Highly efficient and super-bright neurocircuit tracing using vector mixing-based virus cocktail

Mapping the detailed cell-type-specific input networks and neuronal projectomes are essential to understand brain function in normal and pathological states. However, several properties of current tracing systems, including labeling sensitivity, trans-synaptic efficiencies, reproducibility among different individuals and different Cre-driver animals, still remained unsatisfactory. Here, we developed MAP-ENVIVIDERS, a recombinase system-dependent vector mixing-based strategy for highly efficient neurocircuit tracing. MAP-ENVIVIDERS enhanced tracing efficiency of input networks across the whole brain, with over 10-fold improvement in diverse previously poor-labeled input brain regions and particularly, up to 70-fold enhancement in brainstem compared with the current standard rabies-virus-mediated systems. MAP-ENVIVIDERS was over 10-fold more sensitive for cell-type-specific labeling than previous strategies, enabling us to capture individual cell-type-specific neurons with extremely complex axonal branches and presynaptic axonal boutons, both about one order of magnitude than previously reported and considered. MAP-ENVIVIDERS provides powerful tools for deconstructing novel input/output circuitry towards functional studies and disorders-related mechanisms.

[1]  Attila Losonczy,et al.  Rabies Virus CVS-N2cΔG Strain Enhances Retrograde Synaptic Transfer and Neuronal Viability , 2016, Neuron.

[2]  Wenhui Li,et al.  Influenza A Virus Neuraminidase Limits Viral Superinfection , 2008, Journal of Virology.

[3]  Shaoqun Zeng,et al.  Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging , 2014, Nature Communications.

[4]  Alison S. Walker,et al.  A Distance-Dependent Distribution of Presynaptic Boutons Tunes Frequency-Dependent Dendritic Integration , 2018, Neuron.

[5]  H. Büning,et al.  Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities , 2017 .

[6]  Qingming Luo,et al.  Single-cell transcriptomes and whole-brain projections of serotonin neurons in the mouse dorsal and median raphe nuclei , 2019, eLife.

[7]  K. Deisseroth,et al.  Parvalbumin neurons and gamma rhythms enhance cortical circuit performance , 2009, Nature.

[8]  J Montane,et al.  High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency , 2010, Gene Therapy.

[9]  Shaoqun Zeng,et al.  Cell-type-specific and projection-specific brain-wide reconstruction of single neurons , 2018, Nature Methods.

[10]  Hongkui Zeng,et al.  Generation of a whole-brain atlas for the cholinergic system and mesoscopic projectome analysis of basal forebrain cholinergic neurons , 2017, Proceedings of the National Academy of Sciences.

[11]  C. Gilbert,et al.  Axons and Synaptic Boutons Are Highly Dynamic in Adult Visual Cortex , 2006, Neuron.

[12]  Ian R. Wickersham,et al.  Cortical representations of olfactory input by trans-synaptic tracing , 2011, Nature.

[13]  Adi Mizrahi,et al.  Dissecting Local Circuits: Parvalbumin Interneurons Underlie Broad Feedback Control of Olfactory Bulb Output , 2013, Neuron.

[14]  Shaoqun Zeng,et al.  DeepBouton: Automated Identification of Single-Neuron Axonal Boutons at the Brain-Wide Scale , 2019, Front. Neuroinform..

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

[16]  K. Svoboda,et al.  Experience-dependent structural synaptic plasticity in the mammalian brain , 2009, Nature Reviews Neuroscience.

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

[18]  B. Byrne,et al.  Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield , 1999, Gene Therapy.

[19]  J. Trempe,et al.  Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection , 2006, Journal of Virology.

[20]  Partha P. Mitra,et al.  Comparative three-dimensional connectome map of motor cortical projections in the mouse brain , 2016, Scientific Reports.

[21]  N. Plesnila,et al.  Shrinkage-mediated imaging of entire organs and organisms using uDISCO , 2016, Nature Methods.

[22]  Dongmin Lee,et al.  A calcium- and light-gated switch to induce gene expression in activated neurons , 2017, Nature Biotechnology.

[23]  Hildegard Büning,et al.  Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities. , 2017, Human gene therapy methods.

[24]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[25]  Liqun Luo,et al.  Monosynaptic Circuit Tracing with Glycoprotein-Deleted Rabies Viruses , 2015, The Journal of Neuroscience.

[26]  Karl Deisseroth,et al.  A Guide to Creating and Testing New INTRSECT Constructs , 2017, Current protocols in neuroscience.

[27]  Takaki Komiyama,et al.  Subtype-specific plasticity of inhibitory circuits in motor cortex during motor learning , 2015, Nature Neuroscience.

[28]  Michael J. Goard,et al.  Fast Modulation of Visual Perception by Basal Forebrain Cholinergic Neurons , 2013, Nature Neuroscience.

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

[30]  U. Bantel‐Schaal,et al.  Adeno-associated viruses inhibit SV40 DNA amplification and replication of herpes simplex virus in SV40-transformed hamster cells. , 1988, Virology.

[31]  Lief E. Fenno,et al.  Targeting cells with single vectors using multiple-feature Boolean logic , 2014, Nature Methods.

[32]  Martin K. Schwarz,et al.  Dysfunction of Somatostatin-Positive Interneurons Associated with Memory Deficits in an Alzheimer’s Disease Model , 2016, Neuron.

[33]  Wei-Cheng Chang,et al.  Cell type-specific long-range connections of basal forebrain circuit , 2016, eLife.

[34]  Wei-Cheng Chang,et al.  Organization of long-range inputs and outputs of frontal cortex for top-down control , 2016, Nature Neuroscience.

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

[36]  Ralf Bartenschlager,et al.  Analysis of Hepatitis C Virus Superinfection Exclusion by Using Novel Fluorochrome Gene-Tagged Viral Genomes , 2007, Journal of Virology.

[37]  G. Shepherd,et al.  The neocortical circuit: themes and variations , 2015, Nature Neuroscience.

[38]  David V Schaffer,et al.  Viral Vectors for Gene Therapy: Translational and Clinical Outlook. , 2015, Annual review of biomedical engineering.

[39]  David V. Schaffer,et al.  Engineering adeno-associated viruses for clinical gene therapy , 2014, Nature Reviews Genetics.

[40]  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.

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

[42]  Liqun Luo,et al.  Diversity of Transgenic Mouse Models for Selective Targeting of Midbrain Dopamine Neurons , 2015, Neuron.

[43]  Yaron Lipman,et al.  Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes , 2010, Nature communications.

[44]  Bertram Wiedenmann,et al.  Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles , 1985, Cell.

[45]  George Paxinos,et al.  The Mouse Brain in Stereotaxic Coordinates , 2001 .

[46]  C. Gerfen,et al.  GENSAT BAC Cre-Recombinase Driver Lines to Study the Functional Organization of Cerebral Cortical and Basal Ganglia Circuits , 2013, Neuron.

[47]  Lynn W. Enquist,et al.  Circuit-Specific Coinfection of Neurons in the Rat Central Nervous System with Two Pseudorabies Virus Recombinants , 1999, Journal of Virology.

[48]  Karel Svoboda,et al.  Long-Range Neuronal Circuits Underlying the Interaction between Sensory and Motor Cortex , 2011, Neuron.

[49]  Irina A. Kochetkova,et al.  gD-Independent Superinfection Exclusion of Alphaherpesviruses , 2016, Journal of Virology.

[50]  J. Grieger,et al.  Production and characterization of adeno-associated viral vectors , 2006, Nature Protocols.

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

[52]  Edward M. Callaway,et al.  Genetic Dissection of Neural Circuits: A Decade of Progress. , 2018, Neuron.

[53]  Sachie K. Ogawa,et al.  Whole-Brain Mapping of Direct Inputs to Midbrain Dopamine Neurons , 2012, Neuron.

[54]  Charles Rattray,et al.  Themes and variations , 2007, Architectural Research Quarterly.

[55]  Charles R. Gerfen,et al.  Reconstruction of 1,000 Projection Neurons Reveals New Cell Types and Organization of Long-Range Connectivity in the Mouse Brain , 2019, Cell.

[56]  W. Strohl,et al.  Adeno-Associated Virus (AAV) as a Vector for Gene Therapy , 2017, BioDrugs.

[57]  Hongkui Zeng,et al.  Genetic approaches to neural circuits in the mouse. , 2013, Annual review of neuroscience.

[58]  M. Ananth,et al.  Basal Forebrain Cholinergic Circuits and Signaling in Cognition and Cognitive Decline , 2016, Neuron.

[59]  K. Tye,et al.  A light- and calcium-gated transcription factor for imaging and manipulating activated neurons , 2017, Nature Biotechnology.

[60]  Edward M Callaway,et al.  Improved Monosynaptic Neural Circuit Tracing Using Engineered Rabies Virus Glycoproteins. , 2016, Cell reports.

[61]  Liqun Luo,et al.  Presynaptic Partners of Dorsal Raphe Serotonergic and GABAergic Neurons , 2014, Neuron.

[62]  Sen Song,et al.  Increased axonal bouton dynamics in the aging mouse cortex , 2013, Proceedings of the National Academy of Sciences.

[63]  B. Byrne,et al.  Copackaging of multiple adeno-associated viral vectors in a single production step. , 2014, Human gene therapy methods.

[64]  Ruian Xu,et al.  Efficient production of dual recombinant adeno-associated viral vectors for factor VIII delivery. , 2014, Human gene therapy methods.

[65]  Fumitaka Osakada,et al.  Design and generation of recombinant rabies virus vectors , 2013, Nature Protocols.

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

[67]  Naoshige Uchida,et al.  Organization of monosynaptic inputs to the serotonin and dopamine neuromodulatory systems. , 2014, Cell reports.