Extended Data Fig. 7: Neuronal activity triggers Ca²⁺ transients in tanycytes.

a. Top left: GCaMP5g transients in tanycytes after evoking a single action potential (AP) in a synaptically-connected neuron in brain slices superfused with ACSF. Images show Ca²⁺ transients in tanycytes (frame response, orange-fire, arrow) subsequent to injecting current in a patch-clamped neuron (frame AP, neuron is marked by arrow; see also Supplementary Videos 1, 2). Bottom left: Representative traces of GCaMP5g relative fluorescence intensity transients (F/F₀) in tanycytes following an evoked AP (arrows). Top right: NBQX (20 µM) blocked GCaMP5g-encoded Ca²⁺ transients in tanycytes (frame response, orange-fire, arrow) following AP induction in the same neuron (frame AP; see also Supplementary Video 3). Bottom right: Representative traces of GCaMP5g F/F₀ transients in tanycytes following evoked AP (arrows). Time was expressed as frames with inter-frame interval of 300 ms. b. AMPA (100 µM)-induced GCaMP5g-encoded Ca²⁺ transients in tanycytes. Confocal time series (20x) from 300-µm coronal hypothalamic slices from Rax-CreER^T2::PC-G5-tdT mice. Left: Representative frames of GCaMP5g basal activity as F/F₀ transients in tanycytes superfused with ACSF. Middle: AMPA (100 µM)-induced increase in GCaMP5g transients. Right: The effect of AMPA was abolished by NBQX (20 µM; see also Supplementary Videos 4–6). Time was expressed as frames with inter-frame interval of 600 ms. Scale bar = 20 µm. c. PTX (100 µM)-induced GCaMP5g-encoded Ca²⁺ transients in tanycytes. Confocal time series (20x) from Rax-CreER^T2::PC-G5-tdT mice as above. Left: Sequential frames of basal GCaMP5g activity (F/F₀) when tanycytes were superfused with ACSF. Middle: PTX (100 µM)-induced increase in GCaMP5g transients, which were substantially reduced when co-applying TTX (5 µM, right; see also Supplementary Videos 7–9). Scale bars = 20 µm. Time was expressed as frames with an inter-frame interval of 600 ms. d. Top: KCl (50 mM)-induced tanycyte activation. Confocal time series (20x) from 300-µm coronal hypothalamic slices from Rax-CreER^T2::PC-G5-tdT mice. Left: Representative frames of GCaMP5g basal activity (F/F₀,) in tanycytes superfused with ACSF. Right: KCl (50 mM)-induced increase in GCaMP5g-encoded Ca²⁺ events in tanycytes (F/F₀, trace; see also Supplementary Videos 10, 11). Scale bars = 20 µm. Time was expressed as frames with an inter-frame interval of 600 ms.