Primary culture in microfluidic chips
Microfluidic chips were realized as described in . The design used for network reconstruction comprises two culture chambers each connected to two reservoirs and separated by a series of 500 μM-long asymmetrical micro-channels (3 μM high, tapering from 15 μM to 3 μM) . E16 embryos (Swiss mice, Janvier, France) were micro-dissected in GBSS (without CaCl2 and MgCl2) + 0.1% glucose (Merck), digested with papain (20U/mL) and mechanically dissociated in DMEM containing DNAse. 120.103 cortical cells and 45.103 hippocampal cells were seeded in the chamber on contact with the wide and narrow sides of micro-channels, respectively. Cells were cultured in DMEM glutamax (Invitrogen) supplemented with 10% FBS (PAA), N2, streptomycin/penicillin and B27 (Invitrogen). The culture medium was renewed every 3-5 days. Cortical axon entered the micro-channels and reached the hippocampi-containing chamber in 4-5 days. The cortico-hippocampal oriented network was maintained routinely up to 2-3 weeks in vitro.
Oligomeric and fibrillar Aβ peptide preparations
Oligomeric and fibrillar forms of Aβ1-42 (Tocris Bioscience, MN, USA), were produced according to  and controlled by electron microscopy (Additional file 2: Figure S2). Briefly, lyophilized peptides were solubilized at 1 mM in 1, 1, 1, 3, 3, 3,-hexafluoro-2-propanol (HFIP, Sigma Aldrich). After 30 min of incubation at RT, HFIP was evaporated overnight and peptides were dried (Speed Vac, 1 h 4°C). Then, Aβ peptide stock solution were obtained byresolubilizationet 5 mM in dimethylsulfoxide (DMSO, Sigma Aldrich). To obtain oligomers, Aβ stock solution was diluted at 100 μM in phenol-free DMEM-F12 medium (Life technologies), incubated 24 h at 4°C and centrifuged at 20 000 g (10 min; 4°C) before supernatant (soluble Aβ fraction) collection. To obtain fibrils, Aβ stock solution was diluted at 100 μM in HCl 10 mM and then incubated at 4°C for 24 h.
Aβ sample aliquots (10 μM) were allowed to adsorb onto carbon-coated 200-mesh copper grids (EMS, PA, USA) for 2 minutes before blotting off. Then, grids were incubated for 45 seconds in uranyl acetate 2.5% (w/v) to produce a negatively stained protein loaded grid. Images were recorded on a Zeiss 912 omega electron microscope (Carl Zeiss Group, Oberkochen, Germany).
The following compounds were used: Aβ1-42 peptide (1428, Tocris), Aβ25-35 peptide (H-1192, Bachem), control Aβ35-25 peptide (H2964, Bachem), glutamate (G8415, Sigma), MK-801 (0924, Tocris),5 mM NAD+ (Sigma), 50 μM z-VAD-fmk (R&D Systems, Minneapolis, MN, USA) and 50 μM SP 600125 (Sigma). Oligomeric and fibrillar forms of Aβ1-42 were produced according to  and controlled by electron microscopy. To ensure fluidic isolation between compartments, a hydrostatic pressure difference was generated by over-pressurizing the non-treated chamber as described in .
Cultures were fixed (4% PFA, 20 min RT) and immunostained as described in . Primary antibodies included anti-α-tubulin-FITC (F2168, sigma), anti-MAP2 (M4403, Sigma), anti-Synaptophysin (S5768, Sigma), anti-Vglut1, anti-α-synuclein (4179, cell signaling), anti-pTau Thr231 (44746G, Invitrogen). Species-specific secondary antibodies (coupled to Alexa 350, 488, 555) were used (Invitrogen). Images were acquired with an Axio-observer Z1 (Zeiss) fitted with a cooled CCD camera (CoolsnapHQ2, Ropert Scientific) and images were analyzed using ImageJ.
Quantification of axonal fragmentation
For axonal fragmentation analysis and quantification we used fluorescence microscopy (immuno-detection of tubulin), phase contrast, and picture analysis according to a previously described protocol . Briefly, we used a macro developed in NIH ImageJ software that utilizes the Otsu thresholding algorithm, and a particle analyzer algorithm of ImageJ. The total area of axonal regions with circularity greater than 0.9 was determined and normalized by the total axonal area, which was measured from the thresholded image. This ratio, termed fragmentation index, is an indicator of the average axonal fragmentation level and is used in statistical comparisons. Indices of 0.005, 0.083, and 0.157 correspond to <5%, 50%, and >95% fragmentation, respectively.
Quantification of synapse loss
Synaptic disconnection was assessed through fluorescence microscopy by counting α-synuclein clusters affixed to MAP2 positive hippocampal dendrites. Images were all obtained with the same acquisition parameters. The images were similarly processed with ImageJ software before being used for quantification: the brightness/contrast of all control images was optimized manually to eliminate the background and to maximize the signal. The means of the minimum and maximum intensities were then calculated in the control condition, after which these settings were applied to all images. The images of the three α-synuclein/β-tubulin/MAP2 stainings were merged and the resulting image was used to define the zone where hippocampal dendrites were sufficiently innervated by cortical fibres. α-synuclein/MAP2 merges were then used for quantification.
Quantification of Tau phosphorylation
Tau phosphorylation was assessed by counting the number of neurons presenting pTau levels above a fixed threshold. Images were all obtained using the same acquisition parameters. The images were similarly processed with ImageJ software before being used for quantification: the brightness/contrast of all control images was optimized manually to eliminate the background and to maximize the signal. The means of the minimum and maximum intensities were then calculated in the control condition, after which these settings were applied to all images. All pTau images were then processed with the “Lookup Tables, fire” plugin to visualize the intensity of pTau staining with pseudo-colours. The number of neurons above a fixed colour threshold was then counted and normalized by the total number of neurons to have the percentage of hyperphosphorylated tau neurons.
Differences were assessed by ANOVA, followed, when appropriate, by a post-hoc Bonferoni test. For all analysis * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.