Automated four-dimensional long term imaging enables single cell tracking within organotypic brain slices to study neurodevelopment and degeneration.

Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes

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

The E3 ubiquitin ligase IDOL regulates synaptic ApoER2 levels and is important for plasticity and learning.

Neuronal ApoE receptors are linked to learning and memory, but the pathways governing their abundance, and the mechanisms by which they affect the function of neural circuits are incompletely understood. Here we demonstrate that the E3 ubiquitin ligase IDOL determines synaptic ApoER2 protein levels in response to neuronal activation and regulates dendritic spine morphogenesis and plasticity. IDOL-dependent changes in ApoER2 abundance modulate dendritic filopodia initiation and synapse maturation. Loss of IDOL in neurons results in constitutive overexpression of ApoER2 and is associated with impaired activity-dependent structural remodeling of spines and defective LTP in primary neuron cultures and hippocampal slices. IDOL-deficient mice show profound impairment in experience-dependent reorganization of synaptic circuits in the barrel cortex, as well as diminished spatial and associative learning. These results identify control of lipoprotein receptor abundance by IDOL as a post-transcriptional mechanism underlying the structural and functional plasticity of synapses and neural circuits

High-throughput platforms for the screening of new therapeutic targets for neurodegenerative diseases

Despite the recent progress in the understanding of neurodegenerative disorders, a lack of solid fundamental knowledge on the etiology of many of the major neurodegenerative diseases has made it difficult to obtain effective therapies to treat these conditions. Scientists have been looking to carry out more-human-relevant studies, with strong statistical power, to overcome the limitations of preclinical animal models that have contributed to the failure of numerous therapeutics in clinical trials. Here, we identify currently existing platforms to mimic central nervous system tissues, healthy and diseased, mainly focusing on cell-based platforms and discussing their strengths and limitations in the context of the high-throughput screening of new therapeutic targets and drugs.This work had the financial support of Fundação para a Ciência e Tecnologia ( FCT ) through National Funds and, when applicable, co-financed by the FEDER through the PT2020 Partnership Agreement under the 4293 Unit I&D. D.N. Rocha acknowledges FCT for her PhD grant

Multiple Single-Unit Long-Term Tracking on Organotypic Hippocampal Slices Using High-Density Microelectrode Arrays

A novel system to cultivate and record from organotypic brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows for continuous recording of electrical activity of specific individual neurons at high spatial resolution while monitoring at the same time, neuronal network activity. For the first time, the electrical activity patterns of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied during several consecutive weeks at daily intervals. An unsupervised iterative spike-sorting algorithm, based on PCA and k-means clustering, was developed to assign the activities to the single units. Spike-triggered average extracellular waveforms of an action potential recorded across neighboring electrodes, termed ‘footprints’ of single-units were generated and tracked over weeks. The developed system offers the potential to study chronic impacts of drugs or genetic modifications on individual neurons in slice preparations over extended times

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease

Development of Epilepsy-on-a-chip System for High-throughput Antiepileptogenic Drug Discovery

Epilepsy is one of the most common neurological disorders and affects millions of people in the United States. Currently available antiepileptic drugs require continuous administration for suppression of seizures and have not been shown to prevent the development of epilepsy (epileptogenesis). The discovery of antiepileptogenic drug is complicated by the long time course of epileptogenesis in animal models of epilepsy and the requirement of continuous monitoring of epileptiform activity in vivo for the assessment of drug efficacy. In recent years, organotypic hippocampal cultures have been increasingly used as an in vitro model of post-traumatic epilepsy in both basic and translational research. Epileptogenesis in this in vitro model has a compressed time scale and can be monitored by detection of electrographic and biochemical markers of seizure-like activity. However, the lack of a scalable chronic electrical recording platform is a significant bottleneck in high-throughput antiepileptogenic drug discovery using organotypic cultures.In an effort to circumvent the throughput limitations of in vitro antiepileptogenic drug discovery, a hybrid microfluidic-multiple electrode array (µflow-MEA) technology was developed for scalable chronic electrical assay of epileptogenesis in vitro. Specifically, the microfluidic perfusion technique was utilized to miniature the culture platform, which enabled the long-term maintenance of an organotypic culture array on a single device. The integration of the microfluidic perfusion system with a customized planar MEA allowed for parallel continuous recordings. As a proof-of-concept demonstration, a pilot screen of receptor tyrosine kinase (RTK) inhibitor library was performed on µflow-MEA based electrical assay platform. The screen results revealed significant antiepileptogenic effect of cFMS RTK inhibitor.This thesis also provides further validation of the organotypic hippocampal culture model of epilepsy by investigating the influence of culture medium composition on epileptogenesis. We found that epileptogenesis occurred in any culture medium that was capable of supporting neural survival, indicating that culture medium composition has limited influence on epileptogenesis in organotypic hippocampal cultures.It is hoped that the techniques presented in this thesis will accelerate the antiepileptogenic drug discovery and contribute to the development of new therapeutics to treat individuals at risk of epileptogenesis