Mechanical vibration patterns elicit behavioral transitions and habituation in crawling Drosophila larvae.

How animals respond to repeatedly applied stimuli, and how animals respond to mechanical stimuli in particular, are important questions in behavioral neuroscience. We study adaptation to repeated mechanical agitation using the Drosophila larva. Vertical vibration stimuli elicit a discrete set of responses in crawling larvae: continuation, pause, turn, and reversal. Through high-throughput larva tracking, we characterize how the likelihood of each response depends on vibration intensity and on the timing of repeated vibration pulses. By examining transitions between behavioral states at the population and individual levels, we investigate how the animals habituate to the stimulus patterns. We identify time constants associated with desensitization to prolonged vibration, with re-sensitization during removal of a stimulus, and additional layers of habituation that operate in the overall response. Known memory-deficient mutants exhibit distinct behavior profiles and habituation time constants. An analogous simple electrical circuit suggests possible neural and molecular processes behind adaptive behavior.

Plasma myoglobin indicates muscle damage associated with acceleration/deceleration during football.

BACKGROUND: Monitoring muscle damage in athletes assists not only coaches to adjust the training workload but also medical staff to prevent injury. Measuring blood myoglobin concentration can help evaluate muscle damage. The novel portable device utilized in this study allows for easy on-site measurement of myoglobin, providing real-time data on the player's muscle damage. This study investigated the relationship between external load (global positioning system parameters) and internal loads (myoglobin concentration and creatine kinase activity) in 15 male professional football players before and after a match. METHODS: Whole blood samples from participants' fingertips were collected before the match (baseline) and at 2, 16, and 40 h after the match. Myoglobin concentrations were measured using the IA-100 compact immunoassay system. Creatine kinase concentrations were measured in a clinical laboratory, and match loads were monitored using a global positioning system device. RESULTS: The mean myoglobin concentration was significantly higher at 2 h than at the other time points (P<0.05), and decreased to baseline levels within 16 h post-match. The mean creatine kinase concentration increased after the match but did not reach a significant level. Muscle damage monitored by myoglobin after football match-play was strongly associated with acceleration/deceleration metrics rather than the sprint/high-speed running distance. CONCLUSIONS: Our findings indicate that myoglobin is a more sensitive marker of muscle damage than creatine kinase after football match-play. Monitoring myoglobin in athletes can aid in determining their recovery status from the previous training load and help practitioners manage the training load.

Comparative connectomics and escape behavior in larvae of closely related Drosophila species.

Evolution has generated an enormous variety of morphological, physiological, and behavioral traits in animals. How do behaviors evolve in different directions in species equipped with similar neurons and molecular components? Here we adopted a comparative approach to investigate the similarities and differences of escape behaviors in response to noxious stimuli and their underlying neural circuits between closely related drosophilid species. Drosophilids show a wide range of escape behaviors in response to noxious cues, including escape crawling, stopping, head casting, and rolling. Here we find that D. santomea, compared with its close relative D. melanogaster, shows a higher probability of rolling in response to noxious stimulation. To assess whether this behavioral difference could be attributed to differences in neural circuitry, we generated focused ion beam-scanning electron microscope volumes of the ventral nerve cord of D. santomea to reconstruct the downstream partners of mdIV, a nociceptive sensory neuron in D. melanogaster. Along with partner interneurons of mdVI (including Basin-2, a multisensory integration neuron necessary for rolling) previously identified in D. melanogaster, we identified two additional partners of mdVI in D. santomea. Finally, we showed that joint activation of one of the partners (Basin-1) and a common partner (Basin-2) in D. melanogaster increased rolling probability, suggesting that the high rolling probability in D. santomea is mediated by the additional activation of Basin-1 by mdIV. These results provide a plausible mechanistic explanation for how closely related species exhibit quantitative differences in the likelihood of expressing the same behavior.

Nociception in fruit fly larvae.

Nociception, the process of encoding and processing noxious or painful stimuli, allows animals to detect and avoid or escape from potentially life-threatening stimuli. Here, we provide a brief overview of recent technical developments and studies that have advanced our understanding of the Drosophila larval nociceptive circuit and demonstrated its potential as a model system to elucidate the mechanistic basis of nociception. The nervous system of a Drosophila larva contains roughly 15,000 neurons, which allows for reconstructing the connectivity among them directly by transmission electron microscopy. In addition, the availability of genetic tools for manipulating the activity of individual neurons and recent advances in computational and high-throughput behavior analysis methods have facilitated the identification of a neural circuit underlying a characteristic nocifensive behavior. We also discuss how neuromodulators may play a key role in modulating the nociceptive circuit and behavioral output. A detailed understanding of the structure and function of Drosophila larval nociceptive neural circuit could provide insights into the organization and operation of pain circuits in mammals and generate new knowledge to advance the development of treatment options for pain in humans.

Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations resulting in decreased hEAAT1 Cl- channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. We also identified what we believe is a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Recurrent architecture for adaptive regulation of learning in the insect brain.

Dopaminergic neurons (DANs) drive learning across the animal kingdom, but the upstream circuits that regulate their activity and thereby learning remain poorly understood. We provide a synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mushroom body of Drosophila larva. We discover afferent sensory pathways and a large population of neurons that provide feedback from mushroom body output neurons and link distinct memory systems (aversive and appetitive). We combine this with functional studies of DANs and their presynaptic partners and with comprehensive circuit modeling. We find that DANs compare convergent feedback from aversive and appetitive systems, which enables the computation of integrated predictions that may improve future learning. Computational modeling reveals that the discovered feedback motifs increase model flexibility and performance on learning tasks. Our study provides the most detailed view to date of biological circuit motifs that support associative learning.

Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila.

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.

Identification of Inhibitory Premotor Interneurons Activated at a Late Phase in a Motor Cycle during Drosophila Larval Locomotion.

Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). Our knowledge of how CPG networks generate motor patterns in complex nervous systems remains incomplete, despite decades of work in a variety of model organisms. Substrate borne locomotion in Drosophila larvae is driven by waves of muscular contraction that propagate through multiple body segments. We use the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. Our recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. Here, we report on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). We used calcium imaging to search for interneurons that show rhythmic activity and identified GVLIs as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs' wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. We propose that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments.

A multilevel multimodal circuit enhances action selection in Drosophila.

Natural events present multiple types of sensory cues, each detected by a specialized sensory modality. Combining information from several modalities is essential for the selection of appropriate actions. Key to understanding multimodal computations is determining the structural patterns of multimodal convergence and how these patterns contribute to behaviour. Modalities could converge early, late or at multiple levels in the sensory processing hierarchy. Here we show that combining mechanosensory and nociceptive cues synergistically enhances the selection of the fastest mode of escape locomotion in Drosophila larvae. In an electron microscopy volume that spans the entire insect nervous system, we reconstructed the multisensory circuit supporting the synergy, spanning multiple levels of the sensory processing hierarchy. The wiring diagram revealed a complex multilevel multimodal convergence architecture. Using behavioural and physiological studies, we identified functionally connected circuit nodes that trigger the fastest locomotor mode, and others that facilitate it, and we provide evidence that multiple levels of multimodal integration contribute to escape mode selection. We propose that the multilevel multimodal convergence architecture may be a general feature of multisensory circuits enabling complex input-output functions and selective tuning to ecologically relevant combinations of cues.

Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators.

The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.

Discovery of brainwide neural-behavioral maps via multiscale unsupervised structure learning.

A single nervous system can generate many distinct motor patterns. Identifying which neurons and circuits control which behaviors has been a laborious piecemeal process, usually for one observer-defined behavior at a time. We present a fundamentally different approach to neuron-behavior mapping. We optogenetically activated 1054 identified neuron lines in Drosophila larvae and tracked the behavioral responses from 37,780 animals. Application of multiscale unsupervised structure learning methods to the behavioral data enabled us to identify 29 discrete, statistically distinguishable, observer-unbiased behavioral phenotypes. Mapping the neural lines to the behavior(s) they evoke provides a behavioral reference atlas for neuron subsets covering a large fraction of larval neurons. This atlas is a starting point for connectivity- and activity-mapping studies to further investigate the mechanisms by which neurons mediate diverse behaviors.

High-throughput analysis of stimulus-evoked behaviors in Drosophila larva reveals multiple modality-specific escape strategies.

All organisms react to noxious and mechanical stimuli but we still lack a complete understanding of cellular and molecular mechanisms by which somatosensory information is transformed into appropriate motor outputs. The small number of neurons and excellent genetic tools make Drosophila larva an especially tractable model system in which to address this problem. We developed high throughput assays with which we can simultaneously expose more than 1,000 larvae per man-hour to precisely timed noxious heat, vibration, air current, or optogenetic stimuli. Using this hardware in combination with custom software we characterized larval reactions to somatosensory stimuli in far greater detail than possible previously. Each stimulus evoked a distinctive escape strategy that consisted of multiple actions. The escape strategy was context-dependent. Using our system we confirmed that the nociceptive class IV multidendritic neurons were involved in the reactions to noxious heat. Chordotonal (ch) neurons were necessary for normal modulation of head casting, crawling and hunching, in response to mechanical stimuli. Consistent with this we observed increases in calcium transients in response to vibration in ch neurons. Optogenetic activation of ch neurons was sufficient to evoke head casting and crawling. These studies significantly increase our understanding of the functional roles of larval ch neurons. More generally, our system and the detailed description of wild type reactions to somatosensory stimuli provide a basis for systematic identification of neurons and genes underlying these behaviors.

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila.

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

Rich regulates target specificity of photoreceptor cells and N-cadherin trafficking in the Drosophila visual system via Rab6.

Neurons establish specific synaptic connections with their targets, a process that is highly regulated. Numerous cell adhesion molecules have been implicated in target recognition, but how these proteins are precisely trafficked and targeted is poorly understood. To identify components that affect synaptic specificity, we carried out a forward genetic screen in the Drosophila eye. We identified a gene, named ric1 homologue (rich), whose loss leads to synaptic specificity defects. Loss of rich leads to reduction of N-Cadherin in the photoreceptor cell synapses but not of other proteins implicated in target recognition, including Sec15, DLAR, Jelly belly, and PTP69D. The Rich protein binds to Rab6, and Rab6 mutants display very similar phenotypes as the rich mutants. The active form of Rab6 strongly suppresses the rich synaptic specificity defect, indicating that Rab6 is regulated by Rich. We propose that Rich activates Rab6 to regulate N-Cadherin trafficking and affects synaptic specificity.

A combinatorial semaphorin code instructs the initial steps of sensory circuit assembly in the Drosophila CNS.

Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, whereas broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.

A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis.

Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. Here, we identify and characterize a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. We propose that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis.

Tweek, an evolutionarily conserved protein, is required for synaptic vesicle recycling.

Synaptic vesicle endocytosis is critical for maintaining synaptic communication during intense stimulation. Here we describe Tweek, a conserved protein that is required for synaptic vesicle recycling. tweek mutants show reduced FM1-43 uptake, cannot maintain release during intense stimulation, and harbor larger than normal synaptic vesicles, implicating it in vesicle recycling at the synapse. Interestingly, the levels of a fluorescent PI(4,5)P(2) reporter are reduced at tweek mutant synapses, and the probe is aberrantly localized during stimulation. In addition, various endocytic adaptors known to bind PI(4,5)P(2) are mislocalized and the defects in FM1-43 dye uptake and adaptor localization are partially suppressed by removing one copy of the phosphoinositide phosphatase synaptojanin, suggesting a role for Tweek in maintaining proper phosphoinositide levels at synapses. Our data implicate Tweek in regulating synaptic vesicle recycling via an action mediated at least in part by the regulation of PI(4,5)P(2) levels or availability at the synapse.

straightjacket is required for the synaptic stabilization of cacophony, a voltage-gated calcium channel alpha1 subunit.

In a screen to identify genes involved in synaptic function, we isolated mutations in Drosophila melanogaster straightjacket (stj), an alpha(2)delta subunit of the voltage-gated calcium channel. stj mutant photoreceptors develop normal synaptic connections but display reduced "on-off" transients in electroretinogram recordings, indicating a failure to evoke postsynaptic responses and, thus, a defect in neurotransmission. stj is expressed in neurons but excluded from glia. Mutants exhibit endogenous seizure-like activity, indicating altered neuronal excitability. However, at the synaptic level, stj larval neuromuscular junctions exhibit approximately fourfold reduction in synaptic release compared with controls stemming from a reduced release probability at these synapses. These defects likely stem from destabilization of Cacophony (Cac), the primary presynaptic alpha(1) subunit in D. melanogaster. Interestingly, neuronal overexpression of cac partially rescues the viability and physiological defects in stj mutants, indicating a role for the alpha(2)delta Ca(2+) channel subunit in mediating the proper localization of an alpha(1) subunit at synapses.

FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.

To maintain transmitter release during intense stimulation, neurons need to efficiently recycle vesicles at the synapse. Following membrane fusion, vesicles are reshaped and formed from the plasma membrane by bulk or clathrin-mediated endocytosis. Most synapses, including the Drosophila neuromuscular junction (NMJ), can also recycle synaptic vesicles directly by closing the fusion pore, a process referred to as "kiss and run." While the process of clathrin-mediated vesicle retrieval is under intense investigation, the kiss-and-run phenomenon remains much less accepted. To gain better insight into the mechanisms of synaptic vesicle recycling, it is therefore critical not only to identify and characterize novel players involved in the process, but also to develop novel methods to study vesicle recycling. Although in recent years numerous techniques to study vesicle traffic have been developed (see also this volume), in this chapter we outline established procedures that use the fluorescent dye FM 1-43 or related compounds to study vesicle cycling. We describe how FM 1-43 can be used to study and visualize clathrin-mediated or bulk endocytosis from the presynaptic membrane as well as exocytosis of labeled vesicles at the Drosophila NMJ, one of the best-characterized model synapses to study synaptic function in a genetic model system.

Huntingtin-interacting protein 14, a palmitoyl transferase required for exocytosis and targeting of CSP to synaptic vesicles.

Posttranslational modification through palmitoylation regulates protein localization and function. In this study, we identify a role for the Drosophila melanogaster palmitoyl transferase Huntingtin-interacting protein 14 (HIP14) in neurotransmitter release. hip14 mutants show exocytic defects at low frequency stimulation and a nearly complete loss of synaptic transmission at higher temperature. Interestingly, two exocytic components known to be palmitoylated, cysteine string protein (CSP) and SNAP25, are severely mislocalized at hip14 mutant synapses. Complementary DNA rescue and localization experiments indicate that HIP14 is required solely in the nervous system and is essential for presynaptic function. Biochemical studies indicate that HIP14 palmitoylates CSP and that CSP is not palmitoylated in hip14 mutants. Furthermore, the hip14 exocytic defects can be suppressed by targeting CSP to synaptic vesicles using a chimeric protein approach. Our data indicate that HIP14 controls neurotransmitter release by regulating the trafficking of CSP to synapses.