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Vagus nerve stimulation (VNS) is a neuromodulation therapy for a broad and expanding set of neurologic conditions. However, the mechanism through which VNS influences central nervous system circuitry is not well described, limiting therapeutic optimization. VNS leads to widespread brain activation, but the effects on behavior are remarkably specific, indicating plasticity unique to behaviorally engaged neural circuits. To understand how VNS can lead to specific circuit modulation, we leveraged genetic tools including optogenetics and in vivo calcium imaging in mice learning a skilled reach task. We find that VNS enhances skilled motor learning in healthy animals via a cholinergic reinforcement mechanism, producing a rapid consolidation of an expert reach trajectory. In primary motor cortex (M1), VNS drives precise temporal modulation of neurons that respond to behavioral outcome. This suggests that VNS may accelerate motor refinement in M1 via cholinergic signaling, opening new avenues for optimizing VNS to target specific disease-relevant circuitry.

Vagus nerve stimulation (VNS) is a neuromodulation therapy for a broad and rapidly expanding set of neurologic conditions. Classically used to treat epilepsy and depression, VNS has recently received FDA approval for stroke rehabilitation and is under preclinical and clinical investigation for other neurologic indications. Despite benefits across a diverse range of neurological disorders, the mechanism through which VNS influences central nervous system circuitry is not well described, limiting therapeutic optimization. A deeper understanding of the influence of VNS on neural circuits and activity is needed to maximize the use of VNS therapy across a broad range of neurologic conditions.To investigate how VNS can influence the neurons and circuits that underlie behavior, we paired VNS with upper limb movement in mice learning a skilled motor task. We leveraged genetic tools to perform optogenetic circuit dissection, as well as longitudinal in vivo imaging of calcium activity in cortical neurons to understand the effect of VNS on neural function. We found that VNS robustly enhanced motor learning when temporally paired with successful movement outcome, while randomly applied VNS impaired learning. This suggests that temporally-precise VNS may act through augmenting outcome cues, such as reinforcement signals. Within motor cortex, VNS paired with movement outcome selectively modulates the neural population that represents outcome, but not other movement-related neurons, across both acute and behaviorally-relevant timescales. Phasic cholinergic signaling from basal forebrain is required both for VNS-driven improvements in motor learning and the effects on neural activity in M1. These results indicate that VNS enhances motor learning through precisely-timed phasic cholinergic signaling to reinforce outcome, resulting in the recruitment of specific, behaviorally-relevant cortical circuits. A deeper understanding of the mechanisms of VNS on neurons, circuits and behavior provides new opportunities to optimize VNS to treat neurologic conditions.

Summary: Sparse manipulation of neuron excitability during free behavior is critical for identifying neural substrates of behavior. Genetic tools for precise neuronal manipulation exist in the fruit fly, Drosophila melanogaster, but behavioral tools are still lacking to identify potentially subtle phenotypes only detectible using high-throughput and high spatiotemporal resolution. We developed three assay components that can be used modularly to study natural and optogenetically induced behaviors. FlyGate automatically releases flies one at a time into an assay. FlyDetect tracks flies in real time, is robust to severe occlusions, and can be used to track appendages, such as the head. GlobeDisplay is a spherical projection system covering the fly’s visual receptive field with a single projector. We demonstrate the utility of these components in an integrated system, FlyPEZ, by comprehensively modeling the input-output function for directional looming-evoked escape takeoffs and describing a millisecond-timescale phenotype from genetic silencing of a single visual projection neuron type. : Williamson et al. developed a high-throughput device to automatically isolate individual flies and record their behavior with high-speed video. They demonstrate the system’s utility for studying the Drosophila brain using modern genetic tools by discovering behavioral phenotypes that result from manipulation of small groups of neurons. Keywords: neuroscience, Drosophila, behavior, neural substrates, high throughput, neural circuits, high-speed video, visual stimulation, neural activation, escape, machine vision

Most chemical neurotransmission occurs through Ca2+-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca2+ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca2+ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca2+ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H+-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca2+–Calmodulin (CaM)-dependent manner. Ca2+–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca2+-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca2+–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.

Visual projection neurons (VPNs) provide an anatomical connection between early visual processing and higher brain regions. Here we characterize lobula columnar (LC) cells, a class of Drosophila VPNs that project to distinct central brain structures called optic glomeruli. We anatomically describe 22 different LC types and show that, for several types, optogenetic activation in freely moving flies evokes specific behaviors. The activation phenotypes of two LC types closely resemble natural avoidance behaviors triggered by a visual loom. In vivo two-photon calcium imaging reveals that these LC types respond to looming stimuli, while another type does not, but instead responds to the motion of a small object. Activation of LC neurons on only one side of the brain can result in attractive or aversive turning behaviors depending on the cell type. Our results indicate that LC neurons convey information on the presence and location of visual features relevant for specific behaviors. DOI: http://dx.doi.org/10.7554/eLife.21022.001, eLife digest Many animals rely heavily on what they can see to interact with the world around them. But how does the brain use such visual information to guide behavior? Light-sensitive neurons in the eye cannot distinguish between the visual signals associated with, say, an approaching predator or a source of food. Yet the brain can make this distinction. Networks of neurons in the brain perform computations to extract information from a visual scene that indicates the need for a particular behavior, such as an escape response. These networks are found in regions of the brain that communicate closely with the eyes. Cells known as visual projection neurons then relay the output of these networks to more central parts of the brain. By studying visual projection neurons, it is possible to work out what the eye tells the brain, and how the brain uses this information to control behavior. The fruit fly Drosophila is a suitable model organism in which to study these phenomena. This insect shows a range of behavioral responses to visual stimuli, and can be studied using sophisticated genetic tools. Wu, Nern et al. set out to explore how a group of visual projection neurons known as lobula columnar cells help fruit flies respond appropriately to visual stimuli. Experiments revealed that individual subtypes of lobula columnar cells convey information about the presence and general location of specific visual features. Wu, Nern et al. identified a number of lobular columnar subtypes involved in triggering escape responses to specific stimuli – such as walking backwards or taking off in flight – as well as others that can trigger the flies to approach a target. A next step is to map the circuits of neurons that act upstream and downstream of lobula columnar cells. This can help to reveal how these neurons detect specific visual features and how the fly then chooses and executes an appropriate behavior in response. Such studies in flies can provide insights into general principles of how brains use sensory information to guide behavior. DOI: http://dx.doi.org/10.7554/eLife.21022.002

Summary Animals rely on dedicated sensory circuits to extract and encode environmental features. How individual neurons integrate and translate these features into behavioral responses remains a major question. Here, we identify a visual projection neuron type that conveys predator approach information to the Drosophila giant fiber (GF) escape circuit. Genetic removal of this input during looming stimuli reveals that it encodes angular expansion velocity, whereas other input cell type(s) encode angular size. Motor program selection and timing emerge from linear integration of these two features within the GF. Linear integration improves size detection invariance over prior models and appropriately biases motor selection to rapid, GF-mediated escapes during fast looms. Our findings suggest feature integration, and motor control may occur as simultaneous operations within the same neuron and establish the Drosophila escape circuit as a model system in which these computations may be further dissected at the circuit level. Video Abstract

The synaptic v-SNARE n-Syb functions not only in synaptic vesicle exocytosis but also in delivery of protein-degrading enzymes to endosomes that are necessary to prevent protein aggregation and neurodegeneration., Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-syb (neuronal Synaptobrevin) plays a key role during synaptic vesicle exocytosis. In this paper, we report that loss of n-syb caused slow neurodegeneration independent of its role in neurotransmitter release in adult Drosophila melanogaster photoreceptor neurons. In addition to synaptic vesicles, n-Syb localized to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. Our evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescued n-syb–dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. Our findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity.

A vesicular ATPase regulates both vesicle fusion and subsequent acidification of a subset of neuronal lysosomes and autophagosomes., The vesicular adenosine triphosphatase (v-ATPase) is a proton pump that acidifies intracellular compartments. In addition, mutations in components of the membrane-bound v-ATPase V0 sector cause acidification-independent defects in yeast, worm, fly, zebrafish, and mouse. In this study, we present a dual function for the neuron-specific V0 subunit a1 orthologue v100 in Drosophila melanogaster. A v100 mutant that selectively disrupts proton translocation rescues a previously characterized synaptic vesicle fusion defect and vesicle fusion with early endosomes. Correspondingly, V100 selectively interacts with syntaxins on the respective target membranes, and neither synaptic vesicles nor early endosomes require v100 for their acidification. In contrast, V100 is required for acidification once endosomes mature into degradative compartments. As a consequence of the complete loss of this neuronal degradation mechanism, photoreceptors undergo slow neurodegeneration, whereas selective rescue of the acidification-independent function accelerates cell death by increasing accumulations in degradation-incompetent compartments. We propose that V100 exerts a temporally integrated dual function that increases neuronal degradative capacity.

Interindividual differences in neuronal wiring may contribute to behavioral individuality and affect susceptibility to neurological disorders. To investigate the causes and potential consequences of wiring variation in Drosophila melanogaster, we focused on a hemilineage of ventral nerve cord interneurons that exhibits morphological variability. We find that late-born subclasses of the 12A hemilineage are highly sensitive to genetic and environmental variation. Neurons in the second thoracic segment are particularly variable with regard to two developmental decisions, whereas its segmental homologs are more robust. This variability “hotspot” depends on Ultrabithorax expression in the 12A neurons, indicating variability is cell-intrinsic and under genetic control. 12A development is more variable and sensitive to temperature in long-established laboratory strains than in strains recently derived from the wild. Strains with a high frequency of one of the 12A variants also showed a high frequency of animals with delayed spontaneous flight initiation, whereas other wing-related behaviors did not show such a correlation and were thus not overtly affected by 12A variation. These results show that neurodevelopmental robustness is variable and under genetic control in Drosophila and suggest that the fly may serve as a model for identifying conserved gene pathways that stabilize wiring in stressful developmental environments. Moreover, some neuronal lineages are variation hotspots and thus may be more amenable to evolutionary change.

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001, eLife digest In the human brain, 100 billion neurons form 100 trillion connections. Each neuron consists of a cell body with numerous small branch-like projections known as dendrites (from the Greek word for ‘tree’), plus a long cable-like structure called the axon. Neurons receive electrical inputs from neighboring cells via their dendrites, and then relay these signals onto other cells in their network via their axons. The development of the brain relies on new neurons integrating successfully into existing networks. Axon branching helps with this by enabling a single neuron to establish connections with several cells, but it is unclear how individual neurons decide when and where to form branches. Now, Zschätzsch et al. have revealed the mechanism behind this process in the fruit fly, Drosophila. Mutant flies that lack a protein called EGFR produce abnormal numbers of axon branches, suggesting that this molecule regulates branch formation. Indeed in fruit flies, just as in mammals, the developing brain initially produces excessive numbers of branches, which are subsequently pruned to leave only those that have formed appropriate connections. In Drosophila, an uneven distribution of EGFR between branches belonging to the same axon acts as a signal to regulate this pruning process. To examine this mechanism in more detail, high-resolution four-dimensional imaging was used to study brains that had been removed from Drosophila pupae and kept alive in special culture chambers. Axon branching and loss could now be followed in real time, and were found to occur more slowly in brains that lacked EGFR. The receptor controlled the branching of axons by influencing the distribution of another protein called actin, which is a key component of the internal skeleton that gives cells their structure. In addition to providing new insights into a fundamental aspect of brain development, the work of Zschätzsch et al. also highlights the importance of stochastic events in shaping the network of connections within the developing brain. These findings may well be relevant to ongoing efforts to map the human brain ‘connectome’. DOI: http://dx.doi.org/10.7554/eLife.01699.002

We discovered a bimodal behavior in the genetically tractable organism Drosophila melanogaster that allowed us to directly probe the neural mechanisms of an action selection process. When confronted by a predator-mimicking looming stimulus, a fly responds with either a long-duration escape behavior sequence that initiates stable flight or a distinct, short-duration sequence that sacrifices flight stability for speed. Intracellular recording of the descending giant fiber (GF) interneuron during head-fixed escape revealed that GF spike timing relative to parallel circuits for escape actions determined which of the two behavioral responses was elicited. The process was well described by a simple model in which the GF circuit has a higher activation threshold than the parallel circuits, but can override ongoing behavior to force a short takeoff. Our findings suggest a neural mechanism for action selection in which relative activation timing of parallel circuits creates the appropriate motor output.

During Drosophila brain development, a neuron-specific endolysosomal degradation pathway provides a mechanism for continuous guidance receptor turnover and proper connectivity., Axon pathfinding and synapse formation rely on precise spatiotemporal localization of guidance receptors. However, little is known about the neuron-specific intracellular trafficking mechanisms that underlie the sorting and activity of these receptors. Here we show that loss of the neuron-specific v-ATPase subunit a1 leads to progressive endosomal guidance receptor accumulations after neuronal differentiation. In the embryo and in adult photoreceptors, these accumulations occur after axon pathfinding and synapse formation is complete. In contrast, receptor missorting occurs sufficiently early in neurons of the adult central nervous system to cause connectivity defects. An increase of guidance receptors, but not of membrane proteins without signaling function, causes specific gain-of-function phenotypes. A point mutant that promotes sorting but prevents degradation reveals spatiotemporally specific guidance receptor turnover and accelerates developmental defects in photoreceptors and embryonic motor neurons. Our findings indicate that a neuron-specific endolysosomal degradation mechanism is part of the cell biological machinery that regulates guidance receptor turnover and signaling., Author Summary Brain wiring is determined by genetic and environmental factors, nature and nurture. The Drosophila brain is a model for the genetic basis of brain wiring. The fly visual system in particular is thought to be “hard-wired,” i.e., encoded solely by a genetic program. Some key genes encode the guidance receptors that serve as “wiring” and synaptic connectivity signals. However, it is poorly understood how guidance receptors are spatiotemporally regulated to serve as meaningful synapse formation signals. Indeed, many genes required for brain wiring do not encode the guidance receptors themselves, but rather encode parts of the cell biological machinery that governs their spatiotemporal signaling dynamics. For example, the vesicular ATPase is an intracellular sorting and acidification complex involved in regulating guidance receptor turnover and signaling. The protein V100 is a member of this v-ATPase complex, and in this study we show that mutations in the v100 gene cause brain wiring defects specifically in the adult brain. We further describe a V100-dependent intracellular “sort-and-degrade” mechanism that is required in neurons, and find that when this mechanism is perturbed, it leads to progressive build-up of and aberrant signaling by guidance receptors. These data suggest that a v100-dependent neuronal degradation mechanism provides a cell biological basis for guidance receptor turnover and spatiotemporally controlled dynamics during neural circuit formation.

Defective autophagy and lysosomal degradation are hallmarks of numerous neurodegenerative disorders. Vesicular ATPases are intracellular proton pumps that acidify autophagosomes and lysosomes. V0a1 is a key component of the v-ATPase that is only required in neurons in Drosophila melanogaster. We have recently shown that loss of V0a1 in Drosophila photoreceptor neurons leads to slow, adult-onset degeneration.1 Concurrently, Lee et al.2 reported that V0a1 fails to localize to lysosomal compartments in cells from Presenilin 1 knock-out cells. Together these two reports suggest that a neuronal V0a1-dependent degradation mechanism may be causally linked to Alzheimer pathology. Indeed, we now show that loss of V0a1 makes Drosophila neurons more susceptible to insult with human Alzheimer-related neurotoxic Aβ and tau proteins. Furthermore, we discuss the potential significance of the discovery of the neuron-specific degradation mechanism in Drosophila for intracellular degradation defects in Alzheimer Disease.

Gradients of secreted small morphogenic molecules control cell proliferation and patterning throughout animal development. Recent years have seen the discovery of surprising roles for morphogens in later developmental processes, including axon pathfinding and synaptogenesis. The latest addition is a role for the TGF-β superfamily morphogen Activin in synaptic patterning of the Drosophila visual system. In contrast to classical instructive and long-range morphogen gradients, Activin acts as a permissive and local motility restriction signal around several hundred individual photoreceptor axon terminals. Activin must therefore act in concert with other instructively attracting and repelling signals as part of a larger genetic program for brain wiring.