Alpha4beta2 Research Articles

Insights into Crystallization of Neuronal Nicotinic α4β2 Receptor in Polarized Lipid Matrices

Obtaining high-resolution 3D structures of membrane proteins through X-ray crystallography remains a longstanding bottleneck in the field of structural biology. This challenge has led to the optimization of purification methods to acquire high-yielding, pure proteins suitable for crystallization. In this study, we performed crystallization screenings of purified human α4β2 nAChR using a polarized in meso method. After reconstituting the detergent-solubilized α4β2 nAChR into the LCP matrix, the samples were incubated in a polarized lipid matrix using the RMP@LMx device developed in our laboratory. The results showed that under these conditions, the α4β2-nAChR-LFC 16 complex gave a mobile fraction >0.8, suggesting that its diffusion in the polarized lipid matrix is favorable for crystal nucleation. Voltages above 70 mV restricted crystal formation due to sample dehydration. Furthermore, a lipid analysis using UPLC-ESI MS/MS revealed a profile necessary for preserving protein integrity and promoting diffusion across the LCP. We harvested a single crystal and subjected it to X-ray diffraction, resulting in reflections comparable to previous studies of the muscle-type nAChR from Torpedo californica. X-ray diffraction of a single crystal gave distinct low-resolution diffractions of protein nature. These findings lay the groundwork for further optimization of membrane protein crystallization in polarized in meso phases.

AKT2 modulates astrocytic nicotine responses in vivo.

A better understanding of nicotine neurobiology is needed to reduce or prevent chronic addiction, ameliorate the detrimental effects of nicotine withdrawal, and increase successful cessation of use. Nicotine binds and activates two astrocyte-expressed nicotinic acetylcholine receptors (nAChRs), α4β2 and α7. We recently found that Protein kinase B-β (Pkb-β or Akt2) expression is restricted to astrocytes in mice and humans. To determine if AKT2 plays a role in astrocytic nicotinic responses, we generated astrocyte-specific Akt2 conditional knockout (cKO) and full Akt2 KO mice for in vivo and in vitro experiments. For in vivo studies, we examined mice exposed to chronic nicotine for two weeks in drinking water (200 μg/mL) and following acute nicotine challenge (0.09, 0.2 mg/kg) after 24 hrs. Our in vitro studies used cultured mouse astrocytes to measure nicotine-dependent astrocytic responses. We validated our approaches using lipopolysaccharide (LPS) exposure inducing astrogliosis. Sholl analysis was used to measure glial fibrillary acidic protein responses in astrocytes. Our data show that wild-type (WT) mice exhibit increased astrocyte morphological complexity during acute nicotine exposure, with decreasing complexity during chronic nicotine use, whereas Akt2 cKO mice showed increased astrocyte morphology complexity. In culture, we found that 100μM nicotine was sufficient for morphological changes and blocking α7 or α4β2 nAChRs prevented observed morphologic changes. Finally, we performed conditioned place preference (CPP) in Akt2 cKO mice and found that astrocytic AKT2 deficiency reduced nicotine preference compared to controls. These findings show the importance of nAChRs and Akt2 signaling in the astrocytic response to nicotine.

Cholinergic Control of GnRH Neuron Physiology and Luteinizing Hormone Secretion in Male Mice: Involvement of ACh/GABA Cotransmission.

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.

An integrated approach for early in vitro seizure prediction utilizing hiPSC neurons and human ion channel assays.

Seizure liability remains a significant cause of attrition throughout drug development. Advances in stem cell biology coupled with an increased understanding of the role of ion channels in seizure offer an opportunity for a new paradigm in screening. We assessed the activity of 15 pro-seizurogenic compounds (7 CNS active therapies, 4 GABA receptor antagonists, and 4 other reported seizurogenic compounds) using automated electrophysiology against a panel of 14 ion channels (Nav1.1, Nav1.2, Nav1.6, Kv7.2/7.3, Kv7.3/7.5, Kv1.1, Kv4.2, KCa4.1, Kv2.1, Kv3.1, KCa1.1, GABA α1β2γ2, nicotinic α4β2, NMDA 1/2A). These were selected based on linkage to seizure in genetic/pharmacological studies. Fourteen compounds demonstrated at least one "hit" against the seizure panel and 11 compounds inhibited 2 or more ion channels. Next, we assessed the impact of the 15 compounds on electrical signaling using human-induced pluripotent stem cell neurons in microelectrode array (MEA). The CNS active therapies (amoxapine, bupropion, chlorpromazine, clozapine, diphenhydramine, paroxetine, quetiapine) all caused characteristic changes to electrical activity in key parameters indicative of seizure such as network burst frequency and duration. The GABA antagonist picrotoxin increased all parameters, but the antibiotics amoxicillin and enoxacin only showed minimal changes. Acetaminophen, included as a negative control, caused no changes in any of the parameters assessed. Overall, pro-seizurogenic compounds showed a distinct fingerprint in the ion channel/MEA panel. These studies highlight the potential utility of an integrated in vitro approach for early seizure prediction to provide mechanistic information and to support optimal drug design in early development, saving time and resources.

Kinetic modelling of ligand trapping inside acidic vesicles containing alpha-4 beta-2 nicotinic receptors as an explanation for long-term cessation effect of varenicline (Chantix).

Govind's et. al. 2017 paper proposes that the smoking cessation effect of varenicline (or epibatidine) is caused by these molecules being trapped inside acidic vesicles containing α4β2 nicotinic receptors. These ligands are weak bases with high affinity to these receptors. The trapping would lead to their slower release and longer desensitization of the receptors, thereby reducing nicotine upregulation. Meanwhile, nicotine, with lower pKa and lower binding affinity is not trapped within the vesicles. It is unclear whether the acidity of the vesicles and the presence of receptors is sufficient to explain the biphasic trapping behavior observed empirically. To better understand this phenomenon, we model flux of ligands between cellular compartments using Flick's first law of diffusion and the Nernst-Plank Equation. The ratio of neutral to charged ligands within each compartment is calculated via Henderson-Hasselbach equation. Main parameters in the model include the number of vesicles, receptors per vesicle, ratio of vesicles to non-vesicle receptors, pH, membrane permeability, pKa and dissociation constants of the ligand. The system is solved numerically using MATLAB and optimized against empirical data. The results show the experimentally observed biphasic release of epibatidine, affirming the hypothesis of trapping inside the vesicles. Our model also shows that the main trapping effect is due to the presence of receptors inside vesicles, not those located on the cell surface and the high affinity of these ligands to the receptors. The exact acidity level inside the vesicles is not as important. Our model is also able to predict the much faster nicotine outward flow compared with epibatidine. This model can be used to predict the kinetics of other weak basic ligands like those used in positron emission tomography.

Cytisinicline for Smoking Cessation in Adult Smokers

Cytisinicline is a naturally derived, plant-based medication that has been developed as a treatment for smoking cessation and nicotine dependence. Based on a product available in Europe, over 4 million smokers in Central and Eastern Europe have used this medication and over 10,000 subjects have participated in clinical trials worldwide. Cytisinicline acts as a partial agonist and partial antagonist for the alpha-4 beta-2 nicotinic acetylcholine receptors in the brain, which are the same receptors that are activated by nicotine. Cytisinicline helps to reduce cravings, withdrawal symptoms and the reward and satisfaction associated with smoking by preventing nicotine from binding to these receptors and releasing dopamine. The On-going Research of Cytisinicline for Addiction-1 clinical trial has shown that participants receiving 3.0 mg cytisinicline three times daily had the highest biochemically verified point-prevalence abstinence rate (50%) compared with placebo (10%) at the end of treatment, with no significant safety concerns. These quit results were statistically significant (p<0.001). Cytisinicline demonstrates weak binding to the 5-hydroxytryptamine 3 receptor that causes nausea, consistent with few observed nausea events in participants receiving cytisinicline. No new medications for tobacco dependence have received United States Food and Drug Administration approval since 2006. Cytisinicline represents a promising treatment for those globally who continue to smoke tobacco.

Whole-brain modeling explains the context-dependent effects of cholinergic neuromodulation

Integration and segregation are two fundamental principles of brain organization. The brain manages the transitions and balance between different functional segregated or integrated states through neuromodulatory systems. Recently, computational and experimental studies suggest a pro-segregation effect of cholinergic neuromodulation. Here, we studied the effects of the cholinergic system on brain functional connectivity using both empirical fMRI data and computational modeling. First, we analyzed the effects of nicotine on functional connectivity and network topology in healthy subjects during resting-state conditions and during an attentional task. Then, we employed a whole-brain neural mass model interconnected using a human connectome to simulate the effects of nicotine and investigate causal mechanisms for these changes. The drug effect was modeled decreasing both the global coupling and local feedback inhibition parameters, consistent with the known cellular effects of acetylcholine. We found that nicotine incremented functional segregation in both empirical and simulated data, and the effects are context-dependent: observed during the task, but not in the resting state. In-task performance correlates with functional segregation, establishing a link between functional network topology and behavior. Furthermore, we found in the empirical data that the regional density of the nicotinic acetylcholine α4β2 correlates with the decrease in functional nodal strength by nicotine during the task. Our results confirm that cholinergic neuromodulation promotes functional segregation in a context-dependent fashion, and suggest that this segregation is suited for simple visual-attentional tasks.

Detection of Neonicotinoid Insecticides and Their Metabolites in Human Cerebrospinal Fluid.

Detection of Neonicotinoid Insecticides and Their Metabolites in Human Cerebrospinal Fluid.

Development, validation, and application of an ultra-high performance liquid chromatography-tandem mass spectrometry method for the estimation of Ropanicant in human plasma and urine

SUVN-911 (Ropanicant) is a novel alpha-4 beta-2 nicotinic acetylcholine receptor (α4β2 nAChR) antagonist being developed for the treatment of major depressive disorders (MDD). An Ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the quantification of SUVN-911 in human plasma and urine. Liquid-liquid extraction technique was used to extract the SUVN-911 from human plasma and urine. The separation was performed on an Acquity UPLC® BEH C18 (2.1 × 50 mm, 1.7 µm) column with gradient elution using a mobile phase consisting of 0.01M ammonium acetate and acetonitrile. The QTRAP mass spectrometer which is operated in a positive ionization mode utilizing selective reaction monitoring (SRM) mode has been used for the quantification. The developed method was linear across the standard curve concentrations ranging from 0.020 to 50 ng mL−1 in human plasma and 5 to 5000 ng mL−1 in human urine. The accuracy and precision results were well within the acceptance criteria for experiments performed on different days. The average extraction recovery for plasma and urine were 73.9% (% CV 10.1), and 42.7% (% CV 4.9), respectively, and the average matrix factor and internal standard (IS) normalized mean matrix factor were within the acceptable range of 0.9–1.1. The stability of SUVN-911 in blood, plasma, and urine was established at different storage conditions. The developed and validated UHPLC-MS/MS method was utilized for the quantification of SUVN-911 in plasma and urine samples from the first-in-human (FIH) study.

Safety, Tolerability, and Pharmacokinetics of Ropanicant (SUVN-911), a Novel Alpha4 Beta2 Nicotinic Acetylcholine Receptor (α4β2 nAChR) Antagonist, in Healthy Adult and Elderly Subjects.

Ropanicant hydrochloride (previously known as SUVN-911, hereinafter referred to as ropanicant) is a novel alpha4 beta2 nicotinic acetylcholine receptor (α4β2 nAchR) antagonist being developed for the treatment of major depressive disorder. The objectives of the present studies were to evaluate the safety, tolerability, and pharmacokinetics of ropanicant after single and multiple ascending doses and to evaluate the effect of food, sex, and age on its pharmacokinetics in healthy subjects. Two phase I studies have been conducted for ropanicant. Study 1 is a randomized, double-blind, placebo-controlled, first-in-human study to evaluate the safety, tolerability, and pharmacokinetics of single ascending doses (0.5, 6, 15, 30, and 60 mg) and multiple ascending doses (15, 30, and 45 mg) of ropanicant administered orally for 14 days to healthy male subjects. In Study 2, the effect of food, sex, and age on ropanicant pharmacokinetics was evaluated following a single 30-mg oral dose. Ropanicant at single doses up to 60 mg and multiple doses up to 45 mg once daily was found to be safe and well tolerated in healthy subjects. The most frequently reported adverse events were headache and nausea. Ropanicant exposures were more than dose proportional following single and multiple administrations. Urinary excretion of unchanged ropanicant was low across the doses. Upon multiple dosing, 1.5- to 2.5-fold higher exposures for maximum concentration and 1.6- to 4.0-fold higher exposures for area under the concentration-time curve from time 0-24 h were observed on day 14 as compared with day 1. Sex had an effect on the pharmacokinetics of ropanicant as a 64% higher area under the concentration-time curve from time 0 to 24 h and a 26% higher maximum concentration was observed in female adults when compared with male adults. Plasma exposures were comparable in fasted versus fed conditions and in adult versus elderly subjects. Ropanicant was found to be safe and well tolerated following single and multiple oral administrations in healthy subjects. Ropanicant showed nonlinear pharmacokinetics and accumulation following multiple dosing. Urinary excretion represents an insignificant elimination pathway for ropanicant. Ropanicant pharmacokinetics were sex dependent, and food and age had no effect on itspharmacokinetics. NCT03155503 and NCT03551288.

Varenicline-induced sucidal behavior: Case report and literature review

IntroductionNowdays there are different strategies for the treatment of smoking cessation. The treatment include drugs such as varenicline, which acts as a high-affinity partial agonist for the alpha-4 beta-2 nicotinic acetylcholine receptor subtype (nACh). We report a case of a suicidal behaviour in a 39 year-old woman with no previous history of mental illness, who was brought to the emergency department after intentional intoxication with benzodiazepines. The patient was on 10th day of treatment with varenicline.ObjectivesTo present a case of sucidal behavior that developed in a 39 year-old woman after starting varenicline. Review of literature and total number of cases reported in the european database of suspected adverse drug reactions (EudraVigilance).MethodsWe carried out a literature review in Pubmed electing those articles focused on mental disorders in those patients that have been taking varenicline. Review number of cases suicidal behavior reported by the European database of suspected adverse drug reactions.ResultsA 39-year-old female was brought to the emergency department after voluntary ingestion of Lorazepam 1mg (40 tablets) in a sucide attempt. The family reported the starting of thoughts of suicide after 1 week of treatment. No previous history of mental disorders. The patient reported low mood and drowsiness in the last 5 days not linked to any cause. After 5 days of discontinuation these mood symptoms and sucidal behavior remited.ConclusionsVarenicline is associated with different neuropsychiatric sypmtoms. In patients with or without history of mental disorders we should warn about the symptoms for discontinuation of the treatment.DisclosureNo significant relationships.

Evidence for Alpha7 Nicotinic Receptor Activation During the Cough Suppressing Effects Induced by Nicotine and Identification of ATA-101 as a Potential Novel Therapy for the Treatment of Chronic Cough.

Studies performed in healthy smokers have documented a diminished responsiveness to tussive challenges, and several lines of experimental evidence implicate nicotine as an antitussive component in both cigarette smoke and the vapors generated by electronic cigarettes (eCigs). We set out to identify the nicotinic receptor subtype involved in the antitussive actions of nicotine and to further evaluate the potential of nicotinic receptor-selective agonists as cough-suppressing therapeutics. We confirmed an antitussive effect of nicotine in guinea pigs. We additionally observed that the alpha-4 beta-2 (α 4 β 2)-selective agonist Tc-6683 was without effect on evoked cough responses in guinea pigs, while the α 7-selective agonist PHA 543613 dose-dependently inhibited evoked coughing. We subsequently describe the preclinical evidence in support of ATA-101, a potent and highly selective (α 7) selective nicotinic receptor agonist, as a potential candidate for antitussive therapy in humans. ATA-101, formerly known as Tc-5619, was orally bioavailable and moderately central nervous system (CNS) penetrant and dose-dependently inhibited coughing in guinea pigs evoked by citric acid and bradykinin. Comparing the effects of airway targeted administration versus systemic dosing and the effects of repeated dosing at various times prior to tussive challenge, our data suggest that the antitussive actions of ATA-101 require continued engagement of α 7 nicotinic receptors, likely in the CNS. Collectively, the data provide the preclinical rationale for α 7 nicotinic receptor engagement as a novel therapeutic strategy for cough suppression. The data also suggest that α 7 nicotinic acetylcholine receptor (nAChR) activation by nicotine may be permissive to nicotine delivery in a way that may promote addiction. SIGNIFICANCE STATEMENT: This study documents the antitussive actions of nicotine and identifies the α7 nicotinic receptor subtype as the target for nicotine during cough suppression described in humans. We additionally present evidence suggesting that ATA-101 and other α7 nicotinic receptor-selective agonists may be promising candidates for the treatment of chronic refractory cough.

Effects of nicotinic antagonists on working memory performance in young rhesus monkeys

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.

Nicotine-like discriminative and aversive effects of two α4β2-selective nicotine agonists, ispronicline and metanicotine.

An attempt to determine the receptor selective nature of some of nicotine's behavioral effects was undertaken through the evaluation of the ability of two nicotinic α4β2*-selective receptor agonists to produce nicotine-like effects and modify rates of responding in a discrimination assay and in an aversive stimulus assay. A group of eight rats was trained to discriminate the presence of 1 mg/kg nicotine base. Another group of 4-6 rats was trained to report the aversive effects of nicotine by selecting a lever that produced one food pellet over a second lever that produced two food pellets and an intravenous injection of nicotine. Ispronicline and metanicotine, two α4β2*-selective receptor agonists, increased selection of the nicotine-appropriate lever in a dose-related manner, up to a maximum of approximately 75%. The α4β2*-selective receptor antagonist, dihydro-beta-erythroidine blocked both the discriminative stimulus effects and the rate-suppressing effects of ispronicline, metanicotine, and small, but not large doses of nicotine. The nonselective antagonist, mecamylamine, antagonized the discriminative stimulus effects of each of the three nicotine agonists as well as the rate-decreasing effects of nicotine and metanicotine. Mecamylamine did not modify the rate-decreasing effects of ispronicline. Both ispronicline and metanicotine as well as nicotine were avoided in the drug + food vs. food choice situation. The receptor-selective nature of ispronicline and metanicotine was hereby confirmed in a behavioral assay, as were earlier reports that the discriminative stimulus effects of relatively small doses of nicotine are likely mediated by activity at the α4β2* nicotine receptor.

Protective effects of VMY-2-95 on corticosterone-induced injuries in mice and cellular models

Nicotinic α4β2 receptor antagonists have drawn increasing attention in the development of new antidepressants. In this study, we aimed to investigate the protective effect of VMY-2-95, the new selective antagonist of α4β2 nicotinic acetylcholine receptor (nAChR) on corticosterone (CORT) injured mice and cellular models. Fluoxetine was applied as a positive control, and the effects of VMY-2-95 were investigated with three different doses or concentrations (1, 3, 10 mg/kg in mice, and 0.003, 0.03, 0.1 μmol/L in cells). As a result, VMY-2-95 showed significant antidepressant-like effects in the CORT injured mice by improving neuromorphic function, promoting hippocampal nerve proliferation, and regulating the contents of monoamine transmitters. Meanwhile, VMY-2-95 exhibited protective effects on cell viability, cell oxidant, cell apoptosis, and mitochondrial energy metabolism on corticosterone-impaired SH-SY5Y cells. Also, the PKA–CREB–BDNF signaling pathway was up-regulated by VMY-2-95 both in vitro and in vivo, and pathway blockers were also combined with VMY-2-95 to verify the effects furtherly. Therefore, we preliminarily proved that VMY-2-95 had protective effects in depressed mice and SH-SY5Y cells against injuries induced by corticosterone. This work indicated that the application of VMY-2-95 is a potential pharmacological solution for depression. This study also supported the development of α4β2 nAChR antagonists towards neuropsychiatric dysfunctions.

The nicotinic α7 receptor agonist GTS-21 but not the nicotinic α4β2 receptor agonist ABT-418 attenuate the disrupting effects of anesthetic ketamine on recognition memory in rats

Several lines of evidence indicate that anesthetic doses of the non-competitive N-methyl-D-aspartate receptor antagonist ketamine disrupt memory functions in rodents. The mechanism by which anesthetic ketamine produces its adverse behavioural effects is not yet clarified. The implication of nicotinic acetylcholine receptor as a potential site of action of anesthetic ketamine adverse effects on memory is proposed. We investigated the ability of α4β2 nicotinic receptor agonist ABT-418 (0.01, 0.03, 0.1 mg/kg, i.p.) and α7 nicotinic receptor agonist GTS-21 (0.3, 1, 3 mg/kg, i.p.) to counteract recognition memory deficits produced by acute post-training administration of anesthetic ketamine (100 mg/kg, i.p.) in rats. For this purpose, the novel object recognition test, a behavioural paradigm assessing recognition memory abilities in rodents was used. Post-training acute administration of GTS-21 (3 mg/kg) counteracted anesthetic ketamine-induced performance deficits in the novel object recognition memory task. By contrast, ABT-418 failed to reverse the recognition memory deficits caused by anesthetic ketamine. The present findings propose that an α7 nicotinic receptor component might modulate anesthetic ketamine’s adverse effects on recognition memory.

Molecular Regulation of α3β4 Nicotinic Acetylcholine Receptors by Lupeol in Cardiovascular System.

Cardiovascular disease (CVD) occurs globally and has a high mortality rate. The highest risk factor for developing CVD is high blood pressure. Currently, natural products are emerging for the treatment of hypertension to avoid the side effects of drugs. Among existing natural products, lupeol is known to be effective against hypertension in animal experiments. However, there exists no study regarding the molecular physiological evidence against the effects of lupeol. Consequently, we investigated the interaction of lupeol with α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, we performed a two-electrode voltage-clamp technique to investigate the effect of lupeol on the α3β4 nicotine acetylcholine receptor using the oocytes of Xenopus laevis. Coapplication of acetylcholine and lupeol inhibited the activity of α3β4 nAChRs in a concentration-dependent, voltage-independent, and reversible manner. We also conducted a mutational experiment to investigate the influence of residues of the α3 and β4 subunits on lupeol binding with nAChRs. Double mutants of α3β4 (I37A/N132A), nAChRs significantly attenuated the inhibitory effects of lupeol compared to wild-type α3β4 nAChRs. A characteristic of α3β4 nAChRs is their effect on transmission in the cardiac sympathetic ganglion. Overall, it is hypothesized that lupeol lowers hypertension by mediating its effects on α3β4 nAChRs. The interaction between lupeol and α3β4 nAChRs provides evidence against its effect on hypertension at the molecular-cell level. In conclusion, the inhibitory effect of lupeol is proposed as a novel therapeutic approach involving the antihypertensive targeting of α3β4 nAChRs. Furthermore, it is proposed that the molecular basis of the interaction between lupeol and α3β4 nAChRs would be helpful in cardiac-pharmacology research and therapeutics.

RELATIONSHIP BETWEEN CHOLINERGIC RECEPTOR BINDING AND ATTENTION IN COGNITIVE AGING USING POSITRON EMISSION TOMOGRAPHY AND THE STROOP

Introduction Cholinergic (Ach) neurotransmission has been associated with aspects of brain attention networks. Reductions in Ach are also thought to underlie cognitive symptoms in Alzheimer's Disease (AD). In a previous study using positron emission tomography (PET) imaging to measure nicotinic Ach receptor binding in vivo, our group found that Ach receptor binding was reduced in patients with AD relative to healthy controls. Reductions were observed in areas of the brain thought to mediate attention, including the anterior cingulate cortex/medial prefrontal cortex (ACC/mPFC) and insula. The Stroop is a neuropsychological measure used in research and clinical settings to assess attention and executive function. In a different study of AD patients, we found that performance on the Stroop was associated with metabolic activity in brain networks in the right hemisphere; the Stroop COLOR-WORD condition was associated with metabolic rate in the intraparietal sulcus (IPS), ACC/mPFC, and insula. In the present study, we sought to extend these findings by examining whether Ach binding was associated with performance on the Stroop prior to the onset of dementia. Methods The study included a combined group of healthy older adults or older adults with amnestic mild cognitive impairment (MCI). All participants completed the Stroop. The Stroop included three conditions: two simpler tasks requiring participants to name colors (COLOR) and read words (WORD) and a more complex task in which participants are required to name the color of ink used for a word that names a different color from the ink color (COLOR-WORD). Cholinergic (Ach) receptor binding was quantified using PET imaging, specifically 18F-2-FA PET (2FA), a radiotracer that binds to alpha4beta2 nicotinic receptor subunits. Scanning utilized a bolus plus infusion technique and measurement of nonmetabolized 2FA from plasma to calculate distribution volume (VT/fp) in PET Ach image voxels. Ach images were pre-processed using SPM12, which included motion correction, normalization to Montreal Neurological Institute (MNI) space using a co-registered T1 MRI image, and spatial smoothing of 8?mm full width at half maximum. First, a voxel-based analysis across the entire brain was used to measure the association between voxel VT/fp and performance on the Stroop COLOR-WORD test using a liberal statistical threshold (p Results Seventy-six participants were included in the sample, comprised of 43 healthy older adults and 33 patients with amnestic MCI (age: mean=73.1 (SD=7.7), MMSE: mean=28.3 (SD=2.1), 57 males and 19 females, education: mean=15.7?years (SD=2.5)). The combined group included 19 African-Americans, 48 Caucasians, 6 Hispanics, and 3?Asian/?Pacific Islanders. Performance on the COLOR task was associated with mean VT/fp in the ACC/mPFC (r=.23 p=.04), insula (r=.28, p=.02), and IPS (r=.23 p Conclusions Across the spectrum of cognitive aging, performance on the Stroop was associated with Ach binding in key brain regions involved in attention—the IPS, ACC/mPFC, and insula. These findings suggest that Ach binding, in part, mediates attentional processing. This appears true regardless of whether the task is simple, as in the COLOR and WORD conditions, or more complex, as in the COLOR-WORD condition. Thus, changes in Ach receptor binding may be an important factor in attentional deficits resulting from cognitive aging prior to onset of AD, and Ach neurotransmission could be a potential molecular target for novel pharmaceuticals to address these symptoms. This research was funded by: This work was supported by a VA Career Development grant awarded to Brandon C. Yarns, MD, MS, and VA Merit Review grants awarded to David L. Sultzer, MD, and Rebecca J. Melrose, PhD.

Acute and chronic interactive treatments of serotonin 5HT2C and dopamine D1 receptor systems for decreasing nicotine self-administration in female rats

A variety of neural systems are involved in the brain bases of tobacco addiction. Animal models of nicotine addiction have helped identify a variety of interacting neural systems involved in the pathophysiology of tobacco addiction. We and others have found that drug treatments affecting many of those neurotransmitter systems significantly decrease nicotine self-administration. These treatments include dopamine D1 receptor antagonist, histamine H1 antagonist, serotonin 5HT2C agonist, glutamate NMDA antagonist, nicotinic cholinergic α4β2 partial agonist and nicotinic cholinergic α3β4 antagonist acting drugs. It may be the case that combining treatments that affect different neural systems underlying addiction may be more efficacious than single drug treatment. In the current study, we tested the interactions of the D1 antagonist SCH-23390 and the serotonin 5HT2c agonist lorcaserin, both of which we have previously shown to significantly reduce nicotine self-administration. In the acute interactions study, both SCH-23390 and lorcaserin significantly reduced nicotine self-administration when given alone and had additive effects when given in combination. In the chronic study, each drug alone caused a significant decrease in nicotine self-administration. No additive effect was seen in combination because SCH-23390 given alone chronically was already highly effective. Chronic administration of the combination was not seen to significantly prolong reduced nicotine self-administration into the post-treatment period. This research shows that unlike lorcaserin and SCH-23390 interactions when given acutely, when given chronically in combination they do not potentiate or prolong each other's effects in reducing nicotine self-administration.

Aging Affects Nicotinic Acetylcholine Receptors in Brain.

Aging is a common and inevitable stage in the life cycle of higher organisms. Different organs, including the central nervous system, are affected by aging in different ways. Many processes are involved in aging, and neurodegeneration is one of the aging processes in which the central nervous system is engaged. Brain degeneration during normal aging underlies cognitive disorders experienced by older people. Not all molecular mechanisms associated with age-related neurodegeneration are fully understood; however, there is a whole range of data on the participation of nicotinic acetylcholine receptors in the processes of aging and neurodegeneration. Two main subtypes of nicotinic acetylcholine receptor α7 and α4β2 present in the central nervous system are affected by these processes. The loss of these receptor subtypes during normal aging is one of the reasons for the cognitive impairments. The decrease in nicotinic acetylcholine receptors is also very important for the pathogenesis of age-related neurodegenerative diseases. Thus, the drugs enhancing receptor functions may be considered promising for the treatment of cognitive dysfunction in the aged people. To achieve healthy longevity, the molecular processes that occur during aging should be established. In this regard, the participation and role of nicotinic acetylcholine receptors in the brain aging and degeneration are considered in this review.