Journal: Nature Communications

Article Title: Deconstruction of a hypothalamic astrocyte-white adipocyte sympathetic axis that regulates lipolysis in mice

doi: 10.1038/s41467-022-35258-6

Figure Lengend Snippet: a A representative confocal image of viral transductions of GfaABC 1 D-dependent hM3Dq-mCherry in the ARH. Almost all the mCherry-transduced cells (87 ± 16.6/five sections under ×40 objective, n = 6) are GFAP-positive. b , c Representative images showing colocalizations of mCherry-transduced cells (red) and Alexa fluor 488 anti-GFAP antibody-labeled cells (green). The dotted line in b indicates the region shown in ( c ). Scale bars for a 1 mm, b 200 μm, c 25 μm. ARH, arcuate nucleus of the hypothalamus; ME median eminence, VMH ventromedial hypothalamus, 3rd V third ventricle.

Article Snippet: Vectors used in this study included: AAV vectors for astrocyte expressions of hM3Dq (AAV 5 -GfaABC 1 D-hM3Dq-mCherry, Addgene#92284, titer at 4.1 × 10 13 vg/ml), GCaMP 6f (AAV 5 -GfaABC 1 D-cyto-GCaMP 6f , Addgene#52925, titer at 7.0 × 10 12 vg/ml), and ChR2 (AAV 5 -GFAP104-ChR2-mCherry, Addgene#58892, titter at 3.4 × 10 13 vg/ml).

Techniques: Labeling

Journal: Nature Communications

Article Title: Deconstruction of a hypothalamic astrocyte-white adipocyte sympathetic axis that regulates lipolysis in mice

doi: 10.1038/s41467-022-35258-6

Figure Lengend Snippet: a Schematic illustration of targeting a vector carrying GfaABC 1 D-dependent hM3Dq to the ARH and a second retrograde vector carrying hSyn-dependent GRAB_NE to the iWAT, and of implanting a photometry cannula in the injected iWAT. b Representative traces of real-time wireless photometry monitoring of iWAT NE contents with vehicle (blue) or αMPT (red) treatment. c , d Astrocyte stimulation with J60 via i.p. injection increased the intensity of iWAT GRAB_NE signals in the astrocyte c hM3Dq- but not in d tdTomato-transduced mice. e Representative confocal images of virally transduced iWAT GRAB_NE (green) and Alexa Fluo 647-conjugated anti-TH antibody labeled nerves (red). f , g Astrocyte stimulation increases the firing rates of local sympathetic inputs to iWAT: f Representative traces of nerve spikes at baseline and 30 min post J60 treatment via i.p in astrocyte hM3Dq-transduced mice; d Group data of iWAT nerve firing rates recorded in astrocyte hM3Dq or tdTomato-transduced mice ( n = 6 per group). TH tyrosine hydroxylase. Two-way ANOVA with Sidak post hoc tests; data represent mean ± s.e.m.; * p = 0.0187 < 0.05; ** p = 0.0015 < 0.01; **** p < 0.0001. Scale bars for e 100 μm, and f 10 s and 30 μV. Arrows in c , d , g indicate the J60 i.p. injection time point. ARH arcuate nucleus of the hypothalamus, ME median eminence, iWAT inguinal white adipose tissue.

Article Snippet: Vectors used in this study included: AAV vectors for astrocyte expressions of hM3Dq (AAV 5 -GfaABC 1 D-hM3Dq-mCherry, Addgene#92284, titer at 4.1 × 10 13 vg/ml), GCaMP 6f (AAV 5 -GfaABC 1 D-cyto-GCaMP 6f , Addgene#52925, titer at 7.0 × 10 12 vg/ml), and ChR2 (AAV 5 -GFAP104-ChR2-mCherry, Addgene#58892, titter at 3.4 × 10 13 vg/ml).

Techniques: Plasmid Preparation, Injection, Labeling

Journal: Nature Communications

Article Title: Deconstruction of a hypothalamic astrocyte-white adipocyte sympathetic axis that regulates lipolysis in mice

doi: 10.1038/s41467-022-35258-6

Figure Lengend Snippet: a Schematic illustration of targeting a retrograde vector carrying Cre-dependent hM3Dq-mCherry to inguinal iWAT in dual TH-Cre GCaMP 6f mice. b Sample images showing colocalizations of retrograde traced and hM3Dq-mCherry (red)-transduced TH neurons stained with Alex Fluor 350-conjugated anti-TH antibodies (blue) which also expressed GCaMP 6f (green) in T 13 PG. c , d Ex vivo photometry monitoring was performed at acute isolated T 13 PG from the retrograde traced and hM3Dq-transduced TH-Cre GCaMP 6f mice, and chemogenetic stimulation of the hM3Dq-transduced TH neurons with J60 addition to the circulating aCSF increased the intensity of GCaMP 6f signals in the T 13 PG: c A representative trace of real-time monitoring of T 13 PG GCaMP 6f signals with J60 addition as pointed; and d Group data of T 13 PG GCaMP 6f signals in iWAT injected with hM3Dq- or mCherry ( n = 4 per group). e – i PS of ChR2-transduced T 13 PG excited the sympathetic neural inputs in ex vivo inguinal iWAT: e Illustration of targeting a vector carrying Cre-dependent ChR2 to T 13 PG in dual TH-Cre GCaMP 6f mice; f Ex vivo intact spinal cord-iWAT tissues with a PS fiber placed on exposed T 13 ganglion and a photometry fiber placed on the ACFN entry point in inguinal iWAT in a recording chamber; g – i Representative photometry monitoring of GCaMP 6f signals with PS of T 13 PG g ChR2- and h mCherry-transduced mice, and i Group data of average GCaMP 6f signals from g and h (mCherry, n = 4; ChR2, n = 5). Two-way ANOVA with Sidak post hoc tests; data represent mean ± s.e.m; p = 0.67 ( d , mCherry); * p = 0.04 < 0.05 ( d , rghM3Dq); p = 0.91 ( i , mCherry); **** p < 0.0001 ( i , ChR2); n.s. (not significant). Scale bars for b 50 μm. PG, paravertebral ganglia. Arrow in c indicated the addition of J60 in the circulating aCSFs. Blue dotted lines in g and h indicated the PS duration (20 Hz for 1 s; repeated every 4 s for 30 s).

Article Snippet: Vectors used in this study included: AAV vectors for astrocyte expressions of hM3Dq (AAV 5 -GfaABC 1 D-hM3Dq-mCherry, Addgene#92284, titer at 4.1 × 10 13 vg/ml), GCaMP 6f (AAV 5 -GfaABC 1 D-cyto-GCaMP 6f , Addgene#52925, titer at 7.0 × 10 12 vg/ml), and ChR2 (AAV 5 -GFAP104-ChR2-mCherry, Addgene#58892, titter at 3.4 × 10 13 vg/ml).

Techniques: Plasmid Preparation, Staining, Ex Vivo, Isolation, Injection

Journal: Nature Communications

Article Title: Deconstruction of a hypothalamic astrocyte-white adipocyte sympathetic axis that regulates lipolysis in mice

doi: 10.1038/s41467-022-35258-6

Figure Lengend Snippet: a – c Chemogenetic stimulation of astrocytes in the ARH with J60 via i.p. induced iWAT lipolysis in the astrocyte hM3Dq-transduced mice compared to tdTomato-transduced controls, as evidenced by increased levels of p-HSL and glycerol and plasma NEFA: a Representative western blots of p-HSL and HSL and actin in inguinal iWAT extracted 2 h post a single i.p. injection of J60 in astrocyte tdTomato or hM3Dq-transduced mice; b , c Group data of b iWAT glycerol level and c plasma NEFA ( n = 6 per group). d–f LCFN and ACFN physical denervation blunted the astrocyte-stimulation-induced HSL phosphorylation: d , e Representative blots of p-HSL and HSL and actin in inguinal iWAT extracted 2 h post a single i.p. injection of J60, from astrocyte d hM3Dq- or e tdTomato-transduced mice which received a control Sham or crushing injury to iWAT-supporting LCFN and ACFN; and f Group data of p-HSL over HSL from astrocyte tdTomato or hM3Dq-transduced mice with sham or crushing surgeries respectively (Tdtomato Sham , n = 4; Tdtomato Den , n = 5; hM3Dq Sham , n = 5; hM3Dq Den , n = 5). Data represent mean ± s.e.m; two-tailed Student’s t -tests for b ( p = 0.04) and c ( p = 0.01); two-way ANOVA with Sidak post hoc tests for f (tdTomato Sham to Den , p = 0.48; Sham tdTomato to hM3Dq , p = 0.02; tdTomato Sham to hM3Dq Den , p = 0.99; tdTomato Den to hM3Dq Sham , p = 0.0004; tdTomato Sham to hM3Dq Den , p = 0.99; tdTomato Den to hM3Dq Den , p = 0.65; hM3Dq Sham to Den , p = 0.008; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Vectors used in this study included: AAV vectors for astrocyte expressions of hM3Dq (AAV 5 -GfaABC 1 D-hM3Dq-mCherry, Addgene#92284, titer at 4.1 × 10 13 vg/ml), GCaMP 6f (AAV 5 -GfaABC 1 D-cyto-GCaMP 6f , Addgene#52925, titer at 7.0 × 10 12 vg/ml), and ChR2 (AAV 5 -GFAP104-ChR2-mCherry, Addgene#58892, titter at 3.4 × 10 13 vg/ml).

Techniques: Clinical Proteomics, Western Blot, Injection, Phospho-proteomics, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Deconstruction of a hypothalamic astrocyte-white adipocyte sympathetic axis that regulates lipolysis in mice

doi: 10.1038/s41467-022-35258-6

Figure Lengend Snippet: a Schematic illustration of targeting a vector carrying GfaABC 1 D-hM3Dq to ARH and implanting a photometry fiber over ARH in dual POMC-Cre GCaMP 6f mice. b , c Individual photometry traces showing that astrocyte stimulation with J60 via i.p. increased the intensity of POMC neuron GCaMP 6f signals in the astrocyte b hM3D-transduced mice ( n = 8) compared to c tdTomato-transduced mice ( n = 6). The blue line represents the average of the individual lines. d ADO probe calibration curve using increased volumes of ADO (1 mM) following the detailed instructions for the probes. e Representative traces of real-time recording of the signals of ADO in astrocyte hM3Dq or tdTomato-transduced mice. Mice were treated with J60 via i.p. after a stable baseline. f Averaged traces from e (tdTomato, n = 4; hM3Dq, n = 5). g Astrocyte stimulation with J60 addition in circulating aCSF increased POMC neuron GCaMP 6f signals ( n = 4). h CPT blockade of ADORA 1 diminished the effect of astrocyte stimulation with J60 ( n = 4). i Pre-treatment of brain slices with IP 3 R blockers diminished the effect of astrocyte stimulation ( n = 3). Two-way ANOVA with Tukey post hoc tests; data represent mean ± s.e.m.; **** p < 0.0001. Black arrows point to i.p. injections of J60 in b , c , e , f , and J60 addition in circulating aCSF in ( g )–( i) .

Article Snippet: Vectors used in this study included: AAV vectors for astrocyte expressions of hM3Dq (AAV 5 -GfaABC 1 D-hM3Dq-mCherry, Addgene#92284, titer at 4.1 × 10 13 vg/ml), GCaMP 6f (AAV 5 -GfaABC 1 D-cyto-GCaMP 6f , Addgene#52925, titer at 7.0 × 10 12 vg/ml), and ChR2 (AAV 5 -GFAP104-ChR2-mCherry, Addgene#58892, titter at 3.4 × 10 13 vg/ml).

Techniques: Plasmid Preparation

Journal: Nature Communications

Article Title: Deconstruction of a hypothalamic astrocyte-white adipocyte sympathetic axis that regulates lipolysis in mice

doi: 10.1038/s41467-022-35258-6

Figure Lengend Snippet: a Schematic illustration of targeting a vector carrying GfaABC 1 D-hM3Dq and a second vector carrying Cre-dependent hM4Di to the ARH and of implanting an FSCV carbon fiber electrode in iWAT in POMC-Cre mice. b NE probe calibration curve using increased volumes of NE (1 mM) following the detailed instructions for the probes. c Representative traces of real-time recording of the FSCV signals of iWAT in astrocyte hM3Dq POMC neuron mCherry ( n = 5), astrocyte tdTomato POMC neuron hM4Di ( n = 5), or astrocyte hM3Dq POMC neuron hM4Di ( n = 6)-transduced mice, respectively. Mice were treated with J60 via i.p. after a stable baseline. d Averaged traces from c (Astrocyte hM3Dq POMC hM4Di , n = 6; Astrocyte hM3Dq POMC mCherry , n = 5; Astrocyte tdTomato POMC hM4Di , n = 5). Two-way ANOVA with Sidak post hoc tests; data represent mean ± s.e.m.; ** p = 0.006 < 0.01; *** p = 0.0002 < 0.001; **** p < 0.0001. Red arrows in b indicate the time points of adding NE in different volumes. Black arrows in d point to i.p. injections of J60.

Article Snippet: Vectors used in this study included: AAV vectors for astrocyte expressions of hM3Dq (AAV 5 -GfaABC 1 D-hM3Dq-mCherry, Addgene#92284, titer at 4.1 × 10 13 vg/ml), GCaMP 6f (AAV 5 -GfaABC 1 D-cyto-GCaMP 6f , Addgene#52925, titer at 7.0 × 10 12 vg/ml), and ChR2 (AAV 5 -GFAP104-ChR2-mCherry, Addgene#58892, titter at 3.4 × 10 13 vg/ml).

Techniques: Plasmid Preparation