Evaluation of the anti-nociceptive profile of essential oil from Melissa officinalis L. (lemon balm) in acute and chronic pain models.

ETHNOPHARMACOLOGICAL RELEVANCE: Melissa officinalis L. (Lamiaceae) is a medicinal plant native to Mediterranean regions and found in other parts of the world. Extracts and essential oil from this widely cultivated culinary medicinal herb are used in traditional medicine to manage a variety of disorders that include epilepsy and pain. AIM OF THE STUDY: To assess the anti-nociceptive potentials of Melissa officinalis essential oil (MO) and probe the involvement of adrenergic, opioidergic, serotonergic and potassium adenosine triphosphate (KATP) mechanisms in its anti-nociceptive effects. MATERIAL AND METHODS: We employed formalin-, acetic acid and hot plate-induced nociception to study the acute anti-nociceptive effects of MO. The sciatic nerve injury (CCI) model of neuropathic pain was utilized to study the anti-nociceptive effects of MO on chronic pain. Effects of MO on anxiety, cognitive deficits, oxidative stress and inflammation in the CCI rats were evaluated on elevated plus maze, open field test, novel object recognition, oxidative stress parameters and pro-inflammatory cytokines, respectively. The possible mechanism(s) of MO's anti-nociceptive effects were elucidated using prazosin, yohimbine, propranolol, glibenclimide, naloxone and metergoline, which are acknowledged antagonists for α1-, α2- and ß-adrenergic, potassium adenosine triphosphate (KATP), opioidergic and serotonergic systems, respectively. RESULTS: MO significantly attenuated acetic acid- and formalin-induced nociception; prolonged the mean reaction time of rats on hot plate before and following sciatic nerve chronic injury (CCI). MO ameliorated anxiety, cognitive deficits and oxidative stress, reduced pro-inflammatory cytokine levels and produced a near total restoration of injured sciatic nerves in CCI rats. Naloxone, metergoline and glibenclimide significantly blocked, while prazosin, yohimbine and popranolol failed to block the anti-nociceptive effects of MO in formalin-induced nociception. CONCLUSIONS: MO contains biologically active compounds with potential anti-nociceptive properties that modulate KATP, opioidergic and serotonergic pathways. These support the development of bioactive compounds from MO as anti-nociceptive agents.

Metergoline Shares Properties with Atypical Antipsychotic Drugs Identified by Gene Expression Signature Screen.

Novel approaches are required to find new treatments for schizophrenia and other neuropsychiatric disorders. This study utilised a combination of in vitro transcriptomics and in silico analysis with the BROAD Institute's Connectivity Map to identify drugs that can be repurposed to treat psychiatric disorders. Human neuronal (NT2-N) cells were treated with a combination of atypical antipsychotic drugs commonly used to treat psychiatric disorders (such as schizophrenia, bipolar disorder, and major depressive disorder), and differential gene expression was analysed. Biological pathways with an increased gene expression included circadian rhythm and vascular endothelial growth factor signalling, while the adherens junction and cell cycle pathways were transcriptionally downregulated. The Connectivity Map (CMap) analysis screen highlighted drugs that affect global gene expression in a similar manner to these psychiatric disorder treatments, including several other antipsychotic drugs, confirming the utility of this approach. The CMap screen specifically identified metergoline, an ergot alkaloid currently used to treat seasonal affective disorder, as a drug of interest. In mice, metergoline dose-dependently reduced MK-801- or methamphetamine-induced locomotor hyperactivity confirming the potential of metergoline to treat positive symptoms of schizophrenia in an animal model. Metergoline had no effects on prepulse inhibition deficits induced by MK-801 or methamphetamine. Taken together, metergoline appears a promising drug for further studies to be repurposed as a treatment for schizophrenia and possibly other psychiatric disorders.

Serotonin 5-HT7 receptor is a biomarker poor prognostic factor and induces proliferation of triple-negative breast cancer cells through FOXM1.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer and associated with poor prognosis and shorter survival due to significant genetic heterogeneity, drug resistance and lack of effective targeted therapeutics. Therefore, novel molecular targets and therapeutic strategies are needed to improve patient survival. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce growth stimulatory effects in breast cancer. However, the molecular mechanisms by which 5-HT exerts its oncogenic effects in TNBC still are not well understood. METHODS: Normal breast epithelium (MCF10A) and two TNBC cells (MDA-MB-231, BT-546) and MCF-7 cells (ER +) were used to investigate effects of 5-HT7 receptor. Small interfering RNA (siRNA)-based knockdown and metergoline (5-HT7 antagonist) were used to inhibit the activity of 5-HT7. Cell proliferation and colony formation were evaluated using MTS cell viability and colony formation assays, respectively. Western blotting was used to investigate 5-HT7, FOXM1 and its downstream targets protein expressions. RESULTS: We demonstrated that 5-HT induces cell proliferation of TNBC cells and expression of 5-HT7 receptor and FOXM1 oncogenic transcription factor. We found that expression of 5-HT7 receptor is up-regulated in TNBC cells and higher 5-HT7 receptor expression is associated with poor patient prognosis and shorter patient survival. Genetic and pharmacological inhibition of 5-HT7 receptor by siRNA and metergoline, respectively, suppressed TNBC cell proliferation and FOXM1 and its downstream mediators, including eEF2-Kinase (eEF2K) and cyclin-D1. CONCLUSION: Our findings suggest for the first time that the 5-HT7 receptor promotes FOXM1, eEF2K and cyclin D1 signaling to support TNBC cell proliferation; thus, inhibition of 5-HT7 receptor/FOXM1 signaling may be used as a potential therapeutic strategy for targeting TNBC. 5-HT induces cell proliferation of TNBC cells through 5-HT7 receptor signaling. Also, genetic and pharmacological inhibition of 5-HT7 by RNAi (siRNA) and metergoline HTR7 antagonist, respectively inhibits FOXM1 oncogenic transcription factor and suppresses TNBC cell proliferation.

Conditional approach as cooperation in predator inspection: A role for serotonin?

In guppies (Poecilia reticulata), a small number of individuals break away from a shoal and approach a potential predator, a behavior termed "predator inspection". These animals often employ a "conditional approach" strategy, in which an individual approaches the predator in the first move and subsequently approaches it only if a second individual swims even with it during inspection. This strategy is analogous to the "tit-for-tat" strategy of the Prisoner's Dilemma, suggesting that it could be used to study cooperation. Serotonin is thought to mediate cooperative behavior in other fish species. Exposure to the animated image of a predator in a tank that contained a parallel mirror - mimicking an equally cooperating conspecific - promoted inspection and decreased refuge use, but increased freezing, suggesting that conditional approach is also associated with fear. To understand whether serotonin participates in conditional approach in guppies, we treated animals with either vehicle (Cortland's salt solution), fluoxetine (2.5 mg/kg) or metergoline (1 mg/kg), and tested then in a predator inspection paradigm. Fluoxetine increased the time the animal spent inspecting the predator image, while metergoline decreased it. Fluoxetine also decreased time spent avoiding the predator and increased freezing, while metergoline decreased freezing. These results suggest that phasic increases in serotonin levels promote conditional approach, suggesting a role for this neurotransmitter in cooperation. Preprint: https://doi.org/10.1101/436345; Data and scripts: https://github.com/lanec-unifesspa/TFT.

A macrophage-based screen identifies antibacterial compounds selective for intracellular Salmonella Typhimurium.

Salmonella Typhimurium (S. Tm) establishes systemic infection in susceptible hosts by evading the innate immune response and replicating within host phagocytes. Here, we sought to identify inhibitors of intracellular S. Tm replication by conducting parallel chemical screens against S. Tm growing in macrophage-mimicking media and within macrophages. We identify several compounds that inhibit Salmonella growth in the intracellular environment and in acidic, ion-limited media. We report on the antimicrobial activity of the psychoactive drug metergoline, which is specific against intracellular S. Tm. Screening an S. Tm deletion library in the presence of metergoline reveals hypersensitization of outer membrane mutants to metergoline activity. Metergoline disrupts the proton motive force at the bacterial cytoplasmic membrane and extends animal survival during a systemic S. Tm infection. This work highlights the predictive nature of intracellular screens for in vivo efficacy, and identifies metergoline as a novel antimicrobial active against Salmonella.

Investigation of Antidepressant, Anxiolytic and Sedative Activities of the Aqueous Leaf Extract of Musa sapientum Linn. (Banana; Musaceae).

BACKGROUND: Musa sapientum Linn. (Musaceae) is used in traditional African medicine in the management of mental disorders. This study was conducted to evaluate the central nervous system activities of the aqueous leaf extract of M. sapientum (MS). MATERIALS AND METHODS: MS (50, 100 and 200 mg/kg, p.o.) was administered to separate groups of mice 1 h before behavioural studies. The antidepressant effect was studied using the forced swimming test (FST) and tail suspension test (TST) while the elevated plus maze (EPM) and the hole-board tests were used to evaluate the anxiolytic effect. The probable mechanism of antidepressant-like effect was also investigated. RESULTS: MS (50, 100 and 200 mg/kg) produced significant (P<0.0001) reduction in the duration of immobility with peak effect at 200 mg/kg (79.6%) in FST and 66.9 % in TST respectively when compared with control. The pre-treatment of mice with prazosin (α1-adrenoceptor antagonist, 62.5 µg/kg, i.p.) and sulpiride (dopamine D2 receptor antagonist, 50 mg/kg, i.p.) significantly prevented the antidepressant effect produced by MS in FST. However, pre-treatment of mice with metergoline (5-HT2 receptor antagonist, 4 mg/kg, i.p.) and yohimbine (α2-adrenoceptor antagonist, 1 mg/kg, i.p.) did not prevent the antidepressant effect of MS. In the EPM test, MS did not significantly increase open arm exploration. It also did not significantly increase the number of head dips in the hole-board test. CONCLUSIONS: Results showed that MS had antidepressant activity possibly mediated through α1-adrenergic and D2 dopaminergic receptors, without significant anxiolytic effect.

Molecular basis involved in the blocking effect of antidepressant metergoline on C-type inactivation of Kv1.4 channel.

Voltage-gated potassium channels (VGKCs) are transmembrane ion channels specific for potassium. Currently there are nine kinds of VGKCs. Kv1.4 is one of shaker-related potassium channels. It is a representative alpha subunit of potassium channels that can inactivate A type-currents, leading to N pattern inactivation. Inactivation of Kv channels plays an important role in shaping electrical signaling properties of neuronal and muscular cells. The shape of N pattern inactivation can be modified by removing the N-terminal (NT) domain which results in non-inactivated currents and C pattern inactivation. In a previous work, we have reported the regulatory effect of metergoline on Kv1.4 and Nav1.2 channel activity. In the present study, we constructed a mutant of deleted 61 residues from NT of Kv1.4 channels (Kv1.4 Δ2-61) and found that it induced an outward peak and steady-state currents We also studied the modulation effect of metergoline on the activity of this Kv1.4 Δ2-61 mutant channel without having the N-terminal quick inactivation domain. Our results revealed that treatment with metergoline inhibited NT deleted Kv1.4 mutant channel activity in a concentration-dependent manner which was reversible. Interestingly, metergoline treatment induced little effects on the outward peak current in the deleted Kv1.4 mutant channel. However, metergoline treatment conspicuously inhibited steady state currents of Kv1.4 Δ2-61 channels with acceleration current mode. The acceleration of steady-state current of deleted Kv1.4 mutant channel occurred in a concentration-dependent manner. This means that metergoline can accelerate C pattern inactivation of Kv1.4 Δ2-61 channel by acting as an open state dependent channel blocker. We also performed site-directed mutations in V561A and K532Y, also known as C-type inactivation sites. V561A, K532Y, and V561A + K532Y substitution mutants significantly attenuated the acceleration effect of metergoline on C pattern inactivation of hKv1.4 channel currents. In docking modeling study, predicted binding residues for metergoline were analyzed for six amino acids. Among them, the K532 residue known as the C-type inactivation site was analyzed to be a major site of action. Then various mutants were constructed. K532 substitution mutant significantly abolished the effect of metergoline on Kv1.4 currents among various mutants whereas other changes had slight inhibitory effects. Furthermore, we found that metergoline had specificity for Kv1.4, but not for Kv1.5 currents. In addition, the A type current in rat neuronal cell was inhibited and accelerated of inactivation. This result further shows that metergoline might interact with Lys532 residue and then accelerate C pattern inactivation of Kv1.4 channels with channel type specificity. Taken together, these results demonstrate the molecular basis involved in the effect of metergoline, an ergot alkaloid, on human Kv1.4 channel, providing a novel interaction ligand.

Cortical Circuit Activity Evokes Rapid Astrocyte Calcium Signals on a Similar Timescale to Neurons.

Sensory stimulation evokes intracellular calcium signals in astrocytes; however, the timing of these signals is disputed. Here, we used novel combinations of genetically encoded calcium indicators for concurrent two-photon imaging of cortical astrocytes and neurons in awake mice during whisker deflection. We identified calcium responses in both astrocyte processes and endfeet that rapidly followed neuronal events (∼120 ms after). These fast astrocyte responses were largely independent of IP3R2-mediated signaling and known neuromodulator activity (acetylcholine, serotonin, and norepinephrine), suggesting that they are evoked by local synaptic activity. The existence of such rapid signals implies that astrocytes are fast enough to play a role in synaptic modulation and neurovascular coupling. VIDEO ABSTRACT.

Molecularly imprinted polymers immobilized on 3D printed scaffolds as novel solid phase extraction sorbent for metergoline.

In the present work, a novel solid phase extraction (SPE) sorbent was developed based on molecularly imprinted polymers (MIPs) immobilized on 3D-printed scaffolds using polymer networks as MIP-immobilizing layer. MIPs were produced by precipitation polymerization in acetonitrile (ACN) using methacrylic acid (MAA) as functional monomer, trimethylolpropane trimethacrylate (TRIM) as crosslinker and metergoline as model template which allows final recognition of ergot alkaloid mycotoxins. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) analyses showed an average MIP particle size of 457 ± 145 nm. Functional MIP analysis revealed dissociation constants (KD) of 0.29 and 38.90 µM for high and low affinity binding sites respectively. Subsequently, crosslinking of polymer network building blocks was applied as MIP immobilization method on poly-ε-caprolactone (PCL) which was selected as polymer model. Methodology optimization and subsequent evaluation were first realized on 2D PCL surfaces. Based on analyses such as optical evaluation of MIP availability after immobilization through SEM and depth profilometry, an optimal polymer network building block concentration of 7.5 w/w% was selected. In a final part, transfer of MIP immobilization to 3D PCL scaffolds was successfully realized. Functional analysis showed that the newly developed SPE sorbents were able to rebind 44.87 ± 8.30% of a 1 µM metergoline solution. In conclusion, a new type of SPE sorbent was developed for the detection of metergoline by the use of MIP-functionalized polymer scaffolds. The applied technology opens up future possibilities for the extraction of a broad range of components such as other mycotoxins.

Serotonergic and dopaminergic systems are implicated in antidepressant-like effects of chotosan, a Kampo formula, in mice.

We previously demonstrated that chotosan (CTS), a traditional herbal formula called Kampo medicine, improves diabetes-induced cognitive deficits. In the present study, we investigated the antidepressant-like effects of CTS in mice. The administration of CTS (1.0 g/kg, for 3 days) decreased the immobility time in the forced-swim test, and this decrease was prevented by the prior administration of sulpiride (an antagonist of D2/3 receptors) and WAY100635 (an antagonist of 5-HT1A receptors). None of the treatments tested altered the locomotor activity of mice. These results suggest that CTS exerts antidepressant-like effects through changes in the serotonergic and dopaminergic systems.

Potentials of Mangifera indica in the treatment of depressive-anxiety disorders: possible mechanisms of action.

BACKGROUND: Mangifera indica (Anacardiaceae) is an important herb in the traditional African and Ayurvedic medicines. The stem barks are used in the treatment of hypertension, insomnia, tumour, depression, rheumatism and as a tonic. This study was carried out to investigate antidepressant- and anxiolytic-like effect of the hydroethanol stem bark extract of M. indica (HeMI) in mice. METHODS: HeMI (12.5-100 mg/kg, p.o.) was administered 1 h before subjecting the animal to the forced swim test (FST), tail suspension test (TST) and elevated plus maze tests (EPM). RESULTS: HeMI (12.5-100 mg/kg, p.o.) treatment produced significant reduction in immobility time [F(6.56)=8.35, p<0.001], [F(6,56)=7.55, p<0.001] in the FST and TST, respectively. Moreover, co-administration of sub-therapeutic doses of imipramine or fluoxetine with HeMI (3.125 mg/kg) elicited significant reduction in time spent immobile in the FST. However, pretreatment of mice with parachlorophenylalanine, metergoline, yohimbine or sulpiride abolished the antidepressant-like effect elicited by HeMI. In the EPM, HeMI produced significant [F(5,42)=8.91, p<0.001] increase in open arms exploration by 75.55 % and this effect was blocked by pretreatment of mice with flumazenil or metergoline. CONCLUSIONS: Findings from this study showed antidepressant-like effect of M. indica through interaction with 5-HT2 receptor, α2-adrenoceptor and dopamine D2-receptors. Also, an anxiolytic-like effect through its affinity for 5-HT2 and benzodiazepine receptors. Hence, M. indica could be a potential phytotherapeutic agent in the treatment of mixed anxiety-depressive illness.

Regulation of Human Kv1.4 Channel Activity by the Antidepressant Metergoline.

Metergoline is an ergot-derived psychoactive drug that is a ligand for various serotonin and dopamine receptors. Little is known about the effect of metergoline on different types of receptors and ion channels. Potassium channels are the most diverse group of ion channels. Kv1.4, a shaker family K channel alpha subunit, is one of a family of voltage gated K channels that mediates transient and rapid inactivating A-type currents and N-type inactivation. We demonstrated previously that metergoline inhibited the activity of neuronal voltage-dependent Na(+) channels in Xenopus laevis oocytes (Acta Pharmacol. Sin., 35, 2014, Lee et al.). In this study, we sought to elucidate the regulatory effects underlying metergoline-induced human Kv1.4 channel inhibition. We used the two electrode voltage-clamp (TEVC) technique to investigate the effect of metergoline on human Kv1.4 channel currents in Xenopus laevis oocytes expressing human Kv1.4 alpha subunits. Interestingly, metergoline treatment also induced inhibition of peak currents in human Kv1.4 channels in a concentration-dependent manner. The IC50 of peak currents of hKv1.4 currents was 3.6±0.6 µM. These results indicate that metergoline might regulate the human Kv1.4 channel activity that is expressed in X. laevis oocytes. Further, this regulation of potassium currents by metergoline might be one of the pharmacological actions of metergoline-mediated psychoactivity.

Development of a screening method to identify regulators of MICA shedding.

Immune cells, such as natural killer (NK) cells, recognize virally infected and transformed cells, and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells. Shedding of NKG2D ligands is thought to be a type of counter-mechanism employed by pathogenic cells to evade from NKG2D-mediated immune surveillance. MHC class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. We previously reported that, in soluble form, MICA expression levels are significantly associated with hepatitis virus-induced hepatocellular carcinoma. Here, we report a MICA shedding assay that utilizes membrane-bound MICA tagged at its N-terminus with a nano-luciferase reporter to quantify MICA shedding into culture media. Using this method, we screened a compound library and identified putative regulators of MICA shedding that have the potential to enhance the immune reaction by simultaneously increasing cell surface MICA levels and decreasing soluble MICA levels. This shedding assay may be useful for screening regulators of cell surface molecule shedding.

Affinity sensor based on immobilized molecular imprinted synthetic recognition elements.

An affinity sensor based on capacitive transduction was developed to detect a model compound, metergoline, in a continuous flow system. This system simulates the monitoring of low-molecular weight organic compounds in natural flowing waters, i.e. rivers and streams. During operation in such scenarios, control of the experimental parameters is not possible, which poses a true analytical challenge. A two-step approach was used to produce a sensor for metergoline. Submicron spherical molecularly imprinted polymers, used as recognition elements, were obtained through emulsion polymerization and subsequently coupled to the sensor surface by electropolymerization. This way, a robust and reusable sensor was obtained that regenerated spontaneously under the natural conditions in a river. Small organic compounds could be analyzed in water without manipulating the binding or regeneration conditions, thereby offering a viable tool for on-site application.

Antidepressant and anxiolytic effects of the methanol root extract of Capparis thonningii: involvement of monoaminergic, cholinergic and GABAergic systems.

BACKGROUND: Capparis thonningii Schum. (Capparaceae) is used in traditional African Medicine for the treatment of mood disorders. OBJECTIVE: The study investigates antidepressant and anxiolytic activities of methanol root extract of C. thonningii (CT). METHODS: CT (25-100 mg/kg, p. o.) was administered 1 h before behavioral studies were carried out in mice. Antidepressant effect was investigated using the forced swimming test (FST) and tail suspension test (TST). The anxiolytic effect was evaluated using the elevated-plus maze test (EPM), hole-board test (HBT), and light-dark test. RESULTS: CT (25 and 50 mg/kg) increased swimming activity (P<0.05) by 92.73% and attenuated immobility time by 35.72%, similar to anti-immobility effect of imipramine (33.87%) in FST. In addition, CT (50 mg/kg) significantly (P<0.01) reduced immobility time by 30.24% in TST. -However, the antidepressant-like effect elicited by CT was reversed by metergoline, cyproheptadine, and sulpiride (40.81, 45.93, and 48.52%, respectively) pretreatment but prazosin, and yohimbine failed to reverse this antidepressant-like effect. Similar to diazepam, CT (25 mg/kg) increased duration of open arms exploration (P<0.05) by 43.73% in EPM, number of head-dips (HBT) (90.32%), and time spent in the light compartment by 45.77% in light/dark test indicating anxiolytic-like effect. The anxiolytic-like effect of CT was reversed by flumazenil pretreatment. CONCLUSION: The findings from this study suggest antidepressant-like effect of C. thonningii involving interaction with serotonergic (5-HT2), dopaminergic (D2), noradrenergic (α1 and α2), and muscarinic cholinergic systems; and anxiolytic effect through an interaction with GABAA benzodiazepine receptor.

Metergoline inhibits the neuronal Nav1.2 voltage-dependent Na(+) channels expressed in Xenopus oocytes.

AIM: Metergoline is an ergot-derived psychoactive drug that acts as a ligand for serotonin and dopamine receptors. The aim of this study was to investigate the regulatory effects of metergoline on the neuronal Nav1.2 voltage-dependent Na(+) channels in vitro. METHODS: Xenopus oocytes were injected with cRNAs encoding rat brain Nav1.2 α and ß1 subunits. Voltage-activated Na(+) currents were recorded using two-electrode voltage clamp technique. Drugs were applied though perfusion. RESULTS: Both metergoline and lidocaine reversibly and concentration-dependently inhibited the peak of Na(+) currents with IC50 values of 3.6 ± 4.2 and 916.9 ± 98.8 µmol/L, respectively. Metergoline (3 µmol/L) caused a 6.8 ± 1.2 mV depolarizing shift of the steady-state activation curve of the Na(+) currents, and did not alter the inactivation curve. In contrast, lidocaine (3 µmol/L) caused a 12.7 ± 1.2 mV hyperpolarizing shift of the inactivation curve of the Na(+) currents without changing the steady-state activation curve. Both metergoline and lidocaine produced tonic and use-dependent inhibition on the peak of Na(+) currents. CONCLUSION: Metergoline exerts potent inhibition on the activity of neuronal Nav1.2 channels, which may contribute to its actions on the central nervous system.

Use of potentiometric detection in (ultra) high performance liquid chromatography and modelling with adsorption/desorption binding kinetics.

Observation of a potentiometric sensor's response behaviour after injection in flow injection analysis at different concentrations allowed studying "on" and "off" kinetics of the analyte's adsorption/diffusion behaviour. The alkaloid metergoline was mostly used as an example. k(on) and k(off) rate constant values were measured, and the association constant K(ass), and ΔG values of the analyte-surface interaction were calculated with an adsorption-based model which proved to be fully applicable. k(on) increased by decreasing the sensor dimensions, while koff was unaffected by miniaturization. Increasing acetonitrile concentrations in the running buffer increased k(off), while k(on) was unaffected. The experimentally determined ΔG values of the analyte-surface interaction showed a linear relation to the response of the sensor, in mV. This knowledge was applied to optimize the potentiometric detection of plant alkaloids in (U)HPLC. Sub-micromolar detection limits were obtained with the potentiometric detector/(U)HPLC combination. This is the first time that the response rates and the response itself can be modelled accurately for coated wire potentiometric sensors, and it is the first application of a potentiometric detector in UPLC.

5-HT receptor subtypes as key targets in mediating pigment dispersion within melanophores of teleost, Oreochromis mossambicus.

The presence of distinct class of 5-HT receptors in the melanophores of tilapia (Oreochromis mossambicus) is reported. The cellular responses to 5-HT (5-hydroxytryptamine), 5-HT(1), and 5-HT(2), agonists on isolated scale melanophores were observed with regard to pigment translocation within the cells. It was found that 5-HT exerted rapid and strong concentration dependent pigment granule dispersion within the melanophores. The threshold pharmacological dose of 5-HT that could elicit a measurable response was as low as 4.7×10(-12) M/L. Selective 5-HT(1) and 5-HT(2) agonists, sumatriptan and myristicin were investigated and resulted in dose-dependent pigment dispersion. The dispersing effects were effectively antagonized by receptor specific antagonists. It is suggested that 5-HT-induced physiological effects are mediated via distinct classes of receptors that possibly participate in modulation of pigmentary responses of the fish.

Aptamer-based molecular recognition of lysergamine, metergoline and small ergot alkaloids.

Ergot alkaloids are mycotoxins produced by fungi of the genus Claviceps, which infect cereal crops and grasses. The uptake of ergot alkaloid contaminated cereal products can be lethal to humans and animals. For food safety assessment, analytical techniques are currently used to determine the presence of ergot alkaloids in food and feed samples. However, the number of samples which can be analyzed is limited, due to the cost of the equipment and the need for skilled personnel. In order to compensate for the lack of rapid tests for the detection of ergot alkaloids, the aim of this study was to develop a specific recognition element for ergot alkaloids, which could be further applied to produce a colorimetric reaction in the presence of these toxins. As recognition elements, single-stranded DNA ligands were selected by using an iterative selection procedure named SELEX, i.e., Systematic Evolution of Ligands by EXponential enrichment. After several selection cycles, the resulting aptamers were cloned and sequenced. A surface plasmon resonance analysis enabled determination of the dissociation constants of the complexes of aptamers and lysergamine. Dissociation constants in the nanomolar range were obtained with three selected aptamers. One of the selected aptamers, having a dissociation constant of 44 nM, was linked to gold nanoparticles and it was possible to produce a colorimetric reaction in the presence of lysergamine. This system could also be applied to small ergot alkaloids in an ergot contaminated flour sample.

Cocaine modulates pathways for photic and nonphotic entrainment of the mammalian SCN circadian clock.

Cocaine abuse is highly disruptive to circadian physiological and behavioral rhythms. The present study was undertaken to determine whether such effects are manifest through actions on critical photic and nonphotic regulatory pathways in the master circadian clock of the mouse suprachiasmatic nucleus (SCN). Impairment of SCN photic signaling by systemic (intraperitoneal) cocaine injection was evidenced by strong (60%) attenuation of light-induced phase-delay shifts of circadian locomotor activity during the early night. A nonphotic action of cocaine was apparent from its induction of 1-h circadian phase-advance shifts at midday. The serotonin receptor antagonist, metergoline, blocked shifting by 80%, implicating a serotonergic mechanism. Reverse microdialysis perfusion of the SCN with cocaine at midday induced 3.7 h phase-advance shifts. Control perfusions with lidocaine and artificial cerebrospinal fluid had little shifting effect. In complementary in vitro experiments, photic-like phase-delay shifts of the SCN circadian neuronal activity rhythm induced by glutamate application to the SCN were completely blocked by cocaine. Cocaine treatment of SCN slices alone at subjective midday, but not the subjective night, induced 3-h phase-advance shifts. Lidocaine had no shifting effect. Cocaine-induced phase shifts were completely blocked by metergoline, but not by the dopamine receptor antagonist, fluphenazine. Finally, pretreatment of SCN slices for 2 h with a low concentration of serotonin agonist (to block subsequent serotonergic phase resetting) abolished cocaine-induced phase shifts at subjective midday. These results reveal multiple effects of cocaine on adult circadian clock regulation that are registered within the SCN and involve enhanced serotonergic transmission.