We thank Professor Li Xiangdong and photographer Mr. Cheng Jing for helpful discussions and suggestions. This work was supported by the Exploratory Project (20200101) of the Life Science Experimental Teaching Demonstration Center of China Agricultural University.

1. Construction of the observing/documenting system
NOTE: The materials needed to construct the fly behavior observing/documenting system are shown in Figure 1, and the completed system is shown in Figure 2. The protocol to construct the system and how to use it are described below.
<ol>
	<li>Make a fly behavior observation chamber (FBOC).
	<ol>
		<li>Obtain a small, translucent (not transparent) container to make an FBOC of size diameter (&#216;) 15 mm x 20 mm. Use a translucent plastic bottle cap of size ~&#216;17 mm x 22 mm to make an FBOC, or cut a section of ~17 mm from the thick end of a 5 mL pipette tip to make an FBOC.</li>
		<li>Pour 1% agar into the FBOC to adjust its depth. If food needs to be placed in the FBOC (see for the preparation method of the food), pour the agar to obtain a depth of 12 mm. If food does not need to be placed in the FBOC, pour the agar to a depth of 5 mm to track the whereabouts of fruit flies more easily.</li>
		<li>If using a pipette tip to make an FBOC, place the cut-off pipette tip section in a &#216;35 mm or a &#216;60 mm Petri dish. Pour the 1% agar gel into the Petri dish to a thickness of ~5 mm, wait for the agar to solidify, and seal the bottom of the FBOC. Then, pour the agar gel into the FBOC to the desired thickness.</li>
	</ol>
	</li>
	<li>Make an FBOC base by boring a ~10 mm-deep hole in the center of a piece of a 60 mm x 60 mm x 15 mm ethylamine foam sheet with the same diameter as the FBOC. Insert the FBOC into the hole. 
	NOTE: The FBOC base keeps the FBOC stable and prevents it from tipping over and facilitates the movement of the FBOC to track the flies during observation and videography.</li>
	<li>Make fly food, if necessary, with yeast medium2, artificial diet3, or pure sucrose/glucose (use 1% agar as gelling agent), depending on the purpose of observation.
	<ol>
		<li>To visually determine whether flies are feeding and increase the contrast between flies and their environment, add food dyes (see the Table of Materials) to the food to a final concentration of 12.5 mg/100mL. 
		NOTE: The abdomens of the flies change color immediately after feeding.</li>
		<li>Pour the prepared food into a Petri dish to a height of 8 mm. After solidification, use a razor blade to cut out a piece of food of size 8 mm x 4 mm x 8 mm, and place it on a piece of plastic (such as candy wrapper).</li>
		<li>Cut the food into a quadrangular pyramid or quadrangular frustum pyramid, as shown in Figure 3, to allow observation and recording of fly behavior from different angles as the flies land on the food randomly. Use the plastic under the food is to prevent the dye in the food from diffusing into the agar in the FBOC. Use tweezers to place the food in the center of the FBOC.</li>
	</ol>
	</li>
	<li>In some behavioral observations, ensure that the flies are starved in advance. Pour 1% agar gel (1 g agar/100 mL water, 600 &#181;L of propionic acid) into a clean empty bottle to a thickness of 1-2 cm, and place at room temperature for 1-2 h. Transfer flies to the bottle and place at 25 &#176;C for &#8805;36 h. 
	NOTE: Flies can absorb water from the agar gel, so there is no need to add water from time to time4,5.</li>
	<li>Transfer one or more flies into the FBOC using an aspirator. If using an aspirator is difficult, chill and inactivate the flies in crushed ice, sort them on an ice pack, and transfer them to the FBOC as described previously6. 
	NOTE: The use of freezing greatly facilitates the transfer of flies; the chilled flies can regain consciousness within 1 min, much faster than those anesthetized with CO2. Although chilling could have detrimental effects on the behavior of flies, e.g., increased copulation latency of flies from 5 min7 to 40 min8, it does not change fly behavior (such as courtship behavior). Hence, the chilling method may be used to transfer flies for general observation (such as teaching experiments) and videography. However, if the observations are to be used in a scientific report, it is strongly recommended to not expose the flies to any anesthesia.</li>
	<li>After transferring the flies to the FBOC, cover it with a 30-40 mm UV/clear filter for the camera to form an FBOC complex (Figure 4). Place the FBOC complex under the stereomicroscope for observation. 
	NOTE: To obtain clear and sharp images, it is strongly recommended to use high-quality UV/clear filter with high light transmittance (&#62;98%) and reduced flare. Refer to some suggestions described previously9,10; although there is no need to buy expensive filters, avoid covering the FBOC with glass such as the lid of a Petri dish.</li>
	<li>Illuminate the FBOC. Mount mini LED video lights to flash hot shoe mount stands and place them on the left and right sides of the FBOC (Figure 2). Turn on the LED lights, and set the brightness to 100% and the color temperature to 5000-5600 K. 
	NOTE: The mini LED video lights with dimmable light, 5600 K color temperature can provide uniform, bright, nearly shadow-free illumination. Using the top light source that comes with the stereo microscope, the LED Ring Light illuminator, or fiber optic illuminator did not yield satisfactory results. It is best to use continuous power supply (transformers) for LED video lights.</li>
	<li>Observation and videography of fly behavior
	<ol>
		<li>Turn on the LED video lights, and adjust the stereo zoom microscope until the edge of the FBOC can be clearly seen with the naked eye. Move the FBOC to the center of the field of view.</li>
		<li>Attach the clamp of the universal telescope digital camera adapter to an eyepiece of the stereo microscope, and then attach a compact digital camera to the adapter securely by alternately turning the camera mounting screw and camera fixing screw (Figure 2).</li>
		<li>Turn on the digital camera, and turn the horizontal/vertical fine-tuning knobs until the FBOC edge clearly appears in the center of the bright circular field of view on the camera&#39;s LCD screen. Rotate the mode dial to Aperture-priority auto Mode, press focus on the multi selector, choose Macro close-up, and then press the OK button. Move the zoom switch from the wide-angle end to the telephoto end, and zoom into the circular image until its central portion fills the full LCD screen.&#160;Press the Movie-record button to start recording (press the button again to end recording). 
		NOTE: If the image is too dark or too bright, press the side of the control dial close to the exposure compensation icon (Figure 1), and rotate the dial to alter the exposure value (EV) suggested by the camera to achieve the desired effect. Positive EVs make the image brighter, and negative EVs make the image darker. The image must be uniform, bright, without vignetting.</li>
		<li>Turn the focus knob of the microscope until the flies in the FBOC are clearly visible. Choose the fly behavior of interest for observation or video recording. Turn the zoom knob to zoom in and out to achieve the desired magnification for observation or video recording. 
		&#8203;NOTE: This method of taking images under the microscope through the eyepieces is applicable to any microscope with eyepieces. To take photographs of experimental results, use a camera that can shoot in RAW format, as RAW image files are preferable to JPEGs. Use the camera&#39;s LCD screen as a display to observe the behavior of fruit flies, and ensure that the stereo zoom microscope has at least 5-50x zoom.</li>
	</ol>
	</li>
</ol>
2. Protocols for observation and videography of fly behavior
<ol>
	<li>Preparing the flies
	<ol>
		<li>Culture the flies on cornmeal medium at 25 &#176;C with 60% humidity and a 12 h light/dark cycle. Collect flies within 6 days of hatching for observation (except courtship and fighting behavior). 
		NOTE: Here, the medium was composed of 1000 mL of water, 105 g of cornmeal flour, 75 g of sucrose, 15 g of agar, 40 g of yeast powder, 28 mL of 10% methyl paraben (w/v in 95% ethanol), and 6.25 mL of propionic acid.</li>
	</ol>
	</li>
	<li>Regaining consciousness from anesthesia by chilling
	<ol>
		<li>Chill the flies as described previously.6 Transfer the Drosophila from the ice box to the FBOC using tweezers. Record the fly&#39;s process from inactivity to normal posture on video.</li>
	</ol>
	</li>
	<li>Fly sleep, feeding, excretion, and social behavior
	<ol>
		<li>Starve flies for 36 h. Transfer 4-6 fruit flies to the FBOC with stained food. Observe and record fly behavior on video. 
		NOTE: Flies that remain motionless for more than 5 min display sleep behavior11. Drosophila can sleep on food or on a vertical FBOC wall (the body is perpendicular to the observation chamber wall). Although the body does not move when sleeping, the abdomen can be seen to be undulating. Feeding behavior is manifested when the fly stretches out its proboscis, moves on the food while constantly sucking, and its abdomen turns blue. During group feeding or other activities, fruit flies stretch their feet to touch the bodies of other fruit flies in a friendly manner. This is a social behavior.</li>
	</ol>
	</li>
	<li>Fly grooming behavior
	<ol>
		<li>Chill the flies as described6. Throw the frozen flies into agar powder, and roll to cover them with agar powder. Transfer the flies to the FBOC. Observe and record grooming behavior. 
		NOTE: When the fruit fly regains consciousness from the freezing, it will quickly shake off the agar powder from its body and clean every part of its body with its legs12,13. Grooming behavior can also be seen during feeding, courtship, and other behaviors.</li>
	</ol>
	</li>
	<li>Fly courtship and fighting behavior
	<ol>
		<li>Collect female and male flies as described previously7. To observe the courtship behavior of flies, place a female fly and a male fly into the FBOC to observe and record 6 courtship (successful and failed) behaviors.</li>
		<li>To observe the fighting behavior of flies, place two males in the FBOC. Observe and record their behavior of pushing and shoving each other.</li>
	</ol>
	</li>
	<li>Fly egg-laying behavior
	<ol>
		<li>Prepare female flies as described previously5. Transfer 4 female flies into FBOC with food.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Chyb, S., Gompel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of Drosophila morphology: Wild-type and classical mutants. , (2013).</li><li>Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carnivory+in+the+larvae+of+Drosophila+melanogaster+and+other+Drosophila+species.">Carnivory in the larvae of Drosophila melanogaster and other Drosophila species.</a> Scientific Reports. 8, 15484 (2018).</li><li>Piper, M. D., 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+holidic+medium+for+Drosophila+melanogaster.">A holidic medium for Drosophila melanogaster.</a> Nature Methods. 11, 100-105 (2014).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> Journal of visualized experiments : JoVE. (3), e193 (2007).</li><li>Yang, C. -. H., Belawat, P., Hafen, E., Jan, L. Y., Jan, Y. -. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+egg-laying+site+selection+as+a+system+to+study+simple+decision-making+processes.">Drosophila egg-laying site selection as a system to study simple decision-making processes.</a> Science. 319 (5870), 1679-1683 (2008).</li><li>Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+homemade+tools+to+handle+fruit+flies-Drosophila+melanogaster.">Simple homemade tools to handle fruit flies-Drosophila melanogaster.</a> Journal Of Visualized Experiments: JoVE. (149), e59613 (2019).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> Journal of Visualized Experiments: JoVE. (61), e3795 (2012).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetizing+Drosophila+for+behavioural+studies.">Anesthetizing Drosophila for behavioural studies.</a> Journal of Insect Physiology. 46 (4), 439-442 (2000).</li><li>. My not quite complete protective filter article Available from: <a target="_blank" href="https://www.lensrentals.com/blog/2017/06/the-comprehensive-ranking-of-the-major-uv-filters-on-the-market">https://www.lensrentals.com/blog/2017/06/the-comprehensive-ranking-of-the-major-uv-filters-on-the-market</a> (2017)</li><li>. UV and clear lens protection filters review Available from: <a target="_blank" href="https://www.the-digital-picture.com/Reviews/UV-and-Clear-Lens-Protection-Filters.aspx">https://www.the-digital-picture.com/Reviews/UV-and-Clear-Lens-Protection-Filters.aspx</a> (2013)</li><li>Shaw, P. J., Cirelli, C., Greenspan, R. J., Tononi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlates+of+sleep+and+waking+in+Drosophila+melanogaster.">Correlates of sleep and waking in Drosophila melanogaster.</a> Science. 287 (5459), 1834-1837 (2000).</li><li>Barradale, F., Sinha, K., Lebestky, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Drosophila+grooming+behavior.">Quantification of Drosophila grooming behavior.</a> Journal of Visualized Experiments: JoVE. 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The authors have nothing to disclose.

Light is at the very heart of photography and videography and is the decisive factor for obtaining high-quality images16. Here, two LED video lights with adjustable brightness and color temperature were used as illuminators, and a translucent material was selected to make the FBOC. The LED light panels on both sides provided enough brightness, and the translucent material softened and scattered light, eventually producing uniform, soft, and bright light to illuminate the flies in the FBOC, without...

Drosophila melanogaster is an outstanding model in biological research; however, it is a bad model for photography or videography, as it is too small for a camera or a camcorder and too large for a compound microscope1. Despite excellent research described in the literature, most studies have only provided blurred, unclear images, rather than clear and sharp photographs with clear detail that illustrate the fly behavior being described. Moreover, although fly behaviors have been extensively studied (e.g., courtship and fighting), most of these papers have used illustrations to explain their research to readers.
This paper describes a simple and economical approach. Using this method, not only the various behaviors of Drosophila can be observed, but also all the details that can be observed under a stereo zoom microscope can be recorded clearly and sharply. Of course, this method can also be used to record the morphology of flies, as when they enter a sleep or semi-sleep state, the stationary model allows the user to take a photograph or a stack of photographs with different focal planes to get an extended depth of field photo. These methods can be realized without complicated technology and expensive equipment or even superb manual skills.
The video component of this article shows videos of several typical behaviors of flies. The purpose of showing these videos is twofold: one is to let audiences know what can be captured and present the image quality obtained by using this method; the other is to let new students who are interested in Drosophila, but thus far have not had the opportunity to actually observe the behavior of flies understand the behavior of flies (such as courtship, fighting) through these clear videos rather than illustrations or blurred images.
Supplemental Video: Fruit Fly Behavior: <a href="https://www.jove.com/files/ftp_upload/62533/fly behavior.mp4" target="_blank">Please click here to download this file.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>compact camera, Nikon P310</td>
			<td>Nikon</td>
			<td></td>
			<td>3-5x optical zoom, cam record 1080 P HD video</td>
		</tr>
		<tr>
			<td>ethylamine foam</td>
			<td></td>
			<td></td>
			<td>60 mm x 60 mm x 15 mm</td>
		</tr>
		<tr>
			<td>Food Blue No 1</td>
			<td></td>
			<td>CAS 3844-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>mini LED lights and transformer</td>
			<td>GODOX</td>
			<td>LED-P120</td>
			<td>have 5000-5600 K color temperature</td>
		</tr>
		<tr>
			<td>small container (e.g. bottle cap)</td>
			<td></td>
			<td></td>
			<td>about &#216; 15 mm x 20 mm</td>
		</tr>
		<tr>
			<td>UV / Clear filter</td>
			<td></td>
			<td></td>
			<td>high-quality UV/Clear filter with high transmittance, 30-40 mm</td>
		</tr>
		<tr>
			<td>zoom stereo microscope</td>
			<td></td>
			<td></td>
			<td>5-50x zoom</td>
		</tr>
	</tbody>
</table>

an adjustable high-definition imaging system for behavioral studies of drosophila adults

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but effective method to observe and document the behavior or morphology of flies. Flies were placed in a translucent observation chamber c.a. &#216;15 x 5mm (no food inside) or &#216;15 x 12 mm (with an 8 mm-high piece of food inside). After covering with an ultraviolet (UV)/clear filter with high light transmittance, the chamber was placed under a 5-50x zoom stereo microscope, and mini light-emitting diode (LED) video lights were placed on both sides of the microscope to illuminate the chamber to obtain uniform, soft, bright, and nearly shadow-free light. Then, a compact digital camera with 3-5x optical zoom, which can record 1080 P high-definition or higher resolution video (at a frame rate of &#8805;30 fps), was connected to the eyepiece of microscope through a bracket, and photographs or videos were taken through the eyepiece. By adjusting the zoom knob of the zoom stereo microscope, it was very easy to track the flies and take panoramic or detailed close-up images as needed, while the camera recorded everything under the microscope. Because the flies can stay at any position in the chamber, they can be observed and recorded from all directions. The photographs or videos taken are of good image quality. This method can be used both for scientific research and teaching.

Shoot through a UV filter for clear and sharp images 
Perform a simple experiment to observe the difference between a UV filter and ordinary glass in the laboratory. Take a fly culture vial, remove the stopper, place it under a stereo dissecting microscope, and cover it (alternately) with a UV filter and a Petri dish lid. The photographs taken in these two cases are shown in Figure 5. The photograph taken through the UV filter is clear and sharp, very similar to the pho...

Watch this Scientific Journal Video about An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults at JoVE.com

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but ...

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JoVE Journal

Behavior

2.5K Views.  China Agricultural University. This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

Video: An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

Using a Real-Time Locating System to Measure Walking Activity Associated with Wandering Behaviors Among Institutionalized Older Adults

A real-time locating system (RTLS) can be used to track the walking activity of institutionalized older adults in long-term care who are at risk for wandering behaviors. The benefits of a RTLS are objective and continuous measurements of activity. Self-report methods of activity, especially wandering, by health care staff are vulnerable to floor effects and recall bias, and continuous clinical or research observation over the long-term can be time-consuming and expensive. Health care staff also fail to recognize the onset and/or duration of wandering behaviors, which are associated with a variety of adverse health outcomes in this population but amenable to intervention. RTLS technologies can measure the walking activity of institutionalized residents with cognitive impairment over time with a high degree of accuracy. This is particularly useful for the study of wandering, defined as walking for at least 60 seconds with few (if any) breaks in activity. Wandering is associated with disease progression, hospitalizations, falls and death. Previous work suggests older adults with poor balance ability and high sustained walking activity may be particularly susceptible to poor health outcomes. RTLS&#39;s are used to assess cognitive impairment and factors associated with gait and balance; however, supplemental paper and pencil gait/balance tools may be used to further refine risk profiles. This project discusses the use of a RTLS to measure walking activity and also gait quality and balance ability measures on this population.

This paper discusses the use of a continuous and objective real-time locating system to measure walking activity associated with wandering behaviors, focusing on older adults with cognitive impairment. Walking activity is measured by walking distance, sustained walking distance, and sustained gait speed. Also assessed are gait quality and balance ability.

A real-time locating system (RTLS) can be used to track the walking activity of institutionalized older adults in long-term care who are at risk for ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

Lexical Decision Task for Studying Written Word Recognition in Adults with and without Dementia or Mild Cognitive Impairment

Older adults are slower at recognizing visual objects than younger adults. The same is true for recognizing that a letter string is a real word. People with Alzheimer&#39;s disease (AD) or Mild Cognitive Impairment (MCI) demonstrate even longer responses in written word recognition than elderly controls. Despite the general tendency towards slower recognition in aging and neurocognitive disorders, certain characteristics of words influence word recognition speed regardless of age or neuropathology (e.g., a word&#8217;s frequency of use). We present here a protocol for examining the influence of lexical characteristics on word recognition response times in a simple lexical decision experiment administered to younger and older adults and people with MCI or AD. In this experiment, participants are asked to decide as quickly and accurately as possible whether a given letter string is an actual word or not. We also describe mixed-effects models and principal components analysis that can be used to detect the influence of different types of lexical variables or individual characteristics of participants on word recognition speed.

This article describes how to implement a simple lexical decision experiment to assess written word recognition in neurologically healthy participants and in individuals with dementia and cognitive decline. We also provide a detailed description of reaction time analysis using principal components analysis (PCA) and mixed-effects modeling.

Older adults are slower at recognizing visual objects than younger adults. The same is true for recognizing that a letter string is a real word. People ...

An Experimental Paradigm for Measuring the Effects of Ageing on Sentence Processing

Previous studies have found that older adults have greater difficulties in processing syntactically complex sentences than younger adults. However, the exact regions where the difficulties arise have not been fully identified. In this study, a maze task was implemented to investigate how older adults and younger adults differentially processed two types of sentences with different levels of syntactic complexity, namely subject relative clauses and object relative clauses. Participants were asked to choose between two alternatives at each segment of the sentences. The task required participants to engage in a strictly incremental mode of processing. The reading times for each segment were recorded, allowing the quantification of the difficulty of sentence reading. The task allowed us to identify the exact locations of the processing difficulty and thus facilitate a more accurate assessment of the age-related decline in sentence processing. The results indicate that the effect of ageing was found mainly at the head nouns, but not at other regions of the sentences, a finding which suggests that the maze task is an effective method to identify the exact location of the ageing effect on sentence processing. The implications of this experimental paradigm for investigating the effect of ageing on sentence processing are discussed.

Here, we present a protocol to examine the age-related decline in sentence processing using a maze task that allowed us to localize the processing difficulty at each word of the sentences during reading.

Previous studies have found that older adults have greater difficulties in processing syntactically complex sentences than younger adults. However, the ...

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the underlying neurobiological mechanisms, for which diverse experimental approaches and model systems are needed. The main ingredient of alcoholic beverages is ethanol, which causes adaptive changes in the central nervous system and behavior upon chronic intake. Behavioral sensitization (i.e., escalated responses) in particular represents a key adaptive change underlying addiction. Most ethanol-induced behavioral sensitization studies in animal models have been conducted on the locomotor activating effect of ethanol. A prominent effect of ethanol is behavioral disinhibition. Behavioral sensitization to the disinhibition effect of ethanol, however, is underrepresented. To address this issue, we developed the Flypub assay that allows measuring the escalated increase in disinhibited courtship activities upon recurring ethanol exposure in Drosophila melanogaster.&#160;Here, we report the step-by-step Flypub assay including assembly of ethanol exposure chambers, setup of the assay station, criteria for fly care and collection, ethanol delivery, quantification of disinhibited courtship activities, data processing and statistical analysis. Also provided are how to troubleshoot critical steps, overcome limitations and expand its utility to assess additional ethanol-induced behaviors. The Flypub assay in combination with powerful genetic tools in Drosophila melanogaster will facilitate the task of discovering the mechanism underlying ethanol-induced behavioral sensitization.

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction studies.

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the ...