{"artifact":{"id":"experiment_proposal-f8425957-e660-45c0-98f0-915f752154d7","artifact_type":"experiment_proposal","entity_ids":"[\"h-var-e2b5a7e7db\"]","title":"Experiment Proposal: h-var-e2b5a7e7db","quality_score":0.75,"created_by":"forge_experiment_proposal_generator","provenance_chain":"[]","content_hash":"7078349c0ea50514b27840b48ccbc42b0e6c95ca82dafe8841d219c9b384f016","metadata":{"citations":["19449329","23431156","26282667","40796363","41675057"],"generated_at":"2026-04-28T19:58:06.359076+00:00","intervention":"Bilateral stereotactic injections of AAV9 vectors (titer 1×10^13 vg/mL, 500 nL per side) into ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamic nuclei targeting thalamocortical projection neurons (coordinates: AP -1.58 mm, ML ±1.5 mm, DV -3.4 mm from bregma). Pharmacological validation arm: Ro 25-6981 (0.5 mg/kg, Tocris) or if available EU1180-453 (3 mg/kg, custom synthesis) administered i.p. twice daily for 12 weeks. Drug vehicle: 10% DMSO, 90% saline (0.9% NaCl). Treatment duration: 12 weeks of continuous pharmacological or 4-week viral expression followed by 8 weeks of observation with twice-weekly monitoring.","key_controls":["Wild-type C57BL/6J + AAV9-CamKIIα-mCherry: establishes baseline glymphatic function and AQP4 polarization without tau pathology","Wild-type C57BL/6J + Ro 25-6981 pharmacotherapy: validates pharmacological effects independent of tau transgene","P301S + AAV9-CamKIIα-mCherry + vehicle: disease control, establishes pathology-driven glymphatic impairment","P301S + AAV9-CamKIIα-shRNA-Grin2b + vehicle: validates specificity of GRIN2B knockdown on glymphatic function (negative control)","Conditional GCaMP6s expression: AAV9-GFAP-GCaMP6s in separate cohort (n=8 per genotype) for astrocyte calcium dynamics without viral GRIN2B manipulation","AAV-inactivated heat-inactivated control: validates that serotype and promoter, not transgene, do not alter baseline glymphatic function","Cre-negative littermate controls: for experiments using Cre-dependent systems to verify recombination specificity","Positive allosteric modulator arm: EU1180-453 or CIQ (if commercially available) pharmacotherapy group as pharmacological positive control for GRIN2B enhancement","Age-matched sacrifice timeline: all terminal endpoints collected within 2-hour window to minimize circadian-driven glymphatic variability"],"model_system":"Male P301S MAPT transgenic mice (PS19 line, Jackson Laboratory, B6;C3-Tg(MAPT*P301S)PS5VIle/J) aged 3 months at study initiation. Age- and sex-matched wild-type C57BL/6J mice (Jackson Laboratory) serve as strain controls. Three treatment groups with n=16 per group (power=0.80, α=0.05, two-tailed): (1) P301S + AAV9-CamKIIα-GRIN2B-WT (GRIN2B overexpression), (2) P301S + AAV9-CamKIIα-shRNA-Grin2b (Knockdown control), (3) P301S + AAV9-CamKIIα-mCherry (vector control), (4) WT + AAV9-CamKIIα-mCherry (wild-type control), (5) P301S + vehicle (pharmacology control). Sample size of 16/group accounts for 15% attrition and ensures n≥13 evaluable subjects per group based on power calculation (G*Power 3.1, expected d=1.1, variance from published glymphatic studies).","hypothesis_id":"h-var-e2b5a7e7db","timeline_weeks":24,"_schema_version":1,"null_hypothesis":"Selective enhancement of GRIN2B-containing NMDA receptors in thalamocortical projection neurons does not significantly improve glymphatic tau clearance in P301S tau transgenic mice, as measured by intracerebral radiolabeled tau efflux kinetics, multiphoton-monitored perivascular tau transport velocity, and Gd-DTPA MRI-derived glymphatic clearance rates, compared to vehicle-treated or scramble-vector controls.","primary_readout":"Glymphatic tau clearance rate quantified by: (a) intracerebral injection of [125I]-labeled recombinant human tau (0.5 μCi in 2 μL PBS) into right hippocampus (AP -2.0 mm, ML +1.5 mm, DV -1.8 mm), with serial plasma sampling via tail vein at 0, 1, 2, 4, 6, 12, 24, 48, 72, and 96 hours post-injection; (b) area under the clearance curve (AUC) calculated for each animal; (c) HPLC-based quantification of [125I] activity in cervical lymph nodes collected at terminal sacrifice. Secondary primary: multiphoton microscopy velocity measurement of fluorescently labeled K18-FITC tau (rPeptide) transport along cortical penetrating arterioles in awake, head-fixed mice at 2-hour post-injection timepoint.","estimated_cost_usd":387000.0,"secondary_readouts":["AQP4 polarization index: immunofluorescence colocalization of AQP4 with CD31 (vascular marker) and GLUT1 at cortical penetrating vessels, quantified as Pearson's coefficient and Mander's overlap (n≥40 vessels per animal)","Thalamocortical gamma oscillation power: 64-channel LFP recordings from layer IV cortex and VPL nucleus during active wakefulness, spectral analysis in 30-100 Hz band, normalized to baseline","CSF phospho-tau concentrations: Meso Scale Discovery ELISA for pT181 and pT231 tau species in cisterna magna samples","Astrocytic calcium dynamics: two-photon GCaMP6s imaging in astrocyte-specific (Aldh1l1-CreERT2 × Ai96) P301S mice, calcium event frequency and wave propagation velocity","Dystrophin phosphorylation state: Western blot of pS2831-dystrophin in cortical membrane fractions, normalized to total dystrophin","Synaptic integrity markers: ELISA for cortical PSD-95, synapsin-1, and postsynaptic density protein 95 in synaptoneurosome preparations","Spatial working memory: Morris water maze latency and platform crossing frequency","Object recognition memory: novel object discrimination index at 24-hour retention interval","Tau pathology burden: AT8 and PHF-1 immunoreactivity stereology in entorhinal cortex and hippocampus","Neuronal survival: NeuN+ cell counts in thalamocortical relay nuclei and layer IV cortex","Excitotoxicity markers: cleaved caspase-3 and TUNEL in peri-injection regions"],"expected_effect_size":"Cohen's d = 1.1-1.4 for primary tau clearance outcome, based on: (a) prior reports of 55-70% tau clearance enhancement with gamma-frequency optogenetic stimulation (PMID references from hypothesis), (b) 40-65% AQP4 polarization increase with GluN2B modulation, and (c) 2.5-3.2 fold increase in tau efflux with positive allosteric modulation. Secondary outcomes (AQP4, LFP gamma power): d = 0.8-1.0. Power analysis (G*Power 3.1): for d=1.1, α=0.05, power=0.80, two-group comparison requires n=13 per group; for five-group ANOVA with expected medium effect (f=0.35), n=16 per group provides power=0.85. Effect size inflated by 15% to account for technical variability in glymphatic measurements.","statistical_approach":"Primary analysis: two-way ANOVA (genotype × treatment) on log-transformed AUC tau clearance values, with post-hoc Sidak's multiple comparison test. Secondary outcomes: mixed-effects repeated measures ANOVA for longitudinal CSF and behavioral data (time as repeated factor). Multiplicity correction: Benjamini-Hochberg false discovery rate (FDR) controlling at q<0.05 across 11 secondary readouts. Non-parametric alternatives (Kruskal-Wallis) applied if Shapiro-Wilk normality test fails (p<0.05). Outlier exclusion: Grubbs' test at α=0.05, pre-specified criteria. Alpha level: 0.05 two-tailed for primary outcome; 0.05 FDR-corrected for secondary outcomes. Statistical consultation and pre-registration on OpenScience Framework prior to study initiation.","sample_size_rationale":"Power calculation performed using G*Power 3.1.9.4 (Universität Düsseldorf). Primary outcome assumption: mean AUC difference of 40% between GRIN2B-overexpression and vector control (based on precedent from PMID 23431156 and gamma stimulation literature showing 55-70% clearance enhancement). Expected pooled SD = 25% of mean (conservative estimate from published glymphatic microdialysis studies). For two-tailed t-test with d=1.1, α=0.05, power=0.80: n=14 per group. For five-group ANOVA (f=0.35 medium effect): n=15 per group. Accounting for 15% attrition and exclusions: n=16 per group. Total N=80 animals. Post-hoc power for secondary readouts at n=13 evaluable: 0.72-0.85 depending on expected effect size. Tissue from n=6 per group reserved for future mechanistic assays (proteomics, RNA-seq) contingent on primary outcome significance.","falsification_criterion":"The hypothesis is falsified if: (1) Primary outcome: GRIN2B-overexpression fails to increase tau clearance AUC by ≥25% compared to vector control (Sidak-corrected p<0.05); (2) AQP4 polarization index does not increase by ≥30% in GRIN2B-overexpression group vs. vector control (pre-specified threshold, p<0.05); (3) Thalamocortical gamma power does not increase by ≥20% in GRIN2B-overexpression group; (4) Any evidence of excitotoxicity: cleaved caspase-3+ cell density increases >2-fold in thalamic injection sites; (5) Seizure activity detected in ≥25% of treated animals (EEG criterion: ≥3 episodes of >3 SD spike activity per 24-hour recording period). Partial support criterion: statistically significant improvement in tau clearance (p<0.05) without excitotoxicity would support a mechanism but require replication before full hypothesis acceptance. Mechanistic miss: significant tau clearance improvement without corresponding AQP4 polarization change would indicate an AQP4-independent glymphatic pathway, requiring hypothesis revision."},"created_at":"2026-04-28T12:58:06.370255-07:00","updated_at":"2026-04-28T12:58:06.370255-07:00","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":"5e0cc7d4-f4e1-4164-97e9-4423642ec180","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-e2b5a7e7db","link_type":"derives_from","strength":1.0,"evidence":"{\"source\": \"forge_experiment_proposal_generator\", \"method\": \"generate_experiment_proposal\"}"}],"incoming_links":[],"current_artifact_id":"experiment_proposal-f8425957-e660-45c0-98f0-915f752154d7","is_canonical":true,"supersede_chain":["experiment_proposal-f8425957-e660-45c0-98f0-915f752154d7"]}