Preferential pruning of inhibitory synapses by microglia contributes to alteration of the balance between excitatory and inhibitory synapses in the hippocampus in temporal lobe epilepsy

Abstract

Background: A consensus has formed that neural circuits in the brain underlie the pathogenesis of temporal lobe epilepsy (TLE). In particular, the synaptic excitation/inhibition balance (E/I balance) has been implicated in shifting towards elevated excitation during the development of TLE.

Methods: Sprague Dawley (SD) rats were intraperitoneally subjected to kainic acid (KA) to generate a model of TLE. Next, electroencephalography (EEG) recording was applied to verify the stability and detectability of spontaneous recurrent seizures (SRS) in rats. Moreover, hippocampal slices from rats and patients with mesial temporal lobe epilepsy (mTLE) were assessed using immunofluorescence to determine the alterations of excitatory and inhibitory synapses and microglial phagocytosis.

Results: We found that KA induced stable SRSs 14 days after status epilepticus (SE) onset. Furthermore, we discovered a continuous increase in excitatory synapses during epileptogenesis, where the total area of vesicular glutamate transporter 1 (vGluT1) rose considerably in the stratum radiatum (SR) of cornu ammonis 1 (CA1), the stratum lucidum (SL) of CA3, and the polymorphic layer (PML) of the dentate gyrus (DG). In contrast, inhibitory synapses decreased significantly, with the total area of glutamate decarboxylase 65 (GAD65) in the SL and PML diminishing enormously. Moreover, microglia conducted active synaptic phagocytosis after the formation of SRSs, especially in the SL and PML. Finally, microglia preferentially pruned inhibitory synapses during recurrent seizures in both rat and human hippocampal slices, which contributed to the synaptic alteration in hippocampal subregions.

Conclusions: Our findings elaborately characterize the alteration of neural circuits and demonstrate the selectivity of synaptic phagocytosis mediated by microglia in TLE, which could strengthen the comprehension of the pathogenesis of TLE and inspire potential therapeutic targets for epilepsy treatment.

Keywords: E/I balance; epilepsy; microglia; synapse; synaptic phagocytosis.

FIGURE 1

Kainic acid induces stable spontaneous recurrent seizures in rats. (A) The schematic diagram of experimental design. (B, C) Representative EEG recordings and energy spectra of the control and KA‐induced rats. (D–F) Quantification of the number of HPDs, average seizure duration and spiking amplitude indicates the SRSs in rats 14 days after KA induction. *: vs. control, *p < 0.05, **p < 0.01, ***p < 0.001; unpaired student's t test. Data are expressed as the mean ± SEM. n = 6 rats per group.

FIGURE 2

Kainic acid induces the alteration of excitatory synapses in the rats' hippocampus at different time points. (A) The regions of interest are shown in the hippocampus in the schematic diagram. (B, C) Representative vGluT1 (green) immunostaining from the functional regions of the CA1, CA3 and DG 7, 14, 28, and 63 days after KA induction. Scale bar = 10 μm. (D, E) Quantification of the total area of vGluT1 presents a gradual upwards tendency in the SP of CA1 and CA3 from days 7 to 28, which decreased to the control levels on day 63. (F) Quantification of the total area of vGluT1 reveals no obvious changes in the GCL of the DG at various time points following KA induction. (G–I) Quantification of the total area of vGluT1 presents a gradual upwards tendency in the SR of the hippocampal CA1, SL of CA3 and PML of DG from days 7 to 28, which decreases to the control levels on day 63. *: vs. control, *p < 0.05, **p < 0.01, ***p < 0.001, one‐way ANOVA with Tukey's post hoc test. Data were normalized to the control group. Data are expressed as the mean ± SEM. n = 18 images from six rats per group with three brain sections per rat.

FIGURE 3

Kainic acid induces the alteration of inhibitory synapses in the rats' hippocampus at different time points. (A) The regions of interest are shown in the hippocampus in the schematic diagram. (B, C) Representative GAD65 (red) immunostaining from the functional regions of the CA1, CA3 and DG 7, 14, 28, and 63 days after KA induction. Scale bar = 10 μm. (D, E) Quantification of the total area of GAD65 presents a gradual augmentation in SP of the CA1 and CA3 from days 7 to 28 and decreases to the control levels on day 63 after KA induction. (F) The total area of GAD65 shows no obvious changes in the GCL of DG at various time points following KA induction. (G) Quantification of the total area of GAD65 shows a gradual upwards tendency in the SR of the hippocampal CA1 from days 7 to 28, and decreases to the control levels on day 63 after KA induction. (H, I) Quantification of the total area of GAD65 shows a reduction in the SL of the CA3 and PML of the DG from days 7 to 63 after KA induction. (J–O) The relative content of vGluT1 and GAD65 shows a higher percentage of excitatory synapses compared to the control group in the SR, SL and PML but an inconspicuous propensity in the SP and GCL. *: vs. control, *p < 0.05, **p < 0.01, ***p < 0.001; one‐way ANOVA with Tukey's post hoc test. Data were normalized to the control group. Data are expressed as the mean ± SEM. n = 18 images from six rats per group with three brain sections per rat.

FIGURE 4

Kainic acid‐induced activated microglia conduct phagocytosis in the rats' hippocampus during the phase of recurrent seizures. (A) The schematic diagram shows the resting and activated states of microglia. (B, C) Representative confocal images display IBA1⁺ microglia in the functional regions of the CA1, CA3, and DG of the hippocampus at 7, 14, 28, and 63 days after KA induction. Scale bar = 20 μm. (D–I) Quantification of the numbers of activated microglia indicates a significant rise in the CA1, CA3, and DG of the hippocampus from days 7 to 63 after KA induction compared to the control rats, except the GCL of the DG, which demonstrates no obvious changes. (J–O) Quantification of the number of CD68⁺ microglia illustrates a significant increase in the CA1, CA3, and DG of the hippocampus from days 14 to 63 after KA induction compared to control rats, except the GCL of the DG, which exhibit no obvious changes. (P–U) Correlation analysis between activated microglia and CD68⁺ microglia in the SR, SL, and PML is depicted. The equation is calculated via linear regression analyses. Correlation analyses are performed by computing Pearson's correlation coefficient. *p < 0.05; **p < 0.01; ***p < 0.001; (H, J) one‐way ANOVA with Tukey's post hoc test; D‐G, I, K‐O: Kruskal‐Wallis with Dunn's multiple comparisons test. Data are expressed as the mean ± SEM. The quantification is from a one‐time experiment with three samples per group. Scale bar = 20 μm.

FIGURE 5

Kainic acid‐induced activated microglia preferentially prunes inhibitory synapses in the rats' hippocampus. (A) Representative confocal images are shown for CD68 (red), vGluT1 (green), and IBA1 (cyan) immunoreactive puncta in the hippocampal CA1 63 days after KA induction. (B) Immunostaining results are presented for CD68 (red), GAD65 (green) and IBA1 (cyan) in the hippocampal CA1 63 days after KA induction. High magnification confocal images display colocalization of the CD68 and GAD65 signals. (C) Quantification of the CD68 and vGluT1 immunofluorescent signals within IBA1⁺ microglia denotes no colocalization of lysosomes and excitatory synapses during KA‐induced recurrent seizures. (D) Quantification of CD68 and GAD65 immunofluorescent signals within IBA1⁺ microglia indicates phagocytosis of inhibitory synapses by microglia lysosomes during KA‐induced recurrent seizures. (E–J) Quantification of the number of CD68⁺ microglia with engulfed GAD65 illustrates a significant increase in the CA1 and CA3 of the hippocampus from 14 to 63 days after KA induction compared to control rats, where the PML has an obvious augment on days 28 and 63, whereas the GCL of the DG exhibits no obvious changes. *p < 0.05; **p < 0.01; ***p < 0.001; Kruskal‐Wallis with Dunn's multiple comparisons test. Data are expressed as the mean ± SEM. n = 18 images from six rats per group with three brain sections per rat. Scale bar = 10 μm.

FIGURE 6

The selective pruning of inhibitory synapses by microglia is validated in the hippocampus in mTLE patients. (A, B) A representative Nissl staining of the caudal levels of DG from the hippocampus of a patient with mTLE (A: scale bar = 500 μm; B: scale bar = 100 μm). (C, D) Representative confocal images are depicted for CD68 (red), vGluT1 (green), IBA1 (cyan) and CD68 (red), GAD65 (green), and IBA1 (cyan) immunoreactive puncta in the SR of the hippocampus in TLE patients. An orthogonal view of the confocal image revealed the colocalization of IBA1 (cyan), CD68 (red), and GAD65 (green), suggesting that microglia preferentially prune inhibitory synapses in the hippocampus of mTLE patients (scale bar = 10 μm). (E) Quantification of the number of CD68⁺ microglia with engulfed GAD65 or vGluT1 signals shows a greater number of CD68⁺ microglia with engulfed GAD65 than with engulfed vGluT1 in mTLE patients. (F, G) Quantification of CD68 and vGluT1 immunofluorescent signals in IBA1⁺ microglia indicated no colocalization of lysosomes and excitatory synapses, while quantification of CD68 and GAD65 immunofluorescent signals within IBA1⁺ microglia demonstrated phagocytosis of inhibitory synapses by microglial lysosomes in mTLE patients. ***p < 0.001; Kruskal‐Wallis with Dunn's multiple comparisons test. Data are expressed as the mean ± SEM. n = 18 images from six patients per group with three samples per patient.