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Despite the identification of Aβ plaques and NFTs as biomarkers for Alzheimer's disease (AD) pathology, therapeutic interventions remain elusive, with neither an absolute prophylactic nor a curative medication available to impede the progression of AD presently available. Current approaches focus on symptomatic treatments to maintain AD patients' mental stability and behavioral symptoms by decreasing neuronal degeneration; however, the complexity of AD pathology requires a wide range of therapeutic approaches for both preventive and curative treatments. In this regard, this review summarizes the role of receptors as a potential target for treating AD and focuses on the path of major receptors which are responsible for AD progression. This review gives an overall idea centering on major receptors, their agonist and antagonist and future prospects of viral mimicry in AD pathology. This article aims to provide researchers and developers a comprehensive idea about the different receptors involved in AD pathogenesis that may lead to finding a new therapeutic strategy to treat AD.

To better comprehend the relationship between left/right (L/R) differences and hippocampus functions is necessary knowledge of lateral asymmetry and regional distribution. This research was design to examine hippocampal L/R asymmetry and regional distribution profile of the alpha7 and alpha4 subtypes of nicotinic acetylcholine receptors (nAChRs) in the adult rat. 10-12-week-old twenty-four male wistar rats were randomly selected. After removing the brains, immunohistochemistry, real-time PCR, and western blot methods were applied to distinguish the presence of the receptors in the hippocampus. Outcomes stated that the mentioned receptors expression profile was spatial-dependent. As, the hippocampal dispersal of alpha7 and alpha4 subtypes in the left hippocampus (LH) was remarkably maximum compare with the right hippocampus (RH) (p = 0.001, p = 0.005 respectively). Furthermore, the alpha7 optical density (OD) was not significantly different in the diverse regions in hippocampus of adult rat (p = 0.057), while the maximum OD of the alpha4 was detected in the hippocampal dentate gyrus and CA3 regions of LH (p = 0.007, p = 0.009 respectively) and the minimum OD was in the CA1 of the RH (p = 0.019). In real time PCR evaluation, there is a significantly higher expression of alpha7 and alpha4 in LH compared to RH (p = 0.043, p = 0.049 respectively), also, for western blot (p = 0.042, p = 0.030 respectively). According to present data, the alpha7 and alpha4 nAChR subtypes expression profile demonstrated lateral asymmetry, the uniform regional dispersal for alpha7 and different regional dispersal for alpha4 in the adult rat hippocampus., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: To understand the characteristics of DNA methyltransferase 3a (DNMT3a) in thymoma associated Myasthenia Gravis reveal its transcriptional regulator network as while as analyze the effect of DNMT3a on Rel/ nuclear factor-kappaB family (RelA/RelB) and its downstream autoimmune regulatory factor (Aire)., Methods: Tissues of 30 patients with thymoma, with or without myasthenia gravis (MG), were collected and the DNMT3a protein expression were evaluated through immunohistochemistry. We performed mRNA expression profiling microarray detection and analysis, and integrated the analysis by constructing protein-protein interaction networks and the integration with other database. We identified molecular difference between low and high DNMT3a in the thymoma by heatmap. We also performed PCR validation in thymoma tissues. The DNMT3a-shRNA plasmid was transfected into TEC cells, and these cells were treated with 5-aza-2-deoxycytidine, a blocker of DNMT3a. After the down-regulation of DNMT3a in TEC cells, the transcript and protein levels of RelA, RelB, Aire, and CHRNA3 were evaluated by western blotting. In addition, changes in gene expression profiles were screened through microarray technology. We performed differential gene analysis in the thymoma cohort by heatmap with R (v.4.3.0) software., Results: In 30 matched tissue specimens, the expression of DNMT3a protein in thymoma with MG was lower than that in thymoma. Through mRNA expression profiling analysis, we constructed a co-expression network of DNMT3a and found direct interaction between IKZF1 and DNMT3a, and this co-expression relationship was overlappted with Cistrome DB database. We found up-regulation of 149 mRNAs and repression of 177 mRNAs in thymoma with MG compared with thymoma. Gene ontology and pathway analysis show the involvement of a multitude of genes in the mis-regulation of MG-related pathways. RNA interference significantly reduced the level of mRNA of DNMT3a, which proved that plasmid DNMT3a was effective. In comparison to the control group, the levels of DNMT3a, Aire, and CHRNA3 mRNA and protein in TEC cells transfected with DNMT3a-shRNA interference plasmid were significantly decreased, while the expression level of RelA and RelA/RelB was significantly increased., Conclusions: Our study reveals the DNMT3a-NF-κB pathway has a major effect on MG, and can be used as a marker for diagnosis as well as a target for MG treatment., (Copyright © 2021. Published by Elsevier B.V.)

Evidence in humans suggests a correlation between nicotine smoking and severe respiratory symptoms with COVID-19 infection. In lung tissue, angiotensin-converting enzyme 2 (ACE2) appears to mechanistically underlie viral entry. Here, we investigated whether e-cigarette vapor inhalation alters ACE2 and nicotinic acetylcholine receptor (nAChR) expression in male and female mice. In male lung, nicotine vapor inhalation induced a significant increase in ACE2 mRNA and protein, but surprisingly, these differences were not found in females. Further, both vehicle and nicotine vapor inhalation downregulated α5 nAChR subunits in both sexes, while differences were not found in α7 nAChR subunit expression. Finally, blood ACE2 levels did not differ with exposure, indicating that blood sampling is not a sufficient indicator of lung ACE2 changes. Together, these data indicate a direct link between e-cigarette vaping and increased ACE2 expression in male lung tissue, which thereby reveals an underlying mechanism of increased vulnerability to coronavirus infection in individuals vaping nicotine., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The construction and maturation of the postsynaptic apparatus are crucial for synapse and dendrite development. The fundamental mechanisms underlying these processes are most often studied in glutamatergic central synapses in vertebrates. Whether the same principles apply to excitatory cholinergic synapses, such as those found in the insect central nervous system, is not known. To address this question, we investigated a group of projection neurons in the Drosophila larval visual system, the ventral lateral neurons (LNvs), and identified nAchRα1 ( Dα1 ) and nAchRα6 ( Dα6 ) as the main functional nicotinic acetylcholine receptor (nAchR) subunits in the larval LNvs. Using morphological analyses and calcium imaging studies, we demonstrated critical roles of these two subunits in supporting dendrite morphogenesis and synaptic transmission. Furthermore, our RNA sequencing analyses and endogenous tagging approach identified distinct transcriptional controls over the two subunits in the LNvs, which led to the up-regulation of Dα1 and down-regulation of Dα6 during larval development as well as to an activity-dependent suppression of Dα1 Additional functional analyses of synapse formation and dendrite dynamics further revealed a close association between the temporal regulation of individual nAchR subunits and their sequential requirements during the cholinergic synapse maturation. Together, our findings support transcriptional control of nAchR subunits as a core element of developmental and activity-dependent regulation of central cholinergic synapses., Competing Interests: The authors declare no competing interest.

Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.

Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post-traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real-time PCR of rat adrenal medulla revealed an abundance of mRNAs for α3, α7, β2, and β4 subunits. Whole-cell patch-clamp electrophysiology of chromaffin cells and subtype-selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA-5469, was created that is highly selective for α3β2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA-5469 and the α3β4-selective α-conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either α3β4 nAChRs alone or α3β4 together with the α3β2β4 subtype. Conclusions were derived from observations that acetylcholine-gated currents in some cells were sensitive to inhibition by PeIA-5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional α7 nAChRs was determined using three α7-selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist α-conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of α3β2β4 nAChRs as well as functional α7 nAChRs by rat adrenal chromaffin cells., (© 2020 International Society for Neurochemistry.)

Nicotine improves endotoxic manifestations of hypotension and cardiac autonomic dysfunction in rats. Here, we test the hypothesis that brainstem antiinflammatory pathways of α7/α4β2 nicotinic acetylcholine receptors (nAChRs) modulate endotoxic cardiovascular derangements. Pharmacologic and molecular studies were performed to determine the influence of nicotine or selective α7/α4β2-nAChR ligands on cardiovascular derangements and brainstem neuroinflammation caused by endotoxemia in conscious rats. The i.v. administration of nicotine (50 μg/kg) abolished the lipopolysaccharide (LPS, 10 mg/kg i.v.)-evoked: (i) falls in blood pressure and spectral measure of cardiac sympathovagal balance (ratio of the low-frequency to high-frequency power, LF/HF), (ii) elevations in immunohistochemical protein expressions of NFκB and α4β2-nAChR in medullary neurons of the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), and (iii) decreases in medullary α7-nAChR protein expression. These favorable nicotine influences were replicated in rats treated intracisternally (i.c.) with PHA-543613 (selective α7-nAChR agonist) or 5-iodo-A-85380 (5IA, selective α4β2-nAChRs agonist). Measurement of arterial baroreflex activity by the vasoactive method revealed that nicotine, PHA, or 5IA reversed the LPS depression of reflex bradycardic, but not tachycardic, activity. Moreover, the counteraction by nicotine of LPS hypotension was mostly inhibited after treatment with i.c. methyllycaconitine (MLA, α7-nAChR antagonist) in contrast to a smaller effect for dihydro-β-erythroidine (DHβE, α4β2-nAChR antagonist), whereas the associated increases in LF/HF ratio remained unaltered. The data signifies the importance of brainstem α7, and to a lesser extent α4β2, receptors in the nicotine counteraction of detrimental cardiovascular and neuroinflammatory consequences of endotoxemia., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Organophosphate pesticides are developmental neurotoxicants. We gave diazinon via osmotic minipumps implanted into dams prior to conception, with exposure continued into the second postnatal week, at doses (0.5 or 1 mg/kg/day) that did not produce detectable brain cholinesterase inhibition. We evaluated the impact on acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT) systems in brain regions from adolescence through full adulthood. Diazinon produced deficits in presynaptic ACh activity with regional and sex selectivity: cerebrocortical regions and the hippocampus were affected to a greater extent than were the striatum, midbrain or brainstem, and females were more sensitive than males. Diazinon also reduced nicotinic ACh receptors and 5HT 1A receptors, with the same regional and sex preferences. These patterns were similar to those of diazinon given in a much more restricted period (postnatal day 1-4) but were of greater magnitude and consistency; this suggests that the brain is vulnerable to diazinon over a wide developmental window. Diazinon's effects differed from those of the related organophosphate, chlorpyrifos, with regard to regional and sex selectivity, and more importantly, to the effects on receptors: chlorpyrifos upregulates nicotinic ACh receptors and 5HT receptors, effects that compensate for the presynaptic ACh deficits. Diazinon can thus be expected to have worse neurodevelopmental outcomes than chlorpyrifos. Further, the disparities between diazinon and chlorpyrifos indicate the problems of predicting the developmental neurotoxicity of organophosphates based on a single compound, and emphasize the inadequacy of cholinesterase inhibition as an index of safety., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cannabis is the most frequently used illicit psychoactive substance worldwide; around one in ten users become dependent. The risk for cannabis use disorder (CUD) has a strong genetic component, with twin heritability estimates ranging from 51 to 70%. Here we performed a genome-wide association study of CUD in 2,387 cases and 48,985 controls, followed by replication in 5,501 cases and 301,041 controls. We report a genome-wide significant risk locus for CUD (P = 9.31 × 10 -12 ) that replicates in an independent population (P replication  = 3.27 × 10 -3 , P meta-analysis  = 9.09 × 10 -12 ). The index variant (rs56372821) is a strong expression quantitative trait locus for cholinergic receptor nicotinic α2 subunit (CHRNA2); analyses of the genetically regulated gene expression identified a significant association of CHRNA2 expression with CUD in brain tissue. At the polygenic level, analyses revealed a significant decrease in the risk of CUD with increased load of variants associated with cognitive performance. The results provide biological insights and inform on the genetic architecture of CUD.

Sensitization of motor activity is a behavioural test to evaluate the effects of psychostimulants. Conditioned place preference (CPP) is an associative learning procedure to examine the rewarding properties of drugs. We aimed to assess whether motor sensitization to drugs of abuse can make zebrafish more vulnerable to establishing drug-induced CPP. We first evaluated sensitization of locomotor activity of zebrafish to repeated administrations of nicotine and cocaine during 5 days and after 5 days of withdrawal. After withdrawal, when zebrafish were re-exposed to the same dose of nicotine or cocaine locomotor activity was increased by 103% and 166%, respectively. Different groups of zebrafish were sensitized to nicotine or cocaine and trained on a nicotine-CPP task the day after withdrawal. The nicotine dose selected for sensitization was not effective for developing CPP in naïve zebrafish whereas it elicited CPP in zebrafish that were previously sensitized to nicotine or cocaine. Levels of nicotinic acetylcholine receptor β 2 , α 6 and α 7 subunit, Pitx3, and tyrosine hydroxylase 1 (TH1) mRNAs were increased in the brain of nicotine- and cocaine-sensitized zebrafish. Nicotine-CPP performed with drug-sensitized zebrafish provoked further enhancements in the expression of α 6 and α 7 subunit, Pitx3, and TH1 mRNAs suggesting that the expression of these molecules in the reward pathway is involved in both processes. Our findings indicate that repeated exposures to low doses of drugs of abuse can increase subject's sensitivity to the rewarding properties of the same or different drugs. This further suggests that casual drug intake increases the probability of becoming addict., (Copyright © 2019. Published by Elsevier Inc.)

SIDS occurs in early infancy and predominantly during a sleep period. Abnormalities in nicotine receptor binding and in the expression of the nicotinic acetylcholine receptor (nAChR) subunits α7 and β2 have been reported in the brainstem of SIDS infants. This study focuses on the α3 and α4 nAChR subunits as α3 is important for early postnatal survival while α4 is crucial for nicotine-elicited antinociception and sleep-wake cycle regulation. Tissue from the rostral medulla of infants who died with a known cause of death (eSUDI, n = 7), and from SIDS classified as SIDS I (n = 8) and SIDS II (n = 27), was immunohistochemically stained for the α3 and α4 nAChR subunits and quantified in 9 nuclei comparing amongst these groups. The association with risk factors of sex, cigarette smoke exposure, upper respiratory tract infection (URTI), prone sleeping and bedsharing was also evaluated. Results showed that only α4 changes (increase) were evident in SIDS, occurring in the hypoglossal and cuneate nuclei of SIDS II infants and the nucleus of the spinal trigeminal tract of SIDS I infants. Amongst the SIDS infants, cigarette smoke exposure was only associated with decreased α4 in cribriform fibre tracts, while sex and bedsharing were associated with increases in α3 in the dorsal motor nucleus of the vagus and solitary nucleus, respectively. Combined, these findings suggest that abnormalities in endogenous acetylcholine synthesis and regulation may underlie the altered α3 and α4 nAChR subunit expressions in the SIDS brainstem medulla since the changes were not related to cigarette smoke exposure., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The present study was designed to explore the role of transient receptor potential canonical 3 (TRPC3) in nicotine-induced chronic obstructive pulmonary disease (COPD) and its underlying mechanism. In this study, the expression and localization of α5 nicotinic acetylcholine receptor (α5-nAchR) in lung tissues were determined by western blotting and immunohistochemistry. The quantitative real-time PCR (qRT-PCR) analysis was performed to examine the mRNA expression levels of α5-nAchR and TRPC3 in human airway smooth muscle cells (HASMCs). Cell viability was assessed by CCK-8 assay. Proliferation was detected by cell counting and EdU immunofluorescent staining. Fluorescence calcium imaging was carried out to measure cytosolic Ca 2+ ([Ca 2+ ]cyt) concentration. The results showed that the α5-nAchR and TRPC3 expressions were significantly up-regulated in lung tissues of COPD smokers. Nicotine promoted HASMC proliferation, which was accompanied by elevated α5-nAchR and TRPC3 expressions, basal [Ca 2+ ]cyt, store-operated calcium entry (SOCE) and the rate of Mn 2+ quenching in HASMCs. Further investigation indicated that nicotine-induced Ca 2+ response and TRPC3 up-regulation was reversibly blocked by small interfering RNA (siRNA) suppression of α5-nAChR. The knockdown of TRPC3 blunted Ca 2+ response and HASMC proliferation induced by nicotine. In conclusion, nicotine-induced HASMC proliferation was mediated by TRPC3-dependent calcium entry via α5-nAchR, which provided a potential target for treatment of COPD., (Copyright © 2018. Published by Elsevier B.V.)

Communications across chemical synapses are primarily mediated by neurotransmitters and their postsynaptic receptors. There are diverse molecular systems to localize and regulate the receptors at the synapse. Here, we identify HPO-30, a member of the claudin superfamily of membrane proteins, as a positive regulator for synaptic localization of levamisole-dependent AChRs (LAChRs) at the Caenorhabditis elegans neuromuscular junction (NMJ). The HPO-30 protein localizes at the NMJ and shows genetic and physical association with the LAChR subunits LEV-8, UNC-29, and UNC-38. Using genetic and electrophysiological assays in the hermaphrodite C. elegans , we demonstrate that HPO-30 functions through Neuroligin at the NMJ to maintain postsynaptic LAChR levels at the synapse. Together, this work suggests a novel function for a tight junction protein in maintaining normal receptor levels at the NMJ. SIGNIFICANCE STATEMENT Claudins are a large superfamily of membrane proteins. Their role in maintaining the functional integrity of tight junctions has been widely explored. Our experiments suggest a critical role for the claudin-like protein, HPO-30, in maintaining synaptic levamisole-dependent AChR (LAChR) levels. LAChRs contribute to <20% of the acetylcholine-mediated conductance in adult Caenorhabditis elegans ; however, they play a significant functional role in worm locomotion. This study provides a new perspective in the study of LAChR physiology., (Copyright © 2018 Sharma, Li et al.)

Inhibitory interneurons participate in mnemonic processes. However, defined roles for identified interneuron populations are scarce. A subpopulation of oriens lacunosum-moleculare (OLM) interneurons genetically defined by the expression of the nicotinic receptor α2 subunit has been shown to gate information carried by either the temporoammonic pathway or Schaffer collaterals in vitro. Here we set out to determine whether selective modulation of OLMα2 cells in the intermediate CA1 affects learning and memory in vivo. Our data show that intermediate OLMα2 cells can either enhance (upon their inhibition) or impair (upon their activation) object memory encoding in freely moving mice, thus exerting bidirectional control. Moreover, we find that OLMα2 cell activation inhibits fear-related memories and that OLMα2 cells respond differently to nicotine in the dorsoventral axis. These results suggest that intermediate OLMα2 cells are an important component in the CA1 microcircuit regulating learning and memory processes. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Principal neurons in the ventral cochlear nucleus (VCN) receive powerful ascending excitation and pass on the auditory information with exquisite temporal fidelity. Despite being dominated by ascending inputs, the VCN also receives descending cholinergic connections from olivocochlear neurons and from higher regions in the pontomesencephalic tegmentum. In Mongolian gerbils, acetylcholine acts as an excitatory and modulatory neurotransmitter on VCN neurons, but the anatomical structure of cholinergic innervation of gerbil VCN is not well described. We applied fluorescent immunohistochemical staining to elucidate the development and the cellular localization of presynaptic and postsynaptic components of the cholinergic system in the VCN of the Mongolian gerbil. We found that cholinergic fibers (stained with antibodies against the vesicular acetylcholine transporter) were present before hearing onset at P5, but innervation density increased in animals after P10. Early in development cholinergic fibers invaded the VCN from the medial side, spread along the perimeter and finally innervated all parts of the nucleus only after the onset of hearing. Cholinergic fibers ran in a rostro-caudal direction within the nucleus and formed en-passant swellings in the neuropil between principal neurons. Nicotinic and muscarinic receptors were expressed differentially in the VCN, with nicotinic receptors being mostly expressed in dendritic areas while muscarinic receptors were located predominantly in somatic membranes. These anatomical data support physiological indications that cholinergic innervation plays a role in modulating information processing in the cochlear nucleus., (© 2018 Wiley Periodicals, Inc.)

In rodents, CHRNs are involved in bitter taste transduction of nicotine and ethanol. Currently, it is not clear if CHRNs are expressed in human taste cells and if they play a role in transducing the bitter taste of nicotine and ethanol or in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of CHRNs in HBO cells. Using molecular techniques, we demonstrate that a subset of HBO cells express CHRNs that also co-express TRPM5, T1R3 or T2R38. Exposing HBO cells to nicotine or ethanol acutely or to nicotine chronically induced a differential increase in the expression of CHRN mRNA and protein in a dose- and time-dependent manner. Acutely exposing HBO cells to a mixture containing nicotine plus ethanol induced a smaller increase in CHRN mRNAs relative to nicotine or ethanol treatment alone. A subset of HBO cells responded to nicotine, acetylcholine and ATP with a transient increase in [Ca2+]i. Nicotine effects on [Ca2+]i were mecamylamine sensitive. Brain-derived neurotrophic factor (BDNF) protein was detected in HBO cells using ELISA. Acute nicotine exposure decreased BDNF in HBO cells and increased BDNF release in the medium. CHRNs were also detected in HEK293 cells by RT-PCR. Unlike HBO cells, CHRNs were localized in most of HEK293 cells and majority of HEK293 cells responded to nicotine and ethanol stimulation with a transient increase in [Ca2+]i. BDNF levels in HEK293 cells were significantly higher than in HBO cells but the nicotine induced release of BDNF in the media was a fraction of the BDNF cellular content. We conclude that CHRNs are expressed in TRPM5 positive HBO cells. CHRN mRNA expression is modulated by exposure to nicotine and ethanol in a dose- and time-dependent manner. Nicotine induces the synthesis and release of BDNF in HBO cells.

Mathematical models predicting in vivo pharmacodynamic effects from in vitro data can accelerate drug discovery, and reduce costs and animal use. However, data integration and modeling is non-trivial when more than one drug-target receptor is involved in the biological response. We modeled the inhibition of non-esterified fatty acid release by dual G-protein-coupled receptor 81/109A (GPR81/GPR109A) agonists in vivo in the rat, to estimate the in vivo EC 50 values for 12 different compounds. We subsequently predicted those potency estimates using EC 50 values obtained from concentration-response data in isolated primary adipocytes and cell systems overexpressing GPR81 or GPR109A in vitro. A simple linear regression model based on data from primary adipocytes predicted the in vivo EC 50 better than simple linear regression models based on in vitro data from either of the cell systems. Three models combining the data from the overexpressing cell systems were also evaluated: two piecewise linear models defining logical OR- and AND-circuits, and a multivariate linear regression model. All three models performed better than the simple linear regression model based on data from primary adipocytes. The OR-model was favored since it is likely that activation of either GPR81 or GPR109A is sufficient to deactivate the cAMP pathway, and thereby inhibit non-esterified fatty acid release. The OR-model was also able to predict the in vivo selectivity between the two receptors. Finally, the OR-model was used to predict the in vivo potency of 1651 new compounds. This work suggests that data from the overexpressing cell systems are sufficient to predict in vivo potency of GPR81/GPR109A agonists, an approach contributing to faster and leaner drug discovery., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background/aims: Butyric acid plays an important role in maintaining intestinal health. Butyric acid has received special attention as a short-chain fatty acid, but its role in protecting the intestinal barrier is poorly characterized. Butyric acid not only provides energy for epithelial cells but also acts as a histone deacetylase inhibitor; it is also a natural ligand for G protein-coupled receptor 109A (GPR109A). A GPR109A analog was expressed in Sus scrofa and mediated the anti-inflammatory effects of beta-hydroxybutyric acid. This study investigated the effects of butyrate on growth performance, diarrhea symptoms, and tight junction protein levels in 21-day-old weaned piglets. We also studied the mechanism by which butyric acid regulates intestinal permeability., Methods: Twenty-four piglets that had been weaned at an age of 21 days were divided randomly into 2 equal groups: basal diet group and sodium butyrate + basal diet group. Diarrhea rate, growth performance during 3 weeks of feeding on these diets were observed, the lactulose-mannitol ratio in urine were detected by High Performance Liquid Chromatography, the expression levels of tight junction proteins in the intestinal tract and related signaling molecules, such as GPR109A and Akt, in the colon were examined by quantitative real-time PCR or western blot analyses on day 21. Caco-2 cells were used as a colon cell model and cultured with or without sodium butyrate to assess the expression of tight junction proteins and the activation of related signaling molecules. GPR109A-short hairpin RNA (shRNA) and specific antagonists of Akt and ERK1/2 were used as signaling pathway inhibitors to elucidate the mechanism by which butyric acid regulates the expression of tight junction proteins and the colonic epithelial barrier., Results: The sodium butyrate diet alleviated diarrhea symptoms and decreased intestinal permeability without affecting the growth of early weaned piglets. The expression levels of the tight junction proteins Claudin-3, Occludin, and zonula occludens 1 were up-regulated by sodium butyrate in the colon and Caco-2 cells. GPR109A knockdown using shRNA or blockade of the Akt signaling pathway in Caco-2 cells suppressed sodium butyrate-induced Claudin-3 expression., Conclusions: Sodium butyrate acts on the Akt signaling pathway to facilitate Claudin-3 expression in the colon in a GPR109A-dependent manner., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background: Several studies demonstrate that cigarette smoking has a negative effect on the reproductive health of both genders. The mechanisms by which it alters male gonadic function are not entirely clear. The combustion of cigarette produces a lot of chemical compounds that may be responsible for the negative impact of cigarette smoke on sperm parameters. In particular, the effects on semen of nicotine, a substance present in the tobacco plant and the main constituent of cigarette smoke, have been studied, showing that this alkaloid alters sperm parameters. Recently we investigated the mechanism by which nicotine damages sperm through the evaluation of the expression of nicotinic receptors subunits in human spermatozoa., Conclusion: 8 nAChR subunits found to date in mammals are expressed in human spermatozoa but, in non-smokers subjects, only α7 subunit is translated. Cigarette smoking may stimulate the expression of some subunits, not translated in non-smokers. Therefore, the presence in sperm of other nAChR subunits than α7 could represent a marker for smoking-related sperm damage., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Brain-derived neurotrophic factor (BDNF) promotes maturation of cholinergic neurons. However, how activity-dependent BDNF expression affects specific cholinergic gene expression remains unclear. This study addressed this question by determining mRNA levels of 22 acetylcholine receptor subunits, the choline transporter (CHT), and the choline acetyltransferase (ChAT) in mice deficient in activity-dependent BDNF via promoter IV (KIV) and control wild-type mice. Quantitative RT-PCR revealed significant reductions in nicotinic acetylcholine receptor alpha 5 (CHRNA5) in the frontal cortex and hippocampus and M5 muscarinic acetylcholine receptor (CHRM5) in the hippocampus, but significant increases in M2 muscarinic acetylcholine receptor (CHRM2) in the frontal cortex of KIV mice compared to wild-type mice. Three-week treatments with fluoxetine, phenelzine, duloxetine, imipramine, or an enriched environment treatment (EET) did not affect the altered expression of these genes except that EET increased CHRNA5 levels only in KIV frontal cortex. EET also increased levels of CHRNA7, CHT, and ChAT, again only in the KIV frontal cortex. The imipramine treatment was most prominent among the four antidepressants; it up-regulated hippocampal CHRM2 and frontal cortex CHRM5 in both genotypes, and frontal cortex CHRNA7 only in KIV mice. To the best of our knowledge, this is the first evidence that BDNF deficiency disturbs expression of CHRNA5, CHRM2, and CHRM5. Our results suggest that promoter IV-BDNF deficiency - which occurs under chronic stress - causes cholinergic dysfunctions via these receptors. EET is effective on CHRNA5, while its compensatory induction of other cholinergic genes or drugs targeting CHRNA5, CHRM2, and CHRM5 may become an alternative strategy to reverse these BDNF-linked cholinergic dysfunctions., (© 2017 International Society for Neurochemistry.)

Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, our previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. We used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. To our knowledge, this is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance., (Copyright © 2017 by the Genetics Society of America.)

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and β nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-β-erythroidine, a α4β2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and β4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells., Competing Interests: The authors have declared that no competing interests exist.

Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of fear learning behavior. Moreover, recent studies from our laboratory have shown that acute nicotine impaired fear extinction and safety learning in mice. However, the effects of chronic nicotine and withdrawal on fear extinction are unknown. Therefore, the current experiments were conducted to investigate the effects of chronic nicotine as well as withdrawal from chronic nicotine on contextual fear extinction in mice. C57BL6/J mice were given contextual fear conditioning training and retention testing during chronic nicotine administration. Mice then received contextual fear extinction either during chronic nicotine or during withdrawal from chronic nicotine. Our results showed that contextual fear extinction was impaired both during chronic nicotine administration and subsequent withdrawal. However, it was also observed that the effects of prior chronic nicotine disappeared after 72 h in withdrawal, a timeline that closely matches with the timing of the chronic nicotine-induced upregulation of hippocampal nicotinic acetylcholine receptor (nAChR) density. Additional experiments found that 4 days, but not 1 day, of continuous nicotine administration upregulated hippocampal nAChRs and impaired contextual fear extinction. These effects disappeared following 72 h withdrawal. Overall, these experiments provide a potential link between nicotine-induced upregulation of hippocampal nAChRs and fear extinction deficits observed in patients with anxiety disorders, which may lead to advancements in the pharmacological treatment methods for this disorder., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The majority of smokers begin during adolescence, a developmental period with a high susceptibility to substance abuse. Adolescents are affected differently by nicotine compared to adults, with adolescents being more vulnerable to nicotine's rewarding properties. It is unknown if the age-dependent molecular composition of a younger brain contributes to a heightened susceptibility to nicotine addiction. Nicotine, the principle pharmacological component of tobacco, binds and activates nicotinic acetylcholine receptors (nAChRs) in the brain. The most prevalent is the widely expressed α4-containing (α4*) subtype which mediates reward and is strongly implicated in nicotine dependence. Exposing different age groups of mice, postnatal day (P) 44-86 days old, to a two bottle-choice oral nicotine self-administration paradigm for five days yielded age-specific consumption levels. Nicotine self-administration was elevated in the P44 group, peaked at P54-60 and was drastically lower in the P66 through P86 groups. We also quantified α4* nAChR expression via spectral confocal imaging of brain slices from α4YFP knock-in mice, in which the α4 nAChR subunit is tagged with a yellow fluorescent protein. Quantitative fluorescence revealed age-specific α4* nAChR expression in dopaminergic and GABAergic neurons of the ventral tegmental area. Receptor expression showed a strong positive correlation with daily nicotine dose, suggesting that α4* nAChR expression levels are age-specific and may contribute to the propensity to self-administer nicotine., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Studies with heterologous expression systems have shown that the α4β2 nicotinic acetylcholine receptor (nAChR) subtype can exist in two stoichiometries (with two [(α4)2(β2)3] or three [(α4)3(β2)2] copies of the α subunit in the receptor pentamer) which have different pharmacological and functional properties and are differently regulated by chronic nicotine treatment. However, the effects of nicotine treatment in vivo on native α4β2 nAChR stoichiometry are not well known. We investigated in C57BL/6 mice the in vivo effect of 14-day chronic nicotine treatment and subsequent withdrawal, on the subunit expression and β2/α4 subunit ratio of (3)H-epibatidine labeled α4β2*-nAChR in total homogenates of cortex and thalamus. We found that in basal conditions the ratio of the β2/α4 subunit in the cortex and thalamus is different indicating a higher proportion in receptors with (α4)2(β2)3 subunit stoichiometry in the thalamus. For cortex exposure to chronic nicotine elicited an increase in receptor density measured by (3)H-epibatidine binding, an increase in the α4 and β2 protein levels, and an increase in β2/α4 subunit ratio, that indicates an increased proportion of receptors with the (α4)2(β2)3 stoichiometry. For thalamus we did not find a significant increase in receptor density, α4 and β2 protein levels, or changes in β2/α4 subunit ratio. All the changes elicited by chronic nicotine in cortex were transient and returned to basal levels with an average half-life of 2.8 days following nicotine withdrawal. These data suggest that chronic nicotine exposure in vivo favors increased assembly of α4β2 nAChR containing three β2 subunits. A greater change in stoichiometry was observed for cortex (which has relatively low basal expression of (α4)2(β2)3 nAChR) than in thalamus (which has a relatively high basal expression of (α4)2(β2)3 nAChR)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cholinergic neurons in the medial habenula (MHb) modulate anxiety during nicotine withdrawal although the molecular neuroadaptation(s) within the MHb that induce affective behaviors during nicotine cessation is largely unknown. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their behavioral role as autoreceptors in these neurons has not been described. To test the hypothesis that nAChR signaling in MHb cholinergic neurons could modulate anxiety, we expressed novel "gain of function" nAChR subunits selectively in MHb cholinergic neurons of adult mice. Mice expressing these mutant nAChRs exhibited increased anxiety-like behavior that was alleviated by blockade with a nAChR antagonist. To test the hypothesis that anxiety induced by nicotine withdrawal may be mediated by increased MHb nicotinic receptor signaling, we infused nAChR subtype selective antagonists into the MHb of nicotine naïve and withdrawn mice. While antagonists had little effect on nicotine naïve mice, blocking α4β2 or α6β2, but not α3β4 nAChRs in the MHb alleviated anxiety in mice undergoing nicotine withdrawal. Consistent with behavioral results, there was increased functional expression of nAChRs containing the α6 subunit in MHb neurons that also expressed the α4 subunit. Together, these data indicate that MHb cholinergic neurons regulate nicotine withdrawal-induced anxiety via increased signaling through nicotinic receptors containing the α6 subunit and point toward nAChRs in MHb cholinergic neurons as molecular targets for smoking cessation therapeutics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Rationale: Although nicotine exposure upregulates the α4β2* subtype of nicotinic acetylcholine receptors (nAChRs), the upregulation of nAChRs in non-human primates voluntarily self-administering nicotine has never been demonstrated., Objectives: The objective of the study is to determine if short access to nicotine in a non-human primate model of nicotine self-administration is sufficient to induce nAChRs upregulation., Methods: We combined a nicotine self-administration paradigm with in vivo measure of α4β2* nAChRs using 2-[(18)F]fluoro-A-85380 (2-FA) and positron emission tomography (PET) in six squirrel monkeys. PET measurement was performed before and after intravenous nicotine self-administration (unit dose 10 μg/kg per injection). Monkeys were trained to self-administer nicotine under a fixed-ratio (FR) schedule of reinforcement. Intermittent access (1 h daily per weekday) to nicotine was allowed for 4 weeks and levels of α4β2* nAChRs were measured 4 days later., Results: This intermittent access was sufficient to induce upregulation of α4β2* receptors in the whole brain (31 % upregulation) and in specific brain areas (+36 % in amygdala and +62 % in putamen)., Conclusions: These results indicate that intermittent nicotine exposure is sufficient to produce change in nAChRs expression.

Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression.

G protein-coupled receptor 109A (GPR109A) activation by its ligand nicotinic acid (NA) in immune cells increases Ca(2+) levels, and Ca(2+) induces glutamate release and oxidative stress in central blood pressure (BP)-regulating nuclei, for example, the rostral ventrolateral medulla (RVLM), leading to sympathoexcitation. Despite NA's ability to reach the brain, the expression and function of its receptor GPR109A in the RVLM remain unknown. We hypothesized that NA activation of RVLM GPR109A causes Ca(2+)-dependent l-glutamate release and subsequently increases neuronal oxidative stress, sympathetic activity, and BP. To test this hypothesis, we adopted a multilevel approach, which included pharmacologic in vivo studies along with ex vivo and in vitro molecular studies in rat pheochromocytoma cell line (PC12) cells (which exhibit neuronal phenotype). We present the first evidence for GPR109A expression in the RVLM and in PC12 cells. Next, we showed that RVLM GPR109A activation (NA) caused pressor and bradycardic responses in conscious rats. The resemblance of these responses to those caused by intra-RVLM glutamate and their attenuation by NMDA receptor (NMDAR) blockade (2-amino-5-phosphonopentanoic acid) and enhancement by l-glutamate uptake inhibition (l-trans-pyrrolidine-2,4-dicarboxylic acid, PDC) supported our hypothesis. NA increased Ca(2+), glutamate, nitric oxide and reactive oxygen species (ROS) levels in PC12 cells and increased RVLM ROS levels. The inactive NA analog isonicotinic acid failed to replicate the cardiovascular and biochemical effects of NA. Further, GPR109A knockdown (siRNA) abrogated the biochemical effects of NA in PC12 cells. These novel findings yield new insight into the role of RVLM GPR109A in central BP control., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Nicotinic acetylcholine receptors (nAChRs) are the molecular target of nicotine. nAChRs in the medial habenula (MHb) have recently been shown to play a role in nicotine dependence, but it is not clear which nAChR subtypes or MHb neuron types are most important. To identify MHb nAChRs and/or cell types that play a role in nicotine dependence, we studied these receptors and cells with brain slice electrophysiology using both acute and chronic nicotine application. Cells in the ventroinferior (MHbVI) and ventrolateral MHb (MHbVL) subregions expressed functional nAChRs with different pharmacology. Further, application of nicotine to cells in these subregions led to different action potential firing patterns. The latter result was correlated with a differing ability of nicotine to induce nAChR desensitization. Chronic nicotine caused functional upregulation of nAChRs selectively in MHbVI cells, but did not change nAChR function in MHbVL. Importantly, firing responses were also differentially altered in these subregions following chronic nicotine. MHbVI neurons treated chronically with nicotine exhibited enhanced basal pacemaker firing but a blunted nicotine-induced firing response. MHbVL neurons did not change their firing properties in response to chronic nicotine. Together, these results suggest that acute and chronic nicotine differentially affect nAChR function and output of cells in MHb subregions. Because the MHb extensively innervates the interpeduncular nucleus, an area critical for both affective and somatic signs of withdrawal, these results could reflect some of the neurophysiological changes thought to occur in the MHb to the interpeduncular nucleus circuit in human smokers., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

γ oscillations are associated with higher brain functions such as memory, perception and consciousness. Disruption of γ oscillations occur in various neuro-psychological disorders such as schizophrenia. Nicotinic acetylcholine receptors (nAChR) are highly expressed in the hippocampus, however, little is known about the role on hippocampal persistent γ oscillation. This study examined the effects of nicotine and selective nAChR agonists and antagonists on kainate-induced persistent γ oscillation in rat hippocampal slices. Nicotine enhanced γ oscillation at concentrations of 0.1-10 μM, but reduced it at a higher concentration of 100 μM. The enhancement on γ oscillation can be best mimicked by co-application of α4β2- and α7-nAChR agonist and reduced by a combination of nAChR antagonists, DhβE and MLA. However, these nAChR antagonists failed to block the suppressing role of nicotine on γ. Furthermore, we found that the NMDA receptor antagonist D-AP5 completely blocked the effect of nicotine. These results demonstrate that nicotine modulates γ oscillations via α7 and α4β2 nAChR as well as NMDA activation, suggesting that nAChR activation may have a therapeutic role for the clinical disorder such as schizophrenia, which is known to have impaired γ oscillation and hypo-NMDA receptor function.

β-Hydroxybutyric acid (BHBA) has recently been shown to regulate hormone synthesis and secretion in the hypothalamus. However, little is known about the effects of BHBA-mediated hormone regulation or the detailed mechanisms by which BHBA regulates growth hormone-releasing hormone (GHRH) synthesis and secretion. In the present study, we examined the expression of the BHBA receptor GPR109A in primary hypothalamic cell cultures. We hypothesised that BHBA regulates GHRH via GPR109A and its downstream signals. Initial in vivo studies conducted in rats demonstrated that GHRH mRNA expression in the hypothalamus was strongly inversely correlated with BHBA levels in the cerebrospinal fluid during postnatal development (r = -0.89, P < 0.01). Furthermore, i.c.v. administration of BHBA acutely decreased GHRH mRNA expression in rats. Further in vitro studies revealed a decrease in GHRH synthesis and secretion in primary hypothalamic cells after treatment with BHBA; this effect was inhibited when hypothalamic cells were pretreated with pertussis toxin (PTX). BHBA had no effect on GHRH synthesis and secretion in GT1-7 cells, which do not exhibit cell surface expression of GPR109A. Furthermore, BHBA acutely decreased the transcription of the homeobox gene for Gsh-1 in the hypothalamus in both in vivo and in vitro, and this effect was also inhibited by PTX in vitro. In primary hypothalamic cells, BHBA activated the extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) kinases, as shown by western blot analysis. Moreover, inhibition of ERK1/2 with U0126 attenuated the BHBA-mediated reduction in Gsh-1 expression and GHRH synthesis and secretion. These results strongly suggest that BHBA directly regulates GHRH synthesis and secretion via the GPR109A/ERK1/2 MAPK pathway, and also that Gsh-1 is essential for this function., (© 2015 British Society for Neuroendocrinology.)

Myasthenia gravis (MG) is an autoimmune disease characterized by the formation of pathogenic autoantibodies mostly targeting the nicotinic acetylcholine receptor (AChR). The AChR is composed of two alpha subunits and one subunit of each beta, delta and gamma (fetal AChR), or epsilon (adult AChR), respectively. Serological diagnostics is commonly done by radioimmunoassay (RIA). Here we used an indirect immunofluorescence assay with MG patient sera on transiently transfected HEp-2 cells expressing selected components of the AChR. Our data show that already 12 out of 13 MG patient sera showed autoantibody binding to HEp-2 cells transfected to express the alpha subunit solely. Interestingly, 11 out of 13 patient sera reacted positive with cells transfected to reconstitute the complete fetal AChR, but only 6 out of 13 sera showed positive signals with cells expressing the components of adult AChR. Moreover, there was no strict correlation of the serum concentration required to obtain clear-cut fluorescence signals to the antibody titer as measured by RIA. It will be an interesting topic to further investigate if the optimal serum dilution for indirect immunofluorescence as well as the autoantibody binding preferences to defined AChR subunits and to the adult versus the fetal receptor variant could provide additional predictive value in MG diagnostics.

Rationale: In mouse models for atopic dermatitis (AD) hypothalamus pituitary adrenal axis (HPA) dysfunction and neuropeptide-dependent neurogenic inflammation explain stress-aggravated flares to some extent. Lately, cholinergic signaling has emerged as a link between innate and adaptive immunity as well as stress responses in chronic inflammatory diseases. Here we aim to determine in humans the impact of acute stress on neuro-immune interaction as well as on the non-neuronal cholinergic system (NNCS)., Methods: Skin biopsies were obtained from 22 individuals (AD patients and matched healthy control subjects) before and after the Trier social stress test (TSST). To assess neuro-immune interaction, nerve fiber (NF)-density, NF-mast cell contacts and mast cell activation were determined by immunohistomorphometry. To evaluate NNCS effects, expression of secreted mammal Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP) 1 and 2 (endogenous nicotinic acetylcholine receptor ligands) and their main corresponding receptors were assessed by quantitative RT-PCR., Results: With respect to neuro-immune interaction we found higher numbers of NGF+ dermal NF in lesional compared to non-lesional AD but lower numbers of Gap43+ growing NF at baseline. Mast cell-NF contacts correlated with SCORAD and itch in lesional skin. With respect to the NNCS, nicotinic acetylcholine receptor α7 (α7nAChR) mRNA was significantly lower in lesional AD skin at baseline. After TSST, PGP 9.5+ NF numbers dropped in lesional AD as did their contacts with mast cells. NGF+ NF now correlated with SCORAD and mast cell-NF contacts with itch in non-lesional skin. At the same time, SLURP-2 levels increased in lesional AD skin., Conclusions: In humans chronic inflammatory and highly acute psycho-emotional stress interact to modulate cutaneous neuro-immune communication and NNCS marker expression. These findings may have consequences for understanding and treatment of chronic inflammatory diseases in the future.

Nicotine binds to and activates a family of ligand-gated ion channels, neuronal nicotinic acetylcholine receptors (nAChRs). Chronic nicotine exposure alters the expression of various nAChR subtypes, which likely contributes to nicotine dependence; however, the underlying mechanisms regulating these changes remain unclear. A growing body of evidence indicates that microRNAs (miRNAs) may be involved in nAChR regulation. Using bioinformatics, miRNA library screening, site-directed mutagenesis, and gene expression analysis, we have identified a limited number of miRNAs that functionally interact with the 3'-untranslated regions (3' UTRs) of mammalian neuronal nAChR subunit genes. In silico analyses revealed specific, evolutionarily conserved sites within the 3' UTRs through which the miRNAs regulate gene expression. Mutating these sites disrupted miRNA regulation confirming the in silico predictions. In addition, the miRNAs that target nAChR 3' UTRs are expressed in mouse brain and are regulated by chronic nicotine exposure. Furthermore, we show that expression of one of these miRNAs, miR-542-3p, is modulated by nicotine within the mesocorticolimbic reward pathway. Importantly, overexpression of miR-542-3p led to a decrease in the protein levels of its target, the nAChR β2 subunit. Bioinformatic analysis suggests that a number of the miRNAs play a general role in regulating cholinergic signaling. Our results provide evidence for a novel mode of nicotine-mediated regulation of the mammalian nAChR gene family., (© 2014 Hogan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Human nasal miniorgan cultures (MOC) are a useful tool in ecogenotoxicology. Repetitive exposure to nicotine showed reversible DNA damage, and stable CYP2A6 expression was demonstrated in nasal MOC in previous investigations. The aim of the present study was to evaluate the genotoxic effect of nicotine in nasal MOC after chronic nicotine exposure, and to monitor possible metabolism capacities. MOC were dissected from human nasal mucosa and cultured under standard cell culture conditions. MOC were exposed to nicotine for 3 weeks at concentrations of 1 μM and 1 mM. The concentrations were chosen based on nicotine plasma levels in heavy smokers, and possible concentrations used in topical application of nicotine nasal spray. DNA damage was assessed by the comet assay at days 7, 14 and 21. Concentrations of nicotine and cotinine were analyzed in cell culture medium by gas chromatography/mass spectrometry to determine a possible metabolism of nicotine by MOC. Distinct DNA damage in MOC could be demonstrated after 1 week of exposure to 1 μM and 1 mM nicotine. This effect decreased after 2 and 3 weeks with no statistically relevant DNA migration. No nicotine metabolism could be detected by changes in nicotine and cotinine concentrations in the supernatants. This is the first time genotoxic effects have been evaluated in nasal MOC after chronic nicotine exposure for up to 3 weeks. Genotoxic effects were present after 1 week of culture with a decrease over time. Down-regulation of nicotinic acetylcholine receptors, which are expressed in nasal mucosa, may be a possible explanation. The lack of nicotine metabolism in this model could be explained by the functional loss of CYP2A6 during chronic nicotine exposure. Further investigations are necessary to provide a more detailed description of the underlying mechanisms involved in DNA damage by nicotine., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Pyridines are widely distributed in foods. Nicotinic acid (NA), a carboxylated pyridine derivative, inhibits lipolysis in adipocytes by activation of the orphan NA receptor (HM74A) and is applied to treat hyperlipidemia. However, knowledge on the impact of pyridine derivatives on intestinal lipid metabolism is scarce. This study was performed to identify the structural determinants of pyridines for their effects on fatty acid uptake in enterocyte-like Caco-2 cells and to elucidate the mechanisms of action. The impact of 17 pyridine derivatives on fatty acid uptake was tested. Multiple regression analysis revealed the presence of a methyl group to be the structural determinant at 0.1 mM, whereas at 1 mM, the presence of a carboxylic group and the N-methylation presented further structural characteristics to affect the fatty acid uptake. NA, showing a stimulating effect on FA uptake, and N-methyl-4-phenylpyridinium (MPP), inhibiting FA uptake, were selected for mechanistic studies. Gene expression of the fatty acid transporters CD36, FATP2 and FATP4, and the lipid metabolism regulating transcription factors peroxisome proliferator-activated receptor (PPAR) α and PPARγ was up-regulated upon NA treatment. Caco-2 cells were demonstrated to express the low-affinity NA receptor HM74 of which the gene expression was up-regulated upon NA treatment. We hypothesize that the NA-induced fatty acid uptake might result from NA receptor activation and related intracellular signaling cascades. In contrast, MPP increased transepithelial electrical resistance. We therefore conclude that NA and MPP, both sharing the pyridine motif core, exhibit their contrary effects on intestinal FA uptake by activation of different mechanisms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Importance: Cigarette smoking leads to upregulation of nicotinic acetylcholine receptors (nAChRs) in the human brain, including the common α4β2* nAChR subtype. While subjective aspects of tobacco dependence have been extensively examined as predictors of quitting smoking with treatment, no studies to our knowledge have yet reported the relationship between the extent of pretreatment upregulation of nAChRs and smoking cessation., Objective: To determine whether the degree of nAChR upregulation in smokers predicts quitting with a standard course of treatment., Design, Setting, and Participants: Eighty-one tobacco-dependent cigarette smokers (volunteer sample) underwent positron emission tomographic (PET) scanning of the brain with the radiotracer 2-FA followed by 10 weeks of double-blind, placebo-controlled treatment with nicotine patch (random assignment). Pretreatment specific binding volume of distribution (VS/fP) on PET images (a value that is proportional to α4β2* nAChR availability) was determined for 8 brain regions of interest, and participant-reported ratings of nicotine dependence, craving, and self-efficacy were collected. Relationships between these pretreatment measures, treatment type, and outcome were then determined. The study took place at academic PET and clinical research centers., Main Outcomes and Measures: Posttreatment quit status after treatment, defined as a participant report of 7 or more days of continuous abstinence and an exhaled carbon monoxide level of 3 ppm or less., Results: Smokers with lower pretreatment VS/fP values (a potential marker of less severe nAChR upregulation) across all brain regions studied were more likely to quit smoking (multivariate analysis of covariance, F8,69 = 4.5; P < .001), regardless of treatment group assignment. Furthermore, pretreatment average VS/fP values provided additional predictive power for likelihood of quitting beyond the self-report measures (stepwise binary logistic regression, likelihood ratio χ21 = 19.8; P < .001)., Conclusions and Relevance: Smokers with less upregulation of available α4β2* nAChRs have a greater likelihood of quitting with treatment than smokers with more upregulation. In addition, the biological marker studied here provided additional predictive power beyond subjectively rated measures known to be associated with smoking cessation outcome. While the costly, time-consuming PET procedure used here is not likely to be used clinically, simpler methods for examining α4β2* nAChR upregulation could be tested and applied in the future to help determine which smokers need more intensive and/or lengthier treatment., Trial Registration: clinicaltrials.gov Identifier: NCT01526005.

Chronic nicotine administration increases the density of brain α4β2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine-A (saz-A), a potent partial agonist that desensitizes α4β2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4β2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz-A and varenicline each demonstrated anxiolytic effects in mice, but only saz-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz-A, or drugs like it, may be useful in achieving long-term abstinence from smoking., (© 2014 International Society for Neurochemistry.)

Recent genome-wide association studies (GWASs) have identified 15q25.1 as a lung cancer susceptibility locus. Here, we sought to explore the direct carcinogenic effects of genetic variants in this region on the risk of developing lung adenocarcinoma (ADC). Five common SNPs (rs8034191, rs16969968, rs1051730, rs938682, and rs8042374) spanning the 15q25.1 locus were assayed in a case-control study examining a cohort of 301 lung ADCs and 318 healthy controls. Stratification analysis by gender, smoking status, and tumor, node, metastasis (TNM) classification, was performed. In addition, sections from ADC tissue and normal tissue adjacent to tumors were stained with an anti-CHRNA3 (cholinergic receptor nicotinic α3) antibody by immunohistochemistry in 81 cases. Our results demonstrate that rs8042374, a variant of the CHRNA3 gene, is associated with an increased risk of ADC with an OR of 1.76 (95% CI: 1.17-2.65, p=0.024). This variant was linked to a greater risk of ADC in female nonsmokers (OR (95% CI): 1.81 (1.05-3.12), p=0.032) and female stage I+II cases (OR (95% CI): 1.92 (1.03-3.57), p=0.039). Although located within the same gene, rs938682 showed protective effects for smokers, stage III+IV cases, and male stage III+IV cases. Additionally, the CHRNA3 protein level in ADC tissue was slightly higher than in the surrounding normal lung tissue, based on immunohistochemical analysis. Our results suggest that the CHRNA3 polymorphism functions as a genetic modifier of the risk of developing lung ADC in the Chinese population, particularly in nonsmoking females.

The human auditory system is highly susceptible to environmental and metabolic insults which further affect the biochemical and physiological milieu of the cells that may contribute to progressive, hearing loss with aging. The cochlear nucleus (CN) is populated by morphologically diverse types of neurons with discrete physiological and neurochemical properties. Between the dorsal and the ventral cochlear nucleus (DCN and VCN), the VCN is further sub-divided into the rostral (rVCN) and caudal (cVCN) sub-divisions. Although, information is available on the age related neurochemical changes in the mammalian CN similar reports on human CN is still sparse. The morphometry and semiquantitative analysis of intensity of expression of glial fibrillary acidic protein (GFAP), calcium binding proteins (calbindin, calretinin and parvalbumin), gamma amino butyric acid (GABA) and nicotinic acetyl choline receptor (nAchR) beta 2 immunostaining were carried out in all three sub-divisions of the human CN from birth to 90 years. There was increased GFAP immunoreactivity in decades 2 and 3 in comparison to decade 1 in the CN. But no change was observed in rVCN from decade 4 onwards, whereas intense staining was also observed in decades 5 and 6 in cVCN and DCN. All three calcium binding proteins were highly expressed in early to middle ages, whereas a significant reduction was found in later decades in the VCN. GABA and nAchR beta 2 expressions were unchanged throughout in all the decades. The middle age may represent a critical period of onset and progression of aging changes in the CN and these alterations may add to the deterioration of hearing responses in the old age., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In the thymus, T cell maturation is influenced by cholinergic signaling, and the predominantly expressed receptor is the α3-subunit of nicotinic acetylcholine receptors, encoded by the chrna3 gene. We here determined its cellular distribution utilizing an appropriate eGFP-expressing reporter mouse strain. Neither T cells (CD4, CD8) nor mesenchymal cells (desmin-positive) expressed eGFP. In the thymic medulla, eGFP-positive cells either were scattered or, more frequently, formed small clusters resembling Hassall's corpuscles. Immunolabeling revealed that these cells were indeed terminally differentiated epithelial cells expressing keratin 10 (K10) but neither typical cortical (K8, K18) nor medullary keratins (K5, K14). These labeling patterns reflected those in the epidermis of the skin, where overlap of K10 and eGFP expression was seen in the stratum granulosum, whereas underlying basal cells displayed K5-immunoreactivity. A substantial portion of thymic eGFP-positive cells was also immunoreactive to chromogranin A, a peptide previously reported in epidermal keratinocytes in the stratum granulosum. Its fragment catestatin has multiple biological activities, including suppression of proinflammatory cytokine release from macrophages and inhibition of α3β4 nAChR. The present findings suggest that its thymic production and/or release are under cholinergic control involving nAChR containing the α3-subunit.

Background: Chronic exposure to nicotine elicits physical dependence in smokers, yet the mechanism and neuroanatomical bases for withdrawal symptoms are unclear. As in humans, rodents undergo physical withdrawal symptoms after cessation from chronic nicotine characterized by increased scratching, head nods, and body shakes., Results: Here we show that induction of physical nicotine withdrawal symptoms activates GABAergic neurons within the interpeduncular nucleus (IPN). Optical activation of IPN GABAergic neurons via light stimulation of channelrhodopsin elicited physical withdrawal symptoms in both nicotine-naive and chronic-nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blockade of IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the β4 subunit were upregulated in somatostatin interneurons clustered in the dorsal region of the IPN. Blockade of these receptors induced withdrawal signs more dramatically in nicotine-dependent compared to nicotine-naive mice and activated nonsomatostatin neurons in the IPN., Conclusions: Together, our data indicate that therapeutic strategies to reduce IPN GABAergic neuron excitability during nicotine withdrawal, for example, by activating nicotinic receptors on somatostatin interneurons, may be beneficial for alleviating withdrawal symptoms and facilitating smoking cessation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with β4 or β4β3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe(223) residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6β4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6β4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6β4β3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with β2 or β2β3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6β4* nAChRs.

Prenatal stress in humans is associated with psychiatric problems in offspring such as anxiety, depression, and schizophrenia. These same illnesses are also associated with neuronal nicotinic acetylcholine receptor (nAChR) dysfunction. Despite the known associations between prenatal stress exposure and offspring mental illness, and between mental illness and nAChR dysfunction, it is not known whether prenatal stress exposure impacts neuronal nAChRs. Thus, we tested the hypothesis that maternal stress alters the development of hippocampal alpha4 beta2 (α4β2∗) and alpha7 (α7∗) nicotinic receptor levels in adult offspring. Female Sprague-Dawley rats experienced unpredictable variable stressors two to three times daily during the last week of gestation. At weaning (21 days) the offspring of prenatally stressed (PS) and nonstressed (NS) dams were assigned to same-sex PS or NS groups. In young adulthood (56 days), the brains of offspring were collected and adjacent sections processed for quantitative autoradiography using [125I]-epibatidine (α4β2* nicotinic receptor-selective) and [125I]-α-bungarotoxin (α-BTX; α7* nicotinic receptor-selective) ligands. We found that PS significantly increased hippocampal α4β2* nAChRs of males and females in all subfields analyzed. In contrast, only females showed a trend toward PS-induced increases in α7* nAChRs in the dentate gyrus. Interestingly, NS females displayed a significant left-biased lateralization of α7* nAChRs in the laconosum moleculare of area CA1, whereas PS females did not, suggesting that PS interfered with normal lateralization patterns of α7* nAChRs during development. Taken together, our results suggest that PS impacts the development of hippocampal nAChRs, which may be an important link between PS exposure and risk for neuropsychiatric illness., (Copyright © 2013 Wiley Periodicals, Inc.)

Early nicotine exposure has been associated with many long-term consequences that include neuroanatomical alterations, as well as behavioral and cognitive deficits. To describe the effects of early nicotine exposure in Caenorhabditis elegans, the current study observed spontaneous locomotor activity (i.e., reversals) either in the presence or absence of nicotine. Expression of acr-16 (a nicotinic receptor subunit) and a β-like GABA(A) receptor subunit, gab-1, were also examined with RT-PCR. Worms were exposed to nicotine (30 μM) throughout "zygote formation" (period that includes oocyte maturation, ovulation and fertilization), from hatching to adulthood ("larval development") or across both zygote and larval development. Adult larval-exposed worms only showed an increase in spontaneous behavior when tested on nicotine (p<0.001) but levels of activity similar to controls when tested on plain plates (p>0.30). Larval-exposed worms also showed control levels of acr-16 nicotinic receptor expression (p>0.10) but increased gab-1 expression relative to controls (p<0.01). In contrast, zygote-exposed and zygote- plus larval-exposed worms showed a similar increase in spontaneous behavior on plain plates (p<0.001 and p=0.001, respectively) but control levels of responding when tested on nicotine (p>0.90 for each). However, expression of acr-16 and gab-1 was downregulated in zygote-exposed (p<0.01 and p<0.05, respectively) and significantly upregulated in the zygote- plus larval-exposed worms (p<0.000 for each); most surprising was the over five-fold increase in gab-1 expression. These results suggest that spontaneous motor behavior and receptor expression are differentially modulated by nicotine exposure during larval development and/or zygote formation. As well, these findings demonstrate that C. elegans, as a model system, is also sensitive to nicotine exposure during early development and provides the basis for future research to uncover specific mechanisms by which early nicotine exposure modifies neuronal signaling and alters behavior., (© 2013 Elsevier Inc. All rights reserved.)

The present study investigates in aged mice the working memory (WM) enhancing potential of the selective α4β2* nicotinic receptor agonist S 38232 as compared with the cholinesterase inhibitor donepezil, and their effect on cAMP response element binding protein (CREB) phosphorylation (pCREB) as a marker of neuronal activity. We first showed that aged mice exhibit a WM deficit and an increase of pCREB in the prelimbic cortex (PL) as compared with young mice, whereas no modification appears in the CA1. Further, we showed that systemic administration of S 38232 restored WM in aged mice and alleviated PL CREB overphosphorylation. Donepezil alleviated age-related memory deficits, however, by increasing pCREB in the CA1, while pCREB in PL remained unaffected. Finally, whereas neuronal inhibition by lidocaine infusion in the PL appeared deleterious in young mice, the infusion of Rp-cAMPS (a compound known to inhibit CREB phosphorylation) or S 38232 rescued WM in aged animals. Thus, by targeting the α4β2*-nicotinic receptor of the PL, S 38232 alleviates PL CREB overphosphorylation and restores WM in aged mice, which opens new pharmacologic perspectives of therapeutic strategy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

CHRNA5 gene expression variation may play a role in individual susceptibility to lung cancer. Analysis of CHRNA5 transcripts expressed in normal lung tissue detected the full-length transcript (isoform-1) and four splicing transcripts (isoform-2 to isoform-5), derived from the recognition of other splice sites in exon 5. Isoforms-2, -3 and -4 were found by protein modeling to form a completely folded, potentially functional extracellular domain and were observed at the protein level, whereas isoform-5 lacked a consistent part of the distorted β sandwich and was not seen at the protein level. Only isoform-1 appeared to encode a complete, functional subunit able to fulfill the ion channel function. We previously reported that CHRNA5 expression is associated with genetic polymorphisms at this locus and that three haplotypes in its promoter region show functional regulation in vitro. Analysis of differential allelic expression (DAE) of three single nucleotide polymorphisms (rs503464, rs55853698 and rs55781567) tagging the expression haplotypes of the CHRNA5 promoter indicated statistically significant DAE at rs55853698 and rs55781567, in both normal lung and lung adenocarcinoma. Overall, our findings provide evidence for the presence of multiple CHRNA5 messenger RNA (mRNA) isoforms that may modulate the multimeric nicotine receptor and cis-regulatory variations in the CHRNA5 locus that act in vivo in the control of CHRNA5 mRNA expression, in normal lung tissue and in lung adenocarcinoma.

Introduction: Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout several brain regions. Formation of the α4β2 and α7 subtypes in particular is involved in the organisation of different types of memory. Furthermore, due to their location, these receptors can control the release of various types of neurotransmitters and contribute to synaptic plasticity., Methods: Rats were divided into three groups, an experimental group (E), a sham-operated group, (S) and an intact group (T). In group E, stereotactic guidance was used to induce a chemical lesion with 1 μ/μL of 5,7-dihydroxytryptamine (5,7-DHT) in the anteroventral part of the dorsal raphe nucleus (DRN). In the sham-operated group (S), animals underwent surgery including delivery of the same excipient solution to the same site. The intact group (T) received no treatment whatsoever. Twenty days after surgery, animals in all groups were euthanised by decapitation to evaluate the expression of α4 and α7 nAChRs by means of molecular biology techniques., Results: 5-HT denervation of the rat PFC differentially modified the expression of α4 and α7 receptors: while α4 receptor expression increased, α7 expression decreased., Conclusion: Expression differences observed between the two subtypes may be due to their separate locations. The α4 subtype is found in postsynaptic locations and may be related to adaptive changes in postsynaptic cells, while the location of α7 is presynaptic. This explains why the lesion and the elimination of 5-HT fibres in the CPF would cause a decrease in α7 expression., (Copyright © 2012 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.)