Response to Commentary on "Structural characterization of SLYM - a 4th meningeal membrane" by Julie Siegenthaler and Christer Betsholtz.

Histological studies have for decades documented that each of the classical meningeal membranes contains multiple fibroblast layers with distinct cellular morphology. Particularly, the sublayers of the arachnoid membranes have received attention due to their anatomical complexity. Early studies found that tracers injected into the cerebrospinal fluid (CSF) do not distribute freely but are restricted by the innermost sublayer of the arachnoid membrane. The existence of restrictions on CSF movement and the subdivision of the subarachnoid space into several distinct compartments have recently been confirmed by in vivo 2-photon studies of rodents, as well as macroscopic imaging of pigs and magnetic resonance imaging of human brain. Based on in vivo imaging and immunophenotyping characterization, we identified the structural basis for this compartmentalization of the subarachnoid space, which we term 'Subarachnoid lymphatic-like membrane', SLYM. The SLYM layer engages the subarachnoid vasculature as it approaches the brain parenchyma, demarcating a roof over pial perivascular spaces. Functionally, the separation of pial periarterial and perivenous spaces in the larger subarachnoid space is critical for the maintenance of unidirectional glymphatic clearance. In light of its close apposition to the pial surface and to the brain perivascular fluid exit points, the SLYM also provides a primary locus for immune surveillance of the brain. Yet, the introduction of SLYM, in terms of its anatomic distinction and hence functional specialization, has met resistance. Its critics assert that SLYM has been described in the literature by other terms, including the inner arachnoid membrane, the interlaminate membrane, the outer pial layer, the intermediate lamella, the pial membrane, the reticular layer of the arachnoid membrane or, more recently, BFB2-3. We argue that our conception of SLYM as an anatomically and functionally distinct construct is both necessary and warranted since its functional roles are wholly distinct from those of the overlying arachnoid barrier layer. Our terminology also lends clarity to a complex anatomy that has hitherto been ill-described. In that regard, we also note the lack of specificity of DPP4, which has recently been introduced as a 'selected defining marker' of the arachnoid barrier layer. We note that DPP4 labels fibroblasts in all meningeal membranes as well as in the trabecula arachnoides and the vascular adventitial layers, thus obviating its utility in meningeal characterization. Instead, we report a set of glymphatic-associated proteins that serve to accurately specify SLYM and distinguish it from its adjacent yet functionally distinct membranes.