukey post hoc test (see text for more details).


J Rovenský
email: rovensky.jozef@nurch.sk

M Stančíkova

K Švik

K Bauerová

J Jurčovičová

M Stančíková

K Švík

Prof. Jozef Rovenský

The Effects of β-glucan isolated from Pleurotus ostreatus on Methotrexate Treatment in Rats with Adjuvant Arthritis

Keywords: β-glucan, methotrexate, adjuvant arthritis

Effects of β-glucan

Poly-branched -1,3-(D)-glucans are naturally occurring polysaccharides, with or without -1,6-(D)-glucose side chains, that are integral cell wall constituents in a number of bacteria, plants and yeasts. -glucans from various sources are different in their structure, chemical, physical and biological properties [START_REF] Akramiené | Effects of ßglucans on the immune system[END_REF]. Moreover, they represent the conserved structure -pathogen-associated molecular pattern (PAMP) and are effective biological response modifiers, non-specifically enhancing the host immune system by multiple interactions within innate and adaptive mechanisms [START_REF] Muta | Molecular basis for invertebrate innate immune recognition of (1-->3)-beta-D-glucan as a pathogen-associated molecular pattern[END_REF].

The induction of cellular responses by -glucans is likely to involve their specific interactions with several cell surface receptors, as complement receptor 3, lactosylceramide, selected scavenger receptors, and dectin-1 [START_REF] Ross | Specificity of membrane complement receptor type 3 (CR3) for ß-glucans[END_REF][START_REF] Brown | Dectin-1 is a major ß-glucan receptor on macrophages[END_REF][START_REF] Brown | Immune recognition of fungal beta-glucans[END_REF]. -Glucans increase host immune defense by activating the complement system, enhancing the function of macrophages, leucocytes and natural killer cells. The use of -glucans alone or as vaccine adjuvants for viral and bacterial antigens has been shown in animal models to increase resistance to a variety of bacterial, fungal, protozoan and viral infections [START_REF] Ben-Ami | Immunocompromised hosts: immunopharmacology of modern antifungals[END_REF][START_REF] Kernodle | Prophylactic anti-infective activity of poly-[1-6]-beta-D-glucopyranosyl-[1-3]-beta-D-glucopryanose glucan in a Guinea pig model of staphylococcal wound infection[END_REF][START_REF] Vetvicka | Immunological effects of yeast-and mushroom-derived beta-glucans[END_REF]. -Glucans also show anticarcinogenic activity. Its use as adjuvant to cancer chemotherapy and radiotherapy demonstrated the positive role in the restoration of hematopoiesis following bone marrow injury [START_REF] Weitberg | A phase I/II trial of beta-(1,3)/(1,6) D-glucan in the treatment of patients with advanced malignancies receiving chemotherapy[END_REF][START_REF] Mantovani | Beta-Glucans in promoting health: prevention against mutation and cancer[END_REF][START_REF] Bobek | Effect of pleuran (beta-glucan from Pleurotus ostreatus) on the antioxidant status of the organism and on dimethylhydrazineinduced precancerous lesions in rat colon[END_REF].

β-(1,3/1,6)-D-glucan, is an insoluble polysaccharide isolated from the mushroom Pleurotus ostreatus. It is a safe and potent nutritional supplement with a profound systemic effect that can be described as nonspecific immune stimulation combined with antioxidant activity [START_REF] Bobek | Effect of pleuran (beta-glucan from Pleurotus ostreatus) on the antioxidant status of the organism and on dimethylhydrazineinduced precancerous lesions in rat colon[END_REF][START_REF] Bobek | Effect of oyster mushroom (Pleurotus ostreatus) on pathological changes in dimethylhydrazine-induced rat colon cancer[END_REF][START_REF] Nosál'ová | Effects of pleuran (beta-glucan isolated from Pleurotus ostreatus) on experimental colitis in rats[END_REF]. Recently, Smiderle et al. [START_REF] Smiderle | Anti-inflammatory and analgesic properties in a rodent model of a (1-->3),(1-->6)-linked beta-glucan isolated from Pleurotus pulmonarius[END_REF] described the antiinflammatory and analgesic activity of β-(1,3/1,6)-D-glucan isolated from Pleorutus ostreatus on the acetic acid-induced writhing reaction in mice, a typical model for quantifying inflammatory pain. The authors suggested that the glucan had potent antiinflammatory and analgesic activities, possibly due to the inhibition of pro-inflammatory cytokines. In our previous studies with application of β-(1,3/1,6)-Dglucan isolated from Pleurotus ostreatus to rats with adjuvant arthritis we showed decreased activities of pro-inflammatory cytokines TNF-α, IL-1 and IL-6 in the serum of arthritic rats, decreased oxidative stress and suppressed inflammatory and arthritic signs in rats [START_REF] Bauerová | Study of new ways of supplementary and combinatory therapy of rheumatoid arthritis with immunomodulators Glucomannan and Imunoglukán® in adjuvant arthritis[END_REF][START_REF] Stančíková | The effects of immunostimulatory drugs on rat adjuvant arthritis (in slovak)[END_REF]. Protective antioxidant activity and anti-inflammatory activities of carboxylated (1-3)-beta-D-glucan isolated from Saccharomyces cerevisiae were reported in adjuvant arthritis in Lewis rats [START_REF] Kogan | Antioxidant properties of yeast (1→3)-β-d-glucan studied by electron paramagnetic resonance spectroscopy and its activity in the adjuvant arthritis[END_REF].

Methotrexate is an antifolate that is widely used in the treatment of rheumatic disorders and malignant tumors. The efficacy of methotrexate is often limited by severe side effects, which includes also the development of oxidative stress. Sener et al. [START_REF] Sener | Betaglucan ameliorates methotrexate-induced oxidative organ injury via its antioxidant and immunomodulatory effects[END_REF] showed that -glucan can ameliorate the methotrexate-induced oxidative organ injury (liver or kidney) in rats via its antioxidant and immunostimulatory effects.

Our previous results showed the beneficial effects of β-(1,3/1,6)-D-glucan in rat adjuvant arthritis, the aim of the present study was to evaluate its effect on methotrexate treatment of rats with adjuvant arthritis. Induction of arthritis. The rats were injected with 0.1 ml suspension of heat killed Mycobacterium butyricum (12 mg/ml) in incomplete Freund's adjuvant intradermally at the base of the tail.

Materials and methods

Materials

Treatment. MTX and β-glucan-PO were administered in corresponding doses from day 0 (the day of immunization) to day 28 of the study. MTX was prepared by dilution with sterile saline to yield the desired concentration of 0.5 mg in 0.1 ml saline, and applied twice a week per os (1 mg/kg in total per week). β-glucan-PO was administered orally as suspension in saline every second day in dose 1 mg/kg body mass. The untreated groups received the vehicle (sterile saline) in the same manner daily for 28 days.

The animals were divided into the following five groups of 8 animals: group 1non-arthritic untreated healthy controls; group 2 -untreated rats with AA; group 3 -AA rats treated with β-glucan-PO; group 4 -AA rats treated with MTX; group 5 -AA rats treated with the combination of MTX + β-glucan-PO.

Evaluated Parameters

Body mass of rats was measured at the beginning of study and every week during the study.

Hind paw swelling. The volume of the hind paw swelling was measured with an electronic water plethysmographically (UGO BASILE, Comerio-Varese, Italy) on days 14, 21 and 28.

Arthrogram score. The severity of arthritis was quantified by scoring each paw from 0 to 5, based on increasing levels of swelling and periarticular erythema. The sum of the scores for the limbs was calculated as the arthritic index, with a maximum possible score of 20 per rat. Arthrogram scores were evaluated on days 14, 21 and 28.

Serum albumin levels were measured on days 14, 21 and 28 in the rat serum by spectrophotometric method, using SYS 1 kit (BM/Hitachi, Boehringer Mannheim, Germany) on a Hitachi 911 automatic biochemical analyzer.

Statistical analysis of the results. One-way analysis of variance (ANOVA) was used for statistical analysis of the results, and p<0.05 was considered as the significance limit for all comparisons.

Results

Body weight. In the first 7 days of the treatment, the increment in body weight was similar in all groups of rats (Table 1). However, on day 14, the body mass of arthritic control rats and rats treated with β-glucan-PO alone was significantly lower than that of the healthy controls and arthritic rats treated with MTX and with combination of MTX + β-glucan-PO. The increase of body mass in rats treated with combination MTX + β-glucan-PO was similar to that treated with MTX alone.

Hind paw swelling, arthrogram score. The clinical signs of arthritis reflect both inflammatory and arthritic changes occurring in rats with AA. The volume of the swollen hind paws in arthritic rats was significantly higher compared to healthy controls on days 14, 21 and 28, as supported by the mean value for two hind paws (Table 2). Statistically significant decreases of both hind paw swelling and arthrogram scores were observed in the arthritic rats treated with MTX on post-immunization days 14, 21 and 28 (Table 3). β-glucan-PO administered alone significantly decreased both the hind paw swelling and arthritic score on day 21 and 28. The combination treatment MTX + β-glucan-PO reduced these parameters statistically more significantly than MTX treatment alone (MTX vs. MTX+ β-glucan-PO, P<0.05).

Serum albumin levels.

Serum albumin acts as a negative acute phase reactant in both rat and human arthritis. Lower levels of serum albumin correspond to higher levels of inflammatory activity. The concentration of albumin in the serum of arthritic controls was significantly lower than in healthy controls (HC vs. AA rats, p< 0.001).

Both MTX alone and the combination treatment with MTX + β-glucan-PO significantly inhibited the decrease in serum albumin (Table 4).

Discussion

This experiment was focused on the effect of β-(1,3/1,6)-D-glucan isolated from Pleurotus ostreatus on the inflammatory and arthritic markers in rats with AA during basal treatment with MTX. The treatment was prophylactic, which means that the animals were treated immediately after administration of the adjuvant.

The results of our investigation confirmed the previously reported effect of MTX treatment in rats with AA [START_REF] Welles | Studies on the effect of low dose methotrexate in adjuvant arthritis[END_REF][START_REF] Connolly | Alteration of interleukin-1 production and the acute phase response following medication of adjuvant arthritic rats with cyclosporin-A or methotrexate[END_REF]. Methotrexate at a dose of 1 mg/kg/week suppressed, but did not prevent, arthritis development. In our study, MTX significantly suppressed the hind paw swelling and decreased arthrogram scores. β-glucan-PO alone decreased both the hind paw swelling and the arthrogram on days 21 and 28.

The remarkable finding was that β-glucan-PO potentiated the beneficial effect of MTX; reduction of hind paw swelling and arthrogram scores on days 21 and 28 were more significant compared to the rats treated with MTX alone.

Serum albumin acts as a negative acute-phase reactant in rat arthritis.

Decreased levels of serum albumin reflect the changes in synthesis of this protein in the liver secondary to the activation of hepatic cells by inflammatory cytokines, mainly interleukin-1 [START_REF] Connolly | Alteration of interleukin-1 production and the acute phase response following medication of adjuvant arthritic rats with cyclosporin-A or methotrexate[END_REF]. Our results correlate with the observation that MTX markedly prevents the albumin decrease in AA rats. The combination of MTX with β-glucan-PO had no additional effect compared to MTX alone (Table 4). Systemic administration of β-glucan to rats and mice has been demonstrated to protect against various infections by activation of macrophages and attenuation of proinflammatory cytokine release [START_REF] Nosál'ová | Effects of pleuran (beta-glucan isolated from Pleurotus ostreatus) on experimental colitis in rats[END_REF][START_REF] Cleary | The effect of molecular weight and beta-1,6-linkages on priming of macrophage function in mice by (1,3)-beta-D glucan[END_REF][START_REF] Onderdonk | Anti-infective effect of poly-beta-1-6-glucotriosyl-.beta1-3-glucopyranose glucan in vivo[END_REF][START_REF] Bedirli | Beta-glucan attenuates inflammatory cytokine release and prevents acute lung injury in an experimental model of sepsis[END_REF]. Hetland et al. [START_REF] Hetland | Protective effect of beta-glucan against mycobacterium bovis, BCG infection in BALB/c mice[END_REF] have showed that βglucan reduced growth of Mycobacterium tuberculosis in macrophage cultures and had protective effect against Mycobacterium bovis, BCG infection in BALB/c mice [START_REF] Hetland | Beta-1,3-glucan reduces growth of Mycobacterium tuberculosis in macrophage cultures[END_REF]. Certain microbes, such as fungi and viruses led to generation and activation of autoimmune T cells resulting in a development of a particular autoimmune disease in genetically susceptible individuals. β-(1,3/1,6)-D-glucan, an effective activator of immune system may be beneficial also in humans in preventing or eliminating bacterial infections which are known to induce reactive arthritis.

In our study we tested a food supplement, the commercially used -glucan isolated from Pleurotus ostreatus. This -glucan decreased the arthritis development in rats and had additional beneficial effect to methotrexate treatment.

Table 1. The effect of MTX, β-glucan-PO, and their combination on body mass of rats (g).

Data represent mean value and standard deviation (mean value ± SD) for groups of 8 rats.

Significantly different from arthritic control rats: ** p<0.01, *** p<0.001.

Groups of rats

.

  In this study methotrexate injection solution 10 mg/ml in sterile saline from Medac Company, Hamburg, Germany was used. Beta-(1,3/1,6)-D-glucan is insoluble micronized pure compound isolated from Pleurotus ostreatus (β-glucan-PO). Beta-(1,3/1,6)-D-glucan (Imunoglukan®) was obtained from Pleuran s.r.o. company (Bratislava, Slovakia). Mycobacterium butyricum was purchased from Difco Laboratories Co. Ltd. (Detroit, USA) and incomplete Freund's adjuvant from Sigma-Aldrich Chemie GmbH (Germany).

Animals.

  Male Lewis rats (160 -180 g) obtained from Charles River Wiga, Germany were maintained during the experiment in standard animal facilities that comply with European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. The animals were fed pelleted food (TOP DOVO, Dobrá Voda, Slovak Republic) and had free access to both food and water. The State Veterinary Committee of the Slovak Republic and the Ethics Committee for Control of Animals Experimentation at the National Institute of Rheumatic Diseases approved the experimental protocol and all procedures.

Table 2 . The effect of MTX, β-glucan-PO and their combination on hind paws swelling (mL) in AA rats

 2

		Day 1	Day 7	Day 14	Day 21	Day 28
	Healthy controls	167 ± 6	219 ± 13 224 ± 13 ** 239 ± 14 *** 250 ± 15 ***
	Untreated AA controls 170 ± 8	217 ± 12 186 ± 11	179 ± 15	199 ± 13
	AA rats treated with:
	β-glucan-PO	171 ± 6	217 ± 7	193 ± 10	184 ± 9	212 ±12
	MTX-2	173 ± 6	227 ± 7	228 ± 17 *** 236 ± 14 *** 247 ± 15 ***
	MTX-2+β-glucan-PO	171 ± 6	228 ± 8	231 ± 14 *** 230 ± 13 *** 244 ± 14 ***

Table 3 . The effect of MTX, β-glucan-PO and their combination on the arthrogram score in AA rats.

 3 Data represent mean value and standard deviation (mean value ± SD) for groups of 8 rats.Significantly different from arthritic control rats: *p<0.05, **p<0.01, *** p<0.001.Significantly different from arthritic rats treated with MTX: †p<0.05

	Groups of rats	Day 14	Day 21	Day 28
	Untreated AA controls 13.44 ± 1.81	17.22 ± 1.99	14.89 ± 2.26
	AA rats treated with:
	β-glucan-PO	11.30 ± 1.46	14.25 ± 0.53 *	12.14 ± 2.10 *
	MTX-2	8.50 ± 1.52 **	13.33 ± 3.44 **	11.00 ± 2.53 *
	MTX-2+ β-glucan-PO	8.25 ± 2.25 ***	10.88 ± 2.85 *** †	9.88 ± 2.10 *** †

Table 4 . The effect of MTX, β-glucan-PO and their combination on serum albumin concentrations (g/L) in AA rats.

 4 Data represent mean value and standard deviation (mean value ± SD) for groups of 8 rats.Significantly different from arthritic control rats: * p<0.05, **p<0.01, *** p<0.001.

	Groups of rats	Day 14	Day 21	Day 28
	Healthy controls	42.00 ± 2.70 ***	39.92 ± 2.45 ***	42.15 ± 2.14 ***
	Untreated AA controls 27.68 ± 1.08	30.91 ± 2.10	34.28 ± 1.22
	AA rats treated with:
	β-glucan-PO	28.62 ± 1.26	30.66 ± 1.71	34.41 ± 2.04
	MTX-2	32.01 ± 3.36 **	35.33 ± 1.79 ***	38.03 ± 2.19 **
	MTX-2+ β-glucan-PO	36.01 ± 3.44 ***	36.33 ± 3.39 ***	37.84 ± 2.02 **

This work was supported by APVV grant, number APVV-21-055205