Cocaine abstinence induces emotional impairment and brain region-specific upregulation of the oxytocin receptor binding.

The key problem in treating cocaine addiction is the maintenance of a drug-free state as negative emotional symptoms during abstinence often trigger relapse. The mechanisms underpinning the emotional dysregulation during abstinence are currently not well-understood. There is evidence suggesting a role of the neuropeptide oxytocin in the modulation of drug addiction processes. However, its involvement during long-term abstinence from cocaine use remains unclear. In this study, we aimed to behaviourally characterize a mouse model of long-term cocaine withdrawal and assess the effect of chronic cocaine administration and long-term cocaine abstinence on the central oxytocinergic system and the hypothalamic-pituitary-adrenal axis. Fourteen-day escalating-dose cocaine administration (3 × 15-30 mg/kg/day) and 14-day withdrawal increased plasma corticosterone levels and oxytocin receptor (OTR) binding in piriform cortex, lateral septum and amygdala. A specific cocaine withdrawal-induced increase in OTR binding was observed in the medial septum. These biochemical alterations occurred concomitantly with the emergence of memory impairment, contextual psychomotor sensitization and an anhedonic and anxiogenic phenotype during withdrawal. Our study established a clear relationship between cocaine abstinence and emotional impairment in a novel translationally relevant model of cocaine withdrawal and demonstrated for the first time brain region-specific neuroadaptations of the oxytocin system, which may contribute to abstinence-induced negative emotional state.

Emotional Impairment and Persistent Upregulation of mGlu5 Receptor following Morphine Abstinence: Implications of an mGlu5-MOPr Interaction.

BACKGROUND: A difficult problem in treating opioid addicts is the maintenance of a drug-free state because of the negative emotional symptoms associated with withdrawal, which may trigger relapse. Several lines of evidence suggest a role for the metabotropic glutamate receptor 5 in opioid addiction; however, its involvement during opioid withdrawal is not clear. METHODS: Mice were treated with a 7-day escalating-dose morphine administration paradigm. Following withdrawal, the development of affective behaviors was assessed using the 3-chambered box, open-field, elevated plus-maze and forced-swim tests. Metabotropic glutamate receptor 5 autoradiographic binding was performed in mouse brains undergoing chronic morphine treatment and 7 days withdrawal. Moreover, since there is evidence showing direct effects of opioid drugs on the metabotropic glutamate receptor 5 system, the presence of an metabotropic glutamate receptor 5/µ-opioid receptor interaction was assessed by performing metabotropic glutamate receptor 5 autoradiographic binding in brains of mice lacking the µ-opioid receptor gene. RESULTS: Withdrawal from chronic morphine administration induced anxiety-like, depressive-like, and impaired sociability behaviors concomitant with a marked upregulation of metabotropic glutamate receptor 5 binding. Administration of the metabotropic glutamate receptor 5 antagonist, 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine, reversed morphine abstinence-induced depressive-like behaviors. A brain region-specific increase in metabotropic glutamate receptor 5 binding was observed in the nucleus accumbens shell, thalamus, hypothalamus, and amygdala of µ-opioid receptor knockout mice compared with controls. CONCLUSIONS: These results suggest an association between metabotropic glutamate receptor 5 alterations and the emergence of opioid withdrawal-related affective behaviors. This study supports metabotropic glutamate receptor 5 system as a target for the development of pharmacotherapies for the treatment of opioid addiction. Moreover, our data show direct effects of µ-opioid receptor system manipulation on metabotropic glutamate receptor 5 binding in the brain.

A critical role of striatal A2A R-mGlu5 R interactions in modulating the psychomotor and drug-seeking effects of methamphetamine.

Addiction to psychostimulants is a major public health problem with no available treatment. Adenosine A2A receptors (A2A R) co-localize with metabotropic glutamate 5 receptors (mGlu5 R) in the striatum and functionally interact to modulate behaviours induced by addictive substances, such as alcohol. Using genetic and pharmacological antagonism of A2A R in mice, we investigated whether A2A R-mGlu5 R interaction can regulate the locomotor, stereotypic and drug-seeking effect of methamphetamine and cocaine, two drugs that exhibit distinct mechanism of action. Genetic deletion of A2A R, as well as combined administration of sub-threshold doses of the selective A2A R antagonist (SCH 58261, 0.01 mg/kg, i.p.) with the mGlu5 R antagonist, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (0.01 mg/kg, i.p.), prevented methamphetamine- but not cocaine-induced hyperactivity and stereotypic rearing behaviour. This drug combination also prevented methamphetamine-rewarding effects in a conditioned-place preference paradigm. Moreover, mGlu5 R binding was reduced in the nucleus accumbens core of A2A R knockout (KO) mice supporting an interaction between these receptors in a brain region crucial in mediating addiction processes. Chronic methamphetamine, but not cocaine administration, resulted in a significant increase in striatal mGlu5 R binding in wild-type mice, which was absent in the A2A R KO mice. These data are in support of a critical role of striatal A2A R-mGlu5 R functional interaction in mediating the ambulatory, stereotypic and reinforcing effects of methamphetamine but not cocaine-induced hyperlocomotion or stereotypy. The present study highlights a distinct and selective mechanistic role for this receptor interaction in regulating methamphetamine-induced behaviours and suggests that combined antagonism of A2A R and mGlu5 R may represent a novel therapy for methamphetamine addiction.

Differential regulation of mGlu5 R and ΜOPr by priming- and cue-induced reinstatement of cocaine-seeking behaviour in mice.

The key problem for the treatment of drug addiction is relapse to drug use after abstinence that can be triggered by drug-associated cues, re-exposure to the drug itself and stress. Understanding the neurobiological mechanisms underlying relapse is essential in order to develop effective pharmacotherapies for its prevention. Given the evidence implicating the metabotropic glutamate receptor 5 (mGlu5 R), µ-opioid receptor (MOPr), κ-opioid receptor (ΚOPr) and oxytocin receptor (OTR) systems in cocaine addiction and relapse, our aim was to assess the modulation of these receptors using a mouse model of cue- and priming-induced reinstatement of cocaine seeking. Male mice were trained to self-administer cocaine (1 mg/kg/infusion, i.v.) and were randomized into different groups: (1) cocaine self-administration; (2) cocaine extinction; (3) cocaine-primed (10 mg/kg i.p.); or (4) cue-induced reinstatement of cocaine seeking. Mice undergoing the same protocols but receiving saline instead of cocaine were used as controls. Quantitative autoradiography of mGlu5 R, MOPr, KOPr and OTR showed a persistent cocaine-induced upregulation of the mGlu5 R and OTR in the lateral septum and central amygdala, respectively. Moreover, a downregulation of mGlu5 R and MOPr was observed in the basolateral amygdala and striatum, respectively. Further, we showed that priming- but not cue-induced reinstatement upregulates mGlu5 R and MOPr binding in the nucleus accumbens core and basolateral amygdala, respectively, while cue- but not priming-induced reinstatement downregulates MOPr binding in caudate putamen and nucleus accumbens core. This is the first study to provide direct evidence of reinstatement-induced receptor alterations that are likely to contribute to the neurobiological mechanisms underpinning relapse to cocaine seeking.

In vivo dopaminergic and behavioral responses to acute cocaine are altered in adenosine A(2A) receptor knockout mice.

Adenosine, acting on adenosine A(2A) receptors (A2ARs), regulates addictive processes induced by drugs of abuse. This study investigates the role of A(2A) adenosine receptors in neurochemical and behavioral responses to an acute cocaine challenge. Changes in the extracellular levels of dopamine (DA) in the nucleus accumbens (NAc) of mice lacking A(2A) adenosine receptors and wild type (WT) littermates after an acute cocaine (20 mg/kg) administration were evaluated by in vivo microdialysis studies. Locomotor effects induced by cocaine were measured during the microdialysis procedure. Cocaine-evoked increases in extracellular DA were not sustained in mice lacking A(2A) Rs in comparison with wild-type mice (P < 0.05). Cocaine administration significantly increased ambulatory activity in both genotypes. However, overall locomotor activity was further increased, whereas rest and small local movement measures were significantly attenuated in the A(2A) R knockout mice compared with WT littermates (P < 0.05). Our findings support an important role for adenosine A(2A) R in modulating the acute effects of cocaine, as demonstrated by the decrease in cocaine-evoked dopaminergic transmission in the NAc. Furthermore, the results support an important antagonistic role of A(2A) R in vivo in regulating psychostimulant-induced hyperlocomotion.

Inactivation of adenosine A2A receptor attenuates basal and angiotensin II-induced ROS production by Nox2 in endothelial cells.

Endothelial cells (ECs) express a Nox2 enzyme, which, by generating reactive oxygen species (ROS), contributes to EC redox signaling and angiotensin II (AngII)-induced endothelial dysfunction. ECs also express abundantly an adenosine A(2A) receptor (A(2A)R), but its role in EC ROS production remains unknown. In this study, we investigated the role of A(2A)R in the regulation of Nox2 activity and signaling in ECs with or without acute AngII stimulation. In cultured ECs (SVEC4-10), AngII (100 nm, 30 min) significantly increased Nox2 membrane translocation and association with A(2A)R. These were accompanied by p47(phox), ERK1/2, p38 MAPK, and Akt phosphorylation and an increased ROS production (169 ± 0.04%). These AngII effects were inhibited back to the control levels by a specific A(2A)R antagonist (SCH58261), or adenosine deaminase, or by knockdown of A(2A)R or Nox2 using specific siRNAs. Knockdown of A(2A)R, as determined by Western blotting, decreased Nox2 and p47(phox) expression. In wild-type mouse aorta, SCH58261 significantly reduced acute AngII-induced ROS production and preserved endothelium-dependent vessel relaxation to acetylcholine. These results were further confirmed by using aortas from A(2A)R knock-out mice. In conclusion, A(2A)R is involved in the regulation of EC ROS production by Nox2. Inhibition or blockade of A(2A)R protects ECs from acute AngII-induced oxidative stress, MAPK activation, and endothelium dysfunction.

Adenosine A2A receptor signaling regulation of cardiac NADPH oxidase activity.

Cardiac tissues express constitutively an NADPH oxidase, which generates reactive oxygen species (ROS) and is involved in redox signaling. Myocardial metabolism generates abundant adenosine, which binds to its receptors and plays important roles in cardiac function. The adenosine A2A receptor (A2AR) has been found to be expressed in cardiac myocytes and coronary endothelial cells. However, the role of the A2AR in the regulation of cardiac ROS production remains unknown. We found that knockout of A2AR significantly decreased (39+/-8%) NADPH-dependent O2- production in mouse hearts compared to age (10 weeks)-matched wild-type controls. This was accompanied by a significant decrease in Nox2 (a catalytic subunit of NADPH oxidase) protein expression, and down-regulation of ERK1/2, p38MAPK, and JNK phosphorylation (all P<0.05). In wild-type mice, intraperitoneal injection of the selective A2AR antagonist SCH58261 (3-10 mg/kg body weight for 90 min) inhibited phosphorylation of p47phox (a regulatory subunit of Nox2), which was accompanied by a down-regulated cardiac ROS production (48+/-8%), and decreased JNK and ERK1/2 activation by 54+/-28% (all P<0.05). In conclusion, A2AR through MAPK signaling regulates p47phox phosphorylation and cardiac ROS production by NADPH oxidase. Modulation of A2AR activity may have potential therapeutic applications in controlling ROS production by NADPH oxidase in the heart.

Paracetamol inhibits nitric oxide synthesis in murine spinal cord slices.

Paracetamol is an effective analgesic but its mechanism of action is unclear. We investigated the effect of paracetamol and the analgesic adjuvant caffeine on the activity of NO synthase in mouse spinal cord and cerebellar slices in vitro, by measuring the conversion of [(3)H]arginine to [(3)H]citrulline. Paracetamol (100 microM) had no effect on NO synthase activity in cerebellum, but in the spinal cord both paracetamol (100 microM) and caffeine (30 microM) attenuated glutamate (5 mM)-induced [(3)H]citrulline production and in combination they abolished it. In conclusion paracetamol inhibits spinal cord NO synthesis and this may be related to its analgesic effects.

Modulation of paracetamol antinociception by caffeine and by selective adenosine A2 receptor antagonists in mice.

This study investigated the involvement of adenosine receptors in the interaction between paracetamol and caffeine in mice, using the adenosine A2A receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) and the adenosine A2B receptor antagonist 1-propyl-8-p-sulfophenylxanthine (PSB1115), in the tail immersion and hot-plate tests. Paracetamol (10-200 mg/kg) was antinociceptive in both tests, but, in contrast to previous studies, caffeine (10 mg/kg) was pronociceptive in the tail immersion test, and reduced the effects of paracetamol in both tests. SCH58261 (3 mg/kg) was antinociceptive in both tests and in its presence paracetamol (50 mg/kg) had no further effect. PSB1115 (10 mg/kg) had little effect alone but potentiated the effect of paracetamol (50 mg/kg) in the hot-plate test and abolished it in the tail immersion test. These results suggest that adenosine A2B receptors may be involved in the action of paracetamol in a pathway-dependent manner, and also support the existence of pronociceptive adenosine A2A receptors.

Methamphetamine abstinence induces changes in µ-opioid receptor, oxytocin and CRF systems: Association with an anxiogenic phenotype.

The major challenge in treating methamphetamine addicts is the maintenance of a drug free-state since they experience negative emotional symptoms during abstinence, which may trigger relapse. The neuronal mechanisms underlying long-term withdrawal and relapse are currently not well-understood. There is evidence suggesting a role of the oxytocin (OTR), µ-opioid receptor (MOPr), dopamine D2 receptor (D2R), corticotropin-releasing factor (CRF) systems and the hypothalamic-pituitary-adrenal (HPA)-axis in the different stages of methamphetamine addiction. In this study, we aimed to characterize the behavioral effects of methamphetamine withdrawal in mice and to assess the modulation of the OTR, MOPr, D2R, CRF and HPA-axis following chronic methamphetamine administration and withdrawal. Ten-day methamphetamine administration (2 mg/kg) increased OTR binding in the amygdala, whilst 7 days of withdrawal induced an upregulation of this receptor in the lateral septum. Chronic methamphetamine treatment increased plasma OT levels that returned to control levels following withdrawal. In addition, methamphetamine administration and withdrawal increased striatal MOPr binding, as well as c-Fos(+)/CRF(+) neuronal expression in the amygdala, whereas an increase in plasma corticosterone levels was observed following METH administration, but not withdrawal. No differences were observed in the D2R binding following METH administration and withdrawal. The alterations in the OTR, MOPr and CRF systems occurred concomitantly with the emergence of anxiety-related symptoms and the development of psychomotor sensitization during withdrawal. Collectively, our findings indicate that chronic methamphetamine use and abstinence can induce brain-region specific neuroadaptations of the OTR, MOPr and CRF systems, which may, at least, partly explain the withdrawal-related anxiogenic effects.

Changes in spinal delta and kappa opioid systems in mice deficient in the A2A receptor gene.

A large body of evidence indicates important interactions between the adenosine and opioid systems in regulating pain at both the spinal and supraspinal level. Mice lacking the A(2A) receptor gene have been developed successfully, and these animals were shown to be hypoalgesic. To investigate whether there are any compensatory alterations in opioid systems in mutant animals, we have performed quantitative autoradiographic mapping of mu, delta, kappa, and opioid receptor-like (ORL1) opioid receptors in the brains and spinal cords of wild-type and homozygous A(2A) receptor knock-out mice. In addition, mu-, delta-, and kappa-mediated antinociception using the tail immersion test was tested in wild-type and homozygous A(2A) receptor knock-out mice. A significant reduction in [3H]deltorphin-I binding to delta receptors and a significant increase in [3H]CI-977 binding to kappa receptors was detected in the spinal cords but not in the brains of the knock-out mice. Mu and ORL1 receptor expression were not altered significantly. Moreover, a significant reduction in delta-mediated antinociception and a significant increase in kappa-mediated antinociception were detected in mutant mice, whereas mu-mediated antinociception was unaffected. Comparison of basal nociceptive latencies showed a significant hypoalgesia in knock-out mice when tested at 55 degrees C but not at 52 degrees C. The results suggest a functional interaction between the spinal delta and kappa opioid and the peripheral adenosine system in the control of pain pathways.

An investigation of binding sites for paracetamol in the mouse brain and spinal cord.

Quantitative autoradiography has been used to assess whether [3H]paracetamol (3 microM) binds specifically to any area of the murine brain and spinal cord and to investigate whether paracetamol (1-100 microM) competes for binding to the nociceptin opioid peptide (NOP) receptor or to the nitrobenzylthioinosine (NBTI)-sensitive adenosine transporter in the brains of mice. [3H]paracetamol binding was homogenous and, although there was some indication of specific binding overall, this binding in most individual regions failed to reach statistical significance. However, thoracic segments of the spinal cord were found to have significantly higher specific binding than cervical and lumbar regions. Paracetamol did not significantly compete for binding to the NOP receptor or to the NBTI-sensitive adenosine transporter, showing that it does not mediate its effect via these sites. Although paracetamol did bind specifically to the murine brain and spinal cord, the binding was not region-specific, suggesting binding is not related to any particular neurotransmitter system.

The oxytocin analogue carbetocin prevents priming-induced reinstatement of morphine-seeking: Involvement of dopaminergic, noradrenergic and MOPr systems.

Relapse to illicit drug-seeking following abstinence is a major challenge for the treatment of addiction as no effective pharmacotherapy is available. We have recently shown that activating the central oxytocinergic system prevents emotional impairment and stress-induced reinstatement associated with opioid withdrawal. Here, we investigated whether the oxytocin analogue carbetocin (CBT) is able to reverse morphine-primed reinstatement of conditioned-place preference (CPP) in mice. The mechanism underlining the behavioural effect of CBT was investigated by assessing the involvement of the striatal noradrenergic and dopaminergic systems in CBT reversal of priming- and stress-induced reinstatement of opioid CPP. In addition, given recent evidence suggesting the presence of oxytocin receptor (OTR)-µ-opioid receptor (MOPr) interactions in the brain, we further explored these interactions by carrying out OTR autoradiographic binding in brain of mice lacking MOPr. CBT administration prevented priming-induced reinstatement of morphine CPP. While an acute effect of CBT in enhancing dopamine turnover was observed following stress- and priming-induced reinstatement, CBT significantly decreased striatal noradrenaline turnover only following priming-induced reinstatement. Moreover, a significant brain region- specific increase in OTR binding was observed in MOPr knockout mice, indicating the presence of a possible OTR-MOPr interaction, which may be involved in the modulation of relapse. These results support the oxytocinergic system as a promising target for the prevention of relapse to opioid use and highlight the differential involvement of monoaminergic systems on the effects of OTR stimulation in preventing stress- and priming-induced reinstatement of opioid CPP behaviour.

Impairment of vascular function following BCG immunisation is associated with immune responses to HSP-60 in the cholesterol-fed rabbit.

An immune response to heat shock protein (HSP)-60/65 has recently been implicated in atherogenesis. The aim of this study was to determine whether this effect may be mediated by impairment of endothelial function. Rabbits were injected with bacillus Calmette-Guerin (BCG) vaccine (n=12) or saline (n=12). A further injection of BCG or saline was administered after 2 weeks. After a further 2 weeks, animals were fed either a 0.25-1% cholesterol diet or a chow diet for 16 weeks. Blood cholesterol levels were maintained at 10-12mmol/l by altering the dietary cholesterol content. Plasma levels of anti-mycobacterial antibodies rose following BCG immunisation, but anti-HSP antibodies developed only in the BCG-immunised, cholesterol-fed rabbits. Aortic endothelium from cholesterol-fed, but not chow-fed, rabbits stained positively for HSP-60, independently of the immunisation protocol. Endothelial function was impaired in the BCG immunised, cholesterol-fed rabbits as measured by acetylcholine-mediated relaxation of isolated non-atherosclerotic carotid artery rings (P<0.05). This impairment was positively associated with the level of plasma anti-HSP-60 antibodies (P<0.01). These results suggest that BCG immunisation impairs endothelial responses, at least in part, by immune responses against mycobacterial and vascular HSP.

Quantitative autoradiography of adenosine receptors in brains of chronic naltrexone-treated mice.

1. Manipulation of micro opioid receptor expression either by chronic morphine treatment or by deletion of the gene encoding micro opioid receptors leads to changes in adenosine receptor expression. Chronic administration of the opioid receptor antagonist naltrexone leads to upregulation of micro receptor binding in the brain. 2. To investigate if there are any compensatory alterations in adenosine systems in the brains of chronic naltrexone-treated mice, we carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors in the brains of mice treated for 1 week with naltrexone (8 mg(-1) kg(-1) day(-1)), administered subcutaneously via osmotic minipump. 3. Adjacent coronal brain sections were cut from chronic saline- and naltrexone-treated mice for the determination of binding of [(3)H] D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin ([(3)H] DAMGO), [(3)H]1,3-dipropyl-8-cyclopentylxanthine ([(3)H] DPCPX) or [(3)H] 2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([(3)H] CGS21680) to micro, A(1) and A(2A) receptors, respectively. 4. A significant increase in micro and A(1) receptor binding was detected in chronic naltrexone-treated brains. The changes in micro receptors were significant in several regions, but changes in A(1) were relatively smaller but showed significant upregulation collectively. No significant change in A(2A) receptor binding was detected in chronic naltrexone-treated brains. 5. The results show that blockade of opioid receptors causes upregulation of A(1) receptors, but not A(2A) receptors, by as yet undefined mechanisms.

Quantitative autoradiography of adenosine receptors and NBTI-sensitive adenosine transporters in the brains of mice deficient in the preproenkephalin gene.

There is a large body of evidence indicating important interactions between the adenosine and the opioid systems in regulating pain, opioid dependence and withdrawal. Mice lacking the proenkephalin gene and therefore lacking the endogenous enkephalin peptides have been successfully developed and exhibit decreased locomotor activity, are hyperalgesic and show enhanced anxiety and aggression. In addition, an upregulation of mu and delta receptors was also observed in the brains of knockout mice. To investigate if there are any compensatory alterations in adenosine systems in the brains of mutant mice, we have carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors and nitrobenzylthioinosine (NBTI)-sensitive adenosine transporters in the brains of wild-type and homozygous enkephalin knockout mice. Adjacent coronal brain sections were cut from brains of +/+ and -/- mice for the determination of binding of [(3)H]1,3-dipropyl-8-cyclopentylxanthine ([(3)H]DPCPX), [(3)H]2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([(3)H]CGS21680) or [(3)H]NBTI to A(1) and A(2A) adenosine receptors and NBTI-sensitive adenosine transporters, respectively. A small but significant increase in [(3)H]DPCPX and [(3)H]NBTI binding but no significant change in [(3)H]CGS21680 binding was detected in enkephalin knockout brains. The results provide further evidence of functional interactions in the brain between opioid receptors and A(1) adenosine receptors as well as NBTI-sensitive adenosine transporters but not A(2A) receptors.

Quantitative autoradiography of adenosine receptors and NBTI-sensitive adenosine transporters in the brains and spinal cords of mice deficient in the mu-opioid receptor gene.

There is a large body of evidence indicating important interactions between the adenosine and opioid systems in regulating pain at both the spinal and supraspinal level. Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed and the animals show complete loss of analgesic responses to morphine as well as differences in pain sensitivity. To investigate if there are any compensatory alterations in adenosine systems in mutant animals, we have carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors and nitrobenzylthioinosine (NBTI) sensitive adenosine transporters in the brains and spinal cords of wild type, heterozygous and homozygous mu-opioid receptor knockout mice. Adjacent coronal sections were cut from the brains and spinal cords of +/+, +/- and -/- mice for the determination of binding of [3H]DPCPX, [3H]CGS21680 or [3H]NBTI to A(1) and A(2A) adenosine receptors and NBTI-sensitive adenosine transporters, respectively. A small but significant reduction in [3H]DPCPX and [3H]NBTI binding was detected in mutant mice brains but not in spinal cords. No significant change in A(2A) binding was detected in mu-opioid receptor knockout brains. The results suggest there may be functional interactions between mu-receptors and A(1) adenosine receptors as well as NBTI-sensitive adenosine transporters in the brain but not in the spinal cord.

Characterization of [3H]ZM 241385 binding in wild-type and adenosine A2A receptor knockout mice.

The binding of the adenosine A(2A) receptor antagonist [3H] 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol ([3H]ZM 241385) to mouse brain and spinal cord was investigated. In brain homogenates, single-site binding was observed with a Bmax of 299+/-28 fmol mg(-1) protein and a Kd of 0.75+/-0.08 nM. In autoradiographic studies, there was a high density of specific binding of [3H]ZM 241385 in the striatum, with a very low density in the cortex and no binding elsewhere in the brain or in the spinal cord. All specific binding of [3H]ZM 241385 was lost in genetically modified mice lacking the adenosine A(2A) receptor, confirming the selectivity of this radioligand.

Smoking enhances the proinflammatory effects of nucleotides on cytokine release from human lung.

Nucleotides have effects on immune cells which are complex but generally proinflammatory, and have been suggested to play a role in smoking-related lung diseases. However, there have been no studies directly measuring functional responses to nucleotides in human lungs taken from smokers. We used fragments of post mortem human lung from smokers and non-smokers, incubated them with a range of nucleotides (4-1000 µM) in the presence of lipopolysaccharide (LPS; 10 µg/ml) for 24 hours and measured cytokines (IL-1ß, IFNγ, IL-17, TNFα, IL-6, IL-8, IL-2 and IL-10) in the supernatants using multiplex immunoassays. Although the basal cytokine levels in the smokers were generally higher in the smokers than the non-smokers, there were no significant differences in either the basal release or the LPS-stimulated release of any of the cytokines when lungs from smokers and non-smokers were compared. There were no significant effects of ATP, ADP, AMP, UTP, α,ß-methylene-ATP, P1, P4-diATP, 2-methylthio-ATP or Bz-ATP on the release of cytokines from the lungs. However, the stable ATP analogue ATPγS increased the release of IL-1ß and IFNγ, and the effect was greatly increased in lungs from smokers. In non-smokers but not in smokers ATPγS increased the release of IL-17. Overall these results clearly demonstrate for the first time that in normal human lung a stable ATP analogue can enhance LPS-induced pro-inflammatory cytokine release, and that these effects are greatly altered by a prior history of smoking. This provides strong support for the suggestion that nucleotides are involved in the pathogenesis of smoking-related diseases.

Chronic methamphetamine treatment induces oxytocin receptor up-regulation in the amygdala and hypothalamus via an adenosine A2A receptor-independent mechanism.

There is mounting evidence that the neuropeptide oxytocin is a possible candidate for the treatment of drug addiction. Oxytocin was shown to reduce methamphetamine self-administration, conditioned place-preference, hyperactivity and reinstatement in rodents, highlighting its potential for the management of methamphetamine addiction. Thus, we hypothesised that the central endogenous oxytocinergic system is dysregulated following chronic methamphetamine administration. We tested this hypothesis by examining the effect of chronic methamphetamine administration on oxytocin receptor density in mice brains with the use of quantitative receptor autoradiographic binding. Saline (4ml/kg/day, i.p.) or methamphetamine (1mg/kg/day, i.p.) was administered daily for 10 days to male, CD1 mice. Quantitative autoradiographic mapping of oxytocin receptors was carried out with the use of [(125)I]-vasotocin in brain sections of these animals. Chronic methamphetamine administration induced a region specific upregulation of oxytocin receptor density in the amygdala and hypothalamus, but not in the nucleus accumbens and caudate putamen. As there is evidence suggesting an involvement of central adenosine A2A receptors on central endogenous oxytocinergic function, we investigated whether these methamphetamine-induced oxytocinergic neuroadaptations are mediated via an A2A receptor-dependent mechanism. To test this hypothesis, autoradiographic oxytocin receptor binding was carried out in brain sections of male CD1 mice lacking A2A receptors which were chronically treated with methamphetamine (1mg/kg/day, i.p. for 10 days) or saline. Similar to wild-type animals, chronic methamphetamine administration induced a region-specific upregulation of oxytocin receptor binding in the amygdala and hypothalamus of A2A receptor knockout mice and no genotype effect was observed. These results indicate that chronic methamphetamine use can induce profound neuroadaptations of the oxytocinergic receptor system in brain regions associated with stress, emotionality and social bonding and that these neuroadaptations are independent on the presence of A2A receptors. These results may at least partly explain some of the behavioural consequences of chronic methamphetamine use.