Dissertations / Theses on the topic 'Viral tracings'

As with other sensory modalities, the vestibular system recruits efferent circuitry to transport information from the central nervous system (CNS) to the sensory periphery. This efferent vestibular system (EVS) originates in the brainstem and terminates on vestibular hair cells and afferent fibres in the semicircular canals and otolith organs. Understanding how this central component outputs to the vestibular organs, and mediates motor and vestibular coordination, could potentially impact clinical treatment of vestibular disorders. Previous EVS work has primarily focused on the anatomy, pharmacology, synaptic mechanisms, and peripheral effects of efferent vestibular nucleus (EVN) activation. Although this work is fundamental to understanding this system and its mechanism of action, the behavioural function of the EVS is yet to be ascribed. For this, we need to appreciate the physiology of EVN neurons, and their context of activation within the CNS. In this thesis, I characterise the electrophysiological profile of EVN neurons, and trace their direct monosynaptic circuitry. My methodology includes whole-cell current- and voltage- clamp electrophysiology, and glycoproteindeficient rabies virus tracing techniques. Using these, I enrich understanding of EVN action, and hint at potential functional roles from their CNS partners. The data presented in this thesis provides novel insights into the EVS. EVN neurons are characterised with a homogeneous output, but a heterogeneous synaptic input profile. Inputs to the EVN originate from diverse areas in the brainstem and cortex. These findings suggest that the EVN modulates vestibular end organs in multiple different behavioural contexts. This work forms the basis of subsequent EVS behavioural investigations such as loss of function experiments targeting input regions via optogentic means and subsequent EVS recordings, or silencing of EVN activity and subsequent behavioural testing. Collectively, my results, these future directions, and the existing body of EVS literature, brings us closer than ever to understanding and ascribing a functional role for the EVS.

Brown adipose tissue (BAT) is a critical organ for non-shivering thermogenesis, which is under control of both sympathetic and sensory neural innervation. We utilized both a retrograde sympathetic nerve tract tracer pseudorabies virus and an anterograde sensory tract tracer the H129 strain of herpes simplex virus-1 to locate individual neurons across the neuroaxis that are part of the SNS outflow from brain to interscapular BAT and are part of the sensory input to the brain. We found specific neuronal phenotype of the double-infected neurons distributed from the hindbrain to the forebrain with highest densities in several discrete brain regions: the paraventricular hypothalamus (PVH), lateral hypothalamus (LHA), parabrachial nucleus (PB) and raphe pallidus (RPa). The neuroanatomical reality of the SNS-sensory feedback loops suggests coordinated control of BAT thermogenesis at several sites and indicates plasticity of SNS-sensory crosstalk.

A l’interface des systèmes sensoriels et moteurs, le cortex pariétal postérieur génère des représentations stables de l’espace, et contribue au guidage des mouvements corporels. Nous avons exploré les aires intraparietales médiale (MIP), latérale (LIP) et ventrale (VIP) par électrophysiologie et traçage transneuronal rétrograde. Nous démontrons que les informations vestibulaires de mouvement propre dans MIP viennent directement, par trois synapses, du labyrinthe. Nous décrivons aussi des bases neurales des signaux de position et de mouvement oculaire dans LIP et MIP. Dans VIP, nous montrons que les signaux de position de tête permettent un codage de l’espace centré sur le corps. Enfin, nous dévoilons l’organisation complète des entrées cérébelleuses, nucléaires et corticales, sur MIP, et nucléaires sur LIP. Ces travaux sont un progrès vers la compréhension de la dynamique des représentations spatiales, du mouvement propre et du guidage moteur dans le cortex pariétal postérieur.

Final publication is available at http://dx.doi.org/10.1111/ejn.12882<br>Kyoto University (京都大学)<br>0048<br>新制・論文博士<br>博士(医学)<br>乙第13040号<br>論医博第2115号<br>新制||医||1017(附属図書館)<br>33032<br>京都大学大学院医学研究科医学専攻<br>(主査)教授 渡邉 大, 教授 影山 龍一郎, 教授 髙橋 良輔<br>学位規則第4条第2項該当

The Lateral Habenula (LHb) have been implicated in both reward-seeking behavior and in depressive disorders due to its modulatory effects on dopamine rich areas. Excitatory projections from LHb target GABAergic interneurons of both ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg) and consequently provide strong inhibition on VTA‟s dopaminergic neurons. These reward related signals are provided to LHb from distinct neuronal populations in internal Globus Pallidus (GPi). Here by using a dual viral combination of an adeno-associated helper virus (AAV) and a genetically modified rabies virus that displays specific transsynaptic retrograde spread we are providing anatomical evidence for a strong innervations of the LHb by VGLUT2+ glutaminergic and SOM+ GABAergic GPi neurons. Our results provide the first direct evidence for both an excitatory and an inhibitory projection m, from GPi to the LHb. Given the importance of the LHb as a modulatory nucleus of the dopaminergic system, the definition of its connectivity and function will give valuable insights in the understanding of both reward-seeking behavior and depressive disorders.

Les stimuli nociceptifs sont détectés par des neurones sensoriels spécialisés du système nerveux périphérique appelés nocicepteurs. L’information nociceptive est ensuite traitée dans la corne dorsale de la moelle épinière, qui contient des interneurones locaux et des neurones de projection qui envoient des axones vers le cerveau. Les aires supra-spinales projettent à leur tour vers la moelle épinière, où elles contribuent à la synchronisation des signaux nociceptifs. Une sensibilité à la douleur exagérée et anormale s'accompagne d'altérations du traitement de l’information dans la moelle épinière dans les systèmes de contrôle descendants de la douleur. La connexion entre le cortex somatosensoriel en particulier et la moelle épinière est conservée chez les mammifères, mais très peu de choses sont connues sur son rôle dans la modulation du traitement sensoriel dans la moelle épinière. Un défi majeur dans l’étude des circuits neuronaux est de de marquer et de cibler spécifiquement des groupes ou sous-groupes définis de neurones. Les approches classiques incluent le ciblage de populations neuronales définies génétiquement, i.e. sur la base de l'expression d'un gène marqueur. Cependant, cela ne suffit pas toujours pour définir des groupes de neurones fonctionnellement distincts. Ici, nous décrivons et utilisons des stratégies de marquage génétiques et virales basées sur la connectivité des neurones ainsi que sur l’expression d’un ou de deux gènes marqueurs. En particulier, nous avons utilisé une combinaison de lignées de souris transgéniques et d'injections intra-spinales et corticales de vecteurs viraux recombinants pour identifier et cibler des neurones spécifiques du cortex et de la moelle épinière lombaire. Nous avons identifié une population de neurones pyramidaux dans le cortex somatosensoriel qui projettent directement dans la corne dorsale (neurones S1-CST). Ces neurones établissent un contact direct avec les interneurones exprimant c-maf dans la corne dorsale profonde, qui reçoivent également des contacts directs d’afférents primaires mécano-sensoriels à bas seuil. De plus, la manipulation pharmacogénétique des neurones c-maf a entraîné des modifications dans le traitement des stimulations sensorielles mécaniques. Ces résultats identifient deux éléments d’un circuit qui intègre les informations descendantes du cortex avec des signaux sensoriels périphériques et contribue à la modulation de la perception somatosensorielle<br>Noxious stimuli are sensed by specialized sensory neurons of the peripheral nervous system called nociceptors. The nociceptive information is then processed in the spinal cord dorsal horn, which contains local interneurons and projection neurons that send axons to the brain. Supraspinal areas in turn project downwards to the spinal cord where they contribute to the gating of nociceptive signals. Exaggerated and abnormal pain sensitivity is accompanied by alterations in spinal processing and descending pain control systems. The connection between the somatosensory cortex in particular and the spinal cord is conserved in mammals, but very little is known about its role in modulating spinal sensory processing. A major challenge of studying neuronal circuits is to specifically label and target defined groups or subgroups of neurons. Classical approaches include targeting of genetically defined neuronal populations based on the expression of a marker gene. However, this is not always sufficient to define functionally distinct groups of neurons. Here, we describe and used genetic and viral tageting strategies based on the connectivity pattern of the neurons as well as the expression of one or two marker genes. In particular, we used a combination of transgenic mouse lines and intraspinal and cortical injections of recombinant viral vectors to identify and target specific neurons in the cortex and lumbar spinal cord. We identified a population of pyramidal neurons in the somatosensory cortex that project directly to the spinal dorsal horn (S1-CST neurons). These neurons make direct contacts onto c-maf expressing interneurons in the deep dorsal horn which also receive direct inputs from low threshold mechanosensory primary afferents. Additionnally, pharmacogenetic manipulation of c-maf neurons led to altered processing of mechanical stimuli. These results identify two elements of a circuit that integrates descending inputs from the cortex with peripheral sensory signals and contributes to the modulation of somatosensory perception

Pendant la course, la ventilation augmente pour compenser la demande énergétique accrue. Le substrat, soupçonné neuronal, de cette hyperpnée à l'exercice est néanmoins toujours méconnu. Pour le caractériser, nous avons, chez la souris, examiné les interactions entre i) mouvements des membres et cycles respiratoires, et ii) réseaux neuronaux locomoteur et respiratoire. Tout d’abord, en combinant enregistrements électromyographiques (EMG) du diaphragme combinés au suivi vidéo des membres pendant la course, nous montrons que, pour une large gamme de vitesses sur un tapis roulant, la fréquence respiratoire augmente jusqu'à une valeur fixe, indépendante des vitesses de course. Surtout, les inspirations ne sont pas temporellement synchronisées avec les foulées, indiquant que l'hyperpnée à l'exercice peut opérer sans signaux phasiques provenant des retours sensoriels des membres. Nous avons ensuite cherché à identifier, au sein des centres locomoteurs, les neurones déclencheurs de cette hyperpnée, ainsi que leurs cibles dans les centres respiratoires. En combinant enregistrements EMG, traçages viraux et interférences fonctionnelles, nous montrons d’une part que le principal centre de l'initiation locomotrice (la région locomotrice mésencéphalique, MLR) peut réguler à la hausse la respiration, pendant, et même avant, la course. Cet effet repose sur des projections directes de la MLR vers le générateur inspiratoire principal, le complexe préBötzinger. D'autre part, nous montrons que les circuits locomoteurs de la moelle épinière lombaire ont également une action excitatrice sur l'activité respiratoire. Cette voie ascendante cible néanmoins un autre groupe respiratoire, le noyau rétrotrapézoïde. Ce travail met ainsi en évidence la nature multifonctionnelle des centres locomoteurs, et souligne l'existence de multiples voies neuronales capables d’augmenter la respiration pendant, voire avant, la course<br>During running, ventilation increases to match the augmented energetic demand. Yet the presumed neuronal substrates for this running hyperpnea have remained elusive. To fill this gap, we have, in mice, examined the interactions between i) limb movements and respiratory cycles, and ii) locomotor and respiratory neural networks. First, by combining electromyographic recordings (EMG) of the diaphragm with limb video-tracking in running mice, we show that, for a wide range of trotting speeds on a treadmill, breathing rate increases to a fixed value, irrespective of running speeds. Importantly, breaths are never temporally synchronized to strides, highlighting that exercise hyperpnea can operate without phasic signals from limb sensory feedbacks. We next sought to identify candidate trigger neurons in the locomotor central network, and their partners in respiratory centers. Combining EMG recordings, viral tracing, and activity interference tools, we first show that the prime supraspinal center for locomotor initiation (the mesencephalic locomotor region, MLR) can upregulate breathing during, and even before, running. Indeed, the MLR contacts directly and modulates the main inspiratory generator, the preBötzinger complex. We show that the lumbar locomotor circuits also have an excitatory action onto respiratory activity, but that this ascending drive targets another essential respiratory group, the retrotrapezoid nucleus. This work highlights the multifunctional nature of locomotor command and executive centers, and points to multiple neuronal pathways capable of upregulating breathing during, or possibly even prior to, running

Alveolar macrophages are strategically located at the lung mucosal surface and are part of an intricate cellular network. Here, immune cells, epithelial cells and neurons functionally interact to maintain homeostasis despite a multitude of aggressions and ever-changing environmental conditions. Previous evidence suggest that neuron-derived cues may modulate alveolar macrophages, but also indicate that immune cells influence neuronal activity. Thus, we hypothesized that alveolar macrophages functionally interact with neurons in so-called neuro-immune cell units. Nevertheless, the identity of these units and the nature of this communication remains unknown. Therefore, using advanced imaging techniques, molecular analyses and novel tracing techniques, we set out to define the bi-directional communication between alveolar macrophages and neurons. We found that alveolar macrophages and neurons co-localize. Using a novel tracing technique, we demonstrate that these cells may engage in bi-directional communication. We also show that alveolar macrophages possess the machinery to engage in neuro-immune crosstalk. These results can be easily expanded to fully assess this new neuro-immune cell unit. Furthermore, this versatile new tracing strategy can easily be applied to other neuro-immune cell units, allowing us to unravel this new paradigm in mammalian physiology. Ultimately, this will pave the way for the development of new therapeutic targets.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia<br>Estudos recentes têm indicado que o córtex da insula (IC) é uma região cerebral com um papel preponderante no processamento de emoções, incluindo medo e ansiedade. Acredita-se que esta e outras funções, tais como aprendizagem de sinais que transmitem segurança, previsão de risco e antecipação, são executadas juntamente com a amígdala, uma área que, tal como o IC, se encontra também desregulada em distúrbios de ansiedade. Esta hipótese é também suportada por evidências de estudos convencionais de identificação neuronal, efetuados maioritariamente em primatas não humanos e ratos, que mostram uma vasta conexão anatómica entre o IC e a amígdala. No entanto, uma vez que estes são desprovidos de especificidade quanto ao tipo celular e capacidade de determinar quais os neurónios que comunicam diretamente entre si, a conectividade pormenorizada a um nível celular entre o IC e a amígdala é ainda desconhecida.Considerando o papel do núcleo basomedial da amígdala (BM) na regulação da ansiedade, este estudo teve como objetivo elucidar a arquitetura anatómica detalhada entre o IC e o BM em murganhos, usando a técnica de identificação neuronal mono-trans-sináptica baseada no transporte viral retrógrado. Devido à especificidade do método quanto ao tipo celular e à restrição da dispersão do vírus da raiva, esta estratégia permitiu-nos identificar vias aferentes provenientes de diferentes sub-regiões do IC que comunicam com nerónios excitatórios pós-sinápticos nas porções anterior e posterior do BM (BMA e BMP, respetivamente). Observou-se que a maioria das vias que comunicam com o BM são provenientes da sub-região agranular do IC, particularmente da parte ventral (AIV), e que o BMA e o BMP também recebem fortes sinais provenientes das porções anterior e posterior da sub-região agranular do IC, respetivamente.No decorrer da nossa investigação usando uma técnica de identificação neuronal baseada num transporte viral anterógrado também observámos que neurónios excitatórios das porções anterior e média do IC comunicam especialmente com as partes anterior e média de diversos núcleos da amígdala, incluindo o BM. Em particular, verificámos que a porção anterior da insula projeta especificamente para os núcleos basolateral (BLA) e lateral (La) da amígdala enquanto que a parte média da insula envia fibras para uma variedade de núcleos, incluindo o BLA, LA e o BMA, o qual não constituí o principal alvo dessa porção do IC. Tendo em consideração estes resultados nós propomos que: neurónios excitatórios da parte média da insula provavelmente estabelecem conexões anatómicas com neurónios excitatórios pós-sinápticos no BMA; a porção média da insula poderá apresentar traços de conectividade semelhantes à porção anterior e que a conectividade entre o IC e a amígdala poderá seguir um arranjo antero-posterior. Contudo, estudos futuros, como por exemplo efetuar a identificação dos neurónios a partir da porção posterior do IC, são ainda necessários para providenciar uma visão completa da arquitetura anatómica entre o IC e amígdala, e assim, confirmar as nossas hipóteses.<br>The insular cortex (IC) has been recently considered to have an important role in processing emotional feelings, including fear and anxiety. This and other functions, such as safety learning, risk prediction and anticipation, are believed to be played in concert with amygdala, a brain region that, along with IC, is also dysregulated in anxiety disorders. This hypothesis is also supported by evidence from conventional tracing studies, performed mostly in non-human primates and rats, showing wide reciprocal anatomical connections between IC and amygdala. However, as they lack cell-type specificity and the ability to trace direct synaptic connections, the precise connectivity at cellular level between the IC and amygdala is currently unknown.Since the basomedial amygdala (BM) has also been implicated in anxiety regulation, in this work, we aim to unveil the detailed architecture of connections between the IC and the BM in mice by using the mono trans-synaptic retrograde viral tracing technique. By taking advantage of the cell-type specificity and limited spread of modern rabies viral tracing strategies we identified inputs from different subregions of IC to excitatory post-synaptic neurons in the anterior and posterior part of the basomedial amygdala (BMA and BMP, respectively). We observed that most contributions to BM are from the agranular insular cortex, particularly from the ventral portion (AIV) and that BMA and BMP also receive strong inputs from the most anterior and posterior parts of the agranular insula, respectively.In the course of our investigation, by using an anterograde viral tracing methodology, we also found that excitatory neurons from the anterior and middle parts of IC establish connections particularly with the anterior and middle portions of diverse amygdaloid nuclei, including the BM. More precisely, we found that the anterior insula specifically projects to the basolateral (BLA) and lateral nuclei (La) of amygdala whereas the middle part sent fibers to a variety of nuclei, including BLA, La and BMA which turned not to be the main target of this region. Based on these findings we postulated that: excitatory neurons from the middle insula may establish connections with excitatory post-synaptic neurons in the BMA; the middle insula might present some anterior insular connectivity features and that IC-amygdala connectivity may follow an anterior-posterior arrangement. Nevertheless, further experiments, such as tracing from the posterior part of IC, are still required to provide a complete view on the precise architecture between IC and amygdala and, thus, confirm our latter hypothesis.