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Co-production of the temporally specified lateral antennal lobe neurons. (A) Illustration of two models of production of two different types of neurons (square and triangle) in the lAL lineage after clonal induction (red arrow). The labeled cells are outlined in green. Note the difference of labeled cellular composition in the Nb clones in two models. (B-C) The early (B) and later (C) generated neuroblast clones labeled by dual-expression-control MARCM. The neuroblast clones are dually labeled with GAL4-GH298 (B,C) and LG-GH146 (B,C). B and C show the merged images from B,B and C,C, respectively. Note the disappearance (arrow) or weak labeling (arrowhead) of glomeruli located at the ventral AL labeled by LG-GH146 (C).
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The antennal lobe (AL) is the primary structure in the Drosophila brain that relays odor information from the antennae to higher brain centers. The characterization of uniglomerular projection neurons (PNs) and some local interneurons has facilitated our understanding of olfaction; however, many other AL neurons remain unidentified. Because neuron...
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... lAL lineage. The white circled areas in B,C indicate the AL. The nomenclature of each type neuron is listed at the top of each panel. Scale bars: 20 μm. Nb clones. If a lAL Nb never made the next types of neurons until completion of all earlier types (sequential production), we expect to detect no early-type neurons in such mid-sized Nb clones (Fig. 7A, left panel). Conversely, if it involved orderly but overlapping production of multiple neuron types (simultaneous production), one should detect concurrent decreases in the cell numbers of several neuron types among gradually reduced Nb clones (e.g. Jefferis et al., 2001) (Fig. 7A, right). We hope to determine whether such intermediate ...
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... to detect no early-type neurons in such mid-sized Nb clones (Fig. 7A, left panel). Conversely, if it involved orderly but overlapping production of multiple neuron types (simultaneous production), one should detect concurrent decreases in the cell numbers of several neuron types among gradually reduced Nb clones (e.g. Jefferis et al., 2001) (Fig. 7A, right). We hope to determine whether such intermediate multicellular Nb clones already lost some GH146-positive PNs but still contained cells that are positive for GAL4-GH298 (type A ...
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... PNs (uPNs) and GH298-expressing type A LNs in the GAL80-minus clones were simultaneously labeled with lexAoprCD2::GFP and UAS-mCD8, respectively. Partial reduction in the cell numbers of both types of IAL neurons were observed in all the three cases. When compared with the presence of 35 uPNs and 21 type A LNs in most full-sized lAL Nb clones (Fig. 7B,B,B), the three later-derived Nb clones carried 30 uPNs and 18 type A LNs (data not shown), 21 uPNs and 13 type A LNs (Fig. 7C,C,C), and 12 uPNs and 5 type A LNs (data not shown), respectively. The reduction in uPNs was also evidenced by the presence of fewer GH146-labeled glomeruli in the mid-sized clones (Fig. 7C). Analysis of the ...
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... respectively. Partial reduction in the cell numbers of both types of IAL neurons were observed in all the three cases. When compared with the presence of 35 uPNs and 21 type A LNs in most full-sized lAL Nb clones (Fig. 7B,B,B), the three later-derived Nb clones carried 30 uPNs and 18 type A LNs (data not shown), 21 uPNs and 13 type A LNs (Fig. 7C,C,C), and 12 uPNs and 5 type A LNs (data not shown), respectively. The reduction in uPNs was also evidenced by the presence of fewer GH146-labeled glomeruli in the mid-sized clones (Fig. 7C). Analysis of the remaining glomerular innervation patterns revealed missing of common glomerular targets DM1, VA4 and VA7m. This suggests that, as ...
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... most full-sized lAL Nb clones (Fig. 7B,B,B), the three later-derived Nb clones carried 30 uPNs and 18 type A LNs (data not shown), 21 uPNs and 13 type A LNs (Fig. 7C,C,C), and 12 uPNs and 5 type A LNs (data not shown), respectively. The reduction in uPNs was also evidenced by the presence of fewer GH146-labeled glomeruli in the mid-sized clones (Fig. 7C). Analysis of the remaining glomerular innervation patterns revealed missing of common glomerular targets DM1, VA4 and VA7m. This suggests that, as in the adPN lineage, distinct lAL uPNs were derived in an invariant non-overlapping sequence. Nevertheless, the simultaneous reduction of uPNs and type A LNs in the later-derived Nb clones ...
Citations
... In Drosophila, similar to Ae. aegypti, many GABA-positive somata are observed in the lateral area of the AL neuropil (Wilson and Laurent, 2005). Among cell bodies bordering the ALs, the dorsolateral cluster contains cell bodies of both PNs and LNs, while anterodorsal cluster contains cell bodies of PNs (Wong et al., 2002;Das et al., 2008;Lai et al., 2008). PNs project from AL to the calyx of MB via the medial antennal lobe tract (mALT), while LNs, showing an ipsilateral projection in the AL, spread their neurites into most glomeruli which contain both presynaptic and postsynaptic connections (Wilson and Laurent, 2005). ...
The mosquito Aedes aegypti is an important vector of diseases including dengue, Zika, chikungunya, and yellow fever. Olfaction is a critical modality for mosquitoes enabling them to locate hosts, sources of nectar, and sites for oviposition. GABA is an essential neurotransmitter in olfactory processing in the insect brain, including the primary olfactory center, the antennal lobe. Previous work with Ae. aegypti has suggested that antennal lobe inhibition via GABA may be involved in the processing of odors. However, little is known about GABA receptor expression in the mosquito brain, or how they may be involved in odor attraction. In this context, generating mutants that target the mosquito’s olfactory responses, and particularly the GABAergic system, is essential to achieve a better understanding of these diverse processes and olfactory coding in these disease vectors. Here we demonstrate the potential of a transgenic line using the QF2 transcription factor, GABA-B1QF2−ECFP, as a new neurogenetic tool to investigate the neural basis of olfaction in Ae. aegypti. Our results show that the gene insertion has a moderate impact on mosquito fitness. Moreover, the line presented here was crossed with a QUAS reporter line expressing the green fluorescent protein and used to determine the location of the metabotropic GABA-B1 receptor expression. We find high receptor expression in the antennal lobes, especially the cell bodies surrounding the antennal lobes. In the mushroom bodies, receptor expression was high in the Kenyon cells, but had low expression in the mushroom body lobes. Behavioral experiments testing the fruit odor attractants showed that the mutants lost their behavioral attraction. Together, these results show that the GABA-B1QF2−ECFP line provides a new tool to characterize GABAergic systems in the mosquito nervous system.
... These neurons collect information from the antennal lobe (AL), the first olfactory processing center, and convey it mostly to the LH. Here they synapse onto third-order LH neurons (Ito et al., 1997;Jefferis et al., 2007;Lai et al., 2008;Liang et al., 2013;Okada et al., 2009;Shimizu and Stopfer, 2017). ...
... Given the role these neurons play in innate behavior, we asked whether mPNs show experience-dependent changes after aversive conditioning. To monitor the activity of these neurons, we used flies expressing UAS-GCaMP6s (Chen et al., 2013) under the control of the enhancer trap line MZ699-GAL4 (Ito et al., 1997), which labels 86% of mPNs (Lai et al., 2008) (Figure 1C). These neurons have their dendritic processes AL and target 75% of all AL glomeruli (Strutz et al., 2014). ...
Behavior is often categorized as being innate or learned, with the specific circuits being assigned to one of these categories. In Drosophila, neural circuits mediating an innate behavioral response are considered as being 'hard-wired', as activation of these neuronal pathways leads to stereotyped behaviors. However, only a limited number of studies assessed whether innate behaviors and their underlying neural circuits are plastic or show experience-dependent modulation. Here, we show that experience modulates second- order olfactory neurons involved in innate behavioral responses. We focus on the neural circuit defined by multiglomerular projection neurons (mPNs) that target the lateral horn, a structure relevant in the genesis of innate behavior. We show that mPNs, coding for odor attraction, are bidirectionally modulated after olfactory associative learning: when an olfactory stimulus is paired with an aversive electric shock, the activity of these neurons is decreased, while when the odor is paired with a sucrose-reward they are potentiated. We further show that this modulation requires glutamate and serotonin signaling, and that downstream third-order neurons are consequently affected. The bidirectional nature of the plasticity in these neurons is reflected in behavior: silencing mPN activity leads to odor avoidance, while artificial activation induces approach. While output from the mPNs is not required in aversive olfactory conditioning, silencing these neurons during retrieval of appetitive memories leads to a significant memory impairment. Artificially activating these neurons during odor presentation is sufficient to generate a 3 h appetitive memory. Our study in flies shows that a neural circuit coding for innate odor attraction can contribute to learned behavior, is modulated by olfactory learning and can provide reward-like reinforcement.
... They are small enough and stereotyped enough that complete, validated connectomes are within reach . Much is known about the organization of insect brains already, as anatomists have been able to characterize them at the level of single cells, circuits, and regions, and understand the computational roles and interactions between these components (Hanesch et al., 1989;Lai et al., 2008;Li et al., 2020). Understanding the structure of insect brains also provides insights into general organizational and computational principles of other brains (Haberkern and Jayaraman, 2016;Kim et al., 2017;Takemura et al., 2017a). ...
... Brains inherit a degree of hierarchy and modularity during the course of development from neural stem cells (Molyneaux et al., 2007; K. Ito and Awasaki, 2008;Lai et al., 2008;Sawa, 2010). The fly brain, in particular, is composed of clonal units, densely connected populations of cells derived from a single neural stem cell (Hartenstein et al., 2008;. ...
The structure of neural circuitry plays a crucial role in brain function. Previous studies of brain organization generally had to trade off between coarse descriptions at a large scale and fine descriptions on a small scale. Researchers have now reconstructed tens to hundreds of thousands of neurons at synaptic resolution, enabling investigations into the interplay between global, modular organization, and cell type-specific wiring. Analyzing data of this scale, however, presents unique challenges. To address this problem we applied novel community detection methods to analyze the synapse-level reconstruction of an adult female Drosophila melanogaster brain containing over 20 thousand neurons and 10 million synapses. Using a machine-learning algorithm, we find the most densely connected communities of neurons by maximizing a generalized modularity density measure. We resolve the community structure at a range of scales, from large (on the order of thousands of neurons) to small (on the order of tens of neurons). We find that the network is organized hierarchically and larger-scale communities are composed of smaller-scale structures. Our methods identify well-known features of the fly brain, including its sensory pathways. Moreover, focusing on specific brain regions, we are able to identify subnetworks with distinct connectivity types. For example, manual efforts have identified layered structures in the fan-shaped body. Our methods not only automatically recover this layered structure, but also resolve finer connectivity patterns to downstream and upstream areas. We also find a novel modular organization of the superior neuropil, with distinct clusters of upstream and downstream brain regions dividing the neuropil into several pathways. These methods show that the fine-scale, local network reconstruction made possible by modern experimental methods are sufficiently detailed to identify the organization of the brain across scales, and enable novel predictions about the structure and function of its parts.
Significance Statement
The Hemibrain is a partial connectome of an adult female Drosophila melanogaster brain containing over 20 thousand neurons and 10 million synapses. Analyzing the structure of a network of this size requires novel and efficient computational tools. We applied a new community detection method to automatically uncover the modular structure in the Hemibrain data set by maximizing a generalized modularity measure. This allowed us to resolve the community structure of the fly hemibrain at a range of spatial scales revealing a hierarchical organization of the network, where larger-scale modules are composed of smaller-scale structures. The method also allowed us to identify subnetworks with distinct cell and connectivity structures, such as the layered structures in the fan-shaped body, and the modular organization of the superior neuropil. Thus network analysis methods can be adopted to the connectomes being reconstructed using modern experimental methods to reveal the organization of the brain across scales. This supports the view that such connectomes will allow us to uncover the organizational structure of the brain, which can ultimately lead to a better understanding of its function.
... Thus, the pathways of uni-PNs and multi-PNs are generally segregated in the cockroach brain. Comprehensive morphological analysis performed in moths [9,10] and fruit flies[17,56] also reveals biased innervations between uni-and multi-PNs; mALT predominantly have ...
To represent specific olfactory cues from the highly complex and dynamic odor world in the brain, insects employ multiple parallel olfactory pathways that process odors with different coding strategies. Here, we summarize the anatomical and physiological features of parallel olfactory pathways in the hemimetabolous insect, the cockroach Periplaneta americana. The cockroach processes different aspects of odor stimuli, such as odor qualities, temporal information, and dynamics, through parallel olfactory pathways. These parallel pathways are anatomically segregated from the peripheral to higher brain centers, forming functional maps within the brain. In addition, the cockroach may possess parallel pathways that correspond to distinct types of olfactory receptors expressed in sensory neurons. Through comparisons with olfactory pathways in holometabolous insects, we aim to provide valuable insights into the organization, functionality, and evolution of insect olfaction.
... From a total of about 1250 mosquito preparations, we were successful in obtaining recordings along with morphological identification from 208 PNs and 53 LNs. We found that the distribution of cell bodies around the AL in Aedes aegypti is similar to Drosophila melanogaster [76][77][78] : the anterodorsal cluster contains cell bodies of PNs, while the dorsolateral cluster contains cell bodies of both PNs and LNs. By examining the dendritic innervations within the AL, we found that 201 of 208 recorded PNs were uniglomerular, with dense and complete innervation of the corresponding glomerulus ( Fig. 1b and Supplementary Fig. 1a); this high proportion of uniglomerular PNs may be because of the targeting of mostly dorsal clusters of cell bodies. ...
Among the cues that a mosquito uses to find a host for blood-feeding, the smell of the host plays an important role. Previous studies have shown that host odors contain hundreds of chemical odorants, which are detected by different receptors on the peripheral sensory organs of mosquitoes. But how individual odorants are encoded by downstream neurons in the mosquito brain is not known. We developed an in vivo preparation for patch-clamp electrophysiology to record from projection neurons and local neurons in the antennal lobe of Aedes aegypti . Combining intracellular recordings with dye-fills, morphological reconstructions, and immunohistochemistry, we identify different sub-classes of antennal lobe neurons and their putative interactions. Our recordings show that an odorant can activate multiple neurons innervating different glomeruli, and that the stimulus identity and its behavioral preference are represented in the population activity of the projection neurons. Our results provide a detailed description of the second-order olfactory neurons in the central nervous system of mosquitoes and lay a foundation for understanding the neural basis of their olfactory behaviors.
... The second group consists of PNs that are inhibitory and innervate multiple glomeruli (mPNs). The cell bodies of these GABA releasing mPNs are located in the ventral cluster (Lai et al., 2008). The mPNs extend their axons along the mlALT and bypass the MB to innervate the LH directly (Jefferis et al., 2007;Okada et al., 2009;Tanaka et al., 2012). ...
It is long known that the nervous system of vertebrates can be shaped by internal and external factors. On the other hand, the nervous system of insects was long assumed to be stereotypic, although evidence for plasticity effects accumulated for several decades. To cover the topic comprehensively, this review recapitulates the establishment of the term "plasticity" in neuroscience and introduces its original meaning. We describe the basic composition of the insect olfactory system using Drosophila melanogaster as a representative example and outline experience-dependent plasticity effects observed in this part of the brain in a variety of insects, including hymenopterans, lepidopterans, locusts, and flies. In particular, we highlight recent advances in the study of experience-dependent plasticity effects in the olfactory system of D. melanogaster, as it is the most accessible olfactory system of all insect species due to the genetic tools available. The partly contradictory results demonstrate that morphological, physiological and behavioral changes in response to long-term olfactory stimulation are more complex than previously thought. Different molecular mechanisms leading to these changes were unveiled in the past and are likely responsible for this complexity. We discuss common problems in the study of experience-dependent plasticity, ways to overcome them, and future directions in this area of research. In addition, we critically examine the transferability of laboratory data to natural systems to address the topic as holistically as possible. As a mechanism that allows organisms to adapt to new environmental conditions, experience-dependent plasticity contributes to an animal's resilience and is therefore a crucial topic for future research, especially in an era of rapid environmental changes.
... The somata and neurites of these Kenyon cells form four spatially separable clusters, allowing us to score Kenyon cell clone number by counting the groups of labeled soma or axon tracts (Elkahlah et al., 2020). As hydroxyurea ablation sometimes affects the lNB/BAlc that gives rise to lateral PNs, we included the MZ19 marker to track the lateral DA1 glomerulus (Lai et al., 2008;Stocker et al., 1997). Almost all animals retained at least one PN neuroblast ( Figures S6C, D), while the number of Kenyon cell clonal units varied widely. ...
Animals can discriminate myriad sensory stimuli but can also generalize from learned experience. You can probably distinguish the favorite teas of your colleagues while still recognizing that all tea pales in comparison to coffee. Tradeoffs between detection, discrimination, and generalization are inherent at every layer of sensory processing. During development, specific quantitative parameters are wired into perceptual circuits and set the playing field on which plasticity mechanisms play out. A primary goal of systems neuroscience is to understand how material properties of a circuit define the logical operations--computations--that it makes, and what good these computations are for survival. A cardinal method in biology--and the mechanism of evolution--is to change a unit or variable within a system and ask how this affects organismal function. Here, we make use of our knowledge of developmental wiring mechanisms to modify hard-wired circuit parameters in the Drosophila melanogaster mushroom body and assess the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input number, but not cell number, tunes odor selectivity. Simple odor discrimination performance is maintained when Kenyon cell number is reduced and augmented by Kenyon cell expansion.
... 3a, Left; Supplementary Fig. S3)60 . The other is unilateral neurons (non-AMMC-B1 neurons) that project to the antennal lobe and lateral horn (Supplementary Fig. S3), which resemble olfactory ventral projection neurons63 . R49F09-GAL4 in D. simulans labeled morphologically similar neurons to both AMMC-B1 and non-AMMC-B1 neurons labeled in D. melanogaster R49F09-GAL4(Fig. ...
Acoustic communication signals diversify even on short evolutionary time scales. To understand how the auditory system underlying acoustic communication could evolve, we conducted a systematic comparison of the early stages of the auditory neural circuit involved in song information processing between closely-related fruit-fly species. Male Drosophila melanogaster and D. simulans produce different sound signals during mating rituals, known as courtship songs. Female flies from these species selectively increase their receptivity when they hear songs with conspecific temporal patterns. Here, we firstly confirmed interspecific differences in temporal pattern preferences; D. simulans preferred pulse songs with longer intervals than D. melanogaster. Primary and secondary song-relay neurons, JO neurons and AMMC-B1 neurons, shared similar morphology and neurotransmitters between species. The temporal pattern preferences of AMMC-B1 neurons were also relatively similar between species, with slight but significant differences in their band-pass properties. Although the shift direction of the response property matched that of the behavior, these differences are not large enough to explain behavioral differences in song preferences. This study enhances our understanding of the conservation and diversification of the architecture of the early-stage neural circuit which processes acoustic communication signals.
... They are small enough and stereotyped enough that complete, validated connectomes are within reach [45]. Much is known about the organization of insect brains already, as anatomists have been able to characterize them at the level of single cells, circuits, and regions, and understand the computational roles and interactions between these components [21,31,30]. Understanding the structure of insect brains also provides insights into general organizational and computational principles of other brains [20,29,48]. ...
... Brains inherit a degree of hierarchy and modularity during the course of development from neural stem cells [30,24,34,43]. The fly brain, in particular, is composed of clonal units, densely connected populations of cells derived from a single neural stem cell [22,24]. ...
The structure of neural circuitry plays a crucial role in brain function. Previous studies of brain organization generally had to trade off between coarse descriptions at a large scale and fine descriptions on a small scale. Researchers have now reconstructed tens to hundreds of thousands of neurons at synaptic resolution, enabling investigations into the interplay between global, modular organization, and cell type-specific wiring. Analyzing data of this scale, however, presents unique challenges. To address this problem we applied novel community detection methods to analyze the synapse-level reconstruction of an adult fruit fly brain containing over 20 thousand neurons and 10 million synapses. Using a machine-learning algorithm, we find the most densely connected communities of neurons by maximizing a generalized modularity density measure. We resolve the community structure at a range of scales, from large (on the order of thousands of neurons) to small (on the order of tens of neurons). We find that the network is organized hierarchically and larger-scale communities are composed of smaller-scale structures. Our methods identify well-known features of the fly brain, including its sensory pathways. Moreover, focusing on specific brain regions, we are able to identify subnetworks with distinct connectivity types. For example, manual efforts have identified layered structures in the fan-shaped body. Our methods not only automatically recover this layered structure, but also resolve finer connectivity patterns to downstream and upstream areas. We also find a novel modular organization of the superior neuropil, with distinct clusters of upstream and downstream brain regions dividing the neuropil into several pathways. These methods show that the fine-scale, local network reconstruction made possible by modern experimental methods are sufficiently detailed to identify the organization of the brain across scales, and enable novel predictions about the structure and function of its parts.
... To find animals which exhibit different sleep patterns, we can start from a large set of flies mutagenized by chemicals or transposon elements or a spontaneous event [2,8,32,35,39,58,59,66]. We can also express protein-coding genes or noncoding RNAs (including RNAs artificially designed for the gene knockdown as well as naturally occurring regulatory RNAs) in a subset of cells using binary Gal4/UAS expression systems available in Drosophila [5,21,26,28,49,60,62,67]. ...
... One can apply the hypochlorite treatment to embryos and raise the flies with autoclaved fly food to obtain flies that are more germ-free than those treated with antibiotics [67]. 4. In a biosafety cabinet and with gloves, soak the collected embryos in 70% ethanol for 1 min, 2.5% sodium hypochlorite (Wako) for 2 min, and 70% ethanol for 2 min. ...
... The protocol below describes how to prepare disposable two-choice oviposition assay to determine whether a specific compound functions as a volatile or nonvolatile cue during oviposition site selection. The assay consists of a 57 Â 38 Â 17 mm rectangular transparent polystyrene dish accommodating two 10 Â 38 Â 4 mm oviposition zones filled with 0.75% agar containing 100 mM of sucrose as a nutrient source to instigate oviposition [66][67][68][69][70]. The two zones are separated by a 37 Â 38 Â 4 mm middle zone of 3% agar that is unsuitable for oviposition [71]. ...
Social interactions are generally regulated by pheromones that convey information about the identity, physiological state, and location of an individual. The fruit fly, Drosophila melanogaster
, offers a powerful model system to study the mechanisms through which pheromones modulate social interactions. Most of the fruit fly’s social behavior is demonstrably modulated by pheromones, and many of the chemical compounds composing its pheromonal profile have been characterized. This chapter describes several behavioral bioassays that can be used to determine the function of contact and short-range volatile pheromones in D. melanogaster’s social behavior. The chapter first provides instructions on how to rear flies for pheromonal experimentation and how to generate flies that cannot produce cuticular hydrocarbons. Afterward, protocols on how to determine the function of pheromones in courtship behavior
and mate choice are provided, followed by protocols to determine whether pheromones function as volatile or contact cues during oviposition site selection. Finally, the last section of the chapter gives general advice on how to work with pheromones in the laboratory.Key wordsAggregationCourtship
Drosophila melanogaster
Mate choiceOlfactionOvipositionPheromonesSocial behavior
