Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body.

The arthropod mushroom body is well-studied as an expansion layer representing olfactory stimuli and linking them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their function remains unclear. Here, we identify inputs to visual Kenyon cells using the FlyWire adult whole-brain connectome. Input repertoires are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual neurons presynaptic to Kenyon cells have large receptive fields, while interneuron inputs receive spatially restricted signals that may be tuned to specific visual features. Individual visual Kenyon cells randomly sample sparse inputs from combinations of visual channels, including multiple optic lobe neuropils. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the specific input repertoire to the smaller population of visual Kenyon cells suggests a constrained encoding of visual stimuli.

Drosophila medulla neuroblast termination via apoptosis, differentiation, and gliogenic switch is scheduled by the depletion of the neuroepithelial stem cell pool.

The brain is consisted of diverse neurons arising from a limited number of neural stem cells. Drosophila neural stem cells called neuroblasts (NBs) produces specific neural lineages of various lineage sizes depending on their location in the brain. In the Drosophila visual processing centre - the optic lobes (OLs), medulla NBs derived from the neuroepithelium (NE) give rise to neurons and glia cells of the medulla cortex. The timing and the mechanisms responsible for the cessation of medulla NBs are so far not known. In this study, we show that the termination of medulla NBs during early pupal development is determined by the exhaustion of the NE stem cell pool. Hence, altering NE-NB transition during larval neurogenesis disrupts the timely termination of medulla NBs. Medulla NBs terminate neurogenesis via a combination of apoptosis, terminal symmetric division via Prospero, and a switch to gliogenesis via Glial Cell Missing (Gcm); however, these processes occur independently of each other. We also show that temporal progression of the medulla NBs is mostly not required for their termination. As the Drosophila OL shares a similar mode of division with mammalian neurogenesis, understanding when and how these progenitors cease proliferation during development can have important implications for mammalian brain size determination and regulation of its overall function.

Every cell in the body can be traced back to a stem cell. For instance, most cells in the adult brains of fruit flies come from a type of stem cell known as a neuroblast. This includes neurons and glial cells (which support and protect neurons) in the optic lobe, the part of the brain that processes visual information. The numbers of neurons and glia in the optic lobe are tightly regulated such that when the right numbers are reached, the neuroblasts stop making more and are terminated. But how and when this occurs is poorly understood. To investigate, Nguyen and Cheng studied when neuroblasts disappear in the optic lobe over the course of development. This revealed that the number of neuroblasts dropped drastically 12 to 18 hours after the fruit fly larvae developed in to pupae, and were completely gone by 30 hours in to pupae life. Further experiments revealed that the timing of this decrease is influenced by neuroepithelium cells, the pool of stem cells that generate neuroblasts during the early stages of development. Nguyen and Cheng found that speeding up this transition so that neuroblasts arise from the neuroepithelium earlier, led neuroblasts to disappear faster from the optic lobe; whereas delaying the transition caused neuroblasts to persist for much longer. Thus, the time at which neuroblasts are born determines when they are terminated. Furthermore, Nguyen and Cheng showed that the neuroblasts were lost through a combination of means. This includes dying via a process called apoptosis, dividing to form two mature neurons, or switching to a glial cell fate. These findings provide a deeper understanding of the mechanisms regulating stem cell pools and their conversion to different cell types, a process that is crucial to the proper development of the brain. How cells divide to form the optic lobe of fruit flies is similar to how new neurons arise in the mammalian brain. Understanding how and when stem cells in the fruit fly brain stop proliferating could therefore provide new insights in to the development of the human brain.

Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis.

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.

Mapping model units to visual neurons reveals population code for social behaviour.

The rich variety of behaviours observed in animals arises through the interplay between sensory processing and motor control. To understand these sensorimotor transformations, it is useful to build models that predict not only neural responses to sensory input1-5 but also how each neuron causally contributes to behaviour6,7. Here we demonstrate a novel modelling approach to identify a one-to-one mapping between internal units in a deep neural network and real neurons by predicting the behavioural changes that arise from systematic perturbations of more than a dozen neuronal cell types. A key ingredient that we introduce is 'knockout training', which involves perturbing the network during training to match the perturbations of the real neurons during behavioural experiments. We apply this approach to model the sensorimotor transformations of Drosophila melanogaster males during a complex, visually guided social behaviour8-11. The visual projection neurons at the interface between the optic lobe and central brain form a set of discrete channels12, and prior work indicates that each channel encodes a specific visual feature to drive a particular behaviour13,14. Our model reaches a different conclusion: combinations of visual projection neurons, including those involved in non-social behaviours, drive male interactions with the female, forming a rich population code for behaviour. Overall, our framework consolidates behavioural effects elicited from various neural perturbations into a single, unified model, providing a map from stimulus to neuronal cell type to behaviour, and enabling future incorporation of wiring diagrams of the brain15 into the model.

Hue selectivity from recurrent circuitry in Drosophila.

In the perception of color, wavelengths of light reflected off objects are transformed into the derived quantities of brightness, saturation and hue. Neurons responding selectively to hue have been reported in primate cortex, but it is unknown how their narrow tuning in color space is produced by upstream circuit mechanisms. We report the discovery of neurons in the Drosophila optic lobe with hue-selective properties, which enables circuit-level analysis of color processing. From our analysis of an electron microscopy volume of a whole Drosophila brain, we construct a connectomics-constrained circuit model that accounts for this hue selectivity. Our model predicts that recurrent connections in the circuit are critical for generating hue selectivity. Experiments using genetic manipulations to perturb recurrence in adult flies confirm this prediction. Our findings reveal a circuit basis for hue selectivity in color vision.

Spatial patterning controls neuron numbers in the Drosophila visual system.

Neurons must be made in the correct proportions to communicate with the appropriate synaptic partners and form functional circuits. In the Drosophila visual system, multiple subtypes of distal medulla (Dm) inhibitory interneurons are made in distinct, reproducible numbers-from 5 to 800 per optic lobe. These neurons are born from a crescent-shaped neuroepithelium called the outer proliferation center (OPC), which can be subdivided into specific domains based on transcription factor and growth factor expression. We fate mapped Dm neurons and found that more abundant neural types are born from larger neuroepithelial subdomains, while less abundant subtypes are born from smaller ones. Additionally, morphogenetic Dpp/BMP signaling provides a second layer of patterning that subdivides the neuroepithelium into smaller domains to provide more granular control of cell proportions. Apoptosis appears to play a minor role in regulating Dm neuron abundance. This work describes an underappreciated mechanism for the regulation of neuronal stoichiometry.

Crickets in the spotlight: exploring the impact of light on circadian behavior.

Crickets serve as a well-established model organism in biological research spanning various fields, such as behavior, physiology, neurobiology, and ecology. Cricket circadian behavior was first reported over a century ago and prompted a wealth of studies delving into their chronobiology. Circadian rhythms have been described in relation to fundamental cricket behaviors, encompassing stridulation and locomotion, but also in hormonal secretion and gene expression. Here we review how changes in illumination patterns and light intensity differentially impact the different cricket behaviors as well as circadian gene expression. We further describe the cricket's circadian pacemaker. Ample anatomical manipulations support the location of a major circadian pacemaker in the cricket optic lobes and another in the central brain, possibly interconnected via signaling of the neuropeptide PDF. The cricket circadian machinery comprises a molecular cascade based on two major transcriptional/translational negative feedback loops, deviating somewhat from the canonical model of Drosophila and emphasizing the significance of exploring alternative models. Finally, the nocturnal nature of crickets has provided a unique avenue for investigating the repercussions of artificial light at night on cricket behavior and ecology, underscoring the critical role played by natural light cycles in synchronizing cricket behaviors and populations, further supporting the use of the cricket model in the study of the effects of light on insects. Some gaps in our knowledge and challenges for future studies are discussed.

Serine hydroxymethyl transferase is required for optic lobe neuroepithelia development in Drosophila.

Cell fate and growth require one-carbon units for the biosynthesis of nucleotides, methylation reactions and redox homeostasis, provided by one-carbon metabolism. Consistently, defects in one-carbon metabolism lead to severe developmental defects, such as neural tube defects. However, the role of this pathway during brain development and in neural stem cell regulation is poorly understood. To better understand the role of one carbon metabolism we focused on the enzyme Serine hydroxymethyl transferase (Shmt), a key factor in the one-carbon cycle, during Drosophila brain development. We show that, although loss of Shmt does not cause obvious defects in the central brain, it leads to severe phenotypes in the optic lobe. The shmt mutants have smaller optic lobe neuroepithelia, partly justified by increased apoptosis. In addition, shmt mutant neuroepithelia have morphological defects, failing to form a lamina furrow, which likely explains the observed absence of lamina neurons. These findings show that one-carbon metabolism is crucial for the normal development of neuroepithelia, and consequently for the generation of neural progenitor cells and neurons. These results propose a mechanistic role for one-carbon during brain development.

Coordinated control of neuronal differentiation and wiring by sustained transcription factors.

The large diversity of cell types in nervous systems presents a challenge in identifying the genetic mechanisms that encode it. Here, we report that nearly 200 distinct neurons in the Drosophila visual system can each be defined by unique combinations of on average 10 continuously expressed transcription factors. We show that targeted modifications of this terminal selector code induce predictable conversions of neuronal fates that appear morphologically and transcriptionally complete. Cis-regulatory analysis of open chromatin links one of these genes to an upstream patterning factor that specifies neuronal fates in stem cells. Experimentally validated network models describe the synergistic regulation of downstream effectors by terminal selectors and ecdysone signaling during brain wiring. Our results provide a generalizable framework of how specific fates are implemented in postmitotic neurons.

Matched function of the neuropil processing optic flow in flies and crabs: the lobula plate mediates optomotor responses in Neohelice granulata.

When an animal rotates (whether it is an arthropod, a fish, a bird or a human) a drift of the visual panorama occurs over its retina, termed optic flow. The image is stabilized by compensatory behaviours (driven by the movement of the eyes, head or the whole body depending on the animal) collectively termed optomotor responses. The dipteran lobula plate has been consistently linked with optic flow processing and the control of optomotor responses. Crabs have a neuropil similarly located and interconnected in the optic lobes, therefore referred to as a lobula plate too. Here we show that the crabs' lobula plate is required for normal optomotor responses since the response was lost or severely impaired in animals whose lobula plate had been lesioned. The effect was behaviour-specific, since avoidance responses to approaching visual stimuli were not affected. Crabs require simpler optic flow processing than flies (because they move slower and in two-dimensional instead of three-dimensional space), consequently their lobula plates are relatively smaller. Nonetheless, they perform the same essential role in the visual control of behaviour. Our findings add a fundamental piece to the current debate on the evolutionary relationship between the lobula plates of insects and crustaceans.

Dissection of the Head of a Live Fly for Functional Imaging.

In this protocol, we outline procedures to mount the fly and to open up the head cuticle to expose the optic lobes for in vivo imaging. The fly is first inserted into a custom-made fly chamber in which the fly's head is stabilized on a piece of aluminum foil. Once the fly is mounted in the chamber, its head cuticle is removed, exposing the optic lobe for recording. The brain tissues (above the foil), including the optic lobes, should be bathed in fly saline. Meanwhile, the eyes (below the foil) are kept dry to receive light stimuli during the recording. A considerable level of expertise and hand dexterity is required to handle a small animal such as a fly, especially when opening its head capsule without damaging the brain tissue. This expertise should be gained through mindful repetition of the protocol. With appropriate preparation and skills, the success rate for this procedure can be >95%. Using this protocol, it is possible to record ultraviolet (UV)-sensing photoreceptors, which have long visual fibers that terminate at the medulla (the second optic neuropil). Depending on the visual neurons of interest, some modifications to fly mounting might be needed.

Brain size and behavioral specialization in the jataí stingless bee (Tetragonisca angustula).

Social insects are instructive models for understanding the association between investment in brain size and behavioral variability because they show a relatively simple nervous system associated with a large set of complex behaviors. In the jataí stingless bee (Tetragonisca angustula), division of labor relies both on age and body size differences among workers. When young, both minors and soldiers engage in intranidal tasks and move to extranidal tasks as they age. Minors switch to foraging activities, while soldiers take over defensive roles. Nest defense performed by soldiers includes two different tasks: (1) hovering around the nest entrance for the detection and interception of heterospecific bees (a task relying mostly on vision) and (2) standing at the nest entrance tube for inspection of returning foragers and discrimination against conspecific non-nestmates based on olfactory cues. Here, using different-sized individuals (minors and soldiers) as well as same-sized individuals (hovering and standing soldiers) performing distinct tasks, we investigated the effects of both morphological and behavioral variability on brain size. We found a negative allometric growth between brain size and body size across jataí workers, meaning that minors had relatively larger brains than soldiers. Between soldier types, we found that hovering soldiers had larger brain compartments related to visual processing (the optic lobes) and learning (the mushroom bodies). Brain size differences between jataí soldiers thus correspond to behavioral specialization in defense (i.e., vision for hovering soldiers) and illustrate a functional neuroplasticity underpinning division of labor.

A complete temporal transcription factor series in the fly visual system.

The brain consists of thousands of neuronal types that are generated by stem cells producing different neuronal types as they age. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs)1-6. Here we used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila optic lobe neurons. We verify that tTFs regulate the progression of the series by activating the next tTF(s) and repressing the previous one(s), and also identify more complex mechanisms of regulation. Moreover, we establish the temporal window of origin and birth order of each neuronal type in the medulla and provide evidence that these tTFs are sufficient to explain the generation of all of the neuronal diversity in this brain region. Finally, we describe the first steps of neuronal differentiation and show that these steps are conserved in humans. We find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons. This comprehensive analysis of a temporal series of tTFs in the optic lobe offers mechanistic insights into how tTF series are regulated, and how they can lead to the generation of a complete set of neurons.

Neural organization of the third optic neuropil, the lobula, in the highly visual semiterrestrial crab Neohelice granulata.

The visual neuropils (lamina, medulla, and lobula complex) of malacostracan crustaceans and hexapods have many organizational principles, cell types, and functional properties in common. Information about the cellular elements that compose the crustacean lobula is scarce especially when focusing on small columnar cells. Semiterrestrial crabs possess a highly developed visual system and display conspicuous visually guided behaviors. In particular, Neohelice granulata has been previously used to describe the cellular components of the first two optic neuropils using Golgi impregnation technique. Here, we present a comprehensive description of individual elements composing the third optic neuropil, the lobula, of that same species. We characterized a wide variety of elements (140 types) including input terminals and lobula columnar, centrifugal, and input columnar elements. Results reveal a very dense and complex neuropil. We found a frequently impregnated input element (suggesting a supernumerary cartridge representation) that arborizes in the third layer of the lobula and that presents four variants each with ramifications organized following one of the four cardinal axes suggesting a role in directional processing. We also describe input elements with two neurites branching in the third layer, probably connecting with the medulla and lobula plate. These facts suggest that this layer is involved in the directional motion detection pathway in crabs. We analyze and discuss our findings considering the similarities and differences found between the layered organization and components of this crustacean lobula and the lobula of insects.

The complex synaptic pathways onto a looming-detector neuron revealed using serial block-face scanning electron microscopy.

The ability of locusts to detect looming stimuli and avoid collisions or predators depends on a neuronal circuit in the locust's optic lobe. Although comprehensively studied for over three decades, there are still major questions about the computational steps of this circuit. We used fourth instar larvae of Locusta migratoria to describe the connection between the lobula giant movement detector 1 (LGMD1) neuron in the lobula complex and the upstream neuropil, the medulla. Serial block-face scanning electron microscopy (SBEM) was used to characterize the morphology of the connecting neurons termed trans-medullary afferent (TmA) neurons and their synaptic connectivity. This enabled us to trace neurons over several hundred micrometers between the medulla and the lobula complex while identifying their synapses. We traced two different TmA neurons, each from a different individual, from their synapses with the LGMD in the lobula complex up into the medulla and describe their synaptic relationships. There is not a simple downstream transmission of the signal from a lamina neuron onto these TmA neurons; there is also a feedback loop in place with TmA neurons making outputs as well as receiving inputs. More than one type of neuron shapes the signal of the TmA neurons in the medulla. We found both columnar and trans-columnar neurons connected with the traced TmA neurons in the medulla. These findings indicate that there are computational steps in the medulla that have not been included in models of the neuronal pathway for looming detection.

The pigment-dispersing factor neuronal network systematically grows in developing honey bees.

The neuropeptide pigment-dispersing factor (PDF) plays a prominent role in the circadian clock of many insects including honey bees. In the honey bee brain, PDF is expressed in about 15 clock neurons per hemisphere that lie between the central brain and the optic lobes. As in other insects, the bee PDF neurons form wide arborizations in the brain, but certain differences are evident. For example, they arborize only sparsely in the accessory medulla (AME), which serves as important communication center of the circadian clock in cockroaches and flies. Furthermore, all bee PDF neurons cluster together, which makes it impossible to distinguish individual projections. Here, we investigated the developing bee PDF network and found that the first three PDF neurons arise in the third larval instar and form a dense network of varicose fibers at the base of the developing medulla that strongly resembles the AME of hemimetabolous insects. In addition, they send faint fibers toward the lateral superior protocerebrum. In last larval instar, PDF cells with larger somata appear and send fibers toward the distal medulla and the medial protocerebrum. In the dorsal part of the medulla serpentine layer, a small PDF knot evolves from which PDF fibers extend ventrally. This knot disappears during metamorphosis and the varicose arborizations in the putative AME become fainter. Instead, a new strongly stained PDF fiber hub appears in front of the lobula. Simultaneously, the number of PDF neurons increases and the PDF neuronal network in the brain gets continuously more complex.

Sex-specific and caste-specific brain adaptations related to spatial orientation in Cataglyphis ants.

Cataglyphis desert ants are charismatic central place foragers. After long-ranging foraging trips, individual workers navigate back to their nest relying mostly on visual cues. The reproductive caste faces other orientation challenges, i.e. mate finding and colony foundation. Here we compare brain structures involved in spatial orientation of Cataglyphis nodus males, gynes, and foragers by quantifying relative neuropil volumes associated with two visual pathways, and numbers and volumes of antennal lobe (AL) olfactory glomeruli. Furthermore, we determined absolute numbers of synaptic complexes in visual and olfactory regions of the mushroom bodies (MB) and a major relay station of the sky-compass pathway to the central complex (CX). Both female castes possess enlarged brain centers for sensory integration, learning, and memory, reflected in voluminous MBs containing about twice the numbers of synaptic complexes compared with males. Overall, male brains are smaller compared with both female castes, but the relative volumes of the optic lobes and CX are enlarged indicating the importance of visual guidance during innate behaviors. Male ALs contain greatly enlarged glomeruli, presumably involved in sex-pheromone detection. Adaptations at both the neuropil and synaptic levels clearly reflect differences in sex-specific and caste-specific demands for sensory processing and behavioral plasticity underlying spatial orientation.

Vision: Space and colour meet in the fly optic lobes.

Colour vision involves colour-opponent cells, which are excited and inhibited by different wavelengths. Synaptic interconnections between Drosophila Dm8 cells are required for forming spatio-chromatic receptive fields with a center and surround of opposing polarity which can invert, depending on the stimulus.

The RNA-Binding Protein SBR (Dm NXF1) Is Required for the Constitution of Medulla Boundaries in Drosophila melanogaster Optic Lobes.

Drosophila melanogaster sbr (small bristles) is an orthologue of the Nxf1 (nuclear export factor 1) genes in different Opisthokonta. The known function of Nxf1 genes is the export of various mRNAs from the nucleus to the cytoplasm. The cytoplasmic localization of the SBR protein indicates that the nuclear export function is not the only function of this gene in Drosophila. RNA-binding protein SBR enriches the nucleus and cytoplasm of specific neurons and glial cells. In sbr12 mutant males, the disturbance of medulla boundaries correlates with the defects of photoreceptor axons pathfinding, axon bundle individualization, and developmental neurodegeneration. RNA-binding protein SBR participates in processes allowing axons to reach and identify their targets.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization.

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.