Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn^(2+) into the prefrontal cortex indicated that DAT KO mice have a truncated Mn^(2+) distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn^(2+) transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here

Manufacturing Methods for Magnetic Resonance Microscopy Tools with Application to Neuroscience

Magnetresonanztomographie (MR) ist ein unverzichtbares nicht-invases und hochselektives bildgebendes Verfahren in der Medizin. MR Tomographie wird kommerziell in der klinischen Diagnostik und der Forschung für Gehirnkrankheit, z.B. Epilepsie, Alzheimer und Parkinson, angewandt. In den Neurowissenschaften haben sich Kleintiere als biologische Modelle für die grundlegenden Studien zur diesen Gehirnkrankheiten etabliert. MR Methoden sind ein wertvolles Werkzeug um die Morphologie und den Metabolismus von Kleintieren zu untersuchen. Die Modelle für die Untersuchung von Gehirnkrankheiten schließen Zellen/Zellkulturen und organotypische hippocampale Schnittkulturen (OHSC) mit ein. Obwohl die MR Mikroskopie für die Untersuchung von OHSC schon angewandt wurde fehlt eine effektive Plattform für umfangreiche longitudinale Studien an OHSC wie sie in den Neurowissenschaften üblich sind. Zwei Detektorkonzepte für die MR Mikroskopie inklusive ihrer Auslegung, der Herstellung und der Charakterisierung, werden in dieser Arbeit beschrieben. Beide Konzepte basieren auf Herstellungsmethoden welche hohe Fertigungsgenauigkeiten zulassen und in ihrem Herstellungsvolumen skalierbar sind. Hohle solenoide Mikrospulen welche für hochauflösende Untersuchung von Zell und Zellanhäufungen geeignet sind werden eingeführt. Die Herstellung basiert auf dem automatisierten wickeln von Mikrospulen, eine skalierbare und hochpräzise Fertigungsmethode der Mikrotechnologie. Zudem werde induktiv gekoppelte Ober ächenspulen eingeführt. Diese Oberflächenspulen fokussieren den magnetischen Fluss und werden deshalb Lenz Linsen genannt. Die Lenz Linsen werden mit kabelgebundenen und induktiv gekoppelten Spulen verglichen. Ihre Breitband-Fähigkeit machen sie zu einem idealen Kandidaten für die Nutzung in verschiedensten MR Tomographie Systemen. Die Lenz Linsen wurden für den Einsatz in einer MR kompatiblen Inkubationsplattform ausgelegt, welche in dieser Arbeit entwickelt wurde. Der MR Inkubator erweitert die Funktionalität eines MR Tomographen um neurologische Gewebe (z.B. OHSC) über mehrere Stunden andauernde MR Messungen am Leben zu erhalten. Der MR Inkubator erlaubt longitudinale Studien an OHSC und bietet damit eine Plattform für umfangreiche Studien in den Neurowissenschaften. Die Lenz Linsen wurden zusammen mit dem MR Inkubator für MR Mikroskopie Mes- sung von akuten/ xierten hippocampalen Schnitten und OHSC genutzt. Die Resultate dieser MR Mikoskopie Messungen zeigen dass in OHSC die grobe Zytoarchitektur sicht- bar ist, ohne dass die OHSC während der Messungen sterben. Somit ist das eingeführte System bereit für longitudinale Studien an OHSC, welche bereits für die Aufklärung der Epilepsieprogression begonnen wurden

Quantitative MRI correlates of hippocampal and neocortical pathology in intractable temporal lobe epilepsy

Intractable or drug-resistant epilepsy occurs in over 30% of epilepsy patients, with many of these patients undergoing surgical excision of the affected brain region to achieve seizure control. Advances in MRI have the potential to improve surgical treatment of epilepsy through improved identification and delineation of lesions. However, validation is currently needed to investigate histopathological correlates of these new imaging techniques. The purpose of this work is to investigate histopathological correlates of quantitative relaxometry and DTI from hippocampal and neocortical specimens of intractable TLE patients. To achieve this goal I developed and evaluated a pipeline for histology to in-vivo MRI image registration, which finds dense spatial correspondence between both modalities. This protocol was divided in two steps whereby sparsely sectioned histology from temporal lobe specimens was first registered to the intermediate ex-vivo MRI which is then registered to the in-vivo MRI, completing a pipeline for histology to in-vivo MRI registration. When correlating relaxometry and DTI with neuronal density and morphology in the temporal lobe neocortex, I found T1 to be a predictor of neuronal density in the neocortical GM and demonstrated that employing multi-parametric MRI (combining T1 and FA together) provided a significantly better fit than each parameter alone in predicting density of neurons. This work was the first to relate in-vivo T1 and FA values to the proportion of neurons in GM. When investigating these quantitative multimodal parameters with histological features within the hippocampal subfields, I demonstrated that MD correlates with neuronal density and size, and can act as a marker for neuron integrity within the hippocampus. More importantly, this work was the first to highlight the potential of subfield relaxometry and diffusion parameters (mainly T2 and MD) as well as volumetry in predicting the extent of cell loss per subfield pre-operatively, with a precision so far unachievable. These results suggest that high-resolution quantitative MRI sequences could impact clinical practice for pre-operative evaluation and prediction of surgical outcomes of intractable epilepsy

Reward circuitry is perturbed in the absence of the serotonin transporter

The serotonin transporter (SERT) modulates the entire serotonergic system in the brain and influences both the dopaminergic and norepinephrinergic systems. These three systems are intimately involved in normal physiological functioning of the brain and implicated in numerous pathological conditions. Here we use high-resolution magnetic resonance imaging (MRI) and spectroscopy to elucidate the effects of disruption of the serotonin transporter in an animal model system: the SERT knock-out mouse. Employing manganese-enhanced MRI, we injected Mn^(2+) into the prefrontal cortex and obtained 3D MR images at specific time points in cohorts of SERT and normal mice. Statistical analysis of co-registered datasets demonstrated that active circuitry originating in the prefrontal cortex in the SERT knock-out is dramatically altered, with a bias towards more posterior areas (substantia nigra, ventral tegmental area, and Raphé nuclei) directly involved in the reward circuit. Injection site and tracing were confirmed with traditional track tracers by optical microscopy. In contrast, metabolite levels were essentially normal in the SERT knock-out by in vivo magnetic resonance spectroscopy and little or no anatomical differences between SERT knock-out and normal mice were detected by MRI. These findings point to modulation of the limbic cortical–ventral striatopallidal by disruption of SERT function. Thus, molecular disruptions of SERT that produce behavioral changes also alter the functional anatomy of the reward circuitry in which all the monoamine systems are involved

Histological Correlates of Diffusion-Weighted Magnetic Resonance Microscopy in a Mouse Model of Mesial Temporal Lobe Epilepsy

Mesial temporal lobe epilepsy (MTLE) is the most common type of focal epilepsy. It is frequently associated with abnormal MRI findings, which are caused by underlying cellular, structural, and chemical changes at the micro-scale. In the current study, it is investigated to which extent these alterations correspond to imaging features detected by high resolution magnetic resonance imaging in the intrahippocampal kainate mouse model of MTLE. Fixed hippocampal and whole-brain sections of mouse brain tissue from nine animals under physiological and chronically epileptic conditions were examined using structural and diffusion-weighted MRI. Microstructural details were investigated based on a direct comparison with immunohistochemical analyses of the same specimen. Within the hippocampal formation, diffusion streamlines could be visualized corresponding to dendrites of CA1 pyramidal cells and granule cells, as well as mossy fibers and Schaffer collaterals. Statistically significant changes in diffusivities, fractional anisotropy, and diffusion orientations could be detected in tissue samples from chronically epileptic animals compared to healthy controls, corresponding to microstructural alterations (degeneration of pyramidal cells, dispersion of the granule cell layer, and sprouting of mossy fibers). The diffusion parameters were significantly correlated with histologically determined cell densities. These findings demonstrate that high-resolution diffusion-weighted MRI can resolve subtle microstructural changes in epileptic hippocampal tissue corresponding to histopathological features in MTLE

Neuroimaging Evidence of Major Morpho-Anatomical and Functional Abnormalities in the BTBR T+TF/J Mouse Model of Autism

BTBR T+tf/J (BTBR) mice display prominent behavioural deficits analogous to the defining symptoms of autism, a feature that has prompted a widespread use of the model in preclinical autism research. Because neuro-behavioural traits are described with respect to reference populations, multiple investigators have examined and described the behaviour of BTBR mice against that exhibited by C57BL/6J (B6), a mouse line characterised by high sociability and low self-grooming. In an attempt to probe the translational relevance of this comparison for autism research, we used Magnetic Resonance Imaging (MRI) to map in both strain multiple morpho-anatomical and functional neuroimaging readouts that have been extensively used in patient populations. Diffusion tensor tractography confirmed previous reports of callosal agenesis and lack of hippocampal commissure in BTBR mice, and revealed a concomitant rostro-caudal reorganisation of major cortical white matter bundles. Intact inter-hemispheric tracts were found in the anterior commissure, ventro-medial thalamus, and in a strain-specific white matter formation located above the third ventricle. BTBR also exhibited decreased fronto-cortical, occipital and thalamic gray matter volume and widespread reductions in cortical thickness with respect to control B6 mice. Foci of increased gray matter volume and thickness were observed in the medial prefrontal and insular cortex. Mapping of resting-state brain activity using cerebral blood volume weighted fMRI revealed reduced cortico-thalamic function together with foci of increased activity in the hypothalamus and dorsal hippocampus of BTBR mice. Collectively, our results show pronounced functional and structural abnormalities in the brain of BTBR mice with respect to control B6 mice. The large and widespread white and gray matter abnormalities observed do not appear to be representative of the neuroanatomical alterations typically observed in autistic patients. The presence of reduced fronto-cortical metabolism is of potential translational relevance, as this feature recapitulates previously-reported clinical observations

New MR imaging techniques in epilepsy

This thesis is concerned with the application of three magnetic resonance (MR) techniques in epilepsy: i.) Fluid attenuated inversion recovery prepared (FLAIR) imaging, ii.) diffusion imaging including diffusion tensor imaging (DTI) and iii.) serial and high resolution imaging of the hippocampus. I assessed the clinical value of fast FLAIR in epilepsy in a study involving 128 patients and of 3D FLAIR in a study involving 10 patients. The conspicuity of neocortical lesions and hippocampal sclerosis was increased. New lesions were detected in 5% of patients. The extent of low grade tumours was best assessed on 3D fast FLAIR images. Fast FLAIR was inferior to standard MR techniques for identifying and heterotopia. I applied newly developed, experimental diffusion imaging techniques. In eight studies using different diffusion imaging techniques involving a total of 50 patients and 54 control subjects I investigated the mobility of water molecules in the human epileptic brain in vivo. I used spin echo diffusion imaging in two studies, echo planar imaging (EPI) based DTI in four studies and EPI diffusion imaging in a patient during focal status epilepticus. Finally, in a preliminary study I attempted to use EPI diffusion imaging as a contrast to visualise transient changes associated with frequent lateralizing spikes. Our findings were: i.) diffusion is increased in hippocampal sclerosis suggesting a loss of structural organization and expansion of the extracellular space, ii.) displaying the directionality (anisotropy) of diffusion is superior to standard imaging to visualise tracts, iii.) anisotropy is reduced in the pyramidal tract in patients with hemiparesis and iv.) in the optic radiation in patients with hemianopia after temporal lobectomy suggesting wallerian degeneration, v.) both developmental and acquired structural abnormalities have a lower anisotropy than normal white matter, vi.) diffusion abnormalities in blunt head trauma are widespread and may include regions which are normal on standard imaging, indicating micro structural damage suggestive of diffuse axonal injury, vii.) focal status epilepticus can be associated with a reduced difflision in the affected cortex, viii.) diffusion imaging may be useful as a contrast for event-related (spike triggered) functional MR imaging. With serial MRI I demonstrated hippocampal volume loss in a patient after generalized status epilepticus and with high resolution imaging of an anatomical specimen and a control subject I showed hippocampal layers on MR images. The results presented in this thesis emphasised the flexibility of MR imaging and its ability to demonstrate abnormalities in vivo. FLAIR imaging is now part of the clinical work up of patients with epilepsy. Diffusion imaging has been shown to be superior to standard imaging to visualise tracts which has far-reaching implications for neurological applications. Diffusion imaging also provides an exciting window to study cerebral micro structure in vivo. Serial imaging allows for the first time the visualisation of temporal changes and high resolution imaging has the prospect of demonstrating hippocampal layers in vivo. MR imaging is a constantly progressing technique. It is hoped that this thesis will help to formulate hypotheses for new MR experiments to study the relationship of dysfunction and structural abnormalities