Name: long-term behavioral tracking of freely swimming weakly electric fish
Uploaded: 2014-03-06T13:15:00
Description: Watch this Scientific Journal Video about Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish at JoVE.com

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 s...

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">
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			<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>
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			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:277px;"> </td>
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			<td height="21" style="height:21px;width:216px;">[EOD recording setup]</td>
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		<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">
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			<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 ...

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