Reconstruction of 1,000 Projection Neurons Reveals New Cell Types and Organization of Long-Range Connectivity in the Mouse Brain

Neuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons constitute more than 85 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.

[1]  Christof Koch,et al.  Adult Mouse Cortical Cell Taxonomy by Single Cell Transcriptomics , 2016, Nature Neuroscience.

[2]  K. Svoboda,et al.  The subcellular organization of neocortical excitatory connections , 2009, Nature.

[3]  Giulio Iannello,et al.  TeraStitcher - A tool for fast automatic 3D-stitching of teravoxel-sized microscopy images , 2012, BMC Bioinformatics.

[4]  Mark S. Cembrowski,et al.  The subiculum is a patchwork of discrete subregions , 2018, eLife.

[5]  Moritz Helmstaedter,et al.  High-accuracy neurite reconstruction for high-throughput neuroanatomy , 2011, Nature Neuroscience.

[6]  C. Gerfen,et al.  An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: Immunohistochemical localization of an axonally transported plant lectin,Phaseolus vulgaris leucoagglutinin (PHA-L) , 1984, Brain Research.

[7]  Mark T. Harnett,et al.  Nonlinear dendritic integration of sensory and motor input during an active sensing task , 2012, Nature.

[8]  Kei M. Igarashi,et al.  Parallel Mitral and Tufted Cell Pathways Route Distinct Odor Information to Different Targets in the Olfactory Cortex , 2012, The Journal of Neuroscience.

[9]  György Buzsáki,et al.  Three-dimensional reconstruction of the axon arbor of a CA3 pyramidal cell recorded and filled in vivo , 2007, Brain Structure and Function.

[10]  H Burkhardt,et al.  XuvTools: free, fast and reliable stitching of large 3D datasets , 2009, Journal of microscopy.

[11]  X. Zhuang,et al.  Spatially resolved, highly multiplexed RNA profiling in single cells , 2015, Science.

[12]  N. Spruston,et al.  Target‐specific output patterns are predicted by the distribution of regular‐spiking and bursting pyramidal neurons in the subiculum , 2012, Hippocampus.

[13]  Christopher J. Cronin,et al.  Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH , 2018, Cell.

[14]  Charles R. Gerfen,et al.  Distinct descending motor cortex pathways and their roles in movement , 2017, Nature.

[15]  Dimitri Perrin,et al.  Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging , 2015, Nature Protocols.

[16]  Karel Svoboda,et al.  The Past, Present, and Future of Single Neuron Reconstruction , 2011, Neuroinformatics.

[17]  Lars E. Borm,et al.  Molecular Architecture of the Mouse Nervous System , 2018, Cell.

[18]  Robert A. McGovern,et al.  Divergence of Melanocortin Pathways in the Control of Food Intake and Energy Expenditure , 2005, Cell.

[19]  C. Elias,et al.  Connectivity pattern suggests that incerto-hypothalamic area belongs to the medial hypothalamic system , 2007, Neuroscience.

[20]  Joe Chalfoun,et al.  MIST: Accurate and Scalable Microscopy Image Stitching Tool with Stage Modeling and Error Minimization , 2017, Scientific Reports.

[21]  J. Betley,et al.  Parallel, Redundant Circuit Organization for Homeostatic Control of Feeding Behavior , 2013, Cell.

[22]  M. Deschenes,et al.  Corticothalamic projections from layer V cells in rat are collaterals of long-range corticofugal axons , 1994, Brain Research.

[23]  G. Shepherd,et al.  Three-Dimensional Structure and Composition of CA3→CA1 Axons in Rat Hippocampal Slices: Implications for Presynaptic Connectivity and Compartmentalization , 1998, The Journal of Neuroscience.

[24]  Shaoqun Zeng,et al.  High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level , 2016, Nature Communications.

[25]  M. Jouvet,et al.  Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons , 1990, Brain Research.

[26]  A. Reiner,et al.  Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies , 1992, Journal of Neuroscience Methods.

[27]  Giorgio A Ascoli,et al.  In search of a periodic table of the neurons: Axonal‐dendritic circuitry as the organizing principle , 2016, BioEssays : news and reviews in molecular, cellular and developmental biology.

[28]  G. Shepherd,et al.  General and variable features of varicosity spacing along unmyelinated axons in the hippocampus and cerebellum , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Lydia Ng,et al.  The Allen Brain Atlas: Toward Understanding Brain Behavior and Function Through Data Acquisition, Visualization, Analysis, and Integration , 2018 .

[30]  G. Shepherd.  Corticostriatal connectivity and its role in disease , 2013, Nature Reviews Neuroscience.

[31]  K. Svoboda,et al.  Diverse Modes of Axon Elaboration in the Developing Neocortex , 2005, PLoS biology.

[32]  Brian Zingg,et al.  Inhibitory gain modulation of defense behaviors by zona incerta , 2018, Nature Communications.

[33]  T. Bonhoeffer,et al.  Relationship Between Lateral Inhibitory Connections and the Topography of the Orientation Map in Cat Visual Cortex , 1994, The European journal of neuroscience.

[34]  C. Wilson,et al.  Spontaneous firing patterns and axonal projections of single corticostriatal neurons in the rat medial agranular cortex. , 1994, Journal of neurophysiology.

[35]  Alex M. Thomson,et al.  Neocortical Layer 6, A Review , 2010, Front. Neuroanat..

[36]  Xiaohua Lv,et al.  Reconstruction of Intratelencephalic Neurons in the Mouse Secondary Motor Cortex Reveals the Diverse Projection Patterns of Single Neurons , 2018, Front. Neuroanat..

[37]  E. Kuramoto,et al.  Two types of thalamocortical projections from the motor thalamic nuclei of the rat: a single neuron-tracing study using viral vectors. , 2009, Cerebral cortex.

[38]  V. Caviness,et al.  Interhemispheric neocortical connections of the corpus callosum in the reeler mutant mouse: A study based on anterograde and retrograde methods , 1975, The Journal of comparative neurology.

[39]  Prof. Dr. Valentino Braitenberg,et al.  Anatomy of the Cortex , 1991, Studies of Brain Function.

[40]  K. Svoboda,et al.  Channelrhodopsin-2–assisted circuit mapping of long-range callosal projections , 2007, Nature Neuroscience.

[41]  Allan R. Jones,et al.  A mesoscale connectome of the mouse brain , 2014, Nature.

[42]  Arthur W. Toga,et al.  Neural Networks of the Mouse Neocortex , 2014, Cell.

[43]  Paola Arlotta,et al.  Neuronal Subtype-Specific Genes that Control Corticospinal Motor Neuron Development In Vivo , 2005, Neuron.

[44]  V. Gradinaru,et al.  Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems , 2017, Nature Neuroscience.

[45]  Martin Deschênes,et al.  Motor Cortex Gates Vibrissal Responses in a Thalamocortical Projection Pathway , 2007, Neuron.

[46]  R. Douglas,et al.  Chance or design? Some specific considerations concerning synaptic boutons in cat visual cortex , 2002, Journal of neurocytology.

[47]  Andriy Myronenko,et al.  Point Set Registration: Coherent Point Drift , 2009, IEEE Transactions on Pattern Analysis and Machine Intelligence.

[48]  G. Blasdel,et al.  Termination of afferent axons in macaque striate cortex , 1983, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[49]  M. Greicius,et al.  Resting-state functional connectivity reflects structural connectivity in the default mode network. , 2009, Cerebral cortex.

[50]  S Murray Sherman,et al.  Thalamus plays a central role in ongoing cortical functioning , 2016, Nature Neuroscience.

[51]  Chee-Huat Linus Eng,et al.  Profiling the transcriptome by RNA SPOTs , 2018 .

[52]  R. Lin,et al.  Thalamic afferents of the rat barrel cortex: a light- and electron-microscopic study using Phaseolus vulgaris leucoagglutinin as an anterograde tracer. , 1993, Somatosensory & motor research.

[53]  Hao Wu,et al.  Complete morphologies of basal forebrain cholinergic neurons in the mouse , 2014, eLife.

[54]  Evan Z. Macosko,et al.  Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics , 2016, Cell.

[55]  Kirby G. Vosburgh,et al.  3D Slicer: A Platform for Subject-Specific Image Analysis, Visualization, and Clinical Support , 2014 .

[56]  Zachary T. Nolan,et al.  Subset of Cortical Layer 6b Neurons Selectively Innervates Higher Order Thalamic Nuclei in Mice , 2018, Cerebral cortex.

[57]  Justus M. Kebschull,et al.  The logic of single-cell projections from visual cortex , 2018, Nature.

[58]  C. Wilson,et al.  Projection subtypes of rat neostriatal matrix cells revealed by intracellular injection of biocytin , 1990, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[59]  Ken Sugino,et al.  A Mammalian enhancer trap resource for discovering and manipulating neuronal cell types , 2016, eLife.

[60]  Clémentine Bosch-Bouju,et al.  Motor thalamus integration of cortical, cerebellar and basal ganglia information: implications for normal and parkinsonian conditions , 2013, Front. Comput. Neurosci..

[61]  Kat Christiansen,et al.  The subiculum: the heart of the extended hippocampal system. , 2015, Progress in brain research.

[62]  R. Douglas,et al.  A Quantitative Map of the Circuit of Cat Primary Visual Cortex , 2004, The Journal of Neuroscience.

[63]  E. Kuramoto,et al.  Ventral medial nucleus neurons send thalamocortical afferents more widely and more preferentially to layer 1 than neurons of the ventral anterior-ventral lateral nuclear complex in the rat. , 2015, Cerebral cortex.

[64]  T. Kita,et al.  The Subthalamic Nucleus Is One of Multiple Innervation Sites for Long-Range Corticofugal Axons: A Single-Axon Tracing Study in the Rat , 2012, The Journal of Neuroscience.

[65]  Giulio Iannello,et al.  Automated Neuron Tracing Methods: An Updated Account , 2016, Neuroinformatics.

[66]  J. D. Macklis,et al.  Development, specification, and diversity of callosal projection neurons , 2011, Trends in Neurosciences.

[67]  Nicholas N. Foster,et al.  The mouse cortico-striatal projectome , 2016, Nature Neuroscience.

[68]  Martin Deschênes,et al.  The organization of corticothalamic projections: reciprocity versus parity , 1998, Brain Research Reviews.

[69]  Justus M. Kebschull,et al.  High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA , 2016, Neuron.

[70]  V. Caviness,et al.  Interhemispheric neocortical connections of the corpus callosum in the normal mouse: A study based on anterograde and retrograde methods , 1975, The Journal of comparative neurology.

[71]  A. N. van den Pol,et al.  Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation , 2017, Science.

[72]  Sean L. Hill,et al.  BigNeuron: Large-Scale 3D Neuron Reconstruction from Optical Microscopy Images , 2015, Neuron.

[73]  Tianyi Mao,et al.  Inputs to the Dorsal Striatum of the Mouse Reflect the Parallel Circuit Architecture of the Forebrain , 2010, Front. Neuroanat..

[74]  Martin Parent,et al.  Single‐axon tracing study of corticostriatal projections arising from primary motor cortex in primates , 2006, The Journal of comparative neurology.

[75]  Allan R. Jones,et al.  Shared and distinct transcriptomic cell types across neocortical areas , 2018, Nature.

[76]  M. Witter,et al.  Subicular efferents are organized mostly as parallel projections: A double‐labeling, retrograde‐tracing study in the rat , 1998, The Journal of comparative neurology.

[77]  Marc Tessier-Lavigne,et al.  Three-Dimensional Adipose Tissue Imaging Reveals Regional Variation in Beige Fat Biogenesis and PRDM16-Dependent Sympathetic Neurite Density. , 2018, Cell metabolism.

[78]  K. Svoboda,et al.  Cell Type-Specific Structural Plasticity of Axonal Branches and Boutons in the Adult Neocortex , 2006, Neuron.

[79]  Tianyi Mao,et al.  A comprehensive thalamocortical projection map at the mesoscopic level , 2014, Nature Neuroscience.

[80]  B. Sakmann,et al.  Three-dimensional axon morphologies of individual layer 5 neurons indicate cell type-specific intracortical pathways for whisker motion and touch , 2011, Proceedings of the National Academy of Sciences.

[81]  Rangasami L. Kashyap,et al.  Building Skeleton Models via 3-D Medial Surface/Axis Thinning Algorithms , 1994, CVGIP Graph. Model. Image Process..

[82]  T. Cutforth,et al.  Sensory maps in the olfactory cortex defined by long-range viral tracing of single neurons , 2011, Nature.

[83]  G. Buzsáki,et al.  Axonal morphometry of hippocampal pyramidal neurons semi-automatically reconstructed after in vivo labeling in different CA3 locations , 2011, Brain Structure and Function.

[84]  Nikola T. Markov,et al.  A Weighted and Directed Interareal Connectivity Matrix for Macaque Cerebral Cortex , 2012, Cerebral cortex.

[85]  Brian S. Eastwood,et al.  Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area , 2018, Nature Communications.

[86]  Karen L. Smith,et al.  Robust, globally consistent and fully automatic multi‐image registration and montage synthesis for 3‐D multi‐channel images , 2011, Journal of microscopy.

[87]  Karel Svoboda,et al.  A platform for brain-wide imaging and reconstruction of individual neurons , 2016, eLife.

[88]  E. Kuramoto,et al.  A morphological analysis of thalamocortical axon fibers of rat posterior thalamic nuclei: a single neuron tracing study with viral vectors. , 2012, Cerebral cortex.

[89]  Mark S. Cembrowski,et al.  Dissociable Structural and Functional Hippocampal Outputs via Distinct Subiculum Cell Classes , 2018, Cell.

[90]  Zengcai V. Guo,et al.  Maintenance of persistent activity in a frontal thalamocortical loop , 2017, Nature.

[91]  R. Turner,et al.  Corticostriatal Activity in Primary Motor Cortex of the Macaque , 2000, The Journal of Neuroscience.