Behavioral Phenotyping for Autism Spectrum Disorders in Mice

Yu‐Chi Chang1, Toby B. Cole2, Lucio G. Costa3

1 Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, 2 Center on Human Development and Disability, University of Washington, Seattle, Washington, 3 Department of Neuroscience, University of Parma Medical School, Parma
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.22
DOI:  10.1002/cptx.19
Online Posting Date:  May, 2017
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Abstract

Autism spectrum disorder (ASD) represents a heterogeneous group of disorders characterized by alterations in three behavioral symptom domains: Social interactions, verbal and nonverbal communication, and repetitive behaviors. Increasing prevalence of ASD in recent years suggests that exposure to environmental toxicants may be critical in modulating etiology of this disease. As clinical diagnosis of autism still relies on behavioral evaluation, it is important to be able to assess similar behavioral traits in animal models, to provide biological plausibility of associations between environmental exposures and ASD. Rodents naturally exhibit a large number of behaviors that can be linked to similar behaviors in human. In this unit, behavioral tests are described that are relevant to the domains affected in ASD. For the repetitive domain, the T‐maze spontaneous alternation test and marble burying test are described. For the communication domain, neonatal ultrasonic vocalization and olfactory habituation test toward social and non‐social odor are described. Finally, for the sociability domain, the three‐chambered social preference test and the reciprocal interaction test are presented. © 2017 by John Wiley & Sons, Inc.

Keywords: autism spectrum disorders; behavior; mouse; obsessive‐compulsive disorder (OCD); social behavior; olfactory communication; ultrasonic vocalization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Marble Burying
  • Basic Protocol 2: T‐maze Spontaneous Alternation
  • Basic Protocol 3: Neonatal Ultrasonic Vocalization
  • Basic Protocol 4: Olfactory Habituation Test
  • Support Protocol 1: Collection and Storage of Urine Samples
  • Basic Protocol 5: Three‐Chambered Social Preference Test
  • Basic Protocol 6: Reciprocal Interaction
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Marble Burying

  Materials
  • Mice (at least 6 weeks of age)
  • 70% ethanol (for cleaning)
  • Clean standard polycarbonate mouse cage, 29.75 × 61.5 × 74 inches high (76 × 156 × 188 cm high)
  • Shaved aspen bedding
  • 24 marbles (5/8 inch; 1.58 cm in diameter)
  • Clear acrylic ceiling (sturdy clear acrylic sheet with ventilation holes to keep mice in the cage during the recording session)
  • Paper towel (for cleaning)
  • Camera (Microsoft LifeCam HD‐6000 720p HD Webcam) and mounting tripod
  • Ethovision XT11 software (for videotracking and data collection)
  • Light meter (used in measuring lighting intensity of the testing room)

Basic Protocol 2: T‐maze Spontaneous Alternation

  Materials
  • Mice (at least 6 weeks of age)
  • 70% ethanol (for cleaning)
  • T‐maze (composed of three 30 × 10 cm arms joined by a 10 × 10 cm center)
  • Physical barrier to prevent arm entry (custom‐made guillotine doors, or we have used a square‐shaped water bottle)
  • Paper towels
  • Timer
  • Recording camera and computer
  • Light meter (for measuring lighting intensity of the testing room)
  • Ethovision XT11 (optional)

Basic Protocol 3: Neonatal Ultrasonic Vocalization

  Materials
  • Mouse pups (postnatal day 4, 6, or 8)
  • Chlorine dioxide‐based sterilant (e.g., Clidox; for cleaning)
  • Sound attenuating chamber (to allow recording without interference of ambient noise)
  • Electric heating pad (very important when recording neonatal vocalization; helps maintain body temperature during 5‐min recording session)
  • 500‐ml glass beaker
  • Thermometer
  • Noldus UltraVox XT system for recording and data analysis (system includes a digital microphone able to capture the ultrasonic signal up to 125 kHz)
  • Paper towels (for cleaning)
NOTE: For easy transport, a thick walled Styrofoam box could be used as the sound attenuating chamber to keep out ambient noise. Alternatively, any box with thick walls can be lined with acoustic material for sound proofing effect. When installing the microphone and a heating pad in the box, make sure to fill any gaps with high density foam strips to maximize sound proofing effect.

Basic Protocol 4: Olfactory Habituation Test

  Materials
  • Mice (at least 6 weeks of age; if mice are presented with opposite sex urine as one of the olfactory cues, use 9‐week‐old mice to avoid variability in timing of sexual maturation)
  • Olfactory scent samples:
    • Almond extract (McCormick)
    • Banana extract (McCormick)
    • Pooled urine from age‐ and gender‐matched female and male mice (for urine collection, refer to protocol 5Support Protocol)
  • Distilled water
  • Clean, housing cage setup, including standard polycarbonate mouse cage, 29.75 × 61.5 × 74 in high (75.6 × 156 × 188 cm), lined with corn cob bedding, wire top, and water bottle
  • Camera (Microsoft LifeCam HD‐6000 720p HD Webcam) and mounting tripod
  • Ethovision XT11 software (for data collection; Noldus Information Technologies)
  • Small plastic weighing boats (4.1 × 4.1 × 0.8 cm; VWR, cat. no. 89106‐764)
  • Unscented double‐sided Scor‐Tape, 1/8 in wide (0.32 cm; Scor‐Pal; Scotch tape products should be avoided as most of their tapes come with honey‐like scent)
  • Clean filter paper
  • Various sized pipets (20, 200, and 1000 µl), and tips

Support Protocol 1: Collection and Storage of Urine Samples

  Materials
  • 10 male and 10 female mice (strain and age should match those of mice to be used for the olfactory habituation test)
  • 1.75‐ml Eppendorf tubes
  • Non‐toxic marker or ear tag

Basic Protocol 5: Three‐Chambered Social Preference Test

  Materials
  • Mice at 7 weeks of age
  • Two age‐ and sex‐matched mice of the same strain (i.e., novel mice; keep novel mice in a different housing facility than test mice to ensure all aspects of social approach, e.g., tactical, olfactory, vocalization, stay novel)
  • 70% ethanol (for cleaning)
  • Three‐chambered Plexiglas box
  • Two metal restraining cups, 11 cm high, 10.5 cm diameter solid bottom, and stainless steel bars spaced at 1‐cm intervals (cups are designed to allow non‐aggressive tactical interaction and olfactory communication)
  • Ethovision XT videotracking system

Basic Protocol 6: Reciprocal Interaction

  Materials
  • Mice at 7 weeks of age
  • Age‐ and sex‐matched mice (i.e., novel mice; keep novel mice in a different housing facility than test mice to ensure all aspects of social approach, e.g., tactical, olfactory, vocalization, are novel)
  • 70% ethanol (for cleaning)
  • Non‐toxic marker (such as acrylic marker) for animal identification
  • Clean housing cage setup including standard polycarbonate mouse cage, 29.75 × 61.5 × 74 in high (75.6 × 156 × 188 cm), lined with corn cob bedding, wire top, and water bottle
  • Clear acrylic ceiling (sturdy clear acrylic sheet with ventilation holes to keep mice in cages during recording session)
  • Camera (Microsoft LifeCam HD‐6000 720p HD Webcam) and mounting tripod
  • Ethovision XT11 software for data collection
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Figures

Videos

Literature Cited

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