Name: studying the neural basis of adaptive locomotor behavior in insects
Uploaded: 2011-04-13T11:03:00
Duration: 10 min 19 s
Description: Watch this Scientific Journal Video about Studying the Neural Basis of Adaptive Locomotor Behavior in Insects at JoVE.com

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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Extensor motor neuron pattern.</a> J Neurophysiol. 83, 3337-3350 .</li><li>Tsuchida, Y., Hama, N., Takahata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+telemetry+system+for+underwater+recording+of+electromyogram+and+neuronal+activity+from+non-tethered+crayfish.">An optical telemetry system for underwater recording of electromyogram and neuronal activity from non-tethered crayfish.</a> J Neurosci Methods. 137, 103-109 (2004).</li><li>Wang, H., Ando, N., Kanzaki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+control+of+free+flight+maneuvers+in+a+hawkmoth,+Agrius+convolvuli.">Active control of free flight maneuvers in a hawkmoth, Agrius convolvuli.</a> J Exp Biol. 211, 423-432 (2008).</li></ol>

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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		<th>Comment</th>
		</tr>
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<tr>
<td>2-component epoxy glue</td>
<td>&nbsp;</td>
<td>UHU plus, UHU GmbH, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass screens (diameter 130mm)</td>
<td>&nbsp;</td>
<td>Marata screens, Linos Photonics, G&#246;ttingen, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>dental cement</td>
<td>&nbsp;</td>
<td>ProTemp II, 3M ESPE, Seefeld, Germany</td>
<td>3M Id : 70-2011-0358-0 Catalog Number : 46430</td>
<td>Available through 3M (<a href="http://www.3m.com/">http://www.3m.com/</a>)or dental suppliers</td>
</tr>
<tr>
<td>fluorescent pigments </td>
<td>&nbsp;</td>
<td>Dr. Kremer Farbm&#252;hle, Aichstetten, Germany</td>
<td>Cat.#s: i.e 56200 Fluorescent Pigment Golden Yellow 56350 Fluorescent Pigment Flame Red</td>
<td><a href="http://kremer-pigmente.de/en">http://kremer-pigmente.de/en</a> or <a href="http://www.kremerpigments.com/
">http://www.kremerpigments.com/</a>
</td>
</tr>
<tr>
<td>histoacrylic glue </td>
<td>&nbsp;</td>
<td>3M Vetbond, St.Paul, MN, USA</td>
<td>&nbsp;</td>
<td>supplier: WPI</td>
</tr>
<tr>
<td>coated copper wire</td>
<td>&nbsp;</td>
<td>Elektrisola Eckernhagen</td>
<td>&nbsp;</td>
<td><a href="http://www.elektrisola.com/">http://www.elektrisola.com/</a></td>
</tr>
<tr>
<td>electrode cream </td>
<td>&nbsp;</td>
<td>Marquette Hellige, Freiburg, Germany</td>
<td>&nbsp;</td>
<td>Product is now discontinued, we suggest for example: <a href="www.grasstechnologies.com">www.grasstechnologies.com</a></td>
</tr>
<tr>
<td>pulse generator </td>
<td>&nbsp;</td>
<td>Model MS501, electronics workshop, Zoological Institute, Cologne, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>lock-in-amplifier </td>
<td>&nbsp;</td>
<td>electronics workshop, Zoological Institute, Cologne, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>AD converter </td>
<td>&nbsp;</td>
<td>Micro 1401k II, CED, Cambridge, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>preamplifier </td>
<td>&nbsp;</td>
<td>electronics workshop, Zoological Institute, Cologne, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>high speed video camera </td>
<td>&nbsp;</td>
<td>Marlin F-033C, Allied Vision Technologies, Stadtroda, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>UV LED arrays </td>
<td>&nbsp;</td>
<td>30-50V DC, electronics workshop, Zoological Institute, Cologne, Germany</td>
<td>&nbsp;</td>
<td>&lambda;390-395nm 
Luminance 24cd</td>
</tr>
<tr>
<td>Digitalizing software Spike2 </td>
<td>&nbsp;</td>
<td>Vers.5.05, CED, Cambridge, UK</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>motion tracking software </td>
<td>&nbsp;</td>
<td>(WINanalyze, Vers.1.9, Mikromak service, Berlin, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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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

Research

JoVE Journal

Neuroscience

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

A Simple Flight Mill for the Study of Tethered Flight in Insects

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have shown that flight mills are valuable tools for the experimental study of insect flight behavior, allowing researchers to examine how factors such as age, host plants, or population source can influence an insects' propensity to disperse. Flight mills allow researchers to measure components of flight such as speed and distance flown. Lack of detailed information about how to build such a device can make their construction appear to be prohibitively complex. We present a simple and relatively inexpensive flight mill for the study of tethered flight in insects. Experimental insects can be tethered with non-toxic adhesives and revolve around an axis by means of a very low friction magnetic bearing. The mill is designed for the study of flight in controlled conditions as it can be used inside an incubator or environmental chamber. The strongest points are the very simple electronic circuitry, the design that allows sixteen insects to fly simultaneously allowing the collection and analysis of a large number of samples in a short time and the potential to use the device in a very limited workspace. This design is extremely flexible, and we have adjusted the mill to accommodate different species of insects of various sizes.

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less vulnerable to interference. The neural mechanisms underlying the benefit of sleep for memory can be investigated using polysomnography (PSG). PSG is a combination of physiological recordings including signals from the brain (EEG), eyes (EOG), and muscles (EMG) that are used to classify sleep stages. In this protocol, we describe how PSG can be used in conjunction with behavioral memory assessments, actigraphy, and parent-report to examine sleep-dependent memory consolidation. The focus of this protocol is on early childhood, a period of significance as children transition from biphasic sleep (consisting of a nap and overnight sleep) to monophasic sleep (overnight sleep only). The effects of sleep on memory performance are measured using a visuospatial memory assessment across periods of sleep and wakeful-rest. A combination of actigraphy and parent report is used to assess sleep rhythms (i.e., characterizing children as habitual or non-habitual nappers). Finally, PSG is used to characterize sleep stages and qualities of those stages (such as frequencies and the presence of spindles) during naps. The advantage of using PSG is that it is the only tool currently available to assess sleep quality and sleep architecture, pointing to the relevant brain state that supports memory consolidation. The main limitations of PSG are the length of time it takes to prepare the recording montage and that recordings are typically taken over one sleep bought. These limitations can be overcome by engaging young participants in distracting tasks during application and combining PSG with actigraphy and self/parent-report measures to characterize sleep cycles. Together, this unique combination of methods allows for investigations into how naps support learning in preschool children.

This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy.

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less ...