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[Display omitted] • Misuse of veterinary drug, Clenbuterol as growth promoters in humans and livestock. • A comprehensive discussion on usage of nanosensors to detect clenbuterol in food samples. • The application of nano-sensors for real sample analysis has been discussed. • The limit of detection and recovery percentage values of each nanosensor has been compared. Veterinary drugs which are primarily meant for livestock treatment have now been categorised under potential food contaminant due to its unregulated usage and abuse. Their over usage by animal workers lead to production of contaminated animal-based food products which contain veterinary drug residues. These drugs are also misused as growth promoters to enhance the muscle to fat ratio in human body. This review highlights the misuse of such a veterinary drug; Clenbuterol. In this review, we have comprehensively discussed the usage of nanosensors to detect clenbuterol in food samples. Colorimetric, fluorescent, electrochemical, SERS and electrochemiluminescence are major categories of nanosensors that have been utilized for this purpose. The mechanism through which these nanosensors detect clenbuterol have been discussed in detail. The limit of detection and recovery percentage values of each nanosensor have been compared. This review will impart significant information on various nanosensors for clenbuterol detection in real samples. [ABSTRACT FROM AUTHOR]

• Intra-BLA injections of CORT before memory reactivation impaired memory retrieval and its subsequent expression. • Intra-BLA injections of CORT after memory reactivation impaired fear memory reconsolidation and expression. • Co-injection of the β 2 -adrenergic agonist, clenbuterol, prevented, but the β-adrenergic antagonist, propranolol, potentiated the CORT effects. • The effects of glucocorticoids on fear memory expression are modulated by β-adrenoceptors in the BLA. Glucocorticoid administration, before or after fear memory reactivation, impairs subsequent fear memory expression, but the underlying mechanisms are not well understood. The present study examined the role of basolateral amygdala (BLA) β-adrenoceptors in the effects of intra-BLA corticosterone injection on fear memory in rats. Bilateral cannulae were implanted in the BLA of Wistar male rats. The rats were trained and tested using an inhibitory avoidance task (1 mA footshock for 3 s). Forty-eight hours after training, corticosterone (CORT, 5, 10, or 20 ng/0.5 µl/side) and the β2-adrenoceptor agonist clenbuterol (CLEN, 10 or 20 ng/0.5 µl/side) or the β-adrenoceptor antagonist propranolol (PROP, 250 or 500 ng/0.5 µl/side) were injected into the BLA before or right after memory reactivation (retrieval, Test 1). We performed subsequent tests 2 (Test 2), 5 (Test 3), 7 (Test 4), and 9 (Test 5) days after Test 1. The results demonstrated that CORT injection before Test 1 disrupted memory retrieval and reduced fear expression in Tests 2–5, possibly due to enhanced extinction or impaired reconsolidation. CORT injection after Test 1 also impaired reconsolidation and reduced fear expression in Tests 2–5. CLEN prevented, but PROP exacerbated, the effects of CORT on fear expression. The reminder shock did not recover fear memory in CORT-treated animals, suggesting that reconsolidation, not extinction, was affected. These results indicate that glucocorticoids and β-adrenoceptors in the BLA jointly modulate fear memory reconsolidation and expression. Comprehending the neurobiology of stress and the impact of glucocorticoids on fear memory may lead to new treatments for stress and trauma-induced disorders such as PTSD. [ABSTRACT FROM AUTHOR]

Impaired hippocampal neurogenesis is one of the early pathological features of Alzheimer′s disease. Enhancing adult hippocampal neurogenesis has been pursued as a potential therapeutic strategy for Alzheimer′s disease. Recent studies have demonstrated that environmental novelty activates β2 -adrenergic signaling and prevents the memory impairment induced by amyloid-β oligomers. Here, we hypothesized that β2 -adrenoceptor activation would enhance neurogenesis and ameliorate memory deficits in Alzheimer′s disease. To test this hypothesis, we investigated the effects and mechanisms of action of β2 -adrenoceptor activation on neurogenesis and memory in amyloid precursor protein/presenilin 1 (APP/PS1) mice using the agonist clenbuterol (intraperitoneal injection, 2 mg/kg). We found that β2 -adrenoceptor activation enhanced hippocampal neurogenesis, ameliorated memory deficits, and increased dendritic branching and the density of dendritic spines. These effects were associated with the upregulation of postsynaptic density 95, synapsin 1 and synaptophysin in APP/PS1 mice. Furthermore, β2 -adrenoceptor activation decreased cerebral amyloid plaques by decreasing APP phosphorylation at Thr668. These findings suggest that β2 -adrenoceptor activation enhances neurogenesis and ameliorates memory deficits in APP/PS1 mice.

In this review, we discuss the morphologic influence of clenbuterol on muscle atrophy with particular focus on cast-immobilization used in surgical care. β2-agonists induce muscle hypertrophy, particularly in fast muscles. Similar to these anabolic actions, the β2-agonist clenbuterol attenuates immobilization-induced atrophy in fast muscles. Furthermore, the attenuating effects of this agonist on muscle atrophy may be related to its preventative effect in fast muscle fibers.

Common typical β-agonists mainly include ractopamine (RAC), salbutamol (SAL), and clenbuterol (CLB). In view of the harm to human health causes by the ingestion of animal derived food containing β-agonists, and a series of regulations have been issued to restrict the usage of β-agonists as growth promoters. In this work, a fluorescence immunoassay is developed for the simultaneous detection of typical β-agonists based on blue-green upconversion nanoparticles (UCNPs) combine with magnetic separation. Here, blue-green UCNPs act as a signal amplification source, and magnetic polystyrene microspheres (MPMs) act as an ideal separation medium. Based on a competitive form, capture probe competes (RAC-OVA@MPMs and SAL-OVA@MPMs) with targets to bind corresponding signal probe (anti-RAC antibody@NaYF 4 :Yb, Tm UCNPs and anti-SAL antibody@NaYF 4 :Yb, Er UCNPs). The fluorescence difference values of the competitive immune-complex obtained via magnetic separation at 483 nm and 550 nm are proportional to concentrations of RAC and SAL, respectively. The immunoassay has the wide detection linear range from 0.001 to 100 μg L -1 , and the low limit of detection (LOD) is 5.04 × 10 -4 μg L -1 for RAC, 1.97 × 10 -4 μg L -1 for SAL, respectively. Meanwhile, use of antibody with same recognition ability for SAL and CLB makes that the fluorescence immunoassay can achieve simultaneous detection of three typical β-agonists (RAC, SAL, and CLB). This fluorescence immunoassay has good application value and practicability for simultaneous detection of typical β-agonists in animal derived food., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Pompe disease is caused by a deficiency of the enzyme acid alpha-glucosidase (GAA). People with infantile-onset disease have either a complete or a near-complete enzyme deficiency; people with late-onset Pompe disease (LOPD) retain some residual enzyme activity. GAA deficiency is treated with an intravenous infusion of recombinant human acid alglucosidase alfa, an enzyme replacement therapy (ERT). Alglucosidase alfa and avalglucosidase alfa are approved treatments, but cipaglucosidase alfa with miglustat is not yet approved., Objectives: To assess the effects of enzyme replacement therapies in people with late-onset Pompe disease., Search Methods: We searched the Cochrane Inborn Errors of Metabolism Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched MEDLINE OvidSP, clinical trial registries, and the reference lists of relevant articles and reviews. Date of last search: 21 April 2022., Selection Criteria: We included randomised controlled trials (RCTs) of ERT in people with LOPD of any age., Data Collection and Analysis: Two review authors independently assessed trial eligibility, extracted data, assessed the risk of bias and the certainty of the evidence (using GRADE). We resolved disagreements through discussion and by consulting a third author., Main Results: We included six trials (358 randomised participants) lasting from 12 to 78 weeks. A single trial reported on each comparison listed below. None of the included trials assessed two of our secondary outcomes: need for respiratory support and use of a walking aid or wheelchair. Certainty of evidence was most commonly downgraded for selective reporting bias. Alglucosidase alfa versus placebo (90 participants) After 78 weeks, alglucosidase alfa probably improves the six-minute walk test (6MWT) distance compared to placebo (mean difference (MD) 30.95 metres, 95% confidence interval (CI) 7.98 to 53.92; moderate-certainty evidence) and probably improves respiratory function, measured as the change in per cent (%) predicted forced vital capacity (FVC) (MD 3.55, 95% CI 1.46 to 5.64; moderate-certainty evidence). There may be little or no difference between the groups in occurrence of infusion reactions (risk ratio (RR) 1.21, 95% CI 0.57 to 2.61; low-certainty evidence), quality of life physical component score (MD -1.36 points, 95% CI -5.59 to 2.87; low-certainty evidence), or adverse events (RR 0.94, 95% CI 0.64 to 1.39; low-certainty evidence). Alglucosidase alfa plus clenbuterol versus alglucosidase alfa plus placebo (13 participants) The evidence is very uncertain about the effect of alglucosidase alfa plus clenbuterol compared to alglucosidase alfa plus placebo on: change in 6MWT distance after 52 weeks (MD 34.55 metres, 95% CI-10.11 to 79.21; very low-certainty evidence) and change in % predicted FVC (MD -13.51%, 95% CI -32.44 to 5.41; very low-certainty evidence). This study did not measure infusion reactions, quality of life, and adverse events. Alglucosidase alfa plus albuterol versus alglucosidase alfa plus placebo (13 participants) The evidence is very uncertain about the effect of alglucosidase alfa plus albuterol compared to alglucosidase alfa plus placebo on: change in 6MWT distance after 52 weeks (MD 30.00 metres, 95% CI 0.55 to 59.45; very low-certainty evidence), change in % predicted FVC (MD -4.30%, 95% CI -14.87 to 6.27; very low-certainty evidence), and risk of adverse events (RR 0.67, 95% CI 0.38 to 1.18; very low-certainty evidence). This study did not measure infusion reactions and quality of life. VAL-1221 versus alglucosidase alfa (12 participants) Insufficient information was available about this trial to generate effect estimates measured at one year or later. Compared to alglucosidase alfa, VAL-1221 may increase or reduce infusion-associated reactions at three months, but the evidence is very uncertain (RR 2.80, 95% CI 0.18 to 42.80). This study did not measure quality of life and adverse events. Cipaglucosidase alfa plus miglustat versus alglucosidase alfa plus placebo (125 participants) Compared to alglucosidase alfa plus placebo, cipaglucosidase alfa plus miglustat may make little or no difference to: 6MWT distance at 52 weeks (MD 13.60 metres, 95% CI -2.26 to 29.46); infusion reactions (RR 0.94, 95% CI 0.49 to 1.80); quality of life scores for physical function (MD 1.70, 95% CI -2.13 to 5.53) and fatigue (MD -0.30, 95% CI -2.76 to 2.16); and adverse effects potentially related to treatment (RR 0.83, 95% CI 0.49 to 1.40) (all low-certainty evidence). Cipaglucosidase alfa plus miglustat probably improves % predicted FVC compared to alglucosidase alfa plus placebo (MD 3.10%, 95% CI 1.04 to 5.16; moderate-certainty evidence); however, it may make little or no change in % predicted sniff nasal inspiratory pressure (MD -0.06%, 95% CI -8.91 to 7.71; low-certainty evidence). Avalglucosidase alfa versus alglucosidase alfa (100 participants) After 49 weeks, avalglucosidase alfa probably improves 6MWT compared to alglucosidase alfa (MD 30.02 metres, 95% CI 1.84 to 58.20; moderate-certainty evidence). Avalglucosidase alfa probably makes little or no difference to % predicted FVC compared to alglucosidase alfa (MD 2.43%, 95% CI -0.08 to 4.94; moderate-certainty evidence). Avalglucosidase alfa may make little or no difference to infusion reactions (RR 0.78, 95% CI 0.42 to 1.45), quality of life (MD 0.77, 95% CI -2.09 to 3.63), or treatment-related adverse events (RR 0.92, 95% CI 0.61 to 1.40), all low-certainty evidence., Authors' Conclusions: One trial compared the effect of ERT to placebo in LOPD, showing that alglucosidase alfa probably improves 6MWT and respiratory function (both moderate-certainty evidence). Avalglucosidase alfa probably improves 6MWT compared with alglucosidase alfa (moderate-certainty evidence). Cipaglucosidase plus miglustat probably improves FVC compared to alglucosidase alfa plus placebo (moderate-certainty evidence). Other trials studied the adjunct effect of clenbuterol and albuterol along with alglucosidase alfa, with little to no evidence of benefit. No significant rise in adverse events was noted with all ERTs. The impact of ERT on some outcomes remains unclear, and longer RCTs are needed to generate relevant information due to the progressive nature of LOPD. Alternative resources, such as post-marketing registries, could capture some of this information., (Copyright © 2023 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.)

Abusive use of β-agonists as feed additives for animals and medication is detrimental to human health and food safety. Conventional assays are restricted to a single type of β-agonists detection and cannot match the multiplexing features to perform automated, high throughput, and rapid quantitative analysis in real samples. In this research, we develop a portable automated chip system (PACS) with highly integrated automated devices in conjunction with portable microfluidic chips to provide simultaneous point-of-care testing of multiple β-agonists in the field, simplifying complex manual methods, shortening assay times, and improving sensitivity. Specifically, silicon film is used as reaction substrates for immobilizing the conjugates of β-agonists to increase the sensitivity of the assay result. Then, the PACS with a chemiluminescence imaging detector is established for automatic high-throughput and sensitive detection of Clenbuterol, Ractopamine, and Salbutamol based on the indirect immunoassay. Newly developed chip with high mixing performance can improve the sensitivity of target determination. Multiplex assays were carried out using the developed system for Clenbuterol, Ractopamine, and Salbutamol with a limit of detection of 54 pg mL -1 ,59 pg mL -1 , and 93 pg mL -1 , respectively. Except for sample preparation and coating, the detection in the PACS takes less than 47 min. A satisfactory sample recovery (86.33%-108.12%) was obtained, validating the reliability and practical applicability of this PACS. Meanwhile, the PACS enables sensitive and rapid detection of multiple β-agonists in farms or markets where lacking advanced laboratory facilities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The illegal use of beta-agonists could cause severe problems to human health. In this study, the usage of beta-agonists in 31 cities across China was estimated using wastewater-based epidemiology (WBE). The proposed method is based on solid-phase extraction (SPE) and LC-MS/MS and was developed and validated to determine the concentration of seven beta-agonists in wastewater. A population model based on cotinine (COT), NH 4 -N and the flow volume was constructed to estimate the population equivalents for different wastewater treatment plants (WWTPs). Clenbuterol and ractopamine are banned in China for both animal husbandry and medical use, but were nevertheless detected in some wastewater samples at rates of 6.2 % and 4.7 %, respectively (n = 339). The WBE-based consumption of clenbuterol and ractopamine were compared with the acceptable daily intake (ADI) and the health risks were assessed by their hazard quotients (0.26-6.62 for clenbuterol and 9.27 × 10 -4 -0.05 for ractopamine). Salbutamol, clorprenaline and terbutaline were observed in practically all wastewater samples at concentrations of up to several ng/L, whereas the formoterol and bambuterol concentrations were below the detection limit in all samples. Salbutamol consumption (7.35 ± 4.14 mg/1000 inh/day) was highest among the examined beta-agonists and varied regionally. Beta-agonist consumption based on WBE was higher in some cities than that based on medical survey data, indicating potential illegal use. These results show that WBE can be a straightforward and supplementary method for monitoring beta-agonist usage at the population level and spatially., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Abstract A multiplex lateral flow immunoassay sensor based on highly luminescent green-emitting Au nanoclusters (AuNCs-MLFIA sensor) was successfully established for the simultaneous and quantitative determination of clenbuterol (Clen) and ractopamine (RAC) in swine urine. The antigens of Clen and RAC were dispersed on a nitrocellulose membrane as two test lines, and the Au nanoclusters were synthesized from 6-aza-2-thiothymine and l -arginine to obtain highly green luminescence and ultra-small nanoparticles (Arg/ATT/AuNCs). Free carboxyl groups on Arg/ATT/AuNCs enabled conjugation with biomolecules to afford an indicator for the biosensor. The AuNCs-MLFIA sensor is based on the indirect competition assay and could successfully detect samples within 18 min without sample pretreatment, qualitative results can be obtained by visual inspection under a UV lamp. The limits of detection of Clen and RAC by the naked eye were both 0.25 μg L−1. In addition, the AuNCs-MLFIA sensor allowed quantitative detection combined with a portable fluorescence reader. The half-maximal inhibitory concentrations of Clen and RAC were 0.06 and 0.32 μg L−1, respectively, with detection limits of 0.003 and 0.023 μg L−1. Thirty blind-spiked swine urine samples were analyzed by the AuNCs-MLFIA sensor and liquid chromatography–tandem mass spectrometry, and the results of the two methods showed a significant correlation. The newly developed AuNCs-MLFIA sensor overcomes several limitations of conventional LFIA sensors, including their low sensitivity, limitation to quantify analytes, and single-analyte detection. Graphical abstract Multiplex LFIA sensor based on highly luminescent green-emitting gold nanoclusters for simultaneous and quantitative determination of Clen and RAC in swine urine. Image 1 Highlights • Highly luminescent green-emitting AuNCs were applied to LFIA for the first time. • AuNCs-MLFIA sensor was developed for simultaneous and quantitative determination of Clen and RAC in swine urine. • The developed AuNCs-MLFIA sensor revealed superior in sensitivity than other LFIA sensors. [ABSTRACT FROM AUTHOR]

Clenbuterol (CLB) is harmful to human health when used long term, and it has been listed by the World Anti-Doping Agency (WADA). In this work, a novel zinc-based metal-organic frameworks-reduced graphene oxide-CdTe quantum dots (ZnMOF-RGO-CdTe QDs) hybrid was used to construct an electrochemiluminescence (ECL) sensor for detecting CLB. CdTe QDs, loaded by RGO, exhibited an enhanced ECL signal. In addition, the ZnMOFs catalyzed OH• generation by coreactant H2O2, which further strengthened the ECL signal of the CdTe QDs. The integration of ZnMOFs and RGO-CdTe QDs endowed the sensor with high sensitivity for CLB detection. The intensity of the ECL signal increased as the concentration of CLB increased. The linear range of CLB detection was 3.0 × 10−13 M to 6.0 × 10−10 M, and the detection limit was estimated to be 1.0 × 10−13 M. Furthermore, the sensor displayed a good repeatability and stability. The ZnMOF-RGO-CdTe QD hybrids described in this study provide a foundation for the development of new methods of detecting CLB.ᅟ [ABSTRACT FROM AUTHOR]

Immunochromatographic assay (ICA) is an efficient analytical technique and rapid, convenient, easy-to-use, low-cost, and on-site detection method that has been widely used to evaluate food safety. However, an important issue to be addressed for this method is its matrix effect. In this work, time-resolved fluorescent nanobead ICA (TRFN-ICA) and colloidal gold ICA (CG-ICA) were developed to detect clenbuterol in swine urine. Under optimized working conditions, the limits of detection of TRFN-ICA and CG-ICA were 16 and 68 pg/mL, respectively. The matrix effect on TRFN-ICA and CG-ICA was assessed in 20 swine urine samples. Results indicated that the sensitivity of TRFN-ICA was better than that of CG-ICA, and the detection time of the former was shorter than that of the latter. The matrix effect on TRFN-ICA was more serious than that on CG-ICA. [ABSTRACT FROM AUTHOR]

Graphical abstract With large size Prussian blue nanoparticles (PBNPs) as the labeling substrate to reduce the labeled quantity of antibodies and improve visible signal, a lateral flow biosensor was developed for sensitive and cost-effective detection of clenbuterol. Highlights • MOFs of PBNPs were firstly introduced as novel labels in lateral flow assays (LFAs). • Assay cost was reduced because a fewer antibodies were needed in the PBNP-mAb probe. • Assay performance of the PBNP-based LFA was 5-fold improved for clenbuerol detection. • The work opens new possibilities to choose MOFs as labeling matrices in immunoassays. Abstract Prussian blue nanoparticles (PBNPs), one kind of metal organic frameworks with the general formula of Fe 4 [Fe(CN) 6 ] 3 ·nH 2 O, are increasing attractive due to their excellent properties. Here, with PBNPs as labels, a PBNP-based competitive lateral flow assay (LFA) for efficient and sensitive detection of clenbuterol was developed. With unique control structure, PBNPs are quite larger than most of the commonly used labels, meaning much fewer antibodies are needed in the probe to produce clearly visible results, which will bring a more sensitive dose-response relation between the analyte concentration and signal intensity. Under optimal conditions, the PBNP-based LFA showed a great linear relationship over the range of 0.5–5 ng mL−1 for clenbuterol. The visual limit of detection was 1.0 ng mL−1, 5-fold improvement in sensitivity compared with that of the traditional gold nanoparticles based LFA. In our perception, our work opens new possibilities to choose metal organic frameworks as labeling matrices in immunoassays. [ABSTRACT FROM AUTHOR]

A novel multilayer‐shaped CeO2 with obvious electrocatalytic activity was synthesized using a simple heat treatment method. The structure and morphology of the multilayer‐shaped CeO2 were characterized by X‐ray diffraction, scanning electron microscopy, and transmission electron microscopy. Clenbuterol, a kind of β‐agonist drugs, has recently become a serious issue to human body due to the illegal addition in the food additive for livestocks. The drug residue in the human body may cause muscular pain, cardiac palpitation, glaucoma, prostatitis, dizziness and and even death in old people. Therefore, the detection of clenbuterol (CLB) has a important significance. The as‐prepared CeO2 was found to have unique electrocatalytic oxidation activities towards clenbuterol (CLB). The electrochemical sensor was constructed by fixing stable nafion‐modified multilayer‐shaped CeO2 film on a glassy carbon electrode. Compared with the CeO2‐modified electrode and the nafion‐modified electrode, the nafion‐modified multilayer‐shaped CeO2 electrode showed better electrocatalytic performance for clenbuterol in HClO4 solution. There was a good linear relationship between the oxidation peak current and the concentration of CLB between 0.39∼2.79 μmol/L, and the detection limit was 0.08 μmol/L (R=0.99). The nafion‐modified multilayer‐shaped CeO2 electrode exhibited high stability and reproducibility. [ABSTRACT FROM AUTHOR]

Immunochromatographic assays (ICAs) are most frequently used for on-site rapid screening of clenbuterol. To improve sensitivity, a novel probe with bacteria as signal carriers was developed. Bacteria can load a great deal of gold nanoparticles (AuNPs) on their surface, meaning much fewer antibodies are needed to produce clearly visible results, although low concentrations of antibody could also trigger fierce competition between free analyte and the immobilized antigen. Thus, a limited number of antibodies was key to significantly improved sensitivity. Analytical conditions, including bacterial species, coupling method, and concentration, were optimized. The visual detection limit (VDL) for clenbuterol was 0.1 ng/mL, a 20-fold improvement in sensitivity compared with traditional strips. This work has opened up a new route for signal amplification and improved performance of ICAs. Furthermore, inactivated bacteria could also be environment-friendly and robust signal carriers for other biosensors. [ABSTRACT FROM AUTHOR]

A label-free microcantilever immunosensor based on a competitive immunoassay is reported for the determination of clenbuterol. The immunosensor was fabricated by modifying clenbuterol-ovalbumin on the gold surface of the microcantilever with crossing linkage by L-cysteine and glutaraldehyde. Atomic force microscopy was utilized to characterize the construction of immunosensor and to measure the deflection of the microcantilever. The deflection response of the microcantilever was in negatively proportional to the concentration of clenbuterol from 1.0 × 10-2 to 20 µg/L with a limit of detection of 1.0 × 10-2 µg/L. The fabricated immunosensor was used to determine clenbuterol in pork samples with satisfactory results. In addition, the results were in accordance with those obtained by high-performance liquid chromatography. The reported immunosensor displayed high sensitivity and specificity together with excellent repeatability and reliability. [ABSTRACT FROM AUTHOR]

Equine hair is becoming an increasingly popular biological matrix for doping control of horse sports; one of the reasons for this is the significantly longer detection window hair can offer. Hair analysis opens up the opportunity for longitudinal monitoring of drug exposure which would otherwise not be possible with the more traditional and common biological matrices, such as urine and blood. As such, there is a need for more multi-target screening methods covering a broad range of prohibited substances in equine hair at the required sensitivities for equine doping control. This paper describes a sensitive ultra-high performance liquid chromatography – tandem mass spectrometry (UHPLC–MS/MS) method for the detection of 121 drugs and/or their metabolites in equine hair covering ten classes of prohibited substances with estimated limits of detection between 0.1 and 10 pg/mg. To our knowledge, this is the first report of a screening method in equine hair which can cover such a broad range and well over one hundred prohibited substances in a single analytical run. This method has been validated for its specificity, precision and extraction recovery. Applicability of this method has been demonstrated by: (i) the successful identification of clenbuterol, 2-(1-hydroxyethyl) promazine sulfoxide, acepromazine and tetrahydrozoline in genuine equine mane samples; as well as (ii) the detection of drugs from artificially incurred mane hair samples which have been prepared by soaking blank hair samples in solutions of drug targets. [ABSTRACT FROM AUTHOR]

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5'-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33-97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules. [ABSTRACT FROM AUTHOR]

The use of clenbuterol (CLE) as a growth-promoting agent in livestock is banned in the USA, European Union, and Mexico. Nevertheless, different studies have reported the presence of this agent in meat products, potentially causing intoxication in consumers. In this study, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) is proposed for CLE determination in beef. A simple solid-phase extraction step was used as a cleanup sample method; the chromatographic separation was achieved with anAcquity UPLC HSS T3 column using a mobile-phase formic acid and methanol. We use a XEVO-TQS detector by positive-ion electrospray in the multiple reactions monitoring modes. The mass transitions monitored were m/z 277.08 → 202.95 for CLE and m/z 286.1 → 203.9 for clenbuterol-D9, used as an internal standard. The method was linear over the concentration range of 0.1-1.6 ng/g. Once validated, the method was applied to monitor CLE in beef samples collected at local markets of San Luis Potosí (central region of Mexico). Despite the ban on the use of CLE to grow cattle, we find levels of beta-agonist, with concentrations between 0.6 and 10.4 ng/g. Our method is ideal for routine analysis for quality control and food safety. [ABSTRACT FROM AUTHOR]

A number of performance-enhancing drugs (PEDs) are used illicitly to improve muscle strength by the bodybuilders. The misuse of these drugs is associated with serious adverse effects to different organs. A previously healthy 22-year-old male bodybuilder after taking stanozolol, clenbuterol, and triiodothyronine for 10 days presented to the hospital with symptoms of icteric sclera, progressive dyspnea, intermittent cough, and bloody sputum. He was diagnosed with dilated cardiomyopathy and acute hepatic injury. Rapidly progressive dilated cardiomyopathy and acute hepatic injury among bodybuilders in such a short period of time have not been reported. People using these drugs must monitor liver and cardiac functions regularly, and they should discontinue using PEDs after diagnosis of liver or cardiac abnormalities. Physicians should always consider the possibility of the PED abuse in the context of a young athlete suffering cardiomyopathy or hepatic injury. [ABSTRACT FROM AUTHOR]

Clenbuterol is a long-acting β-agonist that has several pharmacologic actions. In the past, it was misused mainly by bodybuilders and athletes to enhance their performances due to its lipolytic and muscle-enhancing effects. Today, its use has spread among recreation athletes as a weight loss agent. Since toxicity following clenbuterol is a rarely reported syndrome, we present a case of acute toxicity resulting in symptoms of sympathetic activation with haemodynamic instability and ECG signs of myocardial damage. A 34-years-old male, active local football player with no previous medical history, presented at our Emergency Unit with severe chest pain and shortness of breath. He was agitated, tachycardic and hypotensive, there were signs of myocardial damage on ECG. On admission, cardiac enzyme levels were normal [Troponin-T (TnT) 0.02 mcg/l] but started to rise soon afterwards (TnT 0.145 mcg/l), hypotension persisted and pain was worsening. The patient was managed with supportive care, he was given fluids, morphine, midazolam, sodium bicarbonate and vasopressor support. No invasive procedures were needed. He was discharged painless, with normal ECG and laboratory tests after 4 days. As abuse of weight loosing agents and anabolic substances is spreading rapidly, it should be always considered in young patients presenting with sympathomimetic features. [ABSTRACT FROM AUTHOR]

This study proposed the filtration method for removal of inhibitors from real urine samples for immunoassay without centrifuge. Although the inhibitors could not be removed by the physical filtration, the carboxyl group terminated silica effectively removed the inhibitors. In a low pH, antibody formed aggregation due to the protonation. We propose to adjust pH of the sample solution by adding a phosphate buffer solution (pH 7.5). As a result of pretreatment, the SPR immunosensing achieved the SPR signal of 45 mdeg and a low limit of detection with 100 ppq (100 fg mL −1 ). [ABSTRACT FROM AUTHOR]

Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken ( Gallus gallus domesticus ) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant β2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC 50 of the scFv towards the two β2-agonists were; IC 50 salbutamol = ∼0.310 μg/ml, IC 50 clenbuterol = ∼0.076 μg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 μg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 μg/ml currently implemented by WADA and may be of potential use in human doping urinalysis. [ABSTRACT FROM AUTHOR]

Abstract: A recent years’ increase in misusing levels of image‐ and performance‐ enhancing drugs (IPEDs) has been observed. Out of these drugs, beta‐2 agonists have recently emerged for their potential of misuse, especially for slimming and bodybuilding purposes. To this perspective, clenbuterol (‘the size zero pill’) has been reported as being both popular and widely available from the illegal market. All clenbuterol and salbutamol misuse/abuse/dependence/withdrawal/overdose/off‐label spontaneous reports (2006–2016) from the European Medicines Agency (EMA) EudraVigilance (EV) database were collected and analysed by age range, gender, concomitant therapies and source of information. From the EV database, 55 of a total number of 920 ‘suspect’ misuse/abuse/dependence/withdrawal/overdose/off‐label ADRs (e.g. 5.97%; corresponding to 25 of 138 individuals) and 1310 of 62,879 ADRs (e.g. 2.08%; corresponding to 474 of 6923 individuals) were, respectively, associated with clenbuterol (typically ingested in combination with a range of anabolic steroids) and salbutamol. Proportional reporting ratio (PRR) value for misuse/abuse ADRs was higher (PRR = 18.38) for clenbuterol in comparison with salbutamol. Clenbuterol misuse/abuse could be a cause for major concern, especially in vulnerable individuals. [ABSTRACT FROM AUTHOR]

Zilpaterol is a β-adrenergic agonist feed additive approved in the United States to increase weight gain and improve feed efficiency of cattle. A zilpaterol immunochromatographic assay was developed as an economical and user-friendly rapid detection method for zilpaterol and validated using urine and tissue samples derived from animal studies. The assay sensitivity was 1.7-23.2 ng g−1 or mL−1 across a variety of feed and animal matrices and did not cross-react with clenbuterol or ractopamine. No sample pre-treatment of cattle and sheep urine was needed, but horse urine and feed required dilution; skeletal muscle required solvent extraction prior to testing. Of 32 incurred sheep urine samples tested, zilpaterol content was correctly identified in all but 2 samples. Horse urine containing >10 ng mL−1 of incurred zilpaterol residue (n = 48) was correctly identified as zilpaterol positive. The assay correctly identified 0-day withdrawal sheep muscle samples as zilpaterol positive and the control and longer withdrawal day sheep muscle samples as negative. Zilpaterol was demonstrated to be stable in horse urine when stored at −20°C for 7 years. [ABSTRACT FROM AUTHOR]

A novel immunochromatographic assay (ICA) using colored silica nanoparticles (colored SiNPs) as optical directly-detected label was developed for point-of-care detection of clenbuterol (CLE). To our knowledge, this is the first report that using colored SiNPs as nanolabel to develop an ICA. CLE was selected as the model analyte for competitive ICA. The coloration of monodispersed silica nanoparticles (SiNPs) was preceded in water with the presence of aminosilanes and C.I Reactive Violet 5. The purple SiNPs were introduced into a pre-hydrolyzed Stöber system to regrow a silica shell, forming core-shell purple SiNPs. The silica shell prevented dye on SiNPs from leaking. Under appropriate conditions, the core-shell purple SiNPs based ICA was developed to screen for CLE detection by naked eyes at cut-off values of 3 ng/mL, 6 ng/mL and 5 ng/mL in phosphate buffered saline, urine and pork, respectively. The core-shell purple SiNPs based immunoassay system has no cross activity with other nonspecific structural analogues. The results showed that colored SiNPs are promising to be alternative visible labels for the ICA strip. [ABSTRACT FROM AUTHOR]

Previously, we highlighted induction of an integrated stress response (ISR) gene program in skeletal muscle of pigs treated with a beta-adrenergic agonist. Hence we tested the hypothesis that the ER-stress inhibitor, sodium 4-phenylbutyrate (PBA), would inhibit Clenbuterol-mediated muscle growth and reduce expression of genes that are known indicators of an ISR in mice. Clenbuterol (1mg/kg/day) administered to C57BL6/J mice for 21 days increased body weight (p<0.001), muscle weights (p<0.01), and muscle fibre diameters (p<0.05). Co-administration of PBA (100mg/kg/day) did not alter the Clenbuterol-mediated phenotype, nor did PBA alone have any effects compared to that of the vehicle treated mice. Clenbuterol increased skeletal muscle mRNA expression of phosphoserine amino transferase 1 (PSAT1, p<0.001) and cyclophillin A (p<0.01) at day 3, but not day 7. Clenbuterol decreased mRNA expression of activating transcription factor (ATF) 4 and ATF5 at day 3 (p<0.05) and day 7 (p<0.01), X-box binding protein 1 (XBP1) variant 2 mRNA at day 3 only (p<0.01) and DNA damage inducible transcript 3 (DDIT3/CHOP) mRNA at day 7 only (p<0.05). Co-administration of PBA had no effect on Clenbuterol-induced changes in skeletal muscle gene expression. In contrast, treatment of C2C12 myotubes with 5mM PBA (8hr) attenuated the thapsigargin-induced ISR gene program. Prolonged (24-48hr) treatment with PBA caused atrophy (p<0.01), reduced neoprotein synthesis (p<0.0001) and decreased expression of myogenin and fast myosin heavy chain genes (p<0.01), indicating an inhibition of myogenic differentiation. In summary, Clenbuterol did not induce an ISR gene program in mouse muscle. On the contrary, it reduced expression of a number of ISR genes, but it increased expression of PSAT1 mRNA. Co-administration of PBA had no effect on Clenbuterol-mediated muscle growth or gene expression in mice, whereas PBA did inhibit thapsigargin-induced ISR gene expression in cultured C2C12 cells and appeared to inhibit myogenic differentiation, independent of altering ISR gene expression. [ABSTRACT FROM AUTHOR]

Background and Aim: Clenbuterol as a β2-agonist drug was investigated according to the concentration of the drug available in the bodies of goats and according to the level of sensitivity of the instruments used for detection. The objective of the current study was to determine withdrawal times after giving a therapeutic dose that resulted in safe slaughters. Materials and Methods: Five healthy male goats with a mean body weight of 20.64 kg were treated with a single dose of 5.10-3 mg/kg in the BW onto jugular vein. Whole blood samples of approximately 5 mL were taken in a time series at 5, 30, 60, 90, 150, 210, 270, 390, 510, 630, and 750 min. At 24 h posttreatment, all subjects were sacrificed, and 300 g samples of the liver were obtained. The plasma concentration and liver residue of the drug were observed by reverse-phase high- performance liquid chromatography. Results: The drug reached a maximum concentration of 19.233±0.331 µg/mL at 5 min, and the elimination half-life was at 173.25 min. The limit detection was obtained at 0.053 µg/mL. A one-way analysis of variance between all goats showed that elimination of the clenbuterol in their bodies was similar (p=1.00), with a withdrawal time of 1,479.326 min and no residues in the liver (p<0.05). Conclusion: Safe times for slaughter were determined to be at 2 days, 13 h, and 12 min as the 2nd safety factor (SF) time and 3 days, 1 h, and 58 min as the 3rd SF time with the liver organ free from residue. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In this study, a paper-based microfluidic enzyme-linked immunosorbent assay (ELISA) was developed as a screening system for rapid detection of clenbuterol, which is illegally used as a growth promoter for food-producing animals. The microfluidic paper-based analytical device (μPAD) was combined with ELISA and the intrinsic properties of paper allowed the entrapment of antibody through cellulosic fibres, validating to be an alternative to 96-well ELISA microplate for food safety monitoring. Detection of clenbuterol in milk was achieved by measuring the intensity of colour change that was proportional to the analyte concentration with a detection limit of 0.2 ppb. The μPAD effectively reduces the cost, volume of reagents, and time required to run ELISA for food sample testing. [ABSTRACT FROM AUTHOR]

A simple, efficient and sensitive molecularly imprinted electrochemiluminescence sensor (MIECLS) based on reduced graphene oxide (rGO) and upconversion nanoparticles (UCNPs) was developed for determination of clenbuterol (CLB). In this study, rGO generated by electrodeposition of graphene oxide not only acted as carrier for immobilizing UCNPs, but also had a significant impact in boosting electrochemiluminescence (ECL) response of UCNPs thanks to its high conductivity, superior electron transport rate and large specific surface area. UCNPs as an advanced ECL emitter possessed wonderful ECL performance. Furthermore, the introduction of molecularly imprinted polymers (MIP) endowed the ECL sensor a new character of specifically identifying analyte CLB. Under the optimal experimental conditions, the ECL signal was proportional to the logarithm of CLB concentration in the range of 10 nM to 100 µM with a low detection limit of 6.3 nM. The proposed MIECLS combining the advantages of UCNPs-ECL and MIP exhibited good sensitivity, desirable selectivity and favorable stability, indicating enormous potential in the future of food safety detection. [ABSTRACT FROM AUTHOR]

Objectives: Clenbuterol is a brain penetrant β2-adrenoceptor agonist with anti-inflammatory and putative neuroprotective properties. In the present investigation, the effect of clenbuterol was assessed in a rat model of acute brain injury induced by intra-striatal administration of the pro-inflammatory cytokine IL-1β. Methods: Clenbuterol (0.5 mg/kg; i.p.) was administered one hour prior to stereotactically delivered IL-1β (100 ng) into the striatum. Four hours postinjection, rats were anesthetized, blood samples were collected for circulating cytokine and chemokine analysis, and the ipsilateral striatum and liver tissue were harvested for mRNA expression analysis of target genes. Results: Intrastriatal IL-1β provoked an inflammatory response with increased expression of IL-1β and the pro-inflammatory cytokine TNF-α. TNF-α expression was also increased in the liver and circulating concentrations of the chemokine cytokine-induced neutrophil chemoattractant 1 (CINC-1) were raised in response to intrastriatal IL-1β administration. The striatal response was accompanied by NFκB activation and 24 hours postinjection, increased immunoreactivity of the neutrophil marker MBS-2, indicative of cell infiltration and increased TUNEL staining, a cell marker of apoptosis. Treatment with clenbuterol attenuated all IL-1β-induced changes in the striatum including MBS-2 immunoreactivity and TUNEL + staining. Clenbuterol also attenuated IL-1β-induced expression of TNF-α in the liver and the increase in circulating CINC-1 concentrations. Conclusions: The results provide evidence that clenbuterol elicits anti-inflammatory effects, suppresses the peripheral acute phase response and reduces the infiltration of neutrophils and apoptotic response to acute IL-1β-induced brain injury. Suppression of both the central and peripheral response following clenbuterol administration may contribute to its protective properties following brain injury. [ABSTRACT FROM AUTHOR]

Analytical chemistry represents a central aspect of doping controls. Routine sports drug testing approaches are primarily designed to address the question whether a prohibited substance is present in a doping control sample and whether prohibited methods (for example, blood transfusion or sample manipulation) have been conducted by an athlete. As some athletes have availed themselves of the substantial breadth of research and development in the pharmaceutical arena, proactive and preventive measures are required such as the early implementation of new drug candidates and corresponding metabolites into routine doping control assays, even though these drug candidates are to date not approved for human use. Beyond this, analytical data are also cornerstones of investigations into atypical or adverse analytical findings, where the overall picture provides ample reason for follow-up studies. Such studies have been of most diverse nature, and tailored approaches have been required to probe hypotheses and scenarios reported by the involved parties concerning the plausibility and consistency of statements and (analytical) facts. In order to outline the variety of challenges that doping control laboratories are facing besides providing optimal detection capabilities and analytical comprehensiveness, selected case vignettes involving the follow-up of unconventional adverse analytical findings, urine sample manipulation, drug/food contamination issues, and unexpected biotransformation reactions are thematized. [ABSTRACT FROM AUTHOR]

The misuse of β-agonists leads to a potential risk to public health and is forbidden in many countries. We developed a rapid, sensitive and reliable multi-residue detection method for zilpaterol, ractopamine, and clenbuterol in bovine tissues by liquid chromatography-tandem mass spectrometry. Residues were extracted in ethyl acetate after protein precipitation, and then analyzed by the developed method. Good linearities (R² > 0.99) were observed, and the recoveries of zilpaterol, ractopamine, and clenbuterol were 99%, 74%, and 102%, respectively. The limits of quantitation for zilpaterol, ractopamine, and clenbuterol were 1.3, 5.0, and 1.7 ng/g, respectively. The method is also applied successfully to bovine tissues within the Korean National Residue Programme. None of the 3 β-agonists were detected from 50 domestic samples. However, zilpaterol (6.3 ng/g) was quantified in one out of the 50 imported samples. The application of this method will be helpful in quality control analysis of β-agonists residues in bovine tissues. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Clenbuterol (CLB) is a notorious “lean meat essence”, which has harmful effects on human health. In this study, the binding of CLB to two proteins, human serum albumin (HSA) and the human fat mass and obesity associated protein (FTO), have been investigated by spectroscopic and molecular modeling methods. Fluorescence data revealed that CLB could bind to HSA (or FTO) to form a complex. Thermodynamic analysis and molecular modeling indicated that the binding process was a spontaneous molecular interaction procedure, in which hydrophobic interaction played a major role. Results indicated that CLB could lead to some microenvironmental changes of both proteins. [ABSTRACT FROM AUTHOR]

5-Hydroxytryptamine2A (5-HT2A) receptors are enriched in layers I and Va of the rat prefrontal cortex and neocortex and their activation increases the frequency of glutamatergic excitatory post-synaptic potentials/currents (EPSP/Cs) onto layer V pyramidal cells. A number of other G-protein coupled receptors (GPCRs) are also enriched in cortical layers I and Va and either induce (α1-adrenergic and orexin2) or suppress (metabotropic glutamate2 [mGlu2], adenosine A1, μ-opioid) both 5-HT-induced EPSCs and head twitches or head shakes induced by the phenethylamine hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI). Another neurotransmitter receptor also localized to apparent thalamocortical afferents to layers I and Va of the rat prefrontal cortex and neocortex is the β2-adrenergic receptor. Therefore, we conducted preliminary electrophysiological experiments with rat brain slices examining the effects of epinephrine on electrically-evoked EPSPs following bath application of DOI (3 μM). Epinephrine (0.3-10 μM) suppressed the late EPSPs produced by electrical stimulation and DOI. The selective β2-adrenergic receptor antagonist ICI-118,551 (300 nM) resulted in a rightward shift of the epinephrine concentration-response relationship. We also tested the selective β2-adrenergic receptor agonist clenbuterol and the antagonist ICI-118,551 on DOI-induced head twitches. Clenbuterol (0.3-3 mg/kg, i.p.) suppressed DOI (1.25 mg/kg, i.p.)-induced head twitches. This clenbuterol effect appeared to be at least partially reversed by the selective β2-adrenergic receptor antagonist ICI-118,553 (0.01-1 mg/kg, i.p.), with significant reversal at doses of 0.1 and 1 mg/kg. Thus, β2-adrenergic receptor activation reverses the effects of phenethylamine hallucinogens in the rat prefrontal cortex. While Gi/Go-coupled GPCRs have previously been shown to suppress both the electrophysiological and behavioral effects of 5-HT2A receptor activation in the mPFC, the present work appears to extend this suppressant action to a Gs-coupled GPCR. Furthermore, the modulation of 5-HT2A receptor activation-induced glutamate release onto mPFC layer V pyramidal neurons apical dendrites by a range GPCRs in rat brain slices appears to results in behaviorally salient effects of relevance when screening for novel CNS therapeutic drugs. [ABSTRACT FROM AUTHOR]

A fully automated spectrophotometric method based on flow-batch analysis has been developed for the determination of clenbuterol including an on-line solid phase extraction using a molecularly imprinted polymer (MIP) as the sorbent. The molecularly imprinted solid phase extraction (MISPE) procedure allowed analyte extraction from complex matrices at low concentration levels and with high selectivity towards the analyte. The MISPE procedure was performed using a commercial MIP cartridge that was introduced into a guard column holder and integrated in the analyzer system. Optimized parameters included the volume of the sample, the type and volume of the conditioning and washing solutions, and the type and volume of the eluent. Quantification of clenbuterol was carried out by spectrophotometry after in-system post-elution analyte derivatization based on azo-coupling using N- (1-Naphthyl) ethylenediamine as the coupling agent to yield a red-colored compound with maximum absorbance at 500 nm. Both the chromogenic reaction and spectrophotometric detection were performed in a lab-made flow-batch mixing chamber that replaced the cuvette holder of the spectrophotometer. The calibration curve was linear in the 0.075–0.500 mg L −1 range with a correlation coefficient of 0.998. The precision of the proposed method was evaluated in terms of the relative standard deviation obtaining 1.1% and 3.0% for intra-day precision and inter-day precision, respectively. The detection limit was 0.021 mg L −1 and the sample throughput for the entire process was 3.4 h −1 . The proposed method was applied for the determination of CLB in human urine and milk substitute samples obtaining recoveries values within a range of 94.0–100.0%. [ABSTRACT FROM AUTHOR]

Beta‐adrenoceptors (β2‐ARs) have beneficial effects on prefrontal cortex (PFC) working memory, however, the cellular and molecular mechanisms are unclear yet. In this study, we probed the effect of β2‐AR‐selective agonist clenbuterol (Clen) on synaptic transmission in layer 5/6 pyramidal neurons of PFC. Bath application of Clen reduced spontaneous IPSC (sIPSC) frequency without effects on sEPSCs. Clen did not alter the frequency and amplitude of miniature IPSCs (mIPSCs), but exerted heterogeneous effects on evoked IPSCs (eIPSCs) recorded from PFC layer 5/6 pyramidal neurons. Clen decreased the firing rate of action potentials of fast‐spiking GABAergic interneurons. Clen‐induced hyperpolarization of fast‐spiking GABAergic interneurons required potentiation of an inward rectifier K+ channels. Clen‐induced hyperpolarization of fast‐spiking interneurons was dependent on Gs protein rather than cAMP and protein kinase A. Our findings demonstrate that Clen (10 μM) enhances inward rectifier K+ channels via Gs protein to cause membrane hyperpolarization of fast‐spiking GABAergic interneurons resulting in reduction of action potentials firing rate to reduce GABAergic transmission. [ABSTRACT FROM AUTHOR]