Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing “only” while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.
21 Related JoVE Articles!
Flexural Rigidity Measurements of Biopolymers Using Gliding Assays
Institutions: Lawrence University.
Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1
. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4
, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9
Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10
. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11
. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.
Biophysics, Issue 69, Bioengineering, Physics, Molecular Biology, Cellular Biology, microtubule, persistence length, flexural rigidity, gliding assay, mechanics, cytoskeleton, actin
An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila
Institutions: University of Wisconsin-Madison.
has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a "drop tube" to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.
Behavior, Issue 84, Drosophila melanogaster, neuroscience, flight performance, slowpoke mutant flies, wild-type Canton-S flies
In situ Protocol for Butterfly Pupal Wings Using Riboprobes
Institutions: SUNY-University at Buffalo, Yale University.
Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes. In situ hybridizations, a mainstay of developmental biology, are useful to study the spatial and temporal patterns of gene expression in developing tissues at the level of transcription. If antibodies that target the protein products of gene transcription have not yet been developed, and/or there are multiple gene copies of a particular protein in the genome that cannot be differentiated using available antibodies, in situs can be used instead. While an in situ technique for larval wing discs has been available to the butterfly community for several years, the current protocol has been optimized for the larger and more fragile pupal wings.
Developmental Biology, issue 4, hybridization, wing, staining
Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila
Institutions: University of Houston, Michigan State University.
The scaling of body parts is a central feature of animal morphology1-7
. Within species, morphological traits need to be correctly proportioned to the body for the organism to function; larger individuals typically have larger body parts and smaller individuals generally have smaller body parts, such that overall body shape is maintained across a range of adult body sizes. The requirement for correct proportions means that individuals within species usually exhibit low variation in relative trait size. In contrast, relative trait size can vary dramatically among species and is a primary mechanism by which morphological diversity is produced. Over a century of comparative work has established these intra- and interspecific patterns3,4
Perhaps the most widely used approach to describe this variation is to calculate the scaling relationship between the size of two morphological traits using the allometric equation y=bxα, where x and y are the size of the two traits, such as organ and body size8,9
. This equation describes the within-group (e.g., species, population) scaling relationship between two traits as both vary in size. Log-transformation of this equation produces a simple linear equation, log(y) = log(b) + αlog(x) and log-log plots of the size of different traits among individuals of the same species typically reveal linear scaling with an intercept of log(b) and a slope of α, called the 'allometric coefficient'9,10
. Morphological variation among groups is described by differences in scaling relationship intercepts or slopes for a given trait pair. Consequently, variation in the parameters of the allometric equation (b and α) elegantly describes the shape variation captured in the relationship between organ and body size within and among biological groups (see 11,12
Not all traits scale linearly with each other or with body size (e.g., 13,14
) Hence, morphological scaling relationships are most informative when the data are taken from the full range of trait sizes. Here we describe how simple experimental manipulation of diet can be used to produce the full range of body size in insects. This permits an estimation of the full scaling relationship for any given pair of traits, allowing a complete description of how shape covaries with size and a robust comparison of scaling relationship parameters among biological groups. Although we focus on Drosophila
, our methodology should be applicable to nearly any fully metamorphic insect.
Developmental Biology, Issue 56, Drosophila, allometry, morphology, body size, scaling, insect
High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
Institutions: Aix-Marseille Université, Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France.
Escherichia coli (E. coli)
is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli
cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
Bioengineering, Issue 89, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing
Institutions: University Hospital Center and University of Lausanne.
Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.
Immunology, Issue 92, blood culture, bacteriology, identification, antibiotic susceptibility testing, MALDI-TOF MS.
Spatial Separation of Molecular Conformers and Clusters
Institutions: CFEL, DESY, University of Hamburg, University of Hamburg.
Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.
Physics, Issue 83, Chemical Physics, Physical Chemistry, Molecular Physics, Molecular beams, Laser Spectroscopy, Clusters
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa
Institutions: Université J. Fourier.
Effectively determining masses of proteins is critical to many biological studies (e.g.
for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e.
2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.
Chemistry, Issue 79, Chemistry Techniques, Analytical, Mass Spectrometry, Analytic Sample Preparation Methods, biochemistry, Analysis of intact proteins, mass spectrometry, matrix-assisted laser desorption ionization, time of flight, sample preparation
Setting Limits on Supersymmetry Using Simplified Models
Institutions: University College London, CERN, Lawrence Berkeley National Laboratories.
Experimental limits on supersymmetry and similar theories are difficult to set because of the enormous available parameter space and difficult to generalize because of the complexity of single points. Therefore, more phenomenological, simplified models are becoming popular for setting experimental limits, as they have clearer physical interpretations. The use of these simplified model limits to set a real limit on a concrete theory has not, however, been demonstrated. This paper recasts simplified model limits into limits on a specific and complete supersymmetry model, minimal supergravity. Limits obtained under various physical assumptions are comparable to those produced by directed searches. A prescription is provided for calculating conservative and aggressive limits on additional theories. Using acceptance and efficiency tables along with the expected and observed numbers of events in various signal regions, LHC experimental results can be recast in this manner into almost any theoretical framework, including nonsupersymmetric theories with supersymmetry-like signatures.
Physics, Issue 81, high energy physics, particle physics, Supersymmetry, LHC, ATLAS, CMS, New Physics Limits, Simplified Models
Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp
Institutions: The George Washington University, Clarkson University.
The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient's ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in vitro
driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e.
a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.
Bioengineering, Issue 84, oil-flow visualization, vocal fold polyp, three-dimensional flow separation, aerodynamic pressure loadings
Preparation of Drosophila Central Neurons for in situ Patch Clamping
Institutions: Arizona State University .
Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster
an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila
voltage gated potassium channel Shaker1,2
. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila
with in situ
patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila
CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila
CNS. In recent years scientists have been able to conduct in situ
patch clamp recordings from neurons in the adult brain3,4
and ventral nerve cord of embryonic5,6
, and adult Drosophila11,12,13,14
. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ
patch clamp recordings from adult Drosophila
neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila
ventral nerve cord, the flight motoneuron 5 (MN515
), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.
Neuroscience, Issue 68, Molecular Biology, Cellular Biology, Anatomy, Physiology, Patch clamp, in situ patch clamp, Drosophila, electrophysiology, motoneuron, neuron, CNS
Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster
Institutions: University College London - UCL, University of Kent.
When startled adult D. melanogaster
react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster
, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al.
, 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.
Neuroscience, Issue 47, Drosophila melanogaster, electrophysiology, Giant Fiber System, flight muscles, nervous system
The Generation of Higher-order Laguerre-Gauss Optical Beams for High-precision Interferometry
Institutions: University of Birmingham.
Thermal noise in high-reflectivity mirrors is a major impediment for several types of high-precision interferometric experiments that aim to reach the standard quantum limit or to cool mechanical systems to their quantum ground state. This is for example the case of future gravitational wave observatories, whose sensitivity to gravitational wave signals is expected to be limited in the most sensitive frequency band, by atomic vibration of their mirror masses. One promising approach being pursued to overcome this limitation is to employ higher-order Laguerre-Gauss (LG) optical beams in place of the conventionally used fundamental mode. Owing to their more homogeneous light intensity distribution these beams average more effectively over the thermally driven fluctuations of the mirror surface, which in turn reduces the uncertainty in the mirror position sensed by the laser light.
We demonstrate a promising method to generate higher-order LG beams by shaping a fundamental Gaussian beam with the help of diffractive optical elements. We show that with conventional sensing and control techniques that are known for stabilizing fundamental laser beams, higher-order LG modes can be purified and stabilized just as well at a comparably high level. A set of diagnostic tools allows us to control and tailor the properties of generated LG beams. This enabled us to produce an LG beam with the highest purity reported to date. The demonstrated compatibility of higher-order LG modes with standard interferometry techniques and with the use of standard spherical optics makes them an ideal candidate for application in a future generation of high-precision interferometry.
Physics, Issue 78, Optics, Astronomy, Astrophysics, Gravitational waves, Laser interferometry, Metrology, Thermal noise, Laguerre-Gauss modes, interferometry
Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology
Institutions: DNA Medicine Institute, Harvard Medical School, NASA Glenn Research Center, ZIN Technologies.
Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.
Cellular Biology, Issue 93, Point-of-care, prototype, diagnostics, spaceflight, reduced gravity, parabolic flight, flow cytometry, fluorescence, cell counting, micromixing, spiral-vortex, blood mixing
Flying Insect Detection and Classification with Inexpensive Sensors
Institutions: University of California, Riverside, University of California, Riverside, University of São Paulo - USP, ISCA Technologies.
An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect’s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.
Bioengineering, Issue 92, flying insect detection, automatic insect classification, pseudo-acoustic optical sensors, Bayesian classification framework, flight sound, circadian rhythm
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
Visually Mediated Odor Tracking During Flight in Drosophila
Institutions: University of California, Los Angeles.
Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.
Neuroscience, Issue 23, Drosophila, magnet, olfaction, vision, behavior, flight, video
Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae
Institutions: University of California, Irvine (UCI).
Developmental Biology, Issue 2, Drosophila, Imaginal Disks, Dissection Technique
Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions
Institutions: German Sport University Cologne, University of Toronto, Queensland University of Technology, Gilching, Germany.
Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al.
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al.
2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al.
2005), which means that they could be crucial for the success and safety of a mission (Genik et al.
2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.
Neuroscience, Issue 51, EEG, NIRS, electrotomography, parabolic flight, weightlessness, imaging, cognitive performance
Operant Learning of Drosophila at the Torque Meter
Institutions: Free University of Berlin.
For experiments at the torque meter, flies are kept on standard fly medium at 25°C and 60% humidity with a 12hr light/12hr dark regime. A standardized breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly's intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly's control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser.
Neuroscience, Issue 16, operant, learning, Drosophila, fruit fly, insect, invertebrate, neuroscience, neurobiology, fly, conditioning