Materials and Reagents

1. 1.5 ml plastic tubes (Bio-bik, catalog number: CF-0150)

2. 27 G needles for injection (Terumo, catalog number: NN-2719S)

3. 1 ml syringe (Terumo, catalog number: 170215)

4. 50 ml centrifuge tubes (Greiner, catalog number: 227261)

5. Teflon tube (Chiyoda, catalog number: TF-4-10)

6. KimWipes (Crecia, catalog number: S-200)

7. Cotton buds (Suzuran, catalog number: 102046)

8. Toothpicks (Yanagi, catalog number: J-613)

9. Disposable balance tray (Bio-bik, catalog number: AS-DS)

10. Cement solution (GC, Product name: Unifast II liquid 100 g)

11. Cement powder (GC, Product name: Unifast II powder A3 35 g)

12. Saline (Otsuka, catalog number: 3311401A7028)

13. Ketamine (Daiichi-Sankyo, catalog number: S9-019780)

14. Xylazine (Bayer, Product name: Rompun 2% w/v solution for injection 25 ml)

15. Vaseline (Wako, catalog number: 227-01211)

16. 70% ethanol (Shinwa, catalog number: WK2-75)

17. Superglue (Sankyo, Product name: aron alpha A 0.5 g × 5)

18. Kwil-sil (WPI, catalog number: KWIK-SIL)

19. Valeraldehyde (Tokyo Chemical Industry, catalog number: V0001)

20. Mineral oil (Sigma, catalog number: M5310-500 ML)

21. Phosphate-buffered saline (PBS)

    
    

Mouse:

The OSN-specific GCaMP transgenic mouse line, OSN-GCaMP3 (OMP-tTA; TRE-GCaMP3 compound heterozygous bacterial artificial chromosome transgenic mice, 8-16 weeks of age) was used (Iwata et al., 2017; Inagaki et al., 2020). OMP-tTA (Accession# CDB0506T) and TRE-GCaMP3 (Accession# CDB0505T) are available from RIKEN (http://www2.clst.riken.jp/arg/index.html).

Note: Transgenic mouse lines expressing any indicators could be used, but sparsely and brightly labeled lines are preferred so that you can easily distinguish OSN responses. In OSN-GCaMP3 mice, GCaMP3 is expressed in 57.9% of the total OSNs (Iwata et al., 2017). A mouse line based on the Tet-system may be suitable for OE imaging in terms of labeling density and fluorescence intensity.

Equipment

1. Micropipette (Gilson, model: P200 and P1000)

2. Heating pad (Natsume, catalog number: KN-475-3-40)

3. Forceps (KFI, catalog number: 1-9749-32)

4. Fine forceps (Ideal-tek, catalog number: 91-2427)

5. Fine scissors (Mizuho, catalog number: 04-001-13)

6. Fluorescent stereomicroscope (Leica, model: M205 C)

   Note: An epifluorescence microscope is useful for assessing the thickness of the skull above the OE (detailed below).

7. External light source for fluorescence excitation (Leica, model: EL6000)

8. Filter cube (Leica, model: GFP)

9. Head holder for surgery (Narishige, model: SG-4N)

10. Custom-made aluminum nose bar (Figure 1)

    Note: We installed a custom-made nose bar to Narishige SG-4N (Figure 1A), as the original nose bar was too long and prevented surgical access to the OE. The size should be adjusted to your head holder and to make the skull over the OE accessible during surgery (Figure 1C). Any head holders can be used as long as the OE is accessible for surgery.

    
    

    
    Figure 1. Installation of a custom-made nose bar to a head holder. A. An original nose bar of a head holder. B. The design of a custom-made nose bar. C. The custom-made nose bar is installed into SG-4N.

    
    

11. Custom-made aluminum head bar (4 × 22 mm)

    Note: The head bar was designed for a custom-built head holder as described in a previous study (Guo et al., 2014).

12. Custom-built head holder for imaging

    Note: The head holder was built as described previously (Guo et al., 2014). Any head-holding system can be used for this protocol if the OE is accessible for in vivo imaging.

13. Φ1 mm drill tip (Meisinger, catalog number: ST1 HP010)

14. Dental drill (Leutor, model: LP-120)

15. Dust blower (UN, catalog number: UN-1321)

16. Two-photon microscope (Olympus, model: FV1000MPE)

17. Fluoview software (Olympus, model: FV10-ASW)

18. 25× objective lens (Olympus, model: XLPLN25XWMP)

19. Custom-built olfactometer

    Note: The design of the olfactometers has been described elsewhere (Slotnick and Restrepo, 2005; Burton et al., 2019). Briefly, the olfactometer consists of an air pump (AS ONE, catalog number: 1-7482-11), activated charcoal filter (Advantec, model: TCC-A1-S0C0 and 1TS-B), and flowmeters (Kofloc, model: RK-1250)]

Procedure