Article Figures & Data
Figures
- Figure 1.
Experimental seizures trigger microglial process convergence (MPCs). a, Seizure scores after intraperitoneal delivery of pilocarpine (300 mg/kg) and kainic acid (18–22 mg/kg). b–d, Representative images of MPCs in acute slices from mice after kainic acid treatment. c, d, Time-lapse images of boxed regions in b showing converging microglial process foci identified with white arrows. e, Quantitation of MPCs under control, pilocarpine, and kainic acid treatment (n = 9–14 slices each). f, g, Ionotropic glutamate receptor antagonists and action potential blockers fail to block pilocarpine- (f) or kainic acid– (g) induced MPCs (n = 5–9 slices each). h, NMDA receptor antagonism during kainic acid–induced seizures reduces MPCs (n = 8–12 slices each). i–k, Representative images of MPCs in vivo from mice after kainic acid treatment. j, k, Time-lapse images of boxed regions in i showing MPC foci identified with white arrows. l, Quantification of MPCs in vivo after kainic acid–induced seizures (n = 4–5 mice each). **p < 0.01; ***p < 0.001.
- Figure 2.
Transient glutamate treatment mimics seizure-induced MPCs. a–c, Images from a time-lapse sequence showing converging microglial processes in a slice after glutamate (10 mm) treatment for 10 min. b, c, Time-lapse images of boxed regions in a showing converging microglial processes foci identified with white arrows. d, e, Schematic of sites of process convergence events (d) and quantified summary (e) showing increased occurrence after glutamate treatment (red arrowhead in e, n = 3 slices each). f, g, Quantification of the number of process convergence events under different conditions showing a role for NMDA receptors (n = 5–14 slices each). *p < 0.05; ***p < 0.001 compared with control and #p < 0.01 compared with previous condition in the respective graphs.
- Figure 3.
Characteristics of glutamate-induced MPCs. a–d, Schematic representation (a, c) and quantitative summary (b, d) showing the occurrence of MPC events at various glutamate concentrations for 10 min (a, b) and various time points at 1 mm (c, d). n = 6–11 slices each. e, f, Microglia respond from similar distances in all conditions of glutamate exposure tested. Glutamate-induced MPCs show similar features in the different conditions. **p < 0.01; ***p < 0.001 compared with control.
- Figure 4.
Neurons respond functionally to multiple hits of glutamate. Images (a, b) and time series (a′, b′) data showing intracellular calcium transients in neuronal somata (top) and dendrites (bottom) from GCaMP2.2 mouse slices after repeated glutamate (1 mm) treatment in cortical layer II/III.
- Figure 5.
Comparison between calcium reduction–induced and glutamate-induced MPCs. a–c, Quantitative summary of MPC events in slices from wild-type and P2Y12-deficient mice after pilocarpine-induced seizures (a), kainic acid–induced seizures (b) and 10-min glutamate treatment (c) (n = 3–7 slices each). d, Example of a converging microglial (green) process (arrow) terminating on a neuronal dendrite (red) after glutamate treatment in a slice from a double transgenic CX3CR1GFP/+:Thy1YFP/+ mouse. f, g, Schematic representation of sites of convergence foci (f) and quantitative summary data (g) showing sites and number of cells that respond during the different treatments. h–j, Various features of MPCs are similar between the two methods of convergence (n = 6–12 slices each). ***p < 0.001.
- Figure 6.
Fractalkine signaling is necessary and sufficient to trigger MPCs. a, b, Images from a time-lapse sequence (a) and summary schematic (b) showing converging microglial processes in slices from CX3CR1GFP/+ and CX3CR1GFP/GFP after glutamate treatment. c–f, Quantification of MPC numbers in slices from CX3CR1GFP/+ and CX3CR1GFP/GFP after glutamate treatment (c), kainic acid–induced seizures (d), and pilocarpine-induced seizures (e) showing reduced events in CX3CR1GFP/GFP slices as well as under basal conditions (f; n = 7–13 slices each). g–i, Quantified summary of MPC numbers after CX3CL1 (200 ng/mL) application in slices from CX3CR1GFP/+ (g, h) and CX3CR1GFP/GFP (i) mice (n = 6–8 slices each). ***p < 0.001.
- Figure 7.
Glutamate-induced MPCs requires IL-1β. a–c, Quantified summary (a, c) and schematic representation (b) showing that IL-1β (30 ng/mL) increases MPCs in slices from both CX3CR1GFP/+ and CX3CR1GFP/GFP mice (n = 6–17 slices). d, CX3CL1-induced MPCs is blocked by IL-1ra (100 ng/mL), an IL-1β antagonist, in slices from CX3CR1GFP/+ mice (n = 8–9 slices each). e–g, Glutamate-induced MPCs fails to occur in the presence of function-blocking CX3CL1 antibodies and is reduced in the presence of IL-1ra (n = 10–12 slices each). **p < 0.01; ***p < 0.001.
- Figure 8.
Neuroprotective potential of MPCs. a, b, Behavioral seizure scores in aged-matched wild type (n = 7), CX3CR1GFP/+ (n = 11), and CX3CR1GFP/GFP (n = 9) mice treated with kainic acid through time (a) and on average (b). c, Pie chart showing the percentage of mice from the seizure experiments that progressed to at least stage 3 seizures (red) along the Racine scale during the 2 h of seizure monitoring after kainic acid treatment. ***p < 0.001.
- Figure 9.
Model for glutamate-induced MPCs by fractalkine receptor–dependent signaling through IL-1β. Our results suggest a bidirectional mechanism of MPC in which (1) excessive glutamate release (2) activates neuronal NMDA receptors, which results in (3) the release of fractalkine from neuronal membranes that activates microglial fractalkine receptors. (4) Fractalkine receptor activation subsequently triggers IL-1β release from microglia, which in turn increases neuronal excitability to elicit (5) a localized release of ATP at specific dendritic hotspots. Finally, (6) ATP released from these hotspots attracts microglial processes via P2Y12 receptor to converge at the release site.
Movies
- Movie 1.
Microglial process convergence occurs after seizures. Representative time-lapse movies taken from CX3CR1GFP/+ mouse slices showing MPCs events. Slices were generated from the brains of mice 2 h after kainic acid treatment. Several MPCs events are identified (white arrows). This movie is 140 min long and is sped up 120×.
- Movie 2.
Glutamate induces MPCs. Representative time-lapse movie taken from slices excised from a CX3CR1GFP/+ mouse previously exposed to 10 min of 1 mm glutamate. Three MPC events can be seen in different regions of the slice and are identified with white arrows. This movie is 75 min long and is sped up 180×.
- Movie 3.
Glutamate-induced MPCs targets neuronal dendrites. Representative time-lapse movie taken from slices excised from a CX3CR1GFP/+;Thy1YFP/+ mouse previously exposed to 10 min of glutamate. A microglial process (green) convergence event terminating on a labeled dendrite (red) is identified with a white arrow. This movie is 48 min long and is sped up 240×.
- Movie 4.
Microglial process convergence in fractalkine receptor heterozygote (left) and knockout (right) slices after glutamate treatment. Representative time-lapse movie taken from CX3CR1GFP/+ (i.e., CX3CR1+/–, left) and CX3CR1GFP/GFP (i.e., CX3CR1–/–, right) slices showing several MPC events (arrows) after a 10-min glutamate (1 mm) treatment. This movie is 30 min long and is sped up 180×.
- Movie 5.
IL-1β increases MPCs. Representative time-lapse movie taken from CX3CR1GFP/+ showing that IL-1β (30 ng/mL) increases the occurrence of MPCs (arrows). This movie is 70 min long and is sped up 180×.
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