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Abstract

Introduction

Protocol

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Behavior

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

Published: October 6th, 2023

DOI:

10.3791/65940

1Department of Integrative Physiology, 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.

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.

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 ....

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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.......

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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.......

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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 ladder13,14. What makes our ladder walking test.......

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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.).

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NameCompanyCatalog NumberComments
Air puffDAISO
Aluminum sticksCAINZ2 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).
BlutackBostik
PlexiglassCAINZ110 cm x 20 cm, 110 cm x 10 cm, 2 parts each
Screws CAINZ

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