Article Figures & Data
- Figure 1.
Timeline of TMEV infection in mice, acute seizure monitoring, and biochemical, molecular, electrophysiological, and pharmacological experiments reported in this article. Negative numbers on the axis indicate days before TMEV infection. vEEG was conducted for more than 1 mo continuously immediately after infection but is shown only through 15 dpi because we had to exclude several mice from the study owing to loss of the electrode assembly after 15 dpi. Various dosing paradigms of XPro1595 were tested for their effects on TMEV-induced acute seizures as described in detail in Results.
- Figure 2.
Mice implanted with an electrode in the dentate gyrus for 24 h/7 d/wk vEEG show focal as well as generalized seizures after TMEV infection. (A) Total number of acute seizures each day after TMEV infection from all mice that developed acute seizures. Of 16 mice implanted with an electrode in the dentate gyrus and infected with 3 × 105 pfu TMEV, 11 developed acute seizures. The mice had nonconvulsive focal seizures (white bar) as well as spontaneous generalized seizures (black bar) during the first week after TMEV infection; however, nonconvulsive focal seizures continued to occur in some mice after acute behavioral seizures subsided. Although the vEEG was conducted for more than 1 mo postinfection, the seizure data are shown until 15 dpi, as several mice were discontinued from the study after 15 dpi owing to loss of electrode assembly. (B) Representative trace of vEEG recording from a mouse at 5 d after TMEV infection. The vertical lines on the upper vEEG trace indicate baseline EEG during normal mouse behavior (a); nonconvulsive focal electrographic seizure (b); stage 3 behavioral seizure (c); stage 4 behavioral seizure (d); stage 4 behavioral seizure without electrographic activity (e); and normal mouse behavior and the electrographic activity returning to baseline (f). All vertical scale bars represent 500 µV, and the horizontal scale bars represent 30 s in the upper vEEG trace and 1 s in each of the expanded traces below. (C) Location of the electrode in the molecular layer of the dentate gyrus (near the center of the image). The coronal brain slice shown here, stained with 0.1% cresyl violet, was obtained from a TMEV-infected mouse that had seizures.
- Figure 3.
No difference in the properties of mEPSCs of DGCs recorded in brain slices obtained from PBS-injected (control) and TMEV-infected mice during the acute seizure period 3–7 dpi. (A) Representative traces of mEPSCs measured in the DGCs from the control group and TMEV-infected mice at 5 dpi. (B) Cumulative fraction distribution of the amplitude of mEPSCs shows no difference between control and TMEV groups. Average amplitudes of mEPSCs are plotted in the lower panel (control, n = 8; TMEV, n = 10). (C) Cumulative fraction distribution of the interevent interval (IEI) of mEPSCs shows no difference between treatment groups. The lower panel shows the average frequency of mEPSCs. Statistics: Kolmogorov–Smirnov test (cumulative fraction), unpaired t test (average amplitude and frequency).
- Figure 4.
Increase in the levels of TNFα and in a ratio of the protein expression of TNFR1:TNFR2 in the hippocampus of TMEV-infected mice during acute seizure activity period. (A) mRNA levels of TNFα, as measured by RT-qPCR, are significantly increased in TMEV-infected mice at 5 and 14 dpi by 161- and 88-fold, respectively, compared with PBS-infected control mice (n = 4 for TMEV and control). (B) 206- and 35-fold increase in the protein expression levels of TNFα in TMEV-infected mice at 5 and 14 dpi compared with the PBS-injected control mice [control, n = 5; TMEV, n = 8 (1 dpi), 6 (5 dpi), and 5 (14 dpi)]. (C) Representative immunoblot shows the protein expression of TNFR1, TNFR2, and actin in the hippocampus from PBS- and TMEV-infected mice (n = 3). (D) Densitometric analysis of the immunoblots shows the expressions of TNFR1 and TNFR2 normalized to the expression levels of actin [control, n = 5; TMEV, n = 5 (1 dpi), 6 (4 and 14 dpi)] O.D., optical density. Relative expression levels of TNFR1 and TNFR2 (TNFR1:TNFR2) are significantly increased by 1.54- and 2.1-fold at 4 and 14 dpi, respectively, in TMEV-infected mice compared with control mice. (E) mRNA levels of TNFR1 and TNFR2 in the hippocampus of TMEV-infected mice before (1 dpi), during (5 dpi), and after (14 dpi) acute seizures. The ratio of TNFR1 to TNFR2 mRNA is significantly reduced during the acute infection period. Data are shown as mean ± SEM. Statistics: two-way ANOVA followed by Bonferroni posttest; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
- Figure 5.
CNS administration of XPro1595 does not affect TMEV-induced acute seizure frequency and intensity. (A) Slow infusion of XPro1595 (10 mg/kg) at 0, 2, 4, and 6 dpi into the left lateral ventricle using a surgically implanted guide cannula did not reduce average seizure frequency or severity (n = 12, XPro1595; n = 11, vehicle). Each circle represents an individual mouse, and the horizontal line shows average number of seizures per group per acute seizure period (3–8 dpi). (B) Average seizure burden corresponding to each stage of modified Racine scale for generalized tonic-clonic seizures shows no difference between vehicle- and XPro1595-treated TMEV-infected mice. Only those mice that had acute seizures are included in this analysis. (C) The surgical placement of the guide cannula into the left lateral ventricle was confirmed by i.c.v. infusion of 0.1% Evans blue dye at 9 dpi in all TMEV-infected mice treated with either XPro1595 or vehicle. The panel shows an example of a diffusion of the dye into the ventricular system.
- Figure 6.
TMEV-induced acute behavioral seizure susceptibility in WT, TNFα–/–, TNFR2–/–, and TNFR1–/–TNFR2–/– mice. (A) TNFα–/– mice have a significant reduction in the seizure frequency (upper panel), plotted as a total number of seizures per mouse, and in the seizure severity (lower panel) as measured by an average cumulative seizure burden during acute seizure period (3–8 dpi) compared with WT mice. Each circle represents data from an individual mouse, and the horizontal line shows an average number of seizures per group. (B) TNFR2–/– mice show an increase in average seizure frequency as well as severity. Latency to exhibit the first TMEV-induced acute seizure is reduced in TNFR2–/– mice compared with WT mice. (C) Reduced average seizure frequency and severity in TNFR1–/–TNFR2–/– mice. (D) Percentage of total infected mice that show acute behavioral seizures during 3–8 dpi. The data for TNFR1–/– mice are from Kirkman et al. (2010) and are included here for comparison. Statistics: unpaired t test (frequency), Scheirer–Ray–Hare test (severity), Fisher’s exact test (% seized mice), and long-rank test (% seizure free); **, p < 0.01; ***, p < 0.001.
- Figure 7.
Increase in the cell-surface levels of GluA1 and GluA2 subunits of AMPARs in TMEV-infected WT C57BL/6J mice during acute seizures. (A) Representative immunoblots from two mice show the levels of GluA1 in the total as well as the cell-surface fractions of proteins isolated from ipsilateral hippocampus at 5 d postinjection of PBS (control) or TMEV. Data in the first (total) and the third (surface) lanes from the left are from the same mouse, and the second (total) and the fourth (surface) lanes correspond to the other mouse. The surface proteins were isolated from the intracellular proteins by cell-surface biotinylation in acute hippocampal slices. Levels of GluA1 were quantified by densitometry, and data are shown as ratio of surface to total protein, which is significantly increased in TMEV-infected mice (n = 6). PAG is a mitochondrial protein and serves as an intracellular control protein. (B) Similar to GluA1, the ratio of surface/total level for GluA2 is also increased in TMEV-infected mice (n = 6). (C) Ratios of surface/total protein expression for GluA1 and GluA2 are increased by 48% and 33%, respectively (data normalized to control). (D) Approximately 50% decrease in the total expressions of GluA1 and GluA2 in TMEV-infected mice compared with control group. Statistics: unpaired two-tailed t test.
- Figure 8.
Increase in the cell-surface levels of GluA1 and GluA2 subunits of AMPARs in TMEV-infected TNFR2–/– mice during acute seizures. (A) Representative immunoblots from two mice show the levels of GluA1 in the total as well as the cell-surface fractions of proteins isolated from ipsilateral hippocampus at 5 d postinjection of PBS (control) or TMEV. The surface proteins were isolated by cell-surface biotinylation procedure in acute hippocampal slices. Levels of GluA1 were quantified by densitometry, and data are shown as ratio of surface to total protein that is significantly increased in TMEV-infected mice (n = 6). PAG is a mitochondrial protein and serves as an intracellular control protein. (B) Similar to GluA1, the ratio of surface/total level for GluA2 is also increased in TMEV-infected mice (n = 6). (C) Ratios of surface/total protein expression for GluA1 and GluA2 are increased by 44% and 34%, respectively (data normalized to control). (D) Approximately 50% decrease in the total expression of GluA1 and GluA2 in TMEV-infected mice compared with control group. Statistics: unpaired two-tailed t test.
- Figure 9.
Effects of TNFα mediated predominantly through TNFR1 signaling may contribute to hyperexcitation and the generation of TMEV-induced acute seizures. The protein level of TNFα, the ratio of TNFR1:TNFR2 protein expression, cell-surface to total expression of GluA1 and GluA2 subunits of AMPAR, and excitatory synaptic transmission increase in the hippocampus during the development of acute seizures 3–7 days after TMEV infection in C57BL/6J mice. The results described in this article suggest that the opposite effects of TNFα on neuronal transmission mediated via TNFR1 and TNFR2, with the activation of the TNFR1 pathway causing hyperexcitation by synaptic upscaling of AMPAR, could be implicated in causing seizures after TMEV infection.
- Table 1.
Significant increase in the protein levels of various inflammatory mediators in the hippocampus of TMEV-infected mice during acute seizure activity period. Statistics: Two-way ANOVA, Bonferroni posttest, #p<0.05, *p<0.01, †p<0.001; SEM, standard error of the mean. (TNFα, tumor necrosis factor-α; IFNγ, interferon-γ; IL, interleukin; CXCL1, C-X-C motif chemokine ligand 1)
Fold change (relative to PBS-injected control mice) 1 dpi (n = 8) 5 dpi (n = 6) 14 dpi (n = 5) Cytokine Average SEM Average SEM Average SEM TNFα 6.9 0.6 206.2† 14.9 34.8# 7.1 IFNγ 4.5 0.8 21,734.4† 3123.2 99.1 21.6 IL-1β 6.9 1.1 58.2† 5.4 46.6 13.5 IL-10 2.3 0.3 47.2† 6.0 9.6 1.9 IL-12p70 2.5 0.3 3.0# 0.5 6.0† 1.0 IL-4 1.0 0.2 0.8 0.1 1.3 0.3 IL-2 2.4 0.3 14.4† 4.2 4.9 0.7 IL-5 2.0 0.1 10.8* 2.5 2.6 1.2 CXCL1 4.6 0.3 30.0† 4.3 2.4 0.3 Statistics: two-way ANOVA, Bonferroni posttest, †p<0.001; #p<0.05; *p<0.01. CXCL1, C-X-C motif chemokine ligand 1.
- Video 1.
Example of a vEEG recording from a mouse implanted with an electrode in the dentate gyrus. The vEEG recording was obtained at 5 d after TMEV infection. It is noted that the behavioral aspects of the generalized tonic-clonic seizure in this mouse are preceded by an electrographic seizure activity in the hippocampus, and the electrographic seizure stops earlier than the behavioral seizure.
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