Journal: Nature Communications

Article Title: Microglia-neuron interaction at nodes of Ranvier depends on neuronal activity through potassium release and contributes to remyelination

doi: 10.1038/s41467-021-25486-7

Figure Lengend Snippet: A Immunohistostainings of post-mortem human hemispheric white matter tissue (healthy donor) showing microglial cells (blue, resident microglia, TMEM119) contacting nodes of Ranvier (Na V , brown). B , C Immunofluorescent stainings of post-mortem human hemispheric white matter tissue (healthy donor) showing microglial cells (green; B , resident microglia, TMEM119, and C , homeostatic microglia, P2Y12R) contacting nodes of Ranvier (Na V , red). B ii, C ii 3D reconstructions correspond to the boxed area in B i and C i respectively. Scale bars: (2D) A , B , C 10 μm; (3D), B , C 2 μm. A : n = 6 samples, B , C : n = 5 samples.

Article Snippet: Primary antibodies: mouse IgG2a anti-AnkyrinG (clone N106/36; 1:100), mouse IgG2b anti-AnkyrinG (clone N106/65; 1:75), and mouse IgG1 anti-Caspr (1:100), all from Neuromab; mouse IgG1 anti-Pan Na v (clone K58/35; 1:150; Sigma); mouse anti-Calbindin (1:500; Sigma), rabbit anti-Calbindin (1:300; Swant), rabbit anti-Caspr (1:300; Abcam), rat anti-PLP (1:10; kindly provided by Dr. K. Ikenaka, Okasaki, Japan), mouse IgG2b anti-MBP (1:200; SMI99, Sigma), rabbit IgG anti-Iba1 (1:500; Wako), chicken anti-GFAP (1:500; Aves Labs), rat anti-PDGFrα (1:100; BD Biosciences), rabbit IgG anti-TMEM119 (1:100; Sigma), rabbit IgG anti-P2Y12r (1:300; Alomone, human tissue), rabbit anti-P2Y12R (1:300; Anaspec, mouse tissue), chicken anti-GFP (1:250; Millipore), mouse IgG2a anti-iNOS (1:100; BD Biosciences), goat anti-IGF1 (1:50; R&D System), and chicken anti-mCherry (1:1000; Abcam).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Hypothalamic Dysfunction of the Thrombospondin Receptor ?2?-1 Underlies the Overeating and Obesity Triggered by Brain-Derived Neurotrophic Factor Deficiency

doi: 10.1523/JNEUROSCI.1572-13.2014

Figure Lengend Snippet: Cell-surface expression of α2δ-1 is reduced in the VMH of BDNF2L/2LCk-cre mice. A, Representative in situ hybridization showing high α2δ-1 mRNA expression in wild-type VMH (arrows). B, Representative immunolabeling of α2δ-1 protein in wild-type hypothalamus showing high expression in the VMH and minimal expression in the DMH. C, Western blot analysis of total, cell-surface (Surf.), and cytosolic (Cyt.) α2δ-1 protein content in the VMH of BDNF2L/2LCk-cre CM mice compared with WT controls (n = 6). *p = 0.002; **p = 0.02. β-tub, β-Tubulin. D, Representative confocal images of WT VMH coimmunolabeled with anti-α2δ-1 (green) and anti-TrkB (red) showing a high level of colocalization of these two markers.

Article Snippet: The following primary antibodies were used: rabbit anti-α2δ-1 (1:1000; Alomone Labs) and mouse anti-β-tubulin (1:40,000; Sigma-Aldrich).

Techniques: Expressing, In Situ Hybridization, Immunolabeling, Western Blot

Journal: The Journal of Neuroscience

Article Title: Hypothalamic Dysfunction of the Thrombospondin Receptor ?2?-1 Underlies the Overeating and Obesity Triggered by Brain-Derived Neurotrophic Factor Deficiency

doi: 10.1523/JNEUROSCI.1572-13.2014

Figure Lengend Snippet: Selective inhibition of α2δ-1 in the VMH induces hyperphagia and excessive weight gain. Food intake (A) and relative body weight changes (B) in WT mice with chronic delivery of saline (Sal; n = 5) or GP (n = 6) to the VMH are shown. *p = 0.02; **p = 0.04.

Article Snippet: The following primary antibodies were used: rabbit anti-α2δ-1 (1:1000; Alomone Labs) and mouse anti-β-tubulin (1:40,000; Sigma-Aldrich).

Techniques: Inhibition

Journal: The Journal of Neuroscience

Article Title: Hypothalamic Dysfunction of the Thrombospondin Receptor ?2?-1 Underlies the Overeating and Obesity Triggered by Brain-Derived Neurotrophic Factor Deficiency

doi: 10.1523/JNEUROSCI.1572-13.2014

Figure Lengend Snippet: Viral-mediated delivery of α2δ-1 to the VMH of wild-type and BDNF mutant mice. A, Representative image of GFP+ cells in the hypothalamus of a mouse delivered AAV-GFP bilaterally showing accurate targeting of the VMH at a coronal level corresponding to bregma −1.70. B, C, Images of coronal levels from the same animal corresponding to bregma −1.06 mm (B) and −2.60 mm (C) and lacking GFP+ cell bodies indicate the restricted rostrocaudal span of the viral injections. D, Representative images of brain sections before and after being subjected to laser capture microdissection (LCM) to selectively dissect VMH cells. E, Quantitative RT-PCR post hoc analysis of α2δ-1 mRNA expression in cells laser captured from the VMH of WT and BDNF2L/2LCk-cre (CM) mice delivered AAV-GFP or AAV-α2δ-1 to the VMH via stereotaxic surgery. F, Measurements of cell-surface and cytosol content of α2δ-1 in BDNF2L/2LCk-cre mice treated with AAV-α2δ-1 (CM-α2δ-1) relative to control mutants that received AAV-GFP (CM-GFP). Minimal detection of β-tubulin (β-tub), a cytoskeletal protein, in the cell-surface fraction confirms the success of the biotinylation assay. Arc, Arcuate nucleus; 3V, third ventricle; PVN, paraventricular nucleus; AHP, anterior hypothalamic area; MT, mammillotegmental tract; PMN, premammillary nucleus; LH, lateral hypothalamic area. *p < 0.05.

Article Snippet: The following primary antibodies were used: rabbit anti-α2δ-1 (1:1000; Alomone Labs) and mouse anti-β-tubulin (1:40,000; Sigma-Aldrich).

Techniques: Mutagenesis, Laser Capture Microdissection, Quantitative RT-PCR, Expressing, Cell Surface Biotinylation Assay

Journal: The Journal of Neuroscience

Article Title: Hypothalamic Dysfunction of the Thrombospondin Receptor ?2?-1 Underlies the Overeating and Obesity Triggered by Brain-Derived Neurotrophic Factor Deficiency

doi: 10.1523/JNEUROSCI.1572-13.2014

Figure Lengend Snippet: The α2δ-1 rescue in the VMH of BDNF2L/2LCk-cre mutants mitigates their hyperphagia, excessive weight gain, and liver steatosis. A, Weekly food intake of BDNF2L/2LCk-cre conditional mutant mice with VMH delivery of AAV-α2δ-1 (CM-α2δ-1) or AAV-GFP (CM-GFP) and WT mice delivered AAV-α2δ-1 (WT-α2δ-1) or AAV-GFP (WT-GFP). There was a significant effect of time on food intake (F(17,25) = 3.175; p = 0.004, two-way ANOVA with repeated measures; n = 9–12). *p < 0.05, CM-α2δ-1 relative to CM-GFP. B, There was also a significant effect on relative body weight gain (F(18,24) = 53.15; p < 0.00001, two-way ANOVA with repeated measures; n = 9–12). *p < 0.05; #p < 0.1; CM-α2δ-1 relative to CM-GFP. C, Measurement of locomotor activity over 3 d expressed as total beam breaks. *p < 0.01. D, There was a significant effect of treatment on liver (F(3,21) = 70.4; p < 0.001, one-way ANOVA) and total fat (F(3,21) = 17.0; p < 0.001, one-way ANOVA) tissue weights. *p < 0.05. E, Representative liver tissue sections from WT-GFP, CM-GFP, and CM-α2δ-1 mice stained with hematoxylin and eosin (H&E) or Oil Red O, which stains lipids.

Article Snippet: The following primary antibodies were used: rabbit anti-α2δ-1 (1:1000; Alomone Labs) and mouse anti-β-tubulin (1:40,000; Sigma-Aldrich).

Techniques: Mutagenesis, Activity Assay, Staining

Journal: The Journal of Neuroscience

Article Title: Hypothalamic Dysfunction of the Thrombospondin Receptor ?2?-1 Underlies the Overeating and Obesity Triggered by Brain-Derived Neurotrophic Factor Deficiency

doi: 10.1523/JNEUROSCI.1572-13.2014

Figure Lengend Snippet: The α2δ-1 rescue in the VMH of BDNF2L/2LCk-cre mutants normalizes deficits in glucose homeostasis. A, There was a significant effect on fasted circulating levels of glucose (F(3,20) = 15.0; p < 0.001), insulin (F(2,14) = 25.3; p < 0.01), and leptin (F(3,29) = 20.6; p < 0.000001, one-way ANOVA) in WT-GFP, WT-α2δ-1, CM-GFP, and CM-α2δ-1 mice. *p < 0.04. B, Glucose tolerance test showing compromised responses in CM-GFP mice compared with WT-GFP mice (F(1,9) = 5.10; p = 0.05, two-way ANOVA with repeated measures) and improved glucose metabolism in CM-α2δ-1 mice compared with the CM-GFP group (F(1,9) = 5.73; p = 0.04, two-way ANOVA with repeated measures). *p < 0.04, CM-GFP mice compared with CM-α2δ-1 mice. C, There was a significant effect of treatment on area under the curve (AUC) for the glucose tolerance test (F(3,20) = 3.3; p = 0.04, one-way ANOVA). *p < 0.02.

Article Snippet: The following primary antibodies were used: rabbit anti-α2δ-1 (1:1000; Alomone Labs) and mouse anti-β-tubulin (1:40,000; Sigma-Aldrich).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Hypothalamic Dysfunction of the Thrombospondin Receptor ?2?-1 Underlies the Overeating and Obesity Triggered by Brain-Derived Neurotrophic Factor Deficiency

doi: 10.1523/JNEUROSCI.1572-13.2014

Figure Lengend Snippet: Pair-feeding improves but does not normalize glucose homeostasis in BDNF2L/2LCk-cre conditional mutants. A, There was a significant effect of pair-feeding on liver (F(2,23) = 8.2; p = 0.002) and total fat (F(2,23) = 17.4; p = 0.000) tissue weights in naive wild-type (WT Ad Lib) and BDNF2L/2LCk-cre conditional mutant (CM Ad Lib) mice with ad libitum access to food and in naive BDNF2L/2LCk-cre conditional mutants pair-fed to BDNF2L/2LCk-cre mutants treated with AAV α2δ-1 (CM Pair Fed). *p < 0.004. B, Fasted serum levels of glucose (F(2,31) = 10.2; p = 0.000), insulin (F(2,13) = 8.1; p = 0.005), and leptin (F(2,23) = 12.4; p = 0.000) in WT Ad Lib, CM Ad Lib, and CM Pair Fed mice. C, Glucose tolerance test in naive wild-type (WT Ad Lib) and BDNF2L/2LCk-cre conditional mutant (CM Ad Lib) mice with ad libitum access to food and in naive BDNF2L/2LCk-cre conditional mutants pair-fed to BDNF2L/2LCk-cre mutants treated with AAV-α2δ-1. Compromised responses to glucose were observed in CM Ad Lib mice compared with WT Ad Lib mice (F(1,13) = 97.0; p = 0.000, two-way ANOVA with repeated measures) and in CM Pair Fed mice compared with the WT Ad Lib group (F(1,13) = 17.9; p = 0.000, two-way ANOVA with repeated measures). *p < 0.02, WT Ad Lib mice compared with CM Pair Fed mice. D, There was a significant effect of treatment on area under the curve (AUC) for the glucose tolerance test (F(2,19) = 26.6; p = 0.000, one-way ANOVA). *p < 0.01; #p < 0.1.

Article Snippet: The following primary antibodies were used: rabbit anti-α2δ-1 (1:1000; Alomone Labs) and mouse anti-β-tubulin (1:40,000; Sigma-Aldrich).

Techniques: Mutagenesis