CCL2/CCR2-mediated monocyte-macrophages infiltration drives methamphetamine-induced depressive-like behaviors

CCL2/CCR2-mediated monocyte-macrophages infiltration drives methamphetamine-induced depressive-like behaviors | 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 CCL2/CCR2-mediated monocyte-macrophages infiltration drives methamphetamine-induced depressive-like behaviors Chunling Ma, Congcong Hou, Rongji Hui, Wenjing Zhang, Xiang Li, and 7 more This is a preprint; it has not been peer reviewed by a journal.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-7742867/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 Methamphetamine (METH) abuse is frequently associated with persistent depressive symptoms, representing a major contributor to psychiatric comorbidity in substance use disorders; however, the underlying mechanisms remain poorly defined. Here, we show that binge METH exposure induces robust and persistent depressive-like behaviors in mice, accompanied by systemic cytokine elevation and expansion of Ly6C hi inflammatory monocytes in the peripheral circulation. These monocyte-derived macrophages infiltrated the medial prefrontal cortex (mPFC) via the choroid plexus and meninges in a CCR2-dependent manner. Single-cell RNA sequencing of mPFC immune cells identified distinct proinflammatory CCR2 + macrophage subsets enriched for IL-1β expression, which in turn amplified neuronal CCL2 production, establishing a self-sustaining macrophage–microglia crosstalk that perpetuated local inflammation. Pharmacological inhibition or genetic disruption of CCR2 prevented immune infiltration, reduced neuroinflammation, preserved synaptic integrity, and rescued both depressive-like and cognitive deficits. Together, these findings identify CCR2-dependent monocyte infiltration as a key mechanism linking METH exposure to affective dysfunction and highlight the CCL2/CCR2 axis as a potential therapeutic target in substance use–associated mood disorders. Biological sciences/Neuroscience Health sciences/Diseases/Psychiatric disorders/Depression Methamphetamine Depressive behaviors monocyte-macrophages CCL2 CCR2 Microglia Synaptic plasticity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The comorbidity of psychostimulant abuse and affective disorders presents a major challenge in neuroscience. Methamphetamine (METH), one of the most widely abused psychostimulants, is strongly linked to persistent depressive symptoms during withdrawal(1-3), yet the underlying mechanisms remain poorly defined. While early models emphasized monoaminergic dysfunction(4), growing evidence indicates that emotional disturbances induced by METH extend beyond direct dopaminergic and serotonergic toxicity and may involve neuroimmune processes. Recent studies highlight the bidirectional communication between the central nervous system (CNS) and the peripheral immune system in the pathogenesis of depression(5, 6). Interactions among neurons, glial cells, and the peripheral immune system are essential for maintaining key brain functions—including cognition, social behavior, and learning and memory(7-9). Peripheral immune hyperactivation can significantly increase the risk of neuropsychiatric disorders including depression and schizophrenia by compromising blood–brain barrier integrity and driving aberrant microglial polarization(10). The macrophage theory of depression, proposed in the early 1990s, attributed depressive pathology primarily to excessive cytokine production by macrophages(11). Under stress or inflammatory stimulation, peripheral monocytes upregulate cytokine secretion, infiltrate the brain, and promote stress susceptibility and depressive-like phenotypes(12). During this process, Ly6C hi inflammatory monocytes—which express high levels of C-C chemokine receptor 2 (CCR2)—are directionally recruited to injury sites in response to their principal ligand C-C chemokine ligand 2 (CCL2). It can be seen that CCL2-CCR2 is critical for driving monocyte transendothelial migration(13, 14). These infiltrating cells differentiate into macrophages, interact with microglia, and amplify neuroinflammation, thereby altering synaptic integrity in mood-regulating regions such as the medial prefrontal cortex (mPFC) and amygdala(15, 16). Clinical findings further support this mechanism, as elevated peripheral CCL2 levels have been reported in patients with depression(17, 18). Despite this framework, how METH engages the CCL2/CCR2 axis remains unclear. Whether METH directly induces CCL2 release, whether monocyte infiltration is required for METH-related depressive behaviors, and through which anatomical routes these cells access the CNS remain unanswered. Moreover, the distribution, transcriptional states, and functional consequences of infiltrating monocytes within the brain have not been systematically defined. Clarifying these issues is essential to understanding how psychostimulant exposure sustains affective pathology through immune pathways. Here, we established a rodent model of binge METH-induced depressive behavior in male mice to examine whether neuronal CCL2 expression and CCR2-dependent monocyte recruitment contribute to emotional dysregulation. By integrating behavioral paradigms, flow cytometry, single-cell transcriptomics, immunohistochemistry, and genetic manipulation, we systematically evaluated the infiltration routes, transcriptional features, and neuroimmune interactions of peripheral monocytes. We further tested whether pharmacological blockade or genetic deletion of CCR2 could mitigate neuroinflammation, synaptic disruption, and depressive-like phenotypes. This work provides mechanistic insight into METH-associated depression and positions the CCL2/CCR2 axis as a promising therapeutic target for substance abuse comorbid with affective disorders. Materials and methods Animals Male C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and CCR2 knockout (CCR2-KO) mice were obtained from Shanghai Model Organisms Center, Inc. Animals were housed under specific pathogen-free (SPF) conditions. The animal facility was maintained at a constant temperature (22 ± 1 °C), relative humidity of 60%, and a 12-h light/dark cycle with ad libitum access to food and water. Bone marrow-derived macrophage Culture Bone marrow-derived macrophages (BMDMs) were generated from isolated bone marrow cells of mature C57BL/6J mice. Following dissection and disinfection of hind limbs, femurs and tibiae were cleaned of muscle tissue, and marrow was flushed using cold PBS (Thermo Fisher Scientific, Cat#10010023) supplemented with 2% FBS (Thermo Fisher Scientific, Cat#16000-044). The cell suspension was filtered through a 40 μm nylon mesh (Falcon, Cat#352340), subjected to erythrocyte lysis (BD, Cat#555899), and cultured in DMEM (Thermo Fisher Scientific, Cat#11965092) high-glucose medium containing 10% FBS and 20 ng/mL M-CSF (ABclonal, Cat#RP01216) at a density of 2×10 5 cells/mL. METH-Induced Depressive-like Model Mice received intraperitoneal injections of methamphetamine (METH, 10 mg/kg) in a high ambient environment (28 °C). Each animal was injected four times daily at 2-h intervals (0.25 mL per injection). Control mice were administered equal volumes of saline under identical conditions. Tail Suspension Test (TST) For the TST, mice were suspended by the distal fifth of the tail using medical adhesive tape affixed to a horizontal bar, allowing free movement without escape for 6 min. The final 5 min were recorded with a video camera and analyzed using EthoVision XT software (Noldus, Netherlands). Immobility duration and active time were quantified, with prolonged immobility considered indicative of depressive-like behavior. Forced Swim Test (FST) The FST was performed in transparent cylindrical tanks (water depth 15 cm, temperature 25 ± 2 °C). Each mouse was individually placed in water for 6 min, and immobility time was scored during the last 5 min of the trial using video tracking. Locomotor Activity Test (LMT) Spontaneous locomotor activity was assessed in an open-field arena (40 × 40 × 40 cm, uncovered). Mice were placed in the center and allowed to freely explore for 5 min. The total distance traveled was quantified. Sucrose Preference Test (SPT) SPT was conducted in single-housed mice. During the adaptation phase, animals had access to two bottles containing 1.5% sucrose solution for 24 h, followed by one sucrose bottle and one water bottle for an additional 24 h. Bottle positions were alternated every 12 h to avoid side bias. Following a 24-h period of food and water deprivation, the test phase was performed over the next 24 h, during which consumption from the sucrose and water bottles was recorded by weighing. Sucrose preference was expressed as the percentage of sucrose intake relative to total fluid consumption. Novel Object Recognition Test (NORT) The NORT consisted of three stages: habituation, familiarization, and testing. During habituation, mice explored the empty chamber for 10 min twice daily. In the familiarization phase, animals were introduced to two identical objects (A+A) placed symmetrically in the chamber for 5 min. Four hours later, one object was replaced by a novel one (B) for short-term memory assessment. At 24 h, object B was replaced with another novel object (C) for long-term memory evaluation. Exploration time directed toward novel (N) versus familiar (F) objects was recorded. The discrimination index was calculated as N/(N+F) × 100%. Barnes Maze Test The Barnes maze consisted of a circular platform containing 20 holes, only one of which led to a hidden escape box. Mice were acclimated the day prior to testing by being guided to the target box. During training, animals were placed in the central start zone and allowed to search for up to 3 min or until they located the escape box. The maze and escape box were cleaned with 75% ethanol after each trial, and the maze was rotated while keeping the target box spatially constant. Training was conducted twice daily for 4 days. On day 5, the probe test was performed with the escape box removed, and the time spent in the target zone vicinity was recorded. Cytokine array profiling Based on the Cytokine array (RayBiotech, Cat#QAM-INF-1), inflammatory factors were detected in mouse serum, choroid plexus, and meninges. Serum samples were collected, centrifuged, and aliquoted. Choroid plexus and meningeal tissues were isolated, lysed with RIPA buffer containing protease inhibitors, centrifuged, and the supernatant was quantified using the BCA assay, with total protein concentration adjusted uniformly to 1 mg/mL. After background subtraction and inter-array normalization, differential protein analysis was performed using the limma package with screening criteria set at |logFC| > log₂ (1.2) and adjusted p-value < 0.05. Principal component analysis (PCA) was applied to evaluate inter-group sample distribution patterns. Differentially expressed proteins were subjected to hierarchical clustering (based on Euclidean distance and complete linkage method) and visualized via heatmap. Further functional enrichment analysis of differential proteins, including GO terms and KEGG pathways, was conducted using Fisher’s exact test (screening criteria: Count ≥ 2, p < 0.05). The top 10 enriched terms/pathways ranked by Count were selected, and the enrichment factor (enrich factor) was calculated. Single-cell RNA sequencing (scRNA-seq) Single-cell RNA sequencing (scRNA-seq) was performed on the medial prefrontal cortex (mPFC) of mice at 4 days after METH or saline exposure. Single-cell suspensions were prepared and quality-controlled to ensure cell count >5×10⁵, viability ≥75%, diameter 5–40 μm, concentration 700–1200 cells/μL, and absence of debris. Cells were encapsulated using gel beads-in-emulsion (GEMs) for mRNA capture and reverse transcription. cDNA was amplified, and libraries were constructed through fragmentation, end repair, adapter ligation, and index PCR. Libraries were quality-checked using Agilent 2100 Bioanalyzer (peak size ~400 bp) and quantified via Qubit (≥2 nM). Sequencing was conducted on Illumina NovaSeq with PE150. Bioinformatic analysis included raw data QC with fastp, alignment and expression matrix generation with Cell Ranger 3.0, cell filtering based on gene/mitochondrial/ribosomal counts, and doublet removal with DoubletFinder. Dimensionality reduction and clustering were performed using Seurat (LogNormalize, PCA, UMAP). Differential gene expression between clusters was identified with Seurat, and functional enrichment analysis (GO and KEGG) was carried out using clusterProfiler, with results visualized via ggplot2. Flow Cytometry After METH exposure, peripheral blood samples were collected. Single-cell suspensions were prepared by filtering through a 40 µm nylon mesh (Falcon, Cat#352340), followed by erythrocyte lysis (BD, Cat#555899). Cells were washed and resuspended in staining buffer (BD, Cat#554656). BMDMs were seeded in 6-well plates at 5×10 5 cells per well, with medium replenished on day 3. After 7 days of differentiation, BMDM purity was confirmed by flow cytometry based on PE-conjugated anti-mouse CD11b Antibody (Cytek® Biosciences, Cat#50-0112) and FITC-conjugated anti-mouse F4/80 Antibody (TONBO Biosciences, Cat#35-4801) expression. For experimental treatment, mature BMDMs were stimulated with METH for 48 hours prior to subsequent analysis. A total of 1 × 10⁶ cells were incubated with APC-conjugated anti-mouse CD86 Antibody (TONBO Biosciences, Cat#20-0862), APC-conjugated anti-mouse CD11b Antibody (Biolegend, Cat#101212), FITC-conjugated anti-mouse Ly6C Antibody (Biolegend, Cat#128005) , FITC-conjugated anti-mouse NK1.1 Antibody (Cytek® Biosciences, Cat#35-5941), PE-conjugated anti-mouse CD4 Antibody (Cytek® Biosciences, Cat#50-0042) or APC-conjugated anti-mouse CD8 Antibody (Cytek® Biosciences, Cat#25-0081) at 4 °C for 40 min in the dark. After washing, cells were analyzed on a FACSCalibur™ flow cytometer (BD Biosciences, New Jersey, USA). In Vivo Cell Depletion To deplete monocyte/macrophages, mice were intraperitoneally injected with 5 mg/mL Clodronate Liposomes (CL, Liposoma B.V., Cat#CP-005-005) solution on day 4 and day 1 prior to METH administration. The CL were stored at 4 ℃, and the administered concentration was 0.15 mL/10 g body weight. Control mice received intraperitoneal injections of an equal volume of PBS Liposome solution at the same time points. Depletion efficiency was verified by flow cytometry after two injections. To antagonize CCR2, mice received intravenous injections of 200 μL RS504393 working solution (Abmole, Cat#M7224, 0.25 mg/mL) from one day before to six days after METH exposure. The working solution was diluted from a 10 mg/mL DMSO stock stored at −20 °C. Control mice were injected with an equal volume of vehicle solvent. Depletion efficiency was verified by flow cytometry after seven injections. Quantitative RT-PCR Total RNA was extracted with TRIzol reagent (Invitrogen, Cat#15596026CN). cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Cat#RR047A). qPCR was performed with TB Green® Premix Ex Taq™ II (Takara, Cat#RR820A). Gene expression was normalized to GAPDH, and primers are listed in Table 1. Primer Sequences used for qRT-PCR Transmission Electron Microscopy Medial Prefrontal Cortex (mPFC) was fixed in electron microscopy fixative (Servicebio, Cat#G1102) at 4 °C for 3 h, pre-embedded in 1% agarose, and post-fixed with 1% osmium tetroxide (Ted Pella, Cat#18456). After dehydration in graded ethanol, samples were embedded in epoxy resin (SPI, Cat#90529-77-4). Ultrathin sections were cut (Leica UC7, GER), mounted on copper grids, and stained with uranyl acetate and lead citrate. Images were acquired with a HITACHI HT7800 transmission electron microscope (JPN). H&E Staining Brains were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 µm. Sections were stained with hematoxylin and eosin following standard protocols, dehydrated through graded alcohols, cleared in xylene, and mounted with neutral resin. Nissl Staining Sections were processed with a methylene blue-based Nissl staining kit (Solarbio, Cat#G1434) according to manufacturer instructions. Stained sections were differentiated, rinsed, mounted, and examined microscopically. Western Blotting Brain tissues were lysed in RIPA buffer (Solarbio, Cat#R0010) containing PMSF and a phosphatase inhibitor mixture (Servicebio, Cat#G2006), and the supernatant was collected. Equal amounts of protein were separated by SDS-PAGE, transferred to NC membranes (Millipore, Cat#HATF00010), and blocked in 5% BSA (Sigma-Aldrich, Cat#V900933). Membranes were incubated overnight at 4 °C with Rabbit Anti-PSD95 Antibody (HUABIO, Cat# ET1602-20, 1:2000) or Rabbit Anti-β-actin Antibody (ABclonal, Cat#AC026, 1:100000), followed by DyLight™ 680-conjugated Rabbit IgG Antibody (Rockland, Cat#611-144-002, 1:5000). Protein bands were visualized using the Odyssey Imaging System (LI-COR, Nebraska, USA) and quantified with ImageJ software (NIH, Maryland, USA). Immunohistochemistry Following standard anesthesia, mouse brain tissue was carefully extracted and fixed in 4% PFA for 48–72 h. After dehydration and embedding, 5 μm sections were prepared. The procedure included dewaxing, rehydration, antigen retrieval via microwave heating in repair solution, peroxidase blocking, donkey serum blocking, incubation with Mouse Anti-CD11b Antibody (HUABIO, Cat#RT1107, 1:200) at 4 °C overnight, HRP-conjugated goat anti-mouse IgG incubation at 37 °C, DAB development (Servicebio, Cat#G1311), and hematoxylin counterstaining before final dehydration and mounting. Immunofluorescence Paraffin-embedded brain sections underwent deparaffinization and antigen retrieval, followed by blocking in donkey serum. Sections were incubated with Rabbit anti-CD45 Antibody (ABclonal, Cat#A2115, 1:200), Rat anti-CD11b Antibody, (Thermo Fisher, Cat# 14-0112-82, 1:200), Rabbit anti-CCR2 Antibody (Affinity Biosciences, Cat#DF7507, 1:150), Rat anti-F4/80 Antibody (HUABIO, Cat# RT1212, 1:200), Mouse anti-CD11b Antibody (Biolegend, Cat#101201), Rat anti-MCP-1 (CCL2) Antibody(Santa Cruz, Cat#sc-52701, 1:200), Rabbit anti-NeuN Antibody (HUABIO, Cat# ET1602-12, 1:500) or Rabbit anti-Iba-1 Antibody (HUABIO, Cat# ET1705-78, 1:200) overnight at 4 °C, and then with Alexa Fluor™ 594 Donkey anti-Rabbit Antibody (Thermo Fisher, Cat#A-21207, 1:500) , Alexa Fluor™ 488 Donkey anti-Rat Antibody (Thermo Fisher, Cat#A-21208, 1:500) or iFluor™ 647 Conjugated Goat anti-mouse Antibody (HUABIO, Cat# HA1127, 1:500) for 40 minutes. Nuclei were counterstained with DAPI (CST, Cat#8961S). Images were acquired with a laser scanning confocal microscope (Leica, SP8) and analyzed using ImageJ. Enzyme-Linked Immunosorbent Assay (ELISA) Mouse serum or mPFC cells were collected, and mPFC cells were lysed thoroughly using lysis buffer to obtain the supernatant. ELISA kits were used to measure the levels of MCP-1(CCL2) (ABclonal, Cat#RK00381) according to the manufacturer’s instructions. Absorbance was measured using a microplate reader (Tecan, Switzerland). Standard curves were generated using known concentrations of the standards, and sample concentrations were calculated using the four-parameter logistic fit based on the absorbance values. Statistical Analysis Data are expressed as mean ± SEM. GraphPad Prism (9.3.1) was used for analysis. ANOVA and Student’s t test were applied for the statistical analyses. Bonferroni’s post hoc correction was used for multiple comparisons. For data that did not meet the normality assumption, the Mann-Whitney U test was used for two-group comparisons, and the Kruskal-Wallis test was used for multi-group comparisons. A p value < 0.05 was considered statistically significant. Raw data and statistical outputs are provided in Supplementary Material. Results 1. Peripheral monocyte-derived macrophages contribute to METH-induced depressive-like behavior We first established a binge METH exposure paradigm in adult male C57BL/6N mice (10 mg/kg, i.p., four injections within one day at 28 °C) and assessed behavioral tests at days 4, 7 and 14 post-exposures. METH-treated animals displayed robust depressive-like phenotypes: immobility times in both TST and FST were significantly prolonged on days 4 and 7 relative to saline controls, with only partial recovery by day 14 (Fig. 1B–C). Importantly, LMT did not differ between groups at these time points (Fig. 1D), indicating that the observed increases in immobility reflect affective changes rather than generalized motor impairment. To characterize systemic immune alterations that accompany these behavioral changes, we performed cytokine array profiling of serum obtained on day 4. Principal component analysis demonstrated clear separation between saline and METH groups (Fig. 1E). Of the cytokines assayed, levels of 15 cytokines were significantly elevated following METH exposure, including IL-6, TNF-α, TNFR I/II, CCL2, CCL12 and ICAM-1 (Fig. 1F; Supplementary Fig. 1A). By contrast, 24 analytes (for example IL-21, IFN-γ and TCA-3) remained unchanged (Supplementary Fig. 1B). Notably, IL-6 and TNF-α are primary cytokines secreted by monocyte-macrophages; CCL2 and CCL12 mediate monocyte chemotaxis; and ICAM-1 facilitates leukocyte adhesion, suggest an activated myeloid response and a chemotactic milieu favoring monocyte recruitment. Gene Ontology (GO) enrichment linked the differentially expressed cytokines to mononuclear cell migration, monocyte migration and to activation of the JAK–STAT signaling cascade (Fig. 1G), while molecular function annotation highlighted CCR2 chemokine receptor binding (Fig. 1H). KEGG pathway analysis further implicated cytokine–cytokine receptor interactions, TNF signaling and IL-17 signaling (Supplementary Fig. 1C–D). These systemic changes are consistent with our prior work showing METH-driven Th17 skewing and IL-17A production, which may be sustained by macrophage-derived IL-6. Flow cytometric analysis of peripheral blood further supported a myeloid-centric immune response. Total CD11b⁺ myeloid cells were increased in mononuclear cells (Supplementary Fig. 1E), whereas frequencies of NK cells (Supplementary Fig. 1F), CD4⁺ and CD8⁺ T cells (Supplementary Fig. 1G) were not significantly altered. The fraction of CD11b⁺Ly6C⁺ inflammatory monocytes increased after METH exposure, peaking on day 4 and remaining elevated at day 7 (Fig. 1I–K; Supplementary Fig. 1H), indicating a selective expansion/activation of the monocyte compartment. To determine whether METH can directly modulate macrophage polarization, bone marrow-derived macrophages (BMDMs) were treated in vitro (purity >98%, Supplementary Fig. 1I). At 100 μM, METH did not induce polarization by itself but potentiated LPS/IFN-γ-driven M1 polarization (Supplementary Fig. 1J). At higher concentrations (200–800 μM), METH directly increased the proportion of F4/80⁺CD86⁺ M1 macrophages in a dose-dependent manner (Fig. 1L–N), demonstrating a direct pro-polarizing effect under these conditions. Finally, to test the functional contribution of peripheral monocyte-macrophages to behavior, we depleted these cells in vivo using clodronate liposomes (CL). Flow cytometry confirmed efficient depletion of F4/80⁺ cells (Supplementary Fig. 1K). CL pretreatment markedly attenuated METH-induced immobility in both TST and FST (Fig. 1P–Q) without altering locomotion (Fig. 1R). Collectively, these results indicate that peripheral monocyte-derived macrophages are necessary mediators of METH-induced depressive-like behavior. 2. METH exposure induces peripheral monocyte-derived macrophages infiltration into the CNS, exhibiting characteristic CCR2 expression. We next examined whether peripheral monocyte-derived cells infiltrate the CNS following METH exposure. Immunofluorescent co-labeling of CD11b and CD45 in mPFC on day 4 revealed a clear increase in CD11b⁺CD45⁺ cells consistent with peripherally derived macrophages (Fig. 2A–B). Comparable, though less pronounced, infiltration was detectable in the amygdala (Supplementary Fig. 2A), while hippocampal CA1 and CA3 regions showed no evident co-localized CD11b⁺CD45⁺ macrophages (Supplementary Fig. 2B–C). Infiltration of CCR2⁺F4/80⁺ macrophages in mPFC was confirmed by immunofluorescence (Fig. 2C). These data imply regionally selective recruitment of peripheral macrophages to limbic circuits, with the mPFC as a principal locus of invasion. To dissect transcriptional signatures, we performed single-cell RNA sequencing (scRNA-seq) of mPFC tissue on day 4. Analysis revealed 33 clusters, annotated into 11 major cell types including microglia, astrocytes, macrophages, oligodendrocytes, neurons, and others (Fig. 2D–G). Macrophages were further subdivided into 13 subclusters (Fig. 2H–I). Among these, subclusters 2, 5, and 8 were expanded after METH (Fig. 2J-K) and expressed distinct gene sets: subcluster 2 (Adgre4, Gngt2, Pparg, Fam49a), subcluster 5 (S100a8, S100a9, Retnlg, G0s2), and subcluster 8 (Chil3, Fn1, Thbs1, Plac8) (Supplementary Fig. 2D). Subcluster 8 exhibited high Ly6C expression (loge FC = 2.1, padj = 1.1×e⁻²²¹). CCR2 was enriched in subclusters 2, 3, 7, and 8 (subcluster 2：log e FC=0.025， P adj= 0.004；subcluster 3：log e FC=1.3， P adj=2.8e -120 ；subcluster 7：log e FC=1.1， P adj= 1.6e -63 ；subcluster 8：log e FC=1.8， P adj= 6.3e -80 ), and CD44 was widely expressed across multiple subclusters (Fig. 2K), suggesting heterogeneous infiltration states. Ly6C⁺CCR2⁺CD44⁺ (subcluster 8) and Ly6C⁻CCR2⁺CD44⁺ (subclusters 2,3,7) signatures were identified. 3. CCR2 blockade ameliorates METH-induced behavioral and inflammatory outcomes Given the CCR2 enrichment among infiltrating macrophage subsets, we tested the functional role of CCR2 signaling. Pharmacological blockade of CCR2 with RS504393 substantially inhibited METH-induced expansion of circulating CD11b⁺L y 6C hi inflammatory monocytes (Fig. 3A–C) and significantly reduced immobility in both TST and FST (Fig. 3D–E) without altering locomotor activity (Fig. 3F). Complementary genetic experiments using CCR2 knockout (CCR2KO) mice provided convergent evidence: CCR2KO animals failed to mount METH-induced increases in total or inflammatory monocytes (Fig. 3H–I; Supplementary Fig. 3A–B) and did not exhibit METH-associated prolongation of immobility observed in wild-type counterparts (Fig. 3J–K). Locomotor performance remained comparable across genotypes (Fig. 3L), indicating that the protective effect of CCR2 loss was not due to altered activity levels. Notably, METH-induced hyperthermia was unaffected by CCR2 inhibition (Supplementary Fig. 3C–D), indicating that thermoregulatory responses are mechanistically separable from the CCR2-dependent behavioral sequelae. Beyond affective endpoints, CCR2KO mice were also protected from METH-induced cognitive deficits, as evidenced by improved performance in the novel object recognition and Barnes maze tasks (Supplementary Fig. 3E–F). Immunofluorescent analysis further demonstrated that CCR2 deficiency prevented METH-induced accumulation of CD45 hi CD11b⁺ and F4/80⁺CCR2⁺ cells within the mPFC (Fig. 3M–N). Together, these observations indicate that CCR2-dependent monocyte recruitment is required for METH-driven neuroinflammation and associated behavioral dysfunction. 4. Choroid plexus and meninges act as portals for macrophage infiltration To delineate anatomical routes of entry for peripheral macrophages, we evaluated immune surveillance structures—chiefly the choroid plexus and meningeal compartments—using high-throughput cytokine assays and histological assessment. METH markedly increased the number of CD11b⁺ cells in choroid plexus (Fig. 4A). Cytokine profiling identified 21 differentially upregulated analytes in the choroid plexus; among these, CCL2, CCL5 and ICAM-1 (implicated in chemotaxis and leukocyte adhesion) and GM-CSF (a driver of monocyte differentiation) were significantly elevated (Fig. 4B; Supplementary Fig. 4A). Although 19 cytokines did not reach statistical significance, the heatmap indicated a trend toward higher overall expression in the METH group (Supplementary Fig. 4C). The meninges likewise exhibited elevation of 12 cytokines, including CCL2 and GM-CSF (Fig. 4D; Supplementary Fig. 4B), while 28 analytes remained unchanged (Supplementary Fig. 4D). Correlation analysis revealed strong co-expression between CCL2/GM-CSF and other differential cytokines in both compartments (r > 0.7; Fig. 4C, E). Functional enrichment analysis of the choroid plexus-derived cytokines highlighted biological processes such as monocyte proliferation and leukocyte differentiation, myeloid cell migration and CCR chemokine receptor binding (Fig. 4F). KEGG pathway mapping underscored TNF signaling, Th17 differentiation, Th1/Th2 differentiation, T cell receptor signaling, JAK–STAT, IL-17 signaling and cytokine–cytokine receptor interaction among the top pathways (Fig. 4G). Comparable GO and KEGG enrichments were observed for meningeal cytokines, emphasizing myeloid leukocyte migration, monocyte chemotaxis, CCR2 binding and proinflammatory signaling cascades (Fig. 4H–I). Consistent with a CCR2-dependent entry mechanism, CCR2KO mice exhibited a significant reduction in CD11b⁺ cell accumulation within the choroid plexus after METH (Fig. 4J). Taking together, these data indicate that the choroid plexus and meninges constitute permissive portals for CCR2⁺ monocyte/macrophage ingress into the CNS following METH exposure. 5. Neuronal CCL2 drives CCR2⁺ macrophage infiltration and is feedback-regulated Ly6C hi bone marrow monocytes require CCR2 signaling to egress into the circulation and to home to sites of tissue damage in response to chemokine gradients. Given the central role of CCL2 as the principal CCR2 ligand, we interrogated central CCL2 dynamics after METH. ELISA quantification revealed a transient elevation of CCL2 in the mPFC at days 1 and 4 post-METH, with levels returning to baseline by day 7 (Fig. 5A). Immunofluorescent co-labeling demonstrated predominant colocalization of CCL2 with NeuN⁺ neuronal somata rather than glial markers (Fig. 5B), indicating that neurons are a major source of early CCL2 production. To test whether peripheral monocyte presence influences this chemokine response, we examined CCL2 levels following interventions that disrupt CCR2 signaling or monocyte availability. Monocyte depletion (CL), pharmacological CCR2 antagonism (RS504393), or genetic CCR2 ablation all prevented the METH-associated rise in peripheral blood CCL2 (Fig. 5C–E). Furthermore, CCR2 blockade inhibited the METH-induced increase in mPFC CCL2 protein and attenuated neuronal CCL2 immunoreactivity (Fig. 5F–G). These observations support a model in which METH-triggered neuronal CCL2 initiates CCR2⁺ monocyte recruitment into the CNS, and infiltrating CCR2⁺ macrophages subsequently amplifying CCL2 expression, establishing a positive feedback loop that sustains neuroinflammation. 6. CCR2⁺ macrophage infiltration activates microglia and induces neuronal injury Finally, we examined downstream inflammatory mediators, glial responses and neuronal integrity within the mPFC. scRNA-seq analysis revealed high expression of the proinflammatory cytokine IL-1β in peripheral-derived monocyte/macrophage subpopulations (subclusters 2, 3, 7 and 8), with detectable expression in other subsets except 0, 4, and 6 (Fig. 6A). This pattern indicates that infiltrating peripheral cells adopt a strongly proinflammatory profile; resident macrophage populations may also be activated to an M1-like phenotype in response to METH. qRT-PCR confirmed elevated IL-1β transcript levels in the mPFC on day 4; notably, CCR2 knockout normalized IL-1β expression (Fig. 6B). Microglia exhibited the largest transcriptional remodeling among CNS cell types (Supplementary Fig. 6A–B), with upregulation of ribosomal genes and IL-1α (Supplementary Fig. 6C–D). Microglial subclusters expressed proinflammatory and proliferation-linked genes including TNF and CSF1R, and IL-1α was enriched in microglia while IL-1β remained macrophage-specific (Supplementary Fig. 6E). GO and KEGG analyses of microglial changes pointed to enrichment of ribosome biogenesis, oxidative phosphorylation, TNF signaling and Toll-like receptor pathways (Supplementary Fig. 6F–G), consistent with a shift toward a proinflammatory, high-metabolic state that may mediate neuroinflammation via IL-1α. Morphologically, microglia in the mPFC transitioned from a resting, ramified phenotype to activated states at day 4 post-METH: the proportion of ramified cells decreased while intermediate and amoeboid morphologies increased. Importantly, CCR2KO restored microglial morphology toward baseline (Fig. 6C–D), indicating that CCR2-dependent infiltration is upstream of microglial activation. Concomitant with glial activation, neuronal pathology was evident in the mPFC on day 4 following METH exposure. Hematoxylin-and-eosin staining revealed enlarged neuronal soma and a reduced neuronal count, both of which were attenuated in CCR2KO mice treated with METH (Fig. 6E–F). Consistent with these findings, Nissl staining showed a reduction and pallor of Nissl bodies after METH exposure, an effect that was also ameliorated in CCR2KO mice administered METH (Fig. 6G). Ultrastructural analysis by transmission electron microscopy demonstrated synaptic abnormalities after METH: synaptic cleft coupling, fusion of pre- and post-synaptic membranes, focal low-electron-density edematous zones, and adjacent mitochondrial swelling and damage—features that were not observed in CCR2KO mice exposed to METH (Fig. 6H). At the protein level, PSD95 expression-assessed by Western blot-was reduced after METH exposure but was significantly preserved in CCR2KO mice (Fig. 6I–J). Collectively, these data indicate that CCR2⁺ macrophage infiltration precipitates microglial activation, drives a neuroinflammatory cascade, and contributes to synaptic degeneration and neuronal loss in the mPFC, thereby linking peripheral immune recruitment to METH-induced neuropathology and behavioral deficits. Discussion The present study identifies CCR2⁺ peripheral monocytes as critical mediators of METH-induced depressive-like behaviors and delineates the cellular and molecular mechanisms through which these immune cells interact with CNS compartments. By integrating behavioral assays, immunological profiling, transcriptomic analyses, and genetic interventions, we demonstrate that binge METH exposure elicits a systemic myeloid response, promotes CCR2-dependent infiltration of macrophages into CNS—particularly the mPFC—and establishes a neuroinflammatory feedback loop that disrupts synaptic integrity and drives emotional pathology. These findings extend previous models of METH neurotoxicity by highlighting a central role for peripheral immune recruitment in sustaining affective disturbances during withdrawal. Our findings highlight the critical role of the CCL2/CCR2 axis in linking peripheral inflammatory responses to central nervous system (CNS) pathology. CCR2 (C-C chemokine receptor type 2), as the primary receptor for CCL2 (MCP-1), serves as a key regulator of peripheral monocyte migration and infiltration(19, 20). Under homeostatic conditions, CCR2-expressing monocyte-derived macrophages support essential immune surveillance and contribute to tissue repair mechanisms within the central nervous system (CNS). In diverse pathological contexts, however, dysregulated activation and subsequent CNS invasion of these cells can initiate and sustain detrimental neuroinflammatory cascades(21, 22). In experimental models of ischemic stroke, for example, CCR2⁺ macrophages amplify blood–brain barrier injury via robust release of reactive oxygen species and matrix metalloproteinases(23). Similarly, in experimental autoimmune encephalomyelitis (EAE), inflammatory CCR2⁺monocytes-secreted IL-1β mediate direct cytotoxic effects on oligodendrocytes, thereby suppressing remyelination and exacerbating demyelination(24). Of therapeutic relevance, genetic ablation of CCR2 significantly attenuates monocyte trafficking into the CNS and confers protection across multiple neurodegenerative disease models(25, 26). These collective findings underscore the translational potential of modulating CCR2 signalling to re-establish neuroimmune homeostasis and alter disease trajectories in neuroinflammatory and degenerative disorders. Elevated levels of circulating cytokines and chemokines, including CCL2, IL-6, and TNF-α, following METH exposure created a chemotactic environment favorable for monocyte trafficking. These mechanisms may extend to psychiatric disorders such as major depressive disorder (MDD), where increased levels of CCL2, IL-6, IL-12, TNF-α, and IL-1β have been consistently observed. Although not exclusively secreted by M1 macrophages, these inflammatory mediators are closely associated with monocyte activation and M1 polarization(27, 28). Supporting this, macrophages derived from patients with bipolar disorder show enhanced production of IL-1β and TNF-α upon stimulation, underscoring the involvement of monocytes in mood disorders via inflammatory pathways(29). Additionally, patients with Crohn’s disease and comorbid depression exhibit elevated M1 macrophage activity, which appears to facilitate depression through pro-inflammatory cytokine signaling(30, 31). These observations emphasize the central contribution of monocyte-derived macrophages and their inflammatory mediators in the pathogenesis of depression, suggesting common immuno-pathogenic pathways across substance-induced and primary psychiatric conditions. Furthermore, scRNA-seq provided evidence that infiltrating monocyte-derived macrophages retain CCR2 expression and undergo dynamic phenotypic reprogramming within the central nervous system (CNS) microenvironment. CD44 expression profiling further demonstrates its specific expression in infiltrating cells, being absent in resident myeloid cells(32, 33). Based on these distinct molecular features, the Ly6C⁺CCR2⁺CD44⁺ macrophage subset (Subset 8) is identified as recently infiltrated inflammatory monocytes of peripheral origin that have not yet fully differentiated. In contrast, subsets in Clusters 2, 3, and 7 (Ly6C⁻CCR2⁺CD44⁺) appear to represent monocyte-derived macrophages that have undergone functional polarization within the CNS—consistent with Ly6C phenotypic evolution observed in experimental autoimmune encephalomyelitis models(34). In the present study, we demonstrate that mPFC neurons themselves become a key source of CCL2 upon METH exposure, orchestrating the early infiltration of peripheral monocytes into the brain—a role traditionally attributed almost exclusively to activated microglia(35). This aligns with recent evidence indicating that substance use disorders, such as opioid abuse(36), similarly upregulate neuron derived CCL2 and drive neuroinflammatory processes via immune cell infiltration. Notably, the infiltration of monocyte-derived macrophages was region-specific, with the most pronounced accumulation occurring in the mPFC region and neuronal CCL2 expression coincided closely with the trajectory of macrophage infiltration. This finding reframes the conventional unidirectional “immune-to-neuron” paradigm, suggesting region-selective neuronally driven initiation of central immune recruitment may contribute to affective dysregulation. Notably, neuronal production of CCL2 was an early and transient response to METH, but the subsequent amplification of CCL2 expression required CCR2⁺ macrophage infiltration, revealing a self-reinforcing feed-forward loop. Such feedback dynamics may explain the persistence of depressive-like behaviors beyond the acute phase of drug exposure. The current work also has broader implications for the understanding of depression comorbid with substance use disorders. Clinical studies have consistently linked CCL2 to depressive disorder(17), and our results suggest that psychostimulant induced CCL2/CCR2 activation may represent a convergent pathway through which drug exposure and stress converge to exacerbate affective pathology. The choroid plexus and meninges, which are central to cerebrospinal fluid dynamics, constitute critical gateways for the entry of peripheral immune cells into the central nervous system (CNS). Emerging evidence indicates that these compartments form specialized immunogenic niches that permit immune cells to actively participate in brain regulation(37). METH exposure triggers marked upregulation of CCL2, CCL12, IL-1β, TNF-α, and GM-CSF within the meninges and choroid plexus. Functional enrichment analyses reveal significant association with biological processes such as “monocyte proliferation” and “CCR2 chemokine receptor binding”, indicating active recruitment and differentiation of peripheral monocyte-derived macrophages in these compartments. The identification of the choroid plexus and meninges as key entry portals expands current understanding of neuroimmune communication in addiction, consistent with emerging evidence that these border tissues regulate immune surveillance and leukocyte entry into the brain. During inflammation, they markedly upregulate secretion of CCL2, CCL12, macrophage colony-stimulating factor (M-CSF), and granulocyte–macrophage colony-stimulating factor (GM-CSF), establishing a chemotactic gradient that facilitates the recruitment of CCR2⁺ monocytes(38-40). Notably, M-CSF and GM-CSF not only promote monocyte-to-macrophage differentiation but also enhance cell survival and drive a pro-inflammatory M1-like polarization via activation of the STAT3 and STAT5 signalling pathways(41, 42). Similarly, within meningeal perivascular spaces, fibroblasts and tissue-resident macrophages are capable of secreting substantial quantities of chemokines, thereby providing navigational cues that guide the transmigration of peripheral monocyte-derived macrophages across CNS barrier structures(43). This coordinated triad of “chemotaxis–survival–polarization” is likely a central driving mechanism underlying METH-induced neuroinflammation. Moreover, the delineation of choroid plexus and meningeal portals as immune entry routes opens new avenues for exploring how systemic inflammatory states may synergize with drug use to worsen psychiatric outcomes. The functional impact of infiltrating CCR2⁺ macrophages was multifaceted. scRNA-seq revealed that these cells adopt transcriptional profiles enriched for proinflammatory mediators such as IL-1β. Together, these changes promoted a proinflammatory milieu that was accompanied by microglial morphological activation, neuronal injury, synaptic ultrastructural abnormalities, and PSD95 loss in the mPFC. Importantly, depletion of monocytes or disruption of CCR2 signaling prevented these outcomes and normalized both behavior and neuropathology. These findings provide causal evidence that peripheral immune recruitment is not merely correlative but necessary for METH-driven neuroinflammation and depressive phenotypes. Our study positions the CCL2/CCR2 axis as a tractable therapeutic target for comorbid depression in the context of substance abuse. Several limitations warrant consideration. First, our experiments were restricted to male mice, and sex-dependent differences in immune responses may limit the generalizability of these findings. Second, while our study highlights macrophage–microglia crosstalk, the potential involvement of other immune subsets, such as Th17 cells previously implicated in METH-induced pathology, remains incompletely defined. Third, although we focused on the mPFC as the primary locus of infiltration, other regions such as the amygdala may contribute to a circuit-specific manner to the observed behavioral alterations. Future investigations employing cell type–specific manipulations, longitudinal imaging, and circuit-level analyses will be essential to determine how infiltrating immune cells functionally integrate into neural networks over time. Finally, although we identified the choroid plexus and meninges as portals for macrophage entry, the precise mechanisms by which immune cells traverse these interfaces and subsequently accumulate within specific regions such as the mPFC remain to be elucidated. In conclusion, our findings provide mechanistic insight into how binge METH exposure couples systemic immune activation with CNS inflammation through a CCR2-dependent pathway. By recruiting proinflammatory monocytes into mPFC, METH establishes a pathological feedback loop that drives microglial activation, neuronal injury, and synaptic degeneration, culminating in depressive-like behaviors. Targeting the CCL2/CCR2 axis thus offers a promising strategy for mitigating the neuropsychiatric consequences of psychostimulant abuse and for addressing the broader challenge of substance use comorbid with affective disorders. Declarations Acknowledgements This research was funded by the Key Project of National Natural Science Foundation of China (82030057 to CM), the General Project of National Natural Science Foundation of China (82371899 to DW), the Hebei Medical University Postdoctoral Funding Project (30705010078 to RH). Declarations Ethics approval and consent to participate The animal experiments were conducted following the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Local Animal Use Committee of Hebei Medical University (approval no., IACUC-Hebmu-P2020072). Availability of data and materials The scRNA-seq data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Other raw data and statistical results are provided in Supplementary Material. Competing interests The authors declare no competing interests. References Lewis D, Kenneally M, van denHeuvel C, Byard RW. Methamphetamine deaths: Changing trends and diagnostic issues. Medicine, science, and the law. 2021;61(2):130-7. Leung J, Mekonen T, Wang X, Arunogiri S, Degenhardt L, McKetin R. 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Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files Rawdata.pdf Raw data SupplementaryMaterial.docx Supplementary Material Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 19 Dec, 2025 Review # 3 received at journal 19 Nov, 2025 Review # 2 received at journal 15 Nov, 2025 Reviewer # 3 agreed at journal 12 Nov, 2025 Review # 1 received at journal 08 Nov, 2025 Reviewer # 2 agreed at journal 05 Nov, 2025 Reviewer # 1 agreed at journal 05 Nov, 2025 Reviewers invited by journal 05 Nov, 2025 Editor assigned by journal 30 Sep, 2025 Submission checks completed at journal 30 Sep, 2025 First submitted to journal 29 Sep, 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. 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16:46:48","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4613827,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/7479d657a91d90bb6cd74994.docx"},{"id":96248959,"identity":"9fea055b-2ac2-4df5-947a-486243627c84","added_by":"auto","created_at":"2025-11-19 07:29:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2364255,"visible":true,"origin":"","legend":"\u003cp\u003ePeripheral monocyte-derived macrophages contribute to METH-induced depression-like behavior.\u003c/p\u003e\n\u003cp\u003eA: The timeline diagram for METH exposure and depression-like behavioral tests. Created with BioRender.com. B-D: The behavioral tests including tail suspension test (TST), forced swimming test (FST) and locomotion test (LMT) were performed 4, 7 days or 14 days after METH treatments. n=10 per group. E: On the fourth day of withdrawal, peripheral blood was collected for cytokine protein chip detection. In principal component analysis, the red represents the saline subjects (n=6), the blue represents the METH treatment subjects (n=6). F: Cluster analysis of differential cytokines in each group. G: Enrichment of peripheral cytokines and chemokines in GO-BP analysis after METH exposure. H: Enrichment of peripheral cytokines and chemokines in GO-MF analysis after METH exposure. I: Flowchart of Flow Cytometry detection for peripheral blood monocytes polarization in METH-stimulated mice. Created with BioRender.com. J: Representative flow cytometry images. K: The proportion of CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi \u003c/sup\u003elabeled inflammatory monocytes in peripheral blood was measured on the 1st, 4th, and 7th days after METH exposure (n=6-10). L: Schematic diagram of BMDM polarization induced by METH. Created with BioRender.com. M: Representative flow images. N: Proportion of F4/80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e-labeled M1 macrophages in BMDMs stimulated with 100-800 μM METH for 48 h (n=3-6). O: Two doses of CL (0.15 mL/10 g) were injected intraperitoneally 4 d before and 1 d before METH exposure, followed by METH injection (10 mg/kg, 4 times/d), and on day 7, mice were tested for depression-like behaviors. Created with BioRender.com. The effects of CL on the immobility time in the TST (P) and FST (Q), as well as on general mobility (R), were measured in METH-treated mice (n = 10). Data are presented as mean±SEM.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/1a39152a8795ebe9d25aef3a.png"},{"id":96108753,"identity":"4522031e-6fcd-416a-b8ba-52d7de93af86","added_by":"auto","created_at":"2025-11-17 16:46:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3645714,"visible":true,"origin":"","legend":"\u003cp\u003eMETH exposure induces peripheral monocyte-derived macrophages infiltration into the CNS, exhibiting characteristic CCR2 expression.\u003c/p\u003e\n\u003cp\u003eA: Schematic diagram of immunofluorescence for infiltrating macrophages in the medial prefrontal cortex (mPFC). Created with BioRender.com. Representative immunofluorescence images of co-localized CD11b\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003e macrophages (B) and F4/80\u003csup\u003e+\u003c/sup\u003eCCR2\u003csup\u003e+\u003c/sup\u003e macrophages (C) in the mPFC at day 4 after METH exposure; scale bar = 25 μm. D: Schematic illustration of cell type identification in the mouse mPFC based on Single-Cell RNA Sequencing. Created with BioRender.com. E: 2D visualized UMAP plots of scRNA-seq from eight mPFC mice (METH and Saline groups, n=4 each). A total of 121283 cells were divided into 33 clusters by PCA. Each color represents a cluster. F: The clusters were identified as 11 distinct cell types based on the expression of cell type-specific markers. G: The expression of each cell type-specific marker in the cluster is highlighted in red. H: Characteristics of macrophage subsets, featuring a heatmap of differential hypervariable genes and classification into 13 subpopulations based on specific marker expression. I: Two-dimensional visualization t-SNE plot of macrophage subsets. J: t-SNE visualization of macrophage subpopulation distribution in the Saline and METH groups. Subgroups 2, 5, and 8 were increased in the METH group. K: The distribution proportion of each macrophage subgroup in the saline group and the METH group. L: Distribution characteristics of Ly6C, CCR2, CD44 genes in different clusters of macrophages.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/dc5f23df2522948449b9a6d0.png"},{"id":96108757,"identity":"a2e04041-39dd-4bdc-9525-f0dcb4dcd24d","added_by":"auto","created_at":"2025-11-17 16:46:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2118270,"visible":true,"origin":"","legend":"\u003cp\u003eCCR2 blockade ameliorates METH-induced behavioral and inflammatory outcomes. A: Schematic for CCR2-specific antagonist (RS 504393) treatment in mice during METH exposure. Created with BioRender.com. B: Representative flow images of CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e labeled inflammatory monocytes. C: Proportion of CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e inflammatory monocytes in peripheral blood, n = 4-5 per group. D-F: Depression-like behaviors in CCR2-blocked mice were assessed using tail suspension test (TST), forced swim test (FST), and locomotor activity test (LMT) (n = 8-9 per group). G: Schematic for CCR2 knockout mice during METH exposure. Created with BioRender.com. H: Representative flow images of CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e labeled inflammatory monocytes. I: proportion of CD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e inflammatory monocytes in peripheral blood, n = 4-6 per group. Data are presented as mean±SEM. J-L: Depression-like behaviors in CCR2KO mice were assessed using TST, FST, and LMT (n = 9-10 per group). M: Representative images of immunofluorescent labeled CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e macrophages in the mPFC. N: Representative images of F4/80\u003csup\u003e+\u003c/sup\u003eCCR2\u003csup\u003e+\u003c/sup\u003e macrophages labeled with immunofluorescence in the mPFC. Scale bar=50 μm. Data are presented as mean±SEM.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/f63444cf39da2e54f270f056.png"},{"id":96249027,"identity":"e9db0d85-8f3f-49c8-b037-e36e49e15c8f","added_by":"auto","created_at":"2025-11-19 07:29:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2806544,"visible":true,"origin":"","legend":"\u003cp\u003eChoroid plexus and meninges act as portals for macrophage infiltration. A: Representative immunohistochemical images showing CD11b\u003csup\u003e+\u003c/sup\u003e cells in the choroid plexus region. scale bar=50 μm. B: Heatmap of differential cytokines in the choroid plexus caused by METH exposure. C: Correlation analysis of differential cytokines in the choroid plexus. D: Heatmap of differential cytokines in the meninges caused by METH exposure. E: Correlation analysis of differential cytokines in the meninges. F: GO analysis of differential cytokines in choroid plexus after METH exposure. G: KEGG pathway analysis of differential cytokines in choroid plexus after METH exposure. H: GO analysis of differential cytokines in meninges after METH exposure. I: KEGG pathway analysis of differential cytokines in meninges after METH exposure. J: Representative images of CD11b\u003csup\u003e+\u003c/sup\u003e positive cells in the choroid plexus labeled by immunofluorescence. Scale bar=50 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/0e04f2d02ab1b94c066629af.png"},{"id":96108760,"identity":"e5fe131a-9618-4721-b1f6-8c066a82e9b0","added_by":"auto","created_at":"2025-11-17 16:46:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3433306,"visible":true,"origin":"","legend":"\u003cp\u003eNeuronal CCL2 drives CCR2⁺macrophage infiltration and is feedback-regulated. A: Changes in CCL2 expression in the mPFC after METH exposure (n=5-8). B: Representative images of immunofluorescence co-labeled neurons and CCL2 in mPFC, scale bar=25 μm. C: Changes in CCL2 concentration in peripheral blood after monocyte depletion (n=6-8). D: Changes in CCL2 concentration in peripheral blood after blocking the CCR2 (n=8). E: CCL2 concentration in peripheral blood in CCR2KO mice (n=8). F: Blockade of the CCR2 reduced CCL2 levels in the mPFC (n=5-6). G: Representative immunofluorescence images of CCL2 following CCR2 blockade, scale bar=25 μm. Data are presented as mean±SEM.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/8819f51478b6fb3ea5d0ea2a.png"},{"id":96108759,"identity":"64e24e58-a7c6-4714-ae90-f18c1411195d","added_by":"auto","created_at":"2025-11-17 16:46:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4916138,"visible":true,"origin":"","legend":"\u003cp\u003eCCR2⁺ macrophage infiltration activates microglia and induces neuronal injury. A: Distribution characteristics of IL-1β in different clusters of macrophages. B: IL-1β mRNA levels were detected by qRT-PCR following CCR2 blockade (n=6). C: Representative immunofluorescence images of Iba-1 in mPFC following CCR2 blockade. Scale bar = 25 μm. D: Statistical plot of the proportion of microglia in different activation states (n=4-6). E: Representative images of H\u0026amp;E staining of the mPFC, scale bar=50 μm. F: Quantification of neuronal counts in the mPFC (n=6). G: Representative images of Nissl staining in mPFC, scale bar=50 μm. H: Representative images of synapses in transmission electron microscopy of mPFC, scale bar=500 nm. I: Representative images of synaptic protein PSD95 expression. J: Statistical plot of changes in synaptic protein PSD95 expression (n=6). Data are presented as mean ±SEM.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/09218febb1ea82456bf926f2.png"},{"id":96256231,"identity":"a3eec2ec-2786-47c7-8e21-8ed340552418","added_by":"auto","created_at":"2025-11-19 07:49:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19012422,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/1072b028-0eab-4153-b10c-4338d866d9f4.pdf"},{"id":96108752,"identity":"06b0220f-c804-463a-a64c-527ed3481a29","added_by":"auto","created_at":"2025-11-17 16:46:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":77287,"visible":true,"origin":"","legend":"Raw data","description":"","filename":"Rawdata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/342f061dd21b106b5d005bd2.pdf"},{"id":96108756,"identity":"cc46c713-271a-4d5d-8e1d-787acd6b3fb5","added_by":"auto","created_at":"2025-11-17 16:46:48","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4613827,"visible":true,"origin":"","legend":"Supplementary Material","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7742867/v1/5cd0e3e5f2e3c76ab4bf5841.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"CCL2/CCR2-mediated monocyte-macrophages infiltration drives methamphetamine-induced depressive-like behaviors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe comorbidity of psychostimulant abuse and affective disorders presents a major challenge in neuroscience. Methamphetamine (METH), one of the most widely abused psychostimulants, is strongly linked to persistent depressive symptoms during withdrawal(1-3), yet the underlying mechanisms remain poorly defined. While early models emphasized monoaminergic dysfunction(4), growing evidence indicates that emotional disturbances induced by METH extend beyond direct dopaminergic and serotonergic toxicity and may involve neuroimmune processes.\u003c/p\u003e\n\u003cp\u003eRecent studies highlight the bidirectional communication between the central nervous system (CNS) and the peripheral immune system in the pathogenesis of depression(5, 6). Interactions among neurons, glial cells, and the peripheral immune system are essential for maintaining key brain functions\u0026mdash;including cognition, social behavior, and learning and memory(7-9). Peripheral immune hyperactivation can significantly increase the risk of neuropsychiatric disorders including depression and schizophrenia by compromising blood\u0026ndash;brain barrier integrity and driving aberrant microglial polarization(10). The macrophage theory of depression, proposed in the early 1990s, attributed depressive pathology primarily to excessive cytokine production by macrophages(11). Under stress or inflammatory stimulation, peripheral monocytes upregulate cytokine secretion, infiltrate the brain, and promote stress susceptibility and depressive-like phenotypes(12). During this process, Ly6C\u003csup\u003ehi\u003c/sup\u003e inflammatory monocytes\u0026mdash;which express high levels of C-C chemokine receptor 2 (CCR2)\u0026mdash;are directionally recruited to injury sites in response to their principal ligand C-C chemokine ligand 2 (CCL2). It can be seen that CCL2-CCR2 is critical for driving monocyte transendothelial migration(13, 14). These infiltrating cells differentiate into macrophages, interact with microglia, and amplify neuroinflammation, thereby altering synaptic integrity in mood-regulating regions such as the medial prefrontal cortex (mPFC) and amygdala(15, 16). Clinical findings further support this mechanism,\u0026nbsp;as elevated peripheral CCL2 levels have been reported in patients with depression(17, 18).\u003c/p\u003e\n\u003cp\u003eDespite this framework, how METH engages the CCL2/CCR2 axis remains unclear. Whether METH directly induces CCL2 release, whether monocyte infiltration is required for METH-related depressive behaviors, and through which anatomical routes these cells access the CNS remain unanswered. Moreover, the distribution, transcriptional states, and functional consequences of infiltrating monocytes within the brain have not been systematically defined. Clarifying these issues is essential to understanding how psychostimulant exposure sustains affective pathology through immune pathways.\u003c/p\u003e\n\u003cp\u003eHere, we established a rodent model of binge METH-induced depressive behavior in male mice to examine whether neuronal CCL2 expression and CCR2-dependent monocyte recruitment contribute to emotional dysregulation. By integrating behavioral paradigms, flow cytometry, single-cell transcriptomics, immunohistochemistry, and genetic manipulation, we systematically evaluated the infiltration routes, transcriptional features, and neuroimmune interactions of peripheral monocytes. We further tested whether pharmacological blockade or genetic deletion of CCR2 could mitigate neuroinflammation, synaptic disruption, and depressive-like phenotypes. This work provides mechanistic insight into METH-associated depression and positions the CCL2/CCR2 axis as a promising therapeutic target for substance abuse comorbid with affective disorders.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and CCR2 knockout (CCR2-KO) mice were obtained from Shanghai Model Organisms Center, Inc. Animals were housed under specific pathogen-free (SPF) conditions. The animal facility was maintained at a constant temperature (22 \u0026plusmn; 1 \u0026deg;C), relative humidity of 60%, and a 12-h light/dark cycle with ad libitum access to food and water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBone marrow-derived macrophage Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow-derived macrophages (BMDMs) were generated from isolated bone marrow cells of mature C57BL/6J mice. Following dissection and disinfection of hind limbs, femurs and tibiae were cleaned of muscle tissue, and marrow was flushed using cold PBS (Thermo Fisher Scientific, Cat#10010023) supplemented with 2% FBS (Thermo Fisher Scientific, Cat#16000-044). The cell suspension was filtered through a 40\u0026nbsp;\u0026mu;m nylon mesh (Falcon, Cat#352340), subjected to erythrocyte lysis (BD, Cat#555899), and cultured in DMEM (Thermo Fisher Scientific, Cat#11965092) high-glucose medium containing 10% FBS and 20 ng/mL M-CSF (ABclonal, Cat#RP01216) at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETH-Induced Depressive-like Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice received intraperitoneal injections of methamphetamine (METH, 10 mg/kg) in a high ambient environment (28 \u0026deg;C). Each animal was injected four times daily at 2-h intervals (0.25 mL per injection). Control mice were administered equal volumes of saline under identical conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTail Suspension Test (TST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the TST, mice were suspended by the distal fifth of the tail using medical adhesive tape affixed to a horizontal bar, allowing free movement without escape for 6 min. The final 5 min were recorded with a video camera and analyzed using EthoVision XT software (Noldus, Netherlands). Immobility duration and active time were quantified, with prolonged immobility considered indicative of depressive-like behavior.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eForced Swim Test (FST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FST was performed in transparent cylindrical tanks (water depth 15 cm, temperature 25 \u0026plusmn; 2 \u0026deg;C). Each mouse was individually placed in water for 6 min, and immobility time was scored during the last 5 min of the trial using video tracking.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLocomotor Activity Test (LMT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpontaneous locomotor activity was assessed in an open-field arena (40 \u0026times; 40 \u0026times; 40 cm, uncovered). Mice were placed in the center and allowed to freely explore for 5 min. The total distance traveled was quantified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSucrose Preference Test (SPT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPT was conducted in single-housed mice. During the adaptation phase, animals had access to two bottles containing 1.5% sucrose solution for 24 h, followed by one sucrose bottle and one water bottle for an additional 24 h. Bottle positions were alternated every 12 h to avoid side bias. Following a 24-h period of food and water deprivation, the test phase was performed over the next 24 h, during which consumption from the sucrose and water bottles was recorded by weighing. Sucrose preference was expressed as the percentage of sucrose intake relative to total fluid consumption.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNovel Object Recognition Test (NORT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NORT consisted of three stages: habituation, familiarization, and testing. During habituation, mice explored the empty chamber for 10 min twice daily. In the familiarization phase, animals were introduced to two identical objects (A+A) placed symmetrically in the chamber for 5 min. Four hours later, one object was replaced by a novel one (B) for short-term memory assessment. At 24 h, object B was replaced with another novel object (C) for long-term memory evaluation. Exploration time directed toward novel (N) versus familiar (F) objects was recorded. The discrimination index was calculated as N/(N+F) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBarnes Maze Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Barnes maze consisted of a circular platform containing 20 holes, only one of which led to a hidden escape box. Mice were acclimated the day prior to testing by being guided to the target box. During training, animals were placed in the central start zone and allowed to search for up to 3 min or until they located the escape box. The maze and escape box were cleaned with 75% ethanol after each trial, and the maze was rotated while keeping the target box spatially constant. Training was conducted twice daily for 4 days. On day 5, the probe test was performed with the escape box removed, and the time spent in the target zone vicinity was recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine array profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the Cytokine array (RayBiotech, Cat#QAM-INF-1), inflammatory factors were detected in mouse serum, choroid plexus, and meninges. Serum samples were collected, centrifuged, and aliquoted. Choroid plexus and meningeal tissues were isolated, lysed with RIPA buffer containing protease inhibitors, centrifuged, and the supernatant was quantified using the BCA assay, with total protein concentration adjusted uniformly to 1 mg/mL. After background subtraction and inter-array normalization, differential protein analysis was performed using the limma package with screening criteria set at |logFC| \u0026gt; log₂ (1.2) and adjusted p-value \u0026lt; 0.05. Principal component analysis (PCA) was applied to evaluate inter-group sample distribution patterns. Differentially expressed proteins were subjected to hierarchical clustering (based on Euclidean distance and complete linkage method) and visualized via heatmap. Further functional enrichment analysis of differential proteins, including GO terms and KEGG pathways, was conducted using Fisher\u0026rsquo;s exact test (screening criteria: Count \u0026ge; 2, p \u0026lt; 0.05). The top 10 enriched terms/pathways ranked by Count were selected, and the enrichment factor (enrich factor) was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-cell RNA sequencing (scRNA-seq)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell RNA sequencing (scRNA-seq) was performed on the medial prefrontal cortex (mPFC) of mice at 4 days after METH or saline exposure. Single-cell suspensions were prepared and quality-controlled to ensure cell count \u0026gt;5\u0026times;10⁵, viability \u0026ge;75%, diameter 5\u0026ndash;40 \u0026mu;m, concentration 700\u0026ndash;1200 cells/\u0026mu;L, and absence of debris. Cells were encapsulated using gel beads-in-emulsion (GEMs) for mRNA capture and reverse transcription. cDNA was amplified, and libraries were constructed through fragmentation, end repair, adapter ligation, and index PCR. Libraries were quality-checked using Agilent 2100 Bioanalyzer (peak size ~400 bp) and quantified via Qubit (\u0026ge;2 nM). Sequencing was conducted on Illumina NovaSeq with PE150. Bioinformatic analysis included raw data QC with fastp, alignment and expression matrix generation with Cell Ranger 3.0, cell filtering based on gene/mitochondrial/ribosomal counts, and doublet removal with DoubletFinder. Dimensionality reduction and clustering were performed using Seurat (LogNormalize, PCA, UMAP). Differential gene expression between clusters was identified with Seurat, and functional enrichment analysis (GO and KEGG) was carried out using clusterProfiler, with results visualized via ggplot2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter METH exposure, peripheral blood samples were collected. Single-cell suspensions were prepared by filtering through a 40 \u0026micro;m nylon mesh (Falcon, Cat#352340), followed by erythrocyte lysis (BD, Cat#555899). Cells were washed and resuspended in staining buffer (BD, Cat#554656).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBMDMs were seeded in 6-well plates at 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well, with medium replenished on day 3. After 7 days of differentiation, BMDM purity was confirmed by flow cytometry based on PE-conjugated anti-mouse CD11b Antibody (Cytek\u0026reg; Biosciences, Cat#50-0112) and FITC-conjugated anti-mouse F4/80 Antibody (TONBO Biosciences, Cat#35-4801) expression. For experimental treatment, mature BMDMs were stimulated with METH for 48 hours prior to subsequent analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 1 \u0026times; 10⁶ cells were incubated with APC-conjugated anti-mouse CD86 Antibody (TONBO Biosciences, Cat#20-0862), APC-conjugated anti-mouse CD11b Antibody (Biolegend, Cat#101212), FITC-conjugated anti-mouse Ly6C Antibody (Biolegend, Cat#128005) , FITC-conjugated anti-mouse NK1.1 Antibody (Cytek\u0026reg; Biosciences, Cat#35-5941), PE-conjugated anti-mouse CD4 Antibody (Cytek\u0026reg; Biosciences, Cat#50-0042) or APC-conjugated anti-mouse CD8 Antibody (Cytek\u0026reg; Biosciences, Cat#25-0081) at 4 \u0026deg;C for 40 min in the dark. After washing, cells were analyzed on a FACSCalibur\u0026trade; flow cytometer (BD Biosciences, New Jersey, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vivo Cell Depletion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo deplete monocyte/macrophages, mice were intraperitoneally injected with 5 mg/mL Clodronate Liposomes (CL, Liposoma B.V., Cat#CP-005-005) solution on day 4 and day 1 prior to METH administration. The CL were stored at 4\u0026nbsp;℃, and the administered concentration was 0.15 mL/10 g body weight. Control mice received intraperitoneal injections of an equal volume of PBS Liposome solution at the same time points. Depletion efficiency was verified by flow cytometry after two injections.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo antagonize CCR2, mice received intravenous injections of 200\u0026nbsp;\u0026mu;L RS504393 working solution (Abmole, Cat#M7224, 0.25 mg/mL) from one day before to six days after METH exposure. The working solution was diluted from a 10 mg/mL DMSO stock stored at\u0026nbsp;\u0026minus;20 \u0026deg;C. Control mice were injected with an equal volume of vehicle solvent. Depletion efficiency was verified by flow cytometry after seven injections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative RT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted with TRIzol reagent (Invitrogen, Cat#15596026CN). cDNA was synthesized using the PrimeScript\u0026trade; RT Reagent Kit with gDNA Eraser (Takara, Cat#RR047A). qPCR was performed with TB Green\u0026reg; Premix Ex Taq\u0026trade; II (Takara, Cat#RR820A). Gene expression was normalized to GAPDH, and primers are listed in\u0026nbsp;\u003cbr\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003ePrimer Sequences used for qRT-PCR\u003c/p\u003e\n\u003cp\u003e\u003cimg 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zRbs/Db9fr+2zqtXr1wNmb/eYDBQp9ORWY0c8JBtg7ynYGvPjTEqy3KtBvX8/FxlWT5Y8/nmzRupZdSAKhj+6+7urrZdY8xajaFtkvLPP/+4eU1sbWPbFxJ/KLA8z2ujFphVkxm/BtLW0oejSjTZtmy2se3xbmPbc/H27dvWGt3JZKKiKFxg31aj6pfv9fW1ytXoG3Zqak5RVZVev35dSzefzxv35SmvFZ+fX9N1D+DHsZ+Z4eR/joRP8fypqVKrLc8m4VCd1WoIzvCJoD+F9y58g7DRMb6fto5PlrzOc7YTlhVFUWNHojiOTZIkrnOSfW07CNl87Gvbu960dJ5KkqQ2T6sOe03b/hZpmjZ2qGrqCGU7Eob7oKBTVtiJ0KyO3+/c5I+K4LMdxnw2v7CTZNgh0ZZjuN9RFNU6aoXLrfDY7DkL9yfLsrWOWtuWjbXpGtz2eK2m68fa5lyYVRkp6NCWpmltPXtuwv0Kt910XLYs/bLw3x8++96xvse1sst1D+DHCe+39n5n38/+fSS8r4b3OSvM097/LHu/Cu9D4bwwHrCfA3galOQzsW8AOzXxAyI/WLAXvRoCoDTo1R6+NsH6dj/+9a9/te6Pv6/hB7saPvx35e9PGBA1Hb/xghF/Co/TeDeRpjR+/n45huu0Ban+1BRYtaXx8wiDn7CsfX452WlToOZPTWXzmGvQTuHx2i9QdmoK9sJybdon05BX0zE27Zflbye8Nv30yWq4tXB907IP9v+nulYec90D+DHCINYEQ0eGX3j9+3fTPcw05Gnv3fb+GW8YqScMvP17j82He8fTaP50BAAA+I35X2bTNK19sQ6DWPsFtqkSIAyM24R52i/a8/nc7UtY+dUkDIzVULGCx6ONMQAA+ONEUeT6fUha6wRblqVrw3t/fy9jjBv15imcnJxIq74luwzVqdWviNp9K8uy1pkf34bAGAAA/FEmk4mSJHGBblNHd38UoKfq3OYH27bj7WNkWaY0TaUtR+3B9giMAQDAH+X+/j6c1cqOWrPtCEDh6BH+ejbYtpP9bYFdh/7U6jcI4jh2Nc94GgTGAAAALfr9vpIk0dHRUbiokR1P3k7bjCe/y9CfvouLC5VlqclkEi7CIxEYAwCAP8rx8bGm06kbP/yhtr32R3qaxih+KpeXl5rNZmtBcK/Xa/2BpW63qzRNNR6Pt67RxmYdYxvQAAAA/CFGo1Ht12CjKNJisdBgMKj9eFZZlup2uyqKwv2iZhzHurm5Ua/Xq7UTbgup2vJs0gl++KNp+5KUJIkLmP385/P5k3YS/NMQGAMAgD9anuc6OzvTYrEIF+EPQ1MKAAAAgBpjAADwJ/ObKNjmFPhzERgDAAAANKUAAAAAlgiMAQAAAAJjAAAAYInAGAAAAPiWwNj/HfDRaKSiKNZ+dSXP81q6oig0Go2k1WDU/rKqqmrrWn4edl0AAADgqe0cGBdFoU6nozRN3e+AHxwcaH9/X58/f3bpRqORjo6OVJalS3d+fq7pdKqqqnRzc6MsyxRFkYwxjb8AU1WV7u7uZIxRWZaaTqf8HjgAAAC+i50D45OTEyVJotPTUzdvOByqLEvd399Lq+B5Op1qPp/XAt6bmxulaaqvX7+6eZt8/frV/dyh/T3wDx8+hMkAAACAb7ZTYFwUhcqy1PHxcbhI3W7XBbFXV1eKoqjxt7pPT08b5zcJ0718+bL2GgAAAHgqOwXGHz9+lCS9ePEiXFRze3urXq8Xzv5mnz9/1snJSTgbAAAA+GY7BcY/2vv37zUcDsPZAAAAwDfbKTC2TRkeaiPc6/We/LfGR6PRk+cJAAAAWDsFxra29urqKlwkrYZg06qDXlmWKooiTKI8z9eGdQuFw7JNJhMdHBzU5gEAAABPaafAWJKyLNN0Ol0LXjudjm5ubqRVAB3Hsfb392vBcZ7nuru729gcoiiK2pjGNoi26xRF0RhwAwAAAN+iY4wx4cyHVFWlKIpq85qymUwmGo/H7nWSJG7kisFgoNls5qX+P2ma6vT0VKPRSNPptLYsiiKaVAAAAODJPSowBgAAAH43OzelAAAAAH5HBMYAAAAAgTEAAACwRGAMAAAAEBgDAAAASwTGAAAAAIExAAAAsERgDAAAABAYAwAAAEsExgAAAACBMQAAALBEYAwAAAAQGAMAAABLBMYAAAAAgfHz6fV66nQ6a9NkMgmT/pKqqlo7rqIoVBSFJKkoirVj9yebzvKXjUYjVVWlPM9raSRpNBrV0tp5kjSZTNa2E6a1wv3L81x5nquqqlo6K89z9Xq9cHajTsPxhfxtbzpeeWXdtm9qOZ7JZLK2zmAwcGns8djyC8vWn2zatuu6szoOn92nTfI8r+VRFEXreyTcVluZhceh1bURarqG8zxvPHePPQdPdU0BAL4PAuNnslgslKap4jiWMUbGGGVZpvF43Phh/ispikJRFCnLMndskrS/v68vX764dGmayhijsiwlSWVZyhijNE1dOhtw2LTGGB0cHCiKIn3+/NnlpVVQdnt769IZY9TpdHR7e+vSzOdzV9ZRFLl0cRy7ACXPc+3v77u0xhjd3d3p6OhIX79+9bb4fy4vL1WWZWPQ5LPn9urqKlwk7Xi81vX1de1vaDKZNB7PeDyuHY8NEm2a29vbtWDPP0f+tWv1er1amfrnOAwAbRm0Xe+TyURHR0dum8YYnZ+fazwe1/LapcwGg0HjNfL+/ftaujzPG6/ho6Oj2jVsPXQOvuc1BQD4fgiMf6DhcChJax/mv5r9/X2laeqOR5JOT081n89rx/b333+7/31v3rxx/x8eHipJEp2enrp5w+FQZVnq/v7ezZtMJirLUovFws3TKsjza936/X5tuXVyciKtgqyjoyNlWVZLe3FxoSzLGoOiqqr0+vVrRVHUGvBad3d3StNU0+k0XCTtcLy++/t7JUmyFtxptW/j8fjB4xmNRoqiSDc3Ny5Nt9utBb17e3vqdrvute/t27eSpNevX4eLpFVeTcviONbl5WU4W0VRaDweaz6f17Z5c3OjNE1rweS2ZTaZTDSbzR68RrQKgLe5hq2HzsH3vKYAAN+RwbNJ09TEcexeJ0liJJmyLGvp7HxJJk1TY4wxcRy7eWVZmrIsjSQTRZExxrjXdrLsNu368/ncZFlmJJkkSUySJC6tnS+ptp9pmtby9Nl1wmPYxO5ruM58Pnf7+JAoimr7/pAsy1xZ+TYdW5s0TU1ZliZN08Y8LZvGHm94XLscr2XPn103LMNtj0fetbWN8NptEsdxa552n+31EkqSZGNZWruU2bbXyK7X8FOdA9+21xQA4PuixviZzWYz1+Zwb29PxphaDVme525+WZbuMfLNzY3iOFaWZep2u+p2u8qyzDUbODw8dI9skyTRaDRSnucaj8eazWY6OzuTMUb9fl+Xl5cyq8fPdv2qqtx8Y4wWi4V75H16eiq/JtF3d3cnrWoIv9XHjx8lSS9evAgX1VRVpbIstbe3Fy7a2YcPHxRFUTh7o/v7e3W7Xb1582bjo+/r62u9efNG3W5XcRyv1QRue7y+q6srDYdD9ft9RVG09ih/m+Ox+/vy5ctw0Xdzd3enfr/vamTD5hS3t7drtbhNti2zXa6RXa/hpzgHoW2vKQDA90Vg/MziOHZtbJselV9eXmo8HqvT6bgP10+fPkmrx//2Q1yrJhjdbldFUagsSxdwT6dT3d7eajgcurah/iNdG/QOh0P3mPn6+roWtJdlWdvWrvxOWYPBIFy8k6YOYt+b3V4n6ERWFIUODg6kVSDVFPBaNtjR6ty1NacIbXu8b9++bXyU/7OpqqoWoCZJ0ticImTbEtupqcOctW2Z5Q2d+77FLufgKa4pAMB3FlYh4/vxH0fbx7fhI+EoikyWZbV5PnvKyrJ06dqaCZiWR+D2EbD/OD1sVrEt+9g4PA6zIc+2phS2TJqOPzwOrZqCbKutjNqas5iWpgF+Mxc7NeXrl7E/+eW0y/GaoKmLP/n7bvdvE1v+4bFt0rQ/oabyMt41Ek4+29Snif+e2KXMFDQJssLrz+5f0zUQ2uUcNOXXVEbbXlMAgO+PGuMfZDgcKo5j1wnM6vV6jZ19rCRJlOe5Pn36pFevXkmS/vrrL1cLvY1+vy9jjObzucbjsYqi0N7e3tooAtuwHaDOz8/DRTsbDoeKokhnZ2fhojVJkrTWwNqhu7bx7t07SdI///wTLmplm5vY5i5Nj76vrq5q6cxq1Aa/JnCX49XqkX+YZ/go//j4WGpoqqBVzeRkMnG1km01nd9awx+6v79f228F+2jLYFOtsHYsszRNNZvN1s5NyHb+DJtEaFVr7dfubnMOvtc1BQD4/giMf6CLiwuVZVkLRF6/fl0bnqooiloAcXx8rMvLS9eMQt7IC/4HeDiGrM9ur9/vK45jaTVixGw2q21rUx6+LMs0m82eJKCyQ1ZtehyuVdnJG3LMGo1GOjg42Lq9aLfbdaNGPHS8k8nEBZ5Wt9tVkiS1LwZtQfnZ2Zmm02lt+bbHa7+8hOxwZla/31eSJDo6Oqqdy6IodH5+7r7I2Gsv3O5gMNgq6NxWnueumYAvTdPadux+j8fjB4Pjbcvs9PRUURRpf39/Y5Bpr4Fw21VV6fDw0F1r256D73FNAQCeSViFjO8jiqLao1LLfzRrmwb4j1abHqmq4VGyfTxspyzLann7j5T9/P3mCOFj4l0fNfvr2ilsYhE+Vg+PwwrLqy2tfQS/S5qm4wjLL0zr5+GvHx7Pv//9b/e//8g8zD98nL7peP1t+McXNtfwl4XnsqlJgWnYbni+mtKEwmOz2/Kvs6YmJE3LwmNq26Zp2C+1nP/wHKmlPMJt+++9x5yDsFzstOs11XS9AgC+j46xzzUBAACAPxhNKQAAAAACYwAAAGCJwBgAAAAgMAYAAACWCIwBAAAAAmMAAABgicAYAAAAIDAGAAAAlgiMAQAAAAJjAAAAYInAGAAAACAwBgAAAJYIjAEAAAACYwAAAGCJwBgAAAAgMAYAAACWCIwBAAAAAmMAAABgicAYAAAAIDAGAAAAlgiMAQAAAAJjAAAAYInAGAAAACAwBgAAAJYIjAEAAAACYwAAAGCJwBgAAAAgMAYAAACWCIwBAAAAAmMAAABgicAYAAAAIDAGAAAAlgiMAQAAAAJjAAAAYInAGAAAACAwBgAAAJYIjAEAAAACYwAAAGCJwPiZ9Ho9dTqdxqmqqjD5T2MwGGgymYSzv0me52tl0FYeVVXVlk0mExVFoaIoanlqta82Xa/Xk6SN+97r9ZTneTi7xt/2aDRSURRr64THUxSFRqNR47JwqqpKk8lkbb4/+YqiqC3L81yTyWTtGhqNRmt52H0KjUaj1mXWtmUbHm9RFI3prMlkosFgUJsX7rs/2W1bT7G9ULh9tRxr07nI83ztXFh5nq/tfyi8Fqqqaj032+QHANiRwbNJ09TEcVybl2WZmc/ntXk/iyRJjCSTpmm46JtkWWayLDPGGDOfz41/GSZJ4srDLrNpzaoMw3nGGBNFUa1sy7I0ktbK27J5P7TcP/Ysy9bm2TIqy9LNi+PYzdvmWNM0dcecZZmJoqiWl83bHrt/vdjt+/OiKKrlYYwxktbmWZJq+xXatmzt/rWVRZMoitaWJ0lSO2Z/O/4xPNX2fHEcr5VTU9nZa+Ghc+Gz+/bQcp/d3yYP5QcA2B01xj/YcDhUv98PZ/8ULi4uFMdxOPtJvHr1KpwlSTo+Pnb/7+/vK01TDYdDN+/09FTz+VyfP39282yN2s3NjZvX7Xa1jGmaXV1dKcsyzWazxhq+k5MTJUmi09NTN284HKosS93f30urGsPpdKr5fK5ut+vS3dzcKE1Tff36VdryWNuugZOTE2lVcz4ej5VlWS3txcWFsizTly9fpFWNY1mWWiwWLo2WkVVj7WKe58qyzP0f2rZsi6LQeDx+sCx8RVHo/PxcknR9fe3m7+3t1fLwvX37Vnri7VmTyUSz2ezBsquqSkdHRw+eC19VVXr9+rWiKNLV1VW4WHmeazabqSzL2vzFYqEkSdau0YfyAwA8Uhgp4/sJa7+SJKktN17tXVirFUWRqyGzNYKSXO2aXcfmGdZaNuXr14rav8ariYvj2MRxXKsh9dM9hbAW1bL73Faz53vMPtlyUkPts92nh2rikiRZq0ncpO1YfWGNsWXPyUOiKGq8rtr410vTetuW7a5lYbxtJ0myVvtshe8Z63tsb9uy2/Zc+NI0NWVZmjRNG/fbvte29VB+AIDHocb4mc1mM9d+8Pb2tras0+loPp/LGKM0TRVFkaqq0mAwUFmWev/+vYwxWiwWWiwWiqJI7969q9XgvXv3TpJ0dnbmatPs/8YYxXGsf/75R1VVaX9/323bGKPT01Plee62c3Z2ptls5tL46b63u7s7aVU7uYlta/zy5ctwUas8z11tbZIkury8rC3/+PGjJOnFixe1+aHb29vGWtjv4cOHD4qiKJxdU1WVyrLU3t5euKhRVVUu7cnJiabTaW35LmX7LWVxfHzcWnPf5qm3t0vZbXMuQvf39+p2u3rz5o3KslxrIz+bzR681n0P5QcAeBwC42cWx7GMMWuPZ/M8VxRF7tHs6empoijS9fW1bm5uFEWRewxsHR4eukfC9kP+06dP0iq4s3ktFgv3v/3w7Xa7ms/n0mpb1tnZmQsU+/3+d2tK8Vh+J8ZNnaiaOjFZd3d3rjyagqQ/xfX1td68eSOtmomopTlFaFPZ+qqGjpOW/+Wk3++7a/1bfI/tNXXua+On8zvMFUWhg4MDafW+i+P4m5o/PHV+AID/Q2D8A/ntNj9//rxWAxa+Dh0fH+vDhw/SKsjJsmyt9tPX6XTWagVDYRvHH8XW3IWBiG1zmSSJbm5uXK2urWG2bFtkrWq57ReCqqo0nU5dAGNrze0XCnk1pE3tVH29Xm+tPer30u12Hzw39hhtG+iHvH//XlEUubJQUI67lG2v11t7umCfZERRpCzLal/ALi8vtb+/77ZdlqW7lrfx1NuzZRfug21XrtV7o9/vu3MRfiGwT2TSNNXFxYWbf3V1paOjI7ft2Wy29rTIPh3axjb5AQAeh8D4J/Hy5cu1D3o7v02/36+t8+rVK1f76a9nh9oyxihJEje/TVPnoedmg5qwljzU7XaVJImm0+lWgcX19bXKsnS19rZM/C8Utva0rRbO1lSfnJy0Psa2Q3c9FVvb2ZSnP0SZLYsmo9HIlZHtiOaXQ5ZltXV3KduzszOpZVizULXqOOZvez6f71Rz/z22l6apZrNZ4/n02eZK//zzT7iolb/tsizXrpu3b9+u7Y81WQ1R6HsoPwCP5w9P6U/+k6BwWEd/aqrUasuzSfhktFoN61kEQ0T6U9O9A48UNjrG99PWkciS13nOdqizoiha6yRmvE5TtqOYfW07rdl87OskSdw2mjqDJashpyytOuw1bfspNO2DZTvghWXmH4Nl99PvrNeUd5iX8crI72xnt920HV/TkFlZlq2tZ1r2J9TW+c5458Y/F/P5fO2YbFn4kiSprReuY9lOnr5ty9buX7h+eO3616evqQPgpvfM99ie7dTqn8/wPWS8Dnjh+nHQWTX1huLzJQ0dAO22w+08Nj8Ajxfei+09z74f/ftKeJ9qu4eHedr3vGXvNf59xW7Xnxfe4+znEJ4GJflM7BvATk3sm8JO9k1nL3o1BKhhr/TwtQnWt/vxr3/9q3V//H0NP+jVEAw8lg1s7NT0gW+8wOyhtGF+/r7am0vT/jelNw3nQy3nzQZJTXlY4b417b9/ntQyIocN2O3UFgyFefnXjZ330HXiC/dfLcfpl7M/Wf615QvzD9OG6a2n3J4Vnk+1lHPT9aGW920Y7DalNw375b/3HpMfgMcJg1gTjFwTfmH17xH+Ml+Yp72f28+DeMPoNGHg7d/TbT68959G86cNAADAb8z/cpumae3LfhjE2i+gTZUaYWDcJszTfrGfz+duX8LKryZhYKyWL+94HNoYAwCAP04URW4oU62GgfSVZena8N7f38sY0/pjTI9hf8Cp3++7DuB//fVXkKqZ3wG3LMtaZ358GwJjAADwR5lMJkqSxAW6TR3doyhyQfNTdW7zg23bcfYxsixTmqbSFiMoYTcExgAA4I+y7bCWktwoNk2jAjUJR4/w17PBtp3sUJG2pniXUaFOT08Vx7GrecbTIDAGAABo0e/3lSSJjo6OwkWN+v1+Lfi1Q4BuYn94qO23CNp+0Ori4kJlWW41dCW2Q2AMAAD+KMfHx5pOp27874fa9tof7Wkao/ipXF5eajabrQXBvV6v9qNBvm63qzRNNR6Pt67RxmYdYxvQAAAA/CFGo1HtR42iKNJisdBgMKj9eFZZlup2uyqKwv1aahzHurm5Ua/Xq7UTbgup2vJs0gl++KNp+1r9oJMNmP385/P5k3YS/NMQGAMAgD9anuc6OzvTYrEIF+EPQ1MKAAAAgBpjAADwJ/ObKNjmFPhzERgDAAAANKUAAAAAlgiMAQAAAAJjAAAAYInAGAAAANg1MB6NRrXf//7f//3f2uvw11q20ev1NBqNwtmaTCbflC8AAACwi50C44uLC2VZpiiKZIzRf/3Xf8kYoziOlaapbm5uwlU26nQ6tV+MsYqi0Hg8dr8zrlWgDAAAAHwvOwXGT80YoyRJwtn6+PGj4jh2r1+/fq37+/taGgAAAOAp/dDAeFv39/c6ODgIZwMAAABP5qcOjG074729PQ2Hw3AxAAAA8GR+6sD49PTUtTGmAx4AAAC+p2cJjHu93jeNMPHmzRvNZjNVVRUuAgAAAJ7Edw2M7TBsi8XCjTCx68gVAAAAwHP4boHxaDT6pg5zs9nM/X99fa04jtXtdmtpAAAAgKfSMbYR7xZGo5Gm06l7/Z///Ef//d//XUvjK8tyYzDb6/XcOMZxHLva5Mlkog8fPtSC4x12EwAAANjZToHxc5lMJrq/v9fFxUW4CAAAAPguvltTCgAAAOBX8lMGxvf399rb2wtnAwAAAN/NT9mUAgAAAHhuP2WNMQAAAPDcCIwBAAAAAmMAAABgicAYAAAAIDAGAAAAlgiMAQAAAAJjAAAAYInAGAAAACAwBgAAAJYIjAEAAAACYwAAAGCJwPiZ9Ho9dTqdtWkymYRJf0lVVa0dV1EUKopCklQUxdqx+1NRFJpMJmvz/emh7eV57rbXlLaqKjdvm/3x+ctGo5GKolCe59KGc2vT+kajUW25nRcK86iqym3P15TfpmtqMBhsXK7geAaDgdSSZ3i+qqra6liesuz8/RoMBmt52Mkeh9V0TYSarrFdr2mfv2zTOQUA/DgExs9ksVgoTVPFcSxjjIwxyrJM4/H4l/9wLIpCURQpyzJ3bJK0v7+vL1++uHRpmsoYo7IsJUllWcoYozRNXbr5fO7KJooil18cxy6IyfO8cXtHR0e17VnX19e1v9Y2+2ODH5vWGKODgwPt7+/r8+fP0iq48/fTz9cPvHq9nm5vb11aY4w6nY5ub29dGhuMhduLoshtzxoMBo35vX//vpbOqqpKs9msdblWwdvh4aHL7+zsTJ1OR/f397V0g8FA4/G4tu3Dw0NNp1OX5inLbttjtcuSJFGSJO71YrGopWu7JqynvKZ3OacAgB+LwPgHGg6HkvTLfzju7+8rTVN3PJJ0enqq+XxeO7a///7b/e978+aN+7/f79eWWScnJ+7/o6OjrbZn3d/fK0mStSBqm/05OTlRkiQ6PT1184bDocqydMHi69ev3TJft9t1yyaTicqyXAvQjDHq9Xru9eHh4YPb0yq/2Wz2YH6+6+trZVmmsizXajO1Cj7jONbFxYWb1+/3ZYypBal5nms2m7lg0FosFkqSxKV9yrLb5ljb8pKkt2/f1l63XRPWU17T255TAMBPwODZpGlq4jh2r5MkMZJMWZa1dHa+JJOmqTHGmDiO3byyLE1ZlkaSiaLIGGPcaztZdpt2/fl8brIsM5JMkiQmSRKX1s6XVNvPNE1refrsOuExbGL3ddM6WZa5YwvnP7Suzx7vfD5vXa9tf+w68/m8Nn+TOI7dOfNFUVQr6ya7bG+b/EI2fRRFa/toyyDLstr8JvZ62mSXY7G+pexC4bXte+ia2PUaM098DQEAfhxqjJ/ZbDZz7Qz39vZkjFG323XL8zx388uy1Hg8VlVVurm5URzHyrJM3W5X3W5XWZa5x/D+4+8kSTQajZTnucbjsWazmc7OzmSMUb/f1+XlpXuca9evqsrNN6tHz7aJx+npqczqUXLo7u5OWtXwPYddt3d1daXhcKh+v68oilofnTf5+PGjJOnFixfhop1UVaWyLLW3txcuqtl2e9vm5yuKQgcHB9Kq9jSsKf306ZMk6a+//qrNbzKbzR4s/22P5SGPOdaHPHRN7HqNbfJU5QAAeB4Exs8sjmP3CLrpMerl5aXG47E6nY6iKJK8oOXk5MR9aGvVBKPb7aooCpVl6QLu6XSq29tbDYdD167Zb6Jgg97hcOgeT19fX9eC9rIsa9vaVVMHrh+tKSD8FfidytqaSWj1pcqm6wSdv2wwqNVj/rbmFKGmDm8/g03HuotdromnvKa3PacAgOdFYPwD2Nre6XS69oG+WCxqHX6MMS6gGQ6HrnNTVVV6+fKlJOnLly+1jmq2xrfN5eWljo6O1PFGxbBtLv08/LambWxNXtNx2A5QNzc3tWXfwm7Pb/PaJs9zTadTF4CMx2OVQaeuTWz5fv36NVy0E1vz2PRFyGe3Z78IWTc3N+4LzmKxcPl9+PChls62W9WqE5j/ZcgvB/uFy9Zmyqsp9udJ0sXFRa0jpCRFUfRgGT512e1yrJtsc0085TW97TkFAPwcCIx/kOFwqDiOa53KtKqVaupAZiVJojzP9enTJ7169UpaBTU2SNiG7VA1n881Ho9VFIX29vYeDHaa2A5F5+fn4aLvwnZqCh9/axUs+0N83d3d1QJ9Y0zjo/M29gvJ1dVVuEha1fptK0mS2ogNPjt013A4VBRFOjs7C5OsSdNUs9lsLXhrkue5G+3DTmma1mpK+/2+4jjWeDyurdvk7du3ms1mjdeLHdLsKctul2N9yDbXxFNe07ucUwDAj0dg/ANdXFyoLMtakPD69WvXrlirWit/OLfj42NdXl66ZhTyRnLwg8JwDFif3Z4NhrTqXT+bzWrb2pSHL8syzWaznYKdx+p2u0rTVOPxuDaGbVVVOjw8dLXcNtgPnZ+fbxX8WbZmPyyLTqezda2hVudaq/V8o9FIBwcH7lxeXl6qLMsHH6+fnp4qiiLt7+8/GDBeXl6u1aienp6qLMva+W7bx5DddlhzbM+H3dZTld0ux7rJLtfEU17T255TAMBPIOyNh+8jiiKjhlEj/JEgbC96f1SKppEZmkYPCEelyLKsdZQJP/+2USnk9bC3o1KEPe5D/rp2Cnvj27z8/Qz5I3C0bdf29reTX07+Nvz8w3WyLNtqf8KyVcPbJkzTNmpDeGxN2zMN10tb2nD//W37+xTuj59/uCzcR3mjo/j866gtTVgu+oay23SsPn+5vS52uSZ84fb0yGva7HBOAQA/Tscsb/4AAADAH42mFAAAAACBMQAAALBEYAwAAAAQGAMAAABLBMYAAAAAgTEAAACwRGAMAAAAEBgDAAAASwTGAAAAAIExAAAAsERgDAAAABAYAwAAAEsExgAAAACBMQAAALBEYAwAAAAQGAMAAABLBMYAAAAAgTEAAACwRGAMAAAAEBgDAAAASwTGAAAAAIExAAAAsERgDAAAABAYAwAAAEsExgAAAACBMQAAALBEYAwAAAAQGAMAAABLBMYAAAAAgTEAAACwRGAMAAAAEBgDAAAASwTGAAAAAIExAAAAsERgDAAAABAYAwAAAEsExgAAAACBMQAAALBEYAwAAAAQGD+fXq+nTqfTOFVVFSb/aQwGA00mk3D2RoPBwB1br9eTpMY88jyvlUNRFLV0k8lkraz8yVdVVW3ZZDJRURQqiqKWziqKYi2PNr1eT3meh7Nr/G2PRiNVVdW4zmg0WjsG/5j9sgunwWDg5bRePlVVaTQa1dIouPZsHk3noyk/P1247/5kz7P1O5/boijW1gmPJyw7AMCvgcD4mSwWC6VpqjiOZYxxU5Zl+vr1a5j8pzAajTSbzcLZG9kAyR7f7e2tOp2OPnz4UEs3mUx0dHSksixd2vPzc43H49oXhfl87sopiiKXNo5jl64oCkVRpCzL3HJJ2t/f15cvX1xevqurK2kVwG1SFIXKstTl5WW4SPKCtjRN3bYPDg4URZE+f/5cSzsYDHR7e1s7/51OR+/fv6+ls8uSJFGSJO71YrGo5TUej2t5HR4eajqd1vLqdDo6PDx0ac7OztTpdHR/f19LNxqNGvMbj8e1dPZ8hdey73c5tzbADs/t/v5+7dxuW3YAgJ8fgfEPNhwO1e/3w9k/hYuLC8VxHM5uZWsrb25u3Lxut7sWOBVFofF4rPl8rm636+bf3NwoTdPaF4W2sjk5OXH/7+/vK01TDYdDN+/09FTz+XwtOPXFcdwaFFlXV1fKskyz2ayxZv/w8FBJkuj09NTNGw6HKsuyFnxOJhPNZrNacKtVEOzXtr5+/bq23Pf27VtpFfDNZjOVZVlbvlgslCSJ28/BYKA4jnVxceHS9Pt9GWNqx5LnuabT6YP57e3t1c6Xz+7b73RuT05OHjy325YdAOAXYfBs0jQ1cRy710mS1JabZQTpprIs3fwoikyapkaSiaLIRFFkJJkkSUxZlm4dm2ccxy6PLMsa853P50aSyzdNU2NW+ynJxHFs4jh2881q//zXvk3LfEmSmCiKwtkbZVnWuI49Nr+sHpJlmZnP527dTWx5SjJZltWW2fKbz+e1+U2iKGo835skSdK4jj0vm9hrItznJtvkFwqvZetPO7ePKTsAwM+LGuNnNpvNXDvE29vb2rJOp+MeL6dpqiiKVFWVBoOByrLU+/fvZVaP1BeLhaIo0rt372q1su/evZMknZ2duVo7+79ZPab+559/VFWV9vf33baNMTo9PVWe5247Z2dna00pbLqQbe/58uXLcNGa29vbtTapj3V3dyetaqa3dXd3p36/72oh2x6553mu4+NjSVKSJGs1kB8/fpQkvXjxojY/VFWVyrLU3t5euOhRZrPZg8f76dMnSdJff/0VLlqzTX7b+tPO7VOWHQDgxyMwfmZ+u0w/gMjzXFEUucfLp6eniqJI19fXurm5URRFOj8/93JaPsa/vr6WVsGXvIDo48ePLq/FYuH+tx/i3W5X8/lcWm3LOjs7c0FCv9/fqSlFk6ZOSU1sW107fWvHpaYOZ1ptxw9Qm4IiywZZknR8fNz6yD3U1PmwSVMHte8t7EDXxravtVNbgLmN3+ncbuMpyw4A8LwIjH8gvy3u58+f14Ko8HXo+PjYdWq7vr5WlmWtgYBWNdJh56xQ2FZyW7ZmzdbyWbY9qFa1zd1uV71eb60m2tZ6245WTbXSTWwgFAaVto1nkiS1cr6+vtZ4PHZBy3Q6XdsXrYKs6XTq0tnadfvFQ17tuD9PXnvaOI61WCzcl5GwA6Jtr6pVube1uQ3ZJwmb2JpiW/NpXVxc1Dq7aZVf+PTCtkXWqpOc38Z3k9/t3D7UMfYpyw4A8OM9OjD2a0Q2DU8lr8Yo/DAPa5L8/DYtt5MvHObKbsuvXQrX+Zm8fPmy8UN8U9OEfr9fW+fVq1eu5stfz5aNWY108JC23v6bdLtdJUmi6XS6dp5DZ2dnUsuQYbuyQVZYm97m/v7e1djbSQ2P3K+vr2ujKtiy8794DIdDRVHkjmeTNE01m83WgrzHePv2bWsNpx3KzNb2bzMywvn5ucqyXCuDx/idzq28ES5Ctqb6KcsOwM8hjCfs5A+HGT5986emSq22PJuET8Wq1fCP4dMof2r6PMDj7BwY22A1HMKoaXgqyz7ut38tW5MUx7HLbz6fazqdajQaNS73P/A63uPnm5ubWk2YraXza5fsej8j+0HsfymYzWYP1jbFcazRaKS///5b3W7XtSF+9eqV5OWzbU1wkiQ6Ojpyr2ezmcbj8VYf/Hb0g4dqNPv9vpIk0Xg8fpIAyo4sEI7zG8rzXAcHB+FspWm6Ftx++PBhre3ou3fv1oLby8tLlWXZeCP02aYx+/v73xwc27zCcrZlaWue7flou/law+FQcRzr6Ohoq/O8ye90brMsc/ciX6fTcTXVT1l2AH4OYTzhxyb+fc1+wd40fKXVlGcURbX7s42vwiE2oyjS/f29expln77ZNHEcK4qi2vbwDcLeeA9p611flmXjfOP1rm/qeW5WPbv90QziOK6lDZdbdnQGq613e1vv/udkR5GwUxN/dAm/N74dYaKp93yaprVjDl+bYH27H//6179a98ff17Ds5Y180SZJklrebevYnv/hFPL3XxtGKQjzkTeqgL9P/kgD4Ygd//M//+P+D/fZTxcuC8+vGs6V8Ub88KemUQ385eH5tMJybnqPmIbya0sblkXbtsNjbfK7nNvwPamGYzAN21JL2QH4NTTFE5EX//j30HCUnqb7q2nI09437H1r0yg3/vwoimqfL48ZwQftmu/yLbYdwshnh06y6zaduDD4CoPocLkVXlThRWf9DIExAAD4efhffNM0rcUJYTxhKzWa4p8wMG4T5unHVLsMsRkGxmqpYMHj7NSUYtshjHxXV1fuRyyi1SgLmxRFoel0qsPDw3DRGtvByG8TW5blWtubhzqcAQCAP0sURW4oU62Gm/T58YTtw7BtJ+lt2B8z6vf7Ow2xKUlHR0du38qyrHVExrfZKTBus+3wVG/fvl37+VvL9ibf399f+7WuXfjtd+y0TYczAADwZ5hMJkqSxAW6TR3dbTwhb0jUb+UH22VZbt3/J5RlmdI0lbYYPQe72Skw3nZ4Kitf/VyqvQjG47HKsmy8wPzOddt+87E1xbajGQAAwEPsz7pvYz6fazabbd3BNhw9wl8vrLyzHYGbnoA/5PT0VHEcu5pnPI2dAuNdhqfSakzbsPZ2m+YU2zo7O1Mcx2s9zAEAAJ6CHW3HH7FpEzt6hJ0eGl1Kq3WiKGr9LYK2kXkuLi5UluWTjAKEpZ0CY+0wPFVRFI0/gXt+fr7V2KoPsY8hHtvkAgAA/JmOj481nU7dEI0Pte21scZDsc+3uLy8bByestfrtcY63W5XaZpuPawqthD2xttWOGST35vSH5LK7zkZDuH073//u/Y67FXZNFRSW9q2YZ/C/QQAAAiHvLQjRrTFE34MY2OQbWOMtjyb+Onatq9gaEk//6aRM7C9jrEtywEAAP5AeZ7r7Oys1k8Kf6adm1IAAAAAvyNqjAEAwB+rKArt7+9Lq1EjqDX+sxEYAwAAADSlAAAAAJYIjAEAAAACYwAAAGCJwBgAAAAgMAYAAACWCIwBAAAAAmMAAABgicAYAAAAIDAGAAAAlgiMAQAAAAJjAAAAYInAGAAAACAwBgAAAJYIjAEAAAACYwAAAGCJwBgAAAAgMAYAAACWCIwBAAAAAmMAAABgicAYAAAAIDAGAAAAlgiMAQAAAAJjAAAAYInAGAAAACAwBgAAAJYIjAEAAAACYwAAAGCJwBgAAAAgMAYAAACWCIyfSa/XU6fTWZsmk0mY9JdUVdXaceV5rqIoJGntuMO0vsFg4Jb1ej1J0mg0qqVp2l5RFG57vk35+cvCaTAY1PLJ87y2vCiKtf1ScK4Hg4GqqnLHuMv2JpNJbbmfj2+XspCkoijU6XTC2Y0Gg0HjNn3h8Wq17206q7Kz7P60TeFx+MtGo5GqqlKe57U0Wp1jP62C/drlXDSd+03HCAD4NREYP5PFYqE0TRXHsYwxMsYoyzKNx+PGD/VfSZ7niqJIWZa5Y5Oko6MjffnyRZJqx+2nnc/nur+/d3nZAMamvb29VWcVFFpFUTRub39/323P2iY/uyxJEiVJ4l4vFguXZjQa6ejoSGVZuuXn5+eaTqcuLxugHh4eujRnZ2eKoqh2jNtubzweu2XGGB0eHmo8Hrs02rEsrKurK2l13japqkqz2Uzv378PFzlNx9vpdGrH67PbtPtgpWkqY4zKspQkV85pmrrjsOVr0xpjdHBwoCiK9Pnz51p+g8FAt7e3tfLrdDprx2KXbToXk8mk8dyPx+PadQQA+PURGP9Aw+FQktY+1H81R0dHStPUHY8knZ6eaj6fu2N7/fq1t8b/6ff72tvbk1bBYBRFurm5ccu73a4L9qz9/f0Ht6ct82vbL0l6+/attAo+p9Op5vO5ut2uW35zc6M0TfX161dptb04jnVxceHS9Pt9GWNcALXN9vI813Q6dUGitVgslCRJLRjbtixCcRzr8vIynF1zfX2tLMtUluVara1WwedDxxu6u7tTmqaaTqe1+X///XfttfXmzRv3/+HhoZIk0enpqZs3HA5VlmUtEJ9MJprNZrXgVqsg2D4x0JbnoigKjcfjB889AOA3YfBs0jQ1cRy710mSGEmmLMtaOjtfkknT1BhjTBzHbl5ZlqYsSyPJRFFkjDHutZ0su027/nw+N1mWGUkmSRKTJIlLa+dLqu1nmqa1PH12nfAYNomiyGRZFs6uHW+bXba3TX6+sDz8+bac29jybzquNm3bs+frIbuUhZVlWe0a2MTuWxRFa+X4mOMty9KkaerWnc/nYRK3LDym+Xzeuk4oiqLGct2k7Vxsc+4BAL8Paoyf2Ww2c+0U9/b2ZIyp1UTlee7ml2XpHtfe3NwojmNlWaZut6tut6ssy3R7eyutatP8x8Kj0Uh5nms8Hms2m+ns7EzGGPX7fV1eXsqsHkPb9auqcvPN6lGyfex9enpaq2X13d3dSaua2G9hayRfvnwZLqrZdnvb5reN29vbWk1jk0+fPkmS/vrrr3DRzmaz2YPHpx3Kwnd3d6d+v+9qmNuaUxRFoYODA2lVexo2QXjM8V5fX+vNmzfqdruK43itOcUmHz9+lCS9ePEiXFRTVZXKsnRPIb7VNuceAPD7IDB+ZnEcu0fkTe0wLy8vNR6P1el0FEWR5AUhJycnLhjSqglGt9tVURQqy9IF3NPpVLe3txoOh65dc7/fd+vZoHc4HLrHzdfX17WgvSzL2rZ+dk0dwH4nYQe1toBWG8qiqqpawJgkSWtziqurKxc8v3nzprU5Raipw5t1f3/vgviTk5O15hSP0dSxsklT57nHaursCAD4PRAY/wC2tnc6na59QC8Wi1pHKmOMC1CGw6ELJqqqcrWhX758URRFtXXC9pW+y8tLHR0d1T7U7+/va52PjDG1tqNtbKDV1qZ0W7YmcFO7WHnbayo324Hq5uZm6/y20ev1NpanvJrTtg5vu4iiyNXkW7btriTN53MNh8Oty8K6vr52X7o6qy9Qs9mstq41nU5dOvsFzdbayjtef54kXVxcKMsydz1atp22zfPo6MjN34a91u2XRMu29Y3jWIvFwgXeHz58qKWzbZG16tjnf1HcpNfrrZWRbaduOz36bZ4BAL82AuMfZDgcKo5jnZyc1Ob3er2NwVySJMrzXJ8+fdKrV6+kVZASdtTaxAZZ8/lc4/FYRVFob2/vUcGt7Rx1fX0dLlJVVY3DmTWxj9fDR/aWrfm0Qcj5+XmQom7b/LZxcnLSWmOa57nyPFe/31cURa01sLts7/z8XGVZbqwV1g5lYd3f39e++NjANdxOnueaz+e1dGma1sqy3+8rjuO1UTLaXF1drW17l+YUw+FQURTp7OwsXLQmTVPNZrPG87Uruz1qhQHgz0Bg/ANdXFyoLMta0PT69evaMFBFUdQCl+PjY11eXrpmFFoFKQrG+t0UkNrt2eBGq1EBZrNZbVub8rC63a7SNNV4PK4FD1VV6fDwcKtaZ8uWR/hIfDAY1AKiLMs0m80eDDa3ze8h9kvM/v5+LdjK81x3d3euRv/y8rJxv3q93k7lYLd3dHS0FrSGti2LPM9dm2FfmqZrZXF5eblWo3p6eroWrNtjCptMhNq+cJ2dndWGu3vI5eVl4/kMnZ6eKoqitfP1GP1+X0mSrF3fAIDfVNgbD99HFEXGjvjgF7s/EoTtFe+PStHUI75pNIBwVIosy1pHmfDzbxuVQt7IAHZUinCkAJ8dNWDTfodpmkYBMA1l1TYSgZ9mU9pt8vOXN+278crBTm37H+5TU7lts73wfGxKG6aTd5z++faPPcz/P//5j/s/HBXDL8NwmT9iip3sKBb+NvyRLcLrNU3TtfINr3ErPJ9tacP81LDvZstzEV67dgIA/F46xj5PBQAAAP5gNKUAAAAACIwBAACAJQJjAAAAgMAYAAAAWCIwBgAAAAiMAQAAgCUCYwAAAIDAGAAAAFgiMAYAAAAIjAEAAIAlAmMAAACAwBgAAABYIjAGAAAACIwBAACAJQJjAAAAgMAYAAAAWCIwBgAAAAiMAQAAgCUCYwAAAEBSxxhjJKnT6YTLAAAAgEdZhZi/FBcYAwAAAH8ymlIAAAAABMYAAADAEoExAAAAQGAMAAAALBEYAwAAAATGAAAAwBKBMQAAACDp/wdg+UojUDe7TAAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMedial Prefrontal Cortex (mPFC) was fixed in electron microscopy fixative (Servicebio, Cat#G1102) at 4 \u0026deg;C for 3 h, pre-embedded in 1% agarose, and post-fixed with 1% osmium tetroxide (Ted Pella, Cat#18456). After dehydration in graded ethanol, samples were embedded in epoxy resin (SPI, Cat#90529-77-4). Ultrathin sections were cut (Leica UC7, GER), mounted on copper grids, and stained with uranyl acetate and lead citrate. Images were acquired with a HITACHI HT7800 transmission electron microscope (JPN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrains were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 \u0026micro;m. Sections were stained with hematoxylin and eosin following standard protocols, dehydrated through graded alcohols, cleared in xylene, and mounted with neutral resin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNissl Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSections were processed with a methylene blue-based Nissl staining kit (Solarbio, Cat#G1434) according to manufacturer instructions. Stained sections were differentiated, rinsed, mounted, and examined microscopically.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain tissues were lysed in RIPA buffer (Solarbio, Cat#R0010) containing PMSF and a phosphatase inhibitor mixture (Servicebio, Cat#G2006), and the supernatant was collected. Equal amounts of protein were separated by SDS-PAGE, transferred to NC membranes (Millipore, Cat#HATF00010), and blocked in 5% BSA (Sigma-Aldrich, Cat#V900933). Membranes were incubated overnight at 4 \u0026deg;C with Rabbit Anti-PSD95 Antibody (HUABIO, Cat#\u0026nbsp;ET1602-20, 1:2000) or Rabbit Anti-\u0026beta;-actin Antibody (ABclonal, Cat#AC026, 1:100000), followed by DyLight\u0026trade; 680-conjugated Rabbit IgG Antibody (Rockland, Cat#611-144-002, 1:5000). Protein bands were visualized using the Odyssey Imaging System (LI-COR, Nebraska, USA) and quantified with ImageJ software (NIH, Maryland, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing standard anesthesia, mouse brain tissue was carefully extracted and fixed in 4% PFA for 48\u0026ndash;72 h. After dehydration and embedding, 5 \u0026mu;m sections were prepared. The procedure included dewaxing, rehydration, antigen retrieval via microwave heating in repair solution, peroxidase blocking, donkey serum blocking, incubation with Mouse Anti-CD11b Antibody (HUABIO, Cat#RT1107, 1:200) at 4 \u0026deg;C overnight, HRP-conjugated goat anti-mouse IgG incubation at 37 \u0026deg;C, DAB development (Servicebio, Cat#G1311), and hematoxylin counterstaining before final dehydration and mounting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded brain sections underwent deparaffinization and antigen retrieval, followed by blocking in donkey serum. Sections were incubated with Rabbit anti-CD45 Antibody (ABclonal, Cat#A2115, 1:200), Rat anti-CD11b Antibody, (Thermo Fisher, Cat#\u0026nbsp;14-0112-82, 1:200), Rabbit anti-CCR2 Antibody (Affinity Biosciences, Cat#DF7507, 1:150), Rat anti-F4/80 Antibody (HUABIO, Cat#\u0026nbsp;RT1212, 1:200), Mouse anti-CD11b Antibody (Biolegend, Cat#101201), Rat anti-MCP-1 (CCL2) Antibody(Santa Cruz, Cat#sc-52701, 1:200), Rabbit anti-NeuN Antibody (HUABIO, Cat#\u0026nbsp;ET1602-12, 1:500) or Rabbit anti-Iba-1 Antibody (HUABIO, Cat#\u0026nbsp;ET1705-78, 1:200) overnight at 4 \u0026deg;C, and then with Alexa Fluor\u0026trade; 594 Donkey anti-Rabbit Antibody (Thermo Fisher, Cat#A-21207, 1:500) , Alexa Fluor\u0026trade; 488 Donkey anti-Rat Antibody (Thermo Fisher, Cat#A-21208, 1:500) or iFluor\u0026trade; 647 Conjugated Goat anti-mouse Antibody (HUABIO, Cat#\u0026nbsp;HA1127, 1:500) for 40 minutes. Nuclei were counterstained with DAPI (CST, Cat#8961S). Images were acquired with a laser scanning confocal microscope (Leica, SP8) and analyzed using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse serum or mPFC cells were collected, and mPFC cells were lysed thoroughly using lysis buffer to obtain the supernatant. ELISA kits were used to measure the levels of MCP-1(CCL2) (ABclonal, Cat#RK00381) according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured using a microplate reader (Tecan, Switzerland). Standard curves were generated using known concentrations of the standards, and sample concentrations were calculated using the four-parameter logistic fit based on the absorbance values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as mean \u0026plusmn; SEM. GraphPad Prism (9.3.1) was used for analysis. ANOVA and Student\u0026rsquo;s t test were applied for the statistical analyses. Bonferroni\u0026rsquo;s post hoc correction was used for multiple comparisons. For data that did not meet the normality assumption, the Mann-Whitney U test was used for two-group comparisons, and the Kruskal-Wallis test was used for multi-group comparisons. A p value \u0026lt; 0.05 was considered statistically significant. Raw data and statistical outputs are provided in Supplementary Material.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Peripheral monocyte-derived macrophages contribute to METH-induced depressive-like behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first established a binge METH exposure paradigm in adult male C57BL/6N mice (10 mg/kg, i.p., four injections within one day at 28 \u0026deg;C) and assessed behavioral tests at days 4, 7 and 14 post-exposures. METH-treated animals displayed robust depressive-like phenotypes: immobility times in both TST and FST were significantly prolonged on days 4 and 7 relative to saline controls, with only partial recovery by day 14 (Fig. 1B\u0026ndash;C). Importantly, LMT did not differ between groups at these time points (Fig. 1D), indicating that the observed increases in immobility reflect affective changes rather than generalized motor impairment.\u003c/p\u003e\n\u003cp\u003eTo characterize systemic immune alterations that accompany these behavioral changes, we performed cytokine array profiling of serum obtained on day 4. Principal component analysis demonstrated clear separation between saline and METH groups (Fig. 1E). Of the cytokines assayed, levels of 15 cytokines were significantly elevated following METH exposure, including IL-6, TNF-\u0026alpha;, TNFR I/II, CCL2, CCL12 and ICAM-1 (Fig. 1F; Supplementary Fig. 1A). By contrast, 24 analytes (for example IL-21, IFN-\u0026gamma; and TCA-3) remained unchanged (Supplementary Fig. 1B). Notably, IL-6 and TNF-\u0026alpha; are primary cytokines secreted by monocyte-macrophages; CCL2 and CCL12 mediate monocyte chemotaxis; and ICAM-1 facilitates leukocyte adhesion, suggest an activated myeloid response and a chemotactic milieu favoring monocyte recruitment. Gene Ontology (GO) enrichment linked the differentially expressed cytokines to mononuclear cell migration, monocyte migration and to activation of the JAK\u0026ndash;STAT signaling cascade (Fig. 1G), while molecular function annotation highlighted CCR2 chemokine receptor binding (Fig. 1H). KEGG pathway analysis further implicated cytokine\u0026ndash;cytokine receptor interactions, TNF signaling and IL-17 signaling (Supplementary Fig. 1C\u0026ndash;D). These systemic changes are consistent with our prior work showing METH-driven Th17 skewing and IL-17A production, which may be sustained by macrophage-derived IL-6.\u003c/p\u003e\n\u003cp\u003eFlow cytometric analysis of peripheral blood further supported a myeloid-centric immune response. Total CD11b⁺ myeloid cells were increased in mononuclear cells (Supplementary Fig. 1E), whereas frequencies of NK cells (Supplementary Fig. 1F), CD4⁺ and CD8⁺ T cells (Supplementary Fig. 1G) were not significantly altered. The fraction of CD11b⁺Ly6C⁺ inflammatory monocytes increased after METH exposure, peaking on day 4 and remaining elevated at day 7 (Fig. 1I\u0026ndash;K; Supplementary Fig. 1H), indicating a selective expansion/activation of the monocyte compartment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether METH can directly modulate macrophage polarization, bone marrow-derived macrophages (BMDMs) were treated in vitro\u0026nbsp;(purity \u0026gt;98%, Supplementary Fig. 1I). At 100 \u0026mu;M, METH did not induce polarization by itself but potentiated LPS/IFN-\u0026gamma;-driven M1 polarization (Supplementary Fig. 1J). At higher concentrations (200\u0026ndash;800 \u0026mu;M), METH directly increased the proportion of F4/80⁺CD86⁺ M1 macrophages in a dose-dependent manner (Fig. 1L\u0026ndash;N), demonstrating a direct pro-polarizing effect under these conditions. Finally, to test the functional contribution of peripheral monocyte-macrophages to behavior, we depleted these cells in vivo using clodronate liposomes (CL). Flow cytometry confirmed efficient depletion of F4/80⁺ cells (Supplementary Fig. 1K). CL pretreatment markedly attenuated METH-induced immobility in both TST and FST (Fig. 1P\u0026ndash;Q) without altering locomotion (Fig. 1R). Collectively, these results indicate that peripheral monocyte-derived macrophages are necessary mediators of METH-induced depressive-like behavior.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. METH exposure induces peripheral monocyte-derived macrophages infiltration into the CNS, exhibiting characteristic CCR2 expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined whether peripheral monocyte-derived cells infiltrate the CNS following METH exposure. Immunofluorescent co-labeling of CD11b and CD45 in mPFC on day 4 revealed a clear increase in CD11b⁺CD45⁺ cells consistent with peripherally derived macrophages (Fig. 2A\u0026ndash;B). \u0026nbsp;Comparable, though less pronounced, infiltration was detectable in the amygdala (Supplementary Fig. 2A), while hippocampal CA1 and CA3 regions showed no evident co-localized CD11b⁺CD45⁺ macrophages (Supplementary Fig. 2B\u0026ndash;C). Infiltration of CCR2⁺F4/80⁺ macrophages in mPFC was confirmed by immunofluorescence (Fig. 2C). These data imply regionally selective recruitment of peripheral macrophages to limbic circuits, with the mPFC as a principal locus of invasion.\u003c/p\u003e\n\u003cp\u003eTo dissect transcriptional signatures, we performed single-cell RNA sequencing (scRNA-seq) of mPFC tissue on day 4. Analysis revealed 33 clusters, annotated into 11 major cell types including microglia, astrocytes, macrophages, oligodendrocytes, neurons, and others (Fig. 2D\u0026ndash;G). Macrophages were further subdivided into 13 subclusters (Fig. 2H\u0026ndash;I). Among these,\u0026nbsp;subclusters 2, 5, and 8 were expanded after METH (Fig. 2J-K) and expressed distinct gene sets: subcluster 2 (Adgre4, Gngt2, Pparg, Fam49a), subcluster 5 (S100a8, S100a9, Retnlg, G0s2), and subcluster 8 (Chil3, Fn1, Thbs1, Plac8) (Supplementary Fig. 2D). Subcluster 8 exhibited high Ly6C expression (loge FC = 2.1, padj = 1.1\u0026times;e⁻\u0026sup2;\u0026sup2;\u0026sup1;). CCR2 was enriched in subclusters 2, 3, 7, and 8 (subcluster\u0026nbsp;2：log\u003csub\u003ee\u003c/sub\u003e FC=0.025，\u003cem\u003eP\u003c/em\u003e adj= 0.004；subcluster\u0026nbsp;3：log\u003csub\u003ee\u003c/sub\u003e FC=1.3，\u003cem\u003eP\u003c/em\u003e adj=2.8e\u003csup\u003e-120\u003c/sup\u003e；subcluster\u0026nbsp;7：log\u003csub\u003ee\u003c/sub\u003e FC=1.1，\u003cem\u003eP\u003c/em\u003e adj= 1.6e\u003csup\u003e-63\u003c/sup\u003e；subcluster\u0026nbsp;8：log\u003csub\u003ee\u003c/sub\u003e FC=1.8，\u003cem\u003eP\u003c/em\u003e adj= 6.3e\u003csup\u003e-80\u003c/sup\u003e), and CD44 was widely expressed across multiple subclusters (Fig. 2K), suggesting heterogeneous infiltration states. Ly6C⁺CCR2⁺CD44⁺ (subcluster 8) and Ly6C⁻CCR2⁺CD44⁺ (subclusters 2,3,7) signatures were identified.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. CCR2 blockade ameliorates METH-induced behavioral and inflammatory outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the CCR2 enrichment among infiltrating macrophage subsets, we tested the functional role of CCR2 signaling. Pharmacological blockade of CCR2 with RS504393 substantially inhibited METH-induced expansion of circulating \u003cstrong\u003eCD11b⁺L\u003c/strong\u003e\u003cstrong\u003ey\u003c/strong\u003e\u003cstrong\u003e6C\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003ehi\u003c/sup\u003e\u003c/strong\u003e inflammatory monocytes (Fig. 3A\u0026ndash;C) and significantly reduced immobility in both TST and FST (Fig. 3D\u0026ndash;E) without altering locomotor activity (Fig. 3F). Complementary genetic experiments using CCR2 knockout (CCR2KO) mice provided convergent evidence: CCR2KO animals failed to mount METH-induced increases in total or inflammatory monocytes (Fig. 3H\u0026ndash;I; Supplementary Fig. 3A\u0026ndash;B) and did not exhibit METH-associated prolongation of immobility observed in wild-type counterparts (Fig. 3J\u0026ndash;K). Locomotor performance remained comparable across genotypes (Fig. 3L), indicating that the protective effect of CCR2 loss was not due to altered activity levels.\u003c/p\u003e\n\u003cp\u003eNotably, METH-induced hyperthermia was unaffected by CCR2 inhibition (Supplementary Fig. 3C\u0026ndash;D), indicating that thermoregulatory responses are mechanistically separable from the CCR2-dependent behavioral sequelae. Beyond affective endpoints, CCR2KO mice were also protected from METH-induced cognitive deficits, as evidenced by improved performance in the novel object recognition and Barnes maze tasks (Supplementary Fig. 3E\u0026ndash;F). Immunofluorescent analysis further demonstrated that CCR2 deficiency prevented METH-induced accumulation of CD45\u003csup\u003ehi\u003c/sup\u003eCD11b⁺ and F4/80⁺CCR2⁺ cells within the mPFC (Fig. 3M\u0026ndash;N). Together, these observations indicate that CCR2-dependent monocyte recruitment is required for METH-driven neuroinflammation and associated behavioral dysfunction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Choroid plexus and meninges act as portals for macrophage infiltration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delineate anatomical routes of entry for peripheral\u0026nbsp;macrophages, we evaluated immune surveillance structures\u0026mdash;chiefly the choroid plexus and meningeal compartments\u0026mdash;using high-throughput cytokine assays and histological assessment. METH markedly increased the number of CD11b⁺ cells in choroid plexus (Fig. 4A). Cytokine profiling identified 21 differentially upregulated analytes in the choroid plexus; among these, CCL2, CCL5 and ICAM-1 (implicated in chemotaxis and leukocyte adhesion) and GM-CSF (a driver of monocyte differentiation) were significantly elevated (Fig. 4B; Supplementary Fig. 4A). Although 19 cytokines did not reach statistical significance, the heatmap indicated a trend toward higher overall expression in the METH group (Supplementary Fig. 4C). The meninges likewise exhibited elevation of 12 cytokines, including CCL2 and GM-CSF (Fig. 4D; Supplementary Fig. 4B), while 28 analytes remained unchanged (Supplementary Fig. 4D). Correlation analysis revealed strong co-expression between CCL2/GM-CSF and other differential cytokines in both compartments (r \u0026gt; 0.7; Fig. 4C, E).\u003c/p\u003e\n\u003cp\u003eFunctional enrichment analysis of the choroid plexus-derived cytokines highlighted biological processes such as monocyte proliferation and leukocyte differentiation, myeloid cell migration and CCR chemokine receptor binding (Fig. 4F). KEGG pathway mapping underscored TNF signaling, Th17 differentiation, Th1/Th2 differentiation, T cell receptor signaling, JAK\u0026ndash;STAT, IL-17 signaling and cytokine\u0026ndash;cytokine receptor interaction among the top pathways (Fig. 4G). Comparable GO and KEGG enrichments were observed for meningeal cytokines, emphasizing myeloid leukocyte migration, monocyte chemotaxis, CCR2 binding and proinflammatory signaling cascades (Fig. 4H\u0026ndash;I). Consistent with a CCR2-dependent entry mechanism, CCR2KO mice exhibited a significant reduction in CD11b⁺ cell accumulation within the choroid plexus after METH (Fig. 4J). Taking together, these data indicate that the choroid plexus and meninges constitute permissive portals for CCR2⁺ monocyte/macrophage ingress into the CNS following METH exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Neuronal CCL2 drives CCR2⁺ macrophage infiltration and is feedback-regulated\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e bone marrow monocytes require CCR2 signaling to egress into the circulation and to home to sites of tissue damage in response to chemokine gradients. Given the central role of CCL2 as the principal CCR2 ligand, we interrogated central CCL2 dynamics after METH. ELISA quantification revealed a transient elevation of CCL2 in the mPFC at days 1 and 4 post-METH, with levels returning to baseline by day 7 (Fig. 5A). Immunofluorescent co-labeling demonstrated predominant colocalization of CCL2 with NeuN⁺ neuronal somata rather than glial markers (Fig. 5B), indicating that neurons are a major source of early CCL2 production.\u003c/p\u003e\n\u003cp\u003eTo test whether peripheral monocyte presence influences this chemokine response, we examined CCL2 levels following interventions that disrupt CCR2 signaling or monocyte availability. Monocyte depletion (CL), pharmacological CCR2 antagonism (RS504393), or genetic CCR2 ablation all prevented the METH-associated rise in peripheral blood CCL2 (Fig. 5C\u0026ndash;E). Furthermore, CCR2 blockade inhibited the METH-induced increase in mPFC CCL2 protein and attenuated neuronal CCL2 immunoreactivity (Fig. 5F\u0026ndash;G). These observations support a model in which METH-triggered neuronal CCL2 initiates CCR2⁺ monocyte recruitment into the CNS, and infiltrating CCR2⁺ macrophages subsequently amplifying CCL2 expression, establishing a positive feedback loop that sustains neuroinflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. CCR2⁺ macrophage infiltration activates microglia and induces neuronal injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, we examined downstream inflammatory mediators, glial responses and neuronal integrity within the mPFC. scRNA-seq analysis revealed high expression of the proinflammatory cytokine\u0026nbsp;IL-1\u0026beta;\u0026nbsp;in peripheral-derived monocyte/macrophage subpopulations (subclusters 2, 3, 7 and 8), with detectable expression in other subsets except 0, 4, and 6 (Fig. 6A). This pattern indicates that infiltrating peripheral cells adopt a strongly proinflammatory profile; resident macrophage populations may also be activated to an M1-like phenotype in response to METH. qRT-PCR confirmed elevated\u0026nbsp;IL-1\u0026beta;\u0026nbsp;transcript levels in the mPFC on day 4; notably, CCR2 knockout normalized\u0026nbsp;IL-1\u0026beta;\u0026nbsp;expression (Fig. 6B).\u003c/p\u003e\n\u003cp\u003eMicroglia exhibited the largest transcriptional remodeling among CNS cell types (Supplementary Fig. 6A\u0026ndash;B), with upregulation of ribosomal genes and IL-1\u0026alpha;\u0026nbsp;(Supplementary Fig. 6C\u0026ndash;D). Microglial subclusters expressed proinflammatory and proliferation-linked genes including TNF and CSF1R, and IL-1\u0026alpha;\u0026nbsp;was enriched in microglia while IL-1\u0026beta;\u0026nbsp;remained macrophage-specific (Supplementary Fig. 6E). GO and KEGG analyses of microglial changes pointed to enrichment of ribosome biogenesis, oxidative phosphorylation, TNF signaling and Toll-like receptor pathways (Supplementary Fig. 6F\u0026ndash;G), consistent with a shift toward a proinflammatory, high-metabolic state that may mediate neuroinflammation via IL-1\u0026alpha;.\u003c/p\u003e\n\u003cp\u003eMorphologically, microglia in the mPFC transitioned from a resting, ramified phenotype to activated states at day 4 post-METH: the proportion of ramified cells decreased while intermediate and amoeboid morphologies increased. Importantly, CCR2KO restored microglial morphology toward baseline (Fig. 6C\u0026ndash;D), indicating that CCR2-dependent infiltration is upstream of microglial activation.\u003c/p\u003e\n\u003cp\u003eConcomitant with glial activation, neuronal pathology was evident in the mPFC on day 4 following METH exposure. Hematoxylin-and-eosin staining revealed enlarged neuronal soma and a reduced neuronal count, both of which were attenuated in CCR2KO mice treated with METH (Fig. 6E\u0026ndash;F). Consistent with these findings, Nissl staining showed a reduction and pallor of Nissl bodies after METH exposure, an effect that was also ameliorated in CCR2KO mice administered METH (Fig. 6G). Ultrastructural analysis by transmission electron microscopy demonstrated synaptic abnormalities after METH: synaptic cleft coupling, fusion of pre- and post-synaptic membranes, focal low-electron-density edematous zones, and adjacent mitochondrial swelling and damage\u0026mdash;features that were not observed in CCR2KO mice exposed to METH (Fig. 6H). At the protein level, PSD95 expression-assessed by Western blot-was reduced after METH exposure but was significantly preserved in CCR2KO mice (Fig. 6I\u0026ndash;J). Collectively, these data indicate that CCR2⁺ macrophage infiltration precipitates microglial activation, drives a neuroinflammatory cascade, and contributes to synaptic degeneration and neuronal loss in the mPFC, thereby linking peripheral immune recruitment to METH-induced neuropathology and behavioral deficits.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study identifies CCR2⁺ peripheral monocytes as critical mediators of METH-induced depressive-like behaviors and delineates the cellular and molecular mechanisms through which these immune cells interact with CNS compartments. By integrating behavioral assays, immunological profiling, transcriptomic analyses, and genetic interventions, we demonstrate that binge METH exposure elicits a systemic myeloid response, promotes CCR2-dependent infiltration of macrophages into CNS\u0026mdash;particularly the mPFC\u0026mdash;and establishes a neuroinflammatory feedback loop that disrupts synaptic integrity and drives emotional pathology. These findings extend previous models of METH neurotoxicity by highlighting a central role for peripheral immune recruitment in sustaining affective disturbances during withdrawal.\u003c/p\u003e\n\u003cp\u003eOur findings highlight the critical role of the CCL2/CCR2 axis in linking peripheral inflammatory responses to central nervous system (CNS) pathology. CCR2 (C-C chemokine receptor type 2), as the primary receptor for CCL2 (MCP-1), serves as a key regulator of peripheral monocyte migration and infiltration(19, 20). Under homeostatic conditions, CCR2-expressing monocyte-derived macrophages support essential immune surveillance and contribute to tissue repair mechanisms within the central nervous system (CNS). In diverse pathological contexts, however, dysregulated activation and subsequent CNS invasion of these cells can initiate and sustain detrimental neuroinflammatory cascades(21, 22). In experimental models of ischemic stroke, for example, CCR2⁺ macrophages amplify blood\u0026ndash;brain barrier injury via robust release of reactive oxygen species and matrix metalloproteinases(23). Similarly, in experimental autoimmune encephalomyelitis (EAE), inflammatory CCR2⁺monocytes-secreted IL-1\u0026beta; mediate direct cytotoxic effects on oligodendrocytes, thereby suppressing remyelination and exacerbating demyelination(24). Of therapeutic relevance, genetic ablation of CCR2 significantly attenuates monocyte trafficking into the CNS and confers protection across multiple neurodegenerative disease models(25, 26). These collective findings underscore the translational potential of modulating CCR2 signalling to re-establish neuroimmune homeostasis and alter disease trajectories in neuroinflammatory and degenerative disorders.\u003c/p\u003e\n\u003cp\u003eElevated levels of circulating cytokines and chemokines, including CCL2, IL-6, and TNF-\u0026alpha;, following METH exposure created a chemotactic environment favorable for monocyte trafficking. These mechanisms may extend to psychiatric disorders such as major depressive disorder (MDD), where increased levels of CCL2, IL-6, IL-12, TNF-\u0026alpha;, and IL-1\u0026beta; have been consistently observed. Although not exclusively secreted by M1 macrophages, these inflammatory mediators are closely associated with monocyte activation and M1 polarization(27, 28). Supporting this, macrophages derived from patients with bipolar disorder show enhanced production of IL-1\u0026beta; and TNF-\u0026alpha; upon stimulation, underscoring the involvement of monocytes in mood disorders via inflammatory pathways(29). Additionally, patients with Crohn\u0026rsquo;s disease and comorbid depression exhibit elevated M1 macrophage activity, which appears to facilitate depression through pro-inflammatory cytokine signaling(30, 31).\u0026nbsp;These observations emphasize the central contribution of monocyte-derived macrophages and their inflammatory mediators in the pathogenesis of depression, suggesting common immuno-pathogenic pathways across substance-induced and primary psychiatric conditions.\u003c/p\u003e\n\u003cp\u003eFurthermore, scRNA-seq provided evidence that infiltrating monocyte-derived macrophages retain CCR2 expression and undergo dynamic phenotypic reprogramming within the central nervous system (CNS) microenvironment. CD44 expression profiling further demonstrates its specific expression in infiltrating cells, being absent in resident myeloid cells(32, 33). Based on these distinct molecular features, the Ly6C⁺CCR2⁺CD44⁺ macrophage subset (Subset 8) is identified as recently infiltrated inflammatory monocytes of peripheral origin that have not yet fully differentiated. In contrast, subsets in Clusters 2, 3, and 7 (Ly6C⁻CCR2⁺CD44⁺) appear to represent monocyte-derived macrophages that have undergone functional polarization within the CNS\u0026mdash;consistent with Ly6C phenotypic evolution observed in experimental autoimmune encephalomyelitis models(34).\u003c/p\u003e\n\u003cp\u003eIn the present study, we demonstrate that mPFC neurons themselves become a key source of CCL2 upon METH exposure, orchestrating the early infiltration of peripheral monocytes into the brain\u0026mdash;a role traditionally attributed almost exclusively to activated microglia(35). This aligns with recent evidence indicating that substance use disorders, such as opioid abuse(36), similarly upregulate neuron derived CCL2 and drive neuroinflammatory processes via immune cell infiltration. Notably, the infiltration of monocyte-derived macrophages was region-specific, with the most pronounced accumulation occurring in the mPFC region and neuronal CCL2 expression coincided closely with the trajectory of macrophage infiltration. This finding reframes the conventional unidirectional \u0026ldquo;immune-to-neuron\u0026rdquo; paradigm, suggesting region-selective neuronally driven initiation of central immune recruitment may contribute to affective dysregulation. Notably, neuronal production of CCL2 was an early and transient response to METH, but the subsequent amplification of CCL2 expression required CCR2⁺ macrophage infiltration, revealing a self-reinforcing feed-forward loop. Such feedback dynamics may explain the persistence of depressive-like behaviors beyond the acute phase of drug exposure. The current work also has broader implications for the understanding of depression comorbid with substance use disorders. Clinical studies have consistently linked CCL2 to depressive disorder(17), and our results suggest that psychostimulant induced CCL2/CCR2 activation may represent a convergent pathway through which drug exposure and stress converge to exacerbate affective pathology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe choroid plexus and meninges, which are central to cerebrospinal fluid dynamics, constitute critical gateways for the entry of peripheral immune cells into the central nervous system (CNS). Emerging evidence indicates that these compartments form specialized immunogenic niches that permit immune cells to actively participate in brain regulation(37). METH exposure triggers marked upregulation of CCL2, CCL12, IL-1\u0026beta;, TNF-\u0026alpha;, and GM-CSF within the meninges and choroid plexus. Functional enrichment analyses reveal significant association with biological processes such as \u0026ldquo;monocyte proliferation\u0026rdquo; and \u0026ldquo;CCR2 chemokine receptor binding\u0026rdquo;, indicating active recruitment and differentiation of peripheral monocyte-derived macrophages in these compartments. The identification of the choroid plexus and meninges as key entry portals expands current understanding of neuroimmune communication in addiction, consistent with emerging evidence that these border tissues regulate immune surveillance and leukocyte entry into the brain. During inflammation, they markedly upregulate secretion of CCL2, CCL12, macrophage colony-stimulating factor (M-CSF), and granulocyte\u0026ndash;macrophage colony-stimulating factor (GM-CSF), establishing a chemotactic gradient that facilitates the recruitment of CCR2⁺ monocytes(38-40). Notably, M-CSF and GM-CSF not only promote monocyte-to-macrophage differentiation but also enhance cell survival and drive a pro-inflammatory M1-like polarization via activation of the STAT3 and STAT5 signalling pathways(41, 42). Similarly, within meningeal perivascular spaces, fibroblasts and tissue-resident macrophages are capable of secreting substantial quantities of chemokines, thereby providing navigational cues that guide the transmigration of peripheral monocyte-derived macrophages across CNS barrier structures(43). This coordinated triad of \u0026ldquo;chemotaxis\u0026ndash;survival\u0026ndash;polarization\u0026rdquo; is likely a central driving mechanism underlying METH-induced neuroinflammation. Moreover, the delineation of choroid plexus and meningeal portals as immune entry routes opens new avenues for exploring how systemic inflammatory states may synergize with drug use to worsen psychiatric outcomes.\u003c/p\u003e\n\u003cp\u003eThe functional impact of infiltrating CCR2⁺ macrophages was multifaceted. scRNA-seq revealed that these cells adopt transcriptional profiles enriched for proinflammatory mediators such as IL-1\u0026beta;. Together, these changes promoted a proinflammatory milieu that was accompanied by microglial morphological activation, neuronal injury, synaptic ultrastructural abnormalities, and PSD95 loss in the mPFC. Importantly, depletion of monocytes or disruption of CCR2 signaling prevented these outcomes and normalized both behavior and neuropathology. These findings provide causal evidence that peripheral immune recruitment is not merely correlative but necessary for METH-driven neuroinflammation and depressive phenotypes.\u0026nbsp;Our study positions the CCL2/CCR2 axis as a tractable therapeutic target for comorbid depression in the context of substance abuse.\u003c/p\u003e\n\u003cp\u003eSeveral limitations warrant consideration. First, our experiments were restricted to male mice, and sex-dependent differences in immune responses may limit the generalizability of these findings. Second, while our study highlights macrophage\u0026ndash;microglia crosstalk, the potential involvement of other immune subsets, such as Th17 cells previously implicated in METH-induced pathology, remains incompletely defined. Third, although we focused on the mPFC as the primary locus of infiltration, other regions such as the amygdala may contribute to a circuit-specific manner to the observed behavioral alterations. Future investigations employing cell type\u0026ndash;specific manipulations, longitudinal imaging, and circuit-level analyses will be essential to determine how infiltrating immune cells functionally integrate into neural networks over time. Finally, although we identified the choroid plexus and meninges as portals for macrophage entry, the precise mechanisms by which immune cells traverse these interfaces and subsequently accumulate within specific regions such as the mPFC remain to be elucidated.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our findings provide mechanistic insight into how binge METH exposure couples systemic immune activation with CNS inflammation through a CCR2-dependent pathway. By recruiting proinflammatory monocytes into mPFC, METH establishes a pathological feedback loop that drives microglial activation, neuronal injury, and synaptic degeneration, culminating in depressive-like behaviors. Targeting the CCL2/CCR2 axis thus offers a promising strategy for mitigating the neuropsychiatric consequences of psychostimulant abuse and for addressing the broader challenge of substance use comorbid with affective disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Key Project of National Natural Science Foundation of China (82030057 to CM), the General Project of National Natural Science Foundation of China (82371899 to DW), the Hebei Medical University Postdoctoral Funding Project (30705010078 to RH).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDeclarations\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe animal experiments were conducted following the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Local Animal Use Committee of Hebei Medical University (approval no., IACUC-Hebmu-P2020072).\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe scRNA-seq data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Other raw data and statistical results are provided in Supplementary Material.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLewis D, Kenneally M, van denHeuvel C, Byard RW. Methamphetamine deaths: Changing trends and diagnostic issues. Medicine, science, and the law. 2021;61(2):130-7.\u003c/li\u003e\n \u003cli\u003eLeung J, Mekonen T, Wang X, Arunogiri S, Degenhardt L, McKetin R. Methamphetamine exposure and depression-A systematic review and meta-analysis. Drug and alcohol review. 2023;42(6):1438-49.\u003c/li\u003e\n \u003cli\u003eYang M, Yang C, Liu T, London ED. Methamphetamine-associated psychosis: links to drug use characteristics and similarity to primary psychosis. Int J Psychiatry Clin Pract. 2020;24(1):31-7.\u003c/li\u003e\n \u003cli\u003eQuinton MS, Yamamoto BK. 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Theranostics. 2021;11(3):1059-78.\u003c/li\u003e\n\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":"Methamphetamine, Depressive behaviors, monocyte-macrophages, CCL2, CCR2, Microglia, Synaptic plasticity","lastPublishedDoi":"10.21203/rs.3.rs-7742867/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7742867/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMethamphetamine (METH) abuse is frequently associated with persistent depressive symptoms, representing a major contributor to psychiatric comorbidity in substance use disorders; however, the underlying mechanisms remain poorly defined. Here, we show that binge METH exposure induces robust and persistent depressive-like behaviors in mice, accompanied by systemic cytokine elevation and expansion of Ly6C\u003csup\u003ehi\u003c/sup\u003e inflammatory monocytes in the peripheral circulation. These monocyte-derived macrophages infiltrated the medial prefrontal cortex (mPFC) via the choroid plexus and meninges in a CCR2-dependent manner. Single-cell RNA sequencing of mPFC immune cells identified distinct proinflammatory CCR2\u003csup\u003e+\u003c/sup\u003e macrophage subsets enriched for IL-1β expression, which in turn amplified neuronal CCL2 production, establishing a self-sustaining macrophage–microglia crosstalk that perpetuated local inflammation. Pharmacological inhibition or genetic disruption of CCR2 prevented immune infiltration, reduced neuroinflammation, preserved synaptic integrity, and rescued both depressive-like and cognitive deficits. Together, these findings identify CCR2-dependent monocyte infiltration as a key mechanism linking METH exposure to affective dysfunction and highlight the CCL2/CCR2 axis as a potential therapeutic target in substance use–associated mood disorders.\u003c/p\u003e","manuscriptTitle":"CCL2/CCR2-mediated monocyte-macrophages infiltration drives methamphetamine-induced depressive-like behaviors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 16:46:43","doi":"10.21203/rs.3.rs-7742867/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-12-19T10:13:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-19T06:57:25+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-16T01:31:13+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-11-13T01:02:15+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-08T11:52:35+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-11-06T03:09:40+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-11-05T23:40:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-11-05T23:33:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-30T16:29:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-30T16:25:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2025-09-29T13:56:56+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":"17a1f746-7d57-4558-bc34-7f3ea110a04e","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":57514713,"name":"Biological sciences/Neuroscience"},{"id":57514714,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"}],"tags":[],"updatedAt":"2026-05-08T09:21:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 16:46:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7742867","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7742867","identity":"rs-7742867","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}