Article Figures & Data
Figures
- Figure 1.
ECS did not affect density of microglia and did not cause neuronal degeneration in mouse CA1 24 h after seizures. A, Representative MIPs from immunofluorescence image showing CX3CR1-GFP+ microglia in green and NeuN+ neurons in red in CA1 (stratum pyramidale = left of dashed line; stratum radiatum = right of dashed line) of control (top) and ECS (bottom) mice 24 h after seizures. B, Density of microglia in CA1sr microglia per 106 μm3 of CA1sr volume. Control animals had 26.69 ± 2.9 which was not significantly different from ECS animals that had 27.59 ± 2.7. C, Number of FJC-positive cells per field imaged (320 × 240 μm). Control and ECS-exposed animals have significantly lower degenerating cell densities in CA1 than SE-exposed animals: 4.2 ± 0.36 for controls (N = 9) and 3.6 ± 0.54 for ECS animals (N = 5), compared to 16.3 ± 1.5 for SE animals (N = 7). ****p < 0.0001.
- Figure 2.
ECS did not affect spontaneous motility of microglia in mouse CA1sr 24 h after seizures. A, B, Representative time-coded images (t = 0 in red, t = 20 min in green, overlap in yellow) of CA1sr fields (A) or single cells (B) from slices from control (right/top) and ECS (left/bottom) treated animals. C, ECS had no effect on mean EI (area of extensions/area of retractions) after 20 min of imaging under baseline conditions: 1.085 ± 0.041 for control slices and 1.067 ± 0.05 for ECS slices.
- Figure 3.
Unlike SE, ECS did not cause morphologic activation of CA1sr microglia. A, Representative binarized images of individual traced microglia from control, ECS or SE animals. B, SE, but not ECS, significantly decreased the number of 3D Sholl crossings in hippocampal microglia (for radii between 2 and 33 μm). C, SE significantly decreased the total number of branch points per cell, while ECS had no significant effect. D, SE significantly decreased the total filament length per cell, while ECS had no significant effect. E, SE significantly decreased the average number of primary branches, while ECS had no significant effect. *p < 0.05, ****p < 0.0001.
- Figure 4.
ECS increased expression of CCL2 without affecting Tnf or the microglial marker Tmem119. A, Hippocampi were lysed 24 h after seizures, and the ratio of CCL2 to total protein was measured in the lysates by ELISA and BCA assay. ECS and SE similarly induced a significant upregulation of relative CCL2 expression. B, After MACS isolation 24 h after ECS or sham ECS, microglial RNA samples were studied by hydrolysis probe-based qPCR. Relative fold change for each transcript assayed (the microglial marker Tmem119, the pro-inflammatory cytokine Tnf and the chemokine Ccl2) was determined by the 2–ΔΔCt method, normalizing to Actb levels. We observed significantly higher relative expression of Ccl2 mRNA in ECS microglial samples and no significant changes elsewhere. *p < 0.05, ****p < 0.0001.
- Figure 5.
ECS, like SE, potentiated the ATP-responsive motility of microglial processes in acute hippocampal slices. A, B, Representative MIPs of confocal zt-stacks showing the time course of the microglial response (in green) to 3 mM ATP in a patch pipette (in red) in acute hippocampal slices from control (A), ECS (B), and SE (C) animals. D, ECS and SE similarly increased the average process velocity during the microglial response to 3 mM ATP. E, ECS-induced enhancement of microglial responsive motility is concentration dependent: there was a small but significant difference in the responses to 1 mM ATP, while the responses to 10 mM were not significantly different. ****p < 0.0001.
- Figure 6.
ECS had no effect on intrinsic electrophysiological properties of CA1sr microglia. A, Representative photomicrograph showing GFP-labeled microglia superimposed with a 60× DIC image of the hippocampal slice. B, Current density-voltage relation in microglia was unchanged after ECS. Current amplitudes were measured at steady state during 500-ms voltage steps. Data are shown as mean±SEM. C–E, ECS did not affect microglial RMP (C), IR (D), or membrane capacitance (E).
- Figure 7.
ECS enhanced P2X7 current density in CA1sr microglia. A, B, Representative voltage-clamp traces showing the currents induced by local application of 1 mM ATP (black bar) in normal or 0CaMg aCSF (yellow bar), in microglia in slices from sham (A) versus ECS (B) animals. Cells were held at Vm = −60 mV, with 500 ms ramps from −120 to +50 mV every 10 s. C, D, Peak current density (current amplitude/cell capacitance, pA/pF) of 1 mM ATP-evoked currents in normal (C) and divalent cation-free/0CaMg aCSF (D) at Vm = −60 mV. ECS resulted in significantly increased current densities under both recording conditions. Panels on the right represent the average ATP-induced current/voltage relation obtained by subtracting the I/V curve obtained before from that obtained during the ATP application. As is expected for P2X-mediated currents, the I/V relation is linear and reverses around 0 mV. E, Peak current density (current amplitude/cell capacitance, pA/pF) of 1 mM ATP-evoked currents in normal aCSF at Vm = −60 mV, with or without preincubation in the specific P2X7 antagonist BBG. BBG significantly reduced the current density evoked by 1 mM ATP in normal aCSF in ECS cells only. F, Peak current density (pA/pF) of 1 mM ATP-evoked currents in divalent cation-free/0CaMg aCSF at Vm = −60 mV, with or without BBG preincubation. BBG significantly reduced the current density evoked by 1 mM ATP in 0CaMg aCSF in both control and ECS cells. **p < 0.01, ****p < 0.0001.
- Figure 8.
ECS did not change microglial expression of purinergic receptor transcripts. After MACS isolation 24 h after ECS or sham ECS, microglial RNA samples were studied by hydrolysis probe-based qPCR. Relative fold change for each purinergic receptor transcript assayed (the ionotropic receptors P2rx1, P2rx4, and P2rx7 and the metabotropic receptors P2ry6 and P2ry12) was determined by the 2–ΔΔCt method, normalizing to Actb levels. ECS did not have statistically significant effects on the expression of any of the studied genes.
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