This work was generously supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR).

This procedure meets the requirements of the University of Ottawa Animal Care Committee. No conflict of interest is declared. Please refer to the Table of Materials and Reagents for the makes and models of the equipment and materials listed below. Custom written Spike2 and MATLAB scripts, and sample data are provided in the Supplemental File.
1. Aquarium Tank and Isolation Chamber Setup
<ol>
	<li>Anti-vibration floor. Construct an anti-vibration surface (2.1 m x 2.1 m) by stacking rubber pads, acoustic Styrofoam, marine plywood panel, and polyurethane foam pads from the bottom to the top (Figure&#160;1A). Lay four wooden studs (5 cm x 10 cm) on the plywood panel to support the edges of the aquarium tank.</li>
	<li>Floor heater. Lay an electrically shielded heating element over thermally graded foam padding (see Figure&#160;1D bottom). Cover the heating element with a metallic mesh for electrical shielding.</li>
	<li>Spatial tank. Construct a wide and shallow aquarium tank (1.8 m x 1.8 m x 30 cm) using 1.3 cm thick tempered glass panels, L-shaped aluminum frame and aquarium-grade silicone (see Figure 1A). Cover the underside of the tank with a large sheet of white background to provide high imaging contrast (see Protocol 3).</li>
	<li>Divide the aquarium tank into a central arena (1.5 m diameter) and four corner compartments (see Figure 1B) by installing walls (22.5 cm tall) made of acrylic sheets (matte white, 0.64 cm thick).
	<ol>
		<li>Bend four acrylic sheets (22.5 cm x 102.7 cm) by applying heat to create four curved wall sections, and attach them to the tank bottom using silicone caulk to separate the central arena from the four corner compartments. Leave 20 cm space between the curved sections for the gate installation.</li>
		<li>Separate neighboring corner compartments by installing four double walls with 15 cm gaps, which provide extra electrical isolation and places for underwater sensors such as a hydrophone.</li>
	</ol>
	</li>
	<li>Assemble four motorized gates, and install them between the corner compartments and the central arena.
	<ol>
		<li>Assemble four door frames as shown in Figure 1C. Create six wells (0.64 cm deep) on each door frame, embed nylon acorn nuts (0.64 cm diameter thread) and secure them with epoxy.</li>
		<li>Cut four door panels from acrylic and rubber sheets, and create six holes (0.64 cm diameter) on the acrylic and rubber panels for the locking mechanism. Join the acrylic and rubber panels using silicone caulking.</li>
		<li>Install acrylic hinges to join the door panels with the door frames.</li>
		<li>Mount swinging arms on servomotors, and install them on the top of the door frames (see Figure 1C). Make loops with cable ties to link the swinging arms to the door panels.</li>
		<li>Position the gate assemblies on the gaps created between the curved wall sections, and secure them using silicone caulking.</li>
		<li>Connect all servomotors to a servo controller, and connect it to a power source and a computer via an active USB extension cable. Test the gates using control software supplied with the servo controller.</li>
		<li>After the silicone hardens, check for watertightness by locking all gates with nylon screws and filling one compartment at a time.</li>
	</ol>
	</li>
	<li>Isolation chamber. Construct an isolation chamber to surround the aquarium and block external sources of light, sound and electrical noise (see Figure&#160;1D).
	<ol>
		<li>Make three wall panels (2 m x 2 m x 5 cm) and four door panels (1.9 m x 0.95 m x 5 cm). For each panel, join aluminum moldings (5 cm x 2.5 cm) to create a rectangular frame; and rivet a white corrugated plastic panel on the aluminum frame. Fill acoustic fiberglass batts in the panels, and close with a black corrugated plastic panel.</li>
		<li>Install three wall panels on the anti-vibration floor, and install piano hinges to join the four door panels on the wall panels.</li>
		<li>Surround the isolation chamber with aluminum meshes, and ground meshes on all sides to create a Faraday cage.</li>
	</ol>
	</li>
	<li>Humidity control. Install a low-noise exhaust fan (Figure&#160;1F top) to remove excess humidity build-up from heating. Place the exhaust fan at least 2 m away from the recording site, and install an air duct between the isolation chamber and the exhaust fan.</li>
	<li>Routinely monitor and maintain the conditions of the tank water and animals.
	<ol>
		<li>Maintain constant water conditions at 10 cm depth, 100 &#181;S/cm conductivity and pH 7.0 by adding water or salt stock solution (refer to Knudsen33 for the recipe). Add a bag of crushed coral if the pH drops below 6.5.</li>
		<li>Install vertical aquarium filters which can operate from shallow water for cleaning and aerating purposes (Figure&#160;1F bottom). Disconnect the filters and take them out of the central arena during recording sessions.</li>
		<li>Deliver live mealworms on the bottom of the tank by attaching them on suction cups with elastics. Avoid free-floating preys such as blackworms to prevent uncontrolled feeding of stray preys during recording.</li>
	</ol>
	</li>
</ol>
2. EOD Tracking
<ol>
	<li>Electrodes installation. Assemble eight graphite electrodes, and space them equally on the curved wall of the central arena.
	<ol>
		<li>Obtain drawing leads (15 cm in length; Mars Carbon 2&#160;mm type HB) and shave off the outer coating of the leads.</li>
		<li>Cut eight 10 cm segments of coaxial cable (RG-174), wrap the cable core around one end of the graphite rods, and apply heat-shrink tubing over them for strong and stable electrical connection. Attach BNC jack connectors on the opposite ends (Figure&#160;2A left).</li>
		<li>Position the electrodes on the wall by taping, and apply thin strips of masking tape on the electrode surfaces to protect from silicone. Apply silicone caulking to permanently hold the electrodes, and remove all tape before the silicone hardens (Figure&#160;2A right).</li>
	</ol>
	</li>
	<li>Build eight cable assemblies by measuring the distance from each electrode to the amplifier unit, and cutting coaxial cables (RG-54) in lengths. Attach BNC plug connectors on both ends of the cables.</li>
	<li>Use the cable assemblies to wire all electrodes to the amplifier unit. Differentially amplify by pairing two 90&#176;&#160;oriented electrodes (see Figure&#160;2B), and ground all coaxial shielding wires by connecting them to the Faraday cage.</li>
	<li>Set the amplifier gain below the signal saturation limit, and apply a band-pass filter (200 Hz-5 kHz) to remove noise. Digitize the four recording electrode pairs at 40 kS/sec.</li>
	<li>Online signal processing. The instructions are written for the Spike2 software, and the parameter settings are optimized for Gymnotus sp. (see Figure&#160;2C for summary).
	<ol>
		<li>Add a DC remove process (&#964; = 0.1 sec) to all recording channels.</li>
		<li>Add a rectify process to all recordings channels.</li>
		<li>Create a virtual channel by summing all four recording channels.</li>
		<li>Extract a unimodal envelope per EOD pulse by adding a RMS (root-mean-squared, <img alt="" fo:content-width="40px" src="/files/ftp_upload/50962/50962_clip_image002.gif" width="70px" /> ) process (&#964; = 0.25 msec) to the virtual channel, for generating a single peak per EOD cycle to unambiguously determine the pulse timing.</li>
		<li>Create a realmark channel from the virtual channel and record the time and values of the peak amplitudes, after setting an appropriate threshold to capture all EOD pulses without missing a pulse, while avoiding false positives.</li>
		<li>Monitor the instantaneous EOD rate in real-time by setting the channel display option of the realmark channel to an instantaneous frequency mode.</li>
		<li>Monitor the fish movement in real-time by duplicating the realmark channel, and set the display option to a waveform mode.</li>
		<li>Quantify an activity level from the RMS of the EOD amplitude slope by creating a virtual channel from the realmark channel (0.01 sec sampling period), and add slope (&#964; = 0.25 msec) and RMS (&#964; = 0.5 msec) processes.</li>
		<li>Export the realmark channel in the Spike2 software to the MATLAB format.</li>
	</ol>
	</li>
</ol>
3. Synchronized Video Tracking
<ol>
	<li>Create a background scene.
	<ol>
		<li>Hide any object that casts a reflection on the water surface by covering with matte white countertop film.</li>
		<li>Install a matte white corrugated plastic panel 15 cm below the ceiling to hide the camera and the air vent.</li>
		<li>Print grid patterns on a large sheet of white paper for calibrating a camera, and lay it underneath the tank to provide a high-contrast background.</li>
	</ol>
	</li>
	<li>Install light sources.
	<ol>
		<li>Obtain IR LED lights and, remove built-in fans to reduce noise. Drive the LED with a current-regulated DC power supply placed outside of the Faraday cage.</li>
		<li>Install IR LED lights for imaging in darkness, and white LED lights for driving a diurnal light cycle in the test fish. Direct all light sources toward the ceiling to achieve indirect and uniform illumination (Figure&#160;3A).</li>
		<li>Regulate the diurnal light cycle by driving the white LED lights with a timer-controlled switch (e.g. 12 hr on/12 hr off).</li>
	</ol>
	</li>
	<li>Install a camera directly above the aquarium.
	<ol>
		<li>Obtain a NIR-sensitive camera, or remove an IR blocking filter by breaking a thin sheet of tinted glass at the back of the lens assembly. Make sure the viewing angle is wide enough to image the whole central arena.</li>
		<li>Make a small viewing hole in the middle of the ceiling panel, and place the camera directly above the hole.</li>
		<li>Install a white ring guard around the lens if the light sources generate glares.</li>
	</ol>
	</li>
	<li>Make a time-synchronized video recording.
	<ol>
		<li>Place an IR LED at one of the four tank corners to generate time synchronization pulses (1 msec duration, 10 sec period). Add a load-limiting resistor (1 k&#937;) in series, and drive the IR LED from a digital output port of the digitizer hardware.</li>
		<li>Use video recording software bundled with the camera if available. Select the highest recording quality (e.g. lossless compression) and the highest resolutions supported.</li>
		<li>Start the video recording immediately before starting the EOD recording, and stop the video recording immediately after the EOD recording.</li>
		<li>After the recording, convert the image frame numbers to the digitizer time unit by linearly interpolating between the first and the last light pulses captured by the signal digitizer and the video recording.</li>
	</ol>
	</li>
	<li>Automated image tracking 
	The instructions are written for the MATLAB Image processing toolbox, and make use of its functions. A custom MATLAB script is provided with this submission for automated image tracking.&#160;
	<ol>
		<li>Import video. Import a video recording file directly to the MATLAB workspace using "Videoreader.read" function.</li>
		<li>Create a composite background image by combining two image frames. Replace the image region occupied by an animal with an unoccupied image of the same region from another frame (see Figure&#160;3B).</li>
		<li>Specify an image region to track by drawing a circular mask around the central arena to exclude the area outside (Figure&#160;3B bottom), and multiply by a constant (rint) to set a minimum threshold for intensity difference. For example, setting rint = 0.85 will suppress the intensity fluctuations 15% = (1 - rint) below the background.</li>
		<li>Image subtraction. Subtract an image frame (=IMk) from the background image (=IM&#173;0) to obtain the difference image (=&#916;IMk). Use unsigned integer numerical precision to store the image intensity values as non-negative integers.</li>
		<li>Segment the difference image by applying an intensity threshold determined from the graythresh function. Clean the binary image using the bwmorph function, and select the largest blob corresponding to an animal after calculating all blob areas using the regionprops function.</li>
		<li>Determine the centroid and major orientation axis of the largest blob by applying the regionprops function, and rotate the image to align the major axis with the x-axis. Divide the image to the head and tail parts at the centroid (Figure 3D top).</li>
		<li>Determine the major axis of the head part, and rotate the entire image to align with the x-axis (Figure 3D bottom left). Fit bounding-boxes around the head and tail parts parallel to their major axes using the regionprops function.</li>
		<li>Determine the median y-coordinates of the blob at the left, center and right vertical edges of the bounding boxes (green dots in Figure 3D bottom); and assign them to five feature points (head-tip, mid-head, mid-body, mid-tail, tail-tip).</li>
		<li>Process successive frames after cropping an image frame centered at the animal&#39;s centroid determined from its previous frame.</li>
		<li>Manually assign the head orientation for the first frame, and use a dot-product between the orientation vectors from two successive frames to automatically determine the head orientation. Inspect the result, and manually flip the head orientation if incorrectly assigned.</li>
	</ol>
	</li>
	<li>Plot an animal trajectory by joining the head-tips, and smooth using median and average filters (n=3) if it has a jittery appearance. Superimpose the trajectory with a background image, and interpolate fish midlines using the five feature points (see Figure 2E).</li>
	<li>Compute the average EOD rate at each image capture time by resampling the instantaneous EOD rate (100 Hz sampling rate) and averaging (0.0625 sec time window). Plot the trajectory in pseudo-colors determined from the time-matched EOD rate, and superimpose with a background image (see Figure 2F).</li>
</ol>

The authors have nothing to disclose.

Significance of our techniques. In summary, we first described the construction of a large aquarium tank and an isolation chamber to observe spontaneous exploratory behaviors produced by WEF. Next, we demonstrated the technique of recording and tracking the EOD rate and the movement states from unrestrained fish in real-time using multiple electrode pairs. Finally, we described the infrared video recording technique through water in a time-synchronized manner, and the image tracking algorithm to measure the body...

Background. Quantitative experiments on animal behavior (e.g. forced choice, shock avoidance, T-maze, etc.) are typically utilized to investigate specific hypotheses concerning sensory-motor skills, learning and memory formation. However, these restrictive experiments miss much of the richness of natural animal behavior&#160;and are likely to result in oversimplified models of the underlying neural basis of behavior. Experiments under more naturalistic conditions are therefore an important complement by which we can explore more fully a species behavioral repertoire. Experiments involving freely moving animals must, however, address unique technical challenges such as movement-induced recording artifacts. Unlike stimulus-evoked responses, spontaneously occurring exploratory behavior cannot be predicted; thus experimental subjects have to be constantly monitored and tracked over an extended period of time. Specific research questions can be best addressed by carefully selected organisms and available technical tools. For example, optical recording and stimulation techniques such as genetically-encoded calcium sensors1 and optogenetics2 have been successfully applied to freely moving genetic model organisms3-5. Alternatively, miniaturized neural telemetry systems can record and stimulate freely moving small animals6,7.
Electric fish. WEF species generate electric&#160;organ discharges (EODs), which allow them to sense their immediate surroundings or to communicate over greater distances. Temporal patterns of EODs vary under different conditions such as self-movements8,9, sensory stimuli10,11, and social interactions12,13. Pulse-type WEF species produce a train of discrete pulses, as opposed to wave-type species which generate continuous quasi-sinusoidal waveforms. In general, pulse-type species exhibit more variable EOD rate compared to the wave-type species; and animals&#39; EOD rates closely reflect novelty contents of their sensory surroundings10,14. Pulse-type species can immediately shorten the inter-pulse interval (IPI) within a single pulse cycle in respond to a novel sensory perturbation (novelty response10,11,14). The ongoing electric behavior of these fish can be perturbed by uncontrolled sensory stimuli from external sources; and different kinds of stimuli such as vibration, sound, electricity, and light are known trigger novelty responses. Therefore, special precautions must be taken to block or attenuate external sensory stimuli during a long-term observation of free-swimming WEF. In this way, changes in EOD rate and movement trajectories can be specifically attributed to stimuli presented by the experimenter.
Aquarium tank and isolation chamber. We therefore placed multiple layers of vibration absorbing materials under a large aquarium tank (2.1 m x 2.1 m x 0.3 m), and surrounded the tank with an insulated enclosure to block external sources of light, electrical noise, sound and heat flux. EOD rate depends on the surrounding temperature15,16, thus the water temperature was tightly regulated at a tropical range (25&#177;1 &#176;C) for South American WEF species. We constructed a large and shallow (10 cm water depth) tank to observe spatial exploratory behaviors of WEF mainly restricted in two dimensions (Figure&#160;1A). The tank was partitioned into a central arena to observe spatial behaviors, and four corner compartments to separately house individual fish (Figure&#160;1B). Each compartment was built watertight to prevent electrical communication between individuals. Animals&#39; access to the central arena was controlled from the outside by four motorized gates. The gates were placed between the compartments, and they became watertight when locked by nylon wing-nuts. No metallic parts were used underwater since WEF react sensitively to metals.
EOD recording. EODs are generated in a stereotyped manner by activation of single (in Mormyrids) or multiple spatially distributed electric organs (in Gymnotiforms)17,18. Temporal modulations in the EOD rate can reveal higher-level neural activities, since the medullary pacemaker receives direct neural inputs from higher brain regions such as the diencephalic prepacemaker nucleus, which in turn receives axonal projections from the forebrain19. However, the EOD timing must be carefully extracted from a raw waveform recording and not biased by the animal&#39;s movement-induced distortions. The electric field generated by a WEF can be approximated as a dipole; thus EOD pulse amplitudes at recording electrodes depend on the relative distances and orientations between the animal and the electrodes8,20. Animal&#39;s self-movements change the relative geometry between the animal and the electrodes, thus movements cause the EOD amplitudes at different electrodes to vary over time in a volatile manner (see Figure&#160;2B in Jun et al.8). Furthermore, self-movements also change the shape of recorded EOD waveforms, because relative contributions from different set of the electric organs depend on their locations along the body length and their local curvatures introduced by tail bending. The movement-induced distortions in the EOD amplitudes and shapes can lead to inaccurate and unreliable EOD timing measurements. We overcame these problems by spatially averaging multiple EOD waveforms recorded at different locations, and by adding an envelope extraction filter to precisely determine the EOD timing from a free-swimming WEF. In addition, our technique also measures the EOD amplitudes, which indicate whether an animal is resting or actively moving based on the change of the EOD amplitudes over time (see Figures&#160;2E and 2F). We recorded differentially amplified signals from the recording electrode pairs to reduce common-mode noise. Since the EOD pulses are generated at irregular time intervals, the EOD event time-series have a variable sampling rate. The EOD time-series can be converted to a constant sampling rate by interpolation if required by an analytic tool of choice.
Video recording. Although EOD recording can monitor a gross movement activity of an animal, video recording permits direct measurements of an animal&#39;s body position and posture. Near-infrared (NIR) illumination (&#955; = 800~900 nm) permits unperturbed visual observation of freely swimming fish21,22, since WEFs are most active in darkness and their eyes are not sensitive to NIR spectrum23,24. Most digital imaging sensors (e.g. CMOS or CCD) can capture NIR spectrum with the wavelength range between 800-900 nm, after removing an infrared (IR) blocking filter25. Certain high-end consumer-grade webcams offer high-definition, wide viewing angle and good low-light sensitivity, which can produce an image quality comparable to, or superior to professional-grade IR cameras available at much greater costs. In addition, certain consumer-grade webcams are bundled with recording software that permits an extended recording duration by compressing video with no quality loss. Most professional-grade cameras offer time synchronization TTL pulse outputs or trigger TTL pulse inputs26 for aligning the timing between the video with the digitized signals, but this feature is generally absent in consumer-grade webcams. However, the timing between a video recording and a signal digitizer can be accurately matched by concurrently capturing a periodically blinking IR LED with the camera and the signal digitizer. The initial and the final IR pulse timing can be used as two time calibration markers for converting the video frame numbers to the signal digitizer time unit&#160;and vice versa.
Lighting &#38; background. Image capturing through water can be technically challenging due to light reflections at the water surface. The water surface can act as a mirror to reflect a visual scene above water, and obscure visual features underwater; thus the scene above water must be rendered featureless to prevent visual interference. In order to image the whole aquarium, a camera needs to be placed directly above the water; and it should be hidden behind the ceiling over a small viewing hole to prevent its reflection on the water surface. Moreover, the water surface can produce glares and nonuniform illumination if light sources are incorrectly projected. Indirect illumination can achieve uniform brightness over the whole aquarium by aiming the light sources toward the ceiling, such that the ceiling and the surrounding walls can reflect and diffuse the light rays before reaching the water surface. Choose an IR illuminator that matches a spectral response of the camera (e.g. 850 nm peak wavelength). Electrical noise from the light sources can be minimized by using LED lights and placing their DC power supplies outside of the Faraday cage. Place a white background underneath the tank, since fish contrasts well in a white background at NIR wavelengths. Similarly, use of matte white color on the inner surfaces of the isolation chamber provides uniform and bright background illumination.
Video tracking. After a video recording, an automated image tracking algorithm can measure the animal&#39;s body positions and postures over time. The video tracking can be automatically performed by either ready-to-use software (Viewpoint or Ethovision), or user-programmable software (OpenCV or MATLAB&#160;Image processing toolbox). As the first step of image tracking, a valid tracking area needs to be defined by drawing a geometric shape to exclude the area outside (masking operation). Next, an animal&#39;s image needs to be isolated from the background by subtracting a background image from an image containing the animal. The subtracted image is converted to a binary format by applying an intensity threshold, such that the centroid and the orientation axis can be computed from binary morphological operations. In Gymnotiforms27-29 and Mormyrids30-32, the electroreceptor density is the highest near the head region; thus the head position at any moment indicates a location of the highest sensory acuity. The head and tail locations can be automatically determined by applying the image rotation and bounding-box operations. The head and tail ends could be distinguished from one another by manually defining them in the first frame, and by keeping track of their locations from comparing two successive frames.

<table border="0" cellpadding="0" cellspacing="0" width="1020">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
 <td></td>
 <td></td>
 <td></td>
			<td height="21" style="height:21px;width:216px;">[Aquarium construction]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrically shielded floor heater</td>
			<td style="width:277px;">ThermoSoft Corp., IL, USA</td>
			<td style="width:241px;">ThermoTile</td>
			<td>www.thermosoft.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tempered glass panel</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;"> </td>
			<td>.5 inch thick, used for the aquarium construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aquarium grade silicone</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acrylic sheet</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;"> </td>
			<td style="width:287px;">.25 inch thick, matt white</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Natural rubber sheet</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;"> </td>
			<td>.25 inch thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Servomotor</td>
			<td style="width:277px;">HITECHRCD Inc., Korea</td>
			<td style="width:241px;">HS-325HB, 180deg rotation</td>
			<td>www.servocity.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Servomotor arm mount</td>
			<td style="width:277px;">HITECHRCD Inc., Korea</td>
			<td style="width:241px;">56362 Large Spline</td>
			<td>www.servocity.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Servomotor controller (6 chan.)</td>
			<td style="width:277px;">sparkfun.com</td>
			<td style="width:241px;">ROB-09664 </td>
			<td style="width:287px;">Micro Maestro 6-channel USB Servo Controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Active USB extension cable</td>
			<td style="width:277px;">C2G</td>
			<td style="width:241px;">38990</td>
			<td style="width:287px;">12m USB 2.0 A Male to A Female 4-Port Active Extension Cable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Exhaust fan</td>
			<td style="width:277px;">Nutone</td>
			<td style="width:241px;">ILFK120</td>
			<td>www.homedepot.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vertical aquarium filter</td>
			<td style="width:277px;">Tetra, Germany</td>
			<td>Whisper Internal Power Filter - 40i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Crushed coral</td>
			<td style="width:277px;"> </td>
			<td></td>
			<td>Used to increase the pH of the tank water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:277px;"> </td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
 <td></td>
 <td></td>
 <td></td>
			<td height="21" style="height:21px;width:216px;">[EOD recording setup]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graphite Electrodes</td>
			<td style="width:277px;">Staedtler, Germany</td>
			<td>Mars Carbon 2-mm type HB</td>
			<td>Shave the outer coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Physiological Amplifier/Filter</td>
			<td style="width:277px;">Intronix, Canada</td>
			<td style="width:241px;">2015F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coaxial Cable</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;">RG174</td>
			<td>For electrodes assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coaxial Cable</td>
			<td style="width:277px;">generic</td>
			<td style="width:241px;">RG54</td>
			<td>For wiring use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BNC jack connector for RG-174</td>
			<td style="width:277px;">Amphenol Connex</td>
			<td style="width:241px;">112160</td>
			<td>For electrodes assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BNC plug connector for RG-54</td>
			<td style="width:277px;">Amphenol Connex</td>
			<td style="width:241px;">112116</td>
			<td>For wiring use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal digitizer hardware</td>
			<td style="width:277px;">Cambridge Electronic Design, UK</td>
			<td style="width:241px;">Power MKII 1401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal digitizer software</td>
			<td style="width:277px;">Cambridge Electronic Design, UK</td>
			<td style="width:241px;">Spike 2. ver 7</td>
			<td></td>
		</tr>
		<tr height="21">
 <td></td>
 <td></td>
 <td></td>
			<td height="21" style="height:21px;width:216px;">[Visual tracking setup]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">White LED light</td>
			<td style="width:277px;">IKEA, Sweden</td>
			<td style="width:241px;">DIODER 201.194.18</td>
			<td>www.ikea.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Infrared LED light (850 nm)</td>
			<td style="width:277px;">Scene Electronics, China</td>
			<td style="width:241px;">S8100-60-B/C-IR</td>
			<td>Remove built-in fan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">USB webcam</td>
			<td style="width:277px;">Logitech Inc., CA, USA</td>
			<td style="width:241px;">C910</td>
			<td>Remove Infrared blocking filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Motorized camera</td>
			<td style="width:277px;">Logitech Inc., CA, USA</td>
			<td style="width:241px;">Quickcam Orbit</td>
			<td>Remove Infrared blocking filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Video recording software</td>
			<td style="width:277px;">Logitech Inc., CA, USA</td>
			<td>Logitech Quickcam Software</td>
			<td>Download from www.logitech.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matlab</td>
			<td style="width:277px;">Mathworks, MA, USA</td>
			<td style="width:241px;">2012a</td>
			<td>Image processing toolbox</td>
		</tr>
	</tbody>
</table>

long-term behavioral tracking of freely swimming weakly electric fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

EOD tracking results
The recorded EOD waveforms from different electrode pairs varied in amplitudes and shapes as expected from their unique positions and orientations (Figure 2C top). The use of multiple electrode pairs ensured strong signal reception at all possible positions and orientations of WEF within the tank. The envelope waveform (Figure&#160;2C bottom, green trace) always contained a single peak per EOD cycle, which served as a reliable time marker for...

Watch this Scientific Journal Video about Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish at JoVE.com

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

long-term-behavioral-tracking-of-freely-swimming-weakly-electric-fish

Research

JoVE Journal

Neuroscience

12.4K Views.  University of Ottawa. We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal's electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Video: Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

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 ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

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 ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.

Measuring the impacts of environmental contaminants on fish behavior is often subjective and challenging particularly when dealing with sublethal endpoints. We describe methods including video technology to quantify swimming behavior of early life stage white sturgeon (Acipenser transmontanus) during and after 96 hr acute exposures to various concentrations of copper.

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction.  ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...