Disturbing sleep in adolescent mice

Disturbing sleep in female adolescent mice does not increase vulnerability to depression triggers later in life.

Egebjerg et al. Brain Behaviour and Immunity, 2025

Overview

- This study investigates the long-term effects of sleep disturbance (SD) during adolescence on vulnerability to depression triggers later in life, using a female adolescent mouse model.

- The primary objectives were to determine whether SD in adolescence induces lasting behavioral or molecular changes that increase susceptibility to depression-like behaviors after exposure to known triggers such as social isolation (SI) or inflammation (LPS).

- The research aims to clarify if adolescent SD acts as a "second hit" in the development of depression, which is highly relevant given the high prevalence of sleep issues and depression during human adolescence.

Key Concepts

Adolescence as a vulnerable period: Characterized by ongoing neurodevelopment, especially in brain regions involved in mood regulation.

Sleep disturbance (SD): Disruption of normal sleep patterns, which has been linked to structural and functional brain changes.

Two-hit hypothesis: Suggests that a combination of genetic vulnerability and environmental insults (e.g., SD, stress) increases risk for neuropsychiatric disorders like depression.

Microglia and neuroinflammation: Microglial morphology and cytokine expression as markers of neuroinflammatory processes potentially involved in depression.

Gene expression markers: Focus on molecules such as Cx3cr1, Dnmt3b, Htr1a, Bdnf, Ntrk2, and Tnf related to neuroplasticity, methylation, serotonin regulation, and inflammation.

Conclusions

- Adolescent sleep disturbance induces specific molecular alterations in the hippocampus and mPFC but does not lead to persistent behavioral deficits or increased vulnerability to depression-like triggers in adulthood.

- The findings challenge the assumption that SD during adolescence acts as a "first hit" for depression development, at least under the less stressful SD model used.

- The study emphasizes the importance of stress levels associated with SD protocols; less stressful methods may not produce the behavioral phenotypes seen with more stressful deprivation paradigms.

- Results suggest that sleep disturbance alone during adolescence may not be sufficient to predispose individuals to depression triggered by social isolation or inflammation later in life.

Methodology

Research design: Experimental study with longitudinal assessments involving adolescent female mice subjected to SD, followed by adult exposure to depression triggers.

Subjects: Female C57BL/6J mice, starting at post-natal day (P)36.

SD protocol: 7 days of voluntary sleep disturbance for 4 hours daily during light phase (ZT2-6), with novel objects introduced to minimize stress.

Data collection:

- Gene expression: Quantitative PCR (qPCR) analysis of hippocampus and medial prefrontal cortex (mPFC) tissues immediately after SD.

- Microglia morphology: Fractal analysis of Iba-1 stained microglia in cortex.

- Behavioral tests: Open Field Test (OFT) and Tail Suspension Test (TST) conducted post-SD and after adult triggers.

  - Home-cage activity: Monitored via Digital Ventilated Cages (DVC) system during social isolation and post-LPS.

Triggers of depressive behaviour: Social isolation (SI) for 7 days and lipopolysaccharide (LPS) injection (250 µg/kg) to induce inflammation.

Key Findings

Molecular changes: SD acutely decreased hippocampal Cx3cr1 and Dnmt3b expression and mPFC Htr1a expression; no significant changes in Tnf, Dnmt3a, Bdnf, Ntrk2.

Microglia morphology: Slight non-significant trend toward increased fractal dimension; no significant differences in circularity, span ratio, density, or lacunarity.

Behavioral outcomes: No significant differences in home-cage activity, anxiety-like behavior (OFT), or despair-like behavior (TST) following adolescent SD alone.

Vulnerability to triggers:

- SI in adulthood did not exacerbate behavioral deficits in SD mice.

- LPS reduced activity initially but did not differentially affect SD vs. control mice; no increase in depression-like behaviors was observed after SD plus LPS.

Overall pattern: While molecular effects of SD were evident acutely, these did not translate into increased behavioral susceptibility to depression triggers later in life.

Limitations

- The SD protocol was designed to be less stressful by using voluntary wakefulness; thus, it may not replicate stress-inducing SD models that show behavioral effects.

- The study focused solely on female mice; sex differences might influence outcomes.

- The timing of SD during mid-adolescence (P36) may not capture effects occurring earlier or later in development.

- The sample size for some measures may limit detection of subtle effects.

- Potential confounding factors include stress from shipment and housing conditions that could influence neurobiological responses.

miR-137 and sleep

The evolutionarily conserved miRNA-137 targets the neuropeptide hypocretin/orexin and modulates the wake to sleep ratio.

Holm et al. PNAS 2022

Overview

- This study investigates the intracellular mechanisms regulating the neuropeptide hypocretin (Hcrt), also known as orexin, which is essential for sleep–wake stability in vertebrates.

- The research aims to identify microRNAs (miRNAs), particularly miR-137, that target Hcrt mRNA and modulate its expression, influencing sleep and wakefulness.

- The significance lies in understanding posttranscriptional regulation of Hcrt, which has implications for sleep disorders such as narcolepsy and insomnia, and potential therapeutic avenues.

Key Concepts

Hypocretin/Orexin System: A hypothalamic neuropeptide signaling pathway critical for maintaining wakefulness; deficits cause narcolepsy.

MicroRNAs (miRNAs): Small noncoding RNAs (~20–25 nucleotides) that regulate gene expression by binding to target mRNAs, leading to degradation or translational repression.

Evolutionary Conservation: The study emphasizes that miR-137's interaction with Hcrt is conserved across species (mice, humans, zebrafish), indicating a fundamental biological role.

Conclusions

- The study uncovers a conserved posttranscriptional regulatory mechanism where miR-137 modulates Hcrt expression, thereby influencing sleep–wake states.

- Manipulating miR-137 levels affects wakefulness and sleep architecture across species, highlighting its potential as a therapeutic target.

- Human genetic data link MIR137 variants to sleep duration, suggesting relevance for individual differences in sleep patterns.

- These findings deepen understanding of the molecular regulation of sleep and open avenues for novel treatments targeting miRNA pathways.

Methodology

In Silico Prediction: Used algorithms (MicroCosm, TargetScan, DIANA, PITA) to identify miRNAs targeting HCRT mRNA, predicting miR-137, miR-637, and miR-654-5p as candidates.

In Vitro Experiments: Transfected human neuroblastoma cell lines (SK-N-MC, SK-N-DZ) with precursor miRNAs; measured HCRT mRNA levels via qRT-PCR; employed luciferase reporter assays to confirm direct targeting.

In Vivo Studies: Injected miRNA mimics or anti-miRNAs intracerebroventricularly into mouse pups; used viral vectors to inhibit miR-137 specifically in Hcrt neurons; measured Hcrt expression and sleep/wake behavior.

Genetic Association Analysis: Analyzed GWAS data from UK Biobank linking MIR137 locus variants to sleep duration.

Cross-Species Validation: Used zebrafish embryos injected with anti-miR-137 morpholino to assess effects on activity and sleep patterns.

Cytokine Stimulation: Treated human neuroblastoma cells with cytokines (IL-13, TNF, IFNs) to observe effects on miR-137 levels and Hcrt expression.

Key Findings

miR-137 targets HCRT: Confirmed through computational prediction, luciferase assays, and mutational analysis.

Conservation Across Species: The interaction between miR-137 and Hcrt is conserved in mice, humans, and zebrafish.

Regulatory Role of miR-137:

- Endogenous miR-137 levels vary diurnally in mice hypothalamus.

- Downregulation of miR-137 increases Hcrt expression and promotes wakefulness.

- Conversely, overexpression reduces Hcrt levels.

Behavioral Effects:

- Inhibiting miR-137 in Hcrt neurons results in increased wakefulness, longer wake bouts, and reduced NREM sleep during early dark hours.

- Zebrafish with inhibited miR-137 show increased activity and decreased sleep.

Cytokine Influence: IL-13 induces miR-137 upregulation, leading to decreased Hcrt expression.

Genetic Association: SNPs in MIR137 locus are significantly associated with sleep duration in humans.

Limitations

- The extent of Hcrt regulation by miR-137 is partial (~20% change), unlikely to cause severe narcolepsy but may influence subtler sleep phenotypes.

- Overexpression or inhibition experiments may not fully replicate physiological conditions; compensatory mechanisms may occur over time.

- The study primarily establishes correlation and mechanistic potential but does not definitively prove causality in complex human sleep disorders.

- Genetic association data suggest correlation but do not confirm direct functional effects of MIR137 variants on sleep regulation.

- Cross-species findings (zebrafish) support conservation but require further validation in mammals.