Article Figures & Data
Figures
- Fig. 1.
A, CCOs are distinct from the endoplasmic reticulum in cultured hippocampal neurons. Cells were initially stained with Fluo-3 and reconstructed in three dimensions using eight optical sections taken at 1 μm intervals (left). The same cells were later stained with DiO, which labels the endoplasmic reticulum (right). Fluo-3 fluorescence was photobleached before the second staining. Note the absence of DiO in the nucleus and the network of labeling in the soma, especially near the base of the primary dendrites. B, An image taken at a single plane through another cell stained with Fluo-3 (left) and DiO (middle).Right, An amplified-difference image between the Fluo-3 and DiO images. Although some organelles are seen also with the DiO staining, they do not overlap with those stained with Fluo-3.
- Fig. 2.
CCOs are not colocalized with mitochondria.A, Fluo-3-loaded cell in control condition (left), after onset of exposure to the mitochondria uncoupler SF-6847 (30 μm; middle), and after 10 min exposure and 20 min wash (right). Note the increase in nuclear and [Ca]i but also of [Ca]C. Fluorescence in the right image is saturated. B, Double-labeled cell stained with Fluo-3 AM (left) and Rhodamine 6G (center).Right, Amplified difference between theleft and center images. A single plane taken through the same cell indicates that the two dyes do not stain the same organelles. Rhodamine 6G staining was made in Fluo-3-labeled, and subsequently bleached, cells, as above. Staining was made with 1 μm dye incubated for 10 min, followed by extensive wash for 40 min.
- Fig. 3.
Transient disappearance of [Ca]Cfluorescence after exposure to Mn2+. A, A Fluo-3-loaded cell shown from left toright, in control condition, immediately after exposure to a pulse application of high K+ (200 mm)-containing medium, 5 sec after K+, and 1 min later. The frame on the right is a net image, subtracting the image taken 5 sec after onset of exposure to K+ from the image taken 1 min later. A andB are the same cell: A, in control medium; B, in a medium containing Mn2+ (2 mm), replacing Ca2+. C, Another cell, preincubated for 15 min in thapsigargin (1 μm), exposed to Mn2+ as in B. The CCOs are clearly visible in B and are nearly absent inC after washout of K+.
- Fig. 4.
Time course of changes in Fluo-3 fluorescence after a momentary exposure to high K+ in Mn2+-containing medium, as in Figure 4. Mean of five experiments. Note the log scale of the time base.
- Fig. 5.
CCOs respond to chemical stimulation in cultured hippocampal neurons. A, Reactivity of CCOs to caffeine.Images 1 and 2 were taken 20 min apart to illustrate the stability of the detection of CCOs. Image 3, 10 min after perfusion of the cell with 10 mmcaffeine. Note that at this time, only CCOs respond to caffeine, whereas the rest of the cell is nearly at baseline level. Single-plane optical sections, 16-frame average, 63× water immersion objective. Nucleus is out of the plane of focus. B, Responses to topical application of glutamate. B1, Top, Three-dimensional reconstructed neuron with a line drawn through its soma and a CCO in one of its primary dendrites. This cell was scanned at a rate of 0.8 msec/line. Bottom, Raster display of the line scan, from top to bottom, comprising 512 lines, showing net fluorescence relative to baseline fluorescence (DF/F). Glutamate (1 mm) was applied ∼50 msec from the top and is followed by a rise of fluorescence in the calciosome at about the same time as the rise in the soma of the same cell. B2, Three-dimensional display of the rise of Fluo-3 fluorescence, frombottom to top, illustrating the rise of fluorescence in the CCO and the soma. Intensity of fluorescence is coded in the height/color of the plot with redindicating low intensity and green-white indicating high intensity. C, Responses of a cluster of cells to caffeine and glutamate. Image 1, control; image 2, after 15 min of exposure to caffeine (5 mm), illustrating a rise in [Ca]C; image 3, 15 min wash; image 4, net frame of images 2–1, image amplified to enhance detection of differences in the image; image 5, during response to glutamate, (1 mm, 100 msec pulse). Note that nuclear fluorescence is saturated. Image 6, 1 min later; image 7, 20 min later; image 8, net frame of images 7–6, amplified. Note the decrease in CCOs fluorescence after exposure to glutamate, and the later recovery. Cells are three-dimensionally reconstructed.
- Fig. 6.
Cellular calcium responses to a brief application of caffeine (A) and glutamate (B andC). A, Exposure to a pulse application of caffeine (5 mm, 300 msec pulse, n = 11 cells) caused a transient rise in [Ca]i, which initially started in CCOs (see the insert amplificationof the first part of the graph) and was larger in the nucleus than in cytoplasm or the CCOs, and a slower recovery to baseline in the latter than in former two compartments. B, Glutamate (1 mm, 100 msec pulse) caused a much larger rise in nuclear [Ca]i than caffeine, but a similar [Ca]Crise and an undershoot in the fluorescence of the latter compartment was clear (glutamate, 1 mm, 50 msec pulse,C). D, Detailed comparison between CCO responses to glutamate and caffeine, relative to the changes in cytoplasmic [Ca]i, at successive time points before and after application of the drug: a, before application;b, the last nonsignificant point on cytoplasmic curve after drug application; c, the first significant point in the cytosolic response; d, at the peak of the response; e, one-half recovery to baseline;f, four-fifths recovery; g, last significant point; h, first point of recovery in cytoplasm. A significant rise of [Ca]C in response to caffeine is seen before [Ca]i changes. [Ca]C remained elevated in response to caffeine compared with the response to glutamate and to caffeine in cytoplasm.Ordinate, Ratio of net/baseline fluorescence.Abscissa (in A, B, and C), time; (in D), points in time relative to a defined [Ca]i state. Abscissa of Cis in a logarithmic scale.
- Fig. 7.
Cell reactivity to short duration (50 msec, 1–2 mm) exposure to caffeine. The response lasted <1 sec. Note the increase in [Ca]C and decrease in nuclear [Ca]i. The same cells responded to a longer exposure to caffeine with an increase in [Ca]i in all compartments, as shown before for other cells.
- Fig. 8.
Cellular reactivity to prolonged exposure to 5 mm caffeine. Caffeine was perfused in the recording medium (as in Fig. 5C), and changes in Fluo-3 fluorescence were monitored in 15 cells. After an initial rise in [Ca]i, cytosolic and nuclear calcium returned to baseline, whereas [Ca]C continued to rise, and remained high even after removal of caffeine.
- Fig. 9.
A, Thapsigargin enhances [Ca]C. Image 1, control; image 2, after 5 min exposure to the drug (0.5 μm);image 3, after 15 min exposure to thapsigargin. Note the increase in [Ca]c in thapsigargin-treated cell.B, Thapsigargin blocks the undershoot of the [Ca]C response to glutamate. CCO responses in control (as in Fig. 6C) and thapsigargin-pretreated cells (15–20 min; n = 13) to same pulse application of glutamate were compared. Unlike the control cases, in which a clear undershoot lasting at least 10 min is seen, the thapsigargin-treated cells do not return to baseline fluorescence after glutamate. Abscissa scale as in Figure 6C.
- Fig. 10.
Ryanodine blocks cytosolic, but not [Ca]C, reactivity to caffeine. Images were taken before (A), 3 sec after (B), and 6 sec after (C) exposure of the cell to a 300 msec puff of caffeine (5 mm). D–F, Same cell after 15 min of exposure to 10 μm ryanodine (with 2–3 intermediate responses not shown). Single plane/frame images of the cell. Note that the bottom left area, rich in CCOs, increases fluorescence before other cell areas (B), and even after ryanodine, whereas the rest of the cell maintains a low level of fluorescence (E and F).
- Fig. 11.
Differential effects of ryanodine on [Ca]i and [Ca]C. Initially, caffeine (5 mm, applied by pressure) caused a larger change in [Ca]i than in [Ca]C (A). After 10–15 min superfusion of 5 μm ryanodine, the baseline fluorescence was not different, the cytosolic response to caffeine was reduced drastically, and [Ca]C was still transiently elevated by caffeine (B). Only 5 min later, and after two to three more responses to caffeine, did [Ca]C response to caffeine attenuate significantly (C).
- Fig. 12.
Changes in [Ca]C caffeine are different from those of the calcium stores, measured in permeabilized cells. A–C, Control conditions. A, Baseline; B, response to a pulse of caffeine (10 mm, 300 msec); C, 3 min later. The cell was permeabilized for 2–3 min with intracellular medium containing 10 μm digitonin and ATP. [Ca]i was clamped by EGTA at 150 nm. D–F, Same sequence of exposure to caffeine, 5 min after the cell was permeabilized. Contrast-enhanced images.
- Fig. 13.
Nuclear reactivity to caffeine is correlated with the presence of calciosomes. The time course of change in nuclear [Ca]i was measured after application of 10 mmcaffeine. Experiments were conducted in young (A) and mature (B) cultures, and the cells were classified into three groups: cells containing no visible CCOs [group (gr.) 1], few CCOs (gr.2), and many CCOs (gr.3). In the older group, no organelle-free cells were found. In both age groups, a shorter latency to caffeine was seen in CCO-containing neurons. A smaller response was seen in CCO-free cells. C, Time course of appearance of CCOs. Cultures were stained with Fluo-3, and the proportion of cells containing these organelles was registered. (The total number of cells counted in each age group is depicted at the bottom of each column.)
