Midbrain dopamine (DA) neurons play a key role in a wide range of behaviours, from motor control, motivation, reward and reinforcement learning. Disorders of midbrain dopaminergic signaling is involved in a variety of nervous system disorders including Parkinson’s disease, schizophrenia and drug addiction. Understanding the basis of how dopaminergic neuronal activity in the substantia nigra pars compacta (SNc) governs movements, requires a deep appreciation of how afferent inputs of various neurotransmitter systems create a neuronal circuit that precisely modulates DA neuronal excitability. Two brainstem cholinergic neuclei, the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT), have major cholinergic projections to the SNc, despite the fact that the precise mechanisms of cholinergic modulation of DA neuronal activity mediated by nAChRs remain unclear. To dissect out the modulatory roles of the cholinergic system in regulating DAergic neuronal activity in the SNc and locomotion, we employed optogenetics along with electrophysiological and behavioural approaches. My results from whole-cell recordings from lateral and medial SNc DA neurons revealed that lateral DA neurons received predominantly excitatory nAChR mediated cholinergic neurotransmission (monosynaptic nicotinic or disynaptic glutamatergic responses) resulting in greater excitability of DA neurons both at 5 and 15 Hz blue LED light stimulation of cholinergic terminals. However, medial SNc DA neurons received predominantly biphasic current responses that were both inhibitory GABAergic and excitatory nAChR mediated cholinergic neurotransmission. This led to a net inhibition of action potential firing of DA neurons at 5 Hz blue LED light stimulation of cholinergic terminals, while at 15 Hz stimulation there was an initial inhibition followed by a significant increase of the baseline action potential firing frequency. Furthermore, in vivo optogenetic experiments showed that activation of the cholinergic system in the medial SNc resulted in decreased locomotion, while for the lateral SNc led to increased locomotion. Together our findings provide new insights into the role of the cholinergic system in modulating DA neurons in the SNc. The cholinergic inputs to different subregions of the SNc may regulate the excitability of the DA neurons differentially within a tight range from excitation to inhibition which may translate into different kinds of locomotor behaviour. / Graduate

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Le sujet de cette thèse est l'étude quantitative du rôle du morphogène Fgf8 durant la somitogenèse en utilisant des perturbations spatio-temporelles de sa concentration dans un embryon de poisson zèbre en développement. Mon objectif était d'élucider le rôle que joue Fgf8 dans le modèle du «clock and wavefront» de la somitogenèse . A cet effet, j'ai développé des moyens optiques afin de perturber rapidement sa concentration dans un embryon vivant par un éclairage approprié. J’ai montré que le blocage du Fgf8 endogène (en utilisant un morpholino contre Fgf8) ou sa surexpression (en utilisant une approche de photo-activation développée dans le laboratoire d'accueil) affectaient la taille des somites observées dans un embryon de poisson zèbre à 24 hpf. Pour comprendre la raison de ce changement de taille des somitesj’ai construit un système de vidéomicroscopie à intervalle régulier qui m'a permis de suivre l'évolution parallèle de 20-30 embryons de poisson zèbre en temps réel. Après avoir comparé la période de la segmentation, la vitesse d'allongement de la queue, la vitesse de racourcissement du PSM et la distribution spatiale de MAPK phosphorylé, mes résultats montrent que la sur-expression globale de Fgf8 induit un retard subtil dans la période de segmentation et ralentit la vitesse de déplacement du front d'onde, qui est la cause principale de la variation de taille des somites. / The subject of this thesis is the quantitative study of the role of Fgf8 in somitogenesis using spatio-temporal perturbations of its concentration in a developing zebrafish embryo. My goal was to elucidate the role that Fgf8 plays in the clock and wavefront model of somitogenesis. For that purpose, I have developed optical means to quickly perturb its concentration in a live embryo by an appropriate illumination. I have shown that blocking endogenous Fgf8 (using a morpholino against Fgf8) or inducing over-expression of Fgf8 (using a photo-activable approach developed in the host lab) affected the size of somites observed at 24 hpf in a zebrafish embryo. To address the reason for this change in somite size, I have built a time-lapse microscopy set-up that allowed me to monitor the parallel development of 20-30 zebrafish embryos in real time. After comparing the period of segmentation, the velocity of tail elongation, the speed of PSM shortening, and the MAPK pattern of phosphorylation (the target of Fgf8), my results show that global over-expression of Fgf8 induces a subtle delay in the segmentation period and slows down the posterior moving wavefront velocity, which is the major cause of the change in somite size.

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京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23340号 / 生博第458号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 見学 美根子, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The world is a complex and dynamic place. The incredibly dense and constantly changing information stream with which our senses are bombarded must be decomposed, taken in, and processed by any organism hoping to make enough sense of this world in order to survive to the next moment. For complex behaviors, and in particular a great many of those that we often feel define us as a human species, this dense sensory stream must not just be processed, but the important features of the environment must be further distilled and structured into representations that can then be stored long-term to guide future behavior through the joint processes of Learning and Memory. The primary goal of this thesis is to further our understanding of the neurobiological bases - at the subcellular, circuit, and network level - of learning and memory. The hippocampus, one of the most studied systems in the brain by far, is thought to play a central role in learning and memory. Principal cells in the hippocampus become tuned to environmental features, forming persistent representations of an animal’s environment, but the precise mechanisms by which these representations are formed, used, and maintained remain unresolved. By employing a variety of experimental techniques including in vivo two-photon calcium imaging, extracellular electrophysiology, optogenetics, and chemogenetics in awake, behaving mice, we attempted to characterize the subcellular and circuit determinants of place field representations and to connect them to these cells’ role in spatial learning and memory.

The research presented in this dissertation focuses on improvements made to lithium niobate leaky-mode modulators for both holographic video and optogenetic applications. The specific improvements found herein are: (1) characterization of leaky-mode modulators to decrease driver bandwidth to match that of commodity graphics processing units, (2) the implementation of surface relief gratings as input couplers to replace rutile prism coupling, (3) the addition of backside surface relief gratings to create an orthogonal output face for the leaky-mode modulator, and (4) the creation of superimposed surface relief gratings in lithium niobate to enable multiple wavelength coupling at a single input angle. These advancements for leaky-mode modulators open avenues in display technologies and optogenetics. As a display technology, the leaky-mode modulator can not only be used more effectively in holographic monitors, but can stand alone as a transparent near-eye display. In regards to optogenetics, these technologies allow for the creation of a highly advanced light delivery method, with multiple illumination angles through non-mechanical steering, a large output area to probe size ratio, and support for simultaneous multiple wavelength output in both common and disparate locations.

Rationale and objective: Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are disorders of learning and memory that often occur comorbidly. Exposure to trauma-related cues can increase alcohol intake in PTSD patients that are using alcohol to self-medicate. The recurrence of anxiety symptoms with subsequent alcohol use may initiate a destructive cycle where stress and alcohol exposure impair the function of the prefrontal cortex (PFC). While the incidence of these disorders has steadily increased, current therapies and treatments often lack efficacy. Thus, investigation into the underlying neurocircuitry responsible for the establishment and maintenance of these disorders is necessary to develop novel treatment targets. Methods: The present study examined the effects of ethanol exposure on the ability to create new learned associations around previously conditioned fear cues in a rat model. Animals were exposed to fear conditioning followed by chronic intermittent ethanol to translationally model trauma exposure followed by alcohol abuse. Optogenetics was used to inhibit the prelimbic (PrL) or infralimbic (IfL) cortex during fear memory reconsolidation, and fear behaviors were measured during subsequent extinction and spontaneous recovery tests. Results and conclusion Chronic ethanol exposure led to deficits in fear extinction learning and increased freezing during spontaneous recovery, both of which were prevented following inhibition of the PrL, but not the IfL, during memory reconsolidation. These results support the involvement of the PrL in fear learning and memory, and strongly suggest that the PrL could serve as a potential target for the treatment of the learning and memory deficits that occur following exposure to stress and alcohol.