Liqun Luo

All Publications

• Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators. Cell Li, J., Han, S., Li, H., Udeshi, N. D., Svinkina, T., Mani, D. R., Xu, C., Guajardo, R., Xie, Q., Li, T., Luginbuhl, D. J., Wu, B., McLaughlin, C. N., Xie, A., Kaewsapsak, P., Quake, S. R., Carr, S. A., Ting, A. Y., Luo, L. 2020

  Abstract

  Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed invivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

  View details for DOI 10.1016/j.cell.2019.12.029

  View details for PubMedID 31955847

• Shared Cortex-Cerebellum Dynamics in the Execution and Learning of a Motor Task CELL Wagner, M. J., Kim, T., Kadmon, J., Nguyen, N. D., Ganguli, S., Schnitzer, M. J., Luo, L. 2019; 177 (3): 669-+

  View details for DOI 10.1016/j.cell.2019.02.019

  View details for Web of Science ID 000464947700020

• Temporal evolution of cortical ensembles promoting remote memory retrieval. Nature neuroscience DeNardo, L. A., Liu, C. D., Allen, W. E., Adams, E. L., Friedmann, D., Fu, L., Guenthner, C. J., Tessier-Lavigne, M., Luo, L. 2019

  Abstract

  Memories of fearful events can last a lifetime. The prelimbic (PL) cortex, a subregion of prefrontal cortex, plays a critical role in fear memory retrieval over time. Most studies have focused on acquisition, consolidation, and retrieval of recent memories, but much less is known about the neural mechanisms of remote memory. Using a new knock-in mouse for activity-dependent genetic labeling (TRAP2), we demonstrate that neuronal ensembles in the PL cortex are dynamic. PL neurons TRAPed during later memory retrievals are more likely to be reactivated and make larger behavioral contributions to remote memory retrieval compared to those TRAPed during learning or early memory retrieval. PL activity during learning is required to initiate this time-dependent reorganization in PL ensembles underlying memory retrieval. Finally, while neurons TRAPed during earlier and later retrievals have similar broad projections throughout the brain, PL neurons TRAPed later have a stronger functional recruitment of cortical targets.

  View details for PubMedID 30692687

• Single-cell transcriptomes and whole-brain projections of serotonin neurons in the mouse dorsal and median raphe nuclei. eLife Ren, J., Isakova, A., Friedmann, D., Zeng, J., Grutzner, S. M., Pun, A., Zhao, G. Q., Kolluru, S. S., Wang, R., Lin, R., Li, P., Li, A., Raymond, J. L., Luo, Q., Luo, M., Quake, S. R., Luo, L. 2019; 8

  Abstract

  Serotonin neurons of the dorsal and median raphe nuclei (DR, MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed diverse and segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.

  View details for DOI 10.7554/eLife.49424

  View details for PubMedID 31647409

• Genetic Dissection of Neural Circuits: A Decade of Progress NEURON Luo, L., Callaway, E. M., Svoboda, K. 2018; 98 (2): 256–81

  Abstract

  Tremendous progress has been made since Neuron published our Primer on genetic dissection of neural circuits 10 years ago. Since then, cell-type-specific anatomical, neurophysiological, and perturbation studies have been carried out in a multitude of invertebrate and vertebrate organisms, linking neurons and circuits to behavioral functions. New methods allow systematic classification of cell types and provide genetic access to diverse neuronal types for studies of connectivity and neural coding during behavior. Here we evaluate key advances over the past decade and discuss future directions.

  View details for PubMedID 29673479

  View details for PubMedCentralID PMC5912347

• Teneurin-3 controls topographic circuit assembly in the hippocampus. Nature Berns, D. S., DeNardo, L. A., Pederick, D. T., Luo, L. 2018; 554 (7692): 328–33

  Abstract

  Brain functions rely on specific patterns of connectivity. Teneurins are evolutionarily conserved transmembrane proteins that instruct synaptic partner matching in Drosophila and are required for vertebrate visual system development. The roles of vertebrate teneurins in connectivity beyond the visual system remain largely unknown and their mechanisms of action have not been demonstrated. Here we show that mouse teneurin-3 is expressed in multiple topographically interconnected areas of the hippocampal region, including proximal CA1, distal subiculum, and medial entorhinal cortex. Viral-genetic analyses reveal that teneurin-3 is required in both CA1 and subicular neurons for the precise targeting of proximal CA1 axons to distal subiculum. Furthermore, teneurin-3 promotes homophilic adhesion in vitro in a splicing isoform-dependent manner. These findings demonstrate striking genetic heterogeneity across multiple hippocampal areas and suggest that teneurin-3 may orchestrate the assembly of a complex distributed circuit in the mammalian brain via matching expression and homophilic attraction.

  View details for PubMedID 29414938

• Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems. Cell Ren, J., Friedmann, D., Xiong, J., Liu, C. D., Ferguson, B. R., Weerakkody, T., DeLoach, K. E., Ran, C., Pun, A., Sun, Y., Weissbourd, B., Neve, R. L., Huguenard, J., Horowitz, M. A., Luo, L. 2018

  Abstract

  The dorsal raphe (DR) constitutes a major serotonergic input to the forebrain and modulates diverse functions and brain states, including mood, anxiety, and sensory and motor functions. Most functional studies to date have treated DR serotonin neurons as a single population. Using viral-genetic methods, we found that subcortical- and cortical-projecting serotonin neurons have distinct cell-body distributions within the DR and differentially co-express a vesicular glutamate transporter. Further, amygdala- and frontal-cortex-projecting DR serotonin neurons have largely complementary whole-brain collateralization patterns, receive biased inputs from presynaptic partners, and exhibit opposite responses to aversive stimuli. Gain- and loss-of-function experiments suggest that amygdala-projecting DR serotonin neurons promote anxiety-like behavior, whereas frontal-cortex-projecting neurons promote active coping in the face of challenge. These results provide compelling evidence that the DR serotonin system contains parallel sub-systems that differ in input and output connectivity, physiological response properties, and behavioral functions.

  View details for PubMedID 30146164

• Cerebellar granule cells encode the expectation of reward NATURE Wagner, M. J., Kim, T. H., Savall, J., Schnitzer, M. J., Luo, L. 2017; 544 (7648): 96-?

  Abstract

  The human brain contains approximately 60 billion cerebellar granule cells, which outnumber all other brain neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes. Although evidence suggests a role for the cerebellum in cognition, granule cells are known to encode only sensory and motor context. Here, using two-photon calcium imaging in behaving mice, we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed sugar-water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward-omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from the current understanding of these neurons and markedly enriches the contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum.

  View details for DOI 10.1038/nature21726

  View details for Web of Science ID 000398323300040

  View details for PubMedID 28321129

• Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-Cell RNA Sequencing. Cell Li, H., Horns, F., Wu, B., Xie, Q., Li, J., Li, T., Luginbuhl, D. J., Quake, S. R., Luo, L. 2017; 171 (5): 1206–20.e22

  Abstract

  The definition of neuronal type and how it relates to the transcriptome are open questions. Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: different PN classes target dendrites to distinct olfactory glomeruli, while PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomes of most PN classes and unequivocally mapped transcriptomes to specific olfactory function for six classes. Transcriptomes of closely related PN classes exhibit the largest differences during circuit assembly but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Transcription factors and cell-surface molecules are the most differentially expressed genes between classes and are highly informative in encoding cell identity, enabling us to identify a new lineage-specific transcription factor that instructs PN dendrite targeting. These findings establish that neuronal transcriptomic identity corresponds with anatomical and physiological identity defined by connectivity and function.

  View details for PubMedID 29149607

• Thirst-associated preoptic neurons encode an aversive motivational drive. Science (New York, N.Y.) Allen, W. E., DeNardo, L. A., Chen, M. Z., Liu, C. D., Loh, K. M., Fenno, L. E., Ramakrishnan, C., Deisseroth, K., Luo, L. 2017; 357 (6356): 1149–55

  Abstract

  Water deprivation produces a drive to seek and consume water. How neural activity creates this motivation remains poorly understood. We used activity-dependent genetic labeling to characterize neurons activated by water deprivation in the hypothalamic median preoptic nucleus (MnPO). Single-cell transcriptional profiling revealed that dehydration-activated MnPO neurons consist of a single excitatory cell type. After optogenetic activation of these neurons, mice drank water and performed an operant lever-pressing task for water reward with rates that scaled with stimulation frequency. This stimulation was aversive, and instrumentally pausing stimulation could reinforce lever-pressing. Activity of these neurons gradually decreased over the course of an operant session. Thus, the activity of dehydration-activated MnPO neurons establishes a scalable, persistent, and aversive internal state that dynamically controls thirst-motivated behavior.

  View details for PubMedID 28912243

• Molecular and Neural Functions of Rai1, the Causal Gene for Smith-Magenis Syndrome. Neuron Huang, W., Guenthner, C. J., Xu, J., Nguyen, T., Schwarz, L. A., Wilkinson, A. W., Gozani, O., Chang, H. Y., Shamloo, M., Luo, L. 2016; 92 (2): 392-406

  Abstract

  Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), which is associated with diverse neurodevelopmental and behavioral symptoms as well as obesity. RAI1 encodes a nuclear protein but little is known about its molecular function or the cell types responsible for SMS symptoms. Using genetically engineered mice, we found that Rai1 preferentially occupies DNA regions near active promoters and promotes the expression of a group of genes involved in circuit assembly and neuronal communication. Behavioral analyses demonstrated that pan-neural loss of Rai1 causes deficits in motor function, learning, and food intake. These SMS-like phenotypes are produced by loss of Rai1 function in distinct neuronal types: Rai1 loss in inhibitory neurons or subcortical glutamatergic neurons causes learning deficits, while Rai1 loss in Sim1(+) or SF1(+) cells causes obesity. By integrating molecular and organismal analyses, our study suggests potential therapeutic avenues for a complex neurodevelopmental disorder.

  View details for DOI 10.1016/j.neuron.2016.09.019

  View details for PubMedID 27693255

• Viral-genetic tracing of the input-output organization of a central noradrenaline circuit. Nature Schwarz, L. A., Miyamichi, K., Gao, X. J., Beier, K. T., Weissbourd, B., DeLoach, K. E., Ren, J., Ibanes, S., Malenka, R. C., Kremer, E. J., Luo, L. 2015; 524 (7563): 88-92

  Abstract

  Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input-output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The 'tracing the relationship between input and output' method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input-output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

  View details for DOI 10.1038/nature14600

  View details for PubMedID 26131933

• Principles of Neurobiology Luo, L. Garland Science. 2015
• Mapping mesoscale axonal projections in the mouse brain using a 3D convolutional network. Proceedings of the National Academy of Sciences of the United States of America Friedmann, D., Pun, A., Adams, E. L., Lui, J. H., Kebschull, J. M., Grutzner, S. M., Castagnola, C., Tessier-Lavigne, M., Luo, L. 2020

  Abstract

  The projection targets of a neuronal population are a key feature of its anatomical characteristics. Historically, tissue sectioning, confocal microscopy, and manual scoring of specific regions of interest have been used to generate coarse summaries of mesoscale projectomes. We present here TrailMap, a three-dimensional (3D) convolutional network for extracting axonal projections from intact cleared mouse brains imaged by light-sheet microscopy. TrailMap allows region-based quantification of total axon content in large and complex 3D structures after registration to a standard reference atlas. The identification of axonal structures as thin as one voxel benefits from data augmentation but also requires a loss function that tolerates errors in annotation. A network trained with volumes of serotonergic axons in all major brain regions can be generalized to map and quantify axons from thalamocortical, deep cerebellar, and cortical projection neurons, validating transfer learning as a tool to adapt the model to novel categories of axonal morphology. Speed of training, ease of use, and accuracy improve over existing tools without a need for specialized computing hardware. Given the recent emphasis on genetically and functionally defining cell types in neural circuit analysis, TrailMap will facilitate automated extraction and quantification of axons from these specific cell types at the scale of the entire mouse brain, an essential component of deciphering their connectivity.

  View details for DOI 10.1073/pnas.1918465117

  View details for PubMedID 32358193

• Loss of the neural-specific BAF subunit ACTL6B relieves repression of early response genes and causes recessive autism. Proceedings of the National Academy of Sciences of the United States of America Wenderski, W., Wang, L., Krokhotin, A., Walsh, J. J., Li, H., Shoji, H., Ghosh, S., George, R. D., Miller, E. L., Elias, L., Gillespie, M. A., Son, E. Y., Staahl, B. T., Baek, S. T., Stanley, V., Moncada, C., Shipony, Z., Linker, S. B., Marchetto, M. C., Gage, F. H., Chen, D., Sultan, T., Zaki, M. S., Ranish, J. A., Miyakawa, T., Luo, L., Malenka, R. C., Crabtree, G. R., Gleeson, J. G. 2020

  Abstract

  Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.

  View details for DOI 10.1073/pnas.1908238117

  View details for PubMedID 32312822

• LIS1 determines cleavage plane positioning by regulating actomyosin-mediated cell membrane contractility. eLife Moon, H. M., Hippenmeyer, S., Luo, L., Wynshaw-Boris, A. 2020; 9

  Abstract

  Heterozygous loss of human PAFAH1B1 (coding for LIS1) results in the disruption of neurogenesis and neuronal migration via dysregulation of microtubule (MT) stability and dynein motor function/localization that alters mitotic spindle orientation, chromosomal segregation, and nuclear migration. Recently, human induced pluripotent stem cell (iPSC) models revealed an important role for LIS1 in controlling the length of terminal cell divisions of outer radial glial (oRG) progenitors, suggesting cellular functions of LIS1 in regulating neural progenitor cell (NPC) daughter cell separation. Here we examined the late mitotic stages NPCs in vivo and mouse embryonic fibroblasts (MEFs) in vitro from Pafah1b1-deficient mutants. Pafah1b1-deficient neocortical NPCs and MEFs similarly exhibited cleavage plane displacement with mislocalization of furrow-associated markers, associated with actomyosin dysfunction and cell membrane hyper-contractility. Thus, it suggests LIS1 acts as a key molecular link connecting MTs/dynein and actomyosin, ensuring that cell membrane contractility is tightly controlled to execute proper daughter cell separation.

  View details for DOI 10.7554/eLife.51512

  View details for PubMedID 32159512

• Amygdala-Midbrain Connections Modulate Appetitive and Aversive Learning. Neuron Steinberg, E. E., Gore, F., Heifets, B. D., Taylor, M. D., Norville, Z. C., Beier, K. T., Földy, C., Lerner, T. N., Luo, L., Deisseroth, K., Malenka, R. C. 2020

  Abstract

  The central amygdala (CeA) orchestrates adaptive responses to emotional events. While CeA substrates for defensive behaviors have been studied extensively, CeA circuits for appetitive behaviors and their relationship to threat-responsive circuits remain poorly defined. Here, we demonstrate that the CeA sends robust inhibitory projections to the lateral substantia nigra (SNL) that contribute to appetitive and aversive learning in mice. CeA→SNL neural responses to appetitive and aversive stimuli were modulated by expectation and magnitude consistent with a population-level salience signal, which was required for Pavlovian conditioned reward-seeking and defensive behaviors. CeA→SNL terminal activation elicited reinforcement when linked to voluntary actions but failed to support Pavlovian associations that rely on incentive value signals. Consistent with a disinhibitory mechanism, CeA inputs preferentially target SNL GABA neurons, and CeA→SNL and SNL dopamine neurons respond similarly to salient stimuli. Collectively, our results suggest that amygdala-nigra interactions represent a previously unappreciated mechanism for influencing emotional behaviors.

  View details for DOI 10.1016/j.neuron.2020.03.016

  View details for PubMedID 32294466

• Single-Cell Transcriptomes Reveal Diverse Regulatory Strategies for Olfactory Receptor Expression and Axon Targeting. Current biology : CB Li, H., Li, T., Horns, F., Li, J., Xie, Q., Xu, C., Wu, B., Kebschull, J. M., McLaughlin, C. N., Kolluru, S. S., Jones, R. C., Vacek, D., Xie, A., Luginbuhl, D. J., Quake, S. R., Luo, L. 2020

  Abstract

  The regulatory mechanisms by which neurons coordinate their physiology and connectivity are not well understood. The Drosophila olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. Each ORN type expresses a unique olfactory receptor, or a combination thereof, and sends their axons to a stereotyped glomerulus. Using single-cell RNA sequencing, we identified 33 transcriptomic clusters for ORNs and mapped 20 to their glomerular types, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN types. Each ORN type expresses hundreds of transcription factors. Transcriptome-instructed genetic analyses revealed that (1) one broadly expressed transcription factor (Acj6) only regulates olfactory receptor expression in one ORN type and only wiring specificity in another type, (2) one type-restricted transcription factor (Forkhead) only regulates receptor expression, and (3) another type-restricted transcription factor (Unplugged) regulates both events. Thus, ORNs utilize diverse strategies and complex regulatory networks to coordinate their physiology and connectivity.

  View details for DOI 10.1016/j.cub.2020.01.049

  View details for PubMedID 32059767

• Brain Circuit of Claustrophobia-like Behavior in Mice Identified by Upstream Tracing of Sighing. Cell reports Li, P., Li, S. B., Wang, X., Phillips, C. D., Schwarz, L. A., Luo, L., de Lecea, L., Krasnow, M. A. 2020; 31 (11): 107779

  Abstract

  Emotions are distinct patterns of behavioral and physiological responses triggered by stimuli that induce different brain states. Elucidating the circuits is difficult because of challenges in interrogating emotional brain states and their complex outputs. Here, we leverage the recent discovery in mice of a neural circuit for sighing, a simple, quantifiable output of various emotions. We show that mouse confinement triggers sighing, and this "claustrophobic" sighing, but not accompanying tachypnea, requires the same medullary neuromedin B (Nmb)-expressing neurons as physiological sighing. Retrograde tracing from the Nmb neurons identified 12 forebrain centers providing presynaptic input, including hypocretin (Hcrt)-expressing lateral hypothalamic neurons. Confinement activates Hcrt neurons, and optogenetic activation induces sighing and tachypnea whereas pharmacologic inhibition suppresses both responses. The effect on sighing is mediated by HCRT directly on Nmbneurons. We propose that this HCRT-NMB neuropeptide relay circuit mediates claustrophobic sighing and that activated Hcrt neurons are a claustrophobia brain state that directly controls claustrophobic outputs.

  View details for DOI 10.1016/j.celrep.2020.107779

  View details for PubMedID 32553161

• Nurturing Undergraduate Researchers in Biomedical Sciences. Cell Li, J., Luo, L. 2020; 182 (1): 1–4

  Abstract

  Undergraduate researchers are the next-generation scientists. Here, we call for more attention from our community to the proper training of undergraduates in biomedical research laboratories. By dissecting common pitfalls, we suggest how to better mentor undergraduates and prepare them for flourishing careers.

  View details for DOI 10.1016/j.cell.2020.05.008

  View details for PubMedID 32649872

• Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses. eLife Donnelly, K. M., DeLorenzo, O. R., Zaya, A. D., Pisano, G. E., Thu, W. M., Luo, L., Kopito, R. R., Panning Pearce, M. M. 2020; 9

  Abstract

  Emerging evidence supports the hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell to cell through the brain in a manner akin to infectious prions. Here, we show that mutant huntingtin (mHtt) aggregates associated with Huntington disease transfer anterogradely from presynaptic to postsynaptic neurons in the adult Drosophila olfactory system. Trans-synaptic transmission of mHtt aggregates is inversely correlated with neuronal activity and blocked by inhibiting caspases in presynaptic neurons, implicating synaptic dysfunction and cell death in aggregate spreading. Remarkably, mHtt aggregate transmission across synapses requires the glial scavenger receptor Draper and involves a transient visit to the glial cytoplasm, indicating that phagocytic glia act as obligatory intermediates in aggregate spreading between synaptically-connected neurons. These findings expand our understanding of phagocytic glia as double-edged players in neurodegeneration-by clearing neurotoxic protein aggregates, but also providing an opportunity for prion-like seeds to evade phagolysosomal degradation and propagate further in the brain.

  View details for DOI 10.7554/eLife.58499

  View details for PubMedID 32463364

• The Temporal Association Cortex Plays a Key Role in Auditory-Driven Maternal Plasticity. Neuron Tasaka, G. I., Feigin, L., Maor, I., Groysman, M., DeNardo, L. A., Schiavo, J. K., Froemke, R. C., Luo, L., Mizrahi, A. 2020

  Abstract

  Mother-infant bonding develops rapidly following parturition and is accompanied by changes in sensory perception and behavior. Here, we study how ultrasonic vocalizations (USVs) are represented in the brain of mothers. Using a mouse line that allows temporally controlled genetic access to active neurons, we find that the temporal association cortex (TeA) in mothers exhibits robust USV responses. Rabies tracing from USV-responsive neurons reveals extensive subcortical and cortical inputs into TeA. A particularly dominant cortical source of inputs is the primary auditory cortex (A1), suggesting strong A1-to-TeA connectivity. Chemogenetic silencing of USV-responsive neurons in TeA impairs auditory-driven maternal preference in a pup-retrieval assay. Furthermore, dense extracellular recordings from awake mice reveal changes of both single-neuron and population responses to USVs in TeA, improving discriminability of pup calls in mothers compared with naive females. These data indicate that TeA plays a key role in encoding and perceiving pup cries during motherhood.

  View details for DOI 10.1016/j.neuron.2020.05.004

  View details for PubMedID 32473095

• Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors. Neuron Luo, L., Ambrozkiewicz, M. C., Benseler, F., Chen, C., Dumontier, E., Falkner, S., Furlanis, E., Gomez, A. M., Hoshina, N., Huang, W. H., Hutchison, M. A., Itoh-Maruoka, Y., Lavery, L. A., Li, W., Maruo, T., Motohashi, J., Pai, E. L., Pelkey, K. A., Pereira, A., Philips, T., Sinclair, J. L., Stogsdill, J. A., Traunmüller, L., Wang, J., Wortel, J., You, W., Abumaria, N., Beier, K. T., Brose, N., Burgess, H. A., Cepko, C. L., Cloutier, J. F., Eroglu, C., Goebbels, S., Kaeser, P. S., Kay, J. N., Lu, W., Luo, L., Mandai, K., McBain, C. J., Nave, K. A., Prado, M. A., Prado, V. F., Rothstein, J., Rubenstein, J. L., Saher, G., Sakimura, K., Sanes, J. R., Scheiffele, P., Takai, Y., Umemori, H., Verhage, M., Yuzaki, M., Zoghbi, H. Y., Kawabe, H., Craig, A. M. 2020

  Abstract

  The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

  View details for DOI 10.1016/j.neuron.2020.01.008

  View details for PubMedID 32027825

• Skilled reaching tasks for head-fixed mice using a robotic manipulandum. Nature protocols Wagner, M. J., Savall, J., Kim, T. H., Schnitzer, M. J., Luo, L. 2020

  Abstract

  Skilled forelimb behaviors are among the most important for studying motor learning in multiple species including humans. This protocol describes learned forelimb tasks for mice using a two-axis robotic manipulandum. Our device provides a highly compact adaptation of actuated planar two-axis arms that is simple and inexpensive to construct. This paradigm has been dominant for decades in primate motor neuroscience. Our device can generate arbitrary virtual movement tracks, arbitrary time-varying forces or arbitrary position- or velocity-dependent force patterns. We describe several example tasks permitted by our device, including linear movements, movement sequences and aiming movements. We provide the mechanical drawings and source code needed to assemble and control the device, and detail the procedure to train mice to use the device. Our software can be simply extended to allow users to program various customized movement assays. The device can be assembled in a few days, and the time to train mice on the tasks that we describe ranges from a few days to several weeks. Furthermore, the device is compatible with various neurophysiological techniques that require head fixation.

  View details for DOI 10.1038/s41596-019-0286-8

  View details for PubMedID 32034393

• Neocortex-Cerebellum Circuits for Cognitive Processing. Trends in neurosciences Wagner, M. J., Luo, L. 2019

  Abstract

  Although classically thought of as a motor circuit, the cerebellum is now understood to contribute to a wide variety of cognitive functions through its dense interconnections with the neocortex, the center of brain cognition. Recent investigations have shed light on the nature of cerebellar cognitive processing and information exchange with the neocortex. We review findings that demonstrate widespread reward-related cognitive input to the cerebellum, as well as new studies that have characterized the codependence of processing in the neocortex and cerebellum. Together, these data support a view of the neocortex-cerebellum circuit as a joint dynamic system both in classical sensorimotor contexts and reward-related, cognitive processing. These studies have also expanded classical theory on the computations performed by the cerebellar circuit.

  View details for DOI 10.1016/j.tins.2019.11.002

  View details for PubMedID 31787351

• Chinese scientists and US leadership in the life sciences NATURE BIOTECHNOLOGY Holtzman, S. H., Cohen, R., Levin, J. M., Maraganore, J., Albers, J., Aoun, J. E., Audia, J. E., Bailey, M. P., Barrett, P., Bartel, D., Bienamie, J., Blum, R. I., Boger, J., Bollenbacher, J., Bonney, M. W., Booth, B., Bradbury, D. M., Brandicourt, O., Braunwald, E., Carpenter, A. E., Chang, H. E., Cheruvu, P., Christensen, G., Church, G., Clayman, M. D., Cooney, C. L., Cox, G. F., Curley, S. J., Dahiyat, B., DePinho, R. A., Diekman, J., Doerfler, D., Dornbusch, D., Douglas, R. H., Dunsire, D., Ebright, R. H., Eskridge, W., Flesher, G. J., Fong, K., Formela, J., Foster-Cheek, K., Francois, C., Franken, M. P., Franklin, H., Fu, Y., Gage, L., Gardner, P., Gardner, J., Genead, M., Geraghty, J. A., Gerweck, L., Gill, S., Gillis, S., Goeddel, D. V., Goldsmith, M., Graves, K., Gray, M., Grayzel, D., Greene, B., Gregory, R. J., Greve-Philips, C. A., Gros, D., Hamburg, M. A., Hammerschmidt, M., Hao, Y., Hartounian, H., Hasnain, F., Hastings, P. J., Hawkins, E. S., He, W., He, W., Hirzel, A., Hong, Z., Hoppenot, H., Jia, W., Kaplan, J., Keiper, J., King, V., Kolchinsky, P., Koller, D., Laikind, P., Lander, E. S., Langer, R. S., LaVoie, D. L., Lawton, A., Lee, J. J., Leff, J. S., Lepore, J., Leschly, N., Li, Q., Liang, M. H., Lieberman, J., Lim, J., Lindenberg, M., Lippard, S. J., Liu, D. R., Liu, Y., Liu, S., Lodish, H., Loncar, B., Lopatin, U., Love, T. W., Lowe, D., Lowy, I., Lu, H., Luo, L., Mahanthappa, N. K., Martin, P. W., Martini, A., Martucci, W., McArthur, J., McCann, C. M., McCarthy, S., McLachlan, D., Mello, C., Mento, S. J., Meyers, J., Meyers, R., Mills, K., Moch, K. I., Moos, W. H., Narachi, M., Nashat, A., Newell, W. J., Nodelman, O., Olle, B., Osborn, J. E., Oyler, J. V., Pao, W., Patel, N., Perez, R., Peterkin, D., Pompino, A., Pruzanski, M., Quinn, D., Qutub, A., Raab, M. G., Radaelli, M., Rakhit, A., Ramamoorthi, K., Rastetter, W., Reed, J. C., Reinhart, H., Rhodes, J. P., Rieflin, W. J., Rong, L., Rosan, D. E., Rosenblatt, M., Rutter, W. J., Samudio, I., Samuels, C., Sato, V. L., Saunders, B., Scadden, D., Scangos, G., Scarlett, J. A., Schegerin, M., Schimmel, P., Schreiber, S. L., Schubert, C. R., Schulman, A., Shaff, E., Shaffer, R., Sharp, P. A., Sheng, M., Sheng, G., Shenk, T., Shi, F., Silos-Santiago, A., Simonian, N., Slattery, W., Smith, J. A., Stocks, C., Stoffel, M., Su, M., Su, L., Tandon, N., Tang, C. M., Taunton-Rigby, A., Tezapsidis, N., Theuer, C., Thornberry, N. A., Tolar, M., Topol, E., Tormos, W., Trask, A., Truex, S., Tuschl, T., Varmus, H. E., Vasconcelles, M. J., Vounatsos, M., Walbert, T. P., Walsh, C. T., Wang, J., Wang, J., Wang, N., Westphal, C., Wierenga, W., Williams, D. E., Williams, L. T., Winningham, R. E., Wirth, P., Witt, R., Wood, C., Woodhouse, D. J., Wright, R., Wu, Y., Xanthopoulos, K. G., Xiao, C., Xiao, T., Xie, J., Xu, Y., Xu, Z. C., Yakatan, G. J., Yuan, L., Yung, W., Zamore, P. D., Zaydman, M., Zeng, X., Zerhouni, E., Zhang, F., Zhang, Q., Zhang, S., Signatories Statement 2019; 37 (11): 1261–63

  View details for DOI 10.1038/s41587-019-0285-x

  View details for Web of Science ID 000494984400011

  View details for PubMedID 31582856

• Transsynaptic Fish-lips signaling prevents misconnections between nonsynaptic partner olfactory neurons. Proceedings of the National Academy of Sciences of the United States of America Xie, Q., Wu, B., Li, J., Xu, C., Li, H., Luginbuhl, D. J., Wang, X., Ward, A., Luo, L. 2019

  Abstract

  Our understanding of the mechanisms of neural circuit assembly is far from complete. Identification of wiring molecules with novel mechanisms of action will provide insights into how complex and heterogeneous neural circuits assemble during development. In the Drosophila olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of partner projection neurons (PNs). Here, we performed an RNA interference screen for cell surface molecules and identified the leucine-rich repeat-containing transmembrane protein known as Fish-lips (Fili) as a novel wiring molecule in the assembly of the Drosophila olfactory circuit. Fili contributes to the precise axon and dendrite targeting of a small subset of ORN and PN classes, respectively. Cell-type-specific expression and genetic analyses suggest that Fili sends a transsynaptic repulsive signal to neurites of nonpartner classes that prevents their targeting to inappropriate glomeruli in the antennal lobe.

  View details for DOI 10.1073/pnas.1905832116

  View details for PubMedID 31341080

• Functional divergence of Plexin B structural motifs in distinct steps of Drosophila olfactory circuit assembly. eLife Guajardo, R., Luginbuhl, D. J., Han, S., Luo, L., Li, J. 2019; 8

  Abstract

  Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct roles is unclear. We previously reported that Plexin B (PlexB) controls multiple steps during the assembly of the Drosophila olfactory circuit (Li et al., 2018). Here, we systematically mutagenized structural motifs of PlexB and examined the function of these variants in these multiple steps: axon fasciculation, trajectory choice, and synaptic partner selection. We found that the extracellular Sema domain is essential for all three steps, the catalytic site of the intracellular RapGAP is engaged in none, and the intracellular GTPase-binding motifs are essential for trajectory choice and synaptic partner selection, but are dispensable for fasciculation. Moreover, extracellular PlexB cleavage serves as a regulatory mechanism of PlexB signaling. Thus, the divergent roles of PlexB motifs in distinct steps of neural development contribute to its functional versatility in neural circuit assembly.

  View details for DOI 10.7554/eLife.48594

  View details for PubMedID 31225795

• Thirst regulates motivated behavior through modulation of brainwide neural population dynamics SCIENCE Allen, W. E., Chen, M. Z., Pichamoorthy, N., Tien, R. H., Pachitariu, M., Luo, L., Deisseroth, K. 2019; 364 (6437): 253-+

  View details for DOI 10.1126/science.aav3932

  View details for Web of Science ID 000464956600040

• Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs. eLife Henderson, N. T., Le Marchand, S. J., Hruska, M., Hippenmeyer, S., Luo, L., Dalva, M. B. 2019; 8

  Abstract

  Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons.

  View details for PubMedID 30789343

• Suppressing Memories by Shrinking the Vesicle Pool. Neuron Richman, E. B., Luo, L. 2019; 101 (1): 5–7

  Abstract

  The cohesin complex regulates cellular functions spanning cell division and neuronal morphogenesis. Now, Phan etal. uncover a role for the cohesin complex in regulating memory acquisition and the size of the synaptic and dense-core vesicle pool.

  View details for PubMedID 30605657

• Topological Organization of Ventral Tegmental Area Connectivity Revealed by Viral-Genetic Dissection of Input-Output Relations. Cell reports Beier, K. T., Gao, X. J., Xie, S., DeLoach, K. E., Malenka, R. C., Luo, L. 2019; 26 (1): 159

  Abstract

  Viral-genetic tracing techniques have enabled mesoscale mapping of neuronal connectivity by teasing apart inputs to defined neuronal populations in regions with heterogeneous cell types. We previously observed input biases to output-defined ventral tegmental area dopamine (VTA-DA) neurons. Here, we further dissect connectivity in the VTA by defining input-output relations of neurochemically and output-defined neuronal populations. By expanding our analysis to include input patterns to subtypes of excitatory (vGluT2-expressing) or inhibitory (GAD2-expressing) populations, we find that the output site, rather than neurochemical phenotype, correlates with whole-brain inputs of each subpopulation. Lastly, we find that biases in input maps to different VTA neurons can be generated using publicly available whole-brain output mapping datasets. Our comprehensive dataset and detailed spatial analysis suggest that connection specificity in the VTA is largely a function of the spatial location of the cells within the VTA.

  View details for PubMedID 30605672

• Complementary Genetic Targeting and Monosynaptic Input Mapping Reveal Recruitment and Refinement of Distributed Corticostriatal Ensembles by Cocaine. Neuron Wall, N. R., Neumann, P. A., Beier, K. T., Mokhtari, A. K., Luo, L., Malenka, R. C. 2019

  Abstract

  Drugs of abuse elicit powerful experiences that engage populations of neurons broadly distributed throughout the brain. To determine how synaptic connectivity is organized to enable robust communication between populations of drug-activated neurons, we developed a complementary targeting system for monosynaptic rabies virus (RV) tracing that identifies direct inputs to activated versus nonactivated neuronal populations. Analysis of over 100,000 synaptic input neurons demonstrated that cocaine-activated neurons comprise selectively connected but broadly distributed corticostriatal networks. Electrophysiological assays using optogenetics to stimulate activated versus nonactivated inputs revealed stronger synapses between coactivated cortical pyramidal neurons and neurons in the dorsal striatum (DS). Repeated cocaine exposure further enhanced the connectivity specifically between drug-activated neurons in the orbitofrontal cortex (OFC) and coactive DS neurons. Selective chemogenetic silencing of cocaine-activated OFC neurons or their terminals in the DS disrupted behavioral sensitization, demonstrating the utility of this methodology for identifying novel circuit elements that contribute to behavioral plasticity.

  View details for DOI 10.1016/j.neuron.2019.10.032

  View details for PubMedID 31759807

• Mapping Histological Slice Sequences to the Allen Mouse Brain Atlas Without 3D Reconstruction FRONTIERS IN NEUROINFORMATICS Xiong, J., Ren, J., Luo, L., Horowitz, M. 2018; 12

  View details for DOI 10.3389/fninf.2018.00093

  View details for Web of Science ID 000452913600001

• Early adolescent Rai1 reactivation reverses transcriptional and social interaction deficits in a mouse model of Smith-Magenis syndrome. Proceedings of the National Academy of Sciences of the United States of America Huang, W., Wang, D. C., Allen, W. E., Klope, M., Hu, H., Shamloo, M., Luo, L. 2018

  Abstract

  Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), a syndromic autism spectrum disorder associated with craniofacial abnormalities, intellectual disability, and behavioral problems. There is currently no cure for SMS. Here, we generated a genetic mouse model to determine the reversibility of SMS-like neurobehavioral phenotypes in Rai1 heterozygous mice. We show that normalizing the Rai1 level 3-4 wk after birth corrected the expression of genes related to neural developmental pathways and fully reversed a social interaction deficit caused by Rai1 haploinsufficiency. In contrast, Rai1 reactivation 7-8 wk after birth was not beneficial. We also demonstrated that the correct Rai1 dose is required in both excitatory and inhibitory neurons for proper social interactions. Finally, we found that Rai1 heterozygous mice exhibited a reduction of dendritic spines in the medial prefrontal cortex (mPFC) and that optogenetic activation of mPFC neurons in adults improved the social interaction deficit of Rai1 heterozygous mice. Together, these results suggest the existence of a postnatal temporal window during which restoring Rai1 can improve the transcriptional and social behavioral deficits in a mouse model of SMS. It is possible that circuit-level interventions would be beneficial beyond this critical window.

  View details for PubMedID 30275311

• Polina Anikeeva and Liqun Luo CURRENT OPINION IN NEUROBIOLOGY Anikeeva, P., Luo, L. 2018; 50: IV-VI

  View details for PubMedID 29754872

• Functional circuit architecture underlying parental behaviour NATURE Kohl, J., Babayan, B. M., Rubinstein, N. D., Autry, A. E., Marin-Rodriguez, B., Kapoor, V., Miyamishi, K., Zweifel, L. S., Luo, L., Uchida, N., Dulac, C. 2018; 556 (7701): 326-+

  Abstract

  Parenting is essential for the survival and wellbeing of mammalian offspring. However, we lack a circuit-level understanding of how distinct components of this behaviour are coordinated. Here we investigate how galanin-expressing neurons in the medial preoptic area (MPOAGal) of the hypothalamus coordinate motor, motivational, hormonal and social aspects of parenting in mice. These neurons integrate inputs from a large number of brain areas and the activation of these inputs depends on the animal's sex and reproductive state. Subsets of MPOAGal neurons form discrete pools that are defined by their projection sites. While the MPOAGal population is active during all episodes of parental behaviour, individual pools are tuned to characteristic aspects of parenting. Optogenetic manipulation of MPOAGal projections mirrors this specificity, affecting discrete parenting components. This functional organization, reminiscent of the control of motor sequences by pools of spinal cord neurons, provides a new model for how discrete elements of a social behaviour are generated at the circuit level.

  View details for PubMedID 29643503

  View details for PubMedCentralID PMC5908752

• Linking neuronal lineage and wiring specificity NEURAL DEVELOPMENT Li, H., Shuster, S., Li, J., Luo, L. 2018; 13: 5

  Abstract

  Brain function requires precise neural circuit assembly during development. Establishing a functional circuit involves multiple coordinated steps ranging from neural cell fate specification to proper matching between pre- and post-synaptic partners. How neuronal lineage and birth timing influence wiring specificity remains an open question. Recent findings suggest that the relationships between lineage, birth timing, and wiring specificity vary in different neuronal circuits. In this review, we summarize our current understanding of the cellular, molecular, and developmental mechanisms linking neuronal lineage and birth timing to wiring specificity in a few specific systems in Drosophila and mice, and review different methods employed to explore these mechanisms.

  View details for PubMedID 29653548

• Genetic tagging of active neurons in auditory cortex reveals maternal plasticity of coding ultrasonic vocalizations NATURE COMMUNICATIONS Tasaka, G., Guenthner, C. J., Shalev, A., Gilday, O., Luo, L., Mizrahi, A. 2018; 9: 871

  Abstract

  Cortical neurons are often functionally heterogeneous even for molecularly defined subtypes. In sensory cortices, physiological responses to natural stimuli can be sparse and vary widely even for neighboring neurons. It is thus difficult to parse out circuits that encode specific stimuli for further experimentation. Here, we report the development of a Cre-reporter mouse that allows recombination for cellular labeling and genetic manipulation, and use it with an activity-dependent Fos-CreERT2 driver to identify functionally active circuits in the auditory cortex. In vivo targeted patch recordings validate our method for neurons responding to physiologically relevant natural sounds such as pup wriggling calls and ultrasonic vocalizations (USVs). Using this system to investigate cortical responses in postpartum mothers, we find a transient recruitment of neurons highly responsive to USVs. This subpopulation of neurons has distinct physiological properties that improve the coding efficiency for pup USV calls, implicating it as a unique signature in parental plasticity.

  View details for PubMedID 29491360

• A Subpopulation of Striatal Neurons Mediates Levodopa-Induced Dyskinesia NEURON Girasole, A. E., Lum, M. Y., Nathaniel, D., Bair-Marshall, C. J., Guenthner, C. J., Luo, L., Kreitzer, A. C., Nelson, A. B. 2018; 97 (4): 787-+

  Abstract

  Parkinson's disease is characterized by the progressive loss of midbrain dopamine neurons. Dopamine replacement therapy with levodopa alleviates parkinsonian motor symptoms but is complicated by the development of involuntary movements, termed levodopa-induced dyskinesia (LID). Aberrant activity in the striatum has been hypothesized to cause LID. Here, to establish a direct link between striatal activity and dyskinesia, we combine optogenetics and a method to manipulate dyskinesia-associated neurons, targeted recombination in active populations (TRAP). We find that TRAPed cells are a stable subset of sensorimotor striatal neurons, predominantly from the direct pathway, and that reactivation of TRAPed striatal neurons causes dyskinesia in the absence of levodopa. Inhibition of TRAPed cells, but not a nonspecific subset of direct pathway neurons, ameliorates LID. These results establish that a distinct subset of striatal neurons is causally involved in LID and indicate that successful therapeutic strategies for treating LID may require targeting functionally selective neuronal subtypes.

  View details for PubMedID 29398356

• Stepwise wiring of the Drosophila olfactory map requires specific Plexin B levels. eLife Li, J., Guajardo, R., Xu, C., Wu, B., Li, H., Li, T., Luginbuhl, D. J., Xie, X., Luo, L. 2018; 7

  Abstract

  The precise assembly of a neural circuit involves many consecutive steps. The conflict between a limited number of wiring molecules and the complexity of the neural network impels each molecule to execute multiple functions at different steps. Here, we examined the cell-type specific distribution of endogenous levels of axon guidance receptor Plexin B (PlexB) in the developing antennal lobe, the first olfactory processing center in Drosophila. We found that different classes of olfactory receptor neurons (ORNs) express PlexB at different levels in two wiring steps - axonal trajectory choice and subsequent target selection. In line with its temporally distinct patterns, the proper levels of PlexB control both steps in succession. Genetic interactions further revealed that the effect of high-level PlexB is antagonized by its canonical partner Sema2b. Thus, PlexB plays a multifaceted role in instructing the assembly of the Drosophila olfactory circuit through temporally-regulated expression patterns and expression level-dependent effects.

  View details for PubMedID 30136927

• Dynamic salience processing in paraventricular thalamus gates associative learning. Science (New York, N.Y.) Zhu, Y., Nachtrab, G., Keyes, P. C., Allen, W. E., Luo, L., Chen, X. 2018; 362 (6413): 423–29

  Abstract

  The salience of behaviorally relevant stimuli is dynamic and influenced by internal state and external environment. Monitoring such changes is critical for effective learning and flexible behavior, but the neuronal substrate for tracking the dynamics of stimulus salience is obscure. We found that neurons in the paraventricular thalamus (PVT) are robustly activated by a variety of behaviorally relevant events, including novel ("unfamiliar") stimuli, reinforcing stimuli and their predicting cues, as well as omission of the expected reward. PVT responses are scaled with stimulus intensity and modulated by changes in homeostatic state or behavioral context. Inhibition of the PVT responses suppresses appetitive or aversive associative learning and reward extinction. Our findings demonstrate that the PVT gates associative learning by providing a dynamic representation of stimulus salience.

  View details for PubMedID 30361366

• Mapping Histological Slice Sequences to the Allen Mouse Brain Atlas Without 3D Reconstruction. Frontiers in neuroinformatics Xiong, J., Ren, J., Luo, L., Horowitz, M. 2018; 12: 93

  Abstract

  Histological brain slices are widely used in neuroscience to study the anatomical organization of neural circuits. Systematic and accurate comparisons of anatomical data from multiple brains, especially from different studies, can benefit tremendously from registering histological slices onto a common reference atlas. Most existing methods rely on an initial reconstruction of the volume before registering it to a reference atlas. Because these slices are prone to distortions during the sectioning process and often sectioned with non-standard angles, reconstruction is challenging and often inaccurate. Here we describe a framework that maps each slice to its corresponding plane in the Allen Mouse Brain Atlas (2015) to build a plane-wise mapping and then perform 2D nonrigid registration to build a pixel-wise mapping. We use the L2 norm of the histogram of oriented gradients difference of two patches as the similarity metric for both steps and a Markov random field formulation that incorporates tissue coherency to compute the nonrigid registration. To fix significantly distorted regions that are misshaped or much smaller than the control grids, we train a context-aggregation network to segment and warp them to their corresponding regions with thin plate spline. We have shown that our method generates results comparable to an expert neuroscientist and is significantly better than reconstruction-first approaches. Code and sample dataset are available at sites.google.com/view/brain-mapping.

  View details for PubMedID 30618698

• NEUROBIOLOGY A bitter-sweet symphony NATURE Li, J., Luo, L. 2017; 548 (7667): 285–87

  View details for PubMedID 28792928

• The MutAnts Are Here CELL Friedman, D. A., Gordon, D. M., Luo, L. 2017; 170 (4): 601–2

  Abstract

  The development of CRISPR/Cas9-mediated gene knockout in two ant species opens a new window into exploring how social insects use olfactory cues to organize their collective behavior.

  View details for PubMedID 28802035

• Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wu, B., Li, J., Chou, Y., Luginbuhl, D., Luo, L. 2017; 114 (29): 7505–12

  Abstract

  The formation of complex but highly organized neural circuits requires interactions between neurons and glia. During the assembly of the Drosophila olfactory circuit, 50 olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form synaptic connections in 50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information-processing channel. Each compartment is separated from the adjacent compartments by membranous processes from ensheathing glia. Here we show that Thisbe, an FGF released from olfactory neurons, particularly from local interneurons, instructs ensheathing glia to wrap each glomerulus. The Heartless FGF receptor acts cell-autonomously in ensheathing glia to regulate process extension so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes overwrapping of the glomeruli their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation.

  View details for PubMedID 28674010

• Genetic strategies to access activated neurons. Current opinion in neurobiology DeNardo, L., Luo, L. 2017; 45: 121-129

  Abstract

  A major goal of modern neuroscience is to understand how ensembles of neurons participate in neural circuits underlying behavior. The recent explosion of genetically-encoded circuit analysis tools has allowed neuroscientists to characterize molecularly-defined neuronal types with unprecedented detail. However, since neurons defined by molecular expression can be functionally heterogeneous, targeting circuit analysis tools to neurons based on their activity is critical to elucidating the neural basis of behavior. Here we review genetic strategies to access activated neurons and characterize their functional properties, molecular profiles, connectivity, and causal roles in sensory-coding, memory, and valence-encoding. We also discuss future possibilities for improving these strategies and using them to screen brain-wide activity patterns underlying adaptive and maladaptive behaviors.

  View details for DOI 10.1016/j.conb.2017.05.014

  View details for PubMedID 28577429

• Identification of preoptic sleep neurons using retrograde labelling and gene profiling. Nature Chung, S., Weber, F., Zhong, P., Tan, C. L., Nguyen, T. N., Beier, K. T., Hörmann, N., Chang, W., Zhang, Z., Do, J. P., Yao, S., Krashes, M. J., Tasic, B., Cetin, A., Zeng, H., Knight, Z. A., Luo, L., Dan, Y. 2017; 545 (7655): 477-481

  Abstract

  In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep. However, the sleep-active neurons are spatially intermingled with wake-active neurons, making it difficult to target the sleep neurons specifically for circuit analysis. Here we identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, we show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate that several peptide markers (cholecystokinin, corticotropin-releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.

  View details for DOI 10.1038/nature22350

  View details for PubMedID 28514446

• Lineage-dependent spatial and functional organization of the mammalian enteric nervous system SCIENCE Lasrado, R., Boesmans, W., Kleinjung, J., Pin, C., Bell, D., Bhaw, L., McCallum, S., Zong, H., Luo, L., Clevers, H., Berghe, P. V., Pachnis, V. 2017; 356 (6339): 722-726

  View details for DOI 10.1126/science.aam7511

  View details for Web of Science ID 000401508400040

• Global Representations of Goal-Directed Behavior in Distinct Cell Types of Mouse Neocortex NEURON Allen, W. E., Kauvar, I. V., Chen, M. Z., Richman, E. B., Yang, S. J., Chan, K., Gradinaru, V., Deverman, B. E., Luo, L., Deisseroth, K. 2017; 94 (4): 891-?

  Abstract

  The successful planning and execution of adaptive behaviors in mammals may require long-range coordination of neural networks throughout cerebral cortex. The neuronal implementation of signals that could orchestrate cortex-wide activity remains unclear. Here, we develop and apply methods for cortex-wide Ca(2+) imaging in mice performing decision-making behavior and identify a global cortical representation of task engagement encoded in the activity dynamics of both single cells and superficial neuropil distributed across the majority of dorsal cortex. The activity of multiple molecularly defined cell types was found to reflect this representation with type-specific dynamics. Focal optogenetic inhibition tiled across cortex revealed a crucial role for frontal cortex in triggering this cortex-wide phenomenon; local inhibition of this region blocked both the cortex-wide response to task-initiating cues and the voluntary behavior. These findings reveal cell-type-specific processes in cortex for globally representing goal-directed behavior and identify a major cortical node that gates the global broadcast of task-related information.

  View details for DOI 10.1016/j.neuron.2017.04.017

  View details for PubMedID 28521139

• Breathing control center neurons that promote arousal in mice SCIENCE Yackle, K., Schwarz, L. A., Kam, K., Sorokin, J. M., Huguenard, J. R., Feldman, J. L., Luo, L., Krasnow, M. A. 2017; 355 (6332): 1411-1415

  Abstract

  Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBötzinger complex (preBötC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9/Dbx1 double-positive preBötC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain.

  View details for DOI 10.1126/science.aai7984

  View details for Web of Science ID 000397809500040

  View details for PubMedID 28360327

• A Brainstem-Spinal Cord Inhibitory Circuit for Mechanical Pain Modulation by GABA and Enkephalins. Neuron François, A., Low, S. A., Sypek, E. I., Christensen, A. J., Sotoudeh, C., Beier, K. T., Ramakrishnan, C., Ritola, K. D., Sharif-Naeini, R., Deisseroth, K., Delp, S. L., Malenka, R. C., Luo, L., Hantman, A. W., Scherrer, G. 2017; 93 (4): 822-839 e6

  Abstract

  Pain thresholds are, in part, set as a function of emotional and internal states by descending modulation of nociceptive transmission in the spinal cord. Neurons of the rostral ventromedial medulla (RVM) are thought to critically contribute to this process; however, the neural circuits and synaptic mechanisms by which distinct populations of RVM neurons facilitate or diminish pain remain elusive. Here we used in vivo opto/chemogenetic manipulations and trans-synaptic tracing of genetically identified dorsal horn and RVM neurons to uncover an RVM-spinal cord-primary afferent circuit controlling pain thresholds. Unexpectedly, we found that RVM GABAergic neurons facilitate mechanical pain by inhibiting dorsal horn enkephalinergic/GABAergic interneurons. We further demonstrate that these interneurons gate sensory inputs and control pain through temporally coordinated enkephalin- and GABA-mediated presynaptic inhibition of somatosensory neurons. Our results uncover a descending disynaptic inhibitory circuit that facilitates mechanical pain, is engaged during stress, and could be targeted to establish higher pain thresholds. VIDEO ABSTRACT.

  View details for DOI 10.1016/j.neuron.2017.01.008

  View details for PubMedID 28162807

• Gating of social reward by oxytocin in the ventral tegmental area. Science (New York, N.Y.) Hung, L. W., Neuner, S., Polepalli, J. S., Beier, K. T., Wright, M., Walsh, J. J., Lewis, E. M., Luo, L., Deisseroth, K., Dölen, G., Malenka, R. C. 2017; 357 (6358): 1406–11

  Abstract

  The reward generated by social interactions is critical for promoting prosocial behaviors. Here we present evidence that oxytocin (OXT) release in the ventral tegmental area (VTA), a key node of the brain's reward circuitry, is necessary to elicit social reward. During social interactions, activity in paraventricular nucleus (PVN) OXT neurons increased. Direct activation of these neurons in the PVN or their terminals in the VTA enhanced prosocial behaviors. Conversely, inhibition of PVN OXT axon terminals in the VTA decreased social interactions. OXT increased excitatory drive onto reward-specific VTA dopamine (DA) neurons. These results demonstrate that OXT promotes prosocial behavior through direct effects on VTA DA neurons, thus providing mechanistic insight into how social interactions can generate rewarding experiences.

  View details for PubMedID 28963257

• Lineage-dependent spatial and functional organization of the mammalian enteric nervous system. Science (New York, N.Y.) Lasrado, R., Boesmans, W., Kleinjung, J., Pin, C., Bell, D., Bhaw, L., McCallum, S., Zong, H., Luo, L., Clevers, H., Vanden Berghe, P., Pachnis, V. 2017; 356 (6339): 722–26

  Abstract

  The enteric nervous system (ENS) is essential for digestive function and gut homeostasis. Here we show that the amorphous neuroglia networks of the mouse ENS are composed of overlapping clonal units founded by postmigratory neural crest-derived progenitors. The spatial configuration of ENS clones depends on proliferation-driven local interactions of ENS progenitors with lineally unrelated neuroectodermal cells, the ordered colonization of the serosa-mucosa axis by clonal descendants, and gut expansion. Single-cell transcriptomics and mutagenesis analysis delineated dynamic molecular states of ENS progenitors and identified RET as a regulator of neurogenic commitment. Clonally related enteric neurons exhibit synchronous activity in response to network stimulation. Thus, lineage relationships underpin the organization of the peripheral nervous system.

  View details for PubMedID 28522527

• Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons. eLife Mosca, T. J., Luginbuhl, D. J., Wang, I. E., Luo, L. 2017; 6

  Abstract

  Precise coordination of synaptic connections ensures proper information flow within circuits. The activity of presynaptic organizing molecules signaling to downstream pathways is essential for such coordination, though such entities remain incompletely known. We show that LRP4, a conserved transmembrane protein known for its postsynaptic roles, functions presynaptically as an organizing molecule. In the Drosophila brain, LRP4 localizes to the nerve terminals at or near active zones. Loss of presynaptic LRP4 reduces excitatory (not inhibitory) synapse number, impairs active zone architecture, and abolishes olfactory attraction - the latter of which can be suppressed by reducing presynaptic GABAB receptors. LRP4 overexpression increases synapse number in excitatory and inhibitory neurons, suggesting an instructive role and a common downstream synapse addition pathway. Mechanistically, LRP4 functions via the conserved kinase SRPK79D to ensure normal synapse number and behavior. This highlights a presynaptic function for LRP4, enabling deeper understanding of how synapse organization is coordinated.

  View details for PubMedID 28606304

• Rabies screen reveals GPe control of cocaine-triggered plasticity. Nature Beier, K. T., Kim, C. K., Hoerbelt, P., Hung, L. W., Heifets, B. D., DeLoach, K. E., Mosca, T. J., Neuner, S., Deisseroth, K., Luo, L., Malenka, R. C. 2017

  Abstract

  Identification of neural circuit changes that contribute to behavioural plasticity has routinely been conducted on candidate circuits that were preselected on the basis of previous results. Here we present an unbiased method for identifying experience-triggered circuit-level changes in neuronal ensembles in mice. Using rabies virus monosynaptic tracing, we mapped cocaine-induced global changes in inputs onto neurons in the ventral tegmental area. Cocaine increased rabies-labelled inputs from the globus pallidus externus (GPe), a basal ganglia nucleus not previously known to participate in behavioural plasticity triggered by drugs of abuse. We demonstrated that cocaine increased GPe neuron activity, which accounted for the increase in GPe labelling. Inhibition of GPe activity revealed that it contributes to two forms of cocaine-triggered behavioural plasticity, at least in part by disinhibiting dopamine neurons in the ventral tegmental area. These results suggest that rabies-based unbiased screening of changes in input populations can identify previously unappreciated circuit elements that critically support behavioural adaptations.

  View details for PubMedID 28902833

• Cell type-specific long-range connections of basal forebrain circuit ELIFE Do, J. P., Xu, M., Lee, S., Chang, W., Zhang, S., Chung, S., Yung, T. J., Fan, J. L., Miyamichin, K., Luo, L., Dan, Y. 2016; 5

  Abstract

  The basal forebrain (BF) plays key roles in multiple brain functions, including sleep-wake regulation, attention, and learning/memory, but the long-range connections mediating these functions remain poorly characterized. Here we performed whole-brain mapping of both inputs and outputs of four BF cell types - cholinergic, glutamatergic, and parvalbumin-positive (PV+) and somatostatin-positive (SOM+) GABAergic neurons - in the mouse brain. Using rabies virus -mediated monosynaptic retrograde tracing to label the inputs and adeno-associated virus to trace axonal projections, we identified numerous brain areas connected to the BF. The inputs to different cell types were qualitatively similar, but the output projections showed marked differences. The connections to glutamatergic and SOM+ neurons were strongly reciprocal, while those to cholinergic and PV+ neurons were more unidirectional. These results reveal the long-range wiring diagram of the BF circuit with highly convergent inputs and divergent outputs and point to both functional commonality and specialization of different BF cell types.

  View details for DOI 10.7554/eLife.13214

  View details for Web of Science ID 000384421400001

  View details for PubMedCentralID PMC5095704

• Wiring and Molecular Features of Prefrontal Ensembles Representing Distinct Experiences CELL Ye, L., Allen, W. E., Thompson, K. R., Tian, Q., Hsueh, B., Ramakrishnan, C., Wang, A., Jennings, J. H., Adhikari, A., Halpern, C. H., Witten, I. B., Barth, A. L., Luo, L., McNab, J. A., Deisseroth, K. 2016; 165 (7): 1776-1788

  Abstract

  A major challenge in understanding the cellular diversity of the brain has been linking activity during behavior with standard cellular typology. For example, it has not been possible to determine whether principal neurons in prefrontal cortex active during distinct experiences represent separable cell types, and it is not known whether these differentially active cells exert distinct causal influences on behavior. Here, we develop quantitative hydrogel-based technologies to connect activity in cells reporting on behavioral experience with measures for both brain-wide wiring and molecular phenotype. We find that positive and negative-valence experiences in prefrontal cortex are represented by cell populations that differ in their causal impact on behavior, long-range wiring, and gene expression profiles, with the major discriminant being expression of the adaptation-linked gene NPAS4. These findings illuminate cellular logic of prefrontal cortex information processing and natural adaptive behavior and may point the way to cell-type-specific understanding and treatment of disease-associated states.

  View details for DOI 10.1016/j.cell.2016.05.010

  View details for PubMedID 27238022

• Developmental Sculpting of Intracortical Circuits by MHC Class I H2-Db and H2-Kb. Cerebral cortex Adelson, J. D., Sapp, R. W., Brott, B. K., Lee, H., Miyamichi, K., Luo, L., Cheng, S., Djurisic, M., Shatz, C. J. 2016; 26 (4): 1453-1463

  Abstract

  Synapse pruning is an activity-regulated process needed for proper circuit sculpting in the developing brain. Major histocompatibility class I (MHCI) molecules are regulated by activity, but little is known about their role in the development of connectivity in cortex. Here we show that protein for 2 MHCI molecules H2-Kb and H2-Db is associated with synapses in the visual cortex. Pyramidal neurons in mice lacking H2-Kb and H2-Db (KbDb KO) have more extensive cortical connectivity than normal. Modified rabies virus tracing was used to monitor the extent of pyramidal cell connectivity: Horizontal connectivity is greater in the visual cortex of KbDb KO mice. Basal dendrites of L2/3 pyramids, where many horizontal connections terminate, are more highly branched and have elevated spine density in the KO. Furthermore, the density of axonal boutons is elevated within L2/3 of mutant mice. These increases are accompanied by elevated miniature excitatory postsynaptic current frequency, consistent with an increase in functional synapses. This functional and anatomical increase in intracortical connectivity is also associated with enhanced ocular dominance plasticity that persists into adulthood. Thus, these MHCI proteins regulate sculpting of local cortical circuits and in their absence, the excess connectivity can function as a substrate for cortical plasticity throughout life.

  View details for DOI 10.1093/cercor/bhu243

  View details for PubMedID 25316337

• Cell type-specific long-range connections of basal forebrain circuit. eLife Do, J. P., Xu, M., Lee, S. H., Chang, W. C., Zhang, S., Chung, S., Yung, T. J., Fan, J. L., Miyamichi, K., Luo, L., Dan, Y. 2016; 5

  Abstract

  The basal forebrain (BF) plays key roles in multiple brain functions, including sleep-wake regulation, attention, and learning/memory, but the long-range connections mediating these functions remain poorly characterized. Here we performed whole-brain mapping of both inputs and outputs of four BF cell types - cholinergic, glutamatergic, and parvalbumin-positive (PV+) and somatostatin-positive (SOM+) GABAergic neurons - in the mouse brain. Using rabies virus -mediated monosynaptic retrograde tracing to label the inputs and adeno-associated virus to trace axonal projections, we identified numerous brain areas connected to the BF. The inputs to different cell types were qualitatively similar, but the output projections showed marked differences. The connections to glutamatergic and SOM+ neurons were strongly reciprocal, while those to cholinergic and PV+ neurons were more unidirectional. These results reveal the long-range wiring diagram of the BF circuit with highly convergent inputs and divergent outputs and point to both functional commonality and specialization of different BF cell types.

  View details for PubMedID 27642784

• Cas9-triggered chain ablation of cas9 as a gene drive brake. Nature biotechnology Wu, B., Luo, L., Gao, X. J. 2016; 34 (2): 137–38

  View details for PubMedID 26849513

• Organization of the Locus Coeruleus-Norepinephrine System CURRENT BIOLOGY Schwarz, L. A., Luo, L. 2015; 25 (21): R1051-R1056

  Abstract

  The release of the neurotransmitter norepinephrine throughout the mammalian brain is important for modulating attention, arousal, and cognition during many behaviors. Furthermore, disruption of norepinephrine-mediated signaling is strongly associated with several psychiatric and neurodegenerative disorders in humans, emphasizing the clinical importance of this system. Most of the norepinephrine released in the brain is supplied by a very small, bilateral nucleus in the brainstem called the locus coeruleus. The goal of this minireview is to emphasize the complexity of the locus coeruleus beyond its primary definition as a norepinephrine-producing nucleus. Several recent studies utilizing innovative technologies highlight how the locus coeruleus-norepinephrine system can now be targeted with increased accuracy and resolution, in order to better understand its role in modulating diverse behaviors.

  View details for DOI 10.1016/j.cub.2015.09.039

  View details for Web of Science ID 000364262500015

  View details for PubMedID 26528750

• Basal forebrain circuit for sleep-wake control. Nature neuroscience Xu, M., Chung, S., Zhang, S., Zhong, P., Ma, C., Chang, W., Weissbourd, B., Sakai, N., Luo, L., Nishino, S., Dan, Y. 2015; 18 (11): 1641-1647

  View details for DOI 10.1038/nn.4143

  View details for PubMedID 26457552

• Connectivity of mouse somatosensory and prefrontal cortex examined with trans-synaptic tracing. Nature neuroscience DeNardo, L. A., Berns, D. S., DeLoach, K., Luo, L. 2015; 18 (11): 1687-1697

  View details for DOI 10.1038/nn.4131

  View details for PubMedID 26457553

• Control of REM sleep by ventral medulla GABAergic neurons NATURE Weber, F., Chung, S., Beier, K. T., Xu, M., Luo, L., Dan, Y. 2015; 526 (7573): 435-?

  View details for DOI 10.1038/nature14979

  View details for Web of Science ID 000362730200052

  View details for PubMedID 26444238

• NEUROSCIENCE. It takes the world to understand the brain. Science Huang, Z. J., Luo, L. 2015; 350 (6256): 42-44

  View details for DOI 10.1126/science.aad4120

  View details for PubMedID 26430110

  View details for PubMedCentralID PMC4975723

• Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping CELL Beier, K. T., Steinberg, E. E., DeLoach, K. E., Xie, S., Miyamichi, K., Schwarz, L., Gao, X. J., Kremer, E. J., Malenka, R. C., Luo, L. 2015; 162 (3): 622-634

  Abstract

  Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) integrate complex inputs to encode multiple signals that influence motivated behaviors via diverse projections. Here, we combine axon-initiated viral transduction with rabies-mediated trans-synaptic tracing and Cre-based cell-type-specific targeting to systematically map input-output relationships of VTA-DA neurons. We found that VTA-DA (and VTA-GABA) neurons receive excitatory, inhibitory, and modulatory input from diverse sources. VTA-DA neurons projecting to different forebrain regions exhibit specific biases in their input selection. VTA-DA neurons projecting to lateral and medial nucleus accumbens innervate largely non-overlapping striatal targets, with the latter also sending extensive extra-striatal axon collaterals. Using electrophysiology and behavior, we validated new circuits identified in our tracing studies, including a previously unappreciated top-down reinforcing circuit from anterior cortex to lateral nucleus accumbens via VTA-DA neurons. This study highlights the utility of our viral-genetic tracing strategies to elucidate the complex neural substrates that underlie motivated behaviors.

  View details for DOI 10.1016/j.cell.2015.07.015

  View details for Web of Science ID 000358801800020

  View details for PubMedCentralID PMC4522312

• Intact-Brain Analyses Reveal Distinct Information Carried by SNc Dopamine Subcircuits CELL Lerner, T. N., Shilyansky, C., Davidson, T. J., Evans, K. E., Beier, K. T., Zalocusky, K. A., Crow, A. K., Malenka, R. C., Luo, L., Tomer, R., Deisseroth, K. 2015; 162 (3): 635-647

  View details for DOI 10.1016/j.cell.2015.07.014

  View details for Web of Science ID 000358801800021

• A transcriptional reporter of intracellular Ca(2+) in Drosophila. Nature neuroscience Gao, X. J., Riabinina, O., Li, J., Potter, C. J., Clandinin, T. R., Luo, L. 2015; 18 (6): 917-925

  Abstract

  Intracellular Ca(2+) is a widely used neuronal activity indicator. Here we describe a transcriptional reporter of intracellular Ca(2+) (TRIC) in Drosophila that uses a binary expression system to report Ca(2+)-dependent interactions between calmodulin and its target peptide. We found that in vitro assays predicted in vivo properties of TRIC and that TRIC signals in sensory systems depend on neuronal activity. TRIC was able to quantitatively monitor neuronal responses that changed slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis cells to food and arousal. Furthermore, TRIC-induced expression of a neuronal silencer in nutrient-activated cells enhanced stress resistance, providing a proof of principle that TRIC can be used for circuit manipulation. Thus, TRIC facilitates the monitoring and manipulation of neuronal activity, especially those reflecting slow changes in physiological states that are poorly captured by existing methods. TRIC's modular design should enable optimization and adaptation to other organisms.

  View details for DOI 10.1038/nn.4016

  View details for PubMedID 25961791

  View details for PubMedCentralID PMC4446202

• Extremely Sparse Olfactory Inputs Are Sufficient to Mediate Innate Aversion in Drosophila PLOS ONE Gao, X. J., Clandinin, T. R., Luo, L. 2015; 10 (4)

  View details for DOI 10.1371/journal.pone.0125986

  View details for Web of Science ID 000353713100121

  View details for PubMedID 25927233

• Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a Drosophila olfactory circuit. Neuron Ward, A., Hong, W., Favaloro, V., Luo, L. 2015; 85 (5): 1013-1028

  Abstract

  Our understanding of the mechanisms that establish wiring specificity of complex neural circuits is far from complete. During Drosophila olfactory circuit assembly, axons of 50 olfactory receptor neuron (ORN) classes and dendrites of 50 projection neuron (PN) classes precisely target to 50 discrete glomeruli, forming parallel information-processing pathways. Here we show that Toll-6 and Toll-7, members of the Toll receptor family best known for functions in innate immunity and embryonic patterning, cell autonomously instruct the targeting of specific classes of PN dendrites and ORN axons, respectively. The canonical ligands and downstream partners of Toll receptors in embryonic patterning and innate immunity are not required for the function of Toll-6/Toll-7 in wiring specificity, nor are their cytoplasmic domains. Interestingly, both Toll-6 and Toll-7 participate in synaptic partner matching between ORN axons and PN dendrites. Our investigations reveal that olfactory circuit assembly involves dynamic and long-range interactions between PN dendrites and ORN axons.

  View details for DOI 10.1016/j.neuron.2015.02.003

  View details for PubMedID 25741726

  View details for PubMedCentralID PMC4351475

• Improved and expanded Q-system reagents for genetic manipulations NATURE METHODS Riabinina, O., Luginbuhl, D., Marr, E., Liu, S., Wu, M. N., Luo, L., Potter, C. J. 2015; 12 (3): 219-?

  Abstract

  The Q system is a repressible binary expression system for transgenic manipulations in living organisms. Through protein engineering and in vivo functional tests, we report here variants of the Q-system transcriptional activator, including QF2, for driving strong and ubiquitous expression in all Drosophila tissues. Our QF2, Gal4QF and LexAQF chimeric transcriptional activators substantially enrich the toolkit available for transgenic regulation in Drosophila melanogaster.

  View details for DOI 10.1038/NMETH.3250

  View details for Web of Science ID 000350670300020

  View details for PubMedID 25581800

  View details for PubMedCentralID PMC4344399

• Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons. Neuron Lammel, S., Steinberg, E. E., Földy, C., Wall, N. R., Beier, K., Luo, L., Malenka, R. C. 2015; 85 (2): 429-438

  View details for DOI 10.1016/j.neuron.2014.12.036

  View details for PubMedID 25611513

• Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons. Neuron Lammel, S., Steinberg, E. E., Földy, C., Wall, N. R., Beier, K., Luo, L., Malenka, R. C. 2015; 85 (2): 429-438

  Abstract

  Ventral tegmental area (VTA) dopamine (DA) neurons have been implicated in reward, aversion, salience, cognition, and several neuropsychiatric disorders. Optogenetic approaches involving transgenic Cre-driver mouse lines provide powerful tools for dissecting DA-specific functions. However, the emerging complexity of VTA circuits requires Cre-driver mouse lines that restrict transgene expression to a precisely defined cell population. Because of recent work reporting that VTA DA neurons projecting to the lateral habenula release GABA, but not DA, we performed an extensive anatomical, molecular, and functional characterization of prominent DA transgenic mouse driver lines. We find that transgenes under control of the tyrosine hydroxylase, but not the dopamine transporter, promoter exhibit dramatic non-DA cell-specific expression patterns within and around VTA nuclei. Our results demonstrate how Cre expression in unintentionally targeted cells in transgenic mouse lines can confound the interpretation of supposedly cell-type-specific experiments. This Matters Arising paper is in response to Stamatakis et al. (2013), published in Neuron. See also the Matters Arising Response paper by Stuber et al. (2015), published concurrently with this Matters Arising in Neuron.

  View details for DOI 10.1016/j.neuron.2014.12.036

  View details for PubMedID 25611513

• Monosynaptic Circuit Tracing with Glycoprotein-Deleted Rabies Viruses. The Journal of neuroscience : the official journal of the Society for Neuroscience Callaway, E. M., Luo, L. 2015; 35 (24): 8979–85

  View details for PubMedID 26085623

• Intersectional illumination of neural circuit function. Neuron Allen, W. E., Luo, L. 2015; 85 (5): 889–92

  Abstract

  In this issue of Neuron, Madisen et al. (2015) report the construction of several new transgenic mouse lines that apply intersectional genetic tools to achieve high levels of expression and cell-type specificity, providing a useful resource for future studies.

  View details for PubMedID 25741716

• Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain. Nature communications Pearce, M. M., Spartz, E. J., Hong, W., Luo, L., Kopito, R. R. 2015; 6: 6768-?

  View details for DOI 10.1038/ncomms7768

  View details for PubMedID 25866135

• Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain. Nature communications Pearce, M. M., Spartz, E. J., Hong, W., Luo, L., Kopito, R. R. 2015; 6: 6768-?

  Abstract

  The brain has a limited capacity to self-protect against protein aggregate-associated pathology, and mounting evidence supports a role for phagocytic glia in this process. We have established a Drosophila model to investigate the role of phagocytic glia in clearance of neuronal mutant huntingtin (Htt) aggregates associated with Huntington disease. We find that glia regulate steady-state numbers of Htt aggregates expressed in neurons through a clearance mechanism that requires the glial scavenger receptor Draper and downstream phagocytic engulfment machinery. Remarkably, some of these engulfed neuronal Htt aggregates effect prion-like conversion of soluble, wild-type Htt in the glial cytoplasm. We provide genetic evidence that this conversion depends strictly on the Draper signalling pathway, unveiling a previously unanticipated role for phagocytosis in transfer of pathogenic protein aggregates in an intact brain. These results suggest a potential mechanism by which phagocytic glia contribute to both protein aggregate-related neuroprotection and pathogenesis in neurodegenerative disease.

  View details for DOI 10.1038/ncomms7768

  View details for PubMedID 25866135

• Deterministic Progenitor Behavior and Unitary Production of Neurons in the Neocortex CELL Gao, P., Postiglione, M. P., Krieger, T. G., Hernandez, L., Wang, C., Han, Z., Streicher, C., Papusheva, E., Insolera, R., Chugh, K., Kodish, O., Huang, K., Simons, B. D., Luo, L., Hippenmeyer, S., Shi, S. 2014; 159 (4): 775-788

  Abstract

  Radial glial progenitors (RGPs) are responsible for producing nearly all neocortical neurons. To gain insight into the patterns of RGP division and neuron production, we quantitatively analyzed excitatory neuron genesis in the mouse neocortex using Mosaic Analysis with Double Markers, which provides single-cell resolution of progenitor division patterns and potential in vivo. We found that RGPs progress through a coherent program in which their proliferative potential diminishes in a predictable manner. Upon entry into the neurogenic phase, individual RGPs produce ?8-9 neurons distributed in both deep and superficial layers, indicating a unitary output in neuronal production. Removal of OTX1, a transcription factor transiently expressed in RGPs, results in both deep- and superficial-layer neuron loss and a reduction in neuronal unit size. Moreover, ?1/6 of neurogenic RGPs proceed to produce glia. These results suggest that progenitor behavior and histogenesis in the mammalian neocortex conform to a remarkably orderly and deterministic program.

  View details for DOI 10.1016/j.cell.2014.10.027

  View details for Web of Science ID 000344522000011

  View details for PubMedCentralID PMC4225456

• Functional transformations of odor inputs in the mouse olfactory bulb FRONTIERS IN NEURAL CIRCUITS Adam, Y., Livneh, Y., Miyamichi, K., Groysman, M., Luo, L., Mizrahi, A. 2014; 8

  Abstract

  Sensory inputs from the nasal epithelium to the olfactory bulb (OB) are organized as a discrete map in the glomerular layer (GL). This map is then modulated by distinct types of local neurons and transmitted to higher brain areas via mitral and tufted cells. Little is known about the functional organization of the circuits downstream of glomeruli. We used in vivo two-photon calcium imaging for large scale functional mapping of distinct neuronal populations in the mouse OB, at single cell resolution. Specifically, we imaged odor responses of mitral cells (MCs), tufted cells (TCs) and glomerular interneurons (GL-INs). Mitral cells population activity was heterogeneous and only mildly correlated with the olfactory receptor neuron (ORN) inputs, supporting the view that discrete input maps undergo significant transformations at the output level of the OB. In contrast, population activity profiles of TCs were dense, and highly correlated with the odor inputs in both space and time. Glomerular interneurons were also highly correlated with the ORN inputs, but showed higher activation thresholds suggesting that these neurons are driven by strongly activated glomeruli. Temporally, upon persistent odor exposure, TCs quickly adapted. In contrast, both MCs and GL-INs showed diverse temporal response patterns, suggesting that GL-INs could contribute to the transformations MCs undergo at slow time scales. Our data suggest that sensory odor maps are transformed by TCs and MCs in different ways forming two distinct and parallel information streams.

  View details for DOI 10.3389/fncir.2014.00129

  View details for PubMedID 25408637

• Dendrite morphogenesis depends on relative levels of NT-3/TrkC signaling SCIENCE Joo, W., Hippenmeyer, S., Luo, L. 2014; 346 (6209): 626-629

  View details for DOI 10.1126/science.1258996

  View details for Web of Science ID 000343799700047

• Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins ELIFE Mosca, T. J., Luo, L. 2014; 3

  View details for DOI 10.7554/eLife.103726

  View details for Web of Science ID 000343421200002

• Drosophila Strip serves as a platform for early endosome organization during axon elongation NATURE COMMUNICATIONS Sakuma, C., Kawauchi, T., Haraguchi, S., Shikanai, M., Yamaguchi, Y., Gelfand, V. I., Luo, L., Miura, M., Chihara, T. 2014; 5

  Abstract

  Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. Here we show that Drosophila Strip is a key regulator for neuronal morphogenesis. Strip knockdown disturbs the early endosome clustering, and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip-mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that has important roles in neuronal morphogenesis.

  View details for DOI 10.1038/ncomms6180

  View details for Web of Science ID 000343981800004

  View details for PubMedID 25312435

  View details for PubMedCentralID PMC4197811

• SELECTIVE ATTENTION Long-range and local circuits for top-down modulation of visual cortex processing SCIENCE Zhang, S., Xu, M., Kamigaki, T., Johnny Phong Hoang Do, J. P., Chang, W., Jenvay, S., Miyamichi, K., Luo, L., Dan, Y. 2014; 345 (6197): 660-665

  Abstract

  Top-down modulation of sensory processing allows the animal to select inputs most relevant to current tasks. We found that the cingulate (Cg) region of the mouse frontal cortex powerfully influences sensory processing in the primary visual cortex (V1) through long-range projections that activate local γ-aminobutyric acid-ergic (GABAergic) circuits. Optogenetic activation of Cg neurons enhanced V1 neuron responses and improved visual discrimination. Focal activation of Cg axons in V1 caused a response increase at the activation site but a decrease at nearby locations (center-surround modulation). Whereas somatostatin-positive GABAergic interneurons contributed preferentially to surround suppression, vasoactive intestinal peptide-positive interneurons were crucial for center facilitation. Long-range corticocortical projections thus act through local microcircuits to exert spatially specific top-down modulation of sensory processing.

  View details for DOI 10.1126/science.1254126

  View details for Web of Science ID 000339962800036

• Presynaptic partners of dorsal raphe serotonergic and GABAergic neurons. Neuron Weissbourd, B., Ren, J., DeLoach, K. E., Guenthner, C. J., Miyamichi, K., Luo, L. 2014; 83 (3): 645-662

  Abstract

  The serotonin system powerfully modulates physiology and behavior in health and disease, yet the circuit mechanisms underlying serotonin neuron activity are poorly understood. The major source of forebrain serotonergic innervation is from the dorsal raphe nucleus (DR), which contains both serotonin and GABA neurons. Using viral tracing combined with electrophysiology, we found that GABA and serotonin neurons in the DR receive excitatory, inhibitory, and peptidergic inputs from the same specific brain regions. Embedded in this overall similarity are important differences. Serotonin neurons are more likely to receive synaptic inputs from anterior neocortex while GABA neurons receive disproportionally higher input from the central amygdala. Local input mapping revealed extensive serotonin-serotonin as well as GABA-serotonin connectivity with a distinct spatial organization. Covariance analysis suggests heterogeneity of both serotonin and GABA neurons with respect to the inputs they receive. These analyses provide a foundation for further functional dissection of the serotonin system.

  View details for DOI 10.1016/j.neuron.2014.06.024

  View details for PubMedID 25102560

• A molecular basis for classic blond hair color in Europeans. Nature genetics Guenther, C. A., Tasic, B., Luo, L., Bedell, M. A., Kingsley, D. M. 2014; 46 (7): 748-752

  Abstract

  Hair color differences are among the most obvious examples of phenotypic variation in humans. Although genome-wide association studies (GWAS) have implicated multiple loci in human pigment variation, the causative base-pair changes are still largely unknown. Here we dissect a regulatory region of the KITLG gene (encoding KIT ligand) that is significantly associated with common blond hair color in northern Europeans. Functional tests demonstrate that the region contains a regulatory enhancer that drives expression in developing hair follicles. This enhancer contains a common SNP (rs12821256) that alters a binding site for the lymphoid enhancer-binding factor 1 (LEF1) transcription factor, reducing LEF1 responsiveness and enhancer activity in cultured human keratinocytes. Mice carrying ancestral or derived variants of the human KITLG enhancer exhibit significant differences in hair pigmentation, confirming that altered regulation of an essential growth factor contributes to the classic blond hair phenotype found in northern Europeans.

  View details for DOI 10.1038/ng.2991

  View details for PubMedID 24880339

• Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice. Proceedings of the National Academy of Sciences of the United States of America Ali, S. R., Hippenmeyer, S., Saadat, L. V., Luo, L., Weissman, I. L., Ardehali, R. 2014; 111 (24): 8850-8855

  Abstract

  The mammalian heart has long been considered a postmitotic organ, implying that the total number of cardiomyocytes is set at birth. Analysis of cell division in the mammalian heart is complicated by cardiomyocyte binucleation shortly after birth, which makes it challenging to interpret traditional assays of cell turnover [Laflamme MA, Murray CE (2011) Nature 473(7347):326-335; Bergmann O, et al. (2009) Science 324(5923):98-102]. An elegant multi-isotope imaging-mass spectrometry technique recently calculated the low, discrete rate of cardiomyocyte generation in mice [Senyo SE, et al. (2013) Nature 493(7432):433-436], yet our cellular-level understanding of postnatal cardiomyogenesis remains limited. Herein, we provide a new line of evidence for the differentiated α-myosin heavy chain-expressing cardiomyocyte as the cell of origin of postnatal cardiomyogenesis using the "mosaic analysis with double markers" mouse model. We show limited, life-long, symmetric division of cardiomyocytes as a rare event that is evident in utero but significantly diminishes after the first month of life in mice; daughter cardiomyocytes divide very seldom, which this study is the first to demonstrate, to our knowledge. Furthermore, ligation of the left anterior descending coronary artery, which causes a myocardial infarction in the mosaic analysis with double-marker mice, did not increase the rate of cardiomyocyte division above the basal level for up to 4 wk after the injury. The clonal analysis described here provides direct evidence of postnatal mammalian cardiomyogenesis.

  View details for DOI 10.1073/pnas.1408233111

  View details for PubMedID 24876275

• Mosaic Analysis with Double Markers (MADM) in Mice. Cold Spring Harbor protocols Espinosa, J. S., Tea, J. S., Luo, L. 2014; 2014 (2)

  Abstract

  The human brain comprises more than 100 billion neurons, each of which has an elaborate shape and a complex pattern of connections. To untangle this complexity, it is often useful to visualize one neuron at a time. Mosaic analysis with double markers (MADM) is a genetic method for labeling and manipulating individual neurons. This method was developed in mice and it allows simultaneous labeling and gene knockout in clones of somatic cells or isolated single cells in vivo. In MADM, labeling is achieved by using site-specific recombinases to induce the reconstitution of chimeric fluorescent proteins. Here we provide the standard procedure for utilizing MADM to examine lineage analysis, neural circuit tracing, and gene function. ROSA26-MADM is used as an example because the reagents are published and available. As MADM cassettes are introduced onto more chromosomes, genes located on these other chromosomes can be subjected to mosaic analysis in an analogous manner to that described below. We present detailed protocols with troubleshooting guides, as well as applications of the technique in tracing neural circuits, live imaging of developing neurons, and studying mechanisms of neuronal morphogenesis.

  View details for DOI 10.1101/pdb.prot080366

  View details for PubMedID 24492775

• Selective attention. Long-range and local circuits for top-down modulation of visual cortex processing. Science (New York, N.Y.) Zhang, S., Xu, M., Kamigaki, T., Hoang Do, J. P., Chang, W. C., Jenvay, S., Miyamichi, K., Luo, L., Dan, Y. 2014; 345 (6197): 660–65

  Abstract

  Top-down modulation of sensory processing allows the animal to select inputs most relevant to current tasks. We found that the cingulate (Cg) region of the mouse frontal cortex powerfully influences sensory processing in the primary visual cortex (V1) through long-range projections that activate local γ-aminobutyric acid-ergic (GABAergic) circuits. Optogenetic activation of Cg neurons enhanced V1 neuron responses and improved visual discrimination. Focal activation of Cg axons in V1 caused a response increase at the activation site but a decrease at nearby locations (center-surround modulation). Whereas somatostatin-positive GABAergic interneurons contributed preferentially to surround suppression, vasoactive intestinal peptide-positive interneurons were crucial for center facilitation. Long-range corticocortical projections thus act through local microcircuits to exert spatially specific top-down modulation of sensory processing.

  View details for PubMedID 25104383

• Genetic Control of Wiring Specificity in the Fly Olfactory System GENETICS Hong, W., Luo, L. 2014; 196 (1): 17-29

  Abstract

  Precise connections established between pre- and postsynaptic partners during development are essential for the proper function of the nervous system. The olfactory system detects a wide variety of odorants and processes the information in a precisely connected neural circuit. A common feature of the olfactory systems from insects to mammals is that the olfactory receptor neurons (ORNs) expressing the same odorant receptor make one-to-one connections with a single class of second-order olfactory projection neurons (PNs). This represents one of the most striking examples of targeting specificity in developmental neurobiology. Recent studies have uncovered central roles of transmembrane and secreted proteins in organizing this one-to-one connection specificity in the olfactory system. Here, we review recent advances in the understanding of how this wiring specificity is genetically controlled and focus on the mechanisms by which transmembrane and secreted proteins regulate different stages of the Drosophila olfactory circuit assembly in a coordinated manner. We also discuss how combinatorial coding, redundancy, and error-correcting ability could contribute to constructing a complex neural circuit in general.

  View details for DOI 10.1534/genetics.113.154336

  View details for Web of Science ID 000330579100002

  View details for PubMedID 24395823

• Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins. eLife Mosca, T. J., Luo, L. 2014; 3

  Abstract

  Understanding information flow through neuronal circuits requires knowledge of their synaptic organization. In this study, we utilized fluorescent pre- and postsynaptic markers to map synaptic organization in the Drosophila antennal lobe, the first olfactory processing center. Olfactory receptor neurons (ORNs) produce a constant synaptic density across different glomeruli. Each ORN within a class contributes nearly identical active zone number. Active zones from ORNs, projection neurons (PNs), and local interneurons have distinct subglomerular and subcellular distributions. The correct number of ORN active zones and PN acetylcholine receptor clusters requires the Teneurins, conserved transmembrane proteins involved in neuromuscular synapse organization and synaptic partner matching. Ten-a acts in ORNs to organize presynaptic active zones via the spectrin cytoskeleton. Ten-m acts in PNs autonomously to regulate acetylcholine receptor cluster number and transsynaptically to regulate ORN active zone number. These studies advanced our ability to assess synaptic architecture in complex CNS circuits and their underlying molecular mechanisms.

  View details for DOI 10.7554/eLife.03726

  View details for PubMedID 25310239

  View details for PubMedCentralID PMC4194450

• Deterministic progenitor behavior and unitary production of neurons in the neocortex. Cell Gao, P., Postiglione, M. P., Krieger, T. G., Hernandez, L., Wang, C., Han, Z., Streicher, C., Papusheva, E., Insolera, R., Chugh, K., Kodish, O., Huang, K., Simons, B. D., Luo, L., Hippenmeyer, S., Shi, S. H. 2014; 159 (4): 775–88

  Abstract

  Radial glial progenitors (RGPs) are responsible for producing nearly all neocortical neurons. To gain insight into the patterns of RGP division and neuron production, we quantitatively analyzed excitatory neuron genesis in the mouse neocortex using Mosaic Analysis with Double Markers, which provides single-cell resolution of progenitor division patterns and potential in vivo. We found that RGPs progress through a coherent program in which their proliferative potential diminishes in a predictable manner. Upon entry into the neurogenic phase, individual RGPs produce ?8-9 neurons distributed in both deep and superficial layers, indicating a unitary output in neuronal production. Removal of OTX1, a transcription factor transiently expressed in RGPs, results in both deep- and superficial-layer neuron loss and a reduction in neuronal unit size. Moreover, ?1/6 of neurogenic RGPs proceed to produce glia. These results suggest that progenitor behavior and histogenesis in the mammalian neocortex conform to a remarkably orderly and deterministic program.

  View details for PubMedID 25417155

• Dissecting Local Circuits: Parvalbumin Interneurons Underlie Broad Feedback Control of Olfactory Bulb Output NEURON Miyamichi, K., Shlomai-Fuchs, Y., Shu, M., Weissbourd, B. C., Luo, L., Mizrahi, A. 2013; 80 (5): 1232-1245

  Abstract

  In the mouse olfactory bulb, information from sensory neurons is extensively processed by local interneurons before being transmitted to the olfactory cortex by mitral and tufted (M/T) cells. The precise function of these local networks remains elusive because of the vast heterogeneity of interneurons, their diverse physiological properties, and their complex synaptic connectivity. Here we identified the parvalbumin interneurons (PVNs) as a prominent component of the M/T presynaptic landscape by using an improved rabies-based transsynaptic tracing method for local circuits. In vivo two-photon-targeted patch recording revealed that PVNs have exceptionally broad olfactory receptive fields and exhibit largely excitatory and persistent odor responses. Transsynaptic tracing indicated that PVNs receive direct input from widely distributed M/T cells. Both the anatomical and functional extent of this M/T→PVN→M/T circuit contrasts with the narrowly confined M/T→granule cell→M/T circuit, suggesting that olfactory information is processed by multiple local circuits operating at distinct spatial scales.

  View details for DOI 10.1016/j.neuron.2013.08.027

  View details for Web of Science ID 000327919500013

  View details for PubMedID 24239125

  View details for PubMedCentralID PMC3932159

• High-speed laser microsurgery of alert fruit flies for fluorescence imaging of neural activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sinha, S., Liang, L., Ho, E. T., Urbanek, K. E., Luo, L., Baer, T. M., Schnitzer, M. J. 2013; 110 (46): 18374-18379

  Abstract

  Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-µm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12- to 350-µm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca(2+) signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5-20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation.

  View details for DOI 10.1073/pnas.1216287110

  View details for Web of Science ID 000326830900029

  View details for PubMedID 24167298

  View details for PubMedCentralID PMC3832030

• GABAergic projection neurons route selective olfactory inputs to specific higher-order neurons. Neuron Liang, L., Li, Y., Potter, C. J., Yizhar, O., Deisseroth, K., Tsien, R. W., Luo, L. 2013; 79 (5): 917-931

  Abstract

  We characterize an inhibitory circuit motif in the Drosophila olfactory system, parallel inhibition, which differs from feedforward or feedback inhibition. Excitatory and GABAergic inhibitory projection neurons (ePNs and iPNs) each receive input from antennal lobe glomeruli and send parallel output to the lateral horn, a higher center implicated in regulating innate olfactory behavior. Ca(2+) imaging of specific lateral horn neurons as an olfactory readout revealed that iPNs selectively suppressed food-related odor responses, but spared signal transmission from pheromone channels. Coapplying food odorant did not affect pheromone signal transmission, suggesting that the differential effects likely result from connection specificity of iPNs, rather than a generalized inhibitory tone. Ca(2+) responses in the ePN axon terminals show no detectable suppression by iPNs, arguing against presynaptic inhibition as a primary mechanism. The parallel inhibition motif may provide specificity in inhibition to funnel specific olfactory information, such as food and pheromone, into distinct downstream circuits.

  View details for DOI 10.1016/j.neuron.2013.06.014

  View details for PubMedID 24012005

• Specific Kinematics and Motor-Related Neurons for Aversive Chemotaxis in Drosophila CURRENT BIOLOGY Gao, X. J., Potter, C. J., Gohl, D. M., Silies, M., Katsov, A. Y., Clandinin, T. R., Luo, L. 2013; 23 (13): 1163-1172

  Abstract

  Chemotaxis, the ability to direct movements according to chemical cues in the environment, is important for the survival of most organisms. The vinegar fly, Drosophila melanogaster, displays robust olfactory aversion and attraction, but how these behaviors are executed via changes in locomotion remains poorly understood. In particular, it is not clear whether aversion and attraction bidirectionally modulate a shared circuit or recruit distinct circuits for execution.Using a quantitative behavioral assay, we determined that both aversive and attractive odorants modulate the initiation and direction of turns but display distinct kinematics. Using genetic tools to perturb these behaviors, we identified specific populations of neurons required for aversion, but not for attraction. Inactivation of these populations of cells affected the completion of aversive turns, but not their initiation. Optogenetic activation of the same populations of cells triggered a locomotion pattern resembling aversive turns. Perturbations in both the ellipsoid body and the ventral nerve cord, two regions involved in motor control, resulted in defects in aversion.Aversive chemotaxis in vinegar flies triggers ethologically appropriate kinematics distinct from those of attractive chemotaxis and requires specific motor-related neurons.

  View details for DOI 10.1016/j.cub.2013.05.008

  View details for Web of Science ID 000321605600017

  View details for PubMedID 23770185

• Permanent Genetic Access to Transiently Active Neurons via TRAP: Targeted Recombination in Active Populations. Neuron Guenthner, C. J., Miyamichi, K., Yang, H. H., Heller, H. C., Luo, L. 2013; 78 (5): 773-784

  Abstract

  Targeting genetically encoded tools for neural circuit dissection to relevant cellular populations is a major challenge in neurobiology. We developed an approach, targeted recombination in active populations (TRAP), to obtain genetic access to neurons that were activated by defined stimuli. This method utilizes mice in which the tamoxifen-dependent recombinase CreER(T2) is expressed in an activity-dependent manner from the loci of the immediate early genes Arc and Fos. Active cells that express CreER(T2) can only undergo recombination when tamoxifen is present, allowing genetic access to neurons that are active during a time window of less than 12 hr. We show that TRAP can provide selective access to neurons activated by specific somatosensory, visual, and auditory stimuli and by experience in a novel environment. When combined with tools for labeling, tracing, recording, and manipulating neurons, TRAP offers a powerful approach for understanding how the brain processes information and generates behavior.

  View details for DOI 10.1016/j.neuron.2013.03.025

  View details for PubMedID 23764283

  View details for PubMedCentralID PMC3782391

• Linking cell fate, trajectory choice, and target selection: genetic analysis of sema-2b in olfactory axon targeting. Neuron Joo, W. J., Sweeney, L. B., Liang, L., Luo, L. 2013; 78 (4): 673-686

  Abstract

  Neural circuit assembly requires selection of specific cell fates, axonal trajectories, and synaptic targets. By analyzing the function of a secreted semaphorin, Sema-2b, in Drosophila olfactory receptor neuron (ORN) development, we identified multiple molecular and cellular mechanisms that link these events. Notch signaling limits Sema-2b expression to ventromedial ORN classes, within which Sema-2b cell-autonomously sensitizes ORN axons to external semaphorins. Central-brain-derived Sema-2a and Sema-2b attract Sema-2b-expressing axons to the ventromedial trajectory. In addition, Sema-2b/PlexB-mediated axon-axon interactions consolidate this trajectory choice and promote ventromedial axon-bundle formation. Selecting the correct developmental trajectory is ultimately essential for proper target choice. These findings demonstrate that Sema-2b couples ORN axon guidance to postsynaptic target neuron dendrite patterning well before the final target selection phase, and exemplify how a single guidance molecule can drive consecutive stages of neural circuit assembly with the help of sophisticated spatial and temporal regulation.

  View details for DOI 10.1016/j.neuron.2013.03.022

  View details for PubMedID 23719164

• Plum, an Immunoglobulin Superfamily Protein, Regulates Axon Pruning by Facilitating TGF-ß Signaling. Neuron Yu, X. M., Gutman, I., Mosca, T. J., Iram, T., Ozkan, E., Garcia, K. C., Luo, L., Schuldiner, O. 2013; 78 (3): 456-468

  Abstract

  Axon pruning during development is essential for proper wiring of the mature nervous system, but its regulation remains poorly understood. We have identified an immunoglobulin superfamily (IgSF) transmembrane protein, Plum, that is cell autonomously required for axon pruning of mushroom body (MB) γ neurons and for ectopic synapse refinement at the developing neuromuscular junction in Drosophila. Plum promotes MB γ neuron axon pruning by regulating the expression of Ecdysone Receptor-B1, a key initiator of axon pruning. Genetic analyses indicate that Plum acts to facilitate signaling of Myoglianin, a glial-derived TGF-β, on MB γ neurons upstream of the type-I TGF-β receptor Baboon. Myoglianin, Baboon, and Ecdysone Receptor-B1 are also required for neuromuscular junction ectopic synapse refinement. Our study highlights both IgSF proteins and TGF-β facilitation as key promoters of developmental axon elimination and demonstrates a mechanistic conservation between MB axon pruning during metamorphosis and the refinement of ectopic larval neuromuscular connections.

  View details for DOI 10.1016/j.neuron.2013.03.004

  View details for PubMedID 23664613

• Plum, an Immunoglobulin Superfamily Protein, Regulates Axon Pruning by Facilitating TGF-beta Signaling NEURON Yu, X. M., Gutman, I., Mosca, T. J., Iram, T., Oezkan, E., Garcia, K. C., Luo, L., Schuldiner, O. 2013; 78 (3): 456-468

  View details for DOI 10.1016/j.neuron.2013.03.004

  View details for Web of Science ID 000318961700008

• Mosaic Analysis with Double Markers Reveals Cell-Type-Specific Paternal Growth Dominance CELL REPORTS Hippenmeyer, S., Johnson, R. L., Luo, L. 2013; 3 (3): 960-967

  Abstract

  Genomic imprinting leads to preferred expression of either the maternal or paternal alleles of a subset of genes. Imprinting is essential for mammalian development, and its deregulation causes many diseases. However, the functional relevance of imprinting at the cellular level is poorly understood for most imprinted genes. We used mosaic analysis with double markers (MADM) in mice to create uniparental disomies (UPDs) and to visualize imprinting effects with single-cell resolution. Although chromosome 12 UPD did not produce detectable phenotypes, chromosome 7 UPD caused highly significant paternal growth dominance in the liver and lung, but not in the brain or heart. A single gene on chromosome 7, encoding the secreted insulin-like growth factor 2 (IGF2), accounts for most of the paternal dominance effect. Mosaic analyses implied additional imprinted loci on chromosome 7 acting cell autonomously to transmit the IGF2 signal. Our study reveals chromosome- and cell-type specificity of genomic imprinting effects.

  View details for DOI 10.1016/j.celrep.2013.02.002

  View details for Web of Science ID 000321896000036

  View details for PubMedID 23453967

  View details for PubMedCentralID PMC3668097

• Neuroscience. dSarm-ing axon degeneration. Science Yu, X. M., Luo, L. 2012; 337 (6093): 418-419

  View details for DOI 10.1126/science.1226150

  View details for PubMedID 22837513

• The SUMO Protease Verloren Regulates Dendrite and Axon Targeting in Olfactory Projection Neurons JOURNAL OF NEUROSCIENCE Berdnik, D., Favaloro, V., Luo, L. 2012; 32 (24): 8331-8340

  Abstract

  Sumoylation is a post-translational modification regulating numerous biological processes. Small ubiquitin-like modifier (SUMO) proteases are required for the maturation and deconjugation of SUMO proteins, thereby either promoting or reverting sumoylation to modify protein function. Here, we show a novel role for a predicted SUMO protease, Verloren (Velo), during projection neuron (PN) target selection in the Drosophila olfactory system. PNs target their dendrites to specific glomeruli within the antennal lobe (AL) and their axons stereotypically into higher brain centers. We uncovered mutations in velo that disrupt PN targeting specificity. PN dendrites that normally target to a particular dorsolateral glomerulus instead mistarget to incorrect glomeruli within the AL or to brain regions outside the AL. velo mutant axons also display defects in arborization. These phenotypes are rescued by postmitotic expression of Velo in PNs but not by a catalytic domain mutant of Velo. Two other SUMO proteases, DmUlp1 and CG12717, can partially compensate for the function of Velo in PN dendrite targeting. Additionally, mutations in SUMO and lesswright (which encodes a SUMO conjugating enzyme) similarly disrupt PN targeting, confirming that sumoylation is required for neuronal target selection. Finally, genetic interaction studies suggest that Velo acts in SUMO deconjugation rather than in maturation. Our study provides the first in vivo evidence for a specific role of a SUMO protease during neuronal target selection that can be dissociated from its functions in neuronal proliferation and survival.

  View details for DOI 10.1523/JNEUROSCI.6574-10.2012

  View details for Web of Science ID 000305295600024

  View details for PubMedID 22699913

  View details for PubMedCentralID PMC3394434

• Teneurins instruct synaptic partner matching in an olfactory map NATURE Hong, W., Mosca, T. J., Luo, L. 2012; 484 (7393): 201-U82

  Abstract

  Neurons are interconnected with extraordinary precision to assemble a functional nervous system. Compared to axon guidance, far less is understood about how individual pre- and postsynaptic partners are matched. To ensure the proper relay of olfactory information in the fruitfly Drosophila, axons of ∼50 classes of olfactory receptor neurons (ORNs) form one-to-one connections with dendrites of ∼50 classes of projection neurons (PNs). Here, using genetic screens, we identified two evolutionarily conserved, epidermal growth factor (EGF)-repeat containing transmembrane Teneurin proteins, Ten-m and Ten-a, as synaptic-partner-matching molecules between PN dendrites and ORN axons. Ten-m and Ten-a are highly expressed in select PN-ORN matching pairs. Teneurin loss- and gain-of-function cause specific mismatching of select ORNs and PNs. Finally, Teneurins promote homophilic interactions in vitro, and Ten-m co-expression in non-partner PNs and ORNs promotes their ectopic connections in vivo. We propose that Teneurins instruct matching specificity between synaptic partners through homophilic attraction.

  View details for DOI 10.1038/nature10926

  View details for Web of Science ID 000303149900027

  View details for PubMedID 22425994

  View details for PubMedCentralID PMC3345284

• Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice NATURE Mosca, T. J., Hong, W., Dani, V. S., Favaloro, V., Luo, L. 2012; 484 (7393): 237-U122

  Abstract

  Synapse assembly requires trans-synaptic signals between the pre- and postsynapse, but our understanding of the essential organizational molecules involved in this process remains incomplete. Teneurin proteins are conserved, epidermal growth factor (EGF)-repeat-containing transmembrane proteins with large extracellular domains. Here we show that two Drosophila Teneurins, Ten-m and Ten-a, are required for neuromuscular synapse organization and target selection. Ten-a is presynaptic whereas Ten-m is mostly postsynaptic; neuronal Ten-a and muscle Ten-m form a complex in vivo. Pre- or postsynaptic Teneurin perturbations cause severe synapse loss and impair many facets of organization trans-synaptically and cell autonomously. These include defects in active zone apposition, release sites, membrane and vesicle organization, and synaptic transmission. Moreover, the presynaptic microtubule and postsynaptic spectrin cytoskeletons are severely disrupted, suggesting a mechanism whereby Teneurins organize the cytoskeleton, which in turn affects other aspects of synapse development. Supporting this, Ten-m physically interacts with α-Spectrin. Genetic analyses of teneurin and neuroligin reveal that they have differential roles that synergize to promote synapse assembly. Finally, at elevated endogenous levels, Ten-m regulates target selection between specific motor neurons and muscles. Our study identifies the Teneurins as a key bi-directional trans-synaptic signal involved in general synapse organization, and demonstrates that proteins such as these can also regulate target selection.

  View details for DOI 10.1038/nature10923

  View details for Web of Science ID 000303149900034

  View details for PubMedID 22426000

  View details for PubMedCentralID PMC3326183

• Controlling gene expression with the Q repressible binary expression system in Caenorhabditis elegans NATURE METHODS Wei, X., Potter, C. J., Luo, L., Shen, K. 2012; 9 (4): 391-U105

  Abstract

  We established a transcription-based binary gene expression system in Caenorhabditis elegans using the recently developed Q system. This system, derived from genes in Neurospora crassa, uses the transcriptional activator QF to induce the expression of target genes. Activation can be efficiently suppressed by the transcriptional repressor QS, and suppression can be relieved by the nontoxic small molecule quinic acid. We used QF, QS and quinic acid to achieve temporal and spatial control of transgene expression in various tissues in C. elegans. We also developed a split Q system, in which we separated QF into two parts encoding its DNA-binding and transcription-activation domains. Each domain showed negligible transcriptional activity when expressed alone, but expression of both reconstituted QF activity, providing additional combinatorial power to control gene expression.

  View details for DOI 10.1038/NMETH.1929

  View details for Web of Science ID 000302218500024

  View details for PubMedID 22406855

• Extensions of MADM (Mosaic Analysis with Double Markers) in Mice PLOS ONE Tasic, B., Miyamichi, K., Hippenmeyer, S., Dani, V. S., Zeng, H., Joo, W., Zong, H., Chen-Tsai, Y., Luo, L. 2012; 7 (3)

  Abstract

  Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM.

  View details for DOI 10.1371/journal.pone.0033332

  View details for Web of Science ID 000303894900024

  View details for PubMedID 22479386

  View details for PubMedCentralID PMC3314016

• Secreted Semaphorins from Degenerating Larval ORN Axons Direct Adult Projection Neuron Dendrite Targeting NEURON Sweeney, L. B., Chou, Y., Wu, Z., Joo, W., Komiyama, T., Potter, C. J., Kolodkin, A. L., Garcia, K. C., Luo, L. 2011; 72 (5): 734-747

  Abstract

  During assembly of the Drosophila olfactory circuit, projection neuron (PN) dendrites prepattern the developing antennal lobe before the arrival of axons from their presynaptic partners, the adult olfactory receptor neurons (ORNs). We previously found that levels of transmembrane Semaphorin-1a, which acts as a receptor, instruct PN dendrite targeting along the dorsolateral-ventromedial axis. Here we show that two secreted semaphorins, Sema-2a and Sema-2b, provide spatial cues for PN dendrite targeting. Sema-2a and Sema-2b proteins are distributed in gradients opposing the Sema-1a protein gradient, and Sema-1a binds to Sema-2a-expressing cells. In Sema-2a and Sema-2b double mutants, PN dendrites that normally target dorsolaterally in the antennal lobe mistarget ventromedially, phenocopying cell-autonomous Sema-1a removal from these PNs. Cell ablation, cell-specific knockdown, and rescue experiments indicate that secreted semaphorins from degenerating larval ORN axons direct dendrite targeting. Thus, a degenerating brain structure instructs the wiring of a developing circuit through the repulsive action of secreted semaphorins.

  View details for DOI 10.1016/j.neuron.2011.09.026

  View details for PubMedID 22153371

• Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Beier, K. T., Saunders, A., Oldenburg, I. A., Miyamichi, K., Akhtar, N., Luo, L., Whelan, S. P., Sabatini, B., Cepko, C. L. 2011; 108 (37): 15414-15419

  Abstract

  To understand how the nervous system processes information, a map of the connections among neurons would be of great benefit. Here we describe the use of vesicular stomatitis virus (VSV) for tracing neuronal connections in vivo. We made VSV vectors that used glycoprotein (G) genes from several other viruses. The G protein from lymphocytic choriomeningitis virus endowed VSV with the ability to spread transsynaptically, specifically in an anterograde direction, whereas the rabies virus glycoprotein gave a specifically retrograde transsynaptic pattern. The use of an avian G protein fusion allowed specific targeting of cells expressing an avian receptor, which allowed a demonstration of monosynaptic anterograde tracing from defined cells. Synaptic connectivity of pairs of virally labeled cells was demonstrated by using slice cultures and electrophysiology. In vivo infections of several areas in the mouse brain led to the predicted patterns of spread for anterograde or retrograde tracers.

  View details for DOI 10.1073/pnas.1110854108

  View details for Web of Science ID 000294804900082

  View details for PubMedID 21825165

• Using the Q system in Drosophila melanogaster NATURE PROTOCOLS Potter, C. J., Luo, L. 2011; 6 (8): 1105-1120

  Abstract

  In Drosophila, the GAL4/UAS/GAL80 repressible binary expression system is widely used to manipulate or mark tissues of interest. However, complex biological systems often require distinct transgenic manipulations of different cell populations. For this purpose, we recently developed the Q system, a second repressible binary expression system. We describe here the basic steps for performing a variety of Q system experiments in vivo. These include how to generate and use Q system reagents to express effector transgenes in tissues of interest, how to use the Q system in conjunction with the GAL4 system to generate intersectional expression patterns that precisely limit which tissues will be experimentally manipulated and how to use the Q system to perform mosaic analysis. The protocol described here can be adapted to a wide range of experimental designs.

  View details for DOI 10.1038/nprot.2011.347

  View details for Web of Science ID 000294480300002

  View details for PubMedID 21738124

• Mosaic Analysis with Double Markers Reveals Tumor Cell of Origin in Glioma CELL Liu, C., Sage, J. C., Miller, M. R., Verhaak, R. G., Hippenmeyer, S., Vogel, H., Foreman, O., Bronson, R. T., Nishiyama, A., Luo, L., Zong, H. 2011; 146 (2): 209-221

  Abstract

  Cancer cell of origin is difficult to identify by analyzing cells within terminal stage tumors, whose identity could be concealed by the acquired plasticity. Thus, an ideal approach to identify the cell of origin is to analyze proliferative abnormalities in distinct lineages prior to malignancy. Here, we use mosaic analysis with double markers (MADM) in mice to model gliomagenesis by initiating concurrent p53/Nf1 mutations sporadically in neural stem cells (NSCs). Surprisingly, MADM-based lineage tracing revealed significant aberrant growth prior to malignancy only in oligodendrocyte precursor cells (OPCs), but not in any other NSC-derived lineages or NSCs themselves. Upon tumor formation, phenotypic and transcriptome analyses of tumor cells revealed salient OPC features. Finally, introducing the same p53/Nf1 mutations directly into OPCs consistently led to gliomagenesis. Our findings suggest OPCs as the cell of origin in this model, even when initial mutations occur in NSCs, and highlight the importance of analyzing premalignant stages to identify the cancer cell of origin.

  View details for DOI 10.1016/j.cell.2011.06.014

  View details for Web of Science ID 000293013000005

  View details for PubMedID 21737130

  View details for PubMedCentralID PMC3143261

• Site-specific integrase-mediated transgenesis in mice via pronuclear injection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen-Tsai, Y., Luo, L. 2011; 108 (19): 7902-7907

  Abstract

  Microinjection of recombinant DNA into zygotic pronuclei has been widely used for producing transgenic mice. However, with this method, the insertion site, integrity, and copy number of the transgene cannot be controlled. Here, we present an integrase-based approach to produce transgenic mice via pronuclear injection, whereby an intact single-copy transgene can be inserted into predetermined chromosomal loci with high efficiency (up to 40%), and faithfully transmitted through generations. We show that neighboring transgenic elements and bacterial DNA within the transgene cause profound silencing and expression variability of the transgenic marker. Removal of these undesirable elements leads to global high-level marker expression from transgenes driven by a ubiquitous promoter. We also obtained faithful marker expression from a tissue-specific promoter. The technique presented here will greatly facilitate murine transgenesis and precise structure/function dissection of mammalian gene function and regulation in vivo.

  View details for DOI 10.1073/pnas.1019507108

  View details for Web of Science ID 000290439500052

  View details for PubMedID 21464299

  View details for PubMedCentralID PMC3093482

• A Combinatorial Semaphorin Code Instructs the Initial Steps of Sensory Circuit Assembly in the Drosophila CNS NEURON Wu, Z., Sweeney, L. B., Ayoob, J. C., Chak, K., Andreone, B. J., Ohyama, T., Kerr, R., Luo, L., Zlatic, M., Kolodkin, A. L. 2011; 70 (2): 281-298

  Abstract

  Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, whereas broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.

  View details for DOI 10.1016/j.neuron.2011.02.050

  View details for Web of Science ID 000291073700010

  View details for PubMedID 21521614

• Cortical representations of olfactory input by trans-synaptic tracing NATURE Miyamichi, K., Amat, F., Moussavi, F., Wang, C., Wickersham, I., Wall, N. R., Taniguchi, H., Tasic, B., Huang, Z. J., He, Z., Callaway, E. M., Horowitz, M. A., Luo, L. 2011; 472 (7342): 191-196

  Abstract

  In the mouse, each class of olfactory receptor neurons expressing a given odorant receptor has convergent axonal projections to two specific glomeruli in the olfactory bulb, thereby creating an odour map. However, it is unclear how this map is represented in the olfactory cortex. Here we combine rabies-virus-dependent retrograde mono-trans-synaptic labelling with genetics to control the location, number and type of 'starter' cortical neurons, from which we trace their presynaptic neurons. We find that individual cortical neurons receive input from multiple mitral cells representing broadly distributed glomeruli. Different cortical areas represent the olfactory bulb input differently. For example, the cortical amygdala preferentially receives dorsal olfactory bulb input, whereas the piriform cortex samples the whole olfactory bulb without obvious bias. These differences probably reflect different functions of these cortical areas in mediating innate odour preference or associative memory. The trans-synaptic labelling method described here should be widely applicable to mapping connections throughout the mouse nervous system.

  View details for DOI 10.1038/nature09714

  View details for Web of Science ID 000289469100036

  View details for PubMedID 21179085

  View details for PubMedCentralID PMC3073090

• The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting NEURAL DEVELOPMENT Tea, J. S., Luo, L. 2011; 6

  Abstract

  The Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs) pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program.We find that loss of Brahma associated protein 55 kD (Bap55) results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM) and the Tat interactive protein 60 kD (TIP60) ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b.Our results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions.

  View details for DOI 10.1186/1749-8104-6-5

  View details for Web of Science ID 000290529200002

  View details for PubMedID 21284845

  View details for PubMedCentralID PMC3038883

• Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration NEURON Hippenmeyer, S., Youn, Y. H., Moon, H. M., Miyamichi, K., Zong, H., Wynshaw-Boris, A., Luo, L. 2010; 68 (4): 695-709

  Abstract

  Coordinated migration of newly born neurons to their prospective target laminae is a prerequisite for neural circuit assembly in the developing brain. The evolutionarily conserved LIS1/NDEL1 complex is essential for neuronal migration in the mammalian cerebral cortex. The cytoplasmic nature of LIS1 and NDEL1 proteins suggest that they regulate neuronal migration cell autonomously. Here, we extend mosaic analysis with double markers (MADM) to mouse chromosome 11 where Lis1, Ndel1, and 14-3-3ɛ (encoding a LIS1/NDEL1 signaling partner) are located. Analyses of sparse and uniquely labeled mutant cells in mosaic animals reveal distinct cell-autonomous functions for these three genes. Lis1 regulates neuronal migration efficiency in a dose-dependent manner, while Ndel1 is essential for a specific, previously uncharacterized, late step of neuronal migration: entry into the target lamina. Comparisons with previous genetic perturbations of Lis1 and Ndel1 also suggest a surprising degree of cell-nonautonomous function for these proteins in regulating neuronal migration.

  View details for DOI 10.1016/j.neuron.2010.09.027

  View details for Web of Science ID 000285079500009

  View details for PubMedID 21092859

  View details for PubMedCentralID PMC3044607

• Ten years of Nature Reviews Neuroscience: insights from the highly cited NATURE REVIEWS NEUROSCIENCE Luo, L., Rodriguez, E., Jerbi, K., Lachaux, J., Martinerie, J., Corbetta, M., Shulman, G. L., Piomelli, D., Turrigiano, G. G., Nelson, S. B., Joels, M., de Kloet, E. R., Holsboer, F., Amodio, D. M., Frith, C. D., Block, M. L., Zecca, L., Hong, J., Dantzer, R., Kelley, K. W., Craig, A. D. 2010; 11 (10): 718-?

  Abstract

  To celebrate the first 10 years of Nature Reviews Neuroscience, we invited the authors of the most cited article of each year to look back on the state of their field of research at the time of publication and the impact their article has had, and to discuss the questions that might be answered in the next 10 years. This selection of highly cited articles provides interesting snapshots of the progress that has been made in diverse areas of neuroscience. They show the enormous influence of neuroimaging techniques and highlight concepts that have generated substantial interest in the past decade, such as neuroimmunology, social neuroscience and the 'network approach' to brain function. These advancements will pave the way for further exciting discoveries that lie ahead.

  View details for DOI 10.1038/nrn2912

  View details for Web of Science ID 000281928500005

  View details for PubMedID 20852655

  View details for PubMedCentralID PMC3395239

• Patterning Axon Targeting of Olfactory Receptor Neurons by Coupled Hedgehog Signaling at Two Distinct Steps CELL Chou, Y., Zheng, X., Beachy, P. A., Luo, L. 2010; 142 (6): 954-966

  Abstract

  We present evidence for a coupled two-step action of Hedgehog signaling in patterning axon targeting of Drosophila olfactory receptor neurons (ORNs). In the first step, differential Hedgehog pathway activity in peripheral sensory organ precursors creates ORN populations with different levels of the Patched receptor. Different Patched levels in ORNs then determine axonal responsiveness to target-derived Hedgehog in the brain: only ORN axons that do not express high levels of Patched are responsive to and require a second step of Hedgehog signaling for target selection. Hedgehog signaling in the imaginal sensory organ precursors thus confers differential ORN responsiveness to Hedgehog-mediated axon targeting in the brain. This mechanism contributes to the spatial coordination of ORN cell bodies in the periphery and their glomerular targets in the brain. Such coupled two-step signaling may be more generally used to coordinate other spatially and temporally segregated developmental events.

  View details for DOI 10.1016/j.cell.2010.08.015

  View details for Web of Science ID 000281855000017

  View details for PubMedID 20850015

  View details for PubMedCentralID PMC3028148

• 'Fore Brain: A Hint of the Ancestral Cortex CELL Sweeney, L. B., Luo, L. 2010; 142 (5): 679-681

  Abstract

  By combining gene expression profiling with image registration, Tomer et al. (2010) find that the mushroom body of the segmented worm Platynereis dumerilii shares many features with the mammalian cerebral cortex. The authors propose that the mushroom body and cortex evolved from the same structure in the common ancestor of vertebrates and invertebrates.

  View details for DOI 10.1016/j.cell.2010.08.024

  View details for Web of Science ID 000281523200011

  View details for PubMedID 20813256

• Histone Deacetylase Rpd3 Regulates Olfactory Projection Neuron Dendrite Targeting via the Transcription Factor Prospero JOURNAL OF NEUROSCIENCE Tea, J. S., Chihara, T., Luo, L. 2010; 30 (29): 9939-9946

  Abstract

  Compared to the mechanisms of axon guidance, relatively little is known about the transcriptional control of dendrite guidance. The Drosophila olfactory system with its stereotyped organization provides an excellent model to study the transcriptional control of dendrite wiring specificity. Each projection neuron (PN) targets its dendrites to a specific glomerulus in the antennal lobe and its axon stereotypically to higher brain centers. Using a forward genetic screen, we identified a mutation in Rpd3 that disrupts PN targeting specificity. Rpd3 encodes a class I histone deacetylase (HDAC) homologous to mammalian HDAC1 and HDAC2. Rpd3(-/-) PN dendrites that normally target to a dorsolateral glomerulus mistarget to medial glomeruli in the antennal lobe, and axons exhibit a severe overbranching phenotype. These phenotypes can be rescued by postmitotic expression of Rpd3 but not HDAC3, the only other class I HDAC in Drosophila. Furthermore, disruption of the atypical homeodomain transcription factor Prospero (Pros) yields similar phenotypes, which can be rescued by Pros expression in postmitotic neurons. Strikingly, overexpression of Pros can suppress Rpd3(-/-) phenotypes. Our study suggests a specific function for the general chromatin remodeling factor Rpd3 in regulating dendrite targeting in neurons, largely through the postmitotic action of the Pros transcription factor.

  View details for DOI 10.1523/JNEUROSCI.1643-10.2010

  View details for Web of Science ID 000280206500030

  View details for PubMedID 20660276

  View details for PubMedCentralID PMC2924735

• Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain PLOS ONE Li, L., Tasic, B., Micheva, K. D., Ivanov, V. M., Spletter, M. L., Smith, S. J., Luo, L. 2010; 5 (7)

  Abstract

  Proper function of the mammalian brain relies on the establishment of highly specific synaptic connections among billions of neurons. To understand how complex neural circuits function, it is crucial to precisely describe neuronal connectivity and the distributions of synapses to and from individual neurons.In this study, we present a new genetic synaptic labeling method that relies on expression of a presynaptic marker, synaptophysin-GFP (Syp-GFP) in individual neurons in vivo. We assess the reliability of this method and use it to analyze the spatial patterning of synapses in developing and mature cerebellar granule cells (GCs). In immature GCs, Syp-GFP is distributed in both axonal and dendritic regions. Upon maturation, it becomes strongly enriched in axons. In mature GCs, we analyzed synapses along their ascending segments and parallel fibers. We observe no differences in presynaptic distribution between GCs born at different developmental time points and thus having varied depths of projections in the molecular layer. We found that the mean densities of synapses along the parallel fiber and the ascending segment above the Purkinje cell (PC) layer are statistically indistinguishable, and higher than previous estimates. Interestingly, presynaptic terminals were also found in the ascending segments of GCs below and within the PC layer, with the mean densities two-fold lower than that above the PC layer. The difference in the density of synapses in these parts of the ascending segment likely reflects the regional differences in postsynaptic target cells of GCs.The ability to visualize synapses of single neurons in vivo is valuable for studying synaptogenesis and synaptic plasticity within individual neurons as well as information flow in neural circuits.

  View details for DOI 10.1371/journal.pone.0011503

  View details for Web of Science ID 000279715300014

  View details for PubMedID 20634890

  View details for PubMedCentralID PMC2901335

• The Q System: A Repressible Binary System for Transgene Expression, Lineage Tracing, and Mosaic Analysis CELL Potter, C. J., Tasic, B., Russler, E. V., Liang, L., Luo, L. 2010; 141 (3): 536-548

  Abstract

  We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This "Q system" offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications, including (1) intersectional "logic gates" with the GAL4 system for manipulating transgene expression patterns, (2) GAL4-independent MARCM analysis, and (3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila and to any organism conducive to transgenesis.

  View details for DOI 10.1016/j.cell.2010.02.025

  View details for Web of Science ID 000277180800023

  View details for PubMedID 20434990

  View details for PubMedCentralID PMC2883883

• Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe NATURE NEUROSCIENCE Chou, Y., Spletter, M. L., Yaksi, E., Leong, J. C., Wilson, R. I., Luo, L. 2010; 13 (4): 439-U60

  Abstract

  Local interneurons are essential in information processing by neural circuits. Here we present a comprehensive genetic, anatomical and electrophysiological analysis of local interneurons (LNs) in the Drosophila melanogaster antennal lobe, the first olfactory processing center in the brain. We found LNs to be diverse in their neurotransmitter profiles, connectivity and physiological properties. Analysis of >1,500 individual LNs revealed principal morphological classes characterized by coarsely stereotyped glomerular innervation patterns. Some of these morphological classes showed distinct physiological properties. However, the finer-scale connectivity of an individual LN varied considerably across brains, and there was notable physiological variability within each morphological or genetic class. Finally, LN innervation required interaction with olfactory receptor neurons during development, and some individual variability also likely reflected LN-LN interactions. Our results reveal an unexpected degree of complexity and individual variation in an invertebrate neural circuit, a result that creates challenges for solving the Drosophila connectome.

  View details for DOI 10.1038/nn.2489

  View details for Web of Science ID 000276073500013

  View details for PubMedID 20139975

  View details for PubMedCentralID PMC2847188

• The olfactory circuit of the fruit fly Drosophila melanogaster SCIENCE CHINA-LIFE SCIENCES Liang, L., Luo Liqun, L. Q. 2010; 53 (4): 472-484

  View details for DOI 10.1007/s11427-010-0099-z

  View details for Web of Science ID 000277417800011

• The olfactory circuit of the fruit fly Drosophila melanogaster. Science China. Life sciences Liang, L., Luo, L. 2010; 53 (4): 472-484

  Abstract

  The olfactory circuit of the fruit fly Drosophila melanogaster has emerged in recent years as an excellent paradigm for studying the principles and mechanisms of information processing in neuronal circuits. We discuss here the organizational principles of the olfactory circuit that make it an attractive model for experimental manipulations, the lessons that have been learned, and future challenges.

  View details for DOI 10.1007/s11427-010-0099-z

  View details for PubMedID 20596914

• Dendritic tiling through TOR signalling EMBO JOURNAL Hong, W., Luo, L. 2009; 28 (24): 3783-3784

  View details for DOI 10.1038/emboj.2009.353

  View details for Web of Science ID 000272833700001

  View details for PubMedID 20010972

  View details for PubMedCentralID PMC2797063

• Leucine-rich repeat transmembrane proteins instruct discrete dendrite targeting in an olfactory map NATURE NEUROSCIENCE Hong, W., Zhu, H., Potter, C. J., Barsh, G., Kurusu, M., Zinn, K., Luo, L. 2009; 12 (12): 1542-U89

  Abstract

  Olfactory systems utilize discrete neural pathways to process and integrate odorant information. In Drosophila, axons of first-order olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons (PNs) form class-specific synaptic connections at approximately 50 glomeruli. The mechanisms underlying PN dendrite targeting to distinct glomeruli in a three-dimensional discrete neural map are unclear. We found that the leucine-rich repeat (LRR) transmembrane protein Capricious (Caps) was differentially expressed in different classes of PNs. Loss-of-function and gain-of-function studies indicated that Caps instructs the segregation of Caps-positive and Caps-negative PN dendrites to discrete glomerular targets. Moreover, Caps-mediated PN dendrite targeting was independent of presynaptic ORNs and did not involve homophilic interactions. The closely related protein Tartan was partially redundant with Caps. These LRR proteins are probably part of a combinatorial cell-surface code that instructs discrete olfactory map formation.

  View details for DOI 10.1038/nn.2442

  View details for Web of Science ID 000272065600014

  View details for PubMedID 19915565

  View details for PubMedCentralID PMC2826190

• Neuroscience. Brain wiring by presorting axons. Science Miyamichi, K., Luo, L. 2009; 325 (5940): 544-545

  View details for DOI 10.1126/science.1178117

  View details for PubMedID 19644096

• Uncoupling Dendrite Growth and Patterning: Single-Cell Knockout Analysis of NMDA Receptor 2B NEURON Espinosa, J. S., Wheeler, D. G., Tsien, R. W., Luo, L. 2009; 62 (2): 205-217

  Abstract

  N-methyl-D-aspartate receptors (NMDARs) play important functions in neural development. NR2B is the predominant NR2 subunit of NMDAR in the developing brain. Here we use mosaic analysis with double markers (MADM) to knock out NR2B in isolated single cells and analyze its cell-autonomous function in dendrite development. NR2B mutant dentate gyrus granule cells (dGCs) and barrel cortex layer 4 spiny stellate cells (bSCs) have similar dendritic growth rates, total length, and branch number as control cells. However, mutant dGCs maintain supernumerary primary dendrites resulting from a pruning defect. Furthermore, while control bSCs restrict dendritic growth to a single barrel, mutant bSCs maintain dendritic growth in multiple barrels. Thus, NR2B functions cell autonomously to regulate dendrite patterning to ensure that sensory information is properly represented in the cortex. Our study also indicates that molecular mechanisms that regulate activity-dependent dendrite patterning can be separated from those that control general dendrite growth and branching.

  View details for DOI 10.1016/j.neuron.2009.03.006

  View details for Web of Science ID 000265774100009

  View details for PubMedID 19409266

  View details for PubMedCentralID PMC2788338

• A New Family of Odorant Receptors in Drosophila CELL Spletter, M. L., Luo, L. 2009; 136 (1): 23-25

  Abstract

  In the fruit fly Drosophila, not all olfactory sensory neurons express a seven transmembrane odorant receptor, suggesting that other types of odorant receptors might exist. Benton et al. (2009) now present evidence that a family of proteins related to ionotropic glutamate receptors is a previously unrecognized class of odorant receptors.

  View details for DOI 10.1016/j.cell.2008.12.031

  View details for Web of Science ID 000262318400011

  View details for PubMedID 19135885

• MicroRNA Processing Pathway Regulates Olfactory Neuron Morphogenesis CURRENT BIOLOGY Berdnik, D., Fan, A. P., Potter, C. J., Luo, L. 2008; 18 (22): 1754-1759

  Abstract

  The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs [1, 2]. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes [3, 4]. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition (Figure 1A). Here, we identify two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections [5, 6]. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers [7-9]. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendrite mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. Our findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system.

  View details for DOI 10.1016/j.cub.2008.09.045

  View details for Web of Science ID 000261244800025

  View details for PubMedID 19013069

  View details for PubMedCentralID PMC2612040

• Octopamine fuels fighting flies NATURE NEUROSCIENCE Potter, C. J., Luo, L. 2008; 11 (9): 989-990

  Abstract

  The neural basis of aggression is poorly understood. A study in this issue used genetic scalpels to dissect the circuitry of the fly brain and identified a small cluster of octopaminergic neurons that can make a fly fighting mad.

  View details for DOI 10.1038/nn0908-989

  View details for Web of Science ID 000258720000003

  View details for PubMedID 18725900

• Genomic analysis of Drosophila neuronal remodeling: A role for the RNA-binding protein boule as a negative regulator of axon pruning JOURNAL OF NEUROSCIENCE Hoopfer, E. D., Penton, A., Watts, R. J., Luo, L. 2008; 28 (24): 6092-6103

  Abstract

  Drosophila mushroom body (MB) gamma neurons undergo axon pruning during metamorphosis through a process of localized degeneration of specific axon branches. Developmental axon degeneration is initiated by the steroid hormone ecdysone, acting through a nuclear receptor complex composed of USP (ultraspiracle) and EcRB1 (ecdysone receptor B1) to regulate gene expression in MB gamma neurons. To identify ecdysone-dependent gene expression changes in MB gamma neurons at the onset of axon pruning, we use laser capture microdissection to isolate wild-type and mutant MB neurons in which EcR (ecdysone receptor) activity is genetically blocked, and analyze expression changes by microarray. We identify several molecular pathways that are regulated in MB neurons by ecdysone. The most striking observation is the upregulation of genes involved in the UPS (ubiquitin-proteasome system), which is cell autonomously required for gamma neuron pruning. In addition, we characterize the function of Boule, an evolutionarily conserved RNA-binding protein previously implicated in spermatogenesis in flies and vertebrates. boule expression is downregulated by ecdysone in MB neurons at the onset of pruning, and forced expression of Boule in MB gamma neurons is sufficient to inhibit axon pruning. This activity is dependent on the RNA-binding domain of Boule and a conserved DAZ (deleted in azoospermia) domain implicated in interactions with other RNA-binding proteins. However, loss of Boule does not result in obvious defects in axon pruning or morphogenesis of MB neurons, suggesting that it acts redundantly with other ecdyonse-regulated genes. We propose a novel function for Boule in the CNS as a negative regulator of developmental axon pruning.

  View details for DOI 10.1523/JNEUROSCI.0677-08.2008

  View details for Web of Science ID 000256668500004

  View details for PubMedID 18550751

  View details for PubMedCentralID PMC2713105

• Genetic dissection of neural circuits NEURON Luo, L., Callaway, E. M., Svoboda, K. 2008; 57 (5): 634-660

  Abstract

  Understanding the principles of information processing in neural circuits requires systematic characterization of the participating cell types and their connections, and the ability to measure and perturb their activity. Genetic approaches promise to bring experimental access to complex neural systems, including genetic stalwarts such as the fly and mouse, but also to nongenetic systems such as primates. Together with anatomical and physiological methods, cell-type-specific expression of protein markers and sensors and transducers will be critical to construct circuit diagrams and to measure the activity of genetically defined neurons. Inactivation and activation of genetically defined cell types will establish causal relationships between activity in specific groups of neurons, circuit function, and animal behavior. Genetic analysis thus promises to reveal the logic of the neural circuits in complex brains that guide behaviors. Here we review progress in the genetic analysis of neural circuits and discuss directions for future research and development.

  View details for DOI 10.1016/j.neuron.2008.01.002

  View details for Web of Science ID 000254077300005

  View details for PubMedID 18341986

  View details for PubMedCentralID PMC2628815

• Timing neurogenesis and differentiation: Insights from quantitative clonal analyses of cerebellar granule cells JOURNAL OF NEUROSCIENCE Espinosa, J. S., Luo, L. 2008; 28 (10): 2301-2312

  Abstract

  The cerebellum is an excellent model system to study how developmental programs give rise to exquisite neuronal circuits in the adult brain. Here, we describe our findings regarding granule cell neurogenesis and differentiation using the MADM method (mosaic analysis with double markers) in mice. By following the development of individual granule cell clones, we show that (1) granule cell precursors (GCPs) undergo predominantly symmetric division during postnatal development; (2) clonally related granule cells (GCs) exit the cell cycle within a narrow time window and stack their axons in the molecular layer in chronological order from deep to superficial sublayers; and (3) whereas the average GCP proliferation in the external granular layer is progressively slower as development proceeds, there is a rapid expansion of GCPs shortly before clonally related GCs exit the cell cycle. These properties produce GC clones that are distinct, each having a restricted axonal projection, but that are on average similar in cell number. We discuss possible developmental mechanisms and functional implications of these findings.

  View details for DOI 10.1523/JNEUROSCI.5157-07.2008

  View details for Web of Science ID 000253818800002

  View details for PubMedID 18322077

  View details for PubMedCentralID PMC2586640

• piggyBac-based mosaic screen identifies a postmitotic function for cohesin in regulating developmental axon pruning DEVELOPMENTAL CELL Schuldiner, O., Berdnik, D., Levy, J. M., Wu, J. S., Luginbuhl, D., Camille Gontang, A., Luo, L. 2008; 14 (2): 227-238

  Abstract

  Developmental axon pruning is widely used to refine neural circuits. We performed a mosaic screen to identify mutations affecting axon pruning of Drosophila mushroom body gamma neurons. We constructed a modified piggyBac vector with improved mutagenicity and generated insertions in >2000 genes. We identified two cohesin subunits (SMC1 and SA) as being essential for axon pruning. The cohesin complex maintains sister-chromatid cohesion during cell division in eukaryotes. However, we show that the pruning phenotype in SMC1(-/-) clones is rescued by expressing SMC1 in neurons, revealing a postmitotic function. SMC1(-/-) clones exhibit reduced levels of the ecdysone receptor EcR-B1, a key regulator of axon pruning. The pruning phenotype is significantly suppressed by overexpressing EcR-B1 and is enhanced by a reduced dose of EcR, supporting a causal relationship. We also demonstrate a postmitotic role for SMC1 in dendrite targeting of olfactory projection neurons. We suggest that cohesin regulates diverse aspects of neuronal morphogenesis.

  View details for DOI 10.1016/j.devce1.2007.11.001

  View details for Web of Science ID 000253241400012

  View details for PubMedID 18267091

  View details for PubMedCentralID PMC2268086

• Development of continuous and discrete neural maps NEURON Luo, L., Flanagan, J. G. 2007; 56 (2): 284-300

  Abstract

  Two qualitatively different kinds of neural map have been described: continuous maps exemplified by the visual retinotopic map, and discrete maps exemplified by the olfactory glomerular map. Here, we review developmental mechanisms of retinotopic and olfactory glomerular mapping and discuss underlying commonalities that have emerged from recent studies. These include the use of molecular gradients, axon-axon interactions, and the interplay between labeling molecules and neuronal activity in establishing these maps. Since visual retinotopic and olfactory glomerular maps represent two ends of a continuum that includes many other types of neural map in between, these emerging general principles may be widely applicable to map formation throughout the nervous system.

  View details for DOI 10.1016/j.neuron.2007.10.014

  View details for Web of Science ID 000250740700007

  View details for PubMedID 17964246

• Fly MARCM and mouse MADM: Genetic methods of labeling and manipulating single neurons Meeting of the Cajal-Club 2006 Luo, L. ELSEVIER SCIENCE BV. 2007: 220–27

  Abstract

  The Golgi staining method has served neuroscience well for more than a century. In this assay I review recent progresses using genetic methods to recapitulate and extend the Golgi staining method. These methods enable new discoveries on organization and development of neuronal circuits in the fly and mouse brains.

  View details for Web of Science ID 000250952300004

  View details for PubMedID 17408568

• A global double-fluorescent cre reporter mouse GENESIS Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. 2007; 45 (9): 593-605

  Abstract

  The Cre/loxP system has been used extensively for conditional mutagenesis in mice. Reporters of Cre activity are important for defining the spatial and temporal extent of Cre-mediated recombination. Here we describe mT/mG, a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision. We show that reporter expression is nearly ubiquitous, allowing visualization of fluorescent markers in live and fixed samples of all tissues examined. We further demonstrate that mG labeling is Cre-dependent, complementary to mT at single cell resolution, and distinguishable by fluorescence-activated cell sorting. Both membrane-targeted markers outline cell morphology, highlight membrane structures, and permit visualization of fine cellular processes. In addition to serving as a global Cre reporter, the mT/mG mouse may also be used as a tool for lineage tracing, transplantation studies, and analysis of cell morphology in vivo.

  View details for DOI 10.1002/dvg.20335

  View details for Web of Science ID 000250365600008

  View details for PubMedID 17868096

• Lola regulates Drosophila olfactory projection neuron identity and targeting specificity NEURAL DEVELOPMENT Spletter, M. L., Liu, J., Liu, J., Su, H., Giniger, E., Komiyama, T., Quake, S., Luo, L. 2007; 2

  Abstract

  Precise connections of neural circuits can be specified by genetic programming. In the Drosophila olfactory system, projection neurons (PNs) send dendrites to single glomeruli in the antenna lobe (AL) based upon lineage and birth order and send axons with stereotyped terminations to higher olfactory centers. These decisions are likely specified by a PN-intrinsic transcriptional code that regulates the expression of cell-surface molecules to instruct wiring specificity.We find that the loss of longitudinals lacking (lola), which encodes a BTB-Zn-finger transcription factor with 20 predicted splice isoforms, results in wiring defects in both axons and dendrites of all lineages of PNs. RNA in situ hybridization and quantitative RT-PCR suggest that most if not all lola isoforms are expressed in all PNs, but different isoforms are expressed at widely varying levels. Overexpression of individual lola isoforms fails to rescue the lola null phenotypes and causes additional phenotypes. Loss of lola also results in ectopic expression of Gal4 drivers in multiple cell types and in the loss of transcription factor gene lim1 expression in ventral PNs.Our results indicate that lola is required for wiring of axons and dendrites of most PN classes, and suggest a need for its molecular diversity. Expression pattern changes of Gal4 drivers in lola-/- clones imply that lola normally represses the expression of these regulatory elements in a subset of the cells surrounding the AL. We propose that Lola functions as a general transcription factor that regulates the expression of multiple genes ultimately controlling PN identity and wiring specificity.

  View details for DOI 10.1186/1749-8104-2-14

  View details for Web of Science ID 000258981200001

  View details for PubMedID 17634136

  View details for PubMedCentralID PMC1947980

• Cytoplasmic and mitochondrial protein translation in axonal and dendritic terminal arborization NATURE NEUROSCIENCE Chihara, T., Luginbuhl, D., Luo, L. 2007; 10 (7): 828-837

  Abstract

  We identified a mutation in Aats-gly (also known as gars or glycyl-tRNA synthetase), the Drosophila melanogaster ortholog of the human GARS gene that is associated with Charcot-Marie-Tooth neuropathy type 2D (CMT2D), from a mosaic genetic screen. Loss of gars in Drosophila neurons preferentially affects the elaboration and stability of terminal arborization of axons and dendrites. The human and Drosophila genes each encode both a cytoplasmic and a mitochondrial isoform. Using additional mutants that selectively disrupt cytoplasmic or mitochondrial protein translation, we found that cytoplasmic protein translation is required for terminal arborization of both dendrites and axons during development. In contrast, disruption of mitochondrial protein translation preferentially affects the maintenance of dendritic arborization in adults. We also provide evidence that human GARS shows equivalent functions in Drosophila, and that CMT2D causal mutations show loss-of-function properties. Our study highlights different demands of protein translation for the development and maintenance of axons and dendrites.

  View details for DOI 10.1038/nn1910

  View details for Web of Science ID 000247560200012

  View details for PubMedID 17529987

• Comprehensive maps of Drosophila higher offactory centers: Spatially segregated fruit and pheromone representation CELL Jefferis, G. S., Potter, C. J., Chan, A. I., Marin, E. C., Rohlfing, T., Maurer, C. R., Luo, L. 2007; 128 (6): 1187-1203

  Abstract

  In Drosophila, approximately 50 classes of olfactory receptor neurons (ORNs) send axons to 50 corresponding glomeruli in the antennal lobe. Uniglomerular projection neurons (PNs) relay olfactory information to the mushroom body (MB) and lateral horn (LH). Here, we combine single-cell labeling and image registration to create high-resolution, quantitative maps of the MB and LH for 35 input PN channels and several groups of LH neurons. We find (1) PN inputs to the MB are stereotyped as previously shown for the LH; (2) PN partners of ORNs from different sensillar groups are clustered in the LH; (3) fruit odors are represented mostly in the posterior-dorsal LH, whereas candidate pheromone-responsive PNs project to the anterior-ventral LH; (4) dendrites of single LH neurons each overlap with specific subsets of PN axons. Our results suggest that the LH is organized according to biological values of olfactory input.

  View details for DOI 10.1016/j.cell.2007.01.040

  View details for Web of Science ID 000245396200023

  View details for PubMedID 17382886

  View details for PubMedCentralID PMC1885945

• Modeling sporadic loss of heterozygosity in mice by using mosaic analysis with double markers (MADM) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Muzumdar, M. D., Luo, L., Zong, H. 2007; 104 (11): 4495-4500

  Abstract

  The initiation and progression of many human cancers involve either somatic activation of protooncogenes or inactivation of tumor-suppressor genes (TSGs) in sporadic cells. Although sporadic gain-of-function of protooncogenes has been successfully modeled in mice [e.g., Johnson L, Mercer K, Greenbaum D, Bronson RT, Crowley D, Tuveson DA, Jacks T (2001) Nature 410:1111-1116], generating a similar degree of sparseness of TSG loss-of-function remains a challenge. Here, we use mosaic analysis with double markers (MADM) to achieve TSG inactivation and concurrent labeling in sporadic somatic cells of mice, closely mimicking loss of heterozygosity as occurs in human cancers. As proof of principle, we studied the consequence of sporadic loss of p27kip1, a cyclin-dependent kinase inhibitor. MADM-mediated loss of p27kip1 results in mutant cell expansion markedly greater than that observed in conventional p27kip1 knockouts. Moreover, the direct comparison of WT and mutant cells at single-cell resolution afforded by MADM reveals that p27kip1 regulates organ size in vivo by cell-autonomous control of cell cycle exit timing. These studies establish MADM as a high-resolution method for modeling sporadic loss of heterozygosity in mice, providing insights into TSG function.

  View details for DOI 10.1073/pnas.0606491104

  View details for Web of Science ID 000244972700046

  View details for PubMedID 17360552

  View details for PubMedCentralID PMC1810340

• Intrinsic control of precise dendritic targeting by an ensemble of transcription factors CURRENT BIOLOGY Komiyama, T., Luo, L. 2007; 17 (3): 278-285

  Abstract

  Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order , and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. We search for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. We previously identified two POU-domain TFs, acj6 and drifter, as essential players. After testing 13 additional candidates, we identified four TFs (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit.

  View details for Web of Science ID 000244164700029

  View details for PubMedID 17276922

• Graded expression of Semaphorin-1a cell-autonomously directs dendritic targeting of olfactory projection neurons CELL Komiyama, T., Sweeney, L. B., Schuldiner, O., Garcia, K. C., Luo, L. 2007; 128 (2): 399-410

  Abstract

  Gradients of axon guidance molecules instruct the formation of continuous neural maps, such as the retinotopic map in the vertebrate visual system. Here we show that molecular gradients can also instruct the formation of a discrete neural map. In the fly olfactory system, axons of 50 classes of olfactory receptor neurons (ORNs) and dendrites of 50 classes of projection neurons (PNs) form one-to-one connections at discrete units called glomeruli. We provide expression, loss- and gain-of-function data to demonstrate that the levels of transmembrane Semaphorin-1a (Sema-1a), acting cell-autonomously as a receptor or part of a receptor complex, direct the dendritic targeting of PNs along the dorsolateral to ventromedial axis of the antennal lobe. Sema-1a also regulates PN axon targeting in higher olfactory centers. Thus, graded expression of Sema-1a contributes to connection specificity from ORNs to PNs and then to higher brain centers, ensuring proper representation of olfactory information in the brain.

  View details for DOI 10.1016/j.cell.2006.12.028

  View details for PubMedID 17254975

• Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions NEURON Sweeney, L. B., Couto, A., Chou, Y., Berdnik, D., Dickson, B. J., Luo, L., Komiyama, T. 2007; 53 (2): 185-200

  Abstract

  Axon-axon interactions have been implicated in neural circuit assembly, but the underlying mechanisms are poorly understood. Here, we show that in the Drosophila antennal lobe, early-arriving axons of olfactory receptor neurons (ORNs) from the antenna are required for the proper targeting of late-arriving ORN axons from the maxillary palp (MP). Semaphorin-1a is required for targeting of all MP but only half of the antennal ORN classes examined. Sema-1a acts nonautonomously to control ORN axon-axon interactions, in contrast to its cell-autonomous function in olfactory projection neurons. Phenotypic and genetic interaction analyses implicate PlexinA as the Sema-1a receptor in ORN targeting. Sema-1a on antennal ORN axons is required for correct targeting of MP axons within the antennal lobe, while interactions amongst MP axons facilitate their entry into the antennal lobe. We propose that Sema-1a/PlexinA-mediated repulsion provides a mechanism by which early-arriving ORN axons constrain the target choices of late-arriving axons.

  View details for DOI 10.1016/j.neuron.2006.12.022

  View details for Web of Science ID 000245126600006

  View details for PubMedID 17224402

• Wld(S) protection distinguishes axon degeneration following injury from naturally occurring developmental pruning NEURON Hoopfer, E. D., McLaughlin, T., Watts, R. J., Schuldiner, O., O'Leary, D. D., Luo, L. 2006; 50 (6): 883-895

  Abstract

  Axon pruning by degeneration remodels exuberant axonal connections and is widely required for the development of proper circuitry in the nervous system from insects to mammals. Developmental axon degeneration morphologically resembles injury-induced Wallerian degeneration, suggesting similar underlying mechanisms. As previously reported for mice, we show that Wlds protein substantially delays Wallerian degeneration in flies. Surprisingly, Wlds has no effect on naturally occurring developmental axon degeneration in flies or mice, although it protects against injury-induced degeneration of the same axons at the same developmental age. By contrast, the ubiquitin-proteasome system is intrinsically required for both developmental and injury-induced axon degeneration. We also show that the glial cell surface receptor Draper is required for efficient clearance of axon fragments during developmental axon degeneration, similar to its function in injury-induced degeneration. Thus, mechanistically, naturally occurring developmental axon pruning by degeneration and injury-induced axon degeneration differ significantly in early steps, but may converge onto a common execution pathway.

  View details for DOI 10.1016/j.neuron.2006.05.013

  View details for Web of Science ID 000238589500009

  View details for PubMedID 16772170

• Wiring stability of the adult Drosophila olfactory circuit after lesion JOURNAL OF NEUROSCIENCE Berdnik, D., Chihara, T., Couto, A., Luo, L. Q. 2006; 26 (13): 3367-3376

  Abstract

  Neuronal wiring plasticity in response to experience or injury has been reported in many parts of the adult nervous system. For instance, visual or somatosensory cortical maps can reorganize significantly in response to peripheral lesions, yet a certain degree of stability is essential for neuronal circuits to perform their dedicated functions. Previous studies on lesion-induced neuronal reorganization have primarily focused on systems that use continuous neural maps. Here, we assess wiring plasticity in a discrete neural map represented by the adult Drosophila olfactory circuit. Using conditional expression of toxins, we genetically ablated specific classes of neurons and examined the consequences on their synaptic partners or neighboring classes in the adult antennal lobe. We find no alteration of connection specificity between olfactory receptor neurons (ORNs) and their postsynaptic targets, the projection neurons (PNs). Ablating an ORN class maintains PN dendrites within their glomerular borders, and ORN axons normally innervating an adjacent target do not expand. Likewise, ablating PN classes does not alter their partner ORN axon connectivity. Interestingly, an increase in the contralateral ORN axon terminal density occurs in response to the removal of competing ipsilateral ORNs. Therefore, plasticity in this circuit can occur but is confined within a glomerulus, thereby retaining the wiring specificity of ORNs and PNs. We conclude that, although adult olfactory neurons can undergo plastic changes in response to the loss of competition, the olfactory circuit overall is extremely stable in preserving segregated information channels in this discrete map.

  View details for DOI 10.1523/JNEUROSCI.4941-05.2006

  View details for Web of Science ID 000236363400001

  View details for PubMedID 16571743

• Dendritic patterning by Dscam and synaptic partner matching in the Drosophila antennal lobe NATURE NEUROSCIENCE Zhu, H. T., Hummel, T., Clemens, J. C., Berdnik, D., Zipursky, S. L., Luo, L. Q. 2006; 9 (3): 349-355

  Abstract

  In the olfactory system of Drosophila melanogaster, axons of olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons typically target 1 of approximately 50 glomeruli. Dscam, an immunoglobulin superfamily protein, acts in ORNs to regulate axon targeting. Here we show that Dscam acts in projection neurons and local interneurons to control the elaboration of dendritic fields. The removal of Dscam selectively from projection neurons or local interneurons led to clumped dendrites and marked reduction in their dendritic field size. Overexpression of Dscam in projection neurons caused dendrites to be more diffuse during development and shifted their relative position in adulthood. Notably, the positional shift of projection neuron dendrites caused a corresponding shift of its partner ORN axons, thus maintaining the connection specificity. This observation provides evidence for a pre- and postsynaptic matching mechanism independent of precise glomerular positioning.

  View details for Web of Science ID 000235645600014

  View details for PubMedID 16474389

• Development of wiring specificity in the olfactory system CURRENT OPINION IN NEUROBIOLOGY Komiyama, T., Luo, L. Q. 2006; 16 (1): 67-73

  Abstract

  The olfactory system discriminates a large number of odorants using precisely wired neural circuits. It offers an excellent opportunity to study mechanisms of neuronal wiring specificity at the single synapse level. Each olfactory receptor neuron typically expresses only one olfactory receptor from many receptor genes (1000 in mice). In mice, this striking singularity appears to be ensured by a negative feedback mechanism. Olfactory receptor neurons expressing the same receptor converge their axons to stereotypical positions with high precision, a feature that is conserved from insects to mammals. Several molecules have recently been identified that control this process, including olfactory receptors themselves in mice. The second order neurons, mitral cells in mammals and projection neurons in insects, have a similar degree of wiring specificity: studies in Drosophila suggest that projection neuron-intrinsic mechanisms regulate their precise dendritic targeting. Finally, recent studies have revealed interactions of different cell types during circuit assembly, including axon-axon interactions among olfactory receptor neurons and dendro-dendritic interactions of projection neurons, that are essential in establishing wiring specificity of the olfactory circuit.

  View details for DOI 10.1016/j.conb.2005.12.002

  View details for Web of Science ID 000236136200010

  View details for PubMedID 16377177

• A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining NATURE PROTOCOLS Wu, J. S., Luo, L. 2006; 1 (4): 2110-2115

  Abstract

  This protocol describes a basic method for dissection and immunofluorescence staining of the Drosophila brain at various developmental stages. The Drosophila brain has become increasingly useful for studies of neuronal wiring and morphogenesis in combination with techniques such as the 'mosaic analysis with a repressible cell marker' (MARCM) system, where single neurons can be followed in live and fixed tissues for high-resolution analysis of wild-type or genetically manipulated cells. Such high-resolution anatomical study of the brain is also important in characterizing the organization of neural circuits using genetic tools such as GAL4 enhancer trap lines, as Drosophila has been intensively used for studying the neural basis of behavior. Advantages of fluorescence immunostaining include compatibility with multicolor labeling and confocal or multiphoton imaging. This brain dissection and immunofluorescence staining protocol requires approximately 2 to 6 d to complete.

  View details for DOI 10.1038/nprot.2006.336

  View details for Web of Science ID 000251155500055

  View details for PubMedID 17487202

• A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila NATURE PROTOCOLS Wu, J. S., Luo, L. 2006; 1 (6): 2583-2589

  Abstract

  Mosaic analysis with a repressible cell marker (MARCM) is a genetic technique used in Drosophila to label single cells or multiple cells sharing a single progenitor. Labeled homozygous mutant cells can be generated in an otherwise unlabeled heterozygous animal. Mutant or wild-type labeled cells can also be made to express one or more transgenes. Major applications of MARCM include (i) lineage analysis, (ii) investigating gene function in single or small populations of cells and (iii) neuronal circuit tracing. Our laboratory uses MARCM primarily to label and genetically manipulate neurons; however, this protocol can be adapted to any cell of interest. The protocol involves generating two fly stocks with the necessary genetic elements for MARCM analysis and subsequently generating MARCM clones. Labeled clones can be followed in live and fixed tissues for high-resolution analysis of wild-type or genetically manipulated cells.NOTE: In the PDF version of this article initially published online, the first "FRT" and the "Mutation" labels in Figure 1b were transposed. In both the PDF and HTML versions, "mutant" was omitted from the label on the right, which should read "Labeled homozygous mutant daughter cell". The figure has been corrected in all versions of the article.

  View details for DOI 10.1038/nprot.2006.320

  View details for Web of Science ID 000251155700008

  View details for PubMedID 17406512

• Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit CURRENT BIOLOGY Ramaekers, A., Magnenat, E., Marin, E. C., Gendre, N., Jefferis, G. S., Luo, L. Q., Stocker, R. F. 2005; 15 (11): 982-992

  Abstract

  Drosophila larvae possess only 21 odorant-receptor neurons (ORNs), whereas adults have 1,300. Does this suggest that the larval olfactory system is built according to a different design than its adult counterpart, or is it just a miniature version thereof?By genetically labeling single neurons with FLP-out and MARCM techniques, we analyze the connectivity of the larval olfactory circuit. We show that each of the 21 ORNs is unique and projects to one of 21 morphologically identifiable antennal-lobe glomeruli. Each glomerulus seems to be innervated by a single projection neuron. Each projection neuron sends its axon to one or two of about 28 glomeruli in the mushroom-body calyx. We have discovered at least seven types of projection neurons that stereotypically link an identified antennal-lobe glomerulus with an identified calycal glomerulus and thus create an olfactory map in a higher brain center.The basic design of the larval olfactory system is similar to the adult one. However, ORNs and projection neurons lack cellular redundancy and do not exhibit any convergent or divergent connectivity; 21 ORNs confront essentially similar numbers of antennal-lobe glomeruli, projection neurons, and calycal glomeruli. Hence, we propose the Drosophila larva as an "elementary" olfactory model system.

  View details for DOI 10.1016/j.cub.2005.04.032

  View details for Web of Science ID 000229984100019

  View details for PubMedID 15936268

• Mosaic analysis with double markers in mice CELL Zong, H., Espinosa, S., Su, H. H., Muzumdar, M. D., Luo, L. Q. 2005; 121 (3): 479-492

  Abstract

  We describe a method termed MADM (mosaic analysis with double markers) in mice that allows simultaneous labeling and gene knockout in clones of somatic cells or isolated single cells in vivo. Two reciprocally chimeric genes, each containing the N terminus of one marker and the C terminus of the other marker interrupted by a loxP-containing intron, are knocked in at identical locations on homologous chromosomes. Functional expression of markers requires Cre-mediated interchromosomal recombination. MADM reveals that interchromosomal recombination can be induced efficiently in vivo in both mitotic and postmitotic cells in all tissues examined. It can be used to create conditional knockouts in small populations of labeled cells, to determine cell lineage, and to trace neuronal connections. To illustrate the utility of MADM, we show that cerebellar granule cell progenitors are fated at an early stage to produce granule cells with axonal projections limited to specific sublayers of the cerebellar cortex.

  View details for DOI 10.1016/j.cell.2005.02.012

  View details for Web of Science ID 000229031600016

  View details for PubMedID 15882628

• Function and regulation of Tumbleweed (RacGAP50C) in neuroblast proliferation and neuronal morphogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Goldstein, A. Y., Jan, Y. N., Luo, L. Q. 2005; 102 (10): 3834-3839

  Abstract

  Drosophila RacGAP50C and its homologues act as part of a complex with a kinesin-like protein (Pavarotti/Zen-4) that is essential for the formation of the central spindle and completion of cytokinesis [Mishima, M., Kaitna, S. & Glotzer, M. (2002) Dev. Cell 2, 41-54; Somers, W. G. & Saint, R. (2003) Dev. Cell 4, 29-39; Jantsch-Plunger et al. (2000) J. Cell Biol. 149, 1391-1404]. We report here that RacGAP50C corresponds to the tumbleweed (tum) gene previously identified based on its defects in dendrite development of sensory neurons [Gao, F. B., Brenman, J. E., Jan, L. Y. & Jan, Y. N. (1999) Genes Dev. 13, 2549-2561]. Using mushroom body neurogenesis and morphogenesis as a model, we show that Tumbleweed (Tum), Pavarotti, and their association are required for neuroblast proliferation. Tum with a mutation predicted to disrupt the GTPase-activating protein (GAP) activity still largely retains its activity in regulating cell division but is impaired in its activity to limit axon growth. We also provide evidence that Tum and Pavarotti regulate the subcellular localization of each other in postmitotic neurons and that cytoplasmic accumulation of both proteins disrupts axon development in a GAP-dependent manner. Taken together with previous studies of RacGAP50C in regulating cytokinesis, we propose that Tum serves as a scaffolding protein in regulating cell division but acts as a GAP to limit axon growth in postmitotic neurons.

  View details for DOI 10.1073/pnas.0500748102

  View details for Web of Science ID 000227533100057

  View details for PubMedID 15738386

  View details for PubMedCentralID PMC553341

• Developmentally programmed remodeling of the Drosophila olfactory circuit DEVELOPMENT Marin, E. C., Watts, R. J., Tanaka, N. K., Ito, K., Luo, L. Q. 2005; 132 (4): 725-737

  Abstract

  Neural circuits are often remodeled after initial connections are established. The mechanisms by which remodeling occurs, in particular whether and how synaptically connected neurons coordinate their reorganization, are poorly understood. In Drosophila, olfactory projection neurons (PNs) receive input by synapsing with olfactory receptor neurons in the antennal lobe and relay information to the mushroom body (MB) calyx and lateral horn. Here we show that embryonic-born PNs participate in both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the MB calyx. They persist in the adult olfactory circuit and are prespecified by birth order to innervate a subset of glomeruli distinct from larval-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon terminal branches locally during early metamorphosis. Electron microscopy analysis reveals that these PNs synapse with MB gamma neurons in the larval calyx and that these synaptic profiles are engulfed by glia during early metamorphosis. As with MB gamma neurons, PN pruning requires cell-autonomous reception of the nuclear hormone ecdysone. Thus, these synaptic partners are independently programmed to prune their dendrites and axons.

  View details for DOI 10.1242/dev.01614

  View details for Web of Science ID 000227427100010

  View details for PubMedID 15659487

• Development of wiring specificity of the Drosophila olfactory system Joint Meeting of the 14th International Symposium on Olfaction and Taste/38th Annual Meeting of the Japanese-Association-for-the-Study-of-Taste-and-Smell Jefferis, G. S., Marin, E. C., Komiyama, T., Zhu, H. T., Chihara, T., Berdnik, D., Luo, L. Q. OXFORD UNIV PRESS. 2005: I94–I94

  View details for Web of Science ID 000227615800047

  View details for PubMedID 15738213

• Axon retraction and degeneration in development and disease ANNUAL REVIEW OF NEUROSCIENCE Luo, L. Q., O'Leary, D. D. 2005; 28: 127-156

  Abstract

  The selective elimination of axons, dendrites, axon and dendrite branches, and synapses, without loss of the parent neurons, occurs during normal development of the nervous system as well as in response to injury or disease in the adult. The widespread developmental phenomena of exuberant axonal projections and synaptic connections require both small-scale and large-scale axon pruning to generate precise adult connectivity, and they provide a mechanism for neural plasticity in the developing and adult nervous system, as well as a mechanism to evolve differences between species in a projection system. Such pruning is also required to remove axonal connections damaged in the adult, to stabilize the affected neural circuits, and to initiate their repair. Pruning occurs through either retraction or degeneration. Here we review examples of these phenomena and consider potential cellular and molecular mechanisms that underlie axon retraction and degeneration and how they might relate to each other in development and disease.

  View details for DOI 10.1146/annurev.neuro.28.061604.135632

  View details for Web of Science ID 000231235700006

  View details for PubMedID 16022592

• Rho GTPases regulate axon growth through convergent and divergent signaling pathways NEURON Ng, J. L., Luo, L. Q. 2004; 44 (5): 779-793

  Abstract

  Rho GTPases are essential regulators of cytoskeletal reorganization, but how they do so during neuronal morphogenesis in vivo is poorly understood. Here we show that the actin depolymerization factor cofilin is essential for axon growth in Drosophila neurons. Cofilin function in axon growth is inhibited by LIM kinase and activated by Slingshot phosphatase. Dephosphorylating cofilin appears to be the major function of Slingshot in regulating axon growth in vivo. Genetic data provide evidence that Rho or Rac/Cdc42, via effector kinases Rok or Pak, respectively, activate LIM kinase to inhibit axon growth. Importantly, Rac also activates a Pak-independent pathway that promotes axon growth, and different RacGEFs regulate these distinct pathways. These genetic analyses reveal convergent and divergent pathways from Rho GTPases to the cytoskeleton during axon growth in vivo and suggest that different developmental outcomes could be achieved by biases in pathway selection.

  View details for Web of Science ID 000225548400007

  View details for PubMedID 15572110

• Like poles repel: Molecular mechanisms of dendritic tiling CELL Chihara, T., Luo, L. Q. 2004; 119 (2): 148-149

  Abstract

  To cover the entire sensory field once and only once, dendrites of some sensory system neurons avoid crossing other dendrites from the same type of neurons. In this issue of Cell, provide first insight into the molecular mechanisms of dendritic tiling.

  View details for Web of Science ID 000224577200002

  View details for PubMedID 15479631

• Olfactory receptor neuron axon targeting: intrinsic transcriptional control and hierarchical interactions NATURE NEUROSCIENCE Komiyama, T., Carlson, J. R., Luo, L. Q. 2004; 7 (8): 819-825

  Abstract

  From insects to mammals, olfactory receptor neurons (ORNs) expressing a common olfactory receptor target their axons to specific glomeruli with high precision. Here we show in Drosophila that the POU transcription factor Acj6 controls the axon targeting specificity of a subset of ORN classes, as defined by the olfactory receptors that they express. Of these classes, some require Acj6 cell-autonomously, whereas others require Acj6 cell-nonautonomously. Mosaic analyses show that cooperative targeting occurs between axon terminals of the same ORN classes and that there are hierarchical interactions among different ORN classes. We propose that the precision of ORN axon targeting derives from both intrinsic transcriptional control and extensive axon-axon interactions.

  View details for DOI 10.1038/nn1284

  View details for Web of Science ID 000222930800011

  View details for PubMedID 15247920

• Glia engulf degenerating axons during developmental axon pruning CURRENT BIOLOGY Watts, R. J., Schuldiner, O., Perrino, J., Larsen, C., Luo, L. Q. 2004; 14 (8): 678-684

  Abstract

  Developmental axon pruning is widely used in constructing the nervous system. Accordingly, diverse mechanisms are likely employed for various forms of axon pruning. In the Drosophila mushroom bodies (MB), gamma neurons initially extend axon branches into both the dorsal and medial MB axon lobes in larvae. Through a well-orchestrated set of developmental events during metamorphosis, axon branches to both lobes degenerate prior to the formation of adult connections. Here, we analyze ultrastructural changes underlying axon pruning by using a genetically encoded electron microscopic (EM) marker to selectively label gamma neurons. By inhibiting axon pruning in combination with the use of this EM marker, we demonstrate a causal link between observed cellular events and axon pruning. These events include changes in axon ultrastructure, synaptic degeneration, and engulfment of degenerating axon fragments by glia for their subsequent breakdown via the endosomal-lysosomal pathway. Interestingly, glia selectively invade MB axon lobes at the onset of metamorphosis; this increase in cell number is independent of axon fragmentation. Our study reveals a key role for glia in the removal of axon fragments during developmental axon pruning.

  View details for DOI 10.1016/j.cub.2004.03.035

  View details for Web of Science ID 000220989700019

  View details for PubMedID 15084282

• Diverse functions of N-cadherin in dendritic and axonal terminal arborization of olfactory projection neurons NEURON Zhu, H. T., Luo, L. Q. 2004; 42 (1): 63-75

  Abstract

  The cadherin superfamily of cell adhesion molecules have been proposed to play important roles in determining synaptic specificity in developing nervous systems. We examine the function of N-cadherin in Drosophila second order olfactory projection neurons (PNs), each of which must selectively target their dendrites to one of approximately 50 glomeruli. Our results do not support an instructive role for N-cadherin in selecting dendritic targets; rather, N-cadherin is essential for PNs to restrict their dendrites to single glomeruli. Mosaic analyses suggest that N-cadherin mediates dendro-dendritic interactions between PNs and thus contributes to refinement of PN dendrites to single glomeruli. N-cadherin is also essential for the development of PN axon terminal arbors in two distinct central targets: regulating branch stability in the lateral horn and restricting high-order branching in the mushroom body. Although the N-cadherin locus potentially encodes eight alternatively spliced isoforms, transgenic expression of one isoform is sufficient to rescue all phenotypes.

  View details for Web of Science ID 000221458400008

  View details for PubMedID 15066265

• Neuroscience. Calcium and CREST for healthy dendrites. Science Jefferis, G. S., Komiyama, T., Luo, L. 2004; 303 (5655): 179-181

  View details for PubMedID 14715999

• Developmental origin of wiring specificity in the olfactory system of Drosophila DEVELOPMENT Jefferis, G. S., Vyas, R. M., Berdnik, D., Ramaekers, A., Stocker, R. F., Tanaka, N. K., Ito, K., Luo, L. Q. 2004; 131 (1): 117-130

  Abstract

  In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.

  View details for DOI 10.1242/dev.00896

  View details for Web of Science ID 000188553900012

  View details for PubMedID 14645123

• Cellular origins of wiring specificity in the olfactory system of Drosophila. Western Regional Meeting of the American-Federation-for-Medical-Research Vyas, R. M., Jefferis, G., Berdnik, D., Ito, K., Luo, L. LIPPINCOTT WILLIAMS & WILKINS. 2004: S154–S154

  View details for Web of Science ID 000188254600447

• Dendritic development of Drosophila high order visual system neurons is independent of sensory experience BMC NEUROSCIENCE Scott, E. K., Reuter, J. E., Luo, L. Q. 2003; 4

  Abstract

  The complex and characteristic structures of dendrites are a crucial part of the neuronal architecture that underlies brain function, and as such, their development has been a focal point of recent research. It is generally believed that dendritic development is controlled by a combination of endogenous genetic mechanisms and activity-dependent mechanisms. Therefore, it is of interest to test the relative contributions of these two types of mechanisms towards the construction of specific dendritic trees. In this study, we make use of the highly complex Vertical System (VS) of motion sensing neurons in the lobula plate of the Drosophila visual system to gauge the importance of visual input and synaptic activity to dendritic development.We find that the dendrites of VS1 neurons are unchanged in dark-reared flies as compared to control flies raised on a 12 hour light, 12 hour dark cycle. The dendrites of these flies show no differences from control in dendrite complexity, spine number, spine density, or axon complexity. Flies with genetically ablated eyes show a slight but significant reduction in the complexity and overall length of VS1 dendrites, although this effect may be due to a reduction in the overall size of the dendritic field in these flies.Overall, our results indicate no role for visual experience in the development of VS dendrites, while spontaneous activity from photoreceptors may play at most a subtle role in the formation of fully complex dendrites in these high-order visual processing neurons.

  View details for Web of Science ID 000185762300001

  View details for PubMedID 12834538

• Axon pruning during Drosphila metamorphosis: Evidence for local degeneration and requirement of the ubiquitin-proteasome system NEURON Watts, R. J., Hoopfer, E. D., Luo, L. Q. 2003; 38 (6): 871-885

  Abstract

  Axon pruning is widely used for the refinement of neural circuits in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neurodegenerative diseases. However, little is known about the cellular and molecular mechanisms of axon pruning. We use the stereotyped pruning of gamma neurons of the Drosophila mushroom bodies (MB) during metamorphosis to investigate these mechanisms. Detailed time course analyses indicate that MB axon pruning is mediated by local degeneration rather than retraction and that the disruption of the microtubule cytoskeleton precedes axon pruning. In addition, multiple lines of genetic evidence demonstrate an intrinsic role of the ubiquitin-proteasome system in axon pruning; for example, loss-of-function mutations of the ubiquitin activating enzyme (E1) or proteasome subunits in MB neurons block axon pruning. Our findings suggest that some forms of axon pruning during development may share similarities with degeneration of axons in response to injury.

  View details for Web of Science ID 000183691400008

  View details for PubMedID 12818174

• Small GTPase Cdc42 is required for multiple aspects of dendritic morphogenesis JOURNAL OF NEUROSCIENCE Scott, E. K., Reuter, J. E., Luo, L. Q. 2003; 23 (8): 3118-3123

  Abstract

  The study of dendritic development in CNS neurons has been hampered by a lack of complex dendritic structures that can be studied in a tractable genetic system. In an effort to develop such a system, we recently characterized the highly complex dendrites of the vertical system (VS) neurons in the Drosophila visual system. Using VS neurons as a model system, we show here using loss-of-function mutations that endogenous Cdc42, a member of Rho family of small GTPases, is required for multiple aspects of dendritic morphogenesis. Cdc42-mutant VS neurons display normal complexity but increased dendritic length compared with wild type and have defects in dendrite caliber and stereotyped dendritic branch positions. Remarkably, Cdc42 mutant neurons also show a 50% reduction in dendritic spine density. These results demonstrate that Cdc42 is a regulator for multiple aspects of dendritic development.

  View details for Web of Science ID 000182475200005

  View details for PubMedID 12716918

• A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis DEVELOPMENT Reuter, J. E., Nardine, T. M., Penton, A., Billuart, P., Scott, E. K., Usui, T., Uemura, T., Luo, L. Q. 2003; 130 (6): 1203-1213

  Abstract

  Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, we performed a genetic screen using the MARCM system in the mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains approximately 20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. We report mutations that affect these different aspects of morphogenesis and phenotypically characterize a subset. We found that roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. Our results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process.

  View details for DOI 10.1242/dev.00319

  View details for Web of Science ID 000181751300016

  View details for PubMedID 12571111

• From lineage to wiring specificity: POU domain transcription factors control precise connections of Drosophila olfactory projection neurons CELL Komiyama, T., JOHNSON, W. A., Luo, L. Q., Jefferis, G. S. 2003; 112 (2): 157-167

  Abstract

  Axonal selection of synaptic partners is generally believed to determine wiring specificity in the nervous system. However, we have recently found evidence for specific dendritic targeting in the olfactory system of Drosophila: second order olfactory neurons (Projection Neurons) from the anterodorsal (adPN) and lateral (lPN) lineages send their dendrites to stereotypical, intercalating but non-overlapping glomeruli. Here we show that POU domain transcription factors, Acj6 and Drifter, are expressed in adPNs and lPNs respectively, and are required for their dendritic targeting. Moreover, misexpression of Acj6 in lPNs, or Drifter in adPNs, results in dendritic targeting to glomeruli normally reserved for the other PN lineage. Thus, Acj6 and Drifter translate PN lineage information into distinct dendritic targeting specificity. Acj6 also controls stereotypical axon terminal arborization of PNs in a central target, suggesting that the connectivity of PN axons and dendrites in different brain centers is coordinately regulated.

  View details for Web of Science ID 000181191600006

  View details for PubMedID 12553905

• Structure of the vertical and horizontal system neurons of the lobula plate in Drosophila JOURNAL OF COMPARATIVE NEUROLOGY Scott, E. K., Raabe, T., Luo, L. Q. 2002; 454 (4): 470-481

  Abstract

  The lobula plate in the optic lobe of the fly brain is a high-order processing center for visual information. Within the lobula plate lie a small number of giant neurons that are responsible for the detection of wide field visual motion. Although the structure and motion sensitivity of these cells have been extensively described in large flies, the system has not been described systematically in Drosophila. Here, we use the mosaic analysis with a repressible cell marker (MARCM) system to analyze a subset of these cells, the horizontal and vertical systems. Our results suggest that the Drosophila horizontal system is similar to those described in larger flies, with three neurons fanning their dendrites over the lobula plate. We found that there are six neurons in the Drosophila vertical system, a figure that compares with 9-11 neurons in large flies. Even so, the Drosophila vertical system closely resembles the systems of larger flies, with each neuron in Drosophila having an approximate counterpart in large flies. This anatomical similarity implies that the inputs to the vertical system are similarly organized in these various fly species, and that it is likely that the Drosophila neurons respond to motions similar to those sensed by their specific structural counterparts in large flies. Additionally, the similar appearance of vertical system cells in multiple cell clones demonstrates that they share a common developmental lineage. Access to these cells in Drosophila should allow for the use of genetic tools in future studies of horizontal and vertical system function.

  View details for DOI 10.1002/cne.10467

  View details for Web of Science ID 000179558900008

  View details for PubMedID 12455010

• Representation of the glomerular olfactory map in the Drosophila brain CELL Marin, E. C., Jefferis, G. S., Komiyama, T., Zhu, H. T., Luo, L. Q. 2002; 109 (2): 243-255

  Abstract

  We explored how the odor map in the Drosophila antennal lobe is represented in higher olfactory centers, the mushroom body and lateral horn. Systematic single-cell tracing of projection neurons (PNs) that send dendrites to specific glomeruli in the antennal lobe revealed their stereotypical axon branching patterns and terminal fields in the lateral horn. PNs with similar axon terminal fields tend to receive input from neighboring glomeruli. The glomerular classes of individual PNs could be accurately predicted based solely on their axon projection patterns. The sum of these patterns defines an "axon map" in higher olfactory centers reflecting which olfactory receptors provide input. This map is characterized by spatial convergence and divergence of PN axons, allowing integration of olfactory information.

  View details for Web of Science ID 000175082600013

  View details for PubMedID 12007410

• Rac GTPases control axon growth, guidance and branching NATURE Ng, J., Nardine, T., Harms, M., Tzu, J., Goldstein, A., Sun, Y., Dietzl, G., Dickson, B. J., Luo, L. Q. 2002; 416 (6879): 442-447

  Abstract

  Growth, guidance and branching of axons are all essential processes for the precise wiring of the nervous system. Rho family GTPases transduce extracellular signals to regulate the actin cytoskeleton. In particular, Rac has been implicated in axon growth and guidance. Here we analyse the loss-of-function phenotypes of three Rac GTPases in Drosophila mushroom body neurons. We show that progressive loss of combined Rac1, Rac2 and Mtl activity leads first to defects in axon branching, then guidance, and finally growth. Expression of a Rac1 effector domain mutant that does not bind Pak rescues growth, partially rescues guidance, but does not rescue branching defects of Rac mutant neurons. Mosaic analysis reveals both cell autonomous and non-autonomous functions for Rac GTPases, the latter manifesting itself as a strong community effect in axon guidance and branching. These results demonstrate the central role of Rac GTPases in multiple aspects of axon development in vivo, and suggest that axon growth, guidance and branching could be controlled by differential activation of Rac signalling pathways.

  View details for PubMedID 11919635

• Rac function and regulation during Drosophila development NATURE Hakeda-Suzuki, S., Ng, J., Tzu, J., Dietzl, G., Sun, Y., Harms, M., Nardine, T., Luo, L. Q., Dickson, B. J. 2002; 416 (6879): 438-442

  Abstract

  Rac GTPases regulate the actin cytoskeleton to control changes in cell shape. To date, the analysis of Rac function during development has relied heavily on the use of dominant mutant isoforms. Here, we use loss-of-function mutations to show that the three Drosophila Rac genes, Rac1, Rac2 and Mtl, have overlapping functions in the control of epithelial morphogenesis, myoblast fusion, and axon growth and guidance. They are not required for the establishment of planar cell polarity, as had been suggested on the basis of studies using dominant mutant isoforms. The guanine nucleotide exchange factor, Trio, is essential for Rac function in axon growth and guidance, but not for epithelial morphogenesis or myoblast fusion. Different Rac activators thus act in different developmental processes. The specific cellular response to Rac activation may be determined more by the upstream activator than the specific Rac protein involved.

  View details for Web of Science ID 000174607800049

  View details for PubMedID 11919634

• Development of neuronal connectivity in Drosophila antennal lobes and mushroom bodies CURRENT OPINION IN NEUROBIOLOGY Jefferis, G. S., Marin, E. C., Watts, R. J., Luo, L. Q. 2002; 12 (1): 80-86

  Abstract

  Recent advances in the study of the connectivity of Drosophila olfactory system include the demonstration that olfactory receptor neurons project to specific glomeruli according to the receptor type they express, and that their projection neuron partners are prespecified to innervate particular glomeruli by birth order or time. This same theme of sequential generation has been observed in the generation of the three major types of mushroom body neurons.

  View details for Web of Science ID 000173813000010

  View details for PubMedID 11861168

• Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Luo, L. Q. 2002; 18: 601-635

  Abstract

  The actin cytoskeleton plays a major role in morphological development of neurons and in structural changes of adult neurons. This article reviews the myriad functions of actin and myosin in axon initiation, growth, guidance and branching, in morphogenesis of dendrites and dendritic spines, in synapse formation and stability, and in axon and dendrite retraction. Evidence is presented that signaling pathways involving the Rho family of small GTPases are key regulators of actin polymerization and myosin function in the context of different aspects of neuronal morphogenesis. These studies support an emerging theme: Different aspects of neuronal morphogenesis may involve regulation of common core signaling pathways, in particular the Rho GTPases.

  View details for DOI 10.1146/annurev.cellbio.18.031802.150501

  View details for Web of Science ID 000179413400022

  View details for PubMedID 12142283

• Target neuron prespecification in the olfactory map of Drosophila NATURE Jefferis, G. S., Marin, E. C., Stocker, R. F., Luo, L. Q. 2001; 414 (6860): 204-208

  Abstract

  In Drosophila and mice, olfactory receptor neurons (ORNs) expressing the same receptors have convergent axonal projections to specific glomerular targets in the antennal lobe/olfactory bulb, creating an odour map in this first olfactory structure of the central nervous system. Projection neurons of the Drosophila antennal lobe send dendrites into glomeruli and axons to higher brain centres, thereby transferring this odour map further into the brain. Here we use the MARCM method to perform a systematic clonal analysis of projection neurons, allowing us to correlate lineage and birth time of projection neurons with their glomerular choice. We demonstrate that projection neurons are prespecified by lineage and birth order to form synapses with specific incoming ORN axons, and therefore to carry specific olfactory information. This prespecification could be used to hardwire the fly's olfactory system, enabling stereotyped behavioural responses to odorants. Developmental studies lead us to hypothesize that recognition molecules ensure reciprocally specific connections of ORNs and projection neurons. These studies also imply a previously unanticipated role for precise dendritic targeting by postsynaptic neurons in determining connection specificity.

  View details for Web of Science ID 000172029100047

  View details for PubMedID 11719930

• Single neuron labeling and genetic manipulation NATURE NEUROSCIENCE Luo, L. Q., Zong, H. 2001; 4: 1158-1159

  View details for Web of Science ID 000172041500005

  View details for PubMedID 11687823

• Regulating axon branch stability: The role of p190 RhoGAP in repressing a retraction signaling pathway CELL Billuart, P., Winter, C. G., Maresh, A., Zhao, X. S., Luo, L. Q. 2001; 107 (2): 195-207

  Abstract

  Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. p190 inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by p190. Local regulation of p190 could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of p190 by integrin and Src, both implicated in neural plasticity.

  View details for Web of Science ID 000171694800009

  View details for PubMedID 11672527

• Drosophila Rho-associated kinase (Drok) links frizzled-mediated planar cell polarity signaling to the actin cytoskeleton CELL Winter, C. G., Wang, B., Ballew, A., Royou, A., Karess, R., Axelrod, J. D., Luo, L. Q. 2001; 105 (1): 81-91

  Abstract

  Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.

  View details for Web of Science ID 000168063300009

  View details for PubMedID 11301004

• How do dendrites take their shape? NATURE NEUROSCIENCE Scott, E. K., Luo, L. Q. 2001; 4 (4): 359-365

  Abstract

  Recent technical advances have made possible the visualization and genetic manipulation of individual dendritic trees. These studies have led to the identification and characterization of molecules that are important for different aspects of dendritic development. Although much remains to be learned, the existing knowledge has allowed us to take initial steps toward a comprehensive understanding of how complex dendritic trees are built. In this review, we describe recent advances in our understanding of the molecular mechanisms underlying dendritic morphogenesis, and discuss their cell-biological implications.

  View details for Web of Science ID 000168762200015

  View details for PubMedID 11276225

• enok encodes a Drosophila putative histone acetyltransferase required for mushroom body neuroblast proliferation CURRENT BIOLOGY Scott, E. K., Lee, T., Luo, L. Q. 2001; 11 (2): 99-104

  Abstract

  Mushroom bodies in the Drosophila brain are centers for olfactory learning and memory. We have previously shown that the mushroom bodies comprise three types of neurons with distinct axonal projections. These three types of neurons are generated sequentially from common neuroblasts. We report here the identification of a gene that we have named enoki mushroom (enok), which when it is mutated gives rise to mushroom bodies with reduced axonal structures. enok encodes a putative histone acetyltransferase (HAT) of the MYST family, members of which have been implicated as important modulators of transcriptional activity. A single amino acid change in the zinc finger motif of the putative catalytic HAT domain gives the same phenotype as a null allele, and this finding indicates the importance of HAT activity to Enok's function. Further phenotypic analysis demonstrates that the mushroom body defect is due to an arrest in neuroblast proliferation rather than a failure of either cell fate switching or axon branching. Clonal analyses in the wing discs and the ovaries suggest that enok is essential for normal cell proliferation in some, but not all, tissues. Our results provide in vivo evidence for essential functions of a histone acetyltransferase in the construction of the Drosophila brain.

  View details for Web of Science ID 000169076200018

  View details for PubMedID 11231125

• Rho GTPases in neuronal morphogenesis NATURE REVIEWS NEUROSCIENCE Luo, L. Q. 2000; 1 (3): 173-180

  Abstract

  The Rho family of small GTPases act as intracellular molecular switches that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. Recent evidence implicates Rho GTPases in the regulation of neuronal morphogenesis, including migration, polarity, axon growth and guidance, dendrite elaboration and plasticity, and synapse formation. Signalling pathways from membrane receptors to Rho GTPases and from Rho GTPases to the actin cytoskeleton are beginning to be discovered. Mutations in these signalling pathways have been reported in human neurological diseases, which underscores their importance in the development and function of the nervous system.

  View details for Web of Science ID 000165764700015

  View details for PubMedID 11257905

• Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila NEURON Lee, T., Marticke, S., Sung, C., Robinow, S., Luo, L. Q. 2000; 28 (3): 807-818

  Abstract

  Neuronal process remodeling occurs widely in the construction of both invertebrate and vertebrate nervous systems. During Drosophila metamorphosis, gamma neurons of the mushroom bodies (MBs), the center for olfactory learning in insects, undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes. To elucidate the underlying molecular mechanisms, we conducted a genetic mosaic screen and identified one ultraspiracle (usp) allele defective in larval process pruning. Consistent with the notion that USP forms a heterodimer with the ecdysone receptor (EcR), we found that the EcR-B1 isoform is specifically expressed in the MB gamma neurons, and is required for the pruning of larval processes. Surprisingly, most identified primary EcR/USP targets are dispensable for MB neuronal remodeling. Our study demonstrates cell-autonomous roles for EcR/USP in controlling neuronal remodeling, potentially through novel downstream targets.

  View details for Web of Science ID 000166057500018

  View details for PubMedID 11163268

• Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration and axonal transport NATURE CELL BIOLOGY Liu, Z., Steward, R., Lu, L. Q. 2000; 2 (11): 776-783

  Abstract

  Haplo-insufficiency of human Lis1 causes lissencephaly. Reduced Lis1 activity in both humans and mice results in a neuronal migration defect. Here we show that Drosophila Lis1 is highly expressed in the nervous system. Lis1 is essential for neuroblast proliferation and axonal transport, as shown by a mosaic analysis using a Lis1 null mutation. Moreover, it is cell-autonomously required for dendritic growth, branching and maturation. Analogous mosaic analysis shows that neurons containing a mutated cytoplasmic-dynein heavy chain (Dhc64C) exhibit phenotypes similar to Lis1 mutants. These results implicate Lis1 as a regulator of the microtubule cytoskeleton and show that it is important for diverse physiological functions in the nervous system.

  View details for Web of Science ID 000165207500010

  View details for PubMedID 11056531

• Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons JOURNAL OF NEUROSCIENCE Nakayama, A. Y., Harms, M. B., Luo, L. Q. 2000; 20 (14): 5329-5338

  Abstract

  The shape of dendritic trees and the density of dendritic spines can undergo significant changes during the life of a neuron. We report here the function of the small GTPases Rac and Rho in the maintenance of dendritic structures. Maturing pyramidal neurons in rat hippocampal slice culture were biolistically transfected with dominant GTPase mutants. We found that expression of dominant-negative Rac1 results in a progressive elimination of dendritic spines, whereas hyperactivation of RhoA causes a drastic simplification of dendritic branch patterns that is dependent on the activity of a downstream kinase ROCK. Our results suggest that Rac and Rho play distinct functions in regulating dendritic spines and branches and are vital for the maintenance and reorganization of dendritic structures in maturing neurons.

  View details for Web of Science ID 000087995300019

  View details for PubMedID 10884317

• Trio quartet in D. (melanogaster) NEURON Luo, L. Q. 2000; 26 (1): 1-2

  View details for Web of Science ID 000086770500001

  View details for PubMedID 10798384

• Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis NEURON Lee, T. M., Winter, C., Marticke, S. S., Lee, A., Luo, L. Q. 2000; 25 (2): 307-316

  Abstract

  The pleiotropic functions of small GTPase Rho present a challenge to its genetic analysis in multicellular organisms. We report here the use of the MARCM (mosaic analysis with a repressible cell marker) system to analyze the function of RhoA in the developing Drosophila brain. Clones of cells homozygous for null RhoA mutations were specifically labeled in the mushroom body (MB) neurons of mosaic brains. We found that RhoA is required for neuroblast (Nb) proliferation but not for neuronal survival. Surprisingly, RhoA is not required for MB neurons to establish normal axon projections. However, neurons lacking RhoA overextend their dendrites, and expression of activated RhoA causes a reduction of dendritic complexity. Thus, RhoA is an important regulator of dendritic morphogenesis, while distinct mechanisms are used for axonal morphogenesis.

  View details for Web of Science ID 000085549100010

  View details for PubMedID 10719887

• Intracellular signaling pathways that regulate dendritic spine morphogenesis HIPPOCAMPUS Nakayama, A. Y., Luo, L. Q. 2000; 10 (5): 582-586

  Abstract

  Rac is a member of the Rho family of small GTPases and acts as a molecular switch. When GTP-bound, Rac binds specific effectors to induce downstream signaling events, including actin cytoskeletal rearrangements (Hall, Science 1998;279:509-514). Herein we review the recent evidence suggesting that Rac is involved in the morphogenesis of dendritic spines (Luo et al., Nature 1996;379:837-840; Nakayama et al., J Neurosci 2000; 20:5329-5338). In addition, we discuss how Rac activity is regulated by guanine nucleotide exchange factors, which may be further regulated by extracellular factors. Thus, the Rac signal transduction pathway may provide links between extracellular ligands or synaptic activity and the regulation of the actin cytoskeleton in spine morphogenesis.

  View details for Web of Science ID 000165297700008

  View details for PubMedID 11075828

• Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast DEVELOPMENT Lee, T., Lee, A., Luo, L. Q. 1999; 126 (18): 4065-4076

  Abstract

  The mushroom bodies (MBs) are prominent structures in the Drosophila brain that are essential for olfactory learning and memory. Characterization of the development and projection patterns of individual MB neurons will be important for elucidating their functions. Using mosaic analysis with a repressible cell marker (Lee, T. and Luo, L. (1999) Neuron 22, 451-461), we have positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the (gamma) lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the alpha' and beta' lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the alpha and beta lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born gamma neurons retain only their main processes in the peduncle, which then project into the adult gamma lobe without bifurcation. In contrast, the basic axon projections of the later-born (alpha'/beta') larval neurons are preserved during metamorphosis. This study illustrates the cellular organization of mushroom bodies and the development of different MB neurons at the single cell level. It allows for future studies on the molecular mechanisms of mushroom body development.

  View details for Web of Science ID 000082965700009

  View details for PubMedID 10457015

• Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis NEURON Lee, T., Luo, L. Q. 1999; 22 (3): 451-461

  Abstract

  We describe a genetic mosaic system in Drosophila, in which a dominant repressor of a cell marker is placed in trans to a mutant gene of interest. Mitotic recombination events between homologous chromosomes generate homozygous mutant cells, which are exclusively labeled due to loss of the repressor. Using this system, we are able to visualize axonal projections and dendritic elaboration in large neuroblast clones and single neuron clones with a membrane-targeted GFP marker. This new method allows for the study of gene functions in neuroblast proliferation, axon guidance, and dendritic elaboration in the complex central nervous system. As an example, we show that the short stop gene is required in mushroom body neurons for the extension and guidance of their axons.

  View details for Web of Science ID 000079483900010

  View details for PubMedID 10197526

• Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Luo, L. Q., Lee, T., Tsai, L., Tang, G., Jan, L. Y., Jan, Y. N. 1997; 94 (24): 12963-12968

  Abstract

  The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.

  View details for Web of Science ID A1997YJ45600050

  View details for PubMedID 9371783

  View details for PubMedCentralID PMC24246