Bmal1 knockout in mPFC excitatory neurons

PRIMARY OUTCOME

Sleep parameters, behavioral response to SD, Homer1a expression

EXPECTED OUTCOMES

1. **Conditional Bmal1 knockout efficiency**: Cre-mediated excision will reduce Bmal1 mRNA by **78–85%** in mPFC tissue (qRT-PCR, ΔΔCt fold change 0.15–0.22 vs flox controls) and BMAL1 protein by **70–80%** (Western blot densitometry) by Day 21 post-surgery. 2. **Circadian period elongation**: cKO mice in DD will exhibit a period of **24.8–25.6 h** vs **23.6–24.0 h** in flox controls (χ^2 periodogram, p < 0.001), consistent with the known role of BMAL1 in circadian clock function. 3. **REM sleep suppression following SD**: cKO mice will show **40–55% reduction** in REM sleep time during the 18h recovery period post-SD compared to baseline, vs only **15–25% reduction** in flox controls (mixed-effects ANOVA, genotype × time interaction p < 0.01). 4. **NREM delta power reduction**: cKO mice will show a **30–45% decrease** in NREM slow-wave delta power (0.5–4 Hz) during recovery sleep, a marker of impaired sleep homeostatic response (two-way ANOVA, F(1,46) > 8.5, p < 0.005). 5. **Homer1a expression decrease**: cKO mice will show **50–65% lower** Homer1a mRNA levels at ZT12 (peak expression window) compared to flox controls (two-tailed t-test, t(22) = 6.8, p < 0.0001), reflecting disrupted clock-to-synapse signaling. 6. **Depression-like behavioral phenotypes**: cKO mice will show **35–50% increase** in TST immobility time and **25–40% decrease** in sucrose preference index vs flox controls (two-way ANOVA, p < 0.01 for both measures), consistent with anhedonic and despair-like behavior. 7. **Homer1a rescue correlation**: Within cKO mice, Homer1a expression levels will positively correlate with NSF latency (Pearson r = 0.68–0.78, p < 0.01), suggesting HOMER1A may mediate some depression-related phenotypes downstream of BMAL1 loss. ---

SUCCESS CRITERIA

- **Bmal1 excision efficiency ≥ 70%** at both mRNA and protein levels in cKO vs flox control (Student's t-test, p < 0.001; required before proceeding to behavioral endpoints) - **Circadian period disruption ≥ 0.8 h** elongation in DD in cKO vs flox controls (χ^2 periodogram, p < 0.01; confirms cell-autonomous clock disruption in mPFC) - **NREM delta power effect size ≥ 0.75** (Cohen's d) during post-SD recovery comparing cKO to flox controls (confirms sleep homeostatic impairment) - **Homer1a fold change ≤ 0.45** in cKO vs flox at ZT12 (Student's t-test, p < 0.001; confirms clock-to-synapse axis disruption) - **TST immobility increase ≥ 30%** in cKO vs flox with statistical significance (Mann-Whitney U test, p < 0.01; confirms depression-like phenotype) - **Sucrose preference decrease ≥ 20%** in cKO vs flox (two-way ANOVA, p < 0.01; confirms anhedonic phenotype) - **Camk2a-Cre specificity ≥ 85%** — co-localization of eGFP (viral reporter) with CAMK2A immunostaining in mPFC excitatory neurons, without recombination in GABAergic interneurons (verified by parvalbumin/calbindin co-stain), confirming targeting specificity

PROTOCOL

### PHASE 1: Pre-Surgical Preparation & Baseline Assessment (Weeks 1–2) **1.1 Animal Allocation** - 48 male Bmal1^fl/fl^ mice (B6.129S4(Cg)-Arntltm1Weit/J, The Jackson Laboratory, #007562), 10–12 weeks old, 25–30g - 24 age-matched C57BL/6J Cre-negative littermates as wild-type controls - 24 Bmal1^fl/fl^;Camk2a-Cre+ mice for conditional knockout (cKO) group - 24 Bmal1^fl/fl^;Camk2a-Cre− mice for floxed control (flox) group - Group sizes: n=24 per genotype, power=0.80, α=0.05, expected effect size d=0.85 for behavioral assays **1.2 Viral Vector Preparation** - AAV9-Camk2a-Cre-eGFP (titer: 1.0×10^12 vg/mL) for cKO group — Addgene or custom packaged - AAV9-Camk2a-eGFP (titer: 1.0×10^12 vg/mL) for flox control group — same serotype, no Cre - AAV9-Camk2a-scrambled-shRNA-eGFP (titer: 1.0×10^12 vg/mL) for wild-type group — confirm no off-target homology - Store at −80°C, thaw on ice immediately before surgery, keep at 4°C maximum 2 hours **1.3 Baseline Sleep Recording Setup** - Implantable telemetry transmitters (DSI ETA-F10) for EEG/EMG recording - Surgery: bilateral cranial window drilling over mPFC (AP: +1.8 mm, ML: ±0.4 mm from Bregma) - Electrode coordinates: EEG cortical layer 2/3 above mPFC, EMG cervical leads - Post-surgical recovery: 7 days with buprenorphine SR (0.1 mg/kg, SC) and meloxicam (2 mg/kg, SC q24h) **1.4 Baseline Behavioral Testing (Days 8–14)** - Open field test (OFT): 30 min session, ANY-maze tracking for locomotor activity and center time - Elevated plus maze (EPM): 5 min session, latency to open arm, open/closed arm time ratio - Tail suspension test (TST): 6 min session, automated scoring (Ugo Basile), immobility threshold <20% cutoff - sucrose preference test (SPT): 48h 1% sucrose vs water bottle choice, preference index = sucrose/(sucrose+water) --- ### PHASE 2: Viral Delivery & Conditional Knockout Induction (Weeks 3–4) **2.1 Stereotaxic Surgery** - Anesthesia: isoflurane 3% induction, 1.5–2% maintenance in 100% O2, respiratory rate maintained 60–80 bpm - Analgesia: buprenorphine 0.1 mg/kg SC pre-op + meloxicam 2 mg/kg SC post-op - Bilateral mPFC injection: AP +1.8 mm, ML ±0.4 mm, DV −2.5 mm from dura - Injection volume: 200 nL per side, rate 50 nL/min, needle retention 5 min post-injection - Viral construct: AAV9-Camk2a-Cre-eGFP (cKO) or AAV9-Camk2a-eGFP (flox control) - Wound clips removed at day 10 post-surgery **2.2 Validation of Recombination (Day 21 post-surgery)** - GFP expression confirmed via in vivo fluorescence imaging (Bruker Amber) - Test for Cre recombination efficiency: qPCR from punched tissue - Primers: Bmal1 excision forward 5′-GCTTCCTGCAGAGCGATCTT-3′, common reverse 5′-CAGGTAGTGGAGCCTTGGTG-3′ - Expected excision amplicon: 180 bp (excised) vs 320 bp (flox intact) - Homogenize tissue in Trizol, isolate RNA with RNeasy kit, DNase I treatment --- ### PHASE 3: Sleep Deprivation Challenge (Weeks 5–6) **3.1 Sleep Deprivation Protocol** - Gentle handling method: cage tapping, transfer to fresh cage every 30 min - SD duration: 6 hours (ZT0–ZT6, lights on baseline), beginning at light onset - Control groups: home cage recovery for equivalent time in adjacent room - Ambient temperature: 22±1°C, 12:12 LD cycle, lights on 06:00–18:00 **3.2 EEG/EMG Recording Throughout** - Continuous recording: 24h baseline → 6h SD → 18h recovery post-SD - Sampling rate: 500 Hz, bandpass filter 0.1–100 Hz - Sleep stage scoring: semi-automatic with SleepSign, manually validated per 4s epoch (NREM, REM, Wake) - Parameters: total sleep time (TST), REM%, NREM delta power (0.5–4 Hz), sleep onset latency --- ### PHASE 4: Behavioral Phenotyping Post-SD (Weeks 6–7) **4.1 Behavioral Test Battery (72h post-SD recovery)** - **TST**: 6 min, immobility time primary readout - **Forced swim test (FST)**: 6 min in 24°C water, immobility latency and total immobility - **OFT**: 30 min, distance traveled, center:periphery ratio - **Novelty-suppressed feeding (NSF)**: 10 min food-deprived, latency to eat in novel environment - **Chronic mild stress (CMS) paradigm**: 3 weeks optional, 2 stressors/day (randomized: tilted cage, intermittent lighting, moist bedding, novel odor) **4.2 Tissue Collection** - Transcardial perfusion: 0.1 M PBS pH 7.4 → 4% PFA in PBS - Brain sectioning: 40 μm coronal sections on cryostat, collected in 6 series - mPFC microdissection for biochemistry: fresh frozen, stored at −80°C --- ### PHASE 5: Molecular Endpoints (Weeks 7–9) **5.1 qRT-PCR (Homer1a, Bmal1, Per1, Cry1, Camk2a)** -引物序列: - Homer1a: F 5′-ACCAGCAGCAGAGGAGGAA-3′, R 5′-TGTTGGCGGTGATGGTGAG-3′ - Bmal1: F 5′-CTGGAGCTGCTGCGGGATA-3′, R 5′-TGCACACACGGTCGATCTTG-3′ - Per1: F 5′-GCCGAGTCACCGAGGAGTTC-3′, R 5′-CCACCCACAGACACAGCTCTC-3′ - Gapdh: F 5′-AGGTCGGTGTGAACGGATTTG-3′, R 5′-TGTAGACCATGTAGTTGAGGTCA-3′ - Program: 95°C 2 min → (95°C 15s → 60°C 30s → 72°C 30s) × 40 cycles - ΔΔCt method, Gapdh as housekeeping gene, results expressed as fold change vs flox group **5.2 Western Blot** - Protein extraction: RIPA buffer + protease/phosphatase inhibitors - Gel: 10% SDS-PAGE, 20 μg per lane - Primary antibodies: anti-BMAL1 (1:500, Abcam ab3350), anti-HOMER1 (1:1000, Synaptic Systems 160011), anti-β-ACTIN (1:5000, Sigma A5441) - Secondary: HRP-anti-rabbit IgG (1:5000), ECL detection (Bio-Rad Clarity) - Densitometry: Image Lab software, normalized to β-actin **5.3 Immunohistochemistry** - Sections: 40 μm free-floating, antigen retrieval in citrate buffer pH 6.0 - Blocking: 5% donkey serum + 0.3% Triton X-100, 1h at RT - Primary: anti-BMAL1 (1:200), anti-c-Fos (1:500, Synaptic Systems), anti-CAMK2A (1:300) - Secondary: Alexa Fluor 488/594 conjugates (1:500) - Imaging: Zeiss LSM 880 confocal, 20x objective, 1024×1024 px, z-stack 5 μm --- ### PHASE 6: Circadian Rhythm Assessment (Weeks 10–12) **6.1 Running Wheel Analysis** - Individual cages with running wheels (Lafayette 80860), continuous 14-day recording - Parameters: wheel revolutions/30s bin, circadian period (χ^2 periodogram), activity onset phase angle - Constant darkness (DD) challenge for 7 days to assess free-running period **6.2 Gene Expression Circadian Profile** - Harvest mPFC at ZT0, ZT4, ZT8, ZT12, ZT16, ZT20 (n=4 per timepoint per genotype) - qRT-PCR for clock genes (Bmal1, Per1, Cry1, Rev-Erbα) and Homer1a - Cosinor analysis: fit y = M + A·cos(2πt/24 + φ), period = 24h ---

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