Дисертації з теми "Viral tracings"

To understand how the nervous system processes information, a map of the connections among neurons is essential. Viral transsynaptic transmission has gained popularity as a method for labeling neural circuits. In particular, the development of retrograde monosynaptic tracing vectors has enabled visualization of the pre-synaptic inputs onto defined sets of postsynaptic neurons. This system utilized the rabies virus (RABV), in which the glycoprotein gene in the virus was deleted, and re-supplied in trans. In order to build alternative, more flexible tracers, we made recombinant VSV genomes, first developing the use of vesicular stomatitis virus (VSV) for tracing neuronal connections. Viruses encoding several different fluorescent proteins were made, giving brilliantly labeled neurons, bright enough for live imaging and characterization of the detailed morphologies of cells. Expression was very rapid, facilitating identification of neurons both in vivo and in ex vivo applications. In addition, the use of an avian glycoprotein (ASLV-A) allowed specific targeting to cells expressing an avian glycoprotein receptor (TVA). This allowed monosynaptic tracing from defined starter cells. In order to alter the direction and cell type specificity of transmission, we then fitted VSV with a glycoprotein from one of multiple other viruses. Glycoproteins such as the rabies virus glycoprotein (RABV-G) endowed VSV with the ability to spread in a retrograde transsynaptic pattern, while the glycoproteins from viruses such as the lymphocytic choriomeningitis virus (LCMV) gave an anterograde pattern of transsynaptic spread. This anterograde or retrograde spread was observed in all species tested, and even for other non-VSV viruses, such as lentiviruses. We also developed transsynaptic tracing viruses which direct viral spread between defined cell types, instead of from a defined cell type to any upstream of downstream cell. In all, we developed an extensive transsynaptic tracing repertoire for tracing neuronal connections.

Une acquisition sensorielle et une perception précises sont des éléments clés de la survie. Bien que de nombreux paramètres sous-jacents au traitement des informations sensorielles soient connus, plusieurs aspects sont encore mal compris, comme la contribution exacte de chaque structure cérébrale. Ici, nous analysons la contribution cérébelleuse au traitement sensoriel dans le système des vibrisses de la souris. Nous identifions une projection anatomique et physiologique disynaptique des noyaux cérébelleux vers le cortex sensoriel primaire, impliquant notamment le thalamus postérieur médian (POm). La modulation de cette projection cérébelleuse et thalamique de type "driver" induit un déficit dans une tâche de discrimination sensorielle fine, et sa co-activation avec des entrées périphériques induit le recrutement accru des projections du POm dans la couche I du cortex sensoriel. Dans leur ensemble, nos résultats montrent que le cervelet cible aussi des zones corticales non motrices et peut directement moduler le traitement sensoriel par l'intermédiaire d'un noyau thalamique d'ordre élevé, le POm
Accurate sensory acquisition and perception are key features to survival. Though many parameters underlying the processing of sensory information is known, several aspects are still poorly understood, such as the exact contribution of each cerebral structure. Here, we analyze the cerebellar contribution to sensory processing in the mouse whisker system. We identify an anatomical and physiological disynaptic projection from the cerebellar nuclei to the primary sensory cortex, involving notably by the posterior medial thalamus (POm). The modulation of this strong driver-like cerebello-thalamic projection induces an impairment in a fine sensory discrimination task, and its co-activation along with peripheral inputs induces the increased recruitment of POm projections to layer I of sensory cortex. Taken together, our results show that the cerebellum targets non-motor cortical areas and can directly modulate sensory processing through a higher order thalamic nucleus, the POm

Le but de ce travail est l'étude de la connectivité anatomique et fonctionnelle desréseaux neuronaux et le développement des nouveaux outils à cet effet. Car le dernieraspect est une préoccupation majeure de la neuroscience actuelle, nous avonsdeveloppé d'abord un nouveau traceur virale permettant la reconstruction neuronale.Nous avons ensuite appliqué cet et d'autres techniques pour sonder les défauts deconnectivité neuronale dans le syndrome de l'X fragile.Dans la première partie, nous avons discuté les avantages et inconvénients d'unetechnique émergente en utilisant un nouveau type de vecteur viral qui permet uneunique application pour l’étude du cerveau.Dans la deuxième partie, nous avons développé, au départ de ce vecteur viral, unenouvelle variante de faciliter le traçage et reconstruction des caractéristiquesmorphologiques de neurones. Nous avons montré la force de cette varianteantérograde du virus de la rage recombinant glycoprotéine supprimé pour lareconstruction de calcul de toutes les caractéristiques morphologiques clés deneurones: les dendrites, épines, les axones longs envergure dans tous les terminaux ducerveau et les boutons.Dans la troisième partie, nous avons examiné les modifications dans la connectivitédes structures cérébrales dans le syndrome du X fragile (FXS). FXS est le retardmental héréditaire la plus fréquente et la forme génétique la plus fréquente del'autisme, ce qui conduit à l'apprentissage et de la mémoire des déficits, lescomportements répétitifs, des convulsions et une hypersensibilité à des stimulisensoriels (visuels). Une des hypothèses éminents dans le domaine de l'autismesuppose une phénotype de hyper-connectivité locale mais de hypo-connectivité pourles connexions longue portée. Pour tester cette hypothèse dans un modèle de sourisFXS nous avons utilisé l'imagerie par résonance magnétique, pour balayer la totalitédu cerveau et de mesurer la connectivité anatomique et fonctionnel. Cela nous apermis d'identifier des altérations de connectivité dans plusieurs domains. Après nous8avons utilisé des traceurs viraux pour explorer un de ceux domains plus detaillé. Enutilisant le virus de la rage rétrograde à quantifier le nombre de neurones projetantvers ces zones, nous avons confirmé une connectivité d'entrée modifié pour le cortexvisuel primaire, ce qui pourrait contribuer au traitement visuel altéré de l'information.Nous avons découvert une connectivité réduite à longue portée globale anatomique etfonctionnelle entre plusieurs régions du cerveau, l'identification FXS comme unepathologie de la connectivité neuronale, ce qui pourrait expliquer les difficultés deplusieurs stratégies de sauvetage visant des cibles moléculaires sont actuellementconfrontés
The goal of this work was the investigation of the anatomical and functionalconnectivity of neuronal networks and the development of novel tools for thispurpose. Since the latter aspect is a major focus of current neuroscience, we firstsought a novel viral tracer enabling sparse neuronal reconstruction and neuronclassification. We then applied this and other techniques to probe neuronalconnectivity defects in Fragile X Syndrome.In the first part we discussed the merits and drawbacks of a emergingtechnique using a new type of viral vector that allows in a unique manner mapping ofthe input of a given brain area.In the second part we developed, departing from this viral vector, a newvariant to facilitate the tracing and reconstructing of morphologic features of neurons.We showed the strength of this anterograde variant of the recombinant glycoproteindeletedrabies virus for computational reconstruction of all key morphologicalfeatures of neurons: dendrites, spines, long-ranging axons throughout the brain andbouton terminals.In the third part we examined alterations in the wiring of brain structures inthe Fragile X Syndrome (FXS). FXS is the most common inherited mental retardationand most frequent genetic form of autism, leading to learning and memory deficits,repetitive behavior, seizures and hypersensitivity to sensory (e.g. visual) stimuli. Oneof the eminent hypotheses in the autism field assumes a local hyper- connectivityphenotype but hypo-connectivity for long-ranging connections. To test this hypothesisin a FXS mouse model we used magnetic resonance imaging, to scan the entire brainand measure the anatomical and functional connectivity. This allowed us to identifyconnectivity alterations in several areas that we further explored using viral tracers.Using retrograde rabies virus to count the number of neurons projecting to such areaswe confirmed an altered input connectivity to the primary visual cortex, which couldcontribute to the altered visual information processing. We discovered an overallreduced anatomical and functional long-range connectivity between several brainareas, identifying FXS as pathology of neuronal connectivity, which might explain thedifficulties several rescue strategies aiming at molecular targets are currently facing

Neuroscience
Ph.D.
Much of our understanding of the fascinating complexity of neuronal circuits comes from anatomical tracing studies that use dyes or fluorescent markers to highlight pathways that run through the brain and spinal cord. Viral vectors have been utilized by many previous groups as tools to highlight pathways or deliver transgenes to neuronal populations to stimulate growth after injury. In a series of studies, we explore anterograde and retrograde tracing with viral vectors to trace spinal pathways and explore their contribution to behavior in a rodent model. In a separate study, we explore the effect of stimulating intrinsic growth programs on regrowth of corticospinal tract (CST) axons after contusive injury. In the first study, we use self-complimentary adeno associated viral (scAAV) vectors to trace long descending tracts in the spinal cord. We demonstrate clear and bright labeling of cortico-, rubro- and reticulospinal pathways without the need for IH, and show that scAAV vectors transduce more efficiently than single stranded AAV (ssAAV) in neurons of both injured and uninjured animals. This study demonstrates the usefulness of these tracers in highlighting pathways descending from the brain. Retrograde tracing is also a key facet of neuroanatomical studies involving long distance projection neurons. In the next study, we highlight a lentivirus that permits highly efficient retrograde transport (HiRet) from synaptic terminals within the cervical and lumbar enlargements of the spinal cord. By injecting HiRet, we can clearly identify supraspinal and propriospinal circuits innervating MN pools relating to forelimb and hindlimb function. We observed robust labeling of propriospinal neurons, including high fidelity details of dendritic arbors and axon terminals seldom seen with chemical tracers. In addition, we examine changes in interneuronal circuits occurring after a thoracic contusion, highlighting populations that potentially contribute to spontaneous behavioral recovery in this lesion model. In a related study, we use a modified version of HiRet as part of a multi-vector system that synaptically silences neurons to explore the contribution of the rubrospinal tract (RST) and CST to forelimb motor behavior in an intact rat. This system employs Tetanus toxin at the neuronal synapse to prevent release of neurotransmitter via cleavage of vesicle docking proteins, effectively preventing the propagation of action potentials in those neurons. We find that shutdown of the RST has no effect on gross forelimb motor function in the intact state, and that shutdown of a small population of CST neurons in the FMC has a modest effect on grip strength. These studies demonstrate that the HiRet lentivirus is a unique tool for examining neuronal circuitry and its contribution to function. In the final study, we explore stimulation of the Phosphoinositide 3-kinase/Rac-alpha serine/threonine Protein Kinase (PI3K/AKT) growth pathway by antagonizing phosphatase and tensin homolog (PTEN), a major inhibitor, to encourage growth of CST axons after a contusive injury. We use systemic infusions of four distinct PTEN antagonist peptides (PAPs) targeted at different sites of the PTEN protein. We find robust axonal growth and sprouting caudal to a contusion in a subset of animals infused with PAPs targeted to the PTEN enzymatic pocket, including typical morphology of growing axons. Serotonergic fiber growth was unaffected by peptide infusion and did not correlate with CST fiber density. Though some variability was seen in the amount of growth within our animal groups, we find these PTEN antagonist peptides a promising and clinically relevant tool to encourage CST sprouting, and a potentially useful addition to therapies using combinatory strategies to enhance growth. These studies demonstrate that viral tracing is a powerful tool for mapping spinal pathways and elucidating their ability to reform spinal circuits after injury. Viral vectors can be used in both anterograde and retrograde tracing studies to highlight intricacies of neuronal cell bodies, axons and dendritic arbors with a high degree of fidelity. In the injured state, these tools can help identify pathways that contribute to spontaneous recovery of function by highlighting those that reform circuits past an injury site. In the uninjured state, these vectors can contain neuronal silencing methods that help define the contribution of specific pathways to behavior.
Temple University--Theses

One feature of the brain is that different parts of it respond to different stimuli. This means not all brain regions or neurons within those regions are active at a given moment. This feature of the brain gives it the ability to encode and store a wide range of stimuli that are then used to make predictions about a changing external environment. Activation of non-overlapping neural populations is fundamental to the ability to encode a wide range of stimuli to represent a changing environment. To examine the limits of this idea we used genetic tools to label active cell populations following a neutral stimulus presentation or a learned negative association with the same stimulus. The study examined the degree of similarity between these active populations by comparing key features of the active neurons including gene expression and monosynaptic inputs. Another feature of the brain is its ability to store information. In a neural population recently activated by a salient stimulus, molecular processes occur that result in the formation and maintenance of a memory. Collectively these processes are referred to as plasticity, and act on short and long time scales to strengthen the connections between active neurons and weaken the connections between inactive ones. Plasticity processes are not only necessary for the formation and storage of memories but also for wiring up the nervous system during development. A molecule called ZIP has been shown to erase memories months after formation and specifically affects plasticity on longer time scales. However, the effects of ZIP on the developing brain are not well understood and difficult to study using ZIP’s typical delivery method of injection into the brain. To facilitate a developmental study of ZIP’s effects, we made a genetic tool that can specify where and when ZIP is delivered to the brain. Results of the study indicated that males were particularly vulnerable to ZIP during early development while females were unaffected. Together these results provide insight into the limits of information coding potential at the anatomical level and reveal a fundamental difference in plasticity processes in males and females.
10000-01-01

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
Kyoto University (京都大学)
0048
新制・論文博士
博士(医学)
乙第13040号
論医博第2115号
新制||医||1017(附属図書館)
33032
京都大学大学院医学研究科医学専攻
(主査)教授 渡邉 大, 教授 影山 龍一郎, 教授 髙橋 良輔
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
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
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
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.
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.