Automatic Segmentation of Drosophila Neural Compartments Using GAL4 Expression Data Reveals Novel Visual Pathways.
ABSTRACT: Identifying distinct anatomical structures within the brain and developing genetic tools to target them are fundamental steps for understanding brain function. We hypothesize that enhancer expression patterns can be used to automatically identify functional units such as neuropils and fiber tracts. We used two recent, genome-scale Drosophila GAL4 libraries and associated confocal image datasets to segment large brain regions into smaller subvolumes. Our results (available at https://strawlab.org/braincode) support this hypothesis because regions with well-known anatomy, namely the antennal lobes and central complex, were automatically segmented into familiar compartments. The basis for the structural assignment is clustering of voxels based on patterns of enhancer expression. These initial clusters are agglomerated to make hierarchical predictions of structure. We applied the algorithm to central brain regions receiving input from the optic lobes. Based on the automated segmentation and manual validation, we can identify and provide promising driver lines for 11 previously identified and 14 novel types of visual projection neurons and their associated optic glomeruli. The same strategy can be used in other brain regions and likely other species, including vertebrates.
Project description:The anatomical organization of distinct regions in the insect brain often reflects their functions. In the present study, the brain structure of Apolygus lucorum was examined by using immunolabeling and three-dimensional reconstruction. The results revealed the location and volume of prominent neuropils, such as the antennal lobes (AL), optic lobes (OL), anterior optic tubercles (AOTU), central body (CB), lateral accessory lobes (LAL), mushroom lobes, and distinct tritocerebral neuropils. As expected, this brain is similar to that of other insects. One exception, however, is that the antennal lobes were found to be the most prominent neuropils. Their size relative to the entire brain is the largest among all insect species studied so far. In contrast, the calyx, a region getting direct input from the antennal lobe, has a smaller size relative to the brain than that of other species. These findings may suggest that olfaction plays an essential role for A. lucorum.
Project description:Octopamine plays an important role in many behaviors in invertebrates. It acts via binding to G protein coupled receptors located on the plasma membrane of responsive cells. Several distinct subtypes of octopamine receptors have been found in invertebrates, yet little is known about the expression pattern of these different receptor subtypes and how each subtype may contribute to different behaviors. One honey bee (Apis mellifera) octopamine receptor, AmOA1, was recently cloned and characterized. Here we continue to characterize the AmOA1 receptor by investigating its distribution in the honey bee brain. We used two independent antibodies produced against two distinct peptides in the carboxyl-terminus to study the distribution of the AmOA1 receptor in the honey bee brain. We found that both anti-AmOA1 antibodies revealed labeling of cell body clusters throughout the brain and within the following brain neuropils: the antennal lobes; the calyces, pedunculus, vertical (alpha, gamma) and medial (beta) lobes of the mushroom body; the optic lobes; the subesophageal ganglion; and the central complex. Double immunofluorescence staining using anti-GABA and anti-AmOA1 receptor antibodies revealed that a population of inhibitory GABAergic local interneurons in the antennal lobes express the AmOA1 receptor in the cell bodies, axons and their endings in the glomeruli. In the mushroom bodies, AmOA1 receptors are expressed in a subpopulation of inhibitory GABAergic feedback neurons that ends in the visual (outer half of basal ring and collar regions) and olfactory (lip and inner basal ring region) calyx neuropils, as well as in the collar and lip zones of the vertical and medial lobes. The data suggest that one effect of octopamine via AmOA1 in the antennal lobe and mushroom body is to modulate inhibitory neurons.
Project description:The optic lobes of the fruit fly Drosophila melanogaster form a highly wired neural network composed of roughly 130.000 neurons of more than 80 different types. How neuronal diversity arises from very few cell progenitors is a central question in developmental neurobiology. We use the optic lobe of the fruit fly as a paradigm to understand how neuroblasts, the neural stem cells, generate multiple neuron types. Although the development of the fly brain has been the subject of extensive research, very little is known about the lineage relationships of the cell types forming the adult optic lobes. Here we perform a large-scale lineage bioinformatics analysis using the graph theory. We generated a large collection of cell clones that genetically label the progeny of neuroblasts and built a database to draw graphs showing the lineage relationships between cell types. By establishing biological criteria that measures the strength of the neuronal relationships and applying community detection tools we have identified eight clusters of neurons. Each cluster contains different cell types that we pose are the product of eight distinct classes of neuroblasts. Three of these clusters match the available lineage data, supporting the predictive value of the analysis. Finally, we show that the neuronal progeny of a neuroblast do not have preferential innervation patterns, but instead become part of different layers and neuropils. Here we establish a new methodology that helps understanding the logic of Drosophila brain development and can be applied to the more complex vertebrate brains.
Project description:Visual information processing in animals with large image forming eyes is carried out in highly structured retinotopically ordered neuropils. Visual neuropils in Drosophila form the optic lobe, which consists of four serially arranged major subdivisions; the lamina, medulla, lobula and lobula plate; the latter three of these are further subdivided into multiple layers. The visual neuropils are formed by more than 100 different cell types, distributed and interconnected in an invariant highly regular pattern. This pattern relies on a protracted sequence of developmental steps, whereby different cell types are born at specific time points and nerve connections are formed in a tightly controlled sequence that has to be coordinated among the different visual neuropils. The developing fly visual system has become a highly regarded and widely studied paradigm to investigate the genetic mechanisms that control the formation of neural circuits. However, these studies are often made difficult by the complex and shifting patterns in which different types of neurons and their connections are distributed throughout development. In the present paper we have reconstructed the three-dimensional architecture of the Drosophila optic lobe from the early larva to the adult. Based on specific markers, we were able to distinguish the populations of progenitors of the four optic neuropils and map the neurons and their connections. Our paper presents sets of annotated confocal z-projections and animated 3D digital models of these structures for representative stages. The data reveal the temporally coordinated growth of the optic neuropils, and clarify how the position and orientation of the neuropils and interconnecting tracts (inner and outer optic chiasm) changes over time. Finally, we have analyzed the emergence of the discrete layers of the medulla and lobula complex using the same markers (DN-cadherin, Brp) employed to systematically explore the structure and development of the central brain neuropil. Our work will facilitate experimental studies of the molecular mechanisms regulating neuronal fate and connectivity in the fly visual system, which bears many fundamental similarities with the retina of vertebrates.
Project description:This article describes the cellular sources for tyramine and the cellular targets of tyramine via the Tyramine Receptor 1 (AmTyr1) in the olfactory learning and memory neuropils of the honey bee brain. Clusters of approximately 160 tyramine immunoreactive neurons are the source of tyraminergic fibers with small varicosities in the optic lobes, antennal lobes, lateral protocerebrum, mushroom body (calyces and gamma lobes), tritocerebrum and subesophageal ganglion (SEG). Our tyramine mapping study shows that the primary sources of tyramine in the antennal lobe and calyx of the mushroom body are from at least two Ventral Unpaired Median neurons (VUMmd and VUMmx) with cell bodies in the SEG. To reveal AmTyr1 receptors in the brain, we used newly characterized anti-AmTyr1 antibodies. Immunolocalization studies in the antennal lobe with anti-AmTyr1 antibodies showed that the AmTyr1 expression pattern is mostly in the presynaptic sites of olfactory receptor neurons (ORNs). In the mushroom body calyx, anti-AmTyr1 mapped the presynaptic sites of uniglomerular Projection Neurons (PNs) located primarily in the microglomeruli of the lip and basal ring calyx area. Release of tyramine/octopamine from VUM (md and mx) neurons in the antennal lobe and mushroom body calyx would target AmTyr1 expressed on ORN and uniglomerular PN presynaptic terminals. The presynaptic location of AmTyr1, its structural similarity with vertebrate alpha-2 adrenergic receptors, and previous pharmacological evidence suggests that it has an important role in the presynaptic inhibitory control of neurotransmitter release.
Project description:Colour processing at early stages of visual pathways is a topic of intensive study both in vertebrate and invertebrate species. However, it is still unclear how colour learning and memory formation affects an insect brain in the peripheral processing stages and high-order integration centres, and whether associative colour experiences are reflected in plasticity of underlying neuronal circuits. To address this issue, we used Camponotus blandus ants as their proven colour learning and memory capabilities, precisely controllable age and experience, and already known central visual pathways offer unique access to analyse plasticity in neuronal circuits for colour vision in a miniature brain. The potential involvement of distinct neuropils-optic lobes (OLs), mushroom body (MB) input (collar) and output (vertical lobe), anterior optic tubercle (AOTU) and central complex (CX)-in associative colour experiences was assessed by quantification of volumetric and synaptic changes (MB collar) directly after colour conditioning and, 3 days later, after the establishment of long-term memory (LTM). To account for potential effects of non-associative light exposure, we compared neuronal changes in the brain of colour-naive foragers with those of foragers that had been exposed to light in a non-associative way. The results clearly show that the OLs, AOTU, and CX respond with plastic changes after colour learning and LTM formation. This suggests a complex neuronal network for colour learning and memory formation involving multiple brain levels. Such a colour-processing network probably represents an efficient design promoting fast and accurate behavioural decisions during orientation and navigation.
Project description:Haller's rule states that brains scale allometrically with body size in all animals, meaning that relative brain size increases with decreasing body size. This rule applies both on inter- and intraspecific comparisons. Only 1 species, the extremely small parasitic wasp Trichogramma evanescens, is known as an exception and shows an isometric brain-body size relation in an intraspecific comparison between differently sized individuals. Here, we investigated if such an isometric brain-body size relationship also occurs in an intraspecific comparison with a slightly larger parasitic wasp, Nasonia vitripennis, a species that may vary 10-fold in body weight upon differences in levels of scramble competition during larval development. We show that Nasonia exhibits diphasic brain-body size scaling: larger wasps scale allometrically, following Haller's rule, whereas the smallest wasps show isometric scaling. Brains of smaller wasps are, therefore, smaller than expected and we hypothesized that this may lead to adaptations in brain architecture. Volumetric analysis of neuropil composition revealed that wasps of different sizes differed in relative volume of multiple neuropils. The optic lobes and mushroom bodies in particular were smaller in the smallest wasps. Furthermore, smaller brains had a relatively smaller total neuropil volume and larger cellular rind than large brains. These changes in relative brain size and brain architecture suggest that the energetic constraints on brain tissue outweigh specific cognitive requirements in small Nasonia wasps.
Project description:The evolutionary success of ants and other social insects is considered to be intrinsically linked to division of labor among workers. The role of the brains of individual ants in generating division of labor, however, is poorly understood, as is the degree to which interspecific variation in worker social phenotypes is underscored by functional neurobiological differentiation. Here we demonstrate that dimorphic minor and major workers of different ages from three ecotypical species of the hyperdiverse ant genus Pheidole have distinct patterns of neuropil size variation. Brain subregions involved in sensory input (optic and antennal lobes), sensory integration, learning and memory (mushroom bodies), and motor functions (central body and subesophageal ganglion) vary significantly in relative size, reflecting differential investment in neuropils that likely regulate subcaste- and age-correlated task performance. Worker groups differ in brain size and display patterns of altered isometric and allometric subregion scaling that affect brain architecture independently of brain size variation. In particular, mushroom body size was positively correlated with task plasticity in the context of both age- and subcaste-related polyethism, providing strong, novel support that greater investment in this neuropil increases behavioral flexibility. Our findings reveal striking levels of developmental plasticity and evolutionary flexibility in Pheidole worker neuroanatomy, supporting the hypothesis that mosaic alterations of brain composition contribute to adaptive colony structure and interspecific variation in social organization.
Project description:Insects exhibit an elaborate repertoire of behaviors in response to environmental stimuli. The central complex plays a key role in combining various modalities of sensory information with an insect's internal state and past experience to select appropriate responses. Progress has been made in understanding the broad spectrum of outputs from the central complex neuropils and circuits involved in numerous behaviors. Many resident neurons have also been identified. However, the specific roles of these intricate structures and the functional connections between them remain largely obscure. Significant gains rely on obtaining a comprehensive catalog of the neurons and associated GAL4 lines that arborize within these brain regions, and on mapping neuronal pathways connecting these structures. To this end, small populations of neurons in the Drosophila melanogaster central complex were stochastically labeled using the multicolor flip-out technique and a catalog was created of the neurons, their morphologies, trajectories, relative arrangements, and corresponding GAL4 lines. This report focuses on one structure of the central complex, the protocerebral bridge, and identifies just 17 morphologically distinct cell types that arborize in this structure. This work also provides new insights into the anatomical structure of the four components of the central complex and its accessory neuropils. Most strikingly, we found that the protocerebral bridge contains 18 glomeruli, not 16, as previously believed. Revised wiring diagrams that take into account this updated architectural design are presented. This updated map of the Drosophila central complex will facilitate a deeper behavioral and physiological dissection of this sophisticated set of structures.
Project description:Neural networks in vertebrates exhibit endogenous oscillations that have been associated with functions ranging from sensory processing to locomotion. It remains unclear whether oscillations may play a similar role in the insect brain. We describe a novel "whole brain" readout for Drosophila melanogaster using a simple multichannel recording preparation to study electrical activity across the brain of flies exposed to different sensory stimuli. We recorded local field potential (LFP) activity from >2,000 registered recording sites across the fly brain in >200 wild-type and transgenic animals to uncover specific LFP frequency bands that correlate with: 1) brain region; 2) sensory modality (olfactory, visual, or mechanosensory); and 3) activity in specific neural circuits. We found endogenous and stimulus-specific oscillations throughout the fly brain. Central (higher-order) brain regions exhibited sensory modality-specific increases in power within narrow frequency bands. Conversely, in sensory brain regions such as the optic or antennal lobes, LFP coherence, rather than power, best defined sensory responses across modalities. By transiently activating specific circuits via expression of TrpA1, we found that several circuits in the fly brain modulate LFP power and coherence across brain regions and frequency domains. However, activation of a neuromodulatory octopaminergic circuit specifically increased neuronal coherence in the optic lobes during visual stimulation while decreasing coherence in central brain regions. Our multichannel recording and brain registration approach provides an effective way to track activity simultaneously across the fly brain in vivo, allowing investigation of functional roles for oscillations in processing sensory stimuli and modulating behavior.