Table 1.
Target sequences used to design probe sets for NanoString nCounter analysis.
Figure 1.
NanoString analysis of expression changes for selected genes in response to IL4- and IL10-treatment.
Treating rat primary microglia for 6 hr with 20 ng/ml IL4 or 20 ng/ml IL10 resulted in gene expression changes that were specific for IL4 (A), specific for IL10 (B), or common to both treatments (C). mRNA expression was normalized to the housekeeping gene, HPRT1, and shown as mean ± SEM with the number of individual cultures indicated on each bar. [Note the differing Y-axis scales.] One-way ANOVA with Tukey's post-hoc test revealed differences from unstimulated (control) microglia: *p<0.05, **p<0.01, ***p<0.001.
Figure 2.
Effect of microglial activation state and selected channel inhibitors on podosome (podonut) expression.
A. Representative fluorescence micrographs show podonut expression in unstimulated primary rat microglia or 24 hr after treatment with LPS (10 ng/ml), IL4 (20 ng/ml) or IL10 (20 ng/ml). The arrows indicate examples of a single, large donut-shaped ring of podosomes, which we call a ‘podonut’ in the lamellum of unipolar microglia. Cells were stained for filamentous (F-) actin with phalloidin (green) to quantify the proportion of cells with a podonut, and with the nuclear marker, DAPI (blue) to quantify total cell numbers. Scale bar, 50 µm. B. Podosome (podonut) expression is affected by the microglial activation state. The proportion of microglial cells bearing a podonut in the lamellum (sum of 3 randomly selected fields of view at 10× magnification) was normalized to unstimulated cells. C–E. Microglia were unstimulated or stimulated for 24 hr with 20 ng/ml IL4 or IL10, and then exposed for 6 hr to control medium or the KCa2.3 inhibitors, 5 nM tamapin or 7 µM NS8593, which also inhibits TRPM7 channels (see Results and Figs. 6, 7). All graphical data are shown as mean ± SEM with sample size (# of individual cultures) indicated on each bar. A one-way ANOVA with Tukey's post-hoc analysis was used to determine significant differences. *p<0.05; ***p<0.001.
Figure 3.
Migration of primary rat microglia is affected by the activation state, and by KCa2.3 and TRPM7 inhibitors.
A. Microglia in the upper well of Transwells were unstimulated or stimulated with either 20 ng/ml IL4 or 20 ng/ml IL10 for 24 hr. The number of cells that migrated to the underside of the filter was then counted and normalized to control (unstimulated) cells, indicated by the dashed line. B–D. Microglia were unstimulated or stimulated with IL4 or IL10 as in panel A, with or without a channel inhibitor: 100 nM apamin, 5 nM tamapin, 7 µM NS8593 or 10 µM AA-861. E–H. IL4 and IL10 increase the invasion capacity of rat microglia, and TRPM7 is involved. Microglia were plated in the upper wells of Matrigel chambers, with or without stimulation by 20 ng/ml IL4 or 20 ng/ml IL10 for 24 hr. E. Invasion of control (unstimulated) microglia was compared with IL4- and IL10-treated cells. F–H. Microglia were unstimulated or stimulated with IL4 or IL10 as in panel A, with or without simultaneous addition of a channel inhibitor: 100 nM apamin, 7 µM NS8593 or 10 µM AA-861. For each stimulus, the number of cells that had migrated or invaded was normalized to the level without a channel inhibitor. The dashed line in all graphs indicates the level in control (unstimulated) cells. Data are expressed as mean ± SEM with the number of individual cultures indicated on each bar. A one-way ANOVA with Tukey's post-hoc test was used to compare results with and without a channel inhibitor; *p<0.05, **p<0.01, ***p<0.001; or for NS8593 versus AA-861 (†p<0.05).
Figure 4.
Migration of MLS-9 microglial cells is increased by IL4 and IL10, and involves TRPM7, not KCa2.3.
A. Representative fluorescence micrographs show MLS-9 cells (rat microglial cell line), unstimulated or 24 hr after stimulation with LPS (100 ng/ml), IL4 (20 ng/ml) or IL10 (20 ng/ml). Cells were stained for F-actin with phalloidin (green) and the nuclear marker, DAPI (blue). Most cells were bipolar with membrane ruffling at one end (examples shown by arrows). Scale bar, 50 µm. B. MLS-9 microglial cells in the upper well of Transwells were unstimulated, or stimulated (24 hr) with 20 or 100 ng/ml IL4, or with 20 or 100 ng/ml IL10. The number of cells that migrated to the underside of the filter was then counted and normalized to control (unstimulated) cells. The dashed line in both graphs indicates the level in control (unstimulated) cells. C. Cells were stimulated as in panel B, with or without a channel inhibitor: 100 nM apamin, 5 nM tamapin, or 7 µM NS8593. For each stimulus, the number of cells that had migrated was normalized to the level without a channel inhibitor. Data are expressed as mean ± SEM with the number of individual cultures indicated on each bar. In panel B, a two-way ANOVA with Bonferroni post-hoc analysis was used to determine significant differences between stimulation (*) and dose (†). In panel C, A one-way ANOVA with Tukey's post-hoc test was used to determine treatment effects. One symbol, p<0.05; two symbols, p<0.01; three symbols, p<0.001.
Figure 5.
KCNN3 expression and KCa2.3 current inhibition by NS8593 in microglia in differing activation states.
A. Expression of KCNN3 mRNA was quantified using NanoString nCounter analysis in unstimulated primary microglia, and at 6 hr (solid bars) and 24 hr (striped bars) after treatment with 20 ng/ml IL4- or IL10-treatment. B–D. NS8593 inhibits the KCa2.3 current, while AA-861 has no effect. KCa2.3 currents were recorded from MLS-9 microglial cells in the perforated-patch configuration produced by amphotericin B (200 µg/ml) in the pipette. The voltage protocol throughout was a 120 ms-long voltage ramp from −100 to +80 mV from a holding potential of −70 mV. The bath always contained 1 µM TRAM-34 to block KCa3.1 currents. Riluzole (300 µM) was used simply as a tool to activate the KCa2.3 current [21]. B. Upper panel: Representative traces show the current evoked by riluzole with or without 7 µM NS8593. Lower panel: The representative time course (current measured at +80 mV) illustrates KCa2.3 current activation and its inhibition by NS8593. C. Representative currents and time course show that no current was activated when 7 µM NS8593 was present in the bath. D. The current is insensitive to 10 µM AA-861. Note that current activation by riluzole is readily reversible (wash), as we previously showed [21]. E–G. The KCa2.3 current in primary rat microglial cells is inhibited by 7 µM NS8593 under differing activation states. Currents were recorded using the same patch-clamp configuration as described for MLS-9 cells. Upper panels: Representative currents in microglia that were unstimulated (E), or treated for 24 hr with 20 ng/mL IL4 (F) or 20 ng/mL IL10 (G). Lower panels: A representative time course for each cell (current at +80 mV) shows activation by riluzole and inhibition by NS8593.
Figure 6.
NS8593 and AA-861 inhibit the TRPM7 current in primary rat microglia and MLS-9 microglial cells.
TRPM7 currents were recorded in the whole-cell configuration using a Mg2+-free pipette solution. The voltage protocol throughout was a 120 ms-long ramp from −100 to +115 mV, from a holding potential of −10 mV. Graphical data are presented as mean ± SEM for the number of cells indicated on each bar, and were compared using an unpaired t-test: ***p<0.001. A–D. Representative currents from primary rat microglial cells (A, B) and MLS-9 microglial cells (C, D) with Mg2+-free intracellular solution (no MgCl2, 10 mM EGTA). Upper panels: Currents are shown with and without 7 µM NS8593 (A, C) or 10 µM AA-861 (B, D) in the bath. Lower panels: Representative time courses (current measured at +115 mV) show current activation, and inhibition by 7 µM NS8593 (A, C) or 10 µM AA-861 (B, D). [The reversibility of NS8593 is shown in panel C.] E. Comparison of the TRPM7 current amplitude (measured at +115 mV) in primary microglia and MLS-9 cells. F, G. Comparison of TRPM7 current inhibition by 7 µM NS8593 (F) and by 10 µM AA-861 (G) in primary microglia and MLS-9 microglial cells.
Figure 7.
Mg2+-dependence of TRPM7 current block by NS8593.
TRPM7 currents were recorded in MLS-9 microglial cells in the whole-cell configuration with differing free Mg2+ concentrations in the pipette solution. The voltage protocol throughout was a 120 ms-long ramp from −100 to +115 mV, from a holding potential of −10 mV. Graphical data are presented as mean ± SEM for the number of cells indicated on each bar, and were compared using a one-way ANOVA. A–D. Effect of 75 µM and 300 µM intracellular free Mg2+ on TRPM7 currents and their inhibition by NS8593. In panels A and B, upper traces are representative currents with and without 7 µM NS8593, and the lower panels (time course of current at +115 mV) show the rapid onset and reversibility of inhibition by NS8593. TRPM7 current amplitude (at +115 mV; panel C), and percent inhibition by 7 µM NS8593 (panel D) are compared for 75 µM and 300 µM intracellular free Mg2+.
Figure 8.
Effects of IL4- and IL10-treatment on TRPM7 expression, current and block by NS8593 in primary rat microglia.
A. TRPM7 mRNA expression was measured using quantitative real-time RT-PCR at 6 hr (solid bars) and 24 hr (striped bars) in unstimulated rat microglia or after treatment with 20 ng/ml IL4 or 20 ng/ml IL10. Values are expressed as mean expression (normalized to HPRT1) ± SEM, with the number of individual cultures indicated on each bar. *p<0.05 indicates the difference from time-matched control (unstimulated) cells, and was determined using a 2-way ANOVA followed by Bonferroni post-hoc test. B–E. The TRPM7 current and block by NS8593 were not affected by 24 hr treatment with 20 ng/ml IL4 or IL10. Whole-cell recordings were performed on primary rat microglia using Mg2+-free pipette solution. B, C. Representative currents (upper panels) in response to 120 ms-long ramps from −100 to +115 mV, from a holding potential of −10 mV. Time course of the current (lower panels) measured at +115 mV. D, E. Summary of TRPM7 current amplitudes measured at +115 mV (D) and percent inhibition of TRPM7 currents by 7 µM NS8593 (E). Graphical data are presented as mean ± SEM for the number of cells indicated on each bar, and were compared using a one-way ANOVA with Tukey's post-hoc test.