Hypothesis

Morning chronotype shifts the peak of glymphatic clearance away from the early‑morning testosterone surge, reducing the hormone’s ability to tag synapses for removal and thereby lowering sleep‑dependent synaptic pruning and neuroplasticity.

Mechanistic rationale

• Glymphatic influx peaks during deep NREM sleep, driven by slow‑wave activity and arterial pulsatility.
• Testosterone secretion shows a sharp rise between 03:00 and 07:00 h, contingent on ≥3 h of uninterrupted sleep with normal architecture.
• Animal work shows testosterone can up‑regulate aquaporin‑4 polarity in astrocytes, enhancing perivascular CSF‑interstitial exchange.
• In evening chronotypes, the sleep‑associated slow‑wave maximum aligns with the testosterone window, allowing concurrent glymphatic flow and androgen signaling.
• Morning types, forced to wake earlier, truncate the late‑night NREM block; their glymphatic peak occurs before testosterone rises, uncoupling the two processes.

Testable predictions

1. In a forced‑desynchrony protocol, participants with morning preference will exhibit a lower correlation coefficient between slow‑wave power (0.5‑4 Hz) and CSF‑intrathecal tracer clearance rate during the 03:00‑07:00 h interval than evening counterparts (p < 0.05).
2. Intranasal testosterone administered during the early morning will rescue glymphatic clearance efficiency in morning‑type subjects to levels matched with evening types, measured by dynamic contrast‑enhanced MRI of perivascular spaces.
3. Chronic shift work that advances sleep onset will decrease the overlap index (glymphatic peak timing ÷ testosterone peak timing) and predict a decline in hippocampal‑dependent memory performance over 4 weeks, mediated by reduced synaptic pruning markers (e.g., decreased complement C1q immunoreactivity in post‑mortem‑like exosomes).
4. Genetic risk scores for morningness will interact with AQP4 polymorphisms to modulate the amplitude of testosterone‑dependent glymphatic response, detectable as a three‑way interaction in a mixed‑effects model.

Falsifiability

If morning chronotype individuals show no difference in the temporal overlap of glymphatic activity and testosterone secretion, or if testosterone manipulation fails to alter glymphatic metrics, the hypothesis is refuted.

Experimental outline

• Recruit 60 healthy men stratified by chronotype (MEQ score).
• Conduct two overnight sessions: baseline and after a single dose of intranasal testosterone (or placebo) administered at 04:00 h.
• Simultaneously acquire high‑density EEG, arterial spin labeling MRI for CSF influx, and serial saliva/flood blood samples for testosterone and cortisol.
• Compute voxel‑wise glymphatic influx rate (using the two‑point method) and align it to hormonal time series.
• Statistical analysis: linear mixed models with chronotype, treatment, and time as fixed effects, subject as random effect.

Expected outcome

A significant chronotype × treatment interaction indicating that testosterone restores glymphatic coupling specifically in morning types would support the idea that sleep’s ‘autopsy’ requires hormonal timing; disruption of this timing explains why some individuals suffer cognitive and metabolic consequences despite adequate sleep duration.