DOI: 10.5061/dryad.tqjq2bw22
title: Auditory brainstem development of Naked Mole-Rats (Heterocephalus glaber)

description: Life underground often leads to animals having specialized auditory
 systems to accommodate the constraints of acoustic transmission in
 tunnels. Despite living underground, naked mole-rats use a highly vocal
 communication system, implying that they rely on central auditory
 processing. However, little is known about these animals’ central auditory
 system, and whether it follows a similar developmental time course as
 other rodents. Naked mole-rats show slowed development in the hippocampus
 suggesting they have altered brain development compared to other rodents.
 Here, we measured morphological characteristics and voltage-gated
 potassium channel Kv3.3 expression and protein levels at different key
 developmental time points (postnatal days 9, 14, 21, and adulthood) to
 determine whether the auditory brainstem (lateral superior olive (LSO) and
 medial nucleus of the trapezoid body (MNTB)), develops similarly to two
 common auditory rodent model species: gerbils and mice. Additionally, we
 measured the hearing onset of naked mole-rats using auditory brainstem
 response (ABR) recordings at the same developmental timepoints. In
 contrast to other work in naked mole-rats showing that they are highly
 divergent in many aspects of their physiology, we show that naked
 mole-rats have a similar hearing onset, between P9-P14, to many other
 rodents. On the other hand, we show some developmental differences, such
 as a unique morphology and Kv3.3 protein levels in the brainstem.
description: 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.
description: Matlab, FIJI (ImageJ)