Excitation-Inhibition Imbalance Underlies Perioperative Neurocognitive Disorders: A Single-Nucleus Transcriptomic Perspective in Mice Hippocampus

Excitation-Inhibition Imbalance Underlies Perioperative Neurocognitive Disorders: A Single-Nucleus Transcriptomic Perspective in Mice Hippocampus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Excitation-Inhibition Imbalance Underlies Perioperative Neurocognitive Disorders: A Single-Nucleus Transcriptomic Perspective in Mice Hippocampus Changwei Wei, Yuzhu Wang, Di Yang, Zhuang Pan, Jing Wang, Sijie Li, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7067489/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Perioperative neurocognitive disorders (PND), highly prevalent in geriatric surgical populations, constitute a major postoperative clinical challenge associated with prolonged hospital stays and adverse surgical outcomes. Although substantial research efforts have been devoted to investigating etiology, the precise molecular mechanisms of PND remains elusive, thereby hindering the development of effective therapeutic interventions. To address this gap, we conducted single-nucleus RNA sequencing on 119,109 hippocampal cells isolated from 18-month-old PND mice and age-matched controls, alongside performing complementary electrophysiological experiments. We noticed that hippocampal neuronal excitation-inhibition (E/I) imbalance serves as a key mechanism underlying PND, which is associated with dysregulated inhibitory control of excitatory plasticity in PND pathology. Furthermore, we identified PND-specific disease-associated astrocytes—distinct from those in other cognitive disorders and linked to E/I imbalance. We also observed significant changes in oligodendrocytes, oligodendrocyte precursor cells, microglia and border associated macrophages (BAM). These cell types played distinct roles in the pathological process of PND. Our study reveals that E/I imbalance, driven by dysregulated inhibitory control of excitatory plasticity, underpins the pathogenesis of PND, providing new insights for therapeutic interventions. Biological sciences/Neuroscience Biological sciences/Physiology perioperative neurocognitive disorders excitation-inhibition balance inhibitory control of excitatory plasticity inhibitory plasticity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Perioperative neurocognitive disorders (PND) are defined as behavioral, emotional, and cognitive changes that occur after anesthesia and surgery [ 1 ]. PND is a common complication for surgical patients, particularly among the elderly [ 2 ]. PND significantly worsens surgical outcomes, leading to reduced postoperative quality of life and increased medical and social resources consumed [ 3 ]. However, due to the pathological mechanism of PND is still unclear, although many interventions to prevent PND have been studied, effective treatments are still lacking. Therefore, studying the pathogenesis of PND, identifying therapeutic targets, and developing effective therapeutic strategies are of great importance. The most common clinical symptom of PND is recent memory loss. The hippocampus is an important brain region responsible for short-term memory. Many molecular, anatomical, and physiological studies have revealed a wide range of neuronal cell types in different regions of the hippocampus. These diverse neuron types are organized into distinct circuits within the hippocampus to perform specific functions [ 4 ]. Do alterations in these specific neurons and associated neural circuits occur throughout the progression of PND? Are such changes causative factors in the pathogenesis, or do they merely contribute to disease advancement? These critical questions remain unresolved. Additionally, it is unclear whether interactions between neurons and non-neuronal cells are altered in PND. To address these issues, we conducted a systematic study of the hippocampus in a mouse model of PND via high-throughput sequencing. By comparing the hippocampus of 18-month-old mice with PND and non-PND, we hypothesized that the inhibitory control of excitatory plasticity was important in PND pathology. We pointed out significant changes in excitation/inhibition (E/I) ratio of excitatory and inhibitory neurons in PND at multiple levels, including phenotype and intercellular crosstalk at gene levels. These changes are supported by both bulk RNA sequencing data and electrophysiology, and are further echoed by neurological function deficits observed in behavioral tests. In addition, we found disease-associated astrocytes, oligodendrocytes, and microglia that differ from the known features of neurodegeneration and may co-regulates E/I disruption in PND. METHODS Mice Animal experiments were approved by Experimental Animal Ethics Committee of Capital Medical University (approval number: AEEI-2022-238) and performed according to National Research Council’s Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice were raised in specific pathogen free conditions with 12 hours of light and 12 hours of darkness. All mice had free access to food and water, and ample space to move around. All mice were euthanized by inhalation of sevoflurane at the end of the experiment. The PND mice model Following five consecutive days of Morris Water Maze training (Supplementary Materials and Methods), the mice were randomly divided into groups of control and the PND model. For mice model of PND, mice were subjected to exploratory laparotomy as described previously [ 5 ]. We used 5% sevoflurane for anesthesia induction. We performed the surgery under 1%-1.5% sevoflurane anesthesia, when the flippance reflex and claw-toe reflex disappeared in the mice, the corneal reflex was weak, and the breathing was stable (55–65 times per minute) [ 6 ], ensuring the mice without episodes of awareness or excessive brain suppression. We made a midline incision of about 3 cm on the abdomen to expose the operative field. We then explored the liver, stomach, spleen, kidneys, intestines, bladder, muscles, fascia, and skin in sequence. The operation took two hours. The incision was closed with 5 − 0 nylon suture. 0.2% ropivacaine was injected subcutaneously along the incision for postoperative analgesia. Sevoflurane anesthesia was terminated after the surgery, and the mice were allowed to recover from anesthesia before being placed back in their cages. The temperature was maintained at 37°C with a heating pad during the surgery. The control mice were not subjected to any anesthesia or surgery. The details of the Morris water maze test and open field test can be found in the supplementary methods. Mice subjected to the paradigm and showing positive results in the Morris water maze test but negative results in the open field test were identified as PND mice [ 5 ]. The PND mice were used for the subsequent snRNA sequencing and electrophysiological experiments (Supplementary Materials and Methods). Single-Nucleus RNA Data Analysis R (version 4.4.0) were used for Seurat v5, CellChat and Monocle pipeline (Supplementary Materials and Methods). Gene Set Enrichment Analysis (GSEA) were performed via clusterProfiler (Table S4 ). E/I Analysis By searching the keywords "excitatory" and "inhibitory" in database ( https://www.gsea-msigdb.org/gsea/index.jsp ), we found total four paired synapse related GO terms for E/I balance analysis (Table S3 ). The scores of synapse related GO terms were calculated based on GO terms ( https://www.gsea-msigdb.org/gsea/index.jsp ) via GSVA. The score of four paired GO pathways were calculated by GSVA and weighted. The E/I balance at the neural communication levels were calculated via the ratio of signal frequency density sent (output E/I) or received (input E/I) by different neural subsets. The output E/I and input E/I were calculated and weighted via CellChat. The E/I scores of bulk RNA-seq were analyzed via GSVA. The data of bulk RNA-seq are available at https://doi.org/10.5061/dryad.jsxksn0cj . Statistics Analysis Bilateral t tests were used for mice ethology and electrophysiology data. Ethology and electrophysiology data were presented as mean ± standard deviation. Ethology data came from surgical and control mice that used for snRNA-seq analysis ( n = 5 per group). Mice for electrophysiological data ( n = 4 per group, three hippocampal slices per mouse) were independent of mice for snRNA-seq. P < 0.05 was considered statistically significant. RESULTS Transcriptomic dynamics in the hippocampus of PND mice Single-nucleus RNA sequencing was performed on prefrontal cortical tissues from five PND mice and five age-matched controls (Fig. 1 A-C and Fig. S1 A-C, n = 119,145 cells after quality filtering). Unsupervised clustering revealed 18 transcriptionally distinct cell types (Fig. 1 D, E). Comparative analysis revealed largely similar cellular landscapes in control and PND mice, with differences in the proportions of several cell populations (Fig. 1 F-I, Fig. S1 D, E). In 18-month-old mice, communication between hippocampal cells is mainly concentrated between excitatory neurons, followed by excitatory and inhibitory neurons (Fig. 1 J and Fig. S1 F). Inhibitory neurons sent more signals to excitatory neurons than excitatory to inhibitory neurons (Fig. 1 J and Fig. S1 F), indicating that inhibitory neurons were more active in regulation of excitatory neurons than excitatory in regulation of inhibitory neurons in hippocampus. Total frequency and strength of hippocampal intercellular communication increased in PND mice when compared with the control, from which the increase of signals between excitatory neurons and from inhibitory to excitatory neurons are the most (Fig. 1 K and Fig. S1 G). Ubiquitous synaptic alterations in hippocampal excitatory neurons of PND mice Excitatory neurons are the predominant neuronal population within the hippocampus (Fig. 1 F, J). Intriguingly, relative to the control, the PND mice exhibited enhanced frequency but reduced strength of crosstalk between excitatory neurons (Fig. 2 A). Moreover, both frequency and strength of signal changes between excitatory neurons in different brain regions showed inconsistency (Fig. 2 B). There were few overlapping gene phenotypes between different subtypes of excitatory neurons in mice hippocampus (Fig. 2 C), indicating different biological characteristics and different functions of these subtypes. GSEA analysis of differentially expressed genes between the PND and the control also showed heterogeneity changes of excitatory neuron subsets (Fig. S2 A, B). Notably, 75 genes were consistently upregulated (log2FC > 1, P < 0.01) across all excitatory neuronal populations in PND (Fig. 2 D and Table S1 ). GO analysis of these genes revealed significant enrichment of synaptic and synaptic vesicle-associated processes, with Stx3 and Snap25 demonstrating consistent associations across nearly all gene sets (Fig. 2 E). SNAP25 is a specific STX3 partner that has an important role in neurite outgrowth [ 7 ]. Upregulated neurite outgrowth may result in excessive and disorganized synaptic connections, which could alter neuronal communication (Fig. 2 A, B) and disrupt the E/I balance. To verify these ubiquitous synaptic alterations, we analyzed other synaptic vesicle-related pathways and the pathway of SNAP25 complex. As expected, all these pathways were upregulated in PND mice (Fig. 2 F). In the following sections, we aim to explore the reasons for these changes in excitatory neurons. Upregulated inhibitory control of excitatory plasticity in PND hippocampus dominates the changes of excitatory neurons Inhibitory neuron subtypes (PV, VIP, SST, and LAMP5) exhibited minimal overlap in marker genes (Fig. 3 B), indicating distinct functional specializations within hippocampal inhibitory circuits. Pathway profiles showed significant divergence in PND mice compared to the control (Fig. 3 D), demonstrating subtype-specific pathogenic mechanisms in PND development (Fig. 3 C, Table S2 ). Communication changes in the frequency and strength of inhibitory neurons indicated altered inhibitory plasticity in the PND mice (Fig. 3 A, G). Hippocampal inhibitory neurons generally emit significantly more signals to excitatory neurons than vice versa (Fig. 3 E, F). Additionally, signals from inhibitory to excitatory neurons were overall increased in PND mice compared to controls, whereas signals from excitatory to inhibitory neurons were decreased (Fig. 3 G, H). Moreover, signals from PV to both inhibitory and excitatory neurons exhibited the most pronounced increase in PND mice (Fig. 3 A, G). The receptor-ligand signaling mediated by inhibitory neurons targeting excitatory neurons exhibited up-regulation of neurexin family members (Nrxn1, Nrxn2, and Nrxn3) in PV of PND mice (Fig. S2 C). These neurexins regulate inhibitory synaptic transmission [ 8 ], and PV plays a pivotal role in regulating the E/I balance [ 9 ]. These findings highlight the critical role of inhibitory control of excitatory plasticity in the pathogenesis of PND. Dominant role of inhibitory neuron dysfunction in E/I balance disruption during PND pathogenesis Our prior analyses of excitatory and inhibitory neurons have pointed to disturbances in E/I balance in PND hippocampus. First, the ratio of excitatory to inhibitory neuron proportions differed very little between the two groups (Fig. 4 A), indicating that E/I imbalance was not caused by alterations in neuronal subtype ratios. In PND mice, the E/I ratio of postsynaptic potential decreased for inputs onto excitatory neurons (Fig. 4 B, D) but increased for those onto inhibitory neurons (Fig. 4 C, D). The E/I ratio of chemical synaptic transmission was reduced in excitatory neurons and enhanced in inhibitory neurons of PND mice (Fig. 4 E). Further, we quantified the score of all paired excitatory and inhibitory gene ontology (GO) pathways in both excitatory and inhibitory neurons (Table S3 ). Both excitatory and inhibitory synapse related score were downregulated in both excitatory and inhibitory neurons of PND mice when compared with control mice (Fig. 4 F). However, the E/I score of excitatory neurons were downregulated in hippocampus of PND mice while the E/I score of inhibitory neurons were upregulated in hippocampus of PND mice (Fig. 4 F). Both excitatory and inhibitory synapse-related scores were downregulated in the overall neurons in hippocampus of PND mice (Fig. 4 G). However, the E/I ratio of synapse-related score in the overall neurons of PND hippocampus was upregulated (Fig. 4 G). In another aspect, we measured the changes of E/I balance at the neural communication levels. First, compared with the control mice, the output E/I of both excitatory and inhibitory neurons increased in PND mice, but the output E/I of signals emitted by inhibitory neurons increased more than that of excitatory neurons (Fig. 4 K). Then, the input E/I of neurons decreased in PND mice except for SST (Fig. 4 L). Moreover, the input E/I of excitatory neurons decreased more than that of the inhibitory neuron (Fig. 4 L). In excitatory neurons, input E/I ratio in CA1, CA2, and CA3—hubs of hippocampal neural activity—decreased the most in PND mice (Fig. 4 L). Among inhibitory neurons, VIP exhibited the highest output signal ratio and the lowest input signal ratio between PND and the control (Fig. 4 K, L). Subsequently, we verified the E/I balance disturbance in PND using public database. In single-cell RNA sequencing (scRNA-seq) of PND mice hippocampus at one day after surgery (GSE267933) [ 10 ], it was also observed that E/I ratio of postsynaptic potential in PND mice hippocampus was downregulated in excitatory neurons and upregulated in inhibitory neurons when compared with the control (Fig. 4 H). One day after surgery, both excitatory and inhibitory synapse related score were downregulated in excitatory and inhibitory neurons when compared with the control (Fig. 4 I). Similar to our snRNA-seq results, the overall E/I scores of excitatory and inhibitory neurons in the hippocampus were upregulated in PND mice when compared with the control (Fig. 4 I). In an bulk RNA sequencing data of PND mice hippocampus at two days after surgery [ 11 ], it was also observed that both excitatory and inhibitory synapse related score were downregulated in hippocampus as well as the overall E/I scores of excitatory and inhibitory neurons were upregulated in PND mice when compared with the control (Fig. 4 J). Herein, the E/I changes in public databases confirm our conclusions and suggest that E/I perturbation in PND mice hippocampus is a continuous process in the first 3 days after surgery. We further detected the changes of E/I in the hippocampal slices of PND mice by detecting the field excitatory postsynaptic potential (fEPSP) and field inhibitory postsynaptic potential (fIPSP). Consistent with our sequencing results, we observed that fEPSP and fIPSP were generally downregulated while the fEPSP/fIPSP upregulated in PND mice hippocampus compared with the control (Fig. 4 M-P). The reduction of inhibitory drive is known to be more important than the increase of excitatory drive in the process of learning [ 12 ]. Increased activity of inhibitory neurons can inhibit learning ability [ 13 ]. Herein, our analysis demonstrates that inhibitory neuron-dominant E/I imbalance underlies the pathogenesis of PND. PND-specific disease-associated astrocytes crosstalk with the E/I changes Astrocytes exhibited the most prominent proportional expansion among hippocampal cell populations in PND mice (Fig. 1 I). We investigated whether astrocytic dysfunction disrupts the E/I balance in the pathophysiology of PND. We analyzed the trajectories of dynamic astrocytes changes during the course of PND (Fig. 5 A-D). Astrocytes in the control mice were mainly distributed in the initial homeostasis state (state 1, Fig. S3 A). State 3 was the developed state of poseudotime trajectory in 18-month-old mice (Fig. 5 B, F and Fig. S3 C), consistent with previous studies in aging mice [ 14 ],[ 15 ]. State 2 was defined as an intermediate state, which marked by genes upregulated in both state 1 and state 3 (Fig. S3 B). Astrocytes activation score via recognized markers ( Gfap , Vim, Nes and Synm ) demonstrated the activation associated with our poseudotime trajectory (Fig. 5 I). States 4 and 5, characterized by genes enriched in cilium-related and lipid-related pathways, respectively (Fig. 5 G, H), showed specific expansion in PND mice (Fig. 5 B-E) and were defined as disease-associated astrocytes. Cilium acts as antenna receivers of communicating signals changing the neuronal circuit [ 20 ], and dysregulated lipid metabolism in astrocytes is correlated with abnormal firing of neurons [ 17 ], both of which may related to the E/I imbalance of PND. Communications between astrocytes sub-states were generally reduced, with the most significant reduction in PND-specific states 4 and 5 (Fig. 5 J). Interestingly, while changes in signaling from astrocytes to neurons primarily depended on the neuron type, signals originating specifically from state 4 astrocytes were significantly downregulated. (Fig. 5 K). Signals sent by excitatory neurons to astrocytes in states 3, 4, and 5 are reduced in PND mice (Fig. 5 L), which consistent with the downregulation of astrocyte-associated pathways in excitatory neurons observed in pathway analysis of PND mice (Fig. S2 C). While signals sent by inhibitory neurons PV to all states of astrocytes increased in PND mice (Fig. 5 M). Therefore, changes in astrocytes in PND mice may partially affect the E/I balance, and the precise mechanisms require further study. Oligodendrocyte lineage cells and central nervous system (CNS) associated macrophages in PND hippocampus Given that oligodendrocytes are the second most abundant cell type in the mouse hippocampus (Fig. 1 F), we investigated their potential role in regulating E/I balance. Unsupervised clustering classified oligodendrocytes into six clusters (Olig 0–5; Fig. 6 A). Olig 0–4 represent mature oligodendrocytes and Olig 5 represent myelin-forming oligodendrocytes (Fig. 6 C), consisting with the results of developing trajectory analysis (Fig. 6 E). Olig 3 was only present in PND mice, which we defined as disease-associated oligodendrocytes (Fig. 6 B, F). However, the gene signatures of Olig3 (Fig. 6 D) differed from those of disease-associated oligodendrocytes previously defined in Alzheimer’s disease [ 22 ], suggesting distinct molecular and pathological alterations in oligodendrocytes between PND and Alzheimer’s disease. The proportion of Olig 4, a population regulating synaptic homeostasis (Fig. 6 F), was reduced in PND mice (Fig. 6 B). This suggests that the loss of these oligodendrocytes may be related to the imbalanced E/I ratio of synaptic conduction observed in PND. Unsupervised clusters oligodendrocyte precursor cells (OPCs) 4 and microglia 2 were expand in PND mice hippocampus (Fig. 6 H, K). However, these sub-clusters showed no significant association with synaptic regulation (Fig. 6 I and Fig. S3 ), suggesting their limited role in shaping PND phenotypes through E/I balance modulation. Therefore, oligodendrocyte lineage cells and CNS-associated macrophages affect the pathological progression of PND in different ways. DISCUSSION Comparative snRNA-seq analysis of the hippocampus in PND mice versus age-matched 18-month-old controls revealed PND-specific alterations in the molecular landscape. Specifically, we observed changes in the inhibitory control over excitatory plasticity and identified a shift in E/I balance within the PND hippocampus, which underlies its pathophysiology. The E/I balance in neuroscience can refer to the overall level of excitatory and inhibitory activity of individual neurons, specific neural circuits or functional units, or a brain region [ 18 ]. Due to the multi-dimensional nature of neuronal activity, including projection connections and communication between nerve cells, the E/I balance is multi-dimensional. However, for quantification, E/I balance is commonly measured by E/I ratio, which simplifies this concept by reducing it to the ratio of excitatory to inhibitory currents [ 19 ]. The E/I balance regulates the functional state of the nervous system, and its perturbations are associated with brain dysfunction [ 18 ]. In this study, we identified a multi-dimensional E/I shift in the hippocampus of PND mice. First, from the perspectives of synapses, we point out that the E/I score of excitatory synapses was downregulated, while the E/I score of inhibitory synapses was upregulated in the hippocampus of PND mice compared to controls. This resulted in increased overall E/I ratio in the hippocampus of PND mice. Interestingly, although the E/I ratio increased in PND mice, both excitatory and inhibitory synaptic transmission levels, as well as the amplitudes of fEPSP and fIPSP, were reduced in the hippocampus of PND mice. A higher E/I ratio is associated with increased plasticity, whereas a lower E/I ratio is related to increased stability [ 20 ]. All of these may lead to changes in the formation and maintenance of memory, just as the changes in the performance of PND mice in our Morris water maze test. Next, from the perspective of intercellular crosstalk, the output E/I of both excitatory and inhibitory neurons increased in the PND mice, but the output E/I of signals emitted by inhibitory neurons increased more significantly than that of excitatory neurons in PND hippocampus when compared with the control. This shift caused the overall hippocampal E/I balance to tilt toward inhibition. We also investigated the mechanism underlying changes in E/I balance from the perspective of excitatory and inhibitory plasticity. Our data revealed a predominance of inhibitory control of excitatory plasticity in normal mice hippocampus as well as the prominent changes of inhibitory control of excitatory plasticity in the hippocampus of PND mice. The concept of inhibitory control of excitatory plasticity is a novel idea in neurology. It refers to the gating function of inhibition in regulating excitatory plasticity within neurons [ 19 ],[ 21 ]. Changes in inhibition can either enable or suppress excitatory plasticity, thereby modulating the threshold of plasticity and the degree of its induction [ 22 ]. Numerous experimental studies have demonstrated the importance of inhibition in memory recall [ 22 ],[ 23 ]. Inhibitory plasticity is critical for establishing and maintaining E/I equilibrium, achieving discharge rate homeostasis, controlling excitatory plasticity, and shaping network connections [ 19 ],[ 21 ]. In addition to the distinct control of excitatory plasticity of inhibitory neurons, we also noted changes in crosstalk between subsets of inhibitory neurons, which also influence the E/I balance in the PND hippocampus [ 24 ]. Activated inhibitory neurons inhibit other inhibitory neurons and are negatively correlated with learning and memory functions [ 25 ],[ 26 ]. We also analyzed PND-specific disease-associated astrocytes, oligodendrocyte lineage cells and CNS associated macrophages, and demonstrated that they had little effect on the E/I balance in the hippocampus of PND mice. These results emphasize the significance of inhibitory neurons in the mechanism underlying the E/I imbalance in the hippocampus of PND mice. From the perspective of perioperative pathophysiology, stress and internal environment disturbance caused by anesthesia and surgery act on the brain, disturbing the E/I balance. The brain must re-establish this balance during and after the recovery period from anesthesia. If this re-establishment process is hindered by the factors such as stress, surgical trauma, and inflammatory response, or aging, serious systemic disease, or ischemic and hypoxic brain injury, the brain’s reconstruction capacity may be insufficient. This insufficiency can lead to long-term or irrecoverable E/I imbalance, which may manifest as PND-related clinical symptoms. Thus, our study explains why complex surgery, advanced age, American Society of Anesthesiologists (ASA) physical status classification of IV, and a history of cerebrovascular disease without sequelae are independent risk factors for PND [ 1 ]. This study has several limitations. (1) We did not include a group that received anesthesia alone because no clinical evidence shows that general anesthesia affects the incidence of PND [ 27 ]. (2) We conducted the experiment on the third day after surgery—when behavioral changes peaked in significance—based on findings from our earlier research. However, neural activity is continuously ongoing, and the E/I balance is dynamic. Although we integrated scRNA-seq data from the first day after surgery and bulk RNA sequencing data from the second day, we cannot fully capture the dynamic changes in E/I balance throughout the pathological process of PND. (3) We analyzed the E/I ratio through excitatory and inhibitory synaptic scores, excitatory and inhibitory interneuron crosstalk, fEPSP and fIPSP measurements. However, this approach is oversimplification, as circuit activity is not a singular unidimensional phenomenon, and both excitation and inhibition operate on multiple timescales. (4) We focused exclusively on the hippocampus and memory capacity of PND mice, while the cortex may also contribute to PND pathology. (5) The Morris Water maze and open field test may not be ideal behavioral tests not sufficient tests to diagnose cognitive disorders under the PND umbrella. Besides, we did not test other clinical symptoms of PND, such as deficits in orientation and social ability. (6) Our data is primarily descriptive, relying solely on sequencing data without protein staining validation or functional validation through targeted gene manipulation. (7) The intercellular communication analysis was based on gene expression data, and its interpretation must be carefully integrated with anatomical and neural network knowledge. For example, we observed an increase in the frequency and intensity of signal communication between CA1 and DG regions increased in the PND mice. This could reflect the formation of an abnormal neural signal bypass pathway in the mouse hippocampus due to surgical trauma or could simply represent a molecular level artifact. Further studies should incorporate calcium imaging, patch clamp, optogenetics, functional magnetic resonance imaging and other techniques to confirm synaptic connections and neuronal crosstalk. (8) The snRNA technology, while robust for neurons, is less effective in capturing immune cells. This limitation may have led to information loss for immune cell populations in our study. Although we were able to measure microglia and border associated macrophages (BAM), we could not obtain data for other myeloid and lymphoid cells, such as dendritic cells, granulocytes, T cells, and NK cells. For the same reason, we were unable to analyze BAM subpopulations, which are likely biologically diverse. Besides, we did not identify significant disease-associated microglia in the hippocampus of PND mice, which may have been influenced by the limitations of snRNA technology above. In further studies, we plan to isolate immune cells individually for scRNA-seq analysis to overcome these limitations. In conclusion, although various cells in the PND hippocampus showed changes in our snRNA-seq data, we propose that neurons, which constitute the majority of the brain, play a dominant role in brain function and dysfunction in PND. We demonstrated alterations in the inhibitory control of excitatory plasticity as a key aspect of PND pathophysiology. In PND mice, the total levels of both excitation and inhibition of hippocampal neurons were downregulated, while the overall E/I ratio was upregulated. Specifically, the E/I ratio of excitatory neurons was downregulated, whereas the E/I ratio of inhibitory neurons were upregulated in the hippocampus, resulting the shift of total E/I balance and accompanying behavioral deficit. Therefore, we propose that E/I imbalance is a fundamental pathological mechanism underlying PND. Declarations DATA AVAILABILITY Data of this study have been uploaded to National Center for Biotechnology Information (GSE279516). Associated code used in this study is available online (Table S4). ACKNOWLEDGEMENTS Not applicable. AUTHOR CONTRIBUTIONS CW, DY and YW designed experiments. YW performed analysis and computation and created figures. YW and DY drafted the original manuscript. DY, JW (Jing Wang), ZP, DL, JW (Jinxu Wang), YS, SL, LG and XW performed the animal experiments. ZP and DY performed the electrophysiology experiments. SCH, CW, DY, YS and JW (Jinxu Wang) reviewed and revised the manuscript. FUNDING This work was funded by National Natural Science Foundation of China (82471214, 82271210, 82071176 and 82401392) and Beijing Natural Science Foundation (7222074 and 7242055). COMPETING INTERESTS Not applicable. References Dilmen OK, Meco BC, Evered LA, Radtke FM. Postoperative neurocognitive disorders: A clinical guide. J Clin Anesth. 2024;92:111320. Kunicki ZJ, Ngo LH, Marcantonio ER, Tommet D, Feng Y, Fong TG, et al. Six-year cognitive trajectory in older adults following major surgery and delirium. JAMA Intern Med. 2023;183(5):442–50. Zhang L, Liu Y, Luo G, Chen C, Dou C, Du J, et al. Upconversion-mediated optogenetics for the treatment of surgery-induced postoperative neurocognitive dysfunction. ACS Nano. 2024;18(17):11058–69. Vandael D, Jonas P. 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Cortical interneurons that specialize in disinhibitory control. Nature. 2013;503(7477):521–4. Arroyo S, Barati S, Kim K, Aparicio F, Ganguly K. Emergence of preparatory dynamics in VIP interneurons during motor learning. Cell Reports. 2023;42(8):112834. Zhu X, Yang M, Mu J, Wang Z, Zhang L, Wang H, et al. The effect of general anesthesia vs. regional anesthesia on postoperative delirium-a systematic review and meta-analysis. Front Med (Lausanne). 2022;9:844371. Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files TableS1.xls Table S1 Significantly upregulated genes in excitatory neuron subsets in PND compared to the control. TableS2.xls Table S2 Significantly upregulated genes in inhibitory neuron subsets in PND compared to the control. TableS3.xls Table S3 GO sets used for Fig. 4. TableS4.doc Table S4 Codes used in this study. SupplementaryMETHODSlinenumber.doc Supplementary METHODS FigS1180mm.pdf Fig. S1 A After randomization, the performance of mice in PND and the control mice on the fifth day of water maze training was reviewed. Band C Motion trail and statistical results of open field tests on the third day after surgery and anesthesia. D UMAP visualization of the reduced dimensional distribution of different cells in hippocampus of PND and control mice. E Proportion of different cells in hippocampus of surgery and the control mice. F Strength of intercellular communication of PND and control mice hippocampus. G Changes of Strength of intercellular communication between PND and control mice. FigS2180mm.pdf Fig. S2 A Gene Set Enrichment Analysis (GSEA) of different excitatory neurons indicated that, compared to the control, most of the downregulated pathways that significantly changed in PND mice involved crosstalk with other brain cells. B Most of the upregulated pathways in PND excitatory neurons related with regulation of immune and brain barrier systems. C Ligand-receptor signals of excitatory neurons crosstalk. FigS3180mm.pdf Fig. S3 A-C Top25 enrichment pathway of marker genes in astrocytes state 1, 2 and 3. D-I Top25 enrichment pathway of marker genes in subsets of central nervous system associated macrophages. P <0.05 was defined as significant. If there were less than 25 pathways with P <0.05, only pathways with P <0.05 were listed. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 16 Jan, 2026 Review # 3 received at journal 10 Dec, 2025 Reviewer # 3 agreed at journal 06 Nov, 2025 Review # 2 received at journal 26 Oct, 2025 Reviewer # 2 agreed at journal 20 Oct, 2025 Reviewer # 1 agreed at journal 24 Jul, 2025 Reviewers invited by journal 21 Jul, 2025 Editor assigned by journal 14 Jul, 2025 Submission checks completed at journal 14 Jul, 2025 First submitted to journal 11 Jul, 2025 Unknown event 08 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7067489","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":488286536,"identity":"6a4ef594-92c2-4402-9dd5-3c52729a3290","order_by":0,"name":"Changwei Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYBAC/hkQmgdEHPgAFZXAp0XiBpKWgzOI0WIQgcRh5iFKi3Tzs4df22xkGCSSDx62qbGLNjjAfPA2D4NdHk4tMsfMjWXb0ngYJNISDuccS87dcIAt2ZqHIbkYpxaJBDNpyW2HeRikcwwO5zYcAGrhMZPmYTiQ2IBTS/o3oJb/QC35Hw5bgrXwf8OvJSLHTPLjtgMgWxgOM0JsYcOrReJGTpk0479kHjb5ZwYHe4B+mXmYzdhyjkEyTi38M9K3Sf44Y2fPz3P48YcfNXa5fcebH954U2GHUwsIgKODDcEFOxiPeiBg/IFffhSMglEwCkY6AAATV1Ty0iVq/wAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing Chao-Yang Hospital, Capital Medical University","correspondingAuthor":true,"prefix":"","firstName":"Changwei","middleName":"","lastName":"Wei","suffix":""},{"id":488286537,"identity":"d01fffb5-2601-48d1-b91c-8ec260c19732","order_by":1,"name":"Yuzhu Wang","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuzhu","middleName":"","lastName":"Wang","suffix":""},{"id":488286538,"identity":"c49cc715-15a5-445c-a466-79896edf1af8","order_by":2,"name":"Di Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Yang","suffix":""},{"id":488286539,"identity":"8c69717b-c7ff-4ea5-bf70-ced9f4643700","order_by":3,"name":"Zhuang Pan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhuang","middleName":"","lastName":"Pan","suffix":""},{"id":488286540,"identity":"19382b49-21b1-416c-89b8-0005619c9c55","order_by":4,"name":"Jing Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""},{"id":488286541,"identity":"13955ac6-3e53-4ad2-8d23-467986a3a022","order_by":5,"name":"Sijie Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sijie","middleName":"","lastName":"Li","suffix":""},{"id":488286542,"identity":"3734ba84-c621-4653-807b-20d29b56caca","order_by":6,"name":"Dandan Lin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Lin","suffix":""},{"id":488286543,"identity":"42e1cde2-120f-41a7-82aa-56d62910396d","order_by":7,"name":"Jinxu Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinxu","middleName":"","lastName":"Wang","suffix":""},{"id":488286544,"identity":"7caa9cae-4620-438e-806b-9f315c2faf5a","order_by":8,"name":"Yi Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Sun","suffix":""},{"id":488286545,"identity":"6fd8ebb5-8fe2-415c-8971-065d4f3e5680","order_by":9,"name":"Lili Gu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Gu","suffix":""},{"id":488286546,"identity":"6d6cc684-899d-4960-8c9b-9bfa26804549","order_by":10,"name":"Xuyang Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xuyang","middleName":"","lastName":"Wang","suffix":""},{"id":488286547,"identity":"9e9e5464-1f39-4814-b172-fe654b7c5153","order_by":11,"name":"Anshi Wu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Anshi","middleName":"","lastName":"Wu","suffix":""},{"id":488286548,"identity":"811d9555-f47d-4610-a318-1b85aecd6fd2","order_by":12,"name":"Stanley Huang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Stanley","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-07-07 16:35:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7067489/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7067489/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87664470,"identity":"21c85d4e-539a-44bb-aa62-9a4119b5daa4","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":477838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-nucleus RNA sequencing of PND and matched 18-month-old mice hippocampus. A\u003c/strong\u003e Experimental flow of PND paradigm. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e Swimming trajectory and statistical results of Morris water maze test on the third day after surgery and anesthesia. \u003cstrong\u003eD\u003c/strong\u003eClusters and Uniform Manifold Approximation (UMAP) visualization of five18-month-old PND mice and five matched mice hippocampus. \u003cstrong\u003eE\u003c/strong\u003e Marker genes of the 18 clusters of PND and matched mice hippocampus. \u003cstrong\u003eF\u003c/strong\u003eThe proportion of excitatory neurons, inhibitory neurons and other cells in hippocampus of PND (inner ring) and control (outer ring) mice. \u003cstrong\u003eG\u003c/strong\u003e The proportion of subtypes of excitatory neurons in hippocampus of PND (inner ring) and control (outer ring) mice. \u003cstrong\u003eH\u003c/strong\u003e The proportion of subtypes of inhibitory neurons in hippocampus of PND (inner ring) and control (outer ring) mice.\u003cstrong\u003e I \u003c/strong\u003eChanges of cell proportions between PND and control mice in mice hippocampus. \u003cstrong\u003eJ\u003c/strong\u003e Frequency of intercellular communication of PND and control hippocampus. \u003cstrong\u003eK\u003c/strong\u003e Changes of frequency of intercellular communication between PND and control mice. Sub: subiculum; OPCs: oligodendrocyte precursor cells; LAMP5: membrane protein family member 5; VIP: vasoactive intestinal polypeptide; PVALB: parvalbumin; SST: somatostatin.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/469b04789fc9482355be762a.png"},{"id":87664471,"identity":"04656909-beb4-4261-b313-d587c2091896","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterogeneous and changes of excitatory neurons in hippocampus of PND mice. A\u003c/strong\u003e and\u003cstrong\u003e B \u003c/strong\u003eChanges of intercellular communication of subtypes of excitatory neurons between PND and control mice. Positive value represents increased levels in PND mice and negative value represents decreased levels in PND mice compared with the control. \u003cstrong\u003eC \u003c/strong\u003eCompared with the control, downregulated genes enriched pathways in PND of different subsets of excitatory neurons.\u003cstrong\u003e D\u003c/strong\u003e Compared with the control, upregulated genes enriched pathways in PND of different subsets of excitatory neurons.\u003cstrong\u003e E \u003c/strong\u003eIntersection of marker genes in subsets of mice hippocampal excitatory neurons. \u003cstrong\u003eF\u003c/strong\u003e Intersection of differential genes between PND and control mice in subsets mice hippocampal excitatory neurons. (G) Enrichment pathways of 75 intersecting genes in Fig. 2F. (H) Synaptic vesicle-related pathways and the pathway of Snap25 complex in different subsets of excitatory neurons in hippocampus of PND and control mice.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/6e6e7386e53f48e468d3b460.png"},{"id":87664474,"identity":"4d6b01dc-90a1-4b07-bc75-a375f333ff40","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":208028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory control of excitatory plasticity and inhibitory plasticity of PND mice hippocampus. A\u003c/strong\u003e Changes of intercellular communication of subtypes of inhibitory neurons between PND and control mice. \u003cstrong\u003eB \u003c/strong\u003eIntersection of marker genes in subsets of mice hippocampal inhibitory neurons. \u003cstrong\u003eC\u003c/strong\u003e Intersection of differential genes between PND and control mice in hippocampal inhibitory neurons. \u003cstrong\u003eD\u003c/strong\u003eGSEA analysis of different expressed genes between PND and control mice in inhibitory neurons. Main enrichment pathways of marker genes in subsets of inhibitory neurons. \u003cstrong\u003eE \u003c/strong\u003eSignals from inhibitory neurons to excitatory neurons (the inhibitory control of excitatory plasticity) in the hippocampus of PND and the control.\u003cstrong\u003e F\u003c/strong\u003e Signals from excitatory neurons to inhibitory neurons in the hippocampus of PND and the control. \u003cstrong\u003eG \u003c/strong\u003eCompared with the control, the changes of signals from inhibitory neurons to excitatory neurons in the hippocampus of mice in PND mice. Positive value represents increased levels in PND mice and negative value represents decreased levels in PND mice compared with the control. \u003cstrong\u003eH \u003c/strong\u003eCompared with the control, the changes of signals from excitatory neurons to inhibitory neurons in the hippocampus of mice in PND mice.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/ecb10aaffa5a10cbd640e593.png"},{"id":87664469,"identity":"3b4f06f5-21df-4552-83c4-322ba292f30d","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":545198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisturbance of excitation-inhibition (E/I) balance in hippocampus of PND. A \u003c/strong\u003eThe ratio of excitatory and inhibitory neurons in PND and control mice. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e The ratio of enrichment score of excitatory postsynaptic potential (GO:0060079) and inhibitory postsynaptic potential (GO:0060080) in excitatory neurons and inhibitory neurons. \u003cstrong\u003eD\u003c/strong\u003eTotal levels and E/I ratio of enrichment score of excitatory postsynaptic potential and inhibitory postsynaptic potential in excitatory neurons and inhibitory neurons. \u003cstrong\u003eE \u003c/strong\u003eTotal levels and E/I ratio of enrichment score of excitatory chemical synaptic transmission and inhibitory chemical synaptic transmission in excitatory neurons and inhibitory neurons.\u003cstrong\u003e F\u003c/strong\u003e Total levels and E/I ratio of enrichment score of excitatory synapse related score and inhibitory synapse related score in excitatory neurons and inhibitory neurons. \u003cstrong\u003eG\u003c/strong\u003e Total levels and E/I ratio of enrichment score of excitatory synapse related score and inhibitory synapse related score in total neurons. \u003cstrong\u003eH\u003c/strong\u003eTotal levels and E/I ratio of enrichment score of excitatory synapse potential and inhibitory synapse potential in excitatory neurons and inhibitory neurons in database GSE267933. \u003cstrong\u003eI \u003c/strong\u003eTotal levels and E/I ratio of enrichment score of excitatory synapse related score and inhibitory synapse related score in total neurons in database GSE267933. \u003cstrong\u003eJ \u003c/strong\u003eTotal levels and E/I ratio of enrichment score of excitatory synapse related score and inhibitory synapse related score in total neurons in data of PMID: 35899365.\u003cstrong\u003e K\u003c/strong\u003e The ratio of excitatory and inhibitory neuron output in PND and control mice. \u003cstrong\u003eL \u003c/strong\u003eThe ratio of excitatory and inhibitory neuron input in PND and control mice. \u003cstrong\u003eM \u003c/strong\u003efEPSP of hippocampal slices on the third day after surgery in PND and control mice. \u003cstrong\u003eN \u003c/strong\u003eand \u003cstrong\u003eO\u003c/strong\u003e Slope and amplitude of fEPSP and fIPSP of hippocampal slices in PND and control mice.\u003cstrong\u003e P\u003c/strong\u003e fEPSP/fIPSP ratio of slope and amplitude of hippocampal slices in PND and control mice.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/282867a1494f977a7a8291ed.png"},{"id":87665601,"identity":"6363ddb7-6722-4193-b282-3e06972f0d4e","added_by":"auto","created_at":"2025-07-27 11:06:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":287741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTrajectories of astrocytes development in PND and PND-specific disease-associated astrocytes. A \u003c/strong\u003eand \u003cstrong\u003eB\u003c/strong\u003eTrajectories and pseudotime analysis of astrocytes development from non-PND control to PND. \u003cstrong\u003eC\u003c/strong\u003e Semi-supervised clustering of astrocytes states upon pseudotime trajectories.\u003cstrong\u003e D \u003c/strong\u003eCluster analysis of different states of astrocytes in PND and control mice.\u003cstrong\u003e E \u003c/strong\u003eProportion of states of astrocytes in all hippocampal cells in PND and control mice.\u003cstrong\u003e F\u003c/strong\u003e Levels of known markers of reactive astrocytes in states of astrocytes in PND and control mice. \u003cstrong\u003eG\u003c/strong\u003e and \u003cstrong\u003eH\u003c/strong\u003e Top25 enrichment pathway of marker genes in astrocytes state 4 and 5. \u003cstrong\u003eI \u003c/strong\u003eReactive astrocytes gene score in trajectories of PND and control mice.\u003cstrong\u003e J \u003c/strong\u003eChanges in cell communication between astrocytes in PND mice compared with the control. \u003cstrong\u003eK\u003c/strong\u003e Changes in signals sent by astrocytes to excitatory neurons in PND mice compared with the control. \u003cstrong\u003eL\u003c/strong\u003e Changes in signals sent by excitatory neurons to astrocytes in PND mice compared with the control. \u003cstrong\u003eM \u003c/strong\u003eChanges in signals sent by inhibitory neurons to astrocytes in PND mice compared with the control. For J to M, Positive value represents increased levels in PND mice and negative value represents decreased levels in PND mice compared with the control.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/9034995272f3f443685e6e21.png"},{"id":87666336,"identity":"1360b95c-d196-42b9-b01d-8af920dbebff","added_by":"auto","created_at":"2025-07-27 11:22:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":439735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of oligodendrocytes, OPCs and central nervous system (CNS) associated macrophages in PND mice hippocampus.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Clusters and UMAP visualization of oligodendrocytes in surgery and control mice hippocampus. \u003cstrong\u003eB\u003c/strong\u003eProportion of subsets of oligodendrocytes in all hippocampal cells in PND mice and the control. \u003cstrong\u003eC\u003c/strong\u003e Levels of myelin-forming oligodendrocytes related genes in subsets of oligodendrocytes in hippocampus of PND and control mice. \u003cstrong\u003eD\u003c/strong\u003eGenes of disease-related oligodendrocytes defined based on Alzheimer’s disease. \u003cstrong\u003eE \u003c/strong\u003eTrajectories and pseudotime analysis of astrocytes development in PND and control mice. \u003cstrong\u003eF\u003c/strong\u003e Enrichment analysis of marker genes in each subsets of oligodendrocytes. \u003cstrong\u003eG\u003c/strong\u003eClusters and UMAP visualization of OPCs in PND and control mice hippocampus. \u003cstrong\u003eH\u003c/strong\u003e Proportion of subsets of OPCs in all hippocampal cells in PND and control mice. \u003cstrong\u003eI\u003c/strong\u003eEnrichment analysis of marker genes in each subsets of OPCs.\u003cstrong\u003e J\u003c/strong\u003e Clusters and UMAP visualization of CNS associated macrophages in surgery and control mice hippocampus. \u003cstrong\u003eK\u003c/strong\u003e Proportion of subsets of CNS associated macrophages in all hippocampal cells in PND and control mice. \u003cstrong\u003eL\u003c/strong\u003e Marker genes of microglia and border associated macrophages (BAM) in mice hippocampus.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/6394b6376ad9b1c90640d268.png"},{"id":87666620,"identity":"eb5ea0d1-d2cf-4fe0-91c6-3a0df847e174","added_by":"auto","created_at":"2025-07-27 11:30:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3069614,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/3ec55066-41fe-4f9c-b94a-12ca86a2c6dd.pdf"},{"id":87664467,"identity":"ea749a6a-2e16-48f6-86c3-d6efd91e6df2","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e Significantly upregulated genes in excitatory neuron subsets in PND compared to the control.\u003c/p\u003e","description":"","filename":"TableS1.xls","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/fb3903c0ab6947cdc05b8c8e.xls"},{"id":87664465,"identity":"cc4d3199-d877-4b7e-9eda-0a7e993d5db7","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003e Significantly upregulated genes in inhibitory neuron subsets in PND compared to the control.\u003c/p\u003e","description":"","filename":"TableS2.xls","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/9460ba6aa7540e45cadabe9a.xls"},{"id":87664468,"identity":"46890b95-dd2b-4b55-a77d-1eee8db0301a","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":27648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3 \u003c/strong\u003eGO sets used for Fig. 4.\u003c/p\u003e","description":"","filename":"TableS3.xls","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/d810115eaf7cb0b710f71f91.xls"},{"id":87664472,"identity":"3dc59cc0-5b3b-46b7-90a1-970c1581e0ad","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4 \u003c/strong\u003eCodes used in this study.\u003c/p\u003e","description":"","filename":"TableS4.doc","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/0261b067dd02c25ecdba1a0a.doc"},{"id":87664486,"identity":"e853c156-f575-4056-b3b1-e42ddefa21df","added_by":"auto","created_at":"2025-07-27 10:58:17","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":35840,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary METHODS\u003c/p\u003e","description":"","filename":"SupplementaryMETHODSlinenumber.doc","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/48391b4de3b815e9925f04e4.doc"},{"id":87666172,"identity":"977dac21-6700-4c8c-9057-6694f9d148d3","added_by":"auto","created_at":"2025-07-27 11:14:16","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1786400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003eAfter randomization, the performance of mice in PND and the control mice on the fifth day of water maze training was reviewed. \u003cstrong\u003eB\u003c/strong\u003eand \u003cstrong\u003eC\u003c/strong\u003e Motion trail and statistical results of open field tests on the third day after surgery and anesthesia. \u003cstrong\u003eD\u003c/strong\u003e UMAP visualization of the reduced dimensional distribution of different cells in hippocampus of PND and control mice. \u003cstrong\u003eE\u003c/strong\u003e Proportion of different cells in hippocampus of surgery and the control mice. \u003cstrong\u003eF\u003c/strong\u003e Strength of intercellular communication of PND and control mice hippocampus. \u003cstrong\u003eG\u003c/strong\u003e Changes of Strength of intercellular communication between PND and control mice.\u003c/p\u003e","description":"","filename":"FigS1180mm.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/e0f507ea83de079e1217244f.pdf"},{"id":87664480,"identity":"6013deea-6b2f-45e7-aa35-a84e33b604e3","added_by":"auto","created_at":"2025-07-27 10:58:16","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":697630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003eGene Set Enrichment Analysis (GSEA) of different excitatory neurons indicated that, compared to the control, most of the downregulated pathways that significantly changed in PND mice involved crosstalk with other brain cells. \u003cstrong\u003eB \u003c/strong\u003eMost of the upregulated pathways in PND excitatory neurons \u0026nbsp;related with regulation of immune and brain barrier systems. \u003cstrong\u003eC \u003c/strong\u003eLigand-receptor signals of excitatory neurons crosstalk.\u003c/p\u003e","description":"","filename":"FigS2180mm.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/b7775f03de889019206e08ef.pdf"},{"id":87666174,"identity":"d5562e48-445e-4588-af0c-3a8266caa69e","added_by":"auto","created_at":"2025-07-27 11:14:16","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":873654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3\u003c/strong\u003e \u003cstrong\u003eA-C\u003c/strong\u003e Top25 enrichment pathway of marker genes in astrocytes state 1, 2 and 3. \u003cstrong\u003eD-I\u003c/strong\u003e Top25 enrichment pathway of marker genes in subsets of central nervous system associated macrophages. \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 was defined as significant. If there were less than 25 pathways with \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, only pathways with \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 were listed.\u003c/p\u003e","description":"","filename":"FigS3180mm.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7067489/v1/a67f9f43f7ab0f14e0faa3ce.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Excitation-Inhibition Imbalance Underlies Perioperative Neurocognitive Disorders: A Single-Nucleus Transcriptomic Perspective in Mice Hippocampus","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePerioperative neurocognitive disorders (PND) are defined as behavioral, emotional, and cognitive changes that occur after anesthesia and surgery [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. PND is a common complication for surgical patients, particularly among the elderly [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PND significantly worsens surgical outcomes, leading to reduced postoperative quality of life and increased medical and social resources consumed [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, due to the pathological mechanism of PND is still unclear, although many interventions to prevent PND have been studied, effective treatments are still lacking. Therefore, studying the pathogenesis of PND, identifying therapeutic targets, and developing effective therapeutic strategies are of great importance.\u003c/p\u003e\u003cp\u003eThe most common clinical symptom of PND is recent memory loss. The hippocampus is an important brain region responsible for short-term memory. Many molecular, anatomical, and physiological studies have revealed a wide range of neuronal cell types in different regions of the hippocampus. These diverse neuron types are organized into distinct circuits within the hippocampus to perform specific functions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Do alterations in these specific neurons and associated neural circuits occur throughout the progression of PND? Are such changes causative factors in the pathogenesis, or do they merely contribute to disease advancement? These critical questions remain unresolved. Additionally, it is unclear whether interactions between neurons and non-neuronal cells are altered in PND. To address these issues, we conducted a systematic study of the hippocampus in a mouse model of PND via high-throughput sequencing.\u003c/p\u003e\u003cp\u003eBy comparing the hippocampus of 18-month-old mice with PND and non-PND, we hypothesized that the inhibitory control of excitatory plasticity was important in PND pathology. We pointed out significant changes in excitation/inhibition (E/I) ratio of excitatory and inhibitory neurons in PND at multiple levels, including phenotype and intercellular crosstalk at gene levels. These changes are supported by both bulk RNA sequencing data and electrophysiology, and are further echoed by neurological function deficits observed in behavioral tests. In addition, we found disease-associated astrocytes, oligodendrocytes, and microglia that differ from the known features of neurodegeneration and may co-regulates E/I disruption in PND.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cb\u003eMice\u003c/b\u003e\u003c/p\u003e\u003cp\u003e Animal experiments were approved by Experimental Animal Ethics Committee of Capital Medical University (approval number: AEEI-2022-238) and performed according to National Research Council\u0026rsquo;s Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice were raised in specific pathogen free conditions with 12 hours of light and 12 hours of darkness. All mice had free access to food and water, and ample space to move around. All mice were euthanized by inhalation of sevoflurane at the end of the experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe PND mice model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing five consecutive days of Morris Water Maze training (Supplementary Materials and Methods), the mice were randomly divided into groups of control and the PND model. For mice model of PND, mice were subjected to exploratory laparotomy as described previously [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. We used 5% sevoflurane for anesthesia induction. We performed the surgery under 1%-1.5% sevoflurane anesthesia, when the flippance reflex and claw-toe reflex disappeared in the mice, the corneal reflex was weak, and the breathing was stable (55\u0026ndash;65 times per minute) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], ensuring the mice without episodes of awareness or excessive brain suppression. We made a midline incision of about 3 cm on the abdomen to expose the operative field. We then explored the liver, stomach, spleen, kidneys, intestines, bladder, muscles, fascia, and skin in sequence. The operation took two hours. The incision was closed with 5\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon suture. 0.2% ropivacaine was injected subcutaneously along the incision for postoperative analgesia. Sevoflurane anesthesia was terminated after the surgery, and the mice were allowed to recover from anesthesia before being placed back in their cages. The temperature was maintained at 37\u0026deg;C with a heating pad during the surgery. The control mice were not subjected to any anesthesia or surgery.\u003c/p\u003e\u003cp\u003eThe details of the Morris water maze test and open field test can be found in the supplementary methods. Mice subjected to the paradigm and showing positive results in the Morris water maze test but negative results in the open field test were identified as PND mice [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The PND mice were used for the subsequent snRNA sequencing and electrophysiological experiments (Supplementary Materials and Methods).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSingle-Nucleus RNA Data Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eR (version 4.4.0) were used for Seurat v5, CellChat and Monocle pipeline (Supplementary Materials and Methods). Gene Set Enrichment Analysis (GSEA) were performed via clusterProfiler (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eE/I Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBy searching the keywords \"excitatory\" and \"inhibitory\" in database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gsea-msigdb.org/gsea/index.jsp\u003c/span\u003e\u003cspan address=\"https://www.gsea-msigdb.org/gsea/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we found total four paired synapse related GO terms for E/I balance analysis (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The scores of synapse related GO terms were calculated based on GO terms (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gsea-msigdb.org/gsea/index.jsp\u003c/span\u003e\u003cspan address=\"https://www.gsea-msigdb.org/gsea/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) via GSVA. The score of four paired GO pathways were calculated by GSVA and weighted. The E/I balance at the neural communication levels were calculated via the ratio of signal frequency density sent (output E/I) or received (input E/I) by different neural subsets. The output E/I and input E/I were calculated and weighted via CellChat. The E/I scores of bulk RNA-seq were analyzed via GSVA. The data of bulk RNA-seq are available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5061/dryad.jsxksn0cj\u003c/span\u003e\u003cspan address=\"10.5061/dryad.jsxksn0cj\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBilateral t tests were used for mice ethology and electrophysiology data. Ethology and electrophysiology data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Ethology data came from surgical and control mice that used for snRNA-seq analysis (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 per group). Mice for electrophysiological data (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group, three hippocampal slices per mouse) were independent of mice for snRNA-seq.\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eTranscriptomic dynamics in the hippocampus of PND mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSingle-nucleus RNA sequencing was performed on prefrontal cortical tissues from five PND mice and five age-matched controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;119,145 cells after quality filtering). Unsupervised clustering revealed 18 transcriptionally distinct cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Comparative analysis revealed largely similar cellular landscapes in control and PND mice, with differences in the proportions of several cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-I, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD, E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn 18-month-old mice, communication between hippocampal cells is mainly concentrated between excitatory neurons, followed by excitatory and inhibitory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). Inhibitory neurons sent more signals to excitatory neurons than excitatory to inhibitory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF), indicating that inhibitory neurons were more active in regulation of excitatory neurons than excitatory in regulation of inhibitory neurons in hippocampus. Total frequency and strength of hippocampal intercellular communication increased in PND mice when compared with the control, from which the increase of signals between excitatory neurons and from inhibitory to excitatory neurons are the most (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003cb\u003eUbiquitous synaptic alterations in hippocampal excitatory neurons of PND mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExcitatory neurons are the predominant neuronal population within the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, J). Intriguingly, relative to the control, the PND mice exhibited enhanced frequency but reduced strength of crosstalk between excitatory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, both frequency and strength of signal changes between excitatory neurons in different brain regions showed inconsistency (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThere were few overlapping gene phenotypes between different subtypes of excitatory neurons in mice hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating different biological characteristics and different functions of these subtypes. GSEA analysis of differentially expressed genes between the PND and the control also showed heterogeneity changes of excitatory neuron subsets (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotably, 75 genes were consistently upregulated (log2FC\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) across all excitatory neuronal populations in PND (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). GO analysis of these genes revealed significant enrichment of synaptic and synaptic vesicle-associated processes, with \u003cem\u003eStx3\u003c/em\u003e and \u003cem\u003eSnap25\u003c/em\u003e demonstrating consistent associations across nearly all gene sets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eSNAP25 is a specific STX3 partner that has an important role in neurite outgrowth [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Upregulated neurite outgrowth may result in excessive and disorganized synaptic connections, which could alter neuronal communication (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B) and disrupt the E/I balance. To verify these ubiquitous synaptic alterations, we analyzed other synaptic vesicle-related pathways and the pathway of SNAP25 complex. As expected, all these pathways were upregulated in PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In the following sections, we aim to explore the reasons for these changes in excitatory neurons.\u003c/p\u003e\u003cp\u003e\u003cb\u003eUpregulated inhibitory control of excitatory plasticity in PND hippocampus dominates the changes of excitatory neurons\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInhibitory neuron subtypes (PV, VIP, SST, and LAMP5) exhibited minimal overlap in marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating distinct functional specializations within hippocampal inhibitory circuits. Pathway profiles showed significant divergence in PND mice compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), demonstrating subtype-specific pathogenic mechanisms in PND development (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCommunication changes in the frequency and strength of inhibitory neurons indicated altered inhibitory plasticity in the PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, G). Hippocampal inhibitory neurons generally emit significantly more signals to excitatory neurons than vice versa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). Additionally, signals from inhibitory to excitatory neurons were overall increased in PND mice compared to controls, whereas signals from excitatory to inhibitory neurons were decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). Moreover, signals from PV to both inhibitory and excitatory neurons exhibited the most pronounced increase in PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, G). The receptor-ligand signaling mediated by inhibitory neurons targeting excitatory neurons exhibited up-regulation of neurexin family members (Nrxn1, Nrxn2, and Nrxn3) in PV of PND mice (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). These neurexins regulate inhibitory synaptic transmission [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and PV plays a pivotal role in regulating the E/I balance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These findings highlight the critical role of inhibitory control of excitatory plasticity in the pathogenesis of PND.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDominant role of inhibitory neuron dysfunction in E/I balance disruption during PND pathogenesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur prior analyses of excitatory and inhibitory neurons have pointed to disturbances in E/I balance in PND hippocampus. First, the ratio of excitatory to inhibitory neuron proportions differed very little between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), indicating that E/I imbalance was not caused by alterations in neuronal subtype ratios. In PND mice, the E/I ratio of postsynaptic potential decreased for inputs onto excitatory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D) but increased for those onto inhibitory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). The E/I ratio of chemical synaptic transmission was reduced in excitatory neurons and enhanced in inhibitory neurons of PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurther, we quantified the score of all paired excitatory and inhibitory gene ontology (GO) pathways in both excitatory and inhibitory neurons (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Both excitatory and inhibitory synapse related score were downregulated in both excitatory and inhibitory neurons of PND mice when compared with control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). However, the E/I score of excitatory neurons were downregulated in hippocampus of PND mice while the E/I score of inhibitory neurons were upregulated in hippocampus of PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Both excitatory and inhibitory synapse-related scores were downregulated in the overall neurons in hippocampus of PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). However, the E/I ratio of synapse-related score in the overall neurons of PND hippocampus was upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eIn another aspect, we measured the changes of E/I balance at the neural communication levels. First, compared with the control mice, the output E/I of both excitatory and inhibitory neurons increased in PND mice, but the output E/I of signals emitted by inhibitory neurons increased more than that of excitatory neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eK).\u003c/p\u003e\u003cp\u003eThen, the input E/I of neurons decreased in PND mice except for SST (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). Moreover, the input E/I of excitatory neurons decreased more than that of the inhibitory neuron (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). In excitatory neurons, input E/I ratio in CA1, CA2, and CA3\u0026mdash;hubs of hippocampal neural activity\u0026mdash;decreased the most in PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). Among inhibitory neurons, VIP exhibited the highest output signal ratio and the lowest input signal ratio between PND and the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, L).\u003c/p\u003e\u003cp\u003eSubsequently, we verified the E/I balance disturbance in PND using public database. In single-cell RNA sequencing (scRNA-seq) of PND mice hippocampus at one day after surgery (GSE267933) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], it was also observed that E/I ratio of postsynaptic potential in PND mice hippocampus was downregulated in excitatory neurons and upregulated in inhibitory neurons when compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). One day after surgery, both excitatory and inhibitory synapse related score were downregulated in excitatory and inhibitory neurons when compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Similar to our snRNA-seq results, the overall E/I scores of excitatory and inhibitory neurons in the hippocampus were upregulated in PND mice when compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eIn an bulk RNA sequencing data of PND mice hippocampus at two days after surgery [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], it was also observed that both excitatory and inhibitory synapse related score were downregulated in hippocampus as well as the overall E/I scores of excitatory and inhibitory neurons were upregulated in PND mice when compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Herein, the E/I changes in public databases confirm our conclusions and suggest that E/I perturbation in PND mice hippocampus is a continuous process in the first 3 days after surgery.\u003c/p\u003e\u003cp\u003eWe further detected the changes of E/I in the hippocampal slices of PND mice by detecting the field excitatory postsynaptic potential (fEPSP) and field inhibitory postsynaptic potential (fIPSP). Consistent with our sequencing results, we observed that fEPSP and fIPSP were generally downregulated while the fEPSP/fIPSP upregulated in PND mice hippocampus compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eM-P).\u003c/p\u003e\u003cp\u003eThe reduction of inhibitory drive is known to be more important than the increase of excitatory drive in the process of learning [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Increased activity of inhibitory neurons can inhibit learning ability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Herein, our analysis demonstrates that inhibitory neuron-dominant E/I imbalance underlies the pathogenesis of PND.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePND-specific disease-associated astrocytes crosstalk with the E/I changes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAstrocytes exhibited the most prominent proportional expansion among hippocampal cell populations in PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). We investigated whether astrocytic dysfunction disrupts the E/I balance in the pathophysiology of PND.\u003c/p\u003e\u003cp\u003eWe analyzed the trajectories of dynamic astrocytes changes during the course of PND (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Astrocytes in the control mice were mainly distributed in the initial homeostasis state (state 1, Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). State 3 was the developed state of poseudotime trajectory in 18-month-old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, F and Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC), consistent with previous studies in aging mice [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e],[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. State 2 was defined as an intermediate state, which marked by genes upregulated in both state 1 and state 3 (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB). Astrocytes activation score via recognized markers (\u003cem\u003eGfap\u003c/em\u003e, \u003cem\u003eVim, Nes\u003c/em\u003e and \u003cem\u003eSynm\u003c/em\u003e) demonstrated the activation associated with our poseudotime trajectory (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eStates 4 and 5, characterized by genes enriched in cilium-related and lipid-related pathways, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H), showed specific expansion in PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-E) and were defined as disease-associated astrocytes. Cilium acts as antenna receivers of communicating signals changing the neuronal circuit [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and dysregulated lipid metabolism in astrocytes is correlated with abnormal firing of neurons [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], both of which may related to the E/I imbalance of PND.\u003c/p\u003e\u003cp\u003eCommunications between astrocytes sub-states were generally reduced, with the most significant reduction in PND-specific states 4 and 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Interestingly, while changes in signaling from astrocytes to neurons primarily depended on the neuron type, signals originating specifically from state 4 astrocytes were significantly downregulated. (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Signals sent by excitatory neurons to astrocytes in states 3, 4, and 5 are reduced in PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eL), which consistent with the downregulation of astrocyte-associated pathways in excitatory neurons observed in pathway analysis of PND mice (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). While signals sent by inhibitory neurons PV to all states of astrocytes increased in PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eM).\u003c/p\u003e\u003cp\u003eTherefore, changes in astrocytes in PND mice may partially affect the E/I balance, and the precise mechanisms require further study.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOligodendrocyte lineage cells and central nervous system (CNS) associated macrophages in PND hippocampus\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that oligodendrocytes are the second most abundant cell type in the mouse hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), we investigated their potential role in regulating E/I balance.\u003c/p\u003e\u003cp\u003eUnsupervised clustering classified oligodendrocytes into six clusters (Olig 0\u0026ndash;5; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Olig 0\u0026ndash;4 represent mature oligodendrocytes and Olig 5 represent myelin-forming oligodendrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), consisting with the results of developing trajectory analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOlig 3 was only present in PND mice, which we defined as disease-associated oligodendrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, F). However, the gene signatures of Olig3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) differed from those of disease-associated oligodendrocytes previously defined in Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], suggesting distinct molecular and pathological alterations in oligodendrocytes between PND and Alzheimer\u0026rsquo;s disease.\u003c/p\u003e\u003cp\u003eThe proportion of Olig 4, a population regulating synaptic homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), was reduced in PND mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This suggests that the loss of these oligodendrocytes may be related to the imbalanced E/I ratio of synaptic conduction observed in PND.\u003c/p\u003e\u003cp\u003eUnsupervised clusters oligodendrocyte precursor cells (OPCs) 4 and microglia 2 were expand in PND mice hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, K). However, these sub-clusters showed no significant association with synaptic regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eI and Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), suggesting their limited role in shaping PND phenotypes through E/I balance modulation.\u003c/p\u003e\u003cp\u003eTherefore, oligodendrocyte lineage cells and CNS-associated macrophages affect the pathological progression of PND in different ways.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eComparative snRNA-seq analysis of the hippocampus in PND mice versus age-matched 18-month-old controls revealed PND-specific alterations in the molecular landscape. Specifically, we observed changes in the inhibitory control over excitatory plasticity and identified a shift in E/I balance within the PND hippocampus, which underlies its pathophysiology.\u003c/p\u003e\u003cp\u003eThe E/I balance in neuroscience can refer to the overall level of excitatory and inhibitory activity of individual neurons, specific neural circuits or functional units, or a brain region [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Due to the multi-dimensional nature of neuronal activity, including projection connections and communication between nerve cells, the E/I balance is multi-dimensional. However, for quantification, E/I balance is commonly measured by E/I ratio, which simplifies this concept by reducing it to the ratio of excitatory to inhibitory currents [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The E/I balance regulates the functional state of the nervous system, and its perturbations are associated with brain dysfunction [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, we identified a multi-dimensional E/I shift in the hippocampus of PND mice.\u003c/p\u003e\u003cp\u003eFirst, from the perspectives of synapses, we point out that the E/I score of excitatory synapses was downregulated, while the E/I score of inhibitory synapses was upregulated in the hippocampus of PND mice compared to controls. This resulted in increased overall E/I ratio in the hippocampus of PND mice. Interestingly, although the E/I ratio increased in PND mice, both excitatory and inhibitory synaptic transmission levels, as well as the amplitudes of fEPSP and fIPSP, were reduced in the hippocampus of PND mice. A higher E/I ratio is associated with increased plasticity, whereas a lower E/I ratio is related to increased stability [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All of these may lead to changes in the formation and maintenance of memory, just as the changes in the performance of PND mice in our Morris water maze test.\u003c/p\u003e\u003cp\u003eNext, from the perspective of intercellular crosstalk, the output E/I of both excitatory and inhibitory neurons increased in the PND mice, but the output E/I of signals emitted by inhibitory neurons increased more significantly than that of excitatory neurons in PND hippocampus when compared with the control. This shift caused the overall hippocampal E/I balance to tilt toward inhibition.\u003c/p\u003e\u003cp\u003eWe also investigated the mechanism underlying changes in E/I balance from the perspective of excitatory and inhibitory plasticity. Our data revealed a predominance of inhibitory control of excitatory plasticity in normal mice hippocampus as well as the prominent changes of inhibitory control of excitatory plasticity in the hippocampus of PND mice. The concept of inhibitory control of excitatory plasticity is a novel idea in neurology. It refers to the gating function of inhibition in regulating excitatory plasticity within neurons [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e],[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Changes in inhibition can either enable or suppress excitatory plasticity, thereby modulating the threshold of plasticity and the degree of its induction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Numerous experimental studies have demonstrated the importance of inhibition in memory recall [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e],[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Inhibitory plasticity is critical for establishing and maintaining E/I equilibrium, achieving discharge rate homeostasis, controlling excitatory plasticity, and shaping network connections [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e],[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to the distinct control of excitatory plasticity of inhibitory neurons, we also noted changes in crosstalk between subsets of inhibitory neurons, which also influence the E/I balance in the PND hippocampus [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Activated inhibitory neurons inhibit other inhibitory neurons and are negatively correlated with learning and memory functions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e],[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe also analyzed PND-specific disease-associated astrocytes, oligodendrocyte lineage cells and CNS associated macrophages, and demonstrated that they had little effect on the E/I balance in the hippocampus of PND mice. These results emphasize the significance of inhibitory neurons in the mechanism underlying the E/I imbalance in the hippocampus of PND mice.\u003c/p\u003e\u003cp\u003eFrom the perspective of perioperative pathophysiology, stress and internal environment disturbance caused by anesthesia and surgery act on the brain, disturbing the E/I balance. The brain must re-establish this balance during and after the recovery period from anesthesia. If this re-establishment process is hindered by the factors such as stress, surgical trauma, and inflammatory response, or aging, serious systemic disease, or ischemic and hypoxic brain injury, the brain\u0026rsquo;s reconstruction capacity may be insufficient. This insufficiency can lead to long-term or irrecoverable E/I imbalance, which may manifest as PND-related clinical symptoms. Thus, our study explains why complex surgery, advanced age, American Society of Anesthesiologists (ASA) physical status classification of IV, and a history of cerebrovascular disease without sequelae are independent risk factors for PND [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study has several limitations. (1) We did not include a group that received anesthesia alone because no clinical evidence shows that general anesthesia affects the incidence of PND [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. (2) We conducted the experiment on the third day after surgery\u0026mdash;when behavioral changes peaked in significance\u0026mdash;based on findings from our earlier research. However, neural activity is continuously ongoing, and the E/I balance is dynamic. Although we integrated scRNA-seq data from the first day after surgery and bulk RNA sequencing data from the second day, we cannot fully capture the dynamic changes in E/I balance throughout the pathological process of PND. (3) We analyzed the E/I ratio through excitatory and inhibitory synaptic scores, excitatory and inhibitory interneuron crosstalk, fEPSP and fIPSP measurements. However, this approach is oversimplification, as circuit activity is not a singular unidimensional phenomenon, and both excitation and inhibition operate on multiple timescales. (4) We focused exclusively on the hippocampus and memory capacity of PND mice, while the cortex may also contribute to PND pathology. (5) The Morris Water maze and open field test may not be ideal behavioral tests not sufficient tests to diagnose cognitive disorders under the PND umbrella. Besides, we did not test other clinical symptoms of PND, such as deficits in orientation and social ability. (6) Our data is primarily descriptive, relying solely on sequencing data without protein staining validation or functional validation through targeted gene manipulation. (7) The intercellular communication analysis was based on gene expression data, and its interpretation must be carefully integrated with anatomical and neural network knowledge. For example, we observed an increase in the frequency and intensity of signal communication between CA1 and DG regions increased in the PND mice. This could reflect the formation of an abnormal neural signal bypass pathway in the mouse hippocampus due to surgical trauma or could simply represent a molecular level artifact. Further studies should incorporate calcium imaging, patch clamp, optogenetics, functional magnetic resonance imaging and other techniques to confirm synaptic connections and neuronal crosstalk. (8) The snRNA technology, while robust for neurons, is less effective in capturing immune cells. This limitation may have led to information loss for immune cell populations in our study. Although we were able to measure microglia and border associated macrophages (BAM), we could not obtain data for other myeloid and lymphoid cells, such as dendritic cells, granulocytes, T cells, and NK cells. For the same reason, we were unable to analyze BAM subpopulations, which are likely biologically diverse. Besides, we did not identify significant disease-associated microglia in the hippocampus of PND mice, which may have been influenced by the limitations of snRNA technology above. In further studies, we plan to isolate immune cells individually for scRNA-seq analysis to overcome these limitations.\u003c/p\u003e\u003cp\u003eIn conclusion, although various cells in the PND hippocampus showed changes in our snRNA-seq data, we propose that neurons, which constitute the majority of the brain, play a dominant role in brain function and dysfunction in PND. We demonstrated alterations in the inhibitory control of excitatory plasticity as a key aspect of PND pathophysiology. In PND mice, the total levels of both excitation and inhibition of hippocampal neurons were downregulated, while the overall E/I ratio was upregulated. Specifically, the E/I ratio of excitatory neurons was downregulated, whereas the E/I ratio of inhibitory neurons were upregulated in the hippocampus, resulting the shift of total E/I balance and accompanying behavioral deficit. Therefore, we propose that E/I imbalance is a fundamental pathological mechanism underlying PND.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData of this study have been uploaded to National Center for Biotechnology Information (GSE279516). Associated code used in this study is available online (Table S4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCW, DY and YW designed experiments. YW performed analysis and computation and created figures. YW and DY drafted the original manuscript. DY, JW (Jing Wang), ZP, DL, JW (Jinxu Wang), YS, SL, LG and XW performed the animal experiments. ZP and DY performed the electrophysiology experiments. SCH, CW, DY, YS and JW (Jinxu Wang) reviewed and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by National Natural Science Foundation of China (82471214, 82271210, 82071176 and 82401392) and Beijing Natural Science Foundation (7222074 and 7242055).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDilmen OK, Meco BC, Evered LA, Radtke FM. Postoperative neurocognitive disorders: A clinical guide. J Clin Anesth. 2024;92:111320.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKunicki ZJ, Ngo LH, Marcantonio ER, Tommet D, Feng Y, Fong TG, et al. Six-year cognitive trajectory in older adults following major surgery and delirium. 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Front Med (Lausanne). 2022;9:844371.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"perioperative neurocognitive disorders, excitation-inhibition balance, inhibitory control of excitatory plasticity, inhibitory plasticity","lastPublishedDoi":"10.21203/rs.3.rs-7067489/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7067489/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePerioperative neurocognitive disorders (PND), highly prevalent in geriatric surgical populations, constitute a major postoperative clinical challenge associated with prolonged hospital stays and adverse surgical outcomes. Although substantial research efforts have been devoted to investigating etiology, the precise molecular mechanisms of PND remains elusive, thereby hindering the development of effective therapeutic interventions. To address this gap, we conducted single-nucleus RNA sequencing on 119,109 hippocampal cells isolated from 18-month-old PND mice and age-matched controls, alongside performing complementary electrophysiological experiments. We noticed that hippocampal neuronal excitation-inhibition (E/I) imbalance serves as a key mechanism underlying PND, which is associated with dysregulated inhibitory control of excitatory plasticity in PND pathology. Furthermore, we identified PND-specific disease-associated astrocytes\u0026mdash;distinct from those in other cognitive disorders and linked to E/I imbalance. We also observed significant changes in oligodendrocytes, oligodendrocyte precursor cells, microglia and border associated macrophages (BAM). These cell types played distinct roles in the pathological process of PND. Our study reveals that E/I imbalance, driven by dysregulated inhibitory control of excitatory plasticity, underpins the pathogenesis of PND, providing new insights for therapeutic interventions.\u003c/p\u003e","manuscriptTitle":"Excitation-Inhibition Imbalance Underlies Perioperative Neurocognitive Disorders: A Single-Nucleus Transcriptomic Perspective in Mice Hippocampus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-27 10:58:11","doi":"10.21203/rs.3.rs-7067489/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-01-16T16:33:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-12-10T14:39:09+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-11-06T06:52:40+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-26T09:47:21+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-20T12:06:36+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-24T07:41:08+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-07-21T05:37:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-14T11:17:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-14T10:41:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2025-07-11T17:35:44+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-07-08T10:30:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7210c9c4-702c-4ac7-a3b5-2b2d7e18f784","owner":[],"postedDate":"July 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":51829310,"name":"Biological sciences/Neuroscience"},{"id":51829311,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-21T09:43:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-27 10:58:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7067489","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7067489","identity":"rs-7067489","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}