Figure 2: Electrically active neurons utilize more GSH and upregulate GSH peroxidase activity.

(a,b) Synaptic activity does not alter steady-state basal GSH levels in unstressed neurons. Cortical neurons were stimulated with BiC/4-AP for 24 h, after which GSH levels were measured using in vivo MCB labelling (a) or a colourometric assay in cell-free extracts (b) see Methods, n=4 for both. (c) GSH utilization is enhanced by synaptic activity. Neurons were treated ±BiC/4-AP (BiC) for 24 h. Subsequently, cells were treated with BSO+BCNU (±H₂O₂) for 1, 2, 4, 8 or 12 h or left untreated. Thirty minutes before the end of this period of time, cells were loaded with MCB and GS-bimane fluorescence measured (normalized to protein). For each condition, GS-bimane fluorescence was plotted against time and the rate of decline in fluorescence (a measure of rate of GSH utilization) obtained by fitting a line to the data. *P=0.0039, 0.033, 0.021, 0.0029, 2WA-FPh (n=7). n.b., where one asterisk indicates two comparisons, the P value for the comparison closest to the asterisk is shown first. See Supplementary Fig. 2c and 2d for schematic of the experimental protocol and example of an experimental replicate used to obtain the rate of decline in GS-bimane fluorescence. (d) GSH utilization is enhanced by synaptic activity. Neurons were treated ±BiC/4-AP for 24 h. Subsequently, the cells were treated with BSO+BCNU for 4 h, after which GSH levels were determined using the colorimetric method and normalized to protein levels. *P=0.0058, (n=3). (e) Synaptic activity upregulates glutathione peroxidase 2 and 4 mRNA expression. Neurons were treated±BiC/4-AP for 24 h and expression of the indicated Gpx genes analysed. *P=0.017, 0.022 (n=10 Gpx2, n=5 Gpx4, n=9 Gpx1). (f) Glutathione peroxidase enzyme activity is increased by synaptic activity. Neurons were treated±BiC/4-AP for 24 h and GPX enzyme activity measured. *P=0.041 (n=8).