Name: inchworming: a novel motor stereotypy in the btbr t+ itpr3tf/j mouse model of autism
Uploaded: 2014-07-05T16:00:00
Description: Watch this Scientific Journal Video about Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism at JoVE.com

The authors are grateful for the technical and logistical assistance and expertise provided by Rose Tobias, Younghee Ahn, and David N. Ruskin. The work described here was funded by the Alberta Children&#39;s Hospital Foundation and the Alberta Children&#39;s Hospital Research Institute.

All procedures were carried out in accordance with the Canadian Animal Care Committee and approved by the University of Calgary Animal Care Committee. 
1. Animal Preparation
<ol>
	<li>Isolate mice overnight (12 hr) prior to testing. To do this, place all of the BTBR and B6 mice (P35-P40) needed for experimental analysis in cages by themselves, the day before testing.</li>
	<li>Place the isolated mice in the behavioral analysis room 30 min prior to testing so that animals wil...

<ol>
	<li>McFarlane, H. G., Kusek, G. K., Yan, M., Phoenix, J. L., Bolivar, V. J., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autism-like+behavioral+phenotypes+in+BTBR+T+tf/J+mice.">Autism-like behavioral phenotypes in BTBR T+tf/J mice.</a> Genes, Brain, and Behavior. 7, 152-163 (2008).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+mouse+behavioral+tasks+relevant+to+autistic-like+behaviors.">Designing mouse behavioral tasks relevant to autistic-like behaviors.</a> Mental Retardation and Developmental Disabilities Research Reviews. 10 (4), 248-258 (2004).</li><li>Deacon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digging+and+marble+burying+in+mice:+simple+methods+for+in+vivo+identification+of+biological+impacts.">Digging and marble burying in mice: simple methods for in vivo identification of biological impacts.</a> Nature Protocols. 1 (1), 122-124 (2006).</li><li>Thomas, A., Burant, A., Bui, N., Graham, D., Yuva-Paylor, L. A., Paylor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marble+burying+reflects+a+repetitive+and+perseverative+behavior+more+than+novelty-induced+anxiety.">Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety.</a> Psychopharmacology. 204, 361-373 (2009).</li><li>Dudek, B. C., Adams, N., Boice, R., Abbott, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+influences+on+digging+behaviours+in+mice+(Mus+musculus)+in+laboratory+and+seminatural+settings.">Genetic influences on digging behaviours in mice (Mus musculus) in laboratory and seminatural settings.</a> Journal of Comparative Psychology. 97 (3), 249-259 (1983).</li><li>Webster, D., Williams, M. H., Owens, R., Geiger, V., Dewsbury, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digging+behavior+in+12+taxa+of+muriod+rodents.">Digging behavior in 12 taxa of muriod rodents.</a> Animal Learning and Behavior. 9 (2), 173-177 (1981).</li><li>Pobbe, R. H., Pearson, B. L., Defensor, E., Bolivar, V. J., Blanchard, D. C., Blanchard, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+social+behaviours+of+C57BL/6J+versus+BTBR+inbred+mouse+strains+in+the+visible+burrow+system.">Expression of social behaviours of C57BL/6J versus BTBR inbred mouse strains in the visible burrow system.</a> Behavioural Brain Research. 214, 443-449 (2010).</li><li>Lewis, M. H., Tanimura, Y., Lee, L., Bodfish, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+restricted+repetitive+behavior+in+autism.">Animal models of restricted repetitive behavior in autism.</a> Behavioural Brain Research. 176, 66-74 (2007).</li><li>Silverman, J. L., Yang, M., Lord, C., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+phenotyping+assays+for+mouse+models+of+autism.">Behavioural phenotyping assays for mouse models of autism.</a> Nature Reviews. 11, 490-502 (2010).</li><li>Deacon, R., Rawlins, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+lesions,+species-typical+behaviours+and+anxiety+in+mice.">Hippocampal lesions, species-typical behaviours and anxiety in mice.</a> Behavioural Brain Research. 156, 241-249 (2005).</li><li>Pearson, B. L., 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=Motor+and+cognitive+sterotypies+in+the+BTBR+T+tf/J+mouse+model+of+autism.">Motor and cognitive sterotypies in the BTBR T+tf/J mouse model of autism.</a> Genes, Brain, and Behavior. 10, 228-235 (2011).</li><li>Pobbe, R. H., Defensor, E., Pearson, B. L., Bolivar, V. J., Blanchard, D. C., Blanchard, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+and+social+anxiety+in+the+BTBR+T++tf/J+mouse+strain.">General and social anxiety in the BTBR T+ tf/J mouse strain.</a> Behavioural Brain Research. 216 (1), 446-451 (2011).</li></ol>

Inchworming is a novel measure of repetitive behavior that can be used to study many disorders that exhibit stereotypies such as ASD. There are 4 steps that are of critical importance to generating reliable inchworming data from the modified juvenile social interaction protocol: 1) the extended isolation period is essential to the inchworming test and should not be removed from the protocol or shortened. This requirement is specific to testing of inchworming in pairs described here. 2) The video camera needs to be approp...

Although the pathophysiology of autism spectrum disorders (ASD) remains unclear, diagnosis depends upon the presence of three broad symptom categories: abnormal social reciprocity, impairments in communication, and repetitive/stereotypic behaviors1. Most rodent models are unable to simultaneously reproduce all three symptoms in a single animal. Thus, models that can reliably simulate some or all aspects of the disorder hold great value for the development of efficacious preventative and therapeutic strategies. One model that does display all three aspects of ASD is the BTBR mouse1,2.
In combination with ultrasonic vocalizations and social approach assays, the juvenile social interaction test is commonly used to characterize mouse models of pediatric ASD; these tests measure decreased and abnormal social behaviors which represent core features of ASD. In addition to the juvenile social interaction test, many other tests have been used to measure stereotypic behaviors in animal models of ASD. For example, the marble burying test, which examines the tendency of an animal to displace bedding material using the snout and forepaws in effort to cover an object, is thought to measure compulsive stereotypic behaviors that occur in response to aversive stimuli3,4. Accordingly, digging tests have been implemented to quantify this repetitive behavior in ASD animal models and different species of rodents3,5-7. Digging behaviors are defined as obvious directed actions of the snout or paws to displace bedding materials and have been hypothesized to be a sensitive parameter of repetitive motor behavior4.
Here, we report a novel type of digging, which we have termed &#34;inchworming,&#34; in the BTBR strain of mice. The characterization of this novel behavior may provide a robust measure of a repetitive motor behavior that may be used for further validation of rodent models such as ASD, Huntington&#39; Disease or obsessive compulsive behavior8,9. We define &#34;inchworming&#34; as at least one synchronous movement of the fore- and hind-limbs inwards followed by a synchronous movement outwards which leads to the displacement of bedding material. This differs from typical digging behavior in many ways. First, the inchworming behavior is usually observed as a combination of episodes rather than a single isolated event. In contrast, digging may occur as single coordinated movement of either the hind or fore paws3. In addition, inchworming movements tend to occur horizontally across the enclosure floor as opposed to the stationary positions that are characteristic of digging behaviors. Finally, the inchworming behavior described below occurs frequently in the BTBR inbred strain of mice but is rarely identified in the B6 control strain, whereas typical digging behaviors are common in B6 and BTBR strains of mice10. The inchworming test procedures outlined in this study represent modifications of a standard juvenile social interaction protocol1.
Given that marble burying, digging, and juvenile play have established value for measuring abnormal behavior in mouse models of ASD, the inchworming behavior complements these previously defined measurements of stereotypic behaviors. In particular, the inchworming behavior provides a useful measure of lower order motor stereotypies that are commonly identified in ASD8,11. In addition, this new inchworming procedure may provide researchers with an additional valuable tool for the analysis of repetitive motor behaviors relevant to developmental disorders such as ASD.

<table border="1">
 <tbody>
 <tr>
 <td>Material Name </td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>30 cm X 30 cm Plexiglas box with lid</td>
 <td></td>
 <td></td>
 <td>Can be constructed </td>
 </tr>
 <tr>
 <td>IR Camera </td>
 <td>Survshop (Calgary, Alberta)</td>
 <td>Sony CCD Camera DP955V- 30' Infrared Armor Dome </td>
 <td>Find a camera with high-resolution and zoom capabilities</td>
 </tr>
 <tr>
 <td>DVR</td>
 <td>Supercircuits (Austin, Texas)</td>
 <td>BLACK Enterprise-Class 4-Channel H.264 security DVR with DVD Burner</td>
 <td>High data capacity</td>
 </tr>
 <tr>
 <td>Sawdust</td>
 <td></td>
 <td></td>
 <td>Same as bedding material </td>
 </tr>
 </tbody>
 </table>

inchworming: a novel motor stereotypy in the btbr t+ itpr3tf/j mouse model of autism

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T+ Itpr3tf/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as &#34;inchworming,&#34; was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.

The modified version of the juvenile play test described here can be used to observe and analyze the novel repetitive digging behavior termed inchworming. Inchworming is exhibited to a far greater extent by BTBR mice than B6 controls. In a typical 10&#160;min&#160;session, BTBR mice will display the distinct inchworming behavior (synchronous inward and outward movement of the paws in an effort to displace the sawdust bedding) for approximately 5 min, or half of the observation period. Furthermore, the latency to inchworm...

Watch this Scientific Journal Video about Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism at JoVE.com

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T+ Itpr3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder.

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal ...

inchworming-novel-motor-stereotypy-btbr-t-itpr3tfj-mouse-model

Research

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Behavior

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

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Mouse models have become increasingly popular in studies of behavioral neuroscience. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. This protocol describes the Pasta Matrix Reaching Task, which is a skilled motor task for use in mouse models of stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models ...

Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder

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We describe how to implement a battery of behavioral tasks to examine the processing and integration of sensory stimuli in children with ASD. The goal is to characterize individual differences in temporal processing of simple auditory and visual stimuli and relate these to higher order perceptual skills like speech perception.

In addition to impairments in social communication and the presence of restricted interests and repetitive behaviors, deficits in sensory processing are ...

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

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Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals. RRBs are commonly split into lower-order and higher-order components, with lower order RRBs consisting of motor stereotypies and higher order RRBs consisting of perseverative and sequencing behaviors. Higher order RRBs are challenging to model in mice. Current assays for RRBs in mice focus primarily on the lower order components, making basic biomedical research into potential treatments or interventions for higher-order RRBs difficult. Here we describe a new assay, novel object exploration. This assay uses a basic open-field arena with four novel objects placed around the perimeter. The test mouse is allowed to freely explore the arena and the order in which the mouse investigates the novel objects is recorded. From these data, patterned sequences of exploration can be identified, as can the most preferred object for each mouse. The representative data shared here and past results using the novel object exploration assay illustrate that inbred mouse strains do demonstrate different behavior in this assay and that strains with elevated lower order RRBs also show elevated patterned behavior. As such, the novel object exploration assay appears to possess good face validity for higher order RRBs in humans and may be a valuable assay for future studies investigating novel therapeutics for ASD.

Higher order restricted, repetitive behaviors (RRBs) disrupt the lives of affected individuals. These behaviors are challenging to model in rodents, making basic biomedical research into potential treatments or interventions for RRBs difficult. Here we describe novel object exploration as a potential assay for higher order RRBs in mice.

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals.  RRBs are ...

The "Motor" in Implicit Motor Sequence Learning: A Foot-stepping Serial Reaction Time Task

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We introduce the foot-stepping serial reaction time (SRT) task. This modified SRT task, complementing the classic SRT task that involves only finger-pressing movement, better approximates daily sequenced activities and allows researchers to study the dynamic processes underlying discrete response measures and disentangle the explicit process operating in implicit sequence learning.

This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that ...

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor deficits. Motor impairments were manually quantified through an open field grid test to measure the gross motor function and through a ladder test to measure the fine motor function. Here, we discuss a technique for the automated quantification of the video-recorded tests using our custom Capadona Behavioral Video Analysis System: Grid and Ladder Test, or BVAS. Leveraging simple and readily available coding software (see the Table of Materials), this program allows for the tracking of a single animal on both the open field grid and the ladder tests. In open field grid tracking, the code thresholds the video for intensity, tracks the position of the rat over the 3 min duration of the grid test, and analyzes the path. It then computes and returns measurements for the total distance traveled, the maximum velocity achieved, the number of left- and right-handed turns, and the total number of grid lines crossed by the rat. In ladder tracking, the code again thresholds the video for intensity, tracks the movement of the rat across the ladder, and returns calculated measurements including the time it took the rat to cross the ladder, the number of paw slips occurring below the plane of the ladder rungs, and the incidence of failures due to stagnation or reversals. We envision that the BVAS developed here can be employed for the analysis of motor function in a variety of applications, including many injury or disease models.

The current study aimed to automate the quantification of motor deficits in rats. The initial evaluation model assesses motor loss resulting from an intracortical microelectrode implantation in the motor cortex. We report on the development and use of a tracking algorithm using easily adaptable, simple, and readily available coding software.

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor ...

Using the Race Model Inequality to Quantify Behavioral Multisensory Integration Effects

Multisensory integration research investigates how the brain processes simultaneous sensory information. Research on animals (mainly cats and primates) and humans reveal that intact multisensory integration is crucial for functioning in the real world, including both cognitive and physical activities. Much of the research conducted over the past several decades documents multisensory integration effects using diverse psychophysical, electrophysiological, and neuroimaging techniques. While its presence has been reported, the methods used to determine the magnitude of multisensory integration effects varies and typically faces much criticism. In what follows, limitations of previous behavioral studies are outlined and a step-by-step tutorial for calculating the magnitude of multisensory integration effects using robust probability models is provided.

The current study aims to provide a step-by-step tutorial for calculating the magnitude of multisensory integration effects in an effort to facilitate the production of translational research studies across diverse clinical populations.

Multisensory integration research investigates how the brain processes simultaneous sensory information. Research on animals (mainly cats and primates) ...