Liqun Luo
Ann and Bill Swindells Professor and Professor, by courtesy, of Neurobiology
Biology
Bio
Dr. Luo grew up in Shanghai, China, and earned his bachelor's degree in molecular biology from the University of Science and Technology of China. After obtaining his PhD in Brandeis University, and postdoctoral training at the University of California, San Francisco, Dr. Luo started his own lab in the Department of Biology, Stanford University in December 1996. Together with his postdoctoral fellows and graduate students, Dr. Luo studies how neural circuits are assembled during development, and how their architectures enable them to perform specific functions in adults. Dr. Luo is currently the Ann and Bill Swindells Professor in the School of Humanities and Sciences, Professor of Biology, and Professor of Neurobiology by courtesy at Stanford University, and a Howard Hughes Medical Institute Investigator. He teaches neurobiology to Stanford undergraduate and graduate students. His single-author textbook “Principles of Neurobiology” (1st edition 2015; 2nd edition 2020) is widely used for undergraduate and graduate courses across the world.
Dr. Luo has served on the editorial boards of several scientific journals, including Neuron, eLife, and Annual Review of Neuroscience, Cell, and PNAS. He has also served on the Pew Scholar National Committee and Scientific Advisory Committee of Damon Runyon Cancer Research Foundation. He is recipient of the McKnight Technological Innovation in Neuroscience Award, the Society for Neuroscience Young Investigator Award, the Jacob Javits Award from National Institute of Neurological Disorders and Stroke, HW Mossman Award from American Association of Anatomists, the Lawrence Katz Prize, the Pradel Award of National Academy of Sciences, and the Education in Neuroscience award from Society for Neuroscience. Dr. Luo is a Member of the National Academy of Sciences and a Fellow of the American Academy of Arts and Sciences.
Academic Appointments
-
Professor, Biology
-
Professor (By courtesy), Neurobiology
-
Member, Bio-X
-
Faculty Fellow, Sarafan ChEM-H
-
Member, Stanford Cancer Institute
-
Member, Wu Tsai Neurosciences Institute
Honors & Awards
-
Education in Neuroscience Award, Society for Neuroscience (2020)
-
Pradel Award, National Academy of Sciences (2019)
-
The Lawrence C. Katz Prize for Innovative Research in Neuroscience, Duke University (2013)
-
Member, National Academy of Sciences (2012)
-
Fellow, American Academy of Arts and Sciences (2012)
-
Fellow, American Association for the Advancement of Science (2011)
-
H.W.Mossman Award, American Association of Anatomists (2007)
-
Investigator, Howard Hughes Medical Institute (2005)
-
Jacob Javits Award, National Institute of Neurological Disorders and Stroke (2005)
-
Technology Innovation Award in Neuroscience, McKnight Foundation (2002)
-
Young Investigator Award, Society for Neuroscience (2002)
Professional Education
-
B.S., Univ. of Sci. & Tech. of China, Molecular Biology (1986)
-
Ph.D., Brandeis University, Biology (1992)
Patents
-
He Z, Zhai Q, Wang J, Watts R, Hoopfer E, Luo L. "United States Patent 7,012,063 Reducing axon degeneration with proteasome inhibitors", Harvard & Stanford
-
Luo L, Zong H. "United States Patent 7,282,621 Somatic recombination", Stanford
-
Luo L, Tsai RY, Tasic B, Hippenmeyer S, Zong H. "United States Patent 9,125,385 Site-directed integration of transgenes in mammals", Stanford
Current Research and Scholarly Interests
1. Assembly of the fly olfactory circuit
A central question in neural circuit assembly is how neurons connect specifically with their synaptic partners. We are using the fly olfactory circuit to investigate the general principles by which wiring specificity is established during development. The assembly of the fly olfactory circuit requires precise matching between axons from 50 olfactory receptor neuron types and dendrites from 50 projection neuron types. In the past 20 years, we have identified key cellular interactions and molecular mechanisms at specific steps of olfactory circuit assembly. More recently, we have also taken transcriptomic, proteomic, and live imaging approaches to complement genetic analyses of individual genes. We are currently integrating these approaches to deepen our understanding of the combinatorial cell-surface codes that instruct connection specificity.
2. Assembly of neural circuits in the mouse brain
We have studied a broad range of developmental processes in rodent brains using genetic tools we have developed. Some of these studies extend what we are learning in the fly, whereas others explore processes more prevalent in vertebrates. For example, cerebellar Purkinje cells have highly elaborate and planar dendritic trees, each of which receives presynaptic inputs from tens of thousands of granule cells. Our investigations of Purkinje cell dendrite morphogenesis have highlighted the importance of competitive interactions in dendritic growth and branching. Our studies of hippocampal network assembly have revealed that the same cell-surface proteins, teneurin-3 and latrophilin-2, can serve both as ligands and receptors to mediate attraction and repulsion, and these molecules are likely reused in the assembly of multiple nodes of the hippocampal networks. We are investigating the function of these molecules in the assembly of additional circuits as well as how they work both as ligands and receptors.
3. Organization and function of neural circuits
We have used genetic and viral strategies to decipher the organizational principles of the fly and mouse olfactory systems, as well as the input–output architecture of norepinephrine, dopamine, and serotonin systems at the scale of the entire mouse brain. We are now also combining single-cell transcriptomics with activity recording, manipulation, and TRAPing, as well as behavioral analyses, to interrogate the functional organization of a variety of neural circuits. Recent discoveries include the dissection of dorsal raphe serotonin neuron subsystems, reward representation in cerebellar granule cells and shared cortex-cerebellum dynamics, the unit of organization and evolution of the cerebellar nuclei, differential encoding of task variables by prefrontal cortical projection neuron classes, temporal evolution of prefrontal cortical neuron ensembles that promote remote memory retrieval, and neural basis of thirst drive for motivated behavior.
4. Tool development
We continue to develop tools to interrogate neural circuit assembly and organization with increasing precision. The MARCM (mosaic analysis with a repressible cell marker) method in flies and MADM (mosaic analysis with double markers) method in mice allow the visualization and genetic manipulation of isolated single neurons. The Q system further expanded binary expression tools in flies. We recently developed tools to map circuit organization in mammals. The TRIO (tracing the relationship between input and output) and cTRIO (cell-type-specific TRIO) methods allow rabies virus–based input tracing to neurons defined by projection, or by cell type and projection. The TRAP (targeted recombination in active population) method enables genetic access to neurons based on their activity, which in combination with tools for labeling, tracing, recording, and manipulating neurons, offers a powerful approach for understanding how neural circuits process information and generate behavior.
2024-25 Courses
- Introduction to Neuroscience
BIO 102 (Win) -
Independent Studies (14)
- Directed Investigation
BIOE 392 (Aut, Win, Sum) - Directed Reading in Biology
BIO 198 (Aut, Win, Spr, Sum) - Directed Reading in Neurosciences
NEPR 299 (Aut, Win, Spr, Sum) - Directed Study
BIOE 391 (Aut, Win, Spr, Sum) - Graduate Research
BIO 300 (Aut, Win, Spr, Sum) - Graduate Research
CBIO 399 (Aut, Win, Spr, Sum) - Graduate Research
NEPR 399 (Aut, Win, Spr, Sum) - Honors
HUMBIO 194 (Spr) - Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum) - Out-of-Department Advanced Research Laboratory in Bioengineering
BIOE 191X (Aut, Win, Spr, Sum) - Out-of-Department Undergraduate Research
BIO 199X (Aut, Win, Spr, Sum) - Research in Human Biology
HUMBIO 193 (Aut, Win) - Teaching Practicum in Biology
BIO 290 (Aut, Win, Spr, Sum) - Undergraduate Research
BIO 199 (Aut, Win, Spr, Sum)
- Directed Investigation
-
Prior Year Courses
2023-24 Courses
- Exploring Neural Circuits
BIO 222 (Spr)
2022-23 Courses
- Exploring Neural Circuits
BIO 222 (Spr) - Principles of Neurobiology
BIO 154 (Win) - Principles of Neurobiology
BIO 254, NBIO 254 (Win)
2021-22 Courses
- Exploring Neural Circuits
BIO 222 (Spr)
- Exploring Neural Circuits
Stanford Advisees
-
Med Scholar Project Advisor
David Wang -
Doctoral Dissertation Reader (AC)
Jacqueline Bendrick, Minseung Choi, Alex Hao, Dane Kawano, Emmalyn Leonard, Ev Nichols, Massimo Onesto, Marija Pavlovic, Anay Ram Reddy, Abigail Rogers, Adarsh Tantry, Emma Theisen, Yandan Wang, Livia Wyss, Xiaochen Xiong, Junhao Xu -
Postdoctoral Faculty Sponsor
Hui Ji, Cheng Lyu, Lijun Qi -
Doctoral Dissertation Advisor (AC)
URee Chon, Hannah Field, Ellen Gingrich, Zhuoran Li, Jun Song, David Wang, Alina Xiao, Chuanyun Xu -
Doctoral Dissertation Co-Advisor (AC)
Lucas Encarnacion-Rivera, Qilin Zhao -
Doctoral (Program)
Zhuoran Li, Alina Xiao
Graduate and Fellowship Programs
-
Biology (School of Humanities and Sciences) (Phd Program)
All Publications
-
Architectures of neuronal circuits.
Science (New York, N.Y.)
2021; 373 (6559): eabg7285
Abstract
[Figure: see text].
View details for DOI 10.1126/science.abg7285
View details for PubMedID 34516844
- Principles of Neurobiology (2nd edition) Garland Science/CRC Press. 2020
-
Dimensionality reduction simplifies synaptic partner matching in an olfactory circuit.
bioRxiv : the preprint server for biology
2024
Abstract
The distribution of postsynaptic partners in three-dimensional (3D) space presents complex choices for a navigating axon. Here, we discovered a dimensionality reduction principle in establishing the 3D glomerular map in the fly antennal lobe. Olfactory receptor neuron (ORN) axons first contact partner projection neuron (PN) dendrites at the 2D spherical surface of the antennal lobe during development, regardless of whether the adult glomeruli are at the surface or interior of the antennal lobe. Along the antennal lobe surface, axons of each ORN type take a specific 1D arc-shaped trajectory that precisely intersects with their partner PN dendrites. Altering axon trajectories compromises synaptic partner matching. Thus, a 3D search problem is reduced to 1D, which simplifies synaptic partner matching and may generalize to the wiring process of more complex brains.
View details for DOI 10.1101/2024.08.27.609939
View details for PubMedID 39253519
View details for PubMedCentralID PMC11383009
-
Molecular mechanisms of proteoglycan-mediated semaphorin signaling in axon guidance.
Proceedings of the National Academy of Sciences of the United States of America
2024; 121 (31): e2402755121
Abstract
The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.
View details for DOI 10.1073/pnas.2402755121
View details for PubMedID 39042673
-
Molecular and cellular mechanisms of teneurin signaling in synaptic partner matching.
Cell
2024
Abstract
In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.
View details for DOI 10.1016/j.cell.2024.06.022
View details for PubMedID 38996528
-
Embryonically active piriform cortex neurons promote intracortical recurrent connectivity during development.
Neuron
2024
Abstract
Neuronal activity plays a critical role in the maturation of circuits that propagate sensory information into the brain. How widely does early activity regulate circuit maturation across the developing brain? Here, we used targeted recombination in active populations (TRAP) to perform a brain-wide survey for prenatally active neurons in mice and identified the piriform cortex as an abundantly TRAPed region. Whole-cell recordings in neonatal slices revealed preferential interconnectivity within embryonically TRAPed piriform neurons and their enhanced synaptic connectivity with other piriform neurons. In vivo Neuropixels recordings in neonates demonstrated that embryonically TRAPed piriform neurons exhibit broad functional connectivity within piriform and lead spontaneous synchronized population activity during a transient neonatal period, when recurrent connectivity is strengthening. Selectively activating or silencing these neurons in neonates enhanced or suppressed recurrent synaptic strength, respectively. Thus, embryonically TRAPed piriform neurons represent an interconnected hub-like population whose activity promotes recurrent connectivity in early development.
View details for DOI 10.1016/j.neuron.2024.06.007
View details for PubMedID 38964330
-
A cerebellar granule cell-climbing fiber computation to learn to track long time intervals.
Neuron
2024
Abstract
In classical cerebellar learning, Purkinje cells (PkCs) associate climbing fiber (CF) error signals with predictive granule cells (GrCs) that were active just prior (∼150 ms). The cerebellum also contributes to behaviors characterized by longer timescales. To investigate how GrC-CF-PkC circuits might learn seconds-long predictions, we imaged simultaneous GrC-CF activity over days of forelimb operant conditioning for delayed water reward. As mice learned reward timing, numerous GrCs developed anticipatory activity ramping at different rates until reward delivery, followed by widespread time-locked CF spiking. Relearning longer delays further lengthened GrC activations. We computed CF-dependent GrC→PkC plasticity rules, demonstrating that reward-evoked CF spikes sufficed to grade many GrC synapses by anticipatory timing. We predicted and confirmed that PkCs could thereby continuously ramp across seconds-long intervals from movement to reward. Learning thus leads to new GrC temporal bases linking predictors to remote CF reward signals-a strategy well suited for learning to track the long intervals common in cognitive domains.
View details for DOI 10.1016/j.neuron.2024.05.019
View details for PubMedID 38870929
-
Neural landscape diffusion resolves conflicts between needs across time.
Nature
2023
Abstract
Animals perform flexible goal-directed behaviours to satisfy their basic physiological needs1-12. However, little is known about how unitary behaviours are chosen under conflicting needs. Here we reveal principles by which the brain resolves such conflicts between needs across time. We developed an experimental paradigm in which a hungry and thirsty mouse is given free choices between equidistant food and water. We found that mice collect need-appropriate rewards by structuring their choices into persistent bouts with stochastic transitions. High-density electrophysiological recordings during this behaviour revealed distributed single neuron and neuronal population correlates of a persistent internal goal state guiding future choices of the mouse. We captured these phenomena with a mathematical model describing a global need state that noisily diffuses across a shifting energy landscape. Model simulations successfully predicted behavioural and neural data, including population neural dynamics before choice transitions and in response to optogenetic thirst stimulation. These results provide a general framework for resolving conflicts between needs across time, rooted in the emergent properties of need-dependent state persistence and noise-driven shifts between behavioural goals.
View details for DOI 10.1038/s41586-023-06715-z
View details for PubMedID 37938783
View details for PubMedCentralID 4306350
-
Toward building a library of cell type-specific drivers across developmental stages.
Proceedings of the National Academy of Sciences of the United States of America
2023; 120 (35): e2312196120
View details for DOI 10.1073/pnas.2312196120
View details for PubMedID 37590431
-
A neural circuit for male sexual behavior and reward.
Cell
2023
Abstract
Male sexual behavior is innate and rewarding. Despite its centrality to reproduction, a molecularly specified neural circuit governing innate male sexual behavior and reward remains to be characterized. We have discovered a developmentally wired neural circuit necessary and sufficient for male mating. This circuit connects chemosensory input to BNSTprTac1 neurons, which innervate POATacr1 neurons that project to centers regulating motor output and reward. Epistasis studies demonstrate that BNSTprTac1 neurons are upstream of POATacr1 neurons, and BNSTprTac1-released substance P following mate recognition potentiates activation of POATacr1 neurons through Tacr1 to initiate mating. Experimental activation of POATacr1 neurons triggers mating, even in sexually satiated males, and it is rewarding, eliciting dopamine release and self-stimulation of these cells. Together, we have uncovered a neural circuit that governs the key aspects of innate male sexual behavior: motor displays, drive, and reward.
View details for DOI 10.1016/j.cell.2023.07.021
View details for PubMedID 37572660
-
Expansion spatial transcriptomics.
Nature methods
2023
Abstract
Capture array-based spatial transcriptomics methods have been widely used to resolve gene expression in tissues; however, their spatial resolution is limited by the density of the array. Here we present expansion spatial transcriptomics to overcome this limitation by clearing and expanding tissue prior to capturing the entire polyadenylated transcriptome with an enhanced protocol. This approach enables us to achieve higher spatial resolution while retaining high library quality, which we demonstrate using mouse brain samples.
View details for DOI 10.1038/s41592-023-01911-1
View details for PubMedID 37349575
-
Aging Fly Cell Atlas identifies exhaustive aging features at cellular resolution.
Science (New York, N.Y.)
2023; 380 (6650): eadg0934
Abstract
Aging is characterized by a decline in tissue function, but the underlying changes at cellular resolution across the organism remain unclear. Here, we present the Aging Fly Cell Atlas, a single-nucleus transcriptomic map of the whole aging Drosophila. We characterized 163 distinct cell types and performed an in-depth analysis of changes in tissue cell composition, gene expression, and cell identities. We further developed aging clock models to predict fly age and show that ribosomal gene expression is a conserved predictive factor for age. Combining all aging features, we find distinctive cell type-specific aging patterns. This atlas provides a valuable resource for studying fundamental principles of aging in complex organisms.
View details for DOI 10.1126/science.adg0934
View details for PubMedID 37319212
-
Origin of wiring specificity in an olfactory map revealed by neuron type-specific, time-lapse imaging of dendrite targeting.
eLife
2023; 12
Abstract
How does wiring specificity of neural maps emerge during development? Formation of the adult Drosophila olfactory glomerular map begins with patterning of projection neuron (PN) dendrites at the early pupal stage. To better understand the origin of wiring specificity of this map, we created genetic tools to systematically characterize dendrite patterning across development at PN type-specific resolution. We find that PNs use lineage and birth order combinatorially to build the initial dendritic map. Specifically, birth order directs dendrite targeting in rotating and binary manners for PNs of the anterodorsal and lateral lineages, respectively. Two-photon- and adaptive optical lattice light-sheet microscope-based time-lapse imaging reveals that PN dendrites initiate active targeting with direction-dependent branch stabilization on the timescale of seconds. Moreover, PNs that are used in both the larval and adult olfactory circuits prune their larval-specific dendrites and re-extend new dendrites simultaneously to facilitate timely olfactory map organization. Our work highlights the power and necessity of type-specific neuronal access and time-lapse imaging in identifying wiring mechanisms that underlie complex patterns of functional neural maps.
View details for DOI 10.7554/eLife.85521
View details for PubMedID 36975203
-
Context-dependent requirement of G protein coupling for Latrophilin-2 in target selection of hippocampal axons.
eLife
2023; 12
Abstract
The formation of neural circuits requires extensive interactions of cell-surface proteins to guide axons to their correct target neurons. Trans-cellular interactions of the adhesion G protein-coupled receptor latrophilin-2 (Lphn2) with its partner teneurin-3 instruct the precise assembly of hippocampal networks by reciprocal repulsion. Lphn2 acts as a repulsive receptor in distal CA1 neurons to direct their axons to proximal subiculum, and as a repulsive ligand in proximal subiculum to direct proximal CA1 axons to distal subiculum. It remains unclear if Lphn2-mediated intracellular signaling is required for its role in either context. Here, we show that Lphn2 couples to Galpha12/13 in heterologous cells; this coupling is increased by constitutive exposure of the tethered agonist. Specific mutations of Lphn2's tethered agonist region disrupt its G protein coupling and autoproteolytic cleavage, whereas mutating the autoproteolytic cleavage site alone prevents cleavage but preserves a functional tethered agonist. Using an in vivo misexpression assay, we demonstrate that wild-type Lphn2 misdirects proximal CA1 axons to proximal subiculum and that Lphn2 tethered agonist activity is required for its role as a repulsive receptor in axons. By contrast, neither tethered agonist activity nor autoproteolysis was necessary for Lphn2's role as a repulsive ligand in the subiculum target neurons. Thus, tethered agonist activity is required for Lphn2-mediated neural circuit assembly in a context-dependent manner.
View details for DOI 10.7554/eLife.83529
View details for PubMedID 36939320
-
Hypothalamic neurons that mirror aggression.
Cell
2023
Abstract
Social interactions require awareness and understanding of the behavior of others. Mirror neurons, cells representing an action by self and others, have been proposed to be integral to the cognitive substrates that enable such awareness and understanding. Mirror neurons of the primate neocortex represent skilled motor tasks, but it is unclear if they are critical for the actions they embody, enable social behaviors, or exist in non-cortical regions. We demonstrate that the activity of individual VMHvlPR neurons in the mouse hypothalamus represents aggression performed by self and others. We used a genetically encoded mirror-TRAP strategy to functionally interrogate these aggression-mirroring neurons. We find that their activity is essential for fighting and that forced activation of these cells triggers aggressive displays by mice, even toward their mirror image. Together, we have discovered a mirroring center in an evolutionarily ancient region that provides a subcortical cognitive substrate essential for a social behavior.
View details for DOI 10.1016/j.cell.2023.01.022
View details for PubMedID 36796363
-
Loss of Rai1 enhances hippocampal excitability and epileptogenesis in mouse models of Smith-Magenis syndrome.
Proceedings of the National Academy of Sciences of the United States of America
2022; 119 (43): e2210122119
Abstract
Hyperexcitability of brain circuits is a common feature of autism spectrum disorders (ASDs). Genetic deletion of a chromatin-binding protein, retinoic acid induced 1 (RAI1), causes Smith-Magenis syndrome (SMS). SMS is a syndromic ASD associated with intellectual disability, autistic features, maladaptive behaviors, overt seizures, and abnormal electroencephalogram (EEG) patterns. The molecular and neural mechanisms underlying abnormal brain activity in SMS remain unclear. Here we show that panneural Rai1 deletions in mice result in increased seizure susceptibility and prolonged hippocampal seizure duration invivo and increased dentate gyrus population spikes ex vivo. Brain-wide mapping of neuronal activity pinpointed selective cell types within the limbic system, including the hippocampal dentate gyrus granule cells (dGCs) that are hyperactivated by chemoconvulsant administration or sensory experience in Rai1-deficient brains. Deletion of Rai1 from glutamatergic neurons, but not from gamma-aminobutyric acidergic (GABAergic) neurons, was responsible for increased seizure susceptibility. Deleting Rai1 from the Emx1Cre-lineage glutamatergic neurons resulted in abnormal dGC properties, including increased excitatory synaptic transmission and increased intrinsic excitability. Our work uncovers the mechanism of neuronal hyperexcitability in SMS by identifying Rai1 as a negative regulator of dGC intrinsic and synaptic excitability.
View details for DOI 10.1073/pnas.2210122119
View details for PubMedID 36256819
-
In situ cell-type-specific cell-surface proteomic profiling in mice.
Neuron
2022
Abstract
Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed in vivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.
View details for DOI 10.1016/j.neuron.2022.09.025
View details for PubMedID 36220098
-
Scent of a human: The mosquito olfactory system defies dogma to ensure attraction to humans.
Cell
2022; 185 (17): 3079-3081
Abstract
Mosquitoes are strongly attracted to humans, and their bites not only cause intense itch but can beget severe diseases. In this issue of Cell, Herre etal. reveal that non-canonical olfactory circuit organization and coding likely endow mosquitoes with a robust ability to locate human hosts.
View details for DOI 10.1016/j.cell.2022.07.018
View details for PubMedID 35985284
-
Illuminating complexity in serotonin neurons of the dorsal raphe nucleus.
Neuron
2022; 110 (16): 2519-2521
Abstract
The function of serotonin in the mammalian brain has been challenging to unravel. In this issue of Neuron, Paquelet etal. (2022) employ microendoscopy to record over 2,000 dorsal raphe serotonin neurons, yielding new insights into their activity from the single neuron to the population level.
View details for DOI 10.1016/j.neuron.2022.07.013
View details for PubMedID 35981523
-
Transcriptional and functional motifs defining renal function revealed by single-nucleus RNA sequencing.
Proceedings of the National Academy of Sciences of the United States of America
2022; 119 (25): e2203179119
Abstract
Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. Here, we performed a single-nucleus study of the adult Drosophila renal system, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. We identified 11 distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless (fru) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 (Hnf4) in regulating glycogen and triglyceride metabolism. In addition, we identified a number of genes, including Rho guanine nucleotide exchange factor at 64C (RhoGEF64c), Frequenin 2 (Frq2), Prip, and CG1093 that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus datasetallows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, our study provides a comprehensive resource for studying the fly kidney.
View details for DOI 10.1073/pnas.2203179119
View details for PubMedID 35696569
-
Isolation and RNA sequencing of single nuclei from Drosophila tissues.
STAR protocols
2022; 3 (2): 101417
Abstract
Many insect cells are encapsulated within the exoskeleton and cannot be dissociated intact, making them inaccessible to single-cell transcriptomic profiling. We have used single-nucleus RNA sequencing to extract transcriptomic information from multiple Drosophila tissues. Here, we describe procedures for the (1) dissociation of single nuclei, (2) isolation of single nuclei using two popular cell sorters, and (3) preparation of libraries for Smart-seq2 and 10× Genomics. This protocol enables generation of high-quality transcriptomes from single nuclei and can be applied to other species. For complete details on the use and execution of this protocol, please refer to McLaughlin et al. (2021) and Li et al. (2022).
View details for DOI 10.1016/j.xpro.2022.101417
View details for PubMedID 35620068
View details for PubMedCentralID PMC9127693
-
A preoptic neuronal population controls fever and appetite during sickness.
Nature
2022
Abstract
During infection, animals exhibit adaptive changes in physiology and behaviour aimed at increasing survival. Although many causes of infection exist, they trigger similar stereotyped symptoms such as fever, warmth-seeking, loss of appetite and fatigue1,2. Yet exactly how the nervous system alters body temperature and triggers sickness behaviours to coordinate responses to infection remains unknown. Here we identify a previously uncharacterized population of neurons in the ventral medial preoptic area (VMPO) of the hypothalamus that are activated after sickness induced by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid. These neurons are crucial for generating a fever response and other sickness symptoms such as warmth-seeking and loss of appetite. Single-nucleus RNA-sequencing and multiplexed error-robust fluorescence in situ hybridization uncovered the identity and distribution of LPS-activated VMPO (VMPOLPS) neurons and non-neuronal cells. Gene expression and electrophysiological measurements implicate a paracrine mechanism in which the release of immune signals by non-neuronal cells during infection activates nearby VMPOLPS neurons. Finally, we show that VMPOLPS neurons exert a broad influence on the activity of brain areas associated with behavioural and homeostatic functions and are synaptically and functionally connected to circuit nodes controlling body temperature and appetite. Together, these results uncover VMPOLPS neurons as a control hub that integrates immune signals to orchestrate multiple sickness symptoms in response to infection.
View details for DOI 10.1038/s41586-022-04793-z
View details for PubMedID 35676482
-
Transcription factor Acj6 controls dendrite targeting via a combinatorial cell-surface code.
Neuron
2022
Abstract
Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.
View details for DOI 10.1016/j.neuron.2022.04.026
View details for PubMedID 35613619
-
Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly.
Science (New York, N.Y.)
2022; 375 (6584): eabk2432
Abstract
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
View details for DOI 10.1126/science.abk2432
View details for PubMedID 35239393
-
Mating-driven variability in olfactory local interneuron wiring.
Science advances
2022; 8 (7): eabm7723
Abstract
Variations in neuronal connectivity occur widely in nervous systems from invertebrates to mammals. Yet, it is unclear how neuronal variability originates, to what extent and at what time scales it exists, and what functional consequences it might carry. To assess inter- and intraindividual neuronal variability, it would be ideal to analyze the same identified neuron across different brain hemispheres and individuals. Here, using genetic labeling and electron microscopy connectomics, we show that an identified inhibitory olfactory local interneuron, TC-LN, exhibits extraordinary variability in its glomerular innervation patterns. Moreover, TC-LN's innervation of the VL2a glomerulus, which processes food signals and modulates mating behavior, is sexually dimorphic, is influenced by female's courtship experience, and correlates with food intake in mated females. Mating also affects output connectivity of TC-LN to specific local interneurons. We propose that mating-associated variability of TC-LNs regulates how food odor is interpreted by an inhibitory network to modulate feeding.
View details for DOI 10.1126/sciadv.abm7723
View details for PubMedID 35179957
-
An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila.
Journal of visualized experiments : JoVE
2021
Abstract
~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.
View details for DOI 10.3791/62983
View details for PubMedID 34723938
-
Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging.
Cell
2021
Abstract
Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.
View details for DOI 10.1016/j.cell.2021.08.030
View details for PubMedID 34551316
-
Teneurins
CURRENT BIOLOGY
2021; 31 (15): R936-R937
View details for Web of Science ID 000684184900003
-
Gut cytokines modulate olfaction through metabolic reprogramming of glia.
Nature
2021
Abstract
Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival1,2. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms3-5. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. Here we show that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. Our findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies.
View details for DOI 10.1038/s41586-021-03756-0
View details for PubMedID 34290404
-
A neural circuit state change underlying skilled movements.
Cell
2021
Abstract
In motor neuroscience, state changes are hypothesized to time-lock neural assemblies coordinating complex movements, but evidence for this remains slender. We tested whether a discrete change from more autonomous to coherent spiking underlies skilled movement by imaging cerebellar Purkinje neuron complex spikes in mice making targeted forelimb-reaches. As mice learned the task, millimeter-scale spatiotemporally coherent spiking emerged ipsilateral to the reaching forelimb, and consistent neural synchronization became predictive of kinematic stereotypy. Before reach onset, spiking switched from more disordered to internally time-locked concerted spiking and silence. Optogenetic manipulations of cerebellar feedback to the inferior olive bi-directionally modulated neural synchronization and reaching direction. A simple model explained the reorganization of spiking during reaching as reflecting a discrete bifurcation in olivary network dynamics. These findings argue that to prepare learned movements, olivo-cerebellar circuits enter a self-regulated, synchronized state promoting motor coordination. State changes facilitating behavioral transitions may generalize across neural systems.
View details for DOI 10.1016/j.cell.2021.06.001
View details for PubMedID 34214470
-
A genome-wide library of MADM mice for single-cell genetic mosaic analysis.
Cell reports
2021; 35 (12): 109274
Abstract
Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions invivo in health and disease. Yet, MADM can only be applied to <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.
View details for DOI 10.1016/j.celrep.2021.109274
View details for PubMedID 34161767
-
The relationship between birth timing, circuit wiring, and physiological response properties of cerebellar granule cells.
Proceedings of the National Academy of Sciences of the United States of America
2021; 118 (23)
Abstract
Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca2+ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiberGrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.
View details for DOI 10.1073/pnas.2101826118
View details for PubMedID 34088841
-
Reciprocal repulsions instruct the precise assembly of parallel hippocampal networks.
Science (New York, N.Y.)
2021; 372 (6546): 1068-1073
Abstract
Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying the assembly of such parallel networks during development remain largely unknown. Our study shows that, in mice, complementary expression of cell surface molecules teneurin-3 (Ten3) and latrophilin-2 (Lphn2) in the medial and lateral hippocampal networks, respectively, guides the precise assembly of CA1-to-subiculum connections in both networks. In the medial network, Ten3-expressing (Ten3+) CA1 axons are repelled by target-derived Lphn2, revealing that Lphn2- and Ten3-mediated heterophilic repulsion and Ten3-mediated homophilic attraction cooperate to control precise target selection of CA1 axons. In the lateral network, Lphn2-expressing (Lphn2+) CA1 axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a "Ten3Ten3, Lphn2Lphn2" rule instructed by reciprocal repulsions.
View details for DOI 10.1126/science.abg1774
View details for PubMedID 34083484
-
Temporal evolution of single-cell transcriptomes of Drosophila olfactory projection neurons.
eLife
2021; 10
Abstract
Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.
View details for DOI 10.7554/eLife.63450
View details for PubMedID 33427646
-
Single-cell transcriptomes of developing and adult olfactory receptor neurons in Drosophila.
eLife
2021; 10
Abstract
Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of Drosophila olfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.
View details for DOI 10.7554/eLife.63856
View details for PubMedID 33555999
-
Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations.
Cell reports
2021; 35 (6): 109123
Abstract
Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.
View details for DOI 10.1016/j.celrep.2021.109123
View details for PubMedID 33979604
-
Deep posteromedial cortical rhythm in dissociation.
Nature
2020
Abstract
Advanced imaging methods now allow cell-type-specific recording of neural activity across the mammalian brain, potentially enabling the exploration of how brain-wide dynamical patterns give rise to complex behavioural states1-12. Dissociation is an altered behavioural state in which the integrity of experience is disrupted, resulting in reproducible cognitive phenomena including the dissociation of stimulus detection from stimulus-related affective responses. Dissociation can occur as a result of trauma, epilepsy or dissociative drug use13,14, but despite its substantial basic and clinical importance, the underlying neurophysiology of this state is unknown. Here we establish such a dissociation-like state in mice, induced by precisely-dosed administration of ketamine or phencyclidine. Large-scale imaging of neural activity revealed that these dissociative agents elicited a 1-3-Hz rhythm in layer5 neurons of the retrosplenial cortex. Electrophysiological recording with four simultaneously deployed high-density probes revealed rhythmic coupling of the retrosplenial cortex with anatomically connected components of thalamus circuitry, but uncoupling from most other brain regions was observed-including a notable inverse correlation with frontally projecting thalamic nuclei. In testing for causal significance, we found thatrhythmic optogenetic activation of retrosplenial cortex layer5 neurons recapitulated dissociation-like behavioural effects. Local retrosplenial hyperpolarization-activated cyclic-nucleotide-gated potassium channel 1 (HCN1) pacemakers were required for systemic ketamine to induce this rhythm and to elicit dissociation-like behavioural effects. In a patient with focal epilepsy, simultaneous intracranial stereoencephalography recordings from across the brain revealed a similarly localized rhythm in the homologous deep posteromedial cortex that was temporally correlated with pre-seizure self-reported dissociation, and local brief electrical stimulation of this region elicited dissociative experiences. These results identify themolecular, cellular and physiological properties of a conserved deep posteromedial cortical rhythm that underlies states of dissociation.
View details for DOI 10.1038/s41586-020-2731-9
View details for PubMedID 32939091
-
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
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
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
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
-
Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators.
Cell
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
-
Skilled reaching tasks for head-fixed mice using a robotic manipulandum.
Nature protocols
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
-
The Mind of a Mouse.
Cell
2020; 182 (6): 1372–76
Abstract
Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.
View details for DOI 10.1016/j.cell.2020.08.010
View details for PubMedID 32946777
-
Single-Cell Transcriptomes Reveal Diverse Regulatory Strategies for Olfactory Receptor Expression and Axon Targeting.
Current biology : CB
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
-
Nurturing Undergraduate Researchers in Biomedical Sciences.
Cell
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
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
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
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
-
Cerebellar nuclei evolved by repeatedly duplicating a conserved cell-type set.
Science (New York, N.Y.)
2020; 370 (6523)
Abstract
How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei, the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole-central nervous system projection tracing, we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes. This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions. The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human lateral nucleus. Our data suggest a model of brain region evolution by duplication and divergence of entire cell-type sets.
View details for DOI 10.1126/science.abd5059
View details for PubMedID 33335034
-
Differential encoding in prefrontal cortex projection neuron classes across cognitive tasks.
Cell
2020
Abstract
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca2+ activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.
View details for DOI 10.1016/j.cell.2020.11.046
View details for PubMedID 33338423
-
Amygdala-Midbrain Connections Modulate Appetitive and Aversive Learning.
Neuron
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
-
GluD2- and Cbln1-mediated competitive interactions shape the dendritic arbors of cerebellar Purkinje cells.
Neuron
2020
Abstract
The synaptotrophic hypothesis posits that synapse formation stabilizes dendritic branches, but this hypothesis has not been causally tested in vivo in the mammalian brain. The presynaptic ligand cerebellin-1 (Cbln1) and postsynaptic receptor GluD2 mediate synaptogenesis between granule cells and Purkinje cells in the molecular layer of the cerebellar cortex. Here we show that sparse but not global knockout of GluD2 causes under-elaboration of Purkinje cell dendrites in the deep molecular layer and overelaboration in the superficial molecular layer. Developmental, overexpression, structure-function, and genetic epistasis analyses indicate that these dendrite morphogenesis defects result from a deficit in Cbln1/GluD2-dependent competitive interactions. A generative model of dendrite growth based on competitive synaptogenesis largely recapitulates GluD2 sparse and global knockout phenotypes. Our results support the synaptotrophic hypothesis at initial stages of dendrite development, suggest a second mode in which cumulative synapse formation inhibits further dendrite growth, and highlight the importance of competition in dendrite morphogenesis.
View details for DOI 10.1016/j.neuron.2020.11.028
View details for PubMedID 33352118
-
Brain Circuit of Claustrophobia-like Behavior in Mice Identified by Upstream Tracing of Sighing.
Cell reports
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
-
Neocortex-Cerebellum Circuits for Cognitive Processing.
Trends in neurosciences
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
-
Transsynaptic Fish-lips signaling prevents misconnections between nonsynaptic partner olfactory neurons.
Proceedings of the National Academy of Sciences of the United States of America
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
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
2019; 364 (6437): 253-+
View details for DOI 10.1126/science.aav3932
View details for Web of Science ID 000464956600040
-
Shared Cortex-Cerebellum Dynamics in the Execution and Learning of a Motor Task
CELL
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
2019; 22 (3): 460-+
View details for DOI 10.1038/s41593-018-0318-7
View details for Web of Science ID 000459423800016
-
Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs.
eLife
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
-
Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs
ELIFE
2019; 8
View details for DOI 10.7554/eLife.41563
View details for Web of Science ID 000459380600001
-
Temporal evolution of cortical ensembles promoting remote memory retrieval.
Nature neuroscience
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
-
Topological Organization of Ventral Tegmental Area Connectivity Revealed by Viral-Genetic Dissection of Input-Output Relations.
Cell reports
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
-
Suppressing Memories by Shrinking the Vesicle Pool
NEURON
2019; 101 (1): 5-7
View details for DOI 10.1016/j.neuron.2018.12.013
View details for Web of Science ID 000454791500003
-
Topological Organization of Ventral Tegmental Area Connectivity Revealed by Viral-Genetic Dissection of Input-Output Relations
CELL REPORTS
2019; 26 (1): 159-+
View details for DOI 10.1016/j.celrep.2018.12.040
View details for Web of Science ID 000454844700015
-
Suppressing Memories by Shrinking the Vesicle Pool.
Neuron
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
-
Complementary Genetic Targeting and Monosynaptic Input Mapping Reveal Recruitment and Refinement of Distributed Corticostriatal Ensembles by Cocaine.
Neuron
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
-
Single-cell transcriptomes and whole-brain projections of serotonin neurons in the mouse dorsal and median raphe nuclei.
eLife
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
-
Mapping Histological Slice Sequences to the Allen Mouse Brain Atlas Without 3D Reconstruction
FRONTIERS IN NEUROINFORMATICS
2018; 12
View details for DOI 10.3389/fninf.2018.00093
View details for Web of Science ID 000452913600001
-
Mapping Histological Slice Sequences to the Allen Mouse Brain Atlas Without 3D Reconstruction.
Frontiers in neuroinformatics
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 DOI 10.3389/fninf.2018.00093
View details for PubMedID 30618698
View details for PubMedCentralID PMC6297281
-
Dynamic salience processing in paraventricular thalamus gates associative learning
SCIENCE
2018; 362 (6413): 423-+
View details for DOI 10.1126/science.aat0481
View details for Web of Science ID 000450441900042
-
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
2018; 115 (42): 10744–49
View details for DOI 10.1073/pnas.1806796115
View details for Web of Science ID 000447491300066
-
Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems
CELL
2018; 175 (2): 472-+
View details for DOI 10.1016/j.cell.2018.07.043
View details for Web of Science ID 000446321300018
-
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
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
-
Stepwise wiring of the Drosophila olfactory map requires specific Plexin B levels
ELIFE
2018; 7
View details for DOI 10.7554/eLife.39088
View details for Web of Science ID 000443245600001
-
Polina Anikeeva and Liqun Luo
CURRENT OPINION IN NEUROBIOLOGY
2018; 50: IV-VI
View details for PubMedID 29754872
-
Functional circuit architecture underlying parental behaviour
NATURE
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
-
Genetic Dissection of Neural Circuits: A Decade of Progress
NEURON
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
-
Linking neuronal lineage and wiring specificity
NEURAL DEVELOPMENT
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
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
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
-
Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems.
Cell
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
-
Dynamic salience processing in paraventricular thalamus gates associative learning.
Science (New York, N.Y.)
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
-
Stepwise wiring of the Drosophila olfactory map requires specific Plexin B levels.
eLife
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
-
Teneurin-3 controls topographic circuit assembly in the hippocampus.
Nature
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
-
NEUROBIOLOGY A bitter-sweet symphony
NATURE
2017; 548 (7667): 285–87
View details for PubMedID 28792928
-
The MutAnts Are Here
CELL
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 (vol 114, pg 7505, 2017)
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2017; 114 (32): E6731
View details for DOI 10.1073/pnas.1712520114
View details for Web of Science ID 000407129000031
-
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
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
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
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
2017; 356 (6339): 722-726
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 DOI 10.1126/science.aam7511
View details for Web of Science ID 000401508400040
-
Lineage-dependent spatial and functional organization of the mammalian enteric nervous system.
Science (New York, N.Y.)
2017; 356 (6339): 722-726
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 DOI 10.1126/science.aam7511
View details for PubMedID 28522527
-
Global Representations of Goal-Directed Behavior in Distinct Cell Types of Mouse Neocortex
NEURON
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
-
Cerebellar granule cells encode the expectation of reward
NATURE
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
-
Breathing control center neurons that promote arousal in mice
SCIENCE
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
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
-
Thirst-associated preoptic neurons encode an aversive motivational drive.
Science (New York, N.Y.)
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
-
Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons.
eLife
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
-
Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-Cell RNA Sequencing.
Cell
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
-
Rabies screen reveals GPe control of cocaine-triggered plasticity.
Nature
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
-
Gating of social reward by oxytocin in the ventral tegmental area.
Science (New York, N.Y.)
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
-
Molecular and Neural Functions of Rai1, the Causal Gene for Smith-Magenis Syndrome.
Neuron
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
-
Cell type-specific long-range connections of basal forebrain circuit
ELIFE
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
-
Cell type-specific long-range connections of basal forebrain circuit.
eLife
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 PubMedID 27642784
View details for PubMedCentralID PMC5095704
-
Liqun Luo
NEURON
2016; 91 (3): 508-510
View details for DOI 10.1016/j.neuron.2016.07.017
View details for Web of Science ID 000382396100006
-
Wiring and Molecular Features of Prefrontal Ensembles Representing Distinct Experiences
CELL
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
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
-
Cas9-triggered chain ablation of cas9 as a gene drive brake.
Nature biotechnology
2016; 34 (2): 137–38
View details for PubMedID 26849513
-
Organization of the Locus Coeruleus-Norepinephrine System
CURRENT BIOLOGY
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
2015; 18 (11): 1641-1647
Abstract
The mammalian basal forebrain (BF) has important roles in controlling sleep and wakefulness, but the underlying neural circuit remains poorly understood. We examined the BF circuit by recording and optogenetically perturbing the activity of four genetically defined cell types across sleep-wake cycles and by comprehensively mapping their synaptic connections. Recordings from channelrhodopsin-2 (ChR2)-tagged neurons revealed that three BF cell types, cholinergic, glutamatergic and parvalbumin-positive (PV+) GABAergic neurons, were more active during wakefulness and rapid eye movement (REM) sleep (wake/REM active) than during non-REM (NREM) sleep, and activation of each cell type rapidly induced wakefulness. By contrast, activation of somatostatin-positive (SOM+) GABAergic neurons promoted NREM sleep, although only some of them were NREM active. Synaptically, the wake-promoting neurons were organized hierarchically by glutamatergic→cholinergic→PV+ neuron excitatory connections, and they all received inhibition from SOM+ neurons. Together, these findings reveal the basic organization of the BF circuit for sleep-wake control.
View details for DOI 10.1038/nn.4143
View details for PubMedID 26457552
View details for PubMedCentralID PMC5776144
-
Connectivity of mouse somatosensory and prefrontal cortex examined with trans-synaptic tracing.
Nature neuroscience
2015; 18 (11): 1687-1697
Abstract
Information processing in neocortical circuits requires integrating inputs over a wide range of spatial scales, from local microcircuits to long-range cortical and subcortical connections. We used rabies virus-based trans-synaptic tracing to analyze the laminar distribution of local and long-range inputs to pyramidal neurons in the mouse barrel cortex and medial prefrontal cortex (mPFC). In barrel cortex, we found substantial inputs from layer 3 (L3) to L6, prevalent translaminar inhibitory inputs, and long-range inputs to L2/3 or L5/6 preferentially from L2/3 or L5/6 of input cortical areas, respectively. These layer-specific input patterns were largely independent of NMDA receptor function in the recipient neurons. mPFC L5 received proportionally more long-range inputs and more local inhibitory inputs than barrel cortex L5. Our results provide new insight into the organization and development of neocortical networks and identify important differences in the circuit organization in sensory and association cortices.
View details for DOI 10.1038/nn.4131
View details for PubMedID 26457553
View details for PubMedCentralID PMC4624522
-
Control of REM sleep by ventral medulla GABAergic neurons
NATURE
2015; 526 (7573): 435-?
Abstract
Rapid eye movement (REM) sleep is a distinct brain state characterized by activated electroencephalogram and complete skeletal muscle paralysis, and is associated with vivid dreams. Transection studies by Jouvet first demonstrated that the brainstem is both necessary and sufficient for REM sleep generation, and the neural circuits in the pons have since been studied extensively. The medulla also contains neurons that are active during REM sleep, but whether they play a causal role in REM sleep generation remains unclear. Here we show that a GABAergic (γ-aminobutyric-acid-releasing) pathway originating from the ventral medulla powerfully promotes REM sleep in mice. Optogenetic activation of ventral medulla GABAergic neurons rapidly and reliably initiated REM sleep episodes and prolonged their durations, whereas inactivating these neurons had the opposite effects. Optrode recordings from channelrhodopsin-2-tagged ventral medulla GABAergic neurons showed that they were most active during REM sleep (REMmax), and during wakefulness they were preferentially active during eating and grooming. Furthermore, dual retrograde tracing showed that the rostral projections to the pons and midbrain and caudal projections to the spinal cord originate from separate ventral medulla neuron populations. Activating the rostral GABAergic projections was sufficient for both the induction and maintenance of REM sleep, which are probably mediated in part by inhibition of REM-suppressing GABAergic neurons in the ventrolateral periaqueductal grey. These results identify a key component of the pontomedullary network controlling REM sleep. The capability to induce REM sleep on command may offer a powerful tool for investigating its functions.
View details for DOI 10.1038/nature14979
View details for Web of Science ID 000362730200052
View details for PubMedID 26444238
View details for PubMedCentralID PMC4852286
-
NEUROSCIENCE. It takes the world to understand the brain.
Science
2015; 350 (6256): 42-44
View details for DOI 10.1126/science.aad4120
View details for PubMedID 26430110
View details for PubMedCentralID PMC4975723
-
Viral-genetic tracing of the input-output organization of a central noradrenaline circuit.
Nature
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
-
Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping
CELL
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
2015; 162 (3): 635-647
Abstract
Recent progress in understanding the diversity of midbrain dopamine neurons has highlighted the importance--and the challenges--of defining mammalian neuronal cell types. Although neurons may be best categorized using inclusive criteria spanning biophysical properties, wiring of inputs, wiring of outputs, and activity during behavior, linking all of these measurements to cell types within the intact brains of living mammals has been difficult. Here, using an array of intact-brain circuit interrogation tools, including CLARITY, COLM, optogenetics, viral tracing, and fiber photometry, we explore the diversity of dopamine neurons within the substantia nigra pars compacta (SNc). We identify two parallel nigrostriatal dopamine neuron subpopulations differing in biophysical properties, input wiring, output wiring to dorsomedial striatum (DMS) versus dorsolateral striatum (DLS), and natural activity patterns during free behavior. Our results reveal independently operating nigrostriatal information streams, with implications for understanding the logic of dopaminergic feedback circuits and the diversity of mammalian neuronal cell types.
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
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
2015; 10 (4)
Abstract
Innate attraction and aversion to odorants are observed throughout the animal kingdom, but how olfactory circuits encode such valences is not well understood, despite extensive anatomical and functional knowledge. In Drosophila melanogaster, ~50 types of olfactory receptor neurons (ORNs) each express a unique receptor gene, and relay information to a cognate type of projection neurons (PNs). To examine the extent to which the population activity of ORNs is required for olfactory behavior, we developed a genetic strategy to block all ORN outputs, and then to restore output in specific types. Unlike attraction, aversion was unaffected by simultaneous silencing of many ORNs, and even single ORN types previously shown to convey neutral valence sufficed to mediate aversion. Thus, aversion may rely on specific activity patterns in individual ORNs rather than the number or identity of activated ORNs. ORN activity is relayed into the brain by downstream circuits, with excitatory PNs (ePN) representing a major output. We found that silencing the majority of ePNs did not affect aversion, even when ePNs directly downstream of single restored ORN types were silenced. Our data demonstrate the robustness of olfactory aversion, and suggest that its circuit mechanism is qualitatively different from attraction.
View details for DOI 10.1371/journal.pone.0125986
View details for Web of Science ID 000353713100121
View details for PubMedID 25927233
View details for PubMedCentralID PMC4416024
-
Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a Drosophila olfactory circuit.
Neuron
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
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
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
-
Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons.
Neuron
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
2015; 35 (24): 8979–85
View details for PubMedID 26085623
-
Intersectional illumination of neural circuit function.
Neuron
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
- Principles of Neurobiology Garland Science. 2015
-
Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain.
Nature communications
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
-
Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain.
Nature communications
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
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 DOI 10.1016/j.cell.2014.10.027
View details for PubMedID 25417155
View details for PubMedCentralID PMC4225456
-
Deterministic Progenitor Behavior and Unitary Production of Neurons in the Neocortex
CELL
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
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
2014; 346 (6209): 626-629
Abstract
Neurotrophins regulate diverse aspects of neuronal development and plasticity, but their precise in vivo functions during neural circuit assembly in the central brain remain unclear. We show that the neurotrophin receptor tropomyosin-related kinase C (TrkC) is required for dendritic growth and branching of mouse cerebellar Purkinje cells. Sparse TrkC knockout reduced dendrite complexity, but global Purkinje cell knockout had no effect. Removal of the TrkC ligand neurotrophin-3 (NT-3) from cerebellar granule cells, which provide major afferent input to developing Purkinje cell dendrites, rescued the dendrite defects caused by sparse TrkC disruption in Purkinje cells. Our data demonstrate that NT-3 from presynaptic neurons (granule cells) is required for TrkC-dependent competitive dendrite morphogenesis in postsynaptic neurons (Purkinje cells)--a previously unknown mechanism of neural circuit development.
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
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.103726
View details for Web of Science ID 000343421200002
View details for PubMedCentralID PMC4194450
-
Drosophila Strip serves as a platform for early endosome organization during axon elongation
NATURE COMMUNICATIONS
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 (New York, N.Y.)
2014; 345 (6197): 660-5
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 PubMedID 25104383
-
SELECTIVE ATTENTION Long-range and local circuits for top-down modulation of visual cortex processing
SCIENCE
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
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
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
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
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
-
Genetic Control of Wiring Specificity in the Fly Olfactory System
GENETICS
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
View details for PubMedCentralID PMC3872183
-
Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins.
eLife
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
-
Dissecting Local Circuits: Parvalbumin Interneurons Underlie Broad Feedback Control of Olfactory Bulb Output
NEURON
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
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
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
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
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
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
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
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 Web of Science ID 000318961700008
-
Mosaic Analysis with Double Markers Reveals Cell-Type-Specific Paternal Growth Dominance
CELL REPORTS
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
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
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
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
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
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
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
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
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
View details for PubMedCentralID PMC3174680
-
Using the Q system in Drosophila melanogaster
NATURE PROTOCOLS
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
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
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
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
View details for PubMedCentralID PMC3095019
-
Cortical representations of olfactory input by trans-synaptic tracing
NATURE
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
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
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
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
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
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
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
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
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
-
The olfactory circuit of the fruit fly Drosophila melanogaster.
Science China. Life sciences
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
-
The olfactory circuit of the fruit fly Drosophila melanogaster
SCIENCE CHINA-LIFE SCIENCES
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 Web of Science ID 000277417800011
-
Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe
NATURE NEUROSCIENCE
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
-
Dendritic tiling through TOR signalling
EMBO JOURNAL
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
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
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
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
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
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
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
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
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
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
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
CURRENT OPINION IN NEUROBIOLOGY
2008; 18 (1): 1-3
View details for DOI 10.1016/j.conb.2008.05.001
View details for Web of Science ID 000257621700001
-
Development of continuous and discrete neural maps
NEURON
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
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
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
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
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
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
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
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
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
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
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
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
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
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
-
Developmental neuroscience - Two gradients are better than one
NATURE
2006; 439 (7072): 23-24
View details for DOI 10.1038/439023a
View details for Web of Science ID 000234378700019
View details for PubMedID 16397483
-
A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila
NATURE PROTOCOLS
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
-
A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining
NATURE PROTOCOLS
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
-
Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit
CURRENT BIOLOGY
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
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
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
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
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
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
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
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
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
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
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
2004; 303 (5655): 179-181
View details for PubMedID 14715999
-
Developmental origin of wiring specificity in the olfactory system of Drosophila
DEVELOPMENT
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
LIPPINCOTT WILLIAMS & WILKINS. 2004: S154–S154
View details for Web of Science ID 000188254600447
-
Food for thought: a receptor finds its ligand
NATURE NEUROSCIENCE
2003; 6 (11): 1119-1120
View details for DOI 10.1038/nn1103-1119
View details for Web of Science ID 000186229200003
View details for PubMedID 14583748
-
Dendritic development of Drosophila high order visual system neurons is independent of sensory experience
BMC NEUROSCIENCE
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
-
Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila
NEURON
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
-
Rho GTPases in neuronal morphogenesis
NATURE REVIEWS NEUROSCIENCE
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
-
Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration and axonal transport
NATURE CELL BIOLOGY
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
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
2000; 26 (1): 1-2
View details for Web of Science ID 000086770500001
View details for PubMedID 10798384
-
split ends encodes large nuclear proteins that regulate neuronal cell fate and axon extension in the Drosophila embryo
DEVELOPMENT
2000; 127 (7): 1517–29
Abstract
split ends (spen) encodes nuclear 600 kDa proteins that contain RNA recognition motifs and a conserved C-terminal sequence. These features define a new protein family, Spen, which includes the vertebrate MINT transcriptional regulator. Zygotic spen mutants affect the growth and guidance of a subset of axons in the Drosophila embryo. Removing maternal and zygotic protein elicits cell-fate and more general axon-guidance defects that are not seen in zygotic mutants. The wrong number of chordotonal neurons and midline cells are generated, and we identify defects in precursor formation and EGF receptor-dependent inductive processes required for cell-fate specification. The number of neuronal precursors is variable in embryos that lack Spen. The levels of Suppressor of Hairless, a key transcriptional effector of Notch required for precursor formation, are reduced, as are the nuclear levels of Yan, a transcriptional repressor that regulates cell fate and proliferation downstream of the EGF receptor. We propose that Spen proteins regulate the expression of key effectors of signaling pathways required to specify neuronal cell fate and morphology.
View details for Web of Science ID 000086810900017
View details for PubMedID 10704397
-
Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis
NEURON
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
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
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
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
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