High-throughput mapping of single neuron projections by sequencing of barcoded RNA

Neurons transmit information to distant brain regions via long-range axonal projections. In the mouse, area-to-area connections have only been systematically mapped using bulk labeling techniques, which obscure the diverse projections of intermingled single neurons. Here we describe MAPseq (Multiplexed Analysis of Projections by Sequencing), a technique that can map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences ("barcodes"). Axons are filled with barcode mRNA, each putative projection area isdissected, and the barcode mRNA is extracted and sequenced. Applying MAPseq to the locus coeruleus (LC), we find that individual LC neurons have preferred cortical targets. By recasting neuroanatomy, which is traditionallyviewed as a problem of microscopy, as a problem of sequencing, MAPseq harnesses advances in sequencing technology to permit high-throughput interrogation of brain circuits.

[1]  Staci A. Sorensen,et al.  Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation , 2014, Front. Neural Circuits.

[2]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[3]  M. T. Shipley,et al.  Surprisingly rich projection from locus coeruleus to the olfactory bulb in the rat , 1985, Brain Research.

[4]  Ian R. Wickersham,et al.  Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons , 2007, Neuron.

[5]  Peter Room,et al.  Divergent axon collaterals of rat locu Demonstration by a fluorescent double labeling technique , 1981, Brain Research.

[6]  S. Sternson.  Hypothalamic Survival Circuits: Blueprints for Purposive Behaviors , 2013, Neuron.

[7]  S. Nelson,et al.  Molecular taxonomy of major neuronal classes in the adult mouse forebrain , 2006, Nature Neuroscience.

[8]  Geoffrey E. Hinton,et al.  Visualizing Data using t-SNE , 2008 .

[9]  F. Bloom,et al.  Nucleus locus ceruleus: new evidence of anatomical and physiological specificity. , 1983, Physiological reviews.

[10]  Dennis A. Steindler,et al.  Locus coerules neurons have axons that branch to the forebrain and cerebellum , 1981, Brain Research.

[11]  J. Friedman,et al.  Virus-Assisted Mapping of Neural Inputs to a Feeding Center in the Hypothalamus , 2001, Science.

[12]  S. Sara.  The locus coeruleus and noradrenergic modulation of cognition , 2009, Nature Reviews Neuroscience.

[13]  Edward S Boyden,et al.  Rosetta Brains: A Strategy for Molecularly-Annotated Connectomics , 2014, 1404.5103.

[14]  Lindsey L. Glickfeld,et al.  Cortico-cortical projections in mouse visual cortex are functionally target specific , 2013, Nature Neuroscience.

[15]  J. Troge,et al.  Tumour evolution inferred by single-cell sequencing , 2011, Nature.

[16]  J. Eberwine,et al.  Transcriptome Analysis of Single Cells , 2011, Journal of visualized experiments : JoVE.

[17]  Jonathan D. Cohen,et al.  An integrative theory of locus coeruleus-norepinephrine function: adaptive gain and optimal performance. , 2005, Annual review of neuroscience.

[18]  B. Waterhouse,et al.  Evidence for Broad Versus Segregated Projections from Cholinergic and Noradrenergic Nuclei to Functionally and Anatomically Discrete Subregions of Prefrontal Cortex , 2012, Front. Behav. Neurosci..

[19]  Jan Ellenberg,et al.  λN-GFP: an RNA reporter system for live-cell imaging , 2007, Nature Methods.

[20]  C. Cepko,et al.  Widespread dispersion of neuronal clones across functional regions of the cerebral cortex. , 1992, Science.

[21]  T. Kaneko,et al.  In Vivo Transduction of Central Neurons Using Recombinant Sindbis Virus , 2001, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[22]  Charles R. Gerfen,et al.  Targeting Cre Recombinase to Specific Neuron Populations with Bacterial Artificial Chromosome Constructs , 2007, The Journal of Neuroscience.

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

[24]  Ian R. Wickersham,et al.  Retrograde neuronal tracing with a deletion-mutant rabies virus , 2007, Nature Methods.

[25]  George M. Church,et al.  Highly Multiplexed Subcellular RNA Sequencing in Situ , 2014, Science.

[26]  Hysell V. Oviedo,et al.  Long-term Cre-mediated retrograde tagging of neurons using a novel recombinant pseudorabies virus , 2014, Front. Neuroanat..

[27]  F. Bloom,et al.  Efferent projections of nucleus locus coeruleus: Topographic organization of cells of origin demonstrated by three-dimensional reconstruction , 1986, Neuroscience.

[28]  Pavel Osten,et al.  Stereotaxic gene delivery in the rodent brain , 2007, Nature Protocols.

[29]  B. Waterhouse,et al.  Heterogeneous organization of the locus coeruleus projections to prefrontal and motor cortices , 2014, Proceedings of the National Academy of Sciences.

[30]  M. Ehrengruber.  Alphaviral gene transfer in neurobiology , 2002, Brain Research Bulletin.

[31]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[32]  C. Rice,et al.  Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs , 1993, Journal of virology.

[33]  G. Palm,et al.  Density of neurons and synapses in the cerebral cortex of the mouse , 1989, The Journal of comparative neurology.

[34]  David J. Anderson,et al.  Internal States and Behavioral Decision-Making: Toward an Integration of Emotion and Cognition. , 2014, Cold Spring Harbor symposia on quantitative biology.

[35]  James H. Fallon,et al.  Locus coeruleus projections to cortex: Topography, morphology and collateralization , 1982, Brain Research Bulletin.

[36]  Anthony M. Zador,et al.  Sources of PCR-induced distortions in high-throughput sequencing data sets , 2014, bioRxiv.

[37]  Hanchuan Peng,et al.  mGRASP enables mapping mammalian synaptic connectivity with light microscopy , 2011, Nature Methods.

[38]  S. Herculano‐Houzel,et al.  Cellular scaling rules for rodent brains , 2006, Proceedings of the National Academy of Sciences.

[39]  Susana Q. Lima,et al.  PINP: A New Method of Tagging Neuronal Populations for Identification during In Vivo Electrophysiological Recording , 2009, PloS one.

[40]  E. Petricoin,et al.  Laser Capture Microdissection , 1996, Science.

[41]  R. W. Draft,et al.  Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system , 2007, Nature.

[42]  Hassana K. Oyibo,et al.  Sequencing the Connectome , 2012, PLoS biology.

[43]  Simon Hippenmeyer,et al.  Clonally Related Forebrain Interneurons Disperse Broadly across Both Functional Areas and Structural Boundaries , 2015, Neuron.

[44]  Erika Check Hayden,et al.  Technology: The $1,000 genome , 2014, Nature.

[45]  M. Snyder,et al.  High-throughput sequencing technologies. , 2015, Molecular cell.

[46]  Liqun Luo,et al.  Viral-genetic tracing of the input–output organization of a central norepinephrine circuit , 2015, Nature.

[47]  Kai Johnsson,et al.  An engineered protein tag for multiprotein labeling in living cells. , 2008, Chemistry & biology.

[48]  Anthony J. Movshon,et al.  Visual Response Properties of Striate Cortical Neurons Projecting to Area MT in Macaque Monkeys , 1996, The Journal of Neuroscience.

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

[50]  D. Woodward,et al.  The distribution of neocortical projection neurons in the locus coeruleus , 1983, The Journal of comparative neurology.

[51]  Z Josh Huang,et al.  Toward a Genetic Dissection of Cortical Circuits in the Mouse , 2014, Neuron.

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

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

[54]  A. G. Munford.  A Note on the Uniformity Assumption in the Birthday Problem , 1977 .

[55]  R C Lin,et al.  Lateralization and functional organization of the locus coeruleus projection to the trigeminal somatosensory pathway in rat , 1997, The Journal of comparative neurology.

[56]  S. Foote,et al.  Extrathalamic modulation of cortical function. , 1987, Annual review of neuroscience.

[57]  C. Sheridan.  Illumina claims $1,000 genome win , 2014, Nature Biotechnology.