published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

Η νικοτίνη είναι ένα φυσικό συστατικό, αλκαλοειδές που συναντάται στην οικογένεια των φυτών Solanaceae και πήρε το όνομά της από το φυτό Nicotiana tabacum. Είναι ένα δηλητήριο που προσβάλει το ΚΝΣ και το ΠΝΣ. Εισέρχεται στον οργανισμό από τους πνεύμονες, κυρίως, αλλά και από το μάσημα του καπνού ή τσιχλών που την περιέχουν. Έτσι, εισέρχεται στο κυκλοφορικό σύστημα και φτάνει μέχρι τον αιματοεγκεφαλικό φραγμό, τον οποίο διαπερνά για να προχωρήσει στο ΚΝΣ (μέσα σε 10-20 δευτερόλεπτα μετά από την εισπνοή). Εκεί, ενεργοποιεί τους νικοτινικούς υποδοχείς της Ach, δρώντας ως αγωνιστής. Η νικοτίνη διεγείρει τη βιοδιαθεσιμότητα των νευροδιαβιβαστών ντοπαμίνη και σεροτονίνη, με αποτέλεσμα να επιταχύνει τη λειτουργία της καρδιάς και να αυξάνει την πίεση του αίματος. Επίσης, η νικοτίνη δρα στους εγκεφαλικούς μηχανισμούς ανταμοιβής, εμμέσως με την ενεργοποίηση των ενδογενών οπιοειδών (opioids) και άμεσα μέσω μηχανισμών της ντοπαμίνης. Οι μηχανισμοί αυτοί είναι το αίτιο εθισμού προς το κάπνισμα, αλλά και κάθε άλλου είδους εθισμού. Η διακοπή του καπνίσματος είναι μια δύσκολη διαδικασία και ενώ υπάρχουν τρόποι και θεραπείες, δεν βοηθούν όλα τα άτομα που θέλουν να διακόψουν το κάπνισμα. Τα πιο αξιοσημείωτα φάρμακα που χρησιμοποιούνται για την διακοπή του καπνίσματος είναι η βαρενικλίνη και η βουπροπιόνη. Στην παρούσα εργασία, μελετήθηκε η γενετική συσχέτιση πολυμορφισμών που πιθανώς εμπλέκονται στον εθισμό στη νικοτίνη (rs1329650), στην προδιάθεση της έναρξης (rs6265) και διακοπής (rs3025343) του καπνίσματος, αλλά και στην ανταπόκριση στη θεραπεία με βαρενικλίνη (rs2072659). Το πληθυσμιακό δείγμα, τόσο των καπνιστών (101 άτομα), όσο και της ομάδας αναφοράς - μη καπνιστών (75 άτομα) που χρησιμοποιήθηκε, προέρχεται από τον ελληνικό πληθυσμό. Οι μέθοδοι γονοτύπησης που εφαρμόσθηκαν περιλαμβάνουν τον προσδιορισμό αλληλουχίας κατά Sanger, την PCR-RFLP και την ARMS-PCR, σε συνδυασμό με ηλεκτροφόρηση σε αγαρόζη. Μια στατιστική τάση συσχέτισης (p=0,0279) ανιχνεύθηκε για τον πολυμορφισμό rs1329650 μεταξύ των καπνιστών και τον μη καπνιστών για την προδιάθεση στην εξάρτηση στη νικοτίνη. Για τους υπόλοιπους πολυμορφισμούς που μελετήθηκαν, δεν παρατηρήθηκαν στατιστικά σημαντικές συσχετίσεις με τα ερωτήματα που τέθηκαν στην παρούσα εργασία. / Nicotine is a natural ingredient (alkaloid), which is found in the plant’s family Solanaceae. Nicotine is named after the plant Nicotiana tabacum. Nicotine is a poison that affects both the CNS and PNS. It enters the body mainly through the lungs, though chewing tobacco or a nicotine-containing gum also helps on nicotine “administration”. Nicotine comes into the circulatory system, overcomes the blood brain barrier and reaches the CNS (within 10-20 seconds after inhalation), where it activates the nicotinic acetylcholine receptors (nicotine is an agonist). Nicotine stimulates the bioavailability of neurotransmitters dopamine and serotonin and therepy, it accelerates the heart rate and increases the blood pressure. Also, nicotine acts on the brain reward mechanisms, indirectly by the activation of the endogenous opioids and via the mechanisms of dopamine. These mechanisms are the cause of addiction to smoking, but also to any other type of addiction. Quitting smoking is a difficult process and while there are several ways and treatments, they do not help all the people who want to stop smoking. The most notable drugs being used for smoking cessation is varenicline and bupropion. Herein, we study the genetic correlation of polymorphisms that are possibly involved in nicotine addiction (rs1329650), the predisposition of individuals to begin (rs6265) or quit (rs3025343) smoking and response to varenicline treatment (rs2072659). The sample population studied involved two groups; smokers (101 people), and non-smokers (75 people) derived from the Greek population. The genotyping methods applied include Sanger sequencing and PCR-RFLP or ARMS-PCR followed by agarose gel electrophoresis. A statistical trend (p = 0.0279) becomes evident regarding rs1329650, when smokers and non-smokers were compared. The rest polymorphisms studied showed no statistically significant correlation.

Developmental nicotine exposure (DNE) is known to cause deleterious effects in neonatal mammals through nicotine's actions on nicotinic acetylcholine receptors (nAChRs). In this work, we studied how DNE altered the structure and function of the hypoglossal motoneurons (XIIMNs) over the first few days post-parturition. Previous work in XIIMNs demonstrated an increase in cellular excitability (Pilarski et al., 2011), alterations in synaptic transmission among respiratory-related neurons (Wang et al., 2006; Pilarski et al., 2012; Jaiswal et al., 2013), and a reduction in inspiratory drive currents in DNE animals (Pilarski et al., 2011). Here we show that the effects of DNE extend to alterations in the spike-timing precision and reliability of XIIMNs, as well as spike-frequency adaptation. Additionally, simple morphological analysis of XIIMNs following nicotine exposure in utero has revealed a reduction in soma cross-sectional area. We were interested in studying the complete morphology of XIIMNs following DNE to discern its effects on more complex morphological parameters. We advanced this research using a combination of techniques in thin brainstem slices of neonatal rats, including whole cell patch clamp recordings and immunohistochemistry of intracellularly labeled hypoglossal motoneurons. Furthermore, morphological analysis revealed significant differences in the complexity of the dendritic arborization, showing that neurons from DNE animals had shorter dendrites that branched less often. We also used computational analysis to gain insight into mechanisms that may underlie the changes in spike-timing precision and reliability. In a single cell model of XIIMNs, decreases in potassium-dependent conductances such as the calcium-activated potassium current could potentially replicate the alterations seen in vitro. Finally, we also did a systems-level study of the hyoglossus muscle, a tongue retractor, to determine the relation between tongue retraction force and motor unit discharge characteristics. These experiments utilized adult, anesthetized rats to record single motor units, whole muscle electromyography (EMG) activity and tongue retraction force during spontaneous breathing. We determined that during inspiration-related tongue retractions in low and high force conditions, recruitment of motor units plays a crucial role in the control of tongue force output, whereas rate coding of single motor units is present, but appears to play a lesser role. Overall, this study shows that DNE effects the input-output properties of XIIMNs, potentially through changes in intrinsic channel properties; DNE also alters XIIMN morphology, particularly dendritic arborization; and that organization of a tongue retractor muscle depends primarily on recruitment, but also rate coding, to increase force output.

Introduction and Rationale: Sudden infant death syndrome (SIDS) continues to be the leading cause of death in infants 28 days to 12 months old and the third leading cause of infant mortality (CDC, 2014; Task Force on SIDS, 2011a). SIDS has no identifiable cause although many hypotheses have existed and there are many known risk factors. Nicotine use is the second most modifiable risk factor for SIDS and to date there is no national public education campaign. Formal education is needed. Written education materials, such as brochures, are an effective way to increase knowledge and awareness of a health topic. Purpose and Objective: The purpose of this DNP Project was to create an educational brochure about SIDS and nicotine as a significant modifiable risk factor. The brochure is directed towards pregnant women and women with infants less than 12 months of age. The objective was to educate and inform mothers about SIDS, nicotine and its relation to SIDS, and the importance of abstaining from nicotine during and after pregnancy. Methods: The brochure was created using the Health Belief Model (HBM) as a framework. The HBM was used to explain health behavior modifications and was used as a foundation for education interventions. The model has successfully been applied to explain other preventative health behaviors and provide health education in various contexts. Results: The brochure was evaluated by two subject matter experts who have published peer reviewed articles using the HBM. Both subject matter experts found the HBM applied to the brochure appropriately. In their opinion, the content of the brochure should have positive impact for health modification in women who use nicotine and are pregnant or have a child less than 12 months of age. Conclusions: This DNP Project demonstrated SIDS as a current significant health problem and identified maternal nicotine use as the second most modifiable risk factor. Due to lack of current public education, a brochure was created using the HBM as a framework. Subject matter experts stated the brochure was created based on the HBM and will likely a have positive health influence on the intended population.

Nicotinic acetylcholine receptors (nAChR) are neurotransmitter-gated ion channels that exist as a family of subtypes defined by unique subunit compositions. nAChR containing α6 subunits (α6*-nAChR) have attracted interest because α6 subunits are thought to be localized in brain regions implicated in reward, mood and drug dependence. To provide new information necessary toward a more complete understanding of roles of α6*-nAChR in neuropsychiatric health and disease, three lines of investigation were pursued. A set of stably transfected, human, immortalized cell lines were generated that heterologously express nAChR α6 subunits in combination with other nAChR subunits found in reward brain regions (nAChR subunit combinations α6β2, α6β4, α6β2β3, α6β4β3, α6β2β3α5, α6β4β3α5, α6α4β2β3 and α6α4β4β3). The α6α4β2β3 combination may have a functional response to epibatidine that differs from that of the α4β2 nAChR. A unique binding site was identified in cells transfected with the α6β4β3α5 nAChR subunit combination. Messenger RNA fluorescence in situ hybridization (mRNA FISH) studies established regional and celluar distribution of nAChR α6 subunit mRNA in the mouse brain. The third line of study extended this work to examine potential co-expression of nAChR α6 subunits and glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH) as labels of GABAergic and dopaminergic/catecholaminergic neurons respectively, using tandem mRNA FISH and fluorescence immunohistochemistry. nAChR α6 subunit signal in the substantia nigra (SN) and ventral tegmental area (VTA) was congruent with previous studies. Message was also detected in the amydala, dentate gyrus, striatum, zona incerta, and cingulate, entorhinal, perirhinal, piriform, and prelimbic cortices. nAChR α6 mRNA was coexpressed with GAD in the amygdala, dentate gyrus, striatum, SN, VTA and cingulate, entorhinal, prelimbic and prelimbic cortices. TH was exclusively co-localized with nAChR α6 mRNA in the SN and VTA. Findings suggest extended roles for α6*-nAChR in the brain, particularly in the control of GABAergic neuronal activity and/or GABA release. These studies provide new insights into the composition of α6*-nAChR, the localization and cellular origins of nAChR α6 subunit expression. Data collected suggest roles for α6*-nAChR in many brain regions, including those involved in higher order processes involved in drug dependence and reward, and in modulation of inhibitory neurotransmission.

Smoking is a worldwide problem and it is the second major cause of death. People often try to quit, but few succeed mainly because of withdrawal symptoms such as irritability, anxiety, increased appetite, hyperventilation and difficulty concentrating. The overall aim of this project was to study neurochemical changes in the brain following sensitization to nicotine which could give more information about what causes an individual to go from using drugs to abusing the drugs. Therefore, we investigated messenger ribonucleic acid (mRNA) expression of several genes known to be involved in the mesolimbic dopamine pathway in the nucleus accumbens, caudate putamen, prefrontal cortex and medial prefrontal cortex using real-time polymerase chain reaction (real-time PCR). The results showed that in the nucleus accumbens, mRNA expression of gamma-aminobutyric acid (GABA) Aα1 subunit receptor and GABA transporter 3 (GAT-3) were significantly increased following nicotine administration, while in the caudate putamen no difference in expression was observed. In prefrontal cortex, the expression of adrenergic subunit receptor α2A was significantly increased following hexamethonium administration. In medial prefrontal cortex a significant decrease of expression of GAT-1 was shown following nicotine and hexamethonium administration, while a decrease of CART expression only was shown following nicotine administration. Overall, these changes in the GABA system may help to explain the mechanism of nicotine sensitization.

In 1999, the Federal Government sued the tobacco companies in an attempt to recover expenses used to treat diseases caused by smoking. The 2006 verdict required the dissemination of corrective statements intended to inform the public of the dangers of smoking. This study analyzes smokers' perceptions of these statements. Statements were tested from November 5 to December 5, 2007. Results indicate: (1) messages created by public health intervenors and tobacco companies tended to have higher mean scores than those created by the DOJ, (2) statements on nicotine being addictive had significantly higher mean scores than the other two sets tested, (3) that all statements were rated higher in terms of believability than in terms of other factors, and (4) light smokers rated statements higher in terms of motivating and likelihood to quit than did medium or heavy smokers. These results will be used to refine the development of corrective statements.

INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.

INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.