Localization of muscarinic acetylcholine receptor dependent rhythm generating modules in the Drosophila larval locomotor network

This work was supported by the Wellcome Trust through an ISSF award (105621/Z/14/Z) to the University of St Andrews. It was also supported by a Biotechnology and Biological Sciences Research Council (BBSRC) project grant (BB/M021793) awarded to SRP, a CASE studentship awarded to J. M. (BB/M010996/1) and a donation from Kaunas Industrial Water Supply (Kauno Pramoninis Vandentiekis, Kaunas, Lithuania) in support of J. J.Mechanisms of rhythm generation h­­ave been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. Here we explored how muscarinic acetylcholine receptor (mAChR) modulated rhythm generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. We measured fictive motor patterns in isolated CNS preparations using a combination of Ca2+ imaging and electrophysiology while manipulating mAChR signalling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca2+ levels and abolished rhythmic activity. Overall, these results suggest that mAChR signalling plays a role in enabling rhythm generation at multiple sites in the larval CNS. This work furthers our understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm generating networks in an emerging genetically tractable locomotor system.Publisher PDFPeer reviewe

Segment-specific optogenetic stimulation in Drosophila melanogaster with linear arrays of organic light-emitting diodes

This research was financially supported by the EPSRC NSF-CBET lead agency agreement (EP/R010595/1, 1706207), the DARPA-NESD programme (N66001-17-C-4012) and the Leverhulme Trust (RPG-2017-231). C.M. acknowledges funding from the European Commission through a Marie Skłodowska Curie individual fellowship (703387). S.R.P acknowledges funding from the Biology and Biotechnology Research council (BB/M021793). M.C.G. acknowledges funding from the Alexander von Humboldt Stiftung (Humboldt-Professorship).Optogenetics allows light-driven, non-contact control of neural systems, but light delivery remains challenging, in particular when fine spatial control of light is required to achieve local specificity. Here, we employ organic light-emitting diodes (OLEDs) that are micropatterned into linear arrays to obtain precise optogenetic control in Drosophila melanogaster larvae expressing the light-gated activator CsChrimson and the inhibitor GtACR2 within their peripheral sensory system. Our method allows confinement of light stimuli to within individual abdominal segments, which facilitates the study of larval behaviour in response to local sensory input. We show controlled triggering of specific crawling modes and find that targeted neurostimulation in abdominal segments switches the direction of crawling. More broadly, our work demonstrates how OLEDs can provide tailored patterns of light for photo-stimulation of neuronal networks, with future implications ranging from mapping neuronal connectivity in cultures to targeted photo-stimulation with pixelated OLED implants in vivo.Publisher PDFPeer reviewe

High-brightness organic light-emitting diodes for optogenetic control of Drosophila locomotor behaviour

We thank Simone Lenk and Tobias Günther (TU Dresden) for fruitful discussions and technical support with OLED preparation. We are grateful for financial support from the Scottish Funding Council (through SUPA), Human Frontier Science Program (RGY0074/2013), Wellcome Trust Institutional Strategic Support Fund St Andrews and the RS Macdonald Charitable Trust. C.M. acknowledges funding by the European Commission through a Marie Sklodowska-Curie Individual Fellowship (703387).Organic light emitting diodes (OLEDs) are in widespread use in today’s mobile phones and are likely to drive the next generation of large area displays and solid-state lighting. Here we show steps towards their utility as a platform technology for biophotonics, by demonstrating devices capable of optical controlling behaviour in live animals. Using devices with pin OLED architecture, sufficient illumination intensity (0.3 mW.mm-2) to activate channelrhodopsins (ChRs) in vivo was reliably achieved at low operating voltages (5 V). In Drosophila melanogaster third instar larvae expressing ChR2(H134R) in motor neurons, we found that pulsed illumination from blue and green OLEDs triggered robust and reversible contractions in animals. This response was temporally coupled to the timing of OLED illumination. With blue OLED illumination, the initial rate and overall size of the behavioural response was strongest. Green OLEDs achieved roughly 70% of the response observed with blue OLEDs. Orange OLEDs did not produce contractions in larvae, in agreement with the spectral response of ChR2(H134R). The device configuration presented here could be modified to accommodate other small model organisms, cell cultures or tissue slices and the ability of OLEDs to provide patterned illumination and spectral tuning can broaden their utility in optogenetics experiments further.Publisher PDFPeer reviewe

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited

Inexpensive methods for live imaging of central pattern generator activity in the Drosophila larval locomotor system

This work was supported by EPSRC Doctoral Training grant EP/L505079/1) and a European Research Council Grant to MCG (640012) It was also supported by a British Council GREAT Scholarship (to VS) as well as grants from the St Andrews Learning and Teaching Initiative and the McCall-McBain Foundation to SRP. This work was also supported by the open source DIY communities as described throughout the paper.Central pattern generators (CPGs) are neural networks that produce rhythmic motor activity in the absence of sensory input. CPGs produce ‘fictive’ behaviours in vitro which parallel activity seen in intact animals. CPG networks have been identified in a wide variety of model organisms and have been shown to be critical for generating rhythmic behaviours such as swimming, walking, chewing and breathing. Work with CPG preparations has led to fundamental advances in neuroscience; however, most CPG preparations involve intensive dissections and require sophisticated electrophysiology equipment, making export to teaching laboratories problematic. Here we present an integrated approach for bringing the study of locomotor CPGs in Drosophila larvae into teaching laboratories. First, we present freely available genetic constructs that enable educators to express genetically encoded calcium indicators in cells of interest in the larval central nervous system. Next, we describe how to isolate the larval central nervous system and prepare it for live imaging. We then show how to modify standard compound microscopes to enable fluorescent imaging using 3D printed materials and inexpensive optical components. Finally, we show how to use the free image analysis programme ImageJ and freely available features in the signal analysis programme DataView to analyse rhythmic CPG activity in the larval CNS. Comparison of results to those obtained on research equipment shows that signal-to-noise levels are comparable and core features of larval CPG activity can be observed. Overall, this work shows the viability of exporting live imaging experiments to low cost environments and paves the way for new teaching laboratory exercises revolving around optical imaging of CPG activity.Publisher PDFPeer reviewe

Identification of inhibitory premotor interneurons activated at a late phase in a motor cycle during Drosophila larval locomotion

This work was supported by a MEXT/JSPS KAKENHI Grant Numbers, 22115002 (to A.N.) and 221S0003 (to A.N. and Y.I.), and 15H04255 (to A.N.). The work was also supported by Janelia Research Campus (Howard Hughes Medical Institute).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.Publisher PDFPeer reviewe

Organic light-emitting diodes for optogenetic stimulation of Drosophila larvae

We are grateful for financial support from the Scottish Funding Council (through SUPA), Human Frontier Science Program (RGY0074/2013), Wellcome Trust Institutional Strategic Support Fund St Andrews, the RS Macdonald Charitable Trust, and EPSRC via grant EP/J01771X/1. CM acknowledges funding by the European Commission through a Marie Skłodowska Curie individual fellowship (703387).Optogenetics is an emerging method in biology that enables controlling neurons with light. We use organic light-emitting diodes to stimulate neurons in Drosophila larvae and investigate subsequent behavioral changes at different light intensities.Postprin

Imaging fictive locomotor patterns in larval Drosophila.

We have established a preparation in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS with genetically encoded Ca(2+) indicators. The Ca(2+) signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca(2+) signals that progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable intersegmental phase delays compared with segmental contractions during forward and backward crawling behavior, despite being ∼10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca(2+) signals were normally initiated did not eliminate production of Ca(2+) waves. When the brain and subesophageal ganglion (SOG) were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed and abolished when the SOG was removed. This work paves the way to studying the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila with imaging techniques.S.R.P. was supported by a Newton International Fellowship (Royal Society) and a Junior Fellowship (Janelia Research Campus, Howard Hughes Medical Institute). T.G.B. was supported by a Medical Research Council (UK) PhD grant. J.B. was supported by a Henry Dale Fellowship (Royal Society and Wellcome Trust). M.B. was supported by the Isaac Newton Trust.This is the final version of the article. It first appeared from the American Physiological Society via http://dx.doi.org/10.1152/jn.00731.201

Optical mapping of ground reaction force dynamics in freely behaving Drosophila melanogaster larvae

Funding: EPSRC (Doctoral Training grant EP/L505079/1 and grant EP/P030017/1), the European Research Council under the European Union’s Horizon 2020 Framework Programme (FP/2014-202) ERC grant agreement no. 640012 (ABLASE), and the Alexander von Humboldt Foundation via the Humboldt Professorship to MCGDuring locomotion, soft-bodied terrestrial animals solve complex control problems at substrate interfaces, but our understanding of how they achieve this without rigid components remains incomplete. Here, we develop new all-optical methods based on optical interference in a deformable substrate to measure ground reaction forces (GRFs) with micrometre and nanonewton precision in behaving Drosophila larvae. Combining this with a kinematic analysis of substrate-interfacing features, we shed new light onto the biomechanical control of larval locomotion. Crawling in larvae measuring ~1 mm in length involves an intricate pattern of cuticle sequestration and planting, producing GRFs of 1–7 µN. We show that larvae insert and expand denticulated, feet-like structures into substrates as they move, a process not previously observed in soft-bodied animals. These ‘protopodia’ form dynamic anchors to compensate counteracting forces. Our work provides a framework for future biomechanics research in soft-bodied animals and promises to inspire improved soft-robot design.Peer reviewe

Selective Inhibition Mediates the Sequential Recruitment of Motor Pools.

Locomotor systems generate diverse motor patterns to produce the movements underlying behavior, requiring that motor neurons be recruited at various phases of the locomotor cycle. Reciprocal inhibition produces alternating motor patterns; however, the mechanisms that generate other phasic relationships between intrasegmental motor pools are unknown. Here, we investigate one such motor pattern in the Drosophila larva, using a multidisciplinary approach including electrophysiology and ssTEM-based circuit reconstruction. We find that two motor pools that are sequentially recruited during locomotion have identical excitable properties. In contrast, they receive input from divergent premotor circuits. We find that this motor pattern is not orchestrated by differential excitatory input but by a GABAergic interneuron acting as a delay line to the later-recruited motor pool. Our findings show how a motor pattern is generated as a function of the modular organization of locomotor networks through segregation of inhibition, a potentially general mechanism for sequential motor patterns.This work was supported by the Howard Hughes Medical Institute, the HHMI Janelia Visitor Program (MFZ and ML), an Isaac Newton Trust/ISSF Wellcome Trust and a Wellcome Trust grant (092986/Z) to ML.This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.neuron.2016.06.03