Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Summary

Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T⁺ Itpr^3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder.

Abstract

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T⁺ Itpr3^tf/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as "inchworming," was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.

Introduction

Although the pathophysiology of autism spectrum disorders (ASD) remains unclear, diagnosis depends upon the presence of three broad symptom categories: abnormal social reciprocity, impairments in communication, and repetitive/stereotypic behaviors¹. Most rodent models are unable to simultaneously reproduce all three symptoms in a single animal. Thus, models that can reliably simulate some or all aspects of the disorder hold great value for the development of efficacious preventative and therapeutic strategies. One model that does display all three aspects of ASD is the BTBR mouse^1,2.

In combination with ultrasonic voca....

Protocol

All procedures were carried out in accordance with the Canadian Animal Care Committee and approved by the University of Calgary Animal Care Committee.

1. Animal Preparation

1. Isolate mice overnight (12 hr) prior to testing. To do this, place all of the BTBR and B6 mice (P35-P40) needed for experimental analysis in cages by themselves, the day before testing.
2. Place the isolated mice in the behavioral analysis room 30 min prior to testing so that animals will habituate to the novel environment.

2. Equipment Preparation

1. Place the 30 x 30 cm Plexiglas box with ....

Results

The modified version of the juvenile play test described here can be used to observe and analyze the novel repetitive digging behavior termed inchworming. Inchworming is exhibited to a far greater extent by BTBR mice than B6 controls. In a typical 10 min session, BTBR mice will display the distinct inchworming behavior (synchronous inward and outward movement of the paws in an effort to displace the sawdust bedding) for approximately 5 min, or half of the observation period. Furthermore, the latency to inchworm is signif.......

Discussion

Inchworming is a novel measure of repetitive behavior that can be used to study many disorders that exhibit stereotypies such as ASD. There are 4 steps that are of critical importance to generating reliable inchworming data from the modified juvenile social interaction protocol: 1) the extended isolation period is essential to the inchworming test and should not be removed from the protocol or shortened. This requirement is specific to testing of inchworming in pairs described here. 2) The video camera needs to be approp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30 cm X 30 cm Plexiglas box with lid			Can be constructed
IR Camera	Survshop (Calgary, Alberta)	Sony CCD Camera DP955V- 30’ Infrared Armor Dome	Find a camera with high-resolution and zoom capabilities
DVR	Supercircuits (Austin, Texas)	BLACK Enterprise-Class 4-Channel H.264 security DVR with DVD Burner	High data capacity
Sawdust			Same as bedding material