Supplementary Fig. 1 ). (D) Comparison of PIP 3 level in primary hippocampal neurons under control conditions (control, N = 7) or with VPA alone (N = 5), or in seizure-inducing conditions in the absence (N = 4) or presence of VPA (1 mM; N = 5), or in control condition with PI3K inhibitor (10 μM, LY-294002, LY, N = 3) measured by PIP 3 ELISA mass assay. Graphs show means ± SEM, with individual data point illustrated. Statistical analysis was performed by post-hoc Tukey test, following ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). Data for other inositol phosphate species are provided in Supplementary Fig. 4 . " title="... in control condition with PI3K inhibitor (10 μM, LY-294002, LY, N = 3) measured by PIP 3 ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Seizure-inducing conditions in primary rat hippocampal neurons decrease PIP 3 levels but this is restored by VPA. (A) Phosphoinositide synthesis and interconversion can be monitored by the use of radio-labelled myo-inositol (MI) that is incorporated into phosphatidylinositol (PI), then phosphatidylinositol 4 phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP 2 ) by sequential phosphorylation of the inositol ring by 4′ kinases (PI4K) and 5′ kinases (PI5K), respectively. Red circles represent phosphates. PIP 2 is cleaved by phospholipase C (PLC) to produce aqueous inositol trisphosphate (IP 3 ) or is phosphorylated by 3′ kinases (PI3K) to produce PIP 3 . (B) Example of phosphoinositide labelling in primary hippocampal neurons under seizure-inducing conditions (PTZ at 5 mM), imaged following separation by TLC. (C) Comparison of level of PIP 3 in primary hippocampal neurons under control condition (N = 18) and with VPA alone (1 mM, N = 9), or in seizure-inducing conditions in the absence (N = 18) or presence of VPA (1 mM, N = 14) measured by quantification of TLC separated phosphoinositides in comparison with standards ( Supplementary Fig. 1 ). (D) Comparison of PIP 3 level in primary hippocampal neurons under control conditions (control, N = 7) or with VPA alone (N = 5), or in seizure-inducing conditions in the absence (N = 4) or presence of VPA (1 mM; N = 5), or in control condition with PI3K inhibitor (10 μM, LY-294002, LY, N = 3) measured by PIP 3 ELISA mass assay. Graphs show means ± SEM, with individual data point illustrated. Statistical analysis was performed by post-hoc Tukey test, following ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). Data for other inositol phosphate species are provided in Supplementary Fig. 4 .

Techniques Used: Enzyme-linked Immunosorbent Assay, Mass Assay

Figure Legend Snippet: VPA control of epileptiform activity is PIP 3 -dependent. (A) Example trace recording of epileptiform activity (burst discharges) in hippocampal slices induced by application of PTZ (PTZ 2 mM, K + 6 mM) under control conditions (DMSO) and with VPA (1 mM); or (B) with VPA (1 mM) after treatment with PI3K inhibitors (PI-103 (10 μM); LY-294002 (LY, 20 μM)) and following sequestration of PIP 2 with Neomycin (Neo, 100 μM) for 30 min. (C) Summary of PTZ-induced epileptiform discharge data for hippocampal slices with VPA treatment only (control) or with prior application of PI-103 (N = 4), LY-294002 (N = 4), or Neomycin (N = 5), where VPA was applied between 0 and 40 min. (D) Comparison of the mean frequency of epileptiform activity under different conditions, averaged from 20 to 40 min post-treatment. Graphs show means ± SEM, with individual data point illustrated. Statistical analysis was performed by post-hoc Tukey test, following ANOVA (**p < 0.01, ***p < 0.001).

Techniques Used: Activity Assay