Z. Cai would like to acknowledge the financial support from the Kwok Chung Bo Fun Charitable Fund for the establishment of the Kwok Yat Wai Endowed Chair of Environmental and Biological Analysis.

In this study, two 7-week-old ICR male mice weighing between 17-25 g were utilized for the animal behavioral assessment. All housing and experimental procedures were approved by the Hong Kong Baptist University Committee on the Use of Human and Animal Subjects in Teaching and Research. The animal room was maintained at a temperature of 25 &#176;C and a room humidity of 40%-70% on a 12-h light-dark cycle.
1. Cage design
<ol>
	<li>Prepare acrylonitrile butadiene styrene bricks in 3 different dimensions for cage construction: 8 mm x 8 mm x 2 mm, 16 mm x 16 mm x 6 mm, and 32 mm x 16 mm x 6 mm.</li>
	<li>Prepare an acrylonitrile butadiene styrene plate (312 mm x 147 mm x 2 mm) as the cage base.</li>
	<li>Prepare a 239 mm x 107 mm non-transparent acrylic plate with a thickness of 2 mm to be used as the bottom plate.</li>
	<li>Prepare a 239 mm x 107 mm transparent acrylic plate with a thickness of 5 mm to be used as the top plate.</li>
	<li>Prepare a 107 mm x 50 mm transparent acrylic plate with a thickness of 7 mm to be used as the terminal plate.</li>
	<li>Construct 2 opaque side walls by stacking bricks to a height of 54 mm.</li>
	<li>Embed the acrylic plates into the acrylonitrile butadiene styrene-based cage, as illustrated in Figure 1A.</li>
	<li>Prepare a chows&#39; chamber that is constructed by five 90 mm x 50 mm transparent acrylic plates with a thickness of 2 mm, as illustrated in Figure 1B. Among the 5 transparent acrylic plates, use 2 plates for the sides, 1 plate for the top, 1 plate for the bottom, and 1 plate for the terminal.</li>
	<li>Prepare a 60 mm x 50 mm 2 mm transparent acrylic plate as a chow introduction plate, and place it in the chow&#39;s chamber.</li>
</ol>
2. Animal behavioral assessment
<ol>
	<li>House littermates of 2 7-week-old ICR male mice together in a regular cage.</li>
	<li>Provide the littermates of 2 mice with free access to food pellets and tap water for a 1-week adaptation period.</li>
	<li>After 1 week, introduce the littermates of 2 mice with a bottle of ethanol (~40% v/v). 
	NOTE: Make sure that they are only allowed to sniff or inhale the provided aqueous ethanol on an ad libitum basis while drinking is restricted.</li>
	<li>Conduct behavioral experiments using the 9-10-week-old mouse model and the transparent cubicle cage that is depicted in Figure 1A.</li>
	<li>Disassemble all the acrylic plates and acrylonitrile butadiene styrene plates and clean them thoroughly. Start by rinsing them with ultrapure water at least 3 times and then dry them using paper towels. Next, spray them with 75% ethanol, followed by cleaning them with lens paper. Finally, allow them to air dry for at least 15 min.</li>
	<li>Weigh the mice and record their body weights before each replication of the behavioral experiment.</li>
	<li>Freshly prepare a mixture of wasabi and peanut butter by weighing 1 g of commercial wasabi and 4.5 g of peanut butter. Mix them in a zip plastic bag. 
	NOTE: Due to the volatility of the isothiocyanates in wasabi, it is important to store the commercial wasabi in a -20 &#176;C freezer.</li>
	<li>Weigh and provide either two 0.5 g pastes of peanut butter or a mixture of wasabi and peanut butter on the chow introduction plate, as illustrated in Figure 1B, C.</li>
	<li>Place the prepared chow introduction plate in the chows&#39; chamber, as illustrated in Figure 1B, C, to allow the 2 mice to have ad libitum access to food during each video recording session.</li>
	<li>Fill the groove underneath with 30 mL of liquid, either pure water or liquor (~42% v/v ethanol), to facilitate concurrent inhalation, as indicated in Figure 1B, C.</li>
	<li>Begin recording using the cameras on 2 smartphones placed on the phone stands at each terminal. 
	NOTE: The specifications of the videos are as follows: frame width, 1920; frame height, 1080; data rate, 20745 kbps; frame rate, 30.00 frames per second (fps).</li>
	<li>Carefully place the 2 trained littermates of mice into the designed animal behavioral study platform from the top and promptly secure the cage with the top plate. 
	NOTE: Make sure this step is completed within 15 s.</li>
	<li>Record each video for 2-3 min. 
	NOTE: Make sure the entire duration of the experiment, from the preparation of the peanut butter-wasabi mixture to the completion of the video recording, is limited to 5 min.</li>
	<li>Repeat the whole experiment 3 times. 
	NOTE: Make sure each replicate of the experiment is separated for at least 6 h.</li>
	<li>Mimic different scenarios. 
	NOTE: For example, in this work, a pair of mice was used in 4 groups with 4 different scenarios that were mimicked by using the experimental setting described above. These scenarios include Scenario A for the background study, Scenario B for the positive control study, Scenario C for the wasabi-alcohol taste-smell interaction study, and Scenario D for the negative control study. Table 1 provides a summary of these scenarios.</li>
</ol>
3. Image recognition
Similar to many studies on image processing23,24,25, a classification model was attained by training a CNN. The script for DL operations was written in Python v.3.10 on Jupyter Notebook (anaconda3). It is available on the following GitHub repository: git@github.com:TommyNHL/imageRecognitionJove.git. To construct and train the CNN, open-source libraries were used, including, numpy v.1.21.5, seaborn v.0.11.2, matplotlib v.3.5.2, cv2 v.4.6.0, sklearn v.1.0.2, tensorflow v.2.11.0, and keras v.2.11.0. These libraries provided the necessary tools and functionality to develop and train CNN for image recognition.
<ol>
	<li>Export a series of video frame images from the collected video clips to generate a data set for model training by using the provided Jupyter Notebook, named Step1_ExtractingAndSavingVideoFrameImages.ipynb.</li>
	<li>Only select the images with at least 1 mouse consuming the provided paste. Examples of the selected images are provided in Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, Supplementary Figure 4, Supplementary Figure 5, Supplementary Figure 6, and Supplementary Figure 7.</li>
	<li>Perform data augmentation by horizontally flipping the generated images by implementing the script provided in the Jupyter Notebook, named Step2_DataAugmentation.ipynb.</li>
	<li>Reserve the image data from each second replicate for external independent CNN model validation. Use the images from each first and third replicates for internal model training and testing.</li>
	<li>Preprocess the image data used in CNN modeling by running the script in the Jupyter Notebook, named Step3_CNNmodeling_TrainTest.ipynb, including image resizing, black color conversion, and image signal normalization.</li>
	<li>Split the training materials into internal training and testing datasets in an 8:2 fashion randomly.</li>
	<li>Initialize the architecture for CNN. Design the number of outputs of CNN based on the number of scenarios to be examined. 
	NOTE: For example, in this study, the neural network was designated to classify 3 classes. Make sure the script for handling data imbalance on class weight is compiled.</li>
	<li>Find the hyperparameter combination that yields minimal loss on the internal test samples for CNN construction.</li>
	<li>Adopt the optimal hyperparameters combination for constructing CNN architecture.</li>
	<li>Open the provided Jupyter Notebooks Step4_CNNmodel_ExternalValOriginal.ipynb and Step5_CNNmodel_ExternalValFlipped.ipynb. Validate the attained model using the independent (original and flipped) images from the second replicate of the animal behavioral experiment.</li>
	<li>Deploy the attained and validated model for classifying the video frame images generated from the experimental group using Jupyter Notebook Step6_CNNmodel_Application.ipynb. 
	NOTE: For example, it is scenario C in this work.</li>
</ol>
4. Manual mouse grimace scale scoring
NOTE: To validate the insights provided by the CNN model prediction, another method previously developed and validated by Langford et al. was applied16. This method involves scoring the MGS based on the 5 specific mouse facial action units (AUs): orbital tightening, nose bulge, cheek bulge, ears tightening outward, and whisker change. Each AU is assigned a score of 0, 1, or 2, indicating the absence, moderate presence, or obvious presence of the AU, respectively. This scoring system allows for the quantification and scaling of each AU to assess the level of nociception or discomfort experienced by the mice.
<ol>
	<li>Capture 3 video frame images of the littermates ingesting the paste for each video clip. Ensure that each frame is separated by at least 3 seconds.</li>
	<li>Blind code and randomly reorder images from different classes of scenarios in sequence by using the provided template file named &#34;shuffleSlides.pptm&#34; (Supplementary File 1) and running the embedded Macro code.</li>
	<li>Invite at least 10 panelists to score the sample images.</li>
	<li>Train the panelists to score image samples using the MGS. Provide the panelists with training materials that include the original article regarding MGS and its manual15,16.</li>
	<li>Calculate the MGS score of each animal subject in a captured frame by averaging all the score points of the corresponding 5 facial AUs. Present the MGS score as the mean&#160;&#177; standard error of measurement (SEM).</li>
	<li>Determine whether statistically significant differences exist among different classes of scenarios by one-way analysis of variance (ANOVA) with Bonferroni&#39;s multiple comparison post-hoc test. 
	NOTE: A value of P &#60; 0.05 is considered statistically significant.</li>
</ol>

Alcohol Diminishes Wasabi Reactions: Behavioral and AI Analysis in Mice

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The authors declare that there are no conflicts of interest.

The proposed method for studying taste-smell interaction in this work is based on the original method of behavioral coding for facial expression of pain in mice, which was developed by Langford et al.16. Several recently published articles have introduced CNN for automatic mouse face tracking and subsequent MGS scoring21,26,27,28. Applying CNNs offers an advantage over t...

Wasabia japonica, commonly known as wasabi, has gained recognition in food preparation1,2. The intense sensory experience it elicits upon consumption, characterized by tearing up, sneezing, or coughing, is well-known. This distinctive pungency of wasabi can be attributed to a homologous compound of chemosensory isothiocyanates (ITCs). They are volatile organosulfur phytochemicals that can be categorized into &#969;-alkenyl and &#969;-methylthioalkyl isothiocyanates3. Among these compounds, allyl isothiocyanate (AITC) is the most predominated natural ITC product found in plants belonging to the Cruciferae family, such as horseradish and mustard4. Commercial wasabi pastes are commonly prepared from horseradish, making AITC a chemical marker used for quality control of these commercial products5.
Pairing dietary alcoholic beverages with wasabi-infused dishes can be considered an example of cultural disposition6. Subjectively, this combination may complement the spiciness and heat between wasabi and the spirit, enhancing the overall culinary experience. Animal qualitative behavioral assessment (QBA) is a comprehensive whole-animal methodological approach that examines behavioral changes in subjects in response to short-term or long-term external stimuli using numerical terms7. This method encompasses pain tests, motor tests, learning and memory tests, as well as emotion tests specifically designed for rodent models8. However, studies investigating the synergistic sensory evaluation of gustation together with olfaction remain scarce in the literature until now9,10. Most of the studies on chemesthetic sensation are confined to examining individual food and beverage consumption separately11. Consequently, there is a dearth of research on the taste-smell interaction involving the act of sniffing liquor while consuming wasabi.
As the wasabi-induced stinging sensation is believed to be a form of nociception12, animal behavioral assessments are well-suited for evaluating the nociceptive sensory responses in rodent animals8,13,14. A method for assessing nociception in mice, known as the mouse grimace scale (MGS) scoring was developed by Langford et al.15,16. This behavioral study method is a pain-related assessment approach, relying on the analysis of facial expressions exhibited by the experimental mice. The experimental setup is straightforward, involving a transparent cage and 2 cameras for video recording. By incorporating advanced technologies17,18,19 for automatic data capture, quantitative and qualitative behavioral measures can be obtained, enhancing animal welfare during behavioral monitoring20. Consequently, the MGS has the potential to be applied in studying the effects of various external stimuli on animals in an uninterrupted and ad libitum manner. However, the scoring process only involves selecting a few (less than 10) video frame images for evaluation by the panelists, and prior training is necessary. Scoring a large number of sample images can be labor-intensive. To overcome this time-consuming challenge, several studies have employed machine learning techniques for predicting the MGS score21,22. Yet, it is important to note that the MGS is a continuous measure. Therefore, a multi-class classification model would be more suitable for evaluating a logical and categorical problem, such as determining whether the images of mice simultaneously ingesting wasabi and sniffing liquor resemble those of normal mice.
In this study, a methodology for investigating the taste-smell interaction in mice was proposed. This methodology combines animal behavioral studies with a convolutional neural network (CNN) to analyze the facial expressions of the mouse subjects. Two mice were observed thrice under normal behavioral conditions, during the experience of wasabi-induced nociception and while sniffing liquor in a specifically designed cage. The facial expressions of the mice were video-recorded, and the generated frame images were utilized to optimize the architecture of a deep learning (DL) model. The model was then validated using an independent image dataset and deployed to classify the images acquired from the experimental group. To determine the extent of wasabi pungency suppression when the mice simultaneously sniffed liquor during wasabi consumption, the insights provided by artificial intelligence were further corroborated through cross-validation with another data analysis method, the MGS scoring16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absolute ethanol (EtOH)</td>
			<td>VWR Chemicals BDH</td>
			<td>CAS# 64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylonitrile butadiene styrene bricks</td>
			<td>Jiahuifeng Flagship Store</td>
			<td>https://shop.paizi10.com/jiahuifeng/chanpin.html</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylonitrile butadiene styrene plates</td>
			<td>Jiahuifeng Flagship Store</td>
			<td>https://shop.paizi10.com/jiahuifeng/chanpin.html</td>
			<td></td>
		</tr>
		<tr>
			<td>Allyl isothiocyanate (AITC)</td>
			<td>Sigma-Aldrich</td>
			<td>CAS# 57-06-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>CAS# 67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Chinese spirit</td>
			<td>Yanghe Qingci</td>
			<td>https://www.chinayanghe.com/article/45551.html</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial wasabi</td>
			<td>S&#38;B FOODS INC.</td>
			<td>https://www.sbfoods-worldwide.com</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid (FA)</td>
			<td>VWR Chemicals BDH</td>
			<td>CAS# 64-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad</td>
			<td>https://www.graphpad.com</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade acetonitrile (ACN)</td>
			<td>VWR Chemicals BDH</td>
			<td>CAS# 75-05-8</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade methanol (MeOH)</td>
			<td>VWR Chemicals BDH</td>
			<td>CAS# 67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel 2016</td>
			<td>Microsoft</td>
			<td>https://www.microsoft.com&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft PowerPoint 2016</td>
			<td>Microsoft</td>
			<td>https://www.microsoft.com</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q water system</td>
			<td>Millipore</td>
			<td>https://www.merckmillipore.com</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: ICR</td>
			<td>Laboratory Animal Services Centre (The Chinese University of Hong Kong, Hong Kong, China)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Peanut butter</td>
			<td>Skippy</td>
			<td>https://www.peanutbutter.com/peanut-butter/creamy</td>
			<td></td>
		</tr>
		<tr>
			<td>Python v.3.10</td>
			<td>Python Software Foundation</td>
			<td>https://www.python.org&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent acrylic plates</td>
			<td>Taobao Store</td>
			<td>https://item.taobao.com/item.htm?_u=32l3b7k63381&#38;id=60996545797 
			0&#38;spm=a1z09.2.0.0.77572e8dFPM 
			EHU</td>
			<td></td>
		</tr>
	</tbody>
</table>

integration of animal behavioral assessment and convolutional neural network to study wasabi-alcohol taste-smell interaction

The commercial wasabi pastes commonly used for food preparation contain a homologous compound of chemosensory isothiocyanates (ITCs) that elicit an irritating sensation upon consumption. The impact of sniffing dietary alcoholic beverages on the sensation of wasabi spiciness has never been studied. While most sensory evaluation studies focus on individual food and beverages separately, there is a lack of research on the olfactory study of sniffing liquor while consuming wasabi. Here, a methodology is developed that combines the use of an animal behavioral study and a convolutional neural network to analyze the facial expressions of mice when they simultaneously sniff liquor and consume wasabi. The results demonstrate that the trained and validated deep learning model recognizes 29% of the images depicting co-treatment of wasabi and alcohol belonging to the class of the wasabi-negative liquor-positive group without the need for prior training materials filtering. Statistical analysis of mouse grimace scale scores obtained from the selected video frame images reveals a significant difference (P &lt; 0.01) between the presence and absence of liquor. This finding suggests that dietary alcoholic beverages might have a diminishing effect on the wasabi-elicited reactions in mice. This combinatory methodology holds potential for individual ITC compound screening and sensory analyses of spirit components in the future. However, further study is required to investigate the underlying mechanism of alcohol-induced suppression of wasabi pungency.

The main objective of this study is to establish a robust framework for investigating the taste-smell interaction in mice. This framework incorporates the use of artificial intelligence and QBA to develop a predictive classification model. Additionally, the insights obtained from DL are cross-validated with a quantitative MGS assessment for an internal independent analysis. The primary application of this methodology is to examine the extent of suppression of the wasabi-invoked nociception when mice sniff dietary alcohol...

Integration of Animal Behavioral Assessment and Convolutional Neural Network to Study Wasabi-Alcohol Taste-Smell Interaction

This article describes a set of methods for measuring the suppressive ability of sniffing alcoholic beverages on the wasabi-elicited stinging sensation.

The commercial wasabi pastes commonly used for food preparation contain a homologous compound of chemosensory isothiocyanates (ITCs) that elicit an ...

integration-animal-behavioral-assessment-convolutional-neural-network

Research

JoVE Journal

Behavior

301 Views.  Hong Kong Baptist University. This article describes a set of methods for measuring the suppressive ability of sniffing alcoholic beverages on the wasabi-elicited stinging sensation.

Video: Integration of Animal Behavioral Assessment and Convolutional Neural Network to Study Wasabi-Alcohol Taste-Smell Interaction

Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant.  The NICU Network Neurobehavioral Scale ...

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

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 ...

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

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 ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...