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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

Astrocytes in the hyperexcitable zone display altered electrophysiological properties. (A) Representative whole-cell current responses to a voltage step protocol in an astrocyte in a slice from a sham-operated control and (B) a recording obtained from an astrocyte in slice from a lesioned animal. Inset shows current displayed at higher resolution. (C) Representative whole-cell currents from sham-operated (black) and lesioned (blue) astrocytes in response to a linear voltage ramp (−160 to +160 mV). (D) Bar graph showing differences in the mean current amplitude obtained from the voltage step protocol (at −140 mV) in control and lesioned astrocytes. (E) Bar graph showing the input resistance in astrocytes in lesioned animals was significantly higher (90.9 ± 13 MΩ) than that of control animals (19 ± 5 MΩ). (F) The resting membrane potential was significantly depolarized in astrocytes from lesioned animals (−71 ± 1.4 mV) relative to control astrocytes (−77 ± 0.77 mV).
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Figure 3: Astrocytes in the hyperexcitable zone display altered electrophysiological properties. (A) Representative whole-cell current responses to a voltage step protocol in an astrocyte in a slice from a sham-operated control and (B) a recording obtained from an astrocyte in slice from a lesioned animal. Inset shows current displayed at higher resolution. (C) Representative whole-cell currents from sham-operated (black) and lesioned (blue) astrocytes in response to a linear voltage ramp (−160 to +160 mV). (D) Bar graph showing differences in the mean current amplitude obtained from the voltage step protocol (at −140 mV) in control and lesioned astrocytes. (E) Bar graph showing the input resistance in astrocytes in lesioned animals was significantly higher (90.9 ± 13 MΩ) than that of control animals (19 ± 5 MΩ). (F) The resting membrane potential was significantly depolarized in astrocytes from lesioned animals (−71 ± 1.4 mV) relative to control astrocytes (−77 ± 0.77 mV).

Mentions: To examine the contribution of astrocytic glutamate transporter dysfunction in the lesion cortex we first investigated the properties of the astrocytes in the hyperexcitable zone of lesioned cortex. Astrocytes recordings obtained from sham-treated animals, displayed large-amplitude, linear currents typical of passive astrocytes (Zhou et al., 2006), as shown in Figure 3A. Similar results were obtained in 15/15 astrocytes from sham-operated animals. In contrast, 13/16 astrocytes from the hyperexcitable zone of lesioned animals displayed prominent voltage-gated currents (Figure 3B). The inset shows currents at higher resolution. We also analyzed the response to voltage ramps. Large linear currents were elicited from control cells in response to a voltage ramp, as shown in the current-voltage plot in Figure 3C. Currents from astrocytes in the lesioned area were significantly smaller in amplitude and demonstrated pronounced voltage dependance. As shown in Figure 3D, peak inward currents at −140 mV in astrocytes from lesioned animals were significantly decreased compared to control (lesioned −1530 ± 373 pA, n = 16 vs. −5297 ± 471 pA, control, n = 15, p < 0.05). This decrease did not appear to be due to a change in cell coupling as no differences in the number of coupled cells between the two groups of astrocytes were observed (lesioned, n = 23.1± 2.8 vs. sham, n = 20.8 ± 3.4, p > 0.05). Supporting this, connexin 43, the primary connexin expressed in astrocytes, was not significantly reduced in lesioned cortex as assessed by qPCR (data not shown). This difference is attributable to a decrease in leak current in hyperexcitable zone astrocytes. Consistent with a reduced leak current, the input resistance in astrocytes from lesioned animals was significantly higher relative to astrocytes from sham-operated animals (90.9 ± 13 MΩ, lesioned, n = 7 vs. 19 ± 5 MΩ, controls, n = 8, p < 0.05, Figure 3E). The resting membrane potential (RMP) in astrocytes from lesioned slices also was significantly depolarized compared to those in controls (−71 ± 1.4 mV, lesioned, n = 40 vs. −77 ± 0.8 mV, control, n = 28, p < 0.05, Figure 3F).


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)

Astrocytes in the hyperexcitable zone display altered electrophysiological properties. (A) Representative whole-cell current responses to a voltage step protocol in an astrocyte in a slice from a sham-operated control and (B) a recording obtained from an astrocyte in slice from a lesioned animal. Inset shows current displayed at higher resolution. (C) Representative whole-cell currents from sham-operated (black) and lesioned (blue) astrocytes in response to a linear voltage ramp (−160 to +160 mV). (D) Bar graph showing differences in the mean current amplitude obtained from the voltage step protocol (at −140 mV) in control and lesioned astrocytes. (E) Bar graph showing the input resistance in astrocytes in lesioned animals was significantly higher (90.9 ± 13 MΩ) than that of control animals (19 ± 5 MΩ). (F) The resting membrane potential was significantly depolarized in astrocytes from lesioned animals (−71 ± 1.4 mV) relative to control astrocytes (−77 ± 0.77 mV).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Astrocytes in the hyperexcitable zone display altered electrophysiological properties. (A) Representative whole-cell current responses to a voltage step protocol in an astrocyte in a slice from a sham-operated control and (B) a recording obtained from an astrocyte in slice from a lesioned animal. Inset shows current displayed at higher resolution. (C) Representative whole-cell currents from sham-operated (black) and lesioned (blue) astrocytes in response to a linear voltage ramp (−160 to +160 mV). (D) Bar graph showing differences in the mean current amplitude obtained from the voltage step protocol (at −140 mV) in control and lesioned astrocytes. (E) Bar graph showing the input resistance in astrocytes in lesioned animals was significantly higher (90.9 ± 13 MΩ) than that of control animals (19 ± 5 MΩ). (F) The resting membrane potential was significantly depolarized in astrocytes from lesioned animals (−71 ± 1.4 mV) relative to control astrocytes (−77 ± 0.77 mV).
Mentions: To examine the contribution of astrocytic glutamate transporter dysfunction in the lesion cortex we first investigated the properties of the astrocytes in the hyperexcitable zone of lesioned cortex. Astrocytes recordings obtained from sham-treated animals, displayed large-amplitude, linear currents typical of passive astrocytes (Zhou et al., 2006), as shown in Figure 3A. Similar results were obtained in 15/15 astrocytes from sham-operated animals. In contrast, 13/16 astrocytes from the hyperexcitable zone of lesioned animals displayed prominent voltage-gated currents (Figure 3B). The inset shows currents at higher resolution. We also analyzed the response to voltage ramps. Large linear currents were elicited from control cells in response to a voltage ramp, as shown in the current-voltage plot in Figure 3C. Currents from astrocytes in the lesioned area were significantly smaller in amplitude and demonstrated pronounced voltage dependance. As shown in Figure 3D, peak inward currents at −140 mV in astrocytes from lesioned animals were significantly decreased compared to control (lesioned −1530 ± 373 pA, n = 16 vs. −5297 ± 471 pA, control, n = 15, p < 0.05). This decrease did not appear to be due to a change in cell coupling as no differences in the number of coupled cells between the two groups of astrocytes were observed (lesioned, n = 23.1± 2.8 vs. sham, n = 20.8 ± 3.4, p > 0.05). Supporting this, connexin 43, the primary connexin expressed in astrocytes, was not significantly reduced in lesioned cortex as assessed by qPCR (data not shown). This difference is attributable to a decrease in leak current in hyperexcitable zone astrocytes. Consistent with a reduced leak current, the input resistance in astrocytes from lesioned animals was significantly higher relative to astrocytes from sham-operated animals (90.9 ± 13 MΩ, lesioned, n = 7 vs. 19 ± 5 MΩ, controls, n = 8, p < 0.05, Figure 3E). The resting membrane potential (RMP) in astrocytes from lesioned slices also was significantly depolarized compared to those in controls (−71 ± 1.4 mV, lesioned, n = 40 vs. −77 ± 0.8 mV, control, n = 28, p < 0.05, Figure 3F).

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