Name: tracking rats in operant conditioning chambers using a versatile homemade video camera and deeplabcut
Uploaded: 2020-06-15T14:30:00
Description: Watch this Scientific Journal Video about Tracking Rats in Operant Conditioning Chambers Using a Versatile Homemade Video Camera and DeepLabCut at JoVE.com

This work was supported by grants from the Swedish Brain Foundation, the Swedish Parkinson Foundation, and the Swedish Government Funds for Clinical Research (M.A.C.), as well as the Wenner-Gren foundations (M.A.C, E.K.H.C), &#197;hl&#233;n foundation (M.A.C) and the foundation Blanceflor Boncompagni Ludovisi, n&#233;e Bildt (S.F).

All procedures that include animal handling have been approved by the Malm&#246;-Lund Ethical committee for animal research.
1. Building the video camera
NOTE: A list of the components needed for building the camera is provided in the Table of Materials. Also refer to Figure 1, Figure 2, Figure 3, Figure 4, Figur...

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Coulbourn Instruments Available from: <a target="_blank" href="https://www.coulbourn.com/product_p/h39-16.htm">https://www.coulbourn.com/product_p/h39-16.htm</a> (2020)</li><li>Mathis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeepLabCut:+markerless+pose+estimation+of+user-defined+body+parts+with+deep+learning.">DeepLabCut: markerless pose estimation of user-defined body parts with deep learning.</a> Nature Neuroscience. 21 (9), 1281-1289 (2018).</li><li>Nath, T., Mathis, A., Chen, A. C., Patel, A., Bethge, M., Mathis, M. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeepPoseKit,+a+software+toolkit+for+fast+and+robust+animal+pose+estimation+using+deep+learning.">DeepPoseKit, a software toolkit for fast and robust animal pose estimation using deep learning.</a> Elife. 8 (47994), (2019).</li><li>Geuther, B. Q., 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=Robust+mouse+tracking+in+complex+environments+using+neural+networks.">Robust mouse tracking in complex environments using neural networks.</a> Communications Biology. 2 (124), (2019).</li></ol>

This protocol describes how to build an inexpensive and flexible video camera that can be used to record videos from operant conditioning chambers and other behavioral test setups. It further demonstrates how to use DeepLabCut to track a strong light signal within these videos, and how that can be used to aid in identifying brief video segments of interest in video files that cover full test sessions. Finally, it describes how to use the tracking of a rat&#8217;s head to complement the analysis of behaviors during operan...

In the field of behavioral neuroscience, researchers commonly use operant conditioning chambers to assess a wide range of different cognitive and psychiatric features in rodents. While there are several different manufacturers of such systems, they typically share certain attributes and have an almost standardized design1,2,3. The chambers are generally square- or rectangle-shaped, with one wall that can be opened for placing animals inside, and one or two of the remaining walls containing components such as levers, nose-poke openings, reward trays, response wheels and lights of various kinds1,2,3. The lights and sensors present in the chambers are used to both control the test protocol and track the animals&#8217; behaviors1,2,3,4,5. The typical operant conditioning systems allow for a very detailed analysis of how the animals interact with the different operanda and openings present in the chambers. In general, any occasions where sensors are triggered can be recorded by the system, and from this data users can obtain detailed log files describing what the animal did during specific steps of the test4,5. While this provides an extensive representation of an animal&#8217;s performance, it can only be used to describe behaviors that directly trigger one or more sensors4,5. As such, aspects related to how the animal positions itself and moves inside the chamber during different phases of the test are not well described6,7,8,9,10. This is unfortunate, as such information can be valuable for fully understanding the animal&#8217;s behavior. For example, it can be used to clarify why certain animals perform poorly on a given test6, to describe the strategies that animals might develop to handle difficult tasks6,7,8,9,10, or to appreciate the true complexity of supposedly simple behaviors11,12. To obtain such articulate information, researchers commonly turn to manual analysis of videos6,7,8,9,10,11.
When recording videos from operant conditioning chambers, the choice of camera is critical. The chambers are commonly located in isolation cubicles, with protocols frequently making use of steps where no visible light is shining3,6,7,8,9. Therefore, the use of infra-red (IR) illumination in combination with an IR-sensitive camera is necessary, as it allows visibility even in complete darkness. Further, the space available for placing a camera inside the isolation cubicle is often very limited, meaning that one benefits strongly from having small cameras that use lenses with a wide field of view (e.g., fish-eye lenses)9. While manufacturers of operant conditioning systems can often supply high-quality camera setups to their customers, these systems can be expensive and do not necessarily fit chambers from other manufacturers or setups for other behavioral tests. However, a notable benefit over using stand-alone video cameras is that these setups can often interface directly with the operant conditioning systems13,14. Through this, they can be set up to only record specific events rather than full test sessions, which can greatly aid in the analysis that follows.
The current protocol describes how to build an inexpensive and versatile video camera using hobby electronics components. The camera uses a fisheye lens, is sensitive to IR illumination and has a set of IR light emitting diodes (IR LEDs) attached to it. Moreover, it is built to have a flat and slim profile. Together, these aspects make it ideal for recording videos from most commercially available operant conditioning chambers as well as other behavioral test setups. The protocol further describes how to process videos obtained with the camera and how to use the software package DeepLabCut15,16 to aid in extracting video sequences of interest as well as tracking an animal&#8217;s movements therein. This partially circumvents the draw-back of using a stand-alone camera over the integrated solutions provided by operant manufacturers of conditioning systems, and offers a complement to manual scoring of behaviors.
Efforts have been made to write the protocol in a general format to highlight that the overall process can be adapted to videos from different operant conditioning tests. To illustrate certain key concepts, videos of rats performing the 5-choice serial reaction time test (5CSRTT)17 are used as examples.

<table>
	<tbody>
		<tr>
			<td>32 Gb micro SD card with New Our Of Box Software (NOOBS) preinstalled</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>32GB</td>
			<td></td>
		</tr>
		<tr>
			<td>330-Ohm resistor</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>100287</td>
			<td>This article is for a package with mixed resistors, where 330-ohm resistors are included.</td>
		</tr>
		<tr>
			<td>Camera module (Raspberry Pi NoIR camera v.2)</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>100004</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera ribbon cable (Raspberry Pi Zero camera cable stub)</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>MMP-1294</td>
			<td>This is only needed if a Raspberry Pi zero is used. If another Raspberry Pi board is used, a suitable camera ribbon cable accompanies the camera component</td>
		</tr>
		<tr>
			<td>Colored LEDs</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>ADA4203</td>
			<td>This article is for a package with mixed colors of LEDs. Any color can be used.</td>
		</tr>
		<tr>
			<td>Female-Female jumper cables</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>ADA266</td>
			<td></td>
		</tr>
		<tr>
			<td>IR LED module (Bright Pi)</td>
			<td>Pi Supply (https://uk.pi-supply.com)</td>
			<td>PIS-0027</td>
			<td></td>
		</tr>
		<tr>
			<td>microcomputer motherboard (Raspberry Pi Zero board with presoldered headers)</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>102373</td>
			<td>Other Raspberry Pi boards can also be used, although the method for automatically starting the Python script only works with Raspberry Pi zero. If using other models, the python script needs to be started manually.</td>
		</tr>
		<tr>
			<td>Push button switch</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>ADA367</td>
			<td></td>
		</tr>
		<tr>
			<td>Raspberry Pi power supply cable</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>102032</td>
			<td></td>
		</tr>
		<tr>
			<td>Raspberry Pi Zero case</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>102118</td>
			<td></td>
		</tr>
		<tr>
			<td>Raspberry Pi, Mod my pi, camera stand with magnetic fish eye lens and magnetic metal ring attachment</td>
			<td>The Pi hut (https://thpihut.com)</td>
			<td>MMP-0310-KIT</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracking rats in operant conditioning chambers using a versatile homemade video camera and deeplabcut

Operant conditioning chambers are used to perform a wide range of behavioral tests in the field of neuroscience. The recorded data is typically based on the triggering of lever and nose-poke sensors present inside the chambers. While this provides a detailed view of when and how animals perform certain responses, it cannot be used to evaluate behaviors that do not trigger any sensors. As such, assessing how animals position themselves and move inside the chamber is rarely possible. To obtain this information, researchers generally have to record and analyze videos. Manufacturers of operant conditioning chambers can typically supply their customers with high-quality camera setups. However, these can be very costly and do not necessarily fit chambers from other manufacturers or other behavioral test setups. The current protocol describes how to build an inexpensive and versatile video camera using hobby electronics components. It further describes how to use the image analysis software package DeepLabCut to track the status of a strong light signal, as well as the position of a rat, in videos gathered from an operant conditioning chamber. The former is a great aid when selecting short segments of interest in videos that cover entire test sessions, and the latter enables analysis of parameters that cannot be obtained from the data logs produced by the operant chambers.

Video camera performance
The representative results were gathered in operant conditioning chambers for rats with floor areas of 28.5 cm x 25.5 cm, and heights of 28.5 cm. With the fisheye lens attached, the camera captures the full floor area and large parts of the surrounding walls, when placed above the chamber (Figure 7A). As such, a good view can be obtained, even if the camera is placed off-center on the chamber&#8217;s top. This should hold ...

Watch this Scientific Journal Video about Tracking Rats in Operant Conditioning Chambers Using a Versatile Homemade Video Camera and DeepLabCut at JoVE.com

Tracking Rats in Operant Conditioning Chambers Using a Versatile Homemade Video Camera and DeepLabCut

This protocol describes how to build a small and versatile video camera, and how to use videos obtained from it to train a neural network to track the position of an animal inside operant conditioning chambers. This is a valuable complement to standard analyses of data logs obtained from operant conditioning tests.

Operant conditioning chambers are used to perform a wide range of behavioral tests in the field of neuroscience. The recorded data is typically based on ...

By following the methods outlined in this protocol researchers will be able to record and analyze videos of rodents performing complex behavioral tests in operant conditioning chambers. The protocol describes how to build inexpensive video camera and use it together with an open source tracking software. This is an attractive approach for labs on a budget.The method is valuable for research projects that involve operant conditioning in rodents. As video analysis can greatly improve the understanding of behaviors seen in this type of tests. Begin by attaching the metal ring around the opening of the camera stand.Then attach the camera module to the stand using the nuts and bolts that accompany the kit. Open the ribbon cable ports on the camera module and microcomputer by gently pooling on the edges of their plastic lips. Blaze the ribbon cable in the open port on the camera module so that the cables silver connectors face the circuit board.Then lock it in place by pushing in the plastic clip. Repeat the process with a port on the microcomputer. Then attach the fisheye lens to the metal ring on the camera stand.Place the microcomputer in the plastic case and insert the listed micro SD card. Then connect a monitor, keyboard and a mouse to the microcomputer and started by connecting its power supply. Open a terminal window and type sudo apt hyphen, get update.Then press the enter key. Next type sudo apt full hyphen upgrade and press enter. Under the start menu, select preferences and raspberry PI configurations.When the window opens, go to the interfaces tab and enable the camera and I2C Then click okay. Copy supplementary file one onto a USB memory stick. Then transfer it to the microcomputers home PI folder and rename it.Open a terminal window type pseudo nano slash etc, slash RC dot local and press enter. Use the keyboards arrow keys to move the cursor down to the space between fi and exit zero. Then add text to make the computer start the copied script.And the infrared LEDs, whenever it boots. Save the changes by pressing control and X followed by Y and enter. Next solder resistors and female jumper cables onto the legs of two colored LEDs.Solder female jumper cables onto two button switches. Then connect the switches colored LEDs and the listed infrared led module to the computers GPI hoop ends. When connected properly, one led will indicate that the camera is switched on and ready to be used.While the other indicates that the camera is recording a video. The button with the long cables is used to start and stop video recordings. While the button with these short cables is used to switch off the camera.Set the protocol to use the operant chambers house light as an indicator of a specific step in the protocol. Then set the protocol to record all events of interest with timestamps in relation to when this protocol step indicator becomes active. Place the camera on top of the operant chambers and started by connecting it to an electrical outlet via the power supply cable.Use the previously connected button to start and stop video recordings. When the video recordings are finished, connect the camera to a monitor, keyboard mouse and USB storage device and retrieve the video files from its desktop. Use DeeplabCuts frame grabbing function to extract 700 to 900 video frames from one or more of the recorded videos.Make sure that the video frames you select display the animal in different postures, both stationary with the head outside and inside of openings and moving in different directions. Use the labeling toolbox to manually mark the position of the rat's head in each video frame by placing a head label in a central position between the rat's ears. Also label other body parts that may be of interest.In addition, mark a position of the protocol step indicator in each video frame where it is actively shining. Next use the create training data set and train network functions to create a training data set from the labeled video frames and start the training of a neural network. When a neural network has been trained, use it to analyze the gathered videos.This will create a CSV file listing the track positions of the rat's head, other body parts of interest and the protocol step indicator in each video frame. In addition, it will create marked up video files where the track positions are displayed visually. To obtain coordinates of specific points of interest inside the operant chambers, manually mark these as previously described and retrieve the coordinates from the CSV file that is automatically stored under labeled data in the project folder.Note, in which video segments the protocol step indicator is tracked within 60 pixels of the position obtained manually in the previous section and extract the exact starting point for each period where the indicator is active. Use the points where the protocol step indicator becomes active and the timestamps recorded by the operant chambers. To determine which video segments cover specific events of the test protocol such as inter-trial intervals, responses or reward retrievals.Note the video frames they cover any events that are specific interest. Finally perform relevant in-depth analysis of the animal's position and movements, during these events. The camera's fish eye lens should allow it to capture a full view of the inside of most rodent operant conditioning chambers.By using a suitable source of infrared illumination, the camera will also allow video capture in complete darkness. A well-trained network should allow for over 90%accuracy when tracking an animal's head. Accurate tracking is clearly identifiable by markers following an animal throughout its movements and plotted paths appearing smooth.In contrast, inaccurate tracking is characterized by markers that do not reliably stay on target and by jagged plotted paths. As a result of this inaccurate tracking typically causes sudden shifts in calculated movements speeds. By tracking where an animal is located throughout a test session, one can assess how distinct movement patterns relate to performance.As an example, in the five choice serial reaction time test, head movements during the inter-trial interval, can be used to separate emission trials where the animal shows a limited interest in performing a response. From a mission trials where the animal simply fails to notice the brief light cue. In addition investigating head movements can enable the detection and characterization of different attentional strategies.When attempting these procedure, it is important that the protocol step indicator is reliable. And that the neural network is trained with a dives ert of video frames. To ensure an accurate tracking.

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Research

JoVE Journal

Behavior

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

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 ...

Classical Short-Delay Eyeblink Conditioning in One-Year-Old Children

Classical eyeblink conditioning (EBC) refers to the learned association between a conditioned stimulus (an auditory tone) and an unconditioned stimulus (a puff of air to the cornea). Eyeblink conditioning is often used experimentally to detect abnormalities in cerebellar-dependent learning and memory that underlies this type of associative learning. While experiments in adults and older children are relatively simple to administer using commercial equipment, eyeblink conditioning in infants is more challenging due to their poor compliance, which makes correct positioning of the equipment difficult. To achieve conditioning in one-year-old infants, a custom-made or an adapted commercial system can be used to deliver the air puff to the infant's cornea. The main challenge lies in successfully detecting and classifying the behavioral responses. We report that automated blink detection methods are unreliable in this population, and that conditioning experiments should be analyzed using frame-by-frame analysis of supplementary video camera recordings. This method can be applied to study developmental changes in eyeblink conditioning and to examine whether this paradigm can detect children with neurological disorders.

This protocol describes an eyeblink conditioning paradigm appropriate for experiments with one-year-old infants. Commercial or custom-made equipment can be used to deliver the stimuli, and data collection and analysis should be performed on the video recordings.

Classical eyeblink conditioning (EBC) refers to the learned association between a conditioned stimulus (an auditory tone) and an unconditioned stimulus (a ...

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...