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Manganese (Mn) and iron (Fe) are trace elements that are essential for proper growth and physiological functions as both play critical role in a variety of enzymatic reactions. At high concentrations, however, they can be toxic and cause neurodegenerative disorders, particularly Parkinson-like syndromes. Nicotine, on the other hand, has been shown to have neuroprotective effects against various endogenous or exogenous toxins that selectively damage the dopaminergic cells. These cells include neuroblastoma-derived SH-SY5Y cells which express significant dopaminergic activity. However, practically no information on possible neuroprotective effects of nicotine against toxicity induced by trace elements is available. Therefore, in this study we investigated the effects of nicotine on toxicity induced by manganese or iron in these cells. Exposure of SH-SY5Y cells for 24 h to manganese (20 μM) or iron (20 μM) resulted in approximately 30% and 35% toxicity, respectively. Pretreatment with nicotine (1 μM) completely blocked the toxicities of Mn and Fe. The effects of nicotine, in turn, were blocked by selective nicotinic receptor antagonists. Thus, dihydro-beta erythroidine (DHBE), a selective alpha 4-beta 2 subtype antagonist and methyllycaconitine (MLA), a selective alpha7 antagonist, as well as mecamylamine, a non-selective nicotinic antagonist all dose-dependently blocked the protective effects of nicotine against both Mn and Fe. These findings provide further support for the potential utility of nicotine or nicotinic agonists in Parkinson's disease-like neurodegenerative disorders, including those that might be precipitated by trace elements, such as Fe and Mn. Moreover, both alpha4-beta2 and alpha7 nicotinic receptor subtypes appear to mediate the neuroprotective effects of nicotine against toxicity induced by these two trace metals.

Acetylcholine plays a pivotal neuromodulatory role in the brain, influencing neuronal activity and cognitive function. Nicotinic receptors, particularly α7 and α4β2 receptors, modulate firing of dorsolateral prefrontal (dlPFC) excitatory networks that underlie successful working memory function. Minimal work however has been done examining working memory following systemic blockade of nicotinic receptor systems in nonhuman primates, limiting the ability to explore interactions of other neuromodulatory influences with working memory impairment caused by nicotinic antagonism. In this study, we investigated working memory performance after administering three nicotinic antagonists, mecamylamine, methyllycaconitine, and dihydro-β-erythroidine, in rhesus macaques tested in a spatial delayed response task. Surprisingly, we found that no nicotinic antagonist significantly impaired delayed response performance compared to vehicle. In contrast, the muscarinic antagonist scopolamine reliably impaired delayed response performance in all monkeys tested. These findings suggest there are some limitations on using systemic nicotinic antagonists to probe the involvement of nicotinic receptors in aspects of dlPFC-dependent working memory function, necessitating alternative strategies to understand the role of this system in cognitive deficits seen in aging and neurodegenerative disease.

Nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine system have been implicated in ethanol behaviors. In particular, work in genetically engineered mice has demonstrated that α6-containing nAChRs are involved in ethanol consumption and sedation. A limitation of these studies is that the alteration in the receptor was present throughout development. The recently described α6β2 antagonist, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), now makes it possible to test for the involvement of these receptors using a pharmacological approach. The aim of this study was to examine the role of α6β2 nAChRs in ethanol behaviors using a pharmacological approach. Adolescent C57BL/6J mice were treated with bPiDI 30 minutes prior to testing the mice for binge-like ethanol consumption in the drinking-in-the-dark (DID) test, ethanol-induced motor incoordination using the balance beam, and ethanol-induced sedation using the Loss of Righting Reflex (LORR) paradigm. Adolescent animals were chosen because they express a high amount of α6 mRNA relative to adult animals. Control studies were also performed to determine the effect of bPiDI on locomotor activity and ethanol metabolism. Female mice treated with 20 mg/kg bPiDI had reduced locomotor activity compared to saline-treated animals during the first 30 minutes following an acute injection. Pretreatment with the α6β2 antagonist reduced adolescent ethanol consumption but also reduced saccharin consumption. No significant effects were observed on ethanol-induced ataxia, sedation, or metabolism. This study provides evidence that α6β2 nAChRs are involved in locomotor activity as well as ethanol and saccharin consumption in adolescent animals.

In this paper, Dr. Corrigall and collaborators described elegant experiments designed to elucidate the neurobiology of nicotine reinforcement. The nicotinic receptor antagonist dihydro-β-erythroidine (DHβE) was infused in the ventral tegmental area (VTA) or nucleus accumbens (NAC) of rats trained to self-administer nicotine intravenously. Additionally, DHβE was infused in the VTA of rats trained to self-administer food or cocaine, and nicotine self-administration was assessed in rats with lesions to the peduculopontine tegmental nucleus (PPT). A number of key themes emerged from this fundamental study that remain relevant today. The primary finding was that infusions of DHβE in the VTA, but not in the NAC, lowered nicotine self-administration, suggesting that nicotinic receptors in VTA are involved in the reinforcing action of nicotine. This conclusion has been confirmed by subsequent findings, and the nature of the nicotinic receptors has also been elucidated. The authors also reported that DHβE in the VTA had no effect on food or cocaine self-administration, and that lesions to the PPT did not alter nicotine self-administration. Since this initial investigation, the question of whether nicotinic receptors in the VTA are necessary for the reinforcing action of other stimuli, and by which mechanisms, has been extensively explored. Similarly, many groups have further investigated the role of mesopontine cholinergic nuclei in reinforcement. This paper not only contributed in important ways to our understanding of the neurochemical basis of nicotine reinforcement, but was also a key catalyst that gave rise to several research themes central to the neuropharmacology of substance abuse. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co-assembled with β2 subunits to form a novel type of α7β2 nAChR. Interestingly, the α7β2 nAChR exhibits distinctive function and pharmacology from traditional homomeric α7 nAChRs. We review recent advances in probing the distribution, function, pharmacology, pathophysiology, and stoichiometry of the heteromeric α7β2 nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling.

Participation of nicotinic acetylcholine receptors (nAChRs) in functioning of polymorphonuclear neutrophils (PMNs) isolated from inflammatory site of mice and expression of different nAChR subunits were studied. Nicotine and acetylcholine (ACh) modified respiratory burst induced by a chemotactic peptide N-formyl-MLF in neutrophils of male (but not female) mice. Antagonists of nAChRs α-cobratoxin (αCTX), α-conotoxins MII and [A10L]PnIA at concentrations of 0.01-5μM, 0.2μM and 1μM, respectively, eliminated nAChR agonist effects. ACh also affected adhesion of PMNs, this effect was also prevented by αCTX (100nM) and MII (1nM). Neutrophils of female mice after chronic nicotine consumption acquired sensitivity to nAChR agonists. Changes of free intracellular Ca(2+) concentration in neutrophils under the action of nAChR ligands were analyzed. In cells with no Ca(2+) oscillations and relatively low resting level of intracellular Ca(2+), nicotine triggered Ca(2+)-spikes, the lag of the response shortened with increasing nicotine concentration. A nicotinic antagonist caramiphen strongly decreased the effect of nicotine. RT-PCR analysis revealed mRNAs of α2, α3, α4, α5, α6, α7, α9, β2, β3, and β4 nAChR subunits. Specific binding of [(125)I]-α-bungarotoxin was demonstrated. Thus in view of the effects and binding characteristics the results obtained suggest a regulatory role of α7, α3β2 or α6* nAChR types in specific functions of PMNs.

Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace (3)H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 μM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick β2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism. Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid.

Effects of nicotinic receptor agonists (epibatidine and nicotine) on mechano-sensitive bladder afferent nerve (MS-BAN) activity were studied in an in vitro bladder-pelvic afferent nerve preparation. MS-BAN activity was induced by isotonic distention of the bladder at pressures of 10–40 cmH2O. The effect of epibatidine varied according to the concentration, route of administration and the intravesical pressure stimulus. Epibatidine (300–500 nM) administered in the perfusate to the serosal surface of the bladder decreased distension evoked afferent firing by 30%–50% depending on the bladder pressure. However these concentrations also produced an immediate increase in tonic afferent firing in the empty bladder. Lower concentrations (50–100 nM) elicited weaker and more variable effects. The inhibitory effects were blocked by bath application of mecamylamine (150 μM) a nicotinic receptor antagonist. Bath application of nicotine (20 μM) elicited similar effects. Intravesical administration of epibatidine (500 nM) significantly increased MS-BAN firing by 15–30%; while lower concentrations (200–300 nM) were ineffective. This facilitatory effect of epibatidine was blocked by intravesical administration of mecamylamine (250 μM). Electrical stimulation on the surface of the bladder elicited action potentials (AP) in BAN. Bath application of epibatidine (300 nM) or nicotine (20 μM) did not change either the voltage threshold or the area of evoked AP. These results indicate that nicotinic agonists: (1) enhance MS-BAN activity originating at afferent receptors near the urothelium, (2) inhibit MS-BAN activity originating at afferent receptors located at other sites in the bladder, (3) directly excite unidentified afferents, (4) do not alter afferent axonal excitability.

Objective GTS-21, a selective α7 nicotinic acetylcholine receptor agonist, has recently been established as a promising treatment for inflammation. However, the detailed molecular mechanism of GTS-21 in suppressing pro-inflammatory cytokine production is only partially explored. The study aimed to analyze cytokine expression suppressed by GTS-21 with lipopolysaccharide (LPS)-induced inflammation in vitro and to gain insights into the role of Akt/NF-κB signaling pathway in this process. Materials and methods Cell Counting Kit-8 (CCK-8) assay was performed to detect drug cytotoxicity. RAW 264.7 cells were stimulated with LPS and treated with GTS-21. Interleukin (IL)-1β, IL-6, or tumor necrosis factor (TNF)-α production was detected using enzyme-linked immunosorbent assay. Western blot was used to assess the expression patterns of signal transduction protein. Nuclear translocation of nuclear factor (NF)-κB was analyzed by confocal fluorescence microscopy. In addition, α7 nicotinic acetylcholine receptors (α7 nAChR) were detected on RAW264.7, and the α7 nAChR-specific antagonist was adopted to verify whether the effect of GTS-21 was mediated by α7 nAChR. Results The CCK-8 assay showed that GTS-21 did not significantly affect cell proliferation. The production of IL-1β, IL-6, and TNF-α decreased after being treated with GTS-21 in LPS-stimulated RAW 264.7 cells. GTS-21 also suppressed LPS-induced phosphorylation of NF-κBp65, IKKα/β, IκBα, and Akt, as well as NF-κB p65 nuclear translocation. Moreover, α7 nAChR was expressed on the surfaces of RAW264.7 cells, and the α7 nAChR-specific antagonist almost completely prohibited the inhibitory effect of GTS-21 on NF-κB activation. Conclusion These findings indicate that GTS-21 suppresses LPS-induced inflammation by inhibiting the Akt/NF-κB signal pathway through α7 nAChR. GTS-21 has a potential application in inflammatory disease therapy.

Oseltamivir, an anti-influenza virus drug, induces marked hypothermia in normal mice. We have proposed that the hypothermic effect arises from inhibition of the nicotinic acetylcholine receptor function of sympathetic ganglion neurons which innervate the brown adipose tissue (a heat generator). It has been reported that local anesthetics inhibit nicotinic acetylcholine receptor function by acting on its ionic channels, and that bupropion, a nicotinic antagonist, induces hypothermia. In this study, we compared the effects of oseltamivir, procaine and bupropion on body temperature, cardiovascular function and neuromuscular transmission. Intraperitoneal administration of oseltamivir (100mg/kg), procaine (86.6mg/kg) and bupropion (86.7mg/kg) lowered the core body temperature of normal mice. At lower doses (10-30mg/kg oseltamivir, 8.7-26mg/kg procaine and bupropion), when administered subcutaneously, the three drugs antagonized the hypothermia induced by intraperitoneal injection of nicotine (1mg/kg). In anesthetized rats, intravenous oseltamivir (30-100mg/kg), procaine (10mg/kg) and bupropion (10mg/kg) induced hypotension and bradycardia. Oseltamivir alone (100mg/kg) did not inhibit neuromuscular twitch contraction of rats, but at 3-30mg/kg it augmented the muscle-relaxing effect of d-tubocurarine. Similar effects were observed when lower doses of procaine (10-30mg/kg) and bupropion (3-10mg/kg) were administered, suggesting that systemic administration of oseltamivir inhibits muscular nicotinic acetylcholine receptors. These results support the idea that the hypothermic effect of oseltamivir is due to its effects on sympathetic ganglia which innervate the brown adipose tissue, and suggest that oseltamivir may exert non-selective ion channel blocking effects like those of ester-type local anesthetics.

This paper reviews the evidence for nicotine-induced acceleration of the internal clock when timing in the seconds-to-minutes timescale, and proposes an alternative explanation to this evidence: that nicotine reduces the threshold for responses that result in more reinforcement. These two hypotheses were tested in male Wistar rats using a novel timing task. In this task, rats were trained to seek food at one location after 8 s since trial onset and at a different location after 16 s. Some rats received the same reward at both times (group SAME); some received a larger reward at 16 s (group DIFF). Steady baseline performance was followed by 3 days of subcutaneous nicotine administration (0.3 mg/kg), baseline recovery, and an antagonist challenge (mecamylamine, 1.0 mg/kg). Nicotine induced a larger, immediate reduction in latencies to switch (LTS) in group DIFF than in group SAME. This effect was sustained throughout nicotine administration. Mecamylamine pretreatment and nicotine discontinuation rapidly recovered baseline performance. These results support a response-threshold account of nicotinic disruption of timing performance, possibly mediated by nicotinic acetylcholine receptors. A detailed analysis of the distribution of LTSs suggests that anomalous effects of nicotine on LTS dispersion may be due to loss of temporal control of behavior.

The alpha7 nicotinic acetylcholine receptor (nAChR) is a potential target in neuroinflammation. Screening a plant extract library identified Solidago nemoralis as containing methyl-quercetin derivatives that are relatively selective ligands for the alpha7 nAChR. Flavonoids are not known for this activity, so we screened a small library of pure flavonoids to confirm our findings. Some flavonoids, e.g. rhamnetin, displaced a selective alpha7 nAChR radioligand from rat brain membranes whereas similar structures e.g. sakuranetin, did not. To evaluate the contribution of this putative nAChR activity to the known anti-inflammatory properties of these flavonoids, we compared their effects on lipopolysaccharide induced release of inflammatory mediators from BV2 microglia. Both rhamnetin and sakuranetin reduced mediator release, but differed in potency (rhamnetin>sakuranetin) and the Hill slope of their concentration response curves. For rhamnetin the Hill coefficient was >3.0 whereas for sakuranetin the coefficient was 1.0, suggesting that effects of rhamnetin are mediated through more than one mechanism, whereas sakuranetin has a single mechanism. nACHR antagonists decreased the Hill coefficient for rhamnetin toward unity, which suggests that a nAChR-mediated mechanism contributes cooperatively to its overall anti-inflammatory effect. In contrast nAChR antagonists had no effect on the potency or Hill coefficient for sakuranetin, but a concentration of nicotine (1μM) that had no effect alone, significantly increased the Hill coefficient of this flavonoid. In conclusion, the anti-inflammatory effects of rhamnetin benefit cooperatively from a nAChR-mediated mechanism. This action, together with potent free radical scavenging activity, suggests that flavonoids with alpha7 nAChR activity have therapeutic potential in neuroinflammatory conditions.

Fluorescent molecules are regularly utilised to study ligand–receptor interactions. Many ligands for nicotinic receptors have been conjugated with fluorophores to study receptor kinetics, recycling and ligand binding characteristics. These include small agonist molecules, as well as large peptidic antagonists. However, no small molecule antagonists have been investigated using this method. Pinnatoxin F is a newly discovered non-peptidic muscle type nicotinic receptor antagonist produced by the marine dinoflagellate species Vulcanodinium rugosum. This molecule has the potential for conjugation to a fluorophore, allowing subsequent visualisation of interactions with nicotinic receptors. Pinnatoxin F was modified by addition of diaminopolyether spacers, to which a fluorophore (VivoTag® 645) was conjugated. The fluorescent pinnatoxin was then applied to muscle sections from thy1-YFP-H transgenic mice, which express YFP in motor nerves, to allow direct visualization of fluorescent binding at the neuromuscular junction. The addition of both the diaminopolyether spacer and the VivoTag® 645 reduced the potency of pinnatoxin F, as evidenced by a reduction in in vitro neuromuscular blocking activity and in vivo toxicity. Despite this reduced potency, the fluorescent molecule selectively labelled endplate regions in thy1-YFP mouse muscle sections and this labelling was inhibited by pre-exposure of muscle sections to native pinnatoxin F or the nicotinic antagonist α-bungarotoxin. This study proves nicotinic receptor binding activity of pinnatoxin F and is the first example of a fluorophore-conjugated small-molecule antagonist for nicotinic receptors. These results indicate the potential for other small-molecule nicotinic receptor antagonists to be fluorescently labelled and used as probes for specific nicotinic receptor subtypes.

Cocaine abuse is associated with a high prevalence of nicotine dependence. In animals, nicotinic antagonists have been reported to block the development of cocaine behavioral sensitization and to attenuate cocaine place preference or self-administration. In the present study, we have determined: (1) changes in the locomotor responses to nicotine challenge during the first week of withdrawal from daily cocaine pretreatment; and (2) effects of the non-selective nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine given during the first 5 days of cocaine withdrawal on the maintenance of cocaine behavioral sensitization. Male Sprague-Dawley rats were pretreated with daily saline (SI) or cocaine (CI) injections for 14 days. In Experiment 1, separate animals in the SI and CI groups received a single nicotine challenge on day 1, 3, or 7 of withdrawal from their respective pretreatments. The CI group displayed enhanced locomotor responses to nicotine as compared to SI controls on days 3 and 7 of withdrawal, but not day 1. In Experiment 2, SI and CI animals were treated once a day with either saline or mecamylamine during the first 5 days of withdrawal, and were subsequently challenged with single cocaine injections on both withdrawal days 7 and 14. Mecamylamine treatment significantly attenuated expression of cocaine behavioral sensitization on both withdrawal days 7 and 14. Time-dependent changes in nicotinic responses occur during the first week of cocaine withdrawal, and intact nAChR neurotransmission during this period may be necessary for maintenance of cocaine behavioral sensitization.

A cytosine to thymidine (C → T) missense mutation in the signal peptide (SP) sequence (rs2472553) of the nicotinic acetylcholine receptor (nAChR) α2 subunit produces a threonine-to-isoleucine substitution (T22I) often associated with nicotine dependence (ND). We assessed effects on function of α2*-nAChR (‘*’indicates presence of additional subunits) of this mutation, which could alter SP cleavage, RNA/protein secondary structure, and/or efficiency of transcription, translation, subunit assembly, receptor trafficking or cell surface expression. Two-electrode voltage clamp analyses indicate peak current responses to ACh or nicotine are decreased 2.8–5.8-fold for putative low sensitivity (LS; 10:1 ratio of α:β subunit cRNAs injected) α2β2- or α2β4-nAChR and increased for putative high sensitivity (HS; 1:10 α:β subunit ratio) α2β2- (5.7–15-fold) or α2β4- (1.9–2.2-fold) nAChR as a result of the mutation. Agonist potencies are decreased 1.6–4-fold for putative LS or HS α2(T22I)β2-nAChR or for either α2*-nAChR subtype formed in the presence of equal amounts of subunit cRNA, slightly decreased for LS α2(T22I)β4-nAChR, but increased 1.4–2.4-fold for HS α2(T22I)β4-nAChR relative to receptors containing wild-type α2 subunits. These effects suggest that the α2 subunit SP mutation generally favors formation of LS receptor isoforms. We hypothesize that lower sensitivity of human α2*-nAChR to nicotine could contribute to increased susceptibility to ND. To our knowledge this is the first report of a SP mutation having a functional effect in a member of cys-loop family of ligand-gated ion channels.

3,7-Diazabicyclo[3.3.1]nonane (bispidine) based nicotinic acetylcholine receptor (nAChR) ligands have been synthesized and evaluated for nAChRs interaction. Diverse spacer motifs were incorporated between the hydrogen bond acceptor (HBA) part and a variety of substituted (hetero)aryl moieties. Bispidine carboxamides bearing spacer motifs often showed high affinity in the low nanomolar range and selectivity for the α4β2(∗) nAChR. Compounds 15, 25, and 47 with Ki values of about 1 nM displayed the highest affinities for α4β2(∗) nAChR. All evaluated compounds are partial agonists or antagonists at α4β2(∗), with reduced or no effects on α3β4(∗) with the exception of compound 15 (agonist), and reduced or no effect at α7 and muscle subtypes.

Clinical evidence indicates that the nicotinic receptor agonist varenicline improves axial symptoms in patients with spinocerebellar ataxia type 3, but pharmacological evidence in an animal model of olivocerebellar degeneration has not been demonstrated. This study investigated whether varenicline and nicotine were efficacious for attenuating ataxia in rats induced by chemical destruction of the olivocerebellar pathway. Rats were trained to maintain their balance on a rotating rod and walk across a stationary beam; rod and beam performance, locomotor activity, and gait were assessed prior to and after administration of the neurotoxin 3-acetylpyridine (3-AP). The administration of 3-AP led to an 85% loss of neurons in the inferior olive at one week after administration without evidence of recovery over the following 4 weeks. The lesion was accompanied by a 72% decrease in rotorod activity, a 3.1-fold increase in the time required to traverse a stationary beam, a 19% decrease in velocity and 31% decrease in distance moved in the open field, and alterations in both forepaw and hindpaw gait parameters, with a 19% increase in hindpaw stride width. The daily administration of nicotine (0.33 mg free base/kg) improved rotorod performance by 50%, an effect apparent following the first week of administration, and which did not improve further over time. Nicotine also normalized the increased hindpaw stride width induced by the lesion. The ability of nicotine to alleviate both rotorod and gait deficits induced by 3-AP were prevented by the administration of the nicotinic antagonist mecamylamine (0.8 mg free base/kg) prior to the daily administration of nicotine. The effects of varenicline were dose-related and doses of 1.0 and 3.0 mg free base/kg daily improved rotorod performance by approximately 50% following the first week of administration. Further, varenicline did not alter the time required for animals to traverse the stationary beam, but did improve the ability of rats to maintain their balance on the beam by increasing lateral tail movements following 3 weeks of administration at doses of 0.3 and 1.0 mg free base/kg daily. Further, doses of nicotine and varenicline that improved the impaired balance and gait did not affect any measure of locomotor activity in the open field. Results provide evidence that nicotinic agonists are of benefit for alleviating some of the behavioral deficits in olivocerebellar ataxia and warrant further studies to elucidate the specific mechanism(s) involved.

Nicotine has been shown in a variety of studies to improve cognitive function including learning, memory and attention. Nicotine both stimulates and desensitizes nicotinic receptors, thus acting both as an agonist and a net antagonist. The relative roles of these two actions for nicotine-induced cognitive improvement have not yet been fully determined. We and others have found that acute nicotinic antagonist treatment can improve learning and attention. Nicotine acts on a variety of nicotinic receptor subtypes. The relative role and interactions of neuronal nicotinic receptor subtypes for cognition also needs to be better characterized. Nicotine acts on nicotinic receptors in a wide variety of brain areas. The role of some of these areas such as the hippocampus has been relatively well studied but other areas like the thalamus, which has the densest nicotinic receptor concentration are still only partially characterized. In a series of studies we characterized nicotinic receptor actions, anatomic localization and circuit interactions, which are critical to nicotine effects on the cognitive functions of learning, memory and attention. The relative role of increases and decreases in nicotinic receptor activation by nicotine were determined in regionally specific studies of the hippocampus, the amygdala, the frontal cortex and the mediodorsal thalamic nucleus with local infusions of antagonists of nicotinic receptor subtypes (α7 and α4β2). The understanding of the functional neural bases of cognitive function is fundamental to the more effective development of nicotinic drugs for treating cognitive dysfunction.

The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4β2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(β2)2 nAChRs.

Theraphosid spider venoms can block neurotransmission in vertebrate nerve-muscle preparations in vitro, but few of the components involved have been characterized. In this work, we describe the neuromuscular activity of venom from the Brazilian theraphosid Vitalius dubius and report the purification and pharmacological characterization of VdTX-1, a 728 Da toxin that blocks nicotinic receptors. Neuromuscular activity was assayed in chick biventer cervicis preparations and muscle responses to exogenous ACh and KCl were determined before and after incubation with venom or toxin. Changes in membrane resting potential were studied in mouse diaphragm muscle. The toxin was purified by a combination of filtration through Amicon® filters, cation exchange HPLC and RP-HPLC; toxin purity and mass were confirmed by mass spectrometry. Venom caused progressive neuromuscular blockade and muscle contracture; the blockade but not the contracture was reversible by washing. Venom attenuated contractures to exogenous ACh and KCl. Filtration yielded low (LM,5 kDa) and high (HM,5 kDa) fractions, with the latter reproducing the contracture seen in venom but with a slight and progressive twitch blockade. The LM fraction caused reversible blockade and attenuated contractures to ACh, but had no effect on contractures to KCl. VdTX-1 (728 Da) purified from the LM fraction was photosensitive and reduced the E(max) to ACh in biventer cervicis muscle without affecting the EC₅₀; VdTX-1 also abolished carbachol-induced depolarizations. V. dubius venom contains at least two components that affect vertebrate neurotransmission. One component, VdTX-1, blocks nicotinic receptors non-competitively to produce reversible blockade without muscle contracture.

The present study describes our ongoing efforts toward the discovery of drugs that selectively target nAChR subtypes. We exploited knowledge on nAChR ligands and their binding site that were previously identified by our laboratory through virtual screenings and identified benzamide analogs as a novel chemical class of neuronal nicotinic receptor (nAChR) ligands. The lead molecule, compound 1 (4-(allyloxy)-N-(6-methylpyridin-2-yl)benzamide) inhibits nAChR activity with an IC₅₀ value of 6.0 (3.4-10.6) μM on human α4β2 nAChRs with a ∼5-fold preference against human α3β4 nAChRs. Twenty-six analogs of compound 1 were also either synthesized or purchased for structure-activity relationship (SAR) studies and provided information relating the chemical/structural properties of the molecules to their ability to inhibit nAChR activity. The discovery of subtype-selective ligands of nAChRs described here should contribute significantly to our understanding of the involvement of specific nAChR subtypes in normal and pathophysiological states.

The 15q25 gene cluster contains genes that code for the α5, α3, and β4 nicotinic acetylcholine receptor (nAChRs) subunits, and in human genetic studies, has shown the most robust association with smoking behavior and nicotine dependence to date. The limited available animal studies implicate a role for the α5 and β4 nAChR subunits in nicotine dependence and withdrawal; however studies focusing on the behavioral role of the α3β4* nAChR receptor subtype in nicotine dependence are lacking. Because of the apparent role of the α3β4* nAChR subtype in nicotine dependence, the goal of the current study was to better evaluate the involvement of this subtype in nicotine mediated behavioral responses. Using the selective α3β4* nAChR antagonist, α-conotoxin AuIB, we assessed the role of α3β4* nAChRs in acute nicotine, nicotine reward, and physical and affective nicotine withdrawal. Because α5 has also been implicated in nicotine dependence behaviors in mice and can form functional receptors with α3β4*, we also evaluated the role of the α3β4α5* nAChR subtype in nicotine reward and somatic nicotine withdrawal signs by blocking the α3β4* nAChR subtype in α5 nAChR knockout mice with AuIB. AuIB had no significant effect on acute nicotine behaviors, but dose-dependently attenuated nicotine reward and physical withdrawal signs, with no significant effect in affective withdrawal measures. Interestingly, AuIB also attenuated nicotine reward and somatic signs in α5 nAChR knockout mice. This study shows that α3β4* nAChRs mediate nicotine reward and physical nicotine withdrawal, but not acute nicotine behaviors or affective nicotine withdrawal signs in mice. The α5 subunit is not required in the receptor assembly to mediate these effects. Our findings suggest an important role for the α3β4* nAChR subtype in nicotine reward and physical aspects of the nicotine withdrawal syndrome.

The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in different regions of the brain and is associated with cognitive function as well as anxiety. Agonists and positive allosteric modulators (PAMs) of the α7 subtype of nAChRs have been shown to improve cognition. Previously nicotine, which activates both α7 and non-α7 subtypes of nAChRs, has been shown to have an anxiogenic effect in behavioral tests. In this study, we compared the effects of the α7-selective agonist (PNU-282987) and PAM (PNU-120596) in a variety of behavioral tests in Sprague Dawley rats to look at their effects on learning and memory as well as anxiety. We found that neither PNU-282987 nor PNU-120596 improved spatial-learning or episodic memory by themselves. However when cognitive impairment was induced in the rats with scopolamine (1 mg/kg), both PNU-120596 and PNU-282987 were able to reverse this memory impairment and restore it back to normal levels. While PNU-120596 reversed the scopolamine-induced cognitive impairment, it did not have any adverse effect on anxiety. PNU-282987 on the other hand displayed an increase in anxiety-like behavior at a higher dose (10 mg/kg) that was significantly reduced by the serotonin 5-HT1a receptor antagonist WAY-100135. However the α7 receptor antagonist methyllycaconitine was unable to reverse these anxiety-like effects seen with PNU-282987. These results suggest that α7 nAChR PAMs are pharmacologically advantageous over agonists, and should be considered for further development as therapeutic drugs targeting the α7 receptors.

Background Cigarette smoking is associated with symptomatic vasospasm after subarachnoid hemorrhage (SAH). Methods Rat basilar arteries of a normal group and SAH groups (1 hour, 2 days, and 1 week) were removed from the brain and cut into spiral preparations. Results A central nervous system (CNS) nicotinic acetylcholine receptor (nAChR) and autonomic ganglionic nAChR antagonist (mecamylamine) and skeletal muscle nAChR antagonist (gallamine) concentration-dependently attenuated the nicotine-induced contraction. An autonomic ganglionic nAChR antagonist (hexamethonium) did not affect nicotine-induced contractions in normal rats or rats with SAH. The various nAChR antagonists showed no significant differences in their effects between normal and SAH (1 hour, 2 days, and 1 week) rats. An L-type Ca 2+ channel antagonist (nifedipine) attenuated the nicotine-induced contraction in a concentration dependent manner. Inhibition by nifedipine was significantly enhanced in the 1-hour and 2-day SAH groups compared with normal and 1-week SAH groups. Levcromakalim showed a greater attenuation of nicotine-induced contraction in SAH (1 hour, 2 days, and 1 week) than in normal rats. Conclusions Nicotine-induced contraction of the rat basilar artery involved the CNS nAChR subfamily, skeletal muscle nAChR subfamily, and L-type Ca 2+ channel pathways. SAH did not affect any of the subfamilies of nAChR, but the Ca 2+ channel was reduced and the adenosine triphosphate–sensitive K + channel was enhanced by SAH.

Kynurenic acid (KYNA), a major tryptophan metabolite, is a glutamate receptor antagonist, which is also reported to inhibit α7 nicotinic acetylcholine receptors (α7nAChRs). Due to variations in experimental approaches, controversy has arisen regarding the ability of KYNA to directly influence α7nAChR function. Here we summarize current concepts of KYNA neurobiology and review evidence pertaining to the proposed role of KYNA as an endogenous modulator of α7nAChRs and synaptic transmission. As dysfunction of α7nAChRs plays a major role in the pathophysiology of central nervous system disorders, elucidation of KYNA's action on this receptor subtype has significant therapeutic implications.

Nicotine and opioids share several behavioral and rewarding properties. Although both opioids and nicotine have their own specific mechanism of action, there is empirical and experimental evidence of interactions between these drugs. We studied receptor-level interactions of nicotine and morphine at α4β2, α7 and α3(⁎) nicotinic acetylcholine receptors. [(3)H]epibatidine displacement was used to determine if morphine binds competitively to nicotinic acetylcholine receptors. Functional interactions of morphine and nicotine were studied with calcium fluorometry and (86)Rb(+) efflux assays. Morphine displaced [(3)H]epibatidine from nicotinic agonist binding sites in all cell lines studied. The Ki values for morphine were 13.2μM in SH-EP1-hα4β2 cells, 0.16μM and 126μM in SH-SY5Y cells and 43.7μM in SH-EP1-hα7 cells. In SH-EP1-hα4β2 cells expressing α4β2 nicotinic acetylcholine receptors, morphine acted as a partial agonist of (86)Rb(+) efflux comparable to cytisine (with EC50 values of 53.3μM for morphine and 5.38μM for cytisine). The effect of morphine was attenuated concentration-dependently by the nicotinic antagonist mecamylamine. In the SH-SY5Y cell line expressing several subtypes of nicotinic acetylcholine receptors morphine had an inhibitory effect on nicotine induced (86)Rb(+) ion efflux mediated by α3(⁎) nicotinic acetylcholine receptors. These results suggest that morphine acts as a partial agonist at α4β2 nicotinic acetylcholine receptors and as a weak antagonist at α3(⁎) nicotinic acetylcholine receptors.

Nicotinic agonists have been shown in a variety of studies to improve cognitive function. Since nicotinic receptors are easily desensitized by agonists, it is not completely clear to what degree receptor desensitization or receptor activation are responsible for nicotinic agonist-induced cognitive improvement. In the current study, the effect of the neuronal nicotinic cholinergic α4β2 receptor antagonist dihydro-β-erythroidine (DHβE) and the α7 nicotinic receptor antagonist methyllycaconitine (MLA) on attentional function was determined. Adult female Sprague-Dawley rats were trained on the visual signal detection task. They were required to discriminate whether or not a light signal occurred on a trial and respond with a lever press on one side after a signal and the opposite side after the absence of a signal in order to receive a food pellet reinforcer. Acute administration of the α4β2 antagonist DHβE improved attentional function either alone or in reversing the attentional impairment caused by the NMDA glutamate antagonist dizocilpine (MK-801). Acute administration of MLA also significantly attenuated the dizocilpine-induced attentional impairment. In previous research we have shown that the α4β2 nicotinic desensitizing agent and partial agonist sazetidine-A also was effective in reversing dizocilpine-induced attentional impairments on the signal detection task and that low doses of the general nicotinic antagonist mecamylamine improved learning and memory. The current studies indicate that blockade of nicotinic receptors can effectively attenuate attentional impairments. Development of drugs that provide a net decrease in nicotinic receptor activity either through antagonism or desensitization could be worth exploring for beneficial effects for treating cognitive impairments.

Clinical reports suggest a positive association between fat consumption and the incidence of hyperactivity, impulsivity and cognitive abnormalities. To investigate possible mechanisms underlying these disturbances under short-term conditions, we examined in Sprague-Dawley rats the influence of 7-day consumption of a high-fat diet (HFD) compared to chow on anxiety, novelty-seeking and exploratory behaviors and also on acetylcholine (ACh) neurotransmission that may mediate these behaviors. The HFD consumption, which elevated circulating fatty acids but produced no change in caloric intake or body weight, stimulated novelty-seeking and exploration in an open field, while reducing anxiety in an elevated plus maze. Using the Ellman assay to measure ACh esterase (AChE) activity that breaks down ACh, the second experiment showed HFD consumption to significantly reduce AChE activity in the frontal cortex, hypothalamus and midbrain. With measurements of [¹²⁵I]-epibatidine or [¹²⁵I]-bungarotoxin binding to nicotinic ACh receptors (nAChRs) containing β2 or α7 subunits, respectively, the results also showed HFD consumption to increase both β2-nAChR binding in the medial prefrontal cortex and substantia nigra and α7-nAChR binding in the lateral and ventromedial hypothalamus. When treated with an acute dose of the nicotinic antagonist, mecamylamine (0.5 mg/kg, sc), the HFD animals responded with significantly reduced exploratory and novelty-seeking behaviors, whereas the chow-consuming rats exhibited no response. These findings suggest that the exploratory and novelty-seeking behaviors induced by dietary fat may be mediated by enhanced nicotinic cholinergic activity, which is accompanied by increased density of β2-nAChRs in cortical and midbrain regions associated with impulsivity and locomotor activity and of α7-nAChRs in hypothalamic regions associated with arousal and energy balance.

Subtype selective molecules for α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4β2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3β4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4β2 nAChRs and human α3β4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4β2 nAChRs.

In the hippocampus, activation of nicotinic receptors that include α4 and β2 subunits (α4β2*) facilitates memory formation. α4β2* receptors may also play a role in nicotine withdrawal, and their loss may contribute to cognitive decline in aging and Alzheimer's disease (AD). However, little is known about their cellular function in the hippocampus. Therefore, using optogenetics, whole cell patch clamping and voltage-sensitive dye (VSD) imaging, we measured nicotinic excitatory postsynaptic potentials (EPSPs) in hippocampal CA1. In a subpopulation of inhibitory interneurons, release of ACh resulted in slow depolarizations (rise time constant 33.2 ± 6.5 ms, decay time constant 138.6 ± 27.2 ms) mediated by the activation of α4β2* nicotinic receptors. These interneurons had somata and dendrites located in the stratum oriens (SO) and stratum lacunosum-moleculare (SLM). Furthermore, α4β2* nicotinic EPSPs were largest in the SLM. Thus, our data suggest that nicotinic EPSPs in hippocampal CA1 interneurons are predominantly mediated by α4β2* nicotinic receptors and their activation may preferentially affect extrahippocampal inputs in SLM of hippocampal CA1.

Chronic pain is inadequately managed with currently available classes of analgesic drugs. Recently, peptide antagonists of the α9α10 nicotinic acetylcholine receptor were shown to be analgesic. The present study was conducted to characterize a novel small molecule, non-peptide antagonist at nicotinic receptors. The tetrakis-quaternary ammonium compound ZZ-204G was evaluated for functional activity on cloned nicotinic receptors expressed in Xenopus oocytes. In-vivo efficacy was assessed in rat models of tonic inflammatory pain (formalin test), neuropathic pain (chronic constriction nerve injury), and thermal nociception (tail flick test). ZZ-204G was an antagonist at nicotinic receptors inhibiting the α9α10 subtype with an IC₅₀ of 0.51 (0.35-0.72) nM. Antagonist activity at other nicotinic subtypes (α1β1δe, α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, α6/α3β2β3, α6/α3β4 and α7) was 10-1000-fold lower than at the α9α10 subtype. In competition binding assays, the k(i) of ZZ-204G at γ-aminobutyric acid(A), serotonin(3), γ-aminobutyric acid(B), κ- and μ-opioid receptors was 1000- to >10,000-fold lower than at α9α10 nicotinic receptors. Parenteral administration of ZZ-204G dose-dependently decreased nociceptive behaviors (paw flinches) in the formalin test and mechanical hyperalgesia in the chronic constriction nerve injury model of neuropathic pain. ZZ-204G was not antinociceptive in the tail flick assay. Results from the rotarod assay indicated that lower doses of ZZ-204G that were analgesic did not alter motor function. In summary, ZZ-204G represents a prototype small molecule antagonist for α9α10 nicotinic receptors and provides a novel molecular scaffold for analgesic agents with the potential to treat chronic inflammatory or neuropathic pain.

Nicotinic acetylcholine receptors are ligand-gated ion conducting transmembrane channels from the Cys-loop receptor super-family. The α4β2 subtype is the predominant heteromeric subtype of nicotinic receptors found in the brain. Allosteric modulators for α4β2 receptors interact at a site other than the orthosteric site where acetylcholine binds. Many compounds which act as allosteric modulators of the α4β2 receptors have been identified, with both positive and negative effects. Such allosteric modulators either increase or decrease the response induced by agonist on the α4β2 receptors. Here we discuss the concept of allosterism as it pertains to the α4β2 receptors and summarize the important features of allosteric modulators for this nicotinic receptor subtype.

Significant advances in human functional brain imaging offer new opportunities for direct observation of the effects of nicotine, novel nicotinic agonists and nicotinic antagonists on human cognitive and behavioral performance. Careful research over the last decade has enabled investigators to explore the role of nicotinic systems on the functional neuroanatomy and neural circuitry of cognitive tasks in domains such as selective attention, working memory, episodic memory, cognitive control, and emotional processing. In addition, recent progress in understanding functional connectivity between brain regions utilized during cognitive and emotional processes offers new opportunities for examining drug effects on network-related activity. This review will critically summarize available nicotinic functional brain imaging studies focusing on the specific cognitive domains of attention, memory, behavioral control, and emotional processing. Generally speaking, nicotine appears to increase task-related activity in non-smokers and deprived smokers, but not active smokers. By contrast, nicotine or nicotinic stimulation decreases the activity of structures associated with the default mode network. These particular patterns of activation and/or deactivation may be useful for early drug development and may be an efficient and cost-effective method of screening potential nicotinic agents. Further studies will have to be done to clarify whether such activity changes correlate with cognitive or affective outcomes that are clinically relevant. The use of functional brain imaging will be a key tool for probing pathologic changes related to brain illness and for nicotinic drug development.

The acute toxicity of organophosphorus (OP) nerve agents arises from accumulation of acetylcholine (ACh) and overstimulation of ACh receptors. The mainstay of current pharmacotherapy is the competitive muscarinic antagonist, atropine. Nicotinic antagonists have not been used due to the difficulties of administering a dose of a competitive neuromuscular blocker sufficient to antagonise the effects of excessive ACh, but not so much that it paralyses the muscles. An alternative approach would be to use a noncompetitive antagonist whose effects would not be overcome by increasing ACh concentrations. This study demonstrates that the compound 1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium), which blocks open nicotinic ion channels noncompetitively, is able to reverse the neuromuscular paralysis after nerve agent poisoning in vitro and to protect guinea pigs against poisoning by nerve agents when used as part of a therapeutic drug combination including a muscarinic antagonist. In contrast to the oxime HI-6, this compound was equally effective in protecting against poisoning by sarin or tabun. Further studies should identify more effective compounds with this action and optimise doses for protection against nerve agent poisoning in vivo.

Exposure to nicotine causes a broad range of biological and molecular effects on osteoblasts which are known to play a crucial role in bone metabolism and fracture healing. Most effects of nicotine on the osteoblasts are long-term adaptations at the genomic level. To identify the nicotine-regulated genes, the Agilent technologies whole human genome gene expression microarray was performed on RNA samples from osteoblast-like cells, MG-63, exposed to 100 μM nicotine. Repeat and cross-controlled microarray analyses revealed 842 genes whose expression was consistently altered at P

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 μM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.

Recently, we reported that intracerebroventricularly (i.c.v.) administered (±)-epibatidine (a non-selective agonist of nicotinic acetylcholine receptors) elevates plasma noradrenaline and adrenaline through brain nicotinic acetylcholine receptor-mediated mechanisms in rats. In the present study, we characterized the receptors involved in these responses using selective agonists and antagonists of nicotinic acetylcholine receptor subtypes in anesthetized rats. (±)-Epibatidine (5 and 10nmol/animal, i.c.v.) and (-)-nicotine (250 and 500nmol/animal, i.c.v.) both elevated plasma noradrenaline and adrenaline (adrenaline>noradrenaline) but the former was more efficient than the latter. The (±)-epibatidine (5nmol/animal, i.c.v.)-induced elevation of plasma catecholamines was reduced by dihydro-β-erythroidine (a selective antagonist of α4β2 nicotinic acetylcholine receptors) (100 and 300nmol/animal, i.c.v.), while methyllycaconitine (a selective antagonist of α7 nicotinic acetylcholine receptors) (100 and 300nmol/animal, i.c.v.) had no effect on the (±)-epibatidine-induced responses. RJR-2403 (a selective agonist of α4β2 nicotinic acetylcholine receptors) (2.5 and 5μmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline (adrenaline>noradrenaline), while PNU-282987 (a selective agonist of α7 nicotinic acetylcholine receptors) (2.5 and 5μmol/animal, i.c.v.) had no effect. Furthermore, the RJR-2403 (5μmol/animal, i.c.v.)-induced responses were abolished by acute bilateral adrenalectomy. Immunohistochemical procedures demonstrated the expression of α4 and β2 nicotinic acetylcholine receptor subunits on the spinally projecting hypothalamic paraventricular neurons. Taken together, brain α4β2 nicotinic acetylcholine receptors seem to be involved in the secretion of noradrenaline and adrenaline from adrenal medulla in rats.

There is a well-established connection between smoking and depression. Depressed individuals are over-represented among smokers, and ex-smokers often experience increased depressive symptoms immediately after stopping smoking. Nicotine in tobacco binds, activates and desensitizes nicotinic acetylcholine receptors (nAChRs), but it is not known whether activation or desensitization is more important for the effects of nicotine on depressive symptoms. Here we review, based on clinical and preclinical studies of nicotinic drugs, the hypothesis that blockade (rather than activation) of neuronal nAChRs might be important for the effects of nicotinic agents on depressive symptoms. The endogenous neurotransmitter for nAChRs is acetylcholine, and the effects of nicotine on depression-like behaviors support the idea that dysregulation of the cholinergic system might contribute to the etiology of major depressive disorder. Thus, pharmacological agents that limit acetylcholine signaling through neuronal nAChRs might be promising for the development of novel antidepressant medications.

AZ11637326 (5'-(2-fluoro[3,4,5(-3)H3]phenyl)-spiro[1-azabicyclo [2.2.2]octane-3,2'(3'H)-furo[2,3-b]pyridine) is a potent partial agonist at the human alpha7 neuronal nicotinic receptor with sub-nanomolar affinity for the human and rat alpha7 [(125)I]alpha-bungarotoxin binding sites. In a search for novel agonist radioligands and imaging ligands for the alpha7 nicotinic receptor, [(3)H]AZ11637326 was synthesized and its in vitro membrane binding properties were characterized. [(3)H]AZ11637326 bound to halpha7-HEK membranes with high specificity (95%), high affinity (230 pM) and a B(max) of 460 fmol/mg. The rank order of affinity of nicotinic standards determined with [(3)H]AZ11637326 strongly correlated with those determined using the classical alpha7 antagonist [(125)I]alpha-bungarotoxin, indicating that [(3)H]AZ11637326 bound to halpha7-HEK membranes with an alpha7 nicotinic-like pharmacology. The K(i) values for the standards were on average 2.3-fold lower affinity than determined using the prototypical alpha7 nicotinic antagonist [(125)I]alpha-bungarotoxin. Because [(3)H]AZ11637326 specific binding is rapid and reversible, the K(i) values determined using this ligand are more accurate estimates of affinity than those determined using the kinetically sluggish [(125)I]alpha-bungarotoxin. [(3)H]AZ11637326 also bound to a high affinity (510 pM), nicotine-sensitive site on rat hippocampal membranes with an average B(max) of 55 fmol/mg. With rat hippocampal membranes, the nicotine-sensitive fraction of total binding was sub-optimal for a radioligand (~50%), yet the potencies and rank order of the K(i) values for standards were consistent with an alpha7 nicotinic pharmacology. Overall, these studies indicate that [(3)H]AZ11637326 is a useful new in vitro probe of the alpha7 nicotinic receptor agonist site and support its potential utility for in vivo receptor occupancy studies.

The treatment of chronic pain is hampered by various issues including multiple underlying mechanisms contributing to disease pathology and treatment-related toxicity concerns. These can be partially circumvented by combining mechanistically distinct drugs with the aim of selectively potentiating analgesia as opposed to side-effects. This approach has been used to assess the antinociceptive efficacy of the nicotinic acetylcholine (nACh) receptor agonist ABT-594 when combined with the antiepileptic drug gabapentin, the mu-opioid receptor agonist morphine or the antidepressant drug duloxetine in the rat formalin test. Alone, ABT-594 (0.01-0.3 mg/kg) dose-dependently attenuated spontaneous flinching behaviour during first (P1) and second (P2) phase. Similarly, P1, interphase and P2 flinching were variously attenuated by gabapentin (25-200 mg/kg), morphine (0.3-3 mg/kg) and duloxetine (3-60 mg/kg). Remarkably, a completely inactive dose of ABT-594 reduced the dose of gabapentin required to produce antinociception during P1 by 4-8 fold and during P2 by 8-16 fold. This striking potentiation was blocked by mecamylamine and indicative of analgesic synergy. Similar, albeit less consistent results (3-10 fold potency increase) were obtained with morphine/ABT-594. Although a 3 fold increase in P2 antinociceptive potency was obtained with duloxetine in the presence of ABT-594, a corresponding increase in efficacy was lacking. Indeed, a mechanistically relevant reduction in antinociceptive efficacy and potency of duloxetine/ABT-594 occurred during interphase. Thus, activation of the nicotinic cholinergic system differentially modulates the antinociceptive actions of distinct mechanism of action compounds, and provides a novel framework for nACh receptor modulators mediating analgesia in the putative absence of adverse events associated with this mechanism of action.

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (alphabetagammadelta) and neuronal (alpha(7), alpha(3)beta(2), and alpha(4)beta(2)) nicotinic acetylcholine receptors (nAChRs) with highest affinity for alpha(7)-nAChRs. The high resolution (1.5 A) crystal structure revealed haditoxin to be a homodimer, like kappa-neurotoxins, which target neuronal alpha(3)beta(2)- and alpha(4)beta(2)-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain alpha-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain alpha-neurotoxins and kappa-neurotoxins notwithstanding, haditoxin exhibited unique blockade of alpha(7)-nAChRs (IC(50) 180 nm), which is recognized by neither short-chain alpha-neurotoxins nor kappa-neurotoxins. This is the first report of a dimeric short-chain alpha-neurotoxin interacting with neuronal alpha(7)-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.

By linking two or three mecamylamine or 2,2,6,6-tetramethylpiperidine (TMP) molecules together via a linear lipophilic bis-methylene linker or a specially designed conformationally restricted tris-linker, a series of bis- and tris-tertiary amine analogs has been synthesized and evaluated as potent antagonists at nAChRs mediating nicotine-evoked [ 3 H]dopamine release from rat striatal slices. Compounds 7e , 14b and 16 demonstrated high potency in decreasing nicotine-evoked [ 3 H]dopamine release (IC 50 = 2.2, 46, and 107 nM, respectively). The preliminary structure–activity data obtained with these new analogs suggest the importance of the length of the methylene linker in the bis-analog series. Such bis-tertiary amino analogs may provide a new strategy for the design of drugable ligands that have high inhibitory potency against nAChRs mediating nicotine-evoked dopamine release in striatum, which have been suggested to be target receptors of interest in the development of potential smoking cessation therapies.

A set of semi-rigid cyclic and acyclic bis-quaternary ammonium analogs, which were part of a drug discovery program aimed at identifying antagonists at neuronal nicotinic acetylcholine receptors, were investigated to determine structural requirements for affinity at the blood-brain barrier choline transporter (BBB CHT). This transporter may have utility as a drug delivery vector for cationic molecules to access the central nervous system. In the current study, a virtual screening model was developed to aid in rational drug design/ADME of cationic nicotinic antagonists as BBB CHT ligands. Four 3D-QSAR comparative molecular field analysis (CoMFA) models were built which could predict the BBB CHT affinity for a test set with an r(2)

alpha-Conotoxins are peptide neurotoxins isolated from venomous cone snails that display exquisite selectivity for different subtypes of nicotinic acetylcholine receptors (nAChR). They are valuable research tools that have profound implications in the discovery of new drugs for a myriad of neuropharmacological conditions. They are characterized by a conserved two-disulfide bond framework, which gives rise to two intervening loops of extensively mutated amino acids that determine their selectivity for different nAChR subtypes. We have used a multistep synthetic combinatorial approach using alpha-conotoxin ImI to develop potent and selective alpha(7) nAChR antagonists. A positional scan synthetic combinatorial library was constructed based on the three residues of the n-loop of alpha-conotoxin ImI to give a total of 10,648 possible combinations that were screened for functional activity in an alpha(7) nAChR Fluo-4/Ca2+ assay, allowing amino acids that confer antagonistic activity for this receptor to be identified. A second series of individual alpha-conotoxin analogs based on the combinations of defined active amino acid residues from positional scan synthetic combinatorial library screening data were synthesized. Several analogs exhibited significantly improved antagonist activity for the alpha(7) nAChR compared with WT-ImI. Binding interactions between the analogs and the alpha(7) nAChR were explored using a homology model of the amino-terminal domain based on a crystal structure of an acetylcholine-binding protein. Finally, a third series of refined analogs was synthesized based on modeling studies, which led to several analogs with refined pharmacological properties. Of the 96 individual alpha-conotoxin analogs synthesized, three displayedor =10-fold increases in antagonist potency compared with WT-ImI.

International audience; Clothianidin is new neonicotinoid insecticide acting selectively on insect nicotinic acetylcholine receptors (nAChRs). Its effects on nAChRs expressed on cercal afferent/giant interneuron synapses and DUM neurons have been studied using mannitol-gap and whole-cell patch-clamp techniques, respectively. Bath-application of clothianidin-induced dose-dependent depolarizations of cockroach cercal afferent/giant interneuron synapses which were not reversed after wash-out suggesting a strong desensitization of postsynaptic interneurons at the 6th abdominal ganglion (A6). Clothinidin activity on the nerve preparation was characterized by an increased firing rate of action potentials which then ceased when the depolarization reached a peak. Clothianidin responses were insensitive to all muscarinic antagonists tested but were blocked by co-application of specific nicotinic antagonists methyllicaconitine, alpha-bungarotoxin and d-tubocurarine. In a second round of experiment, clothianidin actions were tested on DUM neurons isolated from the A6. There was a strong desensitization of nAChRs which was not affected by muscarinic antagonists, pirenzepine and atropine, but was reduced with nicotinic antagonist alpha-bungarotoxin. In addition, clothianidin-induced currents were completely blocked by methyllicaconitine suggesting that (1) clothianidin acted as a specific agonist of nAChR subtypes and (2) a small proportion of receptors blocked by MLA was insensitive to alpha-bungarotoxin. Moreover, because clothianidin currents were blocked by d-tubocurarine and mecamylamine, we provided that clothianidin was an agonist of both nAChRs: imidacloprid-sensitive nAChR1 and -insensitive nAChR2 subtypes.

The 5-HT(3) receptor antagonist, ondansetron, has been shown to correct the auditory gating deficit in medicated schizophrenia patients. Inhibition of 5-HT(3) receptors releases acetylcholine, the endogenous ligand for nicotinic acetylcholine receptors. The schizophrenia-related auditory gating deficit is modulated, in part, by nicotinic acetylcholine receptors, as is the mouse (DBA/2) model of the deficit. The present study assessed the effects of both acute and chronically administered ondansetron on auditory gating in DBA/2 mice. Auditory gating is defined as a decrease in amplitude of response to the second of a paired identical auditory stimulus presented 0.5 s following an initial auditory stimulus. Acute ondansetron administration at the lowest dose (0.1 mg/kg, IP) tested had no effect, while other doses (0.33 and 1 mg/kg, IP) produced improvements in auditory gating. The improvements were produced through both an increase in response to the first auditory stimulus and a decrease in the response to the second auditory stimulus. Co-administration of an alpha7 nicotinic acetylcholine receptor antagonist, alpha-bungarotoxin, or the alpha4beta2 nicotinic acetylcholine receptor antagonist dihydro-beta-erythroidine, with the 0.33 mg/kg dose of ondansetron blocked the improvement in auditory gating produced by ondansetron alone. There was no difference in response between the chronically injected mice and naive mice. Both showed improved auditory gating, thus, demonstrating no "carry over" effect of daily injections. These data demonstrate that indirect stimulation of nicotinic acetylcholine receptors by ondansetron can improve auditory gating parameters in DBA/2 mice.

Prenatal nicotine exposure (PNE) is correlated with breathing abnormalities in humans and other animals. Despite evidence that this relationship results from alterations in nicotinic acetylcholine receptors (nAChRs), the mechanisms are poorly understood. Here, we hypothesize that PNE blunts nAChR-mediated respiratory-related motor output. We also hypothesize that the PNE-induced changes in nAChRs leads to secondary alterations in glutamatergic neurotransmission. To test these hypotheses, we used an in vitro brainstem-spinal cord preparation and recorded C4 ventral root (C4 VR) nerve bursts from 0 to 4-day-old rats that were exposed to either nicotine (6mgkg(-1)day(-1)) or saline (control) in utero. Nicotine bitartrate, nAChR antagonists, NMDA and AMPA were applied to the brainstem compartment of a "split-bath" configuration, which physically separated the medulla from the spinal cord. Nicotine (0.2 or 0.5microM) increased peak C4 VR burst frequency by over 230% in control pups, but only 140% in PNE animals. The application of nAChR antagonists showed that these effects were mediated by the alpha4beta2 nAChR subtype with no effect on alpha7 nAChRs in either group. We also show that AMPA-mediated excitatory neurotransmission is enhanced by PNE, but NMDA-mediated neurotransmission is unaltered. These data and the work of others suggest that the PNE may functionally desensitize alpha4beta2 nAChRs located on the presynaptic terminals of glutamatergic neurons leading to less neurotransmitter release, which in turn up-regulates AMPA receptors on rhythm generating neurons.

Nicotine has been found in many studies to improve cognitive function. However, some studies have not found this effect and others have seen nicotine-induced impairments. Systemic administration bathes the brain with drugs. However, the brain is quite intricately organized with various regions playing very different roles in the bases of cognitive function. We have examined the role of nicotinic receptors in a variety of brain areas for memory. In the hippocampus and amygdala, local infusions of both alpha7 and alpha4beta2 antagonists methyllyaconitine (MLA) and dihydro-beta-erythroidine (DHbetaE) significantly impair memory. In the current studies we locally infused acute and chronic doses of MLA and DHbetaE into the mediodorsal thalamic nucleus and tested memory function on a 16-arm radial maze. The rats also received systemic nicotine to determine the impact of more generalized nicotine effects. Since nicotinic treatments are being developed for cognitive impairment of schizophrenia, interactions were studied with the antipsychotic drug clozapine. In the acute study, the 6.75 microg/side of DHbetaE improved working memory. Co-administration of MLA reversed the DHbetaE-induced improvement. Chronic DHbetaE infusions into the mediodorsal thalamic nucleus also improved working memory. Systemic nicotine reversed this effect. Clozapine had no significant interaction. Nicotinic alpha4beta2 receptors in the mediodorsal thalamic nucleus appear to play an opposite role with regard to working memory than those in the hippocampus and amygdala. Heterogeneity in response to nicotinic drugs given systemically may be due to anatomically distinct nicotinic systems in the brain and their unique roles in the neural bases of cognitive function.

Historically, the identification of alpha4beta2 nicotinic acetylcholine receptor ligands has been based on high-throughput radioligand binding, rubidium efflux assays and Ca++ flux assays using a fluorometric imaging plate reader (FLIPR). Among other approaches, low-throughput electrophysiological assays in Xenopus oocytes and two channel application "liquid filament" systems for mammalian cells have been commonly used. More recent technical innovations that have been introduced into the field of electrophysiology allow for automated simultaneous multi-channel operation. Here we report the development and optimization of a high-throughput electrophysiological assay for identifying functionally active alpha4beta2 nicotinic receptor ligands using such a system. Characterization of the test system yielded results comparable to those obtained by other investigators using conventional electrophysiological assays. For example, the concentration-response relationships obtained for alpha4beta2 receptor activation by acetylcholine and nicotine were best described by biphasic Hill equations, and the inhibition of alpha4beta2 receptor currents by the nicotinic antagonist dihydro-beta-erythroidine was consistent with previously published results. Functional up-regulation of alpha4beta2 receptors by prolonged exposure to nicotine or lower temperature was also confirmed. Using this methodology we were able to characterize the activation of alpha4beta2 receptors by multiple compounds in a mammalian cell expression system, exemplifying its utility for rapid identification of novel nicotinic ligands within a screening cascade. Our results demonstrate the utility of this electrophysiological tool for the discovery of alpha4beta2 nicotinic acetylcholine receptor ligands with potential applications in numerous clinical indications.

Maximal inhibition (Imax) of the agonist effect is an important pharmacological property of inhibitors that interact with multiple receptor subtypes that are activated by the same agonist and which elicit the same functional response. This report represents the first QSAR study on a set of 66 mono- and bis-quaternary ammonium salts that act as antagonists at neuronal nicotinic acetylcholine receptors mediating nicotine-evoked dopamine release, conducted using multi-linear regression (MLR) and neural network (NN) analysis with the maximal inhibition (Imax) values of the antagonists as target values. The statistical results for the generated MLR model were: r2 = 0.89, rmsd = 9.01, q2 = 0.83 and loormsd = 11.1; the statistical results for the generated NN model were: r2 = 0.89, rmsd = 8.98, q2 = 0.83 and loormsd = 11.2. The maximal inhibition values of the compounds exhibited a good correlation with the predictions made by the QSAR models developed, which provide a basis for rationalizing selection of compounds for synthesis in the discovery of effective and selective second generation inhibitors of nAChRs mediating nicotine-evoked dopamine release.