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The C. elegans drug combination assay evaluates the effects of drug combinations in the nematode Caenorhabditis elegans, serving as a valuable tool to assess the efficacy of pharmaceutical agents and natural compounds. Using C. elegans as a model organism, this method allows for the efficient screening of the combined effects of different drugs and evaluation of synergistic effects in drug combinations, which reduces the risk of developing drug resistance. Combination therapy, involving commercial drugs, new agents, or natural products, broadens treatment effectiveness by targeting multiple pathways, effectively managing complex diseases with minimized side effects. The method focuses on discovering effective drug combinations, such as anthelmintic drugs, streamlining early-stage drug discovery to save time and resources. Additionally, its versatility allows for application across most areas of pharmacology and toxicology, extending its usefulness beyond anthelmintic treatments. In the experiments, synchronized worms are exposed to different drug concentrations to evaluate behavioral changes, mostly alterations in worm locomotion. Concentration-response curves for changes in behavior are generated and EC 50 or IC 50 values determined for the individual drugs. To determine whether the effects of a drug combination are synergistic, additive, or antagonistic, at least three different concentration ratios must be tested. These combinations are then analyzed using specialized drug combination analysis software. This methodology ensures consistent and precise outcomes and evaluates drug impacts on worm behavior parameters crucial for effective pharmacological activity. In conclusion, the C. elegans drug combination assay provides critical insights for developing successful market formulations applicable across a wide range of pharmacological treatments. Its ability to efficiently screen for synergistic, additive, or antagonistic effects makes it a valuable tool for identifying effective therapeutic strategies, potentially reducing drug resistance and improving treatment outcomes in various medical and toxicological fields., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Exposure to inorganic arsenic (iAs) detrimentally affects the structure and function of the central nervous system. In-utero and postnatal exposure to iAs has been connected to adverse effects on cognitive development. Therefore, this investigation explores neurobehavioral and neurochemical effects of 0.05 and 0.10 mg/L iAs exposure during gestation and lactation periods on 90-day-old female offspring rats. The assessment of anxiety- and depressive-like behaviors was conducted through the application of an elevated plus maze and a forced swim test. The neurochemical changes were evaluated in the prefrontal cortex (PFC) through the determination of enzyme activities and α1 GABA A subunit expression levels. Our findings revealed a notable impact of iAs exposure on anxiety and the induction of depressive-like behavior in 90-day-old female offspring. Furthermore, the antioxidant status within the PFC exhibited discernible alterations in exposed rats. Notably, the activities of acetylcholinesterase and glutamate pyruvate transaminase demonstrated an increase, while glutamate oxaloacetate transaminase activity displayed a decrease within the PFC due to the iAs treatment. Additionally, a distinct downregulation in the mRNA expression of the α1GABA A receptor was observed in this neuronal region. These findings strongly suggest that iAs exposure during early stages of rat development causes significant modifications in brain oxidative stress markers and perturbs the activity of enzymes associated with cholinergic and glutamatergic systems. In parallel, it elicits a discernible reduction in the level of GABA receptors within the PFC. These molecular alterations may play a role in the diminished anxiety levels and the depressive-like behavior outlined in the current investigation., 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 © 2024 Elsevier B.V. All rights reserved.)

The biological clock in eukaryotes controls daily rhythms in physiology and behavior. It displays a complex organization that involves the molecular transcriptional clock and the redox oscillator which may coordinately work to control cellular rhythms. The redox oscillator has emerged very early in evolution in adaptation to the environmental changes in O 2 levels and has been shown to regulate daily rhythms in glycerolipid (GL) metabolism in different eukaryotic cells. GLs are key components of lipid droplets (LDs), intracellular storage organelles, present in all living organisms, and essential for energy and lipid homeostasis regulation and survival; however, the cell bioenergetics status is not constant across time and depends on energy demands. Thus, the formation and degradation of LDs may reflect a time-dependent process following energy requirements. This work investigated the presence of metabolic rhythms in LD content along evolution by studying prokaryotic and eukaryotic cells and organisms. We found sustained temporal oscillations in LD content in Pseudomonas aeruginosa bacteria and Caenorhabditis elegans synchronized by temperature cycles, in serum-shock synchronized human embryonic kidney cells (HEK 293 cells) and brain tumor cells (T98G and GL26) after a dexamethasone pulse. Moreover, in synchronized T98G cells, LD oscillations were altered by glycogen synthase kinase-3 (GSK-3) inhibition that affects the cytosolic activity of the metabolic oscillator or by knocking down LIPIN-1, a key GL synthesizing enzyme. Overall, our findings reveal the existence of metabolic oscillations in terms of LD content highly conserved across evolutionary scales notwithstanding variations in complexity, regulation, and cell organization., (© 2024. The Author(s).)

Drug modulation of the α7 acetylcholine receptor has emerged as a therapeutic strategy for neurological, neurodegenerative, and inflammatory disorders. α7 is a homo-pentamer containing topographically distinct sites for agonists, calcium, and drug modulators with each type of site present in five copies. However, functional relationships between agonist, calcium, and drug modulator sites remain poorly understood. To investigate these relationships, we manipulated the number of agonist binding sites, and monitored potentiation of ACh-elicited single-channel currents through α7 receptors by PNU-120596 (PNU) both in the presence and absence of calcium. When ACh is present alone, it elicits brief, sub-millisecond channel openings, however when ACh is present with PNU it elicits long clusters of potentiated openings. In receptors harboring five agonist binding sites, PNU potentiates regardless of the presence or absence of calcium, whereas in receptors harboring one agonist binding site, PNU potentiates in the presence but not the absence of calcium. By varying the numbers of agonist and calcium binding sites we show that PNU potentiation of α7 depends on a balance between agonist occupancy of the orthosteric sites and calcium occupancy of the allosteric sites. The findings suggest that in the local cellular environment, fluctuations in the concentrations of neurotransmitter and calcium may alter this balance and modulate the ability of PNU to potentiate α7., (© 2024. The Author(s).)

There is currently an urgent need for new anthelmintic agents due to increasing resistance to the limited available drugs. The chalcone scaffold is a privileged structure for developing new drugs and has been shown to exhibit potential antiparasitic properties. We synthesized a series of chalcones via Claisen-Schmidt condensation, introducing a novel recoverable catalyst derived from biochar obtained from the pyrolysis of tree pruning waste. Employing microwave irradiation and a green solvent, this approach demonstrated significantly reduced reaction times and excellent compatibility with various functional groups. The result was the generation of a library of functionalized chalcones, exhibiting exclusive (E)-selectivity and high to excellent yields. The chalcone derivatives were evaluated on the free-living nematode Caenorhabditis elegans. The chalcone scaffold, along with two derivatives incorporating a methoxy substituent in either ring, caused a concentration-dependent decrease of worm motility, revealing potent anthelmintic activity and spastic paralysis not mediated by the nematode levamisole-sensitive nicotinic receptor. The combination of both methoxy groups in the chalcone scaffold resulted in a less potent compound causing worm hypermotility at the short term, indicating a distinct molecular mechanism. Through the identification of promising drug candidates, this work addresses the demand for new anthelmintic drugs while promoting sustainable chemistry., (© 2024 Wiley-VCH GmbH.)

The Caenorhabditis elegans egg hatching methodology is a valuable tool for assessing the anthelmintic activity of drugs and compounds and evaluating anthelmintic drug efficacy. Isolated eggs from gravid adults are exposed to different concentrations of selected drugs and the percentage of egg hatching is determined with respect to the control condition. The assay allows the construction of concentration-response curves and determination of EC 50 or EC 90 values for egg hatching inhibition. Also, it allows measurements of inhibition as a function of time of exposure. This approach addresses the urgent need for new anthelmintics, as resistance to current treatments poses a significant challenge in parasitic nematode infection. This resistance not only affects humans but also animals and plants, causing significant economic losses in livestock farming and agriculture. By using the free-living nematode C. elegans as a parasitic model organism, researchers can efficiently screen for potential treatments and assess drug combinations for synergistic effects. Importantly, this assay offers a cost-effective and accessible alternative to traditional methods, eliminating the need for specialized infrastructure, hosts, and trained animal maintenance personnel. Additionally, the methodology closely mimics natural conditions, providing insights into egg development and potential therapeutic targets. This method allows for evaluating the direct negative impact of drugs on egg hatching, which correlates with long-term anthelmintic effects, offering advantages in preventing or reducing the transmission and spread of worm infections by eggs. Overall, this approach represents a significant advancement for anthelmintic discovery, offering both practical applications and avenues for further scientific research. •The C. elegans egg hatching assay is a robust and effective method for assessing the anthelmintic potential of various drugs and compounds, allowing the generation of concentration-response curves.•By leveraging the free-living nematode C. elegans as a parasitic model organism, this method facilitates efficient screening of potential treatments and evaluation of drug combinations.•The method addresses the urgent need for new anthelmintics, offering a cost-effective and accessible alternative to traditional approaches., Competing Interests: 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., (© 2024 The Authors. Published by Elsevier B.V.)

Cholinergic deficit is a characteristic factor of several pathologies, such as myasthenia gravis, some types of congenital myasthenic syndromes, and Alzheimer's Disease. Two molecular targets for its treatment are acetylcholinesterase (AChE) and nicotinic acetylcholine receptor (nAChR). In previous studies, we found that caffeine behaves as a partial nAChR agonist and confirmed that it inhibits AChE. Here, we present new bifunctional caffeine derivatives consisting of a theophylline ring connected to amino groups by different linkers. All of them were more potent AChE inhibitors than caffeine. Furthermore, although some of them also activated muscle nAChR as partial agonists, not all of them stabilized nAChR in its desensitized conformation. To understand the molecular mechanism underlying these results, we performed docking studies on AChE and nAChR. The nAChR agonist behavior of the compounds depends on their accessory group, whereas their ability to stabilize the receptor in a desensitized state depends on the interactions of the linker at the binding site. Our results show that the new compounds can inhibit AChE and activate nAChR with greater potency than caffeine and provide further information on the modulation mechanisms of pharmacological targets for the design of novel therapeutic interventions in cholinergic deficit.

The quinuclidine scaffold has been extensively used for the development of nicotinic acetylcholine receptor (nAChR) agonists, with hydrophobic substituents at position 3 of the quinuclidine framework providing selectivity for α7 nAChRs. In this study, six new ligands ( 4 - 9 ) containing a 3-(pyridin-3-yloxy)quinuclidine moiety (ether quinuclidine) were synthesized to gain a better understanding of the structural-functional properties of ether quinuclidines. To evaluate the pharmacological activity of these ligands, two-electrode voltage-clamp and single-channel recordings were performed. Only ligand 4 activated α7 nAChR. Ligands 5 and 7 had no effects on α7 nAChR, but ligands 6 , 8 , and 9 potentiated the currents evoked by ACh. Ligand 6 was the most potent and efficacious of the potentiating ligands, with an estimated EC 50 for potentiation of 12.6 ± 3.32 μM and a maximal potentiation of EC 20 ACh responses of 850 ± 120%. Ligand 6 increased the maximal ACh responses without changing the kinetics of the current responses. At the single-channel level, the potentiation exerted by ligand 6 was evidenced in the low micromolar concentration range by the appearance of prolonged bursts of channel openings. Furthermore, computational studies revealed the preference of ligand 6 for an intersubunit site in the transmembrane domain and highlighted some putative key interactions that explain the different profiles of the synthesized ligands. Notably, Met276 in the 15' position of the transmembrane domain 2 almost abolished the effects of ligand 6 when mutated to Leu. We conclude that ligand 6 is a novel type I positive allosteric modulator (PAM-I) of α7 nAChR.

Cys-loop receptors integrate a large family of pentameric ligand-gated ion channels that mediate fast ionotropic responses in vertebrates and invertebrates. Their vital role in converting neurotransmitter recognition into an electrical impulse makes these receptors essential for a great variety of physiological processes. In vertebrates, the Cys-loop receptor family includes the cation-selective channels, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors, and the anion-selective channels, GABA A and glycine receptors, whereas in invertebrates, the repertoire is significantly larger. The free-living nematode Caenorhabditis elegans has the largest known Cys-loop receptor family as well as unique receptors that are absent in vertebrates and constitute attractive targets for anthelmintic drugs. Given the large number and variety of Cys-loop receptor subunits and the multiple possible ways of subunit assembly, C. elegans offers a large diversity of receptors although only a limited number of them have been characterized to date. C. elegans has emerged as a powerful model for the study of the nervous system and human diseases as well as a model for antiparasitic drug discovery. This nematode has also shown promise in the pharmaceutical industry search for new therapeutic compounds. C. elegans is therefore a powerful model organism to explore the biology and pharmacology of Cys-loop receptors and their potential as targets for novel therapeutic interventions. In this review, we provide a comprehensive overview of what is known about the function of C. elegans Cys-loop receptors from an electrophysiological perspective., Competing Interests: Competing interestsThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Nicotinic acetylcholine receptors (nAChRs) comprise a family of pentameric ligand-gated ion channels widely distributed in the central and peripheric nervous system and in non-neuronal cells. nAChRs are involved in chemical synapses and are key actors in vital physiological processes throughout the animal kingdom. They mediate skeletal muscle contraction, autonomic responses, contribute to cognitive processes, and regulate behaviors. Dysregulation of nAChRs is associated with neurological, neurodegenerative, inflammatory and motor disorders. In spite of the great advances in the elucidation of nAChR structure and function, our knowledge about the impact of post-translational modifications (PTMs) on nAChR functional activity and cholinergic signaling has lagged behind. PTMs occur at different steps of protein life cycle, modulating in time and space protein folding, localization, function, and protein-protein interactions, and allow fine-tuned responses to changes in the environment. A large body of evidence demonstrates that PTMs regulate all levels of nAChR life cycle, with key roles in receptor expression, membrane stability and function. However, our knowledge is still limited, restricted to a few PTMs, and many important aspects remain largely unknown. There is thus a long way to go to decipher the association of aberrant PTMs with disorders of cholinergic signaling and to target PTM regulation for novel therapeutic interventions. In this review we provide a comprehensive overview of what is known about how different PTMs regulate nAChR., 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 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cannabidiol (CBD), an important terpenoid compound from marijuana with no psychoactive effects, has become of great pharmaceutical interest for several health conditions. As CBD is a multitarget drug, there is a need to establish the molecular mechanisms by which CBD may exert therapeutic as well as adverse effects. The α7 nicotinic acetylcholine receptor (α7 nAChR) is a cation-permeable ACh-gated channel present in the nervous system and in non-neuronal cells. It is involved in different pathological conditions, including neurological and neurodegenerative disorders, inflammation, and cancer. By high-resolution single-channel recordings and confocal microscopy, we here reveal how CBD modulates α7 nAChR ionotropic and metabotropic functions. CBD leads to a profound concentration-dependent decrease of α7 nAChR single-channel activity with an IC 50 in the sub-micromolar range. The inhibition of α7 nAChR activity, which takes place through a membrane pathway, is neither mediated by receptor phosphorylation nor overcome by positive allosteric modulators and is compatible with CBD stabilization of resting or desensitized α7 nAChR conformational states. CBD modulation is complex as it also leads to the later appearance of atypical, low-frequency α7 nAChR channel openings. At the cellular level, CBD inhibits the increase in intracellular calcium triggered by α7 nAChR activation, thus decreasing cell calcium responses. The modulation of α7 nAChR is of pharmacological relevance and should be considered in the evaluation of CBD potential therapeutic uses. Thus, our study provides novel molecular information of CBD multiple actions and targets, which is required to set the basis for prospective applications in human health., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Anthelmintics are used to treat human and veterinary parasitic diseases and to reduce crop and livestock production loss associated with parasitosis. The free-living nematode Caenorhabditis elegans, a model system for anthelmintic drug discovery, has a serotonin (5-HT)-gated chloride channel, MOD-1, which belongs to the Cys-loop receptor family and modulates locomotory and behavioral functions. Since MOD-1 is unique to nematodes, it is emerging as an attractive anthelmintic drug target, but details of MOD-1 function are unclear. Here, we revealed novel aspects of MOD-1 function from the molecular level to the organism level and identified compounds targeting this receptor, which may provide new directions for anthelmintic drug discovery. We used whole-cell current recordings from heterologously expressed MOD-1 to show that tryptamine (Tryp), a weak partial agonist of vertebrate serotonin type 3 (5-HT 3 ) receptors, efficaciously activates MOD-1. A screen for modulators revealed that GABAergic ligands piperazine (PZE) and muscimol reduce 5-HT-elicited currents, thus identifying novel MOD-1 allosteric inhibitors. Next, we performed locomotor activity assays, and we found 5-HT and Tryp rapidly decrease worm motility, which is reversible only at low 5-HT concentrations. Mutants lacking MOD-1 are partially resistant to both drugs, demonstrating its role in locomotion. Acting as an antagonist of MOD-1, we showed PZE reduces the locomotor effects of exogenous 5-HT. Therefore, Tryp- and PZE-derived compounds, acting at MOD-1 through different molecular mechanisms, emerge as promising anthelmintic agents. This study enhances our knowledge of the function and drug selectivity of Cys-loop receptors and postulates MOD-1 as a potential target for anthelmintic therapy., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.

Background and Purpose: The α7 and α4β2* ("*" denotes possibly assembly with another subunit) nicotinic acetylcholine receptors (nAChRs) are the most abundant nAChRs in the mammalian brain. These receptors are the most targeted nAChRs in drug discovery programmes for brain disorders. However, the development of subtype-specific agonists remains challenging due to the high degree of sequence homology and conservation of function in nAChRs. We have developed C(10) variants of cytisine, a partial agonist of α4β2 nAChR that has been used for smoking cessation. The C(10) methyl analogue used in this study displays negligible affinity for α7 nAChR, while retaining high affinity for α4β2 nAChR., Experimental Approach: The structural underpinning of the selectivity of 10-methylcytisine for α7 and α4β2 nAChRs was investigated using molecular dynamic simulations, mutagenesis and whole-cell and single-channel current recordings., Key Results: We identified a conserved arginine in the β3 strand that exhibits a non-conserved function in nAChRs. In α4β2 nAChR, the arginine forms a salt bridge with an aspartate residue in loop B that is necessary for receptor expression, whereas in α7 nAChR, this residue is not stabilised by electrostatic interactions, making its side chain highly mobile. This lack of constrain produces steric clashes with agonists and affects the dynamics of residues involved in agonist binding and the coupling network., Conclusion and Implications: We conclude that the high mobility of the β3-strand arginine in the α7 nAChR influences agonist binding and possibly gating network and desensitisation. The findings have implications for rational design of subtype-selective nAChR agents., (© 2021 The British Pharmacological Society.)

The α9α10 nicotinic acetylcholine receptor (nAChR) plays a fundamental role in inner ear physiology. It mediates synaptic transmission between efferent olivocochlear fibers that descend from the brainstem and hair cells of the auditory sensory epithelium. The α9 and α10 subunits have undergone a distinct evolutionary history within the family of nAChRs. Predominantly in mammalian vertebrates, the α9α10 receptor has accumulated changes at the protein level that may ultimately relate to the evolutionary history of the mammalian hearing organ. In the present work, we investigated the responses of α9α10 nAChRs to choline, the metabolite of acetylcholine degradation at the synaptic cleft. Whereas choline is a full agonist of chicken α9α10 receptors it is a partial agonist of the rat receptor. Making use of the expression of α9α10 heterologous receptors, encompassing wild-type, heteromeric, homomeric, mutant, chimeric, and hybrid receptors, and in silico molecular docking, we establish that the mammalian (rat) α10 nAChR subunit underscores the reduced efficacy of choline. Moreover, we show that whereas the complementary face of the α10 subunit does not play an important role in the activation of the receptor by ACh, it is strictly required for choline responses. Thus, we propose that the evolutionary changes acquired in the mammalian α9α10 nAChR resulted in the loss of choline acting as a full agonist at the efferent synapse, without affecting the triggering of ACh responses. This may have accompanied the fine-tuning of hair cell post-synaptic responses to the high-frequency activity of efferent medial olivocochlear fibers that modulate the cochlear amplifier., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moglie, Marcovich, Corradi, Carpaneto Freixas, Gallino, Plazas, Bouzat, Lipovsek and Elgoyhen.)

Gender bias in Science, Technology, Engineering, and Mathematics (STEM) has been identified since a long time ago. However, gender imbalance in neuroscience has not yet been adequately explored worldwide. Here we report the first study on the development of the careers of men and women neuroscientists in Latin America in relation to family life and their perceptions of obstacles to success. Apart from revealing gender inequality in the neuroscience field, distinctive Latin American traits have become evident, thus providing novel insights into the global comprehension of gender imbalance in the region, which is required for guiding future actions, including the design of public policies in the region., (© 2021 The Authors.)

The serotonin type 3 receptor (5-HT 3 ) is a ligand-gated ion channel that converts the binding of the neurotransmitter serotonin (5-HT) into a transient cation current that mediates fast excitatory responses in peripheral and central nervous systems. Information regarding the activation and modulation of the human 5-HT 3 type A receptor has been based only on macroscopic current measurements because of its low ion conductance. By constructing a high-conductance human 5-HT 3 A receptor, we here revealed mechanistic information regarding the orthosteric activation by 5-HT and by the partial agonist tryptamine, and the allosteric activation by the terpenoids, carvacrol, and thymol. Terpenoids potentiated macroscopic currents elicited by the orthosteric agonist and directly elicited currents with slow-rising phases and submaximal amplitudes. At the single-channel level, activation by orthosteric and allosteric agonists appeared as openings in quick succession (bursts) that showed no ligand concentration dependence. Bursts were grouped into long-duration clusters in the presence of 5-HT and even longer in the presence of terpenoids, whereas they remained isolated in the presence of tryptamine. Kinetic analysis revealed that allosteric and orthosteric activation mechanisms can be described by the same scheme that includes transitions of the agonist-bound receptor to closed intermediate states before opening (priming). Reduced priming explained the partial agonism of tryptamine; however, equilibrium constants for gating and priming were similar for 5-HT and terpenoid activation. Thus, our kinetic analysis revealed that terpenoids are efficacious agonists for 5-HT 3 A receptors. These findings not only extend our knowledge about the human 5-HT 3 A molecular function but also provide novel insights into the mechanisms of action of allosteric ligands, which are of increasing interest as therapeutic drugs in all the superfamily., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration., (© 2020 Natarajan et al.)

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel widely distributed in the central nervous system, mainly in the hippocampus and cortex. The enhancement of its activity by positive allosteric modulators (PAMs) is a promising therapeutic strategy for cognitive deficits and neurodegenerative disorders. With the aim of developing novel scaffolds with PAM activity, we designed and synthesized a series of phosphonate-functionalized 1,4-disubstituted 1,2,3-triazoles using supported copper nanoparticles as the cycloaddition reaction catalyst and evaluated their activity on α7 receptors by single-channel and whole-cell recordings. We identified several triazole derivatives that displayed PAM activity, with the compound functionalized with the methyl phosphonate group being the most efficacious one. At the macroscopic level, α7 potentiation was evidenced as an increase of the maximal currents elicited by acetylcholine with minimal effects on desensitization, recapitulating the actions of type I PAMs. At the single-channel level, the active compounds did not affect channel amplitude but significantly increased the duration of channel openings and activation episodes. By using chimeric and mutant α7 receptors, we demonstrated that the new α7 PAMs share transmembrane structural determinants of potentiation with other chemically nonrelated PAMs. To gain further insight into the chemical basis of potentiation, we applied structure-activity relationship strategies involving modification of the chain length, inversion of substituent positions in the triazole ring, and changes in the aromatic nucleus. Our findings revealed that the phosphonate-functionalized 1,4-disubstituted 1,2,3-triazole is a novel pharmacophore for the development of therapeutic agents for neurological and neurodegenerative disorders associated with cholinergic dysfunction.

Nematode parasitosis causes significant mortality and morbidity in humans and considerable losses in livestock and domestic animals. The acquisition of resistance to current anthelmintic drugs has prompted the search for new compounds for which the free-living nematode Caenorhabditis elegans has emerged as a valuable platform. We have previously synthetized a small library of oxygenated tricyclic compounds and determined that dibenzo[ b,e ]oxepin-11(6H)-one (doxepinone) inhibits C. elegans motility. Because doxepinone shows potential anthelmintic activity, we explored its behavioral effects and deciphered its target site and mechanism of action on C. elegans. Doxepinone reduces swimming rate, induces paralysis, and decreases the rate of pharyngeal pumping required for feeding, indicating a marked anthelmintic activity. To identify the main drug targets, we performed an in vivo screening of selected strains carrying mutations in Cys-loop receptors involved in worm locomotion for determining resistance to doxepinone effects. A mutant strain that lacks subunit genes of the invertebrate glutamate-gated chloride channels (GluCl), which are targets of the widely used antiparasitic ivermectin (IVM), is resistant to doxepinone effects. To unravel the molecular mechanism, we measured whole-cell currents from GluClα1/β receptors expressed in mammalian cells. Glutamate elicits macroscopic currents whereas no responses are elicited by doxepinone, indicating that it is not an agonist of GluCls. Preincubation of the cell with doxepinone produces a statistically significant decrease of the decay time constant and net charge of glutamate-elicited currents, indicating that it inhibits GluCls, which contrasts to IVM molecular actions. Thus, we identify doxepinone as an attractive scaffold with promising anthelmintic activity and propose the inhibition of GluCls as a potential anthelmintic mechanism of action., (Copyright © 2020 Castro, Turani, Faraoni, Gerbino and Bouzat.)

The use of positive allosteric modulators (PAM) of α7 nicotinic receptors is a promising therapy for neurodegenerative, inflammatory and cognitive disorders. Flavonoids are polyphenolic compounds showing neuroprotective, anti-inflammatory and pro-cognitive actions. Besides their well-known antioxidant activity, flavonoids trigger intracellular pathways and interact with receptors, including α7. To reveal how the beneficial actions of flavonoids are linked to α7 function, we evaluated the effects of three representative flavonoids -genistein, quercetin and the neoflavonoid 5,7-dihydroxy-4-phenylcoumarin- on whole-cell and single-channel currents. All flavonoids increase the maximal currents elicited by acetylcholine with minimal effects on desensitization and do not reactivate desensitized receptors, a behaviour consistent with type I PAMs. At the single-channel level, they increase the duration of the open state and produce activation in long-duration episodes with a rank order of efficacy of genistein > quercetin ≥ neoflavonoid. By using mutant and chimeric α7 receptors, we demonstrated that flavonoids share transmembrane structural determinants with other PAMs. The α7-PAM activity of flavonoids results in decreased cell levels of reactive oxygen species. Thus, allosteric potentiation of α7 may be an additional mechanism underlying neuroprotective actions of flavonoids, which may be used as scaffolds for designing new therapeutic agents., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The anthelmintic treatment of nematode infections remains the pillar of worm control in both human and veterinary medicine. Since control is threatened by the appearance of drug resistant nematodes, there is a need to develop novel compounds, among which phytochemicals constitute potential anthelmintic agents. Caenorhabditis elegans has been pivotal in anthelmintic drug discovery and in revealing mechanisms of drug action and resistance. By using C. elegans, we here revealed the anthelmintic actions of three plant terpenoids -thymol, carvacrol and eugenol- at the behavioral level. Terpenoids produce a rapid paralysis of worms with a potency rank order carvacrol > thymol > eugenol. In addition to their paralyzing activity, they also inhibit egg hatching, which would, in turn, lead to a broader anthelmintic spectrum of activity. To identify drug targets, we performed an in vivo screening of selected strains carrying mutations in receptors involved in worm locomotion for determining resistance to the paralyzing effect of terpenoids. The assays revealed that two Cys-loop receptors with key roles in worm locomotion -Levamisole sensitive nicotinic receptor (L-AChR) and GABA(A) (UNC-49) receptor- are involved in the paralyzing effects of terpenoids. To decipher the mechanism by which terpenoids affect these receptors, we performed electrophysiological studies using a primary culture of C. elegans L1 muscle cells. Whole cell recordings from L1 cells demonstrated that terpenoids decrease macroscopic responses of L-AChR and UNC-49 receptor to their endogenous agonists, thus acting as inhibitors. Single-channel recordings from L-AChR revealed that terpenoids decrease the frequency of opening events, probably by acting as negative allosteric modulators. The fact that terpenoids act at different receptors may have important advantages regarding efficacy and development of resistance. Thus, our findings give support to the use of terpenoids as either an alternative or a complementary anthelmintic strategy to overcome the ever-increasing resistance of parasites to classical anthelmintic drugs., Competing Interests: The authors have declared that no competing interests exist.

Amyloid β peptide (Aβ) is a key player in the development of Alzheimer's disease (AD). It is the primary component of senile plaques in AD patients and is also found in soluble forms. Cholinergic activity mediated by α7 nicotinic receptors has been shown to be affected by Aβ soluble forms. To shed light into the molecular mechanism of this effect, we explored the direct actions of oligomeric Aβ 1-40 and Aβ 1-42 on human α7 by fluorescence spectroscopy and single-channel recordings. Fluorescence measurements using the conformational sensitive probe crystal violet (CrV) revealed that in the presence of Aβ α7 undergoes concentration-dependent conformational changes. Exposure of α7 to 100 pM Aβ changes CrV K D towards that of the desensitized state. However, α7 is still reactive to high carbamylcholine (Carb) concentrations. These observations are compatible with the induction of active/desensitized states as well as of a novel conformational state in the presence of both Aβ and Carb. At 100 nM Aβ, α7 adopts a resting-state-like structure which does not respond to Carb, suggesting stabilization of α7 in a blocked state. In real time, we found that Aβ is capable of eliciting α7 channel activity either in the absence or presence of the positive allosteric modulator (PAM) PNU-120596. Activation by Aβ is favored at picomolar or low nanomolar concentrations and is not detected at micromolar concentrations. At high Aβ concentrations, the mean duration of activation episodes elicited by ACh in the presence of PNU-120596 is significantly reduced, an effect compatible with slow open-channel block. We conclude that Aβ directly affects α7 function by acting as an agonist and a negative modulator. Whereas the capability of low concentrations of Aβ to activate α7 could be beneficial, the reduced α7 activity in the presence of higher Aβ concentrations or its long exposure may contribute to the cholinergic signaling deficit and may be involved in the initiation and development of AD.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in neuromuscular transmission. In nematodes, muscle nAChRs are targets of antiparasitic drugs. Bephenium is an anthelmintic compound whose molecular action in the free-living nematode Caenorhabditis elegans , which is a model for anthelmintic drug discovery, is poorly known. We explored the effect of bephenium on C. elegans locomotion and applied single-channel recordings to identify its molecular target, mechanism of action, and selectivity between mammalian and C. elegans nAChRs. As in parasites, bephenium paralyzes C. elegans A mutant strain lacking the muscle levamisole-sensitive nAChR (L-AChR) shows full resistance to bephenium, indicating that this receptor is the target site. Bephenium activates L-AChR channels from larvae muscle cells in the micromolar range. Channel activity is similar to that elicited by levamisole, appearing mainly as isolated brief openings. Our analysis revealed that bephenium is an agonist of L-AChR and an open-channel blocker at higher concentrations. It also activates mammalian muscle nAChRs. Opening events are significantly briefer than those elicited by ACh and do not appear in activation episodes at a range of concentrations, indicating that it is a very weak agonist of mammalian nAChRs. Recordings in the presence of ACh showed that bephenium acts as a voltage-dependent channel blocker and a low-affinity agonist. Molecular docking into homology-modeled binding-site interfaces represent the binding mode of bephenium that explains its partial agonism. Given the great diversity of helminth nAChRs and the overlap of their pharmacological profiles, unraveling the basis of drug receptor-selectivity will be required for rational design of anthelmintic drugs., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Daily exposure to fluoride (F) depends mainly on the intake of this element with drinking water. When administered during gestation and lactation, F has been associated with cognitive deficits in the offspring. However, the mechanisms underlying the neurotoxicity of F remain obscure. In the current study, we investigated the effects of oral exposure to low levels of F during the gestational and lactation periods, on the memory of adult female rat offspring. We also considered a possible underlying neurotoxic mechanism. Our results showed that this exposure reduced step-down latency in the inhibitory avoidance task, and decreased both mRNA expression of the α7 nicotinic receptor (nAChR) and catalase activity in hippocampus. Our data indicates that low F concentrations administrated during gestation and lactation decrease the memory of 90-day-old female offspring. This suggests that the mechanism might be connected with an α7 nAChR deficit in the hippocampus, induced by oxidative stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cholinergic α7 nicotinic receptor gene, CHRNA7 , encodes a subunit that forms the homopentameric α7 receptor, involved in learning and memory. In humans, exons 5-10 in CHRNA7 are duplicated and fused to the FAM7A genetic element, giving rise to the hybrid gene CHRFAM7A Its product, dupα7, is a truncated subunit lacking part of the N-terminal extracellular ligand-binding domain and is associated with neurological disorders, including schizophrenia, and immunomodulation. We combined dupα7 expression on mammalian cells with patch clamp recordings to understand its functional role. Transfected cells expressed dupα7 protein, but they exhibited neither surface binding of the α7 antagonist α-bungarotoxin nor responses to acetylcholine (ACh) or to an allosteric agonist that binds to the conserved transmembrane region. To determine whether dupα7 assembles with α7, we generated receptors comprising α7 and dupα7 subunits, one of which was tagged with conductance substitutions that report subunit stoichiometry and monitored ACh-elicited channel openings in the presence of a positive allosteric α7 modulator. We found that α7 and dupα7 subunits co-assemble into functional heteromeric receptors, which require at least two α7 subunits for channel opening, and that dupα7's presence in the pentameric arrangement does not affect the duration of the potentiated events compared with that of α7. Using an α7 subunit mutant, we found that activation of (α7) 2 (dupα7) 3 receptors occurs through ACh binding at the α7/α7 interfacial binding site. Our study contributes to the understanding of the modulation of α7 function by the human specific, duplicated subunit, associated with human disorders., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inorganic arsenic (iAs) is an important natural pollutant. Millions of individuals worldwide drink water with high levels of iAs. Arsenic exposure has been associated to cognitive deficits. However, the underlying mechanisms remain unknown. In the present work we investigated in female adult offspring the effect of the exposure to low arsenite sodium levels through drinking water during pregnancy and lactation on short- and long-term memory. We also considered a possible underlying neurotoxic mechanism. Pregnant rats were exposed during pregnancy and lactation to environmentally relevant iAs concentrations (0.05 and 0.10 mg/L). In 90-day-old female offspring, short-term memory (STM) and long-term memory (LTM) were evaluated using a step-down inhibitory avoidance task. In addition, we evaluated the α7 nicotinic receptor (α7-nAChR) expression, the transaminases and the oxidative stress levels in hippocampus. The results showed that the exposure to 0.10 mg/L iAs in this critical period produced a significant impairment in the LTM retention. This behavioral alteration might be associated with several events that occur in the hippocampus: decrease in α7-nAChR expression, an increase of glutamate levels that may produce excitotoxicity, and a decrease in the antioxidant enzyme catalase (CAT) activity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The α7 nicotinic receptor is a promising drug target for neurological and inflammatory disorders. Although it is the homomeric member of the family, a novel α7β2 heteromeric receptor has been discovered. To decipher the functional contribution of the β2 subunit, we generated heteromeric receptors with fixed stoichiometry by two different approaches comprising concatenated and unlinked subunits. Receptors containing up to three β2 subunits are functional. As the number of β2 subunits increases in the pentameric arrangement, the durations of channel openings and activation episodes increase progressively probably due to decreased desensitization. The prolonged activation episodes conform the kinetic signature of α7β2 and may have an impact on neuronal excitability. For activation of α7β2 receptors, an α7/α7 binding-site interface is required, thus indicating that the three β2 subunits are located consecutively in the pentameric arrangement. α7-positive allosteric modulators (PAMs) are emerging as novel therapeutic drugs. The presence of β2 in the pentamer affects neither type II PAM potentiation nor activation by an allosteric agonist whereas it impairs type I PAM potentiation. This first single-channel study provides fundamental basis required to decipher the role and function of the novel α7β2 receptor and opens doors to develop selective therapeutic drugs.

Over the past four decades, the patch clamp technique and nicotinic ACh (nACh) receptors have established an enduring partnership. Like all good partnerships, each partner has proven significant in its own right, while their union has spurred innumerable advances in life science research. A member and prototype of the superfamily of pentameric ligand-gated ion channels, the nACh receptor is a chemo-electric transducer, binding ACh released from nerves and rapidly opening its channel to cation flow to elicit cellular excitation. A subject of a Nobel Prize in Physiology or Medicine, the patch clamp technique provides unprecedented resolution of currents through single ion channels in their native cellular environments. Here, focusing on muscle and α7 nACh receptors, we describe the extraordinary contribution of the patch clamp technique towards understanding how they activate in response to neurotransmitter, how subtle structural and mechanistic differences among nACh receptor subtypes translate into significant physiological differences, and how nACh receptors are being exploited as therapeutic drug targets., Linked Articles: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc/., (© 2017 The British Pharmacological Society.)

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels involved in many physiological and pathological processes. In vertebrates, there are seventeen different nAChR subunits that combine to yield a variety of receptors with different pharmacology, function, and localization. The homomeric α7 receptor is one of the most abundant nAChRs in the nervous system and it is also present in non-neuronal cells. It plays important roles in cognition, memory, pain, neuroprotection, and inflammation. Its diverse physiological actions and associated disorders have made of α7 an attractive novel target for drug modulation. Potentiation of the α7 receptor has emerged as a novel therapeutic strategy for several neurological diseases, such as Alzheimer's and Parkinson's diseases, and inflammatory disorders. In contrast, increased α7 activity has been associated with cancer cell proliferation. The presence of different drug target sites offers a great potential for α7 modulation in different pathological contexts. In particular, compounds that target allosteric sites offer significant advantages over orthosteric agonists due to higher selectivity and a broader spectrum of degrees and mechanisms of modulation. Heterologous expression of α7, together with chaperone proteins, combined with patch clamp recordings have provided important advances in our knowledge of the molecular basis of α7 responses and their potential modulation for pathological processes. This review gives a synthetic view of α7 and its molecular function, focusing on how its unique activation and desensitization features can be modified by pharmacological agents. This fundamental information offers insights into therapeutic strategies., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Benzodiazepines are psychoactive drugs and some of them also affect immune cells. We here characterized the inflammatory and infiltrating immune cells in the central nervous system (CNS) during the acute phase of experimental autoimmune encephalomyelitis (EAE) in animals treated with Diazepam. Also, we evaluated the expression of Translocator Protein (18kDa) (TSPO), which is a biomarker of neuroinflammatory diseases. The results indicate that Diazepam exerts protective effects on EAE development, decreasing the incidence of the disease and reducing the number of inflammatory cells in CNS, with a concomitant decrease of TSPO levels in brain tissue and CNS inflammatory CD11b + cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits., (Copyright © 2017 by The Author(s).)