Sublamina-specific organization of the blood brain barrier in the mouse olfactory nerve layer

Astrocytes constitute the main glial component of the mammalian blood brain barrier (BBB). However, in the olfactory bulb (OB), the olfactory nerve layer (ONL) is almost devoid of astrocytes, raising the question which glial cells are part of the BBB. We used mice expressing EGFP in astrocytes and tdTomato in olfactory ensheathing cells (OECs), a specialized type of glial cells in the ONL, to unequivocally identify both glial cell types and investigate their contribution to the BBB in the olfactory bulb. OECs were located exclusively in the ONL, while somata of astrocytes were located in deeper layers and extended processes in the inner sublamina of the ONL. These processes surrounded blood vessels and contained aquaporin-4, an astrocytic protein enriched at the BBB. In the outer sublamina of the ONL, in contrast, blood vessels were surrounded by aquaporin-4-negative processes of OECs. Transcardial perfusion of blood vessels with lanthanum and subsequent visualization by electron microscopy showed that blood vessels enwrapped by OECs possessed intact tight junctions. In acute olfactory bulb preparations, injection of fluorescent glucose 6-NBDG into blood vessels resulted in labeling of OECs, indicating glucose transport from the perivascular space into OECs. In addition, Ca²⁺ transients in OECs in the outer sublamina evoked vasoconstriction, whereas Ca²⁺ signaling in OECs of the inner sublamina had no effect on adjacent blood vessels. Our results demonstrate that the BBB in the inner sublamina of the ONL contains astrocytes, while in the outer ONL OECs are part of the BBB.