Extended Data Fig. 6: Synaptic activation of tanycytes.

a. Image survey of the third ventricle (3V) used to determine the density and distribution of synaptic inputs to vimentin⁺ tanycytes (in magenta). Dorsoventral subsetting of the ventricle wall (dashed lines) helped to identify the positions of α1, α2, β1, and β2 tanycytes. Scale bars = 100 µm. b. Image series to illustrate the image analysis pipeline used to quantify synaptic input onto tanycytes. 1,2: Confocal images captured at 63x primary magnification show vimentin⁺ α-tanycytes (in magenta) and VGLUT2⁺ putative presynapses (in green). Scale bar = 10 µm. 3: ‘Filament’ and ‘spots’ rendering algorithms were superimposed on vimentin (in magenta) and VGLUT2 (in green), respectively. Scale bar = 10 µm. The image shows how VGLUT2⁺ terminals were reconstructed as ‘spots’ and accepted as relevant only within a distance of < 0.5 µm from tanycyte filaments by using the built-in MatLab ‘find-spots-close-to-filaments’ function in Imaris. 4: Magnified view of a subset of VGLUT2⁺ spheres (in green) that were < 0.5 µm to the vimentin signal (in magenta). Scale bar = 2 µm. 5: VGLUT2⁺ inputs contacting β2-tanycytes. Scale bar = 20 µm. 6: Reconstruction of VGLUT2⁺ inputs contacting β2-tanycytes. Scale bar = 5 µm. c. Boxplots showing the probability of α1, α2, β1, and β2 tanycytes receiving VGLUT2⁺ inputs at -1.94 mm (relative to bregma, left) and −2.30 mm (relative to bregma, right). None of the tanycyte subtypes was significantly different; n = 7 mice, n = 50 processes reconstructed/subtype/animal, n = 200 processes/animal in total. d. Boxplots of the number of VGLUT2⁺ terminals on tanycytes at the rostro-caudal locations as above. None of the tanycyte subtypes was significantly different in n = 7 mice, n = 50 processes reconstructed/subtype/animal, n = 200 processes/animal in total. e. Quantitative analysis of VGLUT2⁺ punctae in a distance of < 0.5 µm to reconstructed filaments from both rostral and caudal regions. Data were shown as cumulative plots of VGLUT2⁺ terminals juxtaposing α- and β-tanycytes (pooled) at both rostral and caudal positions relative to bregma (b.) as above; n = 500 filaments were reconstructed/tanycyte subtype from n = 7 male mice (P60-90). Kruskal-Wallis test: KW = 96.040; p < 0.001 for α (caudal) vs. β (caudal); for α (rostral) vs. α (caudal); for β (caudal) vs. β (rostral). f. Left: Topographic view of tanycytes expressing GluA2 in apposition to VGLUT2⁺ terminals in Rax-CreER^T2::Ai14 mice. Open rectangle (‘1’) shows the location of the image series to the right. Scale bar = 5 µm. Right: Image series in left-to-tight order: high-resolution images of VGLUT2⁺ presynapses (‘pre’; in grey), tdTomato⁺ tanycytes (in magenta), GluA2 subunits (‘post’, in cyan), and their overlay. Scale bar = 500 nm. g. Current-clamp trace showing the membrane potential (mV) in tanycytes upon current steps. Vertical and horizontal scales are 20 mV and 50 ms, respectively. h. Boxplots define the resting membrane potential (mV) in α- (n = 23) and β-tanycytes (n = 20); t = 0.624; p = 0.536. i. Boxplots showing the membrane resistance (MΩ) in α (n = 23) and β tanycytes (n = 20); t = 1.980; p = 0.054. j. Boxplots depict the membrane capacitance (pF) in α- (n = 23) and β-tanycytes (n = 20); t = 0.960; p = 0.343. k. Boxplots for the time constant (τ) to dissipate current (ms) in α- (n = 23) and β-tanycytes (n = 20); t = 1.974; p = 0.0552. Open circles in h-k are individual data points; data were analyzed by Student’s t-test throughout. l. Representative trace showing the inward deflection of a tonic current in response to s-AMPA (100 µM) and its reversal upon wash-out. Vertical and horizontal scales are 50 pA and 100 s, respectively. m. top: Failure rate of optogenetically-induced EPSCs in tanycytes. Boxplot depicts means ± s.e.m., with individual data points shown as open circles. Bottom: Distribution of the lag-time of EPSCs (ms) relative to the onset of optical stimuli. Data were binned as indicated.

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