Regional distribution of cellular of molecular pathways in a mouse model of Gulf War illness by Single-Cell RNA Sequencing Analysis

doi:10.21203/rs.3.rs-4674549/v1

Download PDF

Research Article

Regional distribution of cellular of molecular pathways in a mouse model of Gulf War illness by Single-Cell RNA Sequencing Analysis

https://doi.org/10.21203/rs.3.rs-4674549/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Gulf War Illness (GWI) is a chronic multisymptom disorder that affects approximately 25–32% of Gulf War veterans, and the combination of permethrin exposure with stress may have collectively and synergistically contributed to its development. However, the exact underlying pathogenic mechanisms within the brain that links permethrin and stress to the development of GWI remains unclear. In the current study, we investigate the effects and the associated molecular mechanisms within the microglia-neuron interplay in a mouse model of GWI, focusing on how exposure to permethrin may act as a priming agent when followed by stress. Subjecting mice to 14 days of chronic permethrin exposure followed by 7 days of stress resulted in the development of depression-like behavior. This behavioral change coincided with distinct alterations in the microglia phenotype, indicating microglial activation in the hippocampa. We revealed that blocking microglial activation through Gi inhibitory DREADD receptors in microglia effectively prevented the behavioral change associated with permethrin and stress exposure. To elucidate the transcriptional networks impacted within distinct microglia populations linked to depression-like behavior in mice exposed to both permethrin and stress, we conducted a single-cell RNA sequencing analysis using 21,566 single nuclei collected from the hippocampus of mice. For bioinformatics, UniCell Deconvolve was a pre-trained, interpretable, deep learning model used to deconvolve cell type fractions and predict cell identity across spatial datasets. Our bioinformatics analysis identified significant alterations in permethrin exposure followed by stress-associated microglia population, notably pathways related to neuronal development, neuronal communication, and neuronal morphogenesis, all of which are associated with neural synaptic plasticity. Additionally, we observed permethrin exposure followed by stress-mediated changes in signal transduction, including modulation of chemical synaptic transmission, regulation of neurotransmitter receptors, and regulation of postsynaptic neurotransmitter receptor activity, a known contributor to the pathophysiology of depression in a subset of the hippocampal pyramidal neurons in the CA3 subregions. These studies tentatively suggest that permethrin may prime toward a depression-like behavior that can be triggered by psychological stress in mice through microglial activation, resulting in alterations of neural plasticity. This new evidence underscores the significance of the synergistic role of multi-causal factors associated with GWI.

A significant proportion of veterans who have experienced deployment face the debilitating effects of depression upon their return to civilian life (1). Several studies indicate that the prevalence of depression is notably higher among deployed veterans compared to the general population, highlighting the urgent need for targeted interventions and support services to address the mental health needs of this vulnerable population (2). Additionally, factors such as post-traumatic stress disorder, traumatic brain injury, and social isolation further contribute to the development and persistence of depressive symptoms among deployed veterans (3, 4). Understanding the complex interplay between deployment-related stressors and depressive symptomatology is crucial for developing effective prevention and treatment strategies tailored to the unique needs of this demographic. Roughly one-third of US military personnel deployed during the 1991 Gulf War (GW) are impacted by Gulf War Illness (GWI), with clinical research suggesting that immune dysfunction and chronic inflammation play significant roles in the symptoms experienced by affected veterans (5). Both the central nervous system and peripheral immune system are implicated in contributing to the pathogenesis of this illness in rodent models of GWI (6). Hence, a comprehensive understanding of the molecular mechanisms behind these immune responses in GWI is vital to advance the development of biomarkers and therapies addressing the central nervous system symptoms and pathology associated with this condition.

Pyrethroids are synthetic counterparts of pyrethrins, natural compounds found in Chrysanthemum cinerariaefolium (7). Permethrin is a type 1 pyrethroid, is among the chemicals which Gulf War Veterans were exposed to during combat theatre (8). The exposure to permethrin is now recognized as a potential factor in the onset of GWI (9, 10). The primary mechanism of action for permethrin involves binding to voltage-gated sodium channels (VGSCs), leading to prolonged opening and alterations in neuronal firing (11–13). Previous studies have shown that pyrethroids, which can cross the blood-brain barrier (BBB), have the ability to stimulate microglial activation through their interaction with VGSCs present on microglia (14). This interaction can lead to an excessive accumulation of intracellular sodium ions and the release of major pro-inflammatory cytokines, such as tumor necrosis factor alpha (14).

Given that previous studies have suggested that permethrin exposure has been associated with microglial activation, potentially priming them for heightened responsiveness to subsequent stressors, which alone have a limited effect on neuroinflammation and psychiatric symptoms. Thus, we hypothesize that the concurrent exposure to permethrin and such stressors could yield a synergistic effect, with permethrin acting as a priming factor. To investigate this hypothesis and comprehend the underlying molecular mechanisms, we exposed mice to permethrin followed by sub-threshold stress, aiming to elucidate their collective influence on neuroinflammation, its associated neural activity, and ultimately, neurophysiological impairments in a mouse model of GWI.

Animals

Mice, including C57BL/6 (WT), Cx3Cr1^CreEr, and hM4Di (Designer Receptors Exclusively Activated by Designer Drugs, R26-hM4Di/mCitrine), were procured from the Jackson Laboratory. Crossbreeding was conducted between Cx3Cr1^CreEr mice and transgenic mice expressing cre recombinase-inducible hM4Di-DREADD, aiming to establish consistent expression of Gi inhibitory receptors on microglia. Double transgenic mice were identified via PCR, while nontransgenic littermates were employed as age-matched controls. All experiments utilized WT or double homozygous Cx3Cr1^CreEr /hM4Di-DREADD mice. Each mouse was housed individually under a 12-hour light/dark cycle, with lights on from 07:00 to 19:00 h, maintaining a steady temperature of 23°C. They had unrestricted access to food and water. The Institutional Animal Care and Use Committee (approval number: IACUC-2019-0043) at the Icahn School of Medicine at Mount Sinai approved all protocols, which were conducted in compliance with NIH guidelines.

Permethrin pharmacokinetic studies

C57BL/6J mice were administered an acute dose of 200mg/kg permethrin and sacrificed at 0, 2, 4, 18, 24, 28, 30, or 48 hours post-treatment. Additionally, an untreated control group was included to validate analytical detection. The animals were anesthetized using a ketamine-xylazine cocktail, blood was collected via cardiac puncture, and the animals were immediately perfused with ice-cold PBS. Brains were then collected and homogenized in 0.2% formic acid using a Fisher Bead Mill 24 homogenizer (Fisher scientific, PA, USA). Blood samples were diluted in 2% formic acid to achieve a final concentration of 0.2% formic acid in the final sample. All blood and homogenized tissue samples were promptly snap-frozen and stored at -80°C until further processing. The samples were allowed to thaw on ice and conditioned to room temperature before processing. Three internal standards (ISs), 13C6-trans-permethrin (50 µg/mL), 13C6-cis-permethrin (50 µg/mL), and 13C6-3-phenoxybenzoic acid (100 µg/mL), were dissolved in nonane and mixed, then diluted in acetone to make the final concentration of 13C6-trans-permethrin, 13C6-cis-permethrin and 13C6-3-phenoxybenzoic acid was 33 ng/mL, 33 ng/mL and 67 ng/mL, respectively. Briefly, for brain and plasma, a 20 µL IS mixer was added to a 200 µL aliquot of brain homogenate. Then, 0.4 mL acetone: hexane (2/8, vol/vol) was added to each sample and vortex-extracted, followed by centrifugation at 5000 xg for 5 min using a micro-centrifuge. The upper organic phase was transferred to a 2 mL Eppendorf tube. The extraction was repeated two additional times with 0.4 mL acetone: hexane (2/8, vol/vol). For the second extraction, 0.125 mL of concentrated hydrochloric acid was added to increase metabolite partitioning into the organic solvent. The combined organic phases were evaporated to dryness under nitrogen before reconstitution for LC-MS/MS analysis. The residue was reconstituted in 400 µL of acetonitrile (ACN) and centrifuged at 16,500 xg for 10 min. For each sample extract, 1 µL was injected into an UPLC-QqQ/MS system for analysis under dynamic multiple reaction monitoring (dMRM) mode in duplicate. The instrument used for chemical analysis was an Agilent 1290 Infinity II UHPLC (Agilent Technology, CA, USA) hyphenated with 6470 triple quadrupole mass spectrometry with electrospray ionization source (ESI) (Santa Clara, CA, USA). Agilent MassHunter Optimizer (version B.07.00) was used for standard compound-related parameters optimization, and MassHunter Workstation software Data Acquisition (version B.08.00) and Quantitative Analysis (version B.07.01) were used for data processing. The columns used for compound separation were AcquityTM Premier HSS T3 C18 VanGuardTM FIT (2.1 × 100 mm, 1.8 µm). For the chromatographic part, the mobile phase A was water, and mobile phase B was methanol, both mobile phases modified with 10 mM ammonium acetate. The compounds were separated using a gradient program as follows: linear gradient from 40 to 100% B in 3 min, isocratic hold at 100% B for 5 min and then drop to 40% B in 0.5 min and keep for 1min prior to the next injection. The mass spectrometer was operating in the positive ionization mode for permethrin and in the negative ionization mode for 3-PBA using the following source parameters: gas temperature, 300°C; gas flow, 12 L/min; nebulizer, 45 psi; sheath gas temperature, 250°C; sheath gas flow, 11 L/min. WinNonlin professional software version 8.0 (Pharsight, CA, USA) was used to calculate the pharmacokinetic parameters. The peak plasma or brain concentration (Cmax) and the peak time (Tmax) were expressed as the mean ± SEM.

Animal Treatments

As previously established (15, 16), mice aged 7–8 weeks, of the C57/BL6J strain, were randomly allocated to treatment cohorts. They were administered either permethrin (200mg/kg in 5% DMSO in corn oil, Sigma-Aldrich, MO, USA) or vehicle (5% DMSO in corn oil) for a duration of 14 days. This was followed by 7 days of exposure to either unpredictable stress or gentle handling (No stress). To inhibit microglia activation, Cx3Cr1CreEr/hM4Di-DREADD mice were treated with JHU37160 for 14 consecutive days. On the same day, they were administered either permethrin or vehicle. Following this treatment period, mice were exposed to either unpredictable stress or no stress for 7 days. Subsequently, behavioral testing took place on the subsequent day (day 22), followed by euthanasia of the animals on the same day.

Unpredictable stress exposure

The unpredictable stress paradigm involved subjecting animals to two stressors daily, administered separately in the morning and evening sessions. Stressors included: wet bedding for 6 hours, placing cages on an orbital shaker for 20 minutes, exposure to hot air (via a hair dryer) for 10 minutes, tilting cages at a 45° angle for 12 hours, forced restraint for 60 minutes, cold water exposure for 5 minutes, no bedding for 6 hours, overnight water restriction, and exposure to light during the dark cycle.

Behavioral tests

Before the initiation of any behavioral testing, all animals were introduced into the behavioral testing room and given a one-hour period to acclimate in their respective home cages. The behavioral session was recorded via a near-infrared camera and analyzed using ANY-maze™ tracking software (Stoelting Co., IL, USA). Each test employed established protocols, detailed below:

1. Forced Swim Test (FST)

Mice were introduced into a large 4L beaker filled with clean water at approximately 21°C for 6 minutes to assess depressive-like behavior. The total duration of immobility, defined as the absence of movement in all four limbs, was measured using ANY-maze software.

2. Open Field Test

For a period of 10 minutes, mice were positioned within a plastic square enclosure measuring 40 cm × 40 cm × 38 cm to measure anxiety-like and anxiolytic behaviors. The behavioral session was captured on video camera and subsequently analyzed using ANY-maze™ software. Quantification of anxiety-like and anxiolytic behaviors involved measuring the total time spent along the edges and corners of the box, as well as the time spent in the center of the open field.

Immunohistochemistry

Fixed brains were immersed in 4% paraformaldehyde and subsequently dehydrated in PBS. Brain sections (30 µm) underwent washing with PBS, permeabilization with PBS + 0.2% Triton X-100 (PBST), and blocking with 2% normal goat serum in PBST. IBA1 (1:1000, Ab178846, Abcam, MA, USA) was applied and incubated overnight at 4°C in PBST with 2% normal goat serum. Following rinsing, brain sections were treated with Alexa Fluor 568-labeled (1:500 in 2% normal goat serum in PBST) for 1 hour at room temperature. Brain sections were then washed, mounted, and coverslipped on microscope slides. Imaging was conducted using a Zeiss LSM 880 Confocal Microscope (Zeiss, DE, Germany). Microscopy and image analysis were performed at the Microscopy CoRE at the Icahn School of Medicine at Mount Sinai.

Sholl analysis

To assess the complexity of each cell, a Sholl analysis was conducted. The determination of soma volume, branch length, number of terminal points, and number of intersection segments in brain sections from the hippocampus and prefrontal cortex (PFC) was conducted utilizing IMARIS 9.1.2 (Bit Plane Inc, MA, USA). The analysis encompassed the following steps: In 3D analysis, z-stack confocal images underwent processing using AutoQuant X3.1 (Media Cybernetics, MD, USA) to eliminate blurriness, followed by analysis with IMARIS. The surface tool was utilized to reconstruct the cell bodies (somas) of the microglia, whereas the filaments tool was employed to reconstruct the branches. One microglial cell was chosen per total process area, and its cell body and soma were traced along with the surface and filament for each process. The quantification involved determining the number of primary processes and branch tips. Each image selected one microglial cell per 0.045 µm², with concentric circles drawn from the soma at 5 µm spacing to measure the intersections of each cell with each circle. Soma volume, branch length, number of terminal points, and number of intersection segments were quantified, and averages were determined among samples within the same group. The analysis encompassed 4 to 6 mice (7–9 microglial cells per mouse) per group, conducted by a single investigator, while an independent evaluator performed the quantification analysis in a blinded manner, utilizing code to conceal knowledge of the experimental groups.

Single Nuclei Sequencing (scRNA-Seq)

Single nuclei suspesnsions were processed using the 10X Genomics Chromium NEXT Gem Single Cell 3’ v3.1 kit, targeting 6000 cells per sample. The resulting libraries were sequenced on a NovaSeq 6000, targeting 50,000 reads per single nucleus. Sequenced reads were processed and aligned to < Genome > using the 10X Genomics Cellranger toolkit. The resulting raw counts matrices were further preprocessed using the scanpy 1.8.2 package. In brief, raw unique molecular identifier (UMI) count matrices for four experimental samples were passed through cellbender 0.2.0, for the purposes of filtering out empty droplets and reducing background noise stemming from ambient RNA. The relevant parameter settings for each sample for cellebender as follow; Vehicle-no stress; expected cell:10000;Total Droplets Included:20000; False Positive Rate:0.1; Epochs:100, Vehicle-stress; expected cell:12434;Total Droplets Included:15000; False Positive Rate:0.01; Epochs:100, Permethrin-no stress; expected cell:4212;Total Droplets Included:10000; False Positive Rate:0.01; Epochs:100, Permethrin-stress; expected cell:3368;Total Droplets Included:10000; False Positive Rate:0.01; Epochs:100.

Following successful droplet filtering and ambient RNA removal, scrublet 0.2.3 was used with default configuration parameters to detect and remove cellular doublets. Quality control metrics were calculated for each cell and gene across all samples, including the calculation of percentage of count deriving from mitochondrial (mt), hemoglobin (hb), and ribosomal (ribo) genes (Supplementary Fig. 1). On a per-sample basis, outlier cells were identified on the basis of any one of the following metrics falling outside 5-times the median absolute deviation: log1p_total_counts, log1p_n_genes_by_counts, pct_counts_in_top_20_genes, pct_counts_mt, pct_counts_hb, and pct_counts_ribo. Lastly, genes expressed in less than 20 cells were filtered out. In total, 21,566 cells and 21,545 genes remained after filtering. Total counts per cell were normalized to 1e⁴, with data scaled to log-plus-one. The scanpy function sc.pp.highly_variable_genes was used to calculated highly variable genes, with a minimum mean of 0.0125, a maximum mean of 3, and a minimum dispersion of 0.5. The effects of pct_counts_mt and total_counts were regressed out using the scanpy function sc.pp.regress_out. Following this, each genes expression was re-scaled to zero mean and one standard deviation. Dimensionality reduction was initiated utilizing Principle Components Analysis (PCA), retaining the top 75 componnets for downstream analysis. The four samples were integrated utilizing the harmonypy 0.0.5 python package, with convergence after two iterations. The resulting adjusted PCA matrix was used to calculate a nearest neighborhood graph with the function sc.pp.neighbors, with n_neighbors set to 15. UMAP was run using min_dist set to 0.05, followed by unsupervised clustering using leiden with resolution set to 1.0.

1. Cell Type Annotation

UniCell Deconvolve (ucdeconvolve 0.1.2) was utilized to performed label transfer for cell type annotations. In brief, the allen-mouse-cortex prebuilt reference atlas was utilized as a reference atlas to annotate cell types using the function ucd.tl.select with default parameters. The resulting predictions were mapped to each celltype using the function ucd.utils.assign_top_celltypes, with the groupby parameter set to a higher-resolution unsupervised leiden cluster assignment with resolution set to 4.0.

Following cell type assignment, top differentially expressed genes for each celltype were identified using the scanpy function sc.tl.rank_genes_groups with default parameters.

2. Gene Set Enrichment Analysis

Differentially expressed genes were calculated using the scanpy function sc.tl.rank_genes_groups, with groupby set to each sample, use_raw set to True, and reference set to the control sample VEH_NS-18-HIP. For each non-control sample, we extracted the top 500 differentially expressed genes. Subsequently, the gseapy 1.0.3 package was used to query enrichr for the GO_Biological_Process_2021 and GO_Molecular_Function_2021 gene sets, with organism set to mus musculus.

Olink assay

The protein lysates were prepared from flash-frozen brain samples at a concentration of 1mg/mL (n = 7–12 per group) using Protein Extraction Reagent (Thermo Scientific, 89900) supplemented with protease inhibitors (Thermo Scientific, 78440). Subsequently, all samples underwent analysis using the Olink® Target 96 Mouse Exploratory Assay (Olink proteomics, MA, USA, 95380). Following this, the samples were sent to the Mount Sinai Human Immune Monitoring Center for assay execution and data collection. The results obtained for the analyzed protein biomarkers were provided in Normalized Protein eXpression (NPX) units.

Statistical analysis

Statistical analysis was conducted, and all data are presented as mean ± SEM. The data were analyzed using two-way ANOVA followed by Tukey's post hoc analysis (*P < 0.05, **P < 0.01, ***P < 0.001). GraphPad Prism 8 software (GraphPad Software Inc., CA, USA) was utilized for all statistical analyses.

Permethrin is brain-penetrant and bioavailable

To confirm the permeability of permethrin through the blood-brain barrier (BBB) and its potential impact on microglia, we conducted pharmacokinetic analysis by evaluating parameters such as time of maximum concentration (T_max), the maximum drug concentration (C_max), and area under concentration-time curve (AUC). The time courses of permethrin, comprising a combination of cis- and trans-isomers, along with primary metabolite 3-phenoxybenzoic acid (3-PBA), were assessed in both plasma (Fig. 1A-1C) and brain (Fig. 1D-1E) over a 48-hour period following a single injection of 200mg/kg permethrin. We first observed a "double-peak" phenomenon in both plasma and the brain at 2 hrs and 24 hrs (Fig. 1). Notably, the plasma exhibited a distinctly double-peak curve, a characteristic often associated with entero-hepatic recycling (Fig. 1A-1C). This result indicates an increased elimination half-life, contributing to the extended action of permethrin in plasma and brain. The average plasma concentrations of cis- and trans- permethrin reached their peak at 24 hrs (Fig. 1A and 1B). Cis-permethrin displayed a C_max of 12655.89 ± 2646.10nM (Fig. 1A), and trans-permethrin exhibited a C_max of 9025.79 ± 3351.66 nM (Fig. 1B) in the plasma. In contrast, the mean brain concentrations of cis- and trans-permethrin peaked at approximately 2 hrs, indicating penetration of cis- and trans-permethrin into the brain occurs rapidly (Fig. 1C and 1D). For cis-permethrin, the mean C_max in the brain was 20637.85 ± 4786.71 nM (Fig. 1C), and for trans-permethrin, it was 20878.55 ± 3137.16 nM (Fig. 1D), both of which were higher than the concentrations observed in plasma. Additionally, we analyzed 3-PBA, the primary metabolite of permethrin, considering its rapid metabolism in the liver. As anticipated, a high concentration of its C_max (Fig. 1C, 68951.01 ± 12672.51 nM) was observed in the plasma. However, in the brain, the C_max of 3-PBA is significantly lower than in the plasma (Fig. 1D, 668.43 ± 53.35 nM). Notably, there was a noteworthy observation of sustained accumulation of 3-PBA in the brain parenchyma extending beyond 48 hrs. After reaching their peak concentrations, the mean concentration-time profiles of cis-permethrin, trans-permethrin, and 3-PBA indicated a steady decline over a period of 48 hrs. These findings suggest that both cis- and trans-permethrin rapidly entered the brain from peripheral, achieving higher concentrations and indicating a concentrated distribution within the brain.

Permethrin exposure primes the onset of depression-like behavior under stress through microglia activation

To examine the effects of permethrin exposure primes, mice were chronically exposed to permethrin (200 mg/kg) or a vehicle control for 14 days (Fig. 2A). Subsequently, the mice experienced either stress induction or gentle handling (no stress) for 7 days (Fig. 2A). Following this experimental protocol, anxiety-like behaviors were evaluated using the OF (Fig. 2B), while depressive-like behaviors were assessed through the FST (Fig. 2C and 2D) to examine neuropsychological responses. There were no significant changes in the time spent in the center zone, indicative of anxiety-like behavior, across all experimental groups (Fig. 2B). In the FST, neither permethrin exposure nor stress exposure alone led to observable behavioral alterations in immobility time when compared to non-stressed mice treated with the vehicle (Fig. 2C). Interestingly, mice subjected to stress and treated with permethrin exhibited a significant increase in the time spent immobile, in comparison to all other experimental groups (Fig. 2C, *p < 0.05, **p < 0.01, ***p < 0.001). This finding suggest that exposure to permethrin acts as a priming factor in the brain, subsequently leading to depression-like behavior in mice under these specific conditions when stress is introduced.

Exposure to permethrin triggers a rapid influx of sodium ions into microglia cells, leading to increased intracellular sodium accumulation; prolonged exposure for 24 hours activates microglia and results in neuroinflammation by releasing pro-inflammatory cytokines (14). In the exploration of microglial functions within the CNS, chemogenetic G protein-coupled receptors (GPCRs), including Gi-signaling (e.g., hM4Di) and Gq-signaling (e.g., hM3Dq) DREADDs, have been studied (17). Building upon this knowledge, we utilized Cx3cr1^CreER/WT: R26^{LSL − hM4Di/WT} mice to explore the impact of permethrin-induced microglia priming on the observed depression-like behavior. This mouse model is designed to selectively express Gi-inhibitory receptors (Gi-DREADDs) in microglia, controlled by the inducible CX3CR1 promoter. Mice underwent daily treatment with 0.1mg/kg of JHU 37160, a novel DREADD agonist known for selectively activating the Gi inhibitory signaling pathway in microglia, throughout the initial 14-day period of permethrin exposure (Fig. 2A). Following this treatment phase, the mice were then subjected to 7 days of stress, culminating in FST (Fig. 2A). The observed results were consistent with previous behavioral tests (Fig. 2C), indicating that mice exposed to stress and treated with permethrin displayed a notable increase in the time spent immobile (Fig. 2D, ****p < 0.0001). Notably, treatment with JHU 37160 during permethrin exposure effectively blocked depressive like-behavior in the FST (Fig. 2D, ***p < 0.01). These result indicates that microglia activation is a key mediator of the depressive-like behavior following exposure to permethrin followed by stress.

Permethrin exposure, followed by stress, induces regional differences in the pleomorphic response of microglia cells

Pro-inflammatory microglia have been observed to undergo deramification, a process characterized by the retraction of their processes, diminished microglial complexity, and the release of inflammatory cytokine (18). In order to further investigate the morphometric changes in microglial cells resulting from exposure to permethrin followed by stress, we conducted a detailed analysis of morphology encompassing parameters such as soma volume, branch length, the number of terminal points, and the number of intersecting segments. Additionally, we conducted a comparative analysis across the hippocampus and prefrontal cortex (PFC) in brain regions to assess regional differences in microglial alterations.

Interestingly, the volume of microglia in hippocampus significantly decreased in mice subjected to both permethrin treatment and stress compared to mice exposed only to permethrin (Fig. 3A and 3B, **p < 0.01). In addition, the mice subjected to permethrin treatment and stress showed a significant decrease in the branch length of microglial processes (Fig. 3A and 3C, *p < 0.05, **p < 0.01), accompanied by a noticeable reduction in the number of terminal points in hippocampus (Fig. 3A and 3D, *p < 0.05). Sholl analysis further revealed that microglia from the group exposed to both permethrin and stress exhibited fewer and less expansive branches compared to other experimental groups (Fig. 3E). Additionally, quantification of the area under the curve further confirmed the significant decrease in microglial branching caused by permethrin and stress (Fig. 3G and 3F, *p < 0.05, **p < 0.01). However, no discernible morphological alterations were observed in microglia in the PFC, encompassing soma volume (Fig. 3G and 3H), branch length (Fig. 3G and 3I), terminal points (Fig. 3G and 3J), intersection of segments (Fig. 3G and 3K) and area under cover (Fig. 3G and 3L).

These findings underscore the importance of regional specificity in understanding neuroinflammatory responses, particularly in the hippocampus, where permethrin treatment acts as a priming factor, subsequently leading to depression-like behavior in mice under stress conditions.

Single-cell sequencing provides profiling of distinct brain cell populations

Notably, the hippocampus, which plays a crucial role in regulating mood and emotional behavior, demonstrates significant changes in microglial morphology in response to these stressors. Given the hippocampus's involvement in depression-like behavior, these observed alterations in microglia may contribute to the development or exacerbation of depressive symptoms under conditions of permethrin exposure and stress. Therefore, investigating the relationship between microglial changes in the hippocampus and depression-like behavior could provide valuable insights into the mechanisms underlying the link between neuroinflammation and mood disorders. To investigate whether the observed microglial morphometric changes in the hippocampal formation are accompanied by a genetic profile indicative of neuroinflammation, we conducted single-cell RNA sequencing analysis using 21,566 single nuclei collected from the hippocampal formation of mice (Fig. 4–6).

UniCell Deconvolve Base (UCDBase) was employed to produce an initial unbiased cell type annotation, assessing cell type fractions across different conditions as previously reported (19). Subsequently, UniCell Deconvolve Select (UCDSelect) was utilized to transfer annotations from a reference mouse cortex/hippocampus atlas onto the novel dataset (Fig. 4A) We then compared the density distribution of cell types by sample, highlighting condition-specific differences in cell type fractions post-batch correction among all experimental groups. It revealed that all experimental groups exhibited a similar density distribution, indicating a consistent profiling of distinct brain cell populations across all groups (Fig. 4B). The identity of the microglial cluster was confirmed by the expression of canonical microglia marker genes, including inpp5d, Tgfbr1, Apbb1ip. Additionally, the identity of neuronal cells was confirmed by the expression of canonical neuronal marker genes such as Dsp, Olfr538, Cpne4m for DG, Galntl6, Gm2164, Gm2115 for CA1, Tafa1,m zfp804a for CA2, Cce1, Mndal, Slc9a4 for CA3 (Fig. 4C). These results confirm the identity of the identified clusters as microglia and neuronal cells, supported by the expression of canonical marker genes characteristic of each cell type using UCDBase analysis, thereby supporting the accuracy of cell type annotation.

Transcriptome analysis reveals alterations in gene expression specific to microglia following exposure to permethrin, followed by stress

To elucidate the transcriptional networks impacted within distinct microglia populations associated with depression-like behavior in mice exposed to permethrin and/or stress, we conducted a functional enrichment analysis, encompassing biological process (Fig. 5A) and molecular function (Fig. 5B). In Fig. 5A, we compared the biological processes selected based on adjusted p-values of < 0.05. While minimal changes were evident in the stress group, both permethrin exposure alone and in combination with stress showed similar alterations in biological processes. Notably, the permethrin with stress condition exhibited heightened significance and involvement of a greater number of gene sets in the same biological processes compared to permethrin-only group. Specifically, microglia cell population in permethrin with stress group displayed enriched biological processes associated with neuronal development (axon development, axon guidance, and the ephrin receptor signaling pathway), neuronal communication (calcium signaling, synaptic transmission, and glutamate receptor activity), as well as neuronal morphogenesis. In the assessment of molecular functions (Fig. 5B), a consistent trend emerged where minimal alterations were observed in the stress group, whereas both the permethrin-only and permethrin combined with stress groups exhibited similar patterns. Notably, within the permethrin with stress group, distinct molecular activities were noted, including ionotropic glutamate receptor activity and ligand- or voltage-gated calcium channel activity. These findings underscore parallels in biological processes concerning neuronal signaling, ion channel modulation, and synaptic transmission.

Subsequently, we investigated differentially expressed genes (DEGs) to discern variations between permethrin treatment alone and permethrin treatment with stress. In Fig. 5c, notable modifications in gene expression were observed, with 2,949 genes in the permethrin and stress group and 2,354 genes in the permethrin-only group exhibiting significant alterations compared to the vehicle group without stress (adjusted p-value < 0.05, Fig. 5C, left). Moreover, 232 genes were exclusively altered in the permethrin-only group, while 845 genes uniquely changed in the permethrin and stress group, suggesting that the permethrin with stress group exhibited more significant gene alterations. Following, utilizing the absolute Z score, we identified the top 100 genes in the permethrin with stress group (Fig. 5C, middle). It was observed that only 5 genes such as Dlgap1, Trank1, Tafa5, Calr, Rtn1 were exclusively expressed in the permethrin with stress group (Fig. 5C, right). Additionally, 87 genes showed differential expression in both groups, with the permethrin with stress group exhibiting a significantly more pronounced increase or decrease in expression compared to the permethrin-only group. Notably, 6 genes such as Epha6, Lyn, Egfem1, Faah, Klhl3 and Fat2 displayed the most substantial changes, with their expression levels in the permethrin with stress group being 1.4-fold higher or lower than those observed in the permethrin-only group (Fig. 5C, right). Overall, the results highlight the potential synergistic impact of permethrin and stress on microglia gene expression, which could contribute to the underlying mechanisms involved in depression-like behavior observed in the animal model (Fig. 2).

Permethrin exposure followed by stress induces a significant alteration in gene expression patterns across regionally distinct neuronal populations

To investigate how permethrin exposure followed by stress impacts transcriptional networks in distinct neuronal cells, which functions are potentially mediated by microglia, in hippocampal regions including CA1, CA2, CA3, and DG, we first compared the number of DEGs across groups: permethrin + no stress, permethrin + stress, and vehicle + stress (Fig. 6A and 6B). Adjust p-value threshold of 0.05 was used to identify DEGs in CA1, CA2, CA3, and DG of the hippocampus in mice from each group. Interestingly, permethrin exposure followed by stress displayed a significantly higher number of DEGs compared to the other groups in all hippocampal regions (Fig. 6A). Analysis of hippocampal regions CA1, CA2, CA3, and DG following permethrin exposure and subsequent stress revealed a prominent increase of DEGs, with CA1 exhibiting the most notable rise compared to other regions, followed by CA3. (Fig. 6A). Given the observed differences in microglial alterations between the permethrin-exposed groups with and without stress (Fig. 5), we specifically compared gene expression in these groups. Among hippocampal regions, only the CA3 region exhibited a high percentage of unique expression in DEGs in the permethrin-stress group. 62.87% were unique to the permethrin with stress group, while 29.89% were common to both permethrin-exposed groups (with or without stress), and only 7% were specific to the permethrin without stress group (Fig. 6B).

To further explore the functional implications of the observed gene expression alterations in the CA3 region, we conducted Gene Ontology (GO) enrichment analysis, specifically comparing the permethrin + stress and permethrin + no stress groups (p < 0.05, Fig. 6C and 6D). Compared to the permethrin + no stress group, the permethrin + stress group in the CA3 region showed significantly enriched GO terms (Fig. 6C) related to signal transduction, including modulation of chemical synaptic transmission, regulation of neurotransmitter receptors, and regulation of postsynaptic neurotransmitter receptor activity. Notably, even for shared biological processes, permethrin with stress group displayed a greater number of genes and a more significant level of enrichment. We then focused on the molecular function of DEGs using GO enrichment analysis (p < 0.05, Fig. 6D). GO enrichment analysis of DEGs revealed a significant enrichment for molecular function terms (p < 0.05, Fig. 6D). The permethrin + stress group exhibited a notably greater abundance of enriched terms compared to the permethrin + no stress group, including synaptotagmin-1 binding, amino acid sodium symporter activity, and volume-sensitive anion channel activity. (Fig. 6D). Overall, this results indicate that permethrin exposure followed by stress triggered a prominent rise in differentially expressed genes, particularly in CA1 and CA3, with the CA3 region exhibiting distinct patterns suggestive of disrupted neuronal communication.

Exposure to permethrin followed by stress altered protein expression associated with neuroinflammation and synaptic plasticity

To investigate the proteomic alterations in mice subjected to permethrin exposure followed by stress, we examined the abundance of a specific set of 92 proteins associated with essential biological functions using proximity extension assay-based Olink technology. We identified significant alterations in the levels of six proteins specifically within the hippocampi of mice exposed to permethrin followed by stress (Fig. 7).

Specifically, there was a notable increase in tumor Necrosis Factor alpha (TNFα) compared to other experimental groups (*p < 0.05, Fig. 7A). Additionally, Aryl hydrocarbon receptor (Ahr), known for its involvement in regulating microglial activation and neuroinflammation, showed elevated levels in the same mice (**p < 0.01, Fig. 7B). Concurrently, there was a decrease in the expression of Interleukin-1beta (IL-1β) (#,*p < 0.05, Fig. 7C).

Repulsive guidance molecule A (RGMa), renowned for its capacity to inhibit neurite growth and newborn neuron survival in the adult dentate gyrus, demonstrated notable upregulation compared to the other three experimental groups (*p < 0.05; **p < 0.01, Fig. 7C). Additionally, Synaptosomal-associated protein 29 (Snap-29), responsible for impeding SNARE complex disassembly and thereby diminishing synaptic transmission, exhibited heightened expression relative to both the no stress and stress alone groups (**p < 0.01, Fig. 7D). Conversely, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1(Ddah1), a hydrolase responsible for regulating genes associated with the synthesis and transportation of acetylcholine (ACh), exhibited significant decrease in expression in the permethrin followed by stress group (*p < 0.05, Fig. 7E). These findings highlight the multifaceted effects of permethrin exposure followed by stress on both inflammatory responses and synaptic plasticity within the hippocampi of mice, underscoring the complex interplay between microglia activation and neuronal function.

GWI is a complex multi-symptom disorder affecting approximately 33% of veterans who served in the first Gulf War (20, 21). These soldiers were exposed to various pesticides, including permethrin, DEET, and chlorpyrifos, as well as chemical weapons, smoke from oil well fires, and fine particulate matter from sandstorms(22). Upon returning from combat, affected veterans experienced a range of symptoms such as fatigue, anxiety, gastrointestinal distress, and cognitive and psychiatric disturbances (3). High levels of stress reported by many veterans during their service suggest that physical and psychological stress may have synergized with toxin exposure (4). Currently, there is no approved therapeutic for GWI, with most treatments focusing on symptom management (23). Although the exact cause of GWI has not been identified, permethrin exposure is suspected to play a role. Understanding the mechanistic factors involved and developing appropriate animal models is crucial for advancing care for this underserved population. Thus, the mouse model used in our current studies to examine interactions between environmental toxin exposure and mild stress could serve as a valuable model for studying GWI. This model may help elucidate the underlying mechanisms and contribute to the development of targeted treatments for veterans suffering from GWI.

The exposome, encompassing the cumulative impact of stressors or environmental exposures throughout an individual's life, is an emerging area of research with profound implications for neuropsychological health (24). In particular, interactions between various low-level or mild exposures represent a potential hazard which may go un-noticed due to the seemingly innocuous effects of individual low-level exposures (25). In the present study, we investigated the effects of low-level permethrin exposure at levels below the threshold required for acute intoxication for their potential synergistic interactions with 7-days of mild stress exposure. While chronic stress over a 30 day period is associated with psychiatric and changes in mice, 1 week of stress has not been shown to have neuroinflammatory or behavioral effects (Fig. 2 or Fig. 3). We revealed a significant increase in depression-like behavior in mice exposed to both permethrin and stress compared to those exposed to either factor alone or controls (Fig. 2). This suggests that permethrin exposure acts as a priming factor, sensitizing the brain to subsequent stressors and exacerbating depressive symptoms. Importantly, our chemogenetic modulation experiments via microglia expressing DREADD receptors highlight the pivotal role of microglial activation in mediating this depressive-like behavior, as blockade of microglial activation effectively mitigated the observed behavioral changes (Fig. 2). Thus, the ability of neurotoxins to stimulate neuroinflammation and depressive behavior may hold important implications regarding the role of environmental toxin exposure in mediating disease susceptibility.

Consistent with previous research (26), our results demonstrate that permethrin, including both cis- and trans- isomers, is capable of crossing the blood-brain barrier, but reaches higher concentrations in the plasma (Fig. 1). This finding highlights the potential neurotoxicity of permethrin, given its ability to directly interact with CNS components. Exposure to permethrin initiates a rapid and continuous influx of sodium into microglial cells through voltage-gated sodium channels (VGSC) (14). Consequently, there is an aberrant accumulation of intracellular sodium, leading to the activation of microglia and the subsequent upregulation of pro-inflammatory cytokine such as TNFα (14). This pro-inflammatory response is characterized by deramification, a phenomenon defined by the retraction of microglial processes (27). To elucidate these morphometric changes, a detailed analysis encompassing soma volume, branch length, terminal points, and intersecting segments in microglia was conducted (Fig. 3). We revealed significant reductions in microglial volume, branch length, and number of terminal points in the hippocampus of mice exposed to permethrin, followed by stress. We also further confirmed a decrease in microglial branching complexity in the hippocampus following combined exposure through sholl analysis. In contrast, no significant morphological alterations were observed in microglia within the PFC across all parameters analyzed. These findings suggests the regional specificity of neuroinflammatory responses, with the hippocampus being particularly susceptible to permethrin-induced microglial activation under stress conditions. The observed deramification and reduced complexity of microglia in the hippocampus suggest a potential mechanism underlying the development of depression-like behavior following exposure to permethrin and stress.

These morphological alterations are accompanied by transcriptional changes indicative of neuroinflammation, as revealed by sc RNA sequencing analysis (Fig. 4–6). Recent study indicates that UCDBase was established to leverage the entirety of publicly available scRNA-Seq data to construct a unified training corpus for deep learning (19). In addition, UCDSelect complements UCDBase by enabling context-specific deconvolution through transfer learning of UCDBase features, incorporating user-defined cell signatures for tailored analyses (19). Our analysis validated the identified clusters as microglia and neuronal cells through the cell type-specific marker gene (Fig. 4). Functional enrichment analysis indicates a synergistic impact of permethrin and stress on microglia gene expression, with the stress-only group showing minimal changes, while the permethrin-only group exhibits a pattern similar to the permethrin-with-stress group, which displays more pronounced alterations (Fig. 5). Especially, we found that among the top 100 genes, only five, such as Dlgap1, Trank1, Tafa5, Calr, and Rtn1, were exclusively altered in the permethrin-with-stress group. Additionally, through functional analysis, we identified that the microglia cell population in the combination of permethrin and stress induces more significant changes, particularly in genes associated with neuronal development, neuronal communication, and neuronal morphogenesis.

Microglia exert a critical role in maintaining synaptic integrity throughout development and adulthood via dynamic sculpting of the synapse. Aberrant microglial function in the mature brain has been implicated in pathological synaptic loss and dysfunction associated with neuroinflammation, and depression (28, 29). For instance, in depressed patients, the reduction in synapse number and function is linked to microglial engulfment of synapses (30). Depressive-like behaviors observed in several chronic stress models have been linked to the specific removal of postsynaptic dendritic spines in targeted branches, along with atrophy of neurons (31–34). To examine how permethrin exposure followed by stress influences transcriptional networks in distinct neuronal cells, potentially mediated by microglia, within hippocampal regions, including CA1, CA2, CA3, and DG. Interestingly, analysis of hippocampal regions CA1, CA2, CA3, and DG following permethrin exposure and subsequent stress revealed a significant increase in DEGs in neurons, with CA1 exhibiting the most notable rise, followed by CA3 (Fig. 6). The microglia density in the CA3 region of the mouse hippocampus is higher than in both the CA1 region and the DG (35, 36). The CA1 region has a higher microglia density compared to the DG(36). It has been proposed that this variation in microglial density could influence site-specific vulnerability in the hippocampus and that the uneven distribution of microglia may play a role in regulating hippocampal neuronal activity. These findings suggest that the high density of microglia in the CA3 region might be a key factor in the significant alterations in neuronal DEGs observed there following permethrin and stress exposure.

Consistent with these findings, our proteomic analysis uncovers alterations in key proteins involved in neuroinflammation and synaptic plasticity within the hippocampi of mice exposed to permethrin followed by stress (Fig. 7). Notably, we found the upregulation of pro-inflammatory cytokines such as TNFα and Ahr, underscores the dysregulated inflammatory response elicited by permethrin exposure and stress (Fig. 7). The upregulation of TNF-α has been implicated in the activation of microglia, which are key players in neuroinflammatory processes and may contribute to neuronal damage by activating microglia (37). AhR is a critical mediator for peripheral immune functions involving the differentiation of dendritic cells and the tissue-specific proinflammatory gene expression (38, 39). In the brain, AhR is ubiquitously expressed in areas including the cerebral cortex, hippocampus, and cerebellum (40), and is related to its environmental ligands-associated sensorimotor and cognitive abnormalities based on its aggravation of oxidative stress or excitotoxicity in neurons (41). AhR can also directly activate cytokine transcription and AhR increases NOX activity by increasing the expression of p47phox to elicit oxidative stress, which contributes to cytokine transcription such as TNF- α (42). Additionally, we found that the expression of synaptic plasticity protein molecules (RGMa, SNAP-29, and Ddah1) in response to permethrin followed by stress (Fig. 7). SNAP-29 serves as a negative modulator by slowing the disassembly of the SNARE complex and consequently diminishing synaptic transmission in cultured neurons. This effect is associated with the attenuation of both the recycling process of the SNARE-based fusion machinery and the turnover of synaptic vesicles (43). RGMa has been identified to hinder synapse formation by disrupting the expression of presynaptic protein synapsin-1 and postsynaptic protein PSD-95 in cortical neurons. (44). Recent study suggests that RGMa regulates neuronal branching through the RhoA pathway, thereby impacting synaptic plasticity. (45). RGMa can also inhibit axon growth in CNS through its potential suppression of axon growth by inducing the expression of Rho-associated coiled-coil protein kinase in neurons. (46). Additionally, research indicates that mice subjected to an Mg-restricted diet display depression-like behavior and diminished expression of DDAH1, the enzyme responsible for breaking down N, N dimethyl-L-arginine, a significant competitive inhibitor of nitric oxide synthase (47).

In conclusion, our study demonstrates that combined permethrin exposure and stress significantly exacerbate depression-like behaviors in mice through microglial activation and upregulation of pro-inflammatory cytokines, particularly in the hippocampus. Additionally, the observed alterations suggest potential disruptions in neuronal communication mediated by microglia activation, further emphasizing the complexity of GWI pathology. These findings highlight the critical role of microglia in mediating neuroinflammatory responses and underscore the importance of considering both environmental toxins and psychological stressors in understanding GWI.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Any remaining data that support the results of the study will be available from the corresponding author upon reasonable request.

Competing interests

The authors have no conflict of interest to report.

Funding

This research was supported in part by the Department of Veteran Affairs grant number BX004583 and Department of Defense grant number W81XWH-19-1-0513. G.M.P. holds a Senior VA Career Scientist Award. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government.

Authors' contributions

S.N. and E.Y. conceived and designed the study concept, performed all experiments, analyzed and interpreted data, and was instrumental in the writing of the manuscript. U.I. and U.R. performed the immunofluorescence experiment and analyzed data. M.E., D.C., L.S., and R.S. analyzed and interpreted single-cell sequencing data. W.L., Q.W., and J.S. performed pharmacological experiments and analyzed the data. S.M., K.J.T and H.W provided assistance throughout the study. G.M.P. conceived and designed the study concept, and supervised the project. In addition, G.M.P. is the guarantor of this work, as such; he has full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read the final version of the manuscript, revised it critically, and gave final approval of the version submitted.

Acknowledgments

The authors extend their sincere thanks to Vassili Tchaikovsky for his invaluable technical support and unwavering assistance, as well as to Josh Palmieri for their exceptional work in administrative duties.

Inoue C, Shawler E, Jordan CH, Moore MJ, Jackson CA. Veteran and Military Mental Health Issues. StatPearls. Treasure Island (FL) ineligible companies. Disclosure: Evan Shawler declares no relevant financial relationships with ineligible companies. Disclosure: Christopher Jordan declares no relevant financial relationships with ineligible companies. Disclosure: Marlyn Moore declares no relevant financial relationships with ineligible companies. Disclosure: Christopher Jackson declares no relevant financial relationships with ineligible companies.2024.
Black DW, Carney CP, Forman-Hoffman VL, Letuchy E, Peloso P, Woolson RF, et al. Depression in veterans of the first Gulf War and comparable military controls. Ann Clin Psychiatry. 2004;16(2):53–61.
Kuhn E, Drescher K, Ruzek J, Rosen C. Aggressive and unsafe driving in male veterans receiving residential treatment for PTSD. J Trauma Stress. 2010;23(3):399–402.
Carlson EB, Palmieri PA, Vogt D, Macia K, Lindley SE. Development and cross-validation of a veterans mental health risk factor screen. PLoS ONE. 2023;18(2):e0272599.
White RF, Steele L, O'Callaghan JP, Sullivan K, Binns JH, Golomb BA, et al. Recent research on Gulf War illness and other health problems in veterans of the 1991 Gulf War: Effects of toxicant exposures during deployment. Cortex. 2016;74:449–75.
Koo BB, Michalovicz LT, Calderazzo S, Kelly KA, Sullivan K, Killiany RJ, et al. Corticosterone potentiates DFP-induced neuroinflammation and affects high-order diffusion imaging in a rat model of Gulf War Illness. Brain Behav Immun. 2018;67:42–6.
Chrustek A, Holynska-Iwan I, Dziembowska I, Bogusiewicz J, Wroblewski M, Cwynar A et al. Current Research on the Safety of Pyrethroids Used as Insecticides. Med (Kaunas). 2018;54(4).
Soderlund DM. Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances. Arch Toxicol. 2012;86(2):165–81.
Sullivan K, Krengel M, Bradford W, Stone C, Thompson TA, Heeren T, et al. Neuropsychological functioning in military pesticide applicators from the Gulf War: Effects on information processing speed, attention and visual memory. Neurotoxicol Teratol. 2018;65:1–13.
Joshi U, Pearson A, Evans JE, Langlois H, Saltiel N, Ojo J, et al. A permethrin metabolite is associated with adaptive immune responses in Gulf War Illness. Brain Behav Immun. 2019;81:545–59.
Soderlund DM, Clark JM, Sheets LP, Mullin LS, Piccirillo VJ, Sargent D, et al. Mechanisms of pyrethroid neurotoxicity: implications for cumulative risk assessment. Toxicology. 2002;171(1):3–59.
Bradberry SM, Cage SA, Proudfoot AT, Vale JA. Poisoning due to pyrethroids. Toxicol Rev. 2005;24(2):93–106.
Field LM, Emyr Davies TG, O'Reilly AO, Williamson MS, Wallace BA. Voltage-gated sodium channels as targets for pyrethroid insecticides. Eur Biophys J. 2017;46(7):675–9.
Hossain MM, Liu J, Richardson JR. Pyrethroid Insecticides Directly Activate Microglia Through Interaction With Voltage-Gated Sodium Channels. Toxicol Sci. 2017;155(1):112–23.
Ribeiro ACR, Deshpande LS. A review of pre-clinical models for Gulf War Illness. Pharmacol Ther. 2021;228:107936.
Venkatasamy L, Nizamutdinov D, Jenkins J, Shapiro LA. Vagus Nerve Stimulation Ameliorates Cognitive Impairment and Increased Hippocampal Astrocytes in a Mouse Model of Gulf War Illness. Neurosci Insights. 2021;16:26331055211018456.
Parusel S, Yi MH, Hunt CL, Wu LJ. Chemogenetic and Optogenetic Manipulations of Microglia in Chronic Pain. Neurosci Bull. 2023;39(3):368–78.
Butovsky O, Weiner HL. Microglial signatures and their role in health and disease. Nat Rev Neurosci. 2018;19(10):622–35.
Charytonowicz D, Brody R, Sebra R. Interpretable and context-free deconvolution of multi-scale whole transcriptomic data with UniCell deconvolve. Nat Commun. 2023;14(1):1350.
Smith BN, Wang JM, Vogt D, Vickers K, King DW, King LA. Gulf war illness: symptomatology among veterans 10 years after deployment. J Occup Environ Med. 2013;55(1):104–10.
Chao LL, Sullivan K, Krengel MH, Killiany RJ, Steele L, Klimas NG, et al. The prevalence of mild cognitive impairment in Gulf War veterans: a follow-up study. Front Neurosci. 2023;17:1301066.
Brown M. Toxicological assessments of Gulf War veterans. Philos Trans R Soc Lond B Biol Sci. 2006;361(1468):649–79.
Holodniy M, Kaiser JD. Treatment for Gulf War Illness (GWI) with KPAX002 (methylphenidate hydrochloride + GWI nutrient formula) in subjects meeting the Kansas case definition: A prospective, open-label trial. J Psychiatr Res. 2019;118:14–20.
Wild CP. Complementing the genome with an exposome: the outstanding challenge of environmental exposure measurement in molecular epidemiology. Cancer Epidemiol Biomarkers Prev. 2005;14(8):1847–50.
Genuis SJ. Toxicant exposure and mental health–individual, social, and public health considerations. J Forensic Sci. 2009;54(2):474–7.
Omotoso G, Oloyede O, Lawal S, Gbadamosi I, Mutholib N, Abdulsalam F, et al. Permethrin exposure affects neurobehavior and cellular characterization in rats' brain. Environ Anal Health Toxicol. 2020;35(4):e2020022–0.
Vidal-Itriago A, Radford RAW, Aramideh JA, Maurel C, Scherer NM, Don EK, et al. Microglia morphophysiological diversity and its implications for the CNS. Front Immunol. 2022;13:997786.
Werneburg S, Jung J, Kunjamma RB, Ha SK, Luciano NJ, Willis CM, et al. Targeted Complement Inhibition at Synapses Prevents Microglial Synaptic Engulfment and Synapse Loss in Demyelinating Disease. Immunity. 2020;52(1):167–82. e7.
Paolicelli RC, Ferretti MT. Function and Dysfunction of Microglia during Brain Development: Consequences for Synapses and Neural Circuits. Front Synaptic Neurosci. 2017;9:9.
Kang HJ, Voleti B, Hajszan T, Rajkowska G, Stockmeier CA, Licznerski P, et al. Decreased expression of synapse-related genes and loss of synapses in major depressive disorder. Nat Med. 2012;18(9):1413–7.
Moda-Sava RN, Murdock MH, Parekh PK, Fetcho RN, Huang BS, Huynh TN et al. Sustained rescue of prefrontal circuit dysfunction by antidepressant-induced spine formation. Science. 2019;364(6436).
Wohleb ES, Terwilliger R, Duman CH, Duman RS. Stress-Induced Neuronal Colony Stimulating Factor 1 Provokes Microglia-Mediated Neuronal Remodeling and Depressive-like Behavior. Biol Psychiatry. 2018;83(1):38–49.
Duman RS, Aghajanian GK. Synaptic dysfunction in depression: potential therapeutic targets. Science. 2012;338(6103):68–72.
McEwen BS, Eiland L, Hunter RG, Miller MM. Stress and anxiety: structural plasticity and epigenetic regulation as a consequence of stress. Neuropharmacology. 2012;62(1):3–12.
Jinno S, Fleischer F, Eckel S, Schmidt V, Kosaka T. Spatial arrangement of microglia in the mouse hippocampus: a stereological study in comparison with astrocytes. Glia. 2007;55(13):1334–47.
Imai Y, Ibata I, Ito D, Ohsawa K, Kohsaka S. A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem Biophys Res Commun. 1996;224(3):855–62.
Allan SM, Rothwell NJ. Cytokines and acute neurodegeneration. Nat Rev Neurosci. 2001;2(10):734–44.
Esser C, Rannug A, Stockinger B. The aryl hydrocarbon receptor in immunity. Trends Immunol. 2009;30(9):447–54.
Wu D, Li W, Lok P, Matsumura F, Vogel CF. AhR deficiency impairs expression of LPS-induced inflammatory genes in mice. Biochem Biophys Res Commun. 2011;410(2):358–63.
Lin CH, Juan SH, Wang CY, Sun YY, Chou CM, Chang SF, et al. Neuronal activity enhances aryl hydrocarbon receptor-mediated gene expression and dioxin neurotoxicity in cortical neurons. J Neurochem. 2008;104(5):1415–29.
Williamson MA, Gasiewicz TA, Opanashuk LA. Aryl hydrocarbon receptor expression and activity in cerebellar granule neuroblasts: implications for development and dioxin neurotoxicity. Toxicol Sci. 2005;83(2):340–8.
Tanaka M, Fujikawa M, Oguro A, Itoh K, Vogel CFA, Ishihara Y. Involvement of the Microglial Aryl Hydrocarbon Receptor in Neuroinflammation and Vasogenic Edema after Ischemic Stroke. Cells. 2021;10(4).
Pan PY, Cai Q, Lin L, Lu PH, Duan S, Sheng ZH. SNAP-29-mediated modulation of synaptic transmission in cultured hippocampal neurons. J Biol Chem. 2005;280(27):25769–79.
Yoshida J, Kubo T, Yamashita T. Inhibition of branching and spine maturation by repulsive guidance molecule in cultured cortical neurons. Biochem Biophys Res Commun. 2008;372(4):725–9.
Feng Y, Duan C, Luo Z, Xiao W, Tian F. Silencing miR-20a-5p inhibits axonal growth and neuronal branching and prevents epileptogenesis through RGMa-RhoA-mediated synaptic plasticity. J Cell Mol Med. 2020;24(18):10573–88.
Fujita Y, Yamashita T. The roles of RGMa-neogenin signaling in inflammation and angiogenesis. Inflamm Regen. 2017;37:6.
Whittle N, Li L, Chen WQ, Yang JW, Sartori SB, Lubec G, et al. Changes in brain protein expression are linked to magnesium restriction-induced depression-like behavior. Amino Acids. 2011;40(4):1231–48.

No competing interests reported.

suppleFig1.tif

Download PDF

Editorial decision: Revision requested
05 Aug, 2024
Reviews received at journal
02 Aug, 2024
Reviews received at journal
30 Jul, 2024
Reviews received at journal
30 Jul, 2024
Reviewers agreed at journal
15 Jul, 2024
Reviewers agreed at journal
12 Jul, 2024
Reviewers agreed at journal
11 Jul, 2024
Reviewers invited by journal
11 Jul, 2024
Editor assigned by journal
11 Jul, 2024
Submission checks completed at journal
10 Jul, 2024
First submitted to journal
02 Jul, 2024

You are reading this latest preprint version

Regional distribution of cellular of molecular pathways in a mouse model of Gulf War illness by Single-Cell RNA Sequencing Analysis

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Permethrin pharmacokinetic studies

Animal Treatments

Unpredictable stress exposure

Behavioral tests

Immunohistochemistry

Sholl analysis

Single Nuclei Sequencing (scRNA-Seq)

Olink assay

Statistical analysis

Results

Permethrin is brain-penetrant and bioavailable

Permethrin exposure primes the onset of depression-like behavior under stress through microglia activation

Permethrin exposure, followed by stress, induces regional differences in the pleomorphic response of microglia cells

Single-cell sequencing provides profiling of distinct brain cell populations

Exposure to permethrin followed by stress altered protein expression associated with neuroinflammation and synaptic plasticity

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1