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Many animals navigate using a combination of visual landmarks and path integration. In mammalian brains, head direction cells integrate these two streams of information by representing an animal's heading relative to landmarks, yet maintaining their directional tuning in darkness based on self-motion cues. Here we use two-photon calcium imaging in head-fixed Drosophila melanogaster walking on a ball in a virtual reality arena to demonstrate that landmark-based orientation and angular path integration are combined in the population responses of neurons whose dendrites tile the ellipsoid body, a toroidal structure in the centre of the fly brain. The neural population encodes the fly's azimuth relative to its environment, tracking visual landmarks when available and relying on self-motion cues in darkness. When both visual and self-motion cues are absent, a representation of the animal's orientation is maintained in this network through persistent activity, a potential substrate for short-term memory. Several features of the population dynamics of these neurons and their circular anatomical arrangement are suggestive of ring attractors, network structures that have been proposed to support the function of navigational brain circuits. Calcium imaging of the brain of tethered flies walking in a virtual reality arena showed that a population of neurons with dendrites that tile the ‘ellipsoid body’ use information from visual landmarks and the fly's own rotation to compute heading; this suggests insects possess neurons with similarities to ‘head direction cells’ known to contribute to spatial navigation in mammalian brains. How insects know their place How insect brains combine visual landmarks and path integration during navigation has been unknown. Johannes Seelig and Vivek Jayaraman perform calcium imaging of the brain of tethered flies walking in a virtual reality arena and show that a population of neurons with dendrites that tile the 'ellipsoid body' uses information from visual landmarks and the fly's own rotation to compute heading. This is the first evidence for an invertebrate equivalent of the 'head direction' neurons known to contribute to spatial navigation in mammalian brains.

The central complex is a highly conserved insect brain region composed of morphologically stereotyped neurons that arborize in distinctively shaped substructures. The region is implicated in a wide range of behaviors and several modeling studies have explored its circuit computations. Most studies have relied on assumptions about connectivity between neurons based on their overlap in light microscopy images. Here, we present an extensive functional connectome of Drosophila melanogaster’s central complex at cell-type resolution. Using simultaneous optogenetic stimulation, calcium imaging and pharmacology, we tested the connectivity between 70 presynaptic-to-postsynaptic cell-type pairs. We identified numerous inputs to the central complex, but only a small number of output channels. Additionally, the connectivity of this highly recurrent circuit appears to be sparser than anticipated from light microscopy images. Finally, the connectivity matrix highlights the potentially critical role of a class of bottleneck interneurons. All data are provided for interactive exploration on a website. Some of the most evocative discoveries in neuroscience have been those of internal representations, such as neural activity patterns that represent which direction an animal is facing and its place in its surroundings. Understanding how neurons connect to one another to form ‘circuits’ is crucial to understanding how these circuits maintain such representations. Many of the design principles that underlie circuit function in the brains of fruit flies apply to other animals. However, fly brains are easier to study because genetic tools can be used on them to selectively activate and image the activity of specific types of neurons. By activating one type of neuron and imaging the activity of another that may be connected to it, we obtain what is called a functional ‘connectome’: a map of neural connectivity that identifies different pathways that information can flow along. A region of the fly brain called the central complex is involved in many important behaviors, including navigation and sleep. Researchers know about the types of neurons in the region and about how the activity of some of them changes during different behaviors. However, obtaining the connectome of the central complex would make it easier to understand how the central complex works. A technique called optogenetics allows specific types of neurons to be activated one at a time by shining light onto them. By imaging the activity of neurons that might be connected to an optogenetically activated neuron, Franconville et al. have now built an extensive – albeit still incomplete – map of the connections within the central complex of fruit flies. The map reveals two key bottlenecks in the central complex circuit. Firstly, a neuron type in a substructure called the protocerebral bridge controls a lot of the information flowing through the circuit. Secondly, the circuit appears to have very few true ‘output’ neuron types – Franconville et al. identified only one. These results suggest that however complicated the computations performed by the central complex circuit might be, the output of the circuit, which likely guides the fly’s actions, may be much simpler. Franconville et al. have compiled the mapping results into an interactive website that makes the neuroscientific data both freely available and easily explorable. As researchers perform more such experiments, the new data can be added to the map. This information can be used to constrain theories and inspire new ideas about how the central complex does what it does.

Two-photon calcium imaging experiments reveal that ring neurons in the Drosophila central complex represent visual features and show direction-selective orientation tuning, resembling simple cells in mammalian primary visual cortex; future fly studies may enhance our understanding of circuit computations underlying visually guided action selection. Visual recognition in the insect brain Insects can perform complex visual pattern recognition and memory tasks, capabilities that equip them for challenging motor behaviours such as flight. But whether and how the neurons of the fly central complex represent visual features are unknown. Now Johannes Seelig and Vivek Jayaraman have imaged neurons of the Drosophila melanogaster central complex in head-fixed walking and flying flies, and show that ring neurons there represent visual features such as direction-selective orientation tuning, thus resembling so-called simple cells found in the primary visual cortex of mammals. This work lays the foundations for the genetic investigation of the neuronal circuits used for visually guided motor commands. Many animals, including insects, are known to use visual landmarks to orient in their environment. In Drosophila melanogaster , behavioural genetics studies have identified a higher brain structure called the central complex as being required for the fly’s innate responses to vertical visual features 1 and its short- and long-term memory for visual patterns 2 , 3 , 4 . But whether and how neurons of the fly central complex represent visual features are unknown. Here we use two-photon calcium imaging in head-fixed walking and flying flies to probe visuomotor responses of ring neurons—a class of central complex neurons that have been implicated in landmark-driven spatial memory in walking flies 2 , 3 and memory for visual patterns in tethered flying flies 5 . We show that dendrites of ring neurons are visually responsive and arranged retinotopically. Ring neuron receptive fields comprise both excitatory and inhibitory subfields, resembling those of simple cells in the mammalian primary visual cortex. Ring neurons show strong and, in some cases, direction-selective orientation tuning, with a notable preference for vertically oriented features similar to those that evoke innate responses in flies 1 , 2 . Visual responses were diminished during flight, but, in contrast with the hypothesized role of the central complex in the control of locomotion 6 , not modulated during walking. Taken together, these results indicate that ring neurons represent behaviourally relevant visual features in the fly’s environment, enabling downstream central complex circuits to produce appropriate motor commands 6 . More broadly, this study opens the door to mechanistic investigations of circuit computations underlying visually guided action selection in the Drosophila central complex.

Flexible behaviors over long timescales are thought to engage recurrent neural networks in deep brain regions, which are experimentally challenging to study. In insects, recurrent circuit dynamics in a brain region called the central complex (CX) enable directed locomotion, sleep, and context- and experience-dependent spatial navigation. We describe the first complete electron microscopy-based connectome of the Drosophila CX, including all its neurons and circuits at synaptic resolution. We identified new CX neuron types, novel sensory and motor pathways, and network motifs that likely enable the CX to extract the fly’s head direction, maintain it with attractor dynamics, and combine it with other sensorimotor information to perform vector-based navigational computations. We also identified numerous pathways that may facilitate the selection of CX-driven behavioral patterns by context and internal state. The CX connectome provides a comprehensive blueprint necessary for a detailed understanding of network dynamics underlying sleep, flexible navigation, and state-dependent action selection.

Many animals maintain an internal representation of their heading as they move through their surroundings. Such a compass representation was recently discovered in a neural population in the Drosophila melanogaster central complex, a brain region implicated in spatial navigation. Here, we use two-photon calcium imaging and electrophysiology in head-fixed walking flies to identify a different neural population that conjunctively encodes heading and angular velocity, and is excited selectively by turns in either the clockwise or counterclockwise direction. We show how these mirror-symmetric turn responses combine with the neurons’ connectivity to the compass neurons to create an elegant mechanism for updating the fly’s heading representation when the animal turns in darkness. This mechanism, which employs recurrent loops with an angular shift, bears a resemblance to those proposed in theoretical models for rodent head direction cells. Our results provide a striking example of structure matching function for a broadly relevant computation.

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging. Neurons encode information with brief electrical pulses called spikes. Monitoring spikes in large populations of neurons is a powerful method for studying how networks of neurons process information and produce behavior. This activity can be detected using fluorescent protein indicators, or “probes”, which light up when neurons are active. The best existing probes produce green fluorescence. However, red fluorescent probes would allow us to see deeper into the brain, and could also be used with green probes to image the activity and interactions of different neuron types simultaneously. However, existing red fluorescent probes are not as good at detecting neural activity as green probes. By optimizing two existing red fluorescent proteins, Dana et al. have now produced two new red fluorescent probes, each with different advantages. The new protein indicators detect neural activity with high sensitivity and allow researchers to image previously unseen brain activity. Tests showed that the probes work in cultured neurons and allow imaging of the activity of neurons in mice, flies, fish and worms. History has shown that enhancing the techniques used to study biological processes can lead to fundamentally new insights. In the future, Dana et al. would therefore like to make even more sensitive protein indicators that will allow larger networks of neurons deeper in the brain to be imaged.

Many animals rely on an internal heading representation when navigating in varied environments 1 – 10 . How this representation is linked to the sensory cues that define different surroundings is unclear. In the fly brain, heading is represented by ‘compass’ neurons that innervate a ring-shaped structure known as the ellipsoid body 3 , 11 , 12 . Each compass neuron receives inputs from ‘ring’ neurons that are selective for particular visual features 13 – 16 ; this combination provides an ideal substrate for the extraction of directional information from a visual scene. Here we combine two-photon calcium imaging and optogenetics in tethered flying flies with circuit modelling, and show how the correlated activity of compass and visual neurons drives plasticity 17 – 22 , which flexibly transforms two-dimensional visual cues into a stable heading representation. We also describe how this plasticity enables the fly to convert a partial heading representation, established from orienting within part of a novel setting, into a complete heading representation. Our results provide mechanistic insight into the memory-related computations that are essential for flexible navigation in varied surroundings. Two-photon calcium imaging and optogenetic experiments in tethered flying flies, combined with modelling, demonstrate how the correlation of compass and visual neurons underpins plasticity that enables the transformation of visual cues into stable heading representations.

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters. Genetically encoded voltage indicators (GEVIs) allow visualisation of fast action potentials in neurons but most are bright at rest and dimmer during an action potential. Here, the authors engineer electrochromic FRET GEVIs with fast, bright and positive-going fluorescence signals for in vivo imaging.

Using two-color two-photon calcium imaging, the authors identified transformations of representations across synaptically connected pairs of neurons along a visual pathway to the Drosophila central complex. Neural responses to stimuli in the ipsilateral field are modulated by stimuli in the contralateral field, an effect that depends on past stimulus history. Many animals orient using visual cues, but how a single cue is selected from among many is poorly understood. Here we show that Drosophila ring neurons—central brain neurons implicated in navigation—display visual stimulus selection. Using in vivo two-color two-photon imaging with genetically encoded calcium indicators, we demonstrate that individual ring neurons inherit simple-cell-like receptive fields from their upstream partners. Stimuli in the contralateral visual field suppressed responses to ipsilateral stimuli in both populations. Suppression strength depended on when and where the contralateral stimulus was presented, an effect stronger in ring neurons than in their upstream inputs. This history-dependent effect on the temporal structure of visual responses, which was well modeled by a simple biphasic filter, may determine how visual references are selected for the fly's internal compass. Our approach highlights how two-color calcium imaging can help identify and localize the origins of sensory transformations across synaptically connected neural populations.