ROLE OF MAGL INHIBITION IN NICOTINE WITHDRAWAL AND REWARD. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. by Pretal Ishvarlal Patel Muldoon Director: M. Imad Damaj, PhD Professor, Department of Pharmacology and Toxicology Tobacco use is one of the leading causes of preventable deaths worldwide. Nicotine, the main psychoactive component of tobacco, sustains and initiates tobacco addiction. Cessation of nicotine induces a dependence withdrawal syndrome. Recent in vivo studies indicate that the endocannabinoid (EC) system modulates both nicotine reward and withdrawal. The purpose of this proposal is to investigate the role of enhancing endogenous 2-arachidonoylglycerol (2-AG) and by blocking its degradative enzyme, monoacylglycerol lipase (MAGL) enzyme, in nicotine reward and dependence. The selective MAGL inhibitor JZL184 dose-dependently reduced both precipitated and spontaneous somatic and aversive withdrawal signs in mice. These effects were blocked by rimonabant indicating a CB1 receptor mechanism. Furthermore, repeated administration of JZL184 for 6 days did not produce tolerance to the alleviation of withdrawal and the treatment did not induce alterations in CB1 receptor levels or receptor-mediated G-protein activity in various brain regions. In addition, a decrease in 2-AG levels was found in the nucleus accumbens in nicotine-dependent mice undergoing precipitated withdrawal, suggesting that a dysregulation of this EC signaling system occurs during nicotine withdrawal. Lastly, we tested the effectiveness of a combination of low-dose JZL184 and high dose of the FAAH inhibitor PF-3845 on spontaneous nicotine withdrawal. Indeed, the combination of low-dose JZL184 and PF-3845 significantly attenuated nicotine spontaneous withdrawal signs. MAGL inhibition by JZL184 dose-dependently caused a significant blockade of nicotine reward as measured in the mouse conditioned place preference (CPP). In contrast to withdrawal, JZL184’s effect on nicotine CPP was not CB1 mediated. In addition, JZL184 treatment did not cause significant alterations in CB1 receptor levels or receptor-mediated G-protein activity in several brain regions involved in nicotine reward. The effects of JZL184 on nicotine CPP was selective since the drug failed to alter food-induced CPP and LiCl-induced conditioned place aversion in the mouse. Interestingly, active doses of JZL184 did not only cause an increases in 2-AG levels but also induced a concomitant decrease in arachidonic acid (AA) levels in various brain regions suggesting an AA cascade dependent-mechanism. In line of these changes, a cox-2 inhibitor, valdecoxib, dose-dependently blocked nicotine preference.

At the end of the 20th century it was known that cannabinoid drugs interact with two receptors, CB1 and CB2. Subsequent pharmacological studies have confirmed that there are other receptors interacting with cannabinoids. GPR55 is a transmembrane G protein coupled receptor, which together with the receptor GPR18 and GPR119 belong to a group of new cannabinoid receptors and is involved in the function of the endocannabinoid system. In addition to some of cannabinoid substances, it is stimulated primarily phospholipid lysofosfatidylinositolem. LPI-dependent signaling GPR55 plays an important role in the regulation of many physiological and pathological processes, such as pain, inflammation, cell proliferation, or endothelial function. It was found that LPI confers tolerance to ischemic brain damage and has a cytoprotective effect on the pyramidal cells. The aim of the study was to determine whether the application of five ligands induce phosphorylation of protein kinase ERK 1/2, Akt and activate the GTPase RhoA and whether activation of the receptor GPR55 has cytoprotective effect in model cell line PC12, in which hypoxic conditions were simulated by adding CoCl2. For working methods were used SDS-PAGE, Western bloting and colorimetric measurement. Pharmacological studies in recent years have shown...

Le travail réalisé dans le cadre de cette thèse vise à l'approfondissement des connaissances sur l'impact de l'environnement gestationnel sur le développement de la commande respiratoire chez le nouveau-né. La respiration est une activité rythmique et autonome. La maturation des réseaux neuronaux impliqués dans son contrôle commence au cours de la gestation et persiste pendant la période postnatale. Le contrôle respiratoire est donc particulièrement susceptible à des variations de l'environnement intra-utérin. Nous avons étudié l'effet de deux stress gestationnels chroniques distincts : l'exposition à des cannabinoïdes et l'exposition à une hypoxie prénatale. A l'aide de techniques intégrées telles que la pléthysmographie à corps entier, éléctrophysiologiques comme la préparation de tronc cérébrale isolé ou les tranches de bulbes et immunohisto- et neurochimiques, nous avons pu caractériser l'impact des stress prénataux étudiés sur la plasticité respiratoire développementale. Nous avons pu établir un rôle central de l'anandamide dans la modulation de la commande respiratoire, probablement via des mécanismes noradrénergiques. L'exposition prénatale au WIN 55,212-2 induit une hyperventilation basale chez la souris nouveau-née qui semble relever d'altérations périphériques. Elle induit également une accentuation de la réponse centrale à l'hypoxie aigue et affecte l'apparition d'irrégularités respiratoires telles que les apnées. Quant à l'hypoxie prénatale, elle induit une hyperventilation basale d'origine centrale ainsi qu'une augmentation de l'activité catécholaminergique du tronc cérébrale. / Breathing is an autonomic rhythmic activity. The maturation of the neuronal networks responsible for breathing control begins in early gestation and persists throughout the neonatal period. This renders breathing control particularly susceptible to early insults arising from the intra uterine environment. We have studied the effects of two such gestational stress factors, namely prenatal exposure to cannabinoids and to gestational hypoxia. We use a wide array of in vivo and in vitro techniques in order to characterise the effect of the adverse intra uterine environment on the development of respiratory control. These include integrated techniques such as whole body plethysmography, electrophysiological preparations (en bloc and brainstem slice recordings) as well as immunohisto- and neurochemical approaches. We show that the prenatal stress factors induce developmental respiratory plasticity. This translates as heightened basal ventilation, observed following prenatal exposure to both cannabinoids and hypoxia. Comparisons between in vivo and in vitro techniques reveal a peripheral origin for the alterations observed following prenatal exposure to cannabinoids, suggesting a perturbation of the initiation and/or integration of information from the peripheral carotid bodies. Following prenatal hypoxia, respiratory perturbations have a central origin and can thus be observed in reduced preparations such as brainstem slice recordings. Neurochemical and immunohistochemical assays also reveal alterations in the bioaminergic modulation of the respiratory rhythm generator, perhaps underpinning the observed effects on respiratory control.

Cannabis and its derivatives have been used for both medicinal and recreational purposes. The study of this plant led to the discovery of over 60 cannabinoids, found exclusively in cannabis, that contribute to the behavioral effects of cannabis use, the most common is delta-9-tetrahydrocannabinol. Cannabinoid receptors function to increase activity in the mesolimbic dopamine reward system. Dopamine is a neurotransmitter that plays a major role in addition and its regulation plays a crucial role in mental and physical well-being. There is evidence that CB1 receptors are important to the reinforcing effects and the development of physical dependence on opiate drugs. Studies have shown that increased levels of dopamine are consistent with addiction while reduced levels lead to a decline in recreational use. The goal of this research is to design, synthesize and develop potential CB1 receptors that exhibit a neutral cannabinoid antagonist pharmacological profile.

This study was aimed at the development of novel CB1 cannabinoid receptor antago­nists that may have clinical applications for the treatment of cannabinoid and psychostimulant addiction. The rationale for the design for our target was to incorporate a bioisosteric 1,2,3-triazole ring into the vicinal diaryl group revealed in the prototypical antagonist/inverse agonist SR141716 (Rimonabant) that was pre­sumed to interact with a unique region in the CB1 receptors. Based on our prelimi­nary results we identified a novel series of 1,2,3-triazole ester and keto deriva­tives as lead compounds for biological evaluation. Here in the design rationale, syn­thesis and CB1 receptor affinity for a series of 4,5-diaryl-1-substituted-1,2,3-triazoles of ester and ketones is described. These derivatives were synthesized via a one-pot regiospecific click/acylation reaction sequence from 1-azido-2,4-dichlorobenzene and commercially available arylacetylenes. From the structure-activity studies the 5-(4-chlorophenyl) congeners exhibited the most potent CB1 receptor affinities relative to other 5-(substituted-phenyl) moieties. The 1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-propylcarbonyl-1,2,3-triazole (­31a) was found to be the most potent (Ki = 4.6 nM) CB1 receptor ligand of the series and exhibited high CB1 selectivity (CB2/CB1 = 417). The triazole ester 31a was further characterized as a cannabinoid antagonist in locomotor-activity studies by blocking the locomotor-reducing effects of cannabinoid agonist WIN55,212-2. In addition, unlike the prototypical cannabinoid antagonist SR141716A (Rimonabant), the triazole ester 31a did not exhibit increased activity in locomotor activ­ity studies, thus indicating the potential for a neutral antagonist profile.

Un des obstacles majeurs de la recherche en neurosciences est la difficulté d’accéder de manière directe au fonctionnement du cerveau afin de comprendre les mécanismes biologiques à l’origine des dysfonctionnements cérébraux dans les troubles psychiatriques. En tant qu’extension anatomique et développementale du système nerveux central, la rétine pourrait permettre d’offrir un accès indirect aux fonctions neurologiques cérébrales. Ainsi, l’investigation de la fonction rétinienne apporte l’unique opportunité d’étudier de manière objective un réseau neuronal complexe présentant des similarités avec celui du cerveau. Le cannabis est une substance neurotoxique identifiée comme modulant la transmission synaptique cérébrale par l’intermédiaire du système cannabinoïde mais les mécanismes précis à l’origine de ces anomalies sont peu connus. La première partie de ce travail consiste à présenter les bases neurobiologiques et les hypothèses physiopathologiques justifiant l’étude de la fonction rétinienne chez les usagers de cannabis, en se basant sur la présence du système cannabinoïde dans la rétine et son implication dans la régulation de la libération synaptique de neurotransmetteurs. La seconde partie discute l’intérêt de l’étude de la fonction rétinienne dans la recherche en psychiatrie avec des méthodes électrophysiologiques. Enfin, la dernière partie présente les dysfonctions rétiniennes présentes chez les usagers de cannabis, après un usage aigu ou régulier, évaluées par les techniques électrophysiologiques comme l’électrorétinogramme. Toutes ces données renforcent la pertinence de la rétine comme site d’investigation du cerveau et ouvrent éventuellement la perspective au développement de marqueurs fonctionnels / One of major obstacles in neuroscience research is the difficulty of directly accessing the brain function to understand the biological mechanisms underlying brain dysfunctions in psychiatric disorders. As an anatomical and developmental extension of the central nervous system, the retina could afford to offer an indirect access to brain neurological functions. Investigating the retinal function provides the unique opportunity to study in an objective way a complex neuronal network which shares similar properties with the brain. Cannabis is a neurotoxic substance identified as modulating brain synaptic transmission through the cannabinoid system, but the precise mechanisms underpinning these anomalies are poorly understood. The first part of this work is dedicated to present the neurobiological basis and pathophysiological hypotheses justifying the study of retinal function in cannabis users and is based on the presence of the cannabinoid system in the retina and its involvement in the regulation of synaptic neurotransmission. The second part discusses the interest of the study of retinal function with electrophysiological methods in psychiatric research. The last part presents the retinal dysfunctions detected in cannabis users, after acute or regular use, and assessed by electrophysiological techniques such as electroretinogram. All these data reinforce the relevance of the retina as a site of brain investigation and possibly open the prospect for the development of functional markers

Synthetic cannabinoids have become a growing concern in society. The extensive list of synthetic cannabinoids and the abuse rate has drawn the attention by government agencies throughout the world. These synthetic cannabinoids can adopt a number of different structures, while still acting on endogenous cannabinoid (CB1 and CB2) receptors. In addition, due to structural modifications of these synthetic cannabinoids, many of these compounds can bind to CB1 and CB2 receptors with greater affinity causing severe adverse and life-threatening effects. Because of their structural dissimilarity to the phytocannabinoid Δ9-THC, combating the rapid growth and emergence of synthetic cannabinoids with conventional THC-based methods has become an ongoing struggle. The purpose of this research was to develop and validate a robust and reliable method to accurately identify and quantify 17 synthetic cannabinoids in human whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated in accordance to SWGTOX guidelines for quantitative analysis using the following analytes: 4-cyano-CUMYL-BUTINACA, 5F-3,5-ABPFUPPYCA, 5F-ADB-PINACA, 5F- PY-PINACA, ADB-PINACA, APP-PICA, CUMYL-THPINACA, EMB-FUNICACA, JWH-250, MDMB-FUBICA, MEP-CHMICA, MO-CHMINACA, NM2201, PB-22, RCS-8, UR144, and XLR11. With this developed method, total analysis time was 8 minutes with samples eluting from 3.8 to 5.8 minutes. Calibration curves for each analyte had acceptable R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 0.5 – 25 ng/mL was used for all analytes, except for APP-PICA and NM2201 which were quantifiable at 0.1 ng/mL and PB-22 which used a quadratic model. Extraction of analytes using supported liquid extraction (SLE) cartridge improved sample-prep time by more than half, compared to traditional solid phase extraction (SPE) methods. Percent recovery of analytes using SLE was determined to be from 54.92 to 83.36%. Bias and Precision was assessed at 1, 3, 7, and 20 ng/mL for all analytes. All samples had acceptable calculated percent bias and percent coefficient of variation (%CV) within ±20%. No carryover was observed with this method. Matrix effect, using 10 different sources, did not have any interfering effects on detection and quantification of analytes. Ionization suppression and enhancement was observed at various levels, from -4.47 to 76.67%, but had little effect on other validation parameters. Analysis of other commonly encountered drugs (clonazepam, diazepam, (+) methadone, morphine, fentanyl, cocaine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 25I-NBOMe, and phencyclidine (PCP)) does not show any source of interference. The overall development and validation of this method demonstrates a sensitive and reliable way to positively identify 17 different synthetic cannabinoids in human whole blood in rapid time. / 2020-01-31

Novel Psychoactive Substances (NPS) also known as "legal highs" replicate the effects of illegal substances such as ecstasy and cocaine. The most common NPS reported are stimulants and synthetic cannabinoids. Despite the Psychoactive Ban (2016) recent reports identified the UK as having the largest market of NPS use anywhere in Europe. These substances have a short history of consumption and consequently little is known about their effects and health implications. Despite this, the sale of NPS is easily achieved through the internet and street dealers. Increased reports of negative health consequences from NPS consumption and research findings highlighting the willingness of young people to consume drugs without knowing what they are, mean it is vital that we investigate young people's understandings and perceptions of them. At present there are very few in-depth qualitative studies on NPS. A series of 7 focus groups with a range of young people (40=N: aged 16- 24 years) across the Merseyside area were carried out. Research sites included colleges, youth groups, supported living accommodations, and youth drug and alcohol services. Focus group interviews explored participants' perceptions of NPS and were followed up with a few semi structured interviews with selected participants. The direction of the study focused on mainly on synthetic cannabinoids which may reflect the age of the study's population. Using thematic analysis informed by a social constructionist perspective, three main themes were identified around stigma and identity, attractive features of NPS and risk. Findings showed that young people's perceptions of these substances were dependent on their level of experience with illegal substances and NPS. A novel finding was that synthetic cannabinoid use is employed in the normalisation of cannabis use. Local, national and policy recommendations are made on how youth and health services in both educational and specialised services could work more closely and effectively with young people NPS. They also identify a need among young people for specific guidelines on how to use the Internet and Print media in relation to previous knowledge and experience.

Tricomas secretores são apêndices epidérmicos presentes nas maiorias das plantas desempenhando funções diversas, dentre elas a defesa contra herbivoria. Dentre as famílias que apresentam tricomas secretores destaca-se Cannabaceae, que é amplamente estudada por produzir compostos psicoativos e outros utilizados como aromatizantes de cerveja. Recentemente alguns gêneros pertencentes à Ulmaceae foram inseridos em Cannabaceae, como Celtis e Trema. Tais gêneros são os mais numerosos em espécies da família. Considerando que tricomas secretores são os principais sítios de produção de substâncias de interesse econômico, e que são amplamente utilizados como caráter de valor taxonômico, o presente trabalho comparou a distribuição e morfologia de tricomas secretores de duas espécies de Cannabaceae (Celtis pubescens e Trema micrantha) e três espécies afins de Ulmaceae (Ampelocera glabra, Phyllostylon rhamnoides e Ulmus parvifolia). Os dados foram comparados aos existentes na literatura e discutidos em um contexto sistemático. Os tricomas secretores de Trema micranta foram, ainda, analisados em detalhe quanto à ontogenia, análise química do exsudato (in situ e direto) e ultraestrutura. Para tal, amostras de folhas e flores (pistiladas e estaminadas) foram coletadas, fixadas e processadas para análise de microscopia de luz, eletrônica de varredura e de transmissão. Foram encontrados cinco morfotipos diferentes nas espécies estudadas. C. pubescens e T. micrantha apresentaram tricomas secretores capitados e filiformes, que diferiram quanto ao pedúnculo, unisseriado em C. pubescens e bisseriado em T. micrantha. As três espécies de Ulmaceae apresentaram um único morfotipo (capitado com pedúnculo unicelular). Os tricomas de T. micranta iniciam o desenvolvimento com uma divisão anticlinal, semelhante ao que ocorre em Cannabis sativa. Seus tricomas secretam terpenos, alcaloides e compostos fenólicos. Testes químicos comprovaram a presença de alcaloides e os indicadores de canabinoides sugerem que T. micrantha apresenta potencial para produção desses compostos. A morfologia dos tricomas encontrados nas espécies de Cannabaceae é diferente da observada nas espécies de Ulmaceae, o que apoia a atual circunscrição destas famílias. A diversidade morfológica e de termos utilizados torna difíceis a classificação e comparação entre os tipos de tricomas secretores, sendo necessários esforços no sentido de padronizar a caracterização destas estruturas secretoras, em especial nas famílias que compõem o clado urticoide. / Secretory trichomes are epidermal appendages that are present in most plants performing various functions, among them the defense against herbivory. Cannabaceae comprises plants covered with secretory trichomes, and is a well-known family for producing psychoactive compounds and others used as beer flavorings. Recently some genera belonging to Ulmaceae were inserted into Cannabaceae, such as Celtis and Trema, the species-richest genera of the family. As the secretory trichomes are the main plant source of economically important substances and are widely used as taxonomic markers, the present study compared the distribution and morphology of secretory trichomes of two species of Cannabaceae (Celtis pubescens and Trema micrantha) and three related species of Ulmaceae (Ampelocera glabra, Phyllostylon rhamnoides and Ulmus parvifolia). The data were compared to those in the literature and discussed in a systematic context. The secretory trichomes of Trema micranta were also analyzed in detail regarding ontogeny, histolocalization of substances and ultrastructure. For this, samples of leaf and flower (pistillate and staminate) were collected, fixed and processed for analyses of light microscopy, scanning electron microscopy and transmission electron microscopy. For T. micranta chemical tests in situ and direct were carried out; in addition, samples were prepared for ultrastructural study in transmission electron microscopy. Five different morphotypes of secretory trichomes were found in the species studied. C. pubescens and T. micrantha exhibited capitate and filiform secretory trichomes that differed in the stalk, uniseriate in C. pubescens and biseriate in T. micrantha. The three species of Ulmaceae had a single morphotype (capitate with a unicellular stalk). The trichomes of T. micranta originate of a first anticlinal cell division, similar to what occurs in Cannabis sativa. They secrete terpenes, alkaloids and phenolic compounds. Chemical tests have confirmed the presence of alkaloids and cannabinoid indicators suggest that T. micrantha is a putative species for the production of these compounds. The morphology of the trichomes found in the species of Cannabaceae is different from that observed in the species of Ulmaceae, which supports the current circumscription of these families. The diversity of morphology and terms employed makes it difficult to classify and compare the types of secretory trichomes. In this sense, efforts are needed to standardize the characterization of these secretory structures, especially in the families that comprise the urticalean rosids.

by Priscilla, Ka-Yee Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 77-100). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.4 / ABSTRACT --- p.5 / ABSTRACT (Chinese) --- p.7 / PUBLICATION --- p.8 / ABBREVIATION --- p.9 / Chapter CHAPTER 1 --- INTRODUCTION --- p.10 / Chapter 1.1 --- Overview of the study --- p.10 / Chapter 1.2. --- Substantia nigra pars reticulata (SNR) --- p.12 / Chapter 1.2.1 --- SNR and the basal ganglia / Chapter 1.2.2 --- GABA neurotransmission in SNR / Chapter 1.2.3 --- SNR and epilepsy / Chapter 1.3 --- GABAb receptors --- p.18 / Chapter 1.3.1 --- GABA receptors / Chapter 1.3.2 --- GABAb receptors and their classification / Chapter 1.3.3 --- Agonists and antagonists of GABAb receptor / Chapter 1.3.4 --- Distribution of GAB AB receptor / Chapter 1.3.5 --- GABAb receptors in epilepsy and the involvement of SNR / Chapter 1.4 --- Cannabinoid receptors --- p.24 / Chapter 1.4.1 --- Cannabinoid receptors and their classification / Chapter 1.4.2 --- Agonists and antagonists of cannabinoid receptor / Chapter 1.4.3 --- Distribution of cannabinoid receptors / Chapter 1.4.4 --- Cannabinoid receptors in epilepsy and the involvement of SNR / Chapter CHAPTER 2 --- METHODS --- p.31 / Chapter 2.1 --- Brain slice preparation and maintenance --- p.31 / Chapter 2.2 --- Experimental set-up --- p.32 / Chapter 2.2.1 --- Visualization of neurones / Chapter 2.2.2 --- Electrophysiological recordings / Chapter 2.2.3 --- Evoked stimulation / Chapter 2.2.4 --- Drug preparation and administration / Chapter 2.3 --- Identification of GAB A and dopamine neurones --- p.36 / Chapter 2.4 --- Data analysis --- p.37 / Chapter 2.4.1 --- Construction of dose-response curve / Chapter 2.4.2 --- Analysis of synaptic currents / Chapter 2.4.3 --- Statistics / Chapter CHAPTER 3 --- RESULTS --- p.39 / Chapter 3.1 --- Basic characteristics of IPSCs in SNR --- p.39 / Chapter 3.1.1 --- Spontaneous and miniature IPSCs / Chapter 3.1.2 --- Evoked IPSCs / Chapter 3.2 --- GABAb receptors in SNR --- p.42 / Chapter 3.2.1 --- Postsynaptic GABAb receptors in SNR neurones / Chapter 3.2.1.1 --- Baclofen-activated postsynaptic response / Chapter 3.2.1.2 --- Effects of GABAb receptor antagonist on IPSCs / Chapter 3.2.2 --- Presynaptic GABAb receptors / Chapter 3.2.3 --- Effects of GAB A uptake blocker / Chapter 3.3 --- Cannabinoid receptors in SNR --- p.51 / Chapter 3.3.1 --- Postsynaptic cannabinoid receptors in SNR neurones / Chapter 3.3.2 --- Presynaptic action of cannabinoids / Chapter CHAPTER 4 --- DISCUSSION and CONCLUSION --- p.55 / Chapter 4.1 --- General properties of IPSCs --- p.55 / Chapter 4.2 --- GABAb receptors in SNR neurones --- p.58 / Chapter 4.2.1 --- Postsynaptic GABAB receptors in SNR neurones / Chapter 4.2.2 --- GABAb component in spontaneous and evoked IPSCs / Chapter 4.2.3 --- Presynaptic GABAb receptors in SNR / Chapter 4.2.4 --- Role of GABA uptake / Chapter 4.3 --- Cannabinoid receptors in SNR neurones --- p.67 / Chapter 4.3.1 --- Postsynaptic cannabinoid receptors in SNR neurones / Chapter 4.3.2 --- Presynaptic cannabinoid receptors in SNR / Chapter 4.4 --- SNR GABAb and cannabinoid receptors - their role in epilepsy --- p.72 / Chapter 4.5 --- Concluding remarks and future direction --- p.75 / REFERENCES --- p.77