Quantitative analysis of the expression of the glutamate–aspartate transporter and identification of functional glutamate uptake reveal a role for cochlear fibrocytes in glutamate homeostasis

Abstract There are several subtypes of fibrocyte in the spiral ligament and spiral limbus of the cochlea that may contribute to fluid homeostasis. Immunocytochemical data suggest that these fibrocytes possess the glutamate–aspartate transporter, GLAST, as do supporting cells around the hair cells. However, functional glutamate uptake has not been demonstrated in fibrocytes. We used confocal and post-embedding immunogold electron microscopy to confirm that GLAST is expressed in adult fibrocytes of CD-1 mice with a relative expression: spiral limbus fibrocytes>type II>V>IV>I spiral ligament fibrocytes. Because they were sparsely present in most samples, type III fibrocytes were assessed only in one sample where their GLAST levels were similar to type I. Type II, type V and spiral limbus fibrocytes have many fine cellular processes that increase their surface area, those of the latter two coming into direct contact with perilymph, and type V fibrocytes contain the most glutamate. These data imply that glutamate uptake occurs in the fibrocytes. We assessed uptake of d -aspartate (a glutamate analogue) together with GLAST expression immunocytochemically and electrophysiologically. d -Aspartate accumulated into GLAST expressing fibrocytes in vitro and evoked currents blockable by the GLAST inhibitor d,l -threo-β-benzyloxyaspartate (TBOA), similar to those of supporting cells around inner hair cells. Currents were strongest in spiral limbus fibrocytes, progressively lower in type V and type II fibrocytes, and were negligible in type I fibrocytes in accordance with the relative expression levels of GLAST. We conclude that in addition to their known homeostatic functions, fibrocytes, in particular spiral limbus, type II and type V fibrocytes play a role in glutamate homeostasis in the cochlea.