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 the logic of brain wiring using genetic tools. They have developed mosaic marking systems in flies and mice and used them to study how signals are transduced from cell surface receptors to the cytoskeleton, how neuronal processes are pruned, and how neural circuits are organized and built. Dr. Luo is currently a Professor of Biology and an investigator of the Howard Hughes Medical Institute. He teaches neurobiology to Stanford undergraduate and graduate students. He recently published a single-author textbook entitled “Principles of Neurobiology.”
Dr. Luo has served on the editorial boards of several scientific journals, including Neuron, eLife, and Annual Review of Neuroscience. 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, and the Lawrence Katz Prize. Dr. Luo is a Member of the National Academy of Sciences and a Fellow of the American Academy of Arts and Sciences.
Honors & Awards
Technology Innovation Award in Neuroscience, McKnight Foundation (2002)
Young Investigator Award, Society for Neuroscience (2002)
Investigator, Howard Hughes Medical Institute (2005)
Jacob Javits Award, National Institute of Neurological Disorders and Stroke (2005)
H.W.Mossman Award, American Association of Anatomists (2007)
Fellow, American Association for the Advancement of Science (2011)
Fellow, American Academy of Arts and Sciences (2012)
Member, National Academy of Sciences (2012)
The Lawrence C. Katz Prize for Innovative Research in Neuroscience, Duke University (2013)
B.S., Univ. of Sci. & Tech. of China, Molecular Biology (1986)
Ph.D., Brandeis University, Biology (1992)
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. Organization of the olfactory system
The olfactory systems from flies to mammals use a similar organizational principle. Olfactory receptor neurons (ORNs) expressing the same odorant receptor project their axons to the same glomerulus. Projection neurons (PNs) send dendrites to individual glomeruli, and relay olfactory information via their axons to high olfactory centers. Using MARCM (see below) to label individual fly PNs, we found that PN axon terminals exhibit striking stereotypy at the lateral horn according to the glomeruli they send dendrites to. Axon terminals of PNs representing food odors are spatially segregated from those that represent mating pheromones. By contrast, PN axon terminal arborizations in the mushroom body, the olfactory learning and memory center, exhibit much less stereotypy. We are currently using two-photon calcium imaging, optogenetics, and quantitative behavioral assays to identify principles of information processing at the antennal lobe and in higher olfactory centers suggested by previous anatomical studies. We are also investigating how the glomerular map in the mouse olfactory bulb is represented in olfactory cortex using virus-mediated trans-synaptic tracing.
2. Development of wiring specificity in the fly olfactory system
The assembly of the fly olfactory system requires precise glomerular targeting of axons from each of the 50 ORN classes, as well as dendrites of each of the 50 PN classes. We are using this neural circuit as a model to investigate the general principles by which precise wiring specificity arises during development. Our previous studies have shown that PN dendrite patterning precedes ORN axon targeting. PN dendrite targeting relies on global cues in the form of gradients, as well as local cues distributed in a salt-and-pepper fashion on dendrites projecting to different glomeruli. Targeting of ORN axons may use the same molecules as PN dendrite targeting, but via distinct mechanisms including axon-axon interactions and axon-target interactions. We are currently performing systematic genetic studies to identify the cell-surface code ORNs and PNs use to form specific connections at stereotypically organized glomeruli.
3. Developmental neurobiology
In addition to our focus on the olfactory system, we are investigating several other developmental neurobiological problems. These include mechanisms of axon pruning, the roles of neuronal activity in neuronal maturation and incorporation into functional circuits, and cell autonomous functions of genes that are implicated in human neurological disorders. We are using both fly and mouse systems to study these problems.
4. Creating genetic tools
In the process of dissecting the adult organization and developmental assembly of complex neural circuits, we have created several useful genetic tools. The MARCM method (Mosaic Analysis with a Repressible Cell Marker) enables the visualization and genetic manipulation of small populations of cells or single neurons in a mosaic fly. We have developed a new repressible binary expression system, the Q system, which has many applications and is helping us to study several problems described above.
We have also developed a mosaic method in the mouse called MADM (Mosaic Analysis with Double Markers) that allows sparse labeling and genetic manipulation of individual cells or cells that share the same lineage with distinct colors in mosaic animals. We have used MADM as trace lineages and study cell autonomous gene functions in neural developmental processes. We are currently expanding the MADM technique to other mouse chromosomes, and will use MADM to study several developmental neurobiological problems (see above). We have also developed other useful mice, such as a double fluorescent Cre reporter and synapse labeling tools in vivo.
- Exploring Neural Circuits
BIO 222 (Spr)
Independent Studies (10)
- Advanced Research Laboratory in Experimental Biology
BIO 199 (Aut, Win, Spr, Sum)
- Directed Reading in Biology
BIO 198 (Aut, Win, Spr, Sum)
- Directed Reading in Neurosciences
NEPR 299 (Aut, Win, Spr, Sum)
- Graduate Research
BIO 300 (Aut, Win, Spr, Sum)
- Graduate Research
NEPR 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Spr, Sum)
- Out-of-Department Directed Reading
BIO 198X (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Win, Spr, Sum)
- Teaching of Biology
BIO 290 (Aut, Win, Spr)
- Advanced Research Laboratory in Experimental Biology
Prior Year Courses
- Exploring Neural Circuits
BIO 222 (Spr)
- Molecular and Cellular Neurobiology
BIO 154 (Win)
- Molecular and Cellular Neurobiology
BIO 254 (Win)
- Exploring Neural Circuits
BIO 222 (Win)
- Exploring Neural Circuits
Doctoral Dissertation Reader (AC)
Eddy Albarran, Jose Andrade Lopez, Daniel Friedman, Michelle Pang
Postdoctoral Faculty Sponsor
Drew Friedmann, Wei-Hsiang Huang, Justus Kebschull, Hongjie Li, Tongchao Li, Daniel Tyler Pederick, Jing Ren, Mark Wagner, Laura Wilke
Doctoral Dissertation Advisor (AC)
Doctoral Dissertation Co-Advisor (AC)
Postdoctoral Research Mentor
Graduate and Fellowship Programs
Biology (School of Humanities and Sciences) (Phd Program)
Genetic Dissection of Neural Circuits: A Decade of Progress
2018; 98 (2): 256–81
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 DOI 10.1016/j.neuron.2018.03.040
View details for Web of Science ID 000430440900008
View details for PubMedID 29673479
View details for PubMedCentralID PMC5912347
Teneurin-3 controls topographic circuit assembly in the hippocampus.
2018; 554 (7692): 328–33
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 DOI 10.1038/nature25463
View details for PubMedID 29414938
Cerebellar granule cells encode the expectation of reward
2017; 544 (7648): 96-?
The human brain contains approximately 60 billion cerebellar granule cells, which outnumber all other brain neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes. Although evidence suggests a role for the cerebellum in cognition, granule cells are known to encode only sensory and motor context. Here, using two-photon calcium imaging in behaving mice, we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed sugar-water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward-omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from the current understanding of these neurons and markedly enriches the contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum.
View details for DOI 10.1038/nature21726
View details for Web of Science ID 000398323300040
View details for PubMedID 28321129
Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-Cell RNA Sequencing.
2017; 171 (5): 1206–20.e22
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 DOI 10.1016/j.cell.2017.10.019
View details for PubMedID 29149607
Thirst-associated preoptic neurons encode an aversive motivational drive.
Science (New York, N.Y.)
2017; 357 (6356): 1149–55
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 DOI 10.1126/science.aan6747
View details for PubMedID 28912243
Viral-genetic tracing of the input-output organization of a central noradrenaline circuit.
2015; 524 (7563): 88-92
Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input-output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The 'tracing the relationship between input and output' method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input-output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.
View details for DOI 10.1038/nature14600
View details for PubMedID 26131933
- Principles of Neurobiology Garland Science. 2015
- Dendrite morphogenesis depends on relative levels of NT-3/TrkC signaling SCIENCE 2014; 346 (6209): 626-629
Teneurins instruct synaptic partner matching in an olfactory map
2012; 484 (7393): 201-U82
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
Functional circuit architecture underlying parental behaviour
2018; 556 (7701): 326-+
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 DOI 10.1038/s41586-018-0027-0
View details for Web of Science ID 000430285200042
View details for PubMedID 29643503
View details for PubMedCentralID PMC5908752
Linking neuronal lineage and wiring specificity
2018; 13: 5
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 DOI 10.1186/s13064-018-0102-0
View details for Web of Science ID 000429994900001
View details for PubMedID 29653548
View details for PubMedCentralID PMC5899351
Genetic tagging of active neurons in auditory cortex reveals maternal plasticity of coding ultrasonic vocalizations
2018; 9: 871
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 DOI 10.1038/s41467-018-03183-2
View details for Web of Science ID 000426275300002
View details for PubMedID 29491360
View details for PubMedCentralID PMC5830453
A Subpopulation of Striatal Neurons Mediates Levodopa-Induced Dyskinesia
2018; 97 (4): 787-+
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 DOI 10.1016/j.neuron.2018.01.017
View details for Web of Science ID 000425713200008
View details for PubMedID 29398356
- Lineage-dependent spatial and functional organization of the mammalian enteric nervous system SCIENCE 2017; 356 (6339): 722-726
Global Representations of Goal-Directed Behavior in Distinct Cell Types of Mouse Neocortex
2017; 94 (4): 891-?
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 Web of Science ID 000401415100024
View details for PubMedID 28521139
Breathing control center neurons that promote arousal in mice
2017; 355 (6332): 1411-1415
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.
2017; 93 (4): 822-839 e6
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
Molecular and Neural Functions of Rai1, the Causal Gene for Smith-Magenis Syndrome.
2016; 92 (2): 392-406
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
Wiring and Molecular Features of Prefrontal Ensembles Representing Distinct Experiences
2016; 165 (7): 1776-1788
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 Web of Science ID 000378062000026
View details for PubMedID 27238022
Developmental Sculpting of Intracortical Circuits by MHC Class I H2-Db and H2-Kb.
2016; 26 (4): 1453-1463
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
View details for PubMedCentralID PMC4785944
Organization of the Locus Coeruleus-Norepinephrine System
2015; 25 (21): R1051-R1056
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
- Connectivity of mouse somatosensory and prefrontal cortex examined with trans-synaptic tracing. Nature neuroscience 2015; 18 (11): 1687-1697
- Control of REM sleep by ventral medulla GABAergic neurons NATURE 2015; 526 (7573): 435-?
- NEUROSCIENCE. It takes the world to understand the brain. Science 2015; 350 (6256): 42-44
- Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping CELL 2015; 162 (3): 622-634
- Intact-Brain Analyses Reveal Distinct Information Carried by SNc Dopamine Subcircuits CELL 2015; 162 (3): 635-647
A transcriptional reporter of intracellular Ca(2+) in Drosophila.
2015; 18 (6): 917-925
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)
Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a Drosophila olfactory circuit.
2015; 85 (5): 1013-1028
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
Improved and expanded Q-system reagents for genetic manipulations
2015; 12 (3): 219-?
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
Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons.
2015; 85 (2): 429-438
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
Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain.
2015; 6: 6768-?
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-?
- Deterministic Progenitor Behavior and Unitary Production of Neurons in the Neocortex CELL 2014; 159 (4): 775-788
Functional transformations of odor inputs in the mouse olfactory bulb
FRONTIERS IN NEURAL CIRCUITS
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 Web of Science ID 000346507900001
View details for PubMedID 25408637
View details for PubMedCentralID PMC4219419
- Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins ELIFE 2014; 3
Drosophila Strip serves as a platform for early endosome organization during axon elongation
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
SELECTIVE ATTENTION Long-range and local circuits for top-down modulation of visual cortex processing
2014; 345 (6197): 660-665
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.
2014; 83 (3): 645-662
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.
2014; 46 (7): 748-752
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
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)
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
2014; 196 (1): 17-29
Precise connections established between pre- and postsynaptic partners during development are essential for the proper function of the nervous system. The olfactory system detects a wide variety of odorants and processes the information in a precisely connected neural circuit. A common feature of the olfactory systems from insects to mammals is that the olfactory receptor neurons (ORNs) expressing the same odorant receptor make one-to-one connections with a single class of second-order olfactory projection neurons (PNs). This represents one of the most striking examples of targeting specificity in developmental neurobiology. Recent studies have uncovered central roles of transmembrane and secreted proteins in organizing this one-to-one connection specificity in the olfactory system. Here, we review recent advances in the understanding of how this wiring specificity is genetically controlled and focus on the mechanisms by which transmembrane and secreted proteins regulate different stages of the Drosophila olfactory circuit assembly in a coordinated manner. We also discuss how combinatorial coding, redundancy, and error-correcting ability could contribute to constructing a complex neural circuit in general.
View details for DOI 10.1534/genetics.113.154336
View details for Web of Science ID 000330579100002
View details for PubMedID 24395823
Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins.
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
Deterministic progenitor behavior and unitary production of neurons in the neocortex.
2014; 159 (4): 775–88
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
Dissecting Local Circuits: Parvalbumin Interneurons Underlie Broad Feedback Control of Olfactory Bulb Output
2013; 80 (5): 1232-1245
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
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.
2013; 79 (5): 917-931
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
2013; 23 (13): 1163-1172
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.
2013; 78 (5): 773-784
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.
2013; 78 (4): 673-686
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.
2013; 78 (3): 456-468
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
Mosaic Analysis with Double Markers Reveals Cell-Type-Specific Paternal Growth Dominance
2013; 3 (3): 960-967
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
- Neuroscience. dSarm-ing axon degeneration. Science 2012; 337 (6093): 418-419
The SUMO Protease Verloren Regulates Dendrite and Axon Targeting in Olfactory Projection Neurons
JOURNAL OF NEUROSCIENCE
2012; 32 (24): 8331-8340
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
Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice
2012; 484 (7393): 237-U122
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
Controlling gene expression with the Q repressible binary expression system in Caenorhabditis elegans
2012; 9 (4): 391-U105
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
2012; 7 (3)
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
2011; 72 (5): 734-747
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 Web of Science ID 000297971100009
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
To understand how the nervous system processes information, a map of the connections among neurons would be of great benefit. Here we describe the use of vesicular stomatitis virus (VSV) for tracing neuronal connections in vivo. We made VSV vectors that used glycoprotein (G) genes from several other viruses. The G protein from lymphocytic choriomeningitis virus endowed VSV with the ability to spread transsynaptically, specifically in an anterograde direction, whereas the rabies virus glycoprotein gave a specifically retrograde transsynaptic pattern. The use of an avian G protein fusion allowed specific targeting of cells expressing an avian receptor, which allowed a demonstration of monosynaptic anterograde tracing from defined cells. Synaptic connectivity of pairs of virally labeled cells was demonstrated by using slice cultures and electrophysiology. In vivo infections of several areas in the mouse brain led to the predicted patterns of spread for anterograde or retrograde tracers.
View details for DOI 10.1073/pnas.1110854108
View details for Web of Science ID 000294804900082
View details for PubMedID 21825165
Using the Q system in Drosophila melanogaster
2011; 6 (8): 1105-1120
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
2011; 146 (2): 209-221
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
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
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
A Combinatorial Semaphorin Code Instructs the Initial Steps of Sensory Circuit Assembly in the Drosophila CNS
2011; 70 (2): 281-298
Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, whereas broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.
View details for DOI 10.1016/j.neuron.2011.02.050
View details for Web of Science ID 000291073700010
View details for PubMedID 21521614
Cortical representations of olfactory input by trans-synaptic tracing
2011; 472 (7342): 191-196
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
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
Genetic Mosaic Dissection of Lis1 and Ndel1 in Neuronal Migration
2010; 68 (4): 695-709
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
Ten years of Nature Reviews Neuroscience: insights from the highly cited
NATURE REVIEWS NEUROSCIENCE
2010; 11 (10): 718-?
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
Patterning Axon Targeting of Olfactory Receptor Neurons by Coupled Hedgehog Signaling at Two Distinct Steps
2010; 142 (6): 954-966
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
2010; 142 (5): 679-681
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
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
Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain
2010; 5 (7)
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
The Q System: A Repressible Binary System for Transgene Expression, Lineage Tracing, and Mosaic Analysis
2010; 141 (3): 536-548
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
The olfactory circuit of the fruit fly Drosophila melanogaster.
Science China. Life sciences
2010; 53 (4): 472-484
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
Diversity and wiring variability of olfactory local interneurons in the Drosophila antennal lobe
2010; 13 (4): 439-U60
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
Leucine-rich repeat transmembrane proteins instruct discrete dendrite targeting in an olfactory map
2009; 12 (12): 1542-U89
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
- Neuroscience. Brain wiring by presorting axons. Science 2009; 325 (5940): 544-545
Uncoupling Dendrite Growth and Patterning: Single-Cell Knockout Analysis of NMDA Receptor 2B
2009; 62 (2): 205-217
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
2009; 136 (1): 23-25
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
2008; 18 (22): 1754-1759
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
Octopamine fuels fighting flies
2008; 11 (9): 989-990
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
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
Genetic dissection of neural circuits
2008; 57 (5): 634-660
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
Timing neurogenesis and differentiation: Insights from quantitative clonal analyses of cerebellar granule cells
JOURNAL OF NEUROSCIENCE
2008; 28 (10): 2301-2312
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
2008; 14 (2): 227-238
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
Development of continuous and discrete neural maps
2007; 56 (2): 284-300
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
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
View details for PubMedCentralID PMC2096471
A global double-fluorescent cre reporter mouse
2007; 45 (9): 593-605
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
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
2007; 10 (7): 828-837
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
2007; 128 (6): 1187-1203
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
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
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
Intrinsic control of precise dendritic targeting by an ensemble of transcription factors
2007; 17 (3): 278-285
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
2007; 128 (2): 399-410
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 Web of Science ID 000244420500022
View details for PubMedID 17254975
Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions
2007; 53 (2): 185-200
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
2006; 50 (6): 883-895
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
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
2006; 9 (3): 349-355
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
The olfactory system discriminates a large number of odorants using precisely wired neural circuits. It offers an excellent opportunity to study mechanisms of neuronal wiring specificity at the single synapse level. Each olfactory receptor neuron typically expresses only one olfactory receptor from many receptor genes (1000 in mice). In mice, this striking singularity appears to be ensured by a negative feedback mechanism. Olfactory receptor neurons expressing the same receptor converge their axons to stereotypical positions with high precision, a feature that is conserved from insects to mammals. Several molecules have recently been identified that control this process, including olfactory receptors themselves in mice. The second order neurons, mitral cells in mammals and projection neurons in insects, have a similar degree of wiring specificity: studies in Drosophila suggest that projection neuron-intrinsic mechanisms regulate their precise dendritic targeting. Finally, recent studies have revealed interactions of different cell types during circuit assembly, including axon-axon interactions among olfactory receptor neurons and dendro-dendritic interactions of projection neurons, that are essential in establishing wiring specificity of the olfactory circuit.
View details for DOI 10.1016/j.conb.2005.12.002
View details for Web of Science ID 000236136200010
View details for PubMedID 16377177
A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining
2006; 1 (4): 2110-2115
This protocol describes a basic method for dissection and immunofluorescence staining of the Drosophila brain at various developmental stages. The Drosophila brain has become increasingly useful for studies of neuronal wiring and morphogenesis in combination with techniques such as the 'mosaic analysis with a repressible cell marker' (MARCM) system, where single neurons can be followed in live and fixed tissues for high-resolution analysis of wild-type or genetically manipulated cells. Such high-resolution anatomical study of the brain is also important in characterizing the organization of neural circuits using genetic tools such as GAL4 enhancer trap lines, as Drosophila has been intensively used for studying the neural basis of behavior. Advantages of fluorescence immunostaining include compatibility with multicolor labeling and confocal or multiphoton imaging. This brain dissection and immunofluorescence staining protocol requires approximately 2 to 6 d to complete.
View details for DOI 10.1038/nprot.2006.336
View details for Web of Science ID 000251155500055
View details for PubMedID 17487202
A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila
2006; 1 (6): 2583-2589
Mosaic analysis with a repressible cell marker (MARCM) is a genetic technique used in Drosophila to label single cells or multiple cells sharing a single progenitor. Labeled homozygous mutant cells can be generated in an otherwise unlabeled heterozygous animal. Mutant or wild-type labeled cells can also be made to express one or more transgenes. Major applications of MARCM include (i) lineage analysis, (ii) investigating gene function in single or small populations of cells and (iii) neuronal circuit tracing. Our laboratory uses MARCM primarily to label and genetically manipulate neurons; however, this protocol can be adapted to any cell of interest. The protocol involves generating two fly stocks with the necessary genetic elements for MARCM analysis and subsequently generating MARCM clones. Labeled clones can be followed in live and fixed tissues for high-resolution analysis of wild-type or genetically manipulated cells.NOTE: In the PDF version of this article initially published online, the first "FRT" and the "Mutation" labels in Figure 1b were transposed. In both the PDF and HTML versions, "mutant" was omitted from the label on the right, which should read "Labeled homozygous mutant daughter cell". The figure has been corrected in all versions of the article.
View details for DOI 10.1038/nprot.2006.320
View details for Web of Science ID 000251155700008
View details for PubMedID 17406512
Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit
2005; 15 (11): 982-992
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
2005; 121 (3): 479-492
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
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
Developmentally programmed remodeling of the Drosophila olfactory circuit
2005; 132 (4): 725-737
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
Axon retraction and degeneration in development and disease
ANNUAL REVIEW OF NEUROSCIENCE
2005; 28: 127-156
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
2004; 44 (5): 779-793
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
2004; 119 (2): 148-149
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
2004; 7 (8): 819-825
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
2004; 14 (8): 678-684
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
2004; 42 (1): 63-75
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.
2004; 303 (5655): 179-181
View details for PubMedID 14715999
Developmental origin of wiring specificity in the olfactory system of Drosophila
2004; 131 (1): 117-130
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
Dendritic development of Drosophila high order visual system neurons is independent of sensory experience
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
2003; 38 (6): 871-885
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
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
2003; 130 (6): 1203-1213
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
2003; 112 (2): 157-167
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
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
2002; 109 (2): 243-255
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
2002; 416 (6879): 442-447
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 Web of Science ID 000174607800050
View details for PubMedID 11919635
Rac function and regulation during Drosophila development
2002; 416 (6879): 438-442
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
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
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
2001; 414 (6860): 204-208
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
Regulating axon branch stability: The role of p190 RhoGAP in repressing a retraction signaling pathway
2001; 107 (2): 195-207
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
2001; 105 (1): 81-91
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?
2001; 4 (4): 359-365
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
2001; 11 (2): 99-104
Mushroom bodies in the Drosophila brain are centers for olfactory learning and memory. We have previously shown that the mushroom bodies comprise three types of neurons with distinct axonal projections. These three types of neurons are generated sequentially from common neuroblasts. We report here the identification of a gene that we have named enoki mushroom (enok), which when it is mutated gives rise to mushroom bodies with reduced axonal structures. enok encodes a putative histone acetyltransferase (HAT) of the MYST family, members of which have been implicated as important modulators of transcriptional activity. A single amino acid change in the zinc finger motif of the putative catalytic HAT domain gives the same phenotype as a null allele, and this finding indicates the importance of HAT activity to Enok's function. Further phenotypic analysis demonstrates that the mushroom body defect is due to an arrest in neuroblast proliferation rather than a failure of either cell fate switching or axon branching. Clonal analyses in the wing discs and the ovaries suggest that enok is essential for normal cell proliferation in some, but not all, tissues. Our results provide in vivo evidence for essential functions of a histone acetyltransferase in the construction of the Drosophila brain.
View details for Web of Science ID 000169076200018
View details for PubMedID 11231125
Rho GTPases in neuronal morphogenesis
NATURE REVIEWS NEUROSCIENCE
2000; 1 (3): 173-180
The Rho family of small GTPases act as intracellular molecular switches that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. Recent evidence implicates Rho GTPases in the regulation of neuronal morphogenesis, including migration, polarity, axon growth and guidance, dendrite elaboration and plasticity, and synapse formation. Signalling pathways from membrane receptors to Rho GTPases and from Rho GTPases to the actin cytoskeleton are beginning to be discovered. Mutations in these signalling pathways have been reported in human neurological diseases, which underscores their importance in the development and function of the nervous system.
View details for Web of Science ID 000165764700015
View details for PubMedID 11257905
Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila
2000; 28 (3): 807-818
Neuronal process remodeling occurs widely in the construction of both invertebrate and vertebrate nervous systems. During Drosophila metamorphosis, gamma neurons of the mushroom bodies (MBs), the center for olfactory learning in insects, undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes. To elucidate the underlying molecular mechanisms, we conducted a genetic mosaic screen and identified one ultraspiracle (usp) allele defective in larval process pruning. Consistent with the notion that USP forms a heterodimer with the ecdysone receptor (EcR), we found that the EcR-B1 isoform is specifically expressed in the MB gamma neurons, and is required for the pruning of larval processes. Surprisingly, most identified primary EcR/USP targets are dispensable for MB neuronal remodeling. Our study demonstrates cell-autonomous roles for EcR/USP in controlling neuronal remodeling, potentially through novel downstream targets.
View details for Web of Science ID 000166057500018
View details for PubMedID 11163268
Drosophila Lis1 is required for neuroblast proliferation, dendritic elaboration and axonal transport
NATURE CELL BIOLOGY
2000; 2 (11): 776-783
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
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
Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis
2000; 25 (2): 307-316
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
2000; 10 (5): 582-586
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
1999; 126 (18): 4065-4076
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
1999; 22 (3): 451-461
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
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