Hélie-Legoupil, PaulinePaulineHélie-LegoupilKloster, FlorenciaFlorenciaKlosterPareja, JavierJavierParejaVladymyrov, MykhailoMykhailoVladymyrovMapunda, Josephine A.Josephine A.MapundaBouillet, ElisaElisaBouilletOetiker, YannikYannikOetikerSpera, IreneIreneSperaBarcos, SaraSaraBarcosBrenna, AmandineAmandineBrennaOdriozola, AdolfoAdolfoOdriozolaBaert, AlyssaAlyssaBaertFankhauser, ChristophChristophFankhauserHaenni, BeatBeatHaenniProulx, Steven T.Steven T.ProulxZuber, BenoîtBenoîtZuber0000-0001-7725-5579Deutsch, UrbanUrbanDeutschEngelhardt, BrittaBrittaEngelhardt0000-0003-3059-98462025-10-132025-10-132025-10-07https://boris-portal.unibe.ch/handle/20.500.12422/220385Pauline Hélie-Legoupil and Florencia Kloster contributed equally.The glia limitans ensheathes the entire central nervous system (CNS) parenchyma towards the outer surfaces and the perivascular spaces and is formed by a subset of astrocytes strategically localized at these outer parenchymal borders. Barrier properties of the glia limitans during health and neuroinflammation are incompletely understood. By developing an aquaporin-4 (Aqp4)-mRuby3 knock-in reporter mouse that allows for in vivo imaging of the superficial and perivascular glia limitans, we here show that the glia limitans forms a barrier for soluble mediators, beads and immune cells. Combining the Aqp4-mRuby3 reporter strain with additional reporter alleles for vascular, leptomeningeal or myeloid cells ensures precise localization of immune cells to CNS border zones versus the CNS parenchyma allowing to assign functional roles in CNS immune surveillance versus neuropathology. Availability of the Aqp4-mRuby3 reporter mouse will further advance our understanding of the active role of the glia limitans in CNS immune privilege.en600 - Technology::610 - Medicine & health600 - Technology::630 - Agriculturearticle10.48620/917554105730510.1038/s41467-025-63945-7