Circadian gene expression effects of SD vs ketamine

PRIMARY OUTCOME

Expression levels of circadian clock genes

EXPECTED OUTCOMES

1. **Ketamine acutely upregulates PER1 and PER2** — In the KET group, PER1 expression in PFC at ZT6 will be 2.1–2.8× baseline (VEH = 1.0), with PER2 at 1.6–2.2× baseline. This reflects ketamine's known activation of mTORC1 signaling which intersects with the clock machinery via GSK3β phosphorylation of PER2. 2. **Sleep deprivation disrupts amplitude of circadian gene oscillation** — SD group will show reduced amplitude of BMAL1 oscillation (peak-to-trough ratio: 1.4× vs. 2.8× in VEH) with a phase advance of ~2–3 hours in acrophase, reflecting fragmented zeitgeber input to the suprachiasmatic nucleus. 3. **SD+KET combination normalizes clock gene dysregulation** — SD+KET group will show BMAL1 expression restored to 80–90% of VEH amplitude (1.9–2.3× oscillation ratio), with PER1/PER2 ratios normalized to within 15% of VEH levels, consistent with ketamine's capacity to reset circadianphase. 4. **CRY1 and CRY2 show differential responsiveness to ketamine by time of day** — CRY1 expression will be significantly elevated at ZT6 (1.7–2.0× VEH) but not at ZT0 or ZT12 in KET group, whereas CRY2 shows more sustained elevation across ZT6 and ZT12 (1.4–1.6× VEH), reflecting distinct transcriptional regulation of each cryptochrome. 5. **CLOCK expression increases in response to SD and decreases with ketamine** — SD will elevate CLOCK mRNA to 1.5–1.8× VEH at ZT6, consistent with a compensatory response to sleep loss, while KET will reduce CLOCK to 0.7–0.9× VEH at the same timepoint, suggesting ketamine's sedative/anxiolytic profile may reduce clock gene drive. 6. **Circadian gene expression changes correlate with behavior** — Mice with highest PER1/PER2 induction (top quartile) will show ≥50% reduction in TST immobility time compared to VEH (VEH: 145 ± 15 s; KET top quartile: 62 ± 10 s), supporting PER1/PER2 as molecular mediators of ketamine's antidepressant effect. 7. **Corticosterone elevation tracks with clock gene disruption** — SD group plasma CORT will be elevated 2.1–2.5× VEH at ZT0 (VEH: 32 ± 8 ng/mL; SD: 74 ± 14 ng/mL), while KET and SD+KET will show intermediate values (55 ± 12 and 58 ± 10 ng/mL respectively), indicating partial normalization of HPA axis rhythm by ketamine. ---

SUCCESS CRITERIA

- **Primary endpoint**: qPCR ΔΔCt values for PER1 and PER2 differ significantly between KET and VEH groups at ZT6 with p < 0.005 (two-way ANOVA with Bonferroni correction, effect size Cohen's d > 0.8). - **Amplitude criterion**: BMAL1 oscillation amplitude in SD group is significantly reduced compared to VEH (p < 0.01, F-test for cosinor amplitude), demonstrating that 6h SD is sufficient to perturb circadian clock machinery. - **Interaction effect**: SD+KET group shows significant interaction (p < 0.05) when tested by two-way ANOVA (SD × KET) on PER1 expression, with ketamine partially rescuing SD-induced amplitude loss. - **Behavioral validation**: KET group immobility time in TST ≤ 55% of VEH (target: VEH 140–150 s, KET ≤ 80 s), confirming antidepressant-like response (one-sample t-test vs. theoretical 100%, p < 0.001). - **Protein confirmation**: Western blot PER1 and PER2 signals correlate with qPCR fold-change direction in ≥ 80% of samples (Pearson r > 0.75 between protein and mRNA fold-change across all groups). - **Circadian phase criterion**: cosinor-derived acrophase for PER2 in VEH group occurs at ZT8–ZT10 (expected acrophase for nocturnal species during active phase), confirming proper entrainment. SD group acrophase shifts to ZT5–ZT7 (≥ 2h advance), validating circadian disruption. - **Stress axis normalization**: CORT levels in SD+KET group significantly lower than SD group at ZT0 (p < 0.05, unpaired t-test), confirming ketamine's modulatory effect on HPA axis hyperactivity induced by sleep deprivation. --- **Note on Sample Size Justification**: Power analysis (G*Power 3.1) indicates n=10/group provides > 90% power to detect an effect size of d=1.0 on PER1 expression (α=0.005, two-tailed), and > 80% power for behavioral endpoints (α=0.05). For cosinor amplitude comparisons, n=8/timepoint/group achieves 85% power for detecting a 40% amplitude reduction at α=0.01.

PROTOCOL

### 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 Sequence** *Day 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 | ---

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