Habituation/dishabituation test

Habituation/dishabituation test

The test was performed as described before (Ferguson et al., 2000; Kobayakawa et al., 2007) with minor modifications.

1. All animals were housed under a standard 12hr light/dark cycle (light on from 8am to 8pm) with constant temperature (22 ± 2°C) and humidity (50 ± 10%).   Food and water were provided ad libitum.
2. Adult littermates (6w-old male) were kept individually in a cage (26 x 40 x 18cm) for 16- 24hr before testing and moved to a new cage in the morning for the test.
3. We provided a plain filter paper (2 × 2cm) for 5min prior to the experiment. This treatment was to habituate the mice to a filter paper and to evaluate the net curiosity to a novel odor in the following filter presentation.
4. A filter paper spotted with 0.5μl of distilled water was intermittently presented for 3min.
   This operation was repeated three times with 1min intervals, trying to reduce the interference of novel environment exploration.
5. The 1st test odorant was presented for 3min three times with 1min intervals (habituation).
6. 1min after the 3rd presentation of the 1st odor, the 2nd test odorant was presented three times in the same way (dishabituation).  These presentations were performed successively on the same day.
7. The mouse behavior was recorded with a digital video camera.
8. Investigation times (sec) were measured, which were defined as a nasal contact with the filter paper within a 1cm distance, observed during each odorant presentation. Habituation is detected by a decrease in the time spent sniffing the same odor.

In this test, the mice were used only once not to confound the data due to the previous learning. The test was blinded to avoid unconscious bias and to insure the objectivity.

(+)-carvone (435759, Sigma-Aldrich)

(–)-carvone (124931, Sigma-Aldrich) eugenol (A0232, Tokyo-Kasei) vanillin (224-00682, Wako)

4-methyl-thiazole (134-12742, Wako)

References

Ferguson JN, Young LJ, Hearn EF, Matzuk MM, Insel TR, Winslow JT. 2000. Social amnesia in mice lacking the oxytocin gene. Nat. Genet. 25:284-288. DOI: https://doi.org/10.1038/77040, PMID: 10888874

Kobayakawa K, Kobayakawa R, Matsumoto H, Oka Y, Imai T, Ikawa M, Okabe M, Ikeda T, Itohara S, Kikusui T, Mori K, Sakano H. 2007. Innate versus learned odour processing in the mouse olfactory bulb. Nature. 450:503-508. DOI: 10.1038/nature06281, PMID: 17989651

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