Journal: bioRxiv

Article Title: Perinatal IL-1β-induced Inflammation Suppresses Tbr2+ Intermediate Progenitor Cell Proliferation in the Developing Hippocampus accompanied by Long-Term Behavioral Deficits

doi: 10.1101/2020.05.08.084129

Figure Lengend Snippet: Neonatal male Swiss Webster mice were administered PBS or IL-1β from P1 through P5 and euthanized at P10. The numbers of total cycling Tbr2 + progenitors in the SGZ were assessed using the optical fractionator method of stereology. (A) Representative of Tbr2/ Ki67 staining of 40 μm sections of fixed-frozen brains from P10 mice. White arrows indicate Tbr2 + /Ki67 + cells. (B) Quantification of the number of Tbr2 + /Ki67 + cells in the SGZ of P10 control versus IL-1β-treated mice. Results are expressed as mean ± SEM; *p<0.05 by unpaired t-test, n=4 mice per group. (C) Representative image of Tbr2/ Ki67 staining of 40 μm sections of fixed-frozen brains from P60 mice. White arrow indicates Tbr2 + /Ki67 + cell. (D) Quantification of the number of Tbr2 + and Ki67 + cells in the SGZ of P60 control versus IL-1β-treated mice. Results are expressed as mean ± SEM; n=3 mice per group. (E) Timeline of treatment and endpoints for in vivo immunofluorescence staining.

Article Snippet: Sections were encircled with a barrier pen, blocked in TDB superblock for 1 h followed by incubation in rabbit polyclonal anti-Tbr2 (Abcam, Cambridge, MA, catalog # ab23354, diluted at 1:500) or rabbit monoclonal anti-Tbr2 (Abcam, Cambridge, MA, catalog # ab183991, diluted at 1:1000), and rat anti-Ki67 (Vector Laboratories, Burlingame, CA, catalog # Z0311, diluted at 1:1000) at 4°C in a humidified chamber overnight.

Techniques: Staining, In Vivo, Immunofluorescence

Journal: bioRxiv

Article Title: Perinatal IL-1β-induced Inflammation Suppresses Tbr2+ Intermediate Progenitor Cell Proliferation in the Developing Hippocampus accompanied by Long-Term Behavioral Deficits

doi: 10.1101/2020.05.08.084129

Figure Lengend Snippet: Cells were stained for Tbr2 and DAPI and analyzed by flow cytometry. Representative histograms showing cell cycle analysis of (A) untreated NPs versus NPs treated with (B) 500 pg/mL IL-1α and (C) 500 pg/mL IL-1β at 48 hrs post-recovery/ treatment. (D) Percentages of cells in each cell cycle phase were averaged across 3 experiments and expressed as mean ± SEM; *p<0.05 by two-way ANOVA followed by Tukey’s multiple comparisons test. n=3 independent experiments. (E) Analyses of Cyclin D1 and Cyclin B/B1 from untreated NPs versus NPs treated with 500 pg/mL IL-1α at 3 hours post-recovery/ treatment. Data depicted in panel E are representative of 2 independent experiments.

Article Snippet: Sections were encircled with a barrier pen, blocked in TDB superblock for 1 h followed by incubation in rabbit polyclonal anti-Tbr2 (Abcam, Cambridge, MA, catalog # ab23354, diluted at 1:500) or rabbit monoclonal anti-Tbr2 (Abcam, Cambridge, MA, catalog # ab183991, diluted at 1:1000), and rat anti-Ki67 (Vector Laboratories, Burlingame, CA, catalog # Z0311, diluted at 1:1000) at 4°C in a humidified chamber overnight.

Techniques: Staining, Flow Cytometry, Cell Cycle Assay

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: DNA Methylation program in normal and alcohol-induced thinning cortex

doi: 10.1016/j.alcohol.2017.01.006

Figure Lengend Snippet: Comparative phenotypic and DNA methylation dynamics in the embryonic neocortex. (A–C) Representative cortical columns from the Chow E17 frontal neocortex immunostained with DMP markers (5mC, 5hmC, MeCP2) and (D–G) phenotypic neural markers (Ki67, Tbr2, P2Y1, and NeuN) are presented for comparison of the DMP dynamics along the radially progressing corticogenesis of the E17 brain. SVZ/VZ (Subventricular Zone/Ventricular Zone); IZ (Intermediate Zone); SP (Subplate); CP (Cortical plate).

Article Snippet: Stage differentiation markers used were: Ki67 (a marker for cell proliferation; 1:500, rabbit polyclonal, Millipore, Billerica, MA), NeuN (a marker for mature neuron; 1:500, mouse monoclonal, Cell Signaling, Danvers, MA), Tbr2 (T-box brain protein 2, a marker for intermediate neural progenitors, 1:500, rabbit polyclonal, Millipore, Billerica, MA), and P2Y1 (metabotropic G-protein P2 receptor, a marker for specialized neural cells capable of communication; 1:1000, rabbit polyclonal, Millipore, Billerica, MA).

Techniques: DNA Methylation Assay, Comparison

Journal: Alcohol (Fayetteville, N.Y.)

Article Title: DNA Methylation program in normal and alcohol-induced thinning cortex

doi: 10.1016/j.alcohol.2017.01.006

Figure Lengend Snippet: Tbr2-im at the E17 frontal cortex across experimental groups. Structural abnormalities were observed during immunophenotypic investigation. Notably, the thickness of cortical plate (J) as well as the entire thickness of frontal cortex (I) were markedly reduced in Alcohol group frontal cortex as compared to their Chow and PF control cortices (A–C). Fetal alcohol exposure also increased the proportion of SVZ + VZ/entire cortical thickness (K) as compared to controls. Lateral Ventricle (LV) expansion was also observed in E17 alcohol cortices (A–C). (G) Finally, Tbr2-im (a marker for neural progenitor migration) was normally observed as a radially extending fiber ascending from the base of the lateral ventricle up to the pial surface. Alcohol noticeably reduced the Tbr2 immunoreactivity in the CP layer. E16 Chow brains were used as developmental stage controls and more closely resembled the E17 alcohol developmental state than E17 Chow (C–D, G–H). Quantitative measurements among the three groups were analyzed by one-way ANOVA, and the difference between paired groups were compared by Student t-test *p < 0.05, **p < 0.005. N (structural analysis) = Chow (5), PF (5), Alc (5). N (Tbr2-im analysis) = Chow (3), PF (3), Alc (3), E16 Chow (3). SVZ/VZ (Subventricular Zone/Ventricular Zone); IZ (Intermediate Zone); SP (Subplate); CP (Cortical plate).

Article Snippet: Stage differentiation markers used were: Ki67 (a marker for cell proliferation; 1:500, rabbit polyclonal, Millipore, Billerica, MA), NeuN (a marker for mature neuron; 1:500, mouse monoclonal, Cell Signaling, Danvers, MA), Tbr2 (T-box brain protein 2, a marker for intermediate neural progenitors, 1:500, rabbit polyclonal, Millipore, Billerica, MA), and P2Y1 (metabotropic G-protein P2 receptor, a marker for specialized neural cells capable of communication; 1:1000, rabbit polyclonal, Millipore, Billerica, MA).

Techniques: Control, Marker, Migration

Journal: Frontiers in Cellular Neuroscience

Article Title: Prenatal Activation of Toll-Like Receptor-4 Dampens Adult Hippocampal Neurogenesis in An IL-6 Dependent Manner

doi: 10.3389/fncel.2016.00173

Figure Lengend Snippet: Prenatal LPS reduces the number of intermediate neuronal progenitors in the dentate gyrus of the hippocampus. Pregnant dams were injected with either LPS (100 μg/kg, i.p.) or pyrogen-free saline (equi-volume) in the absence or the presence of IL-6Ab (10 μg/kg, i.p.) on gestation day 15. Intermediate neuronal progenitor cells expressing T-box brain protein 2 (Tbr2) were counted in the dentate gyrus of the hippocampus. Micrographs in (A) show immunofluorescent staining of Tbr2 in male adult offspring born to dams given either saline (left panel) or LPS (right panel). The graph bars in (B) show the number of Tbr2 containing cells in the dentate gyrus of adult male offspring. In the absence of the IL-6Ab (-IL-6Ab), LPS injection to pregnant dams (solid bars) resulted in a significant decrease in the number of Tbr2 containing cells in the dentate gyrus of adult male offspring when compared to those born to dams given saline only (open bars). Such long lasting impact of prenatal LPS was absent when IL-6Ab was co-administered with LPS injection during gestation period (+IL-6Ab) (Saline–Saline: n = 5, IL-6Ab–Saline: n = 7, Saline–LPS: n = 5, IL-6Ab–LPS: n = 5). DG = Dentate gyrus. ∗ p < 0.05, ∗∗ p < 0.01. Scale bar = 50 μm.

Article Snippet: Brain sections were incubated with either doublecortin (DCX) antibody made in goat (1:1000, Santa Cruz biotechnology,Paso Robles, CA, USA) or T-box brain protein 2 (Tbr2) antibody made in chicken (Tbr2) (1:1000, EMD Millipore, MA, USA) followed by Alexa Fluor 488 bound secondary antibodies (donkey anti-goat IgG for DCX, or donkey anti-chicken for Tbr2; 1:1000; Life Technologies, La Jolla, CA, USA).

Techniques: Injection, Saline, Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Prenatal Activation of Toll-Like Receptor-4 Dampens Adult Hippocampal Neurogenesis in An IL-6 Dependent Manner

doi: 10.3389/fncel.2016.00173

Figure Lengend Snippet: Impact of glucocorticoid antagonist on the long lasting impact of prenatal LPS on newly born neurons in the dentate gyrus of immune challenged adult offspring. Pregnant dams were injected with either LPS (100 μg/kg, i.p.) or pyrogen-free saline (equi-volume) on gestation day 15. Adult male offspring were subsequently injected with LPS (1 mg/kg, i.p.) in the absence [dimethyl sulfoxide (DMSO)] or the presence of the glucocorticoid receptor antagonist RU486 (dissolved in DMSO). Cell count of doublecortin (A) and T-box brain protein 2 (Tbr2) containing cells (B) was monitored in the dentate gyrus of LPS-injected adult offspring born to dams given either saline (open bars) or LPS (solid bars) on gestation day 15. In vehicle (DMSO)-treated adult offspring, rats born to dams given LPS during pregnancy and given LPS during adulthood showed a significantly decreased number of both doublecortin (DMSO: panel A) and Tbr2 (DMSO: panel B) containing cells in their dentate gyrus when compared to those born to saline-injected dams and given LPS during adulthood. The long lasting impact of prenatal LPS on newly born neurons were absent when RU486 was co-injected with LPS during adulthood (Saline–DMSO: n = 4, Saline–RU486: n = 4, LPS–DMSO: n = 4, LPS–RU486: n = 3). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Brain sections were incubated with either doublecortin (DCX) antibody made in goat (1:1000, Santa Cruz biotechnology,Paso Robles, CA, USA) or T-box brain protein 2 (Tbr2) antibody made in chicken (Tbr2) (1:1000, EMD Millipore, MA, USA) followed by Alexa Fluor 488 bound secondary antibodies (donkey anti-goat IgG for DCX, or donkey anti-chicken for Tbr2; 1:1000; Life Technologies, La Jolla, CA, USA).

Techniques: Injection, Saline, Cell Counting