Figure 1. Intracellular injection of Aβ42 oligomers into Xenopus oocytes evokes increases in cytosolic [Ca2+]. A, Overview of the experimental system, constructed around an Olympus IX71 inverted microscope. Fluorescence excited in the specimen by the 488 nm laser beam was collected through the objective lens and imaged by a Photometrics Cascade 128+ camera. An oocyte loaded with fluo-4 dextran was positioned animal hemisphere down on a coverglass forming the base of the imaging chamber. A two-electrode voltage clamp allowed the membrane potential to be stepped to strongly negative potentials to enhance Ca2+ influx through the plasma membrane. Microinjection into oocytes was performed using a Drummond nanoinjector mounted on a hydraulic micromanipulator. A glass pipette filled with Aβ42 solution was inserted vertically down through the entire oocyte to a pre-established position, with the tip positioned a few micrometers inward from the plasma membrane and centered within the image field. B, Aβ42 oligomers, but not monomer, evoke cytosolic Ca2+ signals. The traces represent the mean time course of Ca2+-dependent fluorescence recorded from oocytes injected with 10 nl of Aβ42 monomer (red trace; n = 3 oocytes) or oligomers (black trace; n = 6 oocytes), both at a concentration of 1 μg/ml. The arrow indicates the time of Aβ injection. Measurements were obtained as the average fluorescence throughout the imaged field and are plotted as the ratio of fluorescence changes at any given time (ΔF) over the mean fluorescence before injection (F0). C–E, Line-scan (kymograph) images illustrating different spatiotemporal patterns of fluorescence Ca2+ signals evoked by Aβ42 oligomer injections. Panels depict fluorescence measured along a line on the video record as the y-axis, with time running left to right along the x-axis. Increasing fluo-4 pseudo-ratio signals (increasing free [Ca2+]) are represented by warmer colors as depicted by the color bar and by increasing height of each pixel. The trace(s) above each panel show fluorescence signals monitored from small regions along the line scan, positioned as marked by the horizontal arrow(s). The timescales of these traces are the same as the calibration bar for the line-scan images, and the amplitudes of the fluorescence ratio changes (F/F0) correspond to the heights of the color bars. Aβ42 oligomers were injected 1 s after the beginning of the record in C. Records in D and E were obtained beginning 2–5 min after injection of Aβ42 oligomers. F, Corresponding line-scan image and fluorescence traces recorded in response to injection of 10 nl of 100 pm IP3. The injection was delivered ∼2.5 s before the beginning of the record. Responses are representative of records in five oocytes. G, Traces of average fluorescence ratio changes (ΔF/F0) from ∼20 × 20 μm regions of interest in individual oocytes, showing Ca2+ signals evoked by 10 nl injections of Aβ42 oligomers at concentrations of 3, 10, and 30 μg/ml, as indicated. Responses are representative, respectively, of records in four, four, and five oocytes.