Potential Synaptic Connectivity of Different Neurons onto Pyramidal Cells in a 3D Reconstruction of the Rat Hippocampus

Most existing connectomic data and ongoing efforts focus either on individual synapses (e.g., with electron microscopy) or on regional connectivity (tract tracing). An individual pyramidal cell (PC) extends thousands of synapses over macroscopic distances (∼cm). The contrasting requirements of high-resolution and large field of view make it too challenging to acquire the entire synaptic connectivity for even a single typical cortical neuron. Light microscopy can image whole neuronal arbors and resolve dendritic branches. Analyzing connectivity in terms of close spatial appositions between axons and dendrites could thus bridge the opposite scales, from synaptic level to whole systems. In the mammalian cortex, structural plasticity of spines and boutons makes these “potential synapses” functionally relevant to learning capability and memory capacity. To date, however, potential synapses have only been mapped in the surrounding of a neuron and relative to its local orientation rather than in a system-level anatomical reference. Here we overcome this limitation by estimating the potential connectivity of different neurons embedded into a detailed 3D reconstruction of the rat hippocampus. Axonal and dendritic trees were oriented with respect to hippocampal cytoarchitecture according to longitudinal and transversal curvatures. We report the potential connectivity onto PC dendrites from the axons of a dentate granule cell, three CA3 PCs, one CA2 PC, and 13 CA3b interneurons. The numbers, densities, and distributions of potential synapses were analyzed in each sub-region (e.g., CA3 vs. CA1), layer (e.g., oriens vs. radiatum), and septo-temporal location (e.g., dorsal vs. ventral). The overall ratio between the numbers of actual and potential synapses was ∼0.20 for the granule and CA3 PCs. All potential connectivity patterns are strikingly dependent on the anatomical location of both pre-synaptic and post-synaptic neurons

Diffusion and Perfusion MRI in Paediatric Posterior Fossa Tumours

Brain tumours in children frequently occur in the posterior fossa. Most undergo surgical resection, after which up to 25% develop cerebellar mutism syndrome (CMS), characterised by mutism, emotional lability and cerebellar motor signs; these typically improve over several months. This thesis examines the application of diffusion (dMRI) and arterial spin labelling (ASL) perfusion MRI in children with posterior fossa tumours. dMRI enables non-invasive in vivo investigation of brain microstructure and connectivity by a computational process known as tractography. The results of a unique survey of British neurosurgeons’ attitudes towards tractography are presented, demonstrating its widespread adoption and numerous limitations. State-of-the-art modelling of dMRI data combined with tractography is used to probe the anatomy of cerebellofrontal tracts in healthy children, revealing the first evidence of a topographic organization of projections to the frontal cortex at the superior cerebellar peduncle. Retrospective review of a large institutional series shows that CMS remains the most common complication of posterior fossa tumour resection, and that surgical approach does not influence surgical morbidity in this cohort. A prospective case-control study of children with posterior fossa tumours treated at Great Ormond Street Hospital is reported, in which children underwent longitudinal MR imaging at three timepoints. A region-of-interest based approach did not reveal any differences in dMRI metrics with respect to CMS status. However, the candidate also conducted an analysis of a separate retrospective cohort of medulloblastoma patients at Stanford University using an automated tractography pipeline. This demonstrated, in unprecedented spatiotemporal detail, a fine-grained evolution of changes in cerebellar white matter tracts in children with CMS. ASL studies in the prospective cohort showed that following tumour resection, increases in cortical cerebral blood flow were seen alongside reductions in blood arrival time, and these effects were modulated by clinical features of hydrocephalus and CMS. The results contained in this thesis are discussed in the context of the current understanding of CMS, and the novel anatomical insights presented provide a foundation for future research into the condition

Advanced MRI techniques in the study of cerebellar cortex

The cerebellum (from the Latin "little brain") is the dorsal portion of the metencephalon and is located in the posterior cranial fossa. Although representing only 10% of the total brain volume, it contains more than 50% of the total number of neurons of the central nervous system (CNS). Its organization resembles the one found in the telencephalon, with the presence of a superficial mantle of gray matter (GM) known as the cerebellar cortex, covering the cerebellar white matter (WM) in which three pairs of deep cerebellar GM nuclei are embedded. The number of studies dedicated to the study of the cerebellum and its function has significantly increased during the last years. Nevertheless, although many theories on the cerebellar function have been proposed, to date we still are not able to answer the question about the exact function of this structure. Indeed, the classical theories focused on the role of the cerebellum in fine-tuning for muscle control has been widely reconsidered during the last years, with new hypotheses that have been advanced. These include its role as sensory acquisition device, extending beyond a pure role in motor control and learning, as well as a pivotal role in cognition, with a recognized cerebellar participation in a variety of cognitive functions, ranging from mood control to language, memory, attention and spatial data management. A huge contribution to our understanding of how the cerebellum participates in all these different aspects of motor and non-motor behavior comes from the application of advanced imaging techniques. In particular, Magnetic Resonance Imaging (MRI) can provide a non-invasive evaluation of anatomical integrity, as well as information about functional connections with other brain regions. This thesis is organized as follows: - In Chapter 1 is presented a general introduction to the cerebellar anatomy and functions, with particular reference to the anatomical organization of cerebellar cortex and its connections with the telencephalon - Chapter 2 will contain a general overview about some of the major advanced MRI methods that can be applied to investigate the anatomical integrity and functional status of the cerebellar cortex - In Chapter 3 will be presented a new method to evaluate the anatomy and integrity of cerebellar cortex using ultra-high field MRI scanners - Chapters 4, 5 and 6 will contain data obtained from the application of some of the previously described advanced imaging techniques to the study of cerebellar cortex in neurodegenerative and neuroinflammatory disorders affecting the CNS

Developing novel non-invasive MRI techniques to assess cerebrospinal fluid-interstitial fluid (CSF-ISF) exchange

The pathological cascade of events in Alzheimer’s disease (AD) is initiated decades prior to the onset of symptoms. Despite intensive research, the relative time-course/interaction of these events is yet to be determined. Recent evidence suggests that impairments to brain clearance (facilitated by the compartmental exchange of cerebrospinal-fluid (CSF) with interstitial-fluid (ISF)), contributes to the build-up of amyloid and tau (AD hallmarks). Therefore, abnormalities in CSF-ISF exchange dynamics, may represent an early driver of downstream events. Clinical evaluation of this hypothesis is hampered due to the lack of non-invasive CSF-ISF exchange assessment techniques. In this thesis, the primary aim was to develop a physiologically relevant, non-invasive CSF-ISF exchange assessment technique that would circumvent the limitations associated with current procedures (primarily their invasiveness). Towards this goal, animal studies were conducted to investigate the feasibility of a contrast enhanced-magnetic resonance imaging (CE-MRI) approach as a potential non-invasive CSF-ISF exchange imaging technique. Another aim of this thesis was to investigate whether the proposed MRI platform could detect abnormalities in CSF-ISF exchange, a condition hypothesised to occur in the early stages of AD. As such, pharmacological intervention studies were conducted to alter CSF-ISF exchange dynamics. CE-MRI, in conjunction with high-level image post-processing, demonstrated high sensitivity to physiological CSF-ISF exchange. This novel, non-invasive platform, captured dynamic, whole-brain infiltration of contrast agent from the blood to the CSF and into the parenchyma, via a pathway named ‘VEntricular-Cerebral TranspORt (VECTOR)’. Additionally, the platform detected significant abnormalities in CSF-ISF exchange following pharmacological intervention, demonstrating the potential of VECTOR in the study of the parenchymal accumulation of aberrant proteins. Development of this platform is a breakthrough step towards the clinical assessment of CSF-ISF exchange abnormalities to study its role in disease onset/progression, an approach that may inform understanding of the causal sequence of pathological events that occurs in AD development

A Pipeline for Automated Assessment of Cell Location in 3D Mouse Brain Image Sets

Mapping the neuronal connectivity of the mouse brain has long been hampered by the laborious and time-consuming process of slicing, staining and imaging the brain tissue. Recent developments in automated 3D fluorescence microscopy, such as serial two- photon tomography (STP) and light sheet fluorescence microscopy, now allow for automated rapid 3D imaging of a complete mouse brain at cellular resolution. In combination with transsynaptic viral tracers, this paves the way for high-throughput brain mapping studies, which could greatly advance our understanding of the function of the brain. Because transsynaptic tracers label synaptically connected cells, the analysis of these whole-brain scans requires detection of fluorescently labelled cells and anatomical segmentation of the data, which are very labour- and time-intensive manual tasks and prevent high-throughput analysis. This thesis presents and validates two software tools to automate anatomical segmentation and cell detection in serial two photon (STP) scans of the mouse brain. Automated mouse atlas propagation (aMAP) segments the scans into anatomical regions by matching a 3D reference atlas to the data using affine and free-form image registration. The fast automated cell counting tool (FACCT) then detects fluorescently labelled cells in the dataset with a novel approach of stepwise data reduction combined with object detection using artificial neuronal networks. The tools are optimised for large datasets and are capable of processing a 2.5TB STP scan in under two days. The performance of aMAP and FACCT is evaluated on STP scans from retrograde connectivity tracing experiments using rabies virus injections in the primary visual corte

Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.This research was supported in part by the Intramural Research Program of NHGRI, NIH, and the Wellcome Trust and by NHMRC Australia grant 366746. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

3D Generalization of brain model to visualize and analyze neuroanatomical data

Neuroscientists present data in a 3D form in order to convey a better real world visualization and understanding of the localization of data in relation to brain anatomy and structure. The problem with the visualization of cortical surface of the brain is that the brain has multiple, deep folds and the resulting structural overlap can hide data interweaved within the folds. On one hand, a 2D representation can result in a distorted view that may lead to incorrect localization and analysis of the data. On the other hand, a realistic 3D representation may interfere with our judgment or analysis by showing too many details. Alternatively, a 3D generalization can be used to simplify the model of the brain in order to visualize the hidden data and smooth some of the details. This dissertation addresses the following research question: Is 3D generalization of a brain model a viable approach for visualizing neuroanatomical data

Fully automated dual-resolution serial optical coherence tomography aimed at diffusion MRI validation in whole mouse brains

An automated dual-resolution serial optical coherence tomography (2R-SOCT) scanner is developed. The serial histology system combines a low-resolution ( 25 mu m / voxel ) 3 x OCT with a high-resolution ( 1.5 mu m / voxel ) 40 x OCT to acquire whole mouse brains at low resolution and to target specific regions of interest (ROIs) at high resolution. The 40 x ROIs positions are selected either manually by the microscope operator or using an automated ROI positioning selection algorithm. Additionally, a multimodal and multiresolution registration pipeline is developed in order to align the 2R-SOCT data onto diffusion MRI (dMRI) data acquired in the same ex vivo mouse brains prior to automated histology. Using this imaging system, 3 whole mouse brains are imaged, and 250 high-resolution 40 x three-dimensional ROIs are acquired. The capability of this system to perform multimodal imaging studies is demonstrated by labeling the ROIs using a mouse brain atlas and by categorizing the ROIs based on their associated dMRI measures. This reveals a good correspondence of the tissue microstructure imaged by the high-resolution OCT with various dMRI measures such as fractional anisotropy, number of fiber orientations, apparent fiber density, orientation dispersion, and intracellular volume fraction

Analysis of spine plasticity in CA1 hippocampal pyramidal neurons employing live cell nanoscopic imaging

In der Großhirnrinde von Säugetieren befindet sich die Mehrheit erregender Synapsen auf Dornfortsätzen, kleinen dendritischen Ausbuchtungen, die in Größe und Form stark variieren. Die Auslösung aktivitätsabhängiger synaptischer Langzeitplastizität geht mit strukturellen Veränderungen dendritischer Dornen einher. Da das beugungsbegrenzte Auflösungsvermögen konventioneller Lichtmikroskope nicht ausreicht um die Morphologie der Dornen verlässlich zu untersuchen, stellte die Elektronenmikroskopie bisher das wichtigste bildgebende Verfahren zur Erforschung von struktureller Plastizität dar, blieb dabei jedoch auf die Betrachtung fixierter Gewebeproben beschränkt. Die Anwendung hochauflösender Laser-Raster-Mikroskopie mit Stimulierter-Emissions-Auslöschung hat es mir möglich gemacht, die Dynamik dendritischer Dornenmorphologie in lebenden Zellen zu studieren. Die N-Methyl-D-Aspartat-Rezeptor-abhängige Langzeitpotenzierung von Pyramidenzellen der Cornu-Ammonis Region 1 des Hippocampus bildete dabei den Mechanismus, welcher plastische Veränderungen hervorrief. Nach Potenzierung exzitatorischer Synapsen durch die lokale Ultraviolett-Photolyse von caged-Glutamat wurde ein starker, vorübergehender Anstieg des Anteils dendritischer Dornen mit sichelförmigen Köpfen und ein leichter, anhaltender Zuwachs an pilzförmigen Dornfortsätzen über einen Zeitraum von 50 Minuten beobachtet. Meine Untersuchungen ergänzen frühere Studien zur Wechselbeziehung zwischen synaptischer Potenzierung und struktureller Plastizität dendritischer Dornen und korrespondieren mit dem aktuellen Kenntnisstand der zu Grunde liegenden molekularen Mechanismen.The majority of excitatory synapses in the cortex of mammalian brains is situated on dendritic spines, small protrusions, heterogeneous in size and shape. The induction of activity-dependent long-term synaptic plasticity has been associated with changes in the ultrastructure of spines, particularly in size, head shape and neck width. Since the dimensions of dendritic spines are at the border of the diffraction-limited resolving power of conventional light microscopes, until recently, electron microscopy on fixed tissue constituted the primary method for investigations on spine morphology. I have employed live cell stimulated emission depletion imaging to analyse spine motility and structural transitions in response to n-methyl-d-aspartate receptor dependent long-term potentiation over time at super-resolution in Cornu Ammonis area 1 pyramidal neurons of the hippocampus. Local induction of long-term potentiation via ultraviolet photolysis of caged glutamate facilitated a strong transient increase in the proportion of spines with curved heads and a subtle persistent growth in the amount of mushroom spines over a time course of 50 minutes. My findings reinforce previous investigations on the relation of synaptic potentiation and spine motility, and are in good agreement with the current knowledge of the molecular mechanisms underlying long-term plasticity

Partial connectomes of labeled dopaminergic circuits reveal non-synaptic communication and axonal remodeling after exposure to cocaine

Dopaminergic (DA) neurons exert profound influences on behavior including addiction. However, how DA axons communicate with target neurons and how those communications change with drug exposure remains poorly understood. We leverage cell type-specific labeling with large volume serial electron microscopy to detail DA connections in the nucleus accumbens (NAc) of the mouse (Mus musculus) before and after exposure to cocaine. We find that individual DA axons contain different varicosity types based on their vesicle contents. Spatially ordering along individual axons further suggests that varicosity types are non-randomly organized. DA axon varicosities rarely make specific synapses (<2%, 6/410), but instead are more likely to form spinule-like structures (15%, 61/410) with neighboring neurons. Days after a brief exposure to cocaine, DA axons were extensively branched relative to controls, formed blind-ended ‘bulbs’ filled with mitochondria, and were surrounded by elaborated glia. Finally, mitochondrial lengths increased by ~2.2 times relative to control only in DA axons and NAc spiny dendrites after cocaine exposure. We conclude that DA axonal transmission is unlikely to be mediated via classical synapses in the NAc and that the major locus of anatomical plasticity of DA circuits after exposure to cocaine are large-scale axonal re-arrangements with correlated changes in mitochondria