The Journal of Neuroscience, October 4, 2006, ():
Impaired Volume Regulation is the Mechanism of Excitotoxic Sensitization to Complement
J. Neurosci. Loo and McNamara
26: 10177
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary fig 1. Correlation between volume measurements done with calcein fluorescence and with cross-sectional area using DIC. Overall, an excellent correlation was obtained with the two independent methods, except in the condition of lethal complement treatment. Neurons undergo swelling/RVD following various non-lethal osmotic perturbations as demonstrated using both calcein fluorescence (left panels) and DIC (right panels) of supp fig 1A (hypotonic), 1B (glutamate) and 1C (sublethal complement). Values are mean ± SEM for 5 cells per condition of a single experiment. Inset : Correlation plot of the recordings done with the calcein and cross-sectional area method. Each point represents the Vmax/V0 for a single cell. For cell swelling and RVD following hypotonic stress, the slope of the line depicting the ratio of the recordings obtained based on calcein and cross-sectional area was 1.05 (inset of supp fig 1A); the equivalent results obtained with these two independent methods supports the conclusion that each of these methods provides a valid measure of cell volume under these conditions. Similarly, following sublethal glutamate and sublethal complement insult, the slope of the line depicting the ratio was 1.02 and 0.94 respectively (inset fig 1B and 1C). By contrast, for lethal complement attack, the results with calcein indicated that the cells underwent massive swelling and could not undergo RVD. In addition, the estimate of cell swelling based upon calcein fluorescence is massive and greatly exceeds the estimates based upon direct measures of cross-sectional area as reflected in the ratio of the two methods exceeding 6 (inset fig 1D). The estimate based upon calcein fluorescence is likely an overestimate caused by loss of dye from the cell via channels of the membrane attack complex. That said, the volume measurements using DIC confirmed the cell swelling and lack of RVD (supp fig 1D, right panel).
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Supplementary fig 2. Excitotoxic insults sensitize cortical neurons to various osmotic perturbations and membrane impermeable osmolytes blocked the enhanced cytotoxicity following excitotoxic sensitization. (A) Cultures were exposed to increasing concentrations of glutamate (0-50 μM) for 10 min and washed with MEM. Following removal of glutamate, cultures were exposed to 60% hypotonic solution for 2 hr and cell death was assayed at the end of 2 hr by counting the number of trypan-blue labeled cells. (B) Membrane impermeable osmolytes blocked the enhanced cytotoxicity following excitotoxic sensitization to hypotonic stress. Cortical neurons were pretreated with glutamate (30 μM) or vehicle for 10 min, washed and incubated with 60% hypotonic solution in the presence or absence of dextran (1 mM) or polyethylene glycol (PEG) (50 mM) for 2 hr. Cell death was assayed by counting the number of trypan blue-labeled cells after 24 hr. Filled bars were with glutamate pretreatment while open bars were with vehicle pretreatment. (C) Cultures were exposed to increasing concentrations of glutamate (0-50 μM) for 10 min and washed with MEM. Following removal of glutamate, proteins of the terminal pathway were added sequentially and incubated for 2 hr. Cell death was assayed at the end of 2 hr by counting the number of trypan-blue labeled cells. Filled and open bars denote glutamate or vehicle pretreatment respectively. (D) Cortical neurons were pretreated with glutamate (30 μM) or vehicle for 10 min, washed, followed by C5b6+C7/C8/C9 (1 μg/ml each) in the presence or absence of dextran (1 mM) OR PEG (50 mM) for 2 hr. Cell death was assayed by counting the number of trypan blue-labeled cells after 24 hr. Filled bars were with glutamate pretreatment while open bars were with vehicle pretreatment. (E) Cortical neurons were pretreated with varying concentrations of glutamate for 10 min, washed with MEM and incubated with glutamate (30 μM) for 2 hr and cell death was assayed at the end of 2 hr by counting the number of trypan-blue labeled cells. Shaded bars were with second glutamate treatment while unshaded bars were with subsequent vehicle treatment. (F) Membrane impermeable osmolytes rescued neurons from enhanced cytotoxicity following excitotoxic sensitization. Cortical neurons were pretreated with glutamate (30 μM) or vehicle for 10 min, washed with MEM and incubated with a second glutamate (30 μM) treatment in the presence or absence of dextran (1 mM) OR PEG (50 mM) for 2 hr. Cell death was assayed by counting the number of trypan blue-labeled cells after 24 hr. Filled bars were with glutamate pretreatment while open bars were with vehicle pretreatment. Values are the means and SEM of the determinations made in six wells of a single experiment; each experiment was performed at least three times, and similar results were obtained.
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Supplementary fig 3. The phenomenon of excitotoxic sensitization is specific to glutamate. (A) Cultures were pretreated with vehicle or 60% hypotonic solution for 10 min, washed with an isotonic solution and subsequently treated with vehicle or 60% hyposmotic solution for 2 hr. (B) Cultures were pretreated with vehicle or complement (1 μg/ml) for 10 min, washed with media, and subsequently treated again with vehicle or complement proteins for 2 hr. (C) Cultures were pretreated with vehicle or 60% hypotonic solution for 10 min, washed with media, and subsequently incubated with vehicle or complement for a further 2 hr before performing LDH assay. (D) Cultures were pretreated with vehicle or complement (1 μg/ml) for 10 min, washed with media, and subsequently incubated with vehicle or 60% hypotonic solution for a further 2 hr before performing LDH assay. Values in this figure are the means and SEM of the determinations made in six wells of a single experiment; each experiment was performed at least three times, and similar results were obtained.
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Supplementary fig 4. Extracellular Ca++ is necessary for excitotoxic sensitization. (A) Removal of Ca++o during excitotoxin exposure abolished sensitization to various volume perturbations. Cells were treated with glutamate (30 μM) for 10 min in the presence (1.8 mM) or absence of Ca++o, followed by exposure to hypotonic solution (60%), terminal pathway proteins (1 μg/ml each) or glutamate (30 μM) for 2 hr. LDH released into the media was assayed. Values are the means and SEM of the determinations made in six wells of a single experiment. (B) Removal of Ca++o during excitotoxin exposure abolished sensitization to hypotonic stress. Cells were treated with glutamate (30 μM) for 10 min in the presence or absence of Ca++o, followed by exposure to hypotonic solution (60%). (C) Removal of Ca++o during excitotoxin exposure abolished sensitization to complement insult. Cells were treated with glutamate (30 μM) for 10 min in the presence or absence of Ca++o, followed by exposure to terminal pathway proteins (1 μg/ml each) while measuring cell volume. (D) Removal of Ca++o during excitotoxin exposure abolished sensitization to glutamate insult. Cells were treated with glutamate (30 μM) for 10 min in the presence or absence of Ca++o, followed by exposure to glutamate (30 μM) while measuring cell volume. Values in panels B, C and D are mean ± SEM for 5 cells per condition of a single experiment.
