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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

Synaptic responses in pyramidal cells in slices from sham and lesioned animals. (A) Representative image of a cresyl-violet stained slice from a lesioned animal showing the microsulcus. Dotted circle indicates the hyperexcitable zone. (B) Sample traces of evoked synaptic responses from a sham operated and lesioned animal. Responses obtained in (top: left to right) sham ACSF, sham after TBOA (30 µM) application, lesion neuron in ACSF and lesion neuron after TBOA (30 µM) application are shown. Bar graphs show quantification of the synaptic current response area following stimulation in sham and lesioned slices before and after TBOA application. Scale bar represents 200 pA and 500 mS.
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Figure 1: Synaptic responses in pyramidal cells in slices from sham and lesioned animals. (A) Representative image of a cresyl-violet stained slice from a lesioned animal showing the microsulcus. Dotted circle indicates the hyperexcitable zone. (B) Sample traces of evoked synaptic responses from a sham operated and lesioned animal. Responses obtained in (top: left to right) sham ACSF, sham after TBOA (30 µM) application, lesion neuron in ACSF and lesion neuron after TBOA (30 µM) application are shown. Bar graphs show quantification of the synaptic current response area following stimulation in sham and lesioned slices before and after TBOA application. Scale bar represents 200 pA and 500 mS.

Mentions: A representative image of a cresyl violet stained cortical brain slice from a lesioned animal 24 days post lesion is shown in Figure 1A. The region adjacent to the microgyrus (a distance of 0.5 mm–2.5 mm, circled in black) has been termed hyperexcitable zone (Jacobs et al., 1996; Luhmann and Raabe, 1996). Neurons from this region display a lower threshold for evoking epileptiform activity and when stimulated have the capacity to generate abnormal electrical activity that spreads to surrounding tissue in the cortex (Jacobs et al., 1996; Luhmann and Raabe, 1996; Hablitz and DeFazio, 1998). The occurrence of these long lasting epileptiform discharges in the lesioned cortex are produced by a complex pattern of excitatory and inhibitory inputs (DeFazio and Hablitz, 2000). More recently, it has been demonstrated that the observed hyperexcitability is due in part to higher levels of extracellular glutamate which can be abrogated by blocking neuronal NMDA receptors (Campbell and Hablitz, 2008). Here we tested the notion that elevated glutamate is due in part to deficient astrocyte mediated glutamate clearance. In the first set of experiments we compared the currents elicited layer 2/3 pyramidal neurons in sham-treated slices when glutamate transporters were blocked with TBOA to those elicited in lesioned cortex neurons in normal ACSF. A brief electrical stimulation (50 us, 100 µA) applied in deeper cortical layers IV/V that was four times the stimulation required to elicit a threshold response was used. This was to ensure that only large current responses were induced, where glutamate transporter function would more likely be challenged (Campbell and Hablitz, 2004, 2008). In slices from sham treated animals, stimulation induced large responses in layer II/III pyramidal neurons with a mean response area of 1327 ± 125 pA*ms (n = 15 slices, Figure 1B). Bath application of TBOA (30 µM) significantly prolonged the response area (5081 ± 872 pA*ms, n = 15 slices, p < 0.001). Using the same stimulation protocol (four times the threshold) we elicited responses in the hyperexcitable zone of the lesioned cortex, which induced current responses with similar response area (3961 ± 872 pA*ms, n = 12 slices, p > 0.05) when compared to the response area in the sham slices in the presence of TBOA. When glutamate uptake was inhibited (TBOA 30 µM) in the lesioned cortex there was a pronounced increase in the response area (59214 ± 13271 pA*ms, n = 12 slices, p < 0.05) of the evoked epileptiform activity. This prominent increase in the response area in lesioned slices was independent of the initial stimulus intensity, since both low and high stimulus evoked events resulted in a marked prolongation of epileptiform activity (data not shown). To more directly assess the role of excess glutamate on neuronal excitability, we isolated NMDA receptor-mediate currents in control and lesioned animals in layer II/III pyramidal neurons (Figure 2). Here, neurons were stepped from a holding potential of −70 mV to +50 mV to remove the Mg2+ block. Stimulation in layer IV/V elicited outward NMDA receptor-mediated currents. Current decay for all 13 sham-operated neurons and 14 lesioned neurons were best fit by a sum of two exponentials. Representative, normalized traces demonstrate that the slow component of the decay (τslow) was significantly increased in the lesioned animals relative to sham-operated littermates (Figure 2A). Here the τslow 279.3 +/− 16.3 in sham vs. 375.8 +/− 36.4 in lesioned animals (n = 13 sham-operated, n = 14 lesion, p = 0.0266, Figure 2B), suggesting prolonged glutamate in the ECS in the lesioned cortex following stimulation. Histograms of τslow indicate a greater number of cells in bins with larger τ values for the lesion group (Figure 2C). Although a trend was observed, there was no significant difference in the fast component of the NMDA decay constant (τfast) between sham-operated and lesioned animals (66.7 +/− 10.3 (n = 13) and 96.9 +/− 14.3 (n = 14)). Application of TBOA to block astrocytic glutamate transporters significantly increased the τslow in both sham and lesioned animals (Figures 2B–D, 484.5 +/− 51.9 (n = 7) vs. 609.3 +/− 76.7 (n = 8), respectively) relative to ACSF. However, here was no statistical difference between the decay kinetics in sham and lesioned animals in the presence of TBOA (Figure 2B, p = 0.2142). There was also no difference observed in the fast component of the NMDA receptor-mediated current decay constant (τfast) between sham-operated and lesioned animals in the presence of TBOA (123.2 +/− 27.2 and 139.0 +/− 29, respectively). Together, these results suggest that the increased excitability in the lesioned cortex is due, in part to elevated glutamate levels resulting from alteration in glutamate transporter function.


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)

Synaptic responses in pyramidal cells in slices from sham and lesioned animals. (A) Representative image of a cresyl-violet stained slice from a lesioned animal showing the microsulcus. Dotted circle indicates the hyperexcitable zone. (B) Sample traces of evoked synaptic responses from a sham operated and lesioned animal. Responses obtained in (top: left to right) sham ACSF, sham after TBOA (30 µM) application, lesion neuron in ACSF and lesion neuron after TBOA (30 µM) application are shown. Bar graphs show quantification of the synaptic current response area following stimulation in sham and lesioned slices before and after TBOA application. Scale bar represents 200 pA and 500 mS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Synaptic responses in pyramidal cells in slices from sham and lesioned animals. (A) Representative image of a cresyl-violet stained slice from a lesioned animal showing the microsulcus. Dotted circle indicates the hyperexcitable zone. (B) Sample traces of evoked synaptic responses from a sham operated and lesioned animal. Responses obtained in (top: left to right) sham ACSF, sham after TBOA (30 µM) application, lesion neuron in ACSF and lesion neuron after TBOA (30 µM) application are shown. Bar graphs show quantification of the synaptic current response area following stimulation in sham and lesioned slices before and after TBOA application. Scale bar represents 200 pA and 500 mS.
Mentions: A representative image of a cresyl violet stained cortical brain slice from a lesioned animal 24 days post lesion is shown in Figure 1A. The region adjacent to the microgyrus (a distance of 0.5 mm–2.5 mm, circled in black) has been termed hyperexcitable zone (Jacobs et al., 1996; Luhmann and Raabe, 1996). Neurons from this region display a lower threshold for evoking epileptiform activity and when stimulated have the capacity to generate abnormal electrical activity that spreads to surrounding tissue in the cortex (Jacobs et al., 1996; Luhmann and Raabe, 1996; Hablitz and DeFazio, 1998). The occurrence of these long lasting epileptiform discharges in the lesioned cortex are produced by a complex pattern of excitatory and inhibitory inputs (DeFazio and Hablitz, 2000). More recently, it has been demonstrated that the observed hyperexcitability is due in part to higher levels of extracellular glutamate which can be abrogated by blocking neuronal NMDA receptors (Campbell and Hablitz, 2008). Here we tested the notion that elevated glutamate is due in part to deficient astrocyte mediated glutamate clearance. In the first set of experiments we compared the currents elicited layer 2/3 pyramidal neurons in sham-treated slices when glutamate transporters were blocked with TBOA to those elicited in lesioned cortex neurons in normal ACSF. A brief electrical stimulation (50 us, 100 µA) applied in deeper cortical layers IV/V that was four times the stimulation required to elicit a threshold response was used. This was to ensure that only large current responses were induced, where glutamate transporter function would more likely be challenged (Campbell and Hablitz, 2004, 2008). In slices from sham treated animals, stimulation induced large responses in layer II/III pyramidal neurons with a mean response area of 1327 ± 125 pA*ms (n = 15 slices, Figure 1B). Bath application of TBOA (30 µM) significantly prolonged the response area (5081 ± 872 pA*ms, n = 15 slices, p < 0.001). Using the same stimulation protocol (four times the threshold) we elicited responses in the hyperexcitable zone of the lesioned cortex, which induced current responses with similar response area (3961 ± 872 pA*ms, n = 12 slices, p > 0.05) when compared to the response area in the sham slices in the presence of TBOA. When glutamate uptake was inhibited (TBOA 30 µM) in the lesioned cortex there was a pronounced increase in the response area (59214 ± 13271 pA*ms, n = 12 slices, p < 0.05) of the evoked epileptiform activity. This prominent increase in the response area in lesioned slices was independent of the initial stimulus intensity, since both low and high stimulus evoked events resulted in a marked prolongation of epileptiform activity (data not shown). To more directly assess the role of excess glutamate on neuronal excitability, we isolated NMDA receptor-mediate currents in control and lesioned animals in layer II/III pyramidal neurons (Figure 2). Here, neurons were stepped from a holding potential of −70 mV to +50 mV to remove the Mg2+ block. Stimulation in layer IV/V elicited outward NMDA receptor-mediated currents. Current decay for all 13 sham-operated neurons and 14 lesioned neurons were best fit by a sum of two exponentials. Representative, normalized traces demonstrate that the slow component of the decay (τslow) was significantly increased in the lesioned animals relative to sham-operated littermates (Figure 2A). Here the τslow 279.3 +/− 16.3 in sham vs. 375.8 +/− 36.4 in lesioned animals (n = 13 sham-operated, n = 14 lesion, p = 0.0266, Figure 2B), suggesting prolonged glutamate in the ECS in the lesioned cortex following stimulation. Histograms of τslow indicate a greater number of cells in bins with larger τ values for the lesion group (Figure 2C). Although a trend was observed, there was no significant difference in the fast component of the NMDA decay constant (τfast) between sham-operated and lesioned animals (66.7 +/− 10.3 (n = 13) and 96.9 +/− 14.3 (n = 14)). Application of TBOA to block astrocytic glutamate transporters significantly increased the τslow in both sham and lesioned animals (Figures 2B–D, 484.5 +/− 51.9 (n = 7) vs. 609.3 +/− 76.7 (n = 8), respectively) relative to ACSF. However, here was no statistical difference between the decay kinetics in sham and lesioned animals in the presence of TBOA (Figure 2B, p = 0.2142). There was also no difference observed in the fast component of the NMDA receptor-mediated current decay constant (τfast) between sham-operated and lesioned animals in the presence of TBOA (123.2 +/− 27.2 and 139.0 +/− 29, respectively). Together, these results suggest that the increased excitability in the lesioned cortex is due, in part to elevated glutamate levels resulting from alteration in glutamate transporter function.

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