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. 2024 Mar 22;10(12):eadk9484.
doi: 10.1126/sciadv.adk9484. Epub 2024 Mar 20.

Termination of convulsion seizures by destabilizing and perturbing seizure memory engrams

Shirong Lai  1   2 , Libo Zhang  3   4 , Xinyu Tu  1 , Xinyue Ma  1 , Yujing Song  1 , Kexin Cao  3   5 , Miaomiao Li  6 , Jihong Meng  6 , Yiqiang Shi  1 , Qing Wu  2 , Chen Yang  1 , Zifan Lan  1 , Chunyue Geoffrey Lau  7 , Jie Shi  3 , Weining Ma  6 , Shaoyi Li  6 , Yan-Xue Xue  3   8 , Zhuo Huang  1   9
Affiliations

Termination of convulsion seizures by destabilizing and perturbing seizure memory engrams

Shirong Lai et al. Sci Adv. .

Abstract

Epileptogenesis, arising from alterations in synaptic strength, shares mechanistic and phenotypic parallels with memory formation. However, direct evidence supporting the existence of seizure memory remains scarce. Leveraging a conditioned seizure memory (CSM) paradigm, we found that CSM enabled the environmental cue to trigger seizure repetitively, and activating cue-responding engram cells could generate CSM artificially. Moreover, cue exposure initiated an analogous process of memory reconsolidation driven by mammalian target of rapamycin-brain-derived neurotrophic factor signaling. Pharmacological targeting of the mammalian target of rapamycin pathway within a limited time window reduced seizures in animals and interictal epileptiform discharges in patients with refractory seizures. Our findings reveal a causal link between seizure memory engrams and seizures, which leads us to a deeper understanding of epileptogenesis and points to a promising direction for epilepsy treatment.

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Figures

Fig. 1.
Fig. 1.. Development and validation of a CSM paradigm in mice.
(A) Schematic illustration of the video EEG recording setup and the odor delivery system. (B) The experimental procedure for seizure conditioning. After electrode implantation, mice underwent simultaneously training with CIS alone (pink) or odor alone (light purple) or CIS paired with a specific odor (CIS + odor; blue). In the test session conducted 2 weeks (2 w) later, mice were exposed to the CIS-paired odor, except that the mice were tested with a novel odor (light blue). (C) Example timelines (left) and statistics (right; averages with error bars indicating SEM) indicating the seizure onsets under pre- and postodor exposure conditions in (B), one mouse per row. The width of the vertical line represents the duration of the seizure onset. (D) Typical EEG traces recorded under pre- and postodor exposure conditions in test sessions [red in (B)] showing the spike wave discharges associated with seizure onsets. (E and F) Graphs depicting the number of seizures (E) and seizure duration (F) during the 15-min test session after applying the odor test illustrated in (B) (each group, n = 10). Dots represent individual animals; data are presented as means ± SD. (G) Timeline of experimental protocol for seizure conditioning. Two weeks after the training session under CIS + odor pairing condition, mice with electrodes previously implanted were tested repeatedly with the CIS-paired odor (three tests, at 1-week intervals). (H) Graphs depicting the number of seizures in (G); data are presented as means ± SEM. (I) Graphs depicting seizure duration in (G) recorded from mice before and after odor exposure during the three test sessions (n = 10). Data are presented as means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 2.
Fig. 2.. Pairing chemogenetic activation of odor-responding engram cells in the DG with CIS created artificial CSM.
(A) Experimental scheme illustrating the neural activity–dependent labeling of engram cells in the hippocampal DG area. A subpopulation of DG neurons responding to the olfactory stimulus expresses ERCreER under the control of the E-SARE promotor. In the presence of 4-OHT, these neurons permanently express mCherry and hM3DG(q), enabling subsequent imaging and manipulation, respectively. (B) Timeline depicting the neural activity–dependent behavioral labeling protocol. i.p., intraperitoneally. (C) Representative microscopic image of DG showing viral injection site and with odor-responding engram cells labeled (red). Blue fluorescence is 4′,6-diamidino-2-phenylindole (DAPI). (D) Timeline for the specificity test of the activity-dependent labeling system. IF, immunofluorescence. (E) Representative microscopic images from mice in (D) showing DG neurons labeled with mCherry (red) and/or c-fos (green). DG neurons activated during lemon-lemon test in (D) expressed both (yellow; merged). (F) Quantifications of the proportion (%) of c-fos+/mCherry+ overlapping neurons among all mCherry+ DG neurons [each group, n = 4 mice; one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons]. Data are presented as means ± SEM. (G) The procedure for seizure conditioning with the chemogenetic activation of odor-responding engram cells in DG. (H and I) Graphs depicting the number of seizures (H) and seizure duration (I) (unequal variance, Kruskal-Wallis test followed by Dunn’s multiple comparisons) recorded during the test session from mice under the three conditions in (G) (each group, n = 15 mice). Dots represent individual animals; data are presented as means ± SD; ***P < 0.001, **P < 0.01, and *P < 0.05.
Fig. 3.
Fig. 3.. Retrieval of CSM led to a protein synthesis–dependent reconsolidation process in the hippocampus.
(A) Timeline for protein detection after retrieval of CSM from the seizure conditioning paradigm. Mice were decapitated before (control) or at 15 min, 1 hour, 2 hours, or 9 hours after the retrieval session. (B to H) Representative Western blot (WB) images and quantification indicating the expression levels of pERK and total ERK (C), pmTOR and mTOR (E), BDNF (G), and EGR 1 (H), in the hippocampus of mice decapitated before odor exposure (control) and at the indicated time points after odor exposure [each group, n = 8 mice; two-way ANOVA followed by Tukey’s post hoc test in (C) and (E); one-way ANOVA followed by Dunnett’s multiple comparisons to preodor in (G and H)]; β-actin served as a loading control. Error bars represent the SEM; *P < 0.05. NS, not significant. (I) Timeline of the experiment. Anisomycin (Ani) or vehicle was administrated immediately (at 5 min) or delayed for 9 hours after the first retrieval of CSM at week 3 (test 1). Mice were tested with reexposure to the CIS-paired odor at week 4 (test 2). (J and K) Graphs depicting number of seizures and seizure duration recorded during the test session from mice with the treatment of immediate (J) or delayed (K) anisomycin or vehicle after the first retrieval [immediate vehicle or anisomycin, n = 8 mice, each; two-tailed Welch’s t tests in (J); two-tailed unpaired t tests in (K)]. Dots represent individual animals; data are presented as means ± SD. *P < 0.05 and **P < 0.01.
Fig. 4.
Fig. 4.. Pharmacological intervention targeting mTOR or ERK pathway within the reconsolidation time window impaired the persistent of CSM.
(A) Timeline of the pharmacological intervention experiments. (B and D) Graphs depicting the number of seizures and seizure duration recorded during the test session from mice with the treatment of immediate (B) or delayed (D) rapamycin (Rap) or vehicle after the first retrieval (immediate vehicle, n = 8; immediate rapamycin, n = 8; delayed vehicle, n = 8; delayed rapamycin, n = 7). (C and E) Representative Western blot images and quantification indicating the expression levels of BDNF, pERK1/2, total ERK1/2, pmTOR, and total mTOR in the hippocampus of mice decapitated 1 hour after the test session with the treatment of immediate (C) or delayed (E) rapamycin or vehicle after the first retrieval (each group, n = 6). (F and H) Graphs depicting the number of seizures and seizure duration with the treatment of immediate (F) or delayed (H) propranolol (Prop) or vehicle after the first retrieval (immediate vehicle, n = 6; immediate propranolol, n = 5; delayed vehicle, n = 7; delayed propranolol, n = 6). (G and I) Representative Western blot images and quantification indicating the expression levels of BDNF, pERK1/2, total ERK1/2, pmTOR, and total mTOR 1 hour after the test session with the treatment of immediate (G) or delayed (I) propranolol or vehicle after the first retrieval (each group, n = 6). β-Actin served as a loading control. Data are presented as means ± SD in (B), (D), (F), and (H); data are presented as means ± SEM in (C), (E), (G), and (I). Two-tailed unpaired t tests in [(B), left], (C), (E), [(F), left], (G), [(H), left], and (I); two-tailed Welch’s t tests in [(B), right], [(F), right], and [(H), right]. *P < 0.05 and **P < 0.01.
Fig. 5.
Fig. 5.. Targeting mTOR pathway within the reconsolidation time window decreases IEDs in patients with refractory seizure.
(A to C) Temporal profile of epileptiform paroxysmal activities represented by IEDs. The IEDs were observed from 9 to 14 hours preceding the seizure event until 9 to 46 hours after the seizure event, normalized to the maximum discharge number of each patient. The administration of drug or placebo after a seizure event is marked as a red or blue dot in the diagram. The green shadow represents recovery period of IED frequency to basal level. The IEDs were observed from 14 hours preceding the seizure event until 46 hours after the seizure event in the 1-hour everolimus treatment group (T1 to T3) (A), from 9 hours preceding the seizure event until 16 hours after the seizure event in the 8-hour delayed everolimus treatment group (T4 to T6) (B), and from 9 hours preceding the seizure event until 9 hours after the seizure event in the negative control (vitamin C treatment) group (C1 to C3) (C). (D to F) Comparison of the percentage change in IEDs before and after administration in 1-hour everolimus treatment group (T1 to T3) (D), the 8-hour delayed everolimus treatment group (T4 to T6) (E), and the negative control group (C1 to C3) (F). Data are presented as individual values of each hour and line at median; [(D) and (E)] Wilcoxon rank sum test; *P < 0.05 and **P < 0.01.
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