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Background: We analysed illicit stimulant use in Finland by comparing three separate datasets collected at the same time. Methods: The data used were wastewater analysis (2014 and 2018), population-based drug surveys (2014 and 2018) and European Web Survey on Drugs (2018, Finnish data). Proportions, prevalence levels and trends of stimulant use as well as their consumption were measured. Factors associated with stimulant use were assessed for past-year stimulant or amphetamine use as an outcome measure in regression analyses. Results: Both population-based drug survey and wastewater data showed that stimulant use has increased in Finland between 2014 and 2018. Disadvantaged socio-demographic background and other substance use were associated with past-year stimulant use, with no geographical variation in Finland. The socio-demographics of those reporting amphetamine use differed between population-based drug survey and web survey. In the web survey, infrequent and occasional users of amphetamine were quite alike, whereas frequent users were more likely to be unemployed or use injection as the route of administration. Conclusion: Analysis of three different data revealed findings that would have been missed and conclusions that could not have been made by using only one dataset. Putting findings from different methods into dialogue raises new questions and opens new interpretations. This analysis emphasises the importance of the prevention of frequent use and associated harm, as well as the impact of versatile drug treatment and harm reduction services on it.

Purpose Drug checking is a popular method to reduce risks of drug use. In many countries, including Finland, legislation restricts implementing drug checking. The aim of this study was to explore whether some benefits of drug checking could be achieved by substance residue analysis. Design/methodology/approach Drug paraphernalia (mostly empty plastic bags) were used in the study. Participants left a sample and information about the former content to a local needle exchange point. After laboratory analysis, participants could return for the results and a short consultation on the substance(s) found. Afterward, participants were asked whether they would still use the batch. Findings Ninety-eight samples were received. In most cases, the samples had originally been sold as amphetamine (n = 39). Overall, laboratory results matched with supposed content in 52 cases, but in 21 cases, the sold content had been altered, in 17 cases, only other psychoactive substances were found and in 8 cases, no traces of psychoactive substances were found. Participants returned for results in two-thirds of the cases. When the laboratory result did not match participants’ expectations, the majority of participants estimated they would not use the same batch (17/25) or would use it in a different way (2/25). Originality/value While reports on drug checking are numerous, studies exploring possibilities to achieve harm-reducing benefits of drug checking by analyzing drug residues are scarce. The results of this pilot study suggest some benefits of drug checking can be achieved by substance residue analysis.

Supplementary Table 1. Detailed methods for measurement of plasma and urine COT and 3HC at each Test Site.

Background: The highly genetically variable enzyme CYP2A6 metabolizes nicotine to cotinine (COT) and COT to trans-3′-hydroxycotinine (3HC). The nicotine metabolite ratio (NMR, 3HC/COT) is commonly used as a biomarker of CYP2A6 enzymatic activity, rate of nicotine metabolism, and total nicotine clearance; NMR is associated with numerous smoking phenotypes, including smoking cessation. Our objective was to investigate the impact of different measurement methods, at different sites, on plasma and urinary NMR measures from ad libitum smokers.Methods: Plasma (n = 35) and urine (n = 35) samples were sent to eight different laboratories, which used similar and different methods of COT and 3HC measurements to derive the NMR. We used Bland–Altman analysis to assess agreement, and Pearson correlations to evaluate associations, between NMR measured by different methods.Results: Measures of plasma NMR were in strong agreement between methods according to Bland–Altman analysis (ratios, 0.82–1.16) and were highly correlated (all Pearson r > 0.96, P < 0.0001). Measures of urinary NMR were in relatively weaker agreement (ratios 0.62–1.71) and less strongly correlated (Pearson r values of 0.66–0.98, P < 0.0001) between different methods. Plasma and urinary COT and 3HC concentrations, while weaker than NMR, also showed good agreement in plasma, which was better than that in urine, as was observed for NMR.Conclusions: Plasma is a very reliable biologic source for the determination of NMR, robust to differences in these analytical protocols or assessment site.Impact: Together this indicates a reduced need for differential interpretation of plasma NMR results based on the approach used, allowing for direct comparison of different studies. Cancer Epidemiol Biomarkers Prev; 24(8); 1239–46. ©2015 AACR.

Wastewater-based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population-normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011-17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data.Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017.Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries.Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of ΔBenzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North-Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge.The analysis of wastewater to quantify drug loads provides near real-time drug use estimates that globally correspond to prevalence and seizure data.

Wastewater-based epidemiology (WBE) has recently become one of the most rapidly evolving disciplines engaged in the measurement of population-level (illicit) drug use. While gaps in the current research base remain and further development is beneficial to improve analyses performed through WBE, the approach has partially transitioned from focussing on analytical obstacles and applications to exploring how WBE data can be combined with different data sets and how to most efficiently utilize the elicited information in cooperation with relevant stakeholders. This cooperation would ideally involve cross-governmental and interdisciplinary actors, including scientists, law enforcement agents, health-care workers and policy makers to achieve the most synergistic outcomes. This article provides a synthesis on how WBE, especially in combination with other sources of information, may have significant implications not only for epidemiological research but for the rapid deployment of the gathered information and actual drug policies.

Wastewater-based surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is used to monitor the population-level prevalence of the COVID-19 disease. In many cases, due to lockdowns or analytical delays, the analysis of wastewater samples might only be possible after prolonged storage. In this study, the effect of storage conditions on the RNA copy numbers of the SARS-CoV-2 virus in wastewater influent was studied and compared to the persistence of norovirus over time at 4 °C, −20 °C, and −75 °C using the reverse-transcription quantitative PCR (RT-qPCR) assays E-Sarbeco, N2, and norovirus GII. For the first time in Finland, the presence of SARS-CoV-2 RNA was tested in 24 h composite influent wastewater samples collected from Viikinmäki wastewater treatment plant, Helsinki, Finland. The detected and quantified SARS-CoV-2 RNA copy numbers of the wastewater sample aliquots taken during 19–20 April 2020 and stored for 29, 64, and 84 days remained surprisingly stable. In the stored samples, the SARS betacoronavirus and SARS-CoV-2 copy numbers, but not the norovirus GII copy numbers, seemed slightly higher when analyzed from the pre-centrifuged pellet—that is, the particulate matter of the influent—as compared with the supernatant (i.e., water fraction) used for ultrafiltration, although the difference was not statistically significant. Furthermore, when wastewater was spiked with SARS-CoV-2, linear decay at 4 °C was observed on the first 28 days, while no decay was visible within 58 days at −20 °C or −75 °C. In conclusion, freezing temperatures should be used for storage when immediate SARS-CoV-2 RNA analysis from the wastewater influent is not possible. Analysis of the particulate matter of the sample, in addition to the water fraction, can improve the detection frequency., Graphical abstract Unlabelled Image, Highlights • Wastewater sample aliquots during prolonged storage were investigated. • SARS-CoV-2 RNA was detected for the first time in wastewater in Helsinki, Finland. • SARS-CoV-2 RNA copy numbers were stable in cold temperatures over one to three months. • SARS-CoV-2 RNA copy number declined over 28 days at 4 °C but not at −20 °C or −75 °C. • The inclusion of particulate matter should be considered in the analysis of SARS-CoV RNA.

No single measure is able to provide a complete picture of population- or community-level drug abuse and its current trends. Therefore, a multi-indicator approach is needed. The aim of this study was to combine wastewater-based epidemiology (WBE) with data from other national indicators, namely driving under the influence of drugs (DUID) statistics, drug seizures, and drug use surveys. Furthermore, drug market size estimates and a comparison of confiscated drugs to drugs actually consumed by users were performed using the WBE approach. Samples for wastewater analysis were collected during one-week sampling periods in 2012, 2014 and 2015, with a maximum of 14 cities participating. The samples were analysed with a validated ultra-high-performance liquid chromatography-mass spectrometric (UHPLC-MS/MS) methodology for various common drugs of abuse. The results were then compared with data from other national indicators available. Joint interpretation of the data shows that the use of amphetamine and MDMA has increased in Finland from 2012 to 2014. A similar trend was also observed for cocaine, although its use remains at a very low level compared to many other European countries. Heroin was practically absent from the Finnish drug market during the study period. The retail market for the most common stimulant drugs were estimated to have been worth EUR 70 million for amphetamine and around EUR 10 million for both methamphetamine and cocaine, in 2014 in Finland.

Publisher's version (útgefin grein), Wastewater-based epidemiology is an efficient way to assess illicit drug use, complementing currently used methods retrieved from different data sources. The aim of this study is to compare stimulant drug use in five Nordic capital cities that include for the first time wastewater samples from Torshavn in the Faroe Islands. Currently there are no published reports that compare stimulant drug use in these Nordic capitals. All wastewater samples were analyzed using solid phase extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry. The results were compared with data published by the European Monitoring Centre for Drugs and Drug Addiction based on illicit drugs in wastewater from over 50 European cities. Confirming previous reports, the results showed high amphetamine loads compared with other European countries. Very little apparent abuse of stimulant drugs was detected in Torshavn. Methamphetamine loads were the highest from Helsinki of the Nordic countries, indicating substantial fluctuations in the availability of the drug compared with previous studies. Methamphetamine loads from Oslo confirmed that the use continues to be high. Estimated cocaine use was found to be in the lower range compared with other cities in the southern and western part of Europe. Ecstasy and cocaine showed clear variations between weekdays and weekends, indicating recreational use. This study further demonstrates geographical trends in the stimulant drug market in five Nordic capitals, which enables a better comparison with other areas of the continent., We wish to acknowledgesupport by COST (EuropeanCooperation inScience and Technology) [Grant number ES1307], as well as the Icelan-dic Research Fund [Grant number 163049-051] and the University ofIceland Research Fund [Grant number HI17060092].

Background: The highly genetically variable enzyme CYP2A6 metabolizes nicotine to cotinine (COT) and COT to trans-3′-hydroxycotinine (3HC). The nicotine metabolite ratio (NMR, 3HC/COT) is commonly used as a biomarker of CYP2A6 enzymatic activity, rate of nicotine metabolism, and total nicotine clearance; NMR is associated with numerous smoking phenotypes, including smoking cessation. Our objective was to investigate the impact of different measurement methods, at different sites, on plasma and urinary NMR measures from ad libitum smokers. Methods: Plasma (n = 35) and urine (n = 35) samples were sent to eight different laboratories, which used similar and different methods of COT and 3HC measurements to derive the NMR. We used Bland–Altman analysis to assess agreement, and Pearson correlations to evaluate associations, between NMR measured by different methods. Results: Measures of plasma NMR were in strong agreement between methods according to Bland–Altman analysis (ratios, 0.82–1.16) and were highly correlated (all Pearson r > 0.96, P < 0.0001). Measures of urinary NMR were in relatively weaker agreement (ratios 0.62–1.71) and less strongly correlated (Pearson r values of 0.66–0.98, P < 0.0001) between different methods. Plasma and urinary COT and 3HC concentrations, while weaker than NMR, also showed good agreement in plasma, which was better than that in urine, as was observed for NMR. Conclusions: Plasma is a very reliable biologic source for the determination of NMR, robust to differences in these analytical protocols or assessment site. Impact: Together this indicates a reduced need for differential interpretation of plasma NMR results based on the approach used, allowing for direct comparison of different studies. Cancer Epidemiol Biomarkers Prev; 24(8); 1239–46. ©2015 AACR.

Estimations of drug use at the national level are generally based on various sources of information, such as drug seizures, socio-scientific studies, toxicological data and hospital records. Nevertheless, all of these approaches have limitations that cannot be overcome, even if conclusions are drawn from combined data retrieved from different sources. Drug epidemiology through wastewater analysis has the potential to provide unique perspectives, internationally comparable data, and up-to-date information on the use of both traditional illicit drugs and new psychoactive substances (NPSs). In Finland, no large-scale studies on regional illicit drug consumption, based on a wastewater approach, have been reported. In this study, 24-h influent composite samples were collected during two 1-week study periods from ten different wastewater treatment plants in May and November–December 2012. The cities included in the study represent the geographical areas throughout Finland and cover 40% of the Finnish population. The samples were analyzed with an in-house validated, ultra high-performance liquid–chromatography mass spectrometric (UHPLC–MS/MS) method for various common illicit drugs and some NPS type stimulant drugs. The results were also compared with available statistics, information on drug seizures and laboratory-confirmed toxicological data, as well as other studies available based on wastewater analysis. The data show that illicit stimulant drug use is more common in the larger cities of Southern Finland. Amphetamine was the most commonly used drug in all 10 cities during both collection periods (excluding the collection period in May in Lappeenranta). Cocaine consumption remains very low in Finland in comparison to other European countries; it was concentrated in the biggest cities in Southern Finland. This study shows interesting temporal and spatial differences in drug use in Finland, as well as the possibilities of using wastewater analytics to reveal local hotspots of NPS consumption.

The abuse of anabolic androgenic steroids (AASs), such as nandrolone, is not only a problem in the world of sports but is associated with the polydrug use of non-athletes. Among other adverse effects, AAS abuse has been associated with long term or even persistent psychiatric problems. We have previously found that nandrolone decanoate treatment could produce prolonged changes in rats' brain reward circuits associated to drug dependence. The aim in this study was to evaluate whether AAS-induced neurochemical and behavioral changes are reversible. The increases in extracellular dopamine (DA) and serotonin (5-HT) concentration, as well as stereotyped behavior and locomotor activity (LMA) evoked by cocaine were attenuated by pretreatment with nandrolone. The recovery period, which was needed for the DA system to return back to the basic level, was fairly long compared to the dosing period of the steroid. In the 5-HT system, the time that system needed to return back to the basal level, was even longer than in the DA system. The attenuation was still seen though there were no detectable traces of nandrolone in the blood samples. Given that accumbal outflow of DA and 5-HT, as well as LMA and stereotyped behavior are all related to reward of stimulant drugs, this study suggests that nandrolone decanoate has significant, long-lasting but reversible effects on the rewarding properties of cocaine.

Previously we have reported that sub-chronic administration of nandrolone modifies reward-related neurochemical effects of psychomotor stimulant drugs of abuse. The aim of the present study was to evaluate whether the ability of nandrolone (19-nortestosterone) to attenuate the effects of amphetamine depends on activation of androgen (AR) or estrogen receptors (ER). We used an in vivo microdialysis technique in fully conscious rats to monitor whether administration of the AR-antagonist flutamide (7 × 50 mg/kg) or the ER-antagonist clomiphene (7 × 20 mg/kg), attenuates nandrolone-induced modulation of dopaminergic and serotonergic effects of acute injections of amphetamine (1 mg/kg). Dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites were measured from the samples using high performance liquid chromatography (HPLC). Blocking the androgen receptors with flutamide abolished the attenuating effect of nandrolone pre-treatment on amphetamine-induced elevation of extracellular DA concentration. Blocking the estrogen receptors with clomiphene did the same but to a lesser extent. In conclusion, the results of this study show that the ability of nandrolone to attenuate the effects of amphetamine depends on activation of androgen receptors or to a lesser extent, on estrogen receptors.

The abuse of anabolic androgenic steroids (AASs) is not only a problem in the world of sports but is associated with the polydrug use of nonathletes. Investigations of the neurochemical effects of AAS have focused in part on the monoaminergic systems, involving, among other things, the development of dependence. We have previously shown that pretreatment with nandrolone decanoate attenuates dose-dependently the increase in extracellular dopamine (DA) concentration evoked by amphetamine and 3,4-methylenedioyxymethamphetamine in the nucleus accumbens (NAc).The aim of this study was to investigate whether the nandrolone pre-exposure modulates the acute neurochemical and behavioral effects of cocaine in rats and whether the effects are long lasting.DA, 5-hydroxytryptamine (5-HT), and their metabolites were measured from samples collected from the NAc by microdialysis. The behavior of the animals was recorded.The present study demonstrates that five injections of nandrolone (5 and 20 mg/kg) inhibited cocaine-evoked DA and 5-HT outflow in the NAc, locomotor activity (LMA), and stereotyped behavior in experimental animals, and that these effects are seen even after elimination of nandrolone from bloodstream.Given that accumbal outflow of DA and 5-HT, as well as LMA and stereotyped behavior, is related to gratification of stimulant drugs, this study suggests that nandrolone, at the doses tested, has a significant effect on the pleasurable properties of cocaine. Furthermore, because neurochemical and behavioral responses were still attenuated after a fairly long recovery period, it seems that nandrolone may induce long-lasting changes in the brains of rat.

Previous studies suggest that brain-derived neurotrophic factor and its receptor TrkB are critically involved in the therapeutic actions of antidepressant drugs. We have previously shown that the antidepressants imipramine and fluoxetine produce a rapid autophosphorylation of TrkB in the rodent brain. In the present study, we have further examined the biochemical and functional characteristics of antidepressant-induced TrkB activation in vivo. We show that all the antidepressants examined, including inhibitors of monoamine transporters and metabolism, activate TrkB rapidly in the rodent anterior cingulate cortex and hippocampus. Furthermore, the results indicate that acute and long-term antidepressant treatments induce TrkB-mediated activation of phospholipase-Cgamma1 (PLCgamma1) and increase the phosphorylation of cAMP-related element binding protein, a major transcription factor mediating neuronal plasticity. In contrast, we have not observed any modulation of the phosphorylation of TrkB Shc binding site, phosphorylation of mitogen-activated protein kinase or AKT by antidepressants. We also show that in the forced swim test, the behavioral effects of specific serotonergic antidepressant citalopram, but not those of the specific noradrenergic antidepressant reboxetine, are crucially dependent on TrkB signaling. Finally, brain monoamines seem to be critical mediators of antidepressant-induced TrkB activation, as antidepressants reboxetine and citalopram do not produce TrkB activation in the brains of serotonin- or norepinephrine-depleted mice. In conclusion, our data suggest that rapid activation of the TrkB neurotrophin receptor and PLCgamma1 signaling is a common mechanism for all antidepressant drugs.

4-Methylaminorex is a potential psychostimulant drug of abuse that exists as four stereoisomers: cis-4R,5S, cis-4S,5R, trans-4S,5S, and trans-4R,5R. The racemic mixture of the cis-isomers has been encountered in illicit samples, but previous animal studies suggest that also the trans-isomers could have similar stimulant-like properties. We tested whether the stereoisomers possess rewarding properties and compared their potency using the conditioned place preference method in rats. Furthermore, the involvement of the brain dopaminergic system in the 4-methylaminorex reward was tested with the dopamine D1- and D2-receptor antagonists SCH 23390 and raclopride administered systemically, or with the neurotoxin 6-hydroxydopamine injected into the nucleus accumbens. All the four isomers induced place preference, with no apparent differences in their potency. SCH 23990 and raclopride attenuated 4-methylaminorex-induced increase in place preference, and 6-hydroxydopamine also tended to be efficacious. These findings indicate that all the four stereoisomers of 4-methylaminorex possess rewarding properties and thus abuse potential; the trans-isomers are at least as potent as the cis-isomers. Furthermore, the brain dopaminergic system appears to be involved in the 4-methylaminorex-reward.

The importance of AMPA-type glutamate receptors has been demonstrated in neuronal plasticity and in adaptation to drugs of abuse. We studied the involvement of AMPA receptors in social interaction and anxiety and found that in several paradigms of agonistic behavior naïve male mice deficient for the GluR-A subunit- containing AMPA receptors are less aggressive than wild-type littermates. GluR-A deficient mice and wild-type littermates exhibited similar basic behavior and reflexes as monitored by observational Irwin's test, but they tended to be less anxious in elevated plus-maze and light-dark tests. Maternal aggression or male-female encounters were not affected which suggests that male hormones are involved in the expression of suppressed aggressiveness. However, testosterone levels and brain monoamines can be excluded and found to be similar between GluR-A deficient and wild-type littermates. The reduced AMPA receptor levels caused by the lack of the GluR-A subunit, and measured by a 30% reduction in hippocampal [3H]-S-AMPA binding, seem to be the reason for suppressed male aggressiveness. When we analyzed mice with reduced number of functional AMPA receptors mediated by the genomic introduced GluR-A(Q582R) channel mutation, we observed again male-specific suppressed aggression, providing additional evidence for GluR-A subunit-containing AMPA receptor involvement in aggression.

We describe a rapid GC/MS assay for amphetamine-type stimulant drugs (ATSs) and structurally related common medicaments in blood, serum, oral fluid and urine samples. The drugs were extracted from their matrices and derivatized with heptafluorobutyric anhydride (HFBA) in a single step, using the following procedure: 100 microl (oral fluid) or 200 microl (blood, serum, urine) of the sample were mixed with 50 microl of alkaline buffer and 500 microl of extraction-derivatization reagent (toluene + HFBA + internal standard), centrifuged, and injected into a GC/MS apparatus. As revealed by the validation data this procedure, with its limit of quantitation being set at 20 ng/ml for oral fluid, 25 ng/ml for blood or 200 ng/ml for urine, is suitable for screening, identification and quantitative determination of the ATSs and related drugs in all the matrices examined. Thus, time-consuming and expensive multiple analyses are not needed, unless specifically required.

Alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Non-Alcohol) rats have well-documented differences in their voluntary ethanol consumption and brain opioidergic systems. The aim of the present study was to investigate whether these rat lines differ in their susceptibility to morphine-induced behavioural and neurochemical sensitization. The rats were given 15 injections of morphine (10 mg/kg, s.c.) or saline every other day. Locomotor activity and release of dopamine in the nucleus accumbens were monitored after a challenge with additional morphine injections (10 mg/kg) 1 and 5 weeks after withdrawal from the repeated treatment. Morphine increased locomotion more in the previously morphine-treated rats than in the saline-treated controls. Furthermore, AA rats were more sensitive to this effect of morphine than ANA rats. Accumbal morphine-induced dopamine release was significantly higher in the morphine-treated AA than ANA rats after the first challenge injection 1 week from withdrawal, but no differences were observed after the second challenge. The brain and plasma concentrations of morphine were similar among the lines suggesting that the differences in the effects of morphine cannot be explained in terms of differential pharmacokinetics of morphine in these lines. These data show that AA rats are more susceptible to morphine-induced behavioural sensitization than ANA rats. Furthermore, it suggests that mesolimbic dopamine has at best only a transient role in the expression of opioid-induced behavioural sensitization. The relationship between the mechanisms underlying the differential sensitivity of these rat lines to the effects of repeated morphine and voluntary ethanol drinking remains to be determined.

4-Methylaminorex is a stimulant drug of abuse that exists as four stereoisomers: cis -4 R ,5 S , cis -4 S ,5 R , trans -4 S ,5 S , and trans -4 R ,5 R. These isomers have previously been shown to differ markedly in various respects. In the present study we assessed the effects of the isomers of 4-methylaminorex (2.5, 5.0, and 10 mg/kg i.p.) on extracellular dopamine and 5-hydroxytryptamine (5-HT) levels in the nucleus accumbens, as well as behavior in the rats simultaneously. The relative concentrations of the isomers in the brain were also measured. The samples were collected by in vivo microdialysis and then analyzed for neurotransmitters with high-performance liquid chromatography/electrochemical detection and for cis - and trans -4-methylaminorex with gas chromatography/mass spectrometry. The behavioral effects of the isomers were assessed from videotapes recorded during the microdialysis experiments. All isomers elevated the extracellular levels of both dopamine and 5-HT, with the exception of trans -4 R ,5 R . The rank order of potency for elevating dopamine was trans -4 S ,5 S > cis -4 S ,5 R ≈ cis -4 R ,5 S > trans -4R,5R, and for elevating 5-HT cis -4 S ,5 R > trans -4 S ,5 S ≈ cis -4 R ,5 S > trans -4 R ,5 R . Analysis of the behavioral data, together with the neurochemical data, suggests that behavioral effects of the isomers of 4-methylaminorex are related to drug-induced dopamine release and, in the case of higher doses of the most efficacious isomers, to 5-HT as well. The brain concentrations of the isomers did not reflect their neurochemical efficacy, which implies that their differences are pharmacodynamic rather than pharmacokinetic.

Rationale: It has previously been demonstrated that the 5-HT3 receptors located in the mesolimbic brain areas are able to modulate the dopaminergic effects of various abused drugs, including cocaine (COC). Objectives: The present experiments investigated the role of 5-HT3 receptors in the actions of selected monoamine uptake inhibitors. Methods: The ability of the 5-HT3 receptor antagonist MDL 72222 (MDL; 0.1 and 1.0 mg/kg) to modify the neurochemical and behavioral changes induced by COC (20 mg/kg), mazindol (MAZ; 10 mg/kg), and methylphenidate (MP; 5.0 or 10, and 20 mg/kg) was assessed with an in vivo microdialysis technique, a conditioned place preference method, and motor activity measurements. Results: MDL robustly attenuated the elevation of extracellular dopamine levels in the nucleus accumbens, acquisition of place preference, and motor activity induced by COC and MAZ, but not those induced by MP, the only drug with no significant effect on 5-HT. In contrast, expression of COC-induced place preference was not attenuated by MDL. Conclusions: These results show that COC- and MAZ-induced reward-related neurochemical and behavioral effects, preferentially those implicated in development of conditioned reward, are modified by the 5-HT3 blockade. In contrast to COC and MAZ, the changes induced by MP, which has less effect on the serotonergic system, remain unchanged. Thus it appears that involvement of a serotonergic component in the mechanism of action of a drug could be a prerequisite for effective antagonism by 5-HT3 receptor blockers.

The 4-methylaminorex (4-MAX) is an amphetamine-related psychostimulant drug that has appeared on the clandestine market with a street name of “U4Euh”. This compound exists as four stereoisomers, trans-4R,5R, trans-4S,5S, cis-4R,5S and cis-4S,5R, of which the cis forms have been classified as Schedule I substances in the US. The increasing variety of designer drugs has highlighted the importance of detection, identification, and quantitative measurement of these drugs, including 4-MAX, in biological samples. In the present study, the isomers of 4-MAX were detected in urine of rats treated with the drugs by some but not all of the on-site immunoassays tested, mainly as amphetamine or methamphetamine. To facilitate identification of 4-MAX by laboratories specialized in drug analysis, the electron-ionization mass spectrum and TLC data for underivatized 4-MAX using a routine laboratory drug-screening procedure is provided. In addition, a GC/MS method is described for the quantitative determination of cis- and trans-4-MAX as tert-butyldimethylsilyl-derivatives in plasma, urine and tissue.

Use of antioxidative agents is required in automated LC assay of microdialysis samples, due to rapid degradation of the monoamine neurotransmitters and their metabolites. Addition of oxalic acid prevented degradation of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid efficiently: after a 24-h incubation at room temperature the decreases in peak heights were less than 10%. The long-term stability of the analytes, however, was still enhanced when acetic acid and l -cysteine were included in the solution. Using this antioxidative solution, the monoamine neurotransmitters and their metabolites could be determined with an automated LC assay even at room temperature.

Drugs such as benzodiazepines, which enhance the effects of inhibitory neurotransmitter gamma-amino butyric acid (GABA), are known to modulate the mesocorticolimbic dopaminergic system, which is considered to mediate the rewarding effects of psychostimulants. The effects of diazepam, a benzodiazepine that binds unspecifically to omega 1- (omega1-) and omega2-receptors, and zolpidem, a nonbenzodiazepine drug that binds preferentially to omega1-receptors, on cocaine- and amphetamine-induced place preference were evaluated in Wistar rats. In tests using the counterbalanced method, neither diazepam (0.2, 1, and 5 mg/kg) nor zolpidem (2.5, 5, and 10 mg/kg) alone induced place preference or place aversion. Diazepam pretreatment prevented both cocaine- and amphetamine-induced (15 and 9 mg/kg, respectively) place preference; however, at doses that were earlier shown to cause sedation and amnesia, zolpidem failed to prevent either cocaine- or amphetamine-induced place preference. These results suggest that diazepam interferes with the rewarding properties of the psychostimulants, whereas zolpidem is less effective in this respect, possibly due to differential distribution of omega1- and omega2-receptors in the brain.

Aims: To examine whether smoking habits, nicotine dependence (ND) and plasma cotinine levels differ by diurnal type. Design: Data originated from the national FINRISK 2007 survey. Regression analyses were calculated to examine the association between diurnal type and smoking status, ND, and nicotine intake. Participants: 7091 FINRISK participants with smoking and diurnal type information and a subset of 1746 ever smokers with detailed smoking, and ND assessments. Measurements: Diurnal type assessed with a six-item sum scale was categorized as morning, intermediate and evening type. Smoking status was determined as current (daily or occasional), former, and never smokers. ND was measured with the Fagerstrom Test for Nicotine Dependence (FTND), the Hooked on Nicotine Checklist (HONC), and the Nicotine Dependence Syndrome Scale (NDSS). For current smokers, plasma cotinine was analyzed as biochemical measurement of nicotine intake. Findings: Evening type was associated with current smoking (OR=1.66, 95% CI 1.40, 1.97). A significant association with diurnal type was seen for FTND among men (beta= -0.46, 95% CI -0.72, -0.21), sexes combined for HONC (beta= -0.31, 95% CI -0.52, -0.11) and NDSS (beta= -0.86, 95% CI -1.43, -0.29) and for cotinine among men (beta= -0.73, 95% CI -1.16, -0.29). Adjustment for depressive symptoms attenuated the association of diurnal type with NDSS to be non-significant. Conclusions: Diurnal type was associated with multiple ND measures and nicotine intake, interestingly more so among men. Evening type persons are at higher risk of dependence, but depressive symptoms attenuates this association clearly.

Gamma-hydroxybutyrate (GHB) is an increasingly popular drug of abuse that causes stimulation, euphoria, anxiolysis or hypnosis, depending on the dose used. Low doses of the drug are used recreationally, and also implicated in drug-facilitated sexual assaults. Because of the unusually steep dose-response curves, accidental GHB overdosing, leading to coma, seizures or death can occur. Being a controlled substance, GHB is often substituted with its non-scheduled precursors gamma-butyrolactone (GBL) and 1,4-butanediol (BD), which are rapidly metabolized into GHB in the body. Here we describe an assay for GHB, GBL and BD in blood and/or urine samples. GHB and BD were extracted from diluted 200 microL aliquots of samples with t-butylmethylether (plus internal standard benzyl alcohol) in test tubes preloaded with NaCl. After acidification and centrifugation the solvent phase was transferred to a test tube preloaded with Na(2)SO(4), incubated for 30 min, centrifuged again, and evaporated in vacuum. The residue was mixed with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) in acetonitrile, and injected into a GC-MS. When analyzing GBL, the salting-out step was omitted, and analysis was performed with a GC-FID apparatus. As revealed by the validation data this procedure is suitable for quantitative determination of GHB and its precursors in blood and/or urine samples.

Misuse of anabolic-androgenic steroids (AASs) is increasing, and appears to have much in common with the use of substances known to induce drug dependence. Moreover, persons who abuse AASs also tend to abuse other psychotropic drugs such as amphetamine or 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). The aim of this study was to investigate whether nandrolone (5 x 5 or 5 x 20 mg/kg) pre-exposure modulates the acute neurochemical and behavioral effects of amphetamine (1mg/kg) and MDMA (5 mg/kg) in rats. Dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites were measured from samples collected from the nucleus accumbens (NAc) by in vivo microdialysis. The behavior of the animals was recorded on videotapes, from which it was later rated. Our results demonstrate that sub-chronic treatments with supraphysiological doses of nandrolone attenuate dose-dependently the increase in extracellular DA concentration evoked by amphetamine or MDMA. The lower dose of nandrolone attenuated MDMA-induced increase in 5-HT-levels, while the higher dose potentiated it. Analysis of the behavioral data suggests that effects of the amphetamine and MDMA are dose-dependently attenuated by AAS-treatment, paralleling DA results. In conclusion, the results of this study show that AAS-pre-treatment is able to modulate the reward-related neurochemical and behavioral effects of amphetamine and MDMA.

1-Benzylpiperazine (also known as ‘Legal X’, ‘Legal E’, or ‘A2’) is a psychoactive compound increasingly encountered on the clandestine market. Previous experimental data suggest that the compound possesses addictive properties. In the present study, we used the conditioned place preference method in the rat to test whether 1-benzylpiperazine possesses rewarding properties. Furthermore, the mechanisms of the 1-benzylpiperazine reward were investigated using selected dopamine and serotonin receptor antagonists. 1-Benzylpiperazine (1.25, 5, and 20 mg/kg) induced dose-dependently place preference. This place preference was attenuated by the antagonists SCH23390 (0.2 mg/kg; dopamine D1-like receptors) and MDL72222 (1.0 mg/kg; serotonin3 receptors), but not by raclopride (0.8 mg/kg; dopamine D2-like receptors) or ketanserin (2 mg/kg; preferentially serotonin2 receptors). Our results show that 1-benzylpiperazine possesses rewarding properties in the rat, which suggests the compound to be susceptible to human abuse. The brain dopaminergic and serotonergic systems appear to be involved in the 1-benzylpiperazine reward.

4-Methylaminorex, a potential psychostimulant drug of abuse, exists as four stereoisomers: cis-4R,5S, cis-4S,5R, trans-4S,5S, and trans-4R,5R, which were shown previously to possess stereospecific effects. This study characterized their pharmacokinetic and tissue distribution profiles, and metabolic turnover to norephedrine and norpseudoephedrine, in male Wistar rats. The rats received each isomer intravenously, intraperitoneally, or orally, followed by blood sample collection via cannula (pharmacokinetic study), or tissue sample collection at predetermined time points (tissue distribution study). The samples were analyzed for cis- and trans-isomers, and when appropriate for norephedrine and norpseudoephedrine, with gas chromatography/mass spectrometry. Trans-4S,5S-, cis-4R,5S-, and cis-4S,5R-isomers behaved comparably kinetically (volume of distribution 1.7-2.3 l/kg, distribution half-life 3.8-7.0 min, elimination half-life 35-42 min, and bioavailability 32-57% intraperitoneally or 4-16% orally), whereas trans-4R,5R-isomer differed from the others, with a longer elimination half-life (118-169 min) and higher bioavailability (100% intraperitoneally or 83% orally). The highest isomer concentrations were observed in the kidney followed most frequently by the liver, brain, muscle, and last by fat and blood. The elimination half-lives of the stereoisomers from the tissues were generally similar to those in blood. No pharmacologically significant amounts of norephedrine or norpseudoephedrine were detected in blood or the brain. In conclusion, differences between the stereoisomers of 4-methylaminorex in the pharmacokinetics and tissue distribution are described. However, these differences are not compatible with, and thus may not account for, the distinct behavioral and neurochemical effects of the stereoisomers demonstrated previously. Furthermore, metabolic turnover to norephedrine and norpseudoephedrine does not seem to contribute significantly to 4-methylaminorex pharmacology.

Alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Non-Alcohol) rats have well-documented differences in their voluntary ethanol consumption and brain opioidergic systems. The aim of the present study was to investigate whether these rat lines differ in their susceptibility to morphine-induced behavioural and neurochemical sensitization. The rats were given 15 injections of morphine (10 mg/kg, s.c.) or saline every other day. Locomotor activity and release of dopamine in the nucleus accumbens were monitored after a challenge with additional morphine injections (10 mg/kg) 1 and 5 weeks after withdrawal from the repeated treatment. Morphine increased locomotion more in the previously morphine-treated rats than in the saline-treated controls. Furthermore, AA rats were more sensitive to this effect of morphine than ANA rats. Accumbal morphine-induced dopamine release was significantly higher in the morphine-treated AA than ANA rats after the first challenge injection 1 week from withdrawal, but no differences were observed after the second challenge. The brain and plasma concentrations of morphine were similar among the lines suggesting that the differences in the effects of morphine cannot be explained in terms of differential pharmacokinetics of morphine in these lines. These data show that AA rats are more susceptible to morphine-induced behavioural sensitization than ANA rats. Furthermore, it suggests that mesolimbic dopamine has at best only a transient role in the expression of opioid-induced behavioural sensitization. The relationship between the mechanisms underlying the differential sensitivity of these rat lines to the effects of repeated morphine and voluntary ethanol drinking remains to be determined.

The acute effects of amphetamine derivatives on extracellular concentration of serotonin (5-HT) and dopamine in the nucleus accumbens were studied with in vivo microdialysis using conscious, freely moving rats. 5-HT, dopamine, and their major metabolites were measured by HPLC with electrochemical detection. Amphetamine (1.0-9.0 mg/kg) elevated dopamine levels considerably, but failed to affect the levels of 5-HT, except at the highest dose administered. 3,4-Methylenedioxyamphetamine (MDA, 1.0-9.0 mg/kg) and 3,4-methylenedioxymethamphetamine (MDMA, 1.0-9.0 mg/kg) elevated both 5-HT and dopamine levels dose dependently. The failure of 2,5-dimethoxy-4-methylamphetamine (DOM, 0.5-1.0 mg/kg) to affect the 5-HT levels suggests that extracellular levels of 5-HT play a minor role in hallucinogenic activity. The strong effects of MDA and MDMA on levels of 5-HT indicate that their actions on serotonergic mechanisms are different from those of the hallucinogens. In addition, methylenedioxyamphetamines may act via dopaminergic mechanisms similar to those of amphetamine.

The effects of a 5-HT3 receptor antagonist MDL 72222 on cocaine- and amphetamine-induced increases in extracellular dopamine in the nucleus accumbens and the dorsal striatum were studied with microdialysis technique using halothane anaesthesized rats. Dopamine and its metabolites were measured by HPLC with electrochemical detection. Cocaine elevated extracellular dopamine in the nucleus accumbens and to a lesser extent in the dorsal striatum, but it did not affect dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid. Pretreatment with MDL 72222 (25-100 micrograms/kg) dose-dependently attenuated cocaine-induced elevation of dopamine in both of the nuclei studied. Amphetamine elevated extracellular dopamine and reduced DOPAC and homovanillic acid equally in the nucleus accumbens and in the dorsal striatum. MDL 72222 also attenuated the amphetamine-induced elevation of extracellular dopamine concentration in both brain areas studied, but first at a dose of 100 micrograms/kg. The different potencies of the interactions of the 5-HT3 receptor antagonist with cocaine and amphetamine could be related to the different mechanisms by which these drugs primarily elevate extracellular dopamine.