Indiana University-Purdue University Indianapolis (IUPUI) / Environmental exposure to lead (Pb) and cannabis use are two of the largest public health issues facing modern society in the United States and around the world. Exposure to Pb in early life has been unequivocally shown to have negative impacts on development, and recent research is mounting showing that it may also predispose individuals for risk of developing substance use disorders (SUD). At the same time, societal and legal attitudes towards cannabis (main psychoactive component delta-9-tetrahydrocannabinol) have been shifting, and many American states have legalized the recreational use of cannabis. It is also the 3rd most widely used drug of abuse in the US, and rates of cannabis use disorder are on the rise. This thesis sets out to establish whether there is a link between early life Pb exposure and later THC-related behavior in C57BL6/J mice, as has been demonstrated for other drugs of abuse. The first aim seeks to answer whether Pb exposure affects physiological THC sensitivity (as measured by the cannabinoid-induced tetrad). The second aim seeks to answer whether Pb exposure affects edible THC self-administration and the development of THC tolerance (also measured by the tetrad). It was hypothesized that Pb exposure would decrease THC sensitivity (Aim 1), would enhance THC self-administration (Aim 2), enhance the development of THC tolerance (Aim 2), and finally that sex-dependent effects of Pb-exposure and THC would be observed (Aims 1 & 2). These hypotheses ended up not being supported, but Aim 1 produced findings indicating that THC sensitivity was increased by Pb exposure, but only in female mice. Future research will hopefully be able to fully explore the implications of these findings.

The etiology of many gestational disorders is still unknown. However, insufficient trans-placental passage of nutrients and wastes due to poor placentation is characteristic of several pathologies and may be due, in part, to altered function of placental mitochondria. Mitochondrial activity is essential in pregnancy because it sustains the metabolic activity of the placenta throughout gestation. Exposure to stressors that perturb processes governing placentation, including maternal drug use, can negatively impact fetal development. Cannabis use is prevalent during pregnancy. The psychoactive constituent, delta-9-tetrahydrocannbinol (THC), can cross the placenta to affect placental and fetal physiology. Importantly, cannabinoid receptors have been reported on trophoblast cells, and on mitochondria which are abundant in placentae. It has been reported that THC may target the mitochondria in various tissue types, including placental tissue, and alter its function. However, few studies have addressed the physiological control of mitochondria within the placenta, an organ that is critical for fetal growth and pregnancy maintenance. I investigated the role of mitochondria in trophoblast differentiation and syncytialization using rotenone, a complex I inhibitor. Subsequently, I investigated the role of THC on two important aspects of placentation – invasion and syncytialization – using placental trophoblast cells HTR8/SVneo and BeWo, respectively. In response to rotenone and THC, there was increased ROS production, oxidative stress, and altered transcriptional markers favouring mitochondrial fragmentation. Treatment with 20µM THC for 48 hours led to reduced mitochondrial respiration, ATP production and loss of mitochondrial membrane polarity. Critically, these THC-induced mitochondrial changes occurred concomitant with evidence of reduced trophoblast invasion and syncytialization. Furthermore, THC exposure reduced levels of human chorionic gonadotropin, human placental lactogen and insulin-like growth factor 2, which are growth factors necessary for fetal development. Placental mitochondrial dysfunction, particularly when THC-induced, may be critical in a range of gestational disorders which have important implications for maternal and fetal/offspring health. / Dissertation / Doctor of Philosophy (Medical Science) / Cannabis is commonly used by pregnant women. Fetal exposure to cannabis and its components can impair fetal growth and neurological development. These negative fetal outcomes may be the result of poor placental formation, due to placental cell exposure to cannabis and its psychoactive component, delta-9-tetrahydrocannabinol (THC). Importantly, THC can also target intracellular organelles, like the mitochondria which are known as the “powerhouses” of the cell. Few studies have investigated the direct effects of THC on placental development. The purpose of this study was to determine how THC exposure to placental cells may alter their function. We found that THC impaired processes that allow placental attachment to the uterus and form a protective barrier, and compromised mitochondrial function, which are important for placental formation. These findings serve to inform scientists and doctors, thus stimulating the creation of new ideas and methods to further explore the impact of THC on pregnancy outcomes.

Over the approximate nine months of its intrauterine existence, the development of the fetus is supported by the human placenta. This transient organ is central to pregnancy success as it facilitates maternal-fetal exchange, immunological tolerance, and hormone production. Villous trophoblasts mediate placental formation by engaging in a continuous turnover process of proliferation, differentiation, fusion, and apoptosis. In doing so, cytotrophoblasts and syncytiotrophoblasts maintain the integrity of the outer placental lining known as the syncytium. Exposure to drugs, however, can compromise placental establishment, which can in turn adversely impact pregnancy and fetal health. Specifically, cannabis is widely used by women of reproductive age and during pregnancy. While maternal cannabis use is linked to poor outcomes such as preterm birth and neurodevelopmental delays in exposed children, the underlying mechanisms are not well-defined. First, we characterized a functionally relevant cell line to model differentiation and fusion. In a comparison of the BeWo and BeWo b30 cell lines, our findings demonstrated that both models similarly undergo fusion. We then explored the implications of exposure to delta-9- tetrahydrocannabinol (∆9-THC) on the immunological roles of villous trophoblasts. We observed that cytotrophoblast differentiation and fusion were associated with localized inflammation due to elevated interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-α) but inhibited interleukin-4 (IL-4) and interleukin-10 (IL-10) production. ∆9-THC exposure impaired this T helper 1/2 cytokine balance through decreased IL-2 and TNF-α as well as increased IL-4 and IL-10 levels. Subsequently, we investigated the effects of ∆9-THC in TNF-α- and IL-10-dominant environments, to represent inflammatory and immunomodulatory microenvironments, respectively. Coincident with inflammation, ∆9-THC attenuated trophoblast fusion and the biosynthesis of steroid hormones, progesterone and cortisol, through perturbed cytochrome P450 regulation. This thesis ultimately lays a foundation for understanding how cannabis use during pregnancy may compromise the fusogenic, immune and endocrine functions of villous trophoblasts in the placenta. / Thesis / Master of Science (MSc) / The human placenta is a pregnancy-specific organ that supports the health of the mother- to-be and fetus. Stem cells known as cytotrophoblasts undergo differentiation and fusion to support the establishment of the syncytium, which creates a boundary that separates the maternal and fetal circulations. In the case of cannabis consumption during pregnancy, its biologically active components can travel to the placenta, cross the syncytium, and enter fetal blood. Our primary objective was to determine how cannabis exposure can impact the formation and maintenance of the syncytium. While maternal use has been linked to short- and long-term consequences for child health, existing research lacks a complete understanding of the underlying mechanisms. We demonstrate that cannabis exposure alters the production of important immune and hormonal factors during cytotrophoblast fusion, which may play a role in mediating poor placental development. Ultimately, it is critical to explore the implications of cannabis use for female reproductive health due to a rising trend in its use.

<p>Environmental exposure to lead (Pb) and cannabis use are two of the largest public health issues facing modern society in the United States and around the world. Exposure to Pb in early life has been unequivocally shown to have negative impacts on development, and recent research is mounting showing that it may also predispose individuals for risk of developing substance use disorders (SUD). At the same time, societal and legal attitudes towards cannabis (main psychoactive component delta-9-tetrahydrocannabinol) have been shifting, and many American states have legalized the recreational use of cannabis. It is also the 3^rdmost widely used drug of abuse in the US, and rates of cannabis use disorder are on the rise. This thesis sets out to establish whether there is a link between early life Pbexposure and later THC-related behavior in C57BL6/J mice, as has been demonstrated for other drugs of abuse. The first aim seeks to answer whether Pbexposure affects physiological THC sensitivity (as measured by the cannabinoid-induced tetrad). The secondaimseeks to answer whether Pbexposure affects edible THC self-administration and the development of THC tolerance (also measured by the tetrad).It was hypothesized that Pbexposure would decrease THC sensitivity (Aim 1), would enhance THC self-administration (Aim 2), enhance the development of THC tolerance (Aim 2), and finally that sex-dependent effects of Pb-exposure and THC would be observed (Aims 1 & 2). These hypotheses ended up not being supported, but Aim 1 produced findings indicating that THC sensitivity was increased by Pbexposure, but only in female mice. Future researchwill hopefully be able to fully explore the implications of these findings.</p>

This thesis describes in the form of a commentary on own original publications research on the problems of cannabinoids, ie. phytocannabinoids and some synthetic cannabinoids, their pharmacokinetics and effects. The work consists of four thematic areas: the pharmacokinetics of delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD) in rats, depending on the route of administration; THC concentration time profile in humans (after inhalation) and implications for transport safety; the pharmacokinetic profile of synthetic cannabinoids in rats; extraction and determination of phytocannabinoids in plant material. The first part of the thesis was to determine pharmacokinetic profiles of THC, CBD and combination thereof (1:1 weight ratio) in rats with respect to administration common in humans, i.e. inhalation, oral and subcutaneous administration. THC, its metabolites (11-hydroxy-tetrahydrocannabinol, 11-OH-THC; 11-nor-delta-9- carboxytetrahydrocannabinol, THCOOH) and CBD concentrations in serum and brains of animals were monitored at the 24 hours experimental interval during the study. Except for inhalation administration, co-administration of CBD inhibited THC metabolism (after both oral and subcutaneous), resulting in an increase in THC concentrations in both serum and brain of the rats relative to...

Depuis quelques décennies, la consommation de cannabis et son usage thérapeutique sont le sujet de nombreux débats. Le cannabis est la drogue illicite la plus consommée au monde et cette consommation se trouve dix fois plus élevée chez les patients atteints de schizophrénie que dans la population générale. L’hypothèse d’une automédication initialement proposée afin d’expliquer la consommation élevée de cannabis chez les patients atteints de schizophrénie est maintenant remise en question. En effet, les rapports indiquant une aggravation des symptômes plutôt qu’une amélioration suite à une consommation à long terme sont de plus en plus nombreux. Sachant que le cannabis peut induire des effets soit plaisants soit aversifs, la question se pose à savoir si une prédominance de la valence motivationnelle positive ou une diminution de la valence négative du cannabis peut expliquer la consommation élevée parmi les individus ayant un diagnostic de schizophrénie? Bien qu’un grand nombre de recherches pré-cliniques aient été menées chez l’animal normal pour évaluer l’effet motivationnel du Δ9-tétrahydrocannabinol (THC) et autres cannabinoïdes synthétiques, aucune n’a abordé cette problématique dans un modèle animal de la schizophrénie. Cette lacune nous a donc amené à étudier la valence motivationnelle du THC et de l’agoniste cannabinoïde WIN55,212-2 (WIN) dans un modèle animal de la schizophrénie: la lésion néonatale de l’hippocampe ventral (NVHL). Dans le premier article, nous présentons les résultats de quatre expériences. Une première avait pour objectif de déterminer si la procédure expérimentale que nous avons utilisée permettait de reproduire des signes distinctifs du modèle animal de la schizophrénie. Par la suite, nous avons évalué i) l’effet d’une dose de WIN sur l’activité locomotrice spontanée et ii) la valence motivationnelle du THC (0.5 mg/kg, i.p) et du WIN (1 mg/kg, i.p) chez les rats adolescents (jour post-natal 28-40, PD28-40) et adultes (PD56) au moyen du paradigme de préférence de place conditionnée (PPC). Tel qu’attendu, la réponse locomotrice à l’amphétamine (0.75 et 1.5 mg/kg) chez les rats NVHL adultes était supérieure à celle des rats contrôles (test distinctif du modèle). Le THC a induit une tendance aversive chez les rats contrôles adultes. Enfin, le WIN a stimulé l’activité locomotrice et induit une aversion significative chez les rats adultes NVHL. Dans un deuxième article, nous avons évalué la valence motivationnelle du THC (0.5 mg/kg), du WIN (1 et 3 mg/kg) et l’effet de l’amphétamine au moyen du paradigme d’autostimulation électrique intracérébrale (ASI). Les résultats montrent que : i) l’effet amplificateur de l’amphétamine sur l’ASI était de plus courte durée chez les rats NVHL; ii) le THC produit une légère atténuation de la récompense chez les rats contrôles tandis que le WIN a produit une atténuation plus prononcée de la récompense chez les rats NVHL, un effet qui a été bloqué par l’antagoniste aux récepteurs CB1, le AM251 (3 mg/kg). Pour la première fois les résultats suggèrent une altération du système endocannabinoïde dans un modèle animal de la schizophrénie. Ils indiquent qu’une exposition aigüe conduit à une prédominance de la valence négative. Bien qu’en apparente contradiction avec les études cliniques, ces résultats soulignent l’importance du contexte socio-environnemental pour expliquer les effets du cannabis chez les patients. De plus ils encouragent les futures études à évaluer cette valence sur un modèle d’exposition chronique. / Over the past few decades, the cannabis consumption and its therapeutic use have been the subject of many debates. Cannabis is the most widely used illicit drug and among patients with a diagnosis of schizophrenia, its consumption is ten times higher than in the general population. The self-medication hypothesis that has been initially proposed to account for the co-morbidity schizophrenia – cannabis is now questioned on the basis of several reports showing that long term cannabis consumption worsen schizophrenia symptoms in patients. Knowing that cannabis can provoke both rewarding and aversive effects in human and in animal, the following question can be raised: can co-morbidity schizophrenia – cannabis be explained by a salient positive or a blunted negative motivational valence of cannabis? Even though many pre-clinical studies have been carried out in normal animals on the motivational effects of Δ9-Tetrahydrocannabinol (THC) or other synthetic cannabinoids, none has measured these effects in an animal model of schizophrenia. On the basis of this, we undertook a series of studies on the motivational valence of THC and the cannabinoid agonist WIN55,212-2 (WIN) in an animal model of schizophrenia : the neonatal ventral hippocampus lesion (NVHL). In the first report, we present the results of four studies. The first was aimed at showing that the experimental procedures that we used reproduced some abnormal features of the animal model. Then we evaluated i) the effect of WIN (1 mg/kg) on spontaneous locomotor activity and ii) the motivational valence of THC (0.5 mg/kg) and WIN (1 mg/kg) in the young (post-natal day 28-40, PD28-40) and adult (PD56) rats with the conditioned place-preference paradigm (CPP). As expected, amphetamine produced a higher locomotor activity in NVHL rats, an effect observed at PD56 and not at PD35 (NVHL usual test). THC tended to induce an aversion in control rats at PD56 while WIN produced a significant aversion at PD56 in NVHL rats only. We also assessed, in a second report, the valence of THC (0.5 mg/kg) and WIN (1 and 3 mg/kg), and amphetamine (0.75 mg/kg) using the brain stimulation reward paradigm. Results show that i) the enhancement effect of amphetamine on reward was shorter in adult NVHL rats; ii) THC induced a weak reward attenuation in control rats while WIN produced a marked dose-dependent attenuation in NVHL rats; this effect of WIN was blocked by AM251 (3 mg/kg), an antagonist at CB1 receptors. For the first time, these results suggest that the endogenous cannabinoid system is altered in this animal model of schizophrenia. They indicate that an acute exposure leads to a predominance of negative valence. Even if this seems contradictory with clinical studies, these results highlight the interconnection between the drug and the socio-environment aspects. In addition, they encourage future studies to evaluate this valence on a chronic exposure paradigm with this animal model of schizophrenia.