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.

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

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

To comprehensively understand the various facet of thyroid hormone involvement in the central nervous system, we investigated spectrum of perspectives, spanning the hair phenotype to molecular adulation at the single-cell level. We are also looking on connecting those different dimension of the vibration to elucidate the mechanism of thyroid hormone in brain development. The mainstream research in the field primarily centers around the molecular biology with numerous researches investigating the impact of thyroid hormone on gene expression.Nowadays, the increasing focus on behavior analysis to uncover the role of thyroid hormones, particularly within the central nervous system. This approach could explain the specific behaviors observed in certain thyroid hormone-related diseases. With the current level of available techniques, such as the rotarod test, researchers often struggle does this behavioral patterns, especially motor performance, in motor mice.This hinders the translation of reside to clinical situations. Our protocol has been designed to assess several motor alteration in mice, facilitating a clear interpretation of behavior observations.

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

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.

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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212 Views.  Gunma University Graduate School of Medicine. To comprehensively understand the various facet of thyroid hormone involvement in the central nervous system, we investigated spectrum of perspectives, spanning the hair phenotype to molecular adulation at the single-cell level. We are also looking on connecting those different dimension of the vibration to elucidate the mechanism of thyroid hormone in brain development. The mainstream research in the field primarily centers around the molecular biology with numerous researches investigating ...

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

Setting Up the Ladder Beam Test to Assess Motor Function in Mouse

Begin by setting up the ladder assembly for the Ladder Beam Test. After connecting the four parallel plexiglass containing the holes, proceed to design the various kinds of ladder rungs by inserting aluminum sticks into the holes on the plexiglass. For the Horizontal Ladder Test insert aluminum sticks into the holes on the plexiglass walls.For the Zigzag Ladder Test, insert the aluminum sticks into every third hole in the horizontal, and vertical directions on the plexiglass walls. In both cases, adjust the widths of the ladder rungs as required by widening or narrowing the distance of the plexiglass walls. Then place the dark chamber in the target site to motivate the animals to move toward the goal.Finally, place the cushion under the ladder rungs for safety.

Assessing Motor Function in the Thyroid Hormone-Related Mouse Model Using the Ladder Beam Test

Before subjecting the mouse to the ladder beam test, perform an initial habituation training on the completely assembled ladder beam test apparatus. To do so, place the mouse at the edge of the ladder designated as the starting point. Using an air puff, encourage the mouse to move forward on the ladder rung towards the goal.Repeat the training until the mouse gets habituated to walk on the ladder rungs. To perform the motor performance test, place the already habituated mouse at the starting point. Record the time the animal takes to cross the ladder, and count the number of foot slips occurring when the mouse fails to place its paws or slips after placing paws on the aluminum sticks.Calculate the average value of time and foot slips per day, and compare the average values between the different animal groups. Both horizontal and zigzag ladder beam tests detected robust impairment in motor coordination in MF-1 mice. They spent a significantly longer time crossing the ladder, as well as made more foot slips when compared to wildtype mice.More importantly, the scores did not improve after three days in MF-1 mice, whereas wildtype mice showed improvement in that period. There was no change in grip strength between wildtype and MF-1 mice.