This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant nos. 18H03379 to N.K., 21K15340 to I.A. and 22J11280 to A.N.).

The animal experimentation protocol in the present study was approved by the Animal Care and Experimentation Committee of Gunma University. All procedures for the care and treatment of animals were performed according to the Japanese Act on the Welfare and Management of Animals and the Guidelines for the Proper Conduct of Animal Experiments issued by the Science Council of Japan. The assembly drawing of the apparatus can be found in Figure 1.
1. Ladder setup
<ol>
	<li>Horizontal ladder test (Figure 2C left)
	<ol>
		<li>Insert aluminum sticks (2 mm diameter) into holes on the plexiglass walls. 
		NOTE: The interval distance varies according to body size depending on the strain or sex of the tested mouse (see Table 1).</li>
		<li>Adjust the width of the ladder rungs by widening or narrowing the distance of the plexiglass walls. 
		NOTE: The width may vary according to the body size depending on the strain or sex of the tested mouse (see Table 1).</li>
	</ol>
	</li>
	<li>Zigzag ladder test (Figure 2C right)
	<ol>
		<li>Insert aluminum sticks (2 mm diameter) into every third hole in the horizontal and vertical directions on the plexiglass walls. 
		NOTE: The interval distance varies by body size depending on the strain or sex of the tested mouse (see Table 1).</li>
		<li>Adjust the width of the ladder rungs by widening or narrowing the distance of the plexiglass walls. 
		&#8203;NOTE: The width may vary depending on the body size, strain, or sex of the tested mouse (see Table 1).</li>
		<li>Place the dark chamber in the target site.</li>
		<li>Place the cushion (can be made of a plastic bag stuffed with buffer materials) 30 cm below the ladder setup for safety.</li>
	</ol>
	</li>
</ol>
2. Habituation (Day 0)
NOTE: It is recommended to take at least 1 day for habituating the mice to the ladder setup.
<ol>
	<li>Place the mouse at the edge of the ladder (starting point).</li>
	<li>Using an air puff, encourage the mouse to move forward on the ladder rung.</li>
	<li>Repeat until the mouse gets habituated to walk on the ladder rungs.</li>
</ol>
3. Measurement of motor performance
<ol>
	<li>Place the mouse at the edge of the ladder (starting point).</li>
	<li>Record the time to complete crossing the ladder and the number of foot slips when the mice fail to place their paws or slip after placement on the aluminum sticks (see Figure 2D). If possible, record the video to check the missteps afterward.</li>
	<li>After a 15 min interval, repeat the measurement (4-5 trials per day).</li>
	<li>Repeat the experiment for 3-4 days for assessing motor learning.</li>
</ol>
4. Analysis
<ol>
	<li>Calculate the average value of time and foot slips per day.</li>
	<li>Compare the average value between the groups.</li>
</ol>

Assessing Motor Performance in TH-Related Murine Models Using the Ladder Beam Test

<ol>
	<li>Koibuchi, N., Chin, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+action+and+brain+development.">Thyroid hormone action and brain development.</a> Trends in Endocrinology and Metabolism. 11 (4), 123-128 (2000).</li><li>Koibuchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+thyroid+hormone+on+functional+organization+in+the+cerebellum.">The role of thyroid hormone on functional organization in the cerebellum.</a> Cerebellum. 12 (3), 304-306 (2013).</li><li>Rastogi, M. V., Lafranchi, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+hypothyroidism.">Congenital hypothyroidism.</a> Orphanet Journal of Rare Diseases. 5, 17 (2010).</li><li>Ishii, S., Amano, I., Koibuchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+thyroid+hormone+in+the+regulation+of+cerebellar+development.">The role of thyroid hormone in the regulation of cerebellar development.</a> Endocrinology and Metabolism (Seoul). 36 (4), 703-716 (2021).</li><li>Dunham, N. W., Miya, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+note+on+a+simple+apparatus+for+detecting+neurological+deficit+in+rats+and+mice.">A note on a simple apparatus for detecting neurological deficit in rats and mice.</a> The Journal of the American Pharmacists Association. 46 (3), 208-209 (1957).</li><li>Jones, B. J., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantiative+measurement+of+motor+inco-ordination+in+naive+mice+using+an+acelerating+rotarod.">The quantiative measurement of motor inco-ordination in naive mice using an acelerating rotarod.</a> The Journal of Pharmacy and Pharmacology. 20 (4), 302-304 (1968).</li><li>Lubrich, C., Giesler, P., Kipp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+behavioral+deficits+in+the+cuprizone+model:+Validity+of+the+rotarod+test+paradigm.">Motor behavioral deficits in the cuprizone model: Validity of the rotarod test paradigm.</a> International Journal of Molecular Sciences. 23 (19), 11342 (2022).</li><li>Courchesne, E., Allen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+and+preparation,+fundamental+functions+of+the+cerebellum.">Prediction and preparation, fundamental functions of the cerebellum.</a> Learning and Memory. 4 (1), 1-35 (1997).</li><li>Ninomiya, 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=Long-term+depression-inductive+stimulation+causes+long-term+potentiation+in+mouse+purkinje+cells+with+a+mutant+thyroid+hormone+receptor.">Long-term depression-inductive stimulation causes long-term potentiation in mouse purkinje cells with a mutant thyroid hormone receptor.</a> The Proceedings of National Academy of Sciences of the United States of America. 119 (45), e2210645119 (2022).</li><li>Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+long-term+depression:+Characterization,+signal+transduction,+and+functional+roles.">Cerebellar long-term depression: Characterization, signal transduction, and functional roles.</a> Physiological Reviews. 81 (3), 1143-1195 (2001).</li><li>Miyata, 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=Deficient+long-term+synaptic+depression+in+the+rostral+cerebellum+correlated+with+impaired+motor+learning+in+phospholipase+c+beta4+mutant+mice.">Deficient long-term synaptic depression in the rostral cerebellum correlated with impaired motor learning in phospholipase c beta4 mutant mice.</a> The European Journal of Neuroscience. 13 (10), 1945-1954 (2001).</li><li>Aiba, 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=Deficient+cerebellar+long-term+depression+and+impaired+motor+learning+in+mglur1+mutant+mice.">Deficient cerebellar long-term depression and impaired motor learning in mglur1 mutant mice.</a> Cell. 79 (2), 377-388 (1994).</li><li>Metz, G. A., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+and+subcortical+lesions+impair+skilled+walking+in+the+ladder+rung+walking+test:+A+new+task+to+evaluate+fore-+and+hindlimb+stepping,+placing,+and+co-ordination.">Cortical and subcortical lesions impair skilled walking in the ladder rung walking test: A new task to evaluate fore- and hindlimb stepping, placing, and co-ordination.</a> The Journal of Neuroscience Methods. 115 (2), 169-179 (2002).</li><li>Metz, G. A., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ladder+rung+walking+task:+A+scoring+system+and+its+practical+application.">The ladder rung walking task: A scoring system and its practical application.</a> Journal of Visualized Experiments. (28), (2009).</li><li>Mendes, C. S., 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=Quantification+of+gait+parameters+in+freely+walking+rodents.">Quantification of gait parameters in freely walking rodents.</a> BMC Biology. 13 (1), 50 (2015).</li><li>Cupido, 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=The+Erasmus+ladder:+A+new+tool+for+the+automated+measurement+of+motor+performance+and+motor+learning+in+mice.">The Erasmus ladder: A new tool for the automated measurement of motor performance and motor learning in mice.</a> Conference: Measuring behavior, Wageningen. , (2005).</li><li>Sathyanesan, A., Kratimenos, P., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+neonatal+purkinje+cell+function+underlies+injury-related+learning+deficits.">Disruption of neonatal purkinje cell function underlies injury-related learning deficits.</a> The Proceedings of National Academy of Sciences of the United States of America. 118 (11), e2017876118 (2021).</li><li>Martins, L. A., Schiavo, A., Xavier, L. L., Mestriner, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+foot+fault+scoring+system+to+assess+skilled+walking+in+rodents:+A+reliability+study.">The foot fault scoring system to assess skilled walking in rodents: A reliability study.</a> Frontiers in Behavioral Neuroscience. 16, 892010 (2022).</li><li>Shimokawa, N., 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=Altered+cerebellum+development+and+dopamine+distribution+in+a+rat+genetic+model+with+congenital+hypothyroidism.">Altered cerebellum development and dopamine distribution in a rat genetic model with congenital hypothyroidism.</a> Journal of Neuroendocrinology. 26 (3), 164-175 (2014).</li><li>Amano, I., 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=Aberrant+cerebellar+development+in+mice+lacking+dual+oxidase+maturation+factors.">Aberrant cerebellar development in mice lacking dual oxidase maturation factors.</a> Thyroid. 26 (5), 741-752 (2016).</li></ol>

The authors have no conflicts of interest to disclose.

Our design drew on the past study by Metz and Whishaw, who reported the utility of &#34;the ladder-rung walking test&#34;13,14. They designed the ladder-rung walking test to assess skilled walking and measure both forelimb and hind limb placing, stepping, and inter-limb coordination by requiring animals to walk from a starting point to a goal on a horizontal ladder13,14. What makes our ladder walking test...

Thyroid hormone (TH) is indispensable for brain development1. In particular, its role in the cerebellum is critical because TH deficiency in early life causes aberrant cerebellar development1,2. For example in congenital hypothyroidism, patients display a series of neurological retardation including cognitive and motor deficits3. To unveil the role of TH in cerebellar functional development, some studies have limited TH deficiency in a cerebellar cell-specific manner4. However, compared to generalized congenital hypothyroid mice, in which all tissues and cells are affected by TH deficiency, such cerebellar-specific models display so subtle ataxia that the conventional behavior tests, such as rotarod, footprint, and balance beam tests, barely detect the differences. Thus, to fully investigate the TH effects on cerebellar function, a new assessment tool is needed to detect a subtle change in the motor coordination of model mice.
The rotarod test is the most common tool for assessing motor coordination, originally developed by Dunham and Miya5 and later applied to an accelerating version by Jones and Roberts6. The latency to fall from the rotating rod is interpreted as the test for motor coordination, and its simplicity and conciseness make it commonly used among behavioral researchers studying motor function7. However, the ease of use of this test is a double-edged sword. Because the rod automatically rotates, mice can cling to and stay on the rotating rod without moving. Furthermore, mice may intend to fall off rather than keep balancing on the rotating rod. In either situation, the validity and reliability of the test are questionable for assessing &#34;pure motor coordination&#34;7. In other words, it does not accurately target the cerebellar function and involves other factors such as muscle strength for gripping.
Instead of the conventional tools for motor coordination assessment, here we present a novel behavior test called the &#34;ladder beam test,&#34; which is recently developed in our laboratory. The horizontal ladder walking test was designed to assess cerebellum-related complex motor abilities: feed-forward prediction and integration of motion8. The test device was composed of four pieces of plexiglass with holes (Figure 1). The four plates were connected in parallel by screws and sticks inserted into the holes on the plates. Two outer plates were used to stabilize the device and two inner plates were used to design the various kinds of ladder rungs (Figure 2C). The width of the rung was adjusted depending on the animal size to minimize the animals&#39; moving backward (Figure 2B). The distance from the start point to the goal was 110 cm. The device was located 60 cm above the bench and a safety cushion was set under the device (Figure 2A). The dark chamber was put near the goal to motivate the animals to move toward the goal (Figure 2A).
We examined the TH effects on cerebellar functional development by using transgenic mice expressing dominant-negative TH receptor (TR) in cerebellar Purkinje cells (Mf-1/FVB mice). In both rotarod and ladder beam tests, we observed cerebellar ataxic phenotype in Mf-1/FVB mice; however, the ladder beam test succeeded in detecting more significant differences than the the rotarod test (Figure 3). In addition, motor learning ability can be more thoroughly assessed in ladder beam test (Figure 3B,C). As a cellular background of such a behavioral phenotype, the induction of long-term depression (LTD) was inhibited and instead, long-term potentiation (LTP) was induced following an LTD-inductive stimulation in Mf-1/FVB Purkinje cells9. LTD is essential for motor coordination and motor learning in the cerebellum10. Many studies have reported motor deficits and inhibition of LTD in knockout or mutated mice for key regulator genes in cerebellar function, however, no studies have ever reported the induction of LTP following an LTD-inductive stimulation11,12. Taken together, this phenomenon may be unique to Mf-1/FVB mice or TH-deficient mice (the same phenomenon was observed in adult-onset hypothyroid mice), suggesting that TH regulates cerebellar function differently from the other key proteins. If so, it is plausible that mice with abnormal TH action do not display cerebellar ataxia in the same way as other model mice. This again emphasizes the need for a specific method for assessing TH effects on cerebellar function. This paper presents a novel protocol to investigate TH effects on cerebellar function using the newly-built ladder beam test.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Air puff</td>
			<td>DAISO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum sticks</td>
			<td>CAINZ</td>
			<td></td>
			<td>2 mm diameter, number of sticks may vary depending on the ladder design. Aproximately 30 sticks may be required to build the horizontal ladder (4 cm interval).</td>
		</tr>
		<tr>
			<td>Blutack</td>
			<td>Bostik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plexiglass</td>
			<td>CAINZ</td>
			<td></td>
			<td>110 cm x 20 cm, 110 cm x 10 cm, 2 parts each</td>
		</tr>
		<tr>
			<td>Screws&#160;</td>
			<td>CAINZ</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a versatile, behavioral method to investigate thyroid hormone effects on cerebellar function

Thyroid hormone (TH) action is essential during the development of the central nervous system, including the cerebellum. In case of TH deficiency in early life such as congenital hypothyroidism, patients display neurological disorders such as cognitive retardation and motor deficits. There are various studies using mouse models with tissue- or cell-specific TH deficiency to investigate the role of TH in the cerebellum. Compared to generalized congenital hypothyroid mice, cerebellar cell-specific TH-deficient mice display milder and subtler ataxic features, making the assessment of motor function difficult when using conventional tests such as the rotarod test. 
Due to the need for an alternative tool to assess motor function in TH-related animal models, we developed a versatile behavioral method called the "ladder beam test," in which we can design the various ladder tests depending on the severity of ataxia in model mice. We utilized transgenic mice expressing a dominant-negative TH receptor specifically in the cerebellar Purkinje cell, a sole output neuron in the cerebellar cortex modulating motor performance. The newly-built ladder beam test successfully detected robust impairments in motor performance in the transgenic mice at a greater level compared to the rotarod test. Disruption of motor learning was also detected in the ladder beam test but not in the rotarod test. The protocol with this novel behavioral apparatus can be applied to other animal models that may show mild ataxic phenotype to examine subtle changes in cerebellar function.

In the rotarod test, Mf-1/FVB mice showed a significant decrease in the latency to fall from the rotating rod over 3 consecutive days compared to the wildtype mice, indicating impaired motor coordination (Figure 3A). However, in terms of within-group results, Mf-1/FVB mice significantly improved their performance from Day 1 to Day 3, suggesting the preservation of motor learning (Figure 3A).
Both hori...

Watch this Scientific Journal Video about A Versatile, Behavioral Method to Investigate Thyroid Hormone Effects on Cerebellar Function at JoVE.com

Author Spotlight: Accurately Assessing Thyroid Hormone-Driven Motor Alterations in Mouse

Here we present a protocol for a versatile behavior test developed recently, the ladder beam test. This test has the advantage of detecting subtle cerebellar ataxia caused by a defect of thyroid hormone action in the central nervous system over the conventional behavior tests assessing motor performance.

A Versatile, Behavioral Method to Investigate Thyroid Hormone Effects on Cerebellar Function

Thyroid hormone (TH) action is essential during the development of the central nervous system, including the cerebellum. In case of TH deficiency in early ...

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297 Views.  Gunma University Graduate School of Medicine. Here we present a protocol for a versatile behavior test developed recently, the ladder beam test. This test has the advantage of detecting subtle cerebellar ataxia caused by a defect of thyroid hormone action in the central nervous system over the conventional behavior tests assessing motor performance. 

Video: Author Spotlight: Accurately Assessing Thyroid Hormone-Driven Motor Alterations in Mouse

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

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Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

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A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

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The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

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Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, the traditional ways to monitor behavioral changes in dogs are complicated and not efficient. In the current clinical evaluation, a new, simple monitoring system was used to assess the effectiveness of a specific diet in positively modulating the intense and restless activity of 24 dogs of different ages and breeds. This protocol describes how to easily and rapidly evaluate improvement in a set of symptoms related to generalized anxiety by using a specific sensor, a mobile phone app, a wireless router, and a computer. The results showed that dogs treated with specific diets showed significant improvement in the times spent active and at rest after 10 days (p &lt; 0.01 and p &lt; 0.05, respectively). These dogs also showed an overall significant improvement in clinical and behavioral symptoms. A specific sensor, along with its related hardware, was demonstrated to successfully monitor behavioral changes relating to movement in dogs.

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Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants socially interact. Electroencephalography (EEG) power is an advantageous technique to assess infant functional neural activation. However, many studies record infant EEG only during one baseline condition. This protocol describes a paradigm that is designed to comprehensively assess infant EEG activity in both social and nonsocial contexts as well as tease apart how different types of social inputs differentially relate to infant EEG. The within-subjects paradigm includes four controlled conditions. In the nonsocial condition, infants view objects on computer screens. The joint attention condition involves an experimenter directing the infant's attention to pictures. The joint attention condition includes three types of social input: language, face-to-face interaction, and the presence of joint attention. Differences in infant EEG between the nonsocial and joint attention conditions could be due to any of these three types of input. Therefore, two additional conditions (one with language input while the experimenter is hidden behind a screen and one with face-to-face interaction) were included to assess the driving contextual factors in patterns of infant neural activation. Representative results demonstrate that infant EEG power varied by condition, both overall and differentially by brain region, supporting the functional nature of infant EEG power. This technique is advantageous in that it includes conditions that are clearly social or nonsocial and allows for examination of how specific types of social input relate to EEG power. This paradigm can be used to assess how individual differences in age, affect, socioeconomic status, and parent-infant interaction quality relate to the development of the social brain. Based on the demonstrated functional nature of infant EEG power, future studies should consider the role of EEG recording context and design conditions that are clearly social or nonsocial.

This novel protocol is designed to assess the neural bases of social interaction in infants. The paradigm is designed to tease apart how various social inputs such as language, joint attention, and face-to-face interaction relate to infant neural activation. Infant EEG power is recorded during both social and nonsocial conditions.

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants ...

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

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) ...

Using a Virtual Reality Walking Simulator to Investigate Pedestrian Behavior

To cross a road successfully, individuals must coordinate their movements with moving vehicles. This paper describes use of a walking simulator in which people walk on a treadmill to intercept gaps between two moving vehicles in an immersive virtual environment. Virtual reality allows for a safe and ecologically varied investigation of gap crossing behavior. Manipulating the initial starting distance can further the understanding of a participant&#8217;s speed regulation while approaching a gap. The speed profile may be assessed for various gap crossing variables, such as initial distance, vehicle size, and gap size. Each walking simulation results in a position/time series that can inform how velocity is adjusted differently depending on the gap characteristics. This methodology can be used by researchers investigating pedestrian behavior and behavioral dynamics while employing human participants in a safe and realistic setting.

This protocol describes use of a walking simulator that serves as a safe and ecologically valid method to study pedestrian behavior in the presence of moving traffic.

To cross a road successfully, individuals must coordinate their movements with moving vehicles. This paper describes use of a walking simulator in which ...