Journal: Cells

Article Title: Regional Variation of Gap Junctional Connections in the Mammalian Inner Retina

doi: 10.3390/cells10092396

Figure Lengend Snippet: List of key resources.

Article Snippet: mouse monoclonal anti-calbindin , Synaptic Systems, Göttingen, German , 214011.

Techniques: Plasmid Preparation, Laser-Scanning Microscopy, Software

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Figure Lengend Snippet: Schematic illustration of rodent postnatal granular cell neurogenesis. (a) The major cell type in the cerebellar cortex is the granule cell (GC). GC precursors (GCPs), originating from the rhombic lip, migrate to the mitotically active external granular layer (EGL). GCPs within the EGL are mitotically active from E15 until the third postnatal week. At the time of birth (P0), GCPs begin to differentiate into mature GCs (around P5) and reach their destination (at P20) by radial migration along the Bergmann glia of the GC into the internal granular layer (IGL) over the molecular layer (ML). In rats, the proliferation of cerebellar interneurons and granule cells that arise from the EGL lasts for over 3 weeks after birth. Proliferative activity of GCPs is regulated by extrinsic Purkinje cell- (PC-) derived factor sonic hedgehog (Shh). Brain-derived neurotrophic factor (BDNF), expressed in GCs, regulates survival and migration of GCPs and stimulates own migration of granule cells from EGL to IGL. (b) Cerebellar neurogenesis of granular cells is characterized by the expression of neuronal and proliferative markers and is orchestrated by neuronal transcription factors: brain-derived neurotrophic factor (BDNF), calbindin 1 (Calb1), chromodomain helicase DNA-binding protein 7 (Chd7), cyclin dependent kinase 2 (CycD2), fibroblast growth factor 8 (FGF8), hypoxanthine-guanine phosphoribosyl-transferase (HPRT), LIM homeobox transcription factor 1 alpha (Lmx1 α ), neuronal differentiation 1/2 (NeuroD 1/2), neuronal nuclei (NeuN), paired box 2/6 (Pax2/Pax6), proliferating cell nuclear antigen (PCNA), prospero homeobox 1 (Prox1), semaphoring 6a (Sema6a), sonic hedgehog signaling molecule (Shh), sex-determining region Y-box 2 (Sox2), and synaptophysin (Syp). (c) Cellular players in cerebellar granular cell neurogenesis.

Article Snippet: Primary antibodies were applied after washing once with PBS; slides were incubated overnight at 4°C with either monoclonal mouse anti-calbindin (1 : 500, Abcam, ab75524) or polyclonal rabbit anti-Pax6 (1 : 200, LifeSpan Bioscience, LS-C179903) with monoclonal mouse anti-PCNA (1 : 500, Abcam, ab29) diluted in antibody diluent (Zymed Laboratories, San Francisco, CA).

Techniques: Migration, Activity Assay, Derivative Assay, Expressing, Binding Assay

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Figure Lengend Snippet: Representative cerebellar paraffin sections colabeled with Pax6, PCNA, and DAPI of rat pups exposed to normoxia (NO) or hyperoxia (HY) compared to rat pups treated with caffeine (NOC, HYC). Examinations were performed at postnatal day 3 (P3 (a)) and P5 (c) or after recovery after 3-day exposure at P15 (b) or after 5-day exposure at P15 (d). Immunofluorescent images indicated Pax6 (red), PCNA (green), and nuclei (blue, DAPI). Three -(P3) and five-day (P5) lasting hyperoxia affected the density of granule cells (Pax6), proliferation capacity (PCNA), and thickness of EGL in the newborn rat cerebellum after acute hyperoxia exposure (P3, P5). Caffeine at P3 was partly effective to inhibit the effects of hyperoxia. EGL: external granular layer; ML: molecular layer; IGL: internal granular layer. Scale bar P3/P5 25 μ m and P3_P15/P5_P15 50 μ m.

Article Snippet: Primary antibodies were applied after washing once with PBS; slides were incubated overnight at 4°C with either monoclonal mouse anti-calbindin (1 : 500, Abcam, ab75524) or polyclonal rabbit anti-Pax6 (1 : 200, LifeSpan Bioscience, LS-C179903) with monoclonal mouse anti-PCNA (1 : 500, Abcam, ab29) diluted in antibody diluent (Zymed Laboratories, San Francisco, CA).

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Figure Lengend Snippet: Quantification of (a) Pax6 and (b) PCNA counts of colabelled positive cells of the cerebellar molecular layer was performed for 3 days' postnatal oxygen exposure (P3) and recovery (P3_P15) and 5 days' postnatal oxygen exposure (P5) and recovery (P5_P15), respectively. Acute hyperoxia exposure (P3 and P5; deep dark gray bars) decreased the number of granular cells (Pax6) and proliferating cells (PCNA). Caffeine (gray bars) was protective for Pax6-positive cells at P3 and P5 and for proliferating capacity at P3 with recovery until P15 (P3_P15). Caffeine with normoxia (light gray bars) did not influence density of granular cells and proliferation. Data are normalized to the level of rat pups exposed to normoxia at each time point (control 100%, white bars), and the 100% values are 38 (P3), 54.4 (P3_P15), 46.6 (P5), and 41.3 (P5_P15) Pax6+ cells per regions of lobules or 33 (P3), 37.1 (P3_P15), 65.7 (P5), and 22.3 (P5_P15) PCNA+ cells per region of lobules, respectively. n = 6-8/group. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 vs. control; # p < 0.05 and ## p < 0.01 vs. hyperoxia (ANOVA, Bonferroni's post hoc test; Kruskal-Wallis, Dunn's post hoc test; Brown-Forsythe, Dunnett's post hoc test).

Article Snippet: Primary antibodies were applied after washing once with PBS; slides were incubated overnight at 4°C with either monoclonal mouse anti-calbindin (1 : 500, Abcam, ab75524) or polyclonal rabbit anti-Pax6 (1 : 200, LifeSpan Bioscience, LS-C179903) with monoclonal mouse anti-PCNA (1 : 500, Abcam, ab29) diluted in antibody diluent (Zymed Laboratories, San Francisco, CA).

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