Antisense knockout of a glutamate transporter reveals a role for müller cells in maintaining neurotransmission in the retina

Purpose. To elucidate the role of glial glutamate transporters in the regulation of retinal function. Methods. We administered antisense oligonucleotides to GLAST, a glutamate transporter which is expressed in retinal Müller cells, into one eye of each rat. Sense oligonucleotides (control) were injected into the other eye over a period of 5 days, and the scotopic flash electroretinograms (ERG) recorded. To assay whether the antisense oligonucleotides caused a reduction in the expression or the activity of GLAST, we have developed a novel assay, whereby the retinas are exposed to D-aspartate, a non-endogenous substrate of glutamate transporters, then immunolabelled with specific antibodies for D-aspartate. Results. We demonstrate that antisense oligonucleotides markedly suppress the b-wave of the ERG whereas the sense oligonucleotide has little effect. Preliminary results suggest that there is a marked reduction in the uptake of D-aspartate into Müller cells in the retinas which have been exposed to the antisense oligonucleotides. Conclusion. Since suppression of glutamate uptake into Müller cells is likely to cause a rise in extracellular glutamate, this result may indicate either a subsequent excitotoxic effect of glutamate on retinal neurones or may reflect a direct perturbation of glutamatergic signalling. To verify whether this reduction in uptake of D-aspartate is due to a decline in expression of GLAST or to decreased transporter activity of GLAST, we are currently generating antibodies against each of the glutamate transporters (GLAST, GLT-1 and EAAC1). Using this multifactorial approach it should be possible to accurately ascribe physiological roles of these, and other transporters, in the mammalian retina.