A total of 35 mice were used for this study. 14 Thy1 mice (B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J, Stock #007612, The Jackson Laboratory, Bar Harbor, ME, USA; 9-16 weeks; body weight 19-31g) of both genders (male, n=4; female, n=10) and 21 male wild type mice (C57BL/6N; 8-10 weeks) were used. All mice received five weight drop-induced closed diffuse TBI events. While Thy1 mice were used to collect odor-evoked electrophysiology data, 10 wild type mice were used to collect immunochemistry data and 11 wild type mice were used to collect Y-maze behavioral data. All mice were born and raised under standard laboratory conditions of a 12-hour light/dark cycle at 22°C with 55% humidity and were fed a standard laboratory diet and water ad libitum.
Repetitive concussion treatment
Mice received a total of five weight drop-induced closed diffuse TBIs, each administered every three days. Closed diffuse TBIs were performed by a weight-drop device (weight 100g, fall height 75cm, angle 90deg), as previously described with a slight modification [22, 32]. Control and TBI mice were anesthetized with 2% Avertine (23 μg/g) IP injection before receiving the weight drop-induced TBI. Sham-injured animals were subjected to the same protocol, but no mass was dropped (Fig. 1). No mice were killed following impact, and mice were monitored for signs of severe injury or skull fracture.
Immunohistochemistry was performed as previously described . Animals were deeply anesthetized with 2% avertine (23 μg/g, i.p.) and then perfused through the ascending aorta with 100 ml of cold 0.1 M phosphate buffer saline (PBS), followed by 100 ml of 4% paraformaldehyde in 0.1 M PBS. The brain was removed, post-fixed overnight and cryoprotected in 30% sucrose in 0.1 M PBS at 4°C. Then cut by cryotome into 30 μm coronal sections, and processed histochemically as free-floating sections. Tissue sections were then rehydrated, blocked with blocking solution (3% H2O2), and incubated with p-Tau (S202/T205) (1:200; Thermofisher, Waltham, MA, USA) and p-Tau (S199) (1:200; Abcam, Cambridge, MA, USA) for 24 hrs. After three times of washing, the slides were processed with Vector ABC Kit (Vector Lab, Burlingame, CA, USA). Immunoreactive signals were developed with DAB chromogen (Thermo Fisher Scientific, Waltham, MA, USA). Slides were photomicrographed under bright field microscopy and analyzed using Image J.
EEG electrodes were implanted after repetitive concussion treatment. Mice were deeply anesthetized with ketamine (120mg/kg, intraperitoneal) and xylazine (6mg/kg, intraperitoneal), and fixed in a stereotaxic apparatus (David Kopf Instruments, Model 902, Tujunga, CA, USA). Sterilized micro-screw electrodes (Asia Bolt, South Korea) were fixed onto the skull surface of the olfactory bulb (anteroposterior, 4.8mm; mediolateral, 1.2mm; dorsoventral, -1.1 mm from bregma), frontal (anteroposterior, 0.5mm; mediolateral, 1.2mm), and parietal cortex (anteroposterior, -3.08mm; mediolateral, 3.75mm), with ground/reference electrodes implanted on the interparietal bone. The electrode coordinates were selected in accordance with the mouse atlas . To secure the electrode positions, dental cement (VertexTM Self-Curing, Vertex-Dental, Netherlands) was applied, along with two polycarbonate nuts (inner diameter 3mm, Nippon Chemi-Con, Japan) which were attached to the caudal edge of the cement for head-fixation during the experiment.
After one week of recovery period, mice were placed in front of the olfactometer while being head fixed in a custom mouse restrainer. Mice were placed so that their nose tip had a 1 cm distance from the olfactometer outlet. Three-channel LFP (the olfactory bulb, the frontal cortex, and the parietal cortex) data were collected during the olfactory oddball paradigm with a Cerebus amplifier (Blackrock Microsystems, UT, USA). All signals were digitized with a 2 kHz sampling rate and bandpass filtered from 0.3 to 500Hz.
Olfactory Oddball Paradigm
The experiment was conducted utilizing the olfactometer setup previously described by Kum et al. (2019) (Fig. 1c). For odor delivery, constant flow of filtered air (1L/min) was delivered, with odor stimuli being diluted by pumped air (200ml/min). Methyl salicylate (Sigma Aldrich, > 99% purity, mineral oil solution, odorant:solvent ratio was 3:1) and ethyl acetate (Sigma Aldrich, > 99.5% purity, distilled water solution, odorant:solvent ratio was 1:1) were selected for the olfactory oddball paradigm; methyl salicylate being the standard odorant and ethyl acetate being the deviant odorant. The standard and deviant odor were presented randomly with a 5:1 ratio. The stimulation period was 2 seconds with an inter-stimulus interval of 20 seconds. After presented, odor stimuli were vacated through the vacuum pump (1.5L/min) (Fig. 1d).
One session was composed of 90 trials (75 standard trials and 15 deviant trials), lasting approximately 33 minutes (Fig. 1d). Each mouse received four sessions of experiment in total, which were divided into two days in order to prevent adaptation to the odor stimuli and over-exhaustion caused by the long duration of head-fixing. Two sessions were given each day with an interval of 30 minutes between each session, and there were three to five days between the two days of experiment. The recording room was ventilated for 30 minutes after each recording session.
The LFP data were processed in Matlab 2019a (Mathworks, MA, USA) with Signal Processing Toolbox (ver. 8.2). First, the signals were bandpass-filtered (1-150 Hz using a 5-th order Butterworth filter, then transformed into time-frequency domain using sliding Hanning window (512 ms length, 100 ms resolution) and fast Fourier transform to obtain amplitude spectrogram. The frequency bands of interest were delta (1.5 – 4 Hz), theta (4 – 8 Hz), beta (12 – 30 Hz), low gamma (40 – 60 Hz), and high gamma (70 – 120 Hz) bands. In case of analysis of resting state, the amplitude spectrum of each channel was obtained from each individual by averaging the spectrogram of 2-min before the olfactory oddball paradigm. Cross-frequency coupling was evaluated using modulation index (MI), which is a statistical measure of phase lock of fast oscillation amplitudes to the relative time window of slow oscillation . The MI was calculated by dividing the Kullback-Leibler distance of actual amplitude distribution of fast oscillations over the phase of slow oscillations (bin number = 30) with uniform distribution. For the analysis of olfactory oddball paradigm, event-related spectral perturbation (ERSP) was obtained by averaging the spectrogram of baseline-corrected signals with temporal and frequency resolutions of 100 ms and 2 Hz. To match the number of trials between standard and deviant trials, only the standard trials right before the deviant trials were used. To evaluate the diminished oscillatory activities in TBI model mice, independent sample t-tests (alpha = 0.05, one-tailed) were performed using ttest2.m function in Matlab 2019a, over the individual mean value in the band-of interests and time-of-interests (degree of freedom = 15).
Olfactory Discrimination using Y-maze
Y maze was performed the subsequent day after the fifth weight drop, PND (12 weeks) as previously described with a modification . The Y maze apparatus was composed of three enclosed arms (35.3 x 6 x 18.4 cm (l × w × h)) constructed of white acrylic. A tray of home bedding (7.2 x 2.9 x 1.3) was placed at the end of one arm, and the same to its adjacent arm with new bedding. Each trial began by placing each individual mouse in the arm with no bedding and was observed for 10 minutes. Total distance (cm), velocity (cm/s), time (s), frequency (count), and latency to start (time) at each arm was recorded by Ethovision Software (Noldus Information Technology, Wageningen, Netherlands).