Journal: PLoS Pathogens

Article Title: SV2 Mediates Entry of Tetanus Neurotoxin into Central Neurons

doi: 10.1371/journal.ppat.1001207

Figure Lengend Snippet: (A) Cultured cortical neurons were exposed to the indicated concentrations of TeNT for 3 minutes in high K + buffer then returned to the incubator for 2 days. mEPSCs and mIPSCs were analyzed and the frequency of mEPSCs was reduced to a greater degree than mIPSCs. mEPSC/mIPSC at 0 pM TeNT, n = 10; mEPSC at 0.5 pM TeNT, n = 10; mIPSC at 0.5 pM TeNT, n = 9; mEPSC/mIPSC at 5 pM TeNT, n = 8; error bars represent SEM. (B) Sample traces of dissociated spinal cord neurons exposed to TeNT (50 pM) in high K + buffer, washed than analyzed after 3 hours. Cultures treated with TeNT exhibited pronounced hyper excitability, as evidenced by high-frequency bursting activity, as compared to control neurons. (C) Representative images of SV2A/B staining with markers for excitatory (vGLUT1/2) or inhibitory (vGAT) boutons in cortical neurons and spinal cord slices. Red boxes indicate the region that was subjected to analysis by immunocytochemistry. (D) Pearson's coefficient for SV2A/B localization with excitatory/inhibitory terminals. In cortical neurons, SV2A localizes to inhibitory terminals while SV2B localizes to excitatory terminals. In contrast, in spinal cord slices, SV2A showed a greater degree of colocalization with markers for inhibitory terminals than excitatory terminals, while SV2B largely localized to excitatory terminals. Error bars represent SEM, SV2A cortical neurons n = 4, SV2B cortical neurons n = 6, spinal cord slice n = 13, *p<0.05, ***p<0.001.

Article Snippet: To isolate GABA A receptor-mediated mIPSCs, bicuculline was replaced with 20 μM CNQX (AMPA receptor antagonist, Tocris).

Techniques: Cell Culture, Activity Assay, Control, Staining, Immunocytochemistry

Journal: Molecular Brain

Article Title: Maternal epileptic seizure induced by Pentylenetetrazol: Apoptotic neurodegeneration and decreased GABA B1 receptor expression in prenatal rat brain

doi: 10.1186/1756-6606-2-20

Figure Lengend Snippet: PTZ induced seizure increased the expression of cleaved caspases-3 in prenatal rat hippocampal neurons . Western blot analyses of the caspases-3 in the primary cultured hippocampal neuronal cells at GD 17.5 from PTZ-induced seizure model during pregnancy. Cells are exposed to different drugs for 20 min as previously explained. Immunoblotting with polyclonal antibody showed processing of caspases-3 with the appearance of active fragment (17 kDa cleaved caspase-3). Detail procedures are mentioned in material and method section. β-actin is taken as loading control in each case. A : Immunoblots of caspases-3 of hippocampal neuronal cells under different treatment conditions. The immunoblots were labeled with an anti caspases-3 antibody B : Density values were expressed as mean ± SEM ( n = 4, mean four rat per group) of the corresponding protein of caspases-3 are presented. The density values on (Y-axis) are expressed as arbitrary units (AU). * P < 0.05 and ** P < 0.01 versus control group.

Article Snippet: Immunoreactions were carried out using a rabbit polyclonal IgG GABA B1 R (Santa Cruz),) or rabbit-derived anti-rat GABA B1 R antibody (Abcam Limited, UK), PKA-α (Santa Cruz), cytochrome C a rabbit polyclonal (Santa Cruz) and cleaved caspase-3 a rabbit polyclonal antibody recognizing 17-kDa active subunit of caspase-3 (Cell signaling) antibodies (1:1000, 24 h, 4°C).

Techniques: Expressing, Western Blot, Cell Culture, Control, Labeling

Journal: Molecular Brain

Article Title: Maternal epileptic seizure induced by Pentylenetetrazol: Apoptotic neurodegeneration and decreased GABA B1 receptor expression in prenatal rat brain

doi: 10.1186/1756-6606-2-20

Figure Lengend Snippet: (a, b) PTZ-induced seizure during pregnancy decreases the mRNA and protein level of GABA B1 R in prenatal rat hippocampal neurons . A : RT-PCR analyses change in mRNA ( 5a ) and protein level ( 5b ) of the GABA B1 R in the primary cultured hippocampal neuronal cells at GD 17.5 from PTZ-induced seizure model during pregnancy. Cells are exposed to different drugs for 20 min as previously explained. Detail procedures are mentioned in material and method section. GAPDH and β-actin is taken as control. In case of Western blot analysis immunoblots were labeled with an anti GABA B1 R antibody. B : Density values were expressed as mean ± SEM ( n = 4, mean four rat per group) of the corresponding mRNA of GABA B1 R are presented. The density values on (Y-axis) are expressed as arbitrary units (AU). * P < 0.05 and ** P < 0.01 versus control group.

Article Snippet: Immunoreactions were carried out using a rabbit polyclonal IgG GABA B1 R (Santa Cruz),) or rabbit-derived anti-rat GABA B1 R antibody (Abcam Limited, UK), PKA-α (Santa Cruz), cytochrome C a rabbit polyclonal (Santa Cruz) and cleaved caspase-3 a rabbit polyclonal antibody recognizing 17-kDa active subunit of caspase-3 (Cell signaling) antibodies (1:1000, 24 h, 4°C).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Control, Western Blot, Labeling

Journal: Molecular Brain

Article Title: Maternal epileptic seizure induced by Pentylenetetrazol: Apoptotic neurodegeneration and decreased GABA B1 receptor expression in prenatal rat brain

doi: 10.1186/1756-6606-2-20

Figure Lengend Snippet: (a, b) PTZ-induced seizure during pregnancy decreases the mRNA level of PKA-α in prenatal rat hippocampal neurons . A : RT-PCR analyses change in mRNA ( 6a ) and protein level ( 6b ) of the PKA-α in the primary cultured hippocampal neuronal cells at GD 17.5 from PTZ-induced seizure model during pregnancy. Cells are exposed to different drugs for 20 min as previously explained. Detail procedures are mentioned in material and method section. GAPDH and β-actin is taken as control. In case of Western blot analysis immunoblots were labeled with an anti PKA antibody. B : Density values were expressed as mean ± SEM ( n = 4, mean four rat per group) of the corresponding mRNA of GABA B1 R and PKA are presented. The density values on (Y-axis) are expressed as arbitrary units (AU). * P < 0.05 and ** P < 0.01 versus control group.

Article Snippet: Immunoreactions were carried out using a rabbit polyclonal IgG GABA B1 R (Santa Cruz),) or rabbit-derived anti-rat GABA B1 R antibody (Abcam Limited, UK), PKA-α (Santa Cruz), cytochrome C a rabbit polyclonal (Santa Cruz) and cleaved caspase-3 a rabbit polyclonal antibody recognizing 17-kDa active subunit of caspase-3 (Cell signaling) antibodies (1:1000, 24 h, 4°C).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Control, Western Blot, Labeling

Journal: iMeta

Article Title: Soyasaponin and vertical microbial transmission: Maternal effect on the intestinal development and health of early chicks

doi: 10.1002/imt2.70044

Figure Lengend Snippet: Dynamic changes in the content of SS in the yolk‐meconium axis and identification of characteristic metabolites in the meconium. (A) Flowchart of the identification process for the dynamic changes of soy saponins. (B) The concentration of SS deposited in the egg yolk (before incubation) following SS supplementation was determined using liquid chromatography‐mass spectrometry (LC‐MS). (C) Nontargeted LC‐MS detection of various SS configurations and their metabolite abundance in meconium (just after hatching). (D) Results of the KEGG topology analysis on differential metabolites in meconium. The size of the dots indicates the number of differential metabolites. (E) Screening of differential metabolites based on KEGG enrichment results combined with variable importance in projection (VIP) determination. (F) Relative mRNA expression of Gamma‐aminobutyric acid (GABA) receptors in the ileum at Day 1 ( n = 8). Data are shown as mean ± SEMs. (G) Detection of GABA content in the serum of breeder chickens and the contents of the yolk sac at embryonic Day 19 (E19) ( n = 8). Data are shown as mean ± SEMs. (H) Flowchart of the chicken embryo injection experiment. (I) Relative embryo weight at E13 and E19 ( n = 8). Data are shown as mean ± SEMs. (J) Relative mRNA expression of genes related to GABA receptors in the D1 intestine ( n = 8). Data are shown as mean ± SEMs. (K) Morphological images of H&E‐stained ileum, bar = 200 μm. H&E, hematoxylin and eosin.

Article Snippet: Following centrifugation at 2000 rpm for 8 min, the resultant supernatant was utilized for GABA content analysis using the γ‐Aminobutyric Acid (GABA) Colorimetric Assay Kit E‐BC‐K852‐M (Elabscience Biotechnology Co., Ltd.), and GAD (EC 4.1.1.15) assay kit (JL‐T0864; Jonlnbio).

Techniques: Concentration Assay, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, Injection, Staining