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Tool Development

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Tool development

We continue to develop tools to interrogate neural circuit assembly and organization with increasing precision. For example, the MARCM (mosaic analysis with a repressible cell marker) method in flies and MADM (mosaic analysis with double markers) method in mice allow the visualization and genetic manipulation of isolated single neurons (see Figs. 1-1, 2-1, 2-2, & 3-1). The Q system further expanded binary expression tools in flies. Our lab first introduced single-cell RNA-sequencing to classify Drosophila neurons and to investigate transcriptome dynamics of specific neuron types across development (Fig. 1-3). Cell-surface proteome profiling enabled the identification of new wiring molecules (Fig. 1-4). Other widely used tools we developed include the Cre reporter mouse mTmG and integrase-mediated transgenesis for producing single-copy transgenes in predetermined loci in mice.

We have recently developed tools to map circuit organization in mammals. The TRIO (tracing the relationship between input and output) and cTRIO (cell-type-specific TRIO) methods allow rabies virus–based input tracing to neurons defined by projection, or by cell type and projection (Fig. 4-1). TrailMap (tissue registration and automated identification of light-sheet microscope acquired projectomes) facilitates mapping of mesoscale axons across the entire mouse brain (Fig. 4-2). The TRAP (targeted recombination in active population) method enables genetic access to neurons based on their activity (Fig. 4-3), which in combination with tools for labeling, tracing, recording, and manipulating neurons, offers a powerful approach for understanding how neural circuits process information and generate behavior.

Selected publications:

Lee T & Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22:451-461.

Zong H, Espinosa JS, Su HH, Muzumdar MD & Luo L (2005) Mosaic analysis with double markers in mice. Cell 121:479-492.

Muzumdar MD, Tasic B, Miyamichi K, Li L & Luo L (2007) A global double-fluorescent Cre reporter mouse. Genesis 45:593-605.

Potter CJ*, Tasic B*, Russler EV, Liang L & Luo L (2010) The Q system: a repressible binary system for transgene expression, lineage tracing and mosaic analysis. Cell 141:536-548.

Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y & Luo L (2011) Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci USA 108:7902-7907.

Guenthner CJ, Miyamichi K, Yang HH, Heller HC & Luo L (2013) Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations. Neuron 78:773-784.

Gao XJ, Riabinina O, Li J, Potter CJ, Clandinin TR, Luo L (2015) A transcriptional reporter of intracellular Ca2+ in Drosophila. Nat Neurosci 18:917-925

Schwarz LA*, Miyamichi K*, Gao XJ, Beier KT, Weissbourd B, DeLoach KE, Ren J, Ibanes S, Malenka RC, Kremer EJ & Luo L (2015). Viral-genetic tracing of the input-output organization of a central noradrenaline circuit. Nature 524:88-92.

Allen WE*, DeNardo LA*, Chen MZ*, Liu CD, Loh KM, Fenno LE, Ramakrishnan C, Deisseroth K & Luo L (2017) Thirst-associated median preoptic neurons encode an aversive motivational drive. Science 357:1149-1155.

Li H*, Horns F*, Wu B, Xie Q, Li J, Li T, Luginbuhl DJ, Quake SR# & Luo L# (2017) Classifying Drosophila olfactory projection neuron subtypes by single-cell RNA sequencing. Cell 171:1206-1220.

DeNardo LA, Liu CD, Allen WE, Adams EL, Friedmann D, Fu L, Guenthner CJ, Tessier-Lavigne M & Luo L (2019) Temporal evolution of cortical ensembles promoting remote memory retrieval. Nat Neurosci 22:460-469.

Li J*, Han S*, Li H, Udeshi ND, Svinkina T, Mani DR, Xu C, Guajardo R, Xie Q, Li T, Luginbuhl DJ, Bing W, McLaughlin CN, Xie A, Kaewsapsak P, Quake SR, Carr SA, Ting AY# & Luo L# (2020) Cell-surface proteomic profiling in the fly brain uncovers new wiring regulators. Cell 180:373-386.

Friedmann D*, Pun A*, Adams EL, Lui JH, Kebschull JM, Grutzner SM, Castagnola C, Tessier-Lavigne M & Luo L (2020) Mapping mesoscale axonal projections in the mouse brain using a 3D convolutional network. Proc Natl Acad Sci USA 117:11068-11075.

* co-first authors; # co-corresponding authors

Fig. 4-1: TRIO and cTRIO for tracing the relationship between input and output of neural circuits. (A) In TRIO, input to B neurons projecting to a specific target site (C1 but not C2) are specifically labeled via glycoprotein deleted rabies virus (RVdG)–mediated retrograde trans-synaptic tracing initiated at target C1 using a virus that expresses the Cre recombinase and transduces from axon terminals (such as canine adenovirus 2, CAV2). (B) In cell-type-specific TRIO (cTRIO), input to a specific type of B neurons (defined by Cre expression) and projecting to a specific target site are specifically labeled via RVdG-medicated retrograde trans-synaptic tracing initiated at target C1 using CAV2 that encodes Cre-dependent Flp recombinase. Adapted from Schwarz et al. (2015) Nature 524:88-92.

Fig. 4-2 – Mapping meso-scale axon projections via TrailMap. TrailMap-extracted serotonergic axons innervating the forebrain are subdivided and color-coded based on their presence in Allen Brain Atlas–defined target regions seen from a dorsal viewpoint, with major subdivisions spatially separated. Midline is represented by a dashed white line. Inset highlights axons in a coronal Z-projection of 500 μm. Scale bar, 200 μm. Adapted from Friedmann et al. (2020) Proc Natl Acad Sci USA 117:11068-11075.

Fig. 4-3 – TRAPing active neurons representing thirst and memory. (A) tdTomato-labeled cells in median preoptic nucleus (MnPO) after TRAPing in mice that were water-satiated (left) or water restricted (right). A specific subset of MnPO neurons become highly activated in thirsty mice. a.c., anterior commissure. Genetic access to these dehydration-activated cells allowed us to investigate their physiological properties and behavioral functions (see Fig. 3-6). Adapted from Allen et al. (2017) Science 357:1149-1155. (B) Systematic comparisons of neurons activated by fear memory at different times after learning (TRAP, here 7 days after learning) and remote fear memory recall (Fos; 28 days after learning) suggest that neuronal ensembles that represent fear memory in the prelimbic cortex evolve over time. Neurons TRAPed during memory recalls at later timepoints (e.g., 7 or 14 days after learning, compared to 1 day after learning) are more likely to be reactivated during, and make larger behavioral contributions to, remote memory recall (28 days after learning). Adapted from DeNardo et al. (2019) Nat Neurosci 22:460-469.