Abstract
Olfaction plays a fundamental role in the various behaviors such as feeding, mating, nursing, and avoidance in mice. Behavioral tests that characterize abilities of odor detection and recognition using genetically modified mice reveal the contribution of target genes to the olfactory processing. Here, we describe the olfactory habituation-dishabituation test for investigating the odor detection threshold in mice.
Keywords: Olfaction, Odor detection threshold, Behavioral test, Mouse, Olfactometer
Background
Olfactory system is a good model for studying the sensory processing in the brain. To characterize abilities of odor detection in genetically modified mice, the olfactory habituation-dishabituation test was performed using either filter paper or cotton scented with a test odor (Kobayakawa et al., 2007; Ferquson et al., 2000). There is a natural tendency of mice preferentially exploring novelty such as a novel odor and a novel object (Bevins and Besheer, 2006). Therefore, there is no training in advance before starting these tests. However, it is difficult to constantly supply the test odor especially at lower concentrations (around the detection threshold), because the odor is diluted by diffusion during the test. Here, we describe a method for the olfactory habituation-dishabituation test using an olfactometer. This method has an advantage in the point that mice are exposed to the odor at a constant concentration during the test, compared with that using the filter paper (Takahashi et al., 2016).
Materials and Reagents
Equipment
Note: The gas cylinder containing clean air is connected to the olfactometer. The olfactometer regulates the gas flow (0.5 L/min), and switches flow between clean air and air with an odor by passing the air through a bottle containing eugenol. The olfactometer, connected to the test cage (25 x 37 x 24 cm), can supply either the clean air or the odor through the gas port (0.5-mm diameter hole) on the wall (2-cm height). The roof of test cage is covered with clear acrylic board (Figure 1). Figure 1. Apparatus of the olfactory habituation-dishabituation test
Software
Procedure
Data analysis
Investigation times in the 1st and 2nd trials are measured manually during the 3-min test using the recorded videos. ‘Investigation time’ is defined as the time when the nose enters the 2.5-cm square area near the gas port (Figure 2B and Video 1). We recommend that the investigation times are measured by blinded analysis. Differences in investigation times between the 1st trial and 2nd trial are calculated as the mean ± SEM (Figures 3A and 3B). P-values are calculated by Welch t-test using Microsoft Excel, in which you click Data Analysis and perform t-test: Two-Sample Assuming Unequal Variances. For multiple pairwise comparisons, P-values are then sequentially evaluated according to the Holm-Bonferroni method (Holm, 1979) to keep an experiment-wise α ≤ 0.05, manually. The formula to evaluate the Holm-Bonferroni method is as follows: α/(n - k + 1) Where, n: number of tests, k: rank number of pair. Example for the Holm-Bonferroni correction: Consider four null hypotheses (H1-4) with unadjusted P-values (p1-4), to be tested at significance level α = 0.05. H1: p1 = 0.01, H2: p2 = 0.005, H3: p3 = 0.03, H4: p4 = 0.04
Notes
Acknowledgments
This protocol was modified from a previous study (Kobayakawa et al., 2007). This work was supported by Grants-in-Aid for Scientific Research on (B) (A.T.), (C) (H.T.), and Innovative Areas (Adaptive circuit shift) (A.T.), and for Challenging Exploratory Research (A.T.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
References
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