No description available.

In the mammalian retina, a small population of retinal ganglion cells are intrinsically photosensitive due to the expression of the photopigment melanopsin. These intrinsically photosensitive retinal ganglion cells (ipRGCs) integrate their own intrinsic light response with that of rod and cone photoreceptors to drive a variety of physiological and behavioural responses to light. Recently, however, a subset of these cells have been shown to project to the dorsal Lateral Geniculate Nucleus (dLGN) of the visual thalamus, where they directly contribute to visual perception. In the case of retinal degenerations (the most common being retinitis pigmentosa which affects up to 1:2000 people worldwide), the death of the rod and cone photoreceptors results in complete visual blindness with no available treatment. At least some ipRGCs survive retinal degeneration and can continue signalling light information to the dLGN, suggesting that these cells could support some form of visual perception. However, to-date little is known about this projection during retinal degeneration. Thus, characterising its anatomy and physiology is key to determining the quality of visual information that is conveyed to the dLGN during retinal degeneration and what prevents these cells supporting behaviourally relevant vision. A subset of ipRGCs target the dLGN and continue signalling light information even at advanced stages of retinal degeneration. However, it is unknown whether all ipRGC subtypes survive following the death of rod and cone photoreceptors, and whether they retain normal dendritic architecture following reorganisation of the remnant neural retina. We set out to answer these questions using the multi-colour labelling technique Brainbow. In doing so, we design and describe a unique methodology and toolset, based on Principal Component Analysis (PCA), to analyse 3-Dimensional (3D) multi-colour images. We then demonstrate its utility by identifying, isolating and reconstructing the 3D morphology of individual ipRGCs from a population of labelled cells in the degenerate retina and quantitatively characterise their dendritic architecture. The results indicate that all known ipRGC subtypes are resilient to the effects of outer photoreceptor degeneration. Melanopsin responses in the dLGN have been shown to support global brightness perception in mice with advanced retinal degeneration. However, to-date, it is unknown whether these cells can encode spatial information. Using in-vitro and in-vivo electrophysiological recordings from mice in advanced stages of retinal degeneration, we demonstrate for the first time that ipRGCs in the retina, and their target neurones in the dLGN, possess discrete spatial receptive fields. These receptive fields are large and lack a centre-surround organisation. The retinotopic organisation of these cells' projections would suggest they could support spatial vision. However the poor temporal resolution of the deafferented melanopsin response is the most significant limitation precluding melanopsin signalling from supporting behaviourally relevant vision under naturalistic viewing conditions. Considering these temporal limitations, we finally investigated if melanopsin could contribute to visual perception at earlier stages of degeneration which is more representative of clinical conditions in humans. Here, vision can rely on both the intrinsic melanopsin-driven light response and residual cone function. Using silent substitution in combination with in-vivo electrophysiological recordings from the dLGN of mice in early-stage degeneration, we identify a number of cone-driven responses which could support normal visual function. However, we were unable to detect a significant and robust contribution of melanopsin signalling to these residual light-responses using our silent substitution stimuli in both retinally degenerate and wildtype mice at these age. However, we did find a significant contribution to the Olivary Pretectal Nucleus (OPN) of visually intact mice at equivalent ages, and to the adult dLGN. Supported by anatomical data, this suggests that there is a specific temporal delay in the maturation of ipRGCs which project to the dLGN during development of the visual system.

La microscopie non linéaire a transformé le domaine de la neurobiologie depuis les années 1990, en permettant d'acquérir des images tridimensionnelles de tissus épais avec une résolution subcellulaire. Cependant, les profondeurs d'imagerie accessibles sont limitées à quelques centaines de micromètres dans des tissus diffusants tels que le tissu cérébral. Au cours des dernières années, plusieurs stratégies ont été développées pour dépasser cette limitation de profondeur et accéder à de plus grands volumes de tissu. Ces avancées récentes ont jusqu'à présent été limitées en terme de modes de contrastes accessibles, et ont souvent été réduites à des approches monochromes. Ce travail de thèse vise à développer des techniques d'imagerie non linéaires de grands volumes et de grande profondeur dotées de diverses possibilités de contrastes, indispensables pour l'étude de tissus complexes tels que le tissu cérébral. Dans un premier chapitre, nous présentons les difficultés associées à l'imagerie de grand volume de tissu cérébral, avec une emphase particulière sur les puissantes stratégies de marquages génétiques dont l'usage à jusqu'à présent été limité à des faibles étendues. Ensuite, nous introduisons la microscopie Chrom-SMP (chromatic serial multiphoton), une méthode développée au cours de cette thèse et consistant à combiner l’excitation deux-photon multicouleurs par mélange de fréquences avec une technique d'histologie automatisée (i.e découpe sériée) pour accéder à plusieurs contrastes non linéaires à travers de grands volumes de tissus ex vivo, allant de plusieurs mm3 à des cerveaux entiers, avec une résolution micrométrique et un coalignement intrinsèque des canaux spectraux. Dans un troisième chapitre, nous explorons le potentiel de cette nouvelle approche pour la neurobiologie. En particulier, nous démontrons l'histologie multicouleur de plusieurs mm3 de tissu "Brainbow" avec une résolution constante dans l’ensemble du volume imagé. Nous illustrons le potentiel de notre approche à travers l'analyse de la morphologie, des interactions et du lignage des astrocytes du cortex cérébral de souris. Nous explorons également l’apport du Chrom-SMP pour le suivi multiplexé de projections neuronales marquées par des traceurs de couleurs distinctes sur de grandes distances. Enfin, nous présentons dans un quatrième chapitre le développement de la microscopie à trois photons multimodale, approche permettant d’augmenter la profondeur d’imagerie sur tissus vivants. / Multiphoton microscopy has transformed neurobiology since the 1990s by enabling 3D imaging of thick tissues at subcellular resolution. However the depths provided by multiphoton microscopy are limited to a few hundreds of micrometers inside scattering tissues such as the brain. In the recent years, several strategies have emerged to overcome this depth limitation and to access larger volumes of tissue. Although these novel approaches are transforming brain imaging, they currently lack efficient multicolor and multicontrast modalities. This work aims at developing large-scale and deep-tissue multiphoton imaging modalities with augmented contrast capabilities. In a first chapter, we present the challenges of high-content large-volume brain imaging, with a particular emphasis on powerful multicolor labeling strategies which have so far been restricted to limited scales. We then introduce chromatic serial multiphoton (Chrom-SMP) microscopy, a method which combines automated histology with multicolor two-photon excitation through wavelength-mixing to access multiple nonlinear contrasts across large volumes, from several mm3 to whole brains, with submicron resolution and intrinsic channel registration. In a third chapter, we explore the potential of this novel approach to open novel experimental paradigms in neurobiological studies. In particular, we demonstrate multicolor volumetric histology of several mm3 of Brainbow-labeled tissues with preserved diffraction-limited resolution and illustrate the strengths of this method through color-based tridimensional analysis of astrocyte morphology, interactions and lineage in the mouse cerebral cortex. We further illustrate the potential of the method through multiplexed whole-brain mapping of axonal projections labeled with distinct tracers. Finally, we develop multimodal three-photon microscopy as a method to access larger depths in live settings.

Le travail effectué au cours de cette thèse a porté sur le développement et la mise en œuvre de nouvelles stratégies en microscopie non linéaire permettant d'augmenter le nombre de signaux non linéaires simultanément observables d'une part, et la vitesse d'acquisition d'autre part. Dans un premier temps, nous avons exploré la possibilité de produire des signaux multiples au moyen de deux trains d'impulsions synchronisés de longueur d'onde centrale distincte. Nous avons montré que cette approche permet d'exciter de façon optimale et simultanée trois protéines fluorescentes respectivement bleue, jaune, et rouge. Une application de cette méthode consiste à imager de grands volumes de tissus marqués avec des transgènes Brainbow dans le but d'étudier la connectivité ou le lignage cellulaire. Plus généralement, nous avons montré que cette approche permet de combiner plusieurs signaux non linéaires tels que la fluorescence, la génération de seconde (SHG) et de troisième (THG) harmoniques, ainsi que le mélange à quatre ondes (FWM). Dans un deuxième temps, nous avons étudié la possibilité d'augmenter la vitesse d'imagerie. Pour cela, nous avons mis en œuvre plusieurs manières de produire des faisceaux de Bessel focalisés afin d'augmenter la profondeur de champ d'un microscope à balayage. Enfin, en vue d'augmenter la vitesse d'acquisition tout en préservant le sectionnement optique, nous avons construit un microscope biphotonique à nappe de lumière de profil spatial programmable. Dans cette géométrie nous avons comparé les propriétés d'imagerie de profils d'excitation de type gaussien et de Bessel pour des applications en biologie du développement.

No description available.

This dissertation explores the connections that exist between Gertrude Steins late nineteenth-century psychological studies at Harvard University, her fin-de-sicle brain research at the Johns Hopkins Medical School, and her early twentieth-century cubist writings. This study is important to neuraesthetic researchers, because it appears that Stein produced a secret series of cubist brain maps from approximately 1912 to 1935, and then published her first explicit brain map in _The Geographical History of America or the Relation of Human Nature to the Human Mind_, in 1936. The cubist brain maps that Stein produced during this period can be conceptualized as evolving, neuraesthetic writing practices that reflect her complex, scientific insights and her varied, artistic associations. One of the primary differences between Steins cubist writings and those of her literary peers is that she deploys the cubist painting strategies of Pablo Picasso, for the purpose of portraying the human central nervous system. In addition to exploring the scientific meanings of Steins multidimensional, performative and introspective cubist puns, my study examines how Stein uses color in her cubist writings, as a means of anticipating the visual effects of future scientific discoveries and connectivity maps, such as the Brainbow system, which uses the fluorescent protein from the jellyfish Aequorea Victoria to label the central nervous systems of genetically modified mice with distinguishable colors. Also, this project examines how Stein uses color words and other simple devices from the English language to illustrate the brains cellular structures, neural networks and neuroanatomical features. This studys primary aim is to explore how Steins dissociative writings function within western culture as neuraesthetic modes of masterpiece creation, brain representation and consciousness translation. Through the serial production of cubist brain maps, Stein posed important questions about the modern science of the reading brain. By developing allegorical methods of brain representation, Stein contributes to the western practice of neuroesthetics by foregrounding the role that creative writing plays in the production of imaginary, laboratory practices and imaginative, brain imaging technologies.

Vascular smooth muscle cell (VSMC) accumulation is a hallmark of atherosclerosis and vascular injury. However, fundamental aspects of proliferation and the phenotypic changes within individual VSMCs, which underlie vascular disease remain unresolved. In particular, it is not known if all VSMCs proliferate and display plasticity, or whether individual cells can switch to multiple phenotypes. To assess whether proliferation and plasticity in disease is a general characteristic of VSMCs or a feature of a subset of cells, multi-colour lineage labelling is used to demonstrate that VSMCs in injury-induced neointimal lesions and in atherosclerotic plaques are oligo-clonal, derived from few expanding cells, within mice. Lineage tracing also revealed that the progeny of individual VSMCs contribute to both alpha Smooth muscle actin (aSma)-positive fibrous cap and Mac-3-expressing macrophage-like plaque core cells. Co-staining for phenotypic markers further identified a double-positive aSma+ Mac3+ cell population, which is specific to VSMC-derived plaque cells. In contrast, VSMC-derived cells generating the neointima after vascular injury generally retained expression of VSMC markers and upregulation of Mac3 was less pronounced. Monochromatic regions in atherosclerotic plaques and injury-induced neointima did not contain VSMC-derived cells expressing a different fluorescent reporter protein, suggesting that proliferation-independent VSMC migration does not make a major contribution to VSMC accumulation in vascular disease. Similarly, VSMC proliferation was examined in an Angiotensin II perfusion model of aortic aneurysm in mice, oligo-clonal proliferation was observed in remodelling regions of the vasculature, however phenotypic changes were observed in a large proportion of VSMCs, suggesting that the majority of VSMCs have some potential to modulate their phenotype. To understand the mechanisms behind the inherent VSMC heterogeneity and observed functionality, the single cell transcriptomic techniques Smart-seq2 and the Chromium 10X system were optimized for use on VSMCs. The work within this thesis suggests that extensive proliferation of a low proportion of highly plastic VSMCs results in the observed VSMC accumulation after injury, and the atherosclerotic and aortic aneurysm models of cardiovascular disease.