Extended Data Fig. 10: Tanycytes link the parabrachial nucleus to feeding.
From: A brainstem–hypothalamus neuronal circuit reduces feeding upon heat exposure
a. Left: Immunohistochemical detection of VEGFA (arrows) under control conditions and after Vegfa-specific RNAi infusion in the 3rd ventricle, with fluorescence intensity quantified in n = 4 mice/group (right). *p < 0.05 (Student’s t-test). Scale bars = 20 μm. b,c. Timeline analysis (facet-wrap) of food intake (b; n = 4/group) and body weight (c, n = 8/group) expressed in grams (g) over 5 days prior acute thermal manipulation of mice injected with control or Vegfa-RNAi. Repeated-measures ANOVA for food intake: interaction (RNAi vs. days): F = 1.457; p = 0.246; days: F = 57.150; p < 0.001; RNAi: F = 1.033; p = 0.349, and for body weight: interaction (RNAi vs. days): F = 3.010; p = 0.004; days: F = 1.427; p = 0.244; RNAi: F = 0.505; p = 0.489. d. Difference in body weight expressed in g after 24h at 25 °C and after being exposed to 40 °C in control vs. Vegfa-RNAi. Data in bar graphs were expressed as means ± s.e.m. with interconnected circles showing changes per animal (n = 8/group). Repeated-measures ANOVA: interaction (RNAi vs. temperature), F = 0.278; p = 0.614; temperature: F = 3.783, p = 0.093; RNAi: F = 0.095; p = 0.767. e. Diurnal activity expressed as the mean distance moved (cm) 20h after exposure to 25 °C or 40 °C. Time intervals were expressed as zeitgeber (ZT), with ZT12 and ZT0 coinciding with the onset of the dark and light phases, respectively (n = 8/group). Repeated-measures ANOVA: interaction (RNAi vs. temperature): F = 0.049; p = 0.8249; interaction (RNAi vs. time): F = 6.927; p < 0.001; interaction (time vs. temperature): F = 2.510; p < 0.001; time: F = 15.210; p < 0.0001; RNAi: F = 12.620; p = 0.001; temperature: F = 3.908; p = 0.058. f. Benchmarking the specificity of GFP (control) and TeLC-GFP expression in tanycytes after injection of AAV particles in the third ventricle (3V). The vimentin immunosignal was color-coded in light grey to enhance visual clarity, and to highlight somatic GFP fluorescence in only the outermost cell layer of the ventricular wall. Scale bars = 20 μm. g. Vimentin+ tanycytes (in magenta) expressed VAMP2 (in cyan). Open rectangle denotes the location of the orthogonal image stack shown in Fig. 4i. Arrows point to tanycyte (T) processes with VAMP2 signal. Scale bar = 2 µm. h. Histochemical controls of the successful transduction of PBN neurons with AAVs expressing either GFP only or a TeLC-GFP fusion construct. Abbreviation: scp, superior cerebellar peduncle. Scale bar = 70 µm. i. Individual bodyweights across the phases of behavioral testing (n = 4; F = 3.504; p = 0.148; repeated-measures one-way ANOVA). j. Facet-wrap timeline plot of cumulative drinking time (s) during 24-h of baseline, in mice exposed to CNO (3 mg kg−1; CNO 1), repeated baseline after TeLC delivery and recombination by 4-hydroxytamoxifen (baseline TeLC), and in mice with AAV2-FLEX-TeLC-GFP recombined in tanycytes and injected with CNO (3 mg/g; ‘CNO #2 TeLC’, in blue), n = 4; repeated-measures two-way ANOVA: interaction: F = 5.281; p < 0.0001; time (24 h): F = 58.510; p < 0.0001; treatment: F = 6.153; p = 0.009; subject: F = 4.936; p < 0.0001. k. Facet-wrap timeline plot of the cumulative distance moved (cm). Groups were identical to (i and j); n = 4; repeated-measures two-way ANOVA: interaction: F = 11.130; p < 0.0001; time (24 h), F = 48.930; p < 0.0001; treatment: F = 47.570; p < 0.0001; subject: F = 3.381; p = 0.0003. Data in facet-wrap plots were expressed as means ± 95% confidence interval of the s.d. throughout. Solid circles represent individual data points at time and treatment.
