Hippocampal subregions are morphologically altered in a KA-induced chronic TLE mouse model

To establish a chronic TLE model, KA was administered intrahippocampally at a dose of 10 µg/kg. Following an eight-week period, brain tissues were harvested for both histological and molecular analyses (see Fig. 1A, B for schematic diagram and timeline). DAPI staining revealed significant structural distortion of the dentate gyrus (DG) and an altered distribution of granule cells in KA-induced mice compared to saline mice. These changes were observed across multiple anterior–posterior levels (AP − 2.4, − 2.6, − 2.8, − 3.0 and − 3.2 mm; white dashed areas in Fig. 1C). A close examination of the DG revealed morphology comparable to that observed in the saline group, albeit with a noticeably sparser distribution of granule cells within the DG layers (Fig. 1D). Interestingly, the morphology of the subiculum revealed a reduction in both volume and complexity, indicating substantial alterations in this region (orange dashed areas in Fig. 1C). These alterations were characterized by disrupted organization and decreased neuronal density within the subiculum, potentially implicating this structure in the pathophysiology of TLE.

Fig. 1

Changes in hippocampal apoptosis and morphology in a chronic kainic acid-induced TLE mouse model. A Schematic showing an intrahippocampal kainic acid injection (IHKA) in a mouse. Scale bar, 250 µm. B Experimental timeline showing the IHKA microinjection model of chronic TLE. C Representative DAPI-stained coronal sections showing hippocampus sections between − 2.4 mm and − 3.2 mm from bregma. The top row represents sections from the KA group, while the bottom row represents sections from the saline group. The white dashed areas represent the dentate gyrus (DG), and the orange dashed areas represent the subiculum (Sub.). Scale bar, 50 \(\upmu\)m. D Representative images of TUNEL-positive cells (red) in the DG and Sub. The schematic diagrams on the top right illustrate the selected regions. Scale bar, 100 µm. E Quantitative cell counts of TUNEL-positive cells across the DG and Sub. n = 3 mice per group, Data presented as mean ± SEM, two-tailed unpaired t test; *P < 0.05, **P < 0.01, n.s, not significant

To quantify the extent of cellular damage and apoptosis, TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assays were performed, revealing a marked increase in TUNEL-positive cells in both the ipsilateral DG and subiculum following KA induction (DG, 74.3 ± 4.6; subiculum, 42.3 ± 5.8, Fig. 1E), compared to saline controls (DG, 9.0 ± 3.3, P < 0.01; subiculum 3.3 ± 1.2, P < 0.01, Fig. 1E). These findings collectively highlight the profound effect of KA administration on hippocampal subregions, particularly the DG and subiculum, in the context of chronic TLE modeling, and offer insights into potential targets for therapeutic intervention.

Projections to the subiculum are degraded in the chronic TLE mouse model

Given the altered morphology and elevated apoptosis in the subiculum, we next tested whether neural projections to subiculum were remodeled in chronic TLE mice. We injected rAAV2-retro-hSyn-EYFP into the subiculum of chronic TLE mice (Fig. 2A, B), and following histological analysis, detected obvious EYFP signals around the injection sites, including subicular neurons and the projecting fibers, in addition to upstream brain regions (Fig. 2C). Subsequently, the overall input patterns of subicular neurons in chronic TLE mice were compared to a saline group. In the saline group, we observed dense projections to subicular neurons from several brain regions: the CA1, the entorhinal cortex (ENT), the anterior thalamic nuclei (ANT), the contralateral and ipsilateral anterior cingulate cortex (ACC), and the basolateral amygdala (BLA) (CA1, 344.7 ± 51.9; ENT, 67.0 ± 15.8; ANT, 178.3 ± 20.5; contra ACC, 10.3 ± 2.1; ips ACC, 95.3 ± 11.6; BLA, 58.7 ± 23.7; Fig. 2C, D).There was a similar pattern of projections received by subicular neurons in both the saline and KA group. However, the signal in both the subiculum and the mapped upstream input areas was dramatically lower in the KA group, including the CA1, ENT, ANT, contralateral and ipsilateral ACC (CA1, 0, P < 0.001; ENT, 25.3 ± 3.3, P < 0.05; ANT, 57.7 ± 24.0, P < 0.01; contra. ACC 9.0 ± 2.4; n.s; ips. ACC 65.0 \(\pm\) 5.9, P < 0.05; BLA, 23.7 \(\pm\) 3.8, n.s; Fig. 2C, D). Based on the findings of retrograde tracing experiments, we delineated the potential input circuitry of the subiculum and characterized how these are altered in a chronic mouse model of TLE (Fig. 2E, F). This suggests that the subiculum is a potential pivotal therapeutic target for TLE, essential for both intervention and understanding of the disease pathology.

Fig. 2

Disrupted circuitry in the subiculum of kainic-acid-induced TLE mouse brains. A, B Schematic representation (A) and experimental timeline (B) showing the strategy of retrograde tracing targeting the Sub. C Coronal mouse brain sections illustrating EYFP fluorescence marking the injection site in the Subiculum (Sub.) and retrogradely traced neurons in the CA1, entorhinal cortex (ENT), anterior thalamic nuclei (ANT), contralateral and ipsilateral anterior cingulate cortex (ACC), and the basolateral amygdala (BLA). Scale bar, 20 µm. D Quantitative cell counts of EYFP-positive neurons across the observed brain regions. n = 3 mice per group, Data presented as mean ± SEM, two-tailed unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. E, F Diagrammatic representation showing the effects of KA-induced remodeling of neural circuit inputs to the Sub., where dashed lines indicate reduced input post-KA injection (F)

Characterization of the KA-induced acute chronic TLE mouse model

Although a second dose is not strictly necessary to induce seizures, it does allow more precise temporal control of the seizure onset, which we required. To evaluate the therapeutic potential of the chemogenetic inhibition of subicular pyramidal neurons, we developed an acute kindling model via a second, lower-dose KA injection after eight weeks (Fig. 3A, B). The severity of epileptic seizures was classified according to the Racine grading scale, and mice exhibiting stage 4 or 5 seizures were categorized as having generalized tonic–clonic seizures. Seizure duration was measured for stage 4 or 5 seizures. Epileptic seizures began within 20 min of KA induction. Video footage of mouse seizures in an open field was initially recorded for up to 8 h following injection in three mice; however, we found that seizures occurred intensively within 2 h after acute induction (Fig. 3C, n = 3). Therefore, subsequent experiments all recorded the seizure status of mice for up to 2 h after acute induction in the chronic TLE models. To determine the optimal kindling dose, two doses of KA (1 µg/kg, and 2 µg/kg) were tested via brain cannulation. A significant difference in the kindling rate was observed between the saline (0.9% NaCl) and KA (1 µg/kg, and 2 µg/kg) group (Fig. 3D–F). In the KA group, seizures were stimulated by KA and behavior was observed. As shown in Fig. 3D–F, the total duration of seizures in the 2 µg/kg KA group was higher than that of the saline group, whereas no difference was observed between the 1 µg/kg group and the saline group. Similarly, a significant difference in seizure frequency was only observed with the 2 µg/kg dose (Fig. 3D, E). These results indicate that 2 µg/kg KA is a sufficient dose to induce kindling.

Fig. 3

Characterization of intrahippocampal kainic acid microinjections as a model for chronic TLE. A Experimental timeline for the characterization of the acute-chronic TLE model. B Representative images showing the cannula position above the hippocampus. Scale bar, 250 µm. C Line plot of seizure intensity over time for three individual mice (#74, #75, #76), showing the seizure duration in minutes for each 30-min interval following KA administration. D–F Seizure characteristics in acute KA-induced groups (1 µg/kg and 2 µg/kg) and saline-induced groups (0.9% NaCl). Quantification of total duration (D) seizure frequency (E) and mean seizure duration (F) within 2 h following administration. Each symbol represents an individual animal, and color coding indicates KA or saline. n = 6 mice per group, data are represented as mean ± SEM, one-way ANOVA with post-hoc Tukey’s test; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. G C-Fos immunofluorescence in the subiculum post-KA or saline induction, illustrating differential activation in ipsilateral vs. contralateral sections. White dashed areas represent the subiculum (Sub.). Scale bar, 50 µm

Immunohistochemical analysis confirmed an increase in c-Fos expression within the subiculum following acute KA stimulation with the 2 µg/kg dose. In the saline-induced group, we observed a significantly lower number of c-Fos positive cells than in the KA-induced group (Fig. 3G). These results indicate successful generation of the acute chronic TLE model. This method employs a robust time-controlled protocol to kindle seizures by administration of KA, thus enabling a relatively precise assessment of the chemogenetic silencing effects on epileptic seizures.

Chemogenetic inhibition of the subiculum alleviates seizure severity in the acute chronic TLE mouse model

We next tested the effect of inhibiting subicular pyramidal neurons in KA-kindled chronic TLE mice. We unilaterally injected AAV9-CaMKII\(\alpha\)-hM4Di-EYFP, which expresses hM4Di and enables neuronal silencing upon CNO administration, into the subiculum of acute chronic TLE mice (Fig. 4A–C). To confirm reliable hM4Di expression in subicular excitatory pyramidal neurons driven by the CaMKII \(\alpha\) promoter, co-localization of EYFP and vGlut1 cells in brain sections containing the subiculum from these mice was assessed (Fig. 4D). A substantial overlap between these two cell populations was observed. A significant proportion of EYFP-positive cells were positive for vGlut1, while a majority of vGlut1-positive cells also exhibited EYFP positivity. These results suggest a high degree of specificity and efficiency in the labeling of subicular pyramidal neurons with hM4Di-EYFP and indicate that the expression of hM4Di is primarily restricted to excitatory neurons.

Fig. 4

Labeling of excitatory neurons in the subiculum of kainic acid TLE model mice. A Schematic showing KA injection sites in the CA1 and AAV9-CaMKIIα-hM4Di-2A-EYFP injection sites in the Sub. B Schematic showing the AAV9-CaMKIIα-hM4Di-2A-EYFP construct. C Representative images of the injection site in the Sub. Scale bar, 250 µm. D High magnification images of EYFP-positive subicular neurons (green) labeled with vGlut1 ISH (red), with merged images indicating co-localization (yellow). Scale bar, 20 µm

Next, we tested whether intraperitoneal (i.p.) CNO delivery suppresses generalized tonic–clonic seizures. Following a three-week recovery period for AAV-mediated expression and subsequent cannulation implantation (Fig. 5A), two successive experimental trials were conducted to evaluate KA-induced seizures, each preceded by saline (1st trial) or CNO (2nd trial) treatment.

Fig. 5

Chemogenetic inhibition of hippocampal subicular excitatory neurons mitigates seizure phenotypes in an acute-chronic TLE model. A Schematic showing the timeline of the chemogenetic experiment. B Comparison of seizure severity indexed by seizure score between control and hM4Di groups. C–E Total seizure duration (C), seizure frequency (D), and mean seizure duration (E) for each mouse across both trials. The saline-treated group is denoted by purple dots, and the CNO-treated by red squares. Lines are drawn between the scores of each mouse for comparison (n = 7 control mice, n = 8 h M4Di mice). Data are presented as mean ± SEM, two-tailed paired t-test when comparing two groups between the saline- and CNO-treated groups, and a two-tailed unpaired t-test when comparing two groups within the same trial phase; *P < 0.05, **P < 0.01, n.s.: not significant

Prior to chemogenetic inhibition, the potential role of subicular excitatory neurons in modulating seizure manifestations was assessed. As shown in Fig. 5B, under saline treated conditions (7 separate vehicle sessions) all animals displayed score 5 seizures, i.e., rearing with loss of balance. Treatment with CNO significantly suppressed seizure severity (P < 0.01). When animals were tested with 1 mg/kg of CNO, the median score was reduced to a 2 (i.e., head nodding). We compared the average duration and frequency of seizures across trials, and noted that, following CNO administration, there was a significant reduction in average seizure duration in the hM4Di group compared to saline-treated group (232.1 \(\pm\) 181.1 vs. 34.4 \(\pm\) 38.7, P < 0.05, Fig. 5C), with a concurrent decrease in seizure frequency (4.5 \(\pm\) 2.7 vs. 0.9 \(\pm\) 1.1, P < 0.01, Fig. 5D). Moreover, hM4Di mice exhibited a significantly shorter average seizure duration in the second trial following CNO administration than in the first trial following saline (48.9 \(\pm\) 19.2 vs. 20.6 \(\pm\) 21.0, P < 0.05, Fig. 5E). Specifically, 7 of 8 mice displayed a reduction in seizure severity after this dose of CNO, and 4 mice were completely protected. However, no significant differences in seizure duration or frequency were observed between the two trials in the control group. Collectively, these results indicate that chemogenetic suppression of hippocampal subicular excitatory neurons alleviate the severity of seizures in a KA-induced acute chronic model of epilepsy.