Targeted expression of tetanus toxin reveals sets of neurons involved in larval locomotion in Drosophila

The Drosophila larva is widely used for studies of neuronal development and function, yet little is known about the neuronal basis of locomotion in this model organism. Drosophila larvae crawl over a plain substrate by performing repetitive waves of forward peristalsis alternated by brief episodes of head swinging and turning. To identify sets of central and peripheral neurons required for the spatial or temporal pattern of larval locomotion, we blocked neurotransmitter release from defined populations of neurons by targeted expression of tetanus toxin light chain (TeTxLC) with the GAL4/UAS system. One hundred fifty GAL4 lines were crossed to a UAS-TeTxLC strain and a motion-analysis system was used to identify larvae with abnormal movement patterns. Five lines were selected that show discrete locomotor defects (i.e., increased turning and pausing) and these defects are correlated with diverse sets of central neurons. One line, 4C-GAL4, caused an unusual circling behavior that is correlated with approximately 200 neurons, including dopaminergic and peptidergic interneurons. Expression of TeTxLC in all dopaminergic and serotonergic but not in peptidergic neurons, caused turning deficits that are similar to those of 4C-GAL4/TeTxLC larvae. The results presented here provide a basis for future genetic studies of motor control in the Drosophila larva.     

Tracheal development in the Drosophila brain is constrained by glial cells

The Drosophila brain is tracheated by the cerebral trachea, a branch of the first segmental trachea of the embryo. During larval stages the cerebral trachea splits into several main (primary) branches that grow around the neuropile, forming a perineuropilar tracheal plexus (PNP) at the neuropile surface. Five primary tracheal branches whose spatial relationship to brain compartments is relatively invariant can be distinguished, although the exact trajectories and branching pattern of the brain tracheae are surprisingly variable. Immunohistochemical and electron microscopic studies demonstrate that all brain tracheae grow in direct contact with the glial cell processes that surround the neuropile. To investigate the effect of glia on tracheal development, embryos and larvae lacking glial cells as a result of a genetic mutation or a directed ablation were analyzed. In these animals, the tracheal branching pattern was highly abnormal. In particular, the number of secondary branches entering the central neuropile was increased. Wild-type larvae possess only two central tracheae, typically associated with the mushroom body and the antennocerebral tract. In larvae lacking glial cells, six to ten tracheal branches penetrate the neuropile in a variable pattern. This finding indicates that glia-derived signals constrained tracheal growth in the Drosophila brain and restrict the number of branches entering the neuropile.     

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.     

Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain

The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are “scaffolded” by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments.     

Morphogenesis and proliferation of the larval brain glia in Drosophila

Glial cells subserve a number of essential functions during development and function of the Drosophila brain, including the control of neuroblast proliferation, neuronal positioning and axonal pathfinding. Three major classes of glial cells have been identified. Surface glia surround the brain externally. Neuropile glia ensheath the neuropile and form septa within the neuropile that define distinct neuropile compartments. Cortex glia form a scaffold around neuronal cell bodies in the cortex. In this paper we have used global glial markers and GFP-labeled clones to describe the morphology, development and proliferation pattern of the three types of glial cells in the larval brain. We show that both surface glia and cortex glia contribute to the glial layer surrounding the brain. Cortex glia also form a significant part of the glial layer surrounding the neuropile. Glial cell numbers increase slowly during the first half of larval development but show a rapid incline in the third larval instar. This increase results from mitosis of differentiated glia, but, more significantly, from the proliferation of neuroblasts.     

Ap-let neurons--a peptidergic circuit potentially controlling ecdysial behavior in Drosophila

Here we describe a novel set of peptidergic neurons conserved throughout all developmental stages in the Drosophila central nervous system (CNS). We show that a small complement of 28 apterous-expressing cells (Ap-let neurons) in the ventral nerve cord (VNC) of Drosophila larvae co-express numerous gene products. The products include the neuroendocrine-specific bHLH regulator called Dimmed (Dimm), four neuropeptide biosynthetic enzymes (PC2, Fur1, PAL2, and PHM), and a specific dopamine receptor subtype (dDA1). For the PC2, Fur1, and PAL2 enzymes, and for the dDA1 receptor, this neuronal pattern represents the vast majority of their total expression in the VNC. In addition, while Dimm and PHM are present in the peritracheal Inka cells in larvae, pupae, and adults, Ap, PC2, Fur1, PAL2, and dDA1 are not. PC2, PAL2, and DA1 receptor expression were all controlled by both dimm and ap. Previous genetic analysis of animals deficient in PC2 revealed an abnormal larval ecdysis phenotype. Together, these data support the hypothesis that the small cohort of Ap-let interneurons regulates larval ecdysis behavior by secretion of an unidentified amidated peptide(s). This hypothesis further predicts that the production of the Ap-let neuropeptide(s) is dependent on each of four specific enzymes, and that a certain aspect(s) of its production and/or release is regulated by dopamine input.     

Nociceptive neurons protect Drosophila larvae from parasitoid wasps

BACKGROUND: Natural selection has resulted in a complex and fascinating repertoire of innate behaviors that are produced by insects. One puzzling example occurs in fruit fly larvae that have been subjected to a noxious mechanical or thermal sensory input. In response, the larvae "roll" with a motor pattern that is completely distinct from the style of locomotion that is used for foraging. RESULTS: We have precisely mapped the sensory neurons that are used by the Drosophila larvae to detect nociceptive stimuli. By using complementary optogenetic activation and targeted silencing of sensory neurons, we have demonstrated that a single class of neuron (class IV multidendritic neuron) is sufficient and necessary for triggering the unusual rolling behavior. In addition, we find that larvae have an innately encoded preference in the directionality of rolling. Surprisingly, the initial direction of rolling locomotion is toward the side of the body that has been stimulated. We propose that directional rolling might provide a selective advantage in escape from parasitoid wasps that are ubiquitously present in the natural environment of Drosophila. Consistent with this hypothesis, we have documented that larvae can escape the attack of Leptopilina boulardi parasitoid wasps by rolling, occasionally flipping the attacker onto its back. CONCLUSIONS: The class IV multidendritic neurons of Drosophila larvae are nociceptive. The nociception behavior of Drosophila melanagaster larvae includes an innately encoded directional preference. Nociception behavior is elicited by the ecologically relevant sensory stimulus of parasitoid wasp attack.     

Tyramine and octopamine have opposite effects on the locomotion of Drosophila larvae

Biogenic amines are believed to play important roles in producing behaviors. Although some biogenic amines have been extensively studied in both vertebrates and invertebrates, little is known about the effects of trace amines like tyramine and octopamine. We investigated how trace amines affect behaviors using quantitative morphometric methods on Drosophila Tbetah(nM18) and iav(N) mutants that have altered levels of tyramine and octopamine. Locomotion of wild-type and mutant third instar larvae was analyzed using Dynamic Image Analysis System (DIAS) software. We found that Tbetah(nM18) mutants, with elevated tyramine levels and reduced octopamine levels, had a severe locomotion phenotype. Mutant larvae spent much more time in pausing episodes than wild-type larvae and displayed a reduction in speed and linear translocation. The locomotion phenotype was partially rescued by feeding Tbetah(nM18) larvae octopamine, an effect that could be nullified with simultaneous feeding of tyramine. Feeding Tbetah(nM18) larvae yohimbine, an agent that inhibits the activity of Drosophila tyramine receptors, also improved some locomotion parameters. Feeding both octopamine and yohimbine further improved rescue efficiency. Simultaneously reducing the octopamine and tyramine levels as in iav(N) larvae, in contrast, led to a less severe behavioral phenotype than that of Tbetah(nM18) mutants. Feeding iav(N) larvae either tyramine or octopamine exerted only a minor improvement in locomotion. These results suggest that tyramine and octopamine have opposite effects on Drosophila larval locomotion regulation and that a balance between the two is important in producing normal behavior.     

Drosophila nociceptors mediate larval aversion to dry surface environments utilizing both the painless TRP channel and the DEG/ENaC subunit, PPK1

A subset of sensory neurons embedded within the Drosophila larval body wall have been characterized as high-threshold polymodal nociceptors capable of responding to noxious heat and noxious mechanical stimulation. They are also sensitized by UV-induced tissue damage leading to both thermal hyperalgesia and allodynia very similar to that observed in vertebrate nociceptors. We show that the class IV multiple-dendritic(mdIV) nociceptors are also required for a normal larval aversion to locomotion on to a dry surface environment. Drosophila melanogaster larvae are acutely susceptible to desiccation displaying a strong aversion to locomotion on dry surfaces severely limiting the distance of movement away from a moist food source. Transgenic inactivation of mdIV nociceptor neurons resulted in larvae moving inappropriately into regions of low humidity at the top of the vial reflected as an increased overall pupation height and larval desiccation. This larval lethal desiccation phenotype was not observed in wild-type controls and was completely suppressed by growth in conditions of high humidity. Transgenic hyperactivation of mdIV nociceptors caused a reciprocal hypersensitivity to dry surfaces resulting in drastically decreased pupation height but did not induce the writhing nocifensive response previously associated with mdIV nociceptor activation by noxious heat or harsh mechanical stimuli. Larvae carrying mutations in either the Drosophila TRP channel, Painless, or the degenerin/epithelial sodium channel subunit Pickpocket1(PPK1), both expressed in mdIV nociceptors, showed the same inappropriate increased pupation height and lethal desiccation observed with mdIV nociceptor inactivation. Larval aversion to dry surfaces appears to utilize the same or overlapping sensory transduction pathways activated by noxious heat and harsh mechanical stimulation but with strikingly different sensitivities and disparate physiological responses.     

Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1

Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders.     

An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.     

Morphogenesis and cellular proliferation pattern in the developing antennal lobe of Drosophila melanogaster

The adult antennal lobe of Drosophila melanogaster emerges from a precursor, the larval antennal lobe. Pulse and pulse-chase labelling of dividing cells in larvae and pupae with bromodeoxyuridine confirmed previous data that some of the interneurons of the adult antennal lobe derive from a lateral neuroblast which starts to divide early in the first larval instar. However, the majority of these interneurons originate from neuroblasts that initiate mitosis at later stages, with a peak of about 10–12 pairs of dividing neuroblasts in the late third larval instar. No clustering of adult antennal lobe neurons according to their birthdates was observed. In contrast to neurons, terminal divisions of glia in the antennal lobe reach their maximum only 12 h after puparium formation.     

Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response

Circadian pacemaker neurons contain a molecular clock that oscillates with a period of approximately 24 hr, controlling circadian rhythms of behavior. Pacemaker neurons respond to visual system inputs for clock resetting, but, unlike other neurons, have not been reported to transmit rapid signals to their targets. Here we show that pacemaker neurons are required to mediate a rapid behavior. The Drosophila larval visual system, Bolwig's organ (BO), projects to larval pacemaker neurons to entrain their clock. BO also mediates larval photophobic behavior. We found that ablation or electrical silencing of larval pacemaker neurons abolished light avoidance. Thus, circadian pacemaker neurons receive input from BO not only to reset the clock but also to transmit rapid photophobic signals. Furthermore, as clock gene mutations also affect photophobicity, the pacemaker neurons modulate the sensitivity of larvae to light, generating a circadian rhythm in visual sensitivity.     

Impairment of central pattern generation in <Emphasis Type="Italic">Drosophila</Emphasis> cysteine string protein mutants

The cysteine string proteins are integral synaptic vesicle proteins, critical for fast, Ca2+ -regulated exocytosis. Drosophila larvae with null mutations for the cysteine string protein (csp) gene have temperature-sensitive impairments of neurotransmission and presynaptic calcium removal at the neuromuscular junction. Using the larval Drosophila preparation to examine central pattern generation, we characterized the temperature sensitivity of locomotor patterns in wildtype and csp mutant larvae. Intraburst frequency of motoneuronal activity reached 100 Hz and was sufficiently high to rescue the temperature-sensitive synaptic failure in the mutant. Nevertheless, we show that deletion of the csp gene resulted in a severe deficiency in the generation of coordinated larval motor rhythms. Csp mutants that could generate patterned motor activity had slower, poorly coordinated rhythms with altered temperature sensitivity. We conclude that the temperature-sensitive paralysis characteristic of csp mutants is not a direct result of synaptic failure at neuromuscular junctions, as might be expected, but is the result of a failure of locomotor circuit operation at a higher integrative level.     

Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons

Behavior is a manifestation of temporally and spatially defined neuronal activities. To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit. I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila. In this method, a temperature-sensitive allele of shibire (shi(ts1)) is overexpressed in neuronal subsets using the GAL4/UAS system. Because the shi gene product is essential for synaptic vesicle recycling, and shi(ts1) is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi(ts1) expressing neurons. When shi(ts1) expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30 degrees C. This temperature-induced paralysis was reversible. After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min. When shi(ts1) was expressed in photoreceptor cells, adults and larvae exhibited temperature-dependent blindness. These observations show that the GAL4/UAS system can be used to express shi(ts1) in a specific subset of neurons to cause temperature-dependent changes in behavior. Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals.     

Optogenetic long-term manipulation of behavior and animal development

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used "slow" ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the "dauer" larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.     

Optical dissection of neural circuits responsible for Drosophila larval locomotion with halorhodopsin

Halorhodopsin (NpHR), a light-driven microbial chloride pump, enables silencing of neuronal function with superb temporal and spatial resolution. Here, we generated a transgenic line of Drosophila that drives expression of NpHR under control of the Gal4/UAS system. Then, we used it to dissect the functional properties of neural circuits that regulate larval peristalsis, a continuous wave of muscular contraction from posterior to anterior segments. We first demonstrate the effectiveness of NpHR by showing that global and continuous NpHR-mediated optical inhibition of motor neurons or sensory feedback neurons induce the same behavioral responses in crawling larvae to those elicited when the function of these neurons are inhibited by Shibire(ts), namely complete paralyses or slowed locomotion, respectively. We then applied transient and/or focused light stimuli to inhibit the activity of motor neurons in a more temporally and spatially restricted manner and studied the effects of the optical inhibition on peristalsis. When a brief light stimulus (1-10 sec) was applied to a crawling larva, the wave of muscular contraction stopped transiently but resumed from the halted position when the light was turned off. Similarly, when a focused light stimulus was applied to inhibit motor neurons in one or a few segments which were about to be activated in a dissected larva undergoing fictive locomotion, the propagation of muscular constriction paused during the light stimulus but resumed from the halted position when the inhibition (>5 sec) was removed. These results suggest that (1) Firing of motor neurons at the forefront of the wave is required for the wave to proceed to more anterior segments, and (2) The information about the phase of the wave, namely which segment is active at a given time, can be memorized in the neural circuits for several seconds.     

Optogenetics in the teaching laboratory: using channelrhodopsin-2 to study the neural basis of behavior and synaptic physiology in Drosophila

Here we incorporate recent advances in Drosophila neurogenetics and "optogenetics" into neuroscience laboratory exercises. We used the light-activated ion channel channelrhodopsin-2 (ChR2) and tissue-specific genetic expression techniques to study the neural basis of behavior in Drosophila larvae. We designed and implemented exercises using inexpensive, easy-to-use systems for delivering blue light pulses with fine temporal control. Students first examined the behavioral effects of activating glutamatergic neurons in Drosophila larvae and then recorded excitatory junctional potentials (EJPs) mediated by ChR2 activation at the larval neuromuscular junction (NMJ). Comparison of electrically and light-evoked EJPs demonstrates that the amplitudes and time courses of light-evoked EJPs are not significantly different from those generated by electrical nerve stimulation. These exercises introduce students to new genetic technology for remotely manipulating neural activity, and they simplify the process of recording EJPs at the Drosophila larval NMJ. Relatively little research work has been done using ChR2 in Drosophila, so students have opportunities to test novel hypotheses and make tangible contributions to the scientific record. Qualitative and quantitative assessment of student experiences suggest that these exercises help convey principles of synaptic transmission while also promoting integrative and inquiry-based studies of genetics, cellular physiology, and animal behavior.