Tobacco dependence is high in women who suffer from anxiety disorders yet little is known about the contributions of nicotinic acetylcholine receptors (nAChRs) on anxiety-like behavior. β2*nAChRs (*denotes assembly with other subunits) are the most abundantly expressed nAChRs in the brain yet little is known about the contributions of β2*nAChRs on anxiety-like behavior in female mice. In this study, antagonism and nicotine effects on anxiety-like behavior was investigated across the life span in 6, 12 and 24-month-old drug-naïve knockout (KO), heterozygous (HET) and a gain of function α6L9S mice and wild type (WT). HET mice showed increased sensitivity to di-hydrobeta-erythroidine compared to WT mice. Aged mice showed decreased locomotor activity and exploratory behavior compared to younger mice. Low doses of nicotine produced anxiolytic-like effects, whilst a high dose of nicotine produced anxiogenic-like effects. Activation of the α6*nAChRs supports an anxiolysis-like phenotype. These results implicate α4β2*nAChRs and α6β2*nAChRs in anxiety-like behavior.

Tobacco dependence is the leading cause of preventable death in the United States. Despite currently available smoking cessation therapies, there is a high rate of relapse in smoking among those attempting to quit. While the somatic signs of nicotine withdrawal (insomnia, increased appetite, weight gain) contribute to the continuation of smoking behavior, it has been hypothesized that the affective signs (depression, anxiety, craving, irritability) are greater motivators of relapse and continued tobacco use. There are few studies that assess the molecular and receptor-mediated mechanisms of nicotine withdrawal; therefore, our studies focus on identifying the nicotinic acetylcholine receptor (nAChR) subtypes and post-receptor calcium-dependent mechanisms involved in nicotine withdrawal behaviors. Using precipitated, spontaneous, and conditioned place aversion (CPA) models, we measured physical and affective signs of nicotine withdrawal in mice. Our data show that major nAChR subtypes have differential roles in nicotine withdrawal. Additionally, our results suggest a behavioral relevance for L-type calcium channels in physical nicotine withdrawal signs, while calcium/calmodulin dependent protein kinase II (CaMKII) appears to be involved in both physical and affective withdrawal behaviors. Additionally, we conducted biochemical studies in the ventral tegmental area (VTA) and nucleus accumbens (NAc) to examine the relationship between altered withdrawal behavioral responses and calcium-dependent molecular mechanisms that contribute to nicotine withdrawal behaviors. Our results suggest an important role for β2-containing nAChRs in nicotine-withdrawal induced decreases in CaMKII and synapsin I function in the NAc. Overall, our studies implicate a critical role for the α4α6β2* nAChR subtype in the behavioral and molecular aspects of nicotine withdrawal, thus aiding in the elucidation of nAChR subunits and mechanisms that contribute to nicotine withdrawal behaviors. The current studies are imperative for generating more successful smoking cessation therapies.

Nicotine is a major psychoactive ingredient in tobacco that is thought to promote smoking behavior via nicotinic acetylcholine receptors (nAChRs) in the brain. Given reports that people smoke to relieve anxiety and that anxiety precipitates relapse, the overarching goal of this dissertation research is to assess beta 2 subunit containing nAChR (beta2*nAChR) contributions to anxiety-like behavior. Nicotine’s activity at beta2*nAChRs is concentration-dependent, with high concentrations facilitating activation followed by rapid desensitization and low concentrations preferentially desensitizing beta2*nAChRs; hence, activation or inhibition of beta2*nAChRs may support smoking behavior. Rodent studies reveal that nicotine affects anxiety-like behavior dose-dependently: low doses promote anxiolysis- and high doses support anxiogenic-like behavior. These pharmacological and genetic studies in mice test the hypothesis that nicotine administration promotes anxiolysis via inactivation of beta2*nAChRs and begin to identify which subunits, namely alpha 4 and alpha 6, work in concert with beta 2 to affect anxiety-like behavior. Low dose nicotine and inhibition of beta2*nAChRs supported anxiolysis-like behavior in a number of tasks with predictive validity for anxiolysis efficacy. These studies further suggest that activation of alpha6beta2*nAChRs is sufficient to produce anxiogenic-like behavior and that inhibition of alpha4beta2*nAChRs supports anxiolysis-like behavior. A secondary goal of these studies is to assess if beta2*nAChRs affect anxiety-like behavior during aging. Dysregulation of cholinergic tone can increase anxiety in the elderly, but little is known regarding beta2*nAChR contributions to anxiety in this population or where in the brain this may take place. These studies show that alpha4beta2*nAChR expression differentially affects anxiety-like behavior in adult and aged mice. With a focus on the lateral septum, a GABA-ergic limbic nucleus thought to regulate anxiety-like responses to external stimuli, a third goal of these studies is to elucidate the neuroanatomical and intracellular underpinnings of anxiety-like behavior that are affected by beta2*nAChR inhibition and expression. Previous studies demonstrate that exposure to stressors reduces phosphorylation of extracellular regulated kinase (ERK) in the lateral septum. In these studies, levels of pERK in the lateral septum were inversely associated with alpha4beta2*nAChR expression as well as anxiogenic-like behavior. In sum, these preclinical studies suggest that inhibition alpha4beta2*nAChRs may support cessation in those who smoke to relieve anxiety.

Neuronal nicotinic acetylcholine receptors modulate both cholinergic and non-cholinergic synaptic transmission. Our research concerns α6 and α4 neuronal nicotinic subunits because they often co-assemble with the β2 subunit, which has abundant expression in the CNS and previous work has demonstrated that β2* nAChRs are involved in nicotine and cocaine reward. α6β2* and α4β2* nAChRs are highly expressed in midbrain, which is known to be critical for the incentive salience associated with natural and artificial (drug) reward. Our goal was to assess the role of α6β2* and α4β2* nAChRs in nicotine and cocaine reward using an unbiased conditioned place preference (CPP) test in mice. Adult male C57BL/6J mice or male mice null for the α6 or α4 nicotinic receptor subunit were used. For CPP: On day 1, pre-conditioning scores were recorded; Days 2-4 mice underwent conditioning, where they were randomly assigned to either the black or the white compartment paired with drug, and the opposite chamber paired with saline; Day 5 was a drug-free test day where post conditioning scores were recorded. α-Conotoxin MII[H9A;L15A], a selective antagonist of α6β2* nAChRs, was given centrally either into the lateral ventricle or the nucleus accumbens on conditioning days, which tested for acquisition of CPP, or it was given only once into the lateral ventricle on test day which tested for expression of CPP. Antagonizing α6*nAChRs resulted in a significant attenuation of both nicotine and cocaine place preference. This was complemented with diminished nicotine and cocaine place preference in α6 KO mice compared to WT littermates. Studies with α4 KO mice showed significantly reduced nicotine place preference scores compared to WT littermates. In contrast, α4 KO and WT mice showed significant place preference for 20mg /kg cocaine, suggesting that the α4 subunit is not required for the reward-like effects of cocaine in our behavioral test. Our results implicate α6β2* and α4β2* nAChR involvement in nicotine and cocaine CPP, but only α6β2* nAChR involvement in cocaine CPP. Lithium conditioned place avoidance and food reward were not altered in α6 KO mice or by α-Conotoxin MII[H9A;L15A], thereby validating the specificity of hedonics of targeting α6* nAChRs in CPP. Our studies suggest that α6β2* and α4β2*nAChR should be further characterized for future nicotine cessation therapies, and α6β2* could provide a new target for treating cocaine addiction.

Novel Ca²⁺ signalling pathways in both endothelial cells and smooth muscle cells of rat small resistance arteries were investigated using a combination of confocal imaging, isometric tension recordings, and electrophysiology to study freshly isolated arteries and cells. We first examined the hypothesis that hyperpolarization could alter endothelial cell Ca²⁺ events. Hyperpolarization evoked by direct opening of K_ATP channels in the smooth muscle with levcromakalim triggered an increase in the frequency of Ca²⁺ events in the endothelium of rat cremaster arterioles. These Ca²⁺ events were discrete in nature, requiring subcellular regions of interest to reliably identify them. Opening of K_ATP channels indirectly through β-adrenoceptor stimulation with isoprenaline, caused a similar increase in the frequency of endothelial cell Ca²⁺ events in rat mesenteric third order arteries. These events also had a similar, focal profile. Pharmacological investigation suggested that the response to isoprenaline was receptor-mediated, and dependent on Ca²⁺ influx and opening of K_ATP channels. The presence of β-adrenoceptors on endothelial cells was confirmed using fluorescently-tagged β-adrenoceptor ligands, which showed punctate labelling in smooth muscle and endothelial cells of rat mesenteric arteries. Freshly isolated endothelial cells also showed Ca²⁺ increases to isoprenaline, although this was not consistently observed. Following on from the observed endothelial cell Ca²⁺ response to hyperpolarization, we tested the hypothesized involvement of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels using the channel inhibitor, ZD7288. Pre-treatment with ZD7288 (1 μM) reduced both the endothelial cell Ca²⁺ response to isoprenaline (in mesenteric arteries) and levcromakalim (in cremaster arterioles). HCN channel subtypes were identified in cremaster arterioles through immunolabelling. We also observed an interesting effect of higher concentrations of ZD7288 to potentially inhibit K⁺ channels, including endothelial cell KCa channels, since hyperpolarization to isoprenaline, levcromakalim or acetylcholine (ACh) was reduced by 10 μM ZD7288, and relaxation to ACh was partially inhibited. ACh-mediated relaxation was also partially inhibited by the clinically used HCN channel blocker, ivabradine (0.3-30 μM). Finally, we identified an interaction of the Ca²⁺-releasing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) with BKCa channels in the smooth muscle. NAADP-mobilised Ca²⁺ has been reported to interact with ryanodine receptors hence we hypothesized an interaction with BK_Ca channels via Ca²⁺ sparks. We found that NAADP-AM relaxed and hyperpolarized rat mesenteric arteries, which was blocked by iberiotoxin (BK_Ca channel inhibitor) and high extracellular [K⁺] (45 mM). Furthermore, NAADP increased paxilline-sensitive K⁺ currents and the frequency and amplitude of spontaneous transient outward currents (STOCs) in freshly isolated vascular smooth muscle cells patched in the whole-cell configuration, further supporting an action at BK_Ca channels. All together these data identify novel Ca²⁺ signalling pathways in resistance arteries that are both activated by and promote hyperpolarization, which is a key determinant of vascular tone.

La Broncho-pneumopathie Chronique Obstructive (BPCO) est une maladie respiratoire grave caractérisée par une inflammation chronique entrainant des lésions irréversibles de l’épithélium respiratoire et du parenchyme pulmonaire. Le principal facteur de risque est le tabagisme mais des études d’association génétique pangénomiques ont montré que certains polymorphismes nucléotidiques simples (SNP) des récepteurs nicotiniques (nAChRs) sont associés à l’incidence de la BPCO. Un de ces polymorphisme est le variant rs16969968 dans le 5ème exon du gène CHRNA5 codant la sous-unité α5. Le but de ce travail a été d’évaluer in vivo l’implication du SNP α5 dans les lésions pulmonaires caractérisant la BPCO et d’étudier l’impact fonctionnel du polymorphisme sur les voies de signalisation mises en jeu en aval des nAChRs. A l’aide de différents modèles in vivo murins et humains, nous avons pu montrer qu’indépendamment du tabagisme, le SNPα5 est associé à une inflammation cellulaire plus marquée, une sécrétion de cytokines pro-inflammatoires, des lésions emphysémateuses, une hyperplasie des cellules mucipares et des cellules Club moins fréquentes par rapport au génotype sauvage. Le SNPα5 est associé à une altération de la perméabilité calcique des cellules épithéliales et une modulation de la voie de signalisation AC3-PKA/C. Cette étude apporte pour la première fois une explication biologique à l’association entre le SNPα5 et la BPCO décrite dans les études d’association génétique pangénomiques à travers un rôle pro-inflammatoire du SNPα5 au niveau pulmonaire. / Chronic Obstructive Pulmonary Disease (COPD) is a critical respiratory disease characterized by a chronic inflammation leading to irreversible epithelial and parenchymal injuries. The main risk factor is tobacco consumption but several genome-wide association studies (GWAS) described some single-associated polymorphisms (SNP) on nicotinic acetylcholine receptors (nACHR) genes associated with COPD incidence. One of these polymorphisms is the rs16969968 variant in the 5th exon of CHRNA5 gene coding the α5 subunit (SNP α5). The aim of this study was to determine in vivo the involvement of SNPα5 in COPD-associated lung injuries and to deciphere the functional impact of the polymorphism on nAChR signaling pathways. Thanks to several in vivo models (mouse and human), we describe here that the SNPα5 is associated, irrespective of the tobacco consumption, to an increased inflammation, pro-inflammatory cytokines secretion, emphysema, goblet cell hyperplasia, and Club cell diminution compared to the wild-type genotype. The SNPα5 is associated with a decreased calcium influx and a modulation of AC3-PKA/C pathway in airway epithelial cells. Our study describe for the first time a biological explanation for the association between SNPα5 and COPD shown in GWAS with a pro-inflammatory role of SNPα5 in the lung.

A vareniclina é uma substância química sintética utilizada para o tratamento de tabagismo; atua como agonista de receptores colinérgicos nicotínicos, em especial, como agonista parcial em receptores α4β2 e α3β4, e como agonista total do receptor α7. Levando em consideração que há uma tendência de ampliação do uso clínico da vareniclina para o tratamento da dependência à diversas substâncias de padrão abusivo e que há poucos estudos relacionados aos seus efeitos sobre o comportamento, cognição e sistema motor, tornam-se necessários mais estudos sobre essa substância. Assim, no presente trabalho foram estudados os efeitos da exposição prolongada (28 30 dias) de ratos à vareniclina, avaliando-se o consumo de água e de ração, o ganho de peso e o comportamento animal, por meio dos testes de campo aberto, labirinto em cruz elevado, interação social, comportamento estereotipado, labirinto de Barnes e esquiva passiva. Ainda foram feitas as avaliações dos níveis de neurotransmissores e seus metabólitos em diferentes estruturas cerebrais, bem como avaliações hematológicas, bioquímicas séricas, urinárias e estudos anatomopatológicos e histopatológicos. Foram utilizadas três doses de vareniclina: 0,03 (dose terapêutica para o ser humano), 0,1 e 0,3 mg/kg, por via oral (gavagem). Os resultados mostraram que a exposição prolongada de ratos à diferentes doses de vareniclina não provocou toxicidade, uma vez que não houve alteração no consumo médio de água e de ração e no ganho de peso avaliados semanalmente. Quanto às avaliações comportamentais, observou-se leve aumento da atividade geral no campo aberto, bem como diminuição do tempo de interação social, não sendo capaz de alterar parâmetros neuroquímicos, hematológicos, bioquímicos séricos, urinários, anatomopatológicos e histopatológicos de ratos expostos à vareniclina. / Varenicline is a synthetic chemical used for the smoking addiction treatment; it acts as an agonist of nicotinic cholinergic receptors, in particular, as a partial agonist of receptors α4β2 and α3β4 and as a full agonist of the α7 receptor. More studies about this substance are necessary, given that its clinical use is increasingly being applied to the treatment of addiction to a variety of abusive drugs. Moreover, there are few studies on vareniciline effects on behavior, cognition and the motor system. Thus, in this study the effects of prolonged (28-30 days) exposure of rats to varenicline were evaluated. It was analyzed the water and food consumption, the weight gain and the animal behavior, through open field, elevated plus maze, social interaction, stereotyped behavior, Barnes maze and passive avoidance tests. The neurotransmitter levels and their metabolites in different brain structures were measured and hematological, serum biochemistry, urinary evaluations and pathological and histological studies were carried out. We used three doses of varenicline: 0.03 (therapeutic dose for humans), 0.1 and 0.3 mg/kg orally (gavage). The results showed that prolonged exposure of rats to different doses of varenicline did not cause toxicity, since there were no changes in average weekly consumption of water or food nor body weight gain, which were measured weekly. As for behavioral assessments, there was a slight increase in overall activity in the open field as well as decreased time of social interaction. Varenicline was not able to change neurochemical, hematological, serum biochemical, urinary, pathology and histopathology parameters of rats.

Nematodes of the genus Ascaris are large gastrointestinal parasites. Ascaris lumbricoides infects ~1 billion people globally; causing malnutrition and general morbidity, and can block the gut or bile duct causing fatal complications. Ascaris suum is a parasite of pigs; in addition to its veterinary significance, it can occasionally be zoonotic, and is a good model of the human parasite. One of the main classes of drugs used to treat parasitic nematode infections are the cholinergic anthelmintics, such as levamisole and pyrantel, which act as agonists of nicotinic acetylcholine receptors at the nematode neuromuscular junction.

α7 nicotinic acetylcholine receptors (nAChR) are highly permeable to Ca2+ and are clinical targets for Alzheimer’s disease and schizophrenia. The aim of this work was to examine α7 nAChR-mediated Ca2+ signalling in neuronal cells using three different methods, and to evaluate the effects of the desensitizing agonist and prototypical smoking-cessation drug sazetidine-A on α7 nAChRs. Initial studies used 96-well plate assays with SH-SY5Y cells to characterize responses evoked by the α7 nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596. This was complemented by live-imaging of cortical cultures, where the compounds evoked robust Ca2+ responses from 12 % of cells. Co- application with Cd2+, ryanodine and xestospongin-C significantly inhibited these responses, suggesting the involvement of voltage-gated Ca2+ channels and Ca2+- induced Ca2+-release. CNQX and MK801 also significantly inhibited α7 nAChR mediated Ca2+ elevations, indicating a role for glutamate release. A high-content screening assay was developed to further examine these phenomena. Exploratory experiments using KCl, AMPA and NMDA validated a protocol that could be used to image Ca2+ elevations in large cell populations. Inconsistent responses to PNU-120596 and PNU2-282987 were also observed, reflecting the scarcity of α7 nAChRs in cortical cultures and the need for assay optimization. Combination with immunofluorescent labelling revealed α7 nAChR mediated Ca2+ elevations in a subpopulation of astrocytes and neurons, some of which were GABAergic. PNU-120596 potentiated the effects of sazetidine-A in SH-SY5Y cells (EC50 0.4 μM) eliciting responses in 14 % of cells in cortical cultures in a methyllycaconitine- sensitive manner, consistent with α7 nAChR activation. Pre-incubation with sazetidine-A concentration-dependently attenuated subsequent α7 nAChR-mediated responses in SH-SY5Y cells (IC50 476 nM) and cortical cultures, suggesting that α7 nAChRs could play a role in the behavioural effects of sazetidine-A. These comparative experiments enhance our understanding of α7 nAChR signalling and provide a new method to study them further.

Pneumonia is the second leading cause of disability-adjusted life-years lost worldwide and the eighth leading cause of death in the United States. Traumatic brain injury (TBI) patients have classically been considered immunosuppressed, but recent research reported that mild head trauma patients have reduced incidence of pneumonia compared to blunt trauma patients. Using our mild TBI model followed by bacterial pneumonia, we investigated the effect of neuronal signaling on innate immune function. To test whether any mild injury primes host immune responses to pneumonia, we generated a mild tail trauma (TT) model. mTBI mice showed protection from bacterial pneumonia while TT mice did not. Using an FDA-approved neurokinin-1 receptor (NK1R) antagonist, aprepitant, we confirmed our previous findings that substance P (SP) is a key mediator of enhanced resistance to pneumonia. Blocking NK1R showed that mTBI-induced release of SP augments pulmonary neutrophil recruitment and microbicidal activity to pulmonary bacterial pathogens. In TT mice, NK1R agonism enhanced the same neutrophil functions, further supporting the hypothesis. No differences were found between mTBI and TT neutrophils’ ability to phagocytose, generate oxidative burst, or acidify phagosomes. However, neutrophils from mTBI mice produced more neutrophil extracellular traps in response to bacterial challenge. These studies show that neurokinin signaling in our model contributes to enhanced bacterial clearance. Cholinergic anti-inflammatory pathway signaling though the α7 nicotinic acetylcholine receptor (α7 nAChR) is also a critical component of improved survival. Blockade of α7 nAChR abrogated the mTBI survival benefit. Mimicking cholinergic signaling using α7 nAChR agonist recapitulated the mTBI reduced pro-inflammatory cytokine production and improved survival. No physiologic differences emerged within 24h following pneumonia, but mTBI and α7 agonist treated mice had significantly lower TNFα in bronchoalveolar fluid, suggesting reduced injurious pulmonary inflammation. However, replacing early TNFα during pneumonia did not increase mortality. Western blot analysis showed downregulation of HMGB1 release in mTBI mice, suggesting that vagal cholinergic signaling reduces late mediators of organ damage. Our experiments show that mTBI enhances resistance to pneumonia by activating the vagus nerve signaling through neurokinin and cholinergic pathways. Translation of these findings could be innovative solutions to fighting or preventing infections.