Meningeal lymphatics-microglia axis regulates synaptic physiology [ligation]

Meningeal lymphatics serve as the primary outlet for cerebrospinal fluid, and their dysfunction is associated with various neurodegenerative conditions. Previous studies have demonstrated that dysfunctional meningeal lymphatics evoke behavioral deficits, but the neural mechanisms underlying those behavioral changes remained elusive. Here, we show that prolonged impairment of meningeal lymphatics alters the balance of cortical excitatory and inhibitory synaptic inputs by reducing inhibitory synapses, accompanied by deficits in novelty recognition tasks. These synaptic and behavioral alterations are mediated by microglia, which exhibit transcriptomic, morphological, and functional alterations as a result of lymphatic dysfunction. Notably, microglial expression of Il6 increases, thereby mediating the reduction in inhibitory synapses via neuronal signaling. Interestingly, improving the function of meningeal lymphatics in aged mice restores the numbers of functional inhibitory synapses and cortical network activity. Our findings suggest that dysfunctional meningeal lymphatics adversely impact cortical circuitry through a microglia−IL-6-dependent mechanism, providing a potential target for the treatment of aging-associated cognitive decline. The subject sham/dCLN-ligated 8-weeks-old C57BL/6J male mice were cardio-perfused with 30 ml of 5 U/ml Heparin-PBS after 4-weeks from dCLN-ligation/sham operation. Brains were harvested, located in heparin-PBS and cut through the sharp razor roughly at the AP=+1.00 mm. The anterior part of the brain faces down to the bottom at the section point and is cut horizontally half and half to remove the olfactory tract and striatum; only the upper chunk was used as a PFC sample. 5 PFC samples were pulled per group, and minced with the sharp razor in RPMI 1640 medium with 2% FBS and digested by collagenase VIII and DNase (in 5 ml RPMI 1640 + 2 % FBS) for 45 minutes and titrated with P1000 pipettes once at 30 minutes after digestion starts, and one more at 45 minutes after. Samples were strained through a 70-um strainer, and 5 ml RPMI 1640 + 10% FBS medium was added to stop digestion and spun down in 450g for 5 minutes. Pellets were resuspended in 14% BSA and spun down in 800 g for 10 minutes with slow acceleration/declaration. Supernatnace was removed carefully, and the pellet was resuspended in MACS buffer (0.5% BSA, 2 mM EDTA containing PBS), followed by spinning down (450g, 5 mins). 50 ul of CD45-bead (Miltenyi, 130-052-301) was added with 450 ul MACS buffer, and CD45+ cells were prepared according to the manufacturer's protocol.