2 μm z step, 400–800 ms exposure, 1 × 1 binning, and deconvolved using the “Classic Maximum Likelihood Estimation” algorithm (Huygens, Scientific Volume Imaging B.V.). Deconvolved z stacks were then subjected to a maximum intensity projection, resulting in two-dimensional (2D) images for processing. Subsequent processing for all images was as performed in three steps as described
below: (1) registration between images was optimized using a 2D cross-correlation algorithm, (2) images were thresholded, and (3) colocalization was calculated as the fraction of area overlapping between the above-threshold structures in each image. Image alignment and colocalization analysis was performed using custom algorithms written in MATLAB (MathWorks). Selleckchem BI-2536 For temperature block experiments, cells were first imaged at 37°C, followed by 2 hr incubation at 20°C, and reimaged. Dinaciclib Glycine stimulation was performed in DIV15–DIV17 neurons, essentially as described by Park et al. (2006). Briefly, neurons were incubated at 37°C for 5 min in E4 solution containing 124 mM NaCl, 2 mM CaCl2, 3 mM KCl, 10 mM HEPES, and 10 mM glucose (pH 7.4). Activity was induced with E4 supplemented with 20 μM bicuculine and 200 μM glycine for 5 min, and finally in E4 with 20 μM bicuculine for 15 min before fixation and
imaging. For picrotoxin (PTX) stimulation, cells were incubated in 100 μM PTX at 37°C for the desired time and fixed. In experiments with NMDA inhibitors, neurons were either treated with 50 μM D-APV for 2 hr or 10 μM memantine for 4 hr and then activation as mentioned in the text. Dynasore was used at 40 μM for 30 min at 37°C prior to activity induction and imaging. To test the pHl:APP (see Figure 5B), we replaced neurons incubated in HEPES with MES to obtain a pH of 5.5, keeping other components in the remaining imaging buffer unchanged (124 mM NaCl, 2 mM CaCl2, 3 mM KCl, 10 mM MES, and 10 mM glucose [pH 5.5]), as described by Park et al. (2006). To label actively recycling endosomes, we loaded neurons with 50 μg/ml Alexa 488 transferrin (Invitrogen) diluted in imaging medium for 1 hr at 37°C, rinsed extensively, and imaged immediately thereafter. Methisazone Sequential isolation and
density gradient centrifugation were performed to fractionate vesicular fractions as described previously by Scott et al. (2011). Briefly, brains from 4- to 6-week-old CD-1 mice, or frontal lobe dissected from postmortem frozen human brains (three AD Braak V/VI and two controls, obtained from the AD Research Center brain bank at UCSD), were homogenized in buffer containing 20 mM HEPES, 40 mM KCl, 5 mM EDTA, 5 mM EGTA, and protease inhibitor (pH 7.2). Tissue lysate was centrifuged at 1,000 × g for 15 min to isolate postnuclear supernatant (S1). S1 was centrifuged at 10,200 × g for 20 min to obtain synaptosome-depleted fraction (S2). Finally, S2 fraction was centrifuged at 100,000 × g for 1 hr at 4° to isolate soluble (S100) and vesicular (P100) fraction.