Axons of retinal ganglion cells (RGCs) send visual information to a number of retinorecipient regions in the brain. In rodents, visual thalamus receives dense innervations from RGC axons and is important for both image-forming and nonimage-forming visual functions. Retinal inputs invade visual thalamus during embryonic development, before the arrival of non-retinal inputs (such as local interneurons and axonal inputs from other brain regions). In this dissertation, I explore how early innervation of RGC axons affects circuitry in visual thalamus and the role of visual experience, neural activity, and molecular cues in the development. While the development of astrocytes in cortex has been well-described, they have been largely overlooked in visual thalamus. Using immunohistochemical, functional, and ultrastructural analysis, I show that astrocytes in visual thalamus reach adult-like morphological properties and functionality at retinogeniculate synapses early in development, by eye-opening and before visual experience. These studies reveal that while experience-dependent visual activity from RGC axons is critical for many aspects of visual thalamus development, astrocytic maturation occurs independent of that information about our visual environment. As with astrocytes, little progress has been made in understanding the development of interneurons in the visual thalamus. Here, I show that retinal inputs interact with thalamic astrocytes to influence the recruitment of GABAergic interneurons into visual thalamus. I found that this interaction between RGC axons and astrocytes is not dependent on neural activity of RGCs. Using transcriptomic analysis, in situ hybridization, and reporter lines, I observed thalamus-projecting RGCs express SHH and astrocytes in visual thalamus express SHH signaling molecules. My results reveal that SHH signaling between RGC axons and astrocytes is critical for astrocytic fibroblast growth factor 15 (FGF15) expression in developing visual thalamus. Ultimately, FGF15 serves as a potent motogen that is essential for thalamic interneuron migration. These data identify a novel morphogen-dependent and activity-independent mechanism that mediates crosstalk between RGCs and astrocytes to facilitate the recruitment of interneurons into the developing visual thalamus. / Doctor of Philosophy / The most dominant sense in human is the sight, which we need to interact with our environment efficiently. The retina takes up the information about our visual world and sends it to the brain, which ultimately puts everything together, for us to see properly. The visual information from the retina goes to the brain via nerves (which are essentially cables/wires of brain cells). These nerves from the retina go to many places in the brain, including a region called visual thalamus, which is the focus of my PhD work. For the past five years, I have been trying to understanding if nerves from the retina play a role in the brain formation during early development. To study this, I have used mice as a model system, as their brain regions that process visual information have very similar structural architecture to those in humans. My research shows that retinal nerves are indeed important for the development of visual thalamus. Here, I show that information from the eye is critical for migration (a process during development where brain cells move from their place of origin to their final location) of cells in visual thalamus. Discoveries made in this dissertation are important because they highlight how different cells in the central nervous system communicate with each other at the level of molecules and how these interactions are important for building circuits that are important for vision.

No / Purpose: Does a physiologically plausible model of the retinal ganglion cell (RGC) receptive field (RF) predict the spatial tuning properties of the Hermann Grid Illusion (HGI)? Methods: The spatial tuning of a single intersection HGI was measured psychophysically in normal observers using a nulling technique at different vertical grid line luminances. We used a model based upon a standard RGC RF, balanced to produce zero response under uniform illumination, to predict the response of the model cell to the equivalent range of stimulus conditions when placed in either the 'street' or the 'intersection' of a single element of a Hermann grid. We determined the equivalent of the nulling luminance required to balance these responses and minimise the HGI. Results: The model and the psychophysical data demonstrated broad spatial tuning with similarly shaped tuning profiles and similar strengths of illusion. The line width at the peak of the model tuning function was around twice the model RGC RF centre size. Modelling the psychophysical functions gave RF centre sizes smaller than expected from human anatomical evidence but similar to that suggested by primate physiological evidence. In the model and psychophysically the strength of the illusion varied with the luminance of the vertical grid line when HGI strength was expressed as a Michelson nulling contrast, but this effect was smaller when HGI strength was expressed as a nulling luminance. Conclusions: The shape, width, height and position of the spatial tuning function of the HGI can be well modelled by a RGC RF based model. The broad tuning of these functions does not appear to require a broad range of different cell sizes either in the retina or later in the visual pathway.

Yes / Purpose.: To establish whether a clinically exploitable relationship exists between surrogate measures of retinal ganglion cell number and functional sampling density and visual contrast sensitivity in healthy young eyes. Methods.: Psychometric functions for contrast detection were measured at 9° eccentricity in superior and inferior visual field from 20 healthy adults (age 23–43, median 26 years). Functions were compared with corresponding localized regions of retinal nerve fiber layer (RNFL) thickness measured by optical coherence tomography, a surrogate of retinal ganglion cell number, and to grating resolution acuity, a psychophysical surrogate of retinal ganglion cell sampling density. Correlations between psychometric function parameters and retinal ganglion cell surrogates were measured by Spearman's rank correlation. Results.: All measures exhibited a 2- to 4-fold variation in our sample. Despite this, correlations between measures were weak. Correlations between psychometric function parameters (threshold, spread) and RNFL thickness ranged in magnitude from 0.05 to 0.19 (P = 0.43–0.85). Grating resolution was sampling limited for 16 of 20 participants in superior visual field, and for 12 of 20 participants in inferior visual field. Correlations between psychometric function parameters and grating resolution acuities ranged in magnitude from 0.05 to 0.36 (P = 0.12–0.85) when all data were considered, and from 0.06 to 0.36 (P = 0.26–0.87) when only sampling-limited data were considered. Conclusions.: Despite considerable variation in both psychometric functions for contrast detection and surrogate measures of retinal ganglion cell number and sampling density among healthy eyes, relationships between these measures are weak. These relationships are unlikely to be exploitable for improving clinical tests in healthy populations.

Les surdités neurosensorielles sont classiquement décrites par une élévation des seuils auditifs généralement corrélée à une dégénérescence des cellules ciliées externes (CCE). Toutefois, des travaux récents sur des modèles animaux ont montré qu’un audiogramme normal pouvait être associé à des atteintes auditives périphériques. Ce travail de thèse a contribué à mieux caractériser chez des modèles murins, ces déficiences supraliminaires cachées liées d’une part, à des altérations des CCE et d’autre part, à la dégénérescence de certaines fibres nerveuses auditives. Dans la première partie de cette thèse, l’évolution des profils auditifs de souris présentant une dégénérescence accélérée des CCE, les souris de souche CD1-RjOrl : SWISS, a été caractérisée. Dans cette étude longitudinale, menée au cours du premier mois postnatal, une progressivité de la déficience auditive a été montrée. Toutefois, une discordance surprenante a été mise en évidence entre des seuils auditifs proches des valeurs normales à haute fréquence combinés à des produits de distorsions acoustiques (PDA) absents. Les courbes d’accord de masquage montrent un décalage des pointes vers les basses fréquences. Ces données indiquent que les CCE de la base ne sont plus fonctionnelles et que la perception des hautes fréquences est perturbée. Les observations en microscopie électronique à balayage ont révélé une conformation anormale de la touffe stéréociliaire des CCE au niveau de la base de la cochlée. Ces données témoignent d’une désorganisation de la tonotopie cochléaire. Dans la deuxième partie de cette thèse, l’effet de l’oxaliplatine sur la fonction auditive et sur la morphologie cochléaire a été décrit chez des souris adultes de souche CBA/J. L’oxaliplatine, un sel de platine utilisé en chimiothérapie, a de nombreux effets secondaires parmi lesquels l’apparition d’une neuropathie périphérique. À la suite d’un traitement avec cette drogue, les souris ne présentent pas d’élévation des seuils auditifs et pas d’altération de la fonction des CCE. Cependant, l’étude histologique révèle une dégénérescence surprenante des fibres auditives du ganglion spiral. Avec des tests électrophysiologiques supplémentaires, une diminution de l’amplitude du potentiel d’action composite a été mise en évidence. Le réflexe du système efférent olivocochléaire médian, évalué par un test de suppression controlatéral, semble également être diminué par le traitement. Les souris traitées avec de l’oxaliplatine constituent donc un modèle animal précieux de surdité cachée, qui demande à être mieux caractérisé. Les résultats de ces études confirment l’insuffisance de l’audiogramme pour détecter des altérations subtiles de la cochlée et montrent la nécessité d’améliorer le diagnostic de ces déficiences supraliminaires. Ainsi, les atteintes cachées des CCE peuvent être détectées par l’absence de PDA associée à des potentiels évoqués auditifs normaux et les neuropathies par des PDA présents associés à des potentiels évoqués auditifs anormaux. La combinaison de ces différents tests fonctionnels et électrophysiologiques permettrait une meilleure prise en charge des patients et une amélioration de leur qualité de vie. / Sensorineural hearing loss are classically described by auditory thresholds elevation usually correlated with outer hair cells (OHC) degeneration. However, recent work on animal models has shown that normal audiogram can be associated with peripheral hearing impairments. This thesis contributed to better characterize, in mouse models, these hidden supraliminal deficiencies related on the one hand, with OHC alterations and on the other, to auditory nerve fibers degeneration. In the first part of this thesis, the auditory profiles evolution of mice exhibiting an OHC accelerated degeneration, the CD1-RjOrl: SWISS strain mice, was characterized. In this longitudinal study, conducted in the first postnatal month, a progressivity of the hearing impairment has been observed. However, a surprising discrepancy was found between high frequency hearing thresholds close to normal values associated with missing distortion product otoacoustic emission (DPOAE). The masking tuning curves dips are shifted toward low frequencies. Those data indicate that basal OHC are no longer functional and the perception of high frequencies is disrupted. Observations in scanning electron microscopy revealed an abnormal conformation of the OHC stereocilia bundles at the cochlea base. These results represent an evidence of a disorganized cochlear tonotopy. In the second part of this thesis, the effect of oxaliplatin on the auditory function and on the cochlear morphology was described in adult CBA/J strain mice. Oxaliplatin, a platinum salt used in chemotherapy, has many side effects including development of peripheral neuropathy. Following one treatment with this drug, mice did not present any hearing threshold elevation or OHC function impairment. However, the histological study reveals a surprising degeneration of the spiral ganglion cells. With additional electrophysiological tests, a decrease in the compound action potential amplitude has been demonstrated. The median olivocochlear efferent system reflex, evaluated by a contralateral suppression test, also seems to be diminished by the treatment. The mice treated with oxaliplatin, therefore constitute a precious animal model of hidden deafness, which needs to be better characterized. The results of these studies confirm the audiogram insufficiency to detect subtle cochlea alterations and reveal the need to improve supraliminal deficiencies diagnosis. Thus, hidden OHC impairments can be detected by the absence of DPOAE associated with normal auditory evoked potentials and neuropathies by the presence of DPOAE associated with abnormal auditory evoked potentials. The combination of these functional and electrophysiological tests would allow better management of patients and an improvement in their quality of life.Keywords: hidden hearing loss, CD1 mice, outer hair cells, masking tuning curves, tonotopy, oxaliplatine, spiral

No description available.

published_or_final_version / Anatomy / Master / Master of Philosophy

In the developing visual system, the axon of a pre-synaptic cell must be guided to a post-synaptic partner. Retinal ganglion cells (RGCs) in the eye are an excellent model to study this process. Multiple classes exist that respond to specific types of light input, and these project to different destinations in the brain that process distinct types of information. The RGC axons that navigate to the lateral geniculate nucleus (LGN) do so in a class-specific manner. Axons from RGCs that mediate non-image forming functions innervate the ventral LGN (vLGN) and the intergeniculate leaflet (IGL). Axons from RGCs that process image-forming information bypass these regions to innervate the dorsal LGN (dLGN). The extracellular protein reelin was identified as a potential factor in RGC axonal targeting of the vLGN and IGL, and the reeler mutant mouse used to study the effects of its functional absence. Anterograde labeling of RGCs and their axons with Cholera toxin B (CTB) revealed reduced patterns of retinal innervation to the vLGN and IGL in mutant mice. Moreover, the absence of functional reelin resulted in axons incorrectly growing into inappropriate regions of the thalamus. We identified these misrouted axons as those of the intrinsically photosensitive RGCs (ipRGCS), a class of RGCs known to project to the affected subnuclei. In contrast to defects in ipRGC targeting, no deficits were seen in retinogeniculate or corticothalamic projections in classes of axons that normally target the dLGN. Immunohistochemistry did not reveal any effects of the absence of the functional reelin on the LGN cytoarchitecture, which is unlike many other brain regions altered in the reeler. In summary, results suggest that intact reelin is required for class-specific retinogeniculate targeting to the vLGN and IGL. The defects are likely to be in targeting and not in neuronal positioning.

L’adhésion, la migration cellulaire et l’environnement stromal sont intimement liés pour garantir l’homéostasie du système immuno-hématopoïétique. Néanmoins, nos connaissances des mécanismes responsables du maintien de ce processus fonctionnel restent fragmentaires. Notre étude a permis de mieux caractériser le stroma ganglionnaire et médullaire dans lesquels nous avons démontré de nouveaux rôles immuno-régulateurs des molécules d’adhésion jonctionnelle JAM-B et JAM-C. Dans la zone T des ganglions lymphatiques, les cellules réticulaires fibroblastiques (FRC) sécrètent des composés de la matrice extracellulaire et des chimiokines homéostatiques, nécessaires à la migration intranodale des lymphocytes T naïfs. La génération de nouveaux anticorps monoclonaux a permis d’identifier une diversité phénotypique et fonctionnelle au sein de la population FRC. L’un d’entre eux reconnaît la Thrombomoduline permettant d’identifier une population de FRC exprimant les protéines JAM-C et PDGFRα. Cette population cellulaire, dénommée FRCDP (Double Positive) sécrète des chimiokines homéostatiques, ce qui la distingue de la population FRCDN (Double Negative). Les souris sauvages traitées avec l’anticorps anti-JAM-C présentent une diminution significative du taux intranodal des chimiokines CXCL12, CCL19, CCL21 affectant la recirculation des cellules T naïves. De façon similaire, les cellules stromales des niches hématopoïétiques fournissent un environnement fonctionnel, nécessaire à l’homéostasie du système hématopoïétique. Les molécules d’adhésion sont connues pour contrôler ces mécanismes. JAM-C est exprimée à la surface des cellules souches hématopoïétiques (CSH) mais son rôle dans l’hématopoïèse reste inconnu. Notre étude montre que la molécule JAM-B est exprimée par l’environnement médullaire et interagit spécifiquement avec JAM-C sur les CSH. Les souris déficientes pour le gène jam-b présentent une diminution du nombre de CSH quiescentes et une réponse accrue aux agents mobilisants, démontrant ainsi que le couple JAM-B/JAM-C est nécessaire au maintien et à la rétention des CSH dans la moelle osseuse. / Homeostasis of the immune and hematopoietic system is dependent of cell adhesion, cell migration and stromal environment. Nevertheless, the molecular mechanisms involved in the crosstalk between hematopoietic and stromal cells have remained elusive. Our studies allowed to better characterize lymph node (LN) and bone marrow (BM) stromal compartments through the demonstration that expression of junctional adhesion molecules (JAM) in these compartments is necessary for the maintenance of immune and hematopoietic homeostasis. In the T cell zone (LN), extracellular matrix and homeostatic chemokines are secreted by fibroblastic reticular cells (FRC) which control naive T cell migration. We have identified new FRC subsets using a monoclonal antibody based approach to identify new cell surface markers of stromal cells. We have found that the FRC population expressing JAM-C, Thrombomodulin and PDGFRα (FRCDP, for Double Positive) secretes homeostatic chemokines such as CCL21, CCL19 and CXCL12. In contrast, FRCDN (Double Negative) that lack JAM-C and Thrombomodulin expression do not. Functionally, we have shown that JAM-C controls the secretion of CCL21, CCL19 and CXCL12 by FRCDP and that anti-JAM-C treated mice exhibit a decrease of intranodal chemokine contents. These results suggest that JAM-C may regulate homeostasis through the control of homeostatic chemokine secretion. We therefore asked the question whether similar function for JAM-C or its ligand JAM-B may be identified in the bone marrow. In the BM, Hematopoietic Stem Cells (HSC) are maintained quiescent and undifferentiated in specific stromal structures called HSC niches. HSC/niche interactions via adhesion molecules and chemokines are known to be active player of HSC homeostasis. Recently, JAM-C expression by HSC has been reported, but its role in hematopoiesis has remained elusive. We have demonstrated that HSC interact with JAM-B expressed by BM stromal cells in a JAM-C dependent manner. Moreover, we have observed a decreased pool of quiescent HSC in jam-b deficient mice. Finally, we have found that jam-b deficient mice exhibit an increase in intramedullary CXCL12 content and an exacerbated response to mobilizing agents. Collectively, these data demonstrate that JAM-B and JAM-C play a dual function in lymph node and bone marrow microenvironments through the regulation of leuko-stromal adhesion and chemokine secretion.

Neste estudo, foram abordados os aspectos quantitativos do gânglio cervical cranial (GCC) esquerdo de preás (Galea spixii spixii) machos, oriundos do criatório da Universidade Federal Rural do Semi-Árido Nordestino de Mossoró-RN. Os seguintes parâmetros foram estimados: volume do GCC, número total e tamanho dos neurônios (uni e binucleados) durante dois períodos do desenvolvimento pós-natal (maturação e envelhecimento). Para tanto, utilizamos quatro grupos etários distintos de animais: neonatos, jovens, adultos e senis. Após a eutanásia dos animais, os GCCs foram fixados (perfusão sistêmica) com solução de formoldeído (4%) em PBS, embebidos em solução de ágar (10%) e seccionados sistemática, uniforme e aleatoriamente, aplicando-se o princípio de amostragem e contagem do fractionator. Os principais resultados desta pesquisa foram: houve diferença significativa entre os grupos etários para os parâmetros: Peso Neonato: 0,00056 ; Jovem: 0,00084; Adulto: 0,00116; Senil: 0,00118. Comprimento Neonato: 1,294 ; Jovem: 2,076; Adulto: 2,304; Senil: 2,082. Largura Neonato: 0,842; Jovem: 1,028; Adulto: 1,062; Senil: 1,174. Volume ganglionar Neonato: 0,34 mm3 ; Jovem: 0,30 mm3; Adulto: 0,39 mm3; Senil: 0,63 mm3 e Volume neuronal médio de neurônios uninucleados Neonato: 2916,90 mm3; Jovem: 3550,00 mm3; Adulto: 7409,00 mm3; Senil: 6701,00 mm3. As principais conclusões deste estudo foram: (i) peso, comprimento e largura do GCC apresentaram aumento significativo de 185%, 60% e 79% respectivamente, durante o período de maturação, enquanto durante o período de envelhecimento, o peso do GCC apresentou queda de 33%, o comprimento um pequeno decréscimo de 10%, entretanto a largura, durante este mesmo período, apresentou um aumento progressivo de mais de 10%, tais alterações relacionadas com a idade refletiram no volume ganglionar, causando hipertrofia; (ii) Essa hipertrofia do GCC de preás é secundária ao desenvolvimento pós-natal (maturação e envelhecimento), aumentando em 85%, acompanhado por um aumento alométrico, da ordem de 6 vezes em relação à massa corporal, aproximadamente 622%; (iii) Há hipertrofia dos neurônios uninucleados que é secundária ao desenvolvimento pós-natal do GCC de preás, aumentando em 154% durante o período de maturação com uma pequena queda de 10% durante o envelhecimento. Finalmente, sugere-se que estudos futuros possam investigar se o tamanho dos territórios de inervação destes gânglios pode desencadear mudanças na micro e macroestrutura dos mesmos, incluindo a possibilidade de ocorrência de neurogênese, que seria estudada por meio de imunomarcadores específicos. / This study aimed at detecting whether post-natal development would exert any effect on the size and number of cranial cervical ganglion (CCG) neurons of male preas (Galea spixii spixii). Twenty left CCGs from twenty preas were harvested from the Animal Facility of the Universidade Federal Rural do Semi-Árido Nordestino, Mossoró- RN and were divided into four different age groups: newborn, young, adult and aged subjects. The following parameters were estimated using Cavalieri\'s principle and optical fractionator and planar rotator , respectively: the volume of CCG, total number and mean volume of uni and binucleate neurons. After euthanasia, the CCGs were perfusion-fixed with a 4% formaldehyde solution in PBS, agar-embedded and, SUR vibrosectioned. The ganglion volume was 0.34 mm3 (newborn), 0.30 mm3 (young), 0.39 mm3 (adult) and 0.63 mm3 (aged animals) (p= 0,012). The mean volume of uninucleate neurons was 2,917 mm3 (newborn), 3,550 mm3 (young), 7,409 mm3 (adult) and 6,701 mm3 (aged animals) (p=0,0001). The main conclusions of this study were: (i) The CCG hypertrophy - 85% - is the result of post-natal development (maturation and ageing), (ii) a 154% increase (hypertrophy) in the volume of uninucleate neurons during the maturation is followed by a 10% atrophy of them during ageing. Future studies may investigate whether the size of the CCG\'s target-organs can affect the structural foundation of CCG and, therefore, add further fuel to the notion that a post-natal neurogenesis may indeed exist in sympathetic ganglia.

Teoricamente, conexões por sinapses elétricas entre neurônios poderiam levar ao aumento da faixa de resposta dinâmica da rede neural. A faixa de resposta dinâmica de uma rede de neurônios pode ser definida como a faixa de valores de intensidade dos estímulos de entrada para a qual o conjunto de neurônios produz resposta antes de atingir a saturação. Em um cenário biológico, propôs-se que junções gap entre células ganglionares da retina aumentariam a faixa dinâmica da retina. O teste experimental dessa proposta apresenta várias dificuldades, o que torna a modelagem computacional uma alternativa metodológica para o estudo do papel das sinapses elétricas na faixa dinâmica da camada ganglionar da retina. O objetivo deste trabalho foi a construção de um modelo biologicamente plausível da camada ganglionar da retina da salamandra, submetida a sinais de entrada realísticos conforme evidências experimentais e com a inclusão de sinapses elétricas conectando suas células, para estudar in silico os possíveis efeitos dessas sinapses elétricas sobre a faixa dinâmica da camada ganglionar. A camada ganglionar foi modelada como uma rede bidimensional cujos neurônios foram modelados pelo formalismo de Hodgkin-Huxley. Cada neurônio recebeu um de dois tipos de entrada sináptica, transiente ou sustentada. Avaliou-se o efeito da inibição pré-sináptica das células ganglionares e o efeito de diferentes padrões de conectividade mediados pelas sinapses elétricas. Os resultados sugerem que o acoplamento elétrico aumenta a sensibilidade do sistema e altera o ponto de saturação, mas não necessariamente aumenta a faixa dinâmica. / Theoretically, connections by electrical synapses between neurons could lead to an increase in their dynamic range. The dynamic range of a network of neurons can be defined as the range of input stimuli values for which the network responds before saturation. In a biological scenario, it is hypothesized that gap junctions between retinal ganglion cells may increase the dynamic range of the retina. However, the experimental testing of this hypothesis presents several difficulties, which makes computational modeling a methodological alternative to study the role of electrical synapses on the dynamic range of the ganglion cell layer of the retina. In this work we constructed a biologically plausible computational model of the ganglion cell layer of the salamander retina. A bidimensional network was built with cells modeled by the Hodgkin-Huxley formalism connected via gap junctions and subject to realistic inputs constrained by experimental evidence, to study in silico the effects of gap junctions on the dynamic range of the model. We studied the effect of different gap junction-mediated connectivity patterns, input type combinations (transient, sustained and mixed between the two) and presynaptic inhibition on the dynamic range. Our results suggest that gap junction coupling increases the network\'s sensitivity and alters the saturation point but not necessarily increases the dynamic range.