Inhibiting astrocyte calcium dynamics terminates status epilepticus via wireless septal stimulation

Tian, Chunxiao; Xue, Qiuying; Wu, Tongrui; Zhao, Jingyu; Tian, Chao; Zhu, Yuxuan; He, Hualin; Liu, Aili; Dong, Lei; Zheng, Yu; Shen, Hui

doi:10.1038/s41598-025-09611-w

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Published: 15 July 2025

Inhibiting astrocyte calcium dynamics terminates status epilepticus via wireless septal stimulation

Chunxiao Tian¹^na1,
Qiuying Xue²^na1,
Tongrui Wu¹^na1,
Jingyu Zhao¹,
Chao Tian³,
Yuxuan Zhu²,
Hualin He²,
Aili Liu²,
Lei Dong⁴,
Yu Zheng⁴ &
…
Hui Shen²

Scientific Reports volume 15, Article number: 25579 (2025) Cite this article

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Abstract

Status epilepticus (SE) is an acute medical emergency with unclear cellular and circuit mechanisms, and it is imperative to develop anti-SE strategies. Here, we observe distinct astrocytic Ca²⁺ activity patterns during SE: generalized seizures (GS) show synchronized activity between CA3 and contralateral S1, while focal seizures (FS) exhibit desynchronization. Additionally, micrometer-scale wireless electrical stimulation (WES) targeting the medial septum and ventral diagonal band of Broca (MS/vDB) effectively terminates SE and suppresses abnormal astrocytic Ca²⁺ activity, comparable to conventional deep brain stimulation (DBS). The effect is linked to MS/vDB GABAergic neuron activation, which suppresses calcium dynamics within the CA3 astrocyte-pyramidal neuron microcircuit. Meanwhile, WES of the MS/vDB effectively suppresses chronic spontaneous seizures induced by pilocarpine within 10 days. This study shows the divergent patterns of astrocytic activity during SE. It also demonstrates that WES of MS/vDB can effectively suppress SE, establishing an advanced therapeutic strategy.

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Introduction

Approximately 1% of the global population has epilepsy¹, a disease with complex pathophysiology that is commonly managed using anti-epileptic drugs to control seizures^2,3. However, over a third of epilepsy patients develop drug tolerance, becoming drug-resistant^4,5. Status epilepticus (SE) is an emergency and sustained state of epileptic seizures characterized by alarmingly high rates of disability and mortality^6,7. However, the underlying mechanism of SE is still unclear, and managing the SE poses significant challenges.

Traditionally, SE is characterized by excessive or hypersynchronous discharges with enhanced neuronal excitability due to excitatory-inhibitory imbalance. The contribution of glial cells to physiological and pathological processes in the central nervous system (CNS) is often overlooked. Among glial cells, astrocytes are predominant in the CNS^8,9. The contribution of astrocytes to SE processes is unclear. Although not electrically excitable, astrocytes display a complex repertoire of calcium dynamics^10,11. Studies indicate that astrocyte Ca²⁺ activity increases during seizures¹², but the relationship between the stereotypical patterns of astrocyte Ca²⁺ activity and SE remains unclear. In kainic acid (KA)-induced SE, heightened excitability originating from the hippocampal, the common source of seizures, propagate extensively throughout the brain via neural circuit^13,14,15. However, the Ca²⁺ activity of astrocytes in the hippocampus and contralateral cortex during SE has not been reported. And, astrocyte Ca²⁺ activity is involved in the regulation of the “tripartite synapse”¹⁶. Cross-communication between astrocytes and neurons is mediated by Ca²⁺ activity, where manipulating astrocytes influences Ca²⁺ activity in neurons^{16,17,18,19,20}. Therefore, decoding the key characteristics of astrocyte Ca²⁺ activity in SE is necessary.

Andreas Horn et al. utilized deep brain stimulation to delineate dysfunctional circuits in the frontal cortex²¹, showcasing that regulating relevant circuits in key brain areas is more effective in the treatment of neurological and psychiatric disorders. Our study designed a micrometer-scale wireless-powered electrical stimulation (WES) device. A critical question is whether such a compact receiving coil device effectively capture sufficient energy to support electrical stimulation therapy for SE at the circuit level in vivo? KA-induced SE generally originates from the hippocampus, a large bilateral structure²². The medial septum and ventral diagonal band of Broca (MS/vDB), which have direct connectivity with the hippocampus and their susceptibility to seizure spread in SE^23,24,25.

Here, we investigate the mechanisms underlying calcium dynamics of astrocytes during SE, and whether micrometer-scale WES targeting the MS/vDB can control SE in vivo by combining optogenetics/chemogenetics and calcium fiber photometry recording to observe the effects on SE control. Our results first indicate that in mice experiencing generalized seizure (GS), exhibiting strong coupling of astrocyte Ca²⁺ activity in the CA3 and S1 brain regions. In mice experiencing focal seizure (FS), the astrocyte Ca²⁺ activity between these two regions was not completely synchronized. And, properly performed WES of the MS/vDB can effectively inhibit GS and FS and terminate SE. Furthermore, we demonstrate that the termination of SE is attributed to the activation of MS/vDB GABAergic neurons, which suppress Ca²⁺ activity in the CA3 astrocyte-pyramidal neuron microcircuit. These results show a characteristic patterns of astrocyte Ca²⁺ activity during SE. Additionally, this work of WES establishes an advanced therapeutic strategy for SE.

Results

High activation of astrocytes during SE of hippocampal origin

Following the implantation of WES devices in the MS/vDB of mice (the sham group), an open-field test was conducted to qualitatively and quantitatively assess exploratory and locomotor activity. We confirm that our implantation strategy does not affect motor nerve function in mice (Supplementary Fig. 2a,b).

We induced SE by intraperitoneal (i.p.) injection of KA in mice, which exhibited seizure characteristics similar to those observed in human patients²⁶. We administered i.p. injections of KA to mice implanted with WES (the sham + KA group). To further validate the implantation strategy of WES, we monitored seizure activity of mice in sham group following i.p. injection of saline, and mice in sham + KA group following i.p. injection of KA within 90 min. We found that in the sham group, the implantation of WES alone did not induce seizures, including GS and FS, nor did it lead to mortality (Supplementary Fig. 3a–c). In sham + KA group, GS and FS could be induced by i.p. injection of KA in mice implanted with the WES device (Supplementary Fig. 3a–c).

To investigate abnormal astrocytic Ca²⁺ activity and its association with behavioral changes during SE, we recorded Ca²⁺ activity in astrocytes from the hippocampal CA3 and distal S1, along with corresponding seizure behaviors. To achieve this, the genetically encoded calcium indicator jGCaMP7f. was expressed in the CA3 region on one side and the S1 region on the opposite side of wild-type mice under the GfaABC1D promoter²⁷ (Fig. 1a). Immunohistochemistry analysis confirmed that jGCaMP7f. virus was expressed in CA3 and S1 S100B⁺ astrocytes, indicating that the population of calcium signals was cell-type specific (Fig. 1b). Subsequently, optical cannulas were implanted to capture calcium signals during the 20 min before and 90 min after KA i.p. (Fig. 1d). Simultaneous recordings of seizure activity (Fig. 1e) and changes in GCaMP fluorescence intensity (ΔF/F) were conducted.

Baseline recordings of calcium signals from CA3 and S1 astrocytes revealed greater amplitude fluctuations in S1 astrocytes compared to CA3 astrocytes (Fig. 1f) when the mice with the WES implanted in the MS/vDB, but without the driving device (Fig. 1c, Supplementary Fig. 1). After i.p. injection of KA, in the sham + KA group, a considerable increase in calcium signals (ΔF/F > 50%) was observed in the CA3 and S1 regions, defining a “wave”. Notably, in the sham + KA group, S1 exhibited an immediate baseline-rising, while CA3 displayed an afterhyperpolarization (AHP)-like event. Within 1.5 h following KA injection, multiple GSs and FSs were observed, along with their corresponding waves in CA3 and S1. During each FS, waves could emerge in CA3 and S1 either individually or simultaneously, with or without the presence of high-frequency oscillations (Fig. 1g). During each GS, waves were observed in both CA3 and S1, accompanied by high-frequency oscillations (Fig. 1h,i). This suggests that astrocytic Ca²⁺ activity in CA3 and S1 exhibits distinct patterns during FS and GS. In our experiments, we found that during the baseline-rising in S1, the i.p. administration of KA resulted in the initial baseline-rising phase of Ca²⁺ activity in S1 lasting approximately 10 min, followed by stabilization at an elevated level (Fig. 1g,j). Therefore, we propose that the initial baseline-rising phase in S1 constitutes a considerable temporal window for modulating SE. Notably, synchronized Ca²⁺ activity of astrocytes in CA3 and S1 was recorded during GS (Fig. 1k,l). Furthermore, synchronized astrocytic Ca²⁺ activity between CA3 and S1 was a key feature during GS (Fig. 1m). In addition, since the hippocampus is a common source of KA-induced SE, we simultaneously recorded Ca²⁺ activity of astrocytes in CA3 on one side and pyramidal neurons (PNs) in CA3 on the other side. Both PNs and astrocytes in CA3 exhibited considerably elevated Ca²⁺ activity and synchronized activation during SE (Supplementary Fig. 4a–c). Notably, synchronized Ca²⁺ activity between PNs and astrocytes in CA3 was also observed in the basal state (Supplementary Fig. 4d).

By examining the key characteristics of calcium signals of astrocytes of GS and FS in SE, these findings lay the groundwork for subsequent exploration of specific WES treatment methods and WES regulation of SE.

WES of the MS/vDB activates CA3 and S1 astrocytes in the basal state

To investigate the potential for preventing SE development, we tested whether pre-stimulation of MS/vDB could reduce susceptibility of SE. Based on our identification of a critical time window for the initial baseline-rising phase in S1 within the sham + KA group, we set the WES stimulation duration to 10 min. Pre-stimulation (WES-base) was applied for 1/6 h to the MS/vDB region (upstream of hippocampal CA3) in the basal state while recording astrocytic calcium signals in CA3 and S1 at various time points. Signals were recorded for 1/6 h before stimulation as a control (−1/6 h and 0 d), during WES-base, and at 1/6, 1, and 12 h post-stimulation to assess short-term effects. Long-term effects were evaluated by recording signals for 1/6 h on days 1, 3, 5, 7, and 10 after WES-base (Fig. 2a).

Firstly, we observed that WES-base initially enhanced and then restored the synchronization of astrocytic Ca²⁺ activity between CA3 and S1 (Fig. 2b,c). Additionally, in CA3 astrocytes, WES-base increased the amplitude while reducing the frequency of Ca²⁺ activity (Fig. 2d), suggesting that WES-base activates CA3 astrocytes while lowering the frequency of Ca²⁺ activity in the basal state. Similarly, in S1 astrocytes, WES-base also increased activation and reduced the frequency of Ca²⁺ activity under basal conditions (Fig. 2e). These findings aligned with prior reports that administering the Adra1a receptor agonist A61603 to mice increased Ca²⁺ activity in astrocytes and decreased frequency²⁸.

Collectively, these findings suggest that WES-base of the MS/vDB in a basal state enhances coordination and Ca²⁺ activity level of astrocytes in CA3 and S1 such that these astrocytes are activated. This demonstrates that the WES is effective in mice in vivo, capable of activating cellular responses and supporting the feasibility of SE interventions.

Ability of WES of MS/vDB to intervene in SE of hippocampal origin

In this study, we aimed to identify the critical time window for WES treatment to inhibit or attenuate SE. Therefore, the i.p. administration of KA was used as the dividing point between the Pre and Mid phases. WES was applied during these phases in the KA-induced SE, referred to as Pre-WES and Mid-WES, respectively.

WES of the MS/vDB was administered prior to KA i.p. injection in the Pre-WES group and immediately after KA i.p. injection in the Mid-WES group (Fig. 3a,c). SE severity was assessed across groups (Fig. 3b,d; Supplementary Fig. 5a,b). In the Mid-WES group, calcium signals in CA3 developed into a persistent flashing pattern, and the mice exhibited FS (Fig. 3e). Mid-WES alleviated SE severity, whereas Pre-WES appeared to exacerbate it (Fig. 3f,g). Regarding astrocytic Ca²⁺ activity in CA3 and S1 during SE, we found that Mid-WES significantly reduced the number of astrocytic calcium waves, whereas Pre-WES increased their number (Fig. 3h). This suggests that Mid-WES effectively suppresses astrocytic Ca²⁺ activity in CA3 and S1 during SE, in contrast, Pre-WES promotes astrocytic Ca²⁺ activity. Compared to the sham + KA group, Mid-WES considerably altered calcium signaling characteristics, including flashing, baseline-rising, and AHP-like events, while the effects of Pre-WES were less pronounced (Supplementary Fig. 6a–d). Notably, Mid-WES also modified the timing relationship between S1 baseline-rising and the latency to the 1st-GS (Supplementary Fig. 6e).

To further assess the impact of WES of MS/vDB on SE severity, we separately analyzed GS and FS. Mid-WES reduced both the acquisition and expression of GS, whereas Pre-WES appeared to enhance them (Fig. 4a,b). To explore the relationship between GS, FS, and astrocytic calcium waves in CA3 and S1, the waves were categorized as follows: wave1 (ΔF/F 50%-100%, no high-frequency oscillations), wave2 (ΔF/F 50%-100%, with high-frequency oscillations), wave3 (ΔF/F ≥ 100%, no high-frequency oscillations), and wave4 (ΔF/F ≥ 100%, with high-frequency oscillations). We further analyzed the combinations of astrocytic calcium waves in CA3 and S1, categorizing wave combinations with an occurrence rate of 15% or lower as “others”. During GS, both CA3 and S1 exhibited calcium waves (Fig. 4c). Except for the Pre-WES group, CA3 and S1 predominantly displayed wave4 patterns during GS (Fig. 4e). This suggests that astrocytic Ca²⁺ activity in CA3 and S1 is strongly coupled during GS and that GS is accompanied by high-frequency oscillations. Furthermore, Pre-WES appeared to alter the calcium signaling characteristics observed during GS. Notably, during GS, the wave in CA3 preceded that in S1, while the wave in S1 occurred before GS onset, the pattern could be modified by Pre-WES (Fig. 4d). To further characterize calcium signaling in CA3 and S1 during GS, we quantified several parameters, including peak time, area under the curve (AUC), peak amplitude, and S1 baseline-rising. Using the baseline-rising of S1 as a reference, we divided the signal into two phases: the “up” phase corresponding to the rising portion and the “down” phase representing the recovery of the baseline. We found that Pre-WES could alter the calcium signaling characteristics of astrocytes in CA3 and S1 during GS (Supplementary Fig. 7a–f). In KA-induced SE, the hippocampus plays a crucial role due to its high excitability. Notably, Mid-WES inhibited the occurrence of wave4 in CA3 (Fig. 4f). Mid-WES alleviated GS expression by reducing the number of GS without affecting their duration, whereas Pre-WES increased GS expression by raising the number of GS without prolonging their duration (Fig. 4g,h).

Regarding FS, Pre-WES promoted FS expression, while Mid-WES alleviated it (Fig. 4i). Astrocyte activation in CA3 and S1 was not fully synchronized during FS (Fig. 4j). WES considerably influenced the combination of calcium waves in CA3 and S1 during FS (Fig. 4k), however, only Pre-WES altered the calcium signaling characteristics of astrocytes in CA3 and S1 (Supplementary Fig. 8a–f).

Regarding the efficacy of Mid-WES in treating SE, we further recorded hippocampal electroencephalographic (EEG) in KA-induced SE to verify the effectiveness of Mid-WES in modulating SE. Following WES implantation in mice, SE was induced by KA i.p. (the sham + KA group), after which the Mid-WES group received a 10-min WES intervention prior to EEG recording. We first observed in the sham + KA group that abnormal hippocampal EEG activity occurred during GS (Fig. 4l), which corresponded to high-amplitude calcium oscillations in astrocytes during GS (Fig. 1h)²⁹. Subsequently, we found that Mid-WES effectively suppressed abnormal EEG activity (Fig. 4l). Additionally, we established a chronic epileptic model using pilocarpine to assess the efficacy of Mid-WES. Following WES implantation in mice, the model was induced by pilocarpine i.p. (the sham + PILO group), after which the PILO-WES group received a 10-min WES intervention prior to video monitoring. We observed the occurrence of chronic spontaneous seizures, including FS and GS, within 10 days following pilocarpine-induced SE³⁰. However, WES effectively suppressed both GS and FS, and consequently inhibited the occurrence of chronic spontaneous seizures (Fig. 4m–o). Therefore, the therapeutic strategy of Mid-WES can effectively mitigate chronic spontaneous seizures.

In conclusion, applying WES to the MS/vDB at different phases differentially influences SE severity. Specifically, Pre-WES of the MS/vDB exacerbates SE by increasing the expression of both FS and GS, while Mid-WES mitigates SE by reducing their expression. Furthermore, WES of the MS/vDB not only affects the expression of FS and GS but also alters the characteristics of astrocytic Ca²⁺ activity in CA3 and S1 during SE progression.

The SE-intervening effects of Mid-WES are mediated by MS/vDB GABAergic neurons

Although we have established the SE-terminating effects of Mid-WES, the underlying mechanisms remain unclear. Since electrical stimulation could activate neurons in vivo³¹, we hypothesized that the SE-terminating effects might result from the activation of MS/vDB neurons. To test this hypothesis, we transduced channelrhodopsin-2 (ChR2) into MS/vDB neurons using a viral vector with a synapsin promoter^32,33. Specifically, we injected the AAV-DIO-ChR2-mCherry virus into the MS/vDB of Vgat-cre mice. Following viral expression, we recorded astrocytic calcium signals in CA3 and S1 (Fig. 5a). Blue-light illumination (60 s at 20 Hz) was applied to the MS/vDB immediately after KA i.p. administration (Fig. 5b). SE severity was then assessed (Fig. 5c,d; Supplementary Fig. 9a,b).

Activation of MS/vDB GABAergic neurons, similar to the effects observed in the Mid-WES group, increased survival in mice and reduced the maximum seizure stage (Fig. 5e,f). Regarding astrocytic Ca²⁺ activity in CA3 and S1 during SE, we found that activation of MS/vDB GABAergic neurons significantly reduced the number of astrocytic calcium waves (Fig. 5g). This suggests that activation of MS/vDB GABAergic neurons effectively suppresses astrocytic Ca²⁺ activity in CA3 and S1 during SE. Additionally, MS/vDB GABAergic neuron activation terminated both the acquisition and expression of GS (Fig. 5h,i). Regarding FS, activation of MS/vDB GABAergic neurons reduced FS expression and altered the wave patterns in CA3 and S1 during FS (Fig. 5j,l). Desynchronization of astrocytic activation between CA3 and S1 was also observed during FS (Fig. 5k). Activation of MS/vDB GABAergic neurons also inhibited the occurrence of wave4 in CA3 (Supplementary Fig. 10a). In terms of calcium signaling characteristics during SE, only the baseline-rising was notably altered in the Vgat-ChR2 group (Supplementary Fig. 10b–k).

In summary, similar to the Mid-WES group, the activation of MS/vDB GABAergic neurons inhibits Ca²⁺ activity in astrocytes and effectively terminates SE by suppressing GS expression and considerably reducing FS expression. Furthermore, similar to the Mid-WES group, MS/vDB GABAergic neuron activation not only influences FS and GS expression but also alters the astrocytic Ca²⁺ activity patterns in CA3 and S1 during SE. These findings further demonstrate that the SE-terminating effects of Mid-WES are mediated by MS/vDB GABAergic neurons.

Astrocyte-neuron interaction in the CA3 microcircuit involves the SE-intervening effects of Mid-WES

Microcircuits and their interactions in generating, propagating, and modulating seizure activity are of great interest³⁴. As a key regulatory cell, astrocytes modulate PNs, which are the primary excitatory neurons playing a crucial role in the regulation of seizure. Therefore, understanding how astrocytes influence PNs is of great significance. We have confirmed that the Mid-WES group can effectively terminate SE. However, whether the inhibition of CA3 astrocytes by the Mid-WES group affects the function of CA3 PNs and thereby contributes to SE termination remains to be determined.

To study the effect of astrocytes on PNs in CA3, we manipulated the activity of astrocytes using chemogenetic approach in vivo³⁵. We unilaterally injected AAV-GfaABC1D-hM3Dq-mCherry viruses into the CA3 region of wild-type mice in the Mid-WES group to chemogenetically activate CA3 astrocytes. Additionally, AAV-CaMKIIα-GCaMP6(f) viruses were injected into the ipsilateral CA3 and contralateral S1 to record calcium signals from PNs (Fig. 6a,b). This virus carries hM3Dq, an engineered excitatory Gicoupled human muscarinic receptor, which can increase intracellular Gq signaling with the existence of clozapine-N-oxide (CNO), and thus increases activity³⁵. After two consecutive days of i.p. injections of CNO (1 mg/kg) in mice, KA i.p. was administered 2 h after the final CNO injection, followed by the immediate application of Mid-WES for 10 min. Calcium signals and seizure behaviors were recorded 90 min after the KA injection (Fig. 6c).

In GfaABC1D-mCherry group mice, calcium signals from CA3 and S1 PNs showed flashing in both regions without the expression of GS (Fig. 6d,f). Additionally, cortical spreading depression (CSD)-like calcium signals were recorded in S1 PNs (Fig. 6d,e). In GfaABC1D-hM3D group mice, GS expression was observed along with high-frequency oscillations, while flashing occurred in both CA3 and S1 (Fig. 6g,h). Activation of CA3 astrocytes did not affect the survival rate of the mice but resulted in an increased maximum seizure stage (Fig. 6i,j). Moreover, CA3 astrocyte activation promoted GS expression (Fig. 6k-m). In the GfaABC1D-hM3D group, the latency to the onset of flashing was prolonged (Fig. 6n). Notably, in the GfaABC1D-mCherry group, flashing occurred later than CSD (Fig. 6o). By activating astrocytes and observing Ca²⁺ activity in PNs, we found that unlike astrocytic Ca²⁺ activity, mice exhibiting CSD in PNs calcium signals no longer experienced GS (Fig. 6d). Furthermore, following the occurrence of flashing, GS did not recur (Fig. 6g). Compared to the GfaABC1D-mCherry group, in the GfaABC1D-hM3D group, the latency of flashing was further prolonged as SE severity increased (Fig. 6n). These findings suggest that CSD and flashing may serve as indicators of SE severity.

In conclusion, the activation of CA3 astrocytes enhances the excitability of CA3 PNs and promotes the expression of GS, which in turn influences the alleviation effect of Mid-WES on GS. Therefore, the interaction between astrocytes and PNs in CA3 plays a role in modulating the resistance to GS and the termination of SE by Mid-WES.

Discussion

SE is associated with high mortality and severe damage to the central nervous system, highlighting the need for new therapeutic targets due to drug resistance. This study explored the calcium signaling characteristics of astrocytes in CA3 and S1 during SE. Notably, we successfully terminated SE and inhibited abnormal astrocytic Ca²⁺ activity in CA3 and S1 during SE through WES, primarily through the activation of MS/vDB GABAergic neurons. Notably, astrocyte-neuron interactions within the CA3 microcircuit play a pivotal role in the SE-modulating effects of Mid-WES.

Calcium signals of parvalbumin (PV) neurons in substantia nigra pars reticulata (SNr) increased during GS and FS³⁶. In our study, Ca²⁺ activity in CA3 and S1 astrocytes increased during SE. Similarly, calcium signals of M1 astrocytes increased during seizure²⁹. However, astrocyte activation levels differ between GS and FS. In KA-induced SE, the hippocampus serves as the primary source. During GS, astrocytes in both CA3 and S1 were activated, whereas FS exhibited desynchronized activation between these regions. In the KA-induced SE, WES applied to the MS/vDB at different time points produced distinct effects. Mid-WES inhibited both the acquisition and expression of GS, alleviated FS expression, but did not shorten duration of each GS. In contrast, Pre-WES promoted the expression of both GS and FS without prolonging duration of each GS. Therefore, WES primarily regulates the occurrence of FS and GS to control the severity of SE, rather than by altering wave characteristics.

Recent studies had shown that activation of GABAergic neurons could reduce seizure duration^25,37, suggesting that the activation of GABAergic neuron played a pivotal role in suppressing seizures. Our optogenetic experiments revealed that the SE-terminating effects of Mid-WES in the MS/vDB were mediated by the activation of MS/vDB GABAergic neurons. From the perspective of SE-terminating effects and changes in astrocytic calcium signaling, Mid-WES of the MS/vDB may exhibit effects similar to the activation of MS/vDB GABAergic neurons. Like Mid-WES, the activation of MS/vDB GABAergic neurons inhibited astrocytic Ca²⁺ activity in CA3 and S1, suppressed FS expression and terminated GS. GABAergic neurons in the MS/vDB projected to the CA3 region of the hippocampus, where they broadly targeted interneurons³⁸. Activation of GABA_B receptors in astrocytes triggered calcium release from intracellular stores^{18,39,40,41,42} and modulated different subtypes of GABAergic neurons, such as parvalbumin-expressing (PV) interneurons⁴⁰. Therefore, we hypothesized that the regulation of astrocytic Ca²⁺ activity in CA3 by the activation of GABAergic neurons in the MS/vDB might have been mediated through projections of neurons and their interactions with astrocytes.

A growing body of research had demonstrated that astrocytes controlled ventral tegmental area (VTA) GABA neuron activity in cocaine reward via tonic inhibition in mice⁴³. Moreover, subcellular inputs of GABA and glutamate led to widespread, long-lasting astrocyte Ca²⁺ responses⁴⁴. Meanwhile, activating astrocytes in S1 could attenuate neocortical seizures in rodent models²⁹. Therefore, regulating astrocyte-neuron interactions may serve as a potential therapeutic target for alleviating seizures. This study demonstrated that during the alleviation of GS and termination of SE by Mid-WES, CA3 astrocytes modulated SE by influencing surrounding PNs. In GS, CA3 astrocytes and PNs exhibited synchronized activation, whereas FS primarily involved both synchronization and some desynchronization, highlighting abnormal synchronization between PNs and astrocytes during SE.

Notably, we observed CSD-like calcium signals in S1 PNs. CSD is a slowly propagating wave of near-complete depolarization of brain cells followed by temporary suppression of neuronal activity⁴⁵. Recent studies had shown that CSD may be a fundamental defense against focal neocortical seizures⁴⁶. Several studies had reported prolonged, high-amplitude Ca²⁺ signals resembling CSD following seizure termination, consistent with a phenomenon of postictal depression⁴⁷. In our study, no seizures were observed after the detection of CSD-like calcium signals in S1 PNs. Therefore, CSD is closely associated with seizures.

In summary, our study reveals that CA3 and S1 astrocytes exhibit distinct calcium signaling responses at different stages (GS and FS). Mid-WES effectively terminates SE and inhibits astrocytic Ca²⁺ activity. This effect is mediated by GABAergic neurons of the MS/vDB and involves astrocytes within the CA3 microcircuit. These findings provide insights into the mechanisms of SE at the brain network level, revealing the characteristics of astrocytic calcium dynamics during SE and highlighting the potential of WES as an innovative treatment strategy.

Methods

Animals

C57BL/6 J mice (wild-type, male, 8–10 weeks old) were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license No. SCXK (Jing) 2021- 0006, Beijing, China). Vgat-Cre mice (stock number:016962) were utilized and genotyped following the protocols provided by Jackson Laboratory. Animals were housed and maintained in a temperature-controlled environment on a 12 h light–dark cycle, with ad libitum food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (Tianjin, China) and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8^th ed.; National Research Council, 2011). All experiments were reported in accordance with ARRIVE guidelines. Following completion of the experiments, all experimental animals were euthanized by decapitation, in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). Every effort was made to minimize suffering and reduce the number of animals used in the experiments.

Device design for micrometer-scale WES

This device is a battery-free electromagnetic induction-coupled microstimulator designed for targeted modulation. It achieves fully wireless and complete implantation without percutaneous leads, allowing freedom of movement. A signal generator (SDG1020, Siglent Technologies, China) outputs continuous sinusoidal signals which drives the transmitting coil via a power amplifier. At the receiving end, a concentric bipolar electrode (FHC, Bowdoin, ME) is connected via soldered leads. Wireless power transmission (WPT) is achieved between the transmitting and receiving ends through electromagnetic coupling.

Viral injection and fiber/WES implantation

The following viruses were used for experimentation: AAV2/9-GfaABC1D-jGCaMP7f. viral vectors (viral titers: 2.15 × 10¹³ particles/mL), AAV2/9-CaMKIIα-GCaMP6(f) viral vectors (viral titers: 2.2 × 10¹² particles/mL), AAV2/9-DIO-ChR2-mCherry (viral titers: 3.1 × 10¹² particles/mL), AAV2/9-GfaABC1D-hM3Dq-mCherry viral vectors (viral titers: 2.2 × 10¹² particles/mL). All control mice were injected with the corresponding control virus and underwent identical procedures. Viruses were purchased from Brainvta (Wuhan, China), except for AAV2/9-GfaABC1D-jGCaMP7f. from OBio Technology (China) and AAV2/9-GfaABC1D-hM3Dq-mCherry from Brain Case (China). All viral constructs were stored at − 80 °C.

Before viral injection, mice were anesthetized with an isoflurane/oxygen mixture (RWD Life Science, China; induction concentration: 2.5%; maintenance concentration: 0.8–1.5%) and mounted on a stereotaxic apparatus (RWD Life Science, China). The virus was delivered at a rate of 3 μL/min via a glass micropipette attached to a 1 μL syringe to the CA3 (AP −1.8 mm; L −2.5 mm; V −1.84 mm), S1 (AP −1.6 mm; L 3.0 mm; V −0.6 mm), and MS/vDB (AP 0.9 mm; L −0.8 mm; V −4.62 mm). Notably, for virus injection to the MS/vDB, the syringe must be tilted 10° to the right; the virus was injected according to this coordinate. After injection, the syringe was left in place for 10 min before withdrawal. Optical cannulas (1.25 mm diameter, Fiblaser Technology, China) were implanted in the CA3 and S1 regions for fiber photometry. An optical cannula was also inserted into the MS/vDB for optogenetic control and light stimulation and a WES coil was implanted in the MS/vDB for electrical stimulation. The virus was expressed for a minimum of 4 weeks. Post-expression, verification of viral expression, and WES coil/cannula placement were conducted after behavioral tests. Analysis only included mice with accurate placement, resulting in ~ 30% of mice being excluded.

Open-field test

C57BL/6 J mice were randomly divided into two groups (control, n = 5; sham, n = 5) that underwent different surgical procedures: (i) vehicle control or (ii) WES coil device implantation. The testing apparatus consisted of open-top boxes measuring 45 cm × 45 cm × 45 cm with a white underside. Each mouse was placed in the open field apparatus for a 5 min session, starting upon insertion into the box. The boxes were cleaned before and after each test session.

Calcium fiber photometry

A multi-channel fiber photometry system (Thinkertech, China) was employed to record neural calcium events in mice expressing GfaABC1D-jGCaMP7f. and CaMKIIα-GCaMP6(f). The mice were placed within a transparent, round glass cylinder with a diameter of 35 cm, allowing for unrestricted movement while carefully monitoring their behavior. The collected GCaMP fluorescence was converted to voltage signals using a digital amplifier (Thinkertech, China). The converted signals were recorded at a sampling rate of 50 Hz for a total of 110 min. The recorded data were further analyzed using MATLAB (version R2020a, MathWorks, USA). For each trial, the change in fluorescence (ΔF/F) was calculated as (F–F₀)/F₀, where F represents the current signal value and F₀ represents the average baseline signal value. The ‘MultiPhotometry. mlapp’ program in MATLAB was employed for signal preprocessing (including baseline correction and extraction of behavior-related signals) and calculation of ΔF/F.

Electrodes implantation and EEG recordings

Mice (sham + KA, n = 5; Mid-WES, n = 5) were anesthetized with an isoflurane/oxygen mixture (RWD Life Science, China; induction concentration: 2.5%; maintenance concentration: 0.8–1.5%) and mounted on a stereotaxic apparatus (RWD Life Science, China) for stereotactic implantation of EEG electrodes (RWD Life Science, China) in CA3 (AP −1.8 mm; L −2.5 mm; V −1.84 mm). Mice were surgically implanted with EEG electrodes and allowed to recover for a week. Baseline recordings were then conducted for 20 min. Following KA i.p., mice that developed SE continued to undergo EEG monitoring for 90 min.

KA-induced SE

Following the initial 20 min of calcium event recording, mice received i.p. injection of KA (K0250, Sigma, USA, dissolved in a 5 mg/mL solution of normal saline) at a dosage of 18 mg/kg of body weight to induce the SE. Throughout the recordings of calcium event, behavior was continuously monitored using a webcam with VGA resolution at a frame rate of 30 fps synchronized with the acquired neuronal data.

Behavioral seizures were evaluated according to a modified five-stage Racine’s scale⁴⁸: Stage 1, facial automatism; Stage 2, infrequent myoclonic jerks, such as head nodding; Stage 3, multiple myoclonic jerks, such as forelimb clonus; Stage 4, tonic–clonic forelimb and back extension with rearing; and Stage 5, tonic–clonic forelimb and back extension with falling. Stages 1–3 were classified as focal seizures (FSs), while stages 4–5 were classified as generalized seizures (GSs). Successful establishment of SE in mice was defined as the occurrence of > 2 behavioral episodes of GS without spontaneous remission. Additionally, the SE state has to last for at least 60 min without resulting in mortality.

Pilocarpine-induced chronic epileptic model

A dose of 300 mg/kg of pilocarpine (MedChemExpress, New Jersey, USA) was intraperitoneally injected 30 min after N-methylscopolamine bromide (1 mg/kg, i.p., Sigma, USA) administration. Mice developed SE and injected with a dose of diazepam (10 mg/kg, i.p., Sigma, USA) after 3 h to limit the duration of SE. Subsequently, all mice were continuously video-recorded during the following 10 days (24 h per day) and their seizure behaviors were scored during the videos. To assess chronic spontaneous seizures over a 10-day period, seizure was classified according to Racine’s scale⁴⁸. Stages 1–3 were classified as FSs, while stages 4–5 were classified as GSs.

Optogenetic stimulation

Blue light (473 nm) was delivered using a 200 μm diameter optical fiber (Thinkertech, China) connected to the laser by a commutator. The optical fiber was cut flat and the laser power was adjusted to 10 mW. Shortly before placing the mouse in the chamber, the cannula cap was removed and the optical fiber was directly inserted. For KA-induced SE, optical stimulation was administered immediately after KA i.p.. For optogenetic activation experiments, 473 nm blue light (20 Hz, 20 ms/pulse, and 1200 pulses) was used.

Chemogenetic activation

For chemogenetic activation of astrocytes in CA3, 1 mg/kg clozapine N-oxide (CNO, MedChemExpress, New Jersey, USA) was intraperitoneally injected in mice. After two consecutive days of CNO i.p. in mice, KA i.p. was performed 2 h after the last CNO i.p., and Mid-WES was immediately applied for 10 min to test the effect of chemogenetic activation of astrocytes in CA3 on seizures. Mice with mCherry were also given CNO to test its effect.

Immunohistochemistry

Mice were anesthetized with an isoflurane/oxygen mixture (RWD Life Science, China; induction concentration: 2.5%; maintenance concentration: 0.8–1.5%) and then transcardially perfused with saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (Sigma-Aldrich). After perfusion, the brains were extracted, post-fixed in 4% PFA overnight, and then subjected to gradient dehydration using 20% and 30% sucrose for 48 h. Coronal sections of 30 μm thickness were obtained using a sliding freezing microtome (Leica). After rinsing with phosphate-buffered saline (PBS), the sections were blocked with 0.3% Triton-X 100 and 3% bovine serum albumin (BSA) for 2 h at room temperature. Subsequently, the sections were incubated overnight at 4 °C with the following primary antibodies: anti-S100B (mouse; 1:100, A19108, ABclonal, China). After thorough washing, the sections were then incubated with Alexa Fluor™ 647 secondary antibody (1:1000, Thermo Fisher Scientific) for 2 h at room temperature. After additional washing steps, nuclei were stained with a DNA-specific fluorescent probe (4′,6-diamidino-2-phenylindole [DAPI], Rahway, NJ, USA) for 20 min at room temperature. Following a final rinse, we mounted the sections on slides, confocal images were acquired by laser confocal microscopy (LSM 800, ZEISS, Jena, Germany).

Statistical analysis

All experiments and data analyses in this study were conducted in a blinded manner. The number of experimental replicates (n) is indicated in the figure legends and represents the number of experimental subjects independently treated in each experimental condition. Statistical analyses were performed using Prism GraphPad 8.0 software with appropriate inferential methods as specified in the figure legends. Results with a two-tailed P value < 0.05 were considered statistically significant.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Nos. 62027812), the Joint Funds for the STI2030-Major Projects (Grant No.2021ZD0202900), the National Natural Science Foundation of China (62071329 and 62201383), and the National Science Foundation of Tianjin (23JCZDJC00440).

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Chunxiao Tian, Qiuying Xue and Tongrui Wu contributed equally to this work.

Authors and Affiliations

School of Biomedical Engineering, Tianjin Medical University, Tianjin, China
Chunxiao Tian, Tongrui Wu & Jingyu Zhao
School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
Qiuying Xue, Yuxuan Zhu, Hualin He, Aili Liu & Hui Shen
Department of Anesthesiology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
Chao Tian
School of Life Sciences, Tiangong University, Tianjin, China
Lei Dong & Yu Zheng

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Chunxiao Tian
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Contributions

H.S. and Y.Z. initiated and coordinated the project; C.T., T.W., and Q.X. designed the experiments; C.T., Q.X., T.W., and L.D. conducted the experiments; C.T., Q.X., T.W., and J.Z. analyzed the data; T.W., Y.Z., H.H., C.T., and A.L. contributed to data discussion; C.T., Q.X., T.W., L.D., and H.S. wrote the manuscript.

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Correspondence to Hui Shen.

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Tian, C., Xue, Q., Wu, T. et al. Inhibiting astrocyte calcium dynamics terminates status epilepticus via wireless septal stimulation. Sci Rep 15, 25579 (2025). https://doi.org/10.1038/s41598-025-09611-w

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Received: 02 February 2025
Accepted: 30 June 2025
Published: 15 July 2025
DOI: https://doi.org/10.1038/s41598-025-09611-w