Mouse Auditory Brainstem Response Testing

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The Journal of Neuroscience
Feb 2015



The auditory brainstem response (ABR) test provides information about the inner ear (cochlea) and the central pathways for hearing. The ABR reflects the electrical responses of both the cochlear ganglion neurons and the nuclei of the central auditory pathway to sound stimulation (Zhou et al., 2006; Burkard et al., 2007). The ABR contains 5 identifiable wave forms, labeled as I-V. Wave I represents the summated response from the spiral ganglion and auditory nerve while waves II-V represent responses from the ascending auditory pathway. The ABR is recorded via electrodes placed on the scalp of an anesthetized animal. ABR thresholds refer to the lowest sound pressure level (SPL) that can generate identifiable electrical response waves.

This protocol describes the process of measuring the ABR of small rodents (mouse, rat, guinea pig, etc.), including anesthetizing the mouse, placing the electrodes on the scalp, recording click and tone burst stimuli and reading the obtained waveforms for ABR threshold values. As technology continues to evolve, ABR will likely provide more qualitative and quantitative information regarding the function of the auditory nerve and brainstem pathways involved in hearing.

Keywords: Mouse (鼠标), Inner ear (内耳), Cochlea (耳蜗), Hearing (听力), ABR (ABR)

Materials and Reagents

  1. 1 ml insulin syringe (Thermo Fisher Scientific, BD, catalog number: 22-253-260 )
  2. Precision Glide Needles (30 G x 1/2) (BD, catalog number: 305106 )
  3. Disposable monopolar needle electrodes for electromyography (Rochester Electro-Medical, catalog number: 016386-0 )
  4. Ketamine hydrochloride
    1. Ketaset (Ketamine HCl Injection) (Drugs, catalog number: NDC-0856-2013-01 )
    2. Henry Schein (100 mg/ml solution) (NSN, catalog number: 304401 )
  5. Xylazine hydrochloride (Sedative and Analgesic solution) (20 mg/ml solution) (LLOYD, AnaSed INJECTION, catalog number: 139-236 )
  6. Sterile bi-distilled water
  7. Povidone-iodine 5% (Betadine) antiseptic-Microbicide solution (purchased from any pharmacy)
  8. Dexamethasone ophthalmic ointment (TobraDex) (Alcon)
  9. 70% Ethanol (Surface disinfectant)
  10. Mixture of ketamine hydrochloride and xylazine hydrochloride (see Recipes)


  1. Disposable monopolar needle electrodes for electromyography (Rochester Electro Medical, catalog number: 016386-0)
  2. Computer with BioSig32 Windows Application Installed
  3. TDT BioSigIII system (TDT)
  4. TD speakers (TDT)
  5. Sound proof chamber (Industrial Acoustics Company, Controlled Acoustical Environment)
  6. Non-Electric Heating Pad (to maintain animal body temperature during the test) (Braintree Scientific, Deltaphase Isothermal Pad, model: 39DP )
  7. CONAIR Moist Electric Heating Pad (to maintain animal body temperature during the recovery) (Amazon)
  8. Rectal Probe for body temperature monitoring (Physitemp Instruments, model: Thermalert TH-5 physitemp )
  9. Clean empty cages
  10. Tweezers style 5 (Ted Pella, catalog number: 5665 )


  1. BioSig32 software (coming with the TDT BioSig III system)


All procedures and animal handling described in this protocol should be done according to approved national ethical guidelines and complied with all protocol requirements of the Institutional Animal Care and Use Committee.

  1. Appropriate site-specific IACUC approvals are required to perform the following procedures.
  2. The onset of hearing in mice is around postnatal day 12 (P12), so the hearing test can be performed on mouse older than P12.
  3. The FVB mouse strain was used in this protocol, but any mouse strain can be used with this protocol.

  1. Preparation
    1. The animal is anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (Ketaset 100 mg/kg) and xylazine hydrochloride (Xyla-Ject 10 mg/kg) using the 1 ml insulin syringe with the precision glide needle.
      1. When working with mice, gloves and lab coat should be worn at all times.
      2. Additionally, for less experienced users, a heavy glove may be worn when injecting older mice to protect from biting.
      3. It is advised to use only ketamine and xylazine solution for anesthesia when testing hearing because this drug combination provides stable ABR thresholds.
    2. After injection the animal is placed in an isolated clean and warm cage (~37 °C) using an electric heating pad or other hearing device.
      Note: No other awake animals should be with an anesthetized mouse.
    3. The mouse will be ready for testing within ~2-5 min. Appropriate levels of anesthesia can be tested with a stimulus such as a toe pinch.
    4. Place the mouse on a preheated non-electric heating pad (~37 °C) in a soundproof chamber.
    5. Monitor the body temperature with a rectal probe throughout recording.
    6. Cover the animal's eyes with a protective ophthalmic ointment to keep the eyes moist during anesthesia. This will also suppress the animal's blink reflex, which can add noise to the recording.
    7. Position the mouse so the speaker is placed 10 cm from its left ear.
      1. Speakers for this setup were calibrated using an ACO Pacific 7017 microphone.
      2. The center of the speaker has to be aligned with the external auditory canal.
        For hearing threshold evaluation, 3 subdermal electrodes were used: Ground, reference, and active electrode, are placed 2-3 mm under the skin of the mouse head (Figures 1-2).
    8. Insert the active electrode subdermally at the forehead (Figure 1 and Figure 2: Red).
      Note: Use clean forceps to lift and tense the skin.
    9. Insert the reference electrode below the pinna of the left ear (Figure 1 and Figure 2: Blue), and the ground electrode below the contralateral (right) ear (Figure 1 and Figure 2: Green).
      Note: Figure 1 shows a picture of the mouse in position of recording with all 3 electrodes placed on the head in the sound-proof chamber and Figure 2 shows the exact position of the electrodes.

      Figure 1. A mouse in position for ABR recording. The mouse is placed 10 cm from the speaker on a heating pad with all 3 electrodes placed in the head, within the sound-proof chamber.

      Figure 2. Position of the 3 electrodes on the mouse head. The blue, the green and the red arrows represent the exact position of the electrodes in the mouse head. A picture of the blue electrode is also included. “F” means forehead, “R” means right ear and “L” means left ear.

    10. Check the impedance: If the impedance is over 5 Ohm, adjust the electrodes in the scalp and retake the measurement. Continue to manipulate the electrodes until the impedance drops below 5 Ohm.
    11. Close the sound-proof chamber.
    12. Sounds are presented and ABRs are recorded in a free field condition as previously described (Akil et al., 2015; Akil et al., 2012; Akil et al., 2006).
      Note: To measure each ear separately, as opposed to a sound free field ABR, a tube with an ear tip can be run from the speaker to the ear canal directly (close field), and the process above repeated.

  2. Recording click auditory brainstem responses
    TDT software (BioSig III) and hardware (RP and TD speakers) are used for ABR recording.
    1. TDT BioSigIII system, produces and records stimuli for 20 ms duration and 31 Hz. For click stimulus, in which the mouse is presented with a wide spectrum click (0.1 ms) in decreasing levels between 90 dB and 10 dB, each new stimulus is recorded 5 dB SPL down from the previous. Each point of measurement is recorded and averaged 510 times and analyzed.
      1. ABR results can be affected by other electronics in the sound-proof chamber - turn off extraneous transmitting electronic devices in the chamber, and ensure that the heating pad under the mouse is non-electric.
      2. Animal temperature - monitor the body temperature with a rectal probe throughout recording and keep the mouse’s body temperature warm using a heating pad (Gold et al., 1985).
    2. Continue recording ABR until waveforms are clearly no longer present.
      1. If the middle ear, cochlea or brainstem is not functioning properly, waveforms will not be recorded (Figure 3 shows a sample of ABR waveforms from a wild-type (normal) and deaf mouse).
      2. The median ABR threshold in wild-type mice can vary depending on the system (TDT or Harvard Apparatus) and the method (close or free field) used to collect it. In general the ABR thresholds are ~ 30 ± 5 dB SPL.

        Figure 3. Representative ABR tracings from wild-type and deaf mice. Wild-type mice present normal ABR waveforms, while the deaf mice present no identifiable ABR responses. ‘‘I-V’’ indicates the location of ABR peaks. The black arrow indicates the ABR threshold (33dB SPL).

    3. Read the result and determine the ABR threshold value, or the lowest level an identifiable waveform can be seen.
      1. The threshold can be determined by watching the first 5 peaks.
      2. All five peaks should be seen within the first 10 milliseconds.
      3. The ABR threshold is determined as the lowest recognizable ABR response (Figure 3: Black arrow).
    4. Save the resulting waveforms for future reference and analysis.

  3. Recording frequency-specific auditory brainstem responses
    1. When recording frequency-specific ABRs, three frequencies are classically tested to examine the degree to which regions of the cochlea are functioning (corresponding to base, mid-turn and apex), though in principle any specific frequency can be measured. Tone burst stimuli of 3 single frequencies (1 ms), including 8 kHz, 16 kHz, and 32 kHz are presented in decreasing levels between 90 dB and 10 dB. Each new stimulus is recorded 5 dB SPL down from the previous presentation. Each point of measurement is recorded and averaged 1,000 times and analyzed.
    2. ABR threshold is determined as the lowest recognizable ABR response similar to what it was described above.
    3. Save the resulting waveforms for future reference and analysis.
      1. In general a mouse will remain under anesthesia for ~30-40 min after the first anesthetic injection.
      2. Due to the duration of click and frequency-specific ABR recordings (~40 min), one fifth of the original dose of the anesthetic should be injected at around 20 min to avoid the likelihood of the mouse waking up during the testing.
      3. If a mouse starts moving, the ABR recording will be disrupted and the mouse must be re-anesthetized with one fifth of the original anesthetic dose.
    4. When finished recording, carefully remove the electrode from the mouse.
    5. When all the tests are finished, dip the electrodes in Providone-iodine solution for about an hour; rinse with 70% ethanol then cap them and place them in a sterile container.
    6. Clean the recording chamber and all surfaces that were in contact with mice with 70% ethanol.

  4. Recovery from anesthesia
    1. Place the mouse in an empty and clean preheated cage during emergence from anesthesia. Do not leave mice unattended until they are fully recovered. Place this cage on a heating pad to maintain the mouse’s body temperature until total recovery from the anesthesia has occurred.
      Note: Multiple mice of the same group can be placed in the same cage (no more than 5 mice).
    2. Return mice to their original cage without heating pad only if they can return themselves to their feet after being placed on their backs.

Representative data

Figure 4. Representative graph of an ABR testing. ABR thresholds (decibels of sound pressure level, dBSPL) were measured in mice with normal ABR (Black, Blue and Green curves) and abnormal ABR (Red curve). The mice with abnormal ABR showed higher ABR thresholds at click stimulus and at tones stimuli (8, 16, and 32 kHz) when compared to mice with normal ABR.


  1. Mixture of ketamine hydrochloride and xylazine hydrochloride
    It contains ketamine hydrochloride (Ketaset 100 mg/kg) and xylazine hydrochloride (Xyla-Ject 10 mg/kg):
    2 ml ketamine (100 mg/ml)
    1 ml xylazine (20 mg/ml)
    5 ml sterile bi-distilled water
    Mix well and place in a rubber sealed vial.
    1. The solution of ketamine and xylazine can be used as long as the ketamine or xylazine are not expired and are sterile.
    2. The solution should be stored at 4 °C when it is not in use and at room temperature when it is in use.


The authors would like to acknowledge the financial support provided by Hearing Research Inc (HRI) and by NIH (NIDCD) Grant #R01DC0130671.


  1. Akil, O., Chang, J., Hiel, H., Kong, J. H., Yi, E., Glowatzki, E. and Lustig, L. R. (2006). Progressive deafness and altered cochlear innervation in knock-out mice lacking prosaposin. J Neurosci 26(50): 13076-13088.
  2. Akil, O., Seal, R. P., Burke, K., Wang, C., Alemi, A., During, M., Edwards, R. H. and Lustig, L. R. (2012). Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy. Neuron 75(2): 283-293.
  3. Akil, O., Sun, Y., Vijayakumar, S., Zhang, W., Ku, T., Lee, C. K., Jones, S., Grabowski, G. A. and Lustig, L. R. (2015). Spiral ganglion degeneration and hearing loss as a consequence of satellite cell death in saposin B-deficient mice. J Neurosci 35(7): 3263-3275.
  4. Burkard, R. F., Eggermont, J. J. and Manuel, D. (eds). (2007). Auditory evoked potentials: basic principles and clinical application. Lippincott Williams & Wilkins, Philadelphi.
  5. Gold, S., Cahani, M., Sohmer, H., Horowitz, M. and Shahar, A. (1985). Effects of body temperature elevation on auditory nerve-brain-stem evoked responses and EEGs in rats. Electroencephalogr Clin Neurophysiol 60(2): 146-153.
  6. Zhou, X., Jen, P. H., Seburn, K. L., Frankel, W. N. and Zheng, Q. Y. (2006). Auditory brainstem responses in 10 inbred strains of mice. Brain Res 1091(1): 16-26.


听觉脑干反应(ABR)测试提供有关内耳(耳蜗)和听觉中枢通路的信息。 ABR反映了耳蜗神经节神经元和中枢听觉通路的核对声音刺激的电响应(Zhou et al。,2006; Burkard et al。,2007)。 ABR包含5个可识别的波形,标记为I-V。波I代表螺旋神经节和听觉神经的总和反应,而波II-V代表上升听觉通路的反应。通过置于麻醉动物头皮上的电极记录ABR。 ABR阈值是指可以产生可识别的电响应波的最低声压级(SPL)。

关键字:鼠标, 内耳, 耳蜗, 听力, ABR


  1. 1ml胰岛素注射器(Thermo Fisher Scientific,BD,目录号:22-253-260)
  2. 精密滑动针(30G×1/2)(BD,目录号:305106)
  3. 用于肌电图的一次性单极针电极(Rochester Electro Medical,目录号:016386-0)
  4. 盐酸氯胺酮
    1. Ketaset(氯胺酮注射液)(药品,目录号:NDC-0856-2013-01)
    2. Henry Schein(100mg/ml溶液)(NSN,目录号:304401)
  5. 甲苯噻嗪盐酸盐(镇静和止痛溶液)(20mg/ml溶液)(LLOYD,AnaSed INJECTION,目录号:139-236)
  6. 无菌双蒸水
  7. 紫环酮碘5%(Betadine)防腐剂 - 杀微生物剂溶液(购自任何药店)
  8. 地塞米松眼用软膏(TobraDex)(Alcon)
  9. 70%乙醇(表面消毒剂)
  10. 盐酸氯胺酮和盐酸赛拉嗪的混合物(参见配方)


  1. 用于肌电图的一次性单极针电极(Rochester Electro Medical,目录号:016386-0)
  2. 安装了BioSig32 Windows应用程序的计算机
  3. TDT BioSigIII系统(TDT)
  4. TD扬声器(TDT)
  5. 隔音室(工业声学公司,受控声学环境)
  6. 非电加热垫(在测试期间保持动物体温)(Braintree Scientific,Deltaphase Isothermal Pad,型号:39DP)
  7. CONAIR潮湿电热垫(在恢复期间保持动物体温)(亚马逊)
  8. 用于体温监测的直肠探头(Physitemp Instruments,型号:Thermalert TH-5 physitemp)
  9. 清洁空笼子
  10. 镊子风格5(Ted Pella,目录号:5665)


  1. BioSig32软件(随TDT BioSig III系统提供)



  1. 执行以下程序需要适当的站点特定IACUC批准。
  2. 小鼠听力开始在出生后第12天(P12),因此可以对P12以上的小鼠进行听力测试。
  3. 在该方案中使用FVB小鼠品系,但是任何小鼠品系可以与该方案一起使用。

  1. 准备
    1. 将动物用腹膜内麻醉 注射盐酸氯胺酮(Ketaset 100 mg/kg)和 ?甲苯噻嗪盐酸盐(Xyla-Ject 10mg/kg) 注射器与精确滑翔针。
      1. 在使用鼠标时,应随时佩戴手套和实验室外套。
      2. 此外,对于经验不足的用户,注射较老的老鼠以防止咬伤时,可能会穿着重的手套。
      3. 建议仅使用氯胺酮和赛拉嗪溶液 麻醉时测试听力因为这种药物组合提供 稳定ABR阈值。
    2. 注射后将动物放入 ?隔离清洁和温暖的笼子(?37°C)使用电加热垫或 其他听力设备 注意:没有其他清醒的动物应该使用麻醉的鼠标。
    3. 鼠标将准备在?2-5分钟内进行测试。适当 可以用诸如脚趾捏的刺激来测试麻醉水平。
    4. 将鼠标放在预热的非电加热垫(?37°C)在隔音室。
    5. 在整个记录过程中用直肠探头监测体温。
    6. 用保护性眼用软膏覆盖动物的眼睛 在麻醉期间保持眼睛湿润。这也将压制 动物的眨眼反射,这会增加记录的噪音。
    7. 放置鼠标,使扬声器放在离左耳10厘米处。
      1. 此设置的扬声器使用ACO Pacific 7017麦克风进行校准。
      2. 扬声器的中心必须与外耳道对齐。
        对于听力阈值评价,使用3个皮下电极: 接地,参考和有源电极,放置在下面2-3mm 皮肤的鼠标头(图1-2)。
    8. 将活性电极以皮下方式插入前额(图1和图2:红色)。
    9. 将参考电极插入左耳耳廓下方 (图1和图2:蓝色),和接地电极下面 对侧(右)耳(图1和图2:绿色)。
      注意: 图1显示了鼠标在所有3的记录位置的图片 ?放置在隔音室内的头上的电极和图2 显示电极的确切位置。

      图1.鼠标 ABR录音的位置。鼠标位于距离扬声器10厘米处 在加热垫上,所有3个电极放置在头部,内 隔音室

      图2. 3个电极的位置 鼠标头。蓝色,绿色和红色箭头代表确切的 电极在鼠标头中的位置。蓝色的图片 电极。 "F"表示前额,"R"表示右耳和 "L"表示左耳。

    10. 检查阻抗:如果阻抗 是超过5欧姆,调整电极在头皮和重新拍摄 测量。继续操作电极,直到阻抗 低于5欧姆。
    11. 关闭隔音室。
    12. 声音 ?并且在自由场条件下记录ABR (Akil等人,2015; Akil等人,2012; Akil等人,2011)中所描述的, ?2006)。
      注意:单独测量每只耳朵,而不是声音 自由场ABR,具有耳塞的管可以从扬声器运行到 耳道直接(近场),以上过程重复。

  2. 记录点击听觉脑干反应
    TDT软件(BioSig III)和硬件(RP和TD扬声器)用于ABR记录
    1. TDT BioSigIII系统,产生并记录20毫秒的刺激 持续时间和31Hz。对于点击刺激,其中提供了鼠标 具有在90dB之间的降低水平的宽频谱点击(0.1ms) 和10 dB,每个新的刺激被记录下来5 dB SPL 以前。记录每个测量点并平均510次
      1. ABR结果可能受到其他影响 电子设备在隔音室 - 关闭外部 传输电子设备在室内,并确保 加热垫下的鼠标是非电的。
      2. 动物温度 - ?在整个记录期间用直肠探针监测体温 并使用加热垫保持鼠标的体温温暖(Gold et al。,1985)。
    2. 继续记录ABR,直到波形明显不再存在。
      1. 如果中耳,耳蜗或脑干不能正常工作, ?波形将不被记录(图3示出了ABR的样本 来自野生型(正常)和聋鼠的波形)。
      2. 中位数 野生型小鼠中的ABR阈值可以根据系统(TDT或 ?哈佛装置)和用于收集的方法(关闭或自由场) ?它。通常,ABR阈值为?30±5dB SPL。

        图3。 来自野生型和聋小鼠的代表性ABR示踪。野生型小鼠 ?呈现正常ABR波形,而聋小鼠呈现没有 可识别的ABR反应。 "I-V"指示ABR峰的位置。 ?黑色箭头表示ABR阈值(33dB SPL)。

    3. 读取结果并确定ABR阈值,或可以看到可识别波形的最低级别。
      1. 阈值可以通过观察前5个峰来确定。
      2. 应在前10毫秒内看到所有五个峰。
      3. ABR阈值被确定为最低可识别ABR反应(图3:黑色箭头)。
    4. 保存结果波形以供将来参考和分析。

  3. 记录特定频率的听觉脑干反应
    1. 当记录频率特定ABR时,三个频率 经典测试以检查耳蜗的区域的程度 ?(对应于基地,中转和顶点),虽然在中 任何特定频率的原理都可以测量。色调爆发刺激3 ?单个频率(1ms),包括8kHz,16kHz和32kHz 以90 dB和10 dB之间的递减电平呈现。每个新 刺激被记录为从以前的演示下来的5dB SPL。每 测量点被记录并平均1000次并分析
    2. ABR阈值被确定为类似于上述描述的最低可识别ABR响应
    3. 保存结果波形以供将来参考和分析。
      1. 一般来说,在第一次麻醉剂注射后约30-40分钟,小鼠将保持麻醉状态。
      2. 由于点击和频率特定ABR记录的持续时间 (约40分钟),麻醉剂原始剂量的五分之一应该 在约20分钟注射以避免小鼠醒来的可能性 ?在测试期间。
      3. 如果鼠标开始移动,ABR录音 ?将被破坏,鼠标必须重新麻醉五分之一 的原始麻醉剂量。
    4. 完成录制后,小心地从鼠标中取出电极。
    5. 当所有测试完成后,将电极浸入 聚维酮碘溶液约1小时;然后用70%乙醇冲洗 ?盖上它们并将它们放置在无菌容器中。
    6. 用70%乙醇清洁记录室和所有与小鼠接触的表面

  4. 从麻醉中恢复
    1. 将鼠标放在一个空的和清洁的预热笼中 从麻醉中出现。不要让老鼠无人看管,直到他们 完全恢复。将这个笼子放在加热垫上保持 小鼠的体温直到从麻醉中恢复完全 发生。
    2. 返回小鼠到他们原来的笼子,没有加热垫,如果他们 可以放在他们的背后自己回到自己的脚。


图4. ABR测试的代表图。在具有正常ABR(黑色,蓝色和绿色曲线)和异常ABR(红色曲线)的小鼠中测量ABR阈值(声压级的分贝,dBSPL) 。与具有正常ABR的小鼠相比,具有异常ABR的小鼠在点击刺激和色调刺激(8,16和32kHz)下显示更高的ABR阈值。


  1. 盐酸氯胺酮和盐酸赛拉嗪的混合物
    它含有氯胺酮盐酸盐(Ketaset 100 mg/kg)和盐酸赛拉嗪(Xyla-Ject 10 mg/kg):
    2ml氯胺酮(100mg/ml) 1ml甲苯噻嗪(20mg/ml) 5ml无菌双蒸水
    混合均匀,放在橡胶密封的小瓶中 注意:
    1. 可以使用氯胺酮和赛拉嗪的溶液,只要氯胺酮或赛拉嗪不过期并且是无菌的。
    2. 溶液在不使用时应保存在4℃,在使用时保存在室温下。




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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Akil, O., Oursler, A. E., Fan, K. and Lustig, L. R. (2016). Mouse Auditory Brainstem Response Testing. Bio-protocol 6(6): e1768. DOI: 10.21769/BioProtoc.1768.
  2. Akil, O., Sun, Y., Vijayakumar, S., Zhang, W., Ku, T., Lee, C. K., Jones, S., Grabowski, G. A. and Lustig, L. R. (2015). Spiral ganglion degeneration and hearing loss as a consequence of satellite cell death in saposin B-deficient mice. J Neurosci 35(7): 3263-3275.