Study Design Overview
Adult male C57BL/6J mice (8–10 weeks, ~22–26g) will be randomly assigned to three groups: (1) Sleep Deprivation (SD), (2) Ketamine (KET), and (3) SD + Ketamine (SD+KET). A vehicle control group (VEH) will be run concurrently. Terminal tissue collection will occur at four circadian time points (ZT0, ZT6, ZT12, ZT18) on day 3 post-intervention. Primary outcome: mRNA expression of PER1, PER2, CRY1, CRY2, BMAL1, and CLOCK in prefrontal cortex and hippocampus.
Phase 1: Animal Acclimation and Baseline Assessment (Days -7 to -1)
...Study Design Overview
Adult male C57BL/6J mice (8–10 weeks, ~22–26g) will be randomly assigned to three groups: (1) Sleep Deprivation (SD), (2) Ketamine (KET), and (3) SD + Ketamine (SD+KET). A vehicle control group (VEH) will be run concurrently. Terminal tissue collection will occur at four circadian time points (ZT0, ZT6, ZT12, ZT18) on day 3 post-intervention. Primary outcome: mRNA expression of PER1, PER2, CRY1, CRY2, BMAL1, and CLOCK in prefrontal cortex and hippocampus.
Phase 1: Animal Acclimation and Baseline Assessment (Days -7 to -1)
1.1 Housing and Entrainment
- Mice (n=10/group, 40 total + 10 VEH = 50 mice) housed in Tecniplast IVC cages (2–4/cage, same-sex) under 12h:12h light:dark cycle (lights on at ZT0, lights off at ZT12).
- Room temperature: 22 ± 1°C; humidity: 50 ± 10%; Teklad 2918 chow and sterile water ad libitum.
- Acclimation period: 7 days prior to any intervention.
- Daily health monitoring and body weight recording (Days -7, -5, -3, -1).
1.2 Baseline Behavioral Screening
- Day -2: Open field test (OFT) — 10 min, to assess exploratory activity and anxiety-like behavior. Equipment: Med Associates Open Field (27.3 × 27.3 cm), infrared beam spacing 2.5 cm. Outcome: total distance traveled, center time, peripheral time.
- Day -1: Light-Dark box test (LDB) — 5 min transition, 5 min recording. Outcome: time in light compartment, latency to first light entry, transitions.
1.3 Randomization
- Mice stratified by baseline OFT total distance and body weight into four groups using simple randomization (random number table): VEH (saline, i.p.), SD (sleep deprivation), KET (ketamine, 10 mg/kg, i.p.), SD+KET.
Phase 2: Intervention (Days 1–3)
Intervention Sequence (Day 1)
2.1 Sleep Deprivation Protocol (Modified Multiple Platform Method)
- Platform apparatus: 12 circular platforms (diameter: 3.5 cm) arranged in 2 rows inside a plastic chamber (45 × 30 × 16 cm) filled with room-temperature water to 1 cm below platform surface.
- Mice (SD and SD+KET groups) placed individually on platforms at ZT0.
- Platforms spaced to allow free movement between them but not sustained sleep (muscle atonia on platform triggers awakening).
- SD session duration: 6 hours (ZT0–ZT6) to target REM sleep predominance.
- VEH and KET groups placed in home cages on bench during SD session.
2.2 Ketamine Administration
- Ketamine HCl (Sigma-Aldrich, K2753) dissolved in sterile 0.9% saline (pH 7.2–7.4) at concentration 1 mg/mL.
- KET and SD+KET groups receive ketamine 10 mg/kg (10 mL/kg volume) via intraperitoneal injection at ZT6 (immediately after SD period).
- VEH group receives equal volume of sterile saline (10 mL/kg, i.p.).
- SD group receives no injection (handled similarly during SD).
2.3 Recovery Period
- All mice returned to home cages (ZT6–ZT12).
- Cages placed in designated housing rack; standard conditions maintained.
- Ketamine-treated mice monitored for anesthesia onset and recovery (expected: 5–10 min transient ataxia, normal locomotion by ZT9).
Phase 3: Post-Intervention Monitoring (Days 3–5)
3.1 General Health Monitoring
- Body weight and food/water consumption recorded daily (ZT6 ± 1h).
- Clinical observation: grooming, nest building, sociability, vocalizations, gait disturbances.
3.2 Behavioral Testing SequenceDay 3 (24h post-intervention):
- Tail Suspension Test (TST) — 6 min test, immobility time (s) as primary readout. Equipment: Med Associates Tail Suspension apparatus. Mice suspended by tail tip 30 cm from floor; automated infrared detection of passive vs. active movements. This timepoint captures acute antidepressant effect of ketamine.
Day 4 (48h post-intervention):
- Forced Swim Test (FST) — 6 min test, immobility time (s). Equipment: cylindrical tank (height: 40 cm, diameter: 20 cm), water depth: 30 cm, water temperature: 24 ± 1°C. Videotracked (EthoVision XT) for immobility detection. 10 min pre-exposure on Day 3 to reduce novelty stress.
Day 5 (72h post-intervention):
- Open Field Test (OFT) — 10 min, to re-assess exploratory/anxiety profile.
- Splash Test — 5 min, to assess grooming behavior as proxy for motivational state. 10% sucrose solution sprayed onto dorsal coat; grooming latency and duration recorded.
Phase 4: Tissue Collection at Circadian Timepoints (Day 6, Terminal Procedure)
4.1 Tissue Harvest Schedule
Mice will be sacrificed at four circadian timepoints on Day 6:
- Cohort A (ZT0): sacrifice at ZT0 ± 15 min
- Cohort B (ZT6): sacrifice at ZT6 ± 15 min
- Cohort C (ZT12): sacrifice at ZT12 ± 15 min
- Cohort D (ZT18): sacrifice at ZT18 ± 15 min
Within each cohort, 2–3 mice per experimental group harvested per timepoint to control for within-group variance (n=3–4 per group per timepoint for qPCR; n=2 per group per timepoint for protein/ELISA confirmation).
4.2 Rapid Tissue Dissection
- Mice anesthetized with isoflurane (4% induction, 2% maintenance in O₂) followed by cervical dislocation.
- Brain extracted within 90 seconds and placed in ice-cold RNase-free PBS (pH 7.4).
- PFC dissected: medial prefrontal cortex (Bregma +1.7 to +0.7 mm) using mouse brain atlas (Paxinos).
- Hippocampus dissected: dorsal hippocampus (Bregma -1.2 to -2.5 mm).
- Hypothalamus collected as secondary region for circadian pacemaker correlation.
- Tissue flash-frozen in liquid N₂ within 3 min of sacrifice; stored at -80°C.
4.3 Blood Collection (Trunk Blood)
- Collected in EDTA-coated microtainer tubes during decapitation.
- Plasma separated by centrifugation (2,000 × g, 15 min, 4°C) and stored at -80°C for corticosterone measurement (CORT ELISA, Arbor Assays, K003-H1).
Phase 5: Gene Expression Analysis (Days 7–14)
5.1 RNA Extraction
- Tissue homogenized in QIAzol Lysis Reagent (Qiagen, 79306) using Precellys 24 tissue lyser (3 × 20 sec at 6,500 rpm, 4°C).
- Total RNA extracted using miRNeasy Mini Kit (Qiagen, 217004) per manufacturer's protocol.
- DNase I treatment on-column to remove genomic DNA.
- RNA quality assessed: Agilent 4200 TapeStation; RNA Integrity Number (RIN) > 8.0 required for downstream analysis.
5.2 cDNA Synthesis
- 1 μg total RNA reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814).
- Reaction: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, hold at 4°C.
- cDNA stored at -20°C.
5.3 qPCR (Quantitative Real-Time PCR)
- Predesigned TaqMan gene expression assays (Applied Biosystems):
| Gene | Assay ID | Amplicon Length |
|------|----------|-----------------|
| PER1 | Mm00504313_m1 | 71 bp |
| PER2 | Mm01332833_m1 | 68 bp |
| CRY1 | Mm01221362_m1 | 76 bp |
| CRY2 | Mm01331539_m1 | 63 bp |
| BMAL1 (Arntl) | Mm00500226_m1 | 73 bp |
| CLOCK | Mm01199054_m1 | 67 bp |
| GAPDH ( endogenous control) | Mm99999915_g1 | 107 bp |
- Reaction mix per well (20 μL):
- TaqMan Fast Advanced Master Mix (2×): 10 μL
- Gene-specific TaqMan assay (20×): 1 μL
- cDNA template: 5 μL (equivalent to ~25 ng RNA input)
- Nuclease-free water: 4 μL
- Thermocycling (Applied Biosystems QuantStudio 7 Flex):
- UNG incubation: 50°C, 2 min
- Enzyme activation: 95°C, 2 min
- 40 cycles: 95°C, 15 sec → 60°C, 1 min (data collection)
- Melt curve: 95°C, 15 sec → 60°C, 15 sec → 95°C, 15 sec
- All samples run in triplicate on the same plate; no multiplexing.
5.4 Data Analysis
- Threshold cycle (Cт) values extracted using QuantStudio 7 Flex software (auto baseline, manual threshold at 0.2).
- Relative expression calculated using ΔΔCt method.
- Housekeeping gene stability assessed using NormFinder (Applled Biosystems) and BestKeeper; GAPDH confirmed as stable across conditions.
- Normalization: expression level = 2^(-ΔΔCt) where ΔΔCt = (Ct_gene - Ct_GAPDH)_sample - (Ct_gene - Ct_GAPDH)_VEH_ZT0.
Phase 6: Protein-Level Validation and Secondary Assays (Days 15–21)
6.1 Western Blot (Protein Confirmation)
- Tissue lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease/phosphatase inhibitors.
- Protein concentration determined by BCA assay (Pierce, 23225).
- 30 μg protein/lane run on 10% SDS-PAGE, transferred to PVDF membrane.
- Primary antibodies:
- Anti-PER1 (Abcam, ab34477, 1:500)
- Anti-PER2 (Abcam, ab180655, 1:500)
- Anti-CRY1 (Cell Signaling, 55290S, 1:500)
- Anti-BMAL1 (Bethyl, A302-616A, 1:500)
- Anti-β-ACTIN (Cell Signaling, 4970S, 1:2000, loading control)
- Secondary: HRP-conjugated anti-rabbit (Cell Signaling, 7074P2, 1:5000).
- Signal detected via ECL substrate (SuperSignal West Pico, 34077) and imaged on Bio-Rad ChemiDoc MP.
- Densitometry quantified using ImageLab 6.0 (Bio-Rad).
6.2 Corticosterone ELISA (Stress Axis Validation)
- Plasma corticosterone measured by ELISA (Arbor Assays, K003-H1) per kit protocol.
- Detection range: 16–4000 pg/mL; sensitivity: 12.9 pg/mL.
- Samples run in duplicate; coefficient of variation < 15% required.
6.3 Circadian Rhythm Analysis
- cosinor analysis fit to expression data across four timepoints using R package `cosinor2`:
- 24h period modeled: expression = M + A·cos(2πt/24 + φ)
- Mesor (M), amplitude (A), and acrophase (φ) compared between groups.
- Null hypothesis: no difference in amplitude or acrophase (p < 0.05 rejection criterion).
Controls
| Control Type | Implementation | Purpose |
|---|---|---|
| Vehicle | Saline (0.9%, 10 mL/kg, i.p.) | Baseline for ketamine treatment |
| Naive | Age-matched C57BL/6J mice, no intervention | Confirm behavioral baselines |
| Positive (ketamine efficacy) | Standard dose ketamine (10 mg/kg) historical comparison | Verify antidepressant effect |
| siRNA knockdown (optional, in vitro validation) | PER1/2 siRNA (Ambion, AM16208) in neuronal culture | Confirm specificity of qPCR signal |
| Scrambled siRNA | Non-targeting siRNA (Ambion, AM16210) | Control for off-target effects in vitro |
| qPCR no-template control | Nuclease-free water replacing cDNA | Confirm no contamination |
| qPCR no-RT control | RNA processed without reverse transcriptase | Confirm no genomic DNA |