Auditory brainstem development of Naked Mole-Rats (Heterocephalus glaber)

Brainstem sections were imaged using an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan) with lasers for 405, 543 and 635 nm imaging. High resolution (1024 x 1024 pixel), 20X (UPLSAPO20X, NA 0.75) images were taken for sections displayed in the results at different developmental timepoints. Additional images were taken using a Zeiss (Jena, Germany) LSM 980 with Airyscan2 (1024 x 1024 pixel) at 20X (NA 0.8). The MNTB and LSO were characterized by their morphology, location and position in the trapezoid body and superior olive, and by the shape and size of neurons within these nuclei. Specifically, sections were cut from posterior – anterior and the 7th nerve and cochlear nuclei used as a reference for location of the MNTB and LSO. A montage, covering an extended field of view, was captured by imaging multiple 20X tiles and stitching them together using the Olympus FV10-ASW acquisition software. ABR measurements and analysis methods are described in previous publications. Naked mole-rats were anesthetized with a mixture of ketamine-xylazine (100mg/kg, 10mg/kg respectively) administered via intraperitoneal injections. Once the animals did not respond to toe-pinch, indicating adequate anesthesia, platinum subdermal needle electrodes were placed under the skin. The active electrode was placed between the ears (vertex) with the reference at the nape. A ground electrode was inserted in the foot of the animal. Animals were presented with monaural click stimuli through custom built ear bars. Stimuli were generated and evoked potentials recorded (sampled at 31 kHz and immediately downsampled by half) via an RME Fireface UCX sound card (RME Audio, Haimhausen, DE), controlled with custom built MATLAB software. Sound was generated with TDT MF1 Multi-Field Magnetic Speakers (Tucker-Davis Technologies, FL, USA) with the sound being calibrated prior to presentation using Etymotic ER-7C probe microphones (Etymotic Research Inc, IL, USA). Signals from the electrodes were amplified (10,000x) and digitized using an ISO-80 preamplifer and headstage (World Precision Instruments Sarasota, FL USA). Clicks were presented to both ears at a sound pressure level of 100 dB SPL (peak, re: 1000 Hz tone) attenuated in steps of 10 dB for threshold measurements. Data were averaged over 1000-2600 repetitions per stimuli and filtered with a second order Butterworth filter (cutoffs at 100Hz and 2000 Hz). Peaks in the ABR waveform were examined offline within a latency range of 1-10 ms. The lowest SPL at which an ABR peak was detected was taken as the threshold SPL. For each animal tested, the threshold was determined from the ear with the lower threshold. Several animals, particularly in the youngest age groups, did not have a recognizable ABR waveform for any stimulus SPL and are included as “no signal (N.S.)” datapoints.