雖然焦慮是一種普遍存在之情感性心智活動，迄今仍無充份解釋之實證資料。本研究主要是利用一種焦慮症相關的動物模式，即抬高式T形迷津，探討與焦慮症有關的神經行為機制。整部研究分兩大實驗，分別探討抬高式T形迷津的行為建構動力與破壞依核次級區域在抬高式T形迷津或其他焦慮作業上之行為表現。在實驗一檢驗抬高式T形迷津的行為內涵方面，共有四個實驗：實驗一A探討抬高式T形迷津抑制性躲避行為是否呈現消除現象；實驗一B探討破壞制約害怕神經網路對抬高式T形迷津之抑制性躲避行為的影響，並檢測自發性運動量的改變是否造成干擾效果；實驗一C探討事前暴露經驗對脫逃行為的意義；實驗一D檢測脫逃及抑制性躲避實驗程序互相干擾之可能性。實驗二探討可能涉及抬高式T形迷津或其他焦慮作業的神經機制，針對破壞依核次級區域對焦慮行為的影響進行檢測。此部分包含三個實驗，實驗二A探討依核次級區域受損對傳統焦慮動物模式抬高式十字迷津行為的影響；實驗二B採用已在實驗一建立行為效度的抬高式T形迷津，檢驗破壞依核次級區域後的迷津行為表現，並檢驗依核次級區域受損是否影響受試自發性運動量變化，以致干擾抬高式T形迷津的行為表現。另為深入探討依核的功能角色，實驗二C利用其他嫌惡作業測試破壞依核次級區域對制約躲避電擊行為的影響。實驗一結果顯示抑制性躲避行為是一包含制約害怕及探索行為等多重歷程的行為模式，而脫逃行為對情緒狀態的改變不敏感，且易受抑制性躲避作業的影響。實驗二發現破壞依核殼區同時減抑受試在抬高式十字迷津的危機評估行為、抬高式T形迷津之抑制性躲避行為及制約躲避電擊行為；而破壞依核核區的減抑效果僅見於抬高式T形迷津與制約躲避電擊作業。三個嫌惡作業的結果顯示依核核區與殼區皆涉及制約害怕歷程，但兩區的受損會表現不同焦慮行為，並在抬高式十字迷津之危機評估行為中表現出來。綜合上述二大部分實驗結果，本研究對抬高式T形迷津的行為內涵有更進一步的瞭解，並特別藉依核次級區域破壞的行為測試資料，推估中腦多巴胺系統與傳統理論所指邊緣系統在實證性解釋焦慮具同樣關鍵角色。 / Although anxiety is a well-recognized affective mental reaction, its phenomenon is not fully characterized by the empirical data. By employing a recently developed animal model named the elevated T maze (ETM), the present study investigated the neurobehavioral mechanisms of anxiety. There were two major parts of experiments designed to respectively examine the validity of this task and the involvement of limbic related areas on anxious behavior. Regarding the first part of experiments, Experiment 1A examined the effects of extinction on the inhibitory avoidance of ETM; Experiment 1B evaluated the lesions of six limbic related areas on the measures of inhibitory avoidance and escape; Experiment 1C investigated how pre-exposure experience of stress would affect the ETM behavior; Experiment 1D tested the potential affectiveness derived from different sequences of the test procedure on EMT. The second part of experiments mainly focused on comparing the lesion effects of nucleus accumbens subareas (core and shell) on behavioral measures from three anxiety-related tasks. Elevated plus maze, ETM, and active avoidance were adopted respectively in the experiments of 2A, 2B, and 2C. Results of the first part of experiments indicated 1) inhibitory avoidance of ETM containing fear conditioning and exploration components, and 2) less sensitivity to experimental manipulation for the escape of ETM. In the second part of experiments, the shell lesion significant attenuated the risk assessment behavior of elevated plus maze and inhibitory avoidance of ETM and active avoidance tasks, whereas the core lesion only produced the latter part of impairment. Both core and shell subareas are thus inferred to be involved in the conditioned avoidance, and lesions of these two areas may exert different patterns of anxious behavior. Together, the present study further characterized behavioral components of ETM. With a more systemic work in comparing lesion data of nucleus accumbens over three anxiety-related tasks, it is then suggested that the midbrain dopamine system is as crucial as the traditionally-known limbic system the traditional in terms of providing empirical explanation for the anxiety.

The central theme of development, how cells are organized into functional structures and assembled into whole organisms, is addressed by developmental biology. One important feature of embryonic development is pattern formation, which is the generation of a particular arrangement of cells in three-dimensional space at a given point of time. Central to this work is the model system of the zebrafish, Danio rerio. The aim of the first part of this study was to try to understand how a distinct part of the embryonic brain called midbrain-hindbrain boundary (MHB), a region that acts as an organizer for the adjacent brain regions, is established in vertebrates. spiel-ohne-grenzen (spg) is one mutant which interferes with MHB development. Here, I addressed the role of pou2 in brain development by molecular, phenotypical and functional analysis. By genetic complementation and mapping I could elucidate the molecular nature of this mutant and found that the pou2 gene encoding the POU domain transcription factor is affected in spg mutant embryos. By chromosomal syntenic conservation, phylogenetic sequence comparison, and expression and functional data I imply that pou2 is the orthologue of the mammalian Oct4 (Pou5F1) gene. I find by detailed expression and transplantation analysis that pou2 is cell autonomously required within the neuroectoderm to activate genes of the MHB and hindbrain primordium, like pax2.1, wnt1, gbx2 or krox20. By gain-of-function experiments I demonstrate that pou2 synergizes with Fgf8 signaling in order to activate particularly the hindbrain primordium. Since pou2 is already provided to the embryo by the mother, I generated embryos which lack maternal and zygotic pou2 function (MZspg) to reveal a possible earlier than neuroectodermal role of pou2. In the second part of this work I demonstrate that pou2 is a key factor controlling endoderm differentiation. By expression and gain-of-function analysis I suggest a cell autonomous function for Pou2 in the first step of endodermal differentiation. By gain-of-function experiments involving the gene encoding the HMG transcription factor Casanova (Cas) I show that both Cas and Pou2 are necessary to activate expression of the endodermal differentiation marker sox17 in a mutually dependent way, and that the ability of Cas to ectopically induce sox17 strictly requires Pou2. I conclude that both maternal and zygotic pou2 function is necessary for commitment of endodermal progenitor cells to differentiate into endodermal precursor cells.

The neuromeric concept of brain formation has become a well-established model to explain how order is created in the developing vertebrate central nervous system. The most important feature of neuromeres is their compartmentalization on the cellular level: Each neuromere comprises a lineage-restricted population of cells that does not intermingle with cells from neighboring compartments. The units of the vertebrate hindbrain, the rhombomeres, serve as the best-studied examples of neuromeres. Here, the lineage restriction mechanism has been found to function on the basis of differentially expressed adhesion molecules. To date, hard evidence for the existence of other lineage restricted regions in more anterior parts of the brain is still scarce. The focus of this study is the midbrain-hindbrain boundary (mhb) region, where the juxtaposition of the mesencephalon and metencephalon gives rise to a signaling center, termed the midbrain-hindbrain or isthmic organizer. Evidence for lineage restriction boundaries in the mhb region is still controversial, with some very recent studies supporting the existence of a lineage boundary between the mesencephalon and metencephalon and others rejecting this. Here, I present data strongly supporting the existence of a compartment boundary between the posterior midbrain and anterior hindbrain territory. I base this proposition on cell-tracing experiments with single cell resolution. By connecting the traces to a molecular midbrain marker, I establish a link between cell fate and behavior. In the second part, I present a novel tissue explant method for the zebrafish that has the potential to serve numerous developmental studies, especially imaging of so far inaccessible regions of the embryo.

Why is it that only some people who use drugs actually become addicted? In fact, addiction depends on a complicated process involving a confluence of risk factors related to biology, cognition, behaviour, and personality. Notably, all addictive drugs act on a neural system for reinforcement learning called the midbrain dopamine system, which projects to and regulates the brain's system for cognitive control, called frontal cortex and basal ganglia. Further, the development and expression of the dopamine system is determined in part by genetic factors that vary across individuals such that dopamine related genes are partly responsible for addiction-proneness. Taken together, these observations suggest that the cognitive and behavioral impairments associated with substance abuse result from the impact of disrupted dopamine signals on frontal brain areas involved in cognitive control: By acting on the abnormal reinforcement learning system of the genetically vulnerable, addictive drugs hijack the control system to reinforce maladaptive drug-taking behaviors. The goal of this research was to investigate this hypothesis by conducting a series of experiments that assayed the integrity of the dopamine system and its neural targets involved in cognitive control and decision making in young adults using a combination of electrophysiological, behavioral, and genetic assays together with surveys of substance use and personality. First, this research demonstrated that substance dependent individuals produce an abnormal Reward-positivity, an electrophysiological measure of a cortical mechanism for dopamine-dependent reward processing and cognitive control, and behaved abnormally on a decision making task that is diagnostic of dopamine dysfunction. Second, several dopamine-related neural pathways underlying individual differences in substance dependence were identified and modeled, providing a theoretical framework for bridging the gap between genes and behavior in drug addiction. Third, the neural mechanisms that underlie individual differences in decision making function and dysfunction were identified, revealing possible risk factors in the decision making system. In sum, these results illustrate how future interventions might be individually tailored for specific genetic, cognitive and personality profiles. / Graduate

The neuromeric concept of brain formation has become a well-established model to explain how order is created in the developing vertebrate central nervous system. The most important feature of neuromeres is their compartmentalization on the cellular level: Each neuromere comprises a lineage-restricted population of cells that does not intermingle with cells from neighboring compartments. The units of the vertebrate hindbrain, the rhombomeres, serve as the best-studied examples of neuromeres. Here, the lineage restriction mechanism has been found to function on the basis of differentially expressed adhesion molecules. To date, hard evidence for the existence of other lineage restricted regions in more anterior parts of the brain is still scarce. The focus of this study is the midbrain-hindbrain boundary (mhb) region, where the juxtaposition of the mesencephalon and metencephalon gives rise to a signaling center, termed the midbrain-hindbrain or isthmic organizer. Evidence for lineage restriction boundaries in the mhb region is still controversial, with some very recent studies supporting the existence of a lineage boundary between the mesencephalon and metencephalon and others rejecting this. Here, I present data strongly supporting the existence of a compartment boundary between the posterior midbrain and anterior hindbrain territory. I base this proposition on cell-tracing experiments with single cell resolution. By connecting the traces to a molecular midbrain marker, I establish a link between cell fate and behavior. In the second part, I present a novel tissue explant method for the zebrafish that has the potential to serve numerous developmental studies, especially imaging of so far inaccessible regions of the embryo.

Les neurones dopaminergiques (DAergiques) libèrent non seulement de la DA à partir de leurs terminaisons axonales, mais également dans le mésencéphale au niveau de la substance noire (SN) et l’aire tegmentaire ventrale (ATV). À cet endroit, un mécanisme de libération somatodendritique (STD) de DA a été proposé et impliquerait des senseurs calciques différents de ceux retrouvés du côté axonal. Au niveau axonal, la synaptotagmine 1 (Syt1) est une protéine essentielle à la libération rapide de DA. Toutefois, des études de notre laboratoire sur des knockout conditionnels (cKO) de Syt1 dans les neurones DA démontrent une diminution substantielle de la libération de DA au niveau axonal, mais aussi dans le mésencéphale. Une première hypothèse expliquant cette diminution dans le mésencéphale serait que Syt1 est impliquée dans la libération STD. Cependant, nous observons par microscopie à super-résolution que Syt1 ne se retrouve pas dans le compartiment STD des neurones DAergiques. Une autre possibilité serait la présence de collatérales axonales DAergiques dans le mésencéphale. Par imagerie confocale et électronique, nous observons que le mésencéphale contient plusieurs varicosités axonales asynaptiques et quelques varicosités axonales synaptiques. Enfin, nous avons évalué la plasticité des collatérales axonales DAergiques dans un modèle de lésion partielle des neurones DAergiques induite par la 6-OHDA. Malgré la perte de plus de 40% des neurones DA, la libération de DA dans la SN persiste 14 jours après lésion et s’accompagne d’une augmentation de l’expression axonale de Syt1, suggérant qu’un mécanisme de compensation axonale contribue à la résilience de la libération de DA. / Dopaminergic (DA) neurons not only exhibit a classic vesicular release from their axons in the striatum, but they also release DA in the midbrain in the substantia nigra (SN) and ventral tegmental area (VTA). In this region, somatodendritic (STD) release occurs and it requires different calcium sensors than those found in the axons. Of interest, synaptotagmin 1 (Syt1) has been shown to be implicated in fast DA release in the axons. However, recent research in our lab shows that in mice with conditional knockout (cKO) of Syt1 in DA neurons, there is a substantial decrease of DA release not only in the striatum, but also in the midbrain. Our first hypothesis is that Syt1 is directly involved in STD release. With super-resolution microscopy, we concluded that Syt1 is not localized in the STD compartment of DA neurons. This brings us to our second hypothesis, where local DA axon collaterals contribute to DA release in the midbrain. Through confocal and electron microscopy, we observed that the midbrain contains asynaptic varicosities as well as local DA synapses. In light of these results, we explored the contribution of axonal release to the resilience of SN DA release in a partial 6-OHDA lesion model. We observed that, following a loss of more than 40% of DA neurons, DA release in the SNc persists 14 days after lesion and that this is accompanied by an increase in Syt1 expression in DA axons which suggest that local axonal release is increased to compensate for DA loss.

The term "pattern formation" refers to the process by which order unfolds in development. The present thesis deals with a particular aspect of molecular pattern formation during vertebrate embryogenesis. The model system in the focus of this study is the zebrafish, Danio rerio. In the early developmental phases of the zebrafish, Fibroblast growth factors (Fgfs) are involved in the molecular patterning of various tissues, including two regions of the brain, the forebrain and the midbrain-hindbrain region, affecting cellular processes as diverse as cell proliferation, differentiation, and axonal targeting. The goal of this study was to better understand the mechanisms by which Fgf signaling regulates pattern formation and embryogenesis. I addressed this question on several levels, investigating the extent of intracellular signaling (MAPK activation) relative to sources of Fgf expression, and the transcriptional responses of cells to Fgf signaling during embryogenesis. By a macroarray analysis, I identified putative transcriptional targets of Fgf signaling in late gastrulation, providing a set of molecules that are likely to act as functional players in relaying the patterning information encoded by Fgf signals. Among those are the secreted signaling molecules Chordin and Wnt8, as well as Isthmin, a novel secreted molecule that I found capable to interfere with anterior embryonic patterning. In addition, I identified two ETS domain transcription factors, Erm and Pea3, which constitute bona fide integrators of FgfR signaling. By gain- and loss-of-function studies, I demonstrate that transcript levels of erm and pea3 are tightly regulated by Fgf signaling. Detailed analysis of the expression patterns of erm and pea3 along with other Fgf target genes also provides evidence for a differential read-out of Fgf concentration in the embryo, consistent with a role of Fgf as a vertebrate morphogen. The discovery of novel molecular components downstream of Fgf receptor activity paves a way to characterize previously unknown or underestimated developmental roles of Fgfs in the molecular patterning of the forebrain, the eye and parts of the neural crest.

The term "pattern formation" refers to the process by which order unfolds in development. The present thesis deals with a particular aspect of molecular pattern formation during vertebrate embryogenesis. The model system in the focus of this study is the zebrafish, Danio rerio. In the early developmental phases of the zebrafish, Fibroblast growth factors (Fgfs) are involved in the molecular patterning of various tissues, including two regions of the brain, the forebrain and the midbrain-hindbrain region, affecting cellular processes as diverse as cell proliferation, differentiation, and axonal targeting. The goal of this study was to better understand the mechanisms by which Fgf signaling regulates pattern formation and embryogenesis. I addressed this question on several levels, investigating the extent of intracellular signaling (MAPK activation) relative to sources of Fgf expression, and the transcriptional responses of cells to Fgf signaling during embryogenesis. By a macroarray analysis, I identified putative transcriptional targets of Fgf signaling in late gastrulation, providing a set of molecules that are likely to act as functional players in relaying the patterning information encoded by Fgf signals. Among those are the secreted signaling molecules Chordin and Wnt8, as well as Isthmin, a novel secreted molecule that I found capable to interfere with anterior embryonic patterning. In addition, I identified two ETS domain transcription factors, Erm and Pea3, which constitute bona fide integrators of FgfR signaling. By gain- and loss-of-function studies, I demonstrate that transcript levels of erm and pea3 are tightly regulated by Fgf signaling. Detailed analysis of the expression patterns of erm and pea3 along with other Fgf target genes also provides evidence for a differential read-out of Fgf concentration in the embryo, consistent with a role of Fgf as a vertebrate morphogen. The discovery of novel molecular components downstream of Fgf receptor activity paves a way to characterize previously unknown or underestimated developmental roles of Fgfs in the molecular patterning of the forebrain, the eye and parts of the neural crest.

The central theme of development, how cells are organized into functional structures and assembled into whole organisms, is addressed by developmental biology. One important feature of embryonic development is pattern formation, which is the generation of a particular arrangement of cells in three-dimensional space at a given point of time. Central to this work is the model system of the zebrafish, Danio rerio. The aim of the first part of this study was to try to understand how a distinct part of the embryonic brain called midbrain-hindbrain boundary (MHB), a region that acts as an organizer for the adjacent brain regions, is established in vertebrates. spiel-ohne-grenzen (spg) is one mutant which interferes with MHB development. Here, I addressed the role of pou2 in brain development by molecular, phenotypical and functional analysis. By genetic complementation and mapping I could elucidate the molecular nature of this mutant and found that the pou2 gene encoding the POU domain transcription factor is affected in spg mutant embryos. By chromosomal syntenic conservation, phylogenetic sequence comparison, and expression and functional data I imply that pou2 is the orthologue of the mammalian Oct4 (Pou5F1) gene. I find by detailed expression and transplantation analysis that pou2 is cell autonomously required within the neuroectoderm to activate genes of the MHB and hindbrain primordium, like pax2.1, wnt1, gbx2 or krox20. By gain-of-function experiments I demonstrate that pou2 synergizes with Fgf8 signaling in order to activate particularly the hindbrain primordium. Since pou2 is already provided to the embryo by the mother, I generated embryos which lack maternal and zygotic pou2 function (MZspg) to reveal a possible earlier than neuroectodermal role of pou2. In the second part of this work I demonstrate that pou2 is a key factor controlling endoderm differentiation. By expression and gain-of-function analysis I suggest a cell autonomous function for Pou2 in the first step of endodermal differentiation. By gain-of-function experiments involving the gene encoding the HMG transcription factor Casanova (Cas) I show that both Cas and Pou2 are necessary to activate expression of the endodermal differentiation marker sox17 in a mutually dependent way, and that the ability of Cas to ectopically induce sox17 strictly requires Pou2. I conclude that both maternal and zygotic pou2 function is necessary for commitment of endodermal progenitor cells to differentiate into endodermal precursor cells.