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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

Astrocyte responses to exogenously applied glutamate are reduced in lesioned cortex. (A) Specimen records of glutamate evoked currents from astrocytes in slices from sham-operated (upper) or lesioned (lower) animals. Cells were voltage clamped at −80 mV and responses were evoked by a 500 µM, 0.5 s puff of glutamate. Bathing and puffing solutions contained 50 µM TTX, 200 µM AP5, 200 µM bicuculline, 250 µM CNQX and 100 µM CdCl. (B) Same data as in A but responses averaged across cells are shown. (C) Summary histogram showing that glutamate evoked responses, normalized for cell capacitance, were significantly reduced in the lesioned group.
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Figure 5: Astrocyte responses to exogenously applied glutamate are reduced in lesioned cortex. (A) Specimen records of glutamate evoked currents from astrocytes in slices from sham-operated (upper) or lesioned (lower) animals. Cells were voltage clamped at −80 mV and responses were evoked by a 500 µM, 0.5 s puff of glutamate. Bathing and puffing solutions contained 50 µM TTX, 200 µM AP5, 200 µM bicuculline, 250 µM CNQX and 100 µM CdCl. (B) Same data as in A but responses averaged across cells are shown. (C) Summary histogram showing that glutamate evoked responses, normalized for cell capacitance, were significantly reduced in the lesioned group.

Mentions: Several factors, including alterations in local cortical connections in the dysplastic cortex, differences in threshold for synaptic activation and/or release properties, and changes in the astrocyte morphology could contribute to the differences in STC amplitudes described above. Therefore, we next examined astrocyte responses to exogenously applied glutamate. For these experiments, recordings were obtained in the presence of 500 nM TTX, 100 µM CdCl2, 50 µM CNQX, 20 µM bicuculline and 50 µM AP5 to minimize neuronal activity, release of endogenous glutamate and isolate glutamate transporter currents (Bergles and Jahr, 1997; Grass et al., 2004). Glutamate (500 µM, 500 msec) was pressure applied to astrocytes voltage clamped at −80 mV. The puffer pipette was visually placed in the same focal plane as the voltage clamped cell at a distance that elicited a maximum response. Specimen records from 8 control astrocytes (upper) and 7 astrocytes from the lesioned cortex (lower) are shown superimposed in Figure 5A. The average of these responses across cells is shown in Figure 5B. There was no statistical difference in cell size between the two groups, as measured by cell capacitance (control 21 ± 3 pF and lesion 23 ± 4pF, p > 0.05). The changes in STC current and glutamate uptake currents were nearly 10-fold smaller in lesion animals relative to sham operated animals. Normalizing glutamate transporter current to whole cell capacitance, which measures the amount of current flow per unit area of membrane, we observed transporter currents were significantly less in the hyperexcitable zone relative for that observed in sham operated animals (1.5 ± 0.46 pA/pF, n = 7, lesioned vs. 21.6 ± 4.8 pA/pF, n = 8, control, p < 0.05, Figure 5C). These data suggest that glutamate accumulation during epileptiform discharges in lesioned cortex would be less effectively cleared by astrocytes.


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)

Astrocyte responses to exogenously applied glutamate are reduced in lesioned cortex. (A) Specimen records of glutamate evoked currents from astrocytes in slices from sham-operated (upper) or lesioned (lower) animals. Cells were voltage clamped at −80 mV and responses were evoked by a 500 µM, 0.5 s puff of glutamate. Bathing and puffing solutions contained 50 µM TTX, 200 µM AP5, 200 µM bicuculline, 250 µM CNQX and 100 µM CdCl. (B) Same data as in A but responses averaged across cells are shown. (C) Summary histogram showing that glutamate evoked responses, normalized for cell capacitance, were significantly reduced in the lesioned group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Astrocyte responses to exogenously applied glutamate are reduced in lesioned cortex. (A) Specimen records of glutamate evoked currents from astrocytes in slices from sham-operated (upper) or lesioned (lower) animals. Cells were voltage clamped at −80 mV and responses were evoked by a 500 µM, 0.5 s puff of glutamate. Bathing and puffing solutions contained 50 µM TTX, 200 µM AP5, 200 µM bicuculline, 250 µM CNQX and 100 µM CdCl. (B) Same data as in A but responses averaged across cells are shown. (C) Summary histogram showing that glutamate evoked responses, normalized for cell capacitance, were significantly reduced in the lesioned group.
Mentions: Several factors, including alterations in local cortical connections in the dysplastic cortex, differences in threshold for synaptic activation and/or release properties, and changes in the astrocyte morphology could contribute to the differences in STC amplitudes described above. Therefore, we next examined astrocyte responses to exogenously applied glutamate. For these experiments, recordings were obtained in the presence of 500 nM TTX, 100 µM CdCl2, 50 µM CNQX, 20 µM bicuculline and 50 µM AP5 to minimize neuronal activity, release of endogenous glutamate and isolate glutamate transporter currents (Bergles and Jahr, 1997; Grass et al., 2004). Glutamate (500 µM, 500 msec) was pressure applied to astrocytes voltage clamped at −80 mV. The puffer pipette was visually placed in the same focal plane as the voltage clamped cell at a distance that elicited a maximum response. Specimen records from 8 control astrocytes (upper) and 7 astrocytes from the lesioned cortex (lower) are shown superimposed in Figure 5A. The average of these responses across cells is shown in Figure 5B. There was no statistical difference in cell size between the two groups, as measured by cell capacitance (control 21 ± 3 pF and lesion 23 ± 4pF, p > 0.05). The changes in STC current and glutamate uptake currents were nearly 10-fold smaller in lesion animals relative to sham operated animals. Normalizing glutamate transporter current to whole cell capacitance, which measures the amount of current flow per unit area of membrane, we observed transporter currents were significantly less in the hyperexcitable zone relative for that observed in sham operated animals (1.5 ± 0.46 pA/pF, n = 7, lesioned vs. 21.6 ± 4.8 pA/pF, n = 8, control, p < 0.05, Figure 5C). These data suggest that glutamate accumulation during epileptiform discharges in lesioned cortex would be less effectively cleared by astrocytes.

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