We thank Michael D&uuml;bbert, Oliver Hoffmann, Hans Scharstein, Jan Sydow and Anne Wosnitza for excellent technical assistance. This study was supported by DFG grant Bu 857/8,10 to A.B.

1. The Walking Surface
The black walking surface is made up of two nickel coated brass plates permanently joined side by side and electrically insulated from one another by a 2mm wide strip of 2-component epoxy glue (UHU plus, UHU GmbH, Germany) directly underneath the longitudinal axis of the animal. They produce a total surface area of 13.5x15.5cm (Figure 1). The separation of the half-planes for left and right legs allows independent tarsal contact monitoring for a single leg on each side.
<ol>
<li>The plate is lubricated with a glycerin/water mix at a ratio of 95 parts glycerin to 5 parts saturated NaCl-solution to convey slipperiness and electrical conductivity; the viscosity is approximately 430cStokes (Shankar and Kumar, 1994).</li>
<li>The mixture is applied directly onto the surface and is dispersed with a piece of soft tissue to assure an overall thickness of 0.1-0.2mm.</li>
</ol>
2. Optical Stimulation Setup
<ol>
<li>Walking episodes are elicited using two projectors as described by Scharstein (1989) to project a striped pattern onto two round, ground glass screens (diameter 130mm, Marata screens, Linos Photonics, G&ouml;ttingen, Germany) that are positioned at a distance of approx. 70mm from the eyes, and at an angle of 45&deg; at the left and right front end of the plate (Fig.1). The wave length of the striped pattern is kept constant at &lambda;=21&deg;. The contrast frequency of the moving stimuli can be varied between 0.35, 0.72, 1.07 and 1.49Hz. Since the reaction of the animals depends only on the contrast frequency &#969;/&lambda; (&#969; = angular velocity, &lambda; = pattern wave length) if the pattern contrast is high enough (Fermi and Reichhardt, 1963), we did not vary &lambda; throughout the experiments. The luminance of the bright stripes on the side of the animal is approximately 15cd/m2 and the contrast 0.8. The compound eye of the stick insect (Carausius morosus) has 24 ommatidia in the dorso-ventral axis (Friza, 1928) and a few more in the rostro-caudal axis. As the divergence angle of the stick insect eye is on average 6.2&deg; (Jander and Volk-Henrichs, 1970) our pattern wavelength was approximately 4 times the angle of the ommatidia. The experiments are set up in a darkened Faraday cage and performed in a darkened room at 20-22&deg;C.</li>
<li>Forward walking is induced by a progressive pattern on both screens where the stripes on each screen move outward, or a single black beam on white background placed between the screens</li>
<li>Turning is induced by stripes moving into the same direction on both screens. We have not found a correlation between the speed of the moving stripes and the angle of the turn. Once luminance of the striped pattern is set to induce walking, it usually does not require further adjustment, but it can be adjusted for different animals according to need by the voltage of the halogen lamps in the projectors.</li>
</ol>
3. Preparing the Experimental Animal
<ol>
<li>Female adult stick insects (Carausius morosus) are glued (dental cement, ProTempII, 3M ESPE, Seefeld, Germany) ventral side down onto a thin balsa stick (3x5x100mm, WxHxL), which is inserted in a brass tube that has multiple connectors. The animal is held down for approximately a minute until the glue is at least partly hardened.</li>
<li>The head and legs protrude from the front and side of the dowel to allow their free movement. The area of the coxae of all legs as well as the major part of the abdomen has to be left free of glue.</li>
<li>Insect head, thorax, and the distal ends of each femur and tibia are marked with fluorescent pigments (Dr. Kremer Farbm&uuml;hle, Aichstetten, Germany) mixed with dental cement for later video tracking.</li>
</ol>
4. Placement of EMG Electrodes
Muscle activity of different leg muscles such as, in this example, the protractor and retractor coxae muscles, which are located in the thorax and cause protraction (forward movement) and retraction (backward movement) of the leg, is recorded by means of EMG electrodes implanted into the thorax. All EMG recordings are differentially amplified. The signal was amplified 100x in a preamplifier (electronics workshop, Zoological Institute, Cologne), band-pass filtered, (100Hz-2000Hz), further amplified (10x), and imported through an AD converter into spike2 (Vers.5.05, CED, Cambridge, UK).
<ol>
<li>The electrodes are made from coated copper wires (Elektrisola, Eckernhagen, Germany; 47&mu;m outer diameter). For one electrode, two wires are first smeared with glue, then, the two sticky wires are twisted around each other</li>
<li>At one end the twisted wire ends are soldered to a mini-plug. At the other end the wires are cut to provide clean ends without insulation for the insertion into the animal. </li>
<li>The EMG electrodes are plugged into the small connector at the end of the brass tube.</li>
<li>A minuten pin is used to punch small holes into the cuticle</li>
<li>The cut ends of the electrode are then separated and inserted into a muscle at approximately 1mm distance from each other, which, in our experience, gives a good signal to noise ratio. Care should be taken not to insert the electrodes too deep into the muscle in order to avoid its damage. Figure 2 shows the location of the electrodes in the thorax of the animal. Figure 3 shows an animal with the electrode wires in place.</li>
<li>We use a small drop of histoacrylic glue (3M Vetbond, St.Paul, MN, USA) to hold the wires in place. </li>
<li>A ground wire is inserted into the abdomen.</li>
</ol>
5. Recording of the Tarsal Contact
Tarsal contact is always recorded together with EMG traces. Since the slippery surface is split into two halves, a maximum of two contralateral tarsal contacts can be registered at the same time. For this purpose, the leg is used as a "switch" to close a circuit between the plate and the amplifier. Current can be applied to the plates separately through two sockets at the base of each plate. We can generate two square wave signals with 2-4mV amplitudes with a pulse generator (Model MS501, electronics workshop, Zoological Institute, Cologne) that are phase-delayed by 90&deg;. The signals are attenuated by 60dB (/1000) and applied to the two halves of the slippery surface. In this experiment we only use one of the two signals. It is simultaneously fed into a lock-in-amplifier as a reference signal. The 2-phase lock-in-amplifier (electronics workshop, Zoological Institute, Cologne) selectively amplifies only signals of the same frequency and phase as the provided reference signal. A second channel only detects the 90&deg; phase delayed signal. Noise signals at frequencies other than the reference frequency are rejected and do not affect the measurement. The amplifier output signal is fed into an AD converter (Micro 1401k II, CED, Cambridge, UK) and digitalized using Spike2. Between touch-down and lift-off of the tarsus onto and from the slippery surface, current flows from the plate through tarsus and tibia into the copper wire (see below: 5.3-5.8). The chosen amplifier has a high input resistance (1M&Omega;) and the signal voltage is very small in order to avoid affecting the walking behavior of the animal. The current passing through tarsus and tibia is between 2 and 4nA. The signal lead time during touch-down, between the entry into the lubricant and the surface contact is less than 1ms. The contact signal transition during lift-off is less steep and more delayed. This is due to the delayed tearing of the lubricant from the tarsus by a capillary rise effect and due to movement of the leg without complete lift-off in swing phase.
<ol>
<li>The brass tube with the balsa stick to which the animal is glued is inserted into a micromanipulator to allow height adjustment of the animal at between 7 and 12mm above the slippery surface, which corresponds to the height during free walking.</li>
<li>In order to measure current flow whenever the insect tarsus of the monitored leg is on the ground, a wire (47&mu;m diameter) is cut into approximately 15cm long pieces.</li>
<li>The insulation of a copper is removed by burning it away with alcohol.</li>
<li>A loop is formed with one end of the wire and slid over the tarsus of the animal</li>
<li>The loop is tied at the distal end of the tibia to provide good contact between animal and wire.</li>
<li>The wire is then fixed on the thorax of the animal with a drop of two-component glue (ProTempII, see above), and connected to the differential amplifier through an alligator clamp.</li>
<li>To improve conductivity of the wire at its ends, a drop of electrode cream is applied (Marquette Hellige, Freiburg, Germany) on the areas of contact.</li>
<li>The signal strength is then tested on the monitor. Due to the low-pass filter properties of the lock-in-amplifier (10ms time constant) and the gradual lift-off/touch-down of the tarsus, the signal is not an exact square signal. One therefore has to set suitable thresholds close to the transition point to determine the exact timing of tarsal contact and manually check each event determined by the digitalization software. Based on the tarsal contact, we defined the time when the tarsus of a given leg was on the ground as stance and the time when it was in the air as swing phase.</li>
<li>The trigger signal for the camera is recorded simultaneously with the EMG trace and the tarsal contact signal, and allows a precise frame by frame correlation of behavior and recorded traces.</li>
</ol>
6. Optical Recording of Leg Movements for Digital Analysis
The stepping frequency of stick insects on slippery surfaces can reach 3-4Hz (Graham and Cruse, 1981).
<ol>
<li>Before recording walking sequences, a mirror is placed at a 45&deg;angle behind the animal to have an additional visual monitor for touch-down. The camera is externally triggered and pictures are fed into a PC through a FireWire interface.</li>
<li>We record walking sequences from above with a high speed video camera (Marlin F-033C, Allied Vision Technologies, Stadtroda, Germany) at 100fps to analyze the leg movements digitally, and correlate them with EMG activity and tarsal contact.</li>
<li>During the recording of walking sequences, the animal is illuminated with blue LED arrays (30-50V DC, luminance 24cd, Electronics Workshop, Zoological Institute, University of Cologne), which are adjusted in intensity to illuminate the fluorescent markers. According to the literature (Schlegtendal, 1934), stick insects do not possess color vision and we have not observed an adverse effect of the UV light illumination on the turning response. A yellow filter in front of the camera lens suppresses the short wavelength of the activation light and gives a higher contrast for the video recordings.</li>
<li>The video files can be analyzed using motion tracking software (WINanalyze, Vers.1.9, Mikromak service, Berlin, Germany).</li>
</ol>
7. Recording of Walking Sequences
<ol>
<li>As a control, first a walking sequence with an intact animal is recorded.</li>
<li>The velocity of the striped pattern is set, and pattern and the video recording are started approximately at the same time.</li>
<li>If the animal does not start locomotion spontaneously, it can be stimulated at the abdomen with a brush (B&auml;ssler and Wegener, 1983), or by a puff of air to the antennae. In this case, animals touched on the left side of the abdomen tend to turn left and vice versa. The striped pattern is kept moving until the animal stops walking or until after 3 minutes of continuous recordings.</li>
<li>In order to test how far the muscular activity in one leg is dependent on sensory input from neighboring legs, the animal is induced to autotomize its legs. The site of autotomy closes up naturally within seconds. An example of an animal with autotomized front and hind legs is shown in Figure 3.</li>
<li>For example, for experiments with animals having only one middle leg, we induce autotomy of the pro- and metathoracic legs by pinching the proximal femur with a pair of forceps (Schmidt and Grund, 2003) or cut the legs at the level of the coxae after recording from the intact animal. After that a minimum of 30min for recovery time is given to the animal.</li>
<li>Then we record new walking sequences as before. In reduced preparations, we often need to induce walking using paint brush strokes.</li>
</ol>
8. Representative Results:
<img src="/files/ftp_upload/2629/2629fig1.jpg" alt="Figure 1" /> 
Figure 1. Experimental setup; A: photograph of a stick insect tethered above the slippery surface, marked with fluorescent pigments for leg tracking and wired for EMG recording and tarsal contact measurement. B: wiring diagram for dual leg tarsal contact recording during walking on the slippery surface.
<img src="/files/ftp_upload/2629/2629fig2.jpg" alt="Figure 2" /> 
Figure 2. Positions for the placement of EMG electrodes in the protractor and retractor muscles in the thorax of the stick insect middle leg. ML: middle leg; FL: front leg
<img src="/files/ftp_upload/2629/2629fig3.jpg" alt="Figure 3" /> 
Figure 3. Stick insect with EMG wires in the pro- and retractor muscles of the middle leg in place; front and hind legs have been removed to study the effect of the presence of neighboring legs on the kinematics and muscle activity of the middle legs in the reduced preparation.
<img src="/files/ftp_upload/2629/2629fig4.jpg" alt="Figure 4" /> 
Figure 4. Example trace of retractor EMG activity with reference signal of tarsal contact in an inside middle leg during turning. At the beginning of the trace (first blue box) the retractor is active during stance, while the leg has ground contact ("closed circuit"), indicating that the leg makes forward steps; after a short switch (second blue box), the retractor is active during swing, while the leg is in the air ("open circuit", third blue box), showing the occurrence of backwards directed steps.
<img src="/files/ftp_upload/2629/2629fig5.jpg" alt="Figure 5" /> 
Figure 5. Screen shot of a recording from the protractor (top trace) and retractor (middle trace) muscles and the simultaneously recorded tarsal contact trace (bottom trace). Note the alternating activity in the EMG traces which corresponds to the activity of the two muscles in the step cycle, that is protractor activity just before and during swing, when the tarsal contact trace signals loss of ground contact, and retractor activity after and during touch-down of the tarsus, when leg is in stance. The insert shows the matching video file corresponding to this recording that is used to verify the behavior of the animal.

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No conflicts of interest declared.

We have described a setup that allows the optically induced generation of turning behavior and permits to a large degree the uncoupling of neuronally generated walking activity from the passive effects caused by the mechanical displacement of the other walking limbs on the ground. Potential information flow between the legs through the nervous system about ground reaction forces or tarsal contact, on the other hand, is still possible and allows the experimenter to study the influence of such information in the reduced preparation. Major advantages of the slippery surface setup include that the animals show a very high tendency to walk, and contrary to walking or stepping on a treadwheel, the animal can perform swing and stance phase movements in all the directions of natural walking. In addition, the degrees of freedom for all the legs allow the animal to perform curve walking whether it is an intact or semi-intact preparation. Because the legs cannot be passively moved simply by the forward movement of the animal or the movement of the substrate underneath, every movement reflects the motor output of that leg (Cruse, 1976; Graham and Wendler, 1981). The setup is highly suitable to investigate the neuronal basis of adaptive behaviors such as turning or forward vs. backward walking, because one can combine electrophysiological recordings of motor activity with the analysis of limb movement kinematics.
We used the stick insect's optomotor response to elicit walking. The responses of the animals to the rotating stripe pattern show their readiness to perform curve walking while tethered over the slippery surface. Most surprisingly for us, single legs in single-leg preparations qualitatively show the same moving pattern as in the intact animal. We thus have reason to believe that the control of curve walking can function largely without coordinating sensory input from neighboring legs. It will be important to test in further experiments whether the activity of the motor neurons of the removed legs is also influenced by the optomotor pattern. The setup can easily be modified to allow the study of other tasks such as straight forward and backward walking by placing a single stripe in front of the animal or gently pulling the antennae.
The precise measurement of ground contact allows us to correlate muscle activity and leg position. The high time resolution of this electric contact signal is better than 1ms and leads us to a new insight into the timing of the switch from swing to stance phase. The resolution is worse for the stance to swing transition due to the delay in shearing of the conducting lubricant and a lack of need for a complete lift-off during protraction on the slippery surface. Nevertheless, the knowledge on the precise swing to stance transition is a particularly useful first step if we want to understand the mechanisms that control muscle timing and the coordinated activities of leg muscles in different behavioral contexts (see also: B&uuml;schges et al., 2008; B&uuml;schges &amp; Gruhn 2008).
As an example, we used the retractor and protractor coxae muscle of the middle leg and precisely correlated its activity with the switching from swing to stance phase while we simultaneously monitored the behavioral context in which the leg was used. For this purpose, we induced walking and recorded the muscle activity continuously. A given leg can be an inside or an outside leg, depending on the turning direction. In the stepping middle leg, acting as an inside leg in the functional sense, it can be observed that retractor and protractor muscles can both work as functional stance muscles because the leg intermittently produces backward steps in addition to forward directed steps (see Fig.4).
The electromyograms (EMGs) from both muscles were rectified and normalized to the time of touch-down and the latency of the first muscle spikes was calculated. Interestingly, the latencies of both muscles with respect to lift-off and touch-down depend on the function of the muscle as respective swing or stance muscle (see Fig.4) and not on the muscle itself, and show only minor alterations in the timing of activity onset. Most explanations for the change of state from swing to stance assume that sensory signals of tarsal contact trigger the start of stance. The interesting question of how the short latencies between touch-down and muscle activation in the stick insect are brought about and on what sensory information they depend can now be addressed with the modified setup.
In summary, we show a slippery surface setup that allows us to reliably elicit straight and curve walking in stationary stick insects. Kinematics, muscle activity and the timing of tarsal touch-down and lift-off can be monitored and correlated in the two different behavioral contexts at the same time. This gives us an excellent tool to study the detailed connection between muscle activity and behavioral context for a single leg as well as in the intact animal and the underlying mechanisms.

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studying the neural basis of adaptive locomotor behavior in insects

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch &amp; Pfl&uuml;ger, 1995; B&ouml;hm et al., 1997; Gruhn &amp; Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

Watch this Scientific Journal Video about Studying the Neural Basis of Adaptive Locomotor Behavior in Insects at JoVE.com

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

studying-the-neural-basis-of-adaptive-locomotor-behavior-in-insects

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.7K Views.  University of Cologne. We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate. 

Video: Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

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 &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

Behavior

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, acceleration and forces involved in the locomotory behavior of nematodes suspended in a column of water in response to single wavelengths of light. We demonstrate how to evaluate the locomotion of a microscopic organism using Single Wavelength Shadow Imaging (SWSI) using two different examples.
The first example is a systematic and statistically viable study of the average descent of C. elegans in a column of water. For this study, we used living and dead wildtype C. elegans. When we compared the velocity and direction of nematode active movement with the passive descent of dead worms within the gravitational field, this study showed no difference in descent-times. The average descent was 1.5 mm/sec &#177; 0.1 mm/sec for both the live and dead worms using 633 nm coherent light.
The second example is a case study of select individual C. elegans changing direction during the descent in a vertical water column. Acceleration and force are analyzed in this example. This case study demonstrates the scope of other physical properties that can be evaluated using SWSI while evaluating the behavior using single wavelengths in an environment that is not accessible with traditional microscopes. Using this analysis we estimated an individual nematode is capable of thrusting with a force in excess of 28 nN.
Our findings indicate that living nematodes exert 28 nN when turning, or moving against the gravitational field. The findings further suggest that nematodes passively descend in a column of water, but can actively resist the force of gravity primarily by turning direction.

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

The technique presented here measures the path of freely swimming microscopic species using single wavelength exposure. C. elegans are used to demonstrate shadow imaging as an inexpensive alternative to costly microscopes. This technique can be adapted to accommodate various orientations, environments and species to measure direction, speed, acceleration and forces.

This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, ...

Engineering

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving insects.

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight insect behavior. This technology enables the development of an insect-machine hybrid system using a living insect platform described in this protocol. Moreover, this protocol presents the system configuration and free flight experimental procedures for evaluating the function of the flight muscles in an untethered insect. For demonstration, we targeted the third axillary sclerite (3Ax) muscle to control and achieve left or right turning of a flying beetle. A thin silver wire electrode was implanted on the 3Ax muscle on each side of the beetle. These were connected to the outputs of a wireless backpack (i.e., a neuromuscular electrical stimulator) mounted on the pronotum of the beetle. The muscle was stimulated in free flight by alternating the stimulation side (left or right) or varying the stimulation frequency. The beetle turned to the ipsilateral side when the muscle was stimulated and exhibited a graded response to an increasing frequency. The implantation process and volume calibration of the 3 dimensional motion capture camera system need to be carried out with care to avoid damaging the muscle and losing track of the marker, respectively. This method is highly beneficial to study insect flight, as it helps to reveal the functions of the flight muscle of interest in free flight.

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor sources is an essential task for organism survival. Insects are well-studied organisms with regard to odor tracking, and their behavioral strategies have been applied to mobile robots for evaluation. This "bottom-up" approach is a fundamental way to develop biomimetic robots; however, the biological analyses and the modeling of behavioral mechanisms are still ongoing. Therefore, it is still unknown how such a biological system actually works as the controller of a robotic platform. To answer this question, we have developed an insect-controlled robot in which a male adult silkmoth (Bombyx mori) drives a robot car in response to odor stimuli; this can be regarded as a prototype of a future insect-mimetic robot. In the cockpit of the robot, a tethered silkmoth walked on an air-supported ball and an optical sensor measured the ball rotations. These rotations were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can manipulate any parameter of the robot, which enables the evaluation of the odor-tracking capability of insects and provides useful suggestions for robotic odor-tracking. Furthermore, these manipulations are non-invasive ways to alter the sensory-motor relationship of a pilot insect and will be a useful technique for understanding adaptive behaviors.

The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform.

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

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Rozdziały w tym wideo

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

C. elegans Tracking and Behavioral Measurement

Neural Circuit Recording from an Intact Cockroach Nervous System

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates