Methods
Animals, Housing and Ethics
Statement
12 male Mongolian gerbils, purchased from Janvier (Le Genest-Saint-Isle,
France) were housed in standard animal racks (Bio A.S. Vent Light,
Zoonlab, Emmendingen, Germany) in groups of 3 to 4 animals with free
access to water and food at a room temperature of 20 to
25◦C under a 12h/12h dark/light circle. At the
beginning of the experiments, the animals were 12 weeks old and had been
at least two weeks in the animal facility for habituation. The use and
care of the animals was approved by the state of Bavaria
(Regierungspräsidium Mittelfranken, Ansbach, Germany, No.
54-2532.1-02/13).
Experimental protocol
The animals were investigated twice with the behavioral paradigm of the
gap prepulse inhibition of the acoustic startle response (GPIAS) to
evaluate possible behavioral indications of a tinnitus percept and with
auditory brainstem response (ABR) audiometry under anesthesia to obtain
the hearing thresholds. The respective first measurements were performed
five days before a monaural acoustic noise trauma (2 kHz, 115 dB SPL, 75
min; anesthetized) or sham trauma (2 kHz, 65 dB SPL, 75 min;
anesthetized). The second ABR measurements were obtained 4 days after
that trauma, and GPIAS was obtained immediately before sacrificing the
animals (CO2 euthanasia) 13 days after the trauma. The
brains of the sacrificed animals were extracted, cryo-sliced,
immunostained and immunohistologically evaluated.
Behavioral measurements
For the GIPAS measurements for tinnitus assessment, a custom-made
open-source setup was used as detailed in (Gerum et al. , 2019).
In short, the animals were placed in an acrylic glass restrainer tube,
closed with a wire mesh at the front side and a cap at the back end, and
placed on a sensor platform fixed to a vibration-damped table. Movements
of the sensor platform were registered using a 3D acceleration sensor.
Two loudspeakers were placed at a distance of 10 cm in front of the
animal. One presenting the 115 dB SPL startle stimulus (Neo 25 S,
SinusLive, noise burst 20ms, flattened with 5 ms sin2ramps) and the other the 60 dB SPL spectral noise background (CantonPlus
XS.2). Spectral noise was centered at 1 to 16 kHz in octave steps with
one octave bandwidth, with and without a gap of silence of 50 ms
(flanked by 20 ms sin2 ramps, 10ms complete silence)
starting 100 ms before the startle stimulus.
During the two measurements, animals were given 15 min of habituation in
darkness in the tube. Prior to the actual measurement, five habituation
stimuli were presented to “level” the startle responses. Each stimulus
was repeated 30 times (15 with and 15 without gap), summing up to 120
stimuli, which took roughly 30 min.
The complete evaluation of the GPIAS measurements were performed using
custom-made Python programs. The GPIAS effect was quantified by
calculating the median from the full combinatorial startle amplitudes as
a response to gap and no gap pre-stimulus (for details see Schillinget al. , 2017). Statistics on the mean GPIAS results were
performed with Statistica 14 (TIBCO Software GmbH, Munich, Germany; cf.
below).
Brainstem audiometry
For the frequency-specific ABR measurements the animals were
anesthetized with a ketamine-xylocaine solution (ketamine 500 mg/kg,
xylazine 25 mg/kg). The animals were placed on a remote-controlled
heating pad set to 37°C. Individual audiograms of both ears were
obtained for stimulation frequencies between 1 and 16 kHz in octave
steps for stimulation intensities ranging from 0 to 90 dB SPL in 5 dB
steps (6 ms duration with 2 ms sine-square ramps). For each ear,
stimulus, and intensity, 300 repetitions were presented. The complete
measurement of one ear took around 30 min. Three silver wires were used
as electrodes and were placed subcutaneously retroaural above the bulla
of the tested ear (recording electrode), central between both ears
(reference electrode) and at the basis of the tail (ground electrode).
The signal was recorded differentially between recording and reference
electrode and filtered (bandpass filter 400 to 2000 Hz) via a Neuroamp
401 amplifier (JHM, Mainaschaff, Germany).
Monaural acoustic noise and sham
trauma
Animals were put under deep ketamine-xylocaine anesthesia (cf. above).
One ear (pseudorandomly selected) was plugged with foam (3M™ earplugs
1110, 3M, Neuss, Germany) adding at least 20 dB attenuation in the given
frequency range (Stuermer & Scheich, 2000). The animals were placed on
a remote-controlled heating pad set to 37°C with the non-plugged ear
towards a speaker (CantonPlus XS 2). The acoustic noise trauma (2 kHz,
115 dB SPL, 75 min) or sham trauma (2 kHz, 65 dB SPL, 75 min) was only
performed on that ear.
Brain extraction, cryo-slicing and immunofluorescence
staining
The sacrificed animals were decapitated and the brains were extracted
carefully. They were placed in a 2% paraformaldehyde / 2% sucrose
solution on a rocking shaker for 8 hours at 4°C. The tissue was then
washed with phosphate-buffered saline (PBS) three times over one minute
each, and then transferred into a 10% sucrose solution on a rocking
shaker for 8 hours at 4°C. The incubation was finalized with 15%
sucrose solution and finally 30% sucrose solution on a rocking shaker
for 8 hours each at 4°C, washing was performed with PBS two times for
one minute each. After drying, the tissue was shock-frosted in liquid
nitrogen at ‑196°C and cur on a cryotome in 18 µm thick slices, which
were transferred onto object slides and air-dried for 30 min at ambient
temperature. Until staining, the slides were stored at -20°C in a
fridge,
After defrosting and drying, every 10th object slide
was used and fixated with -20°C cold methanol for three minutes and
dried for 10 minutes. After that the slices were permeabilized with 2 %
horse-serum in 0.1 % Triton X-100 in PBS solution over 2.5 hours. Then
the incubation with Wisteria Floribunda
Lectin-Fluorescein-5-isothiocyanate (WFA-FITC) started, with WFA-FITC
1:200 in 0.5 % horse-serum/PBS over 5 hours at room temperature. It was
stopped with washing with PBS three times over five minutes each.
Finally, the slides were mounted with IS Mounting-Medium PI.
Microscopy and
densitometry
Immunostained tissue slices were investigated with a BZ 9000 Keyence
fluorescence microscope (4x magnification) and single pictures were
combined to a full slice picture with the BZ-II software. We used a
1360x1024 pixel resolution, green fluorescence, exposure adjustments:
shadow 11, highlight 165, gamma 0.7, exposure time 1/1.1 sec.
The combined slice-pictures were evaluated with ImageJ Fiji (version
1.53) and only the slices containing the primary auditory cortex were
evaluated. For marker-intensity reference, always the brainstem of the
same slide was used. The appropriate slices and cortical / subcortical
areas were identified using anatomical markers based on a Mongolian
gerbil brain atlas (plates 28-33 in Radtke-Schuller et al. ,
2016). The areas of interest ipsi and contralateral to the trauma ear
were marked and their luminance was calculated using the inbuilt program
function. The luminance values of cortical areas were divided by the
reference brainstem area values (luminance ratio). Examples of the brain
slices of animals with trauma and behavioral signs of tinnitus, with
trauma but without behavioral signs of tinnitus and sham trauma without
tinnitus are depicted in Figure 1 .