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      A Student’s Guide to Neural Circuit Tracing

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          Abstract

          The mammalian nervous system is comprised of a seemingly infinitely complex network of specialized synaptic connections that coordinate the flow of information through it. The field of connectomics seeks to map the structure that underlies brain function at resolutions that range from the ultrastructural, which examines the organization of individual synapses that impinge upon a neuron, to the macroscopic, which examines gross connectivity between large brain regions. At the mesoscopic level, distant and local connections between neuronal populations are identified, providing insights into circuit-level architecture. Although neural tract tracing techniques have been available to experimental neuroscientists for many decades, considerable methodological advances have been made in the last 20 years due to synergies between the fields of molecular biology, virology, microscopy, computer science and genetics. As a consequence, investigators now enjoy an unprecedented toolbox of reagents that can be directed against selected subpopulations of neurons to identify their efferent and afferent connectomes. Unfortunately, the intersectional nature of this progress presents newcomers to the field with a daunting array of technologies that have emerged from disciplines they may not be familiar with. This review outlines the current state of mesoscale connectomic approaches, from data collection to analysis, written for the novice to this field. A brief history of neuroanatomy is followed by an assessment of the techniques used by contemporary neuroscientists to resolve mesoscale organization, such as conventional and viral tracers, and methods of selecting for sub-populations of neurons. We consider some weaknesses and bottlenecks of the most widely used approaches for the analysis and dissemination of tracing data and explore the trajectories that rapidly developing neuroanatomy technologies are likely to take.

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          A mesoscale connectome of the mouse brain.

          Comprehensive knowledge of the brain's wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.
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            Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes.

            Understanding how neural information is processed in physiological and pathological states would benefit from precise detection, localization, and quantification of the activity of all neurons across the entire brain, which has not, to date, been achieved in the mammalian brain. We introduce a pipeline for high-speed acquisition of brain activity at cellular resolution through profiling immediate early gene expression using immunostaining and light-sheet fluorescence imaging, followed by automated mapping and analysis of activity by an open-source software program we term ClearMap. We validate the pipeline first by analysis of brain regions activated in response to haloperidol. Next, we report new cortical regions downstream of whisker-evoked sensory processing during active exploration. Last, we combine activity mapping with axon tracing to uncover new brain regions differentially activated during parenting behavior. This pipeline is widely applicable to different experimental paradigms, including animal species for which transgenic activity reporters are not readily available.
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              Clades of Adeno-associated viruses are widely disseminated in human tissues.

              The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors.
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                Author and article information

                Contributors
                Journal
                Front Neurosci
                Front Neurosci
                Front. Neurosci.
                Frontiers in Neuroscience
                Frontiers Media S.A.
                1662-4548
                1662-453X
                27 August 2019
                2019
                : 13
                : 897
                Affiliations
                [1] 1Neurobiology of Vital Systems Node, Faculty of Medicine and Health Sciences, Macquarie University , Sydney, NSW, Australia
                [2] 2The School of Physiology, Pharmacology and Neuroscience, University of Bristol , Bristol, United Kingdom
                [3] 3CNRS, Hindbrain Integrative Neurobiology Laboratory, Neuroscience Paris-Saclay Institute (Neuro-PSI), Université Paris-Saclay , Gif-sur-Yvette, France
                Author notes

                Edited by: Vaughan G. Macefield, Baker Heart and Diabetes Institute, Australia

                Reviewed by: Patrice G. Guyenet, University of Virginia, United States; Eberhard Weihe, University of Marburg, Germany; Alice McGovern, The University of Melbourne, Australia

                *Correspondence: Simon McMullan, simon.mcmullan@ 123456mq.edu.au

                These authors have contributed equally to this work

                This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Neuroscience

                Article
                10.3389/fnins.2019.00897
                6718611
                31507369
                98509adc-c50e-47c6-a2e3-043ad1002c92
                Copyright © 2019 Saleeba, Dempsey, Le, Goodchild and McMullan.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 31 May 2019
                : 12 August 2019
                Page count
                Figures: 4, Tables: 0, Equations: 0, References: 245, Pages: 19, Words: 0
                Funding
                Funded by: National Health and Medical Research Council 10.13039/501100000925
                Categories
                Neuroscience
                Review

                Neurosciences
                neuroanatomy,viral tracers,anterograde tracer,retrograde tracers,synaptic contacts,connectome analysis

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