A High-Resolution Anatomical Framework of the Neonatal Mouse Brain for Managing Gene Expression Data

This study aims to provide a high-resolution atlas and use it as an anatomical framework to localize the gene expression data for mouse brain on postnatal day 0 (P0). A color Nissl-stained volume with a resolution of 13.3 × 50 × 13.3 μ3 was constructed and co-registered to a standard anatomical space defined by an averaged geometry of C57BL/6J P0 mouse brains. A 145 anatomical structures were delineated based on the histological images. Anatomical relationships of delineated structures were established based on the hierarchical relations defined in the atlas of adult mouse brain (MacKenzie-Graham et al., 2004) so the P0 atlas can be related to the database associated with the adult atlas. The co-registered multimodal atlas as well as the original anatomical delineations is available for download at http://www.loni.ucla.edu/Atlases/. The region-specific anatomical framework based on the neonatal atlas allows for the analysis of gene activity within a high-resolution anatomical space at an early developmental stage. We demonstrated the potential application of this framework by incorporating gene expression data generated using in situ hybridization to the atlas space. By normalizing the gene expression patterns revealed by different images, experimental results from separate studies can be compared and summarized in an anatomical context. Co-displaying multiple registered datasets in the atlas space allows for 3D reconstruction of the co-expression patterns of the different genes in the atlas space, hence providing better insight into the relationship between the differentiated distribution pattern of gene products and specific anatomical systems

The INCF Digital Atlasing Program: Report on Digital Atlasing Standards in the Rodent Brain

The goal of the INCF Digital Atlasing Program is to provide the vision and direction necessary to make the rapidly growing collection of multidimensional data of the rodent brain (images, gene expression, etc.) widely accessible and usable to the international research community. This Digital Brain Atlasing Standards Task Force was formed in May 2008 to investigate the state of rodent brain digital atlasing, and formulate standards, guidelines, and policy recommendations.

Our first objective has been the preparation of a detailed document that includes the vision and specific description of an infrastructure, systems and methods capable of serving the scientific goals of the community, as well as practical issues for achieving
the goals. This report builds on the 1st INCF Workshop on Mouse and Rat Brain Digital Atlasing Systems (Boline et al., 2007, _Nature Preceedings_, doi:10.1038/npre.2007.1046.1) and includes a more detailed analysis of both the current state and desired state of digital atlasing along with specific recommendations for achieving these goals

Digital Atlases as a Framework for Data Sharing

Digital brain atlases are useful as references, analytical tools, and as a data integration framework. As a result, they and their supporting tools are being recognized as potentially useful resources in the movement toward data sharing. Several projects are connecting infrastructure to these tools which facilitate sharing, managing, and retrieving data of different types, scale, and even location. With these in place, we have the ability to combine, analyze, and interpret these data in a manner not previously possible, opening the door to examine issues in new and exciting ways, and potentially leading to speedier discovery of answers as well as new questions about the brain. Here we discuss recent efforts in the use of digital mouse atlases for data sharing

MBAT: A scalable informatics system for unifying digital atlasing workflows

Abstract Background Digital atlases provide a common semantic and spatial coordinate system that can be leveraged to compare, contrast, and correlate data from disparate sources. As the quality and amount of biological data continues to advance and grow, searching, referencing, and comparing this data with a researcher's own data is essential. However, the integration process is cumbersome and time-consuming due to misaligned data, implicitly defined associations, and incompatible data sources. This work addressing these challenges by providing a unified and adaptable environment to accelerate the workflow to gather, align, and analyze the data. Results The MouseBIRN Atlasing Toolkit (MBAT) project was developed as a cross-platform, free open-source application that unifies and accelerates the digital atlas workflow. A tiered, plug-in architecture was designed for the neuroinformatics and genomics goals of the project to provide a modular and extensible design. MBAT provides the ability to use a single query to search and retrieve data from multiple data sources, align image data using the user's preferred registration method, composite data from multiple sources in a common space, and link relevant informatics information to the current view of the data or atlas. The workspaces leverage tool plug-ins to extend and allow future extensions of the basic workspace functionality. A wide variety of tool plug-ins were developed that integrate pre-existing as well as newly created technology into each workspace. Novel atlasing features were also developed, such as supporting multiple label sets, dynamic selection and grouping of labels, and synchronized, context-driven display of ontological data. Conclusions MBAT empowers researchers to discover correlations among disparate data by providing a unified environment for bringing together distributed reference resources, a user's image data, and biological atlases into the same spatial or semantic context. Through its extensible tiered plug-in architecture, MBAT allows researchers to customize all platform components to quickly achieve personalized workflows

Developmental, Physiological, and Transcriptomic Analyses of Neurons involved in the Generation of Mammalian Breathing

Breathing is a rhythmic motor behavior with obvious physiological importance: breathing movements are essential for respiration, which sustains homeostasis and life itself in a wide array of animals including humans and all mammals. The breathing rhythm is produced by interneurons of the brainstem preBötzinger complex (preBötC) whose progenitors express the transcription factor Dbx1. However, the cellular and synaptic neural mechanisms underlying respiratory rhythmogenesis remain unclear. The first chapter of this dissertation examines a Dbx1 transgenic mouse line often exploited to study the neural control of breathing. It emphasizes the cellular fate of progenitors that express Dbx1 at different times during development. I couple tamoxifen-inducible Dbx1 Cre-driver mice with Cre-dependent reporters, then show that Dbx1-expressing progenitors give rise to preBötC neurons and glia. Further, I quantify the temporal assemblage of Dbx1 neurons and glia in the preBötC and provide practical guidance on breeding and tamoxifen administration strategies to bias reporter protein expression toward neurons (or glia), which can aid researchers in targeting studies to unravel their functions in respiratory neurobiology. The second chapter of this dissertation exploits the mouse model characterized in the first chapter and then focuses on mechanisms of respiratory rhythmogenesis. The breathing cycle consists of inspiratory and expiratory phases. Inspiratory burst-initiation and burst-sustaining mechanisms have been investigated by many groups. Here, I specifically investigate the role of short-term synaptic depression in burst termination and the inspiratory-expiratory phase transition using rhythmically active medullary slice preparations from Dbx1 Cre-driver mice coupled with channelrhodopsin reporters. I demonstrate the existence of a post- inspiratory refractory period that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1-derived preBötC neurons. I show that postsynaptic factors cannot account for the refractory period, and that presynaptic vesicle depletion most likely underlies the refractory period. The third chapter of this dissertation focuses on transcriptomic analysis of Dbx1 preBötC neurons, and differences in gene expression between Dbx1-derived and non- Dbx1-derived preBötC neurons. I analyze and quantify the expression of over 20,000 genes, and make the raw data publicly available for further analysis. I argue that this full transcriptome approach will enable our research group (and others) to devise physiological studies that target specific subunits and isoforms of ion channels and integral membrane proteins to examine the role(s) of Dxb1- derived neurons and glia at the molecular level of breathing behavior. In addition to predictable gene candidates (such as ion channels, etc) this transcriptome analysis delivers unanticipated novel gene candidates that can be investigated in future respiratory physiology experiments. Knowing the site (preBötC) and cell class (Dbx1) at the point of origin of respiration, this dissertation provides tools and specific investigations that advance understanding of the neural mechanisms of breathing

Neuroanatomical and Morphological Properties of Neurons that Generate Inspiratory Related Breathing Rhythm and Influence Respiratory Motor Pattern in Mice

The relationship between neuron morphology and function is a perennial issue in neuroscience. Information about synaptic integration, network connectivity, and the specific roles of neuronal subpopulations can be obtained through morphological analysis of key neurons within any given microcircuit. Breathing is essential behavior for humans and all mammals, yet the neural microcircuit that governs respiration is not completely understood. The respiratory neural microcircuit resides within the ventral respiratory column located in the medulla. Within the respiratory column, the site of respiratory rhythm generation is the bilaterally distributed preBötzinger complex (preBötC). Rhythm-generating neurons in the preBötC are derived from a single genetic line, i.e., precursor cells expressing the transcription factor Developing brain homeobox-1 (Dbx1). An analysis of over 40 dendritic morphological features of rhythmogenic Dbx1 preBötC neurons and putatively premotor Dbx1 neurons in the intermediate reticular formation, revealed these two populations are similar except reticular neurons have a larger dendritic diameter, which may contribute to a greater passive transmembrane conductance. Both populations showed commissural axon projections and reticular formation neurons show premotor-like projections to the XII motor nucleus. These morphological data provide additional evidence supporting bilateral synchronization the preBötC through Dbx1 neurons, and demonstrate that Dbx1 preBötC neuron connectivity includes recurrent interconnections. On the molecular level, the ion channels that mediate rhythm-generating whole-cell ion currents have not been not identified, and were investigated using principally an anatomical approach. The nonspecific cation current, ICAN, underlies robust inspiratory drive potentials in the preBötC and the persistent sodium current, INaP may play a role in the production of robust bursts when respiration is challenged in such cases as anoxia or hypoxia. The leading candidate for ion channels that contribute to ICAN belong to the transient membrane receptor (Trp) ion channel superfamily and the leading ion channel candidate for INaP is Nav1.6. I determined the presence of Trpc3 ion channels and Nav1.6 ion channels on Dbx1 preBötC neurons (as well as their expression in neighboring non-Dbx1 preBötC neurons). Finally, breathing behavior involves periodic sighs, which are slower than normal eupneic breathing but critical for lung function. I examined receptor expression for bomebsin-like peptides neuromedin B (NMB) and gastrin releasing peptide (GRP), which are important for sigh behavior. I show that NMB and GRP receptors are expressed in Dbx1 preBötC neurons and are not expressed by glia in the preBötC, as posited by some because of the low frequency of sigh breaths. These advances in morphological and anatomical knowledge can be used to design targeted in vitro and in vivo experiments to further explore their role in respiratory rhythm and pattern generation

A Biological Global Positioning System: Considerations for Tracking Stem Cell Behaviors in the Whole Body

Many recent research studies have proposed stem cell therapy as a treatment for cancer, spinal cord injuries, brain damage, cardiovascular disease, and other conditions. Some of these experimental therapies have been tested in small animals and, in rare cases, in humans. Medical researchers anticipate extensive clinical applications of stem cell therapy in the future. The lack of basic knowledge concerning basic stem cell biology-survival, migration, differentiation, integration in a real time manner when transplanted into damaged CNS remains an absolute bottleneck for attempt to design stem cell therapies for CNS diseases. A major challenge to the development of clinical applied stem cell therapy in medical practice remains the lack of efficient stem cell tracking methods. As a result, the fate of the vast majority of stem cells transplanted in the human central nervous system (CNS), particularly in the detrimental effects, remains unknown. The paucity of knowledge concerning basic stem cell biology—survival, migration, differentiation, integration in real-time when transplanted into damaged CNS remains a bottleneck in the attempt to design stem cell therapies for CNS diseases. Even though excellent histological techniques remain as the gold standard, no good in vivo techniques are currently available to assess the transplanted graft for migration, differentiation, or survival. To address these issues, herein we propose strategies to investigate the lineage fate determination of derived human embryonic stem cells (hESC) transplanted in vivo into the CNS. Here, we describe a comprehensive biological Global Positioning System (bGPS) to track transplanted stem cells. But, first, we review, four currently used standard methods for tracking stem cells in vivo: magnetic resonance imaging (MRI), bioluminescence imaging (BLI), positron emission tomography (PET) imaging and fluorescence imaging (FLI) with quantum dots. We summarize these modalities and propose criteria that can be employed to rank the practical usefulness for specific applications. Based on the results of this review, we argue that additional qualities are still needed to advance these modalities toward clinical applications. We then discuss an ideal procedure for labeling and tracking stem cells in vivo, finally, we present a novel imaging system based on our experiments

Pharmacokinetics during therapeutic hypothermia in neonates:from pathophysiology to translational knowledge and physiologically-based pharmacokinetic (PBPK) modeling

Introduction: Perinatal asphyxia (PA) still causes significant morbidity and mortality. Therapeutic hypothermia (TH) is the only effective therapy for neonates with moderate to severe hypoxic-ischemic encephalopathy after PA. These neonates need additional pharmacotherapy, and both PA and TH may impact physiology and, consequently, pharmacokinetics (PK) and pharmacodynamics (PD). Areas covered: This review provides an overview of the available knowledge in PubMed (until November 2022) on the pathophysiology of neonates with PA/TH. In vivo pig models for this setting enable distinguishing the effect of PA versus TH on PK and translating this effect to human neonates. Available asphyxia pig models and methodological considerations are described. A summary of human neonatal PK of supportive pharmacotherapy to improve neurodevelopmental outcomes is provided. Expert opinion: To support drug development for this population, knowledge from clinical observations (PK data, real-world data on physiology), preclinical (in vitro and in vivo (minipig)) data, and molecular and cellular biology insights can be integrated into a predictive physiologically-based PK (PBPK) framework, as illustrated by the I-PREDICT project (Innovative physiology-based pharmacokinetic model to predict drug exposure in neonates undergoing cooling therapy). Current knowledge, challenges, and expert opinion on the future directions of this research topic are provided.</p

REWIRED METABOLISM IN APOE4 MICROGLIA: IMPLICATIONS FOR INFLAMMATION AND NEURODEGENERATION

Among the earliest changes to occur in Alzheimer’s disease, metabolic dysfunction and chronic neuroinflammation are now known to be major driving forces in disease progression. The paradigm of ‘immunometabolism’ seeks to bridge these two facets, positing that metabolic transformations are indispensable in determining the response of immune cells, such as microglia – the brain’s resident immune population. Proinflammatory stimulation of microglia leads to a shift away from mitochondrial respiration towards a dramatic upregulation of the glycolysis pathway for energy production. This glycolytic burst provides microglia with a rapid supply of ATP, but comes at a cost, as utilizing glucose to produce energy via glycolysis is less efficient than mitochondrial respiration. Microglia thus must switch back to mitochondrial respiration and ramp down glycolysis in order to effectively respond to anti-inflammatory signals and restore the tissue to homeostasis. A breakdown in this process is evident in the genetics of late-onset Alzheimer’s disease (LOAD), as the majority of LOAD genetic risk factors are highly or specifically expressed in microglia. Many of these risk factors – including the strongest, the ε4 allele of apolipoprotein E (APOE4) - are thought to integrate metabolic inputs with downstream inflammatory signaling. ApoE is the chief lipoprotein in the brain, responsible for trafficking lipids and thereby governing its bioenergetics. APOE4 microglia display a heightened production of pro-inflammatory cytokines coinciding with chronic neuroinflammation in APOE4 brains. This dissertation seeks to unify these two aspects of APOE4’s risk through the concept of immunometabolism. An integrative multi-omic approach enabled us to systematically dissect the role of APOE genotype in the microglial response to aging, inflammation, and amyloid pathology. In response to each of these challenges, a distinct metabolic response was revealed in APOE4 microglia, characterized by an increased reliance on glycolysis and impaired capacity to engage mitochondrial respiration. These changes were accompanied by alterations to the TCA cycle that altogether indicated a predisposition to proinflammatory immunometabolism in APOE4 microglia. Hypoxia-inducible factor 1α (HIF1α) was uncovered as driving mechanism behind not only this metabolic rewiring, but also having the potential to link APOE4 to downstream neurodegenerative phenotypes. Taken together, our findings point to a reprogramming of central metabolism according to APOE genotype such that APOE4 microglia are predisposed to pro-inflammatory and neurodegenerative pathways. Seeking to correct these metabolic impairments may represent a novel therapeutic avenue for the personalized treatment of Alzheimer’s disease