Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Cytotoxicity of intracellular A?42 amyloid oligomers involves Ca 2+ release from the ER by stimulated production of inositol trisphosphate

doi: 10.1523/JNEUROSCI.4367-12.2013

Figure Lengend Snippet: 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 in to 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 µm inward from the plasma membrane and centered within the image field. B, Aβ42 oligomers, but not monomers, 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−1). 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 (Fo). C, D, E, Linescan (kymograph) images illustrating different spatio-temporal 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 linescan, positioned as marked by the horizontal arrow(s). The timescales of these traces are the same as the calibration bar for the linescan images, and the amplitudes of the fluorescence ratio changes (F/Fo) correspond to the heights of the color bars. Aβ42 oligomers were injected 1s 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 linescan image and fluorescence traces recorded in response to injection of 10 nl of 100 pM IP3. The injection was delivered about 2.5 s before the beginning of the record. Responses are representative of records in 5 oocytes. G, Traces of average fluorescence ratio changes (ΔF/Fo) 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−1, as indicated. Responses are representative, respectively, of records in 4, 4 and 5 oocytes.

Article Snippet: Spatio-temporal patterns of Ca 2+ signals evoked by intracellular Aβ42 oligomers and IP 3 Aβ42 oligomer injections evoked distinct spatio-temporal patterns of Ca 2+ signals, as illustrated in as linescan (kymograph) images, derived by measuring fluorescence along a single line within the imaging field and displaying its evolution over time as a pseudocolored representation utilizing the kymograph function in MetaMorph. shows an example where the Ca 2+ fluorescence signal increased slowly and monotonically at several 'hot-spots' along the linescan.

Techniques: Injection, Construct, Inverted Microscopy, Fluorescence, Imaging, Membrane, Clinical Proteomics, Microinjection, Transferring, Concentration Assay