Tonotopic Labeling of Auditory Neurons

For auditory stimulation, mice at P16–P30 were housed in a custom soundproof box, and received sound stimulation as illustrated in Figure 1A. After three quiet hours, continuous sound was delivered for 6 h at ∼90 dB using BioSigRP software (Tucker-Davis Technologies). A 15-kHz tone of 20-ms duration with 2-ms rise and fall time was presented at a rate of 41 Hz (leaving 4.4-ms gaps). The sound was produced by a PC sound card, processed by RZ6 Multi-I/O Processer (Tucker-Davis Technologies), and delivered via a speaker (Ignite) mounted directly above the animals. The sound was paused for no more than 5 min after the first 3 h to administer an intraperitoneal injection of 160 mg/kg Tamoxifen or 50 mg/kg 4-OHT. Tamoxifen was first dissolved in absolute EtOH, then diluted 1/10 by volume with sunflower oil followed by 2-h sonication or overnight nutation, and stored at 4°C in the dark for up to one week. 4-OHT was first dissolved in absolute EtOH at 20 mg/ml, and diluted with Chen oil (a 1:4 mixture of castor oil:sunflower seed oil) to 10 mg/ml for injection (Guenthner et al., 2013 (link)). After sound stimulation, mice were kept in quiet for another 3 h before returning to their home cage, and killed 4–5 d later for experiments. This protocol has been shown to produce tonotopic labeling in DCN and VCN from TRAP mice crossed with the Ai14 reporter line (Guenthner et al., 2013 (link)). The time line is consistent with the time window determined by the pharmacodynamics of 4-OHT in mice (Robinson et al., 1991 (link)).