Hypothesis

During sleep, the glymphatic system not only clears metabolic waste but also transports complement cascade proteins (C1q, C3) to synapses, tagging weakened connections for microglial phagocytosis. This nocturnal synaptic triage shapes network efficiency; disruption of glymphatic complement delivery impairs selective pruning, leading to accumulation of maladaptive synapses and cognitive rigidity.

Mechanistic Rationale

The glymphatic flux peaks in deep NREM when interstitial volume expands and AQP4-dependent CSF influx drives solute movement1. Complement proteins are synthesized by astrocytes and neurons and released into the extracellular space during wakefulness2. Their diffusion is limited, but convective flow via perivascular channels can rapidly distribute them throughout the parenchyma. Sleep-associated reduction in noradrenergic tone opens these channels, allowing complement to reach synapses where it binds to anionic surfaces, initiating the classical pathway and marking targets for microglial CR3-mediated engulfment3. Concurrently, neuronal activity-dependent release of "eat me" signals (e.g., phosphatidylserine) tags synapses that have undergone weakening during the day. Thus, the sleeping brain couples waste clearance with a selective tagging system that decides which architectures persist.

Testable Predictions

1. Pharmacological enhancement of AQP4 polarization during sleep will increase CSF-borne complement levels in the interstitial space and accelerate removal of weakly activated synapses, measurable by a decrease in spine density of low-activity excitatory neurons.
2. Genetic knockout of astrocytic C1q will blunt the sleep-dependent decline in spine density despite normal glymphatic influx, resulting in preserved but dysfunctional synapses and impaired performance on reversal learning tasks.
3. Chronic sleep fragmentation will reduce perivascular complement flux, leading to an accumulation of C3-tagged synapses and heightened cortical theta power, indicative of network rigidity.
4. Acute administration of a complement inhibitor during the early night will mimic sleep-deprivation effects on synaptic pruning, even when glymphatic flow is intact.

Experimental Approach

• Use Thy1-GCaMP mice to identify low-activity cortical columns during wakefulness.
• Apply intrathecal delivery of an AQP4-targeting agonist (e.g., TGN-020) or vehicle before sleep onset.
• Measure CSF complement concentration via microdialysis and correlate with two-photon imaging of spine turnover over successive nights.
• Assess reversal learning in a touchscreen paradigm; predict impaired flexibility in complement-deficient or AQP4-modulated groups.
• Validate with sleep-fragmentation models (e.g., gentle handling) and quantify perivascular tracer flux alongside complement immunohistochemistry.