Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

Bile acids and the gut microbiota: metabolic interactions and impacts on disease.

Despite decades of bile acid research, diverse biological roles for bile acids have been discovered recently due to developments in understanding the human microbiota. As additional bacterial enzymes are characterized, and the tools used for identifying new bile acids become increasingly more sensitive, the repertoire of bile acids metabolized and/or synthesized by bacteria continues to grow. Additionally, bile acids impact microbiome community structure and function. In this Review, we highlight how the bile acid pool is manipulated by the gut microbiota, how it is dependent on the metabolic capacity of the bacterial community and how external factors, such as antibiotics and diet, shape bile acid composition. It is increasingly important to understand how bile acid signalling networks are affected in distinct organs where the bile acid composition differs, and how these networks impact infectious, metabolic and neoplastic diseases. These advances have enabled the development of therapeutics that target imbalances in microbiota-associated bile acid profiles.

Current Challenges and Recent Developments in Mass Spectrometry-Based Metabolomics.

High-resolution mass spectrometry (MS) has advanced the study of metabolism in living systems by allowing many metabolites to be measured in a single experiment. Although improvements in mass detector sensitivity have facilitated the detection of greater numbers of analytes, compound identification strategies, feature reduction software, and data sharing have not kept up with the influx of MS data. Here, we discuss the ongoing challenges with MS-based metabolomics, including de novo metabolite identification from mass spectra, differentiation of metabolites from environmental contamination, chromatographic separation of isomers, and incomplete MS databases. Because of their popularity and sensitive detection of small molecules, this review focuses on the challenges of liquid chromatography-mass spectrometry-based methods. We then highlight important instrumentational, experimental, and computational tools that have been created to address these challenges and how they have enabled the advancement of metabolomics research.

Caloric restriction disrupts the microbiota and colonization resistance.

Diet is a major factor that shapes the gut microbiome1, but the consequences of diet-induced changes in the microbiome for host pathophysiology remain poorly understood. We conducted a randomized human intervention study using a very-low-calorie diet (NCT01105143). Although metabolic health was improved, severe calorie restriction led to a decrease in bacterial abundance and restructuring of the gut microbiome. Transplantation of post-diet microbiota to mice decreased their body weight and adiposity relative to mice that received pre-diet microbiota. Weight loss was associated with impaired nutrient absorption and enrichment in Clostridioides difficile, which was consistent with a decrease in bile acids and was sufficient to replicate metabolic phenotypes in mice in a toxin-dependent manner. These results emphasize the importance of diet-microbiome interactions in modulating host energy balance and the need to understand the role of diet in the interplay between pathogenic and beneficial symbionts.

Improved methods for biomarker analysis of the big five mycotoxins enables reliable exposure characterization in a population of childbearing age women in Rwanda.

Of the five agriculturally important mycotoxins, AFB1, FB1, DON, ZEA and OTA, a well-characterized biomarker of exposure in blood is only available for aflatoxin. Working with a population of 139 women of childbearing age in Rwanda, we undertook a comprehensive assessment of their dietary mycotoxin exposure. Using high-resolution LC-MS/MS with stable isotope dilution analysis, the albumin-aflatoxin adduct was quantitated in plasma. Similarly, AFM1, AFB1, AFG1, FB1 and B2, OTA, zearalenone, α-zearalenol, deoxynivalenol, deoxynivalenol-15-glucuronide and deoxynivalenol-3-glucuronide were quantitated in urine. AFB1-Lys was detected in plasma from 81% of the women, indicative of exposures 1-2 orders of magnitude above current guidance. Zearalenone and/or α-zearalenol were detected in the urine of 61% of the women, the majority of whom had estimated exposures 2-5 times the PMTDI, with one third more than an order of magnitude above. Urinary deoxynivalenol or the two glucuronide conjugates were found in 77% of the participants. Of these, 60% were below the PMTDI, 28% were twice and 12% were >10x the PMTDI. Fumonisin B1 (30%) and ochratoxin A (71%) were also detected in urine. Exposures observed in these Rwandan women raise serious food safety concerns and highlight the need for authorities to help manage multiple mycotoxins in their diet.

The gut microbiome: an orchestrator of xenobiotic metabolism.

Microbes inhabiting the intestinal tract of humans represent a site for xenobiotic metabolism. The gut microbiome, the collection of microorganisms in the gastrointestinal tract, can alter the metabolic outcome of pharmaceuticals, environmental toxicants, and heavy metals, thereby changing their pharmacokinetics. Direct chemical modification of xenobiotics by the gut microbiome, either through the intestinal tract or re-entering the gut via enterohepatic circulation, can lead to increased metabolism or bioactivation, depending on the enzymatic activity within the microbial niche. Unique enzymes encoded within the microbiome include those that reverse the modifications imparted by host detoxification pathways. Additionally, the microbiome can limit xenobiotic absorption in the small intestine by increasing the expression of cell-cell adhesion proteins, supporting the protective mucosal layer, and/or directly sequestering chemicals. Lastly, host gene expression is regulated by the microbiome, including CYP450s, multi-drug resistance proteins, and the transcription factors that regulate them. While the microbiome affects the host and pharmacokinetics of the xenobiotic, xenobiotics can also influence the viability and metabolism of the microbiome. Our understanding of the complex interconnectedness between host, microbiome, and metabolism will advance with new modeling systems, technology development and refinement, and mechanistic studies focused on the contribution of human and microbial metabolism.

Microbiota-Mediated Modulation of Organophosphate Insecticide Toxicity by Species-Dependent Interactions with Lactobacilli in a Drosophila melanogaster Insect Model.

Despite the benefits to the global food supply and agricultural economies, pesticides are believed to pose a threat to the health of both humans and wildlife. Chlorpyrifos (CP), a commonly used organophosphate insecticide, has poor target specificity and causes acute neurotoxicity in a wide range of species via the suppression of acetylcholinesterase. This effect is exacerbated 10- to 100-fold by chlorpyrifos oxon (CPO), a principal metabolite of CP. Since many animal-associated symbiont microorganisms are known to hydrolyze CP into CPO, we used a Drosophila melanogaster insect model to investigate the hypothesis that indigenous and probiotic bacteria could affect CP metabolism and toxicity. Antibiotic-treated and germfree D. melanogaster insects lived significantly longer than their conventionally reared counterparts when exposed to 10 µM CP. Drosophila melanogaster gut-derived Lactobacillus plantarum, but not Acetobacterindonesiensis, was shown to metabolize CP. Liquid chromatography tandem-mass spectrometry confirmed that the L. plantarum isolate preferentially metabolized CP into CPO when grown in CP-spiked culture medium. Further experiments showed that monoassociating germfree D. melanogaster with the L. plantarum isolate could reestablish a conventional-like sensitivity to CP. Interestingly, supplementation with the human probiotic Lactobacillus rhamnosus GG (a strain that binds but does not metabolize CP) significantly increased the survival of the CP-exposed germfree D. melanogaster This suggests strain-specific differences in CP metabolism may exist among lactobacilli and emphasizes the need for further investigation. In summary, these results suggest that (i) CPO formation by the gut microbiota can have biologically relevant consequences for the host, and (ii) probiotic lactobacilli may be beneficial in reducing in vivo CP toxicity.IMPORTANCE An understudied area of research is how the microbiota (microorganisms living in/on an animal) affects the metabolism and toxic outcomes of environmental pollutants such as pesticides. This study focused specifically on how the microbial biotransformation of chlorpyrifos (CP; a common organophosphate insecticide) affected host exposure and toxicity parameters in a Drosophila melanogaster insect model. Our results demonstrate that the biotransformation of CP by the gut microbiota had biologically relevant and toxic consequences on host health and that certain probiotic lactobacilli may be beneficial in reducing CP toxicity. Since inadvertent pesticide exposure is suspected to negatively impact the health of off-target species, these findings may provide useful information for wildlife conservation and environmental sustainability planning. Furthermore, the results highlight the need to consider microbiota composition differences between beneficial and pest insects in future insecticide designs. More broadly, this study supports the use of beneficial microorganisms to modulate the microbiota-mediated biotransformation of xenobiotics.

Promising Prebiotic Candidate Established by Evaluation of Lactitol, Lactulose, Raffinose, and Oligofructose for Maintenance of a Lactobacillus-Dominated Vaginal Microbiota.

Perturbations to the vaginal microbiota can lead to dysbiosis, including bacterial vaginosis (BV), which affects a large portion of the female population. In a healthy state, the vaginal microbiota is characterized by low diversity and colonization by Lactobacillus spp., whereas in BV, these species are displaced by a highly diverse population of bacteria associated with adverse vaginal health outcomes. Since prebiotic ingestion has been a highly effective approach to invigorate lactobacilli for improved intestinal health, we hypothesized that these compounds could stimulate lactobacilli at the expense of BV organisms to maintain vaginal health. Monocultures of commensal Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, and Lactobacillus iners, in addition to BV-associated organisms and Candida albicans, were tested for their ability to utilize a representative group of prebiotics consisting of lactitol, lactulose, raffinose, and oligofructose. The disaccharide lactulose was found to most broadly and specifically stimulate vaginal lactobacilli, including the strongly health-associated species L. crispatus, and importantly, not to stimulate BV organisms or C. albicans Using freshly collected vaginal samples, we showed that exposure to lactulose promoted commensal Lactobacillus growth and dominance and resulted in healthy acidity partially through lactic acid production. This provides support for further testing of lactulose to prevent dysbiosis and potentially to reduce the need for antimicrobial agents in managing vaginal health.IMPORTANCE Bacterial vaginosis (BV) and other dysbioses of the vaginal microbiota significantly affect the quality of life of millions of women. Antimicrobial therapy is often poorly effective, causes side effects, and does not prevent recurrences. We report one of very few studies that have evaluated how prebiotics-compounds that are selectively fermented by beneficial bacteria such as Lactobacillus spp.-can modulate the vaginal microbiota. We also report use of a novel in vitro polymicrobial model to study the impact of prebiotics on the vaginal microbiota. The identification of prebiotic lactulose as enhancing Lactobacillus growth but not that of BV organisms or Candida albicans has direct application for retention of homeostasis and prevention of vaginal dysbiosis and infection.

Depletion of serotonin decreases the effects of the kappa-opioid receptor agonist U-69593 on cocaine-stimulated activity.

Treatment with a kappa-opioid receptor agonist for 5 days decreases locomotor activity and reduces activity in response to a cocaine challenge 3 days later. In addition, chronic cocaine increases kappa-opioid receptor density, striatal dynorphin, and dynorphin gene expression in the striatum. The upregulation of kappa-opioid receptors after cocaine treatment occurs predominantly in brain regions that are highly innervated by serotonin. To determine if serotonin plays a role in the effects of kappa-opioid receptor agonists on cocaine-stimulated activity, parachloroamphetamine (PCA), which depleted serotonin by 53%-66%, or saline, was given prior to a five-day treatment with U-69593 or vehicle. Three days later each rat received a single injection of cocaine and locomotor activity was measured. Treatment with PCA had no effect on the ability of U-69593 alone to decrease locomotor activity. Thus, the behavioral effects of U-69593 alone were not dependent upon serotonin. In rats pretreated with saline, U-69593 treatment significantly blocked the locomotor-activating effects of cocaine. Following PCA pretreatment, however, there were no significant differences in locomotor activity in rats challenged with an injection of cocaine after treatment with U-69593 or vehicle. Thus, serotonin depletion prevented the long-lasting blockade of the locomotor-activating effects of cocaine subsequent to repeated administration of U-69593 but did not alter the effects of cocaine in rats that were treated with vehicle. Thus, the effects of PCA on U-69593 are not due to non-specific alterations in cocaine-induced locomotor activity. These findings suggest that serotonin plays an important role in mediating the effects of kappa-opioid receptor agonists on the behavioral response to cocaine.

Chronic cocaine produces decreases in N/OFQ peptide levels in select rat brain regions.

The interaction of opioids and stimulants is well established; however, the mechanisms that underlie the role that opioid receptors play in psychostimulant action are not. Nociceptin/orphaninFQ (N/OFQ), the endogenous agonist at NOP receptors, attenuates the behavioral effects of cocaine. The effects of cocaine on N/OFQ were examined in rats using immunoautoradiographic and RIA techniques. Chronic administration of cocaine decreased N/OFQ in medial regions of the caudate putamen, the nucleus accumbens shell, and the substantia nigra. These studies show that N/OFQ levels are altered by treatment with cocaine. Furthermore, the changes in N/OFQ parallel those seen for kappa-opioid receptors, suggesting that the interactions between cocaine and these systems might be similar.

Intranasal cocaine in humans: effects of sex and menstrual cycle.

Studies have shown that smoked and intravenous cocaine's effects differ in cocaine-dependent women compared to men and across the menstrual cycle. However, this has not been systematically investigated with intranasal cocaine. Thus, a range of intranasal cocaine doses was examined in cocaine-dependent women across the menstrual cycle. Female cocaine users were admitted to the hospital once during the luteal phase and once during the follicular phase of their menstrual cycle; menstrual cycle phase during admissions was counterbalanced. During each admission, an intranasal cocaine dose-response curve (0.06, 0.34, 0.69 and 1.37 mg/kg) was determined during four laboratory sessions. Cocaine produced similar dose-related increases in ratings of "positive" subjective effects, cardiovascular effects and cocaine plasma levels in women in both menstrual cycle phases. To assess sex differences in the effects of intranasal cocaine, the current data were compared to published data collected in men using an identical procedure. Cocaine produced similar dose-related increases in ratings of positive subjective effects, cardiovascular effects and cocaine plasma levels in men and women. Thus, in contrast to studies examining smoked or intravenous cocaine administration, there were no sex differences or menstrual cycle effects on the subjective or cardiovascular response to intranasal cocaine, suggesting that the influence of sex and menstrual cycle on cocaine's effects vary as a function of route of administration.

Response to cocaine, alone and in combination with methylphenidate, in cocaine abusers with ADHD.

Attention deficit hyperactivity disorder (ADHD) is prevalent in adult cocaine abusers. Yet, it remains to be determined how the response to cocaine differs in cocaine abusers with ADHD compared to cocaine abusers without ADHD. Further, since ADHD is commonly treated with stimulants, such as methylphenidate (MPH), it is important to examine whether MPH maintenance alters the response to cocaine in cocaine abusers with ADHD. Thus, the first phase of this study compared the response to cocaine in adult cocaine abusers with ADHD to those without ADHD. The second phase assessed the effects of oral sustained-release methylphenidate (MPH-SR) maintenance (40 and 60 mg) on the response to cocaine only in those with ADHD. Cocaine abusers with ADHD (N=7) and without ADHD (N=7) who were not seeking treatment remained inpatient initially for 1 week, when the effects of cocaine alone were tested (Phase 1). Cocaine abusers with ADHD remained inpatient for an additional 3 weeks, during which the effects of cocaine during oral MPH-SR maintenance were tested (Phase 2). During cocaine fixed dosing sessions, participants received four injections of i.v. cocaine (0, 16 or 48 mg/70 kg), spaced 14 min apart. During cocaine choice sessions, participants had a choice between receiving i.v. cocaine (16 or 48 mg/70 kg) or two tokens, each exchangeable for 2 US dollars. Subjective effects related to ADHD symptoms (e.g. ratings of "Able to Concentrate") were significantly lower in cocaine abusers with ADHD compared to those without ADHD when placebo cocaine was administered. Active cocaine produced similar increases in cardiovascular and positive subjective effects in both groups and there was no difference in cocaine choice between the two groups. These data suggest that the response to cocaine is not different between cocaine abusers with ADHD compared to those without ADHD. When the cocaine abusers with ADHD were maintained on MPH-SR, cardiovascular effects were increased, however, this did not warrant termination of any test session. Maintenance on MPH-SR decreased some of the positive subjective effects of cocaine. Further, maintenance on a high dose of MPH-SR decreased cocaine choice. Thus, oral MPH-SR is safe in combination with repeated cocaine doses and decreases some of the positive and reinforcing effects of cocaine in cocaine abusers with ADHD.

Nicotine treatment produces persistent increases in amphetamine-stimulated locomotor activity in periadolescent male but not female or adult male rats.

Nicotine is a popular addictive drug used among the adolescent population, and it has long been questioned whether nicotine use in adolescence may lead to the use of other psychostimulant drugs. It is not fully understood, however, how nicotine alters behavior and brain neurochemistry in the adolescent age cohort and how this may affect subsequent illicit drug use. In the current study, periadolescent and adult male and female rats were treated with nicotine for 7 days. One day or 30 days after this treatment, the effects of amphetamine on locomotor activity were studied. Sensitization to nicotine occurred in periadolescent female and adult male and female rats, but not in periadolescent male rats over the course of the 7-day treatment period. On day 8 (1 day after treatment with nicotine ended) and on day 37 (30 days after treatment with nicotine ended), nicotine-pretreated periadolescent male rats were sensitized to the locomotor-activating effects of amphetamine. The response to amphetamine of periadolescent female and adult male and female rats was unchanged at either time point after nicotine pretreatment. Thus, adolescent males are more sensitive than adults or females to the stimulant effects of amphetamine after exposure to nicotine, and this effect is long-lasting. These data suggest that nicotine use during adolescence may carry a greater risk than during adulthood and that male adolescent smokers may be particularly vulnerable to the risk of stimulant abuse.

Neurochemical alterations produced by daily nicotine exposure in periadolescent vs. adult male rats.

Chronic treatment with nicotine differentially alters behavior in adolescent rats compared to adult rats. It is not known, however, whether the effects of nicotine on the neurochemical pathways with which it interacts differ in adolescents vs. adults. In the current study, the effects of a 7-day treatment with nicotine on nicotinic, dopaminergic, and serotonergic neurochemistry were examined in the caudate putamen and nucleus accumbens in periadolescent vs. adult male rats. Nicotine treatment increased dopamine transporter densities and decreased serotonin transporter densities in periadolescent rats. There was no change in nicotinic acetylcholine receptor densities or dopamine D1 or D2 receptor densities in nicotine-pretreated periadolescent rats. In adult rats pretreated with nicotine, there was an increase in nicotinic acetylcholine densities, but no change in dopamine transporter, dopamine D1 or D2 receptor, or serotonin transporter densities. Overall, these findings show that periadolescent rats have neurochemical adaptations to nicotine different from adult rats. These alterations may explain, at least in part, the differential behavioral effects of chronic nicotine in adult and adolescent male rats.

Chronic nicotine differentially alters cocaine-induced locomotor activity in adolescent vs. adult male and female rats.

Tobacco use is prevalent in the adolescent population. It is a major concern because tobacco is highly addictive and has also been linked to illicit drug use. There is not much research, however, on the interaction between nicotine and other stimulant drugs in animal models of early adolescence. This study examined the effects of chronic nicotine alone and on cocaine-stimulated activity in male and female periadolescent rats compared to male and female adult rats. During the seven-day nicotine pretreatment period, nicotine increased locomotor activity in all groups compared to vehicle controls. Male and female adult rats and female periadolescent rats developed sensitization to the locomotor-activating effects of nicotine over the 7-day treatment period, while male periadolescent rats did not. All groups treated with nicotine, however, exhibited sensitization to nicotine-induced repetitive motion over the 7-day nicotine treatment period. On day 8, male periadolescent rats pretreated with nicotine were more markedly sensitized to the locomotor-activating effects of cocaine than male adult rats, while female rats pretreated with nicotine were not sensitized to cocaine. In contrast, male and female periadolescent rats, but not adult rats, had increased amounts of repetitive beam breaks induced by cocaine after nicotine pretreatment. Overall, it appears that cross-sensitization to cocaine is greater in periadolescent than in adult rats, and that males are more sensitized than females. Thus, it may be that nicotine use during adolescence carries a greater risk than during adulthood and that male adolescents may be particularly vulnerable to the risk of cocaine abuse after nicotine use. This information should be taken into account so as to help us better understand the development of drug addiction in adolescents compared to adults.

Cocaine differentially alters behavior and neurochemistry in periadolescent versus adult rats.

This study examined whether there are differences in the behavioral and neurochemical effects of cocaine in periadolescent rats compared to adult rats. Periadolescent (postnatal days 28-35) and adult rats were injected with cocaine or vehicle for 7 days. Ten days later (day 17), rats either were challenged with cocaine, or dopamine transporter and receptor and serotonin transporter binding were examined. Adult rats became sensitized to the locomotor-activating effects of cocaine and there were increases in dopamine transporter density in the caudate putamen compared to vehicle-treated adult rats. In addition, serotonin transporter densities were increased in the ventromedial caudate putamen, nucleus accumbens shell, and the olfactory tubercle in cocaine-treated adult rats compared to vehicle-treated adult rats. In contrast, periadolescent rats did not show sensitization to cocaine and there was no effect of cocaine on either dopamine or serotonin transporter densities. These findings suggest that there are different neurochemical and behavioral adaptations to repeated cocaine administration in periadolescent versus adult rats.

Chronic cocaine increases kappa-opioid receptor density: lack of effect by selective dopamine uptake inhibitors.

Continuous infusion of cocaine or the selective dopamine uptake inhibitors GBR 12909 or RTI-117 increases locomotor stimulation, to which partial tolerance occurs. In addition, all three drugs produce significant decreases in tyrosine hydroxylase immunoreactivity in caudate putamen and nucleus accumbens core, suggesting a decreased dopaminergic tone. An interaction between cocaine and opioids has long been documented. Chronic cocaine significantly increases mu and kappa-opioid receptors and treatment with a kappa-opioid agonist markedly reduces the behavioral effects of cocaine. In addition, chronic cocaine, but not GBR 12909, increases prodynorphin gene expression in caudate putamen. To further understand the interaction between cocaine and the kappa-opioid system, the effects of a chronic continuous infusion for 14 days of cocaine or one of the selective dopamine uptake inhibitors GBR 12909 or RTI-117 via osmotic minipump were examined on kappa-opioid receptors using the selective kappa-opioid ligand [3H] U-69593. [3H] U-69593 binding density was significantly increased in caudate putamen, nucleus accumbens shell, claustrum, and endopiriform nucleus after cocaine, while neither GBR 12909 nor RTI-117 had any effect. The increased kappa-opioid receptor densities observed following cocaine are likely not related to dopamine uptake inhibition, since they were not produced by selective dopamine uptake inhibitors. These findings suggest that regulation of kappa-opioid receptors by cocaine may be via inhibition of serotonin or norepinephrine uptake, by a combination of effects on two or three monoamine transporters, or by a mechanism unrelated to transporter inhibition.

Neuronal nitric oxide synthase inhibition decreases cocaine self-administration behavior in rats.

RATIONALE: Inhibitors of nitric oxide synthase (NOS) have been shown to alter behaviors related to cocaine addiction, including its self-administration. However, previous studies have largely used mixed-action NOS inhibitors and have not examined the effects of a neuronal NOS inhibitor on cocaine self-administration. OBJECTIVES: Pretreatment with the neuronal NOS inhibitor 7-nitroindazole (7-NI) was used and its effects on cocaine self-administration were compared with those produced by pretreatment with an indirect dopamine receptor agonist (cocaine) and a D(1)-like dopamine receptor antagonist (SCH 23390). METHODS: Rats were trained to self-administer 1 mg/kg cocaine under a second-order schedule of drug delivery, which measures drug-seeking behavior independently from drug intake. Pretreatment with various doses of 7-NI, cocaine, and SCH 23390 were tested in combination with the training dose of cocaine followed by studies examining the effects of a selected dose of each pretreatment drug in combination with a range of cocaine doses. Other rats were trained under a second-order schedule of food pellet delivery and pretreated with 7-NI, cocaine, or SCH 23390 to determine the behavioral specificity of the effects of these drugs for cocaine-maintained responding. RESULTS: The results demonstrated that 7-NI reduced responses maintained by the cocaine training dose and produced a downward shift in the cocaine dose-response curve. Changes in drug intake were minor by comparison. Cocaine pretreatment produced effects similar to 7-NI, while the changes observed after SCH 23390 pretreatment were different from 7-NI and cocaine. The reductions in cocaine-maintained responding after 7-NI pretreatment were behaviorally specific because there was no effect of 7-NI on food-maintained responding within the dose range examined. CONCLUSIONS: By selectively reducing drug-seeking behavior, these data suggest that 7-NI may enhance the reinforcing effects of cocaine.