<p>Lead (Pb) is one of the most harmful and most abundant neurotoxins in the environment. Despite the extensive movement made to eradicate toxic levels of Pb in the environment, children, predominately in lower socioeconomic areas, are still exposed to varying levels of Pb. Human studies suggest that Pb exposure leads to altered drug consumption in adults by altering underlying neural mechanisms, specifically dopamine (DA) activity. However, there is limited research on this at blood Pb levels below 10 μg/dL, levels often seen in children growing up in neighborhoods located in old industrial and urban areas. To model how early-life low-level Pb exposure effects DA-dependent behaviors associated with addiction in adulthood, we used C57BL/6J mice. Litters were weaned at PND 21 and assigned to either a three-week history of 30 parts per million (ppm) Lead (IV) Acetate exposure or a control condition of 0 ppm Pb in DI drinking water. After the Pb exposure period, mice were switched to regular tap water until they reached adulthood. Afterward, separate animals were tested in one of three experiments: two-bottle choice alcohol preference drinking, alcohol-induced behavioral sensitization (EBS), and cocaine-induced behavioral sensitization (CBS). In experiment 1, our hypothesis was met, and both male and female mice with a prior Pb exposure displayed significantly higher alcohol intake and preference scores over the three-week period than control mice. In experiment 2, there were no differences in EBS and no evidence of EBS in any of the groups. However, there was an increased acute response to 2.0 g/kg EtOH in the Pb-exposed chronic group as compared to the control animals. Lastly, in experiment 3, Pb-exposed animals in the chronic cocaine group were more sensitive to the effects of cocaine (10 mg/kg) across days than the controls, both the acute cocaine groups and both saline control groups. Thus, with these experiments, we concluded that low levels of developmental Pb exposure might be targeting DA in the reward pathway, which is essential for alcohol intake and drug sensitization.</p>

Kiso H, Takahashi K, Saito K, Togo Y, Tsukamoto H, et al. (2014) Interactions between BMP-7 and USAG-1 (Uterine Sensitization-Associated Gene-1) Regulate Supernumerary Organ Formations. PLoS ONE 9(5): e96938. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18545号 / 医博第3938号 / 新制||医||1006(附属図書館) / 31445 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 戸口田 淳也, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background/Aims: Neonatal quinpirole (NQ) treatment to rats increases dopamine D2 (DAD2) receptor sensitivity in adult animals. We investigated if increased DAD2 sensitivity would be passed to the next (F1) generation, and if these animals demonstrated sensorimotor gating deficits and enhanced behavioral responses to nicotine. Methods: Male and female rats were intraperitoneal (IP) administered quinpirole (1 mg/kg) or saline (NS) from postnatal day (P)1–21. Animals were either behaviorally tested (F0) or raised to P60 and mated, creating F1 offspring. Results: Experiment 1 revealed that F1 generation animals that were the offspring of at least one NQ-treated founder increased yawning behavior, a DAD2-mediated behavioral event, in response to acute quinpirole (0.1 mg/kg). F1 generation rats also demonstrated increased striatal β arrestin-2 and decreased phospho-AKT signaling, consistent with increased G-protein independent DAD2 signaling, which was equal to F0 NQ-treated founders, although this was not observed in all groups. RNA-Seq analysis revealed significant gene expression changes in the F1 generation that were offspring of both NQ-treated founders compared to F0 NQ founders and controls, with enrichment in sensitivity to stress hormones and cell signaling pathways. In Experiment 2, all F1 generation offspring demonstrated sensorimotor gating deficits compared to controls, which were equivalent to F0 NQ-treated founders. In Experiment 3, all F1 generation animals demonstrated enhanced nicotine behavioral sensitization and nucleus accumbens (NAcc) brain-derived neurotrophic factor (BDNF) protein. Further, F1 generation rats demonstrated enhanced adolescent nicotine conditioned place preference equivalent to NQ-treated founders conditioned with nicotine. Conclusions: This represents the first demonstration of transgenerational effects of increased DAD2 sensitivity in a rodent model.

La dermatite de contact allergique (DCA) est une réaction exacerbée du système immunitaire cutané vis-à-vis d’un allergène de contact. La prévalence de la DCA étant de 20 % au sein de la population mondiale, il est important d’identifier les composés allergisants. Différentes réglementations européennes, telles que le règlement REACh ou la directive cosmétique, interdisent l’utilisation de test sur l’animal. C’est dans ce contexte que différentes méthodes alternatives ont été développées pour évaluer la sensibilisation cutanée. La stratégie actuelle d’évaluation du potentiel sensibilisant consiste à réaliser un ensemble de tests alternatifs, chacun mimant un évènement clé du mécanisme : l’hapténisation, l’activation des kératinocytes ou des cellules dendritiques.Cependant, ces tests utilisent principalement des monocultures et ne prennent donc pas en compte les interactions cellulaires qui peuvent avoir lieu in vivo. De plus, les évaluations de la pénétration et du métabolisme cutanés sont négligées dans les tests développés.Afin de mimer la fine orchestration des événements intervenant lors de la sensibilisation cutanée, nous proposons un modèle d’épiderme humain reconstruit (RhE) co-cultivé avec la lignée cellulaire THP-1, servant de substitut aux cellules dendritiques. Nous avons caractérisé, et étudié la pertinence de ce modèle à l’aide de molécules chimiques de référence. Ce travail a permis l’identification de biomarqueurs, tels que CD54, IL-8 et CCL3, spécifiques à l’évaluation in vitro de la sensibilisation cutanée des xénobiotiques. / Allergic contact dermatitis is an exacerbated reaction of skin immune system toward contact allergen. The prevalence of DCA being 20 % among the world population, it is important to identify allergens. Different European regulations such as the REACh regulation or the cosmetic directive prohibit the use of the test on animals. It is in this context that different methods have been developed to evaluate skin sensitization. The current sensitization potential assessment strategy consists of a set of alternative tests, each of which reproduce a key event of the mechanism: the haptenation, the activation of keratinocytes or dendritic cells.However, these tests are mainly based on monocultures and therefore do not account for the cellular crosstalk that happen in vivo. In addition, the evaluations of skin penetration and metabolism are neglected in the developed tests.In order to mimic the fine orchestration of the events involved in skin sensitization, we propose a model of reconstructed human epidermis (RhE) co-cultivated with the THP-1 cell line, as a substitute for dendritic cells. We have characterized and studied the relevance of this model using reference chemical molecules. This work has enabled the identification of biomarkers, such as CD54, IL-8 and CCL3, specific to the in vitro evaluation of skin sensitization to xenobiotics.

Les allergies représentent un problème majeur dans le domaine des maladies professionnelles et ont un impact sérieux sur la vie des travailleurs. Les allergies professionnelles sont principalement cutanées et respiratoires ; elles peuvent être causées par des produits chimiques de bas poids moléculaire. Dans le passé, les tests destinés à identifier les produits susceptibles d’entraîner des allergies étaient réalisés sur l’animal. Or, la législation européenne engage à limiter le recours à l’expérimentation animale pour évaluer le pouvoir sensibilisant des substances chimiques, incitant à développer des tests in vitro de substitution. C’est dans ce contexte que nous avons cherché à développer des modèles de cultures cellulaires destinés à identifier les substances sensibilisantes. Un premier modèle utilisant des cellules dendritiques dérivées de moelle osseuse (BMDC) de souris BALB/c a été développé et a donné des résultats prometteurs pour l’identification des produits sensibilisants et leur catégorisation selon leur puissance sensibilisante. De plus, la voie de signalisation Nrf2/Keap1 semble être impliquée dans la réponse de ce modèle cellulaire aux sensibilisants. Dans le but de compléter ce modèle et d’évaluer la capacité des BMDC à activer les lymphocytes T (LT), un modèle de coculture de BMDC et LT a été mis au point avec un sensibilisant de référence avant d’être testé sur un ensemble de produits de référence (sensibilisants cutanés et respiratoires, irritants et non sensibilisants). Les BMDC de notre modèle, exposées à des sensibilisants, se sont révélées capables d’activer les LT en coculture. Enfin, des essais préliminaires utilisant des cellules de souris de souche C57BL6/J dans notre modèle de coculture ont donné des résultats comparables à ceux obtenus avec des cellules issues de la souche BALB/c. Les modèles de cultures cellulaires BMDC et de coculture BMDC-LT sont prometteurs dans le cadre du développement de méthodes de substitution à l’expérimentation animale pour l’évaluation du pouvoir sensibilisant de substances chimiques / Allergies constitute an important issue in the field of occupational health and have a serious impact on the lives of workers. Occupational allergies are mainly contact and respiratory allergies and can be caused by low molecular weight chemicals. In the past, the tests that were used to identify the potential allergens were carried out on animals. However, European legislation has provided the impetus for reducing the use of animal testing to assess the sensitization potential of chemicals and promoted the development of alternative in vitro tests. In this context, we aimed to develop cell culture models to identify sensitizers. A first model using bone marrow derived dendritic cells (BMDC) from BALB/c mice was developed and showed promising results for identifying sensitizers and classify them according to their allergenic potency. Moreover, the Nrf2/Keap1 pathway seems to be involved in the response of this cell model to sensitizers. In order to supplement this model and to assess the functionality of BMDC, a BMDC-T cell (TC) coculture model was developed with a reference sensitizer before being tested on a range of reference sensitizers (cutaneous and respiratory sensitizers, irritants and non-sensitizers). The BMDC of our model, while exposed to sensitizers, were able to activate TC in coculture. Finally, preliminary tests using the cells of C57BL6/J mice in our coculture model showed that similar results to those obtained with cells from the BALB/c strain. The models of BMDC cultures and BMDC-TC coculture are promising for the development of alternative methods to animal experimentation assessing the sensitizing potential of chemicals

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18593号 / 工博第3954号 / 新制||工||1608(附属図書館) / 31493 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 伊藤 紳三郎, 教授 木村 俊作, 教授 辻井 敬亘 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are peptides implicated in the inflammatory response. They are released in increased amounts during inflammation and induce thermal hyperalgesia. Whether these molecules directly affect the sensitivity of primary nociceptive sensory neurons is unknown. This information could provide a link between increased inflammation-induced release of GFLs and their ability to promote inflammatory hyperalgesia. These molecules bind to one of four GFRα receptor subtypes, and this GFL-GFRα complex often translocates to the receptor tyrosine kinase, Ret. The focus of this dissertation was to determine whether GFLs modulate the stimulated-release of calcitonin gene-related peptide (CGRP). Isolated sensory neurons and freshly dissociated spinal cord tissue were used to examine the enhancement in stimulated-release of CGRP, a measure of sensitization. Exposure of isolated sensory neurons to GDNF, neurturin, and artemin, enhanced the capsaicin stimulated-release of immunoreactive CGRP (iCGRP). Sensitization by GFLs occurred in freshly dissociated spinal cord tissue. Persephin, another member of the GFL family, did not enhance stimulated-release of iCGRP. These results demonstrate that specific GFLs are mediators of neuronal sensitivity. The intracellular signaling pathways responsible for this sensitization were also evaluated. Inhibition of the mitogen activated protein kinase (MAPK)/extracellular signal-related kinase 1/2 (Erk 1/2) pathway selectively abolished the enhancement of CGRP release by GDNF. NTN-induced sensitization was abolished by inhibition of the phosphatidylinositol-3-kinase (PI-3K) pathway. Reduction in Ret abolished the GDNF-induced sensitization, but did not fully inhibit NTN or ART-induced sensitization. Inhibition of other cell surface receptors (neural cell adhesion molecule (NCAM), and Integrin β-1) had distinct effects on the sensitization capability of each of the GFLs. Ret and NCAM inhibition in combination abolished ART-induced sensitization. It was necessary to inhibit Ret, NCAM, and Integrin β-1 to prevent the NTN-induced sensitization. These data demonstrate that the GFLs use distinct signaling mechanisms to induce the sensitization of nociceptive sensory neurons. The work presented in this thesis provides the first evidence for these novel and distinct Ret-independent pathways for GFL-induced actions and provides insight into the mechanism of sensory neuronal sensitization in general.

Indiana University-Purdue University Indianapolis (IUPUI) / Sensitization to the locomotor stimulant effects of alcohol (ethanol) is thought to be a heritable risk factor for the development of alcoholism that reflects progressive increases in the positive motivational effects of this substance. However, very little is known about the genetic influences involved in this phenomenon or the extent to which ethanol’s positive motivational effects are altered in parallel to its development. The first goal of this work was to determine the heritability of ethanol-induced locomotor sensitization in mice using short-term behavioral selection. Genetically heterogeneous C57BL/6J (B6) x DBA/2J (D2) F2 mice were generated from B6D2F1 progenitors, phenotyped for the expression of locomotor sensitization, and bred for high (HLS) and low (LLS) expression of this behavior. A secondary goal was to characterize possible line differences in ethanol’s positive motivational effects using a conditioned place preference assay. There were large and significant differences in locomotor sensitization between HLS and LLS lines by the fourth generation. Twenty-two percent of the observed line difference(s) were attributable to genes (h2=.22). However, there were no significant differences in conditioned place preference between lines despite significant line differences in ethanol-stimulated locomotion following repeated exposures. The results of this work have several implications. First, that changes in ethanol sensitivity following repeated exposures are in part genetically regulated highlights the relevance of studies aimed at determining how genes regulate susceptibility to ethanol-induced behavioral and neural adaptations. Additionally, the lack of line differences in ethanol-induced CPP, and the observation that CPP and ethanol sensitization are dissociable, suggests that 1) different genes regulate these two behaviors and 2) the utility of locomotor sensitization as a model of alterations in ethanol’s positive motivational effects is, at best, still unclear. Together these studies provide evidence that genes are capable of regulating alterations in ethanol-induced locomotor behavior but provide little support for ethanol-induced locomotor sensitization as a model for increases in ethanol’s positive subjective effects in mice.

Neonatal administration of quinpirole produces significant increases in D2 receptor sensitivity that persists into adulthood. This phenomenon, known as D2 receptor priming, is consistent with pathology in schizophrenia. Rats were administered quinpirole or saline postnatally and raised to adulthood. In adulthood, rats were administered d-amphetamine sulfate or saline every other day and were placed in a locomotor arena where activity was measured over 7 trials. Results showed that D2-primed rats receiving amphetamine were higher in locomotor activity across all days of testing compared to other groups. This effect was more prominent in males than in females. After sensitization, cerebrospinal fluid was taken via microdialysis from the nucleus accumbens core and was analyzed for dopamine content. Analysis revealed D2 priming produced a 300% increase of dopamine release in the nucleus accumbens core in response to amphetamine compared to controls. These results suggest that increases in D2 sensitivity may lead to increased reaction to amphetamine in psychotic individuals.

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