Minocycline Treatment Reverses Sound Evoked EEG Abnormalities in a Mouse Model of Fragile X Syndrome

Abstract

Fragile X Syndrome (FXS) is a leading known genetic cause of intellectual disability. Many symptoms of FXS overlap with those in autism including repetitive behaviors, language delays, anxiety, social impairments and sensory processing deficits. Electroencephalogram (EEG) recordings from humans with FXS and an animal model, the Fmr1 knockout (KO) mouse, show remarkably similar phenotypes suggesting that EEG phenotypes can serve as biomarkers for developing treatments. This includes enhanced resting gamma band power and sound evoked total power, and reduced fidelity of temporal processing and habituation of responses to repeated sounds. Given the therapeutic potential of the antibiotic minocycline in humans with FXS and animal models, it is important to determine sensitivity and selectivity of EEG responses to minocycline. Therefore, in this study, we examined if a 10-day treatment of adult Fmr1 KO mice with minocycline (oral gavage, 30 mg/kg per day) would reduce EEG abnormalities. We tested if minocycline treatment has specific effects based on the EEG measurement type (e.g., resting versus sound-evoked) from the frontal and auditory cortex of the Fmr1 KO mice. We show increased resting EEG gamma power and reduced phase locking to time varying stimuli as well as the 40 Hz auditory steady state response in the Fmr1 KO mice in the pre-drug condition. Minocycline treatment increased gamma band phase locking in response to auditory stimuli, and reduced sound-evoked power of auditory event related potentials (ERP) in Fmr1 KO mice compared to vehicle treatment. Minocycline reduced resting EEG gamma power in Fmr1 KO mice, but this effect was similar to vehicle treatment. We also report frequency band-specific effects on EEG responses. Taken together, these data indicate that sound-evoked EEG responses may serve as more sensitive measures, compared to resting EEG measures, to isolate minocycline effects from placebo in humans with FXS. Given the use of minocycline and EEG recordings in a number of neurodegenerative and neurodevelopmental conditions, these findings may be more broadly applicable in translational neuroscience.

Keywords: EEG; MMP-9; autism; forebrain; fragile X syndrome; minocycline; sensory hypersensitivity.

FIGURE 1

Overview of experimental procedures. All experiments were conducted on adult male C57BL/6 mice. After surgical implantation of electrodes, mice were given 6 full days to recover before EEG experiments. All mice were recorded on all EEG protocols pre-drug administration. A total of 23 Fmr1 KO mice and 21 WT mice were tested in “pre” group before any drug administration. The mice then received either minocycline (12 KO and 9 WT) or vehicle (11 KO and 12 WT) solution through oral gavage daily for 10 days followed by “post” tests. EEG recordings were never obtained on the same day that oral gavage was administered.

FIGURE 2

Increased resting power was observed in Fmr1 KO mice compared to WT mice in the pre-drug condition. After mice were habituated to the recording area, 5 min resting EEG was recorded in the absence of any overt sensory stimulation. Mean power density was calculated for both genotypes using both auditory and frontal cortex electrodes (A,C). For statistical comparisons, average power in each frequency band (color) was compared using standard frequency cutoffs (B, D). Statistical comparisons are the result of MANCOVA, using the % movement for individual animals as a covariate, in order to isolate genotype effects from potential hyperactivity phenotypes. In general, Fmr1 KO mice showed an increase in raw power, but the most statistically robust differences were in the low and high gamma band ranges. p-values were corrected for multiple comparisons using Bonferroni procedures, and each brain region was run separately (23 Fmr1 KO mice and 21 WT mice; *p < 0.05, **p < 0.01, ***p < 0.001, ^#p < 0.00001).

FIGURE 3

Minocycline treatment reduces resting gamma power in Fmr1 KO mice to WT levels. After Fmr1 KO mice were treated for 10 days with minocycline, mean power density was calculated for both groups using both auditory and frontal cortex electrodes (A,C). The most significant phenotypes in the gamma frequency range were normalized and resting gamma power was similar to WT (Veh) treatment group (B,D). Lower frequency deficits were still present (12 minocycline KO and 12 vehicle WT; *p < 0.05, ***p < 0.01, ****p < 0.001, *****p < 0.0001, ^#p < 0.00001).

FIGURE 4

Minocycline effects on gamma power were no different from vehicle effects in the Fmr1 KO mice. The 10-day minocycline effectiveness was compared against a 10-day vehicle treated group in Fmr1 KO mice. Mean power density was calculated for both drug conditions using both auditory and frontal cortex electrodes (A,C). No statistical significance was observed on this measure for KO mice (B,D). Minocycline is not better or worse than a vehicle control on resting EEG power spectral density (12 minocycline KO and 11 vehicle KO; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^#p < 0.00001).

FIGURE 5

Minocycline reverses phase locking deficits tested with chirp stimuli. (A–F) Colored regions are group mean differences between different genotype and drug conditions. Black-outlined regions are significantly reduced (blue with solid black outline) or increased (red with dotted black outline) as a result of a statistical permutation approach. (A, D) In the AC, there is a decrease in ITPC at beta to low-gamma bands, and an increase in ITPC in the high gamma range in the Fmr1 KO mice. In the FC, there is a decrease in ITPC between beta to high gamma frequencies. (B, E) Ten days of minocycline treatment increases ITPC in Fmr1 KO mice compared to vehicle in the FC, but not in the AC, suggesting a differential response of the two regions to this treatment. (C, F) The normalizing effect of minocycline in Fmr1 KO mice can be seen. In both the AC and FC, there is no difference between the minocycline treated KO and vehicle treated WT groups at beta/low-gamma bands.

FIGURE 6

Minocycline reverses phase locking deficits in the 40 Hz ASSR in the FC. An auditory steady state response is the response to a series of 40 Hz train of clicks (1 s duration). The analysis and interpretation are the same as in Figure 5. Comparing genotype differences in ITPC before drug treatment reveals a robust reduction in the Fmr1 KO mice compared to WT in the FC (D), but not in the AC (A). After treating KO mice with minocycline for 10 days, ITPC values in the FC increased significantly compared to vehicle-treated controls in matching time x frequency regions (E), with no effect in the AC (B). Direct comparison of ASSR in Fmr1 KO mice treated with minocycline to vehicle-treated WT mice show virtually no difference, suggesting a rescue of the phenotype in the FC (F), and again no effect found in the AC (C).

FIGURE 7

Minocycline reduces sound-induced gamma power in Fmr1 KO mice. Trains of 100 ms duration broad band noise were used to generate event related potentials (200 repetitions) for time × frequency analyses. After correcting for baseline (–250 ms through –150 ms) single trial power was averaged over all trials. This allows for the analysis of power that is normalized to individual animal baselines. In the pre-drug condition, Fmr1 KO mice showed increased on-going power after the auditory stimulus in the delta and high gamma range in both AC and FC (A,D). In addition, the FC showed a significant reduction in power the beta to low gamma frequency range (D). Minocycline treatment significantly reduced onset power in Fmr1 KO animals over vehicle treatment in both FC and AC (B,E), but also increased power in the AC in two distinct bands at ∼20 and ∼40 Hz that occurred well after sound onset. Finally, comparing minocycline treated Fmr1 KO mice to vehicle-treated WT group, reveals a shift in power in AC from high gamma in (A), to low gamma in (C). Additionally in the FC, ongoing gamma power and modulation of beta – low gamma power in (D) are significantly reduced (F).