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

Our design drew on the past study by Metz and Whishaw, who reported the utility of "the ladder-rung walking test"^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 ladder^13,14. 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. "The zigzag ladder test" (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 "horizontal ladder test" (Figure 2C right) and stepping on the low rungs is considered a "misstep." Moreover, the ladder built by Cupid et al. can assess motor learning because a rung raised above the stepping surface ("an unexpected object," 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. "Day 0" 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 "the foot fault scoring system of the ladder rung walking test"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 test^19,20. 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.