The Citrus Research and Development Foundation is acknowledged for providing funding. Thanks to Angel Hoyte and Michael Flores for conducting bioassays.

<ol>
	<li>Assembly of a custom-designed, two-port divided T-olfactometer previously described in Mann et al.10 is shown in Figure 1. The olfactometer consists of a 30 cm glass tube that is bifurcated into two separate ports with a Teflon strip forming a T-maze. Each half is analogous to an arm of a 2-choice style olfactometer enabling the test subject to choose between two potential odor fields.
	<ol>
		<li>Assembly and subsequent assays should take place within a temperature-controlled room or environmental chamber.</li>
		<li>Mount glass T-maze olfactometer vertically as shown in Figure 1 and position it under a fluorescent 900 lux light bulb.</li>
		<li>For optimal light diffusion, the assembly should be mounted within a 1.0 x 0.6 x 0.6 m opaque container to maximize uniform light diffusion.</li>
		<li>Connect the olfactometer arms to odor sources that will be housed in solid-phase micro-extraction holding/odor source chamber (SPMEC) (ARS, Inc., Gainesville, FL) through Teflon;-glass tube connectors. Each SPMEC is comprised of a straight glass tube (17.5 cm long x 3.5 cm wide) supported with an inlet and outlet valve for incoming and outgoing air streams (Figure 1).</li>
		<li>Connect the air delivery system to an external source of humidified and carbon-purified air that is under constant pressure (the air delivery system has both an internal carbon filter for air purification and an air bubbler for humidification if external sources are unavailable).</li>
		<li>Dissolve desired chemical samples within appropriate diluent (solvent) and pipette onto a slow release substrate such as a cotton wick (Petty John Packaging, Inc., Concord, NC). The control treatment should consist of a cotton wick impregnated with solvent only.</li>
		<li>To prevent significant contamination of the glassware, envelop the treated and control cotton wicks in laboratory tissue (Kimwipes, Kimberly-Clark, Roswell, GA) and place into the two SPMEC arms (ARS, Inc., Gainesville, FL). In the case of live plant samples, a guillotine volatile collection chamber can be attached to the olfactometer containing treatments in place of the SPMEC (Figure 1).</li>
		<li>Deliver purified and humidified air through the SPMEC (or guillotine volatile chamber) via two pumps connected to an air delivery system (Figure 1). Maintain airflow rate at 0.1 L/min through both arms of the olfactometer.</li>
	</ol>
	</li>
	<li>Employing olfactometer to test arthropod response to putative repellents. In the present description, a hemipteran insect, Asian citrus psyllid, Diaphorina citri, was used as a test arthropod. Response was tested to citrus volatiles alone or in combination with a repellent (dimethyl disulfide) and clean air as odor sources.
	<ol>
		<li>Conduct all experiments at a standardized temperature and relative humidity.</li>
		<li>Prior to all tests, expose test subjects to clean air versus clean air or solvent versus clean air in the T-maze olfactometer to verify the absence of positional bias or an effect of solvent on behavior of the test subject.</li>
		<li>Assign an odor source randomly to one of the arms of the olfactometer at the beginning of each bioassay. Reverse this position after every 10 test subjects assayed, at minimum, to eliminate potential of positional bias.</li>
		<li>Test the response of a minimum of 30 (and up to 120) test subjects per treatment combination.</li>
		<li>Allow test subjects 100-300 sec to exhibit a behavioral response based on previously established response (latency) interval.</li>
		<li>Record if subject enters the treatment arm, control arm or remains in the release port or below the T-maze division.</li>
		<li>Score a treatment or control arm choice when a test subject crosses the division in the T-maze and moves into either olfactometer arm.</li>
		<li>Score a release arm choice when a test subject remains in the release port or below the T-maze division.</li>
		<li>Clean the olfactometer and connecting tubes thoroughly with 2% soap solution and bake the glass components at 200 &deg;F between the use of different odor treatments.</li>
		<li>For assays in which putative repellent treatments are presented in the T-maze olfactometer with or without a chemical treatment versus clean air, compare the number of test subjects remaining at the release point and not entering the olfactometer between treatments by one way analysis of variance (ANOVA) followed by Tukey's HSD test (&alpha; &lt; 0.05). In cases in which the test subjects leave the release arm, compare the number choosing the control arm versus the treatment arm with Chi square (&chi;2) analysis at &alpha; &lt; 0.05.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Resh, R. H., Card&#233;, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Insects. , (2003).</li><li>Aluja, M., Lesky, T. C., Vincent, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biorational tree-fruit pest management. , (2009).</li><li>Rodriquez-Saona, C., Stelinski, L. L., Peshin, R., Dhawan, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior-modifying+strategies+in+IPM:+Theory+and+Practice.">Behavior-modifying strategies in IPM: Theory and Practice.</a> Integrated Pest Management: Innovation - Development Process. , 261-311 (2009).</li><li>Card&#233;, R. T., Millar, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in insect chemical ecology. , (2004).</li><li>Knight, A. L., Stelinski, L. L., Hebert, V., Gut, L., Light, D., Brunner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+dispensers+simultaneously+releasing+pear+ester+and+sex+pheromone+for+disruption+of+codling+moth+(Lepidoptera:+Tortricidae).">Novel dispensers simultaneously releasing pear ester and sex pheromone for disruption of codling moth (Lepidoptera: Tortricidae).</a> Journal of Applied Entomology. 136 (1-2), 79-86 (2012).</li><li>Mann, R. S., Tiwari, S., Smoot, J. M., Rouseff, R. L., Stelinski, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repellency+and+toxicity+of+plant-based+essential+oils+and+their+constituents+against+Diaphorina+citri+Kuwayama+(Hemiptera:+Psyllidae).">Repellency and toxicity of plant-based essential oils and their constituents against Diaphorina citri Kuwayama (Hemiptera: Psyllidae).</a> Journal of Applied Entomology. 136 (1-2), 87-96 (2012).</li><li>Forbes, A. A., Powell, H. Q., Stelinski, L. L., Smith, J. J., Feder, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+sympatric+speciation+across+trophic+levels.">Sequential sympatric speciation across trophic levels.</a> Science. 323 (5915), 776-779 (2009).</li><li>Kennedy, J. 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+visual+responses+of+flying+mosquitos.">The visual responses of flying mosquitos.</a> Proceedings of the Zoological Society of London A. 109 (4), 221-242 (1940).</li><li>Miller, J. R., Roelofs, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained-flight+tunnel+for+measuring+insect+responses+to+wind-borne+sex+pheromones.">Sustained-flight tunnel for measuring insect responses to wind-borne sex pheromones.</a> Journal of Chemical Ecology. 4 (2), 187-198 (1978).</li><li>Mann, R. S., Rouseff, R. L., Smoot, J. M., Castle, W. S., Stelinski, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfur+volatiles+from+Allium+spp.+affect+Asian+citrus+psyllid,+Diaphorina+citri+Kuwayama+(Hemiptera:+Psyllidae),+response+to+citrus+volatiles.">Sulfur volatiles from Allium spp. affect Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), response to citrus volatiles.</a> Bulletin of Entomological Research. 101 (1), 89-97 (2011).</li><li>Onagbola, E. O., Rouseff, R. L., Smoot, J. M., Stelinski, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guava+leaf+volatiles+and+dimethyl+disulfide+inhibit+response+of+Diaphorina+citri+Kuwayama+to+host+plant+volatiles.">Guava leaf volatiles and dimethyl disulfide inhibit response of Diaphorina citri Kuwayama to host plant volatiles.</a> Journal of Applied Entomology. 135 (6), 404-414 (2011).</li><li>Baker, T. C., Linn, C. E., Hummel, H., Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wind+tunnels+in+pheromone+research.">Wind tunnels in pheromone research.</a> Techniques in Pheromone Research. , 75-110 (1984).</li><li>Stelinski, L. L., Rodriguez-Saona, C., Meyer, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+foreign+oviposition+marking+pheromone+in+a+multitrophic+context.">Recognition of foreign oviposition marking pheromone in a multitrophic context.</a> Naturwissenschaften. 96 (5), 585-592 (2009).</li><li>G&#246;k&#231;e, A., Stelinski, L. L., Whalon, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+electrophysiological+responses+of+leafroller+moths+to+selected+plant+extracts.">Behavioral and electrophysiological responses of leafroller moths to selected plant extracts.</a> Environmental Entomology. 34 (6), 1426-1432 (2005).</li><li>Ali, J. G., Alborn, H. 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The author received funding that was provided by the Citrus Research and Development Foundation.

Herein, an assay device and protocol are described for measuring the response of small arthropods (Insecta: Hemiptera: Psyllidae) to odorants. The method involves a choice test, allowing the insect to make a choice between two odorant fields in the case of assays evaluating a putative attractant. Furthermore, the test subject may display three types of behaviors by leaving the release arm and entering either one of two potential odor fields, or remaining in the release arm, in the case of repellency assays. The olfactometer allows for high throughput data collection because it takes advantage of the negative geotactic (tendency to move upward) and positive phototactic (tendency to move toward light) natural behavioral response of many arthropods. Although the present demonstration uses a psyllid insect test example, the assay can be readily adapted to arthropods broadly, both those that use flight or walking as predominant modes of transportation.
Olfactometers designed for measuring the response of insects were initially developed decades ago and have played a major role in elucidating insect attractants and repellents12. The specific designs of such olfactometers vary widely; however, variations on the general theme of the two-choice assay described here, as well as, similar Y-tube assays have been often used to measure arthropod response to chemicals. Larger flight tunnels that may cause sustained flight of insects9 have also resulted in collection of groundbreaking data elucidating the fundamental mechanisms of insect flight and orientation to semiochemicals, as well as, data informing practical use of pest control tools.
It is often necessary to tailor design an olfactometer and associated instrumentation for the biology of the specific arthropod test subject. Those olfactometers that can be used among a general group of insects are more useful than ones specific to a small group; however, sometimes the economic significance of a small group of insects dictates the need for development of a very specific olfactometer and assay technique. The currently described design builds on previously familiar arthropod techniques. It allows for a more standard two-choice assay than Y-tube bioassays in which the experimental arena or choice test takes the form of a Y-shaped glass device7. Typically, one arm of such a Y-tube will receive a treatment odorant, while the other will be left blank13. Variations on such olfactometers may include addition of multiple radiating arms14 and even addition of a soil medium for assaying the behavior of organisms moving through soil15. In developing such olfactometers and associated assays, it is important to consider how closely natural conditions are replicated and thus the true relevance of the assessed behavioral responses in terms of the test subjects' biology. To a large degree, the data collected will only be as useful as the relevance of the behavioral bioassay with respect to the organism's behavioral ecology16.
The currently described olfactometer and behavioral bioassay is specifically designed for a hemipteran insect that tends to initiate flight as short "jumps"16. The vertical orientation of the olfactometer and the light source arrangement facilitate initiation of insect movement and thus subsequent chemically-mediated orientation behavior can be assayed efficiently and with high throughput. This behavioral assay arrangement can likely be used for a broad array of flying or walking arthropod taxa or could be also easily modified to fit the need of non-arthropod organisms.

Arthropods (including insects and arachnids) are often defined as pests, because they either compete with human consumption in agricultural or urban settings or transmit causal pathogens of disease1. A large amount of investment is made into development of control tools for such pests. These tools can vary widely and significant progress has been made in developing biorational tools that either modify or exploit the natural behavior of pests with the use of behavioral attractants or repellents2,3.
Arthropod pests often rely on olfactory signals or cues for mate and/or host location4. Therefore discovery of effective attractants or repellents for pests can often lead to development of control tools. This process of discovery involves several steps. First, the natural behavior of the test subject must be understood. For example, one must ascertain whether one sex of a given species attracts the opposite sex for mate location. If so, one must determine whether this attraction is mediated by a chemical and where that chemical is produced. Further steps include determining when the chemical is produced and its eventual identification. Throughout this procedure, the test subject's (insect or mite) behavioral response to odorants must be tested. Initially, the behavior of the test subject will be tested to the natural material. For example, a male moth's response may be tested to that of the extract of its female conspecific's pheromone gland. Alternatively, an herbivore's response to the natural odors of its host plant may be tested. While these investigations often have a practical focus on development of management tools5,6, they can also be applied to research that addresses more fundamental questions, such as speciation7. Subsequent steps involve testing the arthropod's response to synthetic versions of identified natural products.
Understanding how arthropods respond to chemicals requires the development of appropriate tools to measure their behavior. The data generated by such tools will depend on how well the investigator is able to measure natural behaviors in a controlled setting. Over six decades ago, sustained flight olfactometers and tunnels were developed to measure insect response to odorants8,9. Olfactometers vary significantly in design and are often tailored to the specific insect or problem being investigated. The goal of this article is to describe the design and use of a vertically oriented, T-maze olfactometer for measuring insect response to odorants (Figure 1). The olfactometer's vertical orientation takes advantage of the negative geotaxis (tendency to move up) exhibited by many insects. A light source positioned directly above the olfactometer takes advantage of the positive phototaxis (tendency to move toward source of light) exhibited by many insects. The olfactometer is particularly useful for hemipteran insects that tend not to exhibit movement readily in horizontally-oriented olfactometers and that are heavily influenced by light intensity with respect to orientation. However, this device can be used for a wide range of flying and crawling insects, as well as arachnids. The olfactometer can be employed to evaluate both attractants and repellents. In the case of attractants, the bifurcated odor field (Figure 1) is established with the putative attractant released on one side versus clean air released on the opposite side. A choice is recorded when the insect released at the base of the olfactometer chooses one of the two odor fields. In the case of repellents, a third behavior is possible. In addition to upward movement into either of the two odor fields, lack of upward movement may be recorded indicating a non-responsive stimulus or repelling stimulus from either odor fields.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Equipment</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Two port divided T-olfactometer</td>
 <td>Analytical Research Systems (ARS), Inc. Gainesville, FL</td>
 <td>None(Custom made)</td>
 <td>If unavailable from ARS, Southern Scientific (Gainesville, FL) also currently builds and distributes such equipment.</td>
 </tr>
 <tr>
 <td>Solid-phase micro-extraction holding/odor source chamber with Teflon;-glass tube connectors.</td>
 <td>Analytical Research Systems (ARS), Inc. Gainesville, FL</td>
 <td>RV-R3</td>
 <td>See above comment</td>
 </tr>
 <tr>
 <td>Air delivery system</td>
 <td>Analytical Research Systems (ARS), Inc. Gainesville, FL</td>
 <td>HADS-2AFM2C.4</td>
 <td>See above comment</td>
 </tr>
 <tr>
 <td>Guillotine chamber</td>
 <td>Analytical Research Systems (ARS), Inc. Gainesville, FL</td>
 <td>L3GP3</td>
 <td>See above comment</td>
 </tr>
 </tbody>
</table>

vertical t-maze choice assay for arthropod response to odorants

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.

Attraction of Asian citrus psyllid (Diaphorina citri) adults to the odors of their natal host plant volatiles (citrus) is depicted in Figure 2A. Significantly (&alpha; &lt; 0.05) more adults chose the arm of the T-maze olfactometer receiving odors from living citrus plants as compared with a blank (clean air) control.
An example of repulsion is shown in Figure 2B. In this case, the insects were exposed to one arm of the T-maze receiving citrus volatiles, while the second arm received volatiles from a citrus plant that was treated with the known repellent, dimethyl disulfide11. In this case, three types of behaviors were observed. The number of psyllids arrested at the release arm and not responding did not differ statistically from the number entering the arm receiving the odors from citrus plants alone (Figure 2B). However, no psyllids entered the arm containing the host plant volatile when co-presented with the repellent (Figure 2B).
<img alt="Figure 1" src="/files/ftp_upload/50229/50229fig1.jpg" /> 
 Figure 1. Schematic diagram of vertically oriented T-maze olfactometer joined to air delivery system and odor treatment release devices. The diagram is an adaptation of Figure 1 in Mann et al.10. A test subject is placed individually into the release chamber of the 2-port divided T-olfactometer. It moves towards the odors through a gate valve which allows controlled upward movement into the olfactometer and prevents the insect from crawling back into the release point area. The tube bifurcates 10 cm after the gate value. A positive response to an odor is recorded once the insect moves 0.5 cm into a specific side of the division. The guillotine volatile collection chamber may be used in place of a solid-phase micro-extraction holding/odor source chamber (SPMEC) as an odor source.
<img alt="Figure 2" fo:content-width="3.5in" fo:src="/files/ftp_upload/50229/50229fig2highres.jpg" src="/files/ftp_upload/50229/50229fig2.jpg" /> 
 Figure 2. Response of Asian citrus psyllid (Diaphorina citri) adults to volatiles from citrus host plants versus clean air control (A) or citrus volatiles versus citrus volatiles co-released with a repellent (dimethyl disulfide) (B) in a vertically oriented T-maze olfactometer. Figure 2A: One-way analysis of variance (ANOVA) followed by Tukey's HSD test (&alpha; &lt; 0.05) was performed to compare the number of psyllid making a choice between either arm (2 treatments, n = 120). Columns indicated by different letters are significantly different from one another (&alpha; &lt; 0.05). Figure 2B: One-way analysis of variance (ANOVA) followed by Tukey's HSD test (&alpha; &lt; 0.05) was performed to compare the number of psyllid making a choice between either arm or remaining in the release point (3 treatments, n = 120). Columns indicated by different letters are significantly different from one another (&alpha; &lt; 0.05).

Watch this Scientific Journal Video about Vertical T-maze Choice Assay for Arthropod Response to Odorants at JoVE.com

Vertical T-maze Choice Assay for Arthropod Response to Odorants

A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

vertical-t-maze-choice-assay-for-arthropod-response-to-odorants

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14.6K Views.  University of Florida. A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Video: Vertical T-maze Choice Assay for Arthropod Response to Odorants

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...