RESUMEN
Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.
Asunto(s)
Encéfalo/fisiología , Conectoma/métodos , Red Nerviosa/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , RatonesRESUMEN
Animals exhibit a behavioral response to novel sensory stimuli about which they have no prior knowledge. We have examined the neural and behavioral correlates of novelty and familiarity in the olfactory system of Drosophila. Novel odors elicit strong activity in output neurons (MBONs) of the α'3 compartment of the mushroom body that is rapidly suppressed upon repeated exposure to the same odor. This transition in neural activity upon familiarization requires odor-evoked activity in the dopaminergic neuron innervating this compartment. Moreover, exposure of a fly to novel odors evokes an alerting response that can also be elicited by optogenetic activation of α'3 MBONs. Silencing these MBONs eliminates the alerting behavior. These data suggest that the α'3 compartment plays a causal role in the behavioral response to novel and familiar stimuli as a consequence of dopamine-mediated plasticity at the Kenyon cell-MBONα'3 synapse.
Asunto(s)
Drosophila melanogaster/fisiología , Cuerpos Pedunculados/fisiología , Animales , Neuronas Dopaminérgicas/fisiología , Aprendizaje , Memoria , Cuerpos Pedunculados/citología , Odorantes , OlfatoRESUMEN
Assigning behavioral functions to neural structures has long been a central goal in neuroscience and is a necessary first step toward a circuit-level understanding of how the brain generates behavior. Here, we map the neural substrates of locomotion and social behaviors for Drosophila melanogaster using automated machine-vision and machine-learning techniques. From videos of 400,000 flies, we quantified the behavioral effects of activating 2,204 genetically targeted populations of neurons. We combined a novel quantification of anatomy with our behavioral analysis to create brain-behavior correlation maps, which are shared as browsable web pages and interactive software. Based on these maps, we generated hypotheses of regions of the brain causally related to sensory processing, locomotor control, courtship, aggression, and sleep. Our maps directly specify genetic tools to target these regions, which we used to identify a small population of neurons with a role in the control of walking.
Asunto(s)
Mapeo Encefálico/métodos , Drosophila melanogaster/fisiología , Animales , Conducta Animal , Femenino , Locomoción , Masculino , Programas InformáticosRESUMEN
When navigating in their environment, animals use visual motion cues as feedback signals that are elicited by their own motion. Such signals are provided by wide-field neurons sampling motion directions at multiple image points as the animal maneuvers. Each one of these neurons responds selectively to a specific optic flow-field representing the spatial distribution of motion vectors on the retina. Here, we describe the discovery of a group of local, inhibitory interneurons in the fruit fly Drosophila key for filtering these cues. Using anatomy, molecular characterization, activity manipulation, and physiological recordings, we demonstrate that these interneurons convey direction-selective inhibition to wide-field neurons with opposite preferred direction and provide evidence for how their connectivity enables the computation required for integrating opposing motions. Our results indicate that, rather than sharpening directional selectivity per se, these circuit elements reduce noise by eliminating non-specific responses to complex visual information.
Asunto(s)
Interneuronas/citología , Percepción de Movimiento , Vías Nerviosas , Lóbulo Óptico de Animales no Mamíferos/fisiología , Percepción Visual , Animales , Drosophila melanogaster , Interneuronas/fisiología , Lóbulo Óptico de Animales no Mamíferos/citología , Transmisión SinápticaRESUMEN
Nervous systems combine lower-level sensory signals to detect higher-order stimulus features critical to survival, such as the visual looming motion created by an imminent collision or approaching predator. Looming-sensitive neurons have been identified in diverse animal species. Different large-scale visual features such as looming often share local cues, which means loom-detecting neurons face the challenge of rejecting confounding stimuli. Here we report the discovery of an ultra-selective looming detecting neuron, lobula plate/lobula columnar, type II (LPLC2) in Drosophila, and show how its selectivity is established by radial motion opponency. In the fly visual system, directionally selective small-field neurons called T4 and T5 form a spatial map in the lobula plate, where they each terminate in one of four retinotopic layers, such that each layer responds to motion in a different cardinal direction. Single-cell anatomical analysis reveals that each arm of the LPLC2 cross-shaped primary dendrites ramifies in one of these layers and extends along that layer's preferred motion direction. In vivo calcium imaging demonstrates that, as their shape predicts, individual LPLC2 neurons respond strongly to outward motion emanating from the centre of the neuron's receptive field. Each dendritic arm also receives local inhibitory inputs directionally selective for inward motion opposing the excitation. This radial motion opponency generates a balance of excitation and inhibition that makes LPLC2 non-responsive to related patterns of motion such as contraction, wide-field rotation or luminance change. As a population, LPLC2 neurons densely cover visual space and terminate onto the giant fibre descending neurons, which drive the jump muscle motor neuron to trigger an escape take off. Our findings provide a mechanistic description of the selective feature detection that flies use to discern and escape looming threats.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Percepción de Movimiento/fisiología , Animales , Calcio/análisis , Calcio/metabolismo , Dendritas/fisiología , Femenino , Neuronas Motoras/fisiología , Inhibición Neural , Análisis de la Célula IndividualRESUMEN
Although all sensory circuits ascend to higher brain areas where stimuli are represented in sparse, stimulus-specific activity patterns, relatively little is known about sensory coding on the descending side of neural circuits, as a network converges. In insects, mushroom bodies have been an important model system for studying sparse coding in the olfactory system, where this format is important for accurate memory formation. In Drosophila, it has recently been shown that the 2,000 Kenyon cells of the mushroom body converge onto a population of only 34 mushroom body output neurons (MBONs), which fall into 21 anatomically distinct cell types. Here we provide the first, to our knowledge, comprehensive view of olfactory representations at the fourth layer of the circuit, where we find a clear transition in the principles of sensory coding. We show that MBON tuning curves are highly correlated with one another. This is in sharp contrast to the process of progressive decorrelation of tuning in the earlier layers of the circuit. Instead, at the population level, odour representations are reformatted so that positive and negative correlations arise between representations of different odours. At the single-cell level, we show that uniquely identifiable MBONs display profoundly different tuning across different animals, but that tuning of the same neuron across the two hemispheres of an individual fly was nearly identical. Thus, individualized coordination of tuning arises at this level of the olfactory circuit. Furthermore, we find that this individualization is an active process that requires a learning-related gene, rutabaga. Ultimately, neural circuits have to flexibly map highly stimulus-specific information in sparse layers onto a limited number of different motor outputs. The reformatting of sensory representations we observe here may mark the beginning of this sensory-motor transition in the olfactory system.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Percepción Olfatoria/fisiología , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Aprendizaje/fisiología , Masculino , Mutación/genética , Neuronas/clasificación , Vías Olfatorias/fisiología , Desempeño PsicomotorRESUMEN
The Mushroom Body (MB) is the primary location of stored associative memories in the Drosophila brain. We discuss recent advances in understanding the MB's neuronal circuits made using advanced light microscopic methods and cell-type-specific genetic tools. We also review how the compartmentalized nature of the MB's organization allows this brain area to form and store memories with widely different dynamics.
Asunto(s)
Drosophila/fisiología , Aprendizaje/fisiología , Memoria/fisiología , Cuerpos Pedunculados/fisiología , AnimalesRESUMEN
The extraction of directional motion information from changing retinal images is one of the earliest and most important processing steps in any visual system. In the fly optic lobe, two parallel processing streams have been anatomically described, leading from two first-order interneurons, L1 and L2, via T4 and T5 cells onto large, wide-field motion-sensitive interneurons of the lobula plate. Therefore, T4 and T5 cells are thought to have a pivotal role in motion processing; however, owing to their small size, it is difficult to obtain electrical recordings of T4 and T5 cells, leaving their visual response properties largely unknown. We circumvent this problem by means of optical recording from these cells in Drosophila, using the genetically encoded calcium indicator GCaMP5 (ref. 2). Here we find that specific subpopulations of T4 and T5 cells are directionally tuned to one of the four cardinal directions; that is, front-to-back, back-to-front, upwards and downwards. Depending on their preferred direction, T4 and T5 cells terminate in specific sublayers of the lobula plate. T4 and T5 functionally segregate with respect to contrast polarity: whereas T4 cells selectively respond to moving brightness increments (ON edges), T5 cells only respond to moving brightness decrements (OFF edges). When the output from T4 or T5 cells is blocked, the responses of postsynaptic lobula plate neurons to moving ON (T4 block) or OFF edges (T5 block) are selectively compromised. The same effects are seen in turning responses of tethered walking flies. Thus, starting with L1 and L2, the visual input is split into separate ON and OFF pathways, and motion along all four cardinal directions is computed separately within each pathway. The output of these eight different motion detectors is then sorted such that ON (T4) and OFF (T5) motion detectors with the same directional tuning converge in the same layer of the lobula plate, jointly providing the input to downstream circuits and motion-driven behaviours.
Asunto(s)
Drosophila/fisiología , Percepción de Movimiento/fisiología , Vías Visuales/fisiología , Animales , Conducta Animal/fisiología , Drosophila/citología , Interneuronas/fisiología , Locomoción/fisiología , Neuronas/fisiología , Transducción de Señal , Vías Visuales/citologíaRESUMEN
Animal behaviour arises from computations in neuronal circuits, but our understanding of these computations has been frustrated by the lack of detailed synaptic connection maps, or connectomes. For example, despite intensive investigations over half a century, the neuronal implementation of local motion detection in the insect visual system remains elusive. Here we develop a semi-automated pipeline using electron microscopy to reconstruct a connectome, containing 379 neurons and 8,637 chemical synaptic contacts, within the Drosophila optic medulla. By matching reconstructed neurons to examples from light microscopy, we assigned neurons to cell types and assembled a connectome of the repeating module of the medulla. Within this module, we identified cell types constituting a motion detection circuit, and showed that the connections onto individual motion-sensitive neurons in this circuit were consistent with their direction selectivity. Our results identify cellular targets for future functional investigations, and demonstrate that connectomes can provide key insights into neuronal computations.
Asunto(s)
Conectoma , Drosophila/fisiología , Modelos Biológicos , Percepción de Movimiento/fisiología , Vías Visuales/fisiología , Animales , Femenino , Vías Visuales/citologíaRESUMEN
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
Asunto(s)
Drosophila melanogaster/genética , Genoma , Animales , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Biología Computacional , Mapeo Contig , Secuenciación de Nucleótidos de Alto Rendimiento , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Cromosomas Politénicos , Mapeo RestrictivoRESUMEN
We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.
Asunto(s)
Proteínas Luminiscentes/química , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Animales , Antígenos , Mapeo Encefálico , Drosophila , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas , Conformación ProteicaRESUMEN
Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila melanogaster can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. Here we show that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. We therefore propose that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. Our results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Memoria/fisiología , Odorantes/análisis , Recompensa , Animales , Conducta Apetitiva/fisiología , Señalización del Calcio , Dendritas/fisiología , Dopamina/metabolismo , Neuronas Dopaminérgicas/citología , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/metabolismo , Olfato/genética , Olfato/fisiologíaRESUMEN
We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns.
Asunto(s)
Encéfalo/citología , Ojo Compuesto de los Artrópodos/inervación , Drosophila/anatomía & histología , Vías Nerviosas/citología , Neuronas/citología , Coloración y Etiquetado/métodos , Animales , Ojo Compuesto de los Artrópodos/citología , Drosophila/fisiología , Genotipo , Inmunohistoquímica , Microscopía ConfocalRESUMEN
Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Drosophila melanogaster/fisiología , Animales , Animales Modificados Genéticamente , Conducta Apetitiva/fisiología , Carbohidratos , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Femenino , Aprendizaje/fisiología , Memoria a Largo Plazo/fisiología , Memoria a Corto Plazo/fisiología , Cuerpos Pedunculados/fisiología , Odorantes , Recompensa , Olfato/fisiología , Gusto/fisiologíaRESUMEN
Glia play crucial roles in the development and homeostasis of the nervous system. While the GLIA in the Drosophila embryo have been well characterized, their study in the adult nervous system has been limited. Here, we present a detailed description of the glia in the adult nervous system, based on the analysis of some 500 glial drivers we identified within a collection of synthetic GAL4 lines. We find that glia make up â¼10% of the cells in the nervous system and envelop all compartments of neurons (soma, dendrites, axons) as well as the nervous system as a whole. Our morphological analysis suggests a set of simple rules governing the morphogenesis of glia and their interactions with other cells. All glial subtypes minimize contact with their glial neighbors but maximize their contact with neurons and adapt their macromorphology and micromorphology to the neuronal entities they envelop. Finally, glial cells show no obvious spatial organization or registration with neuronal entities. Our detailed description of all glial subtypes and their regional specializations, together with the powerful genetic toolkit we provide, will facilitate the functional analysis of glia in the mature nervous system. GLIA 2017 GLIA 2017;65:606-638.
Asunto(s)
Sistema Nervioso/citología , Neuroglía/clasificación , Neuroglía/fisiología , Animales , Animales Modificados Genéticamente , Antígenos CD8/genética , Antígenos CD8/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The analysis of genetic mosaics, in which an animal carries populations of cells with differing genotypes, is a powerful tool for understanding developmental and cell biology. In 1990, we set out to improve the methods used to make genetic mosaics in Drosophila by taking advantage of recently developed approaches for genome engineering. These efforts led to the work described in our 1993 Development paper.
Asunto(s)
Pruebas Genéticas/estadística & datos numéricos , Mosaicismo , Animales , Animales Modificados Genéticamente , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/fisiología , Drosophila/genética , Pruebas Genéticas/tendencias , Genoma/fisiología , Ratones , Modelos Biológicos , Recombinación Genética/fisiologíaRESUMEN
The ability to specify the expression levels of exogenous genes inserted in the genomes of transgenic animals is critical for the success of a wide variety of experimental manipulations. Protein production can be regulated at the level of transcription, mRNA transport, mRNA half-life, or translation efficiency. In this report, we show that several well-characterized sequence elements derived from plant and insect viruses are able to function in Drosophila to increase the apparent translational efficiency of mRNAs by as much as 20-fold. These increases render expression levels sufficient for genetic constructs previously requiring multiple copies to be effective in single copy, including constructs expressing the temperature-sensitive inactivator of neuronal function Shibire(ts1), and for the use of cytoplasmic GFP to image the fine processes of neurons.
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Drosophila/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Transgenes , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Datos de Secuencia Molecular , ARN Mensajero/genéticaRESUMEN
Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast--KD, B2, B3, and R--are highly active and nontoxic in Drosophila and that KD, B2, B3, and the widely used FLP recombinase have distinct target specificities. We also show that the KD and B3 recombinases are active in mice.
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ADN Nucleotidiltransferasas/metabolismo , Genoma/genética , Animales , Células CHO , Cricetinae , Cricetulus , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Expresión Génica , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Recombinación Genética/genética , Saccharomyces cerevisiae/enzimologíaRESUMEN
How memories of past events influence behavior is a key question in neuroscience. The major associative learning center in Drosophila, the mushroom body (MB), communicates to the rest of the brain through mushroom body output neurons (MBONs). While 21 MBON cell types have their dendrites confined to small compartments of the MB lobes, analysis of EM connectomes revealed the presence of an additional 14 MBON cell types that are atypical in having dendritic input both within the MB lobes and in adjacent brain regions. Genetic reagents for manipulating atypical MBONs and experimental data on their functions have been lacking. In this report we describe new cell-type-specific GAL4 drivers for many MBONs, including the majority of atypical MBONs that extend the collection of MBON driver lines we have previously generated (Aso et al., 2014a; Aso et al., 2016; Aso et al.,
Asunto(s)
Drosophila , Cuerpos Pedunculados , Animales , Drosophila/genética , Encéfalo , Condicionamiento Clásico , NeuronasRESUMEN
Animals are often bombarded with visual information and must prioritize specific visual features based on their current needs. The neuronal circuits that detect and relay visual features have been well-studied. Yet, much less is known about how an animal adjusts its visual attention as its goals or environmental conditions change. During social behaviors, flies need to focus on nearby flies. Here, we study how the flow of visual information is altered when female Drosophila enter an aggressive state. From the connectome, we identified three state-dependent circuit motifs poised to selectively amplify the response of an aggressive female to fly-sized visual objects: convergence of excitatory inputs from neurons conveying select visual features and internal state; dendritic disinhibition of select visual feature detectors; and a switch that toggles between two visual feature detectors. Using cell-type-specific genetic tools, together with behavioral and neurophysiological analyses, we show that each of these circuit motifs function during female aggression. We reveal that features of this same switch operate in males during courtship pursuit, suggesting that disparate social behaviors may share circuit mechanisms. Our work provides a compelling example of using the connectome to infer circuit mechanisms that underlie dynamic processing of sensory signals.