Homozygous pairing of wild type (WT) (JAX 0006640) or C3 KO (JAX 003641) mice (Jackson Labs) was used to produce experimental mice. Absence of C3 protein in the brains of C3 KO mice was validated using immunoblots. SE inductions were performed on male mice (2−3 months old) because we had previously established the specific spatiotemporal window for complement activation during the latent period of epileptogenesis in male rodents (Schartz et al., 2016, 2018, 2019). Our ongoing and future studies are focusing on determining the profile of complement activation in females and assessing sex as a biological variable in the context of SE and epilepsy. Mice were kept at ambient temperature with a 12-hr light-dark cycle (6:00−18:00) and unlimited access to food and water. Mice were group-housed (3−4/cage) at the Psychological Sciences animal facility at Purdue University or at the Laboratory Animal Research Core in the Dedman Life Sciences Building at Southern Methodist University. All procedures were approved by Purdue Animal Care and Use Committee (Protocol #1309000927) and Southern Methodist University IACUC (Protocol #A21-003-BREA). All animal research followed NIH and Institutional guidelines (Percie du Sert et al., 2020).

Intraperitoneal (i.p.) injections of scopolamine methyl bromide (1 mg/kg; S8502-1G, Sigma-Aldrich, St. Louis, MO, United States) were given 30 min prior to pilocarpine (dissolved in sterile 0.9% saline) to induce SE (325−350 mg/kg, i.p. for dose response, 350 mg/kg for behavior cohorts) (P6503-10G, Sigma-Aldrich). Sham-controls were given saline. Diazepam (10 mg/kg, i.p; Hospira, Inc., Lake Forest, IL, United States) was injected approximately 1 h after SE onset to interrupt seizure activity. Mice were monitored continuously after pilocarpine administration and scored at 1 min intervals for the first 30 min, 2 min intervals between 30−40 min and 5 min intervals thereafter until Diazepam was administered. Seizures were scored according to the Racine scale (Racine, 1972). Modified Racine scale: (1 = rigid posture, mouth moving; 2 = tail clonus; 3 = partial body clonus, head bobbing; 4 = rearing; 4.5 = severe whole body clonic seizures while retaining posture; 5 = rearing and falling; 6 = tonic-clonic seizure with jumping or loss of posture). Mice were classified as SE when they reached a Racine score of ≥4.5. To aid in recovery, mice received 0.9% saline injections (i.p.), and supplementary chocolate Ensure and up to six Kellogg’s Fruit Loops pieces were put in the cages (control and SE mice) along with their rodent chow at 1 and 2 days after SE. Body weight was recorded daily for up to 2 weeks after SE. We injected 71 WT and 70 C3 KO mice with pilocarpine and 36 WT and 30 C3 KO mice with saline. Out of those injected with pilocarpine, 42.2% WT and 45.5% C3 KO mice died from SE-related complications. The final numbers of SE mice used in subsequent experiments were 41 WT and 38 C3 KO. The final numbers of control mice were 36 WT and 30 C3 KO.

Behavioral testing was performed between 2 and 3 weeks post SE. Prior to any behavioral testing, mice were handled for 1−2 min per day for 5 days to acclimate them to the experimenter. For the OF, mice were placed in an empty arena (40 × 40 × 30 cm) for 10 min. During this time all mice were recorded and tracked with Any-Maze V4.99 (Wood Dale, IL) to measure distance travelled, average speed, time freezing, and time spent in the inner and outer zones as indicators of general locomotor activity or anxiety-like behavioral differences between treatment groups.

Novel object recognition (NOR) began 24 h after OF, where mice were allowed to explore two similar objects in the same chamber for 10 min (acquisition trial) (Trial 1), followed by a test trial in which one object was replaced by a novel object and mice were allowed to explore freely for 5 min (Trial 2). The inter-trial interval between acquisition and testing was set at 5 days, based on a pilot study of several intervals, and habituation and testing times (data not shown). Mice were returned to their home cages between trials. All trials took place under red lights. Object placement was counterbalanced to control for potential side bias. The arena and objects were cleaned with 70% ethanol between trials. Object exploration was assessed with Any-maze V4.99 and verified by blinded investigators. Discrimination index (DI) was calculated [(Novel-Familiar)/(Novel + Familiar) × 100]. Mice with DI ≥ ± 25 during acquisition were removed to eliminate mice with innate side preference (Schartz et al., 2018, 2019). This resulted in the exclusion of one WT + SE and one C3 KO + SE mouse from NOR analysis.

Mice were euthanized at either 1 h or at 14 days after SE (cohorts separate from the behavior cohorts) with a lethal dose of Beuthenasia (200 mg/kg i.p.) and transcardially perfused with ice-cold phosphate buffered saline (PBS) for approximately 5 min or until the blood was cleared. Hippocampi were dissected and processed for immunoblotting as previously described (Schartz et al., 2018, 2019). Equal protein concentrations of whole lysates were resolved through SDS-PAGE, transferred to polyvinylidene difluoride membranes (88518, Thermo Scientific, Waltham, MA, United States), blocked with blocking solution [5% milk in 1X Tris buffered saline with 0.1% tween (0.1% 1X TBST)] (2 h RT) and incubated with primary antibodies overnight (4°C) or 2 h (RT). Primary antibodies used were rabbit anti-C3 (1:1K; ab200999, Abcam, Cambridge, United Kingdom), goat anti-C3 (1:100; 855730, MP Biomedical, Santa Ana, CA, United States) rabbit anti-phospho-S6 (1:1K; #5364, Cell Signaling), rabbit anti-S6 (1:1K; #2217, Cell Signaling, Danvers, MA, United States), mouse anti-PSD95 (1:10K; MABN68, Millipore, Burlington, MA, United States), mouse anti-GFAP (1:40K; #3670, Cell Signaling), rabbit anti-synaptophysin (1:1K; ab16659, Abcam), and rabbit anti-actin (1:5K; ab198991, Abcam) or anti-gapdh (1:30K; G9545, Sigma Aldrich) (loading control). After washing in 1X TBST, membranes were incubated in HRP-linked secondary anti-rabbit, mouse, or goat IgG antibodies for 1 h at RT. Membranes were stripped of primary antibodies using stripping buffer (25 mM glycine, pH 2.0, 10% SDS) for 1−2 h at RT, washed in 1X TBST and re-incubated with different primary antibodies. Immunoreactive signal was visualized with enhanced chemiluminescence (32106, Thermo Scientific) and captured on radiography film (BX57, MIDSCI, Fenton, MO, United States). Immunoreactive bands obstructed by bubbles or damaged due to gel breakage were not analyzed. Pixel intensity of the immunoreactive bands was measured using Image J software (NIH), subtracted from the background, normalized to the actin bands of the same sample/lane, and shown as percent change of controls.

Mice were deeply anesthetized with Euthasol (200 mg/kg) and perfused with ice-cold 0.9% saline solution. Brains were post-fixed overnight in 4% paraformaldehyde, cryoprotected in 30% sucrose, frozen in dry ice, and stored at −80°C until used. Coronal brain sections (30 μm) were cut using a Leica CM1950 cryostat and stored in phosphate buffered saline (PBS) + 0.1% Sodium Azide at 4°C (PBS: BP399, Fisher Scientific). Colorimetric IHC was performed following previously described protocols (Schartz et al., 2016, 2018). Briefly, sections were incubated in 1XPBS (5 min), 3% hydrogen peroxide (30 min), 1X PBS with 0.3% Triton (1X PBS-0.3T) (20 min), and in immuno buffer (5% goat serum, 0.3% BSA, 0.3% 1X PBS-0.1T) (1 h) at RT. Incubation with the primary antibody anti-mouse GFAP (1:1000; #3670, Cell Signaling) was done overnight on a rotating platform at 4°C. Following washes in 0.1% 1X PBST (3 × 10 min), sections were incubated in biotinylated goat anti-mouse secondary antibody (1:500; BA-9200, Vector Laboratories) (1 h), washed with 0.1% 1X PBST (3 × 10 min), and incubated with ABC Peroxidase Standard Staining Kit (#32020, ThermoFisher) (30 min). Immunoreactive GFAP signal was visualized using the DAB peroxidase (HRP) Substrate Kit, 3,3’-diaminobenzidine according to manufacturer’s instructions (SK-4100, Vector Laboratories). Sections were mounted on gelatin-coated slides, air dried, dehydrated through increasing alcohol concentrations (50, 70, 95, and 100%), de-fatted in Xylene (2 × 3 min), and cover slipped using Permount mounting media. Images (4-6 sections per brain) were captured using a Nikon Ti2-E Inverted Motorized Microscope.

Sample sizes were determined with G*Power using previously gathered data to calculate the number of mice per group needed to achieve power of 0.8. NOR object preference was analyzed using paired t-tests. Data acquired from OF, DI, and NOR were compared using two-way ANOVA with Tukey’s post hoc test using GraphPad Prism Software version 8.0.1. All immunoblot data were compared using independent t-tests (2 groups) or one-way ANOVA with Sidak’s multiple comparison test (>2 groups). Significance was set at p = 0.05.

The first aim of this study was to confirm induction of SE by pilocarpine in C3 KO and WT mice. We performed SE inductions on 2−3-month-old mice. At this age, C3 KO and WT mice have been shown to have similar spine density and synaptic responses in the hippocampus (Perez-Alcazar et al., 2014; Shi et al., 2015). Therefore, we performed a dose response with either 325 or 350 mg/kg of pilocarpine (Figures 1A, B). Both C3 KO and WT mice reached similar seizure scores according to the Racine scale (Racine, 1972) with 325 mg/kg [F (1, 9) = 0.06, p = 0.81] or 350 mg/kg [F (1, 11) = 1.84, p = 0.20] of pilocarpine (Figure 1A), suggesting that behavioral seizure susceptibility to pilocarpine was not altered by the lack of C3 protein.

To confirm that the initial neural responses to pilocarpine-induced SE were similar between WT and C3 KO, we collected hippocampi from a subset of animals at 1 h during SE. First, we confirmed the lack of C3 protein in the C3 KO hippocampal lysates relative to the WT group using antibodies against C3 (Figure 1B). Next, we determined levels of phosphorylated S6 (P-S6) relative to total S6 (P-S6/T-S6), a protein within the mechanistic target of rapamycin complex 1 pathway that is indicative of increased neuronal activity by chemoconvulsants (Zeng et al., 2009; Brewster et al., 2013; Figure 1C). Similar SE-induced increases in the P-S6/T-S6 ratio were evident in hippocampi of WT mice [F (2, 14) = 5.75, p = 0.02] and C3 KO mice [F (2, 12) = 4.71, p = 0.03], suggesting hippocampal neuronal activation may be similar in both WT and C3 KO animals subjected to pilocarpine-induced SE. Post-hoc analysis showed that the 350 mg/kg dose significantly increased P-S6/TS6 in WT (p = 0.01) and C3 KO (p = 0.02) mice. Thus, hereafter a dose of 350 mg/kg of pilocarpine was used to induce SE in animals used for subsequent behavioral assessments.

To monitor the health and recovery of all WT and C3 KO mice after SE or sham treatment, mice were weighed daily for 2 weeks (Figure 2A). To assess behavioral comorbidities, we evaluated anxiety-like behaviors (Figure 3), and memory (Figure 4) between 2 and 3 weeks after SE. We found that WT and C3 KO mice used for behavioral evaluations developed SE at similar Racine levels following pilocarpine injections (350 mg/kg) (Figure 2B). The time to first seizure (Racine scale 3) (Figure 2C) and SE (Racine scale ≥ 4.5) (Figure 2D) were similar between the WT and C3 KO groups. Mice from all groups had consistent body weight throughout the study (Figure 2E). Two weeks after SE, we determined the protein levels of C3 in hippocampi from a subset of WT mice with or without SE (Figure 2F). Activated C3 is cleaved into smaller fragments that include C3b/iC3b (Schartz et al., 2018). Utilizing an antibody that recognizes all cleavage products of C3, we found that the levels of C3b/iC3b protein were significantly increased in the SE group compared to controls (p = 0.02) (Figure 2F), consistent with our previous findings in rats (Schartz et al., 2018, 2019) and human (Wyatt et al., 2017). Taken together these findings support that SE provokes increases in C3 activation.

To assess potential differences in locomotion and anxiety-like behaviors in the different groups of mice, we compared total distance travelled (Figure 3A), average speed (Figure 3B), time spent immobile (time freezing) (Figure 3C), and time spent around the outer walls zone (Figure 3D) or in the inner zone of the arena (Figure 3E) between WT + C, C3 KO + C, WT + SE and C3 KO + SE mice. WT + C and WT + SE mice showed no significant differences in these measures although WT + SE mice showed a slight but not significant increase in distance travelled (p = 0.09), faster speed (p = 0.08), and less time freezing (p = 0.06) compared to WT + C mice. The locomotion and anxiety-like behaviors were not different between C3 KO + C and C3 KO + SE mice (p > 0.05). There was no effect of genotype or treatment on time spent in the inner or outer zones of the arena. These findings indicate that at 2 weeks after SE, hyperactivity and anxiety-like behaviors were not significantly altered in either WT or C3 KO mice.

We and others previously reported that a single episode of SE results in memory deficits that are evident 2 weeks post-SE induction (Brewster et al., 2013; Schipper et al., 2016; Schartz et al., 2018). Therefore, at this time point we determined recognition memory using NOR (Figure 4). During NOR acquisition, all groups showed similar exploration times of the right or left objects (data not shown), and the DI or total exploration time were not different between the groups (Two-way ANOVA, p > 0.05) (data not shown). During the test trial (Trial 2), WT + C mice spent significantly more time exploring the novel object compared to the familiar one [t (13) = 6.64, p < 0.001], as did C3 KO + C mice [t (13) = 4.60, p < 0.001] (Figure 4B). WT + SE mice did not show exploratory preference for either object [t (8) = 1.34, p = 0.22], suggesting a recognition memory deficit in this mouse group. In contrast, mice from the C3 KO + SE group explored the novel object more than the familiar object [t (11) = 4.45, p = 0.001] (Figure 4B). In addition, we found a significant effect of treatment on DI [F (1, 45) = 5.02, p = 0.03] (Figure 4C), but no effect of genotype [F (1, 45) = 2.55, p = 0.12] and no interaction between treatment and genotype [F (1, 45) = 2.79, p = 0.10]. Interestingly, our data indicate that C3 KO mice spent more time exploring both objects overall [F (1, 45) = 10.76, p = 0.002] (Figure 4D), which may contribute to increased learning.

Given that the main sources of C3 in the brain are astrocytes (Lian et al., 2015; Liddelow et al., 2017) and that C3 can contribute to the modulation of synaptic elements (Stevens et al., 2007; Schafer et al., 2012), we measured the relative protein levels of the pan-reactive astrocytic marker GFAP along with synaptic markers PSD95 and synaptophysin in hippocampal homogenates at 14 days after SE (Figure 5). We found significant increases in the protein levels of GFAP in the WT + SE group compared to WT + C (p = 0.037) but not in C3 KO + SE compared to C3 KO + C or WT + C groups (p > 0.05) (Figures 5A, B). The levels of PSD95 or synaptophysin were not different between the groups. Consistent with these GFAP findings in whole hippocampal lysates, immunohistological analysis showed a denser population of GFAP-positive cells in hippocampi of WT + SE mice compared to WT + C, C3 KO + C, and C3 KO + SE groups (Figure 5C). Taken together these data support that C3 KO mice are protected against SE-induced increases in GFAP suggesting that C3 modulation of astrocyte function may play a role in the associated memory defects (Figure 4).