Increased expression of chondroitin sulfate proteoglycans in dentate gyrus and amygdala causes postinfectious seizures

Abstract

Alterations in the extracellular matrix are common in patients with epilepsy and animal models of epilepsy, yet whether they are the cause or consequence of seizures and epilepsy development is unknown. Using Theiler's murine encephalomyelitis virus (TMEV) infection-induced model of acquired epilepsy, we found de novo expression of chondroitin sulfate proteoglycans (CSPGs), a major extracellular matrix component, in dentate gyrus (DG) and amygdala exclusively in mice with acute seizures. Preventing the synthesis of CSPGs specifically in DG and amygdala by deletion of the major CSPG aggrecan reduced seizure burden. Patch-clamp recordings from dentate granule cells revealed enhanced intrinsic and synaptic excitability in seizing mice that was significantly ameliorated by aggrecan deletion. In situ experiments suggested that dentate granule cell hyperexcitability results from negatively charged CSPGs increasing stationary cations on the membrane, thereby depolarizing neurons, increasing their intrinsic and synaptic excitability. These results show increased expression of CSPGs in the DG and amygdala as one of the causal factors for TMEV-induced acute seizures. We also show identical changes in CSPGs in pilocarpine-induced epilepsy, suggesting that enhanced CSPGs in the DG and amygdala may be a common ictogenic factor and potential therapeutic target.

Keywords: hippocampus; matrix metalloproteinases; minocycline; potassium; resting membrane potential.

Figure 1

Increased deposition of CSPGs in dentate gyrus and amygdala of Theiler’s murine encephalomyelitis virus (TMEV)-infected mice with acute seizures. (A) Comparative images of the coronal brain hemislices obtained from mice treated with saline (Sham) or TMEV (TMEV S−: mice without acute seizures; TMEV S+: mice with acute seizures) at 5 days post-infection (dpi) and stained with the markers for CSPG [wisteria floribunda agglutinin (WFA), in green] and neurons (NeuN, in red). (B) Enlarged views of hippocampus show substantial increase in the level of CSPGs in dentate gyrus (DG) from the TMEV S+ group. A lack of CSPGs in the CA1 region is due to loss of CA1 pyramidal neurons common during acute TMEV-induced seizures. The CSPG expression pattern and neuronal density in the TMEV S– group were comparable to the Sham group. DG-GL = granular layer of DG; DG-ML = molecular layer of DG. (C) Enlarged views of amygdala show a substantial increase in the level of CSPGs in the TMEV S+ group. The CSPG expression pattern in the TMEV S– group was comparable to the Sham group. BLA = basolateral amygdala; CeA = central nucleus of amygdala; LA = lateral amygdala. (D–F) Image analysis shows a significant increase in mean fluorescence intensity of WFA in the DG-GL (D), DG-ML (E) and amygdala (F) of the TMEV S+ group compared with the Sham and TMEV S– groups (one-way ANOVA, Tukey’s multiple comparisons test; n = 10–11 brain slices from 4–5 mice per group; **P < 0.01, ****P < 0.0001). (G–H) Representative high-resolution images of excitatory neurons from the DG-GL (G) and amygdala (H), showing deposition of CSPGs around soma only in the TMEV S+ group. (I) Representative high-resolution images of parvalbumin (PV)-containing inhibitory interneurons from somatosensory cortex (SSCTX), amygdala and DG-GL, showing a classical perineuronal net (PNN) structure surrounding soma and proximal dendrites. The images shown in G–I are maximum intensity projections of z-stack confocal images from brain slices stained with WFA (green), NeuN (red) and PV (blue). In A–C and G–I, scale bars apply to all images in the panel.

Figure 2

Minocycline treatment significantly reduces Theiler’s murine encephalomyelitis virus (TMEV)-induced acute seizures and inhibits the changes in CSPG expression associated with TMEV-induced acute seizures in dentate gyrus and amygdala. (A) Experimental timeline. Mice were sacrificed at 7 days post-infection (dpi) and the brains were either fixed for histology or dissected into several regions and flash-frozen for biochemical analysis. (B) Heat map shows handling-induced seizures observed based on their severity score for each mouse between 3 and 7 dpi. Mice were handled four times a day—during minocycline (MIN) or vehicle (Veh) injections twice daily and during seizure monitoring twice daily—with at least 2 h without disturbance between each handling session. (C) Percentage of total infected mice in each group that remained seizure-free each day. None of the mice developed seizures before 3 dpi. (D) Average number of seizures per day between 3 and 7 dpi plotted for each mouse shows a significant reduction in seizure frequency under minocycline treatment (n = 12, unpaired two-tailed t-test, **P < 0.01). (E) Average cumulative seizure burden (calculated as a mean of the summation of all seizure scores for each mouse up to each dpi) shows a significant reduction in seizure severity from 5 dpi under minocycline treatment (mean ± SEM, n = 12, two-way ANOVA, Šidák’s multiple comparisons test; ***P < 0.01, ****P < 0.0001). (F) Distribution of seizures based on seizure severity score. (G) Mean seizure score at each dpi shows significant reduction in TMEV-infected mice treated with minocycline (mean ± SEM, n = 12, two-way ANOVA, Šidák’s multiple comparisons test; *P < 0.05). (H) Representative micrographs of dentate gyrus (DG) comparing the expression of CSPGs and neuronal density between mice infected intracortically with either TMEV (T) or Sham (S) and treated intraperitoneally with either minocycline (M) or vehicle (V). The brain slices were obtained at 7 dpi. (I–J) Image analysis shows that minocycline treatment significantly reduced the increased deposition of CSPGs [mean fluorescence intensity of wisteria floribunda agglutinin (WFA)] in the granular layer of DG (DG-GL) (I) and molecular layer of DG (DG-ML) (J) from TMEV-infected mice compared with the T + V group. The data from the S + V and S + M groups are combined, as minocycline had no effect on the expression of CSPGs and neuronal density in mice infected with Sham. (K) Representative micrographs of amygdala comparing the expression of CSPGs and neuronal density among four groups of mice. The brains slices were obtained at 7 dpi. (L) Similar to DG, minocycline treatment partially normalizes the expression of CSPGs in amygdala from TMEV-infected mice. Statistics (I–J and L): one-way ANOVA or Brown–Forsythe and Welch ANOVA, Tukey’s or Dunnett’s T3 multiple comparisons test; n = 12–15 brain slices from 5–6 mice (T + V, T + M), n = 8–9 brain slices from 2–3 mice (S + V, S + M); *P < 0.05, ****P < 0.0001. In H and K, scale bars apply to all images in the panel.

Figure 3

Deletion of aggrecan specifically in both dentate granule cells and amygdala significantly reduces Theiler’s murine encephalomyelitis virus (TMEV)-induced acute seizures. (A) Experimental timeline. Acan^fl/flPOMC-cre− mice were injected in amygdala bilaterally with AAV9-hSyn-eGFP to obtain Acan^+/+ mice (control), whereas Acan^fl/flPOMC-cre+ mice were injected in amygdala bilaterally with AAV9-hSyn-eGFP or AAV9-hSyn-eGFP-Cre to obtain DG-Acan^−/− and DG-AMG-Acan^−/− mice, respectively. All mice were infected with TMEV 10–14 days after injections of AAV9 constructs. (B) Heat map shows handling-induced seizures observed based on their severity score for each mouse between 3–8 days post-infection (dpi). Seizures were induced twice daily by handling the mice, with at least 2 h undisturbed between each handling session. (C) Average weight of mice each day during acute TMEV infection period is not different for all three groups of mice. (D) Percentage of total infected mice in each group that remained seizure-free each day. None of the mice developed seizures before 3 dpi. (E) Average number of seizures per day between 3–8 dpi plotted for each mouse shows a significant reduction in seizure frequency in DG-AMG-Acan^−/− mice compared with Acan^+/+ control mice (n = 9–10, one-way ANOVA, Tukey’s multiple comparisons test, **P < 0.01). (F) Average cumulative seizure burden, which is calculated as a mean of the summation of all seizure scores for each mouse up to each dpi, shows a significant reduction in seizure severity between 6–8 dpi in DG-AMG-Acan^−/− mice compared with the other groups (data shown as mean ± SEM, n = 9–10, two-way ANOVA, Bonferroni’s multiple comparisons test; *comparisons between DG-AMG-Acan^−/− and Acan^+/+, ^#comparisons between DG-AMG-Acan^−/− and DG-Acan^−/−; **P < 0.01, ****P < 0.0001, ^#P < 0.05, ^###P < 0.001). (G) Mean seizure score at each dpi shows significant reduction in DG-AMG-Acan^−/− mice (data shown as mean ± SEM, n = 9–10, two-way ANOVA, Šidák’s multiple comparisons test, *P < 0.05, **P < 0.01, ^#P < 0.05). (H) Distribution of seizures based on seizure severity score. (I) Representative micrographs show increased expression of CSPGs in DG at 10 dpi in Acan^+/+ mice, but not in DG-Acan^−/− and DG-AMG-Acan^−/− mice, confirming deletion of the acan gene in dentate granule cells (DGCs) in DG-Acan^−/− and DG-AMG-Acan^−/− mice. (J and K) Image analysis shows significantly increased deposition of CSPGs [mean fluorescence intensity of wisteria floribunda agglutinin (WFA)] in the granular layer of DG (DG-GL) (J) and molecular layer of DG (DG-ML) (K) in Acan^+/+ mice compared with DG-Acan^−/− and DG-AMG-Acan^−/− mice. (L) Representative micrographs of amygdala show increased expression of CSPGs at 10 dpi in Acan^+/+ and DG-Acan^−/− mice, but not in DG-AMG-Acan^−/− mice, confirming deletion of acan gene in amygdala in DG-AMG-Acan^−/− mice. (M) Mean fluorescence intensity of WFA in amygdala is significantly higher in Acan^+/+ and DG-Acan^−/− mice compared with DG-AMG-Acan^−/− mice. Statistics (J, K and M): one-way ANOVA or Brown–Forsythe and Welch ANOVA, Tukey’s or Dunnett’s T3 multiple comparisons test; n = 10–11 brain slices (single slice per mouse); ***P < 0.001, ****P < 0.0001. In I and L, scale bars apply to all images in the panel.

Figure 4

Increased intrinsic and synaptic excitability of dentate granule cells during Theiler’s murine encephalomyelitis virus (TMEV)-induced acute seizures. (A) Representative micrographs of hippocampus from acute brain slices (300-µm thick) used for patch-clamp recordings show an increased level of CSPGs, as stained with wisteria floribunda agglutinin (WFA, green), in dentate gyrus (DG) and degradation of CSPG in the CA1 region due to neuronal loss from TMEV-infected mice with acute seizures. The scale bar applies to both images. (B) Mean resting membrane potential of dentate granule cells (DGCs) is significantly depolarized in the TMEV group (n = 36–37 cells from 4–6 mice, unpaired two-tailed t-test, ***P < 0.001). (C) Mean input resistance of DGCs is significantly increased in the TMEV group (n = 36–37 cells from 4–6 mice, unpaired two-tailed t-test, ****P < 0.0001). (D) Mean membrane capacitance of DGCs shows no difference between both the groups. (E) Representative traces of action potentials recorded from DGCs of sham- and TMEV-infected mice. (F) The number of action potentials induced by injecting currents of varying strength in DGCs are significantly higher in TMEV-infected mice (data shown as mean ± SEM, n = 36–37 cells from 4–6 mice, two-way ANOVA, Šidák’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (G) A significant increase in mean amplitude (top left), but not mean frequency (bottom left), of spontaneous excitatory postsynaptic currents (sEPSC) recorded from DGCs of TMEV-infected mice. Cumulative distributions show a shift toward higher amplitude (top right) and lower interevent interval (bottom right), indicating a higher frequency, of sEPSC from TMEV-infected mice. (H) No change in mean amplitude (top left) and frequency (bottom left) of spontaneous inhibitory postsynaptic currents (sIPSC) from TMEV-infected mice. Cumulative distributions of amplitude (top right) and interevent interval (bottom right) of sIPSC from TMEV-infected mice are comparable to the control mice. (I) A significant increase in mean amplitude (top left) and mean frequency (bottom left) of miniature excitatory postsynaptic currents (mEPSC) from TMEV-infected mice. Cumulative distributions show a shift toward higher amplitude (top right) and lower interevent interval (bottom right), indicating a higher frequency, of mEPSC from TMEV-infected mice. (J) A significant increase in mean amplitude (top left) and mean frequency (bottom left) of miniature inhibitory postsynaptic currents (mIPSC) from TMEV-infected mice. Cumulative distributions show a shift toward higher amplitude (top right) and lower interevent interval (bottom right), indicating a higher frequency, of mIPSC from TMEV-infected mice. Statistics (G–J): bar graphs, n = 23–28 cells from 4–6 mice (spontaneous), n = 17–25 cells from 4–6 mice (miniature), unpaired two-tailed t-test or Welch’s t-test; cumulative fractions, Kolmogorov–Smirnov test; **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Resting membrane potential of dentate granule cells from DG-AMG-Acan^−/− mice is hyperpolarized but there is no change in action potential firing rate and threshold current compared with Acan^+/+ mice during acute Theiler’s murine encephalomyelitis virus infection. (A) Representative micrographs of hippocampus from acute brain slices (300-µm thick) used for patch-clamp recordings show a lack of increase in the level of CSPGs, as stained with wisteria floribunda agglutinin (WFA, green), in dentate gyrus (DG) from TMEV-infected DG-AMG-Acan^−/− mice with acute seizures. The scale bar applies to both the images. (B and C) Mean input resistance (B) and mean membrane capacitance (C) of dentate granule cells (DGCs) show no difference between Acan^+/+ and DG-AMG-Acan^−/− mice. (D) Mean resting membrane potential of the DGCs is significantly lower in DG-AMG-Acan^−/− mice (n = 26–28 cells from 4–6 mice, unpaired two-tailed t-test, *P < 0.05). (E) Representative traces of action potentials recorded from DGCs from Acan^+/+ and DG-AMG-Acan^−/− mice. (F) No difference in number of action potentials induced by injecting currents of varying strength in DGCs between both the groups. (G) Representative traces of voltage recordings in response to stepwise current injections from 0 to 600 pA (2 pA per step) to identify minimum amount of current needed to generate action potentials (APs) consistently (AP threshold current). (H) No difference in the AP threshold current between Acan^+/+ and DG-AMG-Acan^−/− mice.

Figure 6

Decreased synaptic excitability of dentate granule cells from DG-AMG-Acan^−/− mice compared with Acan^+/+ mice during acute Theiler’s murine encephalomyelitis virus (TMEV) infection. (A) No change in mean amplitude (top left) and frequency (bottom left) of spontaneous excitatory postsynaptic currents (sEPSC) between Acan^+/+ and DG-AMG-Acan^−/− mice. Cumulative distributions show a shift toward lower amplitude (top right) and higher interevent interval (bottom right), indicating a lower frequency, of sEPSC from DG-AMG-Acan^−/− mice. (B) A significant increase in mean amplitude (top left), but not mean frequency (bottom left), of spontaneous inhibitory postsynaptic currents (sIPSC) recorded from dentate granule cells (DGCs) of TMEV-infected DG-AMG-Acan^−/− mice. No difference in cumulative distributions of amplitude (top right) and interevent interval (bottom right) of sIPSC between both groups of mice. (C) A significant decrease in mean frequency (bottom left), but not mean amplitude (top left), of miniature excitatory postsynaptic currents (mEPSC) from TMEV-infected DG-AMG-Acan^−/− mice. Cumulative distributions similarly show a shift toward higher interevent interval (bottom right), indicating a lower frequency, without any changes in amplitude (top right) of mEPSC from DG-AMG-Acan^−/− mice. (D) A significant decrease in mean frequency (bottom left), but not mean amplitude (top left), of miniature inhibitory postsynaptic currents (mIPSC) from TMEV-infected DG-AMG-Acan^−/− mice. Cumulative distributions similarly show a shift toward higher interevent interval (bottom right), indicating a lower frequency, without any changes in amplitude (top right) of mIPSC from DG-AMG-Acan^−/− mice. Statistics: bar graphs, n = 17–22 cells from 4–6 mice (spontaneous), n = 20–22 cells from 4–6 mice (miniature), unpaired two-tailed t-test; cumulative fractions, Kolmogorov–Smirnov test; *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure 7

Negatively charged chondroitin sulphate (CS) binds to K ⁺ and reduces the concentration of K⁺ in CS agarose gel. (A) Experimental set up of a patch-clamp electrophysiology rig to measure K⁺ concentration using K⁺-sensitive microelectrode in CS agarose gel prepared and kept in 3 mM KCl external solution. (B) Representative current-clamp recordings obtained from CS agarose gels (0%–5%). The baseline voltage was recorded from external solution above the CS gels. The deviations in the recordings from the baseline correspond to insertions of K⁺-sensitive microelectrode into the gel. (C) Mean changes in voltage recorded from CS agarose gels (0%–5%) show significant difference between 0% (control), 0.5% and 5% CS agarose gels. (D) Concentration of K⁺ ([K⁺]) as calculated from the voltage changes recorded using the Nernst equation shows a significant decrease in 5% CS gel (2.14 ± 0.18 mM) compared with control (3.14 ± 0.08 mM) and 0.5% CS (2.73 ± 0.08 mM) gels. Statistics (C and D): one-way ANOVA, Holm-Šidák’s multiple comparisons test; n = 4; *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure 8

Structural heterogeneity of perineuronal extracellular matrix differentially influences neuronal physiology. Left: Perineuronal extracellular matrix (ECM) around inhibitory interneurons forms a condensed tightly woven lattice-like structure enwrapping soma as well as proximal dendrites. This arrangement of dense ECM in close proximity to the neuronal membrane retards ion diffusion and functions akin to dielectric material or myelin sheath and increases the charge separation between intracellular and extracellular compartments. As a result, it decreases membrane capacitance and allows interneurons to fire at higher frequency. Right: Perineuronal ECM around excitatory neurons is less compact and mostly present around soma. The lower density of ECM allows ionic diffusion and does not increase charge separation across the membrane and therefore, does not affect the membrane capacitance. Instead, it acts akin to sponge attracting cations, such as K⁺, and raises their concentration in the extracellular compartment near the membrane. An increased extracellular concentration of K⁺ ([K⁺]ₒ) consequently causes hyperexcitability by depolarizing the resting membrane potential of excitatory neurons.