The first three postnatal weeks represent a vulnerable window during which sevoflurane exposure disrupts critical period Arc expression and long-term cognitive deficits

We first employed a well-established neonatal exposure paradigm in which mice were exposed to 3% sevoflurane for 2 h daily from postnatal day (P)6 to P8 (Fig. 1a, b). In control mice, hippocampal mRNA expression of Arc gradually increased during the third postnatal week, peaking at P21, followed by a rapid decrease to baseline levels, which is consistent with previous reports [13]. Notably, compared with control conditions, repeated sevoflurane exposure significantly reduced Arc mRNA levels at P21 (Fig. 1c). Western blot analysis confirmed decreased ARC protein levels in both female and male mice at P21 following sevoflurane exposure (Fig. 1d, e). Cognitive performance was assessed in adulthood (P56) using hippocampus-dependent tasks: the Y-maze spontaneous alternation test (working memory), the NOR test with a 24-h retention interval (intermediate-term memory), and the CFC test with a 5-day forgetting index (long-term memory). Compared with control mice, sevoflurane-exposed mice exhibited a significant increase in the forgetting index for both contextual and cued memory in the CFC test (Fig. 1f). In the NOR test, sevoflurane-exposed mice showed a significantly reduced exploration time for the novel object and fewer entries into the 20 mm exploration zone (Fig. 1g). In the Y-maze task, these mice tended toward reduced spontaneous alternation, although the difference did not reach statistical significance (Fig. 1h).

Fig. 1The alternative text for this image may have been generated using AI.

Neonatal sevoflurane exposure induces both a reduction in Arc expression at P21 and cognitive deficits in adults, whereas exposure during the fourth week reduces Arc expression at P30 but does not cause cognitive deficits. a, b, i Experimental design. c–e Molecular analysis following sevoflurane exposure at P6, P7, and P8, comparing the Control and Sevoflurane groups. (c) RT‒qPCR analysis of Arc mRNA expression at P6, P9, P14, P21, and P30. Control vs. Sevoflurane [two-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001; n = 3–6 mice]. d, e Representative Western blot and quantification of ARC protein expression at P21 in female and male mice. Control vs. Sevoflurane [male: two-tailed unpaired t test, P = 0.032; female: two-tailed unpaired t test, P = 0.019; n = 3 mice]. f–h Cognitive behavioral tests in adulthood following sevoflurane exposure at P6, P7, and P8, comparing the Control and Sevoflurane groups. f Forgetting index for both contextual and cued recall in the CFC test. Control vs. Sevoflurane [context: Mann‒Whitney test, P = 0.0447; cue: two-tailed unpaired t test, P = 0.0141; Control: n = 12 mice (7 males, 5 females); Sevoflurane: n = 13 mice (7 males, 6 females)]. g Exploration trajectories and percentage of time and entries toward the novel object between groups [time: two-tailed unpaired t test, P = 0.019; entries: two-tailed unpaired t test, P < 0.0001; n = 11 mice per group (6 males, 5 females)]. h Spontaneous alternation rate in the Y-maze test in sevoflurane-treated vs. Control mice [two-tailed unpaired t test, P = 0.0531; n = 11 mice per group (6 males, 5 females)]. j–k Representative Western blot and quantification of ARC protein expression following sevoflurane exposure on P24–P26, comparing the Sevoflurane and Control groups [two-tailed unpaired t test, P = 0.016; n = 3 mice]. l–n Cognitive behavioral tests in adulthood following sevoflurane exposure at P24, P25, and P26, comparing the Control and Sevoflurane groups. l Spontaneous alternation rate in the Y-maze test. Control vs. Sevoflurane [two-tailed unpaired t test, P = 0.4108; Control: n = 9 male mice, Sevoflurane: n = 10 male mice]. m Forgetting index for both contextual and cued recall in the CFC test. There was no significant difference between the groups [context: Mann‒Whitney test, P = 0.366; cue: Mann‒Whitney test, P = 0.5338; Control: n = 6 male mice; Sevoflurane: n = 7 male mice]. n Exploration trajectories and percentage of time and entries toward the novel object in both groups [time: two-tailed unpaired t test, P = 0.2537; entries: two-tailed unpaired t test, P = 0.9308; Control: n = 9 male mice; Sevoflurane: n = 10 male mice]. Sev, Sevoflurane. ns, P > 0.05. * and **** indicate P < 0.05 and 0.0001, respectively. The data are presented as the mean ± SEM.

To further investigate whether cognitive vulnerability to sevoflurane is confined to early developmental stages, male mice were exposed to 3% sevoflurane for 2 h per day from P24 to P26 (Fig. 1i). We measured ARC protein expression at P30 in these mice. Compared with those in control mice, the ARC protein levels in the hippocampus were reduced (Fig. 1j, k). Subsequent behavioral assessments, including the NOR test, Y-maze spontaneous alternation test, and CFC test, revealed no statistically significant differences between sevoflurane-exposed mice and controls (Fig. 1l–n).

To more accurately simulate clinical exposure in infants, where anesthetic procedures are typically scheduled with extended intervals, we modified the exposure timing in our paradigm. Specifically, neonatal mice were exposed to 3% sevoflurane for 2 h on P6, P11, and P16, which is once per week during the first three postnatal weeks (Fig. S1a). Consistent with the behavioral outcomes, hippocampal ARC expression at P21 was significantly reduced compared with that in the control group (Fig. S1b, c). Behavioral assessments in adulthood revealed that compared with control mice, mice that had repeated exposure to sevoflurane exhibited significant reductions in both the novel object exploration time and frequency in the NOR test (Fig. S1d), along with marked impairments in working memory, as measured by the Y-maze spontaneous alternation test (Fig. S1e). In contrast, mice that received a single 2-h exposure at P7 (Fig. S2a) did not exhibit significant cognitive impairments in adulthood. The performance of these mice in the CFC, NOR, and Y-maze tasks remained comparable to that of the controls (Fig. S2b–d), which aligns with previous findings [16]. Western blot analysis revealed no change in ARC expression at P21 compared with that in the controls (Fig. S2ae–f). These results collectively indicate that the timing and repetition of sevoflurane exposure during a sensitive postnatal period are critical factors in the induction of long-term cognitive impairment. In comparison, the cumulative dose of anesthesia and the proximity of the exposure intervals appear to play smaller roles.

Taken together, these findings establish that the first three postnatal weeks serve as the vulnerable window for repeated sevoflurane exposure that leads to cognitive impairment, which coincides with the critical developmental period of the hippocampus. Notably, sevoflurane exposure significantly reduces ARC expression during the critical period, which is associated with long-term cognitive deficits. Reductions in ARC expression outside this window do not produce detectable behavioral impairments.

Reduced Arc expression during the critical period leads to long-term cognitive impairments

Immunofluorescence analysis revealed that the ARC protein was predominantly localized to the CA1 region of the hippocampus at P21, with significantly lower expression in other hippocampal subregions (Fig. 2a, b). Within the CA1 region, the ARC signal was observed across multiple layers, including dendrite-rich nonsomatic areas. In mice exposed to sevoflurane, a substantial reduction in the fluorescence intensity of ARC was observed specifically in the CA1 region compared with that in control mice (Fig. 2c, d). To identify the cellular sources of ARC expression, we performed double immunofluorescence staining to assess the colocalization of ARC with MAP2, IBA-1, and GFAP. The results revealed strong colocalization of the ARC with MAP2, while minimal overlap was observed with IBA-1 and GFAP (Fig. 2e, f), indicating that sevoflurane predominantly affects ARC expression in CA1 pyramidal neurons. Given the temporal and spatial specificity of ARC expression during the critical period, we stereotaxically injected Arc ASO into the hippocampi of P16 mice (Fig. 2g). This intervention transiently suppressed ARC protein translation and facilitated mRNA degradation at the third postnatal week but did not affect Arc levels at other time points. Compared with those in the scramble group, fluorescence imaging at P21 confirmed a significant reduction in the expression of the ARC protein in CA1 neurons (Fig. 2h). Western blot analysis validated these results, revealing a pronounced decrease in the level of the ARC protein at P21, with recovery to baseline levels by P28 (Fig. 2i, j). Behavioral assessments conducted in adulthood revealed that in the Y-maze spontaneous alternation test, no significant differences were observed between mice with transient Arc knockdown and those injected with scrambled controls (Fig. 2k). In the fear conditioning test, compared with mice injected with scrambled control, Arc ASO-injected mice exhibited an increased forgetting index for both contextual and cued memory (Fig. 2l). In the NOR test, compared with control mice, Arc ASO mice displayed significant reductions in the exploration time and frequency (Fig. 2m). These results indicate that transient disruption of Arc expression using Arc ASO during the critical developmental period is sufficient to induce hippocampus-dependent cognitive deficits comparable to those observed in sevoflurane exposure models.

Fig. 2The alternative text for this image may have been generated using AI.

Transient Arc knockdown during the critical period leads to cognitive deficits in adulthood. a, b Representative immunofluorescence images of ARC (red) in different subregions of the hippocampus (CA1, DG, CA2, and CA3) and magnified views of the CA1 region at P21. DAPI (blue) was used to stain the nuclei. Scale bars = 200 μm, 10 μm or 5 μm. Quantification of mean ARC intensity in various hippocampal subregions [one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001 for CA1 vs. DG/CA2/CA3, P = 0.9573 for DG vs. CA2, P = 0.8679 for DG vs. CA3, P = 0.9934 for CA2 vs. CA3]. c, d Representative immunofluorescence images of the ARC protein (red) and DAPI (blue) in the hippocampal CA1 region at P21 following sevoflurane exposure at P6-8. Scale bars = 10 μm. The mean ARC intensity was quantified and compared between the Control and Sevoflurane groups [two-tailed unpaired t test, P = 0.0482; quantification was based on the average of three fields per mouse; n = 6 mice per group]. e, f Colocalization analysis of ARC (red) with distinct cellular markers in the hippocampal CA1 region at P21. Neurons were labeled with MAP2 (green), microglia with IBA1 (green), and astrocytes with GFAP (green). Representative colocalization images and corresponding 2D histograms of selected ROIs are presented to evaluate the spatial overlap between ARC and each cellular marker. Scale bars = 10 μm [one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001 for MAP-2 vs. IBA-1 and MAP-2 vs. GFAP; n = 6 cells per group]. g Experimental design. h–j Histological analysis following the hippocampal injection of Arc ASO at P16, comparing the Scramble and Arc ASO groups. h Representative immunofluorescence images of ARC protein (red) and DAPI (blue) in the hippocampal CA1 region at P21. Scale bars = 10 μm. i, j Representative Western blot and quantification of ARC protein expression at P21 and P28. Scramble vs. Arc ASO [P21: two-tailed unpaired t test, P = 0.0241; Scramble: n = 3 mice; Arc ASO: n = 4 mice; P28: two-tailed unpaired t test, P = 0.555; n = 4 mice per group]. k‒m Results of the cognitive behavioral tests in adulthood following the hippocampal injection of Arc ASO at P16, comparing the Scramble and Arc ASO groups. k Spontaneous alternation rate in the Y-maze test between the Scramble and Arc ASO groups [Mann‒Whitney test, P = 0.6189; scramble: n = 10 mice (5 males, 5 females); Arc ASO: n = 9 mice (6 males, 3 females)]. l Forgetting index for both contextual and cued recall in the CFC test. Scramble vs. Arc ASO. [context: two-tailed unpaired t test, P = 0.0251; cue: two-tailed unpaired t test, P = 0.0126; Scramble: n = 10 mice (5 males, 5 females), Arc ASO: n = 8 mice (6 males, 2 females)]. m Exploration trajectories and percentage of time and entries toward the novel object in both groups [time: two-tailed unpaired t test, P < 0.0001; entries: two-tailed unpaired t test, P < 0.0001; Scramble: n = 14 mice (7 males, 7 females); Arc ASO: n = 17 mice (8 males, 9 females)]. ROI, region of interest. ASO, antisense oligonucleotide. ns, P > 0.05. * and **** indicate P < 0.05 and 0.0001, respectively. The data are presented as the mean ± SEM.

Sevoflurane exposure disrupts Arc protein upregulation during the critical developmental period via the activation of GSK3β

Extensive studies have demonstrated that repeated sevoflurane exposure leads to persistent hyperactivation of GSK3β [4, 16] and previous studies have shown that GSK3β promotes Arc degradation via the ubiquitin‒proteasome pathway [17, 18]. To determine the activation state of GSK3β in various sevoflurane exposure models at P21, we assessed its phosphorylation level. Compared with control treatment, repeated exposure to sevoflurane from P6 to P8 significantly increased GSK3β phosphorylation at threonine 216 (GSK3β-T216) and upregulated total GSK3β expression in the hippocampus (Fig. 3a, b). In contrast, compared with control treatment, a single sevoflurane exposure did not significantly alter the GSK3β or GSK3β-T216 levels (Fig. S2e-f). Moreover, compared with control mice, male mice exposed to sevoflurane from P24 to P26 presented elevated GSK3β-T216 levels, indicating that repeated exposure during later developmental stages also sustains GSK3β activation (Fig. 3c, d). Together, these results demonstrate an inverse correlation between GSK3β activation and Arc expression, suggesting that repeated sevoflurane exposure may impair Arc stability during the critical period by promoting its degradation through the activation of GSK3β.

Fig. 3The alternative text for this image may have been generated using AI.

Neonatal sevoflurane exposure activates GSK3β, disrupting Arc upregulation through its interaction with the Arc protein during the critical period. a, b Representative Western blot and quantification of GSK3β-T216 expression relative to total GSK3β expression at P21 following sevoflurane exposure at P6–P8, comparing the Control and Sevoflurane groups [total GSK3β: two-tailed unpaired t test, P = 0.0003; GSK3β-T216/Total GSK3β: two-tailed unpaired t test, P = 0.0315; n = 3 mice per group].c, d Representative Western blot and quantification of GSK3β-T216 expression relative to total GSK3β expression at P30 following sevoflurane exposure at P24–P26 in the Control vs. Sevoflurane groups of mice [total GSK3β: two-tailed unpaired t test, P = 0.2921; GSK3β-T216/Total GSK3β: two-tailed unpaired t test, P = 0.04; n = 3 mice per group]. e Immunofluorescence staining of the hippocampal CA1 region showing the distribution of GSK3β (green), ARC (red), DAPI (blue), and GSK3β-Ser9 (far-red) in control and neonatal sevoflurane-exposed mice. Scale bar = 10 μm. Magnified views indicate regions of colocalization (yellow arrows). Scale bar = 1 μm. f Representative Western blot results of the coimmunoprecipitation experiments showing the interaction between ARC and GSK3β at P21 in control and sevoflurane-exposed mice. g Experimental design. h, i Representative Western blot and quantification of ARC protein expression at P21 after daily intraperitoneal injection of CHIR99021 from P14 to P21 following neonatal sevoflurane exposure [one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.1797 for Control + DMSO vs. Sevoflurane + DMSO, P = 0.0249 for Sevoflurane + DMSO vs. Sevoflurane + CHIR; n = 3 mice per group]. CHIR, CHIR99021. DMSO, dimethyl sulfoxide. ns, P > 0.05. *, **, and *** indicate P < 0.05, 0.01, and 0.001, respectively. The data are presented as the mean ± SEM.

To investigate whether GSK3β directly interacts with Arc during this critical period, we performed immunofluorescence staining of CA1 pyramidal neurons at P21. ARC and GSK3β exhibited strong colocalization in the cytoplasm of sevoflurane-exposed neurons, particularly within dendritic regions, while minimal presence in the nuclei was detected. Additionally, the level of inactive GSK3β (phosphorylated at Ser9) decreased in response to sevoflurane exposure, whereas total GSK3β expression increased and this protein colocalized with Arc (Fig. 3e). To confirm the physical association between GSK3β and Arc, we performed coimmunoprecipitation (Co-IP) assays. These experiments revealed a direct interaction between Arc and GSK3β, with the interaction between Arc and GSK3β in sevoflurane-exposed mice being greater than that in control mice (Fig. 3f).

Finally, to test whether pharmacological inhibition of GSK3β can rescue Arc levels, we administered CHIR99021 (a selective GSK3β inhibitor) via daily intraperitoneal injection from P14 to P21 following sevoflurane exposure. While the baseline reduction in Arc expression between the Control + DMSO and Sevoflurane + DMSO groups was not significantly different (likely because of the biological variability and stress associated with repeated daily injections), Western blot analysis revealed that compared with vehicle treatment (DMSO), CHIR99021 treatment significantly restored Arc protein levels (Fig. 3g–i). These results collectively support that repeated sevoflurane exposure activates GSK3β, promoting Arc degradation and impairing its developmental upregulation during the critical period.

Disruption of Arc expression during the critical period promotes redundant synapses and delays morphological maturation

To elucidate the role of Arc in hippocampal function during the critical period, we performed bulk RNA sequencing on hippocampal tissues collected at P21 from mice that received Arc ASOs or Scramble control ASOs at P16 (Fig. 4a). Differential expression analysis revealed that 556 genes were upregulated and 683 genes were downregulated in the Arc ASO group compared with the Scramble group (Fig. 4b). Notably, the expression of several genes associated with neuronal development and synaptic function, such as Ephb3, Pcdha9, Sema3d, Egr2, Grin2a, and Syt7, was significantly altered in the Arc ASO group compared with the Scramble control group (Fig. S3a). In addition, microglia and immune-related genes, such as Mmp12, Itgb2, Fcgr2b, Tnf, and Cst7, were differentially expressed following Arc knockdown (Fig. S3b), suggesting that Arc may influence neuroimmune signaling pathways. Furthermore, the expression of genes involved in myelination and structure, such as Olig2, Sirt2, Opalin, Gpr17, and Ndrg1, also substantially changed compared with that in the Scramble controls (Fig. S3c). KEGG pathway enrichment analysis revealed significant alterations in several key developmental and signaling pathways, including “MAPK signaling,” “axon guidance,” “glutamatergic synapses,” “neuroactive ligand–receptor interactions,” and “lysosomes,” in the Arc ASO group compared with the Scramble group (Fig. 4c). Together, these findings demonstrate that Arc knockdown during the critical period disrupts transcriptional programs essential for neuronal maturation, synaptic connectivity, glial function, and circuit formation.

Fig. 4The alternative text for this image may have been generated using AI.

Transient Arc knockdown during the critical period leads to synaptic redundancy and microglial dysfunction. a Experimental design. b, c Bulk RNA-seq results at P21 following transient Arc knockdown via hippocampal injection of Arc ASOs at P16, comparing the Scramble and Arc ASO groups. b Volcano plots showing differentially expressed genes. Upregulated genes are shown in red, downregulated genes are shown in blue, and genes with no significant change in expression are shown in gray. Scramble vs. Arc ASO. |FC | ≥ 1, P < 0.05. c KEGG pathway enrichment analysis of the DEGs between groups. d–e Representative Western blots and quantification of the expression of the synaptic proteins PSD95, Homer1, SAP-102, and Synaptophysin in the hippocampus at P21 following the injection of Arc ASOs at P16. Scramble vs. Arc ASO [PSD95: two-tailed unpaired t test, P = 0.0329; Homer1: two-tailed unpaired t test, P = 0.0195; SAP-102: two-tailed unpaired t test, P = 0.162; Synaptophysin: two-tailed unpaired t test, P = 0.0247; n = 3 mice per group]. f, g Representative immunofluorescence images and quantification of the colocalization of the presynaptic marker Synaptophysin (red) and the postsynaptic marker Homer1 (green) in the CA1 region of the hippocampus at P21 following the hippocampal injection of Arc ASOs at P16, comparing the Scramble and Arc ASO groups. Scale bar = 5 μm [Synaptophysin spots: two-tailed unpaired t test, P = 0.1313; Homer1 spots: two-tailed unpaired t test, P = 0.0009; colocalized spots: two-tailed unpaired t test, P = 0.0247; quantification was based on the average of six fields per mouse; n = 3 mice per group]. h–i Representative Golgi-stained images and quantification of dendritic spine morphology in CA1 pyramidal neurons at P21 following hippocampal Arc ASO injection at P16. Analyses were performed on secondary apical dendrites. Spines were classified as mushroom, stubby, long, or filopodia-like. Scale bar = 10 μm [Scramble vs. Arc ASO: two-tailed unpaired t test; mushroom, P = 0.002; stubby, P = 0.0738; long, P = 0.1976; filopodia-like, P = 0.0002; n = 29–32 dendritic segments from 3 mice per group]. j–m Analysis of hippocampal microglia at P21 following Arc ASO injection at P16, comparing the Scramble and Arc ASO groups. j Representative skeletonized images of IBA-1-labeled microglia. Scale bar = 20 μm. k Quantification of microglia number, soma area and branching complexity via Sholl analysis in the hippocampal CA1 region, as assessed by IBA-1 immunostaining. Scramble vs. Arc ASO [cell number: two-tailed unpaired t test with Welch’s correction; P = 0.9574; quantification is based on the average of six fields per mouse; n = 3 mice per group; soma area: two-tailed unpaired t test, P = 0.3348; n = 31–52 cells from 3 mice per group; Sholl analysis: two-way ANOVA followed by Tukey’s post hoc test, P = 0.1956, n = 12 cells from 3 mice per group]. l Representative immunofluorescence images showing the merged signals of IBA-1 (red), CD68 (green), Homer1 (far red), and DAPI (blue), a magnified view of CD68 (green)/Homer1 (far red) colocalization, and a 3D-rendered magnified image of Homer1 puncta within CD68 in IBA-1-labeled microglia. White arrows highlight representative examples of synaptic engulfment, demonstrating the localization of Homer1 within microglial CD68-positive compartments. Scale bars = 10 μm or 5 μm. m Quantification of Homer1 puncta localized within the CD68 compartments of microglia. Scramble vs. Arc ASO [Mann‒Whitney test, P < 0.0001; n = 18 cells from 3 mice per group]. n–o Representative Western blots and quantification of CSF1R and C1qa expression at P21 following hippocampal Arc ASO injection at P16 [CSF1R: two-tailed unpaired t test, P = 0.0442; C1qa: two-tailed unpaired t test, P = 0.7185; n = 3 mice per group]. ASO, antisense oligonucleotide. FC, fold change. DEGs, differentially expressed genes. ns, P > 0.05. *, ***, and **** indicate P < 0.05, 0.001, and 0.0001, respectively. The data are presented as the mean ± SEM.

On the basis of the results of the transcriptomic analysis, we investigated how the transient suppression of Arc expression during the critical developmental period affects hippocampal synaptic structure. Western blot analysis revealed significantly higher protein levels of PSD-95, Homer1, and Synaptophysin in the Arc ASO group than in the Scramble group, whereas the protein level of SAP-102 remained unchanged (Fig. 4d, e). Immunofluorescence staining supported these findings, revealing a trend toward increased expression of the presynaptic marker Synaptophysin in the Arc ASO group compared with the scramble control group, although the difference was not statistically significant. In contrast, the expression of the postsynaptic marker Homer1 was significantly upregulated, and the colocalization signal between Synaptophysin and Homer1 markedly increased, suggesting an increase in the synaptic contact density under conditions of Arc reduction (Fig. 4f, g). To determine whether these molecular changes were accompanied by structural remodeling, we performed Golgi staining of the hippocampal CA1 region. The results revealed significant alterations in dendritic spine morphology in the Arc ASO group, with a reduction in mature mushroom-shaped spines and a marked increase in filopodia-like immature spines compared with those in the scramble group (Fig. 4h, i). Given the increased synaptic pruning activity at this developmental stage, these findings may reflect that Arc knockdown affects processes related to synaptic maturation or elimination.

To assess whether this phenotype could be recapitulated by sevoflurane exposure, we performed the same analysis in the CA1 region at P21 in mice treated with sevoflurane. Like those in the Arc ASO group, these mice exhibited increased numbers of filopodia-like and long spines; however, compared with those in the control group, the number of mature mushroom spines were unchanged (Figure. S4a, b). Together, these results suggest that the transient suppression of Arc expression during the critical period induces the accumulation of immature spines, a hallmark also observed in the sevoflurane exposure model.

Arc suppression impairs microglia-mediated synaptic pruning during the critical period

Synaptic pruning is mediated primarily by microglia and preferentially targets immature synapses. To explore whether Arc downregulation directly affects microglial state and function, we systematically evaluated the morphological features and functions of microglia in the hippocampal CA1 region at P21. We found no significant differences in microglial number, soma size, or branching complexity between the Arc ASO and Scramble control groups (Fig. 4j, k). However, the Arc ASO group showed a significant reduction in microglial synaptic engulfment, as determined by the quantified number of Homer1 puncta within CD68-positive compartments (Fig. 4l–m), indicating that Arc knockdown impaired microglia-mediated synaptic clearance during the critical period. Further analysis revealed that CSF1R expression was significantly lower in the Arc ASO group than in the scramble control group (Fig. 4n, o). Similarly, compared with the control group, the sevoflurane-exposed group also exhibited a significant reduction in CSF1R expression (Fig. S4c, d). Since CSF1R signaling is essential for maintaining microglial proliferation and pruning capacity, this reduction supports the notion that microglia may adopt a pruning-deficient state, with impaired synaptic clearance despite unaltered morphology [19, 20].

Given that the Arc protein exhibits a unique “inverse synaptic tagging” property and is selectively enriched at functionally weak or immature synapses, we used STED superresolution microscopy in Thy1-EGFP mice at P21 to examine its synaptic localization [21, 22]. Arc was predominantly localized to dendritic spines with immature morphology (Fig. S5a, b), suggesting that it may serve as a postsynaptic signal to label underdeveloped synapses for clearance. To investigate whether Arc participates in microglial recognition of immature synapses, we examined its spatial colocalization with C1q, a key initiator of the complement-mediated synaptic pruning cascade that preferentially binds to weak or immature synapses [23]. Immunofluorescence staining revealed robust colocalization of Arc and C1q in the hippocampus during the critical period, with some signals were also detected within microglia (Fig. S5c). Notably, the intensity of Arc-C1q colocalization was significantly lower in sevoflurane-exposed mice than in control mice (Fig. S5d), indicating that this interaction may be disrupted by sevoflurane-induced Arc downregulation. To confirm this causal relationship, Arc expression was knocked down, which significantly reduced the number of C1q and Homer1 colocalized puncta, suggesting decreased synaptic localization of C1q (Fig. 5e, f). Moreover, Western blot analysis revealed that Arc knockdown did not affect overall C1q expression levels in microglia (Fig. 4n, o), suggesting that Arc regulates the localization and synaptic targeting of C1q rather than its production.

Fig. 5The alternative text for this image may have been generated using AI.

Transient Arc knockdown during the critical period leads to synaptic loss and increased microglial phagocytic activity in adolescence/adulthood. a Experimental design. b, c Bulk RNA-seq results at P56 following transient Arc knockdown via hippocampal injection of Arc ASOs at P16, comparing the Scramble and Arc ASO groups. b Volcano plots showing genes that were differentially expressed between groups. Upregulated genes are shown in red, downregulated genes are shown in blue, and genes with no significant change in expression are shown in gray. Scramble vs. Arc ASO. |FC | ≥ 1, P < 0.05. c GO pathway enrichment analysis of the DEGs. d, e Representative Western blots and quantification of the expression of the synaptic proteins PSD-95 and Homer1 in the hippocampus of adults following hippocampal injection of Arc ASO at P16 comparing the Scramble and Arc ASO groups [PSD-95: two-tailed unpaired t test, P = 0.043; Homer1: two-tailed unpaired t test, P = 0.675; n = 3 mice per group]. f, g Representative immunofluorescence images and quantification of the colocalization of the postsynaptic marker PSD95 (red) and the presynaptic marker VGLUT1 (green) in the CA1 region of the hippocampus in adults following the injection of Arc ASO at P16. Scramble vs. Arc ASO. Scale bar = 5 μm [PSD95 spots: two-tailed unpaired t test, P = 0.0286; VGLUT spots: two-tailed unpaired t test, P = 0.0157; colocalized spots: two-tailed unpaired t test, P = 0.0791; quantification was based on the average of six fields per mouse; n = 3 mice per group]. h, i Representative immunofluorescence images of IBA-1, CD68, and DAPI staining in the CA1 region of the hippocampus in adult mice following Arc ASO injection at P16. High-magnification 3D-rendered images illustrate the CD68 signal within IBA-1-labeled microglia. Scale bars = 50 μm or 10 μm. The volume of CD68 per cell is shown. Scramble vs. Arc ASO [two-tailed unpaired t test, P < 0.0001; n = 15 cells from 3 mice per group]. j, k Representative Western blots and quantification of the expression of the synaptic proteins PSD-95, Homer1 and Synaptophysin in the hippocampus at P28 following the hippocampal injection of Arc ASOs at P16 comparing the Scramble and Arc ASO groups [PSD-95: two-tailed unpaired t test, P = 0.0367; Homer1: two-tailed unpaired t test, P = 0.0361; Synaptophysin: two-tailed unpaired t test, P = 0.0021; n = 3 mice per group]. l–o Analysis of hippocampal microglia at P28 following Arc ASO injection at P16 comparing the Scramble and Arc ASO groups. l Representative skeletonized images of IBA-1-labeled microglia. Scale bar = 20 μm. m Quantification of microglia number, soma area and branching complexity via Sholl analysis in the hippocampal CA1 region, as assessed by IBA-1 immunostaining. Scramble vs. Arc ASO [cell number: two-tailed unpaired t test, P = 0.0227; quantification was based on the average of six fields per mouse; n = 3 mice per group; soma area: two-tailed unpaired t test, P < 0.0001; n = 22–23 cells from 3 mice per group; Sholl analysis: two-way ANOVA followed by Tukey’s post hoc test; P < 0.0001; n = 12–13 cells from 3 mice per group]. n Representative immunofluorescence staining images showing merged signals of IBA-1 (red), CD68 (green), Homer1 (far red), and DAPI (blue), a magnified view of CD68 (green)/Homer1 (far red) colocalization, and a 3D-rendered magnified image of Homer1 puncta within CD68 in IBA-1-labeled microglia. White arrows highlight representative examples of synaptic engulfment, demonstrating the localization of Homer1 within CD68-positive microglial compartments. Scale bars = 10 μm or 5 μm. o Quantification of Homer1 puncta localized within CD68-positive microglial compartments. Scramble vs. Arc ASO [two-tailed unpaired t test, P < 0.0001; n = 15 cells from 3 mice per group]. ASO, antisense oligonucleotide. FC, fold change. DEGs, differentially expressed genes. ns, P > 0.05. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively. The data are presented as the mean ± SEM.

Taken together, these findings demonstrate that reduced Arc expression impairs microglial synaptic clearance capacity without affecting microglial morphology. Moreover, Arc may act as a postsynaptic signal that identifies immature synapses and facilitates complement-mediated pruning by microglia during the critical period of synaptic development.

Neonatal sevoflurane exposure does not trigger alterations in microglial function or structure before the critical period

To assess whether repeated sevoflurane exposure from P6 to P8 triggers early microglial responses prior to the onset of the critical period, we examined the status of hippocampal microglia at P14 (Fig. S6a). RT‒qPCR analysis revealed that the expression levels of several microglia-related genes (Aif1, Arg1, Cx3cr1, Csfr1, Tmem119, Trem2, Pu.1, and P2ry12) did not significantly differ between the control and sevoflurane-exposed groups (Fig. S6b). Immunofluorescence staining of IBA-1 and CD68 revealed no significant differences in microglial morphology or phagocytic function between the two groups (Fig. S6c). Specifically, there were no changes in microglial density, soma area, or branching complexity (Sholl analysis) (Fig. S6d). Quantitative analysis confirmed that the CD68 volume within microglia was not significantly different (Fig. S6e). Taken together, these findings suggest that sevoflurane exposure during P6–P8 does not elicit detectable microglial activation or structural remodeling by P14, indicating that subsequent changes in function may not result from an acute immune response but rather emerge progressively during the critical period.

Transient Arc knockdown during the critical period leads to abnormal microglial phagocytic activity and synaptic loss in adulthood

Given the presence of long-term cognitive deficits, we sought to characterize the transcriptional and cellular alterations in the adult hippocampus following transient Arc knockdown during the critical period. We conducted bulk RNA-seq on the hippocampi of adult mice (P56) that had undergone transient Arc knockdown during the critical period (Fig. 5a). The results revealed notable transcriptional changes: compared with the Scramble control group, 539 genes were upregulated, and 572 genes were downregulated (Fig. 5b). GO enrichment analysis highlighted significant alterations in pathways such as “immune system process,” “lysosome,” “synapse,” “microglial cell activation involved in immune response,” and “axon” (Fig. 5c). The differentially expressed genes related to microglial function included Cd68, Trem2, C1qc, Spp1, and Csf3r (Fig. S3d). The genes involved in neurotransmission and signaling included Homer1, Grin2c, Grm3, Nptx2, and Gabre (Fig. S3e). Additionally, genes associated with cytoskeletal organization and neuroprotection included S100b, Ralgds, Plek, Mag, and Pdlim4 (Fig. S3f). These transcriptional changes suggest that transient Arc knockdown during the critical period may have long-lasting effects on neuroimmune signaling pathways and the maintenance of synaptic homeostasis.

To validate the transcriptomic signatures suggesting disrupted synaptic homeostasis and immune activation, we next performed histological and biochemical analyses of the adult hippocampus following transient Arc knockdown. Western blot analysis revealed a significant reduction in PSD95 protein levels in the hippocampus, whereas Homer1 expression remained unchanged (Fig. 5d, e). Immunofluorescence analysis confirmed a significant reduction in the number of PSD95- and VGLUT1-positive puncta compared with that in the Scramble control group, and the number of colocalized puncta tended to decrease (Fig. 5f, g). These results indicate a persistent reduction in synaptic integrity in adulthood, aligning with the cognitive impairments observed at this stage. To investigate the functional status of microglia, we analyzed CD68 expression in adult IBA-1⁺ microglia. Compared with the Scramble group, the Arc ASO group exhibited a significant increase in the intracellular volume of CD68 per microglial cell, indicating increased phagocytic activity (Fig. 5h–i). This sustained microglial hyperactivity may contribute to the observed synaptic loss in adulthood. Taken together, these findings reveal that transient Arc knockdown disrupts the development of neuroimmune balance, leading to enduring synaptic loss and aberrant microglial activation in the adult hippocampus.

Transient Arc knockdown during the critical period disrupts adolescent microglial synaptic pruning

Given that adolescence is another critical window for microglia-mediated synaptic pruning that is crucial for the fine-tuning of neural circuits, we evaluated the synaptic and microglial status in the hippocampus at P28 in mice subjected to transient Arc knockdown during the early critical period (Fig. 5a). Compared with those in the Scramble control group, Western blot analysis revealed that the expression of PSD95, Homer1, and Synaptophysin in the hippocampi of Arc ASO mice was significantly lower, whereas the expression of SAP-102 tended to increase (Fig. 5j, k). Compared with those in the scramble control group, the number of IBA-1⁺ microglia was significantly greater in the Arc ASO group, which was accompanied by an enlarged soma area and increased branching complexity, both of which are morphological indicators of an activated state (Fig. 5l, m). Additionally, the number of Homer1⁺ puncta engulfed by CD68⁺ microglia was significantly greater in the Arc ASO group than in the scramble control group, indicating increased synaptic phagocytosis (Fig. 5n, o).

These findings indicate that the synaptic loss and microglial overactivation observed in adulthood begin to emerge as early as adolescence (P28), a period that represents a critical window for the second wave of synaptic pruning. These results further suggest that Arc knockdown during the early critical period may initiate a trajectory of neuroimmune dysregulation that starts in adolescence and persists into adulthood.

Time-specific restoration of Arc during the critical period reverses both the dysregulation of microglial pruning and the impairment of cognition induced by repeated sevoflurane exposure

To determine whether Arc overexpression could alleviate the abnormal phenotypes induced by repeated sevoflurane exposure, we constructed a doxycycline-inducible, neuron-specific Arc overexpression viral vector (ssAAV9-hSyn-Tet-on 3G-Arc), which was injected into the hippocampi of mice at P1. The mice were then subjected to repeated sevoflurane exposure from P6 to P8, and Arc expression was induced during the critical period (P14–P21) via maternal lact