RESUMO
Invertebrate chordates, such as the tunicate Ciona, can offer insight into the evolution of the chordate phylum. Anatomical features that are shared between invertebrate chordates and vertebrates may be taken as evidence of their presence in a common chordate ancestor. The central nervous systems of Ciona larvae and vertebrates share a similar anatomy despite the Ciona CNS having ~180 neurons. However, the depth of conservation between the Ciona CNS and those in vertebrates is not resolved. The Ciona caudal CNS, while appearing spinal cord-like, has hitherto been thought to lack motor neurons, bringing into question its homology with the vertebrate spinal cord. We show here that the Ciona larval caudal CNS does, in fact, have functional motor neurons along its length, pointing to the presence of a spinal cord-like structure at the base of the chordates. We extend our analysis of shared CNS anatomy further to explore the Ciona "motor ganglion", which has been proposed to be a homolog of the vertebrate hindbrain, spinal cord, or both. We find that a cluster of neurons in the dorsal motor ganglion shares anatomical location, developmental pathway, neural circuit architecture, and gene expression with the vertebrate cerebellum. However, functionally, the Ciona cluster appears to have more in common with vertebrate cerebellum-like structures, insofar as it receives and processes direct sensory input. These findings are consistent with earlier speculation that the cerebellum evolved from a cerebellum-like structure, and suggest that the latter structure was present in the dorsal hindbrain of a common chordate ancestor.
RESUMO
The detection of visual motion enables sophisticated animal navigation, and studies on flies have provided profound insights into the cellular and circuit bases of this neural computation. The fly's directionally selective T4 and T5 neurons encode ON and OFF motion, respectively. Their axons terminate in one of the four retinotopic layers in the lobula plate, where each layer encodes one of the four directions of motion. Although the input circuitry of the directionally selective neurons has been studied in detail, the synaptic connectivity of circuits integrating T4/T5 motion signals is largely unknown. Here, we report a 3D electron microscopy reconstruction, wherein we comprehensively identified T4/T5's synaptic partners in the lobula plate, revealing a diverse set of new cell types and attributing new connectivity patterns to the known cell types. Our reconstruction explains how the ON- and OFF-motion pathways converge. T4 and T5 cells that project to the same layer connect to common synaptic partners and comprise a core motif together with bilayer interneurons, detailing the circuit basis for computing motion opponency. We discovered pathways that likely encode new directions of motion by integrating vertical and horizontal motion signals from upstream T4/T5 neurons. Finally, we identify substantial projections into the lobula, extending the known motion pathways and suggesting that directionally selective signals shape feature detection there. The circuits we describe enrich the anatomical basis for experimental and computations analyses of motion vision and bring us closer to understanding complete sensory-motor pathways.
Assuntos
Drosophila melanogaster , Percepção de Movimento , Animais , Drosophila melanogaster/fisiologia , Interneurônios/fisiologia , Percepção de Movimento/fisiologia , Neurônios/fisiologia , Vias Visuais/fisiologiaRESUMO
Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly's delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging.
Assuntos
Conectoma , Drosophila , Animais , Drosophila/fisiologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Volume , Sinapses/fisiologia , Encéfalo/fisiologiaRESUMO
Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. Neurons have presumably optimized the volume of their bioenergetic machinery to ensure these power demands are met, but the relationship between presynaptic power demands and the volume available to the bioenergetic machinery has never been quantified. Here, we estimated the power demands of six motor nerve terminals in female Drosophila larvae through direct measurements of neurotransmitter release and Ca2+ entry, and via theoretical estimates of Na+ entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio (â¼66 nmol·min-1·µl-1) harbor the largest mitochondria packed at the greatest density. If we assume sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation, then these mitochondria are required to produce ATP at a rate of 52 nmol·min-1·µl-1 at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol·min-1·µl-1 of cytosol at rest, rising to 4.36 during activity. These data provide a quantitative framework for presynaptic bioenergetics in situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand.SIGNIFICANCE STATEMENT The remarkable energy demands of the brain are supported by the complete oxidation of its fuel but debate continues regarding a division of labor between glycolysis and oxidative phosphorylation across different cell types. Here, we exploit the neuromuscular synapse, a model for studying neurophysiology, to elucidate fundamental aspects of neuronal energy metabolism that ultimately constrain rates of neural processing. We quantified energy production rates required to sustain activity at individual nerve terminals and compared these with the volume capable of oxidative phosphorylation (mitochondria) and glycolysis (cytosol). We find strong support for oxidative phosphorylation playing a primary role in presynaptic terminals and provide the first in vivo estimates of energy production rates per unit volume of presynaptic mitochondria and cytosol.
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Encéfalo/fisiologia , Metabolismo Energético/fisiologia , Tamanho Mitocondrial/fisiologia , Neurônios Motores/fisiologia , Terminações Pré-Sinápticas/fisiologia , Animais , Drosophila , Feminino , Mitocôndrias/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Understanding the structure and operation of any nervous system has been a subject of research for well over a century. A near-term opportunity in this quest is to understand the brain of a model species, the fruit fly Drosophila melanogaster. This is an enticing target given its relatively small size (roughly 200,000 neurons), coupled with the behavioral richness that this brain supports, and the wide variety of techniques now available to study both brain and behavior. It is clear that within a few years we will possess a connectome for D. melanogaster: an electron-microscopy-level description of all neurons and their chemical synaptic connections. Given what we will soon have, what we already know and the research that is currently underway, what more do we need to know to enable us to understand the fly's brain? Here, we itemize the data we will need to obtain, collate and organize in order to build an integrated model of the brain of D. melanogaster.
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Conectoma , Animais , Encéfalo , Drosophila , Drosophila melanogaster , NeurôniosRESUMO
Stemmata of strepsipteran insects represent the smallest arthropod eyes known, having photoreceptors which form fused rhabdoms measuring an average size of 1.69 × 1.21 × 1.04 µm and each occupying a volume of only 0.97-1.16 µm3. The morphology of the stemmata of the extremely miniaturized first instar larva of Stylops ovinae (Strepsiptera, Stylopidae) was investigated using serial-sectioning transmission electron microscopy (ssTEM). Our 3D reconstruction revealed that, despite different proportions, all three stemmata maintain the same organization: a biconvex corneal lens, four corneagenous cells and five photoreceptor (retinula) cells. No pigment-containing cell-types were found to adjoin the corneagenous cells. Whereas the retinula cells are adapted to the limited space by having laterally bulged median regions, containing mitochondria and the smallest nuclei yet reported for arthropods (1.37 µm3), special adaptations are found in the corneagenous cells which have cell volumes down to 1 µm3. The corneagenous cells lack nuclei and pigment granules and bear only a few mitochondria (up to three) or none at all. Morphological adaptations due to miniaturization are discussed in the context of photoreceptor function and the visual needs of the larva.
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Holometábolos , Imageamento Tridimensional , Animais , Insetos , Larva , Células FotorreceptorasRESUMO
The dogma that the synaptic cleft acidifies during neurotransmission is based on the corelease of neurotransmitters and protons from synaptic vesicles, and is supported by direct data from sensory ribbon-type synapses. However, it is unclear whether acidification occurs at non-ribbon-type synapses. Here we used genetically encoded fluorescent pH indicators to examine cleft pH at conventional neuronal synapses. At the neuromuscular junction of female Drosophila larvae, we observed alkaline spikes of over 1 log unit during fictive locomotion in vivo. Ex vivo, single action potentials evoked alkalinizing pH transients of only â¼0.01 log unit, but these transients summated rapidly during burst firing. A chemical pH indicator targeted to the cleft corroborated these findings. Cleft pH transients were dependent on Ca2+ movement across the postsynaptic membrane, rather than neurotransmitter release per se, a result consistent with cleft alkalinization being driven by the Ca2+/H+ antiporting activity of the plasma membrane Ca2+-ATPase at the postsynaptic membrane. Targeting the pH indicators to the microenvironment of the presynaptic voltage gated Ca2+ channels revealed that alkalinization also occurred within the cleft proper at the active zone and not just within extrasynaptic regions. Application of the pH indicators at the mouse calyx of Held, a mammalian central synapse, similarly revealed cleft alkalinization during burst firing in both males and females. These findings, made at two quite different non-ribbon type synapses, suggest that cleft alkalinization during neurotransmission, rather than acidification, is a generalizable phenomenon across conventional neuronal synapses.SIGNIFICANCE STATEMENT Neurotransmission is highly sensitive to the pH of the extracellular milieu. This is readily evident in the neurological symptoms that accompany systemic acid/base imbalances. Imaging data from sensory ribbon-type synapses show that neurotransmission itself can acidify the synaptic cleft, likely due to the corelease of protons and glutamate. It is not clear whether the same phenomenon occurs at conventional neuronal synapses due to the difficulties in collecting such data. If it does occur, it would provide for an additional layer of activity-dependent modulation of neurotransmission. Our findings of alkalinization, rather than acidification, within the cleft of two different neuronal synapses encourages a reassessment of the scope of activity-dependent pH influences on neurotransmission and short-term synaptic plasticity.
Assuntos
Ácido Glutâmico/metabolismo , Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Animais , Drosophila , Feminino , Concentração de Íons de Hidrogênio , Plasticidade Neuronal/fisiologia , Vesículas Sinápticas/metabolismoRESUMO
Pigment granules, found in different cell types of the retina in insect compound eyes, fulfill important functions. They isolate the individual ommatidia from stray light, regulate the angular sensitivity, and restrict the light that reaches the photoreceptor according to ambient light intensities. Descriptions of pigment cells within the retina are included in ultrastructural eye descriptions, but knowledge of pigment cell types beneath the retina and basal matrix (BM) are relatively limited in insects. In the miniaturized parasitoid wasp Trichogramma evanescens Westwood 1833, a sub-retinal pigment shield is formed by pigment-bearing cells, which appear in two-dimensional TEM sections to form a separate population beneath the BM. By using three-dimensional reconstructions of serial-section transmission electron microscopy, it was possible to reveal that the sub-retinal pigment shield of T. evanescens is not formed by a separate cell type, but by extensions of the lateral rim pigment cells that penetrate gaps in the BM. The reconstruction is supported by evidence from a statistical analysis of pigment granule volumes of all pigment bearing cell types in the retina and rim region. The study reveals the first known case of the participation of lateral rim cells in a sub-retinal pigment shield in an insect eye. As neither pigmented extensions of secondary pigment cells, nor pigment granules in the extensions of the cone cell projections are present above the BM in T. evanescens, the sub-retinal extensions of the lateral rim cells can be seen as a functional adaptation to miniaturization in order to maintain a proximal shielding function.
Assuntos
Olho Composto de Artrópodes/citologia , Olho Composto de Artrópodes/ultraestrutura , Retina/citologia , Retina/ultraestrutura , Animais , Microscopia Eletrônica de Transmissão , Pigmentos da Retina , VespasRESUMO
Visual pathways from the compound eye of an insect relay to four neuropils, successively the lamina, medulla, lobula, and lobula plate in the underlying optic lobe. Among these neuropils, the medulla, lobula, and lobula plate are interconnected by the complex second optic chiasm, through which the anteroposterior axis undergoes an inversion between the medulla and lobula. Given their complex structure, the projection patterns through the second optic chiasm have so far lacked critical analysis. By densely reconstructing axon trajectories using a volumetric scanning electron microscopy (SEM) technique, we reveal the three-dimensional structure of the second optic chiasm of Drosophila melanogaster, which comprises interleaving bundles and sheets of axons insulated from each other by glial sheaths. These axon bundles invert their horizontal sequence in passing between the medulla and lobula. Axons connecting the medulla and lobula plate are also bundled together with them but do not decussate the sequence of their horizontal positions. They interleave with sheets of projection neuron axons between the lobula and lobula plate, which also lack decussations. We estimate that approximately 19,500 cells per hemisphere, about two thirds of the optic lobe neurons, contribute to the second chiasm, most being Tm cells, with an estimated additional 2,780 T4 and T5 cells each. The chiasm mostly comprises axons and cell body fibers, but also a few synaptic elements. Based on our anatomical findings, we propose that a chiasmal structure between the neuropils is potentially advantageous for processing complex visual information in parallel. The EM reconstruction shows not only the structure of the chiasm in the adult brain, the previously unreported main topic of our study, but also suggest that the projection patterns of the neurons comprising the chiasm may be determined by the proliferation centers from which the neurons develop. Such a complex wiring pattern could, we suggest, only have arisen in several evolutionary steps.
Assuntos
Quiasma Óptico/anatomia & histologia , Lobo Óptico de Animais não Mamíferos/anatomia & histologia , Vias Visuais/anatomia & histologia , Animais , Axônios/fisiologia , Drosophila , Microscopia Eletrônica de Varredura , Neurônios/citologia , Neurônios/fisiologia , Quiasma Óptico/fisiologia , Lobo Óptico de Animais não Mamíferos/fisiologia , Vias Visuais/fisiologiaRESUMO
The brain's synaptic networks endow an animal with powerfully adaptive biological behavior. Maps of such synaptic circuits densely reconstructed in those model brains that can be examined and manipulated by genetic means offer the best prospect for understanding the underlying biological bases of behavior. That prospect is now technologically feasible and a scientifically enabling possibility in neurobiology, much as genomics has been in molecular biology and genetics. In Drosophila, two major advances are in electron microscopic technology, using focused ion beam-scanning electron microscopy (FIB-SEM) milling to capture and align digital images, and in computer-aided reconstruction of neuron morphologies. The last decade has witnessed enormous progress in detailed knowledge of the actual synaptic circuits formed by real neurons. Advances in various brain regions that heralded identification of the motion-sensing circuits in the optic lobe are now extending to other brain regions, with the prospect of encompassing the fly's entire nervous system, both brain and ventral nerve cord.
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Drosophila/fisiologia , Neurônios/citologia , Animais , Comportamento Animal/fisiologia , Encéfalo/citologia , Encéfalo/fisiologia , Biologia Computacional , Drosophila/citologia , Drosophila/genética , Expressão Gênica , Genes Reporter , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência , Neuroanatomia , Neurônios/metabolismo , Neurônios/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
The ability of neurons to identify correct synaptic partners is fundamental to the proper assembly and function of neural circuits. Relative to other steps in circuit formation such as axon guidance, our knowledge of how synaptic partner selection is regulated is severely limited. Drosophila Dpr and DIP immunoglobulin superfamily (IgSF) cell-surface proteins bind heterophilically and are expressed in a complementary manner between synaptic partners in the visual system. Here, we show that in the lamina, DIP mis-expression is sufficient to promote synapse formation with Dpr-expressing neurons and that disrupting DIP function results in ectopic synapse formation. These findings indicate that DIP proteins promote synapses to form between specific cell types and that in their absence, neurons synapse with alternative partners. We propose that neurons have the capacity to synapse with a broad range of cell types and that synaptic specificity is achieved by establishing a preference for specific partners.
Assuntos
Proteínas de Drosophila/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Sinapses/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Imunoglobulinas/genética , Proteínas de Membrana/genética , Neurônios/citologia , Lobo Óptico de Animais não Mamíferos/citologia , Mapas de Interação de ProteínasRESUMO
Neurons can maintain stable synaptic connections across adult life. However, the signals that regulate expression of synaptic proteins in the mature brain are incompletely understood. Here, we describe a transcriptional feedback loop between the biosynthesis and repertoire of specific phospholipids and the synaptic vesicle pool in adult Drosophila photoreceptors. Mutations that disrupt biosynthesis of a subset of phospholipids cause degeneration of the axon terminal and loss of synaptic vesicles. Although degeneration of the axon terminal is dependent on neural activity, activation of sterol regulatory element binding protein (SREBP) is both necessary and sufficient to cause synaptic vesicle loss. Our studies demonstrate that SREBP regulates synaptic vesicle levels by interacting with tetraspanins, critical organizers of membranous organelles. SREBP is an evolutionarily conserved regulator of lipid biosynthesis in non-neuronal cells; our studies reveal a surprising role for this feedback loop in maintaining synaptic vesicle pools in the adult brain.
Assuntos
Retroalimentação Fisiológica , Fosfolipídeos/biossíntese , Células Fotorreceptoras de Invertebrados/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Drosophila melanogaster , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Tetraspaninas/metabolismo , Ativação TranscricionalRESUMO
Existing information on insect compound eyes is mainly limited to two-dimensional information derived from histological or ultrathin sections. These allow a basic description of eye morphology, but are limited in z-axis resolution because of the section thickness or intervals between sections, so that accurate volumetric information cannot be generated. Here we use serial-sectioning transmission electron microscopy to present a 3-D reconstruction at ultrastructural level of a complete ommatidium of a miniaturized insect compound eye. Besides the general presentation of the three dimensional arrangement of the different cell types within the ommatidium, the reconstruction allowed volumetric measurements and numerical analyses to be undertaken, revealing new insights into the number, size and distribution of cell organelles in insect ommatidia. Morphological features that can be related to miniaturization, namely the dimensions and displacement of nuclei, reduction of average pigment granule volume and loss of pigment granules in the terminals of the cone cells, the impact of metabolic activity of cell types on miniaturization, as well as maintenance of rhabdomere volume and limits to its miniaturization, are all discussed.
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Olho Composto de Artrópodes/ultraestrutura , Vespas/ultraestrutura , Animais , Tamanho Corporal , Masculino , Microscopia Eletrônica de TransmissãoRESUMO
Understanding the circuit mechanisms behind motion detection is a long-standing question in visual neuroscience. In Drosophila melanogaster, recently discovered synapse-level connectomes in the optic lobe, particularly in ON-pathway (T4) receptive-field circuits, in concert with physiological studies, suggest a motion model that is increasingly intricate when compared with the ubiquitous Hassenstein-Reichardt model. By contrast, our knowledge of OFF-pathway (T5) has been incomplete. Here, we present a conclusive and comprehensive connectome that, for the first time, integrates detailed connectivity information for inputs to both the T4 and T5 pathways in a single EM dataset covering the entire optic lobe. With novel reconstruction methods using automated synapse prediction suited to such a large connectome, we successfully corroborate previous findings in the T4 pathway and comprehensively identify inputs and receptive fields for T5. Although the two pathways are probably evolutionarily linked and exhibit many similarities, we uncover interesting differences and interactions that may underlie their distinct functional properties.
Assuntos
Encéfalo/fisiologia , Drosophila melanogaster/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Percepção de Movimento , Lobo Óptico de Animais não Mamíferos/fisiologia , Animais , Conectoma , Cruzamentos Genéticos , Dendritos/metabolismo , Feminino , Homozigoto , Modelos Neurológicos , Neurônios/metabolismo , Células Fotorreceptoras de Invertebrados/fisiologia , Sinapses/fisiologiaRESUMO
Neurons of the sparsely populated nervous system of the tadpole larva in the tunicate Ciona intestinalis, a chordate sibling, are known from sporadic previous studies but especially two recent reports that document the connectome of both the central and peripheral nervous systems at EM level. About 330 CNS cells comprise mostly ciliated ependymal cells, with â¼180 neurons that constitute about 50 morphologically distinguishable types. The neurons reveal various chordate characters amid many features that are idiosyncratic. Most neurons are ciliated and lack dendrites, some even lack an axon. Synapses mostly form en passant between axons, and resemble those in basal invertebrates; some are dyads and all have heterogenous synaptic vesicle populations. Each neuron has on average 49 synapses with other cells; these constitute a synaptic network of unpredicted complexity.
Assuntos
Neurônios , Animais , Ciona intestinalis , Humanos , Larva , IrmãosRESUMO
Using FIB-SEM we report the entire synaptic connectome of glomerulus VA1v of the right antennal lobe in Drosophila melanogaster. Within the glomerulus we densely reconstructed all neurons, including hitherto elusive local interneurons. The fruitless-positive, sexually dimorphic VA1v included >11,140 presynaptic sites with ~38,050 postsynaptic dendrites. These connected input olfactory receptor neurons (ORNs, 51 ipsilateral, 56 contralateral), output projection neurons (18 PNs), and local interneurons (56 of >150 previously reported LNs). ORNs are predominantly presynaptic and PNs predominantly postsynaptic; newly reported LN circuits are largely an equal mixture and confer extensive synaptic reciprocity, except the newly reported LN2V with input from ORNs and outputs mostly to monoglomerular PNs, however. PNs were more numerous than previously reported from genetic screens, suggesting that the latter failed to reach saturation. We report a matrix of 192 bodies each having >50 connections; these form 88% of the glomerulus' pre/postsynaptic sites.
Assuntos
Antenas de Artrópodes/inervação , Conectoma , Drosophila melanogaster/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Animais , Antenas de Artrópodes/ultraestrutura , Feminino , Rede Nervosa/fisiologia , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
Histamine (HA) is a neurotransmitter in arthropod photoreceptors. It is recycled via conjugation to ß-alanine to form ß-alanylhistamine (carcinine). Conjugation occurs in epithelial glia that surround photoreceptor terminals in the first optic neuropil, and carcinine (CA) is then transported back to photoreceptors and cleaved to liberate HA and ß-alanine. The gene Inebriated (Ine) encodes an Na+/Cl--dependent SLC6 family transporter translated as two protein isoforms, long (P1) and short (P2). Photoreceptors specifically express Ine-P2 whereas Ine-P1 is expressed in non-neuronal cells. Both ine1 and ine3 have significantly reduced head HA contents compared with wild type, and a smaller increase in head HA after drinking 1% CA. Similarly, uptake of 0.1% CA was reduced in ine1 and ine3 mutant synaptosomes, but increased by 90% and 84% respectively for fractions incubated in 0.05% ß-Ala, compared with wild type. Screening potential substrates in Ine expressing Xenopus oocytes revealed very little response to carcinine and ß-Ala but increased conductance with glycine. Both ine1 and ine3 mutant responses in light-dark phototaxis did not differ from wild-type. Collectively our results suggest that Inebriated functions in an adjunct role as a transporter to the previously reported carcinine transporter CarT.
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The brain is a network of neurons and its biological output is behaviour. This is an exciting age, with a growing acknowledgement that the comprehensive compilation of synaptic circuits densely reconstructed in the brains of model species is now both technologically feasible and a scientifically enabling possibility in neurobiology, much as 30â years ago genomics was in molecular biology and genetics. Implemented by huge advances in electron microscope technology, especially focused ion beam-scanning electron microscope (FIB-SEM) milling (see Glossary), image capture and alignment, and computer-aided reconstruction of neuron morphologies, enormous progress has been made in the last decade in the detailed knowledge of the actual synaptic circuits formed by real neurons, in various brain regions of the fly Drosophila It is useful to distinguish synaptic pathways that are major, with 100 or more presynaptic contacts, from those that are minor, with fewer than about 10; most neurites are both presynaptic and postsynaptic, and all synaptic sites have multiple postsynaptic dendrites. Work on Drosophila has spearheaded these advances because cell numbers are manageable, and neuron classes are morphologically discrete and genetically identifiable, many confirmed by reporters. Recent advances are destined within the next few years to reveal the complete connectome in an adult fly, paralleling advances in the larval brain that offer the same prospect possibly within an even shorter time frame. The final amendment and validation of segmented bodies by human proof-readers remains the most time-consuming step, however. The value of a complete connectome in Drosophila is that, by targeting to specific neurons transgenes that either silence or activate morphologically identified circuits, and then identifying the resulting behavioural outcome, we can determine the causal mechanism for behaviour from its loss or gain. More importantly, the connectome reveals hitherto unsuspected pathways, leading us to seek novel behaviours for these. Circuit information will eventually be required to understand how differences between brains underlie differences in behaviour, and especially to herald yet more advanced connectomic strategies for the vertebrate brain, with an eventual prospect of understanding cognitive disorders having a connectomic basis. Connectomes also help us to identify common synaptic circuits in different species and thus to reveal an evolutionary progression in candidate pathways.