Fig. 2: Parabrachial neurons innervate α-tanycytes.

a, Left, VGLUT2⁺ presynapses (black) formed synapse-like contacts on tanycytes (T) along the third ventricle (3V). The outlined region is enlarged on the right. Right, VGLUT2⁺ presynapse (semi-transparent cyan) apposing the soma of a tanycyte (semi-transparent magenta). Scale bars: 2 μm (left), 200 nm (right). b, Schema of transsynaptic labelling. Tanycytes along the third ventricle served as the primary transduction site for WGA–Cre to mark synaptically connected neurons (cyan). LV, lateral ventricle. c, Top, transsynaptic labelling using rAAV8-EF1a-mCherry-IRES-WGA-Cre in Ai14-tdTomato-loxP mice. Vimentin⁺ tanycytes co-expressed mCherry (arrowheads) and were synaptically connected, among others, to local neurons (arrows). Bottom, putative contact between a vimentin⁺ tanycyte process (arrowheads) and an mCherry⁺ transsynaptically labelled neuron (arrow, cyan). Scale bars: 20 µm (top), 6 µm (bottom). d, Transsynaptic labelling by rAAV8-EF1a-mCherry-IRES-WGA-Cre in Tau^mGFP-loxP mice. mCherry⁺ tanycytes were innervated by local NeuN⁺ neurons (mGFP, colour-coded to cyan, arrows in both overview and inset). Arrowheads denote neuronal processes. Scale bars: 40 µm (main image), 2 µm (inset). e, GFP⁺ parabrachial neurons labelled with AAVrg-CAG-GFP injected at the level of ARC. Scale bar, 50 µm. f, Transsynaptic labelling by rAAV8-EF1a-mCherry-IRES-WGA-Cre in Ai14 mice. Parabrachial neurons (tdTomato⁺, cyan) synaptically connected to tanycytes (see also c) resided laterally to the scp. Scale bar, 50 µm. g, Cartoon of the chemogenetic manipulation of parabrachial glutamatergic neurons in Slc17a6-IRES2-FlpO-D mice. h, Transduced neurons expressed mCherry. cFOS⁺ neurons are shown 1.5 h after either saline or CNO (5 mg kg⁻¹, intraperitoneal) injection. Scale bars, 50 µm. i, cFOS activation (grey) in α-tanycytes (green) 1.5 h after administration of saline or CNO. Scale bars, 10 µm. j, Left, mCherry⁺ parabrachial efferents (magenta) from glutamatergic neurons apposed (arrows) vimentin⁺Hoechst 33,421⁺ tanycytes (composite; outlined region is enlarged on the right) co-expressing cFOS. Right, mCherry⁺ parabrachial efferents (arrowheads) apposed vimentin⁺ processes. Scale bars: 8 µm (left), 1 µm (right).