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

N. L. Barnett, D. V. Pow

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number4
Publication statusPublished - 1 Dec 1997
Externally publishedYes

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Amino Acid Transport System X-AG
D-Aspartic Acid
Antisense Oligonucleotides
Synaptic Transmission
Retina
Glutamic Acid
Oligonucleotides
Retinal Neurons
Antibodies
Neuroglia

Cite this

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title = "Antisense knockout of a glutamate transporter reveals a role for m{\"u}ller cells in maintaining neurotransmission in the retina",
abstract = "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 M{\"u}ller 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 M{\"u}ller cells in the retinas which have been exposed to the antisense oligonucleotides. Conclusion. Since suppression of glutamate uptake into M{\"u}ller 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.",
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Antisense knockout of a glutamate transporter reveals a role for müller cells in maintaining neurotransmission in the retina. / Barnett, N. L.; Pow, D. V.

In: Investigative Ophthalmology and Visual Science, Vol. 38, No. 4, 01.12.1997.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Antisense knockout of a glutamate transporter reveals a role for müller cells in maintaining neurotransmission in the retina

AU - Barnett, N. L.

AU - Pow, D. V.

PY - 1997/12/1

Y1 - 1997/12/1

N2 - 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.

AB - 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.

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