Mechanisms Underlying the Neural Computation of Head Direction.

Many animals use an internal sense of direction to guide their movements through the world. Neurons selective to head direction are thought to support this directional sense and have been found in a diverse range of species, from insects to primates, highlighting their evolutionary importance. Across species, most head-direction networks share four key properties: a unique representation of direction at all times, persistent activity in the absence of movement, integration of angular velocity to update the representation, and the use of directional cues to correct drift. The dynamics of theorized network structures called ring attractors elegantly account for these properties, but their relationship to brain circuits is unclear. Here, we review experiments in rodents and flies that offer insights into potential neural implementations of ring attractor networks. We suggest that a theory-guided search across model systems for biological mechanisms that enable such dynamics would uncover general principles underlying head-direction circuit function.

Generation of stable heading representations in diverse visual scenes.

Many animals rely on an internal heading representation when navigating in varied environments1-10. 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 body3,11,12. Each compass neuron receives inputs from 'ring' neurons that are selective for particular visual features13-16; 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 plasticity17-22, 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.

High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.

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.

Neural dynamics for landmark orientation and angular path integration.

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.

Feature detection and orientation tuning in the Drosophila central complex.

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 and its short- and long-term memory for visual patterns. 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 and memory for visual patterns in tethered flying flies. 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. Visual responses were diminished during flight, but, in contrast with the hypothesized role of the central complex in the control of locomotion, 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. More broadly, this study opens the door to mechanistic investigations of circuit computations underlying visually guided action selection in the Drosophila central complex.

Ultrasensitive fluorescent proteins for imaging neuronal activity.

Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.

Independent optical excitation of distinct neural populations.

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

A neural circuit architecture for rapid learning in goal-directed navigation.

Anchoring goals to spatial representations enables flexible navigation but is challenging in novel environments when both representations must be acquired simultaneously. We propose a framework for how Drosophila uses internal representations of head direction (HD) to build goal representations upon selective thermal reinforcement. We show that flies use stochastically generated fixations and directed saccades to express heading preferences in an operant visual learning paradigm and that HD neurons are required to modify these preferences based on reinforcement. We used a symmetric visual setting to expose how flies' HD and goal representations co-evolve and how the reliability of these interacting representations impacts behavior. Finally, we describe how rapid learning of new goal headings may rest on a behavioral policy whose parameters are flexible but whose form is genetically encoded in circuit architecture. Such evolutionarily structured architectures, which enable rapidly adaptive behavior driven by internal representations, may be relevant across species.

Optimization of a GCaMP calcium indicator for neural activity imaging.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior.

Drosophila melanogaster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball's motion is tracked at high resolution and can be treated as a proxy for the fly's own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in horizontal-system neurons correlated with robust optomotor behavior during walking. Our technique allows both behavior and physiology in identified neurons to be monitored in a genetic model organism with an extensive repertoire of walking behaviors.

Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.

A connectome of the Drosophila central complex reveals network motifs suitable for flexible navigation and context-dependent action selection.

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.

Encoding and decoding of overlapping odor sequences.

Odors evoke complex responses in locust antennal lobe projection neurons (PNs)-the mitral cell analogs. These patterns evolve over hundreds of milliseconds and contain information about odor identity and concentration. In nature, animals often encounter many odorants in short temporal succession. We explored the effects of such conditions by presenting two different odors with variable intervening delays. PN ensemble representations tracked stimulus changes and, in some delay conditions, reached states that corresponded neither to the representation of either odor alone nor to the static mixture of the two. We then recorded from Kenyon cells (KCs), the PNs' targets. Their responses were consistent with the PN population's behavior: in some conditions, KCs were recruited that did not fire during single-odor or mixture stimuli. Thus, PN population dynamics are history dependent, and responses of individual KCs are consistent with piecewise temporal decoding of PN output over large sections of the PN population.

The Neuroanatomical Ultrastructure and Function of a Biological Ring Attractor.

Neural representations of head direction (HD) have been discovered in many species. Theoretical work has proposed that the dynamics associated with these representations are generated, maintained, and updated by recurrent network structures called ring attractors. We evaluated this theorized structure-function relationship by performing electron-microscopy-based circuit reconstruction and RNA profiling of identified cell types in the HD system of Drosophila melanogaster. We identified motifs that have been hypothesized to maintain the HD representation in darkness, update it when the animal turns, and tether it to visual cues. Functional studies provided support for the proposed roles of individual excitatory or inhibitory circuit elements in shaping activity. We also discovered recurrent connections between neuronal arbors with mixed pre- and postsynaptic specializations. Our results confirm that the Drosophila HD network contains the core components of a ring attractor while also revealing unpredicted structural features that might enhance the network's computational power.

Busted! A Dope Ring with Activity Clocked at Dawn and Dusk.

Clock neurons generate circadian rhythms in behavioral activity, but the relevant pathways remain poorly understood. In this issue of Neuron, Liang et al. (2019) show that distinct clock neurons independently drive movement-promoting "ring neurons" in Drosophila through dopaminergic relays to support morning and evening locomotor activity.

Visually Guided Behavior and Optogenetically Induced Learning in Head-Fixed Flies Exploring a Virtual Landscape.

Studying the intertwined roles of sensation, experience, and directed action in navigation has been facilitated by the development of virtual reality (VR) environments for head-fixed animals, allowing for quantitative measurements of behavior in well-controlled conditions. VR has long featured in studies of Drosophila melanogaster, but these experiments have typically allowed the fly to change only its heading in a visual scene and not its position. Here we explore how flies move in two dimensions (2D) using a visual VR environment that more closely captures an animal's experience during free behavior. We show that flies' 2D interaction with landmarks cannot be automatically derived from their orienting behavior under simpler one-dimensional (1D) conditions. Using novel paradigms, we then demonstrate that flies in 2D VR adapt their behavior in response to optogenetically delivered appetitive and aversive stimuli. Much like free-walking flies after encounters with food, head-fixed flies exploring a 2D VR respond to optogenetic activation of sugar-sensing neurons by initiating a local search, which appears not to rely on visual landmarks. Visual landmarks can, however, help flies to avoid areas in VR where they experience an aversive, optogenetically generated heat stimulus. By coupling aversive virtual heat to the flies' presence near visual landmarks of specific shapes, we elicit selective learned avoidance of those landmarks. Thus, we demonstrate that head-fixed flies adaptively navigate in 2D virtual environments, but their reliance on visual landmarks is context dependent. These behavioral paradigms set the stage for interrogation of the fly brain circuitry underlying flexible navigation in complex multisensory environments.

Intensity versus identity coding in an olfactory system.

We examined the encoding and decoding of odor identity and intensity by neurons in the antennal lobe and the mushroom body, first and second relays, respectively, of the locust olfactory system. Increased odor concentration led to changes in the firing patterns of individual antennal lobe projection neurons (PNs), similar to those caused by changes in odor identity, thus potentially confounding representations for identity and concentration. However, when these time-varying responses were examined across many PNs, concentration-specific patterns clustered by identity, resolving the apparent confound. This is because PN ensemble representations changed relatively continuously over a range of concentrations of each odorant. The PNs' targets in the mushroom body-Kenyon cells (KCs)-had sparse identity-specific responses with diverse degrees of concentration invariance. The tuning of KCs to identity and concentration and the patterning of their responses are consistent with piecewise decoding of their PN inputs over oscillation-cycle length epochs.

Building a functional connectome of the Drosophila central complex.

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.

Ring attractor dynamics in the Drosophila central brain.

Ring attractors are a class of recurrent networks hypothesized to underlie the representation of heading direction. Such network structures, schematized as a ring of neurons whose connectivity depends on their heading preferences, can sustain a bump-like activity pattern whose location can be updated by continuous shifts along either turn direction. We recently reported that a population of fly neurons represents the animal's heading via bump-like activity dynamics. We combined two-photon calcium imaging in head-fixed flying flies with optogenetics to overwrite the existing population representation with an artificial one, which was then maintained by the circuit with naturalistic dynamics. A network with local excitation and global inhibition enforces this unique and persistent heading representation. Ring attractor networks have long been invoked in theoretical work; our study provides physiological evidence of their existence and functional architecture.