We thank Ravikumar Boyapati for helping with setting up the arena. This work is funded by Wellcome trust DBT Intermediate India Alliance grant (Grant number IA/I/15/2/502074) to P.K. M.S. was funded by a fellowship by the Indian Council of Medical Research (ICMR). A.B. was funded by NCBS-TIFR institutional funds (No. 12P4167) and the Department of Atomic Energy, Government of India (No. 12-R&#38;amp; D-TFR-5.04-0800 and 12-R&#38;amp; D-TFR-5.04-0900).

Adult male flies of the Drosophila melanogaster Canton-S (CS) wild-type strain were&#160;used for the present study.
1. Experimental preparation
<ol>
	<li>Fly rearing
	<ol>
		<li>Collect adult male flies emerging within a 12 h period and maintain them on standard fly media (prepared in house) for 48 h at 25 &#176;C with 75% relative humidity in a 12 h light/dark cycle. 
		NOTE: The Drosophila media composition used was (for 1 L of media) corn flour (80 g), D-glucose (20 g), sugar (40 g), agar (8 g), yeast powder (15 g), propionic acid (4 mL), methyl 4-hydroxybenzoate (1.25 g in 3 mL of ethanol), and orthophosphoric acid (600 &#181;L) (see Table of Materials). The initial feeding period ensures that the flies have access to sufficient food and nutrients before the starvation phase. While both male and female flies elicit the search behavior, but starvation time is more consistent among male flies. Additionally, female flies change their feeding preference after mating11.</li>
	</ol>
	</li>
	<li>Starvation procedure
	<ol>
		<li>After the feeding period, starve the flies of food but with access to water. 
		NOTE: To standardize the hunger state across trials and strains, it is suggested to determine the duration of 90% survival of the population under food-starved conditions. Based on this result, CS flies were starved for 28 h (Figure 1A) as circadian rhythms and other factors can impact the search behavior12. Circadian changes of fly searching activities at different times of a day were also measured (Figure 1B). We noticed reduced relative search activity at night compared to light phase in flies.</li>
		<li>Calculate the food-starvation tolerance by depriving two- day old flies of food. Place 15-20 flies in a vial with soaked tissue paper at the bottom. This serves as a substrate and ensure that the flies have access to water throughout the starvation period.</li>
		<li>Count the number of flies during the food-starvation period at regular intervals of 1 h. The duration at which 90% of the starved flies survived was used as the starvation period (Figure 1A). Use multiple replicates (3-4 for each strain) to minimize the impact of individual variations and provide a more reliable assessment of starvation tolerance. 
		NOTE: The food-starvation tolerance of each strain was determined in order to establish a standardized hunger state among different strains and experiments. By counting the surviving flies at these intervals, one can monitor the survival rate over time and determine how long each strain could tolerate food deprivation before succumbing to starvation.</li>
	</ol>
	</li>
	<li>Procedure for recording the behavior
	<ol>
		<li>Transfer individual starved flies in small tubes (Figure 2A). Do this in batches of 5-6 flies to minimize isolation time. By testing single flies, isolating each fly, any observed changes or actions can be attributed to the specific fly being observed, rather than being influenced by interactions with other flies.</li>
		<li>Use 90 mm Petri dishes as an arena for behavioral assays (bigger petri dishes can be used. There was no difference in behavior in Petri dishes with larger diameter). Illuminate the arena from the bottom by a panel of surface-mounted cool white LEDs (Figure 2B,C). To maintain a uniform visual environment and minimize external distraction, surround the experimental arena by a white polyvinyl chloride pipe (51.5 mm height, 114 mm inner diameter). 
		NOTE: This pipe acts as a barrier, preventing any visual stimuli from outside the arena from influencing the fly&#39;s behavior. By reducing external distractions, one can focus solely on the fly&#39;s interaction with the food source, maintaining consistency throughout the experiment.</li>
		<li>Use light intensity of 320 lux at the center of the arena.</li>
		<li>Position 0.2 &#181;L sugar solution in the center of the arena. 500 mM sucrose solution was used in the reported experiments but this can be varied. Introduce the fly to the arena using a 2 mL micro centrifuge tube (inner diameter 8.7 mm, length reduced to 5 mm by stuffing cotton at the bottom; Figure 2A) housing a single fly inverted over the sugar drop.</li>
		<li>Once the fly starts ingesting the droplet, remove the fly container, giving the fly unrestricted access to the food source.</li>
		<li>Film the 2D position of the arena with an overhead camera. 
		NOTE: Flea3 (Point Grey, 1214 mm lens, see Table of Materials) was used for the present study and recorded at 40 frames per second (fps). However, any camera that provides good contrast with the background can be used. One can record at 30-60 fps, depending on the nature of the experiment. Record in .avi format since it is compatible with the tracking software.</li>
		<li>Record the trial until the time the fly escaped from the arena. The flies were freely walking and there was no lid on the arena.</li>
		<li>By allowing the fly to decide when to stop searching, fly away, or walk to the periphery of the arena, observe the fly&#39;s natural behavior and food search strategy.Wipe the Petri dishes with 70% ethanol between the trials and dry them completely or else use a new Petri dish. 
		NOTE: It is important to conduct all experiments between 2 h and 6 h after lights are on when the flies exhibit consistently high activity levels. This time frame ensures that the flies are in an active state, maximizing the chances of observing their natural food searching behavior and reducing the impact of other factors that may affect their behavior, such as circadian rhythms. The behavioral set-up was housed inside a temperature and humidity- controlled room. The arena was&#160;placed on a vibration- free table. This experimental setup involved isolating and testing individual flies for their response to sugar. A sugar drop was provided to the starved flies and their behavior was video recorded (Figure 2B,C, Video 1). The experiments were conducted during a specific time frame when the flies exhibited consistent high activity levels.</li>
	</ol>
	</li>
	<li>Analyze the trajectories following step 2 to determine the search behavior.</li>
</ol>
2. Analysis of trajectories for local search
<ol>
	<li>Analyze the recorded videos using Ctrax software11 (see Table of Materials). 
	NOTE: The software tracks and converts the fly&#39;s position in the video into x, y coordinates, enabling precise tracking and analysis of its movements. Refer to Supplementary File 1 for details on how to use Ctrax.</li>
	<li>Divide the trajectories into two phases: the initial feeding phase and the search phase. Define the end of feeding and start of walking as flies moving at a speed &#62;4 mm s-1 in three consecutive frames. 
	NOTE: After the feeding was over, the rest of the trajectory was used as the search response of the flies. This study used VirtualDub (see Table of Materials) to remove the feeding phase from the videos before tracking.</li>
	<li>Quantify the search by the following parameters:
	<ol>
		<li>Path length: This parameter represents the distance walked by the fly from its starting point during the food search (in mm).</li>
		<li>Stay time: The time spent by the flies walking during the search (in s). It indicates the duration of the search and fly&#39;s persistence in searching for food.</li>
		<li>Meander: Calculate this as a ratio by dividing the beeline (the distance between the first and the last point of the path) of the path to the total path length, and subtracting from 1. High values of meander indicate more tortuosity in the trajectory.</li>
		<li>Number of returns: Use the developed algorithm to identify and count the number of returns using two concentric circles. 
		NOTE: An inner circle indicating the origin of search, Rin (2.5 mm) and the outer circle indicating the minimum distance Rout (4 mm) that the fly had to move away from the origin. A return was defined as a movement out of the outer circle (Rout) and then coming back into the inner circle (Rin).</li>
		<li>Activity rate: Expressed as a percentage, calculate the activity rate by determining the time during food searching when the fly&#39;s walking speed exceeds 2 mm/s. This parameter reflects the fly&#39;s level of activity and engagement in the food search. It distinguishes active foragers from less active individuals. 
		NOTE: MATLAB and Python were used in the present study for further analysis of the trajectories. The scripts can be accessed here: https://github.com/eagermeagre/sugar_elicited_search. The analysis procedure involves using the Ctrax software to track the fly&#39;s movements and determine parameters such as the duration of food search, total path length, search time, meander, number of returns and activity rate. These parameters provide valuable insights into the fly&#39;s behavior and foraging efficiency during the recorded food searching experiments.</li>
	</ol>
	</li>
</ol>

Analysis of Sugar-Induced Foraging Behavior in Drosophila

<ol>
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The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The current study introduces a straightforward paradigm for investigating sugar-elicited search behavior in Drosophila, first described by Dethier2. This innate behavior allows the flies to engage in a local search for additional food resources following the encounter of a food reward. The most crucial aspect of the experimental protocol involves appropriately motivating the flies. Firstly, the flies must be in a state of hunger, having been deprived of food while still having access to water, to ensure sugar ingestion. In order to have the uniform hunger state across experimental trials the duration at which 90% of the population survives was used as the starvation period. Crucially, the induction of a search response post-feeding necessitates the provision of a food stimulus that is of sufficient quality but not enough to fully satiate the flies. Therefore, standardizing the concentration and amount of sugar, and starvation duration might be time-consuming but is imperative for robust and reliable behavior.
In this study, a 500 mM, 0.2 &#181;L sucrose solution was employed as the stimulus for starved flies. Sugar intake evokes a characteristic local search behavior characterized by increased turning behavior and frequent returns to the location of the sugar drop (Video 1). Conversely, hungry flies that are not provided with any sugar fail to exhibit a search response. Notably, all the behavior-related parameters including path length, stay time, meander, and number of returns were significantly lower in unfed flies. We have previously demonstrated that the ingestion of water alone does not elicit a search response9.
This setup offers a cost-effective and low-maintenance approach for studying this innate behavior. While a backlit arena is used in this study, top lighting can also be employed as long as there is sufficient contrast between the fly and the background. The tracking software used relies on the detection of fly movement against a static background13. The camera and resolution settings can be adjusted based on the specific scale of behavior under investigation. Importantly, this methodology enables the study of various components of foraging behavior, including sensory cue attention during foraging, food- engagement and feeding, locomotor control of search, and decision-making processes associated with exploitation and exploration, among others. Moreover, this paradigm facilitates the investigation of local search, a behavior that is commonly observed across diverse taxa in various ecological contexts6. Studying this behavior in Drosophila, opens avenues for scientific inquiry aimed at understanding the neural pathways involved in foraging. We have studied local search in honey bees and have shown that the behavior bears resemblance to flies7,8.
Recent studies have demonstrated that local search behavior can be triggered by optogenetic activation of various sugar sensory neurons in flies14,15,16. However, it remains unclear to what extent the local searches observed in these studies accurately represent the natural behavior of flies in response to actual sugar intake. Feeding behavior is tightly regulated in flies, and these findings indicate that the activation of pharyngeal sugar receptors initiates the search behavior. Tarsal taste sensilla are responsible for detecting sugar and inducing the proboscis extension reflex, while pharyngeal taste neurons determine whether feeding should proceed17,18. Once ingested, the sugar solution travels through the esophagus to the proventriculus and enters the crop, with its expansion monitored by a recurrent nerve19. Additionally, it is worth noting that some of the aforementioned studies involved the harnessing or confinement of flies, whereas this method allows the animals to freely walk throughout the experiment. The flies in our experiments were sufficiently motivated to stay and search within the arena without the imposition of a lid.
Understanding the intricate interplay between neural pathways, genetic factors, and environmental cues that govern search behavior in flies can shed light on the fundamental principles of information processing, learning, and memory formation. Furthermore, dysregulation of foraging behavior has been implicated in various human disorders, including eating disorders and obesity. The extensive array of neurogenetic tools available in Drosophila provides a valuable resource for investigating the sugar-elicited search behavior and unravelling the neural and genetic mechanisms underlying foraging. In combination with optogenetic manipulation and functional imaging, this paradigm presents a powerful and promising approach20,21,22. However, modifying the setup for real-time manipulation of neuronal activity with optogenetics might be challenging. To monitor the neuronal activity in the brain while a fly is performing the searching behavior a different set-up will be needed such as the tethered fly on a tread ball. Many aspects of foraging behavior, such as feeding regulation and decision-making processes, are highly conserved across species. Therefore, insights gained from studying the neural mechanisms of foraging in flies can provide valuable insights into similar processes in other organisms, including humans.

Food-seeking and foraging behavior is a fundamental survival strategy exhibited by organisms across taxa. Two types of foraging behaviors have been identified in insects: hunger-induced search for food and post-meal local search1. When hungry, insects rely on sensory cues to locate food sources. Upon encountering and consuming a small food patch, they initiate a local search behavior characterized by convoluted paths and circling around the food location.
Sugar-elicited search behavior, a particular form of local search, was first studied over 60 years ago, by American biologist Vincent Dethier in blowflies2. When starved, flies are presented with a small amount of sugar such that it does not satiate them, they begin a local search. Typical search behavior is characterized by a highly tortuous walk with low locomotory and high turning rate and making returns to the location of the sugar drop. Subsequent studies had investigated this behavior in house flies and fruit flies3,4. The initiation, intensity, and duration of the search is regulated by the internal state of the animal (e.g., deprivation and motivation) as well as external factors such as resource availability and quality1,5,6.
Advancements in tracking technologies have provided researchers with valuable tools to capture and analyze behavior within controlled arenas. Here, we present a behavioral paradigm for tracking freely walking flies after sugar ingestion. This simple setup enables the study of sugar-elicited search behavior in Drosophila by capturing and analyzing the fly&#39;s motion in response to concentrated sugar solution provided in an arena. Employing advanced tracking technology and data analysis techniques, the locomotor patterns, spatial exploration, and response dynamics to sugar stimuli have been successfully quantified.
Using this assay, it has been experimentally demonstrated that the sugar-elicited search involves the use of path integration and can be spatially-temporally separated from sugar intake7,8. Furthermore, it has been shown that the behavior can be triggered by the activation of pharyngeal taste neurons9. Recent results show that sugar stimulus is not an innate releasing mechanism but also modulatory, and controls the initiation of the behavior temporally8. Using this paradigm, we have also studied this behavior in honey bees (Apis mellifera)7,8.
The eventual aim of this research is to unravel neural circuits and novel genetic components involved in regulating search behavior through targeted genetic manipulations and neuroimaging techniques. Food search behaviors have proven to be highly effective experimental paradigms for studying navigation and spatial memory in insects. These behaviors provide a unique opportunity to investigate the sensory perception, decision-making processes, and motor coordination involved in the search for rewarding food sources in flies. Additionally, the findings from these studies have broader implications for understanding feeding behaviors in other organisms, including humans, as many fundamental genetic and neural mechanisms are evolutionarily conserved. Dysregulation of feeding behaviors is associated with various neurological and metabolic disorders10. Therefore, the neural and genetic mechanisms underlying search behavior in flies may offer new avenues for understanding and potentially addressing these complex human health challenges.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 mL Eppendorf tube</td>
			<td>Sigma Aldrich</td>
			<td>BR780546</td>
			<td>Used to introduce the fly to the sugar drop</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>SRL</td>
			<td>9002-18-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Azure lens</td>
			<td>https://www.rmaelectronics.com/azure-photonics-azure-1214mm/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Logicool, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corn flour</td>
			<td>locally available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ctrax software</td>
			<td>https://ctrax.sourceforge.net/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>SRL</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Flea3</td>
			<td>Sony</td>
			<td></td>
			<td>https://www.flir.com/products/flea3-usb3/?vertical=machine+vision&#38;segment=iis</td>
		</tr>
		<tr>
			<td>glass tube</td>
			<td>Borosil</td>
			<td></td>
			<td>Used to house the flies individually&#160;</td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td>Used to provide access to water for flies during food starvation</td>
		</tr>
		<tr>
			<td>LED light panel</td>
			<td></td>
			<td></td>
			<td>custom-made in the workshop</td>
		</tr>
		<tr>
			<td>Light Meter</td>
			<td>TENMARS&#160;</td>
			<td>&#160;TM-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl 4-hydroxybenzoate</td>
			<td>Fisher Scientific</td>
			<td>99-76-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Orthophosphotic acid</td>
			<td>SRL</td>
			<td>7664-38-2&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (90 mm)</td>
			<td>Tarsons</td>
			<td>460090</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>SRL</td>
			<td>79-09-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Qualigens</td>
			<td>Q28105</td>
			<td></td>
		</tr>
		<tr>
			<td>Sugar</td>
			<td>locally available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VirtualDub&#160;</td>
			<td>https://www.virtualdub.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>White polyvinyl chloride pipe (67 mm inner diameter &#215; 100 mm height)</td>
			<td></td>
			<td></td>
			<td>custom-made in the workshop</td>
		</tr>
		<tr>
			<td>Yeast powder</td>
			<td>SRL</td>
			<td>REF-34266</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracking sugar-elicited local searching behavior in drosophila

Foraging behavior is essential for the survival of organisms as it enables them to locate and acquire essential food resources. In Drosophila, hunger triggers a distinct search behavior following the consumption of small quantities of a sugar solution. This report presents a simple experimental setup to study sugar-elicited search behavior with the aim of uncovering the underlying mechanisms. Minute quantities of concentrated sugar solution elicit sustained searching behavior in flies. The involvement of path integration in this behavior has been established, as flies utilize their trajectory to return to the sugar location. The most recent findings provide evidence of temporal modulation in the initiation and intensity of the search behavior after sugar intake. We have also used this setup for artificial activation of specific taste-receptor neurons in the pharynx, which elicits the search behavior. The Drosophila neurogenetic toolkit offers a diverse array of tools and techniques that can be combined with the sugar-elicited search behavior paradigm to study the neural and genetic mechanisms underlying foraging. Understanding the neural basis of hunger-driven searching behavior in flies contributes to the field of neurobiology as a whole, offering insights into the regulatory mechanisms that govern feeding behaviors not only in other organisms but also in humans.

The flies needed to be starved for the period estimated by the food-starvation tolerance and the response to sugar was individually tested (Figure 1A and Figure 2A). The behavior was recorded inside a temperature and humidity-controlled room. 0.2 &#181;L of 500 mM sucrose solution was used for the reported experiments. The sugar drop was positioned in the center of the arena and the flies were introduced to the sugar (Figure 2B). The behavior was recorded until flies escaped from the arena. The videos were analyzed to extract x,y coordinates and trajectories of the flies. Several parameters were used to quantify the behavioral response: path length, stay time, meander, number of returns and activity rate.
Sugar intake in starved flies leads to a local search with a meandering path and loops (Figure 3A,C). As a negative control, starved flies were recorded that were not presented with sugar8. These flies when introduced to and empty arena, did not initiate a search and escaped the arena (Figure 3B,D). This group is referred as unfed flies. The parameters of search: path length, stay time, meander and number of returns were significantly lower in unfed flies compared to the flies that were given a sugar drop (Figure 4A-D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65955/65955fig01.jpg" /> 
Figure 1: Starvation curves of wildtype strain CS. (A)&#160;Starvation curve showing survival rate for wild-type CS male and female flies. (B) Circadian changes of fly searching activities at different times of a day. Error bars represent S.E.M. (n = 10). <a href="https://www.jove.com/files/ftp_upload/65955/65955fig01large.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/65955/65955fig02.jpg" /> 
Figure 2: Experimental setup including starvation tolerance calculation, behavior recording procedures, and trajectory analysis. (A) Food-starvation tolerance was determined to establish a standardized hunger state. For behavior recording, individual flies were isolated in small tubes before the experiment. The behavioral arena, a 90 mm Petri dish, was uniformly illuminated from the bottom. A drop of sugar solution was placed in the center and flies were given unrestricted access to the food source. (B) The behavior was recorded until flies escaped from the arena. Video analysis used Ctrax software for tracking and MATLAB/Python for trajectory analysis. (C) Photograph of the experimental setup. <a href="https://www.jove.com/files/ftp_upload/65955/65955fig02large.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/65955/65955fig03.jpg" /> 
Figure 3: Necessity of the sugar intake for initiating local search. (A) Individual trajectories of flies fed 500 mM, 0.2 &#181;L sugar solution. (B) Individual trajectories of flies that were given no sugar. (C) Overlay of search trajectories flies from the control group (n = 11). (D) Overlay of the paths of the flies (n = 11) which were given no sugar. All the trajectories are normalized to the starting point of walking. This figure is adapted from Shakeel and Brockmann8. <a href="https://www.jove.com/files/ftp_upload/65955/65955fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65955/65955fig04.jpg" /> 
Figure 4: Reduction of behavioral parameters in unfed flies. (A-D) Path length, stay time, meander and number of returns were smaller for hungry flies that were not given sugar reward compared to control flies that were given stimulated with sugar. **p &#60; 0.001, ***p &#60; 0.0001, ****p &#60; 0.00001, Wilcoxon Rank Sum Test. This figure is adapted from Shakeel and Brockmann8. <a href="https://www.jove.com/files/ftp_upload/65955/65955fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Search behavior of a fly with real-time trajectory of the path. <a href="https://www.jove.com/files/ftp_upload/65955/Video 1.zip" target="_blank">Please click here to download this Video.</a>
Supplementary File 1: Step-wise instructions for tracking the video file using CTRAX. <a href="https://www.jove.com/files/ftp_upload/65955/Manal et al supplementary 1 ctrax_instructions.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication at JoVE.com

Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication

This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal mechanisms.

Tracking Sugar-Elicited Local Searching Behavior in Drosophila

Foraging behavior is essential for the survival of organisms as it enables them to locate and acquire essential food resources. In Drosophila, hunger ...

tracking-sugar-elicited-local-searching-behavior-in-drosophila

Research

JoVE Journal

Behavior

1.0K Views.  National Centre for Biological Sciences. This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal mechanisms.

Video: Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication

Studying Food Reward and Motivation in Humans

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as hedonics, motivation, and goal value in more objective ways. This is particularly relevant for the understanding of overeating and obesity as well as their potential treatments. In this paper are described a set of measures of food-related motivation using handgrip force as a motivational measure. These methods can be used to examine changes in food related motivation with metabolic (satiety) and pharmacological manipulations and can be used to evaluate interventions targeted at overeating and obesity. However to understand food-related decision making in the complex food environment it is essential to be able to ascertain the reward goal values that guide the decisions and behavioral choices that people make. These values are hidden but it is possible to ascertain them more objectively using metrics such as the willingness to pay and a method for this is described. Both these sets of methods provide quantitative measures of motivation and goal value that can be compared within and between individuals.

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as ...

A Method to Quantify Visual Information Processing in Children Using Eye Tracking

Visual problems that occur early in life can have major impact on a child's development. Without verbal communication and only based on observational methods, it is difficult to make a quantitative assessment of a child's visual problems. This limits accurate diagnostics in children under the age of 4 years and in children with intellectual disabilities. Here we describe a quantitative method that overcomes these problems. The method uses a remote eye tracker and a four choice preferential looking paradigm to measure eye movement responses to different visual stimuli. The child sits without head support in front of a monitor with integrated infrared cameras. In one of four monitor quadrants a visual stimulus is presented. Each stimulus has a specific visual modality with respect to the background, e.g., form, motion, contrast or color. From the reflexive eye movement responses to these specific visual modalities, output parameters such as reaction times, fixation accuracy and fixation duration are calculated to quantify a child's viewing behavior. With this approach, the quality of visual information processing can be assessed without the use of communication. By comparing results with reference values obtained in typically developing children from 0-12 years, the method provides a characterization of visual information processing in visually impaired children. The quantitative information provided by this method can be advantageous for the field of clinical visual assessment and rehabilitation in multiple ways. The parameter values provide a good basis to: (i) characterize early visual capacities and consequently to enable early interventions; (ii) compare risk groups and follow visual development over time; and (iii), construct an individual visual profile for each child.

A method is described to quantify the quality of visual information processing based on reflexive eye movements in response to specific visual modalities. Reaction times and fixation output parameters are used to characterize visual performance in children with and without visual impairments from 6 months of age.

Visual problems that occur early in life can have major impact on a child's development. Without verbal communication and only based on observational ...

Psychophysical Tracking Method to Measure Taste Preferences in Children and Adults

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to adulthood. The method, which is identical for children, adolescents, and adults, is of short duration (&#60; 15 min), does not rely on sustained attention or place demands on memory (which would yield spurious age differences), and minimizes the impact of language development, making this method amenable to the cognitive limitations of pediatric populations. In this whole-mouth tasting method, subjects are asked to taste (without swallowing) pairs of solutions of different sucrose concentrations and to point to the solution they prefer. Each subsequent pair contains the participant&#39;s preceding preferred concentration and an adjacent stimulus concentration. The procedure continues until the subject chooses either a given concentration of sucrose when paired with both a higher and a lower concentration, or the highest or lowest concentration two consecutive times. Subjects are prevented from reaching response criteria on the basis of first or second position bias by the two-series design of the method, which counterbalances the order of solution presentation within each pair between the series (the weaker concentration is presented first in Series 1, second in Series 2). The geometric mean of the two sucrose concentrations chosen in Series 1 and 2 is an estimate of the participant&#39;s most preferred level of sucrose. Sucrose preference as determined with this laboratory-based measure has been shown to be associated with preference for sugars in foods and beverages and with taste receptor genotype, family history of alcoholism, and race/ethnicity, as well as depressive symptomatology among pediatric populations. The method has real-world relevance and has been applied to determine most preferred level of other tastes (e.g., salt), making it a valuable psychophysical tool.

Sweet taste has powerful hedonic appeal among people of all ages, particularly children. Described herein is a reliable and valid method that can be used to determine the level of sweetness most preferred, making it a valuable psychophysical tool for scientists.

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to ...

Eye-Tracking Control to Assess Cognitive Functions in Patients with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with pathological involvement of upper and lower motoneurons, subsequently leading to progressive loss of motor and speech abilities. In addition, cognitive functions are impaired in a subset of patients. To evaluate these potential deficits in severely physically impaired ALS patients, eye-tracking is a promising means to conduct cognitive tests. The present article focuses on how eye movements, an indirect means of communication for physically disabled patients, can be utilized to allow for detailed neuropsychological assessment. The requirements, in terms of oculomotor parameters that have to be met for sufficient eye-tracking in ALS patients are presented. The properties of stimuli, including type of neuropsychological tests and style of presentation, best suited to successfully assess cognitive functioning, are also described. Furthermore, recommendations regarding procedural requirements are provided. Overall, this methodology provides a reliable, easy to administer and fast approach for assessing cognitive deficits in patients who are unable to speak or write such as patients with severe ALS. The only confounding factor might be deficits in voluntary eye movement control in a subset of ALS patients.

Cognitive deficits are common in about one third of patients with amyotrophic lateral sclerosis, a neurological condition leading to progressive impairments in speech and movement abilities. To conduct cognitive tests in patients unable to speak or write a reliable and easy to administer eye-tracking paradigm was developed.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with pathological involvement of upper and lower motoneurons, subsequently leading to ...

Eye Tracking During Visually Situated Language Comprehension: Flexibility and Limitations in Uncovering Visual Context Effects

The present work is a description and an assessment of a methodology designed to quantify different aspects of the interaction between language processing and the perception of the visual world. The recording of eye-gaze patterns has provided good evidence for the contribution of both the visual context and linguistic/world knowledge to language comprehension. Initial research assessed object-context effects to test&#160;theories of modularity in language processing. In the introduction, we describe how subsequent investigations have taken the role of the wider visual context in language processing as a research topic in its own right, asking questions such as how our visual perception of events and of speakers contributes to&#160;comprehension informed by&#160;comprehenders&#39; experience. Among the examined aspects of the visual context are actions, events, a speaker&#39;s gaze, and emotional facial expressions, as well as spatial object configurations. Following an overview of the eye-tracking method and its different applications, we list the key steps of the methodology in the protocol, illustrating how to successfully use it to study visually-situated language comprehension. A final section presents three sets of representative results and illustrates the benefits and limitations of eye tracking for investigating the interplay between the perception of the visual world and language comprehension.

The present article reviews an eye-tracking methodology for studies on language comprehension. To obtain reliable data, key steps of the protocol must be followed. Among these are the correct set-up of the eye tracker (e.g., ensuring good quality of the eye and head images) and accurate calibration.

The present work is a description and an assessment of a methodology designed to quantify different aspects of the interaction between language processing ...

Gaze in Action: Head-mounted Eye Tracking of Children's Dynamic Visual Attention During Naturalistic Behavior

Young children's visual environments are dynamic, changing moment-by-moment as children physically and visually explore spaces and objects and interact with people around them. Head-mounted eye tracking offers a unique opportunity to capture children's dynamic egocentric views and how they allocate visual attention within those views. This protocol provides guiding principles and practical recommendations for researchers using head-mounted eye trackers in both laboratory and more naturalistic settings. Head-mounted eye tracking complements other experimental methods by enhancing opportunities for data collection in more ecologically valid contexts through increased portability and freedom of head and body movements compared to screen-based eye tracking. This protocol can also be integrated with other technologies, such as motion tracking and heart-rate monitoring, to provide a high-density multimodal dataset for examining natural behavior, learning, and development than previously possible. This paper illustrates the types of data generated from head-mounted eye tracking in a study designed to investigate visual attention in one natural context for toddlers: free-flowing toy play with a parent. Successful use of this protocol will allow researchers to collect data that can be used to answer questions not only about visual attention, but also about a broad range of other perceptual, cognitive, and social skills and their development.

Young children do not passively observe the world, but rather actively explore and engage with their environment. This protocol provides guiding principles and practical recommendations for using head-mounted eye trackers to record infants' and toddlers' dynamic visual environments and visual attention in the context of natural behavior.

Young children's visual environments are dynamic, changing moment-by-moment as children physically and visually explore spaces and objects and interact ...

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

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 ...

Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients

Intracranial recordings from patients with intractable epilepsy provide a unique opportunity to study the activity of individual human neurons during active behavior. An important tool for quantifying behavior is eye tracking, which is an indispensable tool for studying visual attention. However, eye tracking is challenging to use concurrently with invasive electrophysiology and this approach has consequently been little used. Here, we present a proven experimental protocol to conduct single-neuron recordings with simultaneous eye tracking in humans. We describe how the systems are connected and the optimal settings to record neurons and eye movements. To illustrate the utility of this method, we summarize results that were made possible by this setup. This data shows how using eye tracking in a memory-guided visual search task allowed us to describe a new class of neurons called target neurons, whose response was reflective of top-down attention to the current search target. Lastly, we discuss the significance and solutions to potential problems of this setup. Together, our protocol and results suggest that single-neuron recordings with simultaneous eye tracking in humans are an effective method to study human brain function. It provides a key missing link between animal neurophysiology and human cognitive neuroscience.

We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search.

Intracranial recordings from patients with intractable epilepsy provide a unique opportunity to study the activity of individual human neurons during ...

Defining the Role Of Language in Infants' Object Categorization with Eye-tracking Paradigms

Assessing infant category learning is a challenging but vital aspect of studying infant cognition. By employing a familiarization-test paradigm, we straightforwardly measure infants&#8217; success in learning a novel category while relying only on their looking behavior. Moreover, the paradigm can directly measure the impact of different auditory signals on the infant categorization across a range of ages. For instance, we assessed how 2-year-olds learn categories in a variety of labeling environments: in our task, 2-year-olds successfully learned categories when all exemplars were labeled&#160;or the first two exemplars were labeled, but they failed to categorize when no exemplars were labeled or only the final two exemplars were labeled. To determine infants&#8217; success in such tasks, researchers can examine both the overall preference displayed by infants in each condition and infants&#8217; pattern of looking over the course of the test phase, using an eye-tracker to provide fine-grained time-course data. Thus, we present a powerful paradigm for identifying the role of language, or any auditory signal, in infants&#8217; object category learning.

Here we present a protocol for familiarization-test paradigms which provide a direct test of infant categorization and help to define the role of language in early category learning.

Assessing infant category learning is a challenging but vital aspect of studying infant cognition. By employing a familiarization-test paradigm, we ...