Picomolar amyloid-β peptides enhance spontaneous astrocyte calcium transients.

Animals Breeding colonies for wild-type

C57BL/6J and α7 nAChR subunit deficient mice [ 23 ] were housed in animal facilities at Columbia University and maintained on a 12-h light/dark cycle. All experimental protocols involving animals were approved by Columbia University and the Institutional Animal Care and Use Committee.

Primary cultures

Astrocyte cultures were prepared from P0–P1 mouse pups as previously described [ 24 , 25 ]. Briefly, meninges-free forebrains were dissected out and dissociated with trypsin, followed by trituration and plating in culture-treated flasks. Culture media: high-glucose Dulbecco’s Modified Eagle Medium plus GlutaMax (Invitrogen), supplemented with 10% heat-inactivated fetal calf serum plus penicillin (100 U/ml) and streptomycin (100 μg/ml). After the cells reached confluence, the flasks were shaken for 2 h on an orbital shaker. The media was replaced, and the sealed flasks were shaken again overnight (~16 h). The following day, the cultures were extensively washed and passaged onto poly-D-lysine-coated glass coverslips. In all experiments, ~12–16 DIV confluent cultures that underwent only one passage were used. Cultures were regularly checked for contamination by other cell types. The standard overnight shaking method for purifying astrocytes removes most remaining neurons/microglia/oligodendrocytes, as previously described [ 24 , 25 ]. When examined by immunocytochemistry for an astrocyte marker, glial fibrillary acid protein (GFAP), the purified cultures were almost completely GFAP+ (data not shown). By light microscopy examinations, no neurons and nominal microglia and oligodendrocytes were present in the purified cultures.

Amyloid-β peptide preparation

Synthetic human Aβ peptides (American Peptide Company) were prepared as previously described [ 9 , 26 , 27 ]. Briefly, lyophilized peptides were dissolved to 1 mM in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to remove any preexisting structures [ 27 ] and dried by SpeedVac to produce peptide films. Prior to use, films were fully dissolved in anhydrous dimethylsulfoxide (DMSO) to 5 mM and briefly bath sonicated at room temperature. For fresh (predominantly monomeric) preparations, the DMSO solution was immediately diluted down to the picomolar concentration range in imaging buffer prior to an experiment. For oligomeric preparations, the DMSO solution was diluted to 100 μM in PBS and incubated overnight at 4°C; the formation of oligomeric Aβ species was confirmed through western blotting with 6E10 monoclonal antibody (Covance), as previously described [ 9 ] ( Supplementary Fig. 1 ).

Spontaneous calcium transient imaging

Confluent astrocyte cultures on glass coverslips were removed into imaging buffer. Buffer composition (in mM): NaCl (140), KCl (5), MgCl 2 (2), CaCl 2 (2), glucose (5), HEPES (10), pH 7.4. Cells were loaded with Fluo-4 AM esters (Molecular Probes) diluted in imaging buffer with Pluronic F-127 (Invitrogen). The astrocyte cultures were readily loaded with Fluo-4 dye and consistently exhibited even, homogenous loading distributions. A Nikon D-Eclipse C1 confocal microscope (488 nm argon laser) was used for all experiments. Time-lapse movies in all experiments were acquired at 0.33 Hz, generally in continuous 10 min blocks for 60 min total. In analyses of spontaneous transients, the first 10-min imaging block was performed under basal control conditions and was used to normalize subsequent data from that experiment. Movies were imported into MetaMorph software (Molecular Devices) for automated segmentation and quantification of cell body regions of interest. The entire imaging field was analyzed to minimize manual selection bias. The quantified fluorescence intensity data were analyzed in Igor Pro (Wavemetrics). All fluorescent data values were normalized to the global minimum value for that trace. Traces were plotted with these normalized data as fold changes. The basic threshold used for the identification of calcium transients was a minimum 40% rise in normalized fluorescent signal intensity. This threshold reliably detected all distinct calcium transient peaks with minimal contamination by noise. Amplitudes were calculated as (peak maximum) – (base minimum). The frequency measure reflects the absolute number of calcium transients per 10 min imaging block. Data were averaged and binned for each 10 min block during an experiment and represented as fold changes from the baseline control.

Calcium wave imaging

Intercellular calcium waves were induced as previously described by deformation of the cell membrane via a light mechanical stimulus using a glass micropipette electrode [ 28 – 30 ]. Glass micropipette electrodes were pulled from thick-walled borosilicate glass tubing, filled with imaging buffer, and mounted on a piezoelectric micromanipulator. With constant monitoring of the resistance, the electrode was slowly lowered toward the cell layer, briefly contacting the cell surface followed by immediate withdrawal. This mechanical stimulus reliably induced intercellular calcium waves in the astrocyte cultures. Movies were imported into Igor Pro software (Wavemetrics). All fluorescent data were normalized to the global maximum value and plotted as a percentage of this value. The radial distribution of intensities was calculated based on the manually-identified wave center initiation point. Distance and time measures were based on a threshold intensity of 50%.

Immunofluorescence

Immunostaining was performed according to standard protocols. Briefly, cultured cells were washed and fixed in paraformaldehyde followed by permeabilization with Triton X-100. Blocking was done in 1% BSA plus 5% normal goat serum. Primary antibodies were diluted in blocking solution. Antibodies used include: GFAP (Affinity Bioreagents), α7 nAChR subunit (Chemicon/Millipore, Santa Cruz Biotechnology). Analyses were performed with ImageJ (NIH) software. α-Bungarotoxin surface labeling Alexa Fluor 594-conjugated α-bungarotoxin (Invitrogen) was diluted in cell culture media to 0.1 μM and applied to cultures for 15 min at 37°C. Cultures were fixed in paraformaldehyde, mounted with Vectashield (Vector Labs), and examined by confocal microscopy.

Western blot

Cell cultures were washed and directly lysed in 2x standard Laemmli sample buffer. Equal amounts of lysate or synthetic Aβ solutions were loaded in tris-glycine or tricine gels for SDS-PAGE. Proteins were transferred onto 0.2 μm nitrocellulose membranes and subsequently blocked in 5% bovine serum albumin. Primary antibodies were diluted in blocking solution. The antibodies used include: α7 nAChR subunit (Chemicon/Millipore, Santa Cruz Biotechnology), β-actin (Abcam), Aβ 6E10 (Covance). Blots were developed with enhanced chemiluminescence (ECL) substrate or scanned using the Odyssey system (Licor) for infrared labeling detection. Digitized blot images were analyzed using ImageJ (NIH) software. ELISA Aβ (x-42) ELISAs (Invitrogen) were performed according to the manufacturer’s protocols. Tissue culture media was collected, supplemented with protease inhibitors (Roche), and immediately used for the ELISAs.

Statistical analyses

All data in figures are presented as means ± SEM. For the calcium imaging experiments, data were normalized to the global minimum fluorescence intensity of that experiment. Figure data are represented as fold-changes from the baseline control imaging block. Data were analyzed by two-tailed Student’s t -test or two-way ANOVA plus post-hoc Tukey’s multiple comparisons test using Prism (GraphPad) software. The threshold for significance was set at p < 0.05 in all analyses.