Hyperglycemia Induces Oxidative Stress and Impairs Axonal Transport Rates in Mice

studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits.-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively.STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged.Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed

Orexin axon density and bouton quantification in the aging rhesus macaque thalamus: a novel methodology

Thesis (M.A.)--Boston UniversitySleep fragmentation and disturbances of normal circadian rhythms are inherent in the aging human population. The rhesus macaque (Mucaca mulatta) exhibits similar disruption in sleep patterns and is a useful model to study these disturbances. Orexin is an excitatory neuropeptide that has been implicated in the regulation of wakefulness and alertness. Specifically, it has projections from its neuronal bodies in the lateral and perifornical hypothalamus to various forebrain and brainstem regions that control arousal state. One of the regions of high innervation by these orexigenic neurons is the thalamus. This study used a semi-quantitative means to determine if there are age dependent changes in Orexin innervation in the thalamus. There was a general trend of decreasing axon density when analyzed with a novel semi-quantitative method (r = -0.515, p = 0.024, df = 17) and approached significance when assessed with a previously published method (r = -0.454, p = 0.0509, df = 17), indicating a decrease with age. Additionally, there was no significant decrease in unilateral bouton counts iii when examined with age using either method. Although the findings in this study point to an age-related decrease in axon terminals, further research should examine the total orexigenic positive content in the thalamus to explain why bouton counts do not seem to change

Assessing Functional Deficits at Optic Neuritis Onset in EAE Mice Using Manganese-Enhanced MRI (MEMRI) and Diffusion fMRI

Optic neuritis: ON) is frequently a first sign of multiple sclerosis: MS), which is an inflammatory demyelinative disease of the central nerve system: CNS), including brain, optic nerve, and spinal cord. Investigating ON provides an approach to improve MS diagnosis and treatment monitoring. Experimental autoimmune encephalomyelitis: EAE) is a widely used animal model of MS and exhibits pathologies similar to the human disease. Magnetic resonance imaging: MRI) is a non-invasive tool to detect disease progress and as a standard diagnose procedure for MS in the clinic. In biological samples, the hydrogen nuclei are used to produce the MR signal due to its abundance in water and fat. As a result of tissue microstructural differences, 1H nuclei exhibit tissue-specific and pathology-specific relaxation and diffusion properties, which are reflected in the resulting MR image contrast. Therefore, the pathologies of MS, such as inflammation, demyelination, and axonal injury can be detected using different MR-related tools, including T1- and T2-weighted imaging, diffusion-weighted imaging, and diffusion tensor imaging, and so on. Importantly, direct non-invasive assessment of functional deficits could be important for understanding pathology mechanisms or provide a useful bio-index to validate treatment strategies. In this dissertation, manganese-enhanced MRI: MEMRI) and diffusion fMRI were introduced to explore the functional deficits, including axonal transport disruption and axon-activity dysfunction, at optic neuritis onset in EAE mice

Pathological Alterations Induced by intraneuronal in Alzheimer’s Disease

Die Alzheimer Demenz (AD) ist neuropathologisch durch das Auftreten von β-Amyloid (Aβ) und Neurofibrillenbündeln, die aus hyperphosphoryliertem Tau Protein bestehen, charakterisiert. Die Ablagerung von Aβ Peptiden wird als ursächlicher pathologischer Mechanismus betrachtet, da familiär auftretende Mutationen in Proteinen die in der Aβ?Kaskade eine Rolle spielen unweigerlich zur AD führen. Im Gegensatz dazu führen Mutationen im Tau Protein zu Fronto-Temporaler Demenz. Die Amyloid-Hypothese geht davon aus, dass die Akkumulation von Aβ42 ursächlich für die Beeinträchtigung von Nervenzellen und Synapsen ist und letztendlich zu Verhaltensdefiziten führt. Für viele Jahre galt das Hauptaugenmerk der Amyloid-Hypothese dem Auftreten extrazellulärer Aβ-Plaques. In einer Vielzahl von Mausmodellen, die auf familiären Mutationen basieren, konnte die Ablagerung dieser Plaques erfolgreich nachgebildet werden, allerdings traten nur schwache Verhaltensdefizite und kein deutlicher Nervenzellverlust auf. Extrazelluläre Plaque-Pathologie korreliert darüber hinaus nicht mit bei AD Patienten beobachteten kognitiven Defiziten und kommt auch bei Kontrollpatienten vor, die keine Anzeichen einer Demenz aufweisen. Kürzlich wurde eine modifizierte Amyloid-Hypothese vorgestellt, in der frühen intrazellulären Aβ-Akkumulationen, im Gegensatz zu extrazellulären Aβ Plaques eine zentrale Rolle in der pathologischen Kaskade zukommt. Allerdings ist das Vorkommen intrazellulärer Aβ Peptide bei der AD noch Gegenstand wissenschaftlicher Diskussion. Die vorliegende Arbeit untersucht das Vorkommen intrazellulärer Aβ Peptide im Hirngewebe von Alzheimer Patienten, sowie deren Rolle im Gegensatz zu extrazellulärer Plaquepathologie in transgenen Mausmodellen der AD. Nervenzellverlust, axonale Pathologie und funktionelle Defizite im Hinblick auf die Regulation der Expression früher Gene (immediate early genes, IEG) werden dabei besonders berücksichtigt. Im Hinblick auf pathologische Veränderungen bestätigt die vorliegende Arbeit die modifizierte Amyloid-Hypothese. Die Ergebnisse unterstützen die Rolle von intraneuronalem Aβ als früher Auslöser der pathologischen Kaskade und zeigen einen deutlichen Zusammenhang zu axonaler Degeneration und Nervenzellverlust auf. Im Gegensatz dazu scheinen extrazelluläre Plaques eher an funktionellen Defiziten wie etwa der Induktion von IEGs bei neuronaler Aktivität, nicht aber am Nervenzellverlust beteiligt zu sein. Durch eine Optimierung des immunhistochemischen Färbeprotokolls konnte eine deutliche Färbung intraneuronaler Aβ Peptide in Nervenzellen des Hippokampus im Hirngewebe von AD Patienten nachgewiesen werden. Darüber hinaus wurde ein Zusammenhang zwischen der Präsenz des ApoE4 Allels, einem bekannten Risikofaktor für die AD, und intraneuronalem Aβ gefunden, was die wichtige Rolle von Aβ Peptiden innerhalb von Nervenzellen bei der Pathologie der AD unterstreicht

Alkoholin palkitseviin ominaisuuksiin ja amfetamiinin myrkkyvaikutuksiin liittyvien hermostollisten järjestelmien funktionaalinen kuvantaminen

Alcohol addiction is one of the most prevalent brain disorders in the world. A major hurdle for reducing alcohol-related harms and developing effective treatments is the poor understanding of neural processes responsible for the development of dependence and addiction. Alcohol has been shown to affect various neurotransmitter systems; however, the mesolimbic dopamine (DA) system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), has been thought to play a key role in producing the reinforcing effects of alcohol. The VTA region has also been suggested to be the anatomical site for the interaction of the dopaminergic system with the opioidergic and γ-aminobutyric acid (GABA) systems. Here, manganese-enhanced magnetic resonance imaging (MEMRI) and behavioral tests were used to study drug-induced alterations in brain activity of the alcohol-preferring AA (Alko Alcohol) and heterogeneous Wistar rats. MEMRI is based on the ability of paramagnetic Mn2+ ions to accumulate in excitable neurons, thus enhancing the T1-weighted signal in activated brain regions. Mn2+ ions can also be transported anterogradely and retrogradely in neurons, released to the synaptic cleft, and taken up by other neurons. These properties allow MEMRI to measure long-term changes in brain activity, as well as map neural pathways involved in acute and long-term drug actions, including drug reward and toxicity. The AA rats exposed to alcohol compared to water controls displayed a widespread and persistent activation in brain regions that have been previously linked with alcohol reinforcement. Similarly, activity in neural pathways originating in the NAc and projecting caudally to the midbrain was enhanced in alcohol drinking rats. Moreover, this alcohol-induced activation was blocked by systemic naltrexone (NLX) administration. Comparison of naïve AA and Wistar rats revealed a lowered basal activity in the caudal linear nucleus (CLi) of AA rats, which was restored by voluntary alcohol drinking. The intra-CLi γ-aminobutyric acid type A receptor (GABAA) agonist muscimol produced a dose-dependent increase in alcohol drinking, blocked by co-administration of the GABAA antagonist bicuculline, suggesting that the CLi GABAergic system is involved in the regulation of alcohol reward. MEMRI was also employed for assessing stimulant-induced toxicity. Methamphetamine and mephedron displayed disparate effects on brain activity, as methamphetamine produced widespread decreases in activity, whereas mephedron increased activity in limited brain areas. Taken together, the use of MEMRI for mapping alcohol- and stimulant-induced alterations in functional brain activity revealed networks and specific pathways that have potential for guiding further translational efforts to develop medications for drug abuse disorders, as well as for evaluating drug-induced toxicity.Alkoholismi on maailman yleisimpiä aivosairauksia. Alkoholin aiheuttamien haittojen vähentämistä ja tehokkaiden hoitomuotojen kehittämistä haittaa se, että päihderiippuvuuden kehittymiseen vaikuttavat aivojen toiminnan muutokset ymmärretään yhä huonosti. Alkoholin vaikutukset syntyvät monien hermoston välittäjäaineiden toiminnan kautta, ja etenkin nk. mesolimbisellä dopamiinijärjestelmällä on arveltu olevan keskeinen rooli alkoholin tuottamassa mielihyvässä. Töissämme käytettiin mangaanitehosteista magneettiresonanssikuvantamista (MRI) selvitettäessä alkoholin ja stimulanttien vaikutuksia alkoholiin mieltyneiden AA-rottien ja normaalien Wistar-rottien aivoissa. Kyseinen kuvantamismenetelmä perustuu siihen, että magneettisia ominaisuuksia omaavat mangaani-ionit kulkeutuvat hermopäätteiden kalsiumkanavien kautta hermosoluihin niiden aktivoituessa. Mangaani-ioneja sisältävien hermosolujen muodostamat radat ja aivoalueet tuottavat mitattavissa olevan signaalin. Mangaani voi myös siirtyä synapsiraon ylitse viereiseen hermosoluun ja tuottaa siten MRI-kuvan aktiivisista hermoverkoista. Viikkoja kestänyt alkoholin juominen johti aivojen aktiivisuusmuutoksiin sellaisilla aivojen alueilla, joiden on aikaisemmin oletettu liittyvän alkoholin positiivisiin vaikutuksiin. Selvitettäessä tarkemmin yksittäisten hermoratojen merkitystä havaittiin, että etuaivojen accumbens-tumakkeen keskiaivoihin lähettävä rata aktivoitui alkoholin juomisen seurauksena, kun taas alkoholismin hoidossa käytettävä lääkeaine, naltreksoni, vähensi alkoholin aiheuttamaa aktivaatiota. Verrattaessa alkoholiin mieltyneitä rottia tavalliseen rottakantaan löydettiin keskiaivoista tumake (kaudaalinen lineaaritumake), jonka aktiivisuus oli ennen alkoholin juomista tavallista alhaisempi, mutta jota alkoholi aktivoi. Kun tähän tumakkeeseen annettiin ainetta, joka salpasi gamma-aminovoihapon (GABA) vastaanottokohdat, alkoholin kulutus lisääntyi huomattavasti. Tämä löydös viittasi keskiaivojen GABA-järjestelmän merkitykseen alkoholin kulutuksen säätelyssä. Kaikkiaan kehittämämme mangaanitehosteinen magneettiresonanssikuvantaminen tuotti uutta tietoa alkoholin juomista säätelevistä hermoradoista ja yksittäisistä aivojen alueista. Tätä tietoa voidaan käyttää hyväksi, kun suunnitellaan ja testataan alkoholismin hoitoon tarkoitettuja lääkeaineita

IRM du manganèse (MEMRI) (couplage à l'imagerie chimique par microsonde synchrotron pour optimiser l'imagerie fonctionnelle du transport neuronal)

Résumé Le manganèse (Mn2+) est un élément essentiel du corps humain. Ses propriétés paramagnétiques permettent son utilisation comme agent de contraste pour l'IRM (Mn-MRI ou MEMRI). Analogue du calcium (Ca2+), il pénètre les neurones essentiellement par les canaux calciques. Il est ensuite transporté le long des microtubules jusqu'aux synapses où il est libéré, puis capturé par les autres neurones. Ainsi, il peut rendre compte du transport axonal antérograde et rétrograde. L'approche MEMRI peut ainsi apporter des informations uniques sur la connectivité fonctionnelle cérébrale. Toutefois, deux problèmes limitent l'emploi de ce puissant outil d'imagerie in vivo : (i) A doses élevées, le Mn2+ est toxique pour l'organisme et peut provoquer une atteinte grave du système nerveux central, appelé manganisme. Les niveaux et les mécanismes de toxicité sont mal connus. (ii) Le mode de transport du manganèse dans l'approche MEMRI est mal connu. Afin d'apporter des éléments de réponse à ces deux problèmes, nous avons entrepris une étude couplant IRM et microscopie synchrotron pour mieux comprendre le comportement du Mn2+ in vivo. Nous avons précisé les distributions cellulaire et sub-cellulaire du Mn et d'autres métaux pour un modèle de cellules de type neuronal (lignée de neuroblastome N2A), pour des cultures primaires de neurones hippocampiques, mais aussi au niveau de coupes d'hippocampe de rats. En parallèle, nous avons étudié les effets du Mn sur le métabolisme cérébral par une technique de RMN-HRMAS du proton. Pour compléter ce travail, nous avons mis en œuvre l'imagerie MEMRI chez les souris KO MAP6 présentant un déficit d'une protéine stabilisatrice des microtubules pour évaluer la connectivité fonctionnelle du tract thalamo-cortical. Mots clés Hippocampe, MAP6, manganèse, métabolisme, métal, neurone, MRI, rongeurs, synchrotron.Abstract Manganese (Mn2+) is an essential element for human body. The paramagnetic properties of Mn2+ permit it use as a contrast agent for MRI (Mn-MRI or MEMRI). Analogue of calcium (Ca2+), it enters neurons primarily by calcium channels. It is then transported along microtubules to the synapse where it is released and then captured by other neurons. Thus, it can account for the anterograde and retrograde axonal transport. The MEMRI approach can provide unique information about cerebral functional connectivity. Two problems limit the use of this powerful tool for in vivo imaging: (i) At high doses, Mn2+ is toxic to the body and can cause serious problem of the central nervous system, called manganism. The level and the mechanisms of toxicity are poorly understood. (ii) The mode of manganese transport in the MEMRI approach is unclear. To address these two issues, we undertook a study coupling MRI and synchrotron microscopy to study the Mn 2+ behavior in vivo. We characterized the cellular and subcellular distributions of Mn and other metals in "pseudo neurons" cell line N2A, primary cultures of hippocampal neurons, andin hippocampal slices from rats. In parallel, we studied the effects of Mn on brain metabolism by proton-HRMAS NMR . In parallel, weevaluated MEMRI in MAP6 KO mice which exhibit a deficit in microtubule stabilizing protein, to assess the functional connectivity of the thalamocortical tract. Key words hippocampus, MAP6, manganese, metabolism, metal, neuron, MRI, rodent, synchrotron.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Interactions olfacto-alimentaires : étude fonctionnelle de la plasticité du système olfactif chez deux modèles murins de l’obésité

Olfaction makes possible both the detection and the processing of odors related to food location and palatability. Interestingly, receptors to anorexigen and orexigen hormones and neuropeptides found in the hypothalamus are also expressed in the main olfactory bulb (MOB), suggesting that feeding state has an impact on odor representation. However, neuronal mechanisms linking olfaction, food intake and metabolic disorders are still unclear. In this PhD thesis, we wanted to further understand mechanisms of the olfactory-feeding crosstalk and tested the effects of obesity on olfactory activity in the MOB. Leptin, a peptidergic hormone produced by adipocytes, is a major regulator of the energy balance and inhibits food intake. Ob/ob mice are deficient in leptin from birth and are widely used as a murine model of obesity. We used a functional neuroimaging technique, Manganese Enhanced Magnetic Resonance Imaging (MEMRI), in association with statistical parametric mapping (SPM) to monitor food odor-evoked spatial activity in the MOB of these mice. Using MEMRI, we found that odor-evoked signal is different, both in terms of intensity and localization between lean and ob/ob mice for the spontaneous activity and in response to food odor. However, this is not the case for a neutral odor. Moreover, leptin impacts both spontaneous activity and food odor-evoked signal intensity in both groups. Interestingly leptin strongly activates deep layers (mitral cell layer and mainly granule cell layer) of the MOB. To pinpoint what cellular/molecular mechanisms can be responsible for these changes in the spatial distribution of activity, we analyzed the effects of the lack of leptin on the composition and activity of the MOB cellular network.We quantified bulbar adult neurogenesis and found that 21 days after BrdU injections, a cell birth marker, ob/ob mice showed an increased number of both new periglomerular and granular cells as compared to control, suggesting that leptin regulates new neuron elimination. We also tested whether an inflammation could be occurring in the MOB. Using RTPCR of different neuronal and glial markers we did not observe strong signs of inflammation. Then we probed changes in local network activity in the deep layers of the MOB by recording local field potentials during a Go/NoGo odor discrimination task. We observed that the power of beta oscillations, the functional marker of the olfactory network between the MOB and the olfactory cortex, is way higher in the MOB of ob/ob mice compared to control mice.La compréhension des mécanismes cérébraux régulant la prise alimentaire est devenue cruciale face à la pandémie mondiale d’obésité. L’ingestion d’aliments est sous le contrôle d’un large réseau de structures cérébrales qui régulent la balance énergétique en régulant l’apport de nourriture et la dépense énergétique. Etant donné l’importance de l’olfaction dans l’appréciation des aliments, il est primordial d’identifier des mécanismes précis liant olfaction et nutrition.Nous avons choisi d’étudier l’activité dans le Bulbe Olfactif principal (BO), la première structure cérébrale qui permet la représentation spatiotemporelle des odeurs. De façon intéressante, des récepteurs aux hormones anorexigènes et orexigènes sont exprimés à la fois dans l’hypothalamus et le BO. Toutefois, l’impact de la leptine (et de l’obésité) sur l’activité du BO in vivo demeure inconnu.Pour évaluer l’action de la leptine et de l’obésité sur la plasticité olfactive, nous avons choisi le modèle murin ob/ob. Nous avons d’abord décrit la répartition spatiale de l’activité dans le BO en réponse à une odeur alimentaire et à une odeur neutre chez ces souris en les comparant à des souris témoins grâce une technique de neuroimagerie fonctionnelle, l’Imagerie par Résonance Magnétique renforcée au Manganèse (MEMRI). Par le développement d’une méthode de comparaison statistique indépendant de l’utilisateur, le SPM (Statistical Parametric Mapping), nous avons mis en évidence une augmentation de l’activité spontanée et de l’activité induite par odeur alimentaire dans le BO des ob/ob. Cependant, l’activité induite par une odeur neutre n’était pas plus élevée. L’injection de leptine a pour résultat d’augmenter le signal MEMRI dans les couches profondes du BO et ne restaure que partiellement l’activité des ob/ob. En quantifiant la neurogenèse dans le BO par injections de BrdU, nous avons mis en évidence une survie accrue des cellules granulaires et périglomerulaires chez les ob/ob.Pour évaluer la dynamique temporelle de l’activité du BO, nous avons enregistré par électrophysiologie les variations de l’activité oscillatoire du potentiel de champ local dans les couches profondes du BO pendant l’apprentissage d’une tâche de discrimination olfactive, le Go/NoGo. L’apparition d’une oscillation lente dite bêta (15-40 Hz) pendant cet apprentissage est un marqueur fonctionnel de l’état du réseau olfactif. Elle est dépendante des retours centrifuges provenant du cortex olfactif, majoritairement du cortex piriforme. Si nous n’avons pas observé de différence importante dans la discrimination olfactive, nous avons en revanche mis en évidence un problème motivationnel chez les animaux ob/ob, qui n’effectuaient la tâche que si la récompense hydrique contenait du sucre. Nos enregistrements montrent une forte augmentation des oscillations bêta chez les souris obèses après apprentissage comparé aux souris témoins