BACKGROUND: Major depression is a pervasive condition that affects every aspect of a patient’s life, and many patients are unable to find symptom alleviation with the current available medications. Ketamine has recently shown promise as a rapid-acting antidepressant, yet its mechanisms are not yet well-understood. OBJECTIVE: We sought to understand the change in depression symptom interrelationships, with particular interest in sleep, in the context of ketamine treatment in depression by completing a network analysis. METHODS: 97 patients with treatment-resistant depression were given ketamine over six treatments, and symptoms were examined via the Quick Inventory of Depressive Symptomology (QIDS-SR-16). Two networks were constructed: one prior to the first treatment, and one prior to the sixth treatment. Each symptom of the inventory formed a node, and partial correlations were used to construct the edges of the network. Centrality indices and network structure was then evaluated and compared. RESULTS: Centrality indices measured were unstable, limiting assertions to node strength, but global network structure was revealed to be changed between the networks. CONCLUSION: The data suggests that ketamine may affect the interrelationships between depressive symptoms, by impacting some symptoms more than others.

The dopaminergic neurons form a brain-wide neuromodulatory system affecting reward, addiction, and motor behaviors. Alterations in dopamine signaling have been associated with many brain disorders, including schizophrenia and Parkinson’s disease. Over the last decade, it has been well-established that the dopaminergic system is capable of significant neurotransmitter phenotypic plasticity (NPP), which is defined as the activity-dependent loss or gain of a specific neurotransmitter usage by neurons. However, most of the related studies have focused on specific regions, providing important but limited insights into NPP. Attaining an unbiased mapping of brain-wide NPP and its relationship with behavioral changes has remained challenging. In this thesis, we first addressed the technical challenges by establishing a whole-brain phenotyping pipeline. Next, we utilized these tools to generate the first brain-wide map of NPP in response to chronic exposure to (R,S)-ketamine, revealing some of the underlying fundamental principles. Here, we describe a whole brain NPP mapping pipeline for studying dopaminergic phenotypic plasticity following chronic (R,S)-ketamine exposure. The pipeline includes 1) an optimized whole brain tissue clearing/immunostaining method for labeling the dopaminergic neurons by utilizing rate-limiting marker tyrosine hydroxylase (TH), 2) high-resolution whole-brain imaging with CLARITY optimized light-sheet microscopy (COLM) and light sheet theta microscopy (LSTM), and 3) a custom python-based data analysis pipeline for quantitative mapping of the brain-wide NPP. Our first key result is that chronic (R,S)-ketamine administration leads to divergent brain-wide changes in the dopaminergic system. Specifically, ten days (but not 1 or 5 days) of daily (R,S)-ketamine (100 mg/kg) administration resulted in a significant decrease in TH+ neurons in regions of the midbrain and a significant increase in areas of the hypothalamus. Second, chronic (R,S)-ketamine treatment also induced an increase in TH+ neuronal projections, including increases within associative cortical brain regions such as the prelimbic area, orbital area, and anterior cingulate area. Decreases in the density of TH+ neuronal projections were observed in the auditory (AUDd) and visual cortices (VISpl). Third, we performed mRNA expression mapping and before-after treatment comparison of the TH+ neuron population within the same animal to reveal that newly gained TH+ neurons are mainly recruited from the neuron pool that contained untranslated TH mRNA. Overall, this thesis provides a first brain-wide quantitative survey of the NPP caused by a specific drug (R,S)-ketamine, extending our fundamental understanding of the extent of adaptability of our brain in response to external stimuli.

Major Depressive Disorder (MDD) is a common and debilitating mental illness primarily characterized by depressed mood and anhedonia, as well as a heterogenous mixture of other somatic symptoms. Existing therapies for MDD act primarily on monoamine transporters and receptors, with only partial success. A more recent advancement in depression treatment is the identification of the N-methyl-d-aspartate receptor (NMDAR) antagonist (R,S)-ketamine, which has shown efficacy in individuals with treatment resistant depression. Studies have raised the possibility of a mu opioid receptor (MOR)-dependent component to the actions of (R,S)- ketamine, through direct activation of MOR or indirect effects of NMDAR antagonism on endogenous opioids. Considering the ongoing global opioid epidemic, in which over prescribing of opioid painkillers and greater availability of illicit opioids has caused significant morbidity and mortality, studies suggesting an opioid component to ketamine’s actions have caused concern. We undertook the current experiments to better understand 1) the contribution of MORs to the behavioral effects of ketamine, and 2) how very low efficacy agonism of MOR may lead to a better separation between the desirable and undesirable effects of opioid drugs. Previous work from collaborators had established that a single dose of (R,S)-ketamine, specific hydroxynorketamine (HNK) metabolites, and the memantine derivative fluoroethylnormemantine (FENM), given one week before a contextual fear conditioning stressor could prevent the development of learned fear and behavioral despair. Moreover, some of these drugs also prevented the development of learned fear and behavioral despair when given shortly after the contextual fear conditioning stressor. We were interested in how (R,S)- ketamine’s reported MOR activity may contribute to these behavioral effects. We began by testing these compounds in cell-based signaling assays to determine their ability to directly activate MORs. We found that the parent (R)- and (S)-ketamine enantiomers could activate MORs with low potency in a high amplification G protein activation assay. In contrast, in low amplification miniG-based assays, the compounds tested showed little to no activation of MOR, suggesting that (R,S)-ketamine activates MOR with low potency and low efficacy. We moved to experiments in mice with the pseudo-irreversible MOR antagonist methocinnamox (MCAM) and attempted to block various behavioral effects of (R,S)-ketamine and the more selective NMDAR antagonist (FENM). We found that MCAM pretreatment blocked effects of (R,S)-ketamine on antinociception, behavioral despair, and learned fear, while other effects such as anesthesia and changes in hippocampal electrophysiology were not prevented by MCAM. With FENM the antinociceptive effects were less potent and less impacted by MCAM; however, the effects on behavioral despair and learned fear were still MORdependent. These results suggest that the protective effects of (R,S)-ketamine and FENM against stress may be an indirect effect of NMDAR antagonism on endogenous opioids. In a separate line of experiments, we used a range of doses of MCAM to antagonize the behavioral effects of morphine, the MOR agonist antidepressant tianeptine, and the MOR partial agonist 7-OH mitragynine. 7-OH mitragynine is a metabolite of mitragynine, the major alkaloid in kratom, which some reports suggest may have antidepressant and anxiolytic properties. Based on fundamentals of pharmacology, we hypothesized that inherent differences in the presence of “spare receptors” or receptor reserve between brain circuits could be revealed by differential inhibition by MCAM across behaviors and agonists. We assessed the inhibitory potency of MCAM against these drugs in tests of antinociception, hyperlocomotion, behavioral despair, respiration, and gastrointestinal motility. We found that MCAM pretreatment more potently inhibited the low efficacy agonist 7-OH mitragynine in the tests of antinociception, behavioral despair, and constipation. These data suggest that in circuits modulating antinociception, behavioral despair, and constipation, differences in receptor reserve likely facilitate the response to low efficacy agonists. However, our data also argue that the wider therapeutic window of G protein biased, low intrinsic efficacy MOR agonists is not solely a result of differences in the number of “spare” MORs regulating the effects of opioids in different circuits.

Selon l'OMS, les troubles dépressifs majeurs (TDM) seront la 2ème cause d'incapacité dans le monde en 2020 et deviendront la 1ère en 2030. Les antidépresseurs classiques ont des effets thérapeutiques retardés et de nombreux patients sont résistants. La kétamine, antagoniste du récepteur N-methyl-D-aspartate (R-NMDA) du L-glutamate, possède un effet antidépresseur rapide chez les patients résistants à un traitement classique. Le mécanisme de cette activité étonnante n'est pas bien compris. En couplant la microdialyse intracérébrale à un test comportemental prédictif d'une activité antidépressive dans un modèle de souris BALB/cJ de phénotype anxieux, nous montrons que cette activité de la kétamine dépend de la balance excitation-inhibition entre les systèmes glutamate/R-NMDA et R-AMPA, GABA/R-GABAA, sérotonine du circuit cortex préfrontal/noyau du raphé. Nos résultats suggèrent également que ce serait la combinaison [kétamine-(2R,6R)-hydroxynorkétamine, son principal métabolite cérébral] qui porterait l'effet antidépresseur. Mes travaux de thèse contribuent à une meilleure compréhension de l'effet rapide antidépresseur de la kétamine. / According to the WHO, major depressive disorder (MDD) will be the second leading cause of disability in the world in 2020 and will become the first in 2030. Conventional antidepressant drugs have delayed therapeutic effects and many patients are resistant. Ketamine, an N-methyl-D-aspartate (NMDA-R) receptor antagonist of L-glutamate, exerts a rapid antidepressant effect in patients who are resistant to standard therapy. The mechanism of this amazing activity is not well understood. By coupling intracerebral microdialysis to a predictive behavioral test of antidepressant activity in a BALB/cJ mouse model with an anxious phenotype, we show that this ketamine activity is dependent on the excitation-inhibition balance between glutamate/NMDA-R and AMPA-R, GABA/GABAA-R, serotonin systems in the prefrontal cortex/raphe nucleus circuit. Our results also suggest that it would be the combination [ketamine-(2R,6R)-hydroxynorketamine, its main brain metabolite] that would carry the antidepressant effect. My thesis work pave the way for the development of new fast-acting antidepressant drugs.

Ketamine and phencyclidine (PCP) are noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) type of ligand-gated glutamate receptors. Both agents have high abuse liability, and may produce dependence. Tolerance to the reinforcing effects of drugs of abuse is widely regarded as a key component of the dependence process. Therefore, the present study was conducted to examine whether tolerance develops to the reinforcing effects of ketamine, and whether PCP and dizocilpine, a noncompetitive NMDA antagonist with negligible abuse liability, produce cross-tolerance to the reinforcing effects of ketamine. Further, identification of the neural mechanisms that underlie tolerance to the reinforcing effects of drugs may yield information regarding drug dependence.

No description available.

This thesis examines the developmental neurotoxic effects of exposure to low-dose ionizing radiation (IR), alone or together with xenobiotics, during a critical period of neonatal brain development in mice. During mammalian brain development there is a period called the brain growth spurt (BGS), which involves extensive growth and maturation of the brain. It is known that neonatal exposure during the BGS to xenobiotics can have a negative impact on neonatal brain development, resulting in impaired cognitive function in the adult mouse. In humans, the BGS starts during the third trimester of pregnancy and continues for approximately 2 years in the child.   The present thesis has identified a defined critical period, during the BGS, when IR can induce developmental neurotoxicity in mice. The observed neurotoxicity was not dependent on sex or strain and manifested as altered neurobehaviour in the adult mouse. Furthermore, fractionated dose exposures appear to be as potent as a higher acute dose. The cholinergic system can be a target system for developmental neurotoxicity of IR, since alterations in adult mouse cholinergic system susceptibility were observed. Co-exposure to IR and nicotine exacerbated the behavioural disturbances and cholinergic system dysfunction. Furthermore, co-exposure with the environmental agent paraquat has indicated that the dopaminergic system can be a potential target.   In this thesis, clinically relevant doses of IR and a sedative/anesthetic agent (ketamine) were shown to interact and exacerbate defects in adult mouse neurobehaviour, learning and memory, following neonatal exposure, at doses where the single agents did not have any impact on the measured variables. This indicates a shift in the dose-response curve for IR, towards lower doses, if exposure occurs during the neonatal brain development. In addition, co-exposed mice, showing cognitive defects, expressed elevated levels of tau protein in the cerebral cortex. Furthermore, exacerbation of neurochemical deviations were observed following co-exposure compared to irradiation alone. Further investigations of neurotoxic effects following fractionated or acute low-dose IR, modelling the clinical situation during repeated CT scans or levels of radiation deposited in non-target tissue during radiotherapy, and possible interaction effects with xenobiotics, is of great importance in the field of radioprotection.

The noncompetitive NMDA receptor antagonist ketamine produces rapid and sustained antidepressant effects in patients suffering from major depressive disorder. However, abuse liability is a concern. To further evaluate the relationship between antidepressant-like and abuse-related effects of NMDA receptor antagonists, this study evaluated the effects of ketamine, MK-801, and phencyclidine in male Sprague-Dawley rats responding under two procedures that have been used to assess antidepressant-like effects [differential-reinforcement-of-low-rate (DRL) 72 s schedule of food reinforcement] and abuse-related drug effects [intracranial self-stimulation (ICSS)]. Under DRL 72 s, ketamine produced an antidepressant-like effect by increasing reinforcers, decreasing responses, and producing a rightward shift in the peak location of the interresponse time (IRT) distributions. Phencyclidine produced a modest antidepressant-like effect by increasing reinforcers and decreasing responses, but did not shift the IRT distributions. In contrast, MK-801 produced a psychostimulant-like effect by decreasing reinforcers, increasing responses, and producing a leftward shift in the peak location of the IRT distributions. The antidepressant-like effects of ketamine on the DRL 72 s procedure do not appear to be mediated by inhibiting the reuptake of serotonin via serotonin transporters or antagonism of 5-HT2 receptors. Additionally, the dissociable effects of ketamine and MK-801 in the DRL 72 s procedure may be mediated by 5-HT2 receptors. Following acute administration, ketamine produced only dose- and time-dependent depression of ICSS and failed to produce an abuse-related facilitation of ICSS at any dose or pretreatment time. Repeated dosing with ketamine produced dose-dependent tolerance to the rate-decreasing effects of ketamine but failed to unmask expression of ICSS facilitation. Termination of ketamine treatment failed to produce withdrawal-associated decreases in ICSS. In contrast, MK-801 and phencyclidine effects produced dose- and time-dependent facilitation of ICSS by MK-801. Taken together, our findings provide further evidence that expression of these antidepressant-like and abuse-related effects of ketamine, phencyclidine, and MK-801 may be related to NMDA receptor affinity.

A depressão é uma doença com alta incidência na população mundial e os antidepressivos atualmente disponíveis não são completamente eficazes. Esses fármacos apresentam uma latência de 2-4 semanas para induzir uma melhora significativa dos sintomas e cerca de 45% dos pacientes não respondem ao tratamento, cujo mecanismo é baseado na facilitação da neurotransmissão monoaminérgica no SNC. Por outro lado, recentemente tem sido demonstrado que a ketamina, antagonista do receptor de glutamato do tipo NMDA induz um efeito antidepressivo rápido e sustentado em animais e pacientes. No entanto, o uso dessa droga para o tratamento da depressão possui diversas limitações e, assim, o entendimento dos mecanismos subjacentes à sua ação antidepressiva pode contribuir para o desenvolvimento de novas e melhores alternativas terapêuticas. Estes mecanismos parecem ser mais complicados do que simplesmente o bloqueio do receptor NMDA, dado que tal bloqueio com o antagonista MK-801, por exemplo, induz efeito tipo-antidepressivo no teste do nado forçado (FST) por até 3 horas, mas sem reproduzir os efeitos prolongados da ketamina. Por isso, a cascata de eventos neuroquímicos iniciada após a administração de ketamina que culmina com a regulação da expressão gênica e síntese de proteínas relacionadas aos processos de plasticidade neural têm sido alvo de grande investigação a fim de se compreender o mecanismo de ação subjacente ao efeito antidepressivo rápido e sustentado dessa droga. A expressão desses genes pode ser modulada por mecanismos epigenéticos, como a metilação do DNA, um processo realizado por DNA metiltransferases (DNMTs), que também tem apresentado grande relevância para a neurobiologia da depressão. Diante disso, o presente estudo teve como objetivo avaliar os efeitos da administração de antagonistas do receptor NMDA, ketamina e MK-801, em doses e protocolos de tratamento que promovam efeito tipo-antidepressivo no FST, sobre a metilação do DNA em estruturas encefálicas importantes para a neurobiologia da depressão, em animais submetidos ou não ao estresse de nado forçado. Para tanto, primeiramente, foram delineados protocolos experimentais para análise do efeito tipo-antidepressivo destas drogas: Em ratos, administração sistêmica aguda de S(+)-ketamina 10 mg/Kg ou MK-801 0,025 mg/Kg 23 horas após a sessão pré-teste e 1 hora ou 7 dias antes da sessão teste do FST, permitiu a análise de um efeito tipo-antidepressivo rápido e sustentado induzido pela ketamina e apenas rápido pelo MK-801. Em seguida, utilizando estes protocolos, avaliou-se os efeitos do estresse do pré-teste do FST e do tratamento com tais antagonistas do receptor NMDA sobre os níveis de metilação global do DNA e expressão de DNMT3a e DNMT3b no córtex frontal, hipocampo ventral e dorsal dos animais. Foram encontradas alterações nas quantificações realizadas, sugerindo que o estresse e o tratamento podem induzir efeitos importantes sobre a metilação do DNA nas estruturas analisadas. Além disso, o tratamento com ketamina ou MK-801 parecem induzir efeitos diferenciais em algumas regiões, o que poderia estar associado aos efeitos também distintos que apresentam sobre a ação antidepressiva / Although depression presents a high incidence in the world population, currently available antidepressants exhibit a latency of 2-4 weeks to induce a significant improvement of symptoms and around 45% of patients do not respond to these drugs. On the other hand, it has been recently shown that ketamine, a NMDA receptor antagonist, induces a rapid and sustained antidepressant effect in animals and patients. However, the use of this drug for depression treatment has several limitations and, thus, the understanding of the mechanisms underlying its antidepressant action could present a significant importance for the development of new and better therapeutic alternatives. These mechanisms appear to be more complex than the initial blockade of the NMDA receptor, since such blockade by MK-801, for example, reduces the immobility time of mice submitted the forced swimming test (FST) for up to 3 hours, without reproducing the sustained effects of ketamine. Therefore, the cascade of neurochemical events that are initiated after ketamine administration that culminate in the regulation of gene expression and syntehsis of proteins related to neuronal plasticity has been the focus of intense investigation. These genes, in turn, can be modulated by epigenetic mechanisms such as DNA methylation, a process performed by DNA methyltransferase (DNMTs), which has also shown a high relevance to the neurobiology of depression and its treatment. Based on that, the present study aimed at investigating the effects induced by ketamine and MK-801, at doses and treatment protocols that promote antidepressant-like effect in the FST, upon DNA methylation in brain structures of animals submitted or not to the forced swim stress. The first experimental protocols were designed for the analysis of acute and sustained drug-induced antidepressant-like effects: In rats, acute systemic administration of S(+)-Ketamine 10 mg/Kg or MK-801 0.025 mg/Kg 23 hours after the pretest session and 1 hour or 7 days before the test session of FST was investigated. Based on these protocols, the effects of stress (FST) and of treatment with NMDA receptor antagonists were investigated on global DNA methylation levels and DNMT3a and Dnmt3b expression in the rat frontal cortex, ventral and dorsal hippocampus. Both, stress and treatment, induced changes in DNA methylation and DNMT3 expression in some of the brain regions analised. In addition, treatment with MK-801 and ketamine seem to induce differential effects in some areas, which could also be associated with different effects that they present on antidepressant action.

A fisiopatologia do choque séptico caracteriza-se por uma produção excessiva de mediadores inflamatórios, dentre eles o óxido nítrico (NO), conduzindo a uma hipotensão prolongada associada a um aumento inicial de vasopressina (AVP) e uma diminuição na fase tardia. A ketamina é um anestésico com propriedades cardioestimulatórias e anti-inflamatórias. O presente trabalho testou a hipótese de que a ketamina, através de suas propriedades anti-inflamatórias no choque séptico, teria uma ação inibitória sobre a síntese do óxido nítrico, favorecendo a liberação de AVP e preservando a função cardiovascular. O choque endotoxêmico foi induzido através de uma injeção i.v. de 1,5 mg/kg de lipopolissacarídeo (LPS) em ratos Wistar adultos machos. Após a injeção de LPS, um grupo de animais foi tratado com ketamina (10 mg/kg) e o grupo controle recebeu salina. A administração de LPS produziu uma queda significativa da pressão arterial média (PAM) (p<0,01) associada a um aumento da freqüência cardíaca (FC) (p<0,01). Essas alterações foram acompanhadas por uma elevação significativa nas concentrações plasmáticas de AVP após duas horas (p<0,01), seguida de queda nas próximas horas, e por uma elevação nas concentrações de NO plasmático (p<0,01). Quando o LPS foi combinado à administração i.v. de ketamina, observou-se uma atenuação da hipotensão (p<0,01) e uma potencialização na liberação de AVP (p<0,01) pelo LPS. No entanto, a produção de NO após a adminstração da ketamina não mostrou diferença em relação ao LPS, indicando não ser esta a via utilizada pela ketamina. Para verificar o papel da ativação simpática na preservação da função cardiovascular pela ketamina no choque endotoxêmico, utilizou-se um inibidor simpático central, a moxonidina (MOXO). O pré-tratamento i.v. com MOXO (50 µg /Kg) atenuou significativamente o aumento da FC produzido pela ketamina (p < 0,05) apenas na segunda e quarta horas, porém com ação não significativa sobre a PAM. Estes dados sugerem um efeito cardioestimulatório da ketamina no choque séptico principalmente por uma potencialização na liberação da AVP e esta parece não se dar pela via do NO / The pathophysiology of septic shock is characterized by excessive production of inflammatory mediators, including nitric oxide (NO), leading to a prolonged hypotension associated with an initial increase of vasopressin (AVP) and a late phase decrease. Ketamine is an anesthetic with cardiostimulatory and anti- inflammatory properties. The present study tested the hypothesis that ketamine, through its anti-inflammatory properties in septic shock, have an inhibitory effect on the synthesis of nitric oxide, promoting the release of AVP and preserving cardiovascular function. Endotoxemic shock was induced by an iv injection of 1.5 mg / kg lipopolysaccharide (LPS) in adult male Wistar rats. After LPS injection, a group of animals was treated with ketamine (10 mg / kg) and the control group received saline. The LPS administration produced a significant decrease in mean arterial pressure (MAP) (p <0.01) associated with an increase in heart rate (HR) (p <0,01). These changes were accompanied by significant increases in plasma AVP after two hours (p <0.01), followed by fall in the coming hours, and plasma NO increasing (p <0.01). When LPS was combined with iv ketamine administration, there was an attenuation of hypotension (p <0.01) and an enhancement in the release of AVP (p <0.01) by LPS. However, the production of NO after ketamine adminstration showed no difference compared to LPS, indicating this is not the route used by ketamine. To verify the role of sympathetic activation in ketamine\'s preservation of cardiovascular function in endotoxemic shock, used a central sympathetic inhibitor, moxonidine (moxo). Pretreatment with moxo iv (50 µg / kg) significantly attenuated the increase in HR produced by ketamine (p <0.05) only in the second and fourth hour, but with no significant action on the MAP. These data suggest that the cardiostimulatory effect of ketamine in septic shock primarily occurs by potentiation of AVP release, and this does not seem to give the NO pathway