The utility of catalyst-free azide-alkyne [3 + 2] cycloaddition for the immobilization of a variety of molecules onto a solid surface and microbeads was demonstrated. In this process, the surfaces are derivatized with aza-dibenzocyclooctyne (ADIBO) for the immobilization of azide-tagged substrates via a copper-free click reaction. Alternatively, ADIBO-conjugated molecules are anchored to the azide-derivatized surface. Both immobilization techniques work well in aqueous solutions and show excellent kinetics under ambient conditions. We report an efficient synthesis of aza-dibenzocyclooctyne (ADIBO), thus far the most reactive cyclooctyne in cycloaddition to azides. We also describe convenient methods for the conjugation of ADIBO with a variety of molecules directly or via a PEG linker.

The ability of the two opioid receptor-like receptor 1 (ORL1) agonists nociceptin (5 nmol i.c.v.) and synthetic (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one hydrochloride (Ro 64-6198; 0.1, 0.3, and 1.0 mg/kg i.p.) and the opioid antagonist naloxone (0.1, 1.0, and 10.0 mg/kg s.c.) to modify ethanolinduced conditioned place preference was examined in NMRI male mice. The ORL1 agonists were found to significantly reduce the acquisition, expression, and ethanolinduced reinstatement of conditioned place preference. Unlike the ORL1 agonists, naloxone at the doses relevant for opioid receptor blockade failed to significantly influence the acquisition of ethanol-induced conditioned place preference. However, naloxone at 1.0 but not 0.1 mg/kg s.c. potently blocked the expression of ethanol-induced conditioned place preference and significantly inhibited ethanol-induced reinstatement of the conditioned place preference after extinction. Separate experiments indicated that nociceptin and Ro 64-6198 are both devoid of reinforcing or aversive properties. Naloxone, however, at 1.0 and 10.0 mg/kg, produced conditioned place aversion, indicating motivational properties of its own. Both nociceptin and Ro 64-6198 reduced locomotor activity after acute administration. However, tolerance developed very quickly to this effect and already after three i.c.v. (or i.p.) injections, there was no significant reduction of locomotor activity. It is concluded that ORL1 agonists can modulate the acquisition, expression, and reinstatement of the conditioned reinforcing effects of ethanol with no reinforcing or aversive properties of their own. This property might be a potential advantage in the treatment of alcoholism compared with nonselective opioid antagonist naltrexone.Relapse to alcohol use after periods of abstinence is a common feature of alcoholism. Animal models have contributed to the identification of factors that may underlie the motivational aspects of ethanol and that contribute to relapse to ethanol taking. Drug-seeking behavior for ethanol can be initiated or maintained not only by the primary, reinforcing effects of the drugs themselves but also by secondary, drug-associated stimuli (for review, see O'Brien, 1975). The most common experimental model used to study relapse to drug seeking has been the reinstatement procedure. After training to make a response to self-administer a drug and the subsequent extinction of the acquired response, an acute noncontingent injection of test drug (or acute stress) results in the reinstatement of responding (Carroll and Comer, 1996). The conditioned place preference (CPP) procedure provides an alternative experimental approach to examine mechanisms underlying relapse. In this procedure a particular stimulus context or test environment is paired with the effects of the drug, without the animal having to learn to make an explicit response to obtain the drug. In the test trial, the animal is allowed to freely move between the test envir...

Effects of the opioid receptor like-1 (ORL-1) receptor agonist Ro 64-6198 (0.1, 0.3, and 1.0 mg/kg intraperitoneally (i.p.)) on operant ethanol self-administration and activation of self-administration by ethanol deprivation were studied in male Wistar rats. Acute administration of Ro 64-6198 caused a dose-dependent reduction of ethanol self-administration. In comparison, the opioid antagonist naltrexone (0.1, 0.3, and 1.0 mg/kg i.p.) inhibited ethanol self-administration at all doses tested. Ethanol deprivation for 10 days significantly increased ethanol self-administration during the first 2 days after deprivation. Daily pretreatment with Ro 64-6198 (0.3 mg/kg) or naltrexone (0.3 mg/kg) during the last 3 days of ethanol deprivation abolished the deprivation-induced increase in ethanol intake. Thus, stimulation of the ORL-1 receptors by Ro 64-6198 reduced the acute reinforcing effects of ethanol and prevented relapse-like behavior in the ethanol-deprivation model in a similar manner as a blockade of opioid receptors by naltrexone. Ro 64-6198 at 0.1 and 0.3 mg/kg doses did not alter self-administration of 0.2% saccharin solution, indicating an apparent selectivity of this compound in modification of ethanol reward. These findings add further support to the idea that Ro 64-6198 and potentially other synthetic ORL-1 receptor agonists are as effective as naltrexone in blocking the actions of ethanol important for its addictive potential in animal experiments, and therefore may have therapeutic value in the treatment of alcoholism. Neuropsychopharmacology (2007) 32, 902-910.

The effects of 5-hydroxytryptamine 1A (5-HT 1A ) receptor ligands on aversive learning were examined in the passive avoidance (PA) task in mice. Anxiety and autonomic functions were investigated using the elevated plus-maze and heart rate measurements. The main findings from this study are as follows. 1) Pretraining administration of the 5-HT 1A receptor agonist 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide] facilitated PA retention at low doses (0.01 and 0.03 mg/kg) but impaired PA retention at higher doses (0.1-1.0 mg/kg), consistent with previous findings in the rat. Serotonin (5-hydroxytryptamine; 5-HT) is a biogenic amine involved in a wide range of physiological functions, including learning and memory (Ö gren, 1985;Buhot et al., 2000). The ascending 5-HT neurons originating from the brainstem raphe nuclei innervate virtually all regions of the rat forebrain, allowing for widespread neuromodulatory actions (Dahlström and Fuxe, 1964) mediated by 14 identified receptor subtypes (Hoyer et al., 2002). Among the 5-HT receptor subtypes implicated in cognitive functions, the 5-HT 1A receptors are of special interest because they are abundant in brain

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BackgroundAlcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-κB) family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics.Methods and FindingsAnalysis of DNA-binding of NF-κB (p65/p50 heterodimer) and the p50 homodimer as well as NF-κB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant κB binding factor in analyzed tissues. NF-κB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA) mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-κB target DNA sites, κB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with κB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex.ConclusionsWe suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-κB, when repeated over years downregulate RELA expression and NF-κB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of κB regulated genes. Alterations in expression of p50 homodimer/NF-κB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.

Our results indicate that nAChRs are involved in the modulation of operant alcohol self-administration and relapse-like alcohol drinking behavior in rats. Our observations support the working hypothesis that systemically active selective ligands for nAChR alpha(3)beta(2)*, beta(3), and/or alpha(6)* receptor subunits might be of therapeutic value for the treatment of alcoholism.

The present study used conventional and quantitative microdialysis to assess glutamatergic and GABAergic neurotransmission in the hippocampal CA3 area of the rat following a moderate-dose ethanol treatment regimen. Male Wistar rats received 3.4 g/kg of ethanol or water for 6 days via gastric gavage. Microdialysis experiments commenced 2 days later. Basal and depolarization-induced glutamate overflow were significantly elevated in ethanol-treated animals. Basal and depolarization-induced gamma-aminobutyric acid (GABA) overflow were unaltered. Quantitative no-net-flux microdialysis was used to determine if changes in dialysate glutamate levels following ethanol administration are due to an increase in release or a decrease in uptake.To confirm the validity of this method for quantifying basal glutamate dynamics, extracellular concentrations of glutamate and the extraction fraction, which reflects changes in analyte clearance, were quantified in response to retro-dialysis of the glutamate uptake blocker trans-pyrrolidine-2,4-dicarboxylic acid (tPDC). tPDC significantly decreased the extraction fraction for glutamate, resulting in augmented extracellular glutamate concentrations. Repeated ethanol administration did not alter the glutamate extraction fraction. However, extracellular glutamate concentrations were significantly elevated, indicating that glutamate release is increased as a consequence of repeated ethanol administration. These data demonstrate that repeated bouts of moderate ethanol consumption alter basal glutamate dynamics in the CA3 region of the dorsal hippocampus. Basal glutamate release is augmented, whereas glutamate uptake is unchanged. Furthermore, they suggest that dysregulation of glutamate transmission in this region may contribute to the previously documented deficits in cognitive function associated with moderate dose ethanol use.