Elsevier

Neuroscience

Volume 162, Issue 4, 15 September 2009, Pages 1307-1321
Neuroscience

Sensory System
Research Paper
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

https://doi.org/10.1016/j.neuroscience.2009.05.036Get rights and content

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.

Section snippets

Animals

CD-1 mice, pigmented guinea pigs and rats have been used in this study. Five-week old CD-1 mice were used to investigate the distribution of GLAST in different fibrocyte types and cochlear regions by immunofluorescence and quantitative immunogold electron microscopy. Relative levels of glutamate were also assessed in CD-1 mouse fibrocytes. The relative amount of GLAST was determined in different cochlear sub-regions, by dot blotting of guinea-pig tissue, which have four cochlear turns, as

Results

The anatomical landmarks relevant to this study are illustrated in Fig. 1A which shows a cross-section of the cochlear duct. Fibrocytes which contain GLAST are located in the lateral wall and on the perilymphatic surface of the spiral limbus. Other regions known to contain GLAST are the spiral ganglion and organ of Corti.

Discussion

The present study was aimed primarily at determining whether lateral wall fibrocytes in the cochlea exhibit a functional glutamate uptake process based on the expression of GLAST. Our data show that (i) the lateral wall expresses the greater proportion of cochlear GLAST compared with spiral ganglion and organ of Corti in guinea pig; (ii) at the cellular level in mice, type II and type V fibrocytes express the most GLAST and this expression is achieved by an increased surface area and not by

Conclusion

Our data show that lateral wall fibrocytes express GLAST in the relative amount type II>type V>type IV>type I>type III, that the fibrocytes possess functional uptake capacity based on GLAST and can accumulate a glutamate analogue. Type V has the highest glutamate levels and is most exposed to perilymph. Spiral limbus fibrocytes also express high levels of GLAST relative to other types and have strong uptake currents. These data all suggest that the fibrocytes perform a role in glutamate

Acknowledgments

Supported by Deafness Research UK. D.J.J. is a Royal Society University Research Fellow.

References (28)

  • D.N. Furness et al.

    Comparative distribution of glutamate transporters and receptors in relation to afferent innervation density in the mammalian cochlea

    J Neurosci

    (2003)
  • D.N. Furness et al.

    Immunocytochemical localization of a high-affinity glutamate-aspartate transporter, GLAST, in the rat and guinea-pig cochlea

    Eur J Neurosci

    (1997)
  • D.N. Furness et al.

    Distribution of the glutamate/aspartate transporter GLAST in relation to the afferent synapses of outer hair cells in the guinea pig cochlea

    J Assoc Res Otolaryngol

    (2002)
  • E. Glowatzki et al.

    The glutamate-aspartate transporter GLAST mediates glutamate uptake at inner hair cell afferent synapses in the mammalian cochlea

    J Neurosci

    (2006)
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