Journal: eLife

Article Title: Metabolomic profiling reveals a differential role for hippocampal glutathione reductase in infantile memory formation

doi: 10.7554/eLife.68590

Figure Lengend Snippet: ( a ) Scheme depicting the glutathione oxidation/reduction cycle and biosynthesis pathway. ( b ) Quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) of reduced (GSH) and oxidized (GSSG) forms of glutathione, of the GSH/GSSG ratio, and of the GSH amino acid precursors glutamate, cysteine, and glycine in hippocampal samples from postnatal day 17 (PN17), PN24, and PN80 (n = 7 rats per group; one experiment). Data are expressed as mean percentage ± s.e.m. of the value in naive adult group (PN80). *p < 0.05; **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). ( c ) Representative examples and densitometric Western blot analyses of glutathione reductase (GR), glutathione peroxidase (GPx – isozyme 1), glutamate-cysteine ligase (catalytic subunit GCLC, and regulatory subunit GCLM), and glutathione synthetase (GSy), carried out with whole-protein extracts of dHC from PN17 (n = 6), PN24 (n = 4–5), and PN80 (n = 4–5) naive rats; two independent experiments. Actin was used as loading control. Data are expressed as mean percentage ± s.e.m. of the value in naive adult group. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). ( d ) Mean activity of GR, GPx, GCL, and GSy (expressed as the quantity, in nmol, of substrate transformed per minute and normalized to the total quantity of proteins in each sample, in mg) assayed in dHC protein extracts from PN17 (n = 6), PN24 (n = 6), and PN80 (n = 6) naive rats; two independent experiments. Data are expressed as mean percentage ± s.e.m. of the value in naive adult group. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). See and for numerical values and detailed statistical information. Figure 6—source data 1. Numerical values. Figure 6—source data 2. Detailed statistical information.

Article Snippet: Primary antibodies against the following proteins were obtained from the indicated suppliers: GR (1/2000, Abcam, cat# ab16801), GPx isozyme 1 (1/1000, Abcam, cat# ab22604), GSy (1/1000, Abcam, cat# ab133592), GCL catalytic subunit (1/1000, Novus Biologicals, cat# NBP2-45830), GCL regulatory subunit (1/1000, Abcam, cat# ab126704), NeuN (1/1000, Abcam, cat# ab104225), GFAP (1/2000, Abcam, cat# ab4674).

Techniques: Liquid Chromatography, Tandem Mass Spectroscopy, Western Blot, Control, Activity Assay, Transformation Assay

Journal: eLife

Article Title: Metabolomic profiling reveals a differential role for hippocampal glutathione reductase in infantile memory formation

doi: 10.7554/eLife.68590

Figure Lengend Snippet: ( a ) Quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) of reduced (GSH) and oxidized (GSSG) forms of glutathione and the GSH/GSSG ratio in whole hippocampus from PN17, PN24, and PN80, in the untrained condition and 1 hr after inhibitory avoidance (IA) training (n = 7 rats per group). Data are expressed as mean percentage ± s.e.m. of the value in naive group at each age. ***p < 0.001 (two-way ANOVA followed by Bonferroni’s multiple comparisons test). ( b ) Representative examples and densitometric Western blot analyses of GR, glutathione peroxidase isozyme 1 (GPx), glutamate-cysteine ligase (catalytic subunit GCLC, and regulatory subunit GCLM), and glutathione synthetase (GSy), carried out with whole-protein extracts of dHC from postnatal day 17 naive rats (PN17-N) and rats trained in IA at PN17 and euthanized 1 hr (Tr-1h) or 24 hr (Tr-24h) after training (n = 5–6 rats per group; two independent experiments). Actin was used as the loading control. Data are expressed as mean percentage ± s.e.m. of the value in PN17 naive rats. p > 0.05 (one-way ANOVA followed by Tukey’s multiple comparisons test). ( c ) Mean activity of GR, GPx, GCL, and GSy (expressed as the quantity, in nmol, of substrate transformed per minute and normalized to the total quantity of proteins in each sample, in mg) assayed in dHC protein extracts obtained from rats trained in IA at PN17 and euthanized 15 min, 1 hr, 24 hr, or 7 days after training (Tr-15min, Tr-1h, Tr-24h, and Tr-7d, respectively) (n = 6 rats per group; two independent experiments). To account for developmental differences, two groups of naive (N) rats were used: PN17 and PN24 (n = 6 rats per group). Data are expressed as mean percentage ± s.e.m of the value in PN17 naive rats. *p < 0.05, ***p < 0.001: significance vs. PN17 naive rats (one-way ANOVA followed by Dunnett’s multiple comparisons test); p > 0.05 for the comparison between PN24-N and Tr-7d groups (two-tailed unpaired Student’s t-test). ( d ) GR activity carried out with dHC protein extracts obtained from PN17 naive rats (PN17-N), from rats exposed to an immediate footshock without IA-context exposure (shock-only) and euthanized 15 min later (SO-15min) or from rats trained in IA at PN17 and euthanized 15 min later (Tr-15min) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN17 naive rats. *p < 0.05, **p < 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons test). ( e ) GR activity assayed in dHC protein extracts obtained from PN24 naive rats (PN24-N) and rats trained in IA at PN24 and euthanized 15 min, 1 hr, or 24 hr after training (Tr-15min, Tr-1h, and Tr-24h, respectively) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN24 naive rats. p > 0.05 (one-way ANOVA followed by Dunnett’s multiple comparisons test). ( f ) GR activity assayed in dHC protein extracts obtained from PN80 naive rats (PN80-N) and rats trained in IA at PN80 and euthanized 15 min, 1 hr, or 24 hr after training (Tr-15min, Tr-1h, and Tr-24h, respectively) (n = 6 rats per group; two independent experiments). GR activity is expressed as mean percentage ± s.e.m of the value in PN80 naive rats. p > 0.05 (one-way ANOVA followed by Dunnett’s multiple comparisons test). ( g ) Representative examples and densitometric Western blot analyses of NeuN and GFAP carried out with whole-protein extracts of fluorescence-activated cell sorting (FACS)-sorted neurons (NeuN+), astrocytes (GFAP+), and unlabeled counter-selected cells from the dHC of PN17 naive rats (n = 4 x 3 rats per group; two independent experiments). Actin was used as the loading control. NeuN intensity values are expressed as mean percentage ± s.e.m. of the value in neurons (NeuN+) group; GFAP intensity values are expressed as mean percentage ± s.e.m. of the value in astrocytes (GFAP+) group. ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons test). GR activity carried out with whole-protein extracts of FACS-sorted neurons (NeuN+), astrocytes (GFAP+), and unlabeled cells from the dHC of PN17 naive rats (PN17-N) and of rats trained in IA at PN17 and euthanized 15 min later (Tr-15min) (n = 5 x 3 rats per group; two independent experiments). GR activity is expressed as mean nmol/min/mg protein± s.e.m. *p < 0.05 (two-way ANOVA followed by Bonferroni’s multiple comparisons test). See , for numerical values and detailed statistical information. Figure 7—source data 1. Numerical values. Figure 7—source data 2. Detailed statistical information.

Article Snippet: Primary antibodies against the following proteins were obtained from the indicated suppliers: GR (1/2000, Abcam, cat# ab16801), GPx isozyme 1 (1/1000, Abcam, cat# ab22604), GSy (1/1000, Abcam, cat# ab133592), GCL catalytic subunit (1/1000, Novus Biologicals, cat# NBP2-45830), GCL regulatory subunit (1/1000, Abcam, cat# ab126704), NeuN (1/1000, Abcam, cat# ab104225), GFAP (1/2000, Abcam, cat# ab4674).

Techniques: Liquid Chromatography, Tandem Mass Spectroscopy, Western Blot, Control, Activity Assay, Transformation Assay, Comparison, Two Tailed Test, Fluorescence, FACS