Cfw 1310m

The HC activity was evaluated using the Etd uptake method as described (Figueroa et al., 2013 (link)). In brief, sub-confluent HeLa cells grown on glass coverslips were washed twice with recording solution [in mM: NaCl (148); KCl (5); CaCl₂ (1.8); MgCl₂ (1); glucose (5); HEPES (5), pH = 7.4] containing 5 μM Etd. Basal fluorescence intensity from the nucleus of each cell was recorded for 5 min, using an Olympus BX 51W1I upright microscope (Olympus America Inc., Center Valley, PA, USA). Next, cells were washed three times with (Ca²⁺/Mg²⁺-free) DCFS and the fluorescence intensities of the nuclei were recorded. At the end of each experiment, the Cx HC blocker La³⁺ was added to confirm HC mediated Etd uptake (Contreras et al., 2002 (link); Retamal et al., 2007 (link)). Dye uptake of each cell was digitally photographed using a CCD monochrome camera (CFW-1310M; Scion; Frederick, MD, USA). Images were captured every 30 s (exposure time = 30 ms, gain = 0.5). Metafluor software (version 6.2R5, Universal Imaging Co., Downingtown, PA, USA) was used for data acquisition and off-line image analysis. The fluorescence intensity of at least 30 cells per experiment was averaged and plotted against time (expressed in minutes). Lastly, the slope, here called Etd uptake rate, was calculated using Microsoft Excel software and expressed as arbitrary units per minutes (AU/min).

The functional state of gap junctions was evaluated as previously described (Martínez and Sáez, 1999 (link)) in neurosphere-derived cells and in co-cultures of neurosphere-derived cells with astrocytes or microglia. Single NPCs were iontophoretically microinjected with a glass micropipette filled with 75 mM LY (5% w/v in 150 mM LiCl). Dye coupling index was calculated as the mean number of cells to which the dye spread occurred in 3 min. All microinjections were performed in ${\text{HCO}}_3^{-}$ free F-12 medium buffered with 10 mM HEPES (pH 7.4) containing 200 μM La³⁺ to avoid cell leakage of the microinjected dye via hemichannels. Dye coupling was assessed in the absence and in the presence of 750 μM octanol. Fluorescent cells were observed using a Nikon inverted microscope equipped with epifluorescence illumination (Xenon arc lamp) and Nikon B filter to LY (excitation wavelength 450–490 nm; emission wavelength above 520 nm) and XF34 filter to DiI fluorescence (Omega Optical, Inc., Brattleboro, VT, USA). Photomicrographs were obtained using a CCD monochrome camera (CFW-1310M; Scion; Frederick, MD, USA). Five to ten experiments were performed for every type of culture and dye coupling was tested by microinjecting a minimum of 10 cells per experiment.

Tissue sections (20 μm) from AD and control brains were fixed with 3% acetic acid for 10 min at rt. Sections were incubated with 3% BSA dissolved in phosphate-buffered saline (PBS) and permeabilized (0.2% Triton X-100/PBS). HS were stained with an anti-HS (10E4, 1:200; AMS Biotechnology,) revealed with an Alexa-555 antibody (Invitrogen), followed by DAPI-labelling (1 μg/mL). Antibodies references are listed in S1 Table. Images were first obtained with a CCD camera (CFW-1310M, Scion Corporation, USA) in a BH-2 epi-fluorescence optical microscope (Olympus). Image acquisition was made by the Scion VisiCapture 2.0 software and processed by using ImageJ. DAPI labelling of nuclei was quantified as previously [18 (link)].