Mice were anesthetized with an intraperitoneal injection of a mixture of Fentanyl (0.05 mg/kg bodyweight (BW), Fentanyl-Hameln, Hameln Pharma plus, Hameln, Germany), Midazolam (5.0 mg/kg BW, Midazolam-hameln®; Hameln Pharma plus), Medetomidin (0.5 mg/kg BW, Sedator®; Albrecht, Aulendorf, Germany) and atropine sulfate (0.2 mg/kg BW, B. Braun, Melsungen, Germany) diluted with water ad. inj. (Ampuwa, Fresenius KABI, Bad Homburg, Germany) to an injection volume of 10 mL/kg BW. Additional doses of anesthetics were administered if needed. The anesthesia was antagonized after the measurements by a subcutaneously administered mixture of Naloxon (1.2 mg/kg BW, Naloxon-hameln®; Hameln Pharma plus), Flumazenil (0.55 mg/kg BW, Flumazenil®; Fresenius KABI), and Atipazemol (2.5 mg/kg BW, Antisedan®; VETOQUINOL GmbH, Ravensburg, Germany) diluted with water ad. inj. (Ampuwa) to an injection volume of 10 mL/kg BW.
The anesthetized mice lay on a pre-warmed resting pad (37°C) in a soundproof chamber (IAC 400-A, Industrial Acoustics Company GmbH, Niederkrüchten, Germany). Auditory brainstem responses (ABRs) in anesthetized mice were evoked by short-duration sound stimuli with the same stimulus parameters for all groups. The ABRs represent the summed activity of neurons in distinct anatomical structures along the ascending auditory pathway recorded from subcutaneous cranial electrodes. A microphone (Bruel & Kjaer 4191, Naerum, Denmark) was used to calibrate and record the acoustic stimuli. ABR thresholds were elicited with click stimuli (100 μs duration with an FFT mean of 5.4 kHz). The stimulus level was increased stepwise from 10 to 100 dB SPL in 5 dB steps. Stimuli were generated with an I-O-card (PCI-6052E, PCI-6251, or PCIe-6259, National Instruments, Austin, Texas, United States) in an IBM compatible computer. The SPL of the stimuli was modulated by custom-made amplifier and attenuator systems (Wulf Elektronik, Frankfurt, Germany). The measured signals were band-pass filtered from 200 Hz to 5 kHz (F1, 6-pole Butterworth hardware Filter, Wulf Elektronik) and amplified by 100,000. The analog/digital (A/D) rate was 20 kHz. Each stimulus had a recording interval of 16 ms and was directly repeated and averaged up to 512 times.
The anesthetized mice lay on a pre-warmed resting pad (37°C) in a soundproof chamber (IAC 400-A, Industrial Acoustics Company GmbH, Niederkrüchten, Germany). Auditory brainstem responses (ABRs) in anesthetized mice were evoked by short-duration sound stimuli with the same stimulus parameters for all groups. The ABRs represent the summed activity of neurons in distinct anatomical structures along the ascending auditory pathway recorded from subcutaneous cranial electrodes. A microphone (Bruel & Kjaer 4191, Naerum, Denmark) was used to calibrate and record the acoustic stimuli. ABR thresholds were elicited with click stimuli (100 μs duration with an FFT mean of 5.4 kHz). The stimulus level was increased stepwise from 10 to 100 dB SPL in 5 dB steps. Stimuli were generated with an I-O-card (PCI-6052E, PCI-6251, or PCIe-6259, National Instruments, Austin, Texas, United States) in an IBM compatible computer. The SPL of the stimuli was modulated by custom-made amplifier and attenuator systems (Wulf Elektronik, Frankfurt, Germany). The measured signals were band-pass filtered from 200 Hz to 5 kHz (F1, 6-pole Butterworth hardware Filter, Wulf Elektronik) and amplified by 100,000. The analog/digital (A/D) rate was 20 kHz. Each stimulus had a recording interval of 16 ms and was directly repeated and averaged up to 512 times.
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