The neuropeptide, corticotropin-releasing factor (CRF), plays a critical role in the central regulation of various stress-related behaviours, including those unique to subjects with prior cocaine experience. The three series of experiments presented in this dissertation explored the role of two neurochemical systems, the teneurin C-terminal associated peptides (TCAP) and the endocannabinoids (eCBs), in several cocaine- and anxiety-related behaviours induced or mediated by CRF. The first series of experiments examined the effects of TCAP-1 on the reinstatement of cocaine seeking and expression of cocaine-induced behavioural sensitization. Repeated (5-day), but not acute, TCAP-1 treatment blocked the reinstatement of cocaine seeking induced by central injections of CRF. TCAP-1 was, however, without effect on footshock- or cocaine-induced reinstatement. Repeated TCAP-1 further interfered with the expression of behavioural sensitization to a CRF, but not a cocaine, challenge. These findings suggest that TCAP-1 normalizes CRF signaling dysregulated by cocaine exposure to interfere in the subsequent effects of CRF on cocaine-related behaviours. A parallel series of experiments investigated the role of eCB signaling at CB1 receptors in the reinstatement of cocaine seeking and cocaine-sensitized locomotion. Pretreatment with the CB1 receptor antagonist, AM251, selectively interfered with CRF-, but not footshock- or cocaine-induced reinstatement. AM251 further blocked the expression of behavioural sensitization induced by challenge injections of both CRF and cocaine. These findings reveal a mediating role for CB1 receptor transmission in the effects of CRF on cocaine-related behaviours. A final series of experiments examined the role of CB1 receptor transmission in the behavioural anxiety induced by central injections of CRF, and by withdrawal from chronic cocaine exposure. AM251, although itself anxiogenic, reversed anxiety induced by CRF and cocaine withdrawal. Furthermore, AM251 elevated plasma corticosterone levels, indicative of increased HPA axis activity, irrespective of CRF treatment or cocaine withdrawal. These findings suggest that CRF- and cocaine withdrawal-induced anxiety are mediated, at least in part, by CB1 receptor transmission, independent of HPA axis regulation. The collective findings are discussed within a framework of CRF-TCAP-eCB interactions, wherein TCAP-1 and AM251 are proposed to act in parallel to modulate amygdalar CRF transmission, and thus regulate the expression of cocaine- and anxiety-related behaviours.

In the early nineties cannabinoid receptors, the main target for Δ9-tetrahydrocannabinol (THC), the psychoactive component of marijuana were identified. Shortly after their endogenous ligands, N-arachidonoylethanolamine (anandamide, AEA) and 2-diacylglycerol (2-AG) were characterized. The enzymes primarily responsible for catalysing the degradation of AEA and 2-AG are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL) respectively. AEA is a member of the N-acylethanolamine (NAE) class of lipids, which depending on the acyl chain length and number of double bonds can act as ligands for a variety of biological targets. Exogenous cannabinoids have long been reported to have analgesic effects, however the clinical usefulness of such substances is limited by their psychoactive effects. Inhibition of endocannabinoid degradation would mean enhancing the therapeutic effects without producing these unwanted side effects. In order to succeed in developing such compounds the pharmacology of the enzymes responsible for the degradation of endocannabinoids has to be thoroughly understood. When the preclinical part of this thesis was planned, FAAH had been well characterized whereas little was known as to the pharmacology of MGL. A series of compounds were tested in this first study aiming to find MGL-selective compounds. Although no compounds showed selectivity for MGL over FAAH, several interesting agents affecting both enzymes were identified. In order to increase the knowledge concerning which patient group would benefit from such treatment strategies it is important to investigate in which pain states the endocannabinoids/NAEs are altered. Thus the general aim of the clinical part of this thesis was to investigate the levels of endocannabinoids/NAEs in the interstitium of the trapezius muscle in women suffering from chronic neck/shoulder pain (CNSP) and chronic wide spread pain (CWP) and in healthy pain-free controls. Furthermore for the CNSP the effect of training, which is a commonly recommended treatment for these patients, on the levels of endocannabinoids/NAEs was also investigated. Microdialysis technique in the trapezius muscle was used for sampling and masspectrometry was used for analysing. Two NAEs, N-palmitoylethanolamine (PEA) and N-stearoylethanolamine (SEA), could be repeatedly measured. The levels of these two lipids were significantly higher in CNSP compared to CON. The result showed also that PEA and SEA mobilize differently in CWP compared to both CNSP and CON. Taken together the results presented in thesis represent an early characterization of the pharmacology of MGL and provides novel information on NAEs in chronic muscle pain.

The neuropeptide, corticotropin-releasing factor (CRF), plays a critical role in the central regulation of various stress-related behaviours, including those unique to subjects with prior cocaine experience. The three series of experiments presented in this dissertation explored the role of two neurochemical systems, the teneurin C-terminal associated peptides (TCAP) and the endocannabinoids (eCBs), in several cocaine- and anxiety-related behaviours induced or mediated by CRF. The first series of experiments examined the effects of TCAP-1 on the reinstatement of cocaine seeking and expression of cocaine-induced behavioural sensitization. Repeated (5-day), but not acute, TCAP-1 treatment blocked the reinstatement of cocaine seeking induced by central injections of CRF. TCAP-1 was, however, without effect on footshock- or cocaine-induced reinstatement. Repeated TCAP-1 further interfered with the expression of behavioural sensitization to a CRF, but not a cocaine, challenge. These findings suggest that TCAP-1 normalizes CRF signaling dysregulated by cocaine exposure to interfere in the subsequent effects of CRF on cocaine-related behaviours. A parallel series of experiments investigated the role of eCB signaling at CB1 receptors in the reinstatement of cocaine seeking and cocaine-sensitized locomotion. Pretreatment with the CB1 receptor antagonist, AM251, selectively interfered with CRF-, but not footshock- or cocaine-induced reinstatement. AM251 further blocked the expression of behavioural sensitization induced by challenge injections of both CRF and cocaine. These findings reveal a mediating role for CB1 receptor transmission in the effects of CRF on cocaine-related behaviours. A final series of experiments examined the role of CB1 receptor transmission in the behavioural anxiety induced by central injections of CRF, and by withdrawal from chronic cocaine exposure. AM251, although itself anxiogenic, reversed anxiety induced by CRF and cocaine withdrawal. Furthermore, AM251 elevated plasma corticosterone levels, indicative of increased HPA axis activity, irrespective of CRF treatment or cocaine withdrawal. These findings suggest that CRF- and cocaine withdrawal-induced anxiety are mediated, at least in part, by CB1 receptor transmission, independent of HPA axis regulation. The collective findings are discussed within a framework of CRF-TCAP-eCB interactions, wherein TCAP-1 and AM251 are proposed to act in parallel to modulate amygdalar CRF transmission, and thus regulate the expression of cocaine- and anxiety-related behaviours.

The endocannabinoid system is a novel pharmacological target in the treatment of metabolic syndrome. Antagonism of the endocannabinoid-1 receptor (CB1R) leads to a transient reduction in food intake, a sustained decrease in body weight and an improvement in metabolic parameters in animal models of obesity. Skeletal muscle is the primary tissue involved in glucose uptake in response to insulin, and insulin sensitivity of skeletal muscle is vital to the maintenance of whole-body euglycemia. Little is known regarding the effects of CB1R antagonism on skeletal muscle glucose transport activity. The purpose of this dissertation was to test the hypothesis that antagonism of the CB1R activates signaling molecules of the insulin signaling pathway to increase glucose transport activity in normal and insulin-resistant skeletal muscle, thereby improving whole-body glucose tolerance. CB1R antagonism with SR141716 directly enhanced basal and insulin-stimulated glucose transport activity in skeletal muscle from lean and obese Zucker while activation of the CB1R with ACEA, decreased glucose transport activity. Key proteins associated with regulation of glucose transport activity were not altered by either CB1R agonism (ACEA) or antagonism (SR141716). Chronic CB1R antagonist treatment (10 mg/kg SR141716 i.p. / 14 days) also enhanced insulin-stimulated glucose transport activity in skeletal muscle of both lean and obese animals, again with no alteration in relevant signaling factors. Plasma free fatty acids (FFAs) were decreased in chronically-treated lean and obese animals and whole-body insulin sensitivity was improved in obese Zucker rats. The enhanced insulin sensitivity seen in chronically-treated obese animals was associated with a dramatic reduction in insulin secretion following a glucose challenge. Acute CB1R antagonism in obese animals also elicited a reduction in insulin secretion following a glucose challenge; however, with no improvement of whole-body insulin sensitivity. Acute CB1R antagonist treatment did not alter skeletal muscle glucose transport activity or circulating FFAs for any animals. These data suggest that although CB1R antagonism directly enhances basal and insulin stimulated glucose transport in skeletal muscle of lean and obese rats, direct action on the skeletal muscle is not responsible for the improvement in insulin-stimulated glucose transport activity and whole-body insulin sensitivity seen in chronically-treated obese animals.

Gastrointestinal (GI) health is an important contributor to one’s overall well-being. In the past decade, the focus of this aspect of health has been on the organisms that inhabit the intestines: gut microbes. A concept central to understanding bacterial behavior for health or disease promotion is biological film (biofilm) formation. The predominant form of growth for bacteria is biofilm formation, an as yet poorly described phenomenon for gut microbes. In order to better understand bacterial behavior in response to nutrients that pass through the GI system, a high throughput system to assess biofilm formation was developed. Gastrointestinal-associated microbes, Escherichia coli and Enterococcus faecalis, were assayed for biofilm formation in 96-well plates after 24 hours of incubation. Nutrients, inter-and intrakingdom signaling molecules such as monosaccharides, calcium, insulin, endocannabinoids, and AI-1, AI-2 like signaling compounds, respectively, were added to cultures in order to test their effects on biofilm formation. Biofilm was quantified spectrophotometrically by the measurement of optical density of each well measured at 580nm following crystal violet staining of adherent biofilm. Values were expressed as means ± standard error of the mean (SEM); differences between means were assessed using t-testing and ANOVA using GraphPad Prism, with mean differences considered significant at P < 0.05. Results showed that biofilm formation by E. coli was enhanced by glucose, galactose, lactose, AI-1 like signaling compound and insulin at 50 and 100µU/ml, while AI-2 like compound and calcium inhibited biofilm formation. Biofilm formation by E. faecalis was also enhanced by AI-1 like compound and insulin at 50µU/ml in RPMI medium and inhibited in cultures grown in BHI medium or with added calcium. We conclude that gastrointestinal-associated microbes are influenced by nutrients as well as various factors, including the culture medium, signaling compounds, as well as host-signaling compounds such as insulin and calcium. This study provides a platform required for future studies involving nutrient effect on biofilm formation.

Background: Recommendations to restrict summer sunlight exposure to prevent skin cancer apparently conflict with requirements to protect bone health through adequate 25-hydroxyvitamin D (25[OH]D) levels, as provided by cutaneous ultraviolet (UV)B exposure. Furthermore, sunlight exposure promotes a feeling of euphoria that is felt to drive further sun-seeking behaviour. Objectives: My principal objective was to examine health risk (DNA damage) and health benefits (25[OH]D gain, and potential cutaneous photoprotection) following low-level summer sunlight exposures in people of light (phototype II) and darker (phototype V) skin. A further objective was to evaluate serum endocannabinoid levels, potential drivers of mood elevation, following these exposures, and to assess for evidence of tanning addiction in a cross-section of psoriasis patients who had received similar low dose UV exposures, as medical phototherapy. Methods: During wintertime, 10 white Caucasians and 6 South Asians aged 18 to 60 years, from Greater Manchester, UK, received a simulated summer's sunlight exposures, specifically 1.3 standard erythemal dose, thrice weekly for 6 weeks, whilst casually dressed. Serum and urine samples and skin colour measurements were taken at baseline, Monday, Wednesday and Friday of week 1 and then weekly, and buttock skin that had received differential UVR exposures was biopsied for immunohistochemical analysis. Phototype II individuals, who are at higher risk of sunburn, were subsequently challenged with 2X minimal erythema dose (MED) UVB on small areas of simulated summer-exposed and photoprotected skin. Separately, a link to an online tanning questionnaire survey was sent to all members of the National Psoriasis Foundation (USA) during my USA field trip. Results: The simulated summer resulted in 50% gain in 25(OH)D for both phototype groups, but significantly more cutaneous DNA damage (cyclobutane pyrimidine dimers, CPD) in phototype II than V (p<0.0001). There was no accumulation of cutaneous CPD after 6 weeks compared with a single UVR exposure in either group, while phototype V individuals had repaired a greater proportion of their CPD 24 hours after final UVR exposure (p<0.0001). Urinary oxidative DNA damage was higher in phototype II throughout the simulated summer (p=0.002) and unaffected by UVR. All individuals had significant skin darkening, and in phototype II, stratum corneum thickness increased significantly (p<0.05). This tanning response provided significant photoprotection against a pro-inflammatory UVB (2X MED) challenge, as shown by reduced erythema and neutrophil influx in skin exposed to the simulated summer than in photoprotected skin (p<0.05 for both). Serum endocannabinoid (2-arachidonoyl glycerol) levels increased significantly during the simulated summer in both phototype groups (p<0.01), peaking at week 2-3. The cross-sectional study of 1,832 psoriasis patients revealed 34% had used sunbeds; 11% of current users fulfilled diagnostic criteria for addictive-like tanning behaviour: female sex, younger age, younger age at psoriasis diagnosis, severe disease and prior phototherapy were significant risk factors for addiction. Discussion: These findings should assist public health guidance on safe sunlight exposure and highlight the need for distinct guidance targeted to different phototype groups. Furthermore, individuals with psoriasis, in particular those who previously received regular UVR exposure, are at high risk of tanning addiction that may be driven by the endocannabinoid system.

Orientadora: Profª. Drª. Tatiana Lima Ferreira / Coorientador: Prof. Dr. Daniel Carneiro Carrettiero / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, São Bernardo do Campo, 2018. / A doença de Alzheimer (DA) é uma doença neurodegenerativa caracterizada por múltiplos déficits cognitivos, como prejuízo da memória operacional, espacial e emocional. As principais características histopatológicas da DA são o surgimento das placas amilóides e dos emaranhados neurofibrilares. Os emaranhados se originam do acúmulo da proteína tau fosforilada (p-tau), que ocorre devido a falhas nas suas vias de degradação, como a via BAG2/HSP70. Esse acúmulo de p-tau no progresso da DA é parte fundamental do surgimento e agravamento de processos neurodegenerativos como perda sináptica e neuronal, relacionada à piora do comprometimento cognitivo. Os compostos canabinóides tem importante papel no progresso da DA, regulando processos cognitivos, inflamatórios e de degradação de p-tau. Uma redução da quantidade de anandamida (ANA) um endocanabinóide, no encéfalo de humanos está correlacionada com o déficit cognitivo associado à DA. Além disso, uma das formas da ANA exercer seu papel neuroprotetor em processos neuropatológicos seria aumentando os níveis de HSP70, chaperona envolvida na degradação de p-tau. A maioria dos estudos que investigo Os animais que receberam STZ tiveram déficits nos testes de reconhecimento de novo objeto (RNO), latência de escape no labirinto em cruz elevada (LCE) e condicionamento de medo ao som e a injeção de ANA recuperou os prejuízos no RNO e no LCE. O condicionamento de medo ao contexto e a inibição pré-pulso não foram alterados pela STZ e/ou ANA. STZ também induziu alargamento ventricular, redução de receptores canabinóides do tipo 1 e das proteínas sinápticas sinaptofisina e sintaxina. ANA preveniu o aumento do ventrículo e a diminuição de sintaxina. Os níveis de SNAP-25 não foram modificados por ANA e/ou STZ. A respeito da via de degradação da ptau, STZ aumentou os níveis de p-tau Ser 396 e diminuiu p-tau Ser199/202, HSP70 e BAG2. A ANA não preveniu nenhum desses efeitos, apenas acentuou a redução de BAG2. A quantidade de tau total permaneceu inalterada pelos tratamentos. Os resultados sugerem que o sistema endocanabinóide pode estar envolvido nos efeitos induzidos pela STZ, pois, embora a ANA não tenha conseguido prevenir algumas alterações cognitivas observadas na DA esporádica, ela evitou parcialmente as deficiências cognitivas induzidas pela STZ e as alterações no volume ventricular e nos marcadores sinápticos. A relação desse sistema com a degradação de p-tau ainda permanece não esclarecida e são necessários mais estudos para melhor compreensão do substrato neurobiológico pelo qual a ANA poderia exercer sua atividade neuroprotetora. .Assim, este estudo fortalece o uso de STZ como modelo de DA esporádica e corrobora o potencial do sistema endocanabinóide como um importante alvo terapêutico para a DA. / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by multiple cognitive deficits, such as impairment of working, spatial and emotional memory. The main histopathological features of AD are the appearance of amyloid plaques and neurofibrillary tangles. The tangles are originated from the accumulation of phosphorylated tau protein (p-tau), which occurs due to failures in its degradation mechanisms, such as the BAG2/HSP70 pathway. This accumulation of p-tau in AD progress is a fundamental part of the onset and aggravation of neurodegenerative processes, including synaptic and neuronal loss, related to the worsening of cognitive impairment. Cannabinoid compounds play an important role in the pathological progression of AD, regulating cognitive and inflammatory processes and p-tau degradation. A reduction in the amount of anandamide (ANA), an endocannabinoid, in the brain of humans is correlated to the cognitive deficit associated with AD. In addition, one of the ways ANA exert its neuroprotective role in neuropathological processes would be by increasing the levels of HSP70, chaperone involved in the degradation of p-tau. Most studies investigating the role of the endocannabinoid system in AD have used transgenic animals, which are more correlated to familial AD, and are not ideal models to mimic sporadic AD, which is the multifactorial form of the disease, corresponding to more than 95% of the cases. The sporadic DA model based on the intracerebroventricular (icv) injection of streptozotocin (STZ) mimics the cognitive, metabolic, synaptic and histopathological changes of the AD. However, little is known about the role the endocannabinoid system in the changes observed in this model. Thus, the objective of the present study was to evaluate the possible preventive effect of ANA on cognitive changes, synaptic loss and alterations in the HSP70/BAG2 pathway of p-tau degradation in a sporadic AD model. Thus, Wistar rats received ANA and/or STZ icv in the same surgery and after 30 days had their cognitive performance evaluated. Afterwards, they were euthanized and their brains were removed for histological analysis and the hippocampus were used for Western blotting analysis of the onditioning tests and ANA injection recovered the impairments of the performance in NOR and EPM. Contextual fear conditioning and pre-pulse inhibition were not altered by STZ and/or ANA. STZ also induced ventricular enlargement and reduced the levels of cannabinoid receptors of type 1 and of the synaptic proteins synaptophysin and syntaxin. ANA prevented ventricular enlargement and the decrease of syntaxin levels. SNAP-25 levels were not modified by ANA and/or STZ. Regarding the degradation pathway of p-tau, STZ increased p-tau Ser 396 and decreased p-tau Ser199 / 202, HSP70 and BAG2. ANA did not prevent any of these effects, only accentuated the reduction of BAG2. The total levels of tau remained unchanged by the treatments. The results suggest that the endocannabinoid system may be involved in the STZ-induced effects because, although ANA failed to prevent some cognitive changes in sporadic AD, it partially inhibited STZ-induced cognitive deficits and changes in ventricular volume and in the synaptic markers. The relationship of this system to the degradation of p-tau remains unclear and further studies are needed to better understand the neurobiological substrate by which ANA could exert its neuroprotective activity. Thus, this study strengthens the use of STZ as a sporadic DA model and corroborates the potential of the endocannabinoid system as an important therapeutic target for AD.

Receptores CB1 e TRPV1 desempenham papéis opostos na modulação da atividade neuronal e, possivelmente, na regulação da resposta ao estresse. A exposição ao estresse reduz a neurotransmissão mediada por receptores CB1, enquanto que a facilitação do sistema endocanabinóide produz efeito tipoantidepressivo. Por outro lado, estudos farmacológicos e de manipulação genética apontam que a diminuição da sinalização mediada por receptores TRPV1 produz efeito tipo-antidepressivo em modelos animais. Evidências científicas apontam que a modulação da neurotransmissão glutamatérgica, dependente de receptores NMDA, esteja envolvida em respostas mediadas por CB1 e TRPV1. Ambos os receptores são amplamente expressos em estruturas cerebrais envolvidas na resposta emocional, incluindo o córtex pré-frontal medial ventral (CPFmv), o que aponta para essa estrutura como importante alvo para os efeitos mediados por receptores CB1, TRPV1 e NMDA. Entretanto, pouco se sabe sobre a interação entre CB1 e TRPV1 corticais na resposta ao estresse. Dessa forma, avaliamos se receptores CB1 e TRPV1 localizados no CPFmv-pré-límbico (PL) podem contribuir, de maneiras opostas, para as mudanças comportamentais induzidas pelo estresse do teste do nado forçado (TNF), um teste preditivo de efeito tipo-antidepressivo. Em um primeiro grupo experimental observou-se que a AEA induz efeito tipo-antidepressivo com uma curva dose-resposta em U, quando administrada no CPFmv-PL. Além disso, o efeito tipo-antidepressivo da AEA foi prevenido pela pré-administração de AM251, um antagonista para receptores CB1. Por outro lado, a maior dose de AEA, que não induz efeito per se, produziu efeito tipo-antidepressivo quando combinada ao bloqueio de receptores TRPV1 por meio do pré-tratamento com SB366791. Corroborando estes dados, observou-se que a administração intra-CPFmv-PL de um bloqueador dual da enzima FAAH e de receptores TRPV1 (AA-5HT), induziu efeito tipo-antidepressivo no teste do nado forçado. Além disso, a administração do inibidor da FAAH (URB597) e do antagonista de receptores TRPV1 (SB366791), em grupos independentes de animais, reduziu o tempo de imobilidade no teste do nado forçado. Ademais, a coadministração de doses subefetivas de URB597 e SB366791 reduziu o tempo de imobilidade no mesmo teste comportamental, evidenciando somação de efeito do bloqueio combinado da FAAH com receptores TRPV1. Em conjunto, esses ii resultados indicam que tanto a facilitação da neurotransmissão mediada por CB1R quanto o bloqueio de TRPV1R no CPFmv-PL promove efeito tipo-antidepressivo, sugerindo que ambos os receptores corticais são importantes na modulação de respostas comportamentais ao estresse e, possivelmente, na neurobiologia da depressão, porém de maneiras opostas. Buscando elucidar os mecanismos pelos quais a AEA, através da ativação de CB1R e TRPV1R, promove seus efeitos comportamentais, o envolvimento da das neurotransmissões glutamatérgica e nitrérgica nos efeitos induzidos pela AEA foi investigado. Nossos resultados demonstraram que a administração de antagonista de receptores glutamatérgicos do tipo NMDA (LY235959) e de inibidor da nNOS (NPA) induziram efeito tipoantidepressivo no teste do nado forçado quando administrados intra-CPFmv-PL. Além disso, a coadministração de doses subefetivas do antagonista NMDA (LY366791) e da AEA intra-CPFmv-PL, induziu efeito tipo-antidepressivo no TNF, mostrando um efeito aditivo da administração conjunta das drogas. Esse resultado sugere que o efeito tipo-antidepressivo da AEA seja facilitado pela inibição da neurotransmissão glutamatérgica mediada por NMDA. Em conjunto, os resultados do presente trabalho indicam um envolvimento de CB1 e TRPV1 do CPFmv-PL na modulação da resposta comportamental ao estresse do nado forçado, porém de maneira contrária. Além disso, o efeito tipo-antidepressivo da AEA parece envolver a diminuição da neurotransmissão glutamatérgica mediada por NMDAR. / CB1 and TRPV1 receptors play opposite roles in the modulation of neuronal activity and, possibly, in the regulation of the stress response. Exposure to stress attenuates CB1 receptor-mediated neurotransmission, while facilitation of the endocannabonoid system produces antidepressant-like effects. On the other hand, genetic and pharmacological blockade of TRPV1 receptor signalling produces antidepressant-like effect in animal models. Scientific evidence suggests NMDA receptor-mediated glutamatergic neurotransmission might be involved in responses mediated by CB1 and TRPV1. Both receptors are widely expressed in brain structures involved in the emotional response, including the ventral medial prefrontal cortex (CPFmv), which points to this structure as an important target to the effects triggered by CB1 and TRPV1. However, little is known about the interaction between cortical CB1 and TRPV1 in response to stress. Therefore, we evaluated whether CB1 and TRPV1 receptors of the vmPFC-PL may contribute, in opposite ways, to stress-induced behavioral changes in the forced swimming test (FST), a predictive test of antidepressant-like effect. In a first experimental group it was observed that AEA induces antidepressant-like effect with a U shape dose-response curve, when administered in the vmPFC-PL. In addition, the antidepressant-like effect of AEA was prevented by pre-administration with AM251, a CB1 receptor antagonist. On the other hand, the higher dose of AEA, which does not induce effect per se, produced an antidepressant-like effect when combined with TRPV1 receptor blockade with SB366791. Corroborating these data, intra-vmPFC-PL administration of a dual blocker of the FAAH enzyme and TRPV1 receptors (AA-5HT), induced antidepressant-like effect in the forced swimming test. In addition, administration of FAAH inhibitor (URB597) and TRPV1 receptor antagonist (SB366791) in independent groups of animals reduced the immobility time in the forced swimming test. Furthermore, co-administration of URB597 and SB366791, in sub-effective doses, reduced the immobility time in the same behavioral test, evidencing synergism of the combined blockade of FAAH with TRPV1 receptors. Taken together, these results indicate that both facilitation of CB1R-mediated neurotransmission and blockade of TRPV1R in vmPFC-PL promotes antidepressantlike effect, suggesting that both cortical receptors are important in modulating iv behavioral responses to stress and possibly in the neurobiology of depression, but in opposite ways. Aiming to elucidate the mechanisms by which AEA, through the activation of CB1R and TRPV1R, promotes its behavioral effects, the involvement of the possible modulation of glutamatergic and nitrergic neurotransmissions by AEA was investigated. Our results demonstrated that administration of NMDA receptor antagonist (LY235959) and nNOS inhibitor (NPA) induced antidepressant-like effect in the forced swimming test when administered intra-CPFmv-PL. In addition, coadministration of NMDA antagonist (LY366791) and AEA intra-CPFmvPL, in subeffective doses, induced antidepressant-like effect in the FST, showing a synergistic effect of these drugs. This result suggests that the antidepressant-like effect of AEA might involve attenuation of cortical NMDA-mediated glutamatergic neurotransmission. Taken together, results of the present study indicate an opposite involvement for CB1 and TRPV1 receptors in the behavioral responses elicited by forced swimming stress. Furthermore, the antidepressant-like effect of AEA probably involves the attenuation of NMDAR-mediated glutamatergic neurotransmission.

A ativação do receptor CB1, leva a modulação de processos intracelulares que muda a resposta celular de acordo com o estímulo, além de estar envolvida em mecanismos de proliferação, diferenciação, movimentação e morte celular. O objetivo desse trabalho foi avaliar a participação desse sistema em processos oxidativo e inflamatório relacionados à neurodegeneração in vitro. Foi utilizado a linhagem de neuroblastoma Neuro2a diferenciada em células dopaminérgicas que foram expostas a três condições: com 6OHDA, H2O2 e LPS e co-tratadas com o agonista do receptor CB1 ACEA e o antagonista/agonista inverso AM251 por 24 horas. Utilizamos parâmetros funcionais de viabilidade celular, produção de espécies reativas de oxigênio e técnica de western blot. O tratamento com ACEA ou ACEA/AM251 produziram um aumento da viabilidade celular nos três modelos de exposição propostos; redução da produção de espécies reativas de oxigênio e ativação da via da proteína ERK1/2, além da inibição da morte celular pela diminuição da expressão da caspase 3. Concluímos que os canabinoides escolhidos foram capazes de proteger as células dopaminérgicas do dano oxidativo e inflamatório através do aumento da sobrevida celular por diminuição da produção de ROS. / The CB1 receptor activation leads to modulation of intracellular processes that change the cellular response according to the stimulus, as well as being involved in mechanisms of proliferation, differentiation, cell movement and death. The present study evaluated the participation of this system in oxidative and inflammatory processes related to neurodegeneration in vitro. We have used the Neuro2A neuroblastoma lineage, which those were differentiated into dopaminergic cells, and exposed to 6OHDA, H2O2 and LPS. They were co-treated with ACEA, CB1 receptor agonist, and AM251, the CB1 receptor antagonist/inverse agonist, for 24 hours. We used functional parameters of cell viability, production of reactive oxygen species and protein analyses by western blot. Treatment with ACEA or ACEA/AM251 produced an increase in cell viability; reduced production of reactive oxygen species and activation of the ERK1/2 protein, in addition to inhibition of cell death by decreasing the expression of caspase 3 in all three models proposed. We concluded that chosen cannabinoids were able to protect dopaminergic cells from oxidative damage and inflammation through the increased cell survival by decreasing the production of ROS.

Dans cette thèse l’expression du récepteur CB1 dans l’hippocampe de souris mutantes ré-exprimant spécifiquement le gène spécifiquement dans certains types cellulaires du cerveau tels que : les neurones glutamatergiques du télencéphale dorsal, et les neurones GABAergiques a été analysé. De plus, dans le but de connaître la distribution anatomique exacte des récepteurs CB1 astrogliaux par rapport aux synapses excitatrices et inhibitrices, j’ai étudié l’expression des récepteurs CB1 dans les astrocytes de souris exprimant le récepteur CB1 seulement dans les astrocytes et une souris mutante ciblée pour exprimer la protéine cytoplasmique hrGFP diffusible dans les cellules astrogliales, ce qui permet une meilleure détection des prolongements astrocytaires. Les conclusions de ce travail de thèse sont les suivantes : la distance la plus commune entre le récepteur CB1 astroglial et la synapse la plus proche est de 400 à 800 nm. La majorité des synapses entourées par des astrocytes immunopositifs pour le récepteur CB1 dans l’hippocampe, est de nature excitatrice. Les souris mutantes ré-exprimant le récepteur CB1 caractérisées dans ce travail de thèse montrent : 1) l’expression du récepteur CB1 dans différents types cellulaires, 2) la réexpression est limitée à une population neuronale particulière ou aux astrocytes, 3) les niveaux endogènes de récepteurs CB1 sont maintenus dans les différents types cellulaires ré-exprimant le récepteur CB1. De façon générale, ces résultats nous montrent que les souris mutantes ré-exprimant le récepteur CB1 sont d’excellents outils pour l’étude fonctionnelle et translationnelle sur le rôle de ce récepteur dans le cerveau sain ou pathologique. / The Cannabinoid Type I receptor protein (CB1) expression in the hippocampus of rescue mice modified to express the gene exclusively in specific brain cell types: such as dorsal telencephalic glutamatergic neurons, or GABAergic neurons have been analyzed. Furthermore, aiming at knowing the exact anatomical distribution of the astroglial CB1 receptors with respect to the excitatory and inhibitory synapses, the CB1 receptor expression in astrocytes of mouse expressing CB1 receptor only in astrocytes and mutant mouse expressing the protein hrGFP into astrocytes (that allows for better detection of the astrocytic processes) have been also investigated. The results showed that the majority of the hippocampal synapses surrounded by CB1 receptor immunopositive astrocytes in the 400-800 nm range are of excitatory nature. Moreover, the CB1 receptor rescue mutant mice characterized in this Doctoral Thesis have proven 1) to express CB1 receptors in specific brain cell types; 2) the re-expression is limited to the particular brain cell populations; 3) the endogenous levels of CB1 receptors are maintained in the brain cell types re-expressing the receptor. Which makes this mutant mice excellent tools for functional and translational investigations on the role of the CB1 receptors in the normal and diseased brain.