Introduction

Ethanol, a potent psychoactive substance capable of inducing neuroadaptive changes that lead to dependence, continues to maintains widespread sociocultural acceptance across numerous populations1,2. While the pathophysiological mechanisms underlying chronic ethanol exposure have been extensively investigated, the acute health consequences of binge drinking remain relatively understudied in comparison. Defined pharmacologically as acute ethanol intoxication achieved through rapid consumption, binge drinking has emerged as the most prevalent form of alcohol use among youth1. It has been reported that excessive alcohol drinking is a leading preventable cause of over 200 diseases and injuries globally, with binge consumption alone accounting for more than 75% of alcohol-attributable social and economic losses while inducing acute neurological impairment and multi-organ dysfunction3,4,5, such as seizures6. But the mechanisms underlying acute alcohol-induced seizures remain to be extensively investigated.

Epilepsy, characterized by recurrent seizures, is fundamentally linked to an imbalance between excitatory glutamatergic and inhibitory GABAergic neurotransmission7,8.

GABAergic interneurons, essential for maintaining excitatory/inhibitory balance, are particularly vulnerable to neuroinflammatory insults, and their dysfunction is implicated in epileptogenesis9. Disruptions in GABAergic signaling may lead to network hyperexcitability, a hallmark of epilepsy10. While GABAergic stimulation can also contribute to seizure generation11. Additionally, it has been reported that mice treated with alcohol (3.5 g/kg, intraperitoneally) show pathological changes in GABAA receptor trafficking and function12,13. Notably, pharmacological interventions targeting GABA receptors, such as diazepam, have been shown to alter microglial morphology and synaptic plasticity in the hippocampal CA1 region, further linking microglial activity to inhibitory circuit integrity 14. Conversely, calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent signaling in excitatory neurons is critical for maintaining neuronal excitability7,15. Intriguingly, microglial activation has been shown to affect not only neuronal excitability but also neuronal inhibition16,17. Pharmacological interventions that target microglia, with minocycline as an example, exhibit regulatory effects on neuronal function in seizure models,18 suggesting their potential to restore neuronal homeostasis.

Under physiological conditions, microglia maintain the central nervous system (CNS) homeostasis through surveillance and neuroprotective functions19. Alcohol exposure triggers microglial activation, characterized by morphological transformation and release of pro-inflammatory mediators20,21. Activated microglia dynamically regulate neuronal activity by altering neurotransmitter receptor trafficking, remodeling perineuronal nets, and influencing neurogenesis-processes critical for synaptic stability and plasticity22. While microglial modulation of glutamatergic synapses has been extensively documented in brain region-specific contexts, their role in shaping GABAergic inhibitory circuits remains underexplored23,24. Recent studies propose that microglia, the resident immune cells of the central nervous system, may act as key mediators in this process by modulating neuronal circuits through interactions with both inhibitory and excitatory neurons25,26. Emerging evidence underscores the pivotal role of neuroinflammation and microglial dysregulation in the pathogenesis of epilepsy, particularly in alcohol-related epileptogenesis27,28.

However, it remains uncertain whether the microglia activation impacts GABAergic synaptic formation or glutamatergic synaptic formation in the hippocampus and its potential contribution to acute alcohol -induced enhancement of seizure susceptibility. Therefore, we established an acute alcohol treatment model to elucidate the impact of microglia activation on the structural plasticity of GABAergic synapses and glutamatergic synapse and its influence on the enhancement of seizure susceptibility induced by acute alcohol treatment using a combination of behavioral and immunohistochemical approaches.

Methods

Animal study

The study was approved by the Institutional Animal Care and Use Committee of Bengbu Medical University (Permit No. 2023–09; Ethics Approval No. 2022–029). All animal experiments were conducted in compliance with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations. At the end of the experiments, mice were euthanized by cervical dislocation under appropriate restraint. This method was performed by trained personnel to ensure immediate death, which was confirmed by cessation of heartbeat and respiration. Cervical dislocation was selected as it is consistent with AVMA guidelines for euthanasia of rodents and does not interfere with subsequent experimental analysis.

Male C57BL/6 J mice, weighing 18–25 g, were supplied by the Animal Experimental Center of Bengbu Medical University. The animals were maintained in a controlled environment with a constant temperature of 23 ± 2 °C, 50% humidity, ad libitum access to food and water, and a 12 h light–dark cycle. Mice were randomly divided into the following experimental groups: Saline, Alcohol, Alcohol + minocycline, and Alcohol + PLX3397. The Saline group and the Alcohol group were intraperitoneally (i.p.) injected with saline or alcohol (3.5 g/kg) respectively. Twenty-four hours after treatment, mice were either subjected to pentylenetetrazol (PTZ)-induced seizure test or sacrificed for immunohistochemistry. In the Alcohol + minocycline group, mice were (i.p.) injected with minocycline (Sigma-Aldrich, M9511, USA, 50 mg/kg) 23 h after alcohol injection. Minocycline was administered 1 h before PTZ-induced seizure test or immunohistochemistry (Fig. 1A). In the Alcohol + PLX3397 group, mice were administered PLX3397 (APExBIO, USA; 290 mg/kg) via medicated diet for 7 consecutive days prior to alcohol injection. The controls were fed with food without PLX3397. The PTZ-induced seizure test or tissue collection for immunohistochemistry was performed 24 h after alcohol injection.

Fig. 1
figure 1

Acute alcohol exposure increases the seizure susceptibility in mice. (A) Scheme of the acute alcohol treatment, behavioral test, and microglial depletion. (B) PTZ-induced seizure onset of saline-treated and alcohol-treated mice. (C) PTZ-induced seizure duration of saline-treated and alcohol-treated mice. Two-sided t test, n = 7 mice per group. Data are presented as mean ± SD, ** p < 0.01.

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PTZ-induced seizure test

The PTZ-induced seizure test was conducted as previously described. PTZ (Sigma-Aldrich, P6500, USA) was administered i.p. to induce acute seizures at a single dose of 50 mg/kg. Mice were continuously monitored via camera for 30 min post-PTZ administration. The latency to seizure onset and the duration of tonic–clonic seizures were recorded29.

Immunohistochemistry

Immunohistochemical analysis was carried out as previously described in the literature30. 24 h after drug treatment, the mice were deeply anesthetized with sodium pentobarbital (200 mg/kg, i.p.) and perfused transcardially with PBS, followed by 4% paraformaldehyde (PFA) in PBS. The brains were extracted and post-fixed in 4% PFA overnight and then transferred to 30% sucrose in PBS at 4 °C. The brains were fully saturated after 36 h, and 30 μm-thick coronal brain sections were cut using a freezing microtome (Leica, CM30503, Germany). The sections were processed as free-floating sections.

After being blocked in PBS containing 5% normal donkey serum (NDS; Jackson ImmunoResearch 017-000-121), 1% bovine serum albumin (BSA; Sigma-Aldrich, USA), and 0.3% Triton X-100 (Sigma-Aldrich, USA) for 2 h at room temperature, brain sections were incubated with primary antibodies: rabbit anti-Iba1 (Proteintech, 10,904-1-AP, USA), rabbit anti-GFAP (Proteintech, 16,825-1-AP, USA), mouse anti-GAD67 (Proteintech, 67,648-1-Ig, USA), mouse anti-NeuN (Proteintech, 66,836-1-Ig, USA), rabbit anti-VGAT (Proteintech, 14,471-1-AP, USA), rabbit anti-VGLUT1 (Proteintech, 55,491-1-AP, USA), mouse anti-GFAP (Proteintech, 60,190–1-Ig, USA) rabbit anti-C3 (Proteintech, 21,337-1-AP, USA) and rabbit anti-CaMKII (Proteintech, 13,730-1-AP, USA) overnight at 4 °C in blocking solution. Brain slices were washed in PBS (3 × 10 min) and incubated with corresponding secondary antibodies [Alexa Fluor 546 anti-rabbit IgG (Thermo Fisher Scientific, A10040, USA), Alexa Fluor 488 anti-mouse IgG (Thermo Fisher Scientific, A21202, USA)] for 1 h at room temperature. After washing with PBS (3 × 15 min), sections were mounted onto glass slides. An Olympus FV1200MPE SHARE laser-scanning confocal microscope with a × 20/0.75 NA objective was used to acquire Z-stack images. Imaging parameters (gain, threshold, black levels) were kept constant throughout. Maximum intensity projections were generated from Z-stacks and analyzed using ImageJ (NIH, USA) to quantify cell number (GAD67+ cells and C3+/GFAP+ cell), fluorescence intensity (Iba1, GFAP, CaMKII, and NeuN), and puncta number (VGAT and VGlut1).

All behavioral test, image acquisition, and data quantification were conducted in a blinded manner.

Statistical analysis

Data are expressed as mean ± SD. Significance was set at p < 0.05. GraphPad Prism 8.0 (GraphPad Software, USA) was used for analysis. Two-group comparisons used Student’s t-test; multi-group comparisons used one-way ANOVA with post hoc tests.

Software and algorithms

Acquired images were viewed and analyzed using both Olympus OlyVIA software (Version 3.1; Evident Life Science, Japan; https://www.olympus-lifescience.com/en/software/olymp-us-ov/) and ImageJ Fiji (Version 1.54 g; National Institutes of Health, USA; https://imagej.net/software/fiji/). Statistical analysis and graph generation were conducted using GraphPad Prism (Version 8.0; GraphPad Software, USA; https://www.graphpad.com). Figures were assembled and prepared for publication using Adobe Illustrator (Version 2025; Adobe Inc., USA; https://www.adobe.com/products/illustrator.html).

Results

Microglia mediate alcohol-induced enhancement of seizure susceptibility

In rodent models, acute alcohol exposure has been shown to modulate the function of specific neuronal populations31, and seizure activity is closely linked to abnormal neuronal firing patterns32,33. In this study, mice were administered i.p. either a 50% ethanol solution (3.5 g/kg, dissolved in normal saline) or an equivalent volume of normal saline (Fig. 1A). 24 h later, PTZ-induced seizure test are performed. The alcohol-treated group exhibited significantly accelerated seizure onset and prolonged seizure duration compared to the control group (Fig. 1B, C).

To elucidate the cellular mechanisms underlying alcohol-induced enhancement of seizures susceptibility, we examined the role of microglia, which are resident immune cells in the CNS that exhibit heightened responsiveness following alcohol exposure. Immunohistochemical analysis revealed that acute alcohol exposure robustly activated microglia in the hippocampal CA1 region, as evidenced by increased Iba1 fluorescence intensity and cell area 24 h after alcohol treatment (Fig. 2A–C). Interestingly, alcohol treatment did not induce significant changes in astrocyte reactivity assessed by GFAP and complement C3 immunostaining (Fig. 2D, E). To confirm the role of microglia in alcohol-induced seizure susceptibility, we administered the microglial inhibitor minocycline 23 h after alcohol or saline treatment. Immunohistochemical analysis revealed that minocycline treatment significantly reduced microglial activation (Fig. 3A, B) without affecting astrocytes (Fig. 3C, D) or neurons (Fig. 3E, F). Mice treated with minocycline and alcohol exhibited delayed seizure onset (Fig. 3G) and reduced seizure duration (Fig. 3H) compared to mice treated with alcohol. To confirm the role of microglia in alcohol induced enhancement of seizure susceptibility, an additional cohort of mice was treated with the CSF1R inhibitor PLX3397 for 7 days prior to alcohol or saline injection and PTZ test. Similarly, PLX3397 treatment effectively suppressed microglial activation (Fig. 3I, J), and mice treated with PLX3397 and alcohol showed delayed seizure onset (Fig. 3K) and reduced seizure duration (Fig. 3L) compared to mice treated with alcohol only. Overall, these results suggest that alcohol-induced enhancement of seizure susceptibility is dependent on microglia activation.

Fig. 2
figure 2

Microglia activation by exposure to alcohol in the hippocampal CA1 regions. (A) Representative images of Iba1 immunostaining, binary and skeleton in the hippocampal CA1 region. (B) Quantification of relative Iba1 intensity. (C) Quantification of the normalized Iba1 area in the hippocampal CA1 region. Two-sided t-test, n = 7 mice per group. The CA1 subregions shown in the upper left panel include the stratum oriens (SO), stratum pyramidale (SPy), stratum radiatum (SR), and stratum lacunosum moleculare (SLM). (D) Representative images of GFAP and C3 immunohistostaining in the hippocampal CA1 region. (E) Quantification of the the proportion of C3⁺/GFAP⁺ astrocytes in the hippocampal CA1 region. Two-sided t-test, n = 5–6 mice per group, scale bars, 10 μm and 100 μm (in magnification). Data are presented as mean ± SD, ** p < 0.01, ns, not significant.

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Fig. 3
figure 3

Microglial depletion restores alcohol-induced enhancement of seizure susceptibility. (A) Representative images of Iba1 immunostaining in the hippocampal CA1 region. (B) Quantification of relative Iba1 intensity. One-way ANOVA with Tukey multiple comparison test, n = 7 mice per group. (C) Representative images of GFAP immunostaining in the hippocampal CA1 region. (D) Quantification of relative GFAP intensity. One-way ANOVA with Tukey multiple comparison test, n = 7 mice per group. (E) Representative images of NeuN immunostaining in the hippocampal CA1 region. (F) Quantification of relative NeuN intensity. One-way ANOVA with Tukey multiple comparison test, n = 7 mice per group. (G) PTZ-induced seizure onset in saline-treated and alcohol-treated mice with or without minocycline. (H) PTZ-induced seizure duration in saline- or alcohol-treated mice. One-way ANOVA with Tukey multiple comparison test, n = 7 mice per group (I) Representative images of Iba1 immunostaining in the hippocampal CA1 region of saline- or alcohol- treated mice fed with or without PLX3397. (J) Quantification of relative Iba1 intensity. One-way ANOVA with Tukey multiple comparison test. n = 5–7 mice per group. (K) PTZ-induced seizure onset in saline- or alcohol-treated mice fed with or without PLX3397. (L) PTZ-induced seizure duration in saline- or alcohol-treated mice fed with or without PLX3397. One-way ANOVA with Tukey multiple comparison test, n = 5–7 mice per group, scale bars, 100 μm. Data are presented as mean ± SD, *p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significant difference.

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Dysregulation of synapse formation mediated by microglial activation contributes to the alcohol-induced enhancement of seizure susceptibility

It has been reported that the effect of microglia on synaptic plasticity depends on the specific brain region and cell type considered34,35. The hippocampus is involved in initiating and spreading seizure activities29. Most of the stratum lacunosum moleculare (SLM) neurons are GABAergic interneurons36. Our investigation revealed a significant increase in glutamic acid decarboxylase 67 (GAD67, marker for GABAergic interneurons)-positive cells in the hippocampal CA1 area of mice treated with alcohol (Fig. 4A, B). Surprisingly, the expression of Ca2+/calmodulin-activated kinase II (CaMKII), a glutamatergic neuron marker decreased significantly (Fig. 4A, C).

Fig. 4
figure 4

Microglial depletion rescues alcohol-induced enhancement of GABAergic inhibitory synapse formation and impairment of glutamatergic excitatory synapses formation. (A) Representative images of GAD67 and CaMKII immunohistochemical staining in the hippocampal CA1. (B) Quantification of the GAD67-positive cell abundance in the hippocampal CA1. (C) Quantification of relative CaMKII intensity in the hippocampal CA1. One-way ANOVA with Tukey multiple comparison test was performed with n = 7 mice per group and scale bar 100 μm. (D) Representative images of VGAT immunohistostaining. (E) Quantification of the number of VGAT puncta in hippocampal CA1 area. One-way ANOVA with Tukey’s multiple comparisons test, n = 7 mice per group. (F) Representative images of VGlut1 immunohistostaining in the hippocampal CA1 area. (G) Quantification of the number of VGlut1 puncta in the hippocampal CA1 area. Two-way ANOVA with Tukey’s multiple comparisons test, n = 7 mice per group. Scale bars, 100 μm, 50 μm and 10 μm (in magnification). Data are presented as mean ± SD, *p < 0.05, **p < 0.01, *** p < 0.001.

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To further explored the impact of microglia activation on neurons and synapses, minocycline treatment, which inhibited microglial activation, mitigated increased GAD67 expression (Fig. 4A, B) and restored CaMKⅡ expression in mice treated with alcohol (Fig. 4A, C). To gain deeper insights into synaptic alterations, we further quantified the puncta number of vesicular GABA transporter (VGAT, a marker for inhibitory synapses) and vesicular glutamate transporter 1 (VGlut1, marker for excitatory synapses). Alcohol exposure significantly increased VGAT puncta density and decreased VGlut1 puncta density in the CA1 region, and the minocycline treatment reversed the alcohol-induced changes in VGAT and VGlut1 puncta density (Fig. 4D–G). These findings suggest that microglia activation can upregulate GABAergic synapse formation and downregulate glutamatergic synapse formation.

Discussion

The present study reveals the pivotal role of microglia in acute alcohol-induced seizures by modulating synapse formation. PTZ-induced behavioral test demonstrated that intraperitoneal administration of alcohol significantly enhanced seizure susceptibility. Pharmacological inhibition of microglia with either minocycline or PLX3397 fully reversed this phenotype, indicating that microglial activation serves as a central driver of susceptibility to alcohol-induced seizures. We further observed that alcohol exposure, via microglial activation, upregulated GABAergic synapse formation and downregulated glutamatergic synapse formation. These findings provide direct relevance for the cause of acute alcohol-induced seizures.

Recent studies have shown that microglia can affect local circuit function through synaptic pruning and neurotransmitter regulation37,38. Similar to the LPS-induced neuroinflammation model, the present study found that alcohol exposure triggered a proinflammatory phenotype of microglia and specifically promoted the formation of GABAergic inhibitory synapses36. This phenomenon may result from the response of microglia to the concentration of GABA in the microenvironment: acetaldehyde produced by alcohol metabolism activates microglia Toll-like receptor 4 (TLR4), which in turn releases proinflammatory factors such as IL-1β and TNF-α, which have been shown to enhance the survival of GABAergic interneurons and promote their synaptic connections39. In addition, microglia may indirectly amplify the dominance of inhibitory signals by engulfing excitatory synapses, such as glutamatergic terminals, contributing to the observed suppression of excitatory markers and synapses (CaMKII, VGlut1) in the hippocampal CA1 region, thereby lowering seizure threshold40,41.

Critically, these microglia-derived inflammatory mediators (e.g., IL-1β, TNF-α) are established modulators of neuronal excitability and synaptic function42. IL-1β is linked to balancing GABAergic and glutamatergic systems, consistent with our observations of altered GAD67+ cells, CaMKII, VGAT+, and VGlut1+ puncta in alcohol-treat mice. GABA regulates IL-1β production in macrophages, suggesting a bidirectional interplay underlying these changes43. TNF-α similarly promotes synaptic incorporation of GABA receptors and internalization of AMPA receptors, providing a mechanistic link between microglial activation and the Excitatory/Inhibitory (E/I) imbalance in alcohol-induced seizure susceptibility44,45,46. Further studies about cytokine in the hippocampal microenvironment following alcohol exposure may provide potential new targets for therapeutic intervention.

The reversal effect of microglial inhibitors (minocycline and PLX3397) on the epilepsy phenotype highlights the importance of microglia as a therapeutic target. Unlike PLX3397-mediated microglial depletion, minocycline is more suitable for acute intervention by inhibiting microglial activation rather than eliminating the cell population47. It is important to consider that minocycline might have direct effects on neurons, such as modulating neuronal excitability or synaptic function through mechanisms independent of microglia48. Our data showing that minocycline did not alter NeuN intensity (Fig. 3E, F) or CaMK Ⅱ intensity (Fig. 4A, C). Cao et al. also treated mice with the same minocycline dosage (50 mg/kg) to specific inhibit microglial activation25.

Traditionally, epilepsy has been attributed to reduced inhibition, enhanced excitation, or both contribute to the etiology of epilepsy49. However, we showed an increase in the immunoreactivity of GAD67 and vGAT1 with reduction of vGLUT1 reactivity in our study. More recent work has led to a shift in focus toward the potential role of overactive inhibitory interneurons in the initiation of seizures. A growing body of evidence indicates that interneurons exhibit heightened activity immediately before seizure onset50,51. Furthermore, several studies provide compelling support for a causal contribution of interneurons to seizure genesis50,52,53. These findings collectively support an emerging “GABAergic initiation hypothesis”, suggesting that synchronous activation of inhibitory interneurons may actively precipitate seizure events54.

The role of interneurons in seizure induction is both significant and subject to ongoing debate55,56. Through GABAergic synapses, interneurons release GABA onto excitatory neurons. This typically leads to chloride influx, suppression of neuronal firing, and consequently, a reduction in seizure propensity57,58,59,60. Conversely, GABAergic signaling may also facilitate seizure genesis11. Activation of GABA receptors results not only in chloride influx but also in bicarbonate ion (HCO₃⁻) efflux61,62.Under certain conditions, the influx of chloride along with simultaneous bicarbonate efflux can convert the action of GABA from inhibitory to excitatory, thereby promoting epileptic discharge activity11. Additionally, GABAergic interneurons are also intimately involved in the synchronization of neuronal spiking; consequently, an increase in inhibitory activity may potentially contribute to certain forms of pathological synchronization63.

Although this study identifies an acute effect of alcohol on the microglia-inhibitory circuit, the kinetic characteristics of alcohol metabolism (e.g., time to peak plasma concentration, gradient in brain distribution) may further modulate magnitude of the observed neuronal and synaptic dysregulation. For example, high doses of alcohol (> 3 g/kg) may trigger synaptic remodelling by over-activation of microglia, leading to irreversible neural network damage, while low doses of alcohol (1–2 g/kg) may only cause reversible synaptic remodeling64,65. In addition, chronic alcohol exposure may induce chronic seizure susceptibility by altering the homeostatic regulatory capacity of microglia through epigenetic modifications, such as histone acetylation66. Future studies need to combine multi-time point sampling and single-cell sequencing technology to analyze the dynamic evolution of microglia functional state after alcohol exposure.