The endogenous arachidonate-based lipids that activate cannabinoid receptors have been well characterized in mammals. In plants only 12-18 carbon fatty acid ethanolamides have been identifi ed so far and have been shown to modulate a number of physiological processes including seed and seedling development. However, since moss plants contain arachidonic acid, we hypothesized the occurrence of arachidonate-based metabolites in their tissues. Using selective lipidomics approach, we identifi ed the presence of anandamide or arachidonylethanolamide (a 20C polyunsaturated fatty acid ethanolamide) and its precursors, in Physcomitrella patens that were previously not reported in plants. Comprehensive lipid profi les for protonema and gametophyte tissues of moss also revealed the occurrence of other saturated and unsaturated fatty acid ethanolamides and a distinct phospholipid and galactolipid composition. Further studies showed that anandamide, like abscisic acid, inhibits the growth of gametophytes more severely than saturated fatty acid ethanolamides. Our current studies are focused on understanding the physiological and developmental role of polyunsaturated fatty acid ethanolamides in nonseed plants. In conclusion, discovery of anandamide in moss provided us with an exciting possibility to identify fatty acid ethanolamide metabolic pathway in early land plants and elucidate receptor-mediated endocannabinoid signaling responses in plants that is akin to mammals.

Le Système Nerveux Central (SNC) est un acteur majeur de la régulation de l’homéostasie énergétique, intégrant différents signaux nerveux, hormonaux ou nutritionnels. Le métabolisme lipidique joue un rôle essentiel notamment dans la détection des signaux lipidiques, et les enzymes y participant sont donc fortement impliquées dans la régulation de ces signaux et leur expression est cruciale au bon équilibre énergétique. La Lipoprotéine Lipase (LPL), enzyme clé de l'hydrolyse des triglycérides, nous est apparue comme une cible de choix dans la mesure où elle est exprimée dans différentes structures cérébrales comme l'hippocampe ou l'hypothalamus. L'hypothalamus a été identifié depuis de nombreuses années comme un centre de régulation de la prise alimentaire et donc de l'équilibre entre entrées et dépenses d'énergie. Ainsi, il est apparu comme légitime que de s'intéresser plus précisément au rôle de la LPL hypothalamique et son implication dans la régulation de l'homéostasie énergétique.L'objet de cette thèse a donc été d'étudier, dans un premier temps, les effets d'une délétion de LPL dans le VMH, réalisée grâce à une injection d'un AAV2/9 exprimant la Cre recombinase chez des souris LPL lox/lox âgées de 8 semaines. La diminution de l'activité LPL dans le VMH conduit au développement d'une obésité au bout de 3 semaines post-injection, ainsi qu'au développement d'une intolérance au glucose, d'une résistance à l'insuline ainsi qu'une diminution de l'activité locomotrice.Ce phénotype est dû à une diminution transitoire de la quantité de céramides synthétisées par l’enzyme CerS1 au sein de l'hypothalamus durant les semaines qui suivent l'injection et qui perturbent la signalisation homéostatique. Il apparait également que le système endocannabinoïde pourrait être impliqué dans la mise en place de ce phénotype. Les caractéristiques de ce phénotype rappelant celles d'un état de torpeur, nous avons cherché dans la deuxième partie de ce travail de thèse, à reproduire celui-ci pour pouvoir étudier plus précisément les liens et les conséquences entre torpeur et lipases cérébrales. Nous avons donc exposés les animaux à 4°C pendant 4 heures et étudié les répercussions de ce stress thermique sur les gènes des lipases centrales ainsi que ceux du rythme circadien : nous avons pu mettre en évidence une modification du rythme circadien. Nous avons également exposé des animaux délétés en LPL hypothalamique et pu établir que cette délétion centrale en LPL modifie la thermogenèse du tissu adipeux brun ainsi et favorise le développement du tissu adipeux beige. Ce travail de thèse a donc permis de mettre en lumière pour la première fois l'implication de la LPL hypothalamique dans la régulation de l'homéostasie énergétique ainsi que son rôle dans la réponse adaptative à une exposition aiguë au froid. / The Central Nervous System (CNS) is a major actor in the energy balance regulation, integrating different nervous, hormonal or nutritional signals. The lipid metabolism plays an essential role especially in the detection of lipid signals. So, the enzymes taking part in it are involved in the regulation of these signals and their expression is crucial to the energy balance. The Lipoprotein Lipase (LPL), the key enzyme in triglycerides hydrolysis appeared to us as a target of choice as it is expressed in different brain structures like the hypothalamus or the hippocampus.The hypothalamus has long been known as a regulation center of food intake and so of the balance between entrance and expenditure of energy. It seemed interesting to focus more precisely on the role of hypothalamic LPL and its implication in the regulation of energy homeostasis.This dissertation’s main objective was to identify the effects of LPL deletion in the VMH, achieved by injection of an AAV2/9 expressing the Cre-recombinase in LPL lox/lox mice aged of 8 weeks. The decrease of LPL activity in the VMH leads to obesity development around 3 weeks post-injection and to the development of glucose intolerance, resistance to insulin and a decrease in locomotor activity.This phenotype is probably due to a transient decrease of ceramides synthesized by the CerS1 enzyme in the hypothalamus during the weeks post-injection and which disrupts the homeostatic signalling. The endocannabinoid system also seems to be involved in the onset of this phenotype.As the characteristics of this phenotype were reminiscent of a torpor state, we tried in a second part of work to reproduce it to study more precisely the links between torpor and brain lipases. We exposed animals to 4°C for 4 hours and studied the repercussions of this thermic stress on the central lipases genes and on circadian rhythm genes: we were able to highlight a modulation of the circadian rhythm. We also exposed to the cold VMH-LPL-depleted mice and established that this depletion in VMH-LPL modifies the thermogenesis of brown adipose tissue and so promotes the development of beige adipocytes.This work highlights for the first time the implication of hypothalamic LPL in the regulation of energy homeostasis and its role in adaptive response to cold exposure.

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Dopamine cells within the ventral tegmental area of the brain are involved in motivation and reward. Drugs of abuse target these dopamine cells altering their activity and plasticity resulting in addiction. While dopamine cell activity is primarily involved in addiction, the GABA neurons in the VTA have also been shown to have an indirect role. By decreasing the activity of the inhibitory GABA inputs onto dopamine neurons abusive drugs can indirectly increase dopamine cell activity resulting in addictive behaviors. However, although GABA neurons are important in the perception of reward, much less is known about how the excitatory inputs to these cells are regulated and possibly altered by drugs of abuse. Using transgenic mice expressing GFP attached to the GAD promoter, GABA cells were located and patched using whole cell voltage clamp and EPSCs were measured. High frequency stimulation induced LTD of the excitatory inputs to GABA neurons. The endocannabinoid analogue R- methanandamide also induced LTD at these excitatory synapses. These results suggest that endocannabinoids could potentially regulate the activity of GABA cells and as a result the activity of dopamine neurons. The endocannabinoid receptor involved is likely CB1, but not TRPV1 as only the CB1 antagonist AM-251 blocked this high frequency stimulus-induced LTD. Future research could then determine if the pathways involved in this LTD could potentially be altered by drugs of abuse contributing to addiction.

Chlorpyrifos is a widely used organophosphate insecticide, functioning through the inhibition of acetylcholinesterase. Recent studies report negative long-lasting biochemical and behavioral effects at levels without acetylcholinesterase inhibition. Our lab studies have identified the endocannabinoid system as a target for OP low-dose neurotoxicity. This thesis identifies the proteins and their associated neurotransmitter systems in the hippocampus that have been affected by low dose developmental exposure to the OP insecticide CPF. Male rat pups were treated from postnatal day 10 (PND) - PND16 with either corn oil (vehicle), 0.75 mg/kg of CPF, or 0.02 mg/kg of PF-04457845, a specific fatty acid amide hydrolase (FAAH) inhibitor. On PND38, rats were sacrificed for hippocampal extraction, and shotgun proteomics was used for protein expression. DAVID and Ingenuity Pathway Analysis software detected differentially expressed proteins such as Neuroligin-2 and Synaptotagmin 2, and disrupted signaling pathways such as ephrin B signaling, synaptogenesis signaling, and glutamate receptor signaling. Taken together, our data suggests that CPF reduces glutaminergic signaling pathways, greatly reducing long-term potentiation, prohibiting proper synapse formation, and therefore disrupting the proper functioning of the hippocampus.

In non-pathological states the central nervous system maintains a degree of immunological privilege. When illness or injury occur, this privilege can be lost and the immune system drives pathology in the brain and spinal cord. More so, resident immune cells, the microglial, act as major effectors of this response. Cerebral ischemia, or stroke, is the fourth leading cause of death in developed nations. After the initial ischemia, the inflammatory response propagates further injury and cell death. Another affliction of the central nervous system, chronic pain and persistent use of the opioid analgesic, morphine, leads to tolerance and ineffectiveness of the drug. Currently, only one in three patients receive adequate pain relief from their pharmacological regiment. This loss of efficacy in morphine is also driven by an inflammatory response. Thus, a way to quell inflammation in both disease states could lead to better treatments for both disorders. The endogenous cannabinoid system has two known receptors, CB1 and CB2. Both of these receptors have been intimately linked to inflammation and the activation or antagonism of the receptors can impart desired outcomes in modulating the immune response. Primarily the CB1 receptor expression is on presynaptic terminals of neurons to modulate neuronal firing. The CB2 receptor's expression predominates on immunological cells including microglial. However, some degree of expression exists with reports of neuronal CB2 receptors and immunological CB1 receptors. This makes pharmacological therapies targeted at both receptors ideal candidates in treating not only stroke and but also preventing the induction of morphine tolerance. In the studies described here, we sought to investigate the role of the endogenous cannabinoid system in both stroke and as a way to prevent the induction of morphine tolerance. The results showed that CB1 -/- CB2 -/- receptor mice were able to maintain greater blood flow during cerebral ischemia. More so, CB1 antagonism in a permanent occlusion of cerebral vessels showed a protective effect independent of the serotonin receptor. Lastly, a CB2 agonist was able to limit the degree of tolerance that developed from chronic morphine therapy and also prevent hyperalgesia in addition to showing a reduction in pro-inflammatory cytokines. Acutely, this same agonist was found to antagonize the morphine receptor but this could be avoided if morphine was administered before the CB2 agonist. In brief, the studies at hand show that the endogenous cannabinoid system can attenuate inflammation in central nervous system injury and shows great promise as a future therapeutic for clinical use. / Physiology

While there is currently active debate over possible therapeutic applications of marijuana and cannabis-based compounds, consistently their primary drawbacks have been the psychoactive properties, dependence, and abuse potential. Prolonged administration of ∆9-tetrahydrocannabinol (THC), the primary psychoactive constituent in marijuana, demonstrates both tolerance and physical withdrawal in both preclinical and clinical studies. Repeated THC administration also produces CB1 receptor adaptations in the form of reduced activation of receptors, along with a downregulation of membrane surface receptors, in many brain regions involved in THC-associated behaviors. The increased need for drug to maintain therapeutic effects, and a withdrawal syndrome following discontinuation of use, are common risk factors in drugs of abuse. Recently, compounds have been developed that prolong the availability of the major naturally occurring endogenous cannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), through inhibition of their catabolic breakdown by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The overall objectives of this research are to elucidate the physiologic roles of these two endogenous ligands and to determine if either can produce beneficial therapeutic effects without negative cannabis-like CNS effects. Therefore, we tested the impact of acute and prolonged blockade of FAAH and MAGL on a variety of cannabinoid-mediated behaviors and on precipitated cannabinoid withdrawal. Despite that acute blockade of FAAH and MAGL produce similar efficacy in reducing nociceptive responses, and both can reduce THC-induced somatic withdrawal, sustained blockade of these enzymes leads to remarkably different adaptations in CB1 receptor functioning. Namely, prolonged elevations in brain 2-AG leads to marked antinociceptive tolerance, cross-tolerance to exogenous cannabinoid agonists, and physical dependence. In contrast, sustained elevations in brain anandamide continues to dampen pain responses without apparent signs of physical withdrawal, loss of CB1 receptor activation as measured by [35S]GTPγS, or receptor downregulation as measured by [3H]CP,55940. These results suggest that chronic 2-AG elicits greater compensatory changes in CB1 receptor functions than anandamide. With similar efficacy in most therapeutic endpoints tested, and evidence of reduced impact on long-term function of the endocannabinoid system, these results distinguish FAAH as a more promising therapeutic target to treat pain and other conditions than MAGL.

Cannabinoids, such as the marijuana derivative Δ9-THC, are known to have CBl receptor-mediated anticonvulsant effects in several animal models of seizures and epilepsy, including the rat pilocarpine model of acquired epilepsy. However, the distribution of CBl receptor expression and function in brains of epileptic rats has not been characterized. Therefore, this dissertation was initiated to evaluate the effect of epileptogenesis on the distribution and function of the endogenous CBI receptor system in the rat pilocarpine model, a well-established model of acquired temporal lobe epilepsy. Using immunohistochemistry, we demonstrated that chronically epileptic rats exhibit a unique, long-term, and specific redistribution of hippocampal CBl receptors when compared to controls, with concurrent layer-specific increases and decreases in CBl receptor expression within the hippocampus. In addition, studies in this dissertation demonstrated using [3H] WIN55,212-2 autoradiography and agonist-stimulated [35S]GTPγS autoradiography that this CBl receptor-specific reorganization results in corresponding functional changes manifested by alterations in CBl receptor binding and G-protein activation. These regionally selective changes were dependent on NMDA receptor activation during the initial insult of pilocarpine-induced status epilepticus (SE), and were independent of seizure suppression produced with phenobarbital administration in epileptic rats. Furthermore, time-course studies utilizing these techniques demonstrate that within a week following SE, a widespread loss of CBl receptor expression and function occurs throughout the hippocampus. The subsequent redistribution of CBl receptors that occurs temporally correlates with the emergence of spontaneous recurrent seizures, and is still observed up to 1 year following SE. Overall, the reorganization of cannabinoid receptors in epilepsy implicates the endocannabinoid system in modulating neuroexcitability in the epileptic state. This CBl receptor redistribution represents an essentially permanent neuronal plasticity change associated with epileptogenesis, and could account for the anticonvulsant effect of cannabinoids observed in this model.