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Expression of ionotropic glutamate receptors in the retina of the rdta transgenic mouse.

Liu LO, Laabich A, Hardison A, Cooper NG - BMC Neurosci (2001)

Bottom Line: The NMDA receptor subunits (NR1, NR2A/B) and the GluR1; AMPA receptor subunit (GluR1) were examined in immunolabeled western blots.The results demonstrate that the amounts of NR1 and NR2A/B receptor subunits are significantly increased in crude synaptic membrane fractions isolated from retinae of the rdta mice when compared to their normal, littermate controls.Because the rods are lost at an early stage in development, it is likely that these results are indicative of synaptic reorganization in the retina.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Science, University of Louisville School of Medicine, Louisville, Kentucky, USA. 10liu002@gwise.louisville.edu

ABSTRACT

Background: The expression of retinal CaMKII is up-regulated in the retina of the rdta mouse in which rod photoreceptors are genetically ablated. As ionotropic glutamate receptors are known substrates of CAMKII, this study set out to determine if the protein levels of ionotropic glutamate receptors in the rdta mouse retina are also affected.

Results: The NMDA receptor subunits (NR1, NR2A/B) and the GluR1; AMPA receptor subunit (GluR1) were examined in immunolabeled western blots. The results demonstrate that the amounts of NR1 and NR2A/B receptor subunits are significantly increased in crude synaptic membrane fractions isolated from retinae of the rdta mice when compared to their normal, littermate controls. The GluR1 receptor subunit and its phosphorylation are simultaneously increased in retinae of the rdta mice.

Conclusions: These data indicate that the NMDA receptors and AMPA (GluR1) receptors are altered in the retinae of rdta mice that lack rod photoreceptors. Because the rods are lost at an early stage in development, it is likely that these results are indicative of synaptic reorganization in the retina.

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Back -phosphorylation of GluR1 in the retina of the rdta mice and their littermate controls. (A) Ten micrograms of SPM protein from the retinae of the rdta mice and their littermate controls were back-phosphorylated by endogenous CaMKII in the presence of Ca2+/CaM. The samples were then electrophoresed by SDS-PAGE and subsequently transferred to nitrocellular membrane. The phosphorylated bands were visualized by autoradiography. The in vitro phosphorylation was higher in the littermate controls than in the rdta mice. (B) The same blots were then incubated with anti-GluR1 antibody and visualized by alkaline phosphatase conjugated secondary antibody and NBI/BICP substrate. The molecular weight of GluR1 is indicated. The density for GluR1 was elevated in the rdta mice relative to the littermate controls. (C) GluR1s were immunoprecipitated after the back-phosphorylation and subjected to SDS-PAGE. The gel was dried and the image was visualized by autoradiography. These data indicate that the phosphorylation of GluR1 is increased in vivo in the retina of the rdta mice relative to their littermate controls.
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Figure 4: Back -phosphorylation of GluR1 in the retina of the rdta mice and their littermate controls. (A) Ten micrograms of SPM protein from the retinae of the rdta mice and their littermate controls were back-phosphorylated by endogenous CaMKII in the presence of Ca2+/CaM. The samples were then electrophoresed by SDS-PAGE and subsequently transferred to nitrocellular membrane. The phosphorylated bands were visualized by autoradiography. The in vitro phosphorylation was higher in the littermate controls than in the rdta mice. (B) The same blots were then incubated with anti-GluR1 antibody and visualized by alkaline phosphatase conjugated secondary antibody and NBI/BICP substrate. The molecular weight of GluR1 is indicated. The density for GluR1 was elevated in the rdta mice relative to the littermate controls. (C) GluR1s were immunoprecipitated after the back-phosphorylation and subjected to SDS-PAGE. The gel was dried and the image was visualized by autoradiography. These data indicate that the phosphorylation of GluR1 is increased in vivo in the retina of the rdta mice relative to their littermate controls.

Mentions: Because GluR1 is a known substrate of CaMKII in vitro and in vivo [34,36,37,38,39,40] and because CaMKII expression/activity is up-regulated in the retinae of the rdta mice [34], GluR1 phosphorylation might also be increased in the rodless retina. To test this notion, the SPM proteins isolated from the retinae of the rdta and their littermate controls mice were back-phosphorylated in the presence of Ca2+/calmodulin and γ-32P-ATP (Figure 4A). The bands densities in the rdta mice were low; and a long film exposure was necessary to detect the signal (Figure 4A). The relative position of γ-32P-ATP labeled GluR1 as indicated was confirmed by immunoblotting with GluR1 antibodies (Figure 4B). It was found that γ-32P-ATP labeling of GluR1 by this procedure was greater in the retinae of control mice than in the retina of the rdta mice (Figure 4A), whereas the expression of GluR1 was increased in the rdta mice relative to their littermate controls (Figure 4B). These data indicate that GluR1 phosphorylation in vivo was enhanced in the rdta retinae relative to their littermate controls. This result was confirmed using an additional technique. GluR1 was immunoprecipitated with anti-GluR1 antibody and then subjected to in vitro phosphorylation (Figure 4C). Together, these data indicate that in vivo, the amount of GluR1 and the phosphorylation of GluR1 are simultaneously enhanced in retinal synaptic fractions isolated from the retinae of rdta mice.


Expression of ionotropic glutamate receptors in the retina of the rdta transgenic mouse.

Liu LO, Laabich A, Hardison A, Cooper NG - BMC Neurosci (2001)

Back -phosphorylation of GluR1 in the retina of the rdta mice and their littermate controls. (A) Ten micrograms of SPM protein from the retinae of the rdta mice and their littermate controls were back-phosphorylated by endogenous CaMKII in the presence of Ca2+/CaM. The samples were then electrophoresed by SDS-PAGE and subsequently transferred to nitrocellular membrane. The phosphorylated bands were visualized by autoradiography. The in vitro phosphorylation was higher in the littermate controls than in the rdta mice. (B) The same blots were then incubated with anti-GluR1 antibody and visualized by alkaline phosphatase conjugated secondary antibody and NBI/BICP substrate. The molecular weight of GluR1 is indicated. The density for GluR1 was elevated in the rdta mice relative to the littermate controls. (C) GluR1s were immunoprecipitated after the back-phosphorylation and subjected to SDS-PAGE. The gel was dried and the image was visualized by autoradiography. These data indicate that the phosphorylation of GluR1 is increased in vivo in the retina of the rdta mice relative to their littermate controls.
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Related In: Results  -  Collection

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Figure 4: Back -phosphorylation of GluR1 in the retina of the rdta mice and their littermate controls. (A) Ten micrograms of SPM protein from the retinae of the rdta mice and their littermate controls were back-phosphorylated by endogenous CaMKII in the presence of Ca2+/CaM. The samples were then electrophoresed by SDS-PAGE and subsequently transferred to nitrocellular membrane. The phosphorylated bands were visualized by autoradiography. The in vitro phosphorylation was higher in the littermate controls than in the rdta mice. (B) The same blots were then incubated with anti-GluR1 antibody and visualized by alkaline phosphatase conjugated secondary antibody and NBI/BICP substrate. The molecular weight of GluR1 is indicated. The density for GluR1 was elevated in the rdta mice relative to the littermate controls. (C) GluR1s were immunoprecipitated after the back-phosphorylation and subjected to SDS-PAGE. The gel was dried and the image was visualized by autoradiography. These data indicate that the phosphorylation of GluR1 is increased in vivo in the retina of the rdta mice relative to their littermate controls.
Mentions: Because GluR1 is a known substrate of CaMKII in vitro and in vivo [34,36,37,38,39,40] and because CaMKII expression/activity is up-regulated in the retinae of the rdta mice [34], GluR1 phosphorylation might also be increased in the rodless retina. To test this notion, the SPM proteins isolated from the retinae of the rdta and their littermate controls mice were back-phosphorylated in the presence of Ca2+/calmodulin and γ-32P-ATP (Figure 4A). The bands densities in the rdta mice were low; and a long film exposure was necessary to detect the signal (Figure 4A). The relative position of γ-32P-ATP labeled GluR1 as indicated was confirmed by immunoblotting with GluR1 antibodies (Figure 4B). It was found that γ-32P-ATP labeling of GluR1 by this procedure was greater in the retinae of control mice than in the retina of the rdta mice (Figure 4A), whereas the expression of GluR1 was increased in the rdta mice relative to their littermate controls (Figure 4B). These data indicate that GluR1 phosphorylation in vivo was enhanced in the rdta retinae relative to their littermate controls. This result was confirmed using an additional technique. GluR1 was immunoprecipitated with anti-GluR1 antibody and then subjected to in vitro phosphorylation (Figure 4C). Together, these data indicate that in vivo, the amount of GluR1 and the phosphorylation of GluR1 are simultaneously enhanced in retinal synaptic fractions isolated from the retinae of rdta mice.

Bottom Line: The NMDA receptor subunits (NR1, NR2A/B) and the GluR1; AMPA receptor subunit (GluR1) were examined in immunolabeled western blots.The results demonstrate that the amounts of NR1 and NR2A/B receptor subunits are significantly increased in crude synaptic membrane fractions isolated from retinae of the rdta mice when compared to their normal, littermate controls.Because the rods are lost at an early stage in development, it is likely that these results are indicative of synaptic reorganization in the retina.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Science, University of Louisville School of Medicine, Louisville, Kentucky, USA. 10liu002@gwise.louisville.edu

ABSTRACT

Background: The expression of retinal CaMKII is up-regulated in the retina of the rdta mouse in which rod photoreceptors are genetically ablated. As ionotropic glutamate receptors are known substrates of CAMKII, this study set out to determine if the protein levels of ionotropic glutamate receptors in the rdta mouse retina are also affected.

Results: The NMDA receptor subunits (NR1, NR2A/B) and the GluR1; AMPA receptor subunit (GluR1) were examined in immunolabeled western blots. The results demonstrate that the amounts of NR1 and NR2A/B receptor subunits are significantly increased in crude synaptic membrane fractions isolated from retinae of the rdta mice when compared to their normal, littermate controls. The GluR1 receptor subunit and its phosphorylation are simultaneously increased in retinae of the rdta mice.

Conclusions: These data indicate that the NMDA receptors and AMPA (GluR1) receptors are altered in the retinae of rdta mice that lack rod photoreceptors. Because the rods are lost at an early stage in development, it is likely that these results are indicative of synaptic reorganization in the retina.

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