🧬 Experiment Proposal
~$187,500 USD~14 weeks
AIMS
- Test whether Transcranial LIFUS (0.67 MHz fundamental frequency, 40 Hz pulse repetition frequency, 720 mW/cm² ISPTA, 100 ms burst/500 ms interval, 16.7% duty cycle, 30 min/day for 4 weeks). Ultrasound delivered via single-element focused transducer (1 MHz center frequency, 10 mm focal depth, 12 mm aperture) positioned stereotaxically over hippocampus (AP -2.0 mm, ML ±1.5 mm from bregma). Closed-loop EEG monitoring triggers stimulation only during endogenous gamma suppression (<40% baseline power). Sham group receives identical transducer positioning with 0 mW/cm² output. Spadin (5 mg/kg, i.p.) administered 30 min before each LIFUS session in antagonist group. Diphtheria toxin (20 ng/μL, 100 nL) injected stereotaxically into bilateral hippocampus of CCK-Cre;DTR;5xFAD mice 2 weeks prior to LIFUS for interneuron ablation validation. changes Hippocampal local field potential gamma power (30-80 Hz) during active LIFUS and 30-min post-stimulation epochs, recorded via 16-channel silicone probe (NeuroNexus A1x16-3mm-50-177) implanted in CA1 stratum radiatum. Spectral power computed via Welch's method (4 s windows, 50% overlap), primary metric: mean gamma power (30-80 Hz) normalized to baseline (%) across 4 weekly sessions. Secondary LFP metric: theta-gamma coupling strength (modulation index from phase-amplitude coupling analysis at theta phase × gamma amplitude). in 5xFAD mice (APP Swedish/Florida/Iowa transgenes on B6/SJL background), both sexes, 4-5 months age at intervention onset (early amyloid pathology stage). Total n=64 distributed across 4 groups: (1) 5xFAD + active LIFUS, (2) 5xFAD + sham LIFUS, (3) 5xFAD + LIFUS + spadin (TREK-1 antagonist), (4) CCK-Cre;5xFAD + LIFUS with DTR-mediated CCK interneuron ablation. Littermate WT mice (n=16) serve as positive controls for baseline gamma values. Sample size: n=16 per group, balanced for sex (8M/8F), determined by a priori power analysis (G*Power 3.1) targeting d=1.2, α=0.05, power=0.90 for primary outcome..
HYPOTHESES
- Closed-loop LIFUS-mediated TREK-1 activation in CCK interneurons does not restore hippocampal gamma oscillation power or reduce amyloid/tau pathology in 5xFAD mice beyond sham stimulation levels.
PROTOCOL SUMMARY
Transcranial LIFUS (0.67 MHz fundamental frequency, 40 Hz pulse repetition frequency, 720 mW/cm² ISPTA, 100 ms burst/500 ms interval, 16.7% duty cycle, 30 min/day for 4 weeks). Ultrasound delivered via single-element focused transducer (1 MHz center frequency, 10 mm focal depth, 12 mm aperture) positioned stereotaxically over hippocampus (AP -2.0 mm, ML ±1.5 mm from bregma). Closed-loop EEG monitoring triggers stimulation only during endogenous gamma suppression (<40% baseline power). Sham group receives identical transducer positioning with 0 mW/cm² output. Spadin (5 mg/kg, i.p.) administered 30 min before each LIFUS session in antagonist group. Diphtheria toxin (20 ng/μL, 100 nL) injected stereotaxically into bilateral hippocampus of CCK-Cre;DTR;5xFAD mice 2 weeks prior to LIFUS for interneuron ablation validation.
PREDICTED OBSERVATIONS
Cohen's d = 1.15 for primary gamma power restoration (5xFAD active vs 5xFAD sham). Based on Martorell et al. 2019 (PMID 31076275) showing ~65-70% gamma power reduction in 5xFAD vs WT and ~55-70% recovery with 40 Hz entrainment. Power calculation: for two-sided t-test, d=1.15, α=0.05, power=0.90 requires n=15/group; inflated to n=16 for 10% attrition buffer. Secondary readouts (plaque burden, behavior) expected d=0.85-1.0 based on Martorell 2019 and Adaikkan 2019 (PMID 31578527).
FALSIFICATION CRITERIA
The hypothesis is falsified if: (1) Active LIFUS fails to increase hippocampal gamma power above sham by >2 SD of sham group mean (i.e., effect size <0.5 Cohen's d) at week 4; (2) Spadin co-administration abolishes >80% of gamma restoration effect, confirming TREK-1 dependence; (3) CCK interneuron ablation prevents gamma restoration, confirming CCK-specific mechanism; (4) Amyloid burden does not decrease >15% vs sham at week 4 (Bonferroni-adjusted threshold p<0.003 for plaque outcome). Critical negative result: sham group shows equivalent or greater gamma power than active group, indicating failure to activate TREK-1 or engage CCK interneuron circuit. Mechanistic falsification: if CCK interneuron firing rates do not decrease (contrary to model predicting disinhibition), or if pyramidal cell dendritic excitability does not increase, the circuit mechanism is invalidated regardless of gamma power changes.