Re-defining the Glia limitans layer of the olfactory nervous system

The glia of the olfactory nerve, Olfactory Ensheathing cells (OECs), are thought to be part of both the peripheral and central nervous systems (PNS and CNS). However, the olfactory PNS-CNS border is not well defined. Here, we aimed to define this border by determining the cellular constituents of the olfactory nervous system glia limitans, the layer of glial cells which constitutes a physiological and immunological border between PNS and CNS. To understand which cells formed the glia limitans in the olfactory system, we compared the olfactory bulb glia limitans to the glia limitans in other nervous system regions. We analysed cryostat sections from S100βDsRed mice, in which all glial cells fluoresce red. We then defined which cells formed the immune barrier in the olfactory bulb by intranasally inoculating mice with Burkholderia pseudomallei bacteria and analysed how cells in the olfactory glia limitans reacted to the bacteria. Finally, we compared OECs and SCs in their interactions with astrocytes in two- and three-dimensional cultures to understand if they differed in their interactions. We found that the olfactory nervous system glia limitans was morphologically similar to the glia limitans elsewhere, with a comparable degree of intermingling between peripheral glia (OECs/SCs) and astrocytes. We showed that astrocytes in the olfactory glia limitans constituted a defined immunological barrier to bacterial infection. We also found that OECs and SCs interacted similarly with astrocytes in vitro in 2D cultures. However in 3D co-cultures we observed subtle differences in the interactions between OECs and SCs with astrocytes. Thus, we suggest that the olfactory nervous system glia limitans is similar to the glia limitans elsewhere, and consists of astrocytes with OECs being mainly restricted to the PNS.