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. 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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.

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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>

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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 ...

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JoVE Journal

Behavior

7.4K Views.  California Institute of Technology. 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. 

Video: Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

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 this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

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 test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

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 drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

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 expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

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

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

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

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

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 acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

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 decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

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 incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure 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

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 in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat&#8217;s paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson&#8217;s disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

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

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson&#8217;s Disease.

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 ...

A System for Tracking the Dynamics of Social Preference Behavior in Small Rodents

Exploring the neurobiological mechanisms of social behavior requires behavioral tests that can be applied to animal models in an unbiased and observer-independent manner. Since the beginning of the millennium, the three-chamber test has been widely used as a standard paradigm to evaluate sociability (social preference) and social novelty preference in small rodents. However, this test suffers from multiple limitations, including its dependence on spatial navigation and negligence of behavioral dynamics. Presented and validated here is a novel experimental system that offers an alternative to the three-chamber test, while also solving some of its caveats. The system requires a simple and affordable experimental apparatus and publicly available open-source analysis system, which automatically measures and analyzes multiple behavioral parameters at individual and population levels. It allows detailed analysis of the behavioral dynamics of small rodents during any social discrimination test. We demonstrate the efficiency of the system in analyzing the dynamics of social behavior during the social preference and social novelty preference tests as performed by adult male mice and rats. Moreover, we validate the ability of the system to reveal modified dynamics of social behavior in rodents following manipulations such as whisker trimming. Thus, the system allows for rigorous investigation of social behavior and dynamics in small rodent models and supports more accurate comparisons between strains, conditions, and treatments.

Described here is a novel automated experimental system that offers an alternative to the three-chamber test and also solves several caveats. This system supplies multiple behavioral parameters that enable rigorous analysis of small rodent behavioral dynamics during the social preference and social novelty preference tests.