Volumetric Manganese Enhanced Magnetic Resonance Imaging in mice (mus musculus)

The present doctoral thesis introduces a method for semi-automatic volumetric analysis of the hippocampus and other distinct brain regions in laboratory mice. The method of volumetric manganese enhanced magnetic resonance imaging (vMEMRI) makes use of the paramagnetic property of the manganese ion, Mn2+, which results in a positive contrast enhancement of specific brain areas on the MR image and enables a more detailed image of brain morphology. The chemical similarity of Mn2+ to Calcium leads to an accumulation of Mn2+ in excited cells and consequentially an enhanced signal in certain brain regions in an activity dependent manner. However, one major drawback for vMEMRI is the toxicity of Mn2+. Therefore, the aims of the thesis have been: (1) Establishment of a MEMRI protocol in mice (2) Optimization of a Mn2+ application procedure to reduce toxic side effects (3) Development of an automatized method to determine hippocampal volume (4) Validation of vMEMRI analysis (5) Application of volumetric analysis in mouse models of psychopathology This thesis splits into 3 studies. Study 1 deals with Mn2+ toxicity and introduces an application method that considerably reduces the toxic side effects of Mn2+. Study 2 validates vMEMRI as a method to reliably determine hippocampal volume and explores its utilization it in animals with genetically and chemically modified hippocampi. Study 3 displays the application vMEMRI in a mouse model of a psychiatric disorder. Study 1 shows that a single application of Mn2+ in dosages used in current MEMRI studies leads to considerable toxic side effects measurable with physiological, behavioral and endocrine markers. In contrast, a fractionated application of a low dose of Mn2+ is proposed as an alternative to a single injection of a high dose. Repeated application of low dosages of 30 mg/kg Mn2+ showed less toxic side effects compared to the application schemes with higher dosages of 60 mg/kg. Additionally, the best vMEMRI signal contrast was seen for an injection protocol of 30 mg/kg 8 times with an inter-injection interval of 24 h (8x30/24 protocol). The impact of the 8x30/24 application protocol on longitudinal studies was tested by determining whether learning processes are disturbed. Mice were injected with the 8x30/24 protocol 2 weeks prior to receiving a single footshock. Manganese injected mice showed less contextual freezing to the shock context and a shock context reminder one month after shock application. Furthermore, mice showed increased hyperarousal and no avoidance of shock context related odors. This impairment in fear conditioning indicates a disturbed associative learning of Mn2+ injected mice. Therefore, it was investigated whether Mn2+ application shows a specific disturbance of hippocampus dependent learning. Mice were subjected to habitual and spatial learning protocols 12 h after each injection in a water cross-maze. There was no impairment in learning protocols which allowed for hippocampus-independent habitual learning. However, Mn2+ injected mice were specifically impaired in the hippocampus-dependent spatial learning protocol. Furthermore, it was shown that only mice with higher Mn2+ accumulation showed this impairment. Altogether, the results of this chapter argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects. However, the treatment procedure has to be further improved to allow for an analysis of hippocampus-dependent learning processes as well. Because of the potential side effects, the vMEMRI method was applied as a final experiment in study 2 and 3. Study 2 introduces the method of vMEMRI, which allows, for the first time, an in vivo semi-automatic detection of hippocampal volume. Hippocampal volume of mice with genetically altered adult neurogenesis and those with chemically lesioned hippocampi could be analyzed with vMEMRI. Even the highly variable differences in hippocampal volume of these animals could be detected with vMEMRI. vMEMRI data correlated with manually obtained volumes and are in agreement with previously reported histological findings, indicating the high reliability of this method. Study 3 investigates the ability of vMEMRI to detect even small differences in brain morphology by examining volumetric changes of the hippocampus and other brain structures in a mouse model of PTSD supplemented with enriched housing conditions. It was shown, that exposure to a brief inescapable foot shock led to a volume reduction in both the left hippocampus and right central amygdala two months later. Enriched housing decreased the intensity of trauma-associated contextual fear independently of whether it was provided before or after the shock. vMEMRI analysis revealed that enriched housing led to an increase in whole brain volume, including the lateral ventricles and the hippocampus. Furthermore, the enhancement of hippocampal volume through enriched housing was accompanied by the amelioration of trauma-associated PTSD-like symptoms. Hippocampal volume gain and loss was mirrored by ex vivo ultramicroscopic measurements of the hippocampus. Together, these data demonstrate that vMEMRI is able to detect small changes in hippocampal and central amygdalar volumes induced by a traumatic experience in mice. In conclusion, vMEMRI proves to be very reliable and able to detect small volumetric differences in various brain regions in living mice. vMEMRI opens up a great number possibilities for future research determining neuroanatomical structure, volumes and activity in vivo as well as the ability to repeatedly determine such characteristics within each subject, given an improvement of the Mn2+ treatment protocols to minimize potential toxic side effects

Manganese-Enhanced Magnetic Resonance Imaging: Overview and Central Nervous System Applications With a Focus on Neurodegeneration

Manganese-enhanced magnetic resonance imaging (MEMRI) rose to prominence in the 1990s as a sensitive approach to high contrast imaging. Following the discovery of manganese conductance through calcium-permeable channels, MEMRI applications expanded to include functional imaging in the central nervous system (CNS) and other body systems. MEMRI has since been employed in the investigation of physiology in many animal models and in humans. Here, we review historical perspectives that follow the evolution of applied MRI research into MEMRI with particular focus on its potential toxicity. Furthermore, we discuss the more current in vivo investigative uses of MEMRI in CNS investigations and the brief but decorated clinical usage of chelated manganese compound mangafodipir in humans

Manganese-Enhanced Magnetic Resonance Imaging: Overview and Central Nervous System Applications With a Focus on Neurodegeneration

Manganese-enhanced magnetic resonance imaging (MEMRI) rose to prominence in the 1990s as a sensitive approach to high contrast imaging. Following the discovery of manganese conductance through calcium-permeable channels, MEMRI applications expanded to include functional imaging in the central nervous system (CNS) and other body systems. MEMRI has since been employed in the investigation of physiology in many animal models and in humans. Here, we review historical perspectives that follow the evolution of applied MRI research into MEMRI with particular focus on its potential toxicity. Furthermore, we discuss the more current in vivo investigative uses of MEMRI in CNS investigations and the brief but decorated clinical usage of chelated manganese compound mangafodipir in humans

Manganese Enhanced MRI for Use in Studying Neurodegenerative Diseases

MRI has been extensively used in neurodegenerative disorders, such as Alzheimer’s disease (AD), frontal-temporal dementia (FTD), mild cognitive impairment (MCI), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS). MRI is important for monitoring the neurodegenerative components in other diseases such as epilepsy, stroke and multiple sclerosis (MS). Manganese enhanced MRI (MEMRI) has been used in many preclinical studies to image anatomy and cytoarchitecture, to obtain functional information in areas of the brain and to study neuronal connections. This is due to Mn2+ ability to enter excitable cells through voltage gated calcium channels and be actively transported in an anterograde manner along axons and across synapses. The broad range of information obtained from MEMRI has led to the use of Mn2+ in many animal models of neurodegeneration which has supplied important insight into brain degeneration in preclinical studies. Here we provide a brief review of MEMRI use in neurodegenerative diseases and in diseases with neurodegenerative components in animal studies and discuss the potential translation of MEMRI to clinical use in the future

Manganese-Enhanced Magnetic Resonance Imaging: Overview and Central Nervous System Applications With a Focus on Neurodegeneration

Manganese-enhanced magnetic resonance imaging (MEMRI) rose to prominence in the 1990s as a sensitive approach to high contrast imaging. Following the discovery of manganese conductance through calcium-permeable channels, MEMRI applications expanded to include functional imaging in the central nervous system (CNS) and other body systems. MEMRI has since been employed in the investigation of physiology in many animal models and in humans. Here, we review historical perspectives that follow the evolution of applied MRI research into MEMRI with particular focus on its potential toxicity. Furthermore, we discuss the more current in vivo investigative uses of MEMRI in CNS investigations and the brief but decorated clinical usage of chelated manganese compound mangafodipir in humans

Behavioral and neuronal responses of two mouse species, Mus musculus and Mus spicilegus, to con- and heterospecific olfactory signals

The behavioral and neuronal responses of the males of two closely related species of mice (Mus musculus  wagneri, M. spicilegus) to the urine odors of estrus con- and heterospecific females were studied. In two-choice odor tests males significantly longer investigated odor of conspecific females in comparison with heterospecific ones. To investigate neuronal activation in the main and accessory olfactory bulbs (MOB and AOB), one of the methods of functional tomography – manganese-enhanced MRI ( ME-MRI) – was used. There was a significant increase in Mn2+ accumulation in the dorsal section of the posterior part of the MOB in male M. m. wagneri and M. spicilegus exposed to odor of conspecific females compared with the control group males (odor not exposed). There was a local significant increase in manganese accumulation in the dorsal region of the anterior part of the MOB in the case of the exposure of odor of a heterospecific female. The exposure of heptanone-2 to mice resulted not only in an increase in Mn2+ accumulation in certain zones, but also in a significant decrease in the accumulation of Mn2+ in the rest of the olfactory bulbs. A significant increase in the accumulation of MRI contrast in AOB was observed in males only in the case of female urine-conspecific odor exposure. The results support the previously stated assumption of a significant difference in chemical communication systems in two species of mice. A comparison of these results and results obtained previously demonstrated the absence of any differences in behavioral and neuronal responses to con- and heterospecific odors of the house mouse subspecies allopatric (M. m. wagneri) and sympatric (M. m. musculus) to M. spicilegus. This fact does not allow us to assume the effect of the mechanism of “reinforcement” in the process of evolution in the formation of precopulatory reproductive isolation between the sympatric species M. spicilegus and M. musculus

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

Investigating energy homeostasis using in vivo imaging techniques

Obesity is a major growing cause of death with no effective long-term treatment apart from surgical procedures such as Roux-en-Y gastric bypass (RYGB). The sustained weight loss following surgery is thought to be due in part to the increased levels of circulating anorexigenic gut hormones such as peptide YY3-36 (PYY3-36). Peripheral administration of PYY3-36 suppresses appetite in rodents and man and represents a potential therapy for obesity however its effects on feeding are transient. Here I have characterized a long-lasting PYY3-36 analogue; PYY3-36 latrotoxin (PYY3-36 (LT)) which demonstrated greater longevity than PYY3-36 in acute feeding studies and that produced a 10% reduction in bodyweight following once daily peripheral administration in a one month chronic feeding study performed on diet induced obese mice. Manganese enhanced MRI (MEMRI) has been used previously to assess the effects of nutritional status and gut hormones on brainstem and hypothalamic neuronal activity in vivo. I revealed through the MEMRI technique that the anorexigenic hormone pancreatic polypeptide (PP) modulates hypothalamic neuronal activity in fasted mice. Following this work, I performed more in-depth MEMRI experiments that revealed differential temporal effects of PYY3-36 and PYY3-36 (LT) on hypothalamic neuronal activity in fasted mice after treatment that corresponded to the effects seen with these peptides in feeding stuides. I also performed pharmacological MEMRI studies with the food additive monosodium glutamate (MSG) and confirmed its excitatory effect on neurons of the arcuate nucleus of the hypothalamus (ARC) following peripheral administration to mice. Roux-en-Y gastric bypass has been shown to increase energy expenditure possibly via a brown adipose tissue (BAT) mediated mechanism. I performed positron emission tomography-computed tomography (PET-CT) experiments on a rodent model of RYGB. I observed no differences in the activity of BAT of RYGB animals compared to shamoperated controls, indicating means other than increased BAT activity account for the weight loss seen in this bariatric model. The work in this thesis demonstrates that long-lasting PYY3-36 analogues represent a viable obesity therapy. Furthermore, I have further developed both MEMRI and PET-CT techniques for the study of energy balance

The association between physical activity parameters and olfactory function

Le système olfactif a un impact significatif sur la qualité de vie. Une diminution de l'odorat peut influencer la santé physique et mentale des individus, or l’activité physique (AP) pourrait améliorer la fonction olfactive. Cette thèse vise à déterminer l'association entre AP et l'olfaction et si la durée, la fréquence et le volume de l’AP d’intensité moyenne à élevée pouvaient être liés à la fonction olfactive chez l'adulte. Les données (n = 3527) ont été extraites de l'enquête américaine National Health and Nutrition Examination Survey (NHANES) de 2013-2014. L'étude comprenait des participants qui ont rempli à la fois le questionnaire autodéclaré sur le niveau d’AP et des tests d'odeur (chocolat, fraise, raisin, oignon, fumée, gaz naturel, cuir et savon). L'association entre l'olfaction et les paramètres d’AP a été évaluée à l'aide de corrélations et de régressions logistiques, rapportées ici sous forme de d'intervalles de confiance (IC) à 95 % et de rapport de cotes (RC). La valeur p≤0,05 était considérée comme statistiquement significative. En analysant l'association entre les paramètres de l'AP et le score olfactif total, il a été montré une corrélation positive avec la durée, la fréquence de l'AP modérée et la fréquence de l'AP vigoureuse, ainsi que le volume total de l'AP (tous p≤ 0,05). La durée, (intervalles de 10 min/jour), une fréquence (jour/semaine) et un volume (METs*h/semaine) plus élevés d'AP modérée étaient associés à une plus grande capacité à détecter les odeurs de raisin [1,002< RCs <1,047, IC95 % (1,000 -1,082), p=0,007]. La fréquence des AP modérées était positivement associée à la capacité à identifier les odeurs de fumée [RC =1,074, IC 95% (1,019-1,131), p=0,008] et de cuir [RC =1,060, IC 95% (1,019-1,103), p= 0,004]. De plus, la fréquence des AP vigoureuses était positivement associée à la détection de l'odeur de raisin [RC = 1,002, IC 95 % (1,000-1,005), p = 0,028]. D'après ces résultats, les paramètres du PA sont liés à la capacité de détection de certaines odeurs, principalement pour l’AP modérée. Cependant, il faut considérer que l'effet constant de l'AP sur les chances de détecter correctement les odeurs est son meilleur de 7.4%. Par conséquent, ces résultats pourront être utilisés par d'autres chercheurs et kinésiologues pour explorer comment améliorer les troubles olfactifs à l'avenir. En ce sens, l'AP d'intensité modérée pourrait particulièrement être recommandée pour la prévention et le traitement des certains troubles olfactifs.The olfactory system has a significant impact on the quality of life. A decrease in the sense of smell may influence individuals’ physical and mental health, and physical activity (PA) might improve olfactory function. This thesis aimed to determine the association between PA and olfaction and whether the duration, frequency, and volume of moderate to vigorous PA could be related to the olfactory function in adults. The data (n=3527) were extracted from the U.S. National Health and Nutrition Examination Survey (NHANES) 2013-2014. The study included participants who completed both self-reported PA questionnaire and smell tests (chocolate, strawberry, grape, onion, smoke, natural gas, leather, and soap). The association between olfaction and PA parameters was assessed using correlations and logistic regressions, reported here as Odds-Ratio (OR) and 95% confidence intervals (CI). P-value ≤0.05 was considered statistically significant. In analyzing the association between PA parameters and total smell score showed a positive correlation with the duration, frequency of moderate and frequency of vigorous PA, as well as PA total volume (all p≤ 0.05). Higher duration (bouts of 10min/day), frequency (day/week), and volume (METs*h/week) of moderate PA was associated with a higher ability to detect the smells of grapes [1.002<ORs<1.047, CI95% (1.000-1.082), p=0.007]. The frequency of moderate PA was positively associated with the capacity to identify smoke [OR =1.074, CI 95% (1.019-1.131), p=0.008] and leather [OR =1.060, CI 95% (1.019-1.103), p=0.004] odors. Furthermore, the frequency of vigorous PA was positively associated with the detection of grape smell [OR =1.002, CI 95% (1.000-1.005), p=0.028]. As a result of these findings, PA parameters are associated with the ability to detect some odors, mostly moderate PA. However, it should be considered that the constant effect of PA on the chance of correctly detecting the smells is his best at 7.4%. Therefore, these results could be used by other researchers and kinesiologists to improve olfactory disorders in the future. Moderate-intensity PA could especially be recommended for the prevention and treatment of some olfactory disorders