The glutathione cycle shapes synaptic glutamate activity

Abstract

Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one-third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate and could influence synaptic excitability. We find that drugs that inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory postsynaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter.

Keywords: acivicin; glutamate; glutathione; mEPSC; neurotransmission.

Fig. 1.

Blocking efflux of glutamate from the glutathione cycle decreases mEPSC. (A) Schematic representation of the glutathione cycle and inhibition of GGT by acivicin, which is upstream of the liberation of glutamate. Details appear in SI Appendix, Figs. S1 and S2. (B) Representative mEPSC traces in primary cortical neurons treated 24 h with vehicle, 25 μM acivicin, and/or 5 μM PGA. Acivicin decreased mEPSC frequency, which can be recovered by pyroglutamate. (C) Distribution of mEPSC frequency in cortical neurons treated with acivicin. (D) Distribution of mEPSC amplitude in cortical neurons treated with acivicin. (E) Cumulative probability plots of mEPSC frequency. Acivicin, which decreases efflux of glutamate from the glutathione cycle, decreased mEPSC frequency, a reflection of presynaptic drive. Pyroglutamate restores glutamate and presynaptic drive (see also SI Appendix, Fig. S2A) (n.s., not significant). (F) Cumulative probability plots of mEPSC amplitude. Acivicin decreased mEPSC amplitude to a smaller degree than frequency. (^#P < 0.01, ^‡P < 0.0001 by Steel–Dwass all pairs test.)

Fig. 2.

Efflux of glutamate from the glutathione cycle can increase mEPSC. (A) Schematic representation of the glutathione cycle and inhibition of GCL by BSO, which shuttles free glutamate into glutathione. Details appear in SI Appendix, Figs. S1 and S3. (B) Representative mEPSC traces in primary cortical neurons treated 24 h with vehicle or 200 μM BSO. (C) Distribution of mEPSC frequency in cortical neurons treated with BSO. (D) Distribution of mEPSC amplitude in cortical neurons treated with BSO. (E) Cumulative probability plots of mEPSC frequency. BSO, which increases efflux of glutamate from the glutathione cycle, increased mEPSC frequency, a reflection of presynaptic drive (see also SI Appendix, Fig. S4). (F) Cumulative probability plots of mEPSC amplitude in cortical neurons treated with BSO. (^‡P < 0.0001 by Steel–Dwass all pairs test.)

Fig. 3.

The glutathione cycle supports excitatory transmission, but to a smaller degree than glutamine-derived glutamate. (A) Scheme of glutamate-glutamine cycling and blockade of system A glutamine transporters by MeAIB. Details appear in SI Appendix, Fig. S4. (B) Representative traces of mEPSC recordings in primary cortical neurons treated with 25 μM acivicin (24 h) and/or 25 mM MeAIB (2 h). (C and D) Distribution of mEPSC frequency and amplitude in cortical neurons treated with acivicin and/or MeAIB. (E and F) Cumulative probability plots of mEPSC frequency and amplitude. MeAIB decreased mEPSC frequency to a greater degree than acivicin, with both treatments having additive effects for presynaptic drive frequency. (^‡P < 0.0001 by Steel–Dwass all pairs test.)

Fig. 4.

The glutathione cycle can rescue restrictions of glutamine-derived glutamate. (A) Scheme of glutamate-glutamine cycling and blockade of system A glutamine transporters by MeAIB. Details appear in SI Appendix, Fig. S4. (B) Representative traces of mEPSC recordings in primary cortical neurons treated with 25 μM BSO (24 h) and/or 25 mM MeAIB (2 h). (C and D) Distribution of mEPSC frequency and amplitude in cortical neurons treated with BSO and/or MeAIB. (E and F) Cumulative probability plots of mEPSC frequency and amplitude. MeAIB, which restricts glutamine-derived glutamate, inhibits mEPSC frequency (blue). Decreases in mEPSC frequency induced by MeAIB were rescued by pretreatment with BSO (green), which increased mEPSC frequency (red). BSO also improved the decrease in mEPSC amplitudes by MeAIB. (^‡P < 0.0001 by Steel–Dwass all pairs test.)