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

Summary

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.

Abstract

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.

Introduction

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 development^1,2. 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 "pure motor coordination"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 "ladder beam test," 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' 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 stimulation^11,12. 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.

Protocol

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

1. Horizontal ladder test (Figure 2C left)
   1. 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).
   2. 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).
2. Zigzag ladder test (Figure 2C right)
   1. 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).
   2. Adjust the width of the ladder rungs by widening or narrowing the distance of the plexiglass walls.
      ​NOTE: The width may vary depending on the body size, strain, or sex of the tested mouse (see Table 1).
   3. Place the dark chamber in the target site.
   4. Place the cushion (can be made of a plastic bag stuffed with buffer materials) 30 cm below the ladder setup for safety.

2. Habituation (Day 0)

NOTE: It is recommended to take at least 1 day for habituating the mice to the ladder setup.

1. Place the mouse at the edge of the ladder (starting point).
2. Using an air puff, encourage the mouse to move forward on the ladder rung.
3. Repeat until the mouse gets habituated to walk on the ladder rungs.

3. Measurement of motor performance

1. Place the mouse at the edge of the ladder (starting point).
2. 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.
3. After a 15 min interval, repeat the measurement (4-5 trials per day).
4. Repeat the experiment for 3-4 days for assessing motor learning.

4. Analysis

1. Calculate the average value of time and foot slips per day.
2. Compare the average value between the groups.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Air puff	DAISO
Aluminum sticks	CAINZ		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).
Blutack	Bostik
Plexiglass	CAINZ		110 cm x 20 cm, 110 cm x 10 cm, 2 parts each
Screws	CAINZ