Since the success in GK knockdown depends upon the efficiency of adenoviral transduction, time-course studies were carried out in order to determine the time point at which there was a maximal infection rate. We analyzed EGFP fluorescence levels in rat brain slices at 18, 24 and 48 h post-i.c.v. injection of the recombinant adenovirus and compared them with levels of the tanycyte marker, vimentin, by immunostaining (Fig. 1). At 12 h post-injection, a faint signal from EGFP was detected in a few α-tanycytes (data not shown). At 18 h after i.c.v. injection, EGFP expression was detected in ventricular cells with elongated processes, which due to their location, correspond to α-tanycytes (Fig. 1A, arrow), which are vimentin-positive (Fig. 1B,C). High fluorescence was detected in the proximal region and basal processes of vimentin-positive cells at 24 h after injection (Fig. 1D–F). High levels of fluorescence were still observed for at least 48 h post-i.c.v. injection in ependymal cells (1), α-tanycytes, (2), β1-tanycytes (3) and β2-tanycytes (4) (Fig. 1G). Higher magnification images are shown in Fig. 1H and I. Importantly, EGFP fluorescence was limited to tanycytes, and no transduction of other areas was observed. EGFP was detected in the cell bodies and processes of cells lining the infundibular recess, which according to their location, correspond to α and β1-tanycytes. Particularly, the colocalization of Ad-EGFP with vimentin (shown in yellow) became more evident in β2-tanycytes (Fig. 1G–I).Figure 1Time-course of adenoviral transduction in the basal hypothalamus. (A–L) Frontal sections (40 µm) from the hypothalamus at 18 h (A–C), 24 h (D–F) and 48 h (G–I) following injection of adenoviral particles (Ad-shGK-EGFP) into the 3V. EGFP fluorescence (green) and immunoreactivity for vimentin (red) and colocalization of EGFP and vimentin (yellow) are shown. Arrows show the processes of transduced tanycytes. (G) Panoramic view of the basal hypothalamus, showing tanycyte transduction. (I) Higher magnification image showing that Ad-EGFP transduction occurs in β2-tanycytes. AN, arcuate nucleus; 3V, third ventricle; ME, median eminence. Scale bars (A–F and H) 50 µm, (G) 100 µm, (I) 20 µm.