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Slow cortical rhythm (SCR) is a rhythmic alternation of UP and DOWN states during sleep and anesthesia. SCR-associated slow waves reflect homeostatic sleep functions. Adenosine accumulating during prolonged wakefulness and sleep deprivation (SD) may play a role in the delta power increment during recovery sleep. NREM sleep is a local, use-dependent process of the brain. In the present study, direct effect of adenosine on UP and DOWN states was tested by topical application to frontal, somatosensory and visual cortices, respectively, in urethane-anesthetized rats. Local field potentials (LFPs) were recorded using an electrode array inserted close to the location of adenosine application. Multiple unit activity (MUA) was measured from layer V-VI in close proximity of the recording array. In the frontal and somatosensory cortex, adenosine modulated SCR with slow kinetics on the LFP level while MUA remained mostly unaffected. In the visual cortex, adenosine modulated SCR with fast kinetics. In each region, delta power increment was based on the increased frequency of state transitions as well as increased height of UP-state associated slow waves. These results show that adenosine may directly modulate SCR in a complex and region-specific manner which may be related to the finding that restorative processes may take place with varying duration and intensity during recovery sleep in different cortical regions. Adenosine may play a direct role in the increment of the slow wave power observed during local sleep, furthermore it may shape the region-specific characteristics of the phenomenon.

The effect of intracerebellar microinfusion of antisense oligodeoxynucleotide to Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and other naturally occurring cannabinoid receptor (CB(1)) mRNA on Delta(9)-THC-induced motor impairment was investigated in mice. Delta(9)-THC (15-30 microgram/microliter intracerebellar) resulted in a significant motor impairment in a dose-related manner. The intracerebellar pretreatment with antisense oligodeoxynucleotide (3.0 microgram/100 nl/12 h; six administrations/mouse) virtually abolished Delta(9)-THC (15 and 25 microgram/1 microliter intracerebellar)-induced motor impairment. However, intracerebellar pretreatment with the mismatched oligodeoxynucleotide in exactly the same manner as the antisense was completely ineffective in altering the Delta(9)-THC-induced motor impairment. These results strongly suggest the involvement of CB(1) receptor in the expression of Delta(9)-THC-induced motor impairment. The intracerebellar microinfusion of adenosine A(1)-selective agonist, N(6)-cyclohexyladenosine (CHA) (4 ng/100 nl) significantly enhanced Delta(9)-THC-induced motor impairment, suggesting a cerebellar A(1) adenosinergic modulation of motor impairment. A pretreatment with the antisense and the mismatched oligodeoxynucleotide also markedly attenuated and did not alter, respectively, the cerebellar A(1) adenosinergic modulation (enhancement) of Delta(9)-THC-induced motor impairment. There was no change in the normal motor coordination due to intracerebellar pretreatment with antisense and its mismatch, in the presence as well as absence of intracerebellar CHA indicating the selectivity of interactions with Delta(9)-THC. The Delta(9)-THC-induced motor incoordination was also significantly enhanced dose-dependently by systemic (i.p.) ethanol administration suggesting behavioral synergism between the two psychoactive drugs. Pretreatment (intracerebellar) with pertussis toxin (PTX) markedly attenuated Delta(9)-THC- and Delta(9)-THC+CHA-induced motor incoordination suggesting coupling of CB(1) receptor to PTX-sensitive G-protein (G(i)/G(o)). These data suggested co-modulation by cerebellar cannabinoid and adenosine system of Delta(9)-THC-induced motor impairment. Conversely, the results in the present study also suggested co-modulation by cerebellar adenosine A(1) and CB(1) receptors of ethanol-induced motor impairment, thereby indicating a possible common signal transduction pathway in the expression of motor impairment produced by Delta(9)-THC as well as ethanol.

Cerebral ischemia is often followed by a period of delayed hypoperfusion that may contribute to tissue injury. We tested the hypothesis that augmentation of interstitial adenosine can improve tissue perfusion under this condition. 10 min global ischemia was produced in two groups of isoflurane-anesthetized newborn pigs by occlusion of subclavian and brachiocephalic arteries, and changes in local cortical blood flow and cortical interstitial purine metabolites were measured using the combined hydrogen clearance-microdialysis technique. In one group, the dialysis probe was perfused with artificial cerebrospinal fluid buffer containing nitrobenzyl-thioinosine (NBTI, 100 μmol/1), a competitive inhibitor of adenosine transport. In the untreated group ( n = 9), baseline cortical blood flow (39 ± 3 ml/min/100 g) was depressed by 51 ± 5% and 42 ± 6% at 40 and 60 min, respectively, of postischemic reperfusion. NBTI increased baseline interstitial adenosine levels 2.4-fold which increased baseline cortical blood flow 1.5-fold to 60 ± 4 ml/min/ 100 g, and increased both absolute adenosine levels as well as adenosine as a percentage of total purine metabolites throughout ischemia and reperfusion. As a result, the extent of postischemic hypoperfusion was significantly lessened in NBTI-treated animals ( n = 9 ), with reductions in cortical blood flow of only 28 ± 3% and 24 ± 5% at 40 and 60 min of reperfusion, respectively. These results indicate that inhibition of adenosine transport by NBTI elevates interstitial adenosine concentration during and following cerebral ischemia, and concomitantly improves cortical perfusion in the post-ischemic period. The latter effect may contribute to the documented neuroprotective efficacy of adenosinergic therapy in cerebral ischemia.

Many epidemiological studies have suggested a high correlation between the use of tobacco and ethanol, the two most frequently abused psychoactive drugs. Recently, we reported behavioral interactions between (-)-nicotine, (-)-cotinine and ethanol within the CNS. The present report is a confirmation and an extension of that study. Using a 2 g/kg ethanol-induced motor incoordination (EIMI) as the test response, possible behavioral interactions between (-)-nicotine, (-)-cotinine and ethanol and between (-)-nicotine, (-)-cotinine and adenosine agonist + ethanol in the cerebellum were investigated. (-)-Nicotine, 0.625, 1.25 and 5 ng intracerebellarly (ICB) significantly attenuated EIMI in a dose-related manner. Likewise, ICB injection of 1.25, 2.5, and 5 ng (-)-cotinine, a major metabolite of nicotine, significantly attenuated EIMI after the same i.p. dose of ethanol as in case of (-)-nicotine but less markedly compared to (-)-nicotine. No change in normal motor coordination was observed when the highest dose of (-)-nicotine or (-)-cotinine was injected ICB followed by saline control, suggesting selectivity of their behavioral interactions with ethanol. The attenuation of EIMI by (-)-nicotine and (-)-cotinine was blocked by ICB hexamethonium (1 microgram) and trimethaphan (100 ng), the purported nicotinic-cholinergic antagonists. Finally, the ICB injection of adenosine agonists, N6-cyclohexyladenosine (CHA) or 5'-N-ethylcarboxamidoadenosine (NECA), produced marked accentuation of EIMI which was significantly antagonized by ICB (-)-nicotine and (-)-cotinine. The data obtained in the present study suggested, for the first time, a cerebellar adenosinergic-nicotinic cholinergic interaction and modulation of EIMI. The data also suggested participation of cerebellar nicotinic-cholinergic receptors in EIMI.

In recent years misuse of methylphenidate (MPH) has been reported. The main pharmacological target of methylphenidate is the dopaminergic system. Adenosine is a neuromodulator that influences the dopaminergic neurotransmission, but studies on MPH and adenosine are still lacking. In this study, adult mice were acutely treated with MPH (5 mg/kg, i.p.) and to model misuse, they received an acute overdosage (50 mg/kg, i.p). The involvement of adenosine A1 receptors in anxiety-related behavior and locomotor and exploratory activity was examined. The administration of methylphenidate (5 and 50 mg/kg) 30 min before the exposure to open field arena did not modify locomotor activity. The anxiolytic-like behavior was observed with both doses of MPH as revealed by the increase on the number of entries and the time spent in the open arms in the elevated plus-maze. Pre treatment with selective adenosine A1 receptor antagonist (DPCPX 1 mg/kg, i.p.) did not prevent anxiolytic effect caused by MPH 50 mg/kg. Immunoblotting of frontal cortex and hippocampal extracts revealed that MPH 50 mg/kg increased 88% adenosine A1 receptor density in the frontal cortex. Extracts from hippocampus did not reveal any differences in the adenosine A1 receptor density. Our findings ruled out the participation of adenosine A1 receptors on the MPH-triggered anxiolytic effects. However, the density of adenosine A1 receptors increased in a brain area strictly involved in the MPH-mediated effects. Thus, the adenosinergic system may play a role in the methylphenidate actions in the central nervous system.

Electrophysiological recordings were made from rat hippocampal slices exposed to in vitro ischemic conditions in which the superfused medium is hypoxic and lacking glucose. Under these conditions, the evoked population spike recorded in CA1 is initially depressed and then transiently returns prior to an anoxic depolarization. This transient return in synaptic function under ischemic-like conditions also occurs if the population spike is inhibited by pretreatment with adenosinergic agonists or with the γ-aminobutyric acid (GABA)B agonist, baclofen.

Dysfunction of adenosinergic systems has been implicated in the development of multiple sclerosis in humans and experimental autoimmune encephalomyelitis (EAE) in animals. Caffeine, a non-selective antagonist of adenosine receptors, has been shown to provide protection against myelin oligodendroglia glycoprotein (MOG)-induced EAE in mice. In this study, we showed that chronic caffeine similarly imparts neuroprotection against EAE induced in rats by guinea pig spinal cord homogenates (GPSCH). GPSCH-induced EAE is characterized by extensive tissue inflammation with a typical chronic disease course. We showed that caffeine decreases the incidence of EAE and attenuates EAE pathology at behavioral, histological (inflammatory cell infiltration and demyelination) and neurochemical (expression of inflammatory cytokines) levels. The attenuation of GPSCH-induced pathology by chronic caffeine treatment was observed at doses of 10 and 30 mg/kg and during both peak and recovery phases of EAE. Furthermore, it was showed that chronic treatment with caffeine up-regulated A1 receptor and TGF-beta mRNAs and suppressed interferon-gamma mRNA in EAE rats. Together with previous reports, our data demonstrates that chronic treatment with caffeine exerts a neuroprotective effect against EAE, possibly through an A(1) receptor-mediated shift from Th1 to Th2 cell function, and provides a neurobiological basis for epidemiological investigation into the possible relationship between caffeine consumption and development of multiple sclerosis in humans.

The identity of the receptors involved in mediating the depressant actions of adenosine and its analogs on the spontaneous firing of rat cerebral cortical neurons was elucidated by evaluating the effects of selective A1 and A2 receptor agonists and antagonists. The A1 agonist N6-cyclopentyladenosine (CPA) and the A2 agonist CGS 21680 both depressed cortical neuronal firing. At low doses (0.001-0.01 mg/kg) the A1 antagonist DPCPX blocked the effects of CPA, but not those of CGS 21680. At higher doses, it antagonized both agonists. The A2 antagonist CGS 15943 (0.01-0.1 mg/kg) selectively blocked the actions of CGS 21680. At 1 mg/kg it also antagonized the responses of some neurons to CPA. These results suggest that the depressant actions of adenosinergic agonists on the firing of rat cerebral cortical neurons involve both A1 and A2 receptors.

The relationships of adenosine A1 receptor agonists and antagonists in combination with angiotensin II (AT II) and its analogue sarmesin in PTZ (pentylenetetrazol) kindling in ICR mice were studied. The occurrence of clonic and tonic seizures and the latency to their onset in PTZ kindled mice was determined. Combination of adenosinergic and AT II-ergic agents significantly reduced the incidence of clonic seizures in PTZ kindling without changing the latency. Pretreatment with 8-p-Sulfophenyl-theophylline (8-p-SPT) reversed the anti-convulsant effect of sarmesin.

Neuronal dysfunction in the frontal cortex has been reported in the etiology of mental disorders, including schizophrenia. The adenosine A(1) receptor system, as well as the dopaminergic system, are important in the control of cortical neuronal activity. We hypothesize that neuroexcitability in early life is critical to the normal development of the brain, and neurosteroids are factors that modulate neuroexcitability during the development period. In this study, we treated neonatal rats with a neurosteroid, pregnenolone (10 microg/g) from postnatal day (PD) 3 until PD 7. In pregnenolone-treated male and female rats, adenosine A(1) receptor density, the amount of dopamine (DA), serotonin (5-HT) and their metabolites in the fronto-parietal cortex and behavioral responses in the open field were examined pre- and post-puberty. A decrease in K(d) values for the adenosine A(1) receptor binding assay using [(3)H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX), increased formation of DA metabolites and hyper-locomotor activity in the open field were found in pregnenolone-treated rats compared with controls in pre- and post-puberty. An increase in 5-HT metabolites was found in the pregnenolone-treated rats in pre-puberty, but not post-puberty. These effects of pregnenolone were not different between males and females. However, correlations between horizontal and vertical activities in the open field were disrupted only in pregnenolone-treated females. The present results indicate that pregnenolone treatment during the neonatal period influences the cortical dopaminergic and adenosinergic systems as well as behavioral responses in the open field.

Previous studies from our laboratory have provided strong evidence that brain adenosine modulates acute ethanol (i.p.)-induced motor incoordination (MI) through receptor mediated mechanism(s). Recently, we have reported the involvement of the striatum in ethanol-induced MI as well as the striatal adenosinergic modulation of the ethanol-induced motor deficit. The present study was thus designed to further characterize the modulatory effect of striatal adenosine on ethanol-induced MI and to look for its functional correlation with chloride flux within the rat striatum. Intrastriatal microinfusion of adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), significantly accentuated and attenuated, respectively, the motor incoordinating effect of ethanol while having no effect on the normal motor coordination in saline-treated control animals. These data confirmed the role of striatal adenosine in ethanol-induced MI. The selectivity of interactions between adenosine A1 agonist and antagonist and ethanol was further confirmed by the study in which neither intrastriatal CHA nor DPCPX significantly altered the MI induced by sodium pentobarbital. Previously, we have shown that intrastriatal Ro15-4513 not only significantly attenuated ethanol-induced MI but also blocked its accentuation by intrastriatal CHA. It is well known that Ro15-4513 antagonizes many, but not all, CNS effects of ethanol by blocking the ethanol potentiation of GABA-stimulated uptake of chloride. Therefore, experiments using striatal microsac preparations were carried out to investigate the possible modulation of chloride conductance by CHA and its relationship to ethanol. High concentrations of CHA (10 and 100 nM) increased the total chloride uptake by the striatal microsacs. Corresponding to the ethanol-adenosine interaction observed behaviorally, a much lower concentration (1 nM) of CHA, being ineffective itself, significantly enhanced the stimulatory action of ethanol on chloride uptake. This effect was blocked by either Ro15-4513 (100 nM) or DPCPX (10 nM). The modulatory effect of GABA and/or ethanol on chloride influx was also evaluated, and the results supported the appropriateness to use striatal microsac preparations in the present study. Overall, the data suggested a functional interaction between ethanol and striatal adenosine and further supported the hypothesis that striatal adenosine might, in part, modulate ethanol-induced MI through its effect on chloride conductance through chloride channels coupled to GABA-benzodiazepine receptor complex.

As an extension of our previous work pertaining to brain adenosinergic modulation of ethanol-induced motor incoordination, the effect of direct intracerebellar administration of the A1-selective adenosine agonist, N6-cyclohexyladenosine (CHA) on ethanol-induced motor incoordination was evaluated. Marked accentuation of ethanol-induced motor impairment by CHA was observed. No change in the normal motor coordination was noted when CHA administration was followed by saline instead of ethanol. Intracerebellar cAMP or its analog, 8-(4-chlorophenylthio)-cAMP, significantly inhibited ethanol's motor impairment in a dose-related manner as well as abolished CHA's accentuating effect on ethanol-induced motor incoordination. These observations suggested a possible involvement of cAMP in the adenosinergic modulation and in the expression of ethanol-induced motor incoordination. Further support was provided by the observation of a marked accentuation and attenuation in a dose-related manner of ethanol-induced motor impairment as well as CHA's accentuation of ethanol's motor impairment by intracerebellar miconazole and forskolin, respectively. However, equimolar intracerebellar doses of miconazole and forskolin (inhibitor and stimulator of adenylyl cyclase, respectively) failed to significantly alter ethanol-induced motor incoordination probably due to their mutual functional antagonism. The expression of adenosinergic modulation and that of ethanol-induced motor impairment most likely involved Gi protein-coupled receptor(s) (such as adenosine receptors). The involvement of receptors linked to pertussis toxin-sensitive G-proteins was suggested because intracerebellar pertussis toxin pretreatment markedly inhibited ethanol-induced motor incoordination as well as CHA's accentuation of ethanol's motor impairment. Finally, cAMP, unlike its antagonism to CHA's accentuation, failed to antagonize the accentuation of ethanol-induced motor impairment by intracerebellar GABA(A) agonist (+)-muscimol. This indicated selectivity of cAMP participation in G protein coupled receptor (such as adenosine)-mediated response and not in ionic channel coupled receptor (such as GABA(A))-mediated mechanism. Overall, the data suggested a possible involvement of cerebellar adenylyl cyclase-cAMP signalling pathway in the adenosinergic modulation of ethanol's ataxia.

Caffeine and other methylxanthines elevate reinforcement threshold for electrical brain stimulation with an order of potency suggesting that the effect is mediated by antagonism of adenosine A2 receptors. The purpose of this study was to evaluate further the possible mechanism by which caffeine and other methylxanthines elevate reinforcement thresholds for ICSS. Drugs known to affect adenosinergic transmission in predictable ways, adenosine receptor agonists and antagonists and benzodiazepine agonists and inverse agonists, were tested to determine their effect upon reinforcement threshold. Both the selective A1 adenosine agonist, R(-)-PIA, and the non-selective A1/A2 adenosine agonist NECA failed to alter reinforcement thresholds, as did CGS 15943, a potent non-xanthine non-selective adenosine receptor antagonist. Chlordiazepoxide, a benzodiazepine agonist, lowered reinforcement thresholds and FG 7142, a benzodiazepine inverse agonist, elevated reinforcement thresholds, perhaps corresponding to their anxiolytic and anxiogenic subjective effects in humans. However, another benzodiazepine agonist, midazolam and another inverse agonist, beta-CCE, did not alter reinforcement thresholds. These results fail to support a general role for adenosinergic systems in the threshold-elevating effect of methylxanthines.

Chronic administration of caffeine (s.c. for a period of 14 days in escalating doses of 10-70 mg/kg) increased the sensitivity of rat cerebral cortical neurons to the inhibitory action of microiontophoretically applied adenosine. The sensitivity of spontaneously firing rat cerebral cortical neurons in caffeine-treated animals was compared with that of saline-treated controls using the same multiple-barrel micropipettes tested on the same day. Adenosine sensitivity was determined by the I.T50 method. The I.T50 value for 134 neurons in the caffeine-treated rats of 130.77 +/- 4.33 (S.E.M.) was significantly (P less than 0.001) different to that of 136 neurons in the saline-treated control rats (222.16 +/- 6.68), indicating a supersensitivity to adenosine in neurons which had been chronically exposed to caffeine.

The adenosine receptor antagonist, caffeine, transiently induced proto-oncogene c-fos mRNA in mouse brain in a dose-dependent fashion. In situ hybridization revealed that caffeine-induced c-fos expression was high in caudate-putamen and olfactory tubercle at both subconvulsive and convulsive doses. The pattern of c-fos mRNA distribution following caffeine administration differs from that reported after seizures induced by electroconvulsive shock (ECS) or other chemical convulsants, and closely parallels the distribution of adenosine A2 receptors. Furthermore, the potent adenosine A2 receptor agonist, 5'-N-ethylcarboxamide adenosine (NECA) blocked caffeine-induced c-fos expression whereas the adenosine A1 receptor ligand, N6-cyclohexyladenosine (CHA), had no effect. This study suggests that the caffeine-induced expression of c-fos mRNA may be mediated by the adenosine A2 receptor in mouse brain.