Hypothesis

Daily intake of a defined prebiotic mixture enriches gut bacteria that produce butyrate and propionate, which in turn enhance astrocytic AQP4 polarization and microglial phagocytic specificity via HDAC inhibition and GPR109A activation. This shifts the glymphatic system from bulk waste clearance to an active triage mechanism that tags and removes synapses bearing maladaptive activity‑dependent markers (e.g., elevated PSD‑95 phosphorylation, reduced BDNF signaling) while sparing those encoding adaptive engrams. Consequently, subjects experience reduced anxiety‑ and depression‑like behaviors due to a nightly synaptic ‘editing’ that favors resilient circuits.

Mechanistic Rationale

• Butyrate acts as an HDAC inhibitor in astrocytes, increasing transcription of Aqp4 and promoting perivascular water channel localization, thereby boosting glymphatic inflow during NREM sleep.1
• Propionate binds GPR109A on microglia, biasing their phenotype toward a surveillance state that upregulates complement component C1q and the phagocytic receptor CR3, facilitating selective tagging of weakened synapses for removal.2
• Elevated SCFA levels also stabilize the blood‑brain barrier and reinforce circadian clock genes in the suprachiasmatic nucleus, preserving the temporal window for glymphatic flux.
• The combined effect creates a scenario where the brain’s nocturnal ‘autopsy’ preferentially eliminates synaptic configurations linked to heightened threat sensitivity and negative affect, while preserving those associated with reward and stress buffering.

Testable Predictions

1. Participants receiving the prebiotic will show a significant increase in fecal butyrate and propionate concentrations compared with placebo (p < 0.01).
2. DTI‑ALPS analysis will reveal heightened glymphatic flow specifically during slow‑wave sleep in the prebiotic group, correlating with SCFA levels (r > 0.4).
3. Phosphorylated PSD‑95 and reduced BDNF immunoreactivity in extracellular vesicles isolated from CSF will decrease after four weeks of prebiotic supplementation, indicating selective synaptic pruning.
4. Changes in synaptic marker ratios will mediate the relationship between SCFA rise and reductions in HAM‑A and HAM‑D scores (mediation effect > 30 %).
5. Microbiome sequencing will demonstrate enrichment of Faecalibacterium prausnitzii and Eubacterium rectale, taxa previously linked to SCFA synthesis and improved sleep metrics.3

Experimental Design

• Design: Double‑blind, placebo‑controlled, parallel‑group trial (n = 80 adults with moderate anxiety/depression).
• Intervention: 10 g/day of a galacto‑oligosaccharide/inulin blend for 8 weeks.
• Controls: Isocaloric maltodextrin placebo.
• Measurements (baseline, week 4, week 8):
  • Stool SCFA quantification (GC‑MS).
  • CSF collection via lumbar puncture for synaptic extracellular vesicle analysis (Western blot for p‑PSD‑95, BDNF).
  • MRI DTI‑ALPS index during overnight polysomnography.
  • Sleep architecture (PSG) – % N3, REM latency.
  • Clinical scales: HAM‑A, HAM‑D, PSQI.
  • 16S rRNA sequencing of feces.
• Analysis: Mixed‑effects models for longitudinal data; mediation analysis to test SCFA → glymphatic flow → synaptic marker change → symptom improvement pathway.

Potential Outcomes and Falsifiability

If the prebiotic fails to elevate SCFAs, does not alter DTI‑ALPS, or does not produce selective synaptic marker changes despite any symptom improvement, the hypothesis would be falsified. Conversely, observing the predicted cascade would support the notion that sleep‑dependent glymphatic triage is mechanistically modifiable via the gut‑brain axis, positioning targeted prebiotics as a precision tool for synaptic remodeling in affective disorders.

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