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The mechanism for human labour remains poorly understood, restricted by difficulties in human pregnancy experimentation, and this limits our ability to manage complications of parturition including spontaneous preterm birth and induction of labour. The studies presented in this thesis utilise metabolomic and phosphoproteomic techniques to improve our understanding of the pathways involved in the trigger for labour in humans. The first study in the thesis compared mass spectrometry metabolomics analysis of maternal and cord plasma at the time of delivery, and the results indicate that the trigger for spontaneous labour in humans may involve endocannabinoid, sphingolipid and ceramide pathways. The second study presented investigates this further, comparing the metabolic profiles of maternal, cord artery, and cord vein blood sampled from women during low-risk pregnancies, including comparisons of metabolite concentrations between different labour onsets and delivery. This study indicates that there are inherent differences in fetal and maternal signals between women who spontaneously labour and women who have an induction of labour. The third study assessed potential associations between the maternal metabolic profile measured in the second trimester with gestational age at delivery within the Born in Bradford cohort study (analysis sample n = 7440). This study indicates a metabolic profile of dyslipidaemia may be associated with shorter gestational age at delivery, and that non-pathological human labour may involve pathways related to inflammation and vascular function. The final study used phosphoproteomics analysis of fresh myometrium in vitro, and the findings indicate that there may be a myometrial phenotype associated with failed induction of labour and failure to progress. Taken together, these results provide novel leads to follow in order to progress our quest for a full understanding of the trigger for spontaneous labour in humans.

The skin barrier provides protection from external threats, and defects in the skin barrier are involved in several skin diseases such as atopic dermatitis, eczema, and psoriasis. Growing evidence suggests that endocannabinoids (EC) have a role in the biological processes of the skin including skin barrier formation. The EC system involvement in other biological barriers such as intestinal barrier, bronchial barrier and blood brain barrier has been shown to involve regulation of the tight junction (TJ) formation; this leads to the hypothesis that the EC system may also be involved in skin barrier regulation through regulation of epidermal TJ. The aim of this study was to explore the role of EC in keratinocyte differentiation and TJ functionality, two important processes in skin barrier formation, and explore the modulation of the EC system by the anti-inflammatory omega-3 (n-3) polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In order to address this aim, HaCaT keratinocytes, primary normal human epidermal keratinocytes (NHEK) and human skin samples were used as models. UVR and histamine were used to disrupt the barrier. Healthy volunteers were given EPA and DHA as oral supplementation and donated UVR-treated skin. EC and related N-acyl ethanolamines (NAE) and monoacyl glycerols (MAG) were analysed by UPLC/ESI-MS/MS in cells and skin samples. Enzymes (NAPE-PLD, 12-LOX, and 15-LOX2) and Langerhans cells (LC) were analysed by IHC. TJ functionality was assessed by transepithelial electrical resistance (TEER) and FITC-dextran permeability assays. Localisation of TJ proteins was assessed by IF, and protein and gene expression by western blot and RT-qPCR, respectively. EC and NAE were detected in cells and human skin samples. N-palmitoyl ethanolamine (PEA) was effective on TJ and two different paracellular pathways were investigated: the pore pathway responsible for the passage of small ions and leak pathway responsible for the passage of large molecules. TEER and FITC-Dextran assays revealed that PEA increased TJ integrity (p=0.0419) but had no effect on permeability in NHEK. PEA appears to mediate the pore pathway, supported by increased expression of claudin-4 protein (p=0.020) and claudin-4 gene expression (p=0.0011). PEA did not affect the expression of occludin and zonula occluden-1, responsible for the leak pathway. UVR did not alter the production of EC and NAE. However, increases in PEA (p=0.035), N-oleoyl ethanolamine (OEA) (p=0.022) and N-vaccenoyl ethanolamine (VEA) (p=0.024) levels in primary NHEK were observed overtime in response to histamine; this shows that NAE are involved in histamine-induced skin barrier damage. Interestingly, PEA (p=0.0123) was also able to repair the damaging effect of histamine on TJ, as shown by increased TEER. EPA and DHA had differential effects on EC in UVR-challenged skin. DHA overall decreased EC production and NAPE-PLD expression, while EPA showed an opposite effect and increased 15-LOX2. Only DHA reduced the migration of LC caused by UVR, while EPA reduced the effect of UVR on epidermal thickness. Overall, this study showed that EC and NAE are involved in skin barrier formation through TJ; PEA was the most potent species affecting TJ functionality acting through regulation of the TJ pore pathway. Production of EC and NAE are differentially regulated by EPA and DHA, and DHA could be used to modulate the EC system. Altering skin EC may lead to a development of potential barrier therapies by improving the integrity of epidermal TJ.

Human skin has distinct lipid metabolism and production of bioactive lipid mediators that can be modulated by nutritional supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFA), of which eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids exert anti-inflammatory effects. The aims of this project were to gain better understanding of their individual mechanisms in human epidermis and dermis. HaCaT keratinocytes, 46BR.1N fibroblasts, primary human epidermal keratinocytes and dermal fibroblasts were treated with EPA or DHA for 72h and then sham-irradiated or exposed to 15 mJ/cm2 ultraviolet radiation (UVR). Viability was measured by the MTT assay. The expression of cyclooxygenase-2 (COX-2), microsomal prostaglandin synthase-1 (mPGES-1) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) proteins was explored by western blotting. Human skin explants (n=4 donors) were cultured for 3 or 6 days and supplemented with EPA, DHA or vehicle. Culture media were collected to evaluate tissue damage and PUFA cytotoxicity (lactate dehydrogenase assay). Epidermal and dermal lipid profiles were assessed by gas chromatography and liquid chromatography coupled to tandem mass spectrometry. Primary keratinocytes were treated with fatty acids and various lipid mediators for 48h. Their effect was determined by the scratch assay and transepithelial electrical resistance. UVR upregulated COX-2 in HaCaT and primary epidermal keratinocytes, but did not affect mPGES-1 and 15-PGDH protein expression. UVR upregulated COX-2 and mPGES-1 in 46BR.1N fibroblasts but had no effect on 15-PGDH expression. The same UVR dose did not alter the expression of COX-2, mPGES-1 and 15-PGDH in primary dermal fibroblasts. Only EPA attenuated COX-2 expression in HaCaT and primary keratinocytes and either EPA or DHA had any effect in 46BR.1N and primary fibroblasts. Skin explants showed initial post-biopsy tissue damage. EPA and DHA supplementation augmented cellular levels of the corresponding fatty acids in both epidermis and dermis to a different extent. Increased uptake of DHA in the dermis was accompanied by reduced arachidonic acid levels. EPA treatment stimulated the production of PGE3 and various HEPE in epidermis, while DHA treatment caused high levels of HDHA species in dermis. N-3 PUFA and their derivatives delayed wound healing, cell migration and epidermal barrier permeability, while n-6 PUFA lipids showed the opposite effect. Overall, these findings suggest that EPA and DHA differently affect skin cells and skin, with EPA preference in epidermis and DHA in the dermis. These results highlight the importance of differential skin responses that could be important in skin health and disease.

Bioactive compounds from Cannabis sativa have been used for millennia to alleviate the symptoms of a range of diseases. The physiological basis of effects such as analgesia, stimulation of hunger and reduction of inflammation was established in the late 20th century with the discovery of cannabinoid receptors but efforts to synthesise safe and potent drugs targeting these proteins have so far failed. The major barrier to research in this area is the instability of the receptors outside of biological settings, rendering elucidation of the binding sites by traditional means difficult. Certain small molecules can interact with the cannabinoid type 1 receptor (CB1) at locations distinct to the primary ligand docking site. Such allosteric modulation of the endocannabinoid system offers significant advantages over using orthosteric drugs and in this research a range of indole based structures were synthesised and tested in an attempt to improve the activity and drug-like nature of a lead compound. A partial structure-activity relationship was established, including the description of the most potent allosteric enhancer of CB1 so far reported. Efforts were also undertaken to investigate the allosteric binding environments using photoactivatable ligands based on a CB1 inhibitor. In combination with mutation studies and computer modelling this technique could allow the rational design of allosteric modulators, a task which is not trivial at present. Two photoactivatable compounds were synthesised and shown to interact with the receptor, with a method for isolating covalently labelled peptide fragments from other biomolecules demonstrated using “click chemistry” and a modified Wang resin. This work may find application in future investigations aiming to produce allosteric pharmaceuticals targeting CB1. Furthermore, the techniques described may be applied to study the binding site of a recently described allosteric endocannabinoid or could potentially be adapted to look at secondary binding domains in other G protein-coupled receptors.

The effects of endocannabinoids on human, mouse and rabbit bone cells were investigated.  At high concentrations anandamide and 2-arachidonyl glycerol (2-AG) inhibited human osteoclast formation with no effects at lower concentrations. The inhibition was not attenuated by antagonists for the CB1, CB2 or TRPV1 receptors, indicating a non-receptor mediated effect.  Conversely, anandamide and 2-AG increased mouse osteoclast formation.  The effect of anandamide was enhanced in cells from fatty acid amide hydrolase (FAAH)-null mice and abolished in cells from CB1/2 knockout mice.  The effect of 2-AG was not eliminated in CB1/2 knockout cells, indicating a non-CB1/CB2 action.  The CB1 antagonist, AM251, and the CB2 antagonist, AM630, both inhibited mouse osteoclast formation.  These effects were not rescued in the CB1/2-knockout mouse cells.  Both anandamide and 2-AG stimulated actin ring formation and osteoclast activity in human and rabbit osteoclast.  This was prevented in the presence of AM630 but not AM251, indicating a CB2-mediated response.  The endocannabinoids and the cannabinoid receptor antagonists do not have a regulatory action on osteoblast activity. The effects of the novel cyclooxygenase-2 (COX-2) metabolite of 2-AG, prostaglandin E2-glycerol ester (PGE2-G), on human osteoclasts were examined.  Treatment with PGE2-G inhibited formation and ERK phosphorylation of human osteoclasts.  These effects were attenuated by a selective EP4 antagonist and mimicked by PGE2 alone, indicating that PGF2-G is rapidly metabolised into PGE2 in human osteoclast cultures.  However, PGE2-G treatment elevated intracellular calcium levels in human osteoclasts, through a phospholipase C (PLC)- and IP3- dependent mechanism, indicative of a G-protein coupled receptor effect.  This was not mimicked by PGE2, or prevented by the EP4 antagonist, but blocked by a putative PGE2-G receptor antagonist, PDA-94 indicating that PDA-94 may be a PGE2-G receptor antagonist.

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with neuroprotective and cardioprotective properties. It is thought that some of the actions of EPA may be attributed to its elongated metabolite, the PUFA docosapentaenoic acid (DPA). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are bioactive PUFA ubiquitously expressed in neural tissues. EPA and AA can be converted by cyclooxygenase (COX) to prostanoids and by lipoxygenase (LOX) to hydroxy fatty acids. PUFA can also be converted to ethanolamides in the brain. These mediators are involved in physiological and pathological processes in many bodily systems. The purpose of this study was to examine the production of eicosanoids, hydroxy fatty acids and fatty acid ethanolamides in young and aged rat brain following EPA or DPA enriched diets. The effects of specific PUFA on human platelet eicosanoid production were also investigated as these mediators play a role in adhesion and aggregation. Liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) assays were developed and used to measure lipid mediators in rat brain and human platelets. Ageing in rat brain was accompanied with several changes in the prostanoid and hydroxy fatty acid profiles. Supplementing the diet with EPA or DPA at a daily dose of 200 mg/kg for 8 weeks prevented these changes and decreased levels of PGE2. DPA changed the profile of hydroxy fatty acids synthesised in the brain tissue of young animals. This study has shown that levels of eicosapentaenoylethanolamide (EPA-EA) increase in the brain as a result of ageing and that this is accompanied by an increase in levels of anandamide. Feeding aged animals EPA or DPA further increased the levels of EPA-EA but prevented any change in the level of anandamide. Niacin is used to treat hypercholesterolaemia although it is associated with an unpleasant PGD2 mediated skin flush. This exploratory study has shown that human platelets treated with niacin did not show any changes in their prostanoid and hydroxy fatty acid profiles. Platelets treated with EPA showed increased production of TXB3 and 12-HEPE. Niacin augmented the effects of EPA on human platelet mediator synthesis. Overall, this study has demonstrated that EPA can change brain and platelet lipid mediator synthesis and has provided evidence that could explain some of the neuroprotective and cardioprotective actions of this PUFA.

Here we demonstrate that both exogenous and endogenous cannabinoids affect different aspects of learning and memory in the rat.  For example, the potent CB1 receptor agonist, WIN-2 was able to delay-dependently impair short-term memory (STM) sparing reference memory (RM).  This demonstrates that it is the STM but  not RM processes that are more sensitive to the effects of cannabinoids. In addition, given that cannabinoids were able to hinder the recruitment of hippocampal firing correlates that are crucial for correct performance of a STM task, suppress hippocampal principal cell firing during the encoding phase of a STM task, reduce spontaneous bursting and disrupt synchronous firing of hippocampal principal cells respectively, confirm that they do alter the neurophysiology of the hippocampus.  These cannabinoid induced alterations in hippocampal neuronal activity may well explain the observed deficits across numerous other working memory (WM) and STM tasks. The results also revealed that cannabinoid-induced deficits in learning and memory are brought about due to an interaction between cannabinoid and cholinergic systems.  Although endocannabinoids failed to produce impairments in STM under normal physiological conditions, STM deficits were observed when anadamide levels were pharmacologically elevated beyond normal physiological levels.  Moreover, results demonstrate that the endocannabinoid system is involved in behavioural flexibility (i.e. reversal learning) and modulation of acquisition and/or consolidation of certain spatial elements that are necessary to perform an operant conditioning risk. Overall, the results in this thesis show that cannabinoid induced deficits in learning and memory are produced as a result of their direct effects on hippocampal processing.  The exact mechanisms that mediate these cannabinoid-induced deficits in memory are yet unclear and remain to be determined.

Environmental light information is the main stimulus that synchronizes an organisms’ circadian rhythms, which are approximately 24-hour oscillations in physiology and behavior. In mammals, circadian rhythms are driven by a central pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. A neuromodulatory system within the SCN, the endocannabinoid system (ECS), plays a role in clock function; however, little is known about the bidirectional effects each system exerts on one another. Therefore, we investigated the temporal and spatial architecture of the ECS in the SCN and examined the effects of acute administration of exogenous cannabinoids on circadian behavior. To investigate the temporal molecular landscape of ECS components in the SCN we excised the SCN of male and female C57BL/6J mice that were housed under 12:12 light/dark and constant dark conditions. SCN tissue was then processed to examine ECS gene expression across the 24-hour day. Data from these experiments suggest that a considerable number of ECS genes are under control of the circadian clock, and further suggests that they have a time-of-day specific role in SCN clock function. Out of six ECS receptors we examined, only one was detected, cannabinoid receptor 1 (Cnr1/CB1), which is the primary ECS receptor in the central nervous system, along with several genes involved in synthesis, degradation, and transport of endocannabinoids. Next, we investigated the spatial layout of the ECS in the SCN with RNAscope in situ hybridization. Analysis of these data shows most of the inspected ECS components are in a higher percentage of cells in male compared to female animals. All genes were present in both neurons and astrocytes, but only two genes, Mgll and fatty acid binding protein 7 (Fabp7), exhibited higher levels in astrocytes. We also characterized ECS components in cells expressing the neuropeptides Vip, Avp, or neither neuropeptide; there were equal or the highest levels of expression in the neuronal category which lacked Vip or Avp. These results suggest that endocannabinoids are synthesized, degraded, and signal through multiple cell types within the SCN, but primarily signal through neuronally located CB1. We then tested the effects of the exogenous cannabinoids CP 55,940 (a CB1 receptor agonist) and cannabidiol (CBD) (a constituent of marijuana) on circadian wheel-running behavior. Acute injections of CP 55,940 early in the active period paired with a light pulse 30 minutes after intraperitoneal injections reduces the phase shifting effects on clock output in male animals, but not in females. Increasing doses of CP 55,940 increased the amount of wheel-running suppression on the day of injection but did so in both males and females. This suggests that the sex difference in phase delay effects are circadian clock specific. No effects of CBD administration on photic phase shifts were observed. These behavioral data imply that cannabinoids mainly act through CB1 to affect the circadian clock. Taken together, our results support a time-of-day dependent modulation of the ECS in SCN clock function, where endocannabinoid signaling is regulated via neurons and astrocytes. Additionally, our data support the idea that cannabinoids primarily act on the circadian clock by signaling through CB1 and that a sex difference in circadian behavior occurs with its activation.

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally. Cutaneous leishmaniasis (CL) is the most common form, characterized by chronic skin lesions. Currently, there are no approved vaccines for human use. We have generated centrin knock out Leishmania (L.) mexicana (LmexCen-/-) mutants using CRISPR/Cas9. Centrin is a cytoskeletal protein required only for intracellular amastigote replication in Leishmania. Here, we investigated the safety, immunogenicity, and efficacy of LmexCen-/- parasites in vitro and in vivo. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected macrophages and dendritic cells,compared to LmexWT. Furthermore, LmexCen-/- parasites are safe in susceptible mouse models and efficacious against challenge with LmexWT in genetically different BALB/c and C57BL/6 mice. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens compared to the controls. Overall, we demonstrate that LmexCen-/- parasites are a promising candidate vaccine against CL in pre-clinical models.Next, we explored the metabolic drivers of these vaccine-mediated immunological profiles. Metabolomics are emerging as a useful tool to uncover unknown networks that govern immune regulation and determine functional specialization. We analyzed the metabolic changes occurring after immunization with LmexCen-/- and compared them with LmexWT infection. Our results show enriched aspartate metabolism and pentose phosphate pathway (PPP) in ears immunized with LmexCen-/- parasites. These pathways are both known to promote M1 polarization in macrophages, and PPP in particular induces nitric oxide production in macrophages cultured with LmexCen-/-, suggesting a shift to a pro-inflammatory phenotype following immunization. Furthermore, immunized mice showed enriched taurine/hypotaurine metabolism in their ears, and linoleic acid in their lymph nodes, important to enhance macrophage and T cell responses against Leishmania. Uncovering the unique metabolic signatures of vaccination and disease is invaluable to identify novel markers of vaccine efficacy and understand the correlates of protective immunity.Metabolomics can also be used to investigate interesting physiological phenomena in CL and identify the molecular mechanisms responsible. In particular, CL is known to cause painless lesions, indicating that Leishmania infection modulates pain signals within the infected area. Through mass spectrometry analysis, we found elevated levels of caffeine metabolites in L. mexicana-infected compared to uninfected macrophages. Similar results were obtained at the lesion site during chronic infection. These caffeine metabolites are anti-inflammatory and display analgesic properties. Furthermore, we found enriched arachidonic acid (AA) metabolism during infection in vivo. AA is a metabolite of endocannabinoids, well established anti-inflammatory and analgesic mediators. We also observed significantly higher transcript levels of enzymes involved in endocannabinoid biosynthesis, and significantly lower expression of enzymes important for their degradation in infected mice. This study provides the first evidence of metabolic pathways with a potential molecular basis for the analgesia experienced by CL patients. Overall, furthering our understanding of leishmaniasis, as well as the metabolic and immunological changes that affect resistance and susceptibility, can have broadimplications for the development of novel preventive and therapeutic strategies to fill a worldwide critical need.

The endocannabinoid (eCB) system is a complex lipid signaling system consistent of the endogenously produced ligands, 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (AEA), which signal along the cannabinoid receptors [cannabinoid receptor subtype-1 (CB1R), -2 (CB2R) and others] and the enzymes which produce and degrade 2-AG and AEA. The eCB system is expressed throughout the human body controlling a wide variety of homeostatic functions. It was previously reported that blockade of host peripheral CB1R signaling exacerbated metrics of infection with the soil-transmitted nematode Nippostrongylus brasiliensis (N. brasiliensis). Therefore, I hypothesized that host CB1R signaling is protective in host-helminth interactions using N. brasiliensis as a model for hookworm infection in rodents. This study identified that peripheral blockade of CB1R, but not CB2R, lead to significant increases in lung tissue damage, lung hemorrhaging, and lung eosinophilia in response to N. brasiliensis infection with no changes in parasite burden. Because N. brasiliensis can produce eCBs at a quantifiable levels, I developed novel enzyme assays to quantitate enzyme activity for the biosynthetic and degradative eCB enzymatic machinery, including a novel assay to determine the rate of AEA metabolism which does not require the use of radioactive materials. These methods identified that the enzyme alpha/beta hydrolase domain containing-6 contributes to ~55% of all monoacylglycerol metabolism in the lungs of rodents. Furthermore, when these methods were applied to a mouse model of diet induced obesity (DIO) in the large intestine, I found that the eCB system of the colon is severely dysregulated in response to DIO and that mice lacking intestinal epithelial CB1R signaling have exacerbated metrics of diet-induced gut-barrier dysfunction in the large intestine. Furthermore, the RNA expression/regulation in the colon mucosa of the DIO mice lacking intestinal epithelial CB1R signaling is dysregulated with a decrease in tight-junction protein RNA expression and an increase in the inflammatory profile. These studies suggest a potential role for the eCB system in several disease models in mucosal tissues providing novel methods for quantitation of eCB metabolism and evidence that CB1R signaling has a strong role in inflammatory processes.

Exercise has a myriad of benefits for brain and body health. Wheel running is an animal model of physical activity, and artificial selection for increased levels of running in rodents can reveal how genes and the environment affect exercise physiology. I examined endocannabinoids (eCBs), neuroanatomy, and reward behavior in four replicate lines of mice bred for high voluntary wheel running (High Runners; HR), compared to four non-selected Control (C) lines. First, I examined the eCBs 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (AEA) and eCB analogs docosahexaenoylglycerol (DHG), oleoylethanolamide (OEA), and docosahexaenoylethanolamide (DHEA) in the upper small-intestinal epithelium of male and female HR and C mice, housed with or without wheel access for six days. I found a significant 3-way interaction of linetype, wheel access, and sex for 2-AG and DHG. When compared to C mice, lines of HR mice had lower concentrations of 2-AG. Also, jejunal epithelial 2-AG was significantly correlated with circulating 2-AG (data from a prior study in the same mice). Second, I measured five key brain region volumes and cell densities of female HR and C mice, either given or not given wheel access for 10 weeks from weaning. The red nucleus and the hippocampus were significantly larger in HR compared to C brains, but no difference was observed for the basolateral amygdala, nucleus accumbens or ventral pallidum. The cell densities of each region were not statistically different between HR and C mice. Chronic wheel access did not affect the volume or cell density of any region. Third, I used a behavioral test of conditioned place preference (CPP) with methylphenidate and cocaine stimuli, as well as wheel access, in three separate studies to evaluate the extent to which genetic predisposition for exercise reward is associated with increased drug or exercise reward. Both HR and C mice displayed significant CPP for cocaine and methylphenidate, but with no statistical difference between linetypes for either drug. Neither HR nor C mice conditioned to wheel access. Altogether, I identified underlying traits that may contribute to the increased ability and/or motivation of HR mice for running ~3x more per day than C lines.

Abstract: The major endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) mediate their effects through their actions on cannabinoid receptors (CBRs), mainly via CB1R and CB2Rs. The predominant catabolic regulators that degrade AEA and 2-AG are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL also known as MGLL), respectively. Although the eCB signalling pathway is present from very early stages of development, little is known about their mechanisms that underlie embryonic growth, and the onset of locomotion and motor neuron development. In my thesis, I sought to investigate the role of the eCB system in zebrafish embryonic development by pharmacologically inhibiting the CB1 and CB2 receptors (with AM251 and AM630, respectively) for the first two days of post-fertilization (dpf). Selective inhibition of CB1R and CB2Rs upon treatment with AM251 and AM630 resulted in embryos with axial malformations, shorter trunks, pericardial edema and reduced heart rates, particularly at higher concentrations at 2 dpf. Locomotion studies at 5 dpf revealed a change in their activity, swim velocity, the number of swim bouts and the cumulative duration of swim bouts. The morphological and locomotor deficits observed upon inhibition of the CBRs during the first 48 hours of development led me to examine in more detail the role of CB1R and CB2R during development. Therefore, I further investigated the role of the CBRs by pharmacologically inhibiting CB1R and CB2R (with AM251 and AM630, respectively) in either the first or second day of development. Motor neuron morphology and neuromuscular outputs were then examined and quantified in 5 dpf larvae. Although blocking both CB1R and CB2R resulted in gross morphological deficits and reductions in heart rate, the effects of CB2Rs inhibition were more pronounced when compared to CB1Rs. Blocking CB1Rs from 0 to 24 h post-fertilization (hpf) resulted in an increase in the number of secondary and tertiary branches of primary motor neurons, whereas blocking CB2Rs had the opposite effect. Both treatments resulted in reduced levels of swimming. Additionally, blocking CB1Rs resulted in greater instances of non-inflated and partially inflated swim bladders compared with AM630 treatment, suggesting that at least some of the deficits in locomotion may result from an inability to adjust buoyancy. Finally, I studied the effects of inhibiting the enzymes (FAAH and MAGL) that break down the eCBs, in the early part of development. My study analyzed the functional roles of increased accumulation of AEA and 2-AG levels upon inhibition of FAAH and MAGL, and examined their effects on development. In vivo application of a dual FAAH/MAGL inhibitor, JZL 195, resulted in a reduction in primary and secondary motor neuronal axon branching, as well as in a reduction of nicotinic acetylcholine receptors (nAChRs) expression at neuromuscular junctions (NMJs). Application of the specific inhibitor of the FAAH enzyme, URB 597, also resulted in a decrease in motor neuron branching of the primary motor neurons only. However, secondary motor neuron branching and nAChR expression remained unaffected following URB 597 treatment in the first 24 hrs of development. Furthermore, I also confirmed that the effects of JZL 195 and URB 597 were mediated through CB1Rbecause co-treatment with the selective CB1R inhibitor, AM 251, prevented the aberrant branching of motor neurons and reduced nAchRs expression. Interestingly, JZL 184, a selective MAGL inhibitor, showed no effects throughout the study when examining motor neuron development and nAChRs expression at NMJs. Locomotion studies revealed that any disruption of FAAH or MAGL activity in the first 24 hrs of development by JZL 195, URB 597 and JZL 184 can reduce larval swimming activity. Although CB1R inhibition with AM 251 attenuated JZL 195 and URB 597 induced behavioural changes, it was unable to block the effects of JZL 184. Therefore, my study strongly indicates that accumulation of AEA preferentially acting through CB1R at early life is responsible for motor neuronal changes associated locomotor deficiency.

In pregnancy, linoleic acid (LA), obtained from the maternal diet, is vital for fetal development. LA is metabolised into a number of fatty acid modulators that regulate the endocannabinoid system (ECS), and are key for normal metabolic homeostasis. We are currently consuming elevated levels of LA in Western societies, including during pregnancy. We investigated if elevated maternal linoleic acid is detrimental to the mother and her fetus during pregnancy and early life, and the mechanism for this. The primary aims of this study were to investigate if elevated maternal LA consumption during pregnancy alters: 1) the maternal microbiome; 2) the maternal cardiac ECS; 3) the fetal cardiac ECS at embryo (E) day 20; and 4) cardiovascular development, function and the cardiac ECS in offspring at postnatal (PN) day 40. Effects of LA exposure on an in vitro H9c2 cardiomyoblast model (viability, respiration, glycolytic metabolism) were also assessed. A secondary aim was to test whether any of the latter postnatal effects are reversed with a low LA diet. Female Wistar Kyoto rats were fed a diet high in LA (HLA - 6.21% of energy intake) or a control LA diet (LLA - 1.44% of energy) for 10 weeks prior to mating and throughout pregnancy. At E0, E10 and E20, maternal faeces were collected, and the microbiome analysed by 16S sequencing. At E20 and PN40 maternal and offspring rats were sacrificed, body and organ weights recorded, and mRNA extracted from heart tissue to assess transcription of endocannabinoid signalling targets using real time PCR. Hearts from PN40 rats were also assessed for coronary reactivity, and ventricular diastolic and systolic pressure-volume relationships. In addition, effects of elevated LA exposure on cell viability and mitochondrial respiration were assessed in isolated cardiomyocytes. The current results showed that throughout pregnancy there was an increased abundance of Cyanobacteria and Lentisphaerae, and decrease Verrucomicrobia in the microbiome of HLA mothers. Over time, HLA and LLA diets decreased the abundance of Actinobacteria and Tenericutes. There was an interaction effect for Verrucomicrobia in which pregnancy and diet interacted in influencing its abundance. In terms of effects of LA in H9c2 cardiomyoblasts, LA exposure reduced cell viability at concentrations between 300 and 1000 μM. However, there were no effects of an acute exposure to LA on mitochondrial respiration. Just prior to birth, an elevated maternal LA diet increased CB2 expression in maternal hearts whereas in her E20 offspring cardiac CB2 expression declined in both sexes. FAAH mRNA expression was unaltered by diet, and appeared higher in female vs. male hearts. There were no effects of a high maternal LA diet on mRNA expression of endocannabinoid receptor GPR18, DAGL alpha and DAGL beta in E20 offspring hearts. We further assessed cardiovascular development/function in adolescent rats. Data demonstrated that there were no changes in heart mass or heart: body weight ratios. Within the heart, no changes in resting or baseline coronary flow rate, or peak coronary reactivity were detected. However, there was a decline in coronary flow repayment during reactive hyperaemia in female hearts that was significant for the LLA maternal / offspring HLA (LHF) compared with LLA maternal/offspring (LLF) group. A trend of declining coronary flow repayment (after 30 s occlusion) was also observed in HLA maternal/offspring LLA (HLM) and HLA maternal/offspring HLA (HHM) compared to LLM and LHM hearts, though this trend did not achieve statistical significance. In terms of left ventricular diastolic mechanics, there was evidence of an increased diastolic stiffness in HHF hearts (an effect not evident in males), together with an unexpected increase in diastolic compliance in HLM/HHM hearts (counter to changes in HHF vs. other groups). Thus, there is evidence of sexually dimorphic effects of LA on coronary and diastolic function in developing hearts. Systolic pressure development was subtly modified, with a trend to reduced contractility (declining slopes of pressure-volume relationships, increased volume required to generate 100 mmHg pressure) in HL and HH diets in both sexes, though this also failed to achieve statistical significance. Molecular analysis of PN40 hearts demonstrated CB2, LepR, Notch1, NPPA and TGFB1A mRNAs were all expressed in higher levels in HLM vs. LHM groups (with NPPA also elevated vs. LLM). Expression changes in these mRNAs were not evident in females. Collectively, these changes provide further evidence of a sexually dimorphic effect of maternal diet on myocardial phenotype of offspring. In conclusion, a maternal diet high in linoleic acid alters the maternal microbiome and cardiac ECS signalling. This is associated with distinct ECS signalling changes, and alterations in cardiac metabolism and functionality at embryonic and early postnatal stages in offspring hearts. Male and female offspring demonstrated different pathophysiology, with data at these early life stages suggesting that female offspring of mothers consuming a diet high in LA may in particular exhibit altered cardiovascular makeup and function, and potentially greater risk developing cardiovascular disease later in life.

The endocannabinoid (eCB) system is an important regulator of food intake and energy conservation both within the central nervous system and in key metabolically relevant peripheral organs. Recent findings have shown the peripheral eCB system to be an important contributor in regulating energy homeostasis. With the increasing obesity epidemic, we sought to determine whether this system is altered during maternal obesity which might explain some predispositions for later weight gain. Notably, these studies were met with unexpected high neonate mortality, emphasizing how detrimental obesity can be and the further need for therapeutic targets to curtail this disease. Thus, we examined the eCB system in the context of obesity. Previous evidence shows an increase in eCB signaling in the proximal small intestine during obesity. We further examined these findings and determined that these changes occur in the same region as enteroendocrine I-cells, which secrete satiation factor cholecystokinin (CCK). Indeed, I-cells were shown to express cannabinoid receptor subtype 1 (CB1Rs). Using pharmacological agents or increased endogenous signaling (as seen during diet-induced obesity), we showed that CB1R signaling controls CCK secretion. This suggests that CB1R signaling in the proximal small intestine controls satiation through a CCK dependent mechanism. Furthermore, we examined the role of CB1Rs on glucose regulatory peptides glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP1), which are produced by enteroendocrine K- and L-cells, respectively, and both express CB1Rs. CB1R activation inhibited incretin secretion, an effect that was restored by co-administering a peripherally-restricted CB1R antagonist. Lastly, through the use of transgenic mouse models, we demonstrate that CB1R on enteroendocrine K- cells control GIP release, but CB1R on enteroendocrine L-cells do not, suggesting an alternative CB1R dependent, intestinal independent mechanism for GLP1. This body of work begins to elucidate eCB signaling in the small intestine as regulators of enteroendocrine incretin release, which regulate both food intake (CCK) and energy homeostasis (GIP, GLP1).

Stress is a risk factor for neuropsychiatric disorders such as post-traumatic stress disorder and depression, yet not all individuals who are exposed to stress develop such disorders. Several factors influence susceptibility versus resilience to the effects of stress, including coping strategy biological sex. A growing body of research in humans has demonstrated that active coping strategies – defined as using available resources to problem solve – are positively correlated with resilience. In rodents, resilience to a potent acute stressor can be achieved through active coping, such as controlling the termination of a stressor, but only in males. During controllable stress males engage a stress mitigating pathway between the prelimbic (PL) and dorsal raphe nucleus (DRN), but this pathway isn’t engaged by control in females or when stress is uncontrollable in both sexes. Thus, neural activity within the ventromedial prefrontal cortex (vmPFC) is a critical determinant of stressor-induced anxiety. The mechanism that engage vmPFC excitability are not well understood. Therefore, the goals of the dissertation were 1) determine if eCBs in the PL promote neuronal excitability and behavioral resilience 2) test if ES and IS result in differential activation PL afferents, and will specifically test if ES results in greater activation PL-inputs from action-outcome associated regions, while IS leads to greater engagement of stress/fear inputs to the PL, and 3) identify network-wide patterns of activation and test the hypothesis that the stress and action-outcome networks are differentially activated as a function of stressor controllability and/or sex. We’ve demonstrated that augmenting eCBs in the PL increased excitability through a CB1 and GABA receptor dependent mechanism and was sufficient to block the stress induced decrease in social exploration. Regarding goal 2, PL inputs from the orbitofrontal cortex and DRN were activated in response to stress per se, but were not sensitive to stressor controllability and did not differ between males and females. PL afferents from the basolateral amygdala and mediodorsally thalamus were not sensitive to stress. Lastly, we quantified Fos expression in response to controllable and uncontrollable stress in male and female rats in 24 brain regions associated with stress, action-outcome learning, and showing sex differences in response to stress. Using interregional correlations, we found differences in functional connectivity as a function of stressor controllability and sex when considering all 24 regions and when considering only stress associated regions. Females showed greater overall functional connectivity compared with males, and IS resulted in greater overall connectivity than ES. We also reveal potentially important nodes in functional connectivity networks using centrality measures to identify network hubs. The findings of this research emphasize the need to study differences between males and females across all realms of neuroscience, particularly in relation to disorders of stress and anxiety.

The endocannabinoid (eCB) system is a key modulator of central brain processes that control feeding, however recent evidence points to peripheral mechanisms by which eCB signaling may modulate feeding in various disease states. This body of work provides novel mass spectrometric analyses for components of the eCB system, while describing the pitfalls of currently used techniques. Using the described methods, a role for increased small intestinal eCB ligands signaling via peripheral cannabinoid type 1 receptor (CB1R) in the control of hyperphagia is discussed in the context of obesity. These findings were enhanced by further scrutiny of the intestinal eCB system, which revealed that corn-oil induced secretion of an intestine-derived satiation peptide, cholecystokinin (CCK), is blunted by CB1R activation; activation occurred via pharmacological agonists or enhanced eCB levels following diet induced obesity. Further, administration of a CCKA receptor antagonist, devazepide, blocked the anorexigenic effects of peripheral CB1R restriction, suggesting that peripheral CB1R act via intestine derived CCK to block satiation by impaired gut-brain signaling. Additionally, helminth infection was used to evaluate intestinal and lung eCB levels in a model of hookworm infection. For the first time, it was observed that the helminth Nippostrongylus brasiliensis produces endocannabinoids. Together, this body of work provides novel insight to the varied roles that intestinal and peripheral eCBs have during obesity in the modulation of feeding behaviors and immune responses.

Drug addiction is a widespread condition affecting 8-10% of the population nationwide. Although men and women are both affected by drug addiction, sex-differences in responsiveness to drugs of abuse are apparent, with women showing a greater sensitivity than men. For over 30 years, the ovarian hormone estradiol has been recognized as a key biological factor contributing to the development of drug addiction in females, with little understanding of the neurobiological underpinnings. Here we propose a neural mechanism through which estradiol primes females to respond more strongly to drugs of abuse. Estradiol can act at intracellular or membrane-localized estrogen receptors to influence cellular function. Exclusively in females, a subset of these membrane estrogen receptors are coupled to group I metabotropic glutamate receptors (mGluRs). We demonstrate that it is through group I mGluRs that estradiol structurally remodels reward circuits in the nucleus accumbens (NAc), an effect associated with heightened sensitivity to drugs of abuse in females. In the nervous system, both estradiol and group I mGluRs can influence the activity of the endogenous cannabinoid (endoCB) system, an emerging mediator of neural plasticity and behavioral responses to drugs of abuse. Interestingly, we find that estradiol enhances the activity of the endoCB system in the female NAc by rapidly altering concentrations of the two best known endoCBs and increasing cannabinoid receptor expression. Further we demonstrate that activation of the endoCB system is critical for estradiol to induce structural plasticity in the NAc and facilitate behavioral responses to repeated drug exposure. Collectively, our results suggest that estradiol, group I mGluRs and the endoCB system may be linked through a serial pathway that is principally important in female drug addiction. These findings begin to elucidate a neural mechanism underlying female vulnerability to addiction that contributes to sex differences in drug abuse and may actually provide putative therapeutic targets that are particularly effective in treating drug abuse in women.

(i) The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anti-cancer effects. Fatty acid amide hydrolase (FAAH) metabolizes AEA and decreases its anti-tumorigenic activity. In this study, we have analyzed the role of FAAH inhibition in non-small cell lung cancer (NSCLC). We have shown that FAAH and CB1 receptor which is activated by AEA are expressed in lung adenocarcinoma patient samples and NSCLC cell lines A549 and H460. Since the synthetic analogue of anandamide (Met-F-AEA) did not possess significant anti-tumorigenic effects, we used Met-F-AEA in combination with FAAH inhibitor URB597 which significantly reduced EGF (epidermal growth factor)-induced proliferative and chemotactic activities in vitro when compared to anti-tumorigenic activity of Met-F-AEA alone. Further analysis of signaling mechanisms revealed that Met-F-AEA in combination with URB597 inhibits activation of EGFR and its downstream signaling ERK, AKT and NF-kB. In addition, it inhibited MMP2 secretion and stress fiber formation. We have also shown that the Met-F-AEA in combination with URB597 induces G0/G1 cell cycle arrest by downregulating cyclin D1 and CDK4 expressions, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, the combination treatment inhibited tumor growth in a xenograft nude mouse model system. Tumors derived from Met-F-AEA and URB597 combination treated mice showed reduced EGFR, AKT and ERK activation and MMP2/MMP9 expressions when compared to Met-F-AEA or URB597 alone. Taken together, these data suggest in EGFR overexpressing NSCLC that the combination of Met-F-AEA with FAAH inhibitor resulted in superior therapeutic response compared to individual compound activity alone. (ii) JWH-015, a cannabinoid receptor 2 (CB2) agonist has tumor regressive property in various cancer types. However, the underlying mechanism by which it acts in lung cancer is still unknown. Tumor associated macrophage (TAM) intensity has positive correlation with tumor progression. Also, macrophages recruited at the tumor site promote tumor growth by enhancing epithelial to mesenchymal (EMT) progression. In this study, we analyzed the role of JWH-015 on EMT and macrophage infiltration by regulation of EGFR signaling. JWH-015 inhibited EMT in NSCLC cells A549 and also reversed the mesenchymal nature of CALU-1 cells by downregulation of EGFR signaling targets like ERK and STAT3. Also, in vitro co-culture experiments of A549 with M2 polarized macrophages provided evidence that JWH-015 decreased migratory and invasive abilities which was proved by reduced expression of FAK, VCAM1 and MMP2. Furthermore, it decreased macrophage induced EMT in A549 by attenuating the mesenchymal character by downregulating EGFR and its targets. These results were confirmed in an in vivo subcutaneous syngenic mouse model where JWH-015 blocks tumor growth and also inhibits macrophage recruitment and EMT at the tumor site which was regulated by EGFR pathway. Finally, JWH-015 reduced metastatic lesions in a tail vein syngenic mouse model. These data confer the crosstalk between CB2 and EGFR receptors, leading to anti-proliferative and anti-tumorigenic effects, thus enhancing our understanding of the therapeutic efficacy of JWH-015 in NSCLC.

BACKGROUND: Endocannabinoids and temperament traits have been linked to both physical activity and body mass index (BMI) however no study has explored how these factors interact in females. The aims of this cross-sectional study were to 1) examine differences among distinct BMI groups on daytime physical activity and time spent in moderate-vigorous physical activity (MVPA), temperament traits and plasma endocannabinoid concentrations; and 2) explore the association and interaction between MVPA, temperament, endocannabinoids and BMI. METHODS: Physical activity was measured with the wrist-worn accelerometer Actiwatch AW7, in a sample of 189 female participants (43 morbid obese, 30 obese, and 116 healthy-weight controls). The Temperament and Character Inventory-Revised questionnaire was used to assess personality traits. BMI was calculated by bioelectrical impedance analysis via the TANITA digital scale. Blood analyses were conducted to measure levels of endocannabinoids and endocannabinoid-related compounds. Path-analysis was performed to examine the association between predictive variables and MVPA. RESULTS: Obese groups showed lower MVPA and dysfunctional temperament traits compared to healthy-weight controls. Plasma concentrations of 2-arachidonoylglyceryl (2-AG) were greater in obese groups. Path-analysis identified a direct effect between greater MVPA and low BMI (b = -0.13, p = .039) and high MVPA levels were associated with elevated anandamide (AEA) levels (b = 0.16, p = .049) and N-oleylethanolamide (OEA) levels (b = 0.22, p = .004), as well as high Novelty seeking (b = 0.18, p

Obesity and diabetes are clearly established independent risk factors for renal disease. Therapeutic targets have been investigated for their role in treating obesity and diabetic associated renal damage. The endocannabinoid system is an important endogenous lipid signalling system known to mediate glucose and lipid metabolism, inflammation and energy storage. Specifically, diabetes mellitus and obesity induces alterations in the expression of cannabinoid receptor 1 (CB1), cannabinoid receptor 2 (CB2) and putative cannabinoid receptor G-protein coupled receptor 55 (GPR55) in a tissue specific manner. Renal expression and function of these receptors, particularly within the pathophysiological context of obesity and diabetes related renal damage is poorly understood. The research presented in this thesis examines the renal expression and function of CB1, CB2 and GPR55. The significant aim of this PhD candidature was to examine the expression of cannabinoid receptors in the kidney in obese and diabetic conditions. Subsequent studies sought to evaluate the actions of selective manipulation of the receptors by synthetic compounds on markers of renal damage and structure in an animal model of diet induced obesity (DIO).

Abstract: Introduction: Trans 11-18:1 (vaccenic acid, VA) is the predominant ruminant-derived trans fatty acid (TFA) in the food chain and is also the precursor to endogenous synthesis of cis-9, trans-11 conjugated linoleic acid (CLA). Literature documents numerous health benefits associated with CLA, which is a minor component of natural TFA. A few recent studies have reported hypolipidemic properties of VA. However, independent bioactive properties of VA during dyslipidemia and insulin resistance remain elusive. Objectives: To determine whether VA can enhance the putative hypolipidemic effects of CLA. To investigate whether enriching dairy fat with VA can favourably modulate whole body energy use/storage. Methods and Results: Hypolipidemic effects of dietary supplementation with CLA on dyslipidemia were enhanced by the addition of VA in a rat model of metabolic syndrome (the JCR:LA-cp rat). VA was also found to reduce total body fat (-6%) while stimulating adipose tissue re-distribution (reduced mesenteric fat (-17%) while increasing inguinal fat mass (29%)). VA fortification increased metabolic rate concomitantly with an increased preference for whole body glucose utilization for oxidation and increased insulin sensitivity (lower HOMA-IR index (-59%)). Additionally, VA re-equilibrated intestinal and hepatic lipid homeostasis (reduced TG secretion) while exerting differential transcriptional regulation in both organs. Targeted lipidomic analyses revealed that VA can regulate concentrations of endocannabinoids in a tissue-specific manner. Conclusions: VA has hypolipidemic effects independent of CLA and can favorably modulate whole body energy metabolism and lipid homeostasis when added to a dairy-derived diet. The regulatory effect of VA on the endocannabinoid system may have potential therapeutic applications. Based on these results, enriching dairy products with VA (either naturally or by fortification) may maximize the health value of dairy-derived fats. Findings also support the need for nutrition guidelines focused on eliminating industrial forms of TFA from processed foods, as opposed to all TFA per se.

Animal studies point to an implication of the endocannabinoid system on executive functions. In humans, several studies have suggested an association between acute or chronic use of exogenous cannabinoids (Δ9-tetrahydrocannabinol) and executive impairments. However, to date, no published reports establish the relationship between endocannabinoids, as biomarkers of the cannabinoid neurotransmission system, and executive functioning in humans. The aim of the present study was to explore the association between circulating levels of plasma endocannabinoids N-arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) and executive functions (decision making, response inhibition and cognitive flexibility) in healthy subjects. One hundred and fifty seven subjects were included and assessed with the Wisconsin Card Sorting Test; Stroop Color and Word Test; and Iowa Gambling Task. All participants were female, aged between 18 and 60 years and spoke Spanish as their first language. Results showed a negative correlation between 2-AG and cognitive flexibility performance (r = -.37; p

Excessive ethanol consumption, characteristic of alcohol use disorders (AUDs), is associated with widespread neurodegeneration and cognitive and behavioral impairments that may contribute to the chronic and relapsing nature of alcoholism. Therefore, identifying novel targets that can afford neuroprotection will undoubtedly aid current treatment strategies for AUDs. The cannabinoids have been shown to provide neuroprotection in a variety of preclinical models of neurodegeneration; however minimal data is available regarding the use of cannabinoid-based pharmacotherapies for treating ethanol-induced neurodegeneration. Therefore, the current dissertation examined the overarching hypothesis: the cannabinoids are a therapeutic strategy to afford neuroprotection in the context of ethanol-induced neurodegeneration. Importantly, this overarching hypothesis was approached with translational considerations in mind. Specifically, the use of many cannabinoids in the clinic is hindered due to multiple unfavorable pharmacokinetic/pharmacodynamic profiles, including high first pass metabolism and untoward psychoactivity. Therefore, the studies herein were designed to circumvent these PK/PD obstacles. The first set of studies examined whether transdermal delivery of the phytocannabinoid, cannabidiol (CBD), could attenuate binge ethanol induced neurodegeneration. Transdermal CBD afforded neuroprotection in the entorhinal cortex and neuroprotection was similar in magnitude as intraperitoneal administration. The second set of studies found that binge ethanol treatment transiently down-regulated the main CNS cannabinoid receptor, CB1R. Interestingly, these changes were not accompanied by alterations in one of the major endogenous ligands, anandamide (AEA), or other related n-acylethanolamides (NAEs). The latter finding is in contrast to other literature reports demonstrating that endocannabinoid content is substantially elevated in response to a CNS insult. Nevertheless, studies were carried out to determine if administration of the AEA and NAE catabolism inhibitor, URB597, could attenuate binge ethanol induced neurodegeneration. URB597 failed to produce neuroprotection in the entorhinal cortex and dentate gyrus of the hippocampus. However, additional studies found that URB597 failed to elevate AEA in the entorhinal cortex, and in general the biological activity of URB597 was impaired by ethanol exposure. Therefore, with further drug discovery/development efforts, it may be feasible to optimize such treatment strategies. In conclusion, the studies within the current dissertation demonstrated the feasibility of using some cannabinoid-based agents to prevent ethanol-induced neurodegeneration.

AbstractInvestigating the Role of Physiological Systems Outside the Central Nervous System on Huntington's Disease PathogenesisBy Jill LarimoreHuntington's disease (HD) is a devastating neurodegenerative disease for which there is no cure. Classically, HD is thought to result from central nervous system (CNS) dysfunction because its hallmark pathology is neurodegeneration of brain regions important for controlling movement. However, co-occurring pathologies, including severe weight loss and peripheral immune system activation, might contribute to HD progression. Given that the mutant Huntingtin protein (mHtt) is ubiquitously expressed and directly affects cells outside the CNS, an integrated understanding of mHtt-induced pathogenesis is needed. In this dissertation, I sought to understand how both central nervous and peripheral physiological systems contribute to HD pathogenesis by taking advantage of the endocannabinoid system, in which the two main cannabinoid receptors, CB1 and CB2, have distinct roles. In chapter 2, I investigated the role of CB1, which is primarily expressed in the CNS and whose expression is one of the earliest genetic changes to occur in HD-vulnerable neurons, on HD progression. We found that early genetic deletion of CB1 receptors in R6/2 mice accelerated the onset of symptoms and further decreased lifespan compared to CB1-receptor-expressing R6/2 mice, suggesting CB1 plays a functional role in HD progression. In chapter 2.1, I investigated the effects of animal weight on HD progression in BACHD mice, an overweight HD model. Specifically, using ANCOVA as a robust statistical tool, I found that changes in weight induced by deletion of CB1 expression significantly influenced HD behavior. In chapter 3, we investigated the peripheral immune system, showing that genetic deletion of CB2 in a slow-progressing HD mouse model accelerated the onset and increased the severity of motor deficits. Treatment of mice with a CB2-receptor agonist extended lifespan and suppressed motor deficits, synapse loss, and CNS inflammation, while a peripherally restricted CB2-receptor antagonist blocked these effects. CB2 regulates blood interleukin-6 levels, and interleukin-6-neutralizing antibodies partially rescued motor deficits and weight loss in HD mice. These findings support a causal link between CB2-receptor signaling in peripheral immune cells and the onset and severity of HD-induced neurodegeneration. Together, these results demonstrate that mHtt can significantly impact peripheral physiological systems, and identify novel therapeutic approaches for treating HD.

My research utilized a long-term artificial selection experiment in which mice (Mus domesticus) are selectively bred on the basis of voluntary exercise (high levels of voluntary wheel running: four replicate High Runner or HR lines) to address how a specific motivated behavior is generated in the brain, and how this may affect the performance of other motivated behaviors and organismic processes (e.g., reproduction). I studied the endocannabinoid system, one signaling system involved in control of motivated behaviors via the perception of natural rewards. The endocannabinoid system is thought to play a role in voluntary wheel running in mice. In support of this, we found that HR mice, as compared to four non-selected Control lines, have a differential and sex-specific response to two drugs that affect the endocannabinoid system (Rimonabant (SR141716); WIN 55,212-2).Secondly, I studied whether the HR and Control linesdiffer with respect to life history characteristics of dams and litters over a 10-generation span. I found that HR did not differ from Control in most measures of reproductive output or measures of maternal care.Thirdly, I measured oxytocin receptor densities in several brain areas that reflect either neural dopaminergic connections or strong involvement in mouse social or parental behavior (or both). I did not find statistical differences between postpartum HR and Control dams. Lastly, I allowed HR and Control mice wheel access for 6 days, after which, wheel access was blocked with screen mesh and a coffee-cup lid-barrier for one additional day. I found no statistical linetype difference in the proportion of mice that broke through the barrier. This indicates that HR and Control mice may be equally motivated to run on wheels; however, these results may have been influenced by confounding features of the experimental design. In sum, these studies suggest that HR mice have sex-specifically diverged from Control mice in aspects of reward-related signaling; however, selection does not seem to influence the performance or regulation of reproduction in females, nor motivation break through a barrier for wheel-access in either sex.