{"artifact":{"id":"experiment_proposal-68ac0c5a-2ffe-4adc-bb4b-4e67725ff20e","artifact_type":"experiment_proposal","entity_ids":"[\"CCK\", \"Alzheimer's disease\", \"h-var-a4975bdd96\"]","title":"Experiment Proposal: Closed-loop transcranial focused ultrasound to restore hippocampal gamma oscillations via cholecystokinin interneuron neuromodulation in Alzheimer's disease","quality_score":0.78,"created_by":"forge_experiment_proposal_generator","provenance_chain":"[{\"artifact_id\": \"hypothesis-h-var-a4975bdd96\", \"relation\": \"derived_from\", \"timestamp\": \"2026-04-28T20:06:46.186650+00:00\"}]","content_hash":"5400b6044f1343e3fdaada919f044effc44af907f9e4b86818702b01a1cc04bd","metadata":{"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.."],"citations":["31076275","31578527","35151204","36450248","37384704","38642614","39964974"],"generator":"forge_experiment_proposal_generator","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."],"est_cost_usd":187500.0,"generated_at":"2026-04-28T20:02:00+00:00","key_controls":["Sham LIFUS (identical surgical/positioning procedure, 0 mW/cm² output) to control for stress/anesthesia/handling effects","WT littermates (no amyloid pathology) to establish full gamma restoration ceiling and control for age-related gamma decline","Spadin-only group (no LIFUS, TREK-1 antagonist alone) to confirm specificity of mechanosensitive channel activation","CCK-Cre;DTR;5xFAD with DTX but no LIFUS to control for ablation-induced circuit changes independent of ultrasound","AAV9-hM4Di-mCherry injection in CCK-Cre;5xFAD + CNO (chemogenetic inhibition) as alternative CCK suppression control","Heat-killed LIFUS (identical parameters but subthreshold intensity, 50 mW/cm²) to control for non-specific tissue effects","Open-loop LIFUS (continuous 40 Hz, not closed-loop) to dissect contribution of feedback control mechanism","Age-matched 5xFAD with behavioral training only (no LIFUS) to control for environmental enrichment effects on gamma","PIEZO1 conditional knockout in CCK+ neurons to test upstream mechanotransduction requirement","Interleukin-1β ELISA on hippocampal tissue to confirm anti-inflammatory component of gamma restoration"],"model_system":"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.","artifact_path":"experiment-proposals/proposal_h_var_a4975bdd96_9d7150f3.json","hypothesis_id":"h-var-a4975bdd96","pmid_validity":{"31076275":true,"31578527":true,"35151204":true,"36450248":true,"37384704":true,"38642614":true,"39964974":true},"_schema_version":1,"primary_readout":"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).","hypothesis_title":"Closed-loop transcranial focused ultrasound to restore hippocampal gamma oscillations via cholecystokinin interneuron neuromodulation in Alzheimer's disease","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.","est_duration_weeks":14,"secondary_readouts":["Amyloid-β plaque burden: thioflavin-S stereology in dorsal hippocampus, expressed as % area occupied (3 sections/mouse, 200 μm spacing)","6E10 immunohistochemistry in CA1 stratum lacunosum-moleculare, plaques counted per mm²","CSF/hippocampal interstitial fluid Aβ42/40 ratio via microdialysis (2 h collection, 1 μL/min, 35 kDa MWCO membrane)","Tau phosphorylation at S396 (PHF-1 antibody) via ELISA of hippocampal homogenate","In vivo calcium imaging of CCK interneurons: AAV9-CaMKII-GCaMP8m injection in CCK-Cre;5xFAD mice, gradient-index lens implant, fiber photometry during LIFUS","Microglial morphological analysis: Iba1 Sholl analysis, branch length, endpoints per cell (30 cells/mouse, 3 sections)","CCK interneuron electrophysiology: whole-cell patch-clamp from identified CCK+ neurons in acute hippocampal slices 24 h after final LIFUS session, measure resting membrane potential, input resistance, firing frequency at 200 pA current injection","TREK-1 expression: qPCR (Kcnk2) and western blot from microdissected CCK+ cells via RiboTag sorting","Novel object recognition: 48 h inter-trial interval, discrimination index as primary behavioral readout","Contextual fear conditioning: % freezing during context re-exposure 24 h after training","Place navigation task: Morris water maze (hidden platform, 60 s max, 4 trials/day × 5 days)","Hippocampal volume: ex vivo MRI (9.4T) for CA1 subfield volumetry","CSF biomarkers: p-tau181, neurogranin via ELISA from cisterna magna tap"],"dataset_dependencies":[],"statistical_approach":"Linear mixed-effects model (lme4 package, R) for primary and secondary readouts with fixed effects: genotype × treatment × time, random intercept for mouse (repeated measures), sex as covariate. Post hoc pairwise comparisons with Tukey HSD. Primary threshold: two-sided α=0.05. Multiplicity correction: Benjamini-Hochberg FDR (5% level) across 14 secondary readouts. Mixed-effects model accommodates missing data from attrition. Pre-registration of primary/secondary hierarchy and statistical plan on OSF prior to data collection. Effect sizes reported as Cohen's d with 95% CI. Sensitivity analysis excludes outliers >3 SD from group mean.","sample_size_rationale":"Power analysis performed in G*Power 3.1. For two-group comparison of gamma power (primary outcome): expected d=1.15 based on Martorell 2019 (Cell) showing ~65% baseline deficit and 55-70% recovery with gamma entrainment. Using α=0.05, power=0.90 yields n=15 per group; inflated to n=16 for anticipated 7% attrition (lost implants, mortality). Total N=80 mice across 5 groups. This exceeds the n=12-15 per group typical for 5xFAD behavioral studies (Jankowsky 2005, Oakley 2006) and aligns with published LIFUS studies in AD models (e.g., 16-20/group in Bobola 2023, Transl Neurodegener). Stratification by sex ensures balanced representation across groups.","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.","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)."},"created_at":"2026-04-28T13:06:46.212494-07:00","updated_at":"2026-04-28T13:06:46.212494-07:00","version_number":3,"parent_version_id":null,"version_tag":null,"changelog":null,"is_latest":1,"lifecycle_state":"active","superseded_by":null,"deprecated_at":null,"deprecated_reason":null,"dependencies":null,"market_price":0.5,"origin_type":"internal","origin_url":null,"lifecycle_changed_at":null,"citation_count":0,"embed_count":0,"derivation_count":0,"support_count":0,"contradiction_count":0,"total_usage":0.0,"usage_score":0.5,"usage_computed_at":null,"quality_status":null,"contributors":[],"answers_question_ids":null,"deprecated_reason_detail":null,"deprecated_reason_code":null,"commit_sha":null,"commit_submodule":null,"last_mutated_at":"2026-05-16T14:51:34.657673-07:00","disputed_at":null,"gap_id":null,"mission_id":null,"intrinsic_priority":null,"effective_priority":null,"artifact_id":"c4fa1304-bd9b-44b4-9ce9-bf6faa993930","artifact_dir":null,"primary_filename":null,"accessory_filenames":null,"folder_layout_version":1,"migrated_to_folder_at":null,"hypothesis_id":null,"authorship":{"kind":"human","contributors":[{"role":"author","actor_ref":"forge_experiment_proposal_generator"}]},"epistemic_tier":"T3_provisional","created_by_agent_id":null},"outgoing_links":[{"target_artifact_id":"h-var-a4975bdd96","link_type":"derives_from","strength":1.0,"evidence":"{\"source\": \"forge_experiment_proposal_generator\", \"method\": \"generate_experiment_proposal\"}"}],"incoming_links":[],"current_artifact_id":"experiment_proposal-68ac0c5a-2ffe-4adc-bb4b-4e67725ff20e","is_canonical":true,"supersede_chain":["experiment_proposal-68ac0c5a-2ffe-4adc-bb4b-4e67725ff20e"]}