Name: automated analysis of a nematode population-based chemosensory preference assay
Uploaded: 2017-07-13T14:00:00
Duration: 9 min 44 s
Description: Watch this Scientific Journal Video about Automated Analysis of a Nematode Population-based Chemosensory Preference Assay at JoVE.com

Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work is supported by the Howard Hughes Medical Institute, with which P.W.S. is an investigator.

<ol>
	<li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+nervous+system+of+the+nematode+Caenorhabditis+elegans.">The structure of the nervous system of the nematode Caenorhabditis elegans.</a> Philos Trans R Soc Lond B Biol Sci. 314, 1-340 (1986).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7 (5), 729-742 (1991).</li><li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74 (3), 515-527 (1993).</li><li>Hilliard, M. A., Bargmann, C. I., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+responds+to+chemical+repellents+by+integrating+sensory+inputs+from+the+head+and+the+tail.">C. elegans responds to chemical repellents by integrating sensory inputs from the head and the tail.</a> Curr Biol. 12 (9), 730-734 (2002).</li><li>Hilliard, M. A., Bergamasco, C., Arbucci, S., Plasterk, R. H., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worms+taste+bitter:+ASH+neurons,+QUI-1,+GPA-3+and+ODR-3+mediate+quinine+avoidance+in+Caenorhabditis+elegans.">Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans.</a> EMBO J. 23 (5), 1101-1111 (2004).</li><li>Zariwala, H. A., Miller, A. C., Faumont, S., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step+response+analysis+of+thermotaxis+in+Caenorhabditis+elegans.">Step response analysis of thermotaxis in Caenorhabditis elegans.</a> J Neurosci. 23 (10), 4369-4377 (2003).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc Natl Acad Sci U S A. 70 (3), 817-821 (1973).</li><li>Wicks, S. R., de Vries, C. J., van Luenen, H. G., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHE-3,+a+cytosolic+dynein+heavy+chain,+is+required+for+sensory+cilia+structure+and+function+in+Caenorhabditis+elegans.">CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans.</a> Dev Biol. 221 (2), 295-307 (2000).</li><li>Jansen, G., Weinkove, D., Plasterk, R. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+G-protein+subunit+gpc-1+of+the+nematode+C.+elegans+is+involved+in+taste+adaptation.">The G-protein subunit gpc-1 of the nematode C. elegans is involved in taste adaptation.</a> EMBO J. 21 (5), 986-994 (2002).</li><li>Srinivasan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blend+of+small+molecules+regulates+both+mating+and+development+in+Caenorhabditis+elegans.">A blend of small molecules regulates both mating and development in Caenorhabditis elegans.</a> Nature. 454 (7208), 1115-1118 (2008).</li><li>Choe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascaroside+signaling+is+widely+conserved+among+nematodes.">Ascaroside signaling is widely conserved among nematodes.</a> Curr Biol. 22 (9), 772-780 (2012).</li><li>Ezcurra, M., Tanizawa, Y., Swoboda, P., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+sensitizes+C.+elegans+avoidance+behaviours+through+acute+dopamine+signalling.">Food sensitizes C. elegans avoidance behaviours through acute dopamine signalling.</a> EMBO J. 30 (6), 1110-1122 (2011).</li><li>Ward, A., Liu, J., Feng, Z., Xu, X. Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-sensitive+neurons+and+channels+mediate+phototaxis+in+C.+elegans.">Light-sensitive neurons and channels mediate phototaxis in C. elegans.</a> Nat Neurosci. 11 (8), 916-922 (2008).</li><li>Kunitomo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+memory-dependent+synaptic+plasticity+of+a+taste+circuit+regulates+salt+concentration+chemotaxis+in+Caenorhabditis+elegans.">Concentration memory-dependent synaptic plasticity of a taste circuit regulates salt concentration chemotaxis in Caenorhabditis elegans.</a> Nat Commun. 4, 2210 (2013).</li><li>Snow, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+anterograde+intraflagellar+transport+motors+cooperate+to+build+sensory+cilia+on+C.+elegans+neurons.">Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons.</a> Nat Cell Biol. 6 (11), 1109-1113 (2004).</li><li>Tobin, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+expression+of+TRPV+channel+proteins+defines+their+sensory+functions+and+subcellular+localization+in+C.+elegans+neurons.">Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons.</a> Neuron. 35 (2), 307-318 (2002).</li><li>Waggoner, L. E., Zhou, G. T., Schafer, R. W., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+alternative+behavioral+states+by+serotonin+in+Caenorhabditis+elegans.">Control of alternative behavioral states by serotonin in Caenorhabditis elegans.</a> Neuron. 21 (1), 203-214 (1998).</li><li>Sawin, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetic and cellular analysis of modulated behaviors in Caenorhabditis elegans. , (1996).</li><li>Ramot, D., Johnson, B. E., Berry, T. L., Carnell, L., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parallel+worm+tracker:+A+platform+for+measuring+average+speed+and+drug-induced+paralysis+in+nematodes.">The parallel worm tracker: A platform for measuring average speed and drug-induced paralysis in nematodes.</a> PLoS One. 3 (5), e2208 (2008).</li><li>Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+behavioral+analysis+in+C.+elegans.">High-throughput behavioral analysis in C. elegans.</a> Nat Methods. 8 (7), 592-598 (2011).</li></ol>

The authors have nothing to disclose.

A critical step in the protocol is ensuring that the assay plates have a consistent level of dryness across different experimental days. Different dryness levels will result in different diffusion rates of the solution into the agar and consequently, variations in behavioral outcome. Thus, assay plates should always be made fresh on the afternoon before experiments. The number of worms tested per assay should also be regulated for ease of comparison between treatments. For reference, a wild-type worm lays 4-10 eggs/h on average yielding &#62;360 worms per assay if the worm synchronization protocol above is followed17. If certain mutant strains are egg-laying defective, pick more gravid adult worms for egg-laying to reach the target number of progeny. Another important step in the protocol is to handle worms gently during the washing process and worm placement. Worms are sensitive to mechanical stimuli, which elicit stress responses such as reversals and egg-laying inhibition18. Furthermore, care should be taken to define the ROI accurately and to determine the optimal thresholding constant value for specific lighting conditions before proceeding with the video analysis. It is also recommended that the calibration and thresholding processes be repeated if a lengthy period of time has elapsed since the last experiment was run.
A limitation of this method is that it is not suitable for assaying small worm populations. However, if the appropriate controls to determine the influence of the presence of food on the sensory behavior are performed, then utilizing food to restrict the worms&#39; spatial exploratory location as in the retention assay is also possible with this setup. In addition, this method is not intended for studying stimulus gradient navigation due to the close proximity and small amounts of the control and experimental solution drops used.
In the future, programming software that enables multi-worm tracking and single-worm feature extraction can be integrated into this system19,20. Recording subtle behavior parameters of single worm behavior such as reversal speeds and amplitude of body bends will provide a more detailed picture of an individual worm&#39;s chemosensory behavior in the context of a population-based assay. The assay can also be modified to study habituation by boring holes through the ROI using syringe needles with the appropriate gauge size and filling the holes with agar infused with the experimental compound or control buffer. This will ensure a more consistent surface concentration of the compound over a longer period of recording time as is necessary during habituation studies. Another potential application of this method is to conduct comparative behavior studies across different nematode species. In addition, the behavior chamber can be modified in multiple ways to study behavioral responses to multimodal stimuli. For optogenetic applications, high-intensity LED arrays can be attached next to the camera mount to selectively activate neurons of interest during the assay. Heating elements, cooling systems, and temperature sensors can also be added to the setup to study the effects of temperature on sensory behaviors. Furthermore, odor delivery systems can be installed inside the chamber to investigate interactions between odorsensory and chemosensory modalities.

Foraging animals must integrate inputs from multiple sensory modalities and select appropriate behavioral strategies in order to successfully navigate their environment. Understanding how external sensory inputs are received and transduced into neural information to guide action selection is a central goal in the field of neurobiology. The genetically tractable nematode, C. elegans, is an attractive model organism in which to study the neural mechanisms underlying sensory biology and multimodal integration. Although C. elegans has only 302 neurons, it can detect and discriminate between a wide variety of environmental stimuli including soluble compounds, volatile odorants, and ambient temperature1,2,3,4,5,6,7. The nematode C. elegans relies heavily on its chemosensory apparatus to localize food sources and to alert itself to potential threats. Thus, behavioral assays designed to screen the responses of wild-type and mutant C. elegans to chemical stimuli play a crucial role in dissecting the genetic, cellular, and neural mechanisms underlying C. elegans&#39; remarkable sensory capabilities.
To assay the response to soluble compounds, three types of assays have been described &#8211; the drop test, the chemotaxis assay, and the retention assay. In the drop test, a small drop of the compound is placed at the tail of a moving worm and the worm&#39;s decision to reverse or move forward once the liquid reaches the anterior sensory apparatus is scored4. The drop test requires little experimental preparation and is useful when the sample size of worms is small, as in the case of laser-operated worms. However, as only one worm can be assayed at a time and the experimenter must be present throughout the duration of the assay, the drop test can be time consuming. The drop test is also vulnerable to variations in drop delivery between each worm within a sample, which may influence the overall results of the assay. Another limitation of the drop test is that it can only be used to assay the worm&#39;s response to aversive compounds as it is not possible to discriminate between an attractive or neutral effect of the compound from the worm&#39;s forward movement.
The chemotaxis assay for soluble compounds generally involves dividing an agar plate into four quadrants, with the experimental solution mixed into the agar of two opposing quadrants and the control solution mixed into the other two quadrants8,9. At the start of the assay, a drop of buffer containing worms is placed at the center of the plate and the number of worms in each quadrant is scored at different time points. The chemotaxis assay provides greater statistical power compared to the drop test as large numbers of worms are tested in each assay. However, one limitation of this method is that preparation of the chemotaxis assay plates requires large quantities of the experimental compound. This will make it difficult to conduct large-scale behavior screens if a complicated purification process with limited yields is required to obtain the compound of interest, as in the case of the ascaroside signaling molecules10. In addition, the manual counting of worms throughout the assay is susceptible to errors and the perturbation of the plates during the counting process might affect the results.
Unlike the two aforementioned methods, the retention assay utilizes machine vision, which minimizes error during the scoring process and reduces experimenter&#39;s interference during the assay11. Computerized analysis of video recordings of worm behavior can also potentially reveal subtler behavioral dynamics that will be missed when scoring is only performed at a few discrete time points. In the retention assay, two solutions spots are added on opposite sides of a small circular bacterial food patch followed by the placement of a small number of worms in the middle of the food patch. The worms&#39; behavior is then video recorded, analyzed, and a preference index value is calculated based on the total number of worm pixels in each solution region. Although the presence of an attractive food patch enables smaller populations of worms to be used in each assay, food has previously been shown to sensitize avoidance behaviors to soluble repellants12. Furthermore, worms exhibit a photophobic response to short-wavelength light and the use of microscope light sources that emit white light in the behavior recording setup might affect behavior13.
The purpose of the method discussed in this article is to record and analyze C. elegans&#39; preference for soluble compounds using a population-based assay. To this end, the current method integrates and improves upon aspects from all three of the previously discussed methods. It enables large populations of worms to be tested and requires only small amounts of the experimental solution to be used in each assay. In addition, the assay is conducted within a custom-built enclosed behavior chamber with infrared LED backlighting to minimize the effects of short-wavelength light on behavior. Each chamber can also be outfitted with multiple microscope cameras, which increases experimental throughput without compromising bench space. Finally, video analysis software outputs the preference index value for each video as well as an accompanying worm occupancy plot to visualize population behavior dynamics over time. The chamber setup and assay protocol can be further modified to study multimodal behavior responses such as the effect of odorants or temperature on chemosensory behaviors.
This article describes the construction of the behavior chamber and the assay protocol. It also demonstrates the utility of this method in assaying the response of wild-type worms and chemosensory defective mutants to the known soluble repellant, copper ions4. Finally, the video analysis process using the provided software is detailed.

<table border="0" cellpadding="0" cellspacing="0" width="840">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Aluminum T-slotted framing extrusions</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td style="width:119px;">47065T101</td>
			<td style="width:359px;">Single profile, 1&#34; size, solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brackets</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td>47065T236</td>
			<td>1&#34; long for 1&#34; high single profile extrusions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Compact end-feed fasteners</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td>47065T139</td>
			<td>1&#34; (single), pack of 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Twist-in solid panel holders</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td>47065T251</td>
			<td>For 1&#34; high extrusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic end caps</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td>47065T91</td>
			<td>For 1&#34; high extrusion</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Optically clear cast acrylic sheet</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td style="width:119px;">8560K211</td>
			<td>3/16&#34; thick, 12&#34; x 12&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vinyl-coated polyester fabric</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td style="width:119px;">88505K57</td>
			<td>0.027&#34; thick, 61&#34; width, black</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brass grommets</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td>9604K22</td>
			<td>Trade size 0, 0.545&#34; outer diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Steel washers</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td>90107A029</td>
			<td>1/4&#34; screw size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rounded head screws</td>
			<td style="width:147px;">McMaster-Carr</td>
			<td>90272A546</td>
			<td>1/4&#34;-20 thread size, 1-1/2&#34; long</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Standard operating backlight</td>
			<td style="width:147px;">Smart Vision Lights</td>
			<td style="width:119px;">See local vendor</td>
			<td>8&#34;x8&#34;, infrared 850nm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">IVP-C1 Variable Control Pot</td>
			<td style="width:147px;">Smart Vision Lights</td>
			<td style="width:119px;">See local vendor</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">T1 Power Supply</td>
			<td style="width:147px;">Smart Vision Lights</td>
			<td style="width:119px;">See local vendor</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Dino lite Pro AM4113T</td>
			<td style="width:147px;">Dino-Lite Digital Microscope</td>
			<td style="width:119px;">See local vendor</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">MS09B microscope stand</td>
			<td style="width:147px;">Dino-Lite Digital Microscope</td>
			<td>See local vendor</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated analysis of a nematode population-based chemosensory preference assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

Figure 3A shows the preference index values obtained for different genotype and treatment pairings. A preference index value of 1 indicates strong attraction to the solution placed in the experimental ROI while a preference index value of -1 indicates strong repulsion. A preference index of 0.02 was obtained when the M13 buffer solution was placed in both the control and experimental ROIs, demonstrating that there is no spatial bias towards either ROI. N2 worms strongly avoided the copper ions resulting in a preference index of -0.67, which corroborates previous findings that copper ions are a strong repellant (Supplementary Movie 1)4. osm-3 mutants, which lacks proper formation of the distal segments of sensory cilia, showed a significantly decreased avoidance of copper ions (PI = -0.19)15. ocr-2 mutants, which are defective in many ASH-mediated nociceptive responses including copper avoidance, also exhibit significantly decreased avoidance and even some mild attraction to copper ions (PI = 0.19) (Supplementary Movie 2)16.
Figure 3B shows representative worm occupancy plots, which indicate the density of worms in the control versus the experimental ROIs over time in each video. The darker the color of the plotting area, the greater the number of worm pixels in the ROI. The occupancy plot for N2 worms treated with the M13 buffer control shows that the number of worms in both ROIs remains similar throughout the assay. However, the occupancy plot for N2 worms treated with copper ions indicates that the worms strongly and consistently avoid the experimental ROI throughout the assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55963/55963fig1.jpg" /> 
Figure 1: Photo and Schematic Representation of Behavior Chamber Design. (A) Front view of behavior chamber setup (left) and corresponding schematic representation of chamber frame (right). Opaque polyester sheets enclose the chamber on all faces except the bottom face, which allows light through from the infrared backlight panel. The numbers 1, 2, and 3 correspond to the extrusion layers in (B). (B) Schematic of top view of extrusion layers comprising the behavior chamber frame. This includes the top layer (1), the middle camera mount assembly layer (2), and the bottom stage layer (3). <a href="http://cloudflare.jove.com/files/ftp_upload/55963/55963fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55963/55963fig2.jpg" /> 
Figure 2: Solution-placement and Plate-alignment Templates. (A) The solution-placement template has four quadrants and number alignment markers. The control solution is placed in the upper left and bottom right quadrants while the experimental solution is placed in the upper right and bottom left quadrants. (B) The plate-alignment template has only number alignment markers to minimize occlusion of the worms in the field of view during video recording and analysis. <a href="http://cloudflare.jove.com/files/ftp_upload/55963/55963fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55963/55963fig3.jpg" /> 
Figure 3: Example Data Collected Using this Assay. (A) Preference Index (PI) values for wild-type N2 and mutant C. elegans in response to M13 buffer and 10 mM CuCl2. N2 worms showed no preference between control and experimental ROIs when M13 control solution was placed in both ROIs but strongly avoided the experimental ROI when copper ions were placed in it (PI = 0.02 and -0.67, respectively). osm-3 (p802) mutants and ocr-2 (ak47) mutants showed significantly decreased avoidance of copper ions compared to N2 (PI = -0.1 and 0.2, respectively). N= 6 assays for each genotype-treatment pairing, &#62;360 worms per assay, error bars indicate &#177; 1 S.E.M., ***p &#60;0.001, one-way ANOVA followed by post-hoc Tukey Honestly Significant Difference (HSD) test. (B) Representative worm occupancy plots for N2 worms with M13 buffer in both ROIs (top) and N2 worms with M13 buffer in the control ROI and copper ions in the experimental ROI (bottom). The color scale below each occupancy plot represents the number of worm pixels. <a href="http://cloudflare.jove.com/files/ftp_upload/55963/55963fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Movie 1: Behavioral Response of N2 worms to 10 mM Copper Chloride. Worms are attracted to the control quadrants with M13 buffer (upper left and bottom right) but strongly avoid the quadrants containing copper ions dissolved in M13 buffer (upper right and bottom left). Video was recorded at 1 frame per second and sped up 15x. <a href="http://cloudflare.jove.com/files/ftp_upload/55963/Supplementary_Video_1.mp4">Please click here to download this file.</a>
Supplementary Movie 2: Behavioral Response of ocr-2 (ak47) Worms to 10 mM Copper Chloride. Worms roam to a roughly equal extent in both the control quadrants with M13 buffer (upper left and bottom right) and the quadrants containing copper ions dissolved in M13 buffer (upper right and bottom left) throughout the recording duration. The mutants exhibit a slight preference for the quadrants containing copper ions at the start of the recording. Video was recorded at 1 frame per second and sped up 15x. <a href="http://cloudflare.jove.com/files/ftp_upload/55963/Supplementary_video_2.mp4">Please click here to download this file.</a>
Supplemental File: Solution-placement and Plate-alignment Templates. <a href="http://cloudflare.jove.com/files/ftp_upload/55963/JoVE_template.pptx">Please click here to download this file.</a>

Watch this Scientific Journal Video about Automated Analysis of a Nematode Population-based Chemosensory Preference Assay at JoVE.com

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Research

JoVE Journal

Behavior

Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with ...

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...