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

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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 different from theirs is the versatility; the experimenter can change the ladder design and thus the complexity of the test by changing the location of the aluminum sticks on the walls with 5 holes in the vertical direction and 100 holes in the horizontal direction. &#34;The zigzag ladder test&#34; (Figure 2C right and Figure 3C) allows the experimenter to examine more complex movements that cannot be evaluated by the rotarod test. Such complex movements require the coordination of limb movements and likely reflect the cerebellar function15. The concern about neuromuscular strength affecting the performance in the zigzag ladder test can be excluded by performing the grip strength test (Figure 3D).
A more advanced tool for assessing cerebellar-specific function is presented by Cupid and colleagues16. The similarity to our model is that their ladder consists of high and low rungs, making it zigzag in the vertical direction. However, mice are required to walk on the higher rungs, which is equivalent to the &#34;horizontal ladder test&#34; (Figure 2C right) and stepping on the low rungs is considered a &#34;misstep.&#34; Moreover, the ladder built by Cupid et al. can assess motor learning because a rung raised above the stepping surface (&#34;an unexpected object,&#34; cued by a sound stimulus) makes mice learn to deal with it. The analysis software is built into the apparatus, allowing the automatic detection and recording of time, foot slips, and even back steps and jumps17. Although the model of Cupid and colleagues is such a handy, useful, and accurate tool for motor function, the apparatus is costly and thus, not always available to every laboratory. In contrast, our ladder beam test apparatus can be easily hand-made (requiring only three steps) and can be modified when building without considerable expense. The cost-effectiveness is another advantage of our ladder beam test.
Before beginning the test, it is important to determine the proper spacing of the ladder rungs depending on the body size, sex, and age of the mice. If the space is too short, there is no difference from the normal walking test or a gait pattern test, for example, the footprint test. If the space is too long, it is difficult for the mice to reach the next rung and walk on the ladder rungs. In either situation, the test would be unable to assess motor coordination and thus, cerebellar function. The ideal space is likely the distance that mice can continuously walk without any unnecessary efforts made by parts of the body other than the cerebellum. Moreover, the width of the ladder rungs should be carefully chosen. If it is too wide compared to the body size of the mice, mice can easily turn around and come back to the starting point. It is preferred to adjust the plexiglass walls on both sides so that they get close to mice but never touch them and disrupt their walking (Figure 2B). Adjusting the parts of the apparatus for each mouse is a critical step in establishing the validity of the test.
Generally, mice prefer dark and narrow conditions. Therefore, the dark chamber located at the goal site should motivate mice to complete the task. In case mice refuse to move forward or suddenly stop in the middle of the ladder, applying air pressure to mice from the back helps them start moving. However, too much air pressure may make mice stressed out or nervous, which may affect the test result. Habituating mice on the novel ladder rungs should be conducted well before starting the experiment. It usually takes 1 day to get mice habituated to a novel condition (being on the elevated ladder rungs). The training session may be cut off before the mice can improve their performance to prevent the training effect. We recommend separating the habituation day and test day to avoid the effect of fatigue in mice after the training session.
Our ladder beam test certainly has a few limitations. First, it is time-consuming to conduct the experiment. &#34;Day 0&#34; should be prepared for the habituation phase. In general, it takes ~15-30 min to get mice habituated and walk on the elevated ladder. If the experimenter has a large group, it may take an entire day to complete the habituation. Even after the habituation, the treated mice, especially those with motor deficits, may take a long time to complete crossing the ladder.
While conducting the test, the experimenters need to watch the mice to track the time and the number of foot slips. Alternatively, video recording is recommended for post experiment analysis. Recorded videos can also be analyzed using &#34;the foot fault scoring system of the ladder rung walking test&#34;18. Second, the throughput performance of the test is quite low. As mentioned earlier, the test requires a lot of time to conduct. In addition, only one mouse can be used per trial. In contrast, the rotarod test has higher throughput performance as the apparatus has 4-5 lanes and allows the experimenter to conduct the test with a group of mice at once. With the automatic tracking system such as the apparatus built by Mendes et al.15, it allows the experimenter to make several parallel lanes of a ladder and conduct the experiment with more than one sample.
Nevertheless, the present study emphasizes the superior part of the ladder beam test to the rotarod test using the transgenic mouse model expressing a dominant-negative TR specifically in the cerebellar Purkinje cells. Previous studies have indicated that congenital hypothyroid mice or transgenic mice with TH deficiency display impairment in motor coordination by the rotarod test19,20. However, since they conducted the rotarod test only for 1 day, the TH effects on motor learning have been neglected. Motor learning is also important in the cerebellar function, requiring the proper synaptic plasticity in Purkinje cells10. The ladder beam test succeeded in detecting the impairment in not only motor coordination but also motor learning in Mf-1/FVB mice, whereas the rotarod test did not (Figure 3).
TH action in the cerebellum has yet to be elucidated. For future studies, the test is expected to be used to assess the cerebellar function of the various animal models of hypothyroidism or TH diseases. Furthermore, this test can be applied to other animal models that may show mild ataxic phenotype to examine subtle changes in cerebellar function.

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><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&amp;nbsp;</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 horizontal and zigzag ladder beam tests detected robust impairment in motor coordination in Mf-1/FVB mice as they spent significantly longer time to complete crossing the ladder and made more foot slips compared to wildtype mice (Figure 3B,C). More importantly, the score did not improve for 3 days in Mf-1/FVB mice, whereas wildtype mice showed improvement (Figure 3B,C).&#160;Unlike the rotarod test, Mf-1/FVB mice failed to exhibit any motor learning in the ladder beam test. These results indicate that the ladder beam test is superior to the conventional rotarod test for assessing not only motor coordination but also motor learning. There was no change in grip strength between wildtype and Mf-1/FVB mice (Figure 3D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65940/65940fig01.jpg" /> 
Figure 1: Assembly drawing of ladder beam test apparatus. 1. Cut out the plexiglass (110 cm x 20 cm, 110 cm x 10 cm). Drill a 2-mm hole (5 in vertical, 100 in horizontal, 1 cm intervals). 2. Insert thick metal sticks into holes at four corners to connect and stabilize the plexiglasses. 3. Inert aluminum sticks into holes to set up the ladder test. Stick adhesive rubber to the inserted part for stabilization. <a href="https://www.jove.com/files/ftp_upload/65940/65940fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65940/65940fig02.jpg" /> 
Figure 2: Experimental apparatus for the ladder beam test. (A) A side-view image of a custom-made apparatus for the ladder beam test. The safety cushion for mice was made of a cardboard box covered with a shock-absorbing plastic bag. (B) Back and top views of the ladder apparatus are shown with an adult-sized mouse. (C) Aluminum sticks can be inserted into holes on the wall to design the ladder test. Horizontal and zigzag ladder tests are shown as examples. (D) An image describes what the foot slip looks like on the ladder rung. <a href="https://www.jove.com/files/ftp_upload/65940/65940fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65940/65940fig03.jpg" /> 
Figure 3. Comparison between the rotarod and the ladder beam tests in assessing motor coordination and motor learning using Mf-1/FVB mice. (A) The results of the rotarod test for 3 consecutive days are shown for each group (wildtype, open circles; Mf-1/FVB mice, filled triangles). Mf-1/FVB mice stayed on the rotating rod for less time than the wildtype mice (**p &#60; 0.01), although both groups improved their performance on Day 3 compared to Day 1 (# p &#60; 0.001). (B) Horizontal ladder beam test. (Upper) Mf-1/FVB mice took much longer times to complete the task than wildtype mice (****p &#60; 0.0001) and no improvement in the performance at Day 3 compared to Day 1, in contrast to wildtype mice (# p &#60; 0.01). (lower) Mf-1/FVB mice made more foot slips than wildtype mice (****p &#60; 0.0001). Only wildtype mice improved performance (# p &#60; 0.01). (C) Zigzag ladder test. Mf-1/FVB mice spent a longer time crossing the ladder than the wildtype mice (***p &#60; 0.001). (Upper) Mf-1/FVB mice spent a longer time completing the task than the wildtype mice (****p &#60; 0.0001). Only wildtype mice improved performance from Day 1 to 3 (#p &#60; 0.01). (D) No change was observed in grip strength between groups (p = 0.8998). Data are expressed as mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/65940/65940fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">C57bl/6J</td>
			<td colspan="2">FVB/NJcl</td>
		</tr>
		<tr>
			<td>Age</td>
			<td>3-6 weeks old</td>
			<td>7 weeks old ~</td>
			<td>3-6 weeks old</td>
			<td>7 weeks old~</td>
		</tr>
		<tr>
			<td rowspan="3">Male</td>
			<td>Horizontal: 1-2 cm</td>
			<td>Horizontal: 3-4 cm</td>
			<td>Horizontal: 2cm</td>
			<td>Horizontal: 4-5 cm</td>
		</tr>
		<tr>
			<td>Zigzag: 2 cm</td>
			<td>Zigzag: 4 cm</td>
			<td>Zigzag: 3 cm</td>
			<td>Zigzag: 4 cm</td>
		</tr>
		<tr>
			<td>Width: 3 cm</td>
			<td>Width: 4 cm</td>
			<td>Width: 4 cm</td>
			<td>Width: 5 cm</td>
		</tr>
		<tr>
			<td rowspan="3">Female</td>
			<td>Horizontal: 1-2 cm</td>
			<td>Horizontal: 3-4 cm</td>
			<td>Horizontal: 2 cm</td>
			<td>Horizontal: 3-4 cm</td>
		</tr>
		<tr>
			<td>Zigzag: 2 cm</td>
			<td>Zigzag: 3 cm</td>
			<td>Zigzag: 3 cm</td>
			<td>Zigzag: 3-4 cm</td>
		</tr>
		<tr>
			<td>Width: 3 cm</td>
			<td>Width: 3-4 cm</td>
			<td>Width: 4 cm</td>
			<td>Width: 4-5 cm</td>
		</tr>
	</tbody>
</table>
Table 1: Recommended intervals and widths for the experiment. The distance of intervals and the width may vary depending on the strain, age, and sex of the tested mouse. The example of C57bl/6J mice and FVB/NJcl mice are shown.

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 ...

a-versatile-behavioral-method-to-investigate-thyroid-hormone-effects

Nanyang Technological University

Research

JoVE Journal

Behavior

313 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.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson&#8217;s disease.

Exercise may alleviate behavioral impairments associated with neurodegenerative diseases. Studies of the mechanisms underlying exercise-mediated effects have increased in disease models, but the neurobiological background accompanying these diseases, specifically aging, is likely not considered. Here, we illustrate measures to increase the likelihood of a successful exercise paradigm in aged rats.

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these ...

Modulating Cognition Using Transcranial Direct Current Stimulation of the Cerebellum

Numerous studies have emerged recently that demonstrate the possibility of modulating, and in some cases enhancing, cognitive processes by exciting brain regions involved in working memory and attention using transcranial electrical brain stimulation. Some researchers now believe the cerebellum supports cognition, possibly via a remote neuromodulatory effect on the prefrontal cortex. This paper describes a procedure for investigating a role for the cerebellum in cognition using transcranial direct current stimulation (tDCS), and a selection of information-processing tasks of varying task difficulty, which have previously been shown to involve working memory, attention and cerebellar functioning. One task is called the Paced Auditory Serial Addition Task (PASAT) and the other a novel variant of this task called the Paced Auditory Serial Subtraction Task (PASST). A verb generation task and its two controls (noun and verb reading) were also investigated. All five tasks were performed by three separate groups of participants, before and after the modulation of cortico-cerebellar connectivity using anodal, cathodal or sham tDCS over the right cerebellar cortex. The procedure demonstrates how performance (accuracy, verbal response latency and variability) could be selectively improved after cathodal stimulation, but only during tasks that the participants rated as difficult, and not easy. Performance was unchanged by anodal or sham stimulation. These findings demonstrate a role for the cerebellum in cognition, whereby activity in the left prefrontal cortex is likely dis-inhibited by cathodal tDCS over the right cerebellar cortex. Transcranial brain stimulation is growing in popularity in various labs and clinics. However, the after-effects of tDCS are inconsistent between individuals and not always polarity-specific, and may even be task- or load-specific, all of which requires further study. Future efforts might also be guided towards neuro-enhancement in cerebellar patients presenting with cognitive impairment once a better understanding of brain stimulation mechanisms has emerged.

Transcranial direct current stimulation (tDCS) over the cerebellum exerts a remote effect on the prefrontal cortex, which can modulate cognition and performance. This was demonstrated using two information-processing tasks of varying complexity, whereby only cathodal tDCS improved performance when the task was difficult, but not easy.

Numerous studies have emerged recently that demonstrate the possibility of modulating, and in some cases enhancing, cognitive processes by exciting brain ...

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).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ataxias and other movement disorders marked by progressive degeneration of the cerebellum. The goal of this protocol is to assess mitochondrial dysfunction in Spinocerebellar ataxia type 1 (SCA1) and assess the efficacy of pharmacological targeting of metabolic respiration via the water-soluble compound succinic acid to slow disease progression. This approach is applicable to other cerebellar diseases and can be adapted to a host of water-soluble therapies.
Ex vivo analysis of mitochondrial respiration is used to detect and quantify disease-related changes in mitochondrial function. With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic booster succinic acid by dissolving this compound directly into the home cage drinking water. The ability of the drug to pass the blood brain barrier can be deduced using high performance liquid chromatography (HPLC). The efficacy of these compounds can then be tested using multiple behavioral paradigms including the accelerating rotarod, balance beam test and footprint analysis. Cytoarchitectural integrity of the cerebellum can be assessed using immunofluorescence assays that detect Purkinje cell nuclei and Purkinje cell dendrites and soma. These methods are robust techniques for determining mitochondrial dysfunction and the efficacy of treatment with water-soluble compounds in cerebellar neurodegenerative disease.

We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases.

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ...

Medicine

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Neuroscience

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

Immunology and Infection

Imaging of Thyroid Hormone Action Indicator Mice to Assess Endocrine Disruption

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that can disturb hormone production or action. Many environmental pollutants are EDCs, representing an emerging threat to both human health and agricultural production. This has led to an increased demand for proper test systems to examine the effects of potential EDCs. However, current methodologies face challenges. Most test systems use endogenous markers regulated by multiple, often complex regulatory processes, making it difficult to distinguish direct and indirect effects. Moreover, in vitro test systems lack the physiological complexity of EDC metabolism and pharmacokinetics in mammals. Additionally, exposure to environmental EDCs usually involves a mixture of multiple compounds, including in vivo generated metabolites, so the possibility of interactions cannot be ignored. This complexity makes EDC characterization difficult. The Thyroid Hormone Action Indicator (THAI) mouse is a transgenic model that carries a TH-responsive luciferase reporter system, enabling the assessment of tissue-specific TH action. One can evaluate the tissue-specific effects of chemicals on local TH action by quantifying luciferase reporter expression in tissue samples. Furthermore, with in vivo imaging, the THAI mouse model allows for longitudinal studies on the effects of potential EDCs in live animals. This approach provides a powerful tool for testing long-term exposure, complex treatment structures, or withdrawal, as it enables the assessment of changes in local TH action over time in the same animal. This report describes the process of in vivo imaging measurements on THAI mice. The protocol discussed here focuses on developing and imaging hyper- and hypothyroid mice, which can serve as controls. Researchers can adapt or expand the treatments presented to meet their specific needs, offering a foundational approach for further investigation.

Author Spotlight: In Vivo Assessment of Thyroid Hormone Disruption Using the THAI Mouse Model

The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that ...

Synthesis and Regulation of Thyroid Hormones

Low blood levels of the thyroid hormones &#8212; triiodothyronine (T3) and thyroxine (T4) &#8212; signal the hypothalamus to release the thyrotropin-releasing hormone (TRH). TRH then reaches the pituitary gland and stimulates the release of thyroid-stimulating hormone(TSH) into the bloodstream.
Upon reaching the thyroid gland, TSH stimulates the follicular cells&#39; active uptake of iodide ions from the blood. The ions diffuse to the apical surface of the cells and are oxidized to iodine. The iodine is then added to tyrosine residues of the thyroglobulin protein stored in the lumen colloid, forming monoiodotyrosine (T1) and diiodotyrosine (T2). The coupling of one molecule of T1 with T2 forms a T3 molecule, whereas two T2 molecules combine together to create T4.
Finally, endocytosis and enzymatic cleavage release the T3 and T4 in the follicular cells. These hormones then cross into the blood vessels for transport to target tissues. An increase in blood levels of T3 and T4 temporarily inhibits the production of TRH and TSH, regulating the level of thyroid hormones in the body.

Synteza i regulacja hormonów tarczycy

Low blood levels of the thyroid hormones &#8212; triiodothyronine (T3) and thyroxine (T4) &#8212; signal the hypothalamus to release the ...

Education

JoVE Core

Anatomy and Physiology

Unit 1

The Endocrine System

KEY TERMS AND CONCEPTS

Functions of Thyroid Hormones

The thyroid hormone (TH) plays a pivotal role in the intricate orchestration of physiological processes, exerting profound effects on development, metabolism, and homeostasis throughout different life stages.
TH is indispensable for the normal development and maturation of the skeletal, muscular, and nervous systems during fetal and childhood growth. It facilitates bone mineral turnover and regulates protein synthesis in developing tissues, contributing significantly to overall growth and maturation.
TH continues its influence upon reaching adulthood, particularly on the reproductive and circulatory systems. Primarily in females, it regulates gonadal hormone secretion, impacting the reproductive system, and stimulates endocrine tissue, contributing to hormonal balance. In the circulatory system, TH promotes normal heart function, facilitates red blood cell synthesis, and enhances tissue oxygenation.
TH binds to various receptors within the cell, including cytoplasmic, nuclear, and mitochondrial receptors. Cytoplasmic receptors store TH and release it when intracellular TH levels decrease, maintaining cellular homeostasis. TH activates glycolysis and ATP production genes in the mitochondria, increasing energy synthesis. Simultaneously, TH indirectly enhances ATP production in the nucleus by controlling the synthesis rate of enzymes involved in the cellular metabolic rate.
The culmination of these actions is known as the calorigenic effect, reflecting an elevated cellular metabolic rate induced by thyroid hormone. This multifaceted role underscores the critical contribution of TH to fundamental physiological processes, ensuring the harmonious functioning of various organ systems and the maintenance of overall cellular homeostasis.