Bmal1 knockout in mPFC excitatory neurons

Validation Score: 0.950 Price: $0.50 depression Bmal1 floxed mutant mice (B6.129S4(Cg)-Arntltm1Weit/J) Status: proposed

What This Experiment Tests

Validation experiment designed to validate causal mechanisms targeting BMAL1,HOMER1A,CAMK2A in Bmal1 floxed mutant mice (B6.129S4(Cg)-Arntltm1Weit/J). Primary outcome: Sleep parameters, behavioral response to SD, Homer1a expression

Description

This experiment used targeted deletion of the core clock gene Bmal1 specifically in CaMK2a-expressing excitatory neurons of the mPFC to investigate the role of the molecular clock in sleep regulation and antidepressant response. Viral-mediated Bmal1 knockout disrupted sleep-wake architecture, elevated slow-wave activity, and completely abolished both the behavioral antidepressant effects and molecular response (Homer1a induction) to sleep deprivation. This demonstrated the essential role of the mPFC molecular clock in mediating SD effects.

TARGET GENE

BMAL1,HOMER1A,CAMK2A

MODEL SYSTEM

Bmal1 floxed mutant mice (B6.129S4(Cg)-Arntltm1Weit/J)

ESTIMATED COST

TIMELINE

0 months

PATHWAY

circadian rhythms,glutamatergic signaling,synaptic plasticity

SOURCE

extracted_from_pmid_41023421

PRIMARY OUTCOME

Sleep parameters, behavioral response to SD, Homer1a expression

Scoring Dimensions

📖 Wiki Pages

CAMK2A Genegene BMAL1 (ARNTL) Genegene BMAL1 Proteinprotein CaMKII Protein (CaMK2A)protein Depression in Neurodegenerationdisease Depression (Major Depressive Disorder)disease CLOCK Genegene

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

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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

Expected Outcomes

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.

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.

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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 axi

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Related Hypotheses (2)

Circadian Rhythm Entrainment of Reactive Astrocytes0.722

CaMKII-Dependent Synaptic Circuit Amplification0.611

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