Nicotine-induced ER Stress and ASM Cell Proliferation is Mediated by α7nAChR and Chaperones-RIC-3 and TMEM35.

Nicotine exposure in the context of smoking or vaping worsens airway function. Although commonly thought to exert effects through the peripheral nervous system, we previously showed airway smooth muscle (ASM) expresses nicotinic acetylcholine receptors (nAChRs), particularly alpha7 subtype (α7nAChR) with functional effects on contractility and metabolism. However, the mechanisms of nAChR regulation and downstream effects in ASM are not fully understood. Using human ASM cells from non-asthmatics vs. mild-moderate asthmatics, we tested the hypothesis that nAChR-specific ER chaperones RIC-3 and TMEM35 promote cell surface localization of α7nAChR with downstream influence on its functionality: effects exacerbated by inflammation. We found that mild-moderate asthma and exposure to pro-inflammatory cytokines relevant to asthma promote chaperone and α7nAChR expression in ASM. Downstream, ER stress was linked to nicotine/α7nAChR signaling, where RIC-3 and TMEM35 regulate nicotine-induced ER stress, Ca2+ regulation and ASM cell proliferation. Overall, our data highlights the importance α7nAChR chaperones in mediating and modulating nicotine effects in ASM towards airway contractility and remodeling.

Alcohol and the dopamine system.

The mesolimbic dopamine pathway plays a major role in drug reinforcement and is likely involved also in the development of drug addiction. Ethanol, like most addictive drugs, acutely activates the mesolimbic dopamine system and releases dopamine, and ethanol-associated stimuli also appear to trigger dopamine release. In addition, chronic exposure to ethanol reduces the baseline function of the mesolimbic dopamine system. The molecular mechanisms underlying ethanol´s interaction with this system remain, however, to be unveiled. Here research on the actions of ethanol in the mesolimbic dopamine system, focusing on the involvement of cystein-loop ligand-gated ion channels, opiate receptors, gastric peptides and acetaldehyde is briefly reviewed. In summary, a great complexity as regards ethanol´s mechanism(s) of action along the mesolimbic dopamine system has been revealed. Consequently, several new targets and possibilities for pharmacotherapies for alcohol use disorder have emerged.

Neuronal Excitability in the Medial Habenula and Ventral Tegmental Area Is Differentially Modulated by Nicotine Dosage and Menthol in a Sex-Specific Manner.

The medial habenula (MHb) has been identified as the limiting factor for nicotine intake and facilitating nicotine withdrawal. However, few studies have assessed MHb neuronal excitability in response to nicotine, and, currently, a gap in knowledge is present for finding behavioral correlates to neuronal excitability in the region. Moreover, no study to date has evaluated sex or nicotine dosage as factors of excitability in the MHb. Here, we utilized an e-vape self-administration (EVSA) model to determine differences between sexes with different nicotine dosages ± menthol. Following this paradigm, we employed patch-clamp electrophysiology to assess key metrics of MHb neuronal excitability in relation to behavioral endpoints. We observed female mice self-administered significantly more than males, regardless of dosage. We also observed a direct correlation between self-administration behavior and MHb excitability with low-dose nicotine + menthol in males. Conversely, a high dose of nicotine ± menthol yields an inverse correlation between excitability and self-administration behavior in males only. In addition, intrinsic excitability in the ventral tegmental area (VTA) does not track with the amount of nicotine self-administered. Rather, they correlate to the active/inactive discrimination of mice. Using fast-scan cyclic voltammetry, we also observed that dopamine release dynamics are linked to reinforcement-related behavior in males and motivation-related behaviors in females. These results point to a sex-specific difference in the activity of the MHb and VTA leading to distinct differences in self-administration behavior. His could lend evidence to clinical observations of smoking and nicotine-use behavior differing between males and females.

Nicotine: From Discovery to Biological Effects.

Nicotine, the primary psychoactive agent in tobacco leaves, has led to the widespread use of tobacco, with over one billion smokers globally. This article provides a historical overview of tobacco and discusses tobacco dependence, as well as the biological effects induced by nicotine on mammalian cells. Nicotine induces various biological effects, such as neoangiogenesis, cell division, and proliferation, and it affects neural and non-neural cells through specific pathways downstream of nicotinic receptors (nAChRs). Specific effects mediated by α7 nAChRs are highlighted. Nicotine is highly addictive and hazardous. Public health initiatives should prioritize combating smoking and its associated risks. Understanding nicotine's complex biological effects is essential for comprehensive research and informed health policies. While potential links between nicotine and COVID-19 severity warrant further investigation, smoking remains a significant cause of morbidity and mortality globally. Effective public health strategies are vital to promote healthier lifestyles.

Electronic nicotine delivery systems (ENDS): A convenient means of smoking?

Electronic nicotine delivery systems (ENDS), which are becoming increasingly popular in many parts of the world, have recently become more sophisticated in terms of their more active content and better controlled vaporisation. This review begins by describing how cigarette smoking led to the development of ENDS as a means of combatting nicotine addiction. ENDS are usually categorised as belonging to one of only three main generations, but a fourth has been added in order to differentiate the latest, most powerful, most advanced and innovative that have improved heating efficiency. Descriptions of the principal substances contained in ENDS are followed by considerations concerning the risk of toxicity due to the presence of albeit low concentrations of such a variety of compounds inhaled over a long time, and the increasingly widespread use of ENDS as a means of smoking illicit drugs. We also review the most widely used pharmacotherapeutic approaches to smoking cessation, and recent epidemiological data showing that ENDS can help some people to stop smoking. However, in order to ensure their appropriate regulation, there is a need for higher-quality evidence concerning the health effects and safety of ENDS, and their effectiveness in discouraging tobacco smoking.

Contribution of the α5 nAChR Subunit and α5SNP to Nicotine-Induced Proliferation and Migration of Human Cancer Cells.

Nicotine in tobacco is known to induce tumor-promoting effects and cause chemotherapy resistance through the activation of nicotinic acetylcholine receptors (nAChRs). Many studies have associated the α5 nicotinic receptor subunit (α5), and a specific polymorphism in this subunit, with (i) nicotine administration, (ii) nicotine dependence, and (iii) lung cancer. The α5 gene CHRNA5 mRNA is upregulated in several types of cancer, including lung, prostate, colorectal, and stomach cancer, and cancer severity is correlated with smoking. In this study, we investigate the contribution of α5 in the nicotine-induced cancer hallmark functions proliferation and migration, in breast, colon, and prostate cancer cells. Nine human cell lines from different origins were used to determine nAChR subunit expression levels. Then, selected breast (MCF7), colon (SW480), and prostate (DU145) cancer cell lines were used to investigate the nicotine-induced effects mediated by α5. Using pharmacological and siRNA-based experiments, we show that α5 is essential for nicotine-induced proliferation and migration. Additionally, upon downregulation of α5, nicotine-promoted expression of EMT markers and immune regulatory proteins was impaired. Moreover, the α5 polymorphism D398N (α5SNP) caused a basal increase in proliferation and migration in the DU145 cell line, and the effect was mediated through G-protein signaling. Taken together, our results indicate that nicotine-induced cancer cell proliferation and migration are mediated via α5, adding to the characterization of α5 as a putative therapeutical target.

Proteomic responses in the human dopaminergic LUHMES cell line to imidacloprid and its metabolites imidacloprid-olefin and desnitro-imidacloprid.

Neonicotinoids (neonics) are amongst the most commonly used class of pesticides globally. In the United States, imidacloprid (IMI) is extensively used for agriculture and in other common applications such as house-hold pest control. Regular exposure to IMI, and several of its known metabolites including IMI-olefin and desnitro-imidacloprid (DN-IMI), has been shown to be harmful to many organisms including mammals, birds, and fish. Studies show that neonics bind human nicotinicacetylcholine receptors (nAChRs) and cause cellular toxicity. In the dopaminergic Lund human mesencephalic (LUHMES) cell line, IMI and other neonics (10-100 µM) have been recently shown to activate intracellular calcium signaling through nAChRs. Thus, we examined proteomic responses of LUHMES cells to a 48-h treatment with 50 µM IMI, IMI-olefin, or DN-IMI. Our findings show differential effects of these neonics on cellular protein expression. Bioinformatic analysis of significantly altered proteins indicates an effect of IMI, IMI-olefin, and DN-IMI on protein synthesis and ribosomal function. These findings suggest a role for protein synthesis and transcriptional regulation in neonic-mediated dopaminergic neurotoxicity.

Chemical Flavorants in Vaping Products Alter Neurobiology in a Sex-Dependent Manner to Promote Vaping-Related Behaviors.

Electronic nicotine delivery systems (ENDS) are distinctly different from combustible cigarettes because of the availability of flavor options. Subjective measures have been used to demonstrate that adults and adolescents prefer flavors for various reasons; (1) they are pleasing and (2) they mask the harshness of nicotine. Despite this, there have been few investigations into the molecular interactions that connect chemical flavorants to smoking or vaping-related behaviors. Here, we investigated the effects of three chemical flavorants (hexyl acetate, ethyl acetate, and methylbutyl acetate) that are found in green apple (GA) ENDS e-liquids but are also found in other flavor categories. We used a translationally relevant vapor self-administration mouse model and observed that adult male and female mice self-administered GA flavorants in the absence of nicotine. Using α4-mCherryα6-GFP nicotinic acetylcholine receptor (nAChR) mice, we observed that mice exposed to GA flavorants exhibited a sex-specific increase (upregulation) of nAChRs that was also brain-region specific. Electrophysiology revealed that mice exposed to GA flavorants exhibited enhanced firing of ventral tegmental area dopamine neurons. Fast-scan cyclic voltammetry revealed that electrically stimulated dopamine release in the nucleus accumbens core is increased in mice that are exposed to GA flavorants. These effects were similarly observed in the medial habenula. Overall, these findings demonstrate that ENDS flavors alone change neurobiology and may promote vaping-dependent behaviors in the absence of nicotine. Furthermore, the flavorant-induced changes in neurobiology parallel those caused by nicotine, which highlights the fact that nonmenthol flavorants may contribute to or enhance nicotine reward and reinforcement.SIGNIFICANCE STATEMENT The impact of flavors on vaping is a hotly debated topic; however, few investigations have examined this in a model that is relevant to vaping. Although a full understanding of the exact mechanism remains undetermined, our observations reveal that chemical flavorants in the absence of nicotine alter brain circuits relevant to vaping-related behavior. The fact that the flavorants investigated here exist in multiple flavor categories of vaping products highlights the fact that a multitude of flavored vaping products may pose a risk toward vaping-dependent behaviors even without the impact of nicotine. Furthermore, as the neurobiological changes have an impact on neurons of the reward system, there exists the possibility that nonmenthol flavorants may enhance nicotine reward and reinforcement.

Antagonising a novel toxin "T14" in Alzheimer's disease: Comparison of receptor blocker versus antibody effects in vitro.

A 14mer peptide, T14, is a possible signaling molecule driving neurodegeneration. Its levels are doubled in the Alzheimer brain, but its effects can be blocked at the target alpha-7 receptor by a cyclised variant, 'NBP14', which has beneficial effects, in a transgenic mouse model, on the behavioral and histochemical profile. Since the antagonism of T14 has evident therapeutic potential, we explore here an alternative method of preventing its action by comparing the efficacy of NBP14 with a proprietorial polyclonal antibody against T14, 'Ab-19', at inhibiting three distinct effects of the peptide in PC12 cells: calcium influx, cell viability and compensatory acetylcholinesterase (AChE) release. None of these three parameters was affected by either blocking agent when applied alone. However, both NBP14 and the Ab-19 exhibited a dose-dependent profile against the actions of T14 in all three scenarios: the least sensitive effect observed was in the lower dose range, for both the antibody and the receptor blocker, in antagonizing T14-triggered release of AChE: this parameter is interpreted as indirect compensation for the T14-induced compromise of cell viability, triggered by the enhanced influx of calcium through the initial binding of the peptide to an allosteric site on the alpha-7 receptor. As such, it is the most delayed and indirect index of T14 action and thus the least relatively impacted by lowest doses of either NBP14 or Ab-19. In all three scenarios however the effects of T14 are successfully offset by either agent and thus offer two potentially very different therapies against Alzheimer's disease.

Cannabidiol as a modulator of α7 nicotinic receptors.

Cannabidiol (CBD), an important terpenoid compound from marijuana with no psychoactive effects, has become of great pharmaceutical interest for several health conditions. As CBD is a multitarget drug, there is a need to establish the molecular mechanisms by which CBD may exert therapeutic as well as adverse effects. The α7 nicotinic acetylcholine receptor (α7 nAChR) is a cation-permeable ACh-gated channel present in the nervous system and in non-neuronal cells. It is involved in different pathological conditions, including neurological and neurodegenerative disorders, inflammation, and cancer. By high-resolution single-channel recordings and confocal microscopy, we here reveal how CBD modulates α7 nAChR ionotropic and metabotropic functions. CBD leads to a profound concentration-dependent decrease of α7 nAChR single-channel activity with an IC50 in the sub-micromolar range. The inhibition of α7 nAChR activity, which takes place through a membrane pathway, is neither mediated by receptor phosphorylation nor overcome by positive allosteric modulators and is compatible with CBD stabilization of resting or desensitized α7 nAChR conformational states. CBD modulation is complex as it also leads to the later appearance of atypical, low-frequency α7 nAChR channel openings. At the cellular level, CBD inhibits the increase in intracellular calcium triggered by α7 nAChR activation, thus decreasing cell calcium responses. The modulation of α7 nAChR is of pharmacological relevance and should be considered in the evaluation of CBD potential therapeutic uses. Thus, our study provides novel molecular information of CBD multiple actions and targets, which is required to set the basis for prospective applications in human health.

Dog and human bladders have different neurogenic and nicotinic responses in inner versus outer detrusor muscle layers.

The aim of this study was to investigate layer and species variations in detrusor muscle strip responses to myogenic, neurogenic, and nicotinic, and muscarinic receptor stimulations. Strips from bladders of 9 dogs and 6 human organ transplant donors were dissected from inner and outer longitudinal muscle layers, at least 1 cm above urethral orifices. Strips were mounted in muscle baths and maximal responses to neurogenic stimulation using electrical field stimulation (EFS) and myogenic stimulation using potassium chloride (KCl, 120 mM) determined. After washing and re-equilibration was completed, responses to nicotinic receptor agonist epibatidine (10 µM) were determined followed by responses to EFS and muscarinic receptor agonist bethanechol (30 µM) in continued presence of epibatidine. Thereafter, strips and full-thickness bladder sections from four additional dogs and three human donors were examined for axonal density and intramural ganglia. In dog bladders, contractions to KCl, epibatidine, and bethanechol were 1.5- to 2-fold higher in the inner longitudinal muscle layer, whereas contractions to EFS were 1.5-fold higher in the outer (both pre- and post-epibatidine). Human bladders showed 1.2-fold greater contractions to epibatidine in the inner layer and to EFS in the outer, yet no layer differences to KCl or bethanechol were noted. In both species, axonal density was 2- to 2.5-fold greater in the outer layer. Dogs had more intramural ganglia in the adventitia/serosa layer, compared with more internal layers and to humans. These findings indicate several layer-dependent differences in receptor expression or distribution, and neurogenic responses in dog and human detrusor muscles, and myogenic/muscarinic differences between dog versus humans.

Peripheral immune challenges elicit differential up-regulation of hippocampal cytokine and chemokine mRNA expression in a mouse model of the 15q13.3 microdeletion syndrome.

The human heterozygous 15q13.3 microdeletion is associated with neuropathological disorders, most prominently with epilepsy and intellectual disability. The 1.5 Mb deletion encompasses six genes (FAN1 [MTMR15], MTMR10, TRPM1, KLF13, OTUD7A, and CHRNA7); all but one (TRPM1) are expressed in the brain. The 15q13.3 microdeletion causes highly variable neurological symptoms, and confounding factors may contribute to a more severe phenotype. CHRNA7 and KLF13 are involved in immune system regulation and altered immune responses may contribute to neurological deficits. We used the Df[h15q13]/+ transgenic mouse model with a heterozygous deletion of the orthologous region (Het) to test the hypothesis that the microdeletion increases innate immune responses compared to wild type (WT). Male and female mice were acutely challenged with the bacteriomimetic lipopolysaccharide (LPS, 0.1 mg/kg, i.p.) or the viral mimetic polyinosinic:polycytidylic acid (Poly(I:C), 5 mg/kg). Hippocampal mRNA expression of pro-inflammatory cytokines and chemokines were determined three hours after injection using quantitative PCR analysis. In controls, expression was not affected by sex or genotype. LPS and Poly(I:C) resulted in significantly increased hippocampal expression of cytokines, chemokines, and interferon-γ (IFNγ), with more robust increases for TNF-α, IL-6, IL-1ß, CXCL1, and CCL2 by LPS, higher induction of IFNγ by Poly(I:C), and similar increases of CCL4 and CCL5 by both agents. Generally, Hets exhibited stronger responses than WT mice, and significant effects of genotype or genotype × treatment interactions were detected for CXCL1 and CCL5, and IL-6, IL-1ß, and CCL4, respectively, after LPS. Sex differences were detected for some targets. LPS but not Poly(I:C), reduced overnight burrowing independent of sex or genotype, suggesting that LPS induced sickness behavior. Thus, mice carrying the microdeletion have an increased innate immune response following a LPS challenge, but further studies will have to determine the extent and mechanisms of altered immune activation and subsequent contributions to 15q13.3 microdeletion associated deficits.

Nicotine patches in patients on mechanical ventilation for severe COVID-19: a randomized, double-blind, placebo-controlled, multicentre trial.

PURPOSE: Epidemiologic studies have documented lower rates of active smokers compared to former or non-smokers in symptomatic patients affected by coronavirus disease 2019 (COVID-19). We assessed the efficacy and safety of nicotine administered by a transdermal patch in critically ill patients with COVID-19 pneumonia. METHODS: In this multicentre, double-blind, placebo-controlled trial conducted in 18 intensive care units in France, we randomly assigned adult patients (non-smokers, non-vapers or who had quit smoking/vaping for at least 12 months) with proven COVID-19 pneumonia receiving invasive mechanical ventilation for up to 72 h to receive transdermal patches containing either nicotine at a daily dose of 14 mg or placebo until 48 h following successful weaning from mechanical ventilation or for a maximum of 30 days, followed by 3-week dose tapering by 3.5 mg per week. Randomization was stratified by centre, non- or former smoker status and Sequential Organ Function Assessment score (< or ≥ 7). The primary outcome was day-28 mortality. Main prespecified secondary outcomes included 60-day mortality, time to successful extubation, days alive and free from mechanical ventilation, renal replacement therapy, vasopressor support or organ failure at day 28. RESULTS: Between November 6th 2020, and April 2nd 2021, 220 patients were randomized from 18 active recruiting centers. After excluding 2 patients who withdrew consent, 218 patients (152 [70%] men) were included in the analysis: 106 patients to the nicotine group and 112 to the placebo group. Day-28 mortality did not differ between the two groups (30 [28%] of 106 patients in the nicotine group vs 31 [28%] of 112 patients in the placebo group; odds ratio 1.03 [95% confidence interval, CI 0.57-1.87]; p = 0.46). The median number of day-28 ventilator-free days was 0 (IQR 0-14) in the nicotine group and 0 (0-13) in the placebo group (with a difference estimate between the medians of 0 [95% CI -3-7]). Adverse events likely related to nicotine were rare (3%) and similar between the two groups. CONCLUSION: In patients having developed severe COVID-19 pneumonia requiring invasive mechanical ventilation, transdermal nicotine did not significantly reduce day-28 mortality. There is no indication to use nicotine in this situation.

Divergent Roles of α5 and ß4 Nicotinic Receptor Subunits in Food Reward and Nicotine-induced Weight Loss in Male Mice.

A major obstacle to successful smoking cessation is the prospect of weight gain. Despite a clear relationship between cigarette smoking and body weight, surprisingly little is known about the physiological and molecular mechanism by which nicotine affects energy homeostasis and food-motivated behaviors. Here we use loss-of-function mouse models to demonstrate that 2 nicotinic acetylcholine receptor (nAChR) subunits encoded by the CHRNA5-CHRNA3-CHRNB4 gene cluster, α5 and ß4, exhibit divergent roles in food reward. We also reveal that ß4-containing nAChRs are essential for the weight-lowering effects of nicotine in diet-induced obese mice. Finally, our data support the notion of crosstalk between incretin biology and nAChR signaling, as we demonstrate that the glycemic benefits of glucagon-like peptide-1 receptor activation partially relies on ß4-containing nAChRs. Together, these data encourage further research into the role of cholinergic neurotransmission in regulating food reward and the translational pursuit of site-directed targeting of ß4-containing nAChRs for treatment of metabolic disease.

The Human-Restricted Isoform of the α7 nAChR, CHRFAM7A: A Double-Edged Sword in Neurological and Inflammatory Disorders.

CHRFAM7A is a relatively recent and exclusively human gene arising from the partial duplication of exons 5 to 10 of the α7 neuronal nicotinic acetylcholine receptor subunit (α7 nAChR) encoding gene, CHRNA7. CHRNA7 is related to several disorders that involve cognitive deficits, including neuropsychiatric, neurodegenerative, and inflammatory disorders. In extra-neuronal tissues, α7nAChR plays an important role in proliferation, differentiation, migration, adhesion, cell contact, apoptosis, angiogenesis, and tumor progression, as well as in the modulation of the inflammatory response through the "cholinergic anti-inflammatory pathway". CHRFAM7A translates the dupα7 protein in a multitude of cell lines and heterologous systems, while maintaining processing and trafficking that are very similar to the full-length form. It does not form functional ion channel receptors alone. In the presence of CHRNA7 gene products, dupα7 can assemble and form heteromeric receptors that, in order to be functional, should include at least two α7 subunits to form the agonist binding site. When incorporated into the receptor, in vitro and in vivo data showed that dupα7 negatively modulated α7 activity, probably due to a reduction in the number of ACh binding sites. Very recent data in the literature report that the presence of the duplicated gene may be responsible for the translational gap in several human diseases. Here, we will review the studies that have been conducted on CHRFAM7A in different pathologies, with the intent of providing evidence regarding when and how the expression of this duplicated gene may be beneficial or detrimental in the pathogenesis, and eventually in the therapeutic response, to CHRNA7-related neurological and non-neurological diseases.

The α7 nAChR allosteric modulator PNU-120596 amends neuroinflammatory and motor consequences of parkinsonism in rats: Role of JAK2/NF-κB/GSk3ß/ TNF-α pathway.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and a leading cause of disability. The current gold standard for PD treatment, L-Dopa, has limited clinical efficacy and multiple side effects. Evidence suggests that activation of α7 nicotinic acetylcholine receptors (α7nAChRs) abrogates neuronal and inflammatory insults. Here we tested whether PNU-120596 (PNU), a type II positive allosteric modulator of α7 nAChR, has a critical role in regulating motor dysfunction and neuroinflammation correlated with the associated PD dysfunction. Neuroprotective mechanisms were investigated through neurobehavioral, molecular, histopathological, and immunohistochemical studies. PNU reversed motor incoordination and hypokinesia induced via the intrastriatal injection of 6-hydroxydopamine and manifested by lower falling latency in the rotarod test, short ambulation time and low rearing incidence in open field test. Tyrosine hydroxylase immunostaining showed a significant restoration of dopaminergic neurons following PNU treatment, in addition to histopathological restoration in nigrostriatal tissues. PNU halted striatal neuroinflammation manifested as a suppressed expression of JAK2/NF-κB/GSk3ß accompanied by a parallel decline in the protein expression of TNF-α in nigrostriatal tissue denoting the modulator anti-inflammatory capacity. Moreover, the protective effects of PNU were partially reversed by the α7 nAChR antagonist, methyllycaconitine, indicating the role of α7 nAChR modulation in the mechanism of action of PNU. This is the first study to reveal the positive effects of PNU-120596 on motor derangements of PD via JAK2/NF-κB/GSk3ß/ TNF-α neuroinflammatory pathways, which could offer a potential therapeutic strategy for PD.

Levo-tetrahydropalmatine inhibits α4ß2 nicotinic receptor response to nicotine in cultured SH-EP1 cells.

Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4ß2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 µM) induced inward currents, co-application of l-THP (3 µM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 µM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4ß2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4ß2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42ß23-nAChR) and cytisine-activated, low-sensitive one (α43ß22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4ß2-nAChR function, which may underlie its inhibition on nicotine addiction.

Role of prediction error and the cholinergic system on memory reconsolidation processes in mice.

The ability to make predictions based on stored information is a general coding strategy. A prediction error (PE) is a mismatch between expected and current events. Our memories, like ourselves, are subject to change. Thus, an acquired memory can become active and update its content or strength by a labilization-reconsolidation process. Within the reconsolidation framework, PE drives the updating of consolidated memories. In the past our lab has made key progresses showing that a blockade in the central cholinergic system during reconsolidation can cause memory impairment, while reinforcement of cholinergic activity enhances it. In the present work we determined that PE is a necessary condition for memory to reconsolidate in an inhibitory avoidance task using both male and female mice. Depending on the intensity of the unconditioned stimulus (US) used during training, a negative (higher US intensity) or positive (lower US intensity/no US) PE on a retrieval session modified the behavioral response on a subsequent testing session. Furthermore, we demonstrated that the cholinergic system modulates memory reconsolidation only when PE is detected. In this scenario administration of oxotremorine, scopolamine or nicotine after memory reactivation either enhanced or impaired memory reconsolidation in a sex-specific manner.

Novel Putative Positive Modulators of α4ß2 nAChRs Potentiate Nicotine Reward-Related Behavior.

The popular tobacco and e-cigarette chemical flavorant (-)-menthol acts as a nonselective, noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs), and contributes to multiple physiological effects that exacerbates nicotine addiction-related behavior. Menthol is classically known as a TRPM8 agonist; therefore, some have postulated that TRPM8 antagonists may be potential candidates for novel nicotine cessation pharmacotherapies. Here, we examine a novel class of TRPM8 antagonists for their ability to alter nicotine reward-related behavior in a mouse model of conditioned place preference. We found that these novel ligands enhanced nicotine reward-related behavior in a mouse model of conditioned place preference. To gain an understanding of the potential mechanism, we examined these ligands on mouse α4ß2 nAChRs transiently transfected into neuroblastoma-2a cells. Using calcium flux assays, we determined that these ligands act as positive modulators (PMs) on α4ß2 nAChRs. Due to α4ß2 nAChRs' important role in nicotine dependence, as well as various neurological disorders including Parkinson's disease, the identification of these ligands as α4ß2 nAChR PMs is an important finding, and they may serve as novel molecular tools for future nAChR-related investigations.

Activation of Neuronal Nicotinic Receptors Inhibits Acetylcholine Release in the Neuromuscular Junction by Increasing Ca2+ Flux through Cav1 Channels.

Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system and in several branches of the central nervous system. Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is the relationship between the nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in calcium transients were recorded using a calcium-sensitive dye. Nicotine hydrogen tartrate salt application (10 µM) decreased the amount of evoked ACh release, while the calcium transient increased in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil, and nitrendipine. These data allow us to suggest that neuronal nicotinic ACh receptor activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels.