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Functional changes in glutamate transporters and astrocyte biophysical properties in a rodent model of focal cortical dysplasia.

Campbell SL, Hablitz JJ, Olsen ML - Front Cell Neurosci (2014)

Bottom Line: Synaptically evoked glutamate transporter currents in astrocytes showed a near 10-fold reduction in amplitude compared to sham operated controls.Astrocyte glutamate transporter currents from lesioned animals were also significantly reduced when challenged exogenously applied glutamate.Significant decreases in astrocyte resting membrane potential and increases in input resistance were observed in lesioned animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama at Birmingham Birmingham, AL, USA.

ABSTRACT
Cortical dysplasia is associated with intractable epilepsy and developmental delay in young children. Recent work with the rat freeze-induced focal cortical dysplasia (FCD) model has demonstrated that hyperexcitability in the dysplastic cortex is due in part to higher levels of extracellular glutamate. Astrocyte glutamate transporters play a pivotal role in cortical maintaining extracellular glutamate concentrations. Here we examined the function of astrocytic glutamate transporters in a FCD model in rats. Neocortical freeze lesions were made in postnatal day (PN) 1 rat pups and whole cell electrophysiological recordings and biochemical studies were performed at PN 21-28. Synaptically evoked glutamate transporter currents in astrocytes showed a near 10-fold reduction in amplitude compared to sham operated controls. Astrocyte glutamate transporter currents from lesioned animals were also significantly reduced when challenged exogenously applied glutamate. Reduced astrocytic glutamate transport clearance contributed to increased NMDA receptor-mediated current decay kinetics in lesioned animals. The electrophysiological profile of astrocytes in the lesion group was also markedly changed compared to sham operated animals. Control astrocytes demonstrate large-amplitude linear leak currents in response to voltage-steps whereas astrocytes in lesioned animals demonstrated significantly smaller voltage-activated inward and outward currents. Significant decreases in astrocyte resting membrane potential and increases in input resistance were observed in lesioned animals. However, Western blotting, immunohistochemistry and quantitative PCR demonstrated no differences in the expression of the astrocytic glutamate transporter GLT-1 in lesioned animals relative to controls. These data suggest that, in the absence of changes in protein or mRNA expression levels, functional changes in astrocytic glutamate transporters contribute to neuronal hyperexcitability in the FCD model.

No MeSH data available.


Related in: MedlinePlus

GLT-1 protein and mRNA expression levels were unchanged in the lesioned animals. (A) A representative Western blot demonstrating no change in GLT-1 expression in the hyperexcitable zone of lesioned tissue relative to tissue obtained from the same region in sham-operated animals. These blots were also stripped and re-probed with gapDH as a loading control and the astrocyte intermediate filament GFAP. (B) Plot of relative GLT-1 expression in lesioned tissue relative to control tissue. GLT-1 expression is normalized to the loading control gapDH. No significant difference was observed. (C) Wide field fluorescent microscopy was used to image the lesion, hyperexcitable zone and surrounding areas. No gross changes in GLT-1 expression were observed (scale bar = 100 µm). (D) Confocal imaging from a z-stack (60 sections, 1 µm/section) from the hyperexcitable region indicating GLT-1 immunoreactivity in a sham operated and lesioned animal (scale bar = 50 µm). (E) mRNA expression of GLT-1 (SLC1A2) was also unchanged in the hyperexcitable zone from lesioned animals when compared to tissue obtained from sham-operated animals (each sample ran in triplicate). (F) GLAST mRNA levels were not significantly different between groups. (G) GFAP expression was significantly elevated in the hyperexcitable zone (1.6 fold) relative to control tissue obtained from the same region.
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Figure 6: GLT-1 protein and mRNA expression levels were unchanged in the lesioned animals. (A) A representative Western blot demonstrating no change in GLT-1 expression in the hyperexcitable zone of lesioned tissue relative to tissue obtained from the same region in sham-operated animals. These blots were also stripped and re-probed with gapDH as a loading control and the astrocyte intermediate filament GFAP. (B) Plot of relative GLT-1 expression in lesioned tissue relative to control tissue. GLT-1 expression is normalized to the loading control gapDH. No significant difference was observed. (C) Wide field fluorescent microscopy was used to image the lesion, hyperexcitable zone and surrounding areas. No gross changes in GLT-1 expression were observed (scale bar = 100 µm). (D) Confocal imaging from a z-stack (60 sections, 1 µm/section) from the hyperexcitable region indicating GLT-1 immunoreactivity in a sham operated and lesioned animal (scale bar = 50 µm). (E) mRNA expression of GLT-1 (SLC1A2) was also unchanged in the hyperexcitable zone from lesioned animals when compared to tissue obtained from sham-operated animals (each sample ran in triplicate). (F) GLAST mRNA levels were not significantly different between groups. (G) GFAP expression was significantly elevated in the hyperexcitable zone (1.6 fold) relative to control tissue obtained from the same region.

Mentions: A 1 mm diameter tissue punch was used to obtain cortical samples 0.7–1 mm thick from the hyperexcitable zone in lesioned animals and comparable areas in sham operated controls. A representative western blot is shown in Figure 6A. GLT-1 immunoreactivity was unchanged in the hyperexcitable zone relative to tissue from sham-operated controls. Quantification from four sets of animals is shown in Figure 6B. These results demonstrate there is no significant difference in GLT-1 protein expression between the hyperexcitable zone of lesioned animals and sham tissue when normalized to a loading control.


Functional changes in glutamate transporters and astrocyte biophysical properties in a rodent model of focal cortical dysplasia.

Campbell SL, Hablitz JJ, Olsen ML - Front Cell Neurosci (2014)

GLT-1 protein and mRNA expression levels were unchanged in the lesioned animals. (A) A representative Western blot demonstrating no change in GLT-1 expression in the hyperexcitable zone of lesioned tissue relative to tissue obtained from the same region in sham-operated animals. These blots were also stripped and re-probed with gapDH as a loading control and the astrocyte intermediate filament GFAP. (B) Plot of relative GLT-1 expression in lesioned tissue relative to control tissue. GLT-1 expression is normalized to the loading control gapDH. No significant difference was observed. (C) Wide field fluorescent microscopy was used to image the lesion, hyperexcitable zone and surrounding areas. No gross changes in GLT-1 expression were observed (scale bar = 100 µm). (D) Confocal imaging from a z-stack (60 sections, 1 µm/section) from the hyperexcitable region indicating GLT-1 immunoreactivity in a sham operated and lesioned animal (scale bar = 50 µm). (E) mRNA expression of GLT-1 (SLC1A2) was also unchanged in the hyperexcitable zone from lesioned animals when compared to tissue obtained from sham-operated animals (each sample ran in triplicate). (F) GLAST mRNA levels were not significantly different between groups. (G) GFAP expression was significantly elevated in the hyperexcitable zone (1.6 fold) relative to control tissue obtained from the same region.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269128&req=5

Figure 6: GLT-1 protein and mRNA expression levels were unchanged in the lesioned animals. (A) A representative Western blot demonstrating no change in GLT-1 expression in the hyperexcitable zone of lesioned tissue relative to tissue obtained from the same region in sham-operated animals. These blots were also stripped and re-probed with gapDH as a loading control and the astrocyte intermediate filament GFAP. (B) Plot of relative GLT-1 expression in lesioned tissue relative to control tissue. GLT-1 expression is normalized to the loading control gapDH. No significant difference was observed. (C) Wide field fluorescent microscopy was used to image the lesion, hyperexcitable zone and surrounding areas. No gross changes in GLT-1 expression were observed (scale bar = 100 µm). (D) Confocal imaging from a z-stack (60 sections, 1 µm/section) from the hyperexcitable region indicating GLT-1 immunoreactivity in a sham operated and lesioned animal (scale bar = 50 µm). (E) mRNA expression of GLT-1 (SLC1A2) was also unchanged in the hyperexcitable zone from lesioned animals when compared to tissue obtained from sham-operated animals (each sample ran in triplicate). (F) GLAST mRNA levels were not significantly different between groups. (G) GFAP expression was significantly elevated in the hyperexcitable zone (1.6 fold) relative to control tissue obtained from the same region.
Mentions: A 1 mm diameter tissue punch was used to obtain cortical samples 0.7–1 mm thick from the hyperexcitable zone in lesioned animals and comparable areas in sham operated controls. A representative western blot is shown in Figure 6A. GLT-1 immunoreactivity was unchanged in the hyperexcitable zone relative to tissue from sham-operated controls. Quantification from four sets of animals is shown in Figure 6B. These results demonstrate there is no significant difference in GLT-1 protein expression between the hyperexcitable zone of lesioned animals and sham tissue when normalized to a loading control.

Bottom Line: Synaptically evoked glutamate transporter currents in astrocytes showed a near 10-fold reduction in amplitude compared to sham operated controls.Astrocyte glutamate transporter currents from lesioned animals were also significantly reduced when challenged exogenously applied glutamate.Significant decreases in astrocyte resting membrane potential and increases in input resistance were observed in lesioned animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama at Birmingham Birmingham, AL, USA.

ABSTRACT
Cortical dysplasia is associated with intractable epilepsy and developmental delay in young children. Recent work with the rat freeze-induced focal cortical dysplasia (FCD) model has demonstrated that hyperexcitability in the dysplastic cortex is due in part to higher levels of extracellular glutamate. Astrocyte glutamate transporters play a pivotal role in cortical maintaining extracellular glutamate concentrations. Here we examined the function of astrocytic glutamate transporters in a FCD model in rats. Neocortical freeze lesions were made in postnatal day (PN) 1 rat pups and whole cell electrophysiological recordings and biochemical studies were performed at PN 21-28. Synaptically evoked glutamate transporter currents in astrocytes showed a near 10-fold reduction in amplitude compared to sham operated controls. Astrocyte glutamate transporter currents from lesioned animals were also significantly reduced when challenged exogenously applied glutamate. Reduced astrocytic glutamate transport clearance contributed to increased NMDA receptor-mediated current decay kinetics in lesioned animals. The electrophysiological profile of astrocytes in the lesion group was also markedly changed compared to sham operated animals. Control astrocytes demonstrate large-amplitude linear leak currents in response to voltage-steps whereas astrocytes in lesioned animals demonstrated significantly smaller voltage-activated inward and outward currents. Significant decreases in astrocyte resting membrane potential and increases in input resistance were observed in lesioned animals. However, Western blotting, immunohistochemistry and quantitative PCR demonstrated no differences in the expression of the astrocytic glutamate transporter GLT-1 in lesioned animals relative to controls. These data suggest that, in the absence of changes in protein or mRNA expression levels, functional changes in astrocytic glutamate transporters contribute to neuronal hyperexcitability in the FCD model.

No MeSH data available.


Related in: MedlinePlus