EGFP fluorescence was detected in cells whose localization and morphological features are suggestive of tanycytes. We evaluated astrocyte transduction in a three dimensional reconstruction of 20 focal planes (Supplementary Fig. S1). Sections of the basal hypothalamus from transduced animals were analyzed by spectral confocal microscopy for detecting EGFP fluorescence (green) and the astrocyte markers, GFAP (blue) and vimentin (red). EGFP expression was detected in the ventricular cells having long cellular processes, which due to their location, are α- and β-tanycytes. The expression of GFAP and vimentin was also detectable in the same region, where a different distribution of both proteins was clearly observed. In agreement with previous results27, GFAP was mainly detected in the subependymal region of the ME. Analysis of the co-expression of EGFP and GFAP at higher magnification revealed no co-expression of both proteins. However, a clear co-distribution of EGFP and vimentin was observed in the proximal region (arrowhead) and processes of tanycytes (arrows). Because adenoviruses can cause gliosis, which favors the incorporation of adenovirus by reactive astrocytes, we next evaluated the colocalization of both glial markers using a focal plane and Pearson’s correlation analysis. Colocalization of EGFP with vimentin was detected in the ventricular area (Fig. 2B,E, arrows) and in several cell processes (Fig. 2B,E, arrowheads). The Rr value estimated for EGFP/vimentin colocalization was 0.47 ± 0.11 in the area of α-tanycytes (Fig. 2B), whereas the Rr value for EGFP/GFAP colocalization in the same region was 0.18 ± 0.02, suggesting that EGFP colocalized to a larger extent with vimentin as compared with GFAP (Fig. 2C). Intense EGFP and vimentin colocalization (yellow) was observed in β-tanycytes (Fig. 2E, arrows) and the long fine processes contacting AN neurons (Fig. 2E, arrowheads). In this region, the Rr value estimated for EGFP/vimentin colocalization was 0.53 ± 0.06, whereas the Rr value estimated for EGFP/GFAP colocalization was 0.01 ± 0.02 (Fig. 2F). Astrocytes located in the proximity of α-tanycytes were strongly positive for GFAP, but not for EGFP (Fig. 2C, asterisks). The absence of transduction was detected also in astrocytes located in the proximity of β1-tanycytes and β2-tanycytes from the ME (Fig. 2F, asterisks). Although other authors have reported the serotype 5 adenoviruses transduces astrocytes, our results suggest that if adenovirus is injected in the 3V, it preferentially transduced tanycytes and not astrocytes. We used the neuronal marker, NeuN, and the tanycyte marker, vimentin, to evaluate neuronal transduction (Fig. 3A–E). EGFP was absent in NeuN-positive neurons (Fig. 3C,E), suggesting that AN neurons were not transduced with the adenovirus. Additionally, we analyzed the EGFP fluorescence in other cerebral regions, such as the cortex, hippocampus, and choroidal plexus, and compared it with that observed in the ventricular walls of the same animals. The intensity of EGFP fluorescence was systematically different in the tanycyte population with most fluorescence detected in β2-tanycytes then α-tanycytes and slightly lower in β1-tanycytes. Similar to β2-tanycytes, EGFP was detected in 3V-ependymal cells with weak signal observed in the choroid plexus and no signal detected in the cortex and hippocampus (Supplementary Fig. S2).Figure 2Distribution of EGFP in hypothalamic glial cells. (A–F) Representative confocal images of one focal plane using anti-vimentin (red) or anti-GFAP (red) antibodies as well as the fluorescence associated with EGFP (green) and the nuclear marker, TOPRO-3 (blue). (A–C) Adenoviral transduction in α-tanycytes. (D–F) Adenoviral transduction of β-tanycytes. (A,C) EGFP detected in apical membranes (arrows) and in long cellular processes in the basal hypothalamus (arrowheads). (B,E) Vimentin showing both proximal (arrows) and distal (arrowheads) tanycyte regions and colocalization with the EGFP. (C,F) GFAP showing astrocyte distribution, the absence of GFAP in the ventricular wall (arrows) and EGFP-positive processes (arrowheads). Scale bars (A–H) 50 µm. Figure 3Co-distribution of EGFP and the neuronal marker, NeuN, in the basal hypothalamus. (A–E) Representative confocal images of one focal plane using anti-vimentin (red) or anti-NeuN (red) antibodies as well as EGFP fluorescence (green) and TOPRO-3 (blue). (A) Image showing EGFP in an α-/β-tanycyte transition region. (B) EGFP detected in the proximal (arrows) and distal processes (arrowheads) of vimentin-positive α-and β-tanycytes. (C) The neuronal marker, NeuN, distribution (red), showing the absence of colocalization with EGFP. (D,E) High magnification image of the area shown in (B and C). Scale bars (A–C) 50 μm; (D,E) 25 μm.