Methods to Measure Olfactory Behavior in Mice

Junhui Zou1, Wenbin Wang1, Yung‐Wei Pan2, Song Lu1, Zhengui Xia2

1 Toxicology Program, Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, 2 Graduate Program, Molecular and Cellular Biology, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.18
DOI:  10.1002/0471140856.tx1118s63
Online Posting Date:  February, 2015
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Abstract

Mice rely on the sense of olfaction to detect food sources, recognize social and mating partners, and avoid predators. Many behaviors of mice, including learning and memory, social interaction, fear, and anxiety are closely associated with their function of olfaction, and behavior tasks designed to evaluate those brain functions may use odors as cues. Accurate assessment of olfaction is not only essential for the study of olfactory system but also critical for proper interpretation of various mouse behaviors, especially learning and memory, emotionality and affect, and sociality. Here we describe a series of behavior experiments that offer multidimensional and quantitative assessments for mouse olfactory function, including olfactory habituation, discrimination, odor preference, odor detection sensitivity, and olfactory memory, with respect to both social and nonsocial odors. © 2015 by John Wiley & Sons, Inc.

Keywords: olfaction; mouse; olfactory discrimination; olfactory learning and memory

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cotton Tip–Based Olfactory Habituation/Dishabituation Test
  • Alternate Protocol 1: Cotton Tip–Based Olfactory Habituation/Dishabituation Test for Fine Odor Discrimination
  • Alternate Protocol 2: Cotton Tip–Based Olfactory Habituation/Dishabituation Test for Discrimination Between Male And Female Urinary Odors
  • Alternate Protocol 3: Cotton Tip–Based Olfactory Habituation/Dishabituation Test for Fine Discrimination of Urinary Odors Between Different Groups of Mice
  • Alternate Protocol 4: Cotton Tip‐Based Olfactory Habituation/Dishabituation Test for Fine Discrimination of Urinary Odors Between Ovariectomized and Estrous Female Urine
  • Basic Protocol 2: Cotton Tip–Based Olfactory Preference Test
  • Alternate Protocol 5: Cotton Tip–Based Olfactory Preference test For Preference Between Male and Female Urinary Odors
  • Alternate Protocol 6: Cotton Tip–Based Olfactory Preference Test for Preference Between Ovariectomized and Estrous Urinary Odors
  • Basic Protocol 3: Cotton Tip–Based Olfactory Detection Threshold Test
  • Basic Protocol 4: Cotton Tip–Based Olfactory Short‐Term Memory Test
  • Basic Protocol 5: Sand‐Digging Task–Based Olfactory Discrimination/Learning Test
  • Alternate Protocol 7: Sand‐Digging Task–Based Olfactory Discrimination/Learning Test With Structurally Similar Odorants
  • Alternate Protocol 8: Sand‐Digging Task–Based Olfactory Discrimination/Learning Test with Multiple Negative Odorants
  • Alternate Protocol 9: Sand‐Digging Task–Based Olfactory Discrimination/Learning Test with Multiple Odorant Pairs
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cotton Tip–Based Olfactory Habituation/Dishabituation Test

  Materials
  • Mice: 3 to 8 months old, housed in regular mouse cages with bedding, nestlets, and food and water bottles placed over the wire tops
  • Odorants
  • Vehicle (plain mineral oil or distilled water)
  • Clean caps of 15‐ml conical tubes
  • Cotton‐tipped wooden applicators (6‐in. length with one end wrapped with cotton; Allegiance, cat. no. C15055‐006)
  • Stopwatch
  • Digital timerDigital video recorder
  • Fans for ventilating room

Alternate Protocol 1: Cotton Tip–Based Olfactory Habituation/Dishabituation Test for Fine Odor Discrimination

  Additional Materials (also see protocol 1)
  • Pairs of structurally similar odorants at the same concentration to assess fine odor discrimination: e.g., (+)limonene and (–)limonene (a pair of enantiomers); or butanol and pentanol (alcohols with one‐carbon difference)

Alternate Protocol 2: Cotton Tip–Based Olfactory Habituation/Dishabituation Test for Discrimination Between Male And Female Urinary Odors

  Additional Materials (also see protocol 1)
  • 10 urine samples from normal male mice between 3 and 8 months of age
  • 10 urine samples from normal female mice between 3 and 8 months of age

Alternate Protocol 3: Cotton Tip–Based Olfactory Habituation/Dishabituation Test for Fine Discrimination of Urinary Odors Between Different Groups of Mice

  Additional Materials (also see protocol 1 and protocol 3)
  • 10 urine samples from normal male mice between 3 and 8 months of age (male group A)
  • 10 urine samples from normal male mice between 3 and 8 months of age (male group B)
  • 10 urine samples from normal female mice between 3 and 8 months of age (female group A)
  • 10 urine samples from normal female mice between 3 and 8 months of age (female group B)

Alternate Protocol 4: Cotton Tip‐Based Olfactory Habituation/Dishabituation Test for Fine Discrimination of Urinary Odors Between Ovariectomized and Estrous Female Urine

  Additional Materials (also see protocol 1 and protocol 3)
  • Five to ten 3 to 7 month old ovariectomized mice with their ovariectomy surgeries performed at 2 months old
  • Estradiol benzoate
  • Progesterone
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Basic Protocol 2: Cotton Tip–Based Olfactory Preference Test

  Additional Materials (also see protocol 1)
  • Two odorants (odorant 1 and 2) and two stopwatches are needed

Alternate Protocol 5: Cotton Tip–Based Olfactory Preference test For Preference Between Male and Female Urinary Odors

  Materials
  • Mice: 3 to 8 months old, housed in regular mouse cages with bedding, nestlets and food and water bottles over the wire tops
  • Odorants
  • Vehicle (plain mineral oil or distilled water)
  • Cotton‐tipped wooden applicators (6 in. in length with one end wrapped with cotton, Allegiance, Cat. No. C15055‐006)
  • Two stopwatches
  • One digital timerDigital video recorder
  • Caps of clean 15‐ml conical tubes

Alternate Protocol 6: Cotton Tip–Based Olfactory Preference Test for Preference Between Ovariectomized and Estrous Urinary Odors

  Materials
  • A cohort of 3 to 7 month old male mice, individually housed and pre‐handled as in protocol 1
  • Superglue
  • Mouse food pellets (Bioserv, product no. F0071)
  • Playground sand (Sarrete), autoclaved before the test
  • 70% and 95% ethanol
  • Odorant solutions: 1 mM isoamyl acetate (IAA) and 1 mM citralva are used as examples in this protocol
  • Lids of two 96‐well culture dishes
  • Lids of two 35‐mm culture dishes
  • Plexiglas sheets of various sizes:
    • 55 × 50 mm, clear
    • 20 × 32 cm, clear
    • 25 × 20 cm taped to 25 × 18 cm, painted to make it opaque (the paint coat must be very thin and completely dried and should not have any residual paint odor)
  • 35‐mm culture dishes

Basic Protocol 3: Cotton Tip–Based Olfactory Detection Threshold Test

  Additional Materials (also see protocol 11)
  • Pairs of structurally similar odorants at the same concentration to assess fine odor discrimination: e.g., (+)limonene and (–)limonene (a pair of enantiomers); or butanol and pentanol (alcohols with one carbon difference)

Basic Protocol 4: Cotton Tip–Based Olfactory Short‐Term Memory Test

  Additional Materials (also see protocol 11)
  • Odorant solutions including one positive odorant and three negative odorants. For example, use IAA at 1 mM as the positive odorants, and use citralva, octanol, benzaldehyde, all at 1 mM, as negative odorants

Basic Protocol 5: Sand‐Digging Task–Based Olfactory Discrimination/Learning Test

  Materials
  • Three pairs of structurally similar odorants, e.g.:
    • Pair 1: (+)Limonene at 10 mM designated as the positive odorant and (–)limonene at the same concentration designated as the negative odorant
    • Pair 2: Butanol and pentanol at 10 mM designated as the positive and negative odorant, respectively
    • Pair 3: (+)Carvone and (−)carvone at 10 mM designated as the positive and negative odorant, respectively
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Figures

Videos

Literature Cited

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