This project involved the study of mechanisms which modulate autonomic transmission within the sympathetic nervous system using the mouse vas deferens as a model tissue. Data was collected using contraction studies, electrophysiological techniques with sharp microelectrodes, and fluorescent calcium imaging of both smooth muscle cells and nerve terminal varicosities. An additional series of experiments was conducted using the PC12 cell line, derived from a phaeochromocytoma of the rat adrenal medulla, for flow cytometry experiments using fluorescence-activated cell sorting. During the course of this project a novel technique for studying the activity of the norepinephrine transporter within a whole organ preparation was developed using the neurotransmitter uptake assay. The uptake of this assay within the nerve terminals of the vas deferens was abolished by desipramine whilst its rate of washout was increased by amphetamine. However, some non-neuronal, peri-nuclear staining which could not be prevented by a range of pharmacological means was also observed. This new technique was then used in other work exploring putative NET regulation by cannabinoids. The modulatory effects of two pharmacological groups were assessed: testosterone and cannabinoids. Testosterone was found to have a rapid, non-genomic effect inhibiting neurotransmission within the vas deferens. This was a postjunctional effect which appeared to involve modulation of the opening of L-type calcium channels on the smooth muscle cells. For the studies of cannabinoids, two broad areas of research were conducted. First the effects of Δ⁹-tetrahydrocannabinol were investigated with regard to the pre-junctional release of neurotransmitters and the effect of THC on calcium dynamics within individual nerve terminal varicosities. Secondly, a surprising novel effect upon the norepinephrine transporter was identified and examined. This inhibitory effect was revealed initially by contraction experiments demonstrating a decrease in the rate of uptake of noradrenaline from the junction. This work demonstrates that there are still novel modes of regulation of sympathetic transmission to be uncovered. The ongoing challenge is to establish their role within physiology and pathophysiology.

The phytocannabinoid cannabidiol (CBD) has a complex pharmacology which is thought to include, but is not limited to, an ability to act as an inverse agonist at the CB1 and CB2 receptors and an antagonist of GPR55. Moreover, is has been shown to reduce infarct size and ameliorate reductions in left ventricular function in vivo. These improvements in the pathogenesis of experimental MI are accompanied by a reduction in inflammatory cell migration to the area at risk. More recently it has been shown that CBD is anti-arrhythmic in acute experimental MI. Thus, it was suggested that the cardioprotective effects of CBD might be due to an anti-inflammatory action. In addition, GPR55 receptor activation is acknowledged to mediate mobilisation of intracellular Ca2+ (Ca2+i) which could potentially be pro-arrhythmic and so CBD, as an antagonist may confer cardioprotection via GPR55. However, the receptors and/or mechanisms responsible for mediating the cardioprotective effects of CBD are get to be determined. The present studies were therefore performed to; (1) better understand the pharmacology of CBD by assessing haemodynamic responses to CBD and other cannabinoids ligands in anaesthetised rats, (2) investigate the receptors involved in the anti-arrhythmic effect of CBD in a rat model of coronary artery occlusion (CAO), and (3) investigate if CBD can alter [Ca2+]i in isolated rat cardiomyocytes. The characterisation of the pharmacology of CBD in vivo showed that; firstly, CB1 receptor activation causes a hypotensive response which can be dose-dependently inhibited by AM251; secondly, both CBD and AM251 alone (a CB1 receptor antagonist and GPR55 agonist) can induce vasodepressor responses and finally, CBD can potentiate the AM251-mediated hypotension when co-administered, suggesting possible cross-talk between the CB1 and GPR55. Results from CAO studies showed that CBD and AM251 each have the capacity to reduce arrhythmias. Moreover, when CBD and AM251 were co-administered the anti-arrhythmic capacity of either alone was potentiated. However, the degree of potentiation was dependent on the order of administration, suggesting that more than one receptor is involved in the summative anti-arrhythmic effects. The investigation of cardiomyocyte [Ca2+]i suggested that AM251 can modulate [Ca2+]i at the level of the cardiomyocyte, while CBD cannot. These data give novel insight into the anti-arrhythmic effects of CBD and, moreover, for the first time demonstrate that AM251 is anti-arrhythmic. In addition, these data suggest a role for GPR55 in increasing [Ca2+]i via AM251.

Although the impact of cannabinoids (CBs) on anxiety has been thoroughly studied, current research paradigms fail to incorporate acute stressors. The present study investigated the synthetic CB HU-210’s anxiolytic potential in an acute stress CD1 male mouse model, where the animals were subject to a 10-minute Forced Swimming (FS) test between treatment and behavioral tests. Surprisingly, HU-210 did not show anxiolytic action in the Open Field (OFT) and Elevated-Plus Maze (EPM) stressed mice as previously reported in the naïve model literature. The combination of acute stress and high HU-210 doses produced severe locomotor impairments in ambulatory movement that were not previously observed in unstressed mice. It is hypothesized that this anxiogenic phenotype results from the summation of exogenous CB treatment and stress-induced endocannabinoid (eCB) release. Subsequently, the impact of the eCB signaling on anxiety behaviors was examined. Systemic administration of KML29, the selective inhibitor of 2-AG degradative enzyme, returned stress-induced anxiety-like behaviors to baseline levels, without significantly affecting locomotion. KML29’s anxiolyticism was abolished when combined with the cannabinoid receptor antagonist AM281, implying this is a CB receptor-mediated process. A GABAA receptor agonist muscimol was co-administered with KML29 in order to pharmacologically investigate the role of GABAergic neurotransmission in this anxiolytic phenomenon, but it did not alter KML29’s effects. Collectively, these findings suggest that exogenous CBs and acute stress act synergistically in an anxiogenic manner, but that enhanced 2-AG signaling in response to stress demonstrates anxiolytic potential.

In this study, the presence and possible function of endocannabinoid ligands in the planarian is investigated. The endocannabinoids ananadamide (AEA) and 2-arachidonoylglycerol (2-AG) and entourage NAE compounds palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and oleoylethanolamide (OEA) were found in Dugesia dorotocephala. Changes in SEA, PEA, and AEA levels were observed over the initial twelve hours of active regeneration. Exogenously applied AEA, 2-AG and their catabolic inhibition effected biphasic changes in locomotor velocity, analogous to those observed in murines. The genome of a close relative, Schmidtea mediterranea, courtesy of the University of Utah S. med genome database, was explored for cannabinoid receptors, none were found. A putative fatty acid amide hydrolase (FAAH) homolog was found in Schmidtea mediterranea.

The primary goal of this study was to investigate the awareness and prevalence of Spice and K2 usage among a population of college students, as well as the demographics of such users. The study also sought to determine whether or not students prefer these products over natural cannabis, in addition to examining the most popular methods of obtainment and the most commonly reported side effects of K2 and Spice usage. Participants consisted of 643 undergraduate students enrolled at the University of North Texas during the fall 2011 semester. Findings indicate that while students exhibit a relatively high awareness of K2 and Spice, usage of these products is not a prevalent occurrence. Implications of the findings are discussed.

Since their introduction to the United States in 2008, synthetic cannabinoids became the most widely used recreational drug behind marijuana, then regressed to an estimated prevalence of less than 1%. Contrary to expectations for a drug declining in use, emergency department presentations and acute poisonings related to the use of synthetic cannabinoids are increasing. Alongside this phenomenon, a growing body of literature is beginning to uncover a relationship between psychosis and synthetic cannabinoid use. A current gap in the literature exists surrounding harm prevention methods and targeted intervention strategies for users of synthetic cannabinoids. To date, no known studies have examined individuals with a history of use of these substances and investigated the reasons they decided to discontinue recreational use. The purpose of the current study was to fill this gap in the literature while also further confirming and expanding existing research on the characterization of synthetic substance use, perceived harm of synthetic cannabinoids, and users’ knowledge about synthetic cannabinoids. Cross sectional survey methods in a non-experimental comparative design was utilized with participants recruited through the online crowd sourcing platform Amazon MTurk. Significant motivating factors for both discontinuation and continuation of synthetic cannabinoid use were found including personal experience, accessibility, preference towards other substance, and questions surrounding the source and purity of the synthetic cannabinoids. It was also found that individuals who currently use synthetic cannabinoids have less general knowledge about the substance class when compared to individuals who have discontinued use. These results suggest that psychoeducational campaigning surrounding general knowledge about the substance class as well as information on the physiological effects of synthetic cannabinoids may be an effective harm reduction method.

Adequate evidence exists in the literature indicating a relatively positive shift with regards to the legal acceptance of cannabis and cannabis-derived products for medicinal purposes in some countries. Concomitantly, scientists are showing renewed interest in cannabis-related research work. Over the years, clinical and preclinical studies have demonstrated the therapeutic significance of cannabinoids for diverse indications. Additionally, efforts are being made to develop cannabis-related products into acceptable prescription products. FDA authorization for the commercial use of four cannabinoid-derived products, available as oral dosage forms is a significant progress already. However, there are certain drawbacks associated with the conventional delivery forms of cannabinoids. These include low oral bioavailability due to hepatic degradation, gastric instability, poor water solubility, and the side effects experienced upon the use of high doses of psychotropic cannabinoids associated with heightened plasma concentrations of the drug. These are however, limitable with the aid of transcutaneous drug delivery. Emerging topical and transdermal strategies could be exploited for the successful development of highly effective delivery systems for cannabinoids. This review discusses the feasibility of delivering therapeutic cannabinoids via skin and provides a comprehensive account of the supporting research studies that have been reported in the literature till date.

In recent years, there has been a rise in the use of New Psychoactive Substances (NPS) that mimic the effects of controlled drugs and licensed medicines, which have become a significant public health concern globally. Synthetic cannabinoids, a rapidly expanding category of NPS, with much higher potency and binding affinity to the cannabinoid receptors than ∆9-tetrahydrocannabinol (THC), have led to serious psychiatric complications and other adverse effects. This project aims to synthesize and analyze four potential metabolites of a synthetic cannabinoid, ADB-5´Br-BUTINACA, to determine if the synthesized metabolites correspond to those produced in human hepatocytes. The metabolites were synthesized by alkylation, amide coupling, and hydrolysis/TFA treatment and analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR). The reaction strategy combined all three steps into a single process, making it significantly easier to carry out and not requiring much expertise. The results suggest that the synthetic approach used in this project was successful in generating the desired potential metabolites, with overall yields varying from 27.5 % to 57.6 % and high purities ranging from 95.6 % to 99.3 %. However, the overall yields were lower than expected due to product loss during the transfer of the solution mixture, the possibility of side reactions, and incomplete conversion. Further optimization of reaction conditions may be necessary to improve the yield of the synthesized metabolites.

Chronic neuropathic pain is a leading cause of disability worldwide and is associated with immense economic burden. Of all chronic neuropathic pain conditions, chemotherapy-induced peripheral neuropathy (CIPN) persists as a monumental public health crisis, as it is the most common comorbidity among those receiving chemotherapy for cancer treatment. CIPN is unique compared to other forms of neuropathic pain in that it is severely dose-limiting, often leading to disruption or cessation of chemotherapeutic treatment and complicating an individual’s cancer prognosis. Current pharmacological treatments for combatting CIPN are widespread yet are all accompanied with the same hindrances – they are limited in therapeutic efficacy when administered chronically and are associated with severe risk for adverse effects. Therefore, there is a clear unmet need for novel pharmacotherapies for CIPN that achieve strong therapeutic efficacy while minimizing the susceptibility to adverse events.Here, we characterize the therapeutic efficacy and pharmacological mechanisms of a novel, plant-derived alkaloid mitragynine (MG), a constituent of the kratom plant (Mitragyna speciosa) in a mouse model of CIPN. Kratom products have emerged in the US in recent years as a popular form of self-treating pain, opioid withdrawal, and symptoms of anxiety and depression, but these intended uses are largely based on anecdotal reports in humans. MG possesses a unique, mixed pharmacological profile combining opioid, adrenergic, and serotonergic properties – resembling the pharmacology of current CIPN pharmacotherapies such as antidepressants. However, the relation of these pharmacological mechanisms of MG to the context of CIPN remain under characterized. Kratom products are also commonly used in combination with cannabis products, which are also used for self-treating pain, and play a significant role in palliative care for terminal cancer patients. Yet, interactions between kratom alkaloids and cannabinoid signaling have yet to be studied in the context of CIPN. Lastly, the basis of potential utility of individual kratom constituents such as MG on anxiety- and depression-like behaviors, which are heavily comorbid in individuals with CIPN, remain understudied. The present studies were conducted to explore the role of the kratom alkaloid MG on both pain and affective behaviors associated with CIPN, at the pharmacological, cellular, and molecular level. To accomplish this, we measured 1. Contributions of opioid and adrenergic signaling mechanisms to the therapeutic efficacy of MG in a mouse model of oxaliplatin-induced mechanical hypersensitivity using pharmacological inhibition. 2. Contributions of cannabinoid signaling to the therapeutic efficacy of MG in a mouse model of oxaliplatin-induced mechanical hypersensitivity and inflammatory pain using pharmacological and genetic approaches. 3. Effects of MG on affective behaviors associated with CIPN using mouse models of the tail-suspension test, elevated zero maze, and conditioned place preference. Overall, the findings from this dissertation support the hypothesis that MG displays therapeutic efficacy against nocifensive behavior of CIPN and pain-related affective behaviors. Opioid, adrenergic, and cannabinoid mechanisms all contribute to the effect of MG on oxaliplatin-induced mechanical hypersensitivity. MG is also capable of normalizing aberrant neurotrophic factor signaling associated with CIPN. Lastly, MG produces anxiolytic effects when repeatedly administered without developing a conditioned place preference, suggesting that it achieves therapeutic efficacy in a model of CIPN without risk of adverse events. / Biomedical Sciences

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