Optical mapping of ground reaction force dynamics in freely behaving larvae.

During 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 larvae. Combining this with a kinematic analysis of substrate-interfacing features, we shed new light onto the biomechanical control of larval locomotion.

STEERING FROM THE REAR: COORDINATION OF CENTRAL PATTERN GENERATORS UNDERLYING NAVIGATION BY ASCENDING INTERNEURONS.

Understanding how animals coordinate movements to achieve goals is a fundamental pursuit in neuroscience. Here we explore how neurons that reside in posterior lower-order regions of a locomotor system project to anterior higher-order regions to influence steering and navigation. We characterized the anatomy and functional role of a population of ascending interneurons in the ventral nerve cord of larvae.

Deformable microlaser force sensing.

Mechanical forces are key regulators of cellular behavior and function, affecting many fundamental biological processes such as cell migration, embryogenesis, immunological responses, and pathological states. Specialized force sensors and imaging techniques have been developed to quantify these otherwise invisible forces in single cells and in vivo. However, current techniques rely heavily on high-resolution microscopy and do not allow interrogation of optically dense tissue, reducing their application to 2D cell cultures and highly transparent biological tissue.

Temporal dynamics of Na/K pump mediated memory traces: insights from conductance-based models of neurons.

Sodium potassium ATPases (Na/K pumps) mediate long-lasting, dynamic cellular memories that can last tens of seconds. The mechanisms controlling the dynamics of this type of cellular memory are not well understood and can be counterintuitive. Here, we use computational modeling to examine how Na/K pumps and the ion concentration dynamics they influence shape cellular excitability.

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

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 larvae. We measured fictive motor patterns in isolated CNS preparations, using a combination of Ca imaging and electrophysiology while manipulating mAChR signaling pharmacologically.

An electrically coupled pioneer circuit enables motor development via proprioceptive feedback in Drosophila embryos.

Precocious movements are widely seen in embryos of various animal species. Whether such movements via proprioceptive feedback play instructive roles in motor development or are a mere reflection of activities in immature motor circuits is a long-standing question. Here we image the emerging motor activities in Drosophila embryos that lack proprioceptive feedback and show that proprioceptive experience is essential for the development of locomotor central pattern generators (CPGs).

Regulation of coordinated muscular relaxation in Drosophila larvae by a pattern-regulating intersegmental circuit.

Typical patterned movements in animals are achieved through combinations of contraction and delayed relaxation of groups of muscles. However, how intersegmentally coordinated patterns of muscular relaxation are regulated by the neural circuits remains poorly understood. Here, we identify Canon, a class of higher-order premotor interneurons, that regulates muscular relaxation during backward locomotion of Drosophila larvae.

Inexpensive Methods for Live Imaging of Central Pattern Generator Activity in the Larval Locomotor System.

Central pattern generators (CPGs) are neural networks that produce rhythmic motor activity in the absence of sensory input. CPGs produce 'fictive' behaviours 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.

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

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.

Narrowband Organic Light-Emitting Diodes for Fluorescence Microscopy and Calcium Imaging.

Fluorescence imaging is an indispensable tool in biology, with applications ranging from single-cell to whole-animal studies and with live mapping of neuronal activity currently receiving particular attention. To enable fluorescence imaging at cellular scale in freely moving animals, miniaturized microscopes and lensless imagers are developed that can be implanted in a minimally invasive fashion; but the rigidity, size, and potential toxicity of the involved light sources remain a challenge. Here, narrowband organic light-emitting diodes (OLEDs) are developed and used for fluorescence imaging of live cells and for mapping of neuronal activity in Drosophila melanogaster via genetically encoded Ca indicators.

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

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 optically controlling behaviour in live animals. Using devices with a pin OLED architecture, sufficient illumination intensity (0.

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.

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.

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.

Whole-central nervous system functional imaging in larval Drosophila.

Understanding how the brain works in tight concert with the rest of the central nervous system (CNS) hinges upon knowledge of coordinated activity patterns across the whole CNS. We present a method for measuring activity in an entire, non-transparent CNS with high spatiotemporal resolution. We combine a light-sheet microscope capable of simultaneous multi-view imaging at volumetric speeds 25-fold faster than the state-of-the-art, a whole-CNS imaging assay for the isolated Drosophila larval CNS and a computational framework for analysing multi-view, whole-CNS calcium imaging data.

Light Activated Escape Circuits: A Behavior and Neurophysiology Lab Module using Drosophila Optogenetics.

The neural networks that control escape from predators often show very clear relationships between defined sensory inputs and stereotyped motor outputs. This feature provides unique opportunities for researchers, but it also provides novel opportunities for neuroscience educators. Here we introduce new teaching modules using adult Drosophila that have been engineered to express csChrimson, a red-light sensitive channelrhodopsin, in specific sets of neurons and muscles mediating visually guided escape behaviors.

Independent optical excitation of distinct neural populations.

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain.

Ultrasensitive fluorescent proteins for imaging neuronal activity.

Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines.

The fundamentals of flying: simple and inexpensive strategies for employing Drosophila genetics in neuroscience teaching laboratories.

Drosophila researchers have developed a powerful suite of genetic techniques for studying the neural basis of animal behavior. Many of these tools can be exported to neuroscience teaching laboratories (Berni et al., 2010; Pulver et al.

Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca(2+)-bound state of the sensors and the engineered protein domain interfaces of the different indicators.

Autonomous circuitry for substrate exploration in freely moving Drosophila larvae.

Background: Many organisms, from bacteria to human hunter-gatherers, use specialized random walk strategies to explore their environment. Such behaviors are an efficient stratagem for sampling the environment and usually consist of an alternation between straight runs and turns that redirect these runs. Drosophila larvae execute an exploratory routine of this kind that consists of sequences of straight crawls, pauses, turns, and redirected crawls.

Why flies? Inexpensive public engagement exercises to explain the value of basic biomedical research on Drosophila melanogaster.

Invertebrate model organisms are powerful systems for uncovering conserved principles of animal biology. Despite widespread use in scientific communities, invertebrate research is often severely undervalued by laypeople. Here, we present a set of simple, inexpensive public outreach exercises aimed at explaining to the public why basic research on one particular invertebrate, the insect Drosophila melanogaster, is valuable.