Fig 2: Effect of PDI knockdown on basal neurotransmission in the dentate gyrus at 7 days after PDI siRNA infusion.(A) Representative paired-pulse responses in control siRNA- and PDI siRNA-infused animals. The inset at top shows the measurement of fEPSP slope (EPSP) and population spike amplitude (PS). Filled circles indicate stimulus artifacts. (B) Quantification of effects of PDI siRNA on IO curve, normalized population spike amplitude ratios and normalized excitability ratio in the dentate gyrus (n = 7, respectively). PDI knockdown does not affect GABAergic or glutamatergic transmission in basal condition.

Fig 3: Effect of PDI knockdown on spontaneous seizure activity in epileptic (6 week-post SE) rats.(A) Representative EEGs for control siRNA- and PDI siRNA-infused epileptic animals. (B–D) Anticonvulsive effect of PDI siRNA on spontaneous seizure activity: (B) the mean seizure frequency, (C) seizure duration and (D) behavioral seizure score (Open circles indicate each individual value. Closed circles indicate mean value. *p < 0.05 vs. control siRNA; n = 5, respectively). PDI knockdown inhibits the spontaneous seizure activity in chronic epileptic rats. (E) Co-immunoprecipitation analyses of NR1 or NR2A interaction with PDI in control siRNA- and PDI siRNA-infused epileptic animals. M, molecular weight marker. (F) The quantitative analyses of co-immunoprecipitation of PDI bound to NR1 and NR2A (*p < 0.05 vs. control siRNA; n = 7, respectively). (G) Western blot data for the amounts of -SH + -SNO on NR1 and NR2A subunits in normal (N), control siRNA- and PDI siRNA-infused epileptic animals. M, molecular weight marker. (H,I) Quantification of effects of PDI siRNA on the amount of -SH + -SNO on NR1 and NR2A subunit and PDI expression in epileptic rats (mean ± S.E.M.; *p < 0.05 vs. normal; n = 7, respectively). PDI siRNA infusion reduces the binding of PDI to NMDAR, the amount of -SH + -SNO of NMDAR subunits and PDI expression level in epileptic animals.

Fig 4: Effect of PDI neutralization on seizure activity and in vitro thiol reductase activity of PDI on NR1 subunit.(A–C) Effect of PDI neutralization on seizure susceptibility in response to PILO. PDI neutralization reduces seizure susceptibility in response to PILO. (A) Representative EEG traces and frequency-power spectral temporal maps in response to PILO. (B,C) Quantification of effect of PDI neutralization on SE induction, latency and total EEG power in response to PILO (mean ± S.E.M.; *p < 0.05 vs. control IgG; n = 10, respectively). (D,E) In vitro thiol reductase activity of PDI on recombinant NR1 protein. As compared to vehicle (Tris), PDI treatment significantly increases the amount of thiols on recombinant NR1 protein. (D) Western blot representing the amount of thiols on recombinant NR1 protein (the upper arrow). TMT antibody also detects the thiols on PDI (the lower arrow). M, molecular weight marker. (E) Quantification of effects of PDI on the amount of thiols on recombinant NR1 protein (mean ± S.E.M.; *p < 0.05 vs. vehicle; n = 7, respectively). (F) Effect of PDI siRNA on PDI activity in vivo (mean ± S.E.M.; *p < 0.05 vs. control siRNA; n = 7, respectively). PDI siRNA inhibits the insulin reduction activity, as compared to control siRNA. (G) Representative double immunofluorescent photo for colocazation of PDI with NR1 in the normal rat hippocampus. PDI is colocalized with NR1 clusters in perikarya and the primary dendrite (panel 1). In the distal dendrites (panel 2–3), PDI immunoreactive structures are observed as continuous tubular (ER-like) or punctuate (vesicle-like) shapes. Some PDI positive structures contain NR1 immunoreactivity (arrows) and attach to NR1 positive puncta (arrow heads). Panels 2 and 3 are high magnification images for rectangles in panels 1–2. Bar = 10 (panel 1), 5 (panel 2) and 2.5 (panel 2) μm.

Fig 5: PDI expression in the hippocampus following SE.(A) In non-SE animals (N), PDI expression is observed in CA1–CA3 pyramidal cells as well as dentate granule cells. Three days (3D) after SE, PDI expression is increased in the same regions. However, PDI expression is decreased 7 days (7D) after SE. Bar = 300 μm. (B) Western blot shows the gradual up-regulation of PDI 3 days after SE. At 7 days after SE, the expression is reduced. M, molecular weight marker. (C) Quantification of PDI expression level based on western blot data (mean ± S.E.M.; *p < 0.05 vs. non-SE; n = 7, respectively).