abstract: Development of effective therapeutic interventions for the treatment of mental health disorders has been a significant driving force in the search to understand the human brain. Current treatments for mental health disorders rely on modulating neurotransmitter systems such as norepinephrine (NE), serotonin (5-HT), dopamine (DA) and γ-aminobutyric acid (GABA) to achieve clinically relevant relief of symptoms. While many medications are available to the clinician that individually target these neural systems, treatment often results in patients reporting unwanted side effects or experiencing incomplete relief. To counter this lack of treatment efficacy, further investigation of other avenues for achieving similar or better outcomes and potentially reach patients refractory to common therapies must be undertaken. One of these potential new target systems is the endogenous cannabinoid system (ECS), which is currently composed of cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). These metabotropic seven transmembrane (7-TM) loop G-protein coupled receptors (GPCR) are responsible for mediating the effects of acute Cannabis ingestion as well as modulating several core functions of the nervous system including emotion, memory, and learning behavior. Due ubiquitous expression of ECS proteins, there is broad overlap between brain regions that show high levels of receptor expression and those thought to be involved in the etiology of a range of mental health disorders including depression, anxiety and schizophrenia. Consequently, modulation of cannabinoid receptor function is a novel and potentially clinically relevant mechanism for influencing the levels of other neuromodulators and neurotransmitters, such as dopamine, that are known to play crucial roles in the progression of mental illness. In addition, characterization of endogenous cannabinoids and cannabinoid receptors with respect to their normal physiological function and possible roles in pathophysiology may provide insight for the development of future ECS-based therapies. / Dissertation/Thesis / Masters Thesis Biology 2019

A problematic connection has been reported between those who use nicotine related products alone or in combination with ADHD medications, like methylphenidate (MPH), in late childhood or early adolescence and the increased likelihood of later marijuana abuse in adulthood. Pre-clinical studies have found that the use of nicotine during the early adolescence period produces enduring changes to the endocannabinoid system in the brain. Since CB agonists, like marijuana, exert their effect through the eCB system, it is possible that early nicotine use may alter the rewarding nature of CB agonists in adulthood. In addition, MPH has also been shown to increase nicotine self-administration and abuse related behaviors of nicotine in rats. Thus, the current study consisted of two experiments looking at the effects of early nicotine and methylphenidate exposure on adult CB-agonist place conditioning in rats. In the first experiment, rats were pre-exposed to either saline or nicotine (0.16, 0.32, or 0.64 mg/kg) from PD 31 to PD 40. On PD 60, rats began a 13-day biased CPP procedure with the CB agonist, CP 55,940 (10, 20 or 30 μg/kg), or vehicle. No significant group differences were found, suggesting that early nicotine exposure does not influence the rewarding nature of CB agonists. Additional individual subgroup comparisons were conducted to determine if any subgroups significantly differed from 0 or no mean change in preference from preconditioning to testing. These analyses revealed that rats pre-exposed to the moderate (0.32 mg/kg) dose of nicotine showed a significant aversion to the high (30 μg/kg) dose of CP 55,940, suggesting that early nicotine exposure may reduce the rewarding nature of CB agonists in adulthood. In the second experiment, rats were pre-exposed to either saline or MPH (0.5, 2, 0r 5 mg/kg) from PD 21 to PD 30. Similar to the first experiment, rats began a 13-day biased CPP procedure on PD 60 with CP 55,940 (10, 20 or 30 μg/kg) or vehicle. Rats conditioned with the moderate (20 μg/kg) dose of CP 55,940 showed a significant preference for the CB agonist as compared to rats conditioned with the high (30 μg/kg) dose of CP 55,940. CP 55,940 exposed rats did not significantly differ from control rats. There was no significant effect of MPH or a MPH x CP 55,940 interaction, suggesting that early MPH exposure does not alter the rewarding nature of CB agonists in adulthood. Together these findings suggest that early nicotine, but not MPH, exposure may influence the rewarding nature of CB agonists in adulthood, suggesting an additional risk factor of early nicotine use. However, future studies should evaluate the effects of persistent nicotine and MPH exposure starting in early adolescence or childhood through adulthood to determine whether the effects of nicotine and MPH are altered if use is continued into adulthood.

A growing body of evidence implicates the importance of the endogenous cannabinoid 2-arachidonyl glycerol (2-AG) in memory regulation. The biosynthesis of 2-AG occurs primarily through the diacylglycerol lipases (DAGL-α and -β), with 2-AG serving as a bioactive lipid to both activate cannabinoid receptors and as a rate limiting precursor for the production of arachidonic acid and subsequent pro-inflammatory mediators. Gene deletion of DAGL-α shows decrements in synaptic plasticity and hippocampal neurogenesis suggesting this biosynthetic enzyme may be important for processes of normal spatial memory. Additionally, 2-AG is elevated in response to pathogenic events such as traumatic brain injury (TBI), suggesting its regulatory role may extend to conditions of neuropathology. As such, this dissertation investigates the in vivo role of DAGL-α and -β to regulate spatial learning and memory in the healthy brain and following neuropathology (TBI). The first part of this dissertation developed a mouse model of learning and memory impairment following TBI, using hippocampal-dependent tasks of the Morris water maze (MWM). We found modest, but distinct differences in MWM performance between left and right unilateral TBI despite similar motor deficits, histological damage, and glial reactivity. These findings suggest that laterality in mouse MWM deficit might be an important consideration when modeling TBI-induced functional consequences. The second part of this dissertation work evaluated DAGL-β as a target to protect against TBI-induced learning and memory deficit given its selective expression on microglia and the role of 2-AG as a precursor for eicosanoid production. The gene deletion of DAGL-β did not protect against TBI-induced MWM or motor deficits, but unexpectedly produced a survival protective phenotype. These findings suggest that while DAGL-β does not contribute to injury-induced memory deficit, it may contribute to TBI-induced mortality. The third and final set of experiments investigated the role of DAGL-α in mouse spatial learning and memory under physiological conditions (given the predominantly neuronal expression of DAGL-α). Complementary pharmacological and genetic manipulations produced task specific impaired MWM performance, as well as impaired long-term potentiation and alterations to endocannabinoid lipid levels. These results suggest that DAGL-α may play a selective role in the integration of new spatial information in the normal mouse brain. Overall, these data point to DAGL-α, but not DAGL-β, as an important contributor to hippocampal-dependent learning and memory. In contrast, DAGL-β may contribute to TBI-induced mortality.

Anandamide (AEA) is an endogenous fatty acid which activates the same cannabinoid receptors as ∆9-tetrahydrocannabinol, the psychoactive substance in marijuana. In vivo, anandamide exerts a number of actions including effects upon pain and inflammation. However, AEA has a short duration of action since it is rapidly metabolised, primarily by the intracellular enzyme fatty acid amide hydrolase (FAAH). The general aim of this thesis has been to identify and characterize compounds capable of preventing the metabolism of anandamide. The chemical approach was based on the endogenous anti-inflammatory compound palmitoylethanolamide (PEA), a compound related to anandamide with the ability to inhibit anandamide degradation by substrate competition, but without the ability to directly activate cannabinoid receptors. A number of compounds were identified as inhibitors of rat brain FAAH in the initial in vitro studies, without having major affinity for the cannabinoid receptors. In particular, palmitoylisopropylamide (PIA) was found to reduce the metabolism of AEA in intact C6 glioma cells with potency similar to the prototypical AEA reuptake inhibitor AM404. This compound was in addition found to exert less effect upon C6 glioma cell proliferation than either AM404 or the closely related uptake inhibitor VDM11. To evaluate if PIA was effective in vivo, a model of mast cell dependent inflammation, oedema of the ear following local injection of compound 48/80, was set up using anaesthetised mice. Initially, a CB2 cannabinoid receptor selective agonist was used to probe the model and demonstrated to produce an anti-oedema effect. In contrast, the compound was inactive in vitro in skin slice preparations. PIA showed a similar pattern, although there was a large variation in responses which affected the significance of the result obtained, as did the vehicle used to dissolve the compound. Taken together, the present data would suggest that PIA can inhibit the degradation of AEA without having deleterious effects upon cell proliferation or affinity for the cannabinoid receptors. Further experimentation is necessary to elucidate the usefulness of this compound in vivo.

The mechanisms underlying cannabinoid impairment of fear memory is not clear. This study investigated the effects of the synthetic cannabinoid HU210 and the endocannabinoid hydrolysis inhibitor JZL 195 on fear memory following contextual fear conditioning (CFC; an animal model of fear). The long-term depression (LTD)-blocking peptide Tat-GluR2 was utilized to investigate whether the expression of cannabinoid-induced LTD (CB-LTD) is required for the cannabinoid impairment of acquisition and consolidation of contextual fear memory. HU210 reduced freezing throughout the test phase of the acquisition protocol, which was not affected by pre-administration of Tat-GluR2. High and moderate doses of HU210 reduced freezing during the first and last half, respectively, of the test phase of the consolidation protocol, which was prevented by pre-treatment with Tat-GluR2. HU210 did not affect freezing during the test phase of the retrieval protocol. Thus, these results suggest that HU210 impairs acquisition and consolidation, but not retrieval of contextual fear memory, and that in vivo CB-LTD expression is required for HU210 impairment of the consolidation, but not acquisition, of contextual fear memory. We also observed that HU210 and JZL 195 do not facilitate the acquisition of contextual fear memory extinction.

Neuroinflammation is a major driving force in the progression of neurodegenerative disorders. Nicotinic acetylcholine receptors, as well as cannabinoid CB2 receptors, have been shown to have strong anti-inflammatory properties when activated. These effects are shown, in vivo, to be a result of stimulation of α7 nAChRs and CB2 cannabinoid receptors. Microglia cells, an immune cell in the brain, are shown to express both of these receptor subtypes. The studies detailed herein, investigated the ability of two compounds, nicotine and Δ9-THC, in modulation of inflammatory processes. Stimulation of these receptors on microglia using nicotine and Δ9-THC blocked the activation of these cells, observed through reductions in pro-inflammatory cytokine production. Reductions in inflammation as well as pathology in the PSAPP mouse model of Alzheimer’s Disease were also observed following nicotine and Δ9-THC administration. These data raise the possibility that α7 nAChRs and CB2 cannabinoid receptors may prove to be viable and effective strategy for reducing neuroinflammation observed in neurodegenerative disease.

Introduction: Tobacco dependence is a chronic disorder that carries the risk of relapse at any time point during abstinence. It is a major health issue in the world and current pharmacotherapies have had limited efficacy. Therefore, development and validation of novel treatments are required. Objective: Investigate the novel neutral cannabinoid-1 receptor antagonist AM4113 on nicotine (main psychoactive ingredient in tobacco)-taking behaviour in animals. Methods: Using the nicotine intravenous- and food control- self administration paradigms, we tested the acute and chronic (10-days) effects of AM4113 on nicotine- and food-taking behaviour. Results: Acute AM4113 treatments (1-, 3-, 10-mg/kg) reduced nicotine self administration. Chronic AM4113 administration (10mg/kg) produced a sustained reduction of nicotine-taking behaviour during the course of the treatment. In the similar food control self administration experiments, AM4113 overall produced no effect. Conclusion: AM4113 can attenuate nicotine-taking behaviour and its effect is sustained under chronic treatment.

Introduction: Tobacco dependence is a chronic disorder that carries the risk of relapse at any time point during abstinence. It is a major health issue in the world and current pharmacotherapies have had limited efficacy. Therefore, development and validation of novel treatments are required. Objective: Investigate the novel neutral cannabinoid-1 receptor antagonist AM4113 on nicotine (main psychoactive ingredient in tobacco)-taking behaviour in animals. Methods: Using the nicotine intravenous- and food control- self administration paradigms, we tested the acute and chronic (10-days) effects of AM4113 on nicotine- and food-taking behaviour. Results: Acute AM4113 treatments (1-, 3-, 10-mg/kg) reduced nicotine self administration. Chronic AM4113 administration (10mg/kg) produced a sustained reduction of nicotine-taking behaviour during the course of the treatment. In the similar food control self administration experiments, AM4113 overall produced no effect. Conclusion: AM4113 can attenuate nicotine-taking behaviour and its effect is sustained under chronic treatment.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 100-108).