Decreased glutamate transport enhances excitability in a rat model of cortical dysplasia

Abstract

Glutamate transporters function to maintain low levels of extracellular glutamate and play an important role in synaptic transmission at many synapses. Disruption of glutamate transporter function or expression can result in increased extracellular glutamate levels. Alterations in glutamate transporter expression have been reported in human epilepsy and animal seizure models. Functional electrophysiological changes that occur when transporter expression is disrupted in chronic epilepsy models have not been examined. Here, we used a freeze-induced model of cortical dysplasia to test the role of glutamate transporters in synaptic hyperexcitability. We report that inhibiting glutamate transporters with the non-selective antagonist, DL-threo-beta-benzylozyaspartic acid (TBOA) preferentially prolongs postsynaptic currents (PSCs) and decreases the threshold for evoking epileptiform activity in lesioned compared to control cortex. The effect of inhibiting uptake is mediated primarily by the glia glutamate transporter (GLT-1) since the selective antagonist dihydrokainate (DHK) mimicked the effects of TBOA. The effect of uptake inhibition is mediated by activation of N-methyl-D-aspartate (NMDA) receptors since D-(-)-2-amino-5-phosphonovaleric acid (APV) prevents TBOA-induced effects. Neurons in lesioned cortex also have a larger tonic NMDA current. These results indicate that chronic changes in glutamate transporters and NMDA receptors contribute to hyperexcitability in cortical dysplasia.

Fig. 1

Evoked postsynaptic currents (PSCs) in layer II/III pyramidal neurons from control and lesioned cortex. Responses to stimuli of increasing intensity are shown. The stimulation electrode was placed in layer IV/V lateral to recording site. A, Specimen records showing averaged traces (n = 10) in a pyramidal cell from control cortex. Intensities were 1 to 5 times threshold (1-5T) stimulation. B, Same recording conditions as in A, but in lesioned cortex. Recordings were done in the hyperexcitable region of lesion cortex (.5 to 1.5 mm from the microsulcus). A holding potential of −70 mV was used in all recordings. Scale bars represent 25 ms and 50 pA in A; 25 ms and 500 pA in B.

Fig. 2

Inhibition of uptake enhances evoked synaptic responses in lesioned but not control cortex. A, Representative averages (n = 10) of evoked responses in control cortex before and during TBOA (30 µM) application. B, Similar to A but in lesioned cortex. Responses are significantly prolonged in presence of TBOA. C, Application of DHK, a specific GLT-1 inhibitor, rolongs responses in lesioned cortex. D, Summary plot showing TBOA and DHK’s effect on the amplitude and area of PSCs in control and lesioned cortex. Comparison of group data from control and lesioned cortex demonstrated that uptake inhibition had a significant effect on the response area but not the amplitude. E, Summary plot showing effect of transporter inhibition on holding currents. Scale bars represent 50 ms and 200 pA in A, B and C. Asterisk (*) indicates p<0.05.

Fig. 3

TBOA depolarizes neurons and increases evoked responses. A, Current clamp recordings in lesioned cortex under control conditions. B, Response to same stimulation recorded in presence of TBOA (30 µM). C, Summary of the changes in response area showing prolongation during TBOA application. D, Summary of effects of TBOA on membrane potential. Asterisk (*) indicates p<0.05.

Fig. 4

Inhibition of uptake decreases the threshold for triggering epileptiform discharges in lesioned cortex. A, Superimposed traces of evoked PSCs in ACSF (black) and during TBOA (5 µM) (grey) application in control animals. B, Superimposed traces of evoked responses in ACSF (black) and during TBOA (5 µM) (grey) application in the lesioned cortex. C, Summary plot showing effect of low concentrations of TBOA on response amplitude and area. Asterisk (*) indicates p<0.05.

Fig. 5

Alteration in uptake increases the probability of observing spontaneous discharges in lesioned cortex. A, Specimen records in control cortex showing lack of epileptiform discharges before (upper two traces) and during (lower two records) TBOA (30 µM) application. B, Upper, two representative traces in lesioned cortex showing lack of spontaneous activity under control conditions, Lower, spontaneous epileptiform discharges detected during application of TBOA (30 µM). C, Summary bar graph of the probability of detecting spontaneous discharges before and during TBOA (30 µM) application. Spontaneous discharges were never detected in control. In the presence of TBOA (30 µM), spontaneous discharges were detected in 2 of 21 experiments in the control cortex (control: P_o = 0 vs TBOA: P_o = 0.09 ± 0.06; n = 21). TBOA caused a significant increase in the probability of detecting a spontaneous discharge from 0.09 ± 0.06 to 0.59 ± 0.1 (n = 22). Scale bar is the same for A and C. Asterisk (*) indicates p<0.05.