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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

Synaptically evoked glutamate transporter currents (STCs) are markedly reduced in astrocytes in lesioned animals. (A) Specimen records showing evoked responses from a L2/3 astrocyte in a slice from a sham-operated animal following stimulation in cortical L4/5. Responses before and after bath application of TBOA are shown superimposed. TBOA effectively blocked the response. Each trace is average of ten consecutive responses. Experiments were performed in the presence of BaCl2 (100 µM) to inhibit a K+ uptake currents. (B) Similar to A, but from an astrocyte in a slice from a lesioned animal. Specimen records show that responses were smaller in amplitude relative to controls. Responses were TBOA sensitive. (C) Summary plots showing differences in STCs between astrocytes in slices from sham-operated and lesioned animals. STCs were significantly smaller in astrocytes from lesioned slices relative to astrocytes from sham-operated animals. (D) TBOA inhibited 92.8 +/− 3.3% (n = 4) of STC mediated currents while 88.6 +/− 6.7% (n = 3) of current was inhibited by DHK.
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Figure 4: Synaptically evoked glutamate transporter currents (STCs) are markedly reduced in astrocytes in lesioned animals. (A) Specimen records showing evoked responses from a L2/3 astrocyte in a slice from a sham-operated animal following stimulation in cortical L4/5. Responses before and after bath application of TBOA are shown superimposed. TBOA effectively blocked the response. Each trace is average of ten consecutive responses. Experiments were performed in the presence of BaCl2 (100 µM) to inhibit a K+ uptake currents. (B) Similar to A, but from an astrocyte in a slice from a lesioned animal. Specimen records show that responses were smaller in amplitude relative to controls. Responses were TBOA sensitive. (C) Summary plots showing differences in STCs between astrocytes in slices from sham-operated and lesioned animals. STCs were significantly smaller in astrocytes from lesioned slices relative to astrocytes from sham-operated animals. (D) TBOA inhibited 92.8 +/− 3.3% (n = 4) of STC mediated currents while 88.6 +/− 6.7% (n = 3) of current was inhibited by DHK.

Mentions: Thus far we have demonstrated that the neuronal response in lesioned animals in the hyperexcitable zone is similar to the response in sham treated animals in the presence of TBOA. We have also demonstrated that the NMDA receptor-mediated currents are significantly slower in lesioned animals, suggesting altered glutamate transport contributes to these differences. Furthermore, we have shown that astrocytes have markedly different electrophysiological properties in the hyperexcitable zone. In the next set of experiments, we examined astrocyte glutamate transporter currents in sham and lesioned animals. Release of glutamate from synaptic terminals can be detected in astrocytes by recording synaptically activated transporter currents (STCs; Mennerick and Zorumski, 1994; Bergles and Jahr, 1997; Diamond et al., 1998). STCs were evoked using a bipolar stimulating electrode located subjacent to the recorded astrocyte. Astrocytes in superficial cortical layers were voltage clamped at −80 mV and recordings were collected in saline containing 100 µM BaCl2 to block KIR currents (Newman, 1993; Olsen et al., 2006). Sample recordings of currents evoked in an astrocyte from a sham-operated animal are shown in Figure 4A. In response to single shock stimulation, an inward current was observed. This response was completely blocked by TBOA (30 µM), identifying the current as a STC. When recordings were made in astrocytes located in the hyperexcitable zone (Figure 4B), responses were significantly smaller in amplitude and duration. STCs in these astrocytes were also blocked by TBOA. As shown in Figure 4C, STC amplitudes in lesioned animals were significantly smaller than those recorded in control astrocytes (568 ± 53 pA, n = 3 control animals, n = 10 slices with one cell per slice vs. 62 ± 24 pA, n = 4 lesioned animals, n = 12 slices with one cell per slice, p < 0.001). Two Na+ dependent glutamate transporters, GLT-1 and GLAST, have been identified in astrocytes with GLT-1 being the predominant transporter expressed in gray matter astrocytes (Rothstein et al., 1996; Tanaka et al., 1997; Regan et al., 2007), and accounting for over 90% of glutamate uptake (Tanaka et al., 1997). TBOA is non-specific, blocking both GLT-1 and GLAST in astrocytes. Therefore we repeated these experiments in sham-operated astrocytes using the GLT-1 specific inhibitor, DHK (300 µM). These data demonstrate that STC amplitudes were reduced by 92.8 +/− 3.3% (n = 4) by TBOA, whereas 88.6 +/− 6.7% (n = 3) of current was inhibited by DHK (Figure 4D). These data indicate that nearly all the synaptically evoked transporter current is mediated by GLT-1.


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)

Synaptically evoked glutamate transporter currents (STCs) are markedly reduced in astrocytes in lesioned animals. (A) Specimen records showing evoked responses from a L2/3 astrocyte in a slice from a sham-operated animal following stimulation in cortical L4/5. Responses before and after bath application of TBOA are shown superimposed. TBOA effectively blocked the response. Each trace is average of ten consecutive responses. Experiments were performed in the presence of BaCl2 (100 µM) to inhibit a K+ uptake currents. (B) Similar to A, but from an astrocyte in a slice from a lesioned animal. Specimen records show that responses were smaller in amplitude relative to controls. Responses were TBOA sensitive. (C) Summary plots showing differences in STCs between astrocytes in slices from sham-operated and lesioned animals. STCs were significantly smaller in astrocytes from lesioned slices relative to astrocytes from sham-operated animals. (D) TBOA inhibited 92.8 +/− 3.3% (n = 4) of STC mediated currents while 88.6 +/− 6.7% (n = 3) of current was inhibited by DHK.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Synaptically evoked glutamate transporter currents (STCs) are markedly reduced in astrocytes in lesioned animals. (A) Specimen records showing evoked responses from a L2/3 astrocyte in a slice from a sham-operated animal following stimulation in cortical L4/5. Responses before and after bath application of TBOA are shown superimposed. TBOA effectively blocked the response. Each trace is average of ten consecutive responses. Experiments were performed in the presence of BaCl2 (100 µM) to inhibit a K+ uptake currents. (B) Similar to A, but from an astrocyte in a slice from a lesioned animal. Specimen records show that responses were smaller in amplitude relative to controls. Responses were TBOA sensitive. (C) Summary plots showing differences in STCs between astrocytes in slices from sham-operated and lesioned animals. STCs were significantly smaller in astrocytes from lesioned slices relative to astrocytes from sham-operated animals. (D) TBOA inhibited 92.8 +/− 3.3% (n = 4) of STC mediated currents while 88.6 +/− 6.7% (n = 3) of current was inhibited by DHK.
Mentions: Thus far we have demonstrated that the neuronal response in lesioned animals in the hyperexcitable zone is similar to the response in sham treated animals in the presence of TBOA. We have also demonstrated that the NMDA receptor-mediated currents are significantly slower in lesioned animals, suggesting altered glutamate transport contributes to these differences. Furthermore, we have shown that astrocytes have markedly different electrophysiological properties in the hyperexcitable zone. In the next set of experiments, we examined astrocyte glutamate transporter currents in sham and lesioned animals. Release of glutamate from synaptic terminals can be detected in astrocytes by recording synaptically activated transporter currents (STCs; Mennerick and Zorumski, 1994; Bergles and Jahr, 1997; Diamond et al., 1998). STCs were evoked using a bipolar stimulating electrode located subjacent to the recorded astrocyte. Astrocytes in superficial cortical layers were voltage clamped at −80 mV and recordings were collected in saline containing 100 µM BaCl2 to block KIR currents (Newman, 1993; Olsen et al., 2006). Sample recordings of currents evoked in an astrocyte from a sham-operated animal are shown in Figure 4A. In response to single shock stimulation, an inward current was observed. This response was completely blocked by TBOA (30 µM), identifying the current as a STC. When recordings were made in astrocytes located in the hyperexcitable zone (Figure 4B), responses were significantly smaller in amplitude and duration. STCs in these astrocytes were also blocked by TBOA. As shown in Figure 4C, STC amplitudes in lesioned animals were significantly smaller than those recorded in control astrocytes (568 ± 53 pA, n = 3 control animals, n = 10 slices with one cell per slice vs. 62 ± 24 pA, n = 4 lesioned animals, n = 12 slices with one cell per slice, p < 0.001). Two Na+ dependent glutamate transporters, GLT-1 and GLAST, have been identified in astrocytes with GLT-1 being the predominant transporter expressed in gray matter astrocytes (Rothstein et al., 1996; Tanaka et al., 1997; Regan et al., 2007), and accounting for over 90% of glutamate uptake (Tanaka et al., 1997). TBOA is non-specific, blocking both GLT-1 and GLAST in astrocytes. Therefore we repeated these experiments in sham-operated astrocytes using the GLT-1 specific inhibitor, DHK (300 µM). These data demonstrate that STC amplitudes were reduced by 92.8 +/− 3.3% (n = 4) by TBOA, whereas 88.6 +/− 6.7% (n = 3) of current was inhibited by DHK (Figure 4D). These data indicate that nearly all the synaptically evoked transporter current is mediated by GLT-1.

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