Targeting neuroinflammation

Abstract

Provided herein are methods that use neuroimmune interactions and their targeting to identify new therapeutic targets for neuropsychiatric and inflammatory diseases.

Description

[0001] Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0002] TARGETING NEUROINFLAMMATION CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No.

[0004] 63 / 778,295, filed on March 26, 2025. The entire contents of the foregoing are incorporated herein by reference.

[0005] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with Government support under Grant No.

[0007] MH130458, MH132632, DA061199, andNS114111 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] Provided herein are methods that use neuroimmune interactions and their targeting to identify new therapeutic targets for neuropsychiatric and inflammatory diseases.

[0010] BACKGROUND

[0011] Neuroimmune communication facilitates tissue adaptations to environmental changes1. Indeed, the immune system engages in bidirectional communication with the brain during psychological stress2 3. Signals from immune cells to the brain can influence the development of neuropsychiatric diseases such as major depressive disorder (MDD)6 9. Thus, while immunotherapy targeting peripheral signals induced by psychological stress may be a viable therapeutic area in neuropsychiatric disorders such as MDD5-4-6 l0. many relationships between behavioral changes and immunoregulatoiy mechanisms in the brain remain to be defined.

[0012] SUMMARY

[0013] Thus, provided herein are methods for identifying a potential candidate neuroimmune target transcript for modifying neuroimmunity in a mammal. The methods comprise: subjecting anon-human animal model to a behavioral stress condition; obtaining first samples comprising cells from the CNS, and second samples immune cells from the animal; determining RNA transcriptomic profiles from the first and second samples, and comparing the RNA transcriptomic profiles from the firstAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0014] and second samples to RNA transcriptomic profiles from first and second samples determined from a control animal that was not subjected to the behavioral stress condition, to identify transcripts altered in the animal model as compared to the control animal; selecting transcripts that are altered in the first samples and second samples, and identifying one or a subset of transcripts that are altered in the first samples that are related to transcripts that are altered in the second samples, thereby identifying potential candidate target transcripts for modifying neuroimmunity in a mammal.

[0015] In some embodiments, the methods further comprise using cell sorting or computational sorting to identify cell subtypes within the first and second samples, and identifying transcripts altered in one or more of the subtypes.

[0016] In some embodiments, computational sorting is performed by clustering based on gene expression.

[0017] In some embodiments, the cells from the CNS comprise astrocytes, microglia, excitatory neurons, inhibitory neurons, oligodendrocyte precursor cells, and oligodendrocytes.

[0018] In some embodiments, the immune cells comprise astrocytes from the CNS. In some embodiments, identifying one or a subset of transcripts that are altered in the first samples that are related to transcripts that are altered in the second samples comprises using a database to identify’ transcripts in the first samples that affect expression of transcripts that are altered in the second samples, or vice versa.

[0019] In some embodiments, the methods further comprise: providing an in vitro model of the CNS cells or immune cells; perturbing the potential candidate target transcript; evaluating an effect of the perturbation on related transcripts, and selecting a potential candidate target transcript that alters the related transcripts as a candidate target.

[0020] In some embodiments, perturbing the candidate target transcript comprises using a genetic or pharmacological tool to reduce or increase expression of the candidate target transcript. In some embodiments, the genetic tool is selected from CRISPR knockout, or administration of an antisense oligonucleotide (ASO), Cre recombinase based deletion, small interfering RNA (siRNA), small hairpin RNA (shRNA). In some embodiments, the pharmacological tool is selected from contactingAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0021] the cells with a small molecule or antibody that binds to the protein product of the candidate transcript.

[0022] In some embodiments, the methods further comprise: subjecting a second nonhuman animal model to a behavioral stress condition in the presence of a perturbation of the candidate transcript, wherein the second non-human animal model is the same as the non-human animal model used in an earlier step; evaluating a parameter of the behavior of the animal in the presence of the perturbation as compared to the parameter of the behavior in the absence of the perturbation; and identifying a transcript that alters the parameter when perturbed as a potential therapeutic candidate.

[0023] In some embodiments, the methods further comprise: obtaining first samples comprising cells from the CNS, and second samples immune cells from the animal; determining RNA transcriptomic profiles from the first and second samples, and comparing the RNA transcriptomic profiles from the first and second samples in the presence of the perturbation of the candidate transcript to RNA transcriptomic profiles from first and second samples determined from a control animal in the absence of a perturbation of the candidate transcript, to determine an effect of the perturbation of the candidate transcript; selecting transcripts that are altered in the first samples and second samples, and identifying one or a subset of transcripts that are altered in the first samples that are related to transcripts that are altered in the second samples, and determining whether the perturbation of the candidate transcript alters related transcripts in the second samples; thereby identifying a potential therapeutic candidate transcript for modifying neuroimmunity in a mammal.

[0024] In some embodiments, the methods further comprise the methods further comprise: obtaining a first human sample comprising human CNS cells and a second human sample comprising human immune cells; perturbing the potential therapeutic candidate transcript in the first and second human samples; determining whether the perturbation of the potential therapeutic candidate transcript alters related transcripts in the second human samples and selecting a potential therapeutic candidate transcript when the perturbation alters the related transcripts as a therapeutic candidate.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use inAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0026] the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety7. In case of conflict, the present specification, including definitions, will control.

[0027] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0028] DESCRIPTION OF DRAWINGS FIGs. la-r. Amygdala astrocyte EGFR signaling limits stress-induced fear behavior, a, Schematic to study neuroimmune interactions in chronic stress, b, Fear behavior of mice exposed to control handling or 18-day restraint stress. n=15 mice per group. Two-way repeated measures ANOVA. c, ELISA of plasma cytokines. 18 days of restraint stress (n=4) or controls (n=5). Two-way ANOVA with multiple t-test. d, Uniform manifold approximation and projection (UMAP) plot of clusters detected by scRNA-seq. e,f, Proportion of cluster per condition (e) and number of cells in cluster 1 (f) from control (n=3) or 18-day stressed (n=3) mice, g, Pathway analysis of cluster 1 cells. n=2,232 cells. h,i, Experimental schematic (h) and fear behavior (i) of mice transduced with sgEgfr or sgRosa26 exposed to 7 days of restraint stress. n=9 per group. Two-way repeated measures ANOVA. j,k, Volcano (j) and GSEA (k) plots analyzing bulk RNA-seq of amygdala astrocytes from sgEgfr (n=3) or sgRosa26 (n=4) mice. 1, Bar plot of all detected PTPR family members in cluster 1 cells, m, Images and quantification of BLA astrocyte PTPRS levels after 18 days restraint stress (n=40 images) or control (n=38 images), n=5 mice per group. Unpaired t-test. n, qPCR of primary astrocytes treated for 24-hrs with corticosterone and either 100 ng / mL IL-lb, 100 ng / mL IL-12, or 50 ng / mL TNFa. n=6 per group, vehicle-treated group normalized to 1. One-sample t-test. o, Qiagen IPA prediction of IL-lb as an upstream regulator by RNA-seq. Fisher's exact test, p, qPCR of AAV- targeted primary astrocytes treated with indicated compounds for 24-hrs. n=6 per group. Mann-Whitney test, q, Volcano plot of bulk RNA-seq data analyzing amygdala neurons from mice analyzed in (j). r, GSEA plot from data in (q) showing leading edge genes. n=3 mice per group. NES, normalized enrichment score. FC, fold change. Data shown as mean±SEM.Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0029] FIGs. 2a-k. Amygdala neuron Nr2f2 expression promotes fear behavior, a, Venn diagram illustrating the overlap between transcription factors upregulated in bulk RNA-seq data of amygdala neurons isolated from mice transduced with AAV targeting astrocyte Egfr relative to Rosa26 control following 7 days of restraint stress and fear conditioning and predicted transcriptional regulators. n=3 mice per group, b, Ranking by p-value of differentially expressed regulators shown in (a), c, Schematic of cell type-specific knockdown strategy used in astrocyte-neuron co-cultures (left) and qPCR expression data of in neurons 7 days following knockdown (right: Nr2f2 n=ll; Cebpg, n=ll; Etsl, n=12). One sample t-test performed relative to Rosa26-targeting control for each gene, d, Experimental schematic for neuron targeting and behavioral analysis, e, Freezing behavior of mice exposed to 7-days of restraint stress following injection of AAV delivering saCas9 under the hSyn promoter and targeting sgRosa26, sgCebpg, or sgEtsl (n=5 mice per group), or sgNr2f2 (n=7) or sgRosa26 (n=8) mice. Two-way repeated measures ANOVA. f-h, Volcano (I), GSEA analysis (g), and StringDB -generated PPI network (h) plots obtained from bulk RNA-seq of amygdala neurons from mice targeted using sgNr2f2 (n=3) or sgRosa26 (n=4) that underwent 7 days of restraint stress and fear conditioning, i j, ATAC-seq analysis showing NR2F2 motif sequence (i) and down regulated cellular compartments following Nr2f2 knockdown in amygdala neurons (j) from mice that underwent 7 days of restraint stress and fear conditioning. n=4 mice per group, k, Quantification of excitatory synapses (white arrowheads) in the BLA of Nr2f2- (n=3) or Rosa26- (n=3) targeted mice by immunostaining. n=13 images per group. Mann- Whitney test. PPI, protein-protein interaction. FDR, false discovery' rate. NES, normalized enrichment score. Data shown as mean±SEM.

[0030] FIGs. 3a-g. Analysis of amygdala subnuclei regulation during chronic stress, a, Subclustered excitatory' neurons that are associated with 18-day restraint stress. n=3 mice per group. Two-way ANOVA with Sidak’s multiple comparisons test, b, Pie chart associating an amy gdala subnucleus w ith each cluster 2 excitatory neuron detected in the amygdala, c, Nearest neighbor analysis of Egfr expression in astrocytes from stressed mice analyzing the top 10% of astrocytes nearest to cluster 2 excitatory' neurons versus all other astrocytes. Unpaired Welch’s t-test. d, Quantification of Nr2f2 gene averaged counts across excitatory neuron clusters, e, Inflammatory’ pathways in cluster 2 excitatory neurons. ND, not detected. D, dorsal.Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0031] V, ventral. M, medial. L, lateral. Data show n as mean±SEM. f, Plot of microglial or monocyte marker genes in microglial cells detected by Stereo-seq. g, Total counts of selected cytokine genes in analyzed amygdala samples. MSN, medium spiny neurons. CeA, central amygdala. BLA, basolateral amygdala. ROI, region of interest. ND, not detected. Data show n as mean±SEM.

[0032] FIGs. 4a-s. Stress-induced neuroimmune interactions are modulated by psychedelics, a, Quantification by FACS of inflammatory monocytes in the meninges or deep cervical lymph nodes (dCLN) 24-hours after mice were exposed to 7- or 18 days of restraint or controls (left). FACS plots of meningeal monocytes (right). n=10 for 18-d stress meninges; n=4 for 18d dCLN, otherwise n=5 per group. Unpaired t-test on log-transformed data, b, Schematic of monocyte adoptive transfer in C57B1 / 6J mice, c, FACS analysis of meningeal monocytes 24h post-stress (Unstressed mice (vehicle, n=7); 7-day stressed mice (vehicle, n=5; monocyte, n=10). Mann-Whitney test. d,e, Behavior of host mice adoptively transferred monocytes. n=5 unstressed mice; n=10 otherwise, (d) EPM data. Unpaired t-test. (e) Fear behavior. n=5-10 mice per group. Two-way repeated measures ANOVA. f, Pathway analysis of (b-e) amygdala astrocyte bulk RNA-seq data, g, Schematic of a-CCR2 antibody i.c.m. injection. h,i, Analysis of EPM (h) and fear behavior (i) in a-CCR2 antibody-treated or isotype-treated group. n=10 mice per group. Unpaired t-test for EPM. Two-way- repeated measures ANOVA for fear behavior. Experiment repeated twice, j, EGFR transcriptional network comparing a-CCR2 antibody-injected mice versus isotype-injected controls. Fisher's exact test, k, Schematic (left), imaging and quantification (right) of biotinylated IL-lb delivered via i.c.m. injection in 18-day stressed mice or controls. n=7 images per group from n=4 mice per group. Mann- Whitney test. 1, GSEA plots from bulk RNA-seq of meningeal monocytes of mice exposed to 18 days of restraint stress or unstressed controls. n=3 mice per group, m, FACS analysis (left) and plots (right) of meningeal inflammatory- monocytes 24-hours after indicated treatment. n=5 per group. One-way ANOVA, Dunnett post-test, n, Schematic (left) and ELISA (right) of meningeal explants from mice in the indicated condition. n=5 mice per group, n=10 for TNFa control, n=4 for IL-lb control. One-way ANOVA, Fisher’s LSD test. Data shown as mean±SEM. o,p, Schematic of photoconversion in the spleen of Kaede mice (left) and number of photoconverted cells (right) (o);

[0033] percentage of photoconverted monocytes in the meninges, dCLN. bone marrow, andAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0034] spleen of stressed and unstressed mice (p). n=3 mice per group. Unpaired t-test. q, Schematic and FACS analysis of monocyte adoptive transfer via i.v. injection in stressed and control NSG mice. (Unstressed mice (vehicle, n=5); 7-day stressed mice (vehicle, n=5; monocyte, n=10). Kruskal-Wallis test, with Dunn post-test, r, Time spent in open arms versus closed arms in EPM. (Unstressed mice (vehicle, n=4); 7-day stressed mice (vehicle. n=5; monocyte, n=9). Unpaired t-test. s, FACS analysis of CCR2+pro-inflammatory meningeal monocytes following i.c.m. injection of a-CCR2 or isotype control. n=5 per group. Unpaired t-test.

[0035] FIGs. 5a-d. Relevance of detected neuroimmune interactions in humans. a,b, qPCR analysis of PTPRS (a) and EGFR (b) expression in primary human astrocytes treated with the indicated compounds for 6-hrs. n=12 (a) or n=6 (b) per group. Mann-Whitney test, c, RNA-seq data from primary' human monocytes treated with the indicated compounds for 6-hrs and shown as volcano plot from the indicated conditions. n=3 per group, d, Prediction of an NR2F2 gene regulatory network in cluster 2 excitatory neurons. In violin plots, Solid horizontal line: median; white box: interquartile range (25th-75th percentile); whiskers: min and max values: dashed line: mean. Data shown as mean±SEM.

[0036] FIGs. 6a-g. Molecular and behavioral changes after 18 days restraint stress, a, Schematic of behavioral paradigm. Tube restraint for 7, 12, or 18 days, followed by cued fear acquisition, with fear retrieval conducted the next day in a novel context with only cue presentation, b, Acquisition of fear behavior in mice exposed to 7 days (n=10), 12 days (n=5), or 18 days (n=15) of stress or controls (n=14). Two-way repeated measures ANOVA. c, Time spent freezing in conditioned fear behavior across trials. n=10 mice per group. Two-way repeated measures ANOVA. d, Time in open / closed arm of elevated plus maze (EPM) in mice exposed to 18 days of stress (n=20) or controls (n=9). Unpaired t-test. e, Cytokines detected by ELISA in the plasma of mice exposed to 18 days of restraint stress (n=4) or controls (n=5). Tw o-w ay ANOVA. f, Changes in corticosterone levels in 18-day stressed mice or control mice, measured at 30-minutes (n=5 per group) and 6-hours (control, n=5; 18d-stress, n=4) after final session of restraint stress concluded. Unpaired t-test. g, Plots of pathways in cluster 0 (left) and cluster 2 (right) cells. Data shown as mean±SEM.Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0037] FIGs. 7a-e. Psychedelic control of the immune response across tissues, a-c, FACS analysis of major cell types in the meninges (a), dCLN (b), and spleen (c) in mice exposed to 18 days of restraint stress and treated with vehicle (n=5), 1 mg / kg psilocybin (n=5), or 10 mg / kg MDMA racemate (n=5). Unpaired t-test. d, qPCR analysis in primary' astrocytes at baseline (left: Illrl, n=4; Htr2a, n=4; Htr2b, n=5, Htr2c, n=6; Nlrk2, n=6) or after a 6-hour treatment with 10 pM psilocybin, 10 pM MDMA, or vehicle (right, n=6 per group). One-way ANOVA per gene, e, Ptprs expression level by qPCR in primary' astrocytes treated with indicated compounds for 6-hrs (vehicle, n=9; psilocybin, n=9; MDMA, n=6). Unpaired t-test. Data shown as mean±SEM.

[0038] FIGs. 8a-b. Flow charts showing exemplary methods described herein in simplified (a) and detailed (b) versions.

[0039] DETAILED DESCRIPTION

[0040] To investigate links between brain-immune communication in neuropsychiatric disorders, we can draw parallels to other brain diseases where tissueresident glial cells in the brain, such as astrocytes, have emerged as key players in central nervous system (CNS) immunoregulation11. Astrocytes also play direct roles in the regulation of psychological stress responses12 15and influence affective behaviors related to neuropsychiatric disorders including compulsion16 17, fear1418 19, and helplessness12,20. Therefore, defining astrocyte immunoregulatory mechanisms in chronic stress provides a link between inflammation and affective behavior and potentially offer new ways to identify therapeutic avenues for treating MDD and other conditions.

[0041] The present study defines mechanisms of astrocyte-neuron cross-talk in the amygdala and their potential regulation by peripheral immune cells in chronic stress and possibly MDD, emphasizing the therapeutic potential of targeting immune mechanisms in neuropsychiatric disorders2’56’42The present data suggest that inflammatory monocytes promote stress-induced fear behavior, with meningeal recruitment influenced by splenic reservoirs but potentially other peripheral tissues such as the skull bone marrow55or gut56. In addition, we detected astrocyte subsets modulated by diverse cues in chronic stress and potentially MDD. Specifically, we uncovered an interplay between astrocyte EGFR expression and NF-KB signalingAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0042] where these pathways may exh i bi t opposing influences on one another, which potentially modifies stress-induced fear behavior.

[0043] The present study also underscores the therapeutic potential of psychedelics beyond psychiatry, including in immunology52,58. By using a phenotypic screen of shared properties among two clinically relevant psychedelics, analogous to studies of social behavior50, we found that both psilocybin and MDMA similarly influenced immune cell abundance in tissues. The present data suggest that brain-body communication may be an underappreciated component of psychedelic therapy.

[0044] In summary, described herein are methods for identifying agents that can modulate a psychedelic-sensitive neuroimmune signaling axis tuned by the recruitment of inflammatory monocytes to the brain meninges, which influences astrocyte-neuron responses in the amygdala and fear behavior. The present methods study neuroimmune interactions and their targeting, e.g., by psychedelics, to lead to new therapeutic targets in both neuropsychiatric and inflammatory diseases.

[0045] FIGs. 8a-b provide flow charts showing exemplary methods as described herein in simplified (a) and detailed (b) versions, which are described in greater detail below.

[0046] Step 1 Identify candidate targets co-dysregulated in CNS and immune system In the present method, a non-human animal model is subjected to behavioral stress. Exemplary non-human animal models include rodents such as mice and rats; rabbits; and non-human primates. In some embodiments, the animal model is a transgenic animal that expresses one or more cell-specific reporter genes, to allow for cell sorting of specific cell types (e.g., astrocytes or specific types of neurons).

[0047] Preferably the behavioral stress is quantifiable, e.g., there are quantifiable parameters that described the animal’s behavior before, during, and after the stress. Exemplary behavioral stresses include forced swim assays, elevated plus maze (EPM), chronic restraint, tail suspension test, and contextual fear conditioning. The stress can be continued for at least 2, 4, 6, 8, or 12 hours, up to 1, 2 or more days, e.g., 3, 5, 7, 9. 12. 14. 18, or 21 days or longer, as appropriate for the type of stress, as known in the art.

[0048] After cessation of the stress (optionally immediately after or 2, 4, 6, 12, or 24 hours or longer after), samples are collected from the animals. The samples includeAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0049] cells from the CNS, as well as immune cells. The samples can also include blood and / or urine samples.

[0050] Methods known in the art can be used to determine the transcriptomic profiles in the cells, including high throughput sequencing, arrays, spatial transcriptomics, and other methods. As an alternative to transcriptomes, proteomes or other cell characteristics (e.g., chromatin accessibility) can be determined and compared.

[0051] The cells in the samples can be grouped, e.g., using computational methods such as clustering based on gene expression (e.g., K-means clustering), or physical means such as cell sorting, e.g., based on reporter expression or cell surface markers. The grouped cells can then be analyzed separately, e.g., to determine and select cell types that are more (or less) affected by the stress conditions (e.g., that have greater numbers or amplitude of transcriptome changes, or that have greater or smaller numbers of cells, as compared to control conditions), and analyze the transcriptomic profile in the cells in one or more of the groups separately.

[0052] One or more transcripts that show a significant amplitude (e.g., change above a selected threshold level, optionally statistical significance) of change (in either direction) in the stress versus control conditions in either the CNS or immune cells can then be selected as a potential candidate and further evaluated for their potential role in neuroimmunity, to determine whether they are related to transcripts in the other cells, and thus co-dysregulated (altered by stress) in CNS and immune system. For example, CNS transcripts can be analyzed to identify those known to interact with immune cells or proteins expressed in immune cells (e.g., ligand-receptor pairs), or those known to be transcription factors that affect expression of immune cells, and so on. Such identification can be made based on reference to a database, pathway analysis, and so on.

[0053] Step 2 - Perturb Potential Candidate Targets

[0054] Once a transcript is identified as a potential candidate, in vitro methods can be used to determine the effect of perturbing the expression of the transcnpt. Methods known in the art can be used to perturb the transcript, including genetic (e g., CRISPR, siRNA, shRNA, ASOs, or other knockout / knockdown methods including Cre recombinase based deletion) or pharmacological (e.g., using a small molecule or antibody that binds to the protein product of the potential candidate). The perturbation can be evaluated in vitro to identify candidate target that, when perturbed, altersAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0055] dysregulation in both the CNS and immune systems, e.g., using in vitro CNS and immune models.

[0056] Step 3 - Evaluate Effect of Perturbation in Vivo

[0057] Once a transcript has been identified as a candidate target, the methods can include evaluating the effects of perturbing the candidate target transcript in an animal model, and the same behavioral assays from Step 1 can be performed in the animal model in the presence and absence of the perturbation, with samples comprising cells from the CNS and immune cells obtained as in Step 1, to validate that the candidate target is a suitable target to alter neuroimmunity. A candidate target can then be selected if the perturbation of that target results in rescue or improvement of the performance of the animal model (e.g., a reduction in fear, in peripheral cytokine levels, etc).

[0058] The candidate targets can thus be identified as targets for treatment of a number of human psychiatric conditions, including major depressive disorder (MDD), anxiety disorder, bipolar disorder, and post-traumatic stress disorder (PTSD). Further investigation can be performed to identify pharmacological and other interventions that alter the target.

[0059] EXAMPLES

[0060] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0061] Methods

[0062] The following materials and methods were used in the Examples below.

[0063] Mice

[0064] Mice were group-housed under a standard light cycle (12h light / dark) (lights on from 7am to 7pm) at 20-23C and humidity (-50%) with ad libitum access to water and food. Genotypes were confirmed by qPCR (Transnetyx). All mice were 2-3 months old at the start of tube restraint, fear conditioning, and stereotaxic injection. Adult male mice 8-10 weeks old and P0-P3 pups were used on a C57B1 / 6J background (The Jackson Laboratory, #000664). M^QNOD.Cg-Prkdsc'dIl2rfniIW,l / Szj (NSG mice, The Jackson Laboratory, #005557) were used for adoptive transfer studies61,62. AldhlllCre~ERT2 +mice63(The Jackson Laboratory, #031008) were bred to B6.Cg-Gt(ROSA)26Sor'm^ #007909) mice64to produce

[0065]

[0066] Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0067] astrocyte reporter mice since this strain does not label neurons or other glial cells63. When Aldhl utdTomato / +reporter mice were used, administration of two doses of 225 mg / kg tamoxifen (Sigma- Aldrich, T5648) diluted in com oil (Sigma-Aldrich, C8267) was administered two days apart before starting the experiment 1-week later.

[0068] TdTomato induction was confirmed by flow cytometry. C57B1 / 6-Gl(ROSA)26Sor'm / ' / / 6A'

[0069]

[0070] . / (The Jackson Laboratory, #007900) females65were bred to C51B\l6-Ccr2eml(lcre'ERT2)PengJ males66(The Jackson Laboratory, #035229) to generate Ccr2DTR +mice. When Ccr2DTR+mice were used, 200mg / kg tamoxifen (Sigma-Aldrich, T5648) diluted in com oil (Sigma-Aldrich, C8267) was administered by oral gavage 4 days before starting the experiment and every 4 days for the duration of the experiment. Mice received doses of 200 mg / kg tamoxifen on days -4, 0, and 4. then received 100 mg / kg tamoxifen every 4 days for the remainder of the experiment. Additionally, 5pg / kg diphtheria toxin (Sigma-Aldrich, #D0564) diluted in IX PBS was administered by intraperitoneal injection 2 days before starting the experiment and every 4 days following the start of the experiment. Depletion of cells expressing Ccr2 was confirmed by flow cytometry. Male Tg(CAG-Kaede)15Kgwa mice were used on a C57B1 / 6J background67. All procedures were approved by the Brigham and Women’s Hospital IACUC.

[0071] Restraint stress

[0072] Tubes were washed with 70% ethanol before every use. 30-40 air holes were drilled using a 1 / 16” drill bit into 50-mL conical tubes (Falcon) for proper ventilation. Mice were placed into tubes and placed horizontally into a tube rack for 6 hours each day for up to 18 consecutive days. Stress sessions were started daily between 9-10 AM. After restraint, mice were returned to their cages and tubes were washed with soap, w ater, and 70% ethanol.

[0073] Contextual fear conditioning

[0074] Fear conditioning w as performed largely as we and others have previously done, with slight modifications6869. Mice were habituated to the testing room 30 minutes- 1 hour each day before the start of fear conditioning and testing of freezing behavior. The testing room was isolated, dimly lit, and kept consistent for each experiment. Trials were recorded for downstream analysis. For fear acquisition, mice were placed in a 22x22cm mouse shuttle box (Maze Engineers) with escape door closed, and black walls scented with 0.1% banana (Shank’s Extracts, #S90159) inAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0075] 70% ethanol. Following a 2-minute habituation period, mice were presented with a white noise tone and chamber light-shock pairings (5 kHz, 70dB), consisting of a 30-second white noise and chamber light co-terminating with a 1 -second 0.7 mA footshock. Intertrial intervals were 30-seconds. Mice remained in the chamber for 30 seconds following the fifth and final trial before being returned to their home cage. The shock chamber was washed with 0.1% banana-containing 70% ethanol between each mouse. Following one day of fear acquisition, mice underwent testing of freezing behavior in response to the CS. Mice were placed in a novel chamber with white walls scented with 0.1% lemon (Shank’s Extracts, S90066) in 70% ethanol. Following a 2-minute habituation, mice received the same visual and auditory' cues as fear acquisition with the same intertrial interval but did not receive a footshock. After the fifth and final trial, mice remained in the chamber for 30 seconds before being returned to their home cage. Analysis of videos was performed using the Noldus Ethovision System (Noldus Information Technology', version 17.0) activity analysis with the activity threshold set at 11. Mice with an activity state below 0.2% were considered freezing, in which the mice exhibited no movement other than breathing. Periodically, automated data analysis was independently validated by manual scoring to confirm accuracy. The freezing ratio was calculated as the time spent freezing divided by the total duration of the period where mice were exposed to the conditioned stimulus (30 seconds). For analysis of psychedelic effects during fear behavior, mice were first subjected to fear acquisition, then 24-hours later, were administered either 1 mg / kg psilocybin or 10 mg / kg MDMA. 2-hours after administration, mice underwent fear extinction, akin to previous studies70.

[0076] Elevated plus maze

[0077] After a 30-minute habituation penod in the behavioral room, mice were placed in the center of the elevated plus maze (EPM), which consisted of two open and two closed arms. The arms measured 35 cm in length, with the maze elevated 61 -cm above the ground, and the closed arms surrounded by 20 cm-high walls with 1-cm-high end plates to prevent mice from falling (Maze Engineers). The room lighting was set to 100 lux, ensuring no shadows on any of the arms. Behavior was recorded over a 5-minute session. After each trial, all arms and the center area were cleaned with 70% ethanol followed by distilled water. The time spent in the open arms and closed arms were automatically analyzed using the Noldus EthoVision XT software (NoldusAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0078] Information Technology, version 17.0). Data are shown as ratio of time in open to closed arms.

[0079] Flow cytometry of tdTomato+cells

[0080] Transgenically labeled tdTomato+cells were sorted largely as previously described24’25,71. Briefly, mice were perfused with IX PBS and the CNS was isolated into 10 mL of enzyme digestion solution consisting of 75 pL Papain suspension (Worthington, #LS003126) diluted in enzyme stock solution (ESS) and equilibrated to 37C. ESS consisted of 10 mL 10X EBSS (Sigma-Aldrich, #E7510), 2.4 mL 30% D(+)-Glucose (Sigma-Aldrich, #G8769), 5.2 mL IM NaHCO3 (VWR, AAJ62495-AP), 200 pL 500 mM EDTA (Thermo Fisher Scientific, #15575020), and 168.2 mL ddH2O, filter-sterilized through a 0.22 pm filter. Samples were shaken at 80rpm for 30-40 minutes at 37C. Enzymatic digestion was stopped with 1 mL of 10X hi ovomucoid inhibitor solution and 20 pL 0.4% DNase (Worthington, #LS002007) diluted in 10 mL inhibitor stock solution (ISS). 10X hi ovomucoid inhibitor stock solution contained 300 mg BSA (Sigma- Aldrich, #A8806), 300 mg ovomucoid trypsin inhibitor (Worthington, #LS003086) diluted in 10 mL IX PBS and filter sterilized using at 0.22 pm filter. ISS contained 50 mL 10X EBSS (Sigma-Aldrich, #E7510), 6 mL 30% D(+)-Glucose (Sigma-Aldrich, #G8769), 13 mL lMNaHCO3 (VWR, #AAJ62495-AP) diluted in 170.4 mL ddH2O and filter-sterilized through a 0.22 pm filter. Tissue was mechanically dissociated using a 5 mL serological pipette and filtered through a 70 pm cell strainer (Fisher Scientific, #22363548) into a fresh 50 mL conical. Tissue w as centrifuged at 500g for 5 minutes and resuspended in 10 mL of 30% Percoll solution (9 mL Percoll (GE Healthcare Biosciences, #17-5445-01), 3 mL 10XPBS, 18 mL ddH2O). Percoll suspension was centrifuged at 500g for 25 minutes with no brakes. Supernatant was discarded and the cell pellet was washed IX with IX PBS, centrifuged at 500g for 5 minutes and prepared for downstream applications. Cells were sorted on a FACS Aria IIu (BD Biosciences). For gating of tdTomato+cells, tdTomato fluorescence was judged against a WT control animal using a yellow-green laser on a FACS Aria IIu.

[0081] Drop-seq

[0082] Drop-seq w as performed as described24,72. A microfluidic mask was fabricated at 125 micrometers in height using soft lithography. Curing agent and PDMS prepolymer (Momentive. #RTV615) were mixed 1 : 10 and degassed in a vacuumAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0083] chamber. The PDMS mixture was poured onto the master mold, further degassed, and baked at 65C for 4 hours. The PDMS replica was punched with a 0.75-mm biopsy punch (Harris Uni-Core) and bonded to a glass slide (75 x 50 x 1.0 mm, Fisher Scientific, 12-550C) using a plasma bonder (Technics Plasma Etcher 500-11). The device was placed on a hot plate at 150C for 10 minutes, baked at 65C for 4 hours, and treated with Aquapel to render it hydrophobic. A cell suspension was prepared using flow cytometry-sorted cells from mouse brain. Cells were counted and resuspended at 250,000 cells / mL (final concentration 125 cells / pL) in PBS and 16% Opti-prep (Sigma- Aldrich, #D1556-250ML). Cell mixture was loaded into a 3 mL syringe (BD Biosciences, #309657) with a 27 gauge needle (BD Biosciences.

[0084] #305109) and connected to the microfluidic device using tubing (Scientific Commodities, #BB31695-PE / 2). Barcoded beads (Fisher Scientific, #NC0927472) were resuspended in lysis buffer consisting of: 57% Opti-prep (Sigma- Aldrich, #D1556-250ML), 2.4% Ficoll PM-400 (GE Healthcare, #17-0300-10), 0.2% Sarkosyl (Teknova, #S3376), 20 mM EDTA pH=8.0, 200 mM Tris-HCl pH=7.4 (Sigma-Aldrich, #T2663-1L), and 50 mM DTT (Sigma-Aldnch, #646563-10X.5ML) at a concentration of 300,000 beads / mL. Beads resuspended in lysis buffer were loaded into a 3 mL syringe with a magnetic mixing disc (V&P Scientific, #772DP-N42-5-2) and gently stirred during encapsulation using a magnetic mixer (V&P Scientific, #710D2). A third syringe was loaded with oil for droplet generation (Biorad, #186-4006). To perform Drop-seq experiments, pumps were run at 1500 pL / hour (cell mixture), 1500 pL / hour (barcoded beads), and 4500 pL / hour (oil) for approximately 15 minutes per sample. Droplets were collected, broken with 1H, 1H, 2H, 2H-Perfluoro-l-octanol (PFO) (Sigma- Aldrich, #370533) added at a ratio of 1:3 PFO:oil, washed with 10 mL 6X SSC (National Diagnostics, #EC-873), and centrifuged for 1 minute at 1000g. Beads at the interface were removed, oil was eliminated, and beads were washed 3X with 6X SSC. Beads were next washed with Maxima H-minus IX RT buffer (Thermo Fisher Scientific, #EP0753). Beads were resuspended in 50 pL of Maxima H-minus IX RT buffer. The following RT mixture was added to each tube of beads: 40 pL 20% Ficoll PM-400, 30 pL 5X Maxima H-minus RT buffer, 2 pL 100 mM dNTPs (Life Technology, #4368813), 5 pL 100 pM template switching oligonucleotide (TSO) primer (IDT), 5 pL RNase inhibitor (Lucigen, #30281-2), 58 pL nuclease free water, and 10 pL Maxima H-minus reverse transcriptase (ThermoAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0085] Fisher Scientific, #EP0753). Beads suspended in RT mixture were incubated at room temperature for 30 minutes on an inverter, followed by 90 minutes at 42C on an inverter. Following reverse transcription, beads were washed IX with 1 mL TE-SDS (10 mM Tns-HCl pH=8.0, 1 mM EDTA pH=8.0, 0.5% SDS), and 2X with TE-TW (10 mM Tns-HCl pH=8.0, 1 mM EDTA pH-8.0. 0.01% Tween-20) and pelleted during each step by centrifuging at 1000g for 1 minute. Beads were washed IX with 10 mM Tris-HCl pH=8.0 and resuspended in exonuclease mix consisting of: 170 pL nuclease free water, 20 pL 1 OX Exonuclease I buffer, and 10 pL Exonuclease I (Thermo Fisher Scientific, #EN0582). Resuspended beads were incubated for 45 minutes at 37C with inversion. After treatment, beads were washed IX with TE-SDS, 2X with TE-TW. and 2X with nuclease free water. Beads were counted and resuspended at a concentration of 80 beads / pL in preparation for PCR. Beads were then added to a PCR tube at a concentration of 2000 beads per tube. PCR mix (25 pL HiFi HotStart Ready Mix (Kapa Biosystems, KK2602); 0.4 pL 100 pM SMART PCR primer (IDT)) was added to each tube and beads were manually mixed before PCR. PCR cycling conditions were: 95C (3 min.); 4 cycles of: 98C (20s), 65C (45s), 72C (3 min); 9 cycles of: 98C (20s), 67C (20s), 72C (3 min); 72C (5 min); 4C hold.

[0086] Following PCR, cDNA samples were purified in a 96-well plate (Biorad, HSP9611) on a magnetic stand (NEB. #S 151 IS) using Agencourt AMPure XP magnetic beads (Beckman Coulter. #A63881) at a 0.6X ratio according to the standard protocol. The sample from each PCR tube was eluted in 10 pL nuclease free water, and technical replicates were pooled following elution. cDNA was run on a Bioanalyzer High Sensitivity DNA chip (Agilent Technologies, #5067-4626) on a 2100 Bioanalyzer (Agilent). For cDNA library tagmentation. 600 pg of cDNA in 5 pL was added to 10 pL of Nextera Tagment DNA buffer and 5 pL of Amplicon Tagment Mix (Illumina, #FC-131-1096). cDNA was tagmented at 55C for 5 minutes, followed by addition of room temperature-equilibrated 5 pL Neutralize Tagment buffer (Illumina, #FC-131-1096). Samples sat at room temperature for 5 minutes followed by addition of 15 pL Nextera PCR Mix (Illumina. #FC-131-1096), 8 pL nuclease free H2O, 1 pL 10 pM New-P5-SMART PCR hybrid oligo (IDT), and 1 pL of 10 pM Nextera indexing oligonucleotide (Invitrogen). Samples were PCRed using the following conditions: 95C (30s); 12 cycles of: 95C (10s), 55C (30s), 72C (30s); 72C (5 min); 4C hold. Tagmented libraries were purified using Agencourt AMPure XP magnetic beads at aAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0087] 0.6X ratio according to the manufacturer’s protocol and eluted in 10 pL nuclease free H2O. Samples were then run on a Bioanalyzer 2100 to assess library size. Libraries were quantified by qPCR using a Library Quantification Kit (Kapa Biosystems, #KK4824). Tagmented libraries were pooled at 5nM concentration and sequenced using aNovaSeq SI at the Broad Institute using a Custom Read 1 primer and 20+80 bp reads. Raw fastq files were processed as we have previously done21,22’24’25’71using the Drop-Seq Resources pipeline72aligned against the mmlO mouse genome assembly. A digital count matrix was then generated for downstream processing. Thresholds for cells included in the analysis were 200-3,000 genes expressed per cell, at least 2 cells expressing a gene, and <50% mitochondrial reads. Statistics per cell for those included in the final dataset were 447 genes. 772 UMIs. and 6.2% mitochondrial reads per cell. Data were normalized by read counts, highly variable genes extracted, data were then filtered and scaled, followed by PC A. Trajectories were created using RNA velocity' in scanpy73by briefly, drawing a force directed graph, determining spliced and unspliced transcripts, developing a pseudotime kernel, and computing a transition matrix that was supenmposed on the UMAP embedding. Differential expression analysis was performed by comparing each cluster to all others using scanpy. tl.rank genes groups, which were then used in downstream analyses such as Qiagen IPA by using the fold change of the cluster of interest relative to all others.

[0088] Harvesting of plasma

[0089] Blood was removed from the aorta with a 21G needle (Becton Dickinson, #305165) and placed in a blood collection tube containing the anti-coagulant heparin (Becton Dickinson, #365965) on ice. Blood was centrifuged at 4C for 10 minutes at 5000g and the upper, clear layer comprising the plasma was transferred to a 1.5-mL Eppendorf tube. Plasma was stored at -80C and used for downstream multiplexed Luminex assays.

[0090] Multiplexed Luminex assays

[0091] The multiplexing analysis was performed using the Luminex 200 system. For some samples, two markers were simultaneously measured in the samples using the Human Circadian / Stress Panel 2-Plex Custom Assay (MilliporeSigma, #HNCSMAG-35K) according to the manufacturer's protocol. The 2-plex consisted of Cortisol and Melatonin. Assay sensitivity is >4.3 pg / mL. For a second set of samples, a mouse high sensitivity 18-plex discovery assay (MilliporeSigma. #FCYTMAG20KPX19BK)Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0092] was used according to the manufacturer's protocol. The 18-plex consisted of GM-CSF, IFN-y, IL-la, IL-ip, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10. IL-12(p70), IL-13, IL-17A, KC / CXCL1, LIX, MCP-1, MIP-2 and TNFa. Assay sensitivity for each marker is >0.06 pg / mL.

[0093] Cloning of AAV vectors

[0094] Constructs were derived from the pAAV-FLEX-SaCas9-U6-sgRNA backbone (Addgene, #124844), a gift from Larry Zweifel74. The FLEX-containing portion was eliminated by PCR-based amplification of saCas9 using the forward primer 5’-aaaGTCGACGGCGCGCCatggccccaaagaagaagcggaa-3’ and the reverse primer 5’-tttgaattcTTAAGCGTAATCTGGAACATCGT-3’, followed by restriction digest using EcoRI-HF (NEB, #R3101S) and Sall-HF (NEB, #R0138S). Successfully cloned plasmids were sequenced by Plasmidsaurus. The plasmid was then digested with Xbal (NEB, #R0145S) and Sall-HF (NEB, #R0138S) in nuclease-free water (Thermo Fisher Scientific, #AM9937) and CutSmart buffer. The linearized plasmid was run on a gel and purified (Qiagen. #28704). To substitute promoters, the GfaABCID and hSyn promoters were amplified by PCR, PCR purified (Qiagen, #28104), and digested with Xbal (NEB, #R0145S), Sall-HF (NEB, #R0138S), and Dpnl (NEB, #R0176S) in CutSmart buffer. The inserts were ligated into the plasmid with T4 DNA ligase (NEB, #M0202S) in nuclease-free water (Thermo Fisher Scientific, #AM9937) and T4 DNA ligase reaction buffer and proper cloning was confirmed with whole-plasmid sequencing (Plasmidsaurus). For cloning CRISPR sgRNAs, the sgRNA oligos were phosphorylated at 37C in PNK (NEB, #M0201S) and ligase buffer. Subsequently, the phosphory lated oligos were placed in a heat block at 100C for 5 minutes and allowed to cool to room temperature for 1-2 hours. The parent plasmids containing the promoters were digested with BsaI-HFv2 (NEB, #R3733S) in CutSmart buffer and PCR purified (Qiagen, #28104) into 50 pL of EB buffer. 4 pL of PCR-purified plasmid w as added to 10 pL of annealed oligo mix with 1 pL of T4 DNA ligase (NEB, #M0202S) and incubated for 2 hours at room temperature. Ligation products were transformed into NEB Stable Competent E. coli (NEB, #C3040H) and plated on LB agarose (Thermo Fisher Scientific, #MP113002221) plates with ampicillin (Thermo Fisher Scientific, #11593027) at 37C overnight. Successful colonies were grown overnight in LB broth (Thermo Fisher Scientific, #50488764) supplemented with ampicillin (Thermo Fisher Scientific. #11593027). Cultures were purifiedAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0095] (Qiaprep Spin Miniprep, #27104) and digested with Hindlll-HF (NEB, #R3104S) and BsaI-HFv2 (NEB, #R3733S) in CutSmart buffer to confirm cloning of sgRNAs. Successful colonies were stored in 50% glycerol (Sigma Aldrich, #G5516) at -80C. All sgRNA oligos were used at a concentration of lOOpM, desalted, and scaled at 25 nmole (Thermo Fisher Scientific, custom standard DNA oligos). sgRNA sequences were designed for saCas9 cutting using CRISPick: mouse GRCm38 reference genome. Sequences were chosen by focusing on those cutting >5% of the gene and targeting the most upstream exon position.

[0096] The genes targeted for knockdown using the pAAV -GfaABC lD-saCas9-U6- sgRNA delivery' system were:

[0097] Sequence SEQ ID NO: Rosa26 F 5’- AAACTCAGAGAGCCTCGGCTAGGTA-3’ 1

[0098] Rosa26 R 5 ’-C ACCTACCTAGCCGAGGCTCTCTGAGTTT-3 ’ 2

[0099] Eg / r F 5 -CACCCATTGCCCTCAACACCGTGGA-3’ 3

[0100] Egfr R 5 ’ -AAACTCC ACGGTGTTGAGGGC AATG-3 ’ 4

[0101] Ptprs F 5’-CACCCATGAATTGTGATTTCGCCCA-3’ 5

[0102] Ptprs R 5 ’ - AAACTGGGCGAAATC AC AATTC ATG-3 ’ 6

[0103]

[0104] The genes targeted for knockdown using the pAAV -hSyn-sciCas9-U6-sgRNA delivery system were: 5 ’-AAACTCAGAGAGCCTCGGCTAGGTA-3’,

[0105] Sequence SEQ ID NO: Rosa26 F : 5 ’ -AAACTCAGAGAGCCTCGGCTAGGTA-3 ’ 7

[0106] Rosa26 R 5’-CACCTACCTAGCCGAGGCTCTCTGAGTTT-3’ 8

[0107] Nr2f2 F 5’-CACCGCTGCTGCTGCTTGTCGCTGC-3‘ 9

[0108] Nr2f2 R 5 -AAACGCAGCGACAAGCAGCAGCAGC-3 ’ 10

[0109] Etsl F 5 ’ -C ACCGAGGATCTTCAAAAGCGTTAA-3 ’ 11

[0110] Etsl R 5’-AAACTTAACGCTTTTGAAGATCCTC-3’ 12

[0111] Cebpg F 5’-CACCGTCGCAGCCAGCCACTACTCC-3’ 13

[0112] Cebpg R 5 -AAACGGAGTAGTGGCTGGCTGCGAC-3 ’ 14

[0113] Slitrk2 F 5 ’ -C ACCAGCTGATAGATGCGATACTGG-3 ’ 15

[0114] Slitrk2 R 5’-AAACCCAGTATCGCATCTATCAGCT-3’. 16

[0115]

[0116] Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0117] AAV production

[0118] To produce AAV, previously described protocols were largely followed75’76. Briefly, pAAV2 / 5 (Addgene, #104964, a gift from Melina Fan), pAdDeltaF6 (Addgene, #112867, a gift from James M. Wilson), pAAV-GfaABClD-saCas9-U6-sgRNA, and pAAV-hSyn-saCas9-U6-sgRNA were maxi prepped (Qiagen, #12863). AAV pro 293T cells (Takara, #632273) were grown to confluency in DMEM (Life Technologies, #11965092) supplemented with 10% FBS (Life Technologies.

[0119] #10438026) and 1% penicillin / streptomycin (Life Technologies, #15140122) in 150mm tissue culture plates (Coming, #353025). Media was changed to DMEM (Life Technologies, #11965092) supplemented with 2% FBS (Life Technologies, #10438026) 2 hours before transfection. For transfection, 11.4-pg of pAAV2 / 5 (Addgene, #104964), 5.7-pg of pAdDeltaF6 (Addgene, #112867), and 2.9-pg of pAAV-GfaABC lD-saCas9-U6-sgRNA or pAAV-hSyn-saCas9-U6-sgRNA were pooled in 500-pL of Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, #31985070). 80 pL of 1 mg / mL PEI MAX (Polysciences, #49553-93-7) in nuclease-free water (Thermo Fisher Scientific, #AM9937) was added to 500-pL of Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, #31985070) and incubated for 5 minutes at room temperature. The PEI and Opti-MEM solution was added to the Opti-MEM solution containing the plasmids, vortexed, and incubated at room temperature for 20 minutes. The transfection solution was then added dropwise to the 293T cells (Takara, #632273), and the plate was gently swirled to mix the solution. After 24 hours, the media was removed and 20-mL of DMEM (Life Technologies, #11965092) supplemented with 10% FBS (Life Technologies, #10438026) and 1% penicillin / streptomycin (Life Technologies #15140122) was added to the plate. The media was collected in a 250-mL solution bottle 48 hours after the media change, stored at 4C, and replaced with another 20-mL of DMEM (Life Technologies, #11965092) supplemented with 10% FBS (Life Technologies, #10438026) and 1% penicillin / streptomycin (Life Technologies, #15140122). The media was collected following another 48 hours and pooled into the 250-mL solution bottle. 2-mL of IX PBS was added to the tissue culture plate, and cells were detached with a cell scraper (Westnet Inc, #229311). The cells were pooled into the 250-mL solution bottle with the collected media, and residual cells were removed with 10-mL of IX PBS and collected into the 250-mL solution bottle. The media and cells were centrifuged for 15Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0120] minutes at 2000g at room temperature and supernatant was collected. 400g of PEG8000 (Millipore Sigma, #P5413) and 146.1g ofNaCl (Millipore Sigma, #SX0420-3) were dissolved in 1 liter of ddH2O. The viral supernatant was removed and incubated with 1 :4 volume of 40% w / v PEG8000, 2.5M NaCl solution for 2 hours on ice or overnight followed by centrifugation at 4000g for 30 minutes at 4C. 9.22g of NaCl (Millipore Sigma, #SX0420-3), 4.85g of Tris base (MP Biomedicals, #819620), and 2.03g of MgC12 6H2O (Macron Fine Chemicals, #5958-04) were dissolved in 1 liter of distilled water to make SAN buffer. 700U of SAN (Arcticzymes, #70910202) was added to 7-mL of SAN buffer, and the supernatant pellet was resuspended in 1-mL of SAN+SAN buffer. The remaining viral pellet was resuspended with 5-mL of SAN+SAN buffer and both suspensions were incubated in a water bath for 1 hour at 37C. The cell suspensions were centrifuged for 10 minutes at 2000g at 4C.

[0121] Supernatant was collected, and an equivalent volume of chloroform (Millipore Sigma, #J67341) was added to each tube. The solution was vortexed for 3 minutes and spun down for 10 minutes at 4000g at 4C. Ammonium sulfate (Millipore Sigma, #A4418) was dissolved in distilled water to make 40% w / v ammonium sulfate solution.

[0122] PEG8000 (Millipore Sigma, #P5413) was dissolved in distilled water to make 40% PEG8000 solution. PEG8000 and ammonium sulfate solution were combined with virus to a final concentration of 10% and 13.2%, respectively. The solution was vortexed and incubated at room temperature for 20 minutes. Following incubation, the solution was centrifuged for 15 minutes at 3000g at 25C, and the clear, bottom layer containing AAV was removed with a syringe. The AAV was transferred to an Amicon Ultra-15 Centrifugal Filter Unit (EMD Millipore, #UFC910024) and centrifuged for 15 minutes at 3000rpm at 4C. 10-mL of IX PBS was added to the Amicon Ultra-15 Centrifugal Filter Unit (EMD Millipore, #UFC910024) once the volume of AAV was at 1 -mb, and the AAV was centrifuged for 15 minutes at 3000rpm at 4C. This process w as repeated a total of 3 times w ith IX PBS. The total volume w as then brought to 200-pL. and the virus was stored at -80C. AAV was titered with the qPCR AAV Titer Kit (Applied Biological Materials, #G931) according to the manufacturer’s protocol. AAV5-CamKIIa-mCherry was a gift from Karl Deisseroth (Addgene viral prep #114469-AAV5).Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0123] Stereotaxic injections

[0124] Intracranial injections were used to deliver concentrated AAV to mice as previously described21 25 71. Mice were anesthetized with 1-3% isoflurane (Covetrus, #11695067772) in oxygen. Heads were shaved and cleaned with ethanol and betadine (Thermo Fisher, #19-027132) and a small incision was made in the skin with a scalpel (Exel International, #29550). Bilateral injections were made targeting the midpoint of the basolateral amygdala with a 5-pL Hamilton syringe (Sigma- Aldrich, #20787) at + / - 3.3 (lateral), -1.6 (posterior), -4.5 (ventral) relative to Bregma using a stereotaxic alignment system (Kopf, #1900) or atlas-integrated robotic stereotaxic system (Neurostar); virus was injected at a rate of 75 nL / min over 4 minutes. After injection, the needle remained at the injection site for 10 minutes before being withdrawn at a rate of 1-mm / minute. Mice were sutured (Ethicon, #1667H) and allowed to recover in a clean cage on a heating pad for 1 hour. Instruments were sterilized between each mouse using a Glass Bead Sterilizer (CellPoint Scientific, #GRM5-1450). Mice recovered for >2 weeks before experimental use. Stereotaxic targeting and knockdown were validated by immunostaining, genomic identification of marker genes, and / or integration with previously published genomic samples. For analysis of BLA coverage by excitatory neurons, AAV targeting either Nr2f2 or Rosa26 was injected into the same mouse in opposing hemispheres, with a 1:10 dilution of AAV5-CaMKIIa: :mCherry. In these experiments, mice underwent restraint stress, fear conditioning, and fear extinction.

[0125] Forced swim test

[0126] Mice were habituated to the testing room 30 minutes- 1 hour each day before the start of the forced swim test. Mice were placed in a 2000-mL glass beaker (Coming, #1000-2L) filled with 1300-mL of water between 20-22C and recorded for 6 minutes. Following the end of the 6-minute duration, mice were returned to their home cage on a heating pad and monitored for 30 minutes. To quantify behavior during the forced swim assay, we trained a model using DeepLabCut77,78to systematically quantify swimming behavior in this assay. Briefly. 10 training videos of mice in the assay between 10-20 seconds were recorded, varying the lighting, angle, mouse coat color, and position of testing apparatus. Approximately 200 frames were manually annotated to identify the forepaws, hindpaws, nose, ears, and base of the tail. A resnet_vl_50 model was then trained for 201,000 iterations, which wasAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0127] judged to be sufficient based on a plateau of rate of error change. Testing accuracy was measured within DeepLabCut and found to be within an error range of 20 pixels from the trained dataset. Next, the last 4 minutes of experimental forced swim videos were analyzed within the Google Colab version of DeepLabCut, and the positions of each body part at each frame were recorded. The .csv files containing the body position coordinates were analyzed in RStudio version 4.3.0 as described79. In brief, data was preprocessed with imputeTS to interpolate values with likelihood values greater than 0.95 and body position pixel coordinates were converted to centimeters. The area of the mice was calculated as a polygon using the coordinates of the left ear, right ear, nose, and tail base. The immobility' time calculated for the forced swim test was calculated using the FSTAnalysis function pre-validated settings in the DLCAnalyzer R package. Mice were considered immobile if the rate of change of their body area was below a floating cutoff of 0.03.

[0128] Immunostaining

[0129] Immunostaining was performed largely as we have done previously21 25'71. Mice were intracardially perfused with ice cold IX PBS and the brain was embedded in OCT (Sakura, #4583) and flash frozen using 2-methylbutane (Sigma- Aldrich, #M32631). Then, the brain was sectioned into 20 pm sections onto SuperFrost Plus slides (Fisher Scientific, #15-188-48), and sections were fixed in PFA (Electron Microscopy Sciences, # 15714-S) freshly diluted to 4% in IX PBS for 10 minutes at room temperature. Sections were then washed with IX PBS and let dry. A hydrophobic boundary was drawn around the tissue (Vector Laboratories, #H-4000) and sections were washed 3X for 5 minutes with 0.3% Triton X-100 (Thermo Fisher Scientific, #NC9356391) in PBS (PBS-T) then blocked in 10% donkey serum or 5% goat serum for 30 minutes at room temperature. Sections were then incubated with primary antibodies diluted in IX blocking buffer overnight at 4C. Following primary antibody incubation, sections were washed 3X with 0.3% PBS-T and incubated with secondary' antibodies diluted in blocking solution for 2 hours at room temperature. For synapse staining, samples were permeabilized in 0.5% PBS-T for 15 minutes prior to blocking, washed using 0.1% PBS-T, and secondaries were used for 1-hour at room temperature. After secondary antibody incubation, sections were washed 3X with 0.3% PBS-T, dried, and coverslips were mounted using Fluoromount-G with DAPI (SouthemBiotech, #0100-20). For immunocytochemistry, isolated cells were platedAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0130] on a p -Plate 96 Well Square (iBidi, 89626) after poly-L-lysine (Sigma Aldrich, #P4707) coating. After 24 hours, the cells were fixed with 4% PFA for 15 minutes, followed by permeabilization with 0.3% PBS-T for 15 minutes. Subsequently, the cells were incubated with a blocking buffer (1% BSA, 10% FBS in PBS) for 1 hour and then stained using the same protocol as tissue immunostaining with Hoechst 33342 (Invitrogen, 1:10,000, #H3570). Primary antibodies used in this study were: rabbit anti-mCherry (Abeam, 1:500. #abl67453), chicken anti-GFAP (Abeam, 1:200, #ab4674), mouse anti-GFAP (Millipore, 1 :500, #MAB360), rabbit anti-EGFR (D38B1) (Cell Signaling, 1:100, #4267), rabbit anti-GFP (Abeam, #ab290, 1:400), chicken anti-GFP (Abeam, 1:200, #abl3970), rabbit anti-Homerl (Synaptic systems, 1:500, #160002). guinea pig anti-VGLUT2 (Sigma, 1:1000. #AB2251-I). rabbit anti-cFos (Cell Signaling, 1:100, #2250S), rabbit anti-PTPRS (Proteintech, 1:100, #13008-1-AP), rabbit anti-IL-lRl (Abeam, 1:100, #abl06278), mouse anti-SlOOb (Millipore Sigma, 1:200, #S2532), rabbit anti-Collagen IV (BioRad, 1:100, #2150-1470), rabbit anti-Ibal (Abeam, 1:100, #abl78846), chicken anti-GFP (Abeam, 1:100, #abl3970), and mouse anti-TUBB3 (BioLegend, 1:500, #801201). Secondary antibodies used in this study were: donkey anti-rabbit IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 555 (Invitrogen, #A31572), donkey anti-rabbit IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen, #A21206), donkey anti-mouse IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen, #A21202), donkey anti-chicken IgY (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen, #A78948), Alexa Fluor 594 Goat anti-Guinea pig IgG (H+L) (Invitrogen, #A11076), goat anti-rabbit IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen, #A11034), goat anti -rabbit IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 594 (Invitrogen, #A11037), goat antimouse IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 594 (Invitrogen, #A11032), and goat anti-chicken IgY (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen, #A11039). all at 1:500 working dilution. Immunostaining of EGFP w as detected from AAV5 hSyn-eNpHR3.0-EYFP (Addgene, #26972, a gift from Karl Deisseroth80). The biotinylated IL-lb was labeled by Oregon Green 488 conjugate of NeutrA vidin biotin-binding protein (Invitrogen, 1:200, #A6374). For quantification of mCherry coverage in excitatory neurons, images of the amygdala w ere captured using a 63X objective lens with z-stack of 3 images at maximum intensity. Regions of interest (ROIs) were uniformly applied in FIJI (ImageJ, v 1.54k) using normalizedAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0131] intensity to discriminate dendritic patterns across all images, and coverage was quantified through the measurement tool in FIJI.

[0132] Confocal imaging and quantification

[0133] Sections were imaged on a Zeiss LSM710 confocal using a 5X, 10X (tile scans), 20X, or 63X objective. For all analyses, the amygdala was identified based on DAPI fluorescence. Imaging was performed using the LSM710 smart setup parameters with each channel acquired in sequence to minimize crosstalk between channels to essentially zero. Quantification of specific markers in brain sections was performed by first quantifying the number of positive cells for the given cell type marker (e.g., GFAP, Ibal) and then counting the absolute number of that type positive for a molecular marker, with a visible cell body in the DAPI channel. For analysis of synapses, VGLUT2 and Homerl puncta co-localization was determined by generating a high density signal mask for the VGLUT2 channel for each image in FIJI, followed by using automated thresholds to isolate only the puncta for both VGLUT2 and Homerl channels, largely as we did before81. These puncta were quantified and the number of overlapping puncta were normalized by the high-density area mask. For quantifying cFos-positive cells, images of the amygdala were acquired using a 2x2 tile scan with a 20x objective lens. The cFos signals were detected by setting an automated threshold, and noise was minimized by filtering out puncta smaller than 10 pixels. The number of cFos-positive puncta was normalized to the total area of the imaged region. These parameters were consistently applied across all experimental groups. To measure the intensity of astrocytic PTPRS and IL-1R1, 5 z-stack images were acquired at 0.5 pm intervals. Regions of interest (ROIs) were then defined based on SI 00b signals using FIJI, and maximal intensity projections were used to quantify signal intensities for each marker within these ROIs. For immunocytochemistry, two 20X images per mouse were captured and quantified using FIJI.

[0134] Isolation of astrocytes from adult mice

[0135] Astrocytes were isolated by magnetic-activated cell sorting (MACS) with the Miltenyi ACSA-2 Microbead Kit (Miltenyi Biotec, #130-097-679) akin to previous protocols82. Briefly, mice were perfused with IX PBS, and using a stainless-steel mouse brain matrix (Kent Scientific, RBMS-205C) and a dissecting microscope, the amygdala was isolated into 500-uL of Hibemate-A (Thermo Fisher Scientific, #A12475-01) containing 7.5-uL of papain suspension (Worthington, #LS003126) andAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0136] B-27 Supplement (Thermo Fisher Scientific, #17504044). Tissue was shaken at 80 rpm for 20 minutes at 37C, mechanically dissociated using a P1000 pipette, and filtered through a 70-um cell strainer (Millipore Sigma, #B AHI 36800070). Cells were resuspended in 90 uL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS and 10 uL of FcR blocking reagent and incubated at 4C for 10 minutes. 10 uL of Anti-ACSA-2 MicroBeads were added, and cells were incubated at 4C for an additional 15 minutes. Cells were washed with 1-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS, centrifuged at 500g for 5 minutes, and resuspended in 500 uL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS. The cell suspension was applied onto an LS column (Miltenyi Biotec, #130-042-401) on a QuadroMACS Separator pre-rinsed with 3-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS. The column was washed 3 times with 3-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS, removed from the separator, and placed onto a 15-mL conical tube. 5-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS was added to the column, and astrocytes were removed with a plunger. Cells were centrifuged at 500g for 5 minutes, resuspended in 100 pL of Picopure extraction buffer (Thermo Fisher Scientific, #KIT0204), and incubated in a heat block at 42C for 30 minutes. Samples were stored at -80C until further processing.

[0137] Isolation of neurons from adult mice

[0138] Neurons were isolated by MACS with the Miltenyi Adult Neuron Isolation Kit (Miltenyi Biotec. #130-126-602) akin to previous protocols82. Briefly, mice were perfused with IX PBS, and using a stainless-steel mouse brain matrix (Kent Scientific, RBMS-205C) and a dissecting microscope, the amygdala was isolated into 500-uL of Hibemate-A (Thermo Fisher Scientific, #A12475-01) containing 7.5-uL of papain suspension (Worthington, #LS003126) and B-27 Supplement (Thermo Fisher Scientific, #17504044). Tissue was shaken at 80 rpm for 20 minutes at 37C, mechanically dissociated using a Pl 000 pipette, and filtered through a 70-um cell strainer (Millipore Sigma, #B AHI 36800070). Cells were centrifuged at 500g for 5 minutes, resuspended in 90-uL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS and 10-nL of Adult Non-Neuronal Cell Biotin- Antibody cocktail, and incubated for 5 minutes at 4C. Cells were washed with 1-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS, centrifuged at 500g for 5 minutes, and resuspended in 90-uL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS and 10-uL of Anti-Biotin Microbeads and incubated for 10 minutes at 4C. 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS was added to aAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0139] total volume of 500-uL, and the cell suspension was applied onto an LS column (Miltenyi Biotec. #130-042-401) pre-rinsed with 3-mL of 0.5% BSA. 2 mM EDTA pH=8.0 in IX PBS. Neurons were collected into a 15-mL conical tube on ice, and the column was washed with 2-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS to collect remaining cells. Cells were centrifuged at 500g for 5 minutes, resuspended in 100-uL of Picopure extraction buffer (Thermo Fisher Scientific, #KIT0204), and incubated in a heat block at 42C for 30 minutes. Samples were stored at -80C until further processing.

[0140] Bulk RNA-seq

[0141] Bulk RNA isolated from sorted cells was processed using the PicoPure kit (Thermo Fisher, #KIT0204) and used as input with the kit (NEB, #E6420) according to the manufacturer’s protocol. Reverse transcription was performed according to the Smart protocol using a template switching oligo. Then, cDNA was amplified and cleaned using Ampure XP beads (Beckman-Coulter, #A63881) and quantified using a Bioanalyzer DNA HS assay (Agilent, #50674626). Libraries were then fragmented, end-repaired, and ligated to Illumina compatible adaptors followed by sample barcoding using NEBNext Multiplex Oligos for Illumina (#E7335S, #E7500S, #E7710S, #E7730S). Samples were selected again using Ampure XP beads. Final libraries were quantified using a Bioanalyzer and a Kapa library quantification kit (Kapa Biosystems, #KK4824). The libraries were then diluted and pooled at 4nM in nuclease-free water (Thermo Fisher Scientific, #AM9937), and run on an Illumina NextSeq550 as 1x75 bp reads with 6-bp index read and de-multiplexed into FASTQ files. The fastq files of each RNA-seq data sample were aligned to the A / u.s musculus GRCm38 transcriptome using STAR (version 2.7.5c)83. The raw sequencing reads were quality assessed using fastp (version 0.23.2)84, fastQC (version 0.11.9), and reported using multiqc (version 1.11)85. Processed RNA-seq data was filtered, removing genes with low read counts (average counts >0.5). Feature counts were tabulated with featureCounts (version 2.0.1)86. Differential expression analysis was done using EdgeR (version 3.42.4)87. Enrichment plots for RNA-Seq data were generated using GSEA88using molecular signatures from canonical pathways:

[0142] KEGG / Reactome / Biocarta (c2.cp.all), Gene ontology7(c5.cp.all), and Hallmark (h.all) in astrocytes, neurons, and monocytes individually. Pathway analysis was performed using IPA (Qiagen), ENRICHR89 9I. and StringDB92. For all IPA analyses, at mostAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0143] 8,000 genes were filtered on log fold change and uploaded for both upregulated and downregulated genes. For ENRICHR analyses, genes were selected based on statistical significance and a common fold change direction. For StringDB analyses, significantly downregulated genes in sgNr2f2 vs. sgRosa26 associated with the GOBP Synaptic Signaling pathway (G0:0099536) were used as input, then PPI networks were clustered with k-means clustering using a resolution of 3 clusters. Cluster 2 is shown. Bulk RNA-seq data of amygdala neurons were obtained as fastq files under the GEO accession numbers GSE16241793; GSE6634594; GSE13026895;

[0144] GSE13852296; GSE18309297; and GSE15179898, and processed according to the parameters described above.

[0145] Primary astrocyte cultures

[0146] Procedures were performed largely as described previously21 25'71. Brains of mice aged P0-P3 were dissected into HBSS (Thermo Fisher Scientific, #14025-134) on ice. The tissue was mechanically dissociated, centrifuged at 500g for 10 minutes at 4C, and resuspended in 0.25% Trypsin-EDTA (Thermo Fisher Scientific, #25200-072) at 37C for 10 minutes. Trypsin was neutralized by adding DMEM / F12+GlutaMAX (Thermo Fisher Scientific, #10565018) supplemented with 10% FBS (Thermo Fisher Scientific, #10438026) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148). Cells were passed through a 70 pm cell strainer. Cells were centrifuged at 500g for 5 minutes at 4C. resuspended in DMEM / F12+GlutaMAX (Thermo Fisher Scientific, #10565018) supplemented with 10% FBS (Thermo Fisher Scientific, #10438026) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148) and cultured in T-75 flasks (Falcon, #353136) pre-coated with poly-L -lysine (Sigma Aldrich, #P4707) for 1 h at 37C and washed with IX PBS. Cells were cultured at 37C in a humidified incubator with 5% CO2, for 7-10 days until confluency was reached. Media was replaced every 2-3 days.

[0147] Microglia were removed by shaking for 30 minutes at 180 rpm and media was replaced. To plate astrocytes, the flask was shaken for 2 hours at 220 rpm, media was removed, and 5-mL of trypsin was added at 37C for 3 minutes. Trypsin was neutralized with DMEM / F12+GlutaMAX (Thermo Fisher Scientific, #10565018) supplemented with 10% FBS (Thermo Fisher Scientific, #10438026) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148). Cells were centrifuged at 500g for 5 minutes at 4C, resuspended in DMEM / F12+GlutaMAXAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0148] (Thermo Fisher Scientific, #10565018) supplemented with 10% FBS (Thermo Fisher Scientific, #10438026) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148), and cultured in a tissue culture-treated plate pre-coated with poly-L-lysine (Sigma Aldrich, #P4707). Astrocytes were plated for at least 5 days before in vitro treatments.

[0149] In vitro stimulation of mouse primary cells

[0150] For astrocytes, neurons, and astrocyte-neuron co-cultures from mice, stimulations were performed for 24 hours with compounds diluted in DMEM / F12+GlutaMAX (Life Technologies, #10565042) supplemented with 10% FBS (Life Technologies, #10438026) and 1% penicillin / streptomycin (Life Technologies, #15140122). For peripheral CD1 lb+cells from mice, stimulations were performed for 6 hours with molecules diluted in DMEM / F12+GlutaMAX (Life Technologies, #10565042) supplemented with 10% FBS (Life Technologies, #10438026), 1% penicillin-streptomycin (Life Technologies, #15140122), 1% sodium pyruvate (Life Technologies, #11360070), 1% HEPES (Life Technologies, #15630106) and 1% MEM non-essential amino acids (Life Technologies,

[0151] #11140050). The following molecules were used to stimulate astrocytes, neurons, and astrocyte-neuron co-cultures: 50 ng / mL TNF (R&D Systems, #410-MT-010), 100 ng / mL IL-ip (R&D Systems, #401-ML-005), 100 ng / mL IL- 12 (PeproTech, #210-12), and 200 ng / mL corticosterone, a physiologic dose chosen based on the serum concentration in chronically stressed mice" (Millipore Sigma, #27840). The following molecules were used to stimulate monocytes: 10-pM psilocybin (NIDA Drug Supply Program), 10-pM MDMA-HC1 racemate (NIDA Drug Supply Program), 20-pM MDL 100907 (Tocris Biotechne. #4173), 200 ng / mL corticosterone (Millipore Sigma, #27840) and 10 ng / mL LPS (InvivoGen, #tlrl-3pelps).

[0152] RNA isolation from primary cells

[0153] Primary' cells were lysed in buffer RLT (Qiagen), and RNA was isolated using the Qiagen RNeasy Mini kit (Qiagen, #74106). cDNA was transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, #4368813). Gene expression was then measured by qPCR using Taqman Fast Universal PCR Master Mix (Life Technologies, #4367846). Taqman probes used in this study are: Gapdh (Mm999999 I5_g 1 ), Illb (Mm0262011 l_sl), Ptprs (Mm00465150_ml), Cebpg (Mm01266786_ml), Etsl (MmOl 175819 ml), Nr2f2 (Mm00772789_ml),Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0154] Cxcr3 (Mm99999054_sl), Cxcr4 (Mm01996749_sl), Cx3crl (Mm02620111_sl), Htr2a (Mm00555764_ml), Htr2b (Mm00434123_ml), Htr2c (Mm00434127_ml), Ntrk2 (Mm00435422_ml), Illrl (Mm00434237_ml), m Actb (Mm()26l9580_gl). qPCR data were analyzed by the ddCt method by normalizing the expression of each gene for each replicate to Gapdh and then to the control group.

[0155] Primary astrocyte-neuron co-cultures

[0156] Procedures were performed largely as described previously21 25 71using the Worthington papain dissociation system (Worthington Biochemical Corporation, #LK003150). Brains of mice aged P0-P3 were dissected into HBSS (Thermo Fisher Scientific, #14175103) supplemented with 1% sodium pyruvate (Invitrogen, #H-4034), 0.1% w / v glucose (Sigma- Aldrich, #G-6152) in nuclease-free water (Thermo Fisher Scientific, #AM9937), and 1% HEPES (Invitrogen, #H-4034) in a petri dish on ice. The meninges and brainstem were removed, and the remaining tissue was placed in a 15-mL conical tube with papain and DNase in 5.35-mL of EBSS and incubated at 37C for 30 minutes. Brains were dissociated with a P1000 pipette, and cells were passed through a 70 pm filter into a 50-mL conical tube. Albumin ovomucoid inhibitor was reconstituted in 32-mL of EBSS, and cells were resuspended in 2.7-rnL EBSS supplemented with 0.3-mL of reconstituted albumin ovomucoid inhibitor and 150-uL of DNase I. The cell solution was layered on top of 5-mL of ovomucoid inhibitor and centrifuged at 100g for 5 minutes at 22C. Cells were resuspended in MACS Neuro Medium (Miltenyi Biotec, #130-093-570) supplemented with 2% MACS NeuroBrew-21 (Miltenyi Biotec, #130-093-566) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148). Cells were plated in tissue culture plates coated with laminin and poly -L -ornithine (Westnet, #354659). Media was changed after 48 hours then changed every 3 days. After 10-14 days of plating, co-cultures were transduced with AAV diluted in in MACS Neuro Medium (Miltenyi Biotec, #130-093-570) supplemented with 2% MACS NeuroBrew-21 (Miltenyi Biotec, #130-093-566) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148). Half of the total volume of media was changed every 3 days following AAV transduction. The following molecules in MACS Neuro Medium (Miltenyi Biotec, #130-093-570) supplemented with 2% MACS NeuroBrew-21 (Miltenyi Biotec, #130-093-566) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148) were used to stimulate co-cultures 7 days after transduction:Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0157] 100 ng / mL IL- 10 (R&D Systems, #401-ML-005) and 200 ng / mL corticosterone (Millipore Sigma, #27840). For qPCR analysis of co-cultures. astrocytes were isolated by MACS as described under “Isolation of astrocytes from adult mice.” The remaining flow-through represented the isolated neuron population. Cells were lysed with RLT (Qiagen) 24 hours after stimulation. Lysed cells were stored at -80C until processing.

[0158] ATAC-seq

[0159] Sequencing libraries were prepared largely as described previously22 24. After isolation of nuclei, transposition was performed using the kit (Illumina, #FC-121- 1030) according to the original protocol100. DNA was then amplified using NEBNext High Fidelity 2X PCR Master Mix (New England Biolabs, #M0541S) for 5 cycles. DNA quantity was then measured using a Viia 7 Real-Time PCR System (Thermo Fisher Scientific) and the number of cycles required to achieve 1 / 3 of maximal SYBR Green fluorescence was determined and libraries were amplified accordingly. TruSeq adaptors (universal: Adl_noMX and barcoded: Ad2.1-Ad2.24) were used according to the standard protocol. Libraries were purified using MiniElute PCR Purification Kit (Qiagen, #28006) followed by double sided Agencourt AMPure XP bead purification (Beckman Coulter, #A63881) to remove primer dimers and large DNA fragments. Libraries were analyzed on a 2100 Bioanalyzer (Agilent Technologies) and High Sensitivity DNA Kit (Agilent Technologies. #5067-4626). Libraries were sequenced on an Illumina NextSeq550 by 35+40 bp paired end sequencing and de-multiplexed into FASTQ files. The fastq files of each ATAC-seq data sample were aligned to the Mus musculus GRCm39 / mmlO mouse genome assembly with BWA mem (version 0.7.17-rl 188). Duplicate reads were removed using Picard MarkDuplicates (version 2.25.2). The raw sequencing reads were quality assessed using fastp (version 0.23.2)84, fastQC (version 0.11.9), and reported using multiqc (version 1.11)83. Peak calling was performed by Macs2 (version 2.2.9.1). Peak differential analysis was done using DiffBind (version 3.10.0), and motif enrichment and annotation was performed with Homer (version 4.10).

[0160] Stereo-seq

[0161] Mouse brains were harvested and immediately flash frozen in Tissue-Tek OCT medium (Sakura Finetek USA Inc., Cat#4583) in 2-methylbutane, and then stored at -80C until used for Stereo-seq. Cryo-sections were cut at the thickness of 10Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0162] pm and mounted on Stereo-seq permeabilization chips (STOmics Americas Ltd, Cat#210CPl 18) or transcriptomics chips (STOmics Americas Ltd, Cat#210CT114). Tissue fixation and the following spatial transcriptomics procedure were performed according to the vendor’s manual and previous publications101,102. Briefly, the tissue section on the Stereo-seq chip (0.5 cm x 0.5 cm) was incubated at 37C for 5 min and subsequently fixed in pre-chilled methanol (Sigma, Cat#34860, precooled for 30 mins at -20C) at -20C for 30 min. Once the fixation was completed, the Stereo-seq chip was removed and the residual methanol was dried out in a chemical hood. The tissue section on the chip was then stained with nucleic acid reagent (Invitrogen, Cat#Q10212, 0.5% v / v) for 5 min and subsequently washed with 0.1X SSC buffer (Ambion, Cat#AM9770; containing 0.05 U / mL RNase inhibitor). The nuclei images were captured using a Zeiss Axio Scan Z1 microscope (at EGFP wavelength) and then followed by incubating tissue section in the permeabilization buffer (STOmics Americas, Cat#l 11KP118) for 12 minutes at 37C. Stereo-seq transcriptomics chip-captured RNAs from the permeabilized tissue were then reverse transcribed for 3 hours at 42C. Then the tissue was removed and the cDNAs were released from the chip using the transcriptomics reagent kit (STOmics Americas, Cat#l 11KT114). After the cDNA yield was size-selected, amplified, and purified, the concentration was quantified by Qubit dsDNA HS assay kit (Invitrogen, Cat#Q32854). 20 ng of cDNA from each sample was used for library construction using the library preparation kit (STOmics Americas, Cat#l 11KL114) and subsequently for DNB (DNA Nano Ball) generation. Finally, the DNBs were sequenced on the DNBSEQTM T7 sequencing platform (Complete Genomics, San Jose, USA) with 50 bp readl and 100 bp read2 (Complete Genomics, Cat# 1000028455). CID (coordinate ID 25bp in length) for each read was mapped to the designed coordinates of the in situ captured chip achieved from the first round of sequencing, allowing 1 base mismatch to correct for sequencing and PCR errors. Each read is also labeled with an MID (unique molecule ID). Reads with MID containing either N bases or more than 2 bases with quality score lower than 10 were filtered out. After CID mapping and MID assignment, retained reads were then aligned to the reference genome of the sample, using STAR83. Mapped reads with MAPQ > 10 were counted and annotated to their corresponding genes. UMI with the same CID and the same gene locus were collapsed, allowing 1 mismatch to correct for sequencing and PCR errors. ThisAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0163] information was then used to generate a CID-containing expression profile matrix. For cell segmentation analysis, nucleic acid staining images from the same section were projected to the Stereo-seq chips (image registration). The process started with summing up the total UMI in each DNB spot which harbor a specific spatial coordinate to generate a spatial density matrix, then the matrix was converted into an image where each pixel corresponds to one DNB and the total UMI of a DNB spot corresponds to the grayscale of the pixel. The nucleic acid staining image was then aligned to the transcriptome image. After image alignment, cell segmentation analysis was performed using Scikit-image package (VO.18.1)103. Briefly, the background of the staining image was removed with a global threshold approach, then the watershed algorithm was applied to obtain single cell segmentations. The number of markers required for the watershed algorithm were obtained through Gaussian-weighted local threshold binarization with block size of 41 and offset of 0.003. For each of the segmented cells, UMI from all DNB within the corresponding segmentation were aggregated per-gene and then summed to generate a cell by gene matrix for downstream analysis. The centroid of each cell was determined using ream (https: / / github.com / LudvigOlsen / rearrr). The analysis workflow^ with detailed documentation is publicly available at https: / / github.com / STOmics / SAW. The SAW software version applied in this study was v6.0.2. Data from cells segmented using the watershed algorithm from an entire coronal section taken from each biological replicate were aggregated together. Cells were included based on 50-1200 genes expressed and <5% mitochondrial reads. A gene was included only if at least 5 cells expressed it. Next, cell types w ere called using ingest104against a reference cell type atlas of the mouse brain36. Thereafter, cells localized to the amygdala were extracted for each tissue section. The amygdala location was determined by H&E staining, the presence of excitatory neuron markers (Slcl7a7, Slcl7ct6), inhibitory neuron markers (Slc32al), BLA neuron marker11'5 113(Etvl), and central amygdala marker114(Tac2~). In the selection of the amygdala, w e also guided the ROI selection by two external references: (1) the Allen Reference Atlas, judged by the Allen Atlas coronal section matching the experimental histological section most closely. (2) Unsupervised low-resolution regional clustering of 100 pm x 100 pm spots to approximate medial, central, basal / lateral amygdala regions. ROI selection of the amygdala was intended to be broad and therefore included the medial amygdala, central amygdala,Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0164] basomedial amygdala, basolateral amygdala, and lateral amygdala, as detected within a given section. From this analysis, most cell types were classified as inhibitory neurons, excitatory neurons, astrocytes, microglia, and oligodendrocytes. We did detect 4 cells classified as vascular smooth muscle, endothelial cells, pericytes, and perivascular macrophages, which were excluded from downstream analysis. The capture sensitivity of the Stereo-seq method, our thresholding parameters, and the analysis of only a single brain slice per animal likely led to the underestimation of other cell types in the brain, including endothelial cells, and even classes of immune cells that are recruited to the left amygdala during chronic stress5. Amygdala cell ty pe objects were then subsetted and validated for known marker genes Gpc5 (astrocytes), Slcl7a7 (excitatory neurons), Slcl7a6 (excitatory neurons), Slc32al (inhibitory’ neurons), Cst3 (microglia), and / / ? / (oligodendrocytes). To analyze astrocyte nearest neighbors to excitatory neuron cluster 2, the distance (d) between x-y coordinates of each cluster 2 excitatory neuron and astrocyte within a biological replicate from stressed mice was measured using < / =^((x2 - xi)2+ (y2 - y i)2). The top 10% of astrocytes with the shortest distance to the excitatory neuron were considered nearest neighbors. Trajectories were created using RNA velocity in scanpy73by briefly, drawing a force directed graph, determining spliced and unspliced transcripts, developing a pseudotime kernel, and computing a transition matrix that w as projected on the low-dimensional embedding. Differential expression analysis was performed by comparing each cluster to all others using scanpy .tl.rank genes groups , which were then used in dow nstream analyses such as Qiagen IPA by using the fold change of the cluster of interest relative to all others.

[0165] Isolation of meningeal cells

[0166] Animals were euthanized and blood was removed by cutting the right atrium. Following mouse perfusion, the head was severed, and the skin w as removed from the head. A cut w as made around the cranium (from the foramen magnum to the frontal bone) to dissect the bones of the calvarium. The calvarium was then transferred to a dissection scope and the meninges were removed. Tissue was incubated for 30 minutes at 37C in 500-pL RPMI-1640 (Life Technologies, #11875119) supplemented with 0.5 mg / mL collagenase P (Sigma-Aldrich, #11213865001), 0.5 mg / mL of dispase (Worthington, #LS02104), and 125 U / mL of DNase I (Sigma-Aldrich, #10104159001). After incubation, samples were washed with 1-mL of 0.5% BSA,Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0167] 2mM EDTA in IX PBS. Cells were centrifuged at 500g for 10 minutes, resuspended in 1-mL ice-cold 0.5% BSA. 2mM EDTA in IX PBS. and gently dissociated using a wide-bore 1-mL pipette tip. Finally, the cell suspension was filtered through a 40-pm FlowMi filter (Sigma- Aldrich, #BAH136800040-50EA) and prepared for downstream applications.

[0168] Isolation of mouse deep cervical lymph node cells

[0169] Deep cervical lymph nodes were isolated following mouse perfusion and mechanically dissociated. Cells were centrifuged at 1500rpm for 5 minutes and prepared for downstream applications.

[0170] Isolation of mouse splenic cells

[0171] Spleens were isolated following mouse perfusion and mechanically dissociated. Red blood cells were lysed with ACK lysing buffer (Life Technology, #A10492-01) for 5 min, washed with IX PBS and prepared for downstream applications.

[0172] FACS

[0173] Immune cell populations were analyzed largely as described previously21,23-25,113. Splenic, deep cervical lymph node, and meningeal cell suspensions were stimulated with 50 ng / mL phorbol 12-myristate 13-acetate (PMA, Sigma- Aldrich, #P8139), 1 pM lonomycin (Sigma- Aldrich, #I39O9-1ML), GolgiStop (BD Biosciences, #554724, 1:500) and GolgiPlug (BD Biosciences, #555029, 1:500) diluted in complete RPMI (Life Technologies, #11875119) containing 10% FBS, 1% penicillin / streptomycin, 50 pM 2-mercaptoethanol (Sigma-Aldrich, #M6250), and 1% non-essential amino acids (Life Technologies, #11140050). After 4 hours, cell suspensions were washed with 0.5% BSA. 2 mM EDTA in IX PBS and incubated with an FCyR-blocking antibody, surface antibodies and a live / dead fixable aqua stain (Thermo Fisher Scientific, #L34966) on ice. After 30 min, cells were washed with 0.5% BSA, 2mM EDTA in IX PBS and fixed according to the manufacturer's protocol of an intracellular labeling kit (eBiosciences, #00-5523-00). The FCyR-blocking antibody used in this study was: CD16 / CD32 (BD Biosciences, #553141. 1:200). Surface antibodies used in this study were: BUV661 anti-mouse CD45 (BD Biosciences, #612975, 1:100), BV650 anti-mouse CD3 (BD Biosciences, #740530, 1:100), PE-Cy7 anti-mouse CD4 (BD Biosciences, #561099, 1:100), BV786 antimouse CDllb (BD Biosciences, #740861. 1:100). BV570 anti-mouse Ly6CAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0174] (Biolegend, #128029, 1:100), BUV805 anti-mouse CD8a(BD Biosciences, #612898, 1:100), BUV563 anti-mouse Ly6G (BD Biosciences, #612921, 1:100), BUV737 antimouse CDllc (BD Biosciences, #612797, 1:100), BV750 anti-mouse NK1.1 (BD Biosciences, #746876, 1:100), and BV605 anti-mouse CD19 (Biolegend, #115539, 1:100). Intracellular antibodies were: Alexa Fluor 488 anti-GFP (Biolegend, #338007, 1:100), BV421 anti-mouse TNF (BD Biosciences, #563387, 1:100), and FITC antimouse CD192 (CCR2) (BioLegend, #150608. 1:100). Cells were counted at a20X dilution on a LSRFortessa (BD Biosciences). FACS was performed on a Symphony A5 (BD Biosciences) with BD FACSDIVA software (v.8.0.1). Compensation was performed on single-stained beads and all samples were gated against unstained controls within experiments. Flow cytometry data was analyzed using FlowJo software (FlowJo LLC, version 10.10.0). Cell populations were gated on SSC and FSC singlets followed by exclusion of dead cells, selection of CD45+cells, then gated as follows: B cells were gated as CD45 CD3 CD19+; CD4+T cells were gated as CD45+CD3+CD4+; CD8+T cells were gated as CD45+CD3+CD8+; dendritic cells were gated as CD45 CD3 CD1 lc+; macrophages were gated as CD45 CD3 CD1 lc‘ CD1 lb+Ly6C Ly6G"; neutrophils were gated as CD45 CD3 CD1 Ic'CDl lb+Ly6C Ly6G+; and pro-inflammatory monocytes were gated as CD45+CD3 CDllc" CDllb+Ly6C+.

[0175] In vivo photoconversion of CAGAae' / emice

[0176] Photoconversion of the spleen was performed largely as previously described36following 18 days of restraint stress as mice were briefly anesthetized. The surrounding tissue was covered with sterile aluminum foil and a small opening was made in the peritoneal cavity. The spleen was extended and exposed to 405-nm violet light using a 200 mW Electra Pro portable laser (Laserglow7Technologies) for a total time of 90 seconds over the whole spleen surface with continuous application of PBS on the exposed spleen. After careful returning of the spleen to the peritoneal cavity, the peritoneum was closed with absorbable sutures (Ethicon, #1667H) and the skin was closed with surgical staples (Braintree Scientific, #EZC-KIT). Mice were sacrificed 36-hours later for FACS analysis.

[0177] Adoptive transfer of monocytes

[0178] The skin and muscle of the legs were removed from mice following euthanasia. Bone marrow was isolated from C57B1 / 6J mice by flushing PBS throughAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0179] the head of the femur and tibia with a 25-gauge needle (BD Biosciences, #305125). Red blood cells were lysed with ACK lysing buffer (Life Technologies, #A10492-01) for 5 min, washed with IX PBS, and centrifuged at 500g for 5 minutes at 4C.

[0180] Monocytes were then isolated by MACS with the Miltenyi Monocyte Isolation Kit (Miltenyi Biotec, #130-100-629). Cells were resuspended in 175 pL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS with 25 pL of FcR Blocking Reagent and 50 pL of Monocyte Biotin -Antibody Cocktail. Cells were incubated at 4C for 5 minutes and subsequently washed with 10-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS. The samples were then centrifuged at 500g for 10 minutes, and cell pellet was resuspended in 400 pL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS. 100-pL of anti-Biotin Microbeads were added to each sample, and cells were incubated at 4C for 10 minutes. The cell suspension was applied onto an LS column (Miltenyi Biotec, #130-042-401) on a QuadroMACS Separator pre-rinsed with 3-rnL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS and collected into a 15-mL conical tube. The column was washed 3 times with 3-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS and flowthrough was collected. Cells were centrifuged at 500g for 5 minutes at 4C and resuspended in IX sterile PBS. Mice were intravenously injected with 200-uL of PBS as a control or 106monocytes in 200pL of PBS with an insulin syringe (BD Biosciences, #329410) 18-hours prior to the first day of restraint. Subsequently, IX PBS or monocytes were administered to the mice each day 2-hours following the end of the 6-hour restraint period for 7 consecutive days. Mice were subjected to behavioral testing following the 7 day restraint period.

[0181] Intra-cisterna magna injections

[0182] Injections were carried out largely as previously described116. Briefly, mice were anesthetized with 1-3% isoflurane (Covetrus, #11695067772) in oxygen. Heads were shaved and cleaned with ethanol and betadine (Thermo Fisher, #19-027132) and a small opening was made in the skin at the back of the neck with surgical scissors. The head of the mouse was placed on a support box and angled down to expose the muscle around the cistema magna. Muscle tissue blocking the cistema magna was gently pulled away with fine-tip forceps. The tip of a 25-gauge Hamilton syringe needle was bent to a 45-degree angle and an injection of 6 pL of antibody was made into the cistema magna with a 5 pL Hamilton syringe (Sigma- Aldrich, #20787) at a concentration of 4 pg / pL. After injection, mice were sutured (Ethicon, #1667H) andAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0183] allowed to recover in a clean cage on a heating pad for 1 hour. Instruments were sterilized between each mouse using a glass bead sterilizer (CellPoint Scientific, #GRM5-1450). Evans Blue (Thermo Scientific, #A16774.09) was dissolved to 3% in DPBS and administrated to mice viai.c.m. injection. Following 18 days of restraint stress, 24 pg of isotype control (R&D Systems, #MAB0061) or a-CCR2 (R&D Systems, #MAB55381) antibody were administered to mice via i.c.m. injection as previously described116; after fear acquisition mice underwent fear extinction 48-h later. Prior to injection of IL-1 b, similar to previous studies117mouse IL-lb (R&D Systems, #201-LB-025) was biotinylated by EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher, #A39256) following the manufacturer's instructions and then purified using a Pierce C18 Spin Columns, 7KMWC0 (Thermo Fisher. #89870). The biotinylated IL-lb was administered into the cistema magna of anesthetized 18-d stressed or control mice at a concentration of 250 ng / mL in 5pL IX DPBS. Mice were sacrificed one day after injection.

[0184] Isolation of monocytes from adult mice across tissues

[0185] To process the meninges, the head was severed, and the skin was removed from the head. A cut was made around the cranium (from the foramen magnum to the frontal bone) to dissect the bones of the calvarium. The calvarium was then transferred to a dissection scope and the meninges were removed. Tissue was incubated for 30 minutes at 37C in 500 pL RPMI-1640 (Life Technologies, #11875119) supplemented with 0.5 mg / mL collagenase P (Sigma- Aldrich, #11213865001), 0.5 mg / mL of dispase (Worthington, #LS02104), and 125 U / mL of DNase I (Sigma-Aldrich, #10104159001). After incubation, samples were washed with 1-mL of 0.5% BSA, 2mM EDTA in IX PBS. Cells were centrifuged at 500g for 10 minutes, resuspended in 1-mL ice-cold 0.5% BSA, 2mM EDTA in IX PBS, and gently dissociated using a wide-bore 1-mL pipette tip. Finally, the cell suspension was filtered through a 40 pm FlowMi filter (Sigma- Aldrich, #BAH136800040-50EA). Monocytes were isolated by MACS with the Miltenyi Monocyte Isolation Kit (Miltenyi Biotec. #130-100-629). Cells were centrifuged at 1500rpm for 5 minutes and resuspended in 175 uL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS with 25 uL of FcR Blocking Reagent and 50 uL of Monocyte Biotin-Antibody Cocktail. Cells were incubated at 4C for 5 minutes and subsequently washed with 10-mL of 0.5% BSA. 2 mM EDTA pH=8.0 in IX PBS. The samples were then centrifuged at 500gAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0186] for 10 minutes, and cell pellet was resuspended in 400 uL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS. 100-uL of Anti-Biotin Microbeads were added to each sample, and cells were incubated at 4C for 10 minutes. The cell suspension was applied onto an LS column (Miltenyi Biotec, #130-042-401) on a QuadroMACS Separator prerinsed with 3-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS and collected into a 15-mL conical tube. The column was washed 3 times with 3-mL of 0.5% BSA. 2 mM EDTA pH=8.0 in IX PBS and flowthrough was collected. Monocytes were centrifuged at 500g for 5 minutes, resuspended in 100 pL of Picopure extraction buffer (Thermo Fisher Scientific, #KIT0204), and incubated in a heat block at 42C for 30 minutes. Samples were stored at -80C until processing.

[0187] Primary splenic CDllb+cell culture

[0188] CD1 lb+cells were isolated by MACS with the Miltenyi CD 1 lb Microbeads (Miltenyi Biotec, #130-049-601). In brief, spleens were isolated following mouse perfusion and mechanically dissociated. Red blood cells were lysed with ACK lysing buffer (Life Technology. #A10492-01) for 5 min and washed with IX PBS. Cells were resuspended in 90 uL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS and 10 uL of CD1 lb Microbeads and incubated at 4C for 10 minutes. Cells were washed with 1-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS, centrifuged at 500g for 5 minutes, and resuspended in 500-uL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS. The cell suspension was applied onto an LS column (Miltenyi Biotec, #130-042-401) on a QuadroMACS Separator pre-rinsed with 3-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS. The column was washed 3 times with 3-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS, removed from the separator, and placed onto a 15-mL conical tube. 5-mL of 0.5% BSA, 2 mM EDTA pH=8.0 in IX PBS was added to the column, and CDllb+cells were removed with a plunger. Cells were centrifuged at 500g for 5 minutes and resuspended in DMEM / F12+GlutaMAX (Life Technologies, #10565042) supplemented with 10% FBS (Life Technologies, #10438026), 1% penicillin-streptomycin (Life Technologies, #15140122), 1% sodium pyruvate (Life Technologies, #11360070), 1% HEPES (Life Technologies. #15630106) and 1% MEM non-essential amino acids (Life Technologies, #11140050). Cells were stimulated for 6 hours after plating.Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0189] Administration of psychedelics

[0190] Similar to previous studies, 1 mg / kg psilocybin47(NIDA Drug Supply Program) or 10 mg / kg MDMA-HC1 racemate50’31(NIDA Drug Supply Program) were administered via intraperitoneal injection at a dose of 1 mg / kg or 10 mg / kg, respectively in IX PBS 24-hours after the final day of restraint stress, or as otherwise indicated. Control mice were treated with equal volume of vehicle (IX PBS). Mice were monitored for 6-hours following drug administration. We analyzed psilocybin and MDMA because of their use in clinical trials48,49, and because the effects of both are thought to be mediated by complementary activation of serotonin signaling118, either through binding of serotonin receptors, (primarily psilocybin119) or by limiting serotonin reuptake (MDMA46). Of note, MDMA bioavailability at 10 mg / kg peaks at -2000 ng / mL120, a concentration within range of its affinity for 5-HT1 and 5-HT2 receptors121,122, and it also is reported to bind 5-HT2B receptors123,124, hence in these studies it may also activate serotonin receptors. Although we did not directly examine other transporters or neurotransmitter systems, except for TrkB125expression, it is possible these mechanisms play a role as well123425 l27. Please also note that the doses of psilocybin and MDMA we used in mice ( 1 mg / kg and 10 mg / kg) roughly correspond to doses that have shown clinical benefit in trials for humans48,49(i.e., 25 mg psilocybin corresponds to 0.32-0.42 mg / kg, and 180 mg MDMA corresponds to 2.25-3 mg / kg, in average weight human males and females, respectively). Finally, we note that while we sometimes use the term “psychedelic” for semantic simplicity, psilocybin is considered a “classical” psychedelic45while MDMA is considered a consciousness-altering drug126or empathogenic psychedelic50.

[0191] Meningeal explants

[0192] Meningeal explants were performed largely as described previously116.

[0193] Briefly, animals were euthanized, and blood was removed by cutting the right atrium. Following mouse perfusion, the head was severed, and the skin was removed from the head. A cut w as made around the cranium (from the foramen magnum to the frontal bone) to dissect the bones of the calvarium. The skull was placed in a 24-well plate with 1-mL of DMEM (Life Technologies, #11965092) and incubated at 32C for 30 minutes at 150rpm. Media was removed and stored in a 1.5-rnL tube at -80C (IL-lb). The skull was subsequently incubated in 1-mL of 50 ng / mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, #P8139) and 1 pM lonomycin (Sigma-Aldrich,Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0194] I3909-1ML) diluted in complete RPMI (Life Technologies, #11875119) containing 10% FBS, 1% penicillin / streptomycin. 50 pM 2-mercaptoethanol (Sigma- Aldrich, #M6250), and 1% non-essential amino acids (Life Technologies, #11140050) at 32C for 2 hours at 150rpm. Media was removed and stored in a 1.5-mL tube at -80C until processing and analysis (TNFa).

[0195] Administration of nifedipine

[0196] Nifedipine (Sigma-Aldrich, #N7634) was prepared as a stock solution in DMSO (Sigma- Aldrich, #472301) at 50 mg / mL. This stock was diluted 20-fold in com oil and sonicated for 30 minutes after vortexing. Working dilutions were administered via intraperitoneal injection at a dose of 30 mg / kg in 300-uL of com oil (Sigma- Aldrich, #C8267) 30-minutes prior to administration of psychedelics. A second dose of nifedipine (Sigma-Aldrich, #N7634) was delivered 3-hours after the initial psychedelic dosing. Control mice were treated with equal volume of vehicle (300 pL of com oil for nifedipine, and 100 pL of IX PBS for psychedelics). Samples were processed for FACS 6-hours following the initial nifedipine treatment.

[0197] Fluorescent imaging of vascularity

[0198] Mice were anesthetized with isoflurane and heads shaved as described above to visualize the skin clearly on top of the head. Vascularity was measured by in vivo fluorescent imaging. IVISense Vascular 750 (Revvity, #NEV10011EX) was reconstituted in PBS according to the manufacturer’s protocol and 100-uL of suspension was intravenously administered by tail vein injection 30-minutes prior to administration of psychedelics. Mice that underwent 18 days of restraint stress w ere then administered IX PBS, 1 mg / kg psilocybin, or 10 mg / kg MDMA following the tracer injection described above. Fluorescence was captured with ex. 750-nm and em.

[0199] 790-nm at high sensitivity and 5-s exposure time using the In-Vivo Xtreme Optical / X-ray imaging system (Bruker) 30-minutes and 6-hours after injection. MI SE v7 software (Bruker) w as used to quantitate net fluorescent signal intensity within the area of the mouse skull by manually selecting an ROI (region of interest) comprising the signal within the head of the mouse and subtracting the median background intensity from the ROI.

[0200] Chemical sympathectomy

[0201] Sympathectomy was performed in a manner similar to previous studies128. In brief, 6-hydroxydopamine hydrobromide (Tocris Bioscience, #2547) was dissolved inAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0202] 0.1% sodium ascorbate (Sigma Aldrich, # PI I R.1279) immediately before administration and protected from light. 6-hydroxy dopamine hydrobromide was administered via intraperitoneal injection at a dose of lOOmg / kg for 3 consecutive days starting 24 hours after the final day of tube restraint. Tissues were taken for processing 4 days after the final 6-hydroxy dopamine hydrobromide treatment.

[0203] Whole-mount immunostaining of peripheral organs

[0204] These procedures were performed largely as we and others have done previously for peripheral neurons129,130. Briefly, organs were harvested and post-fixed in PFA (Electron Microscopy Sciences, #15714-S) freshly diluted to 4% in IX PBS for 2 days, then transferred to IX PBS until processing. Organs were then washed 3X for 15 minutes with ice cold IX PBS at 4C. Organs were then incubated in blocking solution (5% donkey serum / 1% PBS-T) overnight in the cold. Organs were then incubated in primary antibodies diluted in blocking solution for 2 days overnight with rotation at 4C. Organs were then washed with 1% Triton X-100 (Thermo Fisher Scientific, #NC9356391) in PBS (PBS-T) 3X for 2 hours at room temperature, then incubated with secondary antibodies diluted in blocking solution overnight at 4C. Organs were washed 3X for 2 hours at room temperature and serially dehydrated in MeOH in PBS (50%, 80%, 100%). Organs were then cleared in a mixture of 2:1 BABB and imaged on a Leica DMi8 Widefield Fluorescence Microscope at 5X magnification. The primary antibody used in this analysis was rabbit anti-tyrosine hydroxylase (Millipore, AB152, 1:100). The secondary antibody used in this analysis was goat anti -rabbit Alexa Fluor 488 (Thermo Fisher, #A-11008, 4pg / mL).

[0205] ImmGen data browser analysis

[0206] Ultra-low input mouse and human RNA-seq data were obtained from the ImmGen Data Browser131and accessed from skyline plots. Cell types from multiple tissues that were analyzed in mouse ImmGen data are show n in the following order per gene plot: DC_4+_Sp, DC_8+_Sp, DC_pDC_Sp, GN BM, GN_Sp, GN_Thio_PC, Mo_6C-II-_Bl, M0 6C+II- BI, MF_102+480+_PC,

[0207] MF 226+II+4801O PC, MF Alv Lu, MF AT, MF Fem PC. MF microglia CNS, MF PC, MF_pIC_Alv_Lu, MF RP Sp, MMP2 150+48+ BM, MMP3 48+ BM, MMP4 135+ BM, LTHSC 34- BM, LTHSC 34+ BM, MC BM, STHSC 150-_BM. Cell types accessed from human data were all derived from the blood and each cell class is listed in the legend associated with the plot. For mice, the minimumAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0208] reported value of normalized expression in the data browser was 0.1, while in humans it was 1.

[0209] Primary human astrocyte cell culture

[0210] Human astrocytes (ScienCell, #1800) were cultured largely as described previously21-25’71. Cells were centrifuged at 500g for 5 minutes at 4C, resuspended in DMEM / F12+GlutaMAX (Thermo Fisher Scientific. #10565018) supplemented with 10% FBS (Thermo Fisher Scientific. #10438026) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148) and cultured in T-75 flasks (Falcon, #353136) pre-coated with poly -L -lysine (Sigma Aldrich, #P4707) for 1 h at 37C and washed with IX PBS. Cells were cultured at 37C in a humidified incubator with 5% CO2, for 7-10 days until confluency was reached. Media was replaced every 2-3 days. To plate astrocytes, the flask was shaken for 2 hours at 220 rpm, media removed, and 5-mL of trypsin was added at 37C for 3 minutes. Trypsin was neutralized with DMEM / F12+GlutaMAX (Thermo Fisher Scientific, #10565018) supplemented with 10% FBS (Thermo Fisher Scientific. #10438026) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148). Cells were centrifuged at 500g for 5 minutes at 4C, resuspended in DMEM / F12+GlutaMAX (Thermo Fisher Scientific, #10565018) supplemented with 10% FBS (Thermo Fisher Scientific, #10438026) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148), and cultured in a tissue culture-treated plate pre-coated with poly-L-lysine (Sigma Aldrich. #P4707). Astrocytes were plated for 5 days before in vitro treatments. The following molecules in DMEM / F12+GlutaMAX (Thermo Fisher Scientific, #10565018) supplemented with 10% FBS (Thermo Fisher Scientific, #10438026) and 1% penicillin / streptomycin (Thermo Fisher Scientific, #15140148) were used to stimulate human astrocytes for 6 hours: 50 ng / mL IL- i (Sigma Aldrich, #H6291), 50 ng / mL IL-6 (Sigma Aldrich, #H7416), 50 ng / mL TNFa (Sigma Aldrich, #SRP3177), and 200 ng / mL cortisol (Sigma Aldrich, #0106) or 1 pg / mL IL-lb (for EGFR). Following stimulation, astrocytes were treated with buffer RLT (Qiagen) and stored at -80C. Taqman probes used in this study are: GAPDH (Hs02786624 gl). PTPRS (Hs01548375 ml), and EGFR (Hs01076090_ml).

[0211] Primary human monocyte cell culture

[0212] Primary human monocytes (iQ Biosciences, #IQB-Hul-M10) were cultured largely as described previously132. Cells were centrifuged and resuspended in 10-mLAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0213] of RPMI-1640 (Life Technologies, #11875119) supplemented with 10% FBS (Life Technologies, #10438026) and 1% penicillin-streptomycin (Life Technologies, #15140122). Cells were stimulated 24 hours after plating. Stimulations were performed for 6 hours with the following molecules in RPMI (Life Technologies, #11875119) containing 10% FBS and 1% penicillin / streptomycin: 10 pM psilocybin (NIDA Drug Supply Program), 10 pM MDMA-HC1 racemate (NIDA Drug Supply Program), 200 ng / mL cortisol (Sigma Aldrich, #C-106), and 100 ng / mL LPS (InvivoGen, #tlrl-3pelps).

[0214] 10X genomics single-nucleus RNA-seq

[0215] Flash frozen amygdala from sex, age, and race matched patients was obtained from the NIH NeuroBioBank. Patient recruitment at the NIH NeuroBioBank is approved programmatically through the IRB of each institution providing patient samples. The tissue was processed using the 10X Chromium Nuclei Isolation Kit (10X Genomics, #PN-1000494). In brief, 25-mg of flash frozen amygdala was placed in a 1.5-mL DNA LoBind Tube (Eppendorf. #022431021) with 200 uL of Lysis Reagent supplemented with 0.2 uL of Reducing Agent B and 2.0-uL of Surfactant A. Tissue was dissociated with a plastic pestle and 300-uL of Lysis Reagent supplemented with 0.3 uL of Reducing Agent B and 3.0 uL of Surfactant A was added once tissue was homogenized. Homogenized tissue was incubated on ice for 10 minutes. Dissociated tissue was transferred to a pre-chilled Nuclei Isolation Column and centrifuged at 4C for 20 seconds at 16000g. The column was discarded, and nuclei were centrifuged at 4C for an additional 3 minutes at 500g. The pellet was resuspended in 500 uL of Debris Removal Reagent supplemented with 0.5 uL of Reducing Agent B and centrifuged at 4C for 10 minutes at 700g. Supernatant was discarded, and nuclei were resuspended in 875 uL of IX PBS supplemented with 100 uL of 10% BSA and 25 uL of RNase Inhibitor. Nuclei were centrifuged at 4C for 5 minutes at 500g, resuspended again in 875 uL of IX PBS supplemented with 100 uL of 10% BSA and 25 uL of RNase Inhibitor, and centrifuged at 4C for 5 minutes at 500g. The nuclei were resuspended in 100 uL of Resuspension buffer and counted with aNexcelom Cell Counter. Equal numbers of nuclei were pooled for control and MDD patients, respectively, centrifuged at 4C for 5 minutes at 500g, and resuspended in diluted nuclei buffer containing 20X Nuclei Buffer, DTT, RNase Inhibitor, and Nuclease-free Water. Nuclei were then encapsulated using the 10X Genomics kitAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0216] (10X Genomics, #PN-1000283) with Chip J (10X Genomics, #1000234) and the Chromium X Controller according to the manufacturer’s protocol. GEMs were incubated following formation to produce reverse transcribed cDNA and quenched following the end of the incubation period. The GEMs were then broken with recovery agent. Reverse transcribed cDNA was purified with Dynabeads and SPRIselect beads (Beckman Coulter, #B23317). The purified cDNA was then amplified and used for library construction. Samples were indexed with Dual Index Plate TT Set A (10X Genomics, #PN-1000215). Final libraries were quantified with the Bioanalyzer DNA HS assay kit (Agilent, #50674626) and by qPCR with the Kapa library quantification kit (Kapa Biosystems, #KK4824). The libraries were then diluted and pooled at 4nM in nuclease-free water (Thermo Fisher Scientific, #AM9937), run on an Illumina NextSeq550, and de-multiplexed into FASTQ files. The following read lengths were used for the run: Read 1 = 28bp, i7 index = lObp, i5 index = lObp, Read 2= 44bp. Raw .fastq files for each of the MDD and control lanes were uploaded and processed on the 10X Genomics Cloud CLI using the 3’ Gene Expression pipeline using standard settings and cellranger-7.1.0. Briefly, intron mode was used to align samples against GRCh38-2020-A, and the top 10,000 barcodes were selected for downstream analysis based on the input number of nuclei per experiment. Each sample was then merged using Cell Ranger Aggr v7.1.0, and the Loupe Browser file was output. Overall, and before filtering, per cell we detected 31,787 reads. 1,345 median UMIs, and 883 median genes. Using 10X Genomics Loupe Browser, only cells expressing 500-20,000 UMI and <15% mitochondrial reads were included. Using K-means clustering based on transcriptional clustering parameters of 8 clusters, we could distinguish major amygdala cell types. One cluster of approximately 110 cells did not express clear marker genes of a know n cell population so it was excluded from downstream analyses, as w ell as any duplicate barcodes between the MDD and control samples. From there, we focused on the 7 remaining cell type clusters, which were identified using known marker genes for each cell type: oligodendrocytes (MOG), astrocytes (A QP4), OPCs (PDGFRA), excitatory neurons (SLC17A7), inhibitory neurons (GAD1), and microglia (P2RY12). Based on these markers, specific cell type populations w ere subclustered, such as astrocytes or excitatory neurons. During astrocyte subclustering, we detected a cluster expressing high levels of the endothelial cell marker gene CLDN5, hence all cells within this cluster wereAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0217] removed from the analysis of subclustered astrocytes, and astrocytes were reclustered. To develop the EGFR signaling score for astrocytes, we compiled together a list of common genes known to be elevated by EGFR signaling from the pathways Gene Ontology70007173, KEGGN00279, and KEGGN00284; the genes are: CBLC, SHC1, SPRY1, EPGN, TGFB1, NUP62, SOCS4, PIK3C2A, GRB2, DUSP3, RNF126, MTOR, HBEGF, ITGA1, SLC30A10, EGFR. CEACAM1, STAM2, RAB7A, MAP2K1, BRAE BCAR3. DAB2IP. VIL1, ERRFI1. SPRY2, RNF115, ABL1. NEUS, PTPN12, GAREM1. RAFI, CHMP6, EPS15L1. CNOT9, FER, ZGPAT, CBL, RPS6KB1, ZFYVE28, PLAUR, IQGAP1, CCDC88A, CBLB, ARHGEF7, NCK2, HIP1, MAPI, ADAMI 7, VPS25, GABI, PTPRJ, DGKD, SHKBP1, SH3KBP1, GPER1, CDH13. These features were combined and the “Feature Avg” was taken per cluster in the subclustered astrocytes with endothelial cells removed. Statistics were determined for EGFR pathw ay signature score by' performing pre-ranked GSEA on the log2(FC) within cluster 2 astrocytes relative to all other astrocyte clusters. For excitatory neurons, the “Feature Sum” otNR2F2 and SLITRK2 was taken in the subclustered excitatory' neurons. For Qiagen 1PA analyses, the log fold change of differentially expressed genes of a cell type subcluster relative to all other cell type subclusters 'as used as input.

[0218] Immunostaining of human tissue

[0219] Patient recruitment at the NIH NeuroBioBank is approved programmatically through the IRB of each institution providing patient samples and consent is given as described above. For staining of human amygdala tissue, flash-frozen samples obtained from the NIH NeuroBiobank were fixed in 4% PFA at 4°C overnight and subsequently incubated in 30% sucrose for 24 hours. Tissues were washed 3X for 5 minutes each in DPBS, then embedded in OCT. Brain sections (20 pm) were obtained using a cryostat and placed onto SuperFrost Plus slides (Fisher Scientific, #15-188-48). Sections were incubated in blocking solution (5% goat serum in 0.3% PBS-T) for 2 hours at room temperature. Primary antibodies, diluted in the same blocking solution, were applied within a hydrophobic boundary drawn around the tissue and incubated at 4°C overnight. The following day, sections were washed 3X in 0.3% PBS-T at room temperature, then incubated with secondary antibodies diluted in blocking solution for 2 hours at room temperature. After five final washes in DPBS, sections were mounted using Fluoromount-G with DAPI (SouthemBiotech, #0100-Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0220] 20). Confocal images were captured with a Zeiss LSM880 using a 63X objective, with an 8-plane z-stack at 1 pm intervals. Maximal intensity’ of GFAP-positive cells was quantified using FIJI. The primary' antibodies used were mouse anti-EGFR antibody (Abeam, 1:100, #ab30) and rabbit anti-GFAP (Abeam, 1:200, #ab68428), with corresponding secondary' antibodies: goat anti-rabbit IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 594 (Invitrogen, #A11037) and goat anti-mouse IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 488 (Invitrogen, #A11029).

[0221] Statistics

[0222] Statistical analyses were performed using GraphPad Prism 10. For analyses of gene expression represented by volcano plots, the control group is listed second (Fig. Ij, Iq, 2f, 5c) Hence, gene expression is shown as the experimental group relative to the control group. For t-tests, all tests are two-tailed unless explicitly specified otherwise. For elevated plus maze data, unpaired two-tailed t-tests were used (Figs.

[0223] 4d, 4h, Figs. 6d, 4p). For fear conditioning and extinction data (Figs, lb, li, 2e, 4e, 4i Figs. 6b, 6c). two-way repeated measures ANOVA was used; Fig. 6b was analyzed using the interaction statistic, while other panels were analyzed using the group statistic. Independent experiments were grouped together. Analyses of mouse plasma were performed by two-way ANOVA to determine differences as a function of condition followed by unpaired two-tailed t-tests to determine significance by group (Fig. 1c) Plasma analyses of all other cytokines were analyzed by two-way ANOVA by condition (Fig. 6e). The presented representative confocal images were replicated at least three times (Figs. Im), except for Fig. 2k, Fig. 4k, which were replicated twice. All figures were replicated at least three times, excluding Fig. 1c, which was repeated twice. For mouse tdTomato+scRNA-seq data (Fig. le), the cluster fold changes in response to stress relative to control are: 0.48 (Cluster 0); 1.96 (Cluster 1); 2.39 (Cluster 2); 1.05 (Cluster 3); 1.32 (Cluster 4); 1.04 (Cluster 5); 0.98 (Cluster 6); 106.41 (Cluster 7); and 1.44 (Cluster 8). For human astrocyte snRNA-seq data (Fig.

[0224] 5g), the cluster fold changes in MDD relative to control are: 0.12 (Cluster 1); 7.55 (Cluster 2); 0.40 (Cluster 3); 1.72 (Cluster 4); 0.01 (Cluster 5); 4.60 (Cluster 6). For human excitatory’ neuron snRNA-seq data (Fig.5j), the cluster fold changes in MDD relative to control are: 0.92 (Cluster 1); 5.27 (Cluster 2); 0.48 (Cluster 3); 47.83 (Cluster 4); 3.09 (Cluster 5); 1.07 (Cluster 6); 0.14 (Cluster 7); 0.24 (Cluster 8); notAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0225] detected in MDD (Cluster 9). Significance levels if not explicitly indicated in Figures is as follows: *P < 0.05, **P <0.01, ***P< 0.001, NS: P>0.05.

[0226] Example 1. Amygdala astrocyte subsets limit fear

[0227] To define neuroimmune interactions dysregulated over time in response to chronic psychological stress (Fig. la), we subjected mice to periods of chronic restraint stress followed by behavioral testing using contextual fear conditioning and elevated plus maze (Fig. 6a). Although we did not detect stress-induced changes in task acquisition across groups (Fig. 6b), mice that underwent 18 days of restraint stress showed elevated fear behavior relative to controls (Fig. lb, Figs. 6c, d). After 18 days of chronic stress, we also detected elevated plasma levels of corticosterone and the cytokines IL-lb, IL-12, TNFaand the chemokine MIP2 (Fig. 1c, Figs. 6e,f), suggesting potential links between chronic stress, peripheral inflammatory responses, fear behavior, and neuroimmune interactions.

[0228] Multiple astrocyte populations that influence local inflammatory signals in the nervous system have recently been described"'21 25. Moreover, astrocytes have emerged as direct targets of elevated corticosterone signaling in the context of chronic stress1214. However, the processes by which astrocytes integrate inflammatory cues to shape behavior in response to chronic stress remains largely unknown. To begin to address this question, we subjected AldhllltdTomato / +mice that inducibly expressed a tdTomato reporter in Aldhlll+ cells (as an astrocyte reporter) to 18 days of restraint stress, then performed single-cell RNA-sequencing of tdTomato+ cells using Drop-seq. We captured 11,700 reporter cells from AldhllltdTomato / +mice subdivided across nine clusters, associated with either control or chronically stressed mice (Fig. Id).

[0229] We detected multiple clusters modulated by chronic stress, some of which were overrepresented in stress conditions (clusters 1-5, 7, and 8) while others were overrepresented in controls (clusters 0 and 6) (Fig. le). We focused our attention on cluster 1 because it was the most numerous population of cells expanded in response to 18 days of restraint stress (Fig. 1g). Pathway analyses suggested that cluster 1 cells w ere transcriptionally regulated by signaling from the glucocorticoid receptor (Fig. Ih), a key transcriptional target of stress-induced corticosterone, which controls behavioral phenotypes when activated by psychological stress in astrocytes1214. We next performed a pseudotime analysis using RNA velocity, which suggested cluster 1Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0230] was associated with late-stage pseudotime and potentially linked with chronic stress responses.

[0231] In addition to glucocorticoid receptor activation, pathway analyses of cluster 1 cells pointed to a significant downregulation of signaling by the epidermal growth factor receptor (EGFR) family21(Fig. 1g). Consistent with these data, analysis of differentially expressed genes within cluster 1 cells revealed Egfr as significantly downregulated. Of note, cluster 2, which was maximally expanded in response to chronic stress, but less numerous than cluster 1, was also marked by significantly lower Egfr expression together with dow nregulation of pathways protective during chronic stress; cluster 0 cells that were associated with control mice w ere marked by elevated IL-10 signals and decreased inflammatory pathways (Fig. 6g). Together, these data suggest low Egfr expression levels might be linked with stress-induced behavioral changes.

[0232] Because multiple brain areas play important roles in stress-induced fear behavior26, we examined astrocyte Egfr expression across the brain from a previously published mouse brain atlas27to identify regions potentially influenced by astrocyte EGFR signaling. Among all brain areas re-examined, the amygdala harbored the lowest astrocyte Egfr expression, which we independently validated by immunostaining. These data suggest that in response to 18 days of restraint stress, the amygdala may be more susceptible to behavioral changes controlled by Egfr-dependent mechanisms in astrocytes. The amygdala is a limbic area composed of multiple sub-nuclei that appraises environmental threats28but is sensitive to immune cell-derived cues released during psychological stress exposure5-29, which can enhance fear behavior70.

[0233] We previously linked Egfr+astrocytes to anti-inflammatory signaling in autoimmunity21’31, so we hypothesized that Egfr+astrocytes might act as negative regulators of local inflammatory responses triggered by chronic stress that may activate amygdala circuits. We therefore tested this hypothesis after 7 days of restraint stress, a timepoint where there were otherwise no significant changes in fear behavior or plasma cytokine levels (Figs. 6b, c). To study whether Egfr+amygdala astrocytes limit fear behavior, w e used an astrocyte-specific AAV-based CRISPR / Cas9 system to knockdown (KD) Egfr or, as a control, target the inert Rosa26 locus (Fig. Ih). Mice with genetic inactivation of Egfr in amygdala astrocytes showed increased fearAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0234] behavior following 7 days of restraint stress, suggesting Egfr+astrocytes restrict stress-induced behavioral changes (Fig. li).

[0235] Analysis of Egfr KD amygdala astrocytes by bulk RNA-seq revealed increased activation of inflammatory’ pathways, consistent with known antiinflammatory' properties of EGFR in astrocytes21,31, as well as Nptxl and Fos, genes related to astrocyte stabilization of amygdala fear memories32(Figs. lj,k). Astrocyte Egfr loss-of-function also pointed to increased transcriptional signals associated with receptor protein tyrosine phosphatases (Fig. Ik), which are induced by NF-kB signaling33and engage in heterotypic cell-cell interactions34.

[0236] Investigation of our scRNA-seq dataset of tdTomato+cells isolated from AldhllltdTomato / +mice exposed to 18 days of restraint stress or controls (Fig. If) identified Ptprs as the most significantly upregulated of all receptor protein tyrosine phosphatase family members in cluster 1 cells linked with chronic stress (Fig. 11), although Ptprs and other family members were differentially represented across clusters. First, we detected increased PTPRS expression after 18 days of restraint stress in astrocytes located in the basolateral amygdala (BLA), the main input subnucleus of the amygdala28(Fig. Im). To define cytokines that might influence astrocyte expression of receptor protein ty rosine phosphatases during chronic stress, we quantified Ptprs expression in primary astrocytes treated with corticosterone and either IL- lb, IL- 12, or TNFa, which are cytokines we found upregulated in the plasma of mice after 18 days of stress (Fig. 1c). The combination of corticosterone and IL- lb increased Ptprs expression (Fig. In) consistent with elevated IL-lb activity7in Egfr KD amygdala astrocytes isolated from mice that underw ent 7 days of restraint stress (Fig. Io), although multiple factors can potentially modulate astrocyte Ptprs expression in vivo. Likewise, Egfr KD in primary astrocytes boosted Ptprs expression in response to IL-lb and corticosterone co-treatment (Fig. Ip). These data suggest EGFR loss-of-function in the presence of IL-lb and corticosterone leads to elevated Ptprs expression in astrocytes.

[0237] In addition to other receptors, PTPRS binds Slitrk2, a single-pass transmembrane receptor expressed on neurons that influences the stability of neuron contacts35. Amygdala neurons analyzed by bulk RNA-seq from mice where Egfr was KD in astrocytes showed upregulation of Slitrk2 (Fig. Iq) and increased transcriptional responses linked to pathways for neuroinflammation, CREP signaling,Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0238] neurotrophin signaling, and to a lesser extent, synaptogenesis, as well as receptor protein tyrosine phosphatase activity (Fig. Ir). We therefore hypothesized that in the amygdala, astrocyte cross-talk with neurons may influence stress-induced fear behavior.

[0239] Example 2. NR2F2+amygdala neurons drive fear

[0240] To define potential mechanisms controlled by astrocyte-neuron communication, we analyzed transcription factors expressed in amygdala neurons isolated from astrocyte Egfr KD or control mice. These analyses revealed 5 upregulated transcription factors also predicted to act as transcriptional regulators (Fig. 2a). We focused on the 3 most significantly expressed in neurons which were NR2F2, ETS 1, and C / EBPg (Fig. 2b). To test whether astrocyte-neuron PTPRS-Slitrk2 signaling affected the expression of the transcriptional regulators predicted by our RNA-seq analy ses, we performed in vitro co-cultures of primary' astrocytes and neurons using AAV -based KD of astrocyte Ptprs and neuron Slitrk2 or non-targeting controls (Fig. 2c). KD of both astrocyte Ptprs and neuron Slitrk2, but not each separately, decreased Nr2f2 expression in neurons (Fig. 2c). These data suggest that this neuronal transcriptional program is regulated by astrocyte-neuron cross-talk potentially relevant during chronic stress.

[0241] To test if these transcriptional programs controlled fear behavior, we bilaterally targeted the amygdala to KD Nr2f2. Cebpg, or Etsl, and subjected mice to 7 days of restraint stress (Fig. 2d) since we had detected the transcriptional signatures of these factors after 1-week of stress (Figs.2a, b) Consistent with our in vitro and genomic studies (Figs.2a-c), inactivation of Nr2f2 but not Etsl or Cebpg decreased stress-induced fear behavior (Fig. 2e). Using bulk RNA-seq and ATAC-seq, we uncovered downregulated activation of pathways linked to chronic stress, fear behavior, and synaptic domains in Nr2f2 KD mice (Figs. 2f-j) . Consistent with this, in validation studies by immunostaining, we detected fewer VGLUT27HOMERU puncta in the BLA of Nr2f2 KD mice (Fig. 2k), together with decreased excitatory neuron coverage in the BLA. Overall, these data suggest that in the context of chronic stress and fear, amygdala astrocytes potentially influence NR2F2 transcriptional programs in neurons.Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0242] Example 3. Regulation of amygdala responses

[0243] We next sought to infer the molecular cues modulating astrocyte-neuron interactions in amygdala subnuclei during chronic stress. Because we found cellular evidence that chronic stress and fear conditioning synergize to modulate astrocyte and neuron function, we performed spatial transcriptomics using Stereo-seq of mice that underwent 18 days of restraint stress followed by fear conditioning. We assigned cell types using an atlas of the mouse nervous system27recently applied to the amygdala36, and spatially defined the amygdala based on anatomical and cell type markers in conjunction with immunohistochemistry. This analysis detected Slcl7af and Slcl 7a6+excitatory' neurons, inhibitory' neurons, astrocytes, oligodendrocytes, and microglia, which were evenly distributed across samples.

[0244] Because we found NR2F2 influenced fear behavior and excitatory BLA neuron features (Figs. 2e, 2k), we subclustered Slc af and Slcl7a6+excitatory' amygdala neurons, and identified that cluster 2 excitatory neurons were associated with a late-stage pseudotime trajectory after 18 days of restraint stress and fear conditioning (Fig. 3a). Anatomically, most neurons linked with the stress-associated subset of cluster 2 excitatory neurons were confined to either the BLA28or the nucleus of the lateral olfactory' tract, an amygdala subnucleus interconnected with the BLA that amplifies olfactory cue-driven fear behavior37such as those present in our contextual fear conditioning paradigm (Fig. 3b). Consistent with our genetic inactivation and genomic studies, cluster 2 excitatory neurons were localized near astrocytes expressing undetectable levels of Egfr (Fig. 3c) and also expressed significantly higher levels of Nr2f2 relative to other excitatory' neuron clusters (Fig.

[0245] 3d)

[0246] Analysis of cluster 2 excitatory neurons revealed activation of pathways predicted to be driven by7several of the same cytokines and chemokines we detected in plasma of 18-day stressed mice including IL-lb and IL-12 (Figs. 1c, 3e). However, we did not directly detect peripheral immune cells in the amygdala in our spatial transcriptomic studies, perhaps due to transcriptional dropout of these and other less abundant cell types and the analysis of single sections per animal. We also did not detect markers of peripheral monocytes, such as Ccr2, in the cells we classified as microglia (Fig. 3f), consistent with chronic stress studies focused on the nucleus accumbens, where peripheral myeloid cells are observed to remain within theAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0247] vasculature6,38. Finally, we directly quantified in our amygdala spatial transcriptomic data the expression of cytokines related to each activated pathway we detected, but found negligible to undetectable expression of 1112a, 1112b, Illa, Illb, and 116 (Fig.

[0248] 3g). Based on the prediction of excitatory amygdala neuron transcriptional responses related to IL-lb and IL-12, coupled with our plasma cytokine studies, and recent reports of neural circuit regulation by peripheral immunity during chronic stress2’5’6’38, we hypothesized the neuro-glial interactions we detected in the amygdala might be tuned by peripheral immune cells.

[0249] Example 4. Psychedelics tune neuroinunune interactions

[0250] Multiple populations of peripheral immune cells influence behavior during chronic stress2’5’6,39Beyond the brain vasculature, immune cells within the brain meninges also control complex behaviors40-42. Hence, we first evaluated by FACS the abundance of immune cell subsets in the meninges, deep cervical lymph nodes (dCLN), and spleen from chronically stressed or control mice. We observed an increase in several meningeal immune cell populations from mice that underwent restraint stress for 18 days; however, we didn’t observe significant recruitment to dCLN or spleen and did not detect increases after 7 days of stress.

[0251] We focused on inflammatory monocytes because of recent reports detailing the heterogeneity of monocyte recruitment to the brain and other organs in the context of chronic stress2,6,38’43. We detected increased meningeal monocyte recruitment but depletion of splenic monocytes after 18 days of restraint stress (Fig. 4a). Thus, we examined whether monocyte trafficking from spleen to the meninges could account for these changes. Using mice ubiquitously expressing the photoconvertible fluorescent protein Kaede (CAGKaedemice), we measured splenic cells trafficking to the meninges, bone marrow, and dCLN 36-hours after photoconversion following 18 days of restraint stress (Fig. 4o). In CAGKliedemice, we found that relatively more splenic immune cells were recruited to the meninges compared to other tissues but that the proportion of splenic-derived meningeal immune cells decreased after chronic stress, suggesting meningeal immune cells may also traffic from other tissues (Fig.

[0252] 4o). In addition, we found the relative proportion of splenic monocytes trafficking to the meninges after 18 days of restraint stress w as significantly lower than that of other tissues such as the bone marrow, highlighting additional destinations for monocyte trafficking during chronic stress (Fig. 4p).Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0253] To examine the links between inflammatory monocytes and stress-induced fear behavior, we used multiple orthogonal gain- and loss-of-function approaches. First, we performed daily adoptive transfer of pro-inflammatory' monocytes into C57B1 / 6 hosts undergoing 7 days of restraint stress (Fig. 4b), which we validated accumulated in the meninges (Fig. 4c). Monocyte adoptive transfer led to exacerbated fear behavior and decreased EGFR-related transcriptional responses in amygdala astrocytes of recipient mice (Figs. 4d-f). We validated these findings in a second set of experiments using immunodeficient NSG mice undergoing 7 days of restraint stress (Figs. 4q,r). Third, we acutely ablated meningeal monocytes using intra-cistema magna (i.c.m.) injection of an anti-CCR2 monoclonal antibody after 18 days of restraint stress (Figs. 4g, s). Mice administered an anti-CCR2 antibody i.c.m. showed decreased fear behavior relative to controls and elevated EGFR pathway activity7in amygdala astrocytes (Figs. 4h-j). As a fourth independent validation, we inducibly expressed diphtheria toxin receptor (DTR) in monocytes (Ccr2DTR / +mice) to chronically deplete inflammatory monocytes. Consistent with our other analyses, we detected decreased fear behavior alongside depletion of meningeal and splenic monocytes in Ccr2P1R / +mice; importantly, diphtheria toxin administration did not affect the abundance of other immune cell subsets or amygdala microglia. Systemic monocyte depletion also limited astrocyte pro-inflammatory transcriptional responses linked with IL-1. IL-6, and TNFa, and increased astrocyte EGFR pathway activity.

[0254] In order to determine if meningeal products can infiltrate the amygdala, we injected Evans blue dye via i.c.m. injection and detected dye throughout the surface of the brain. Because IL-lb was predicted to modulate some of the astrocyte responses we detected (Figs. 1c, In-p, 3e, 4f), we analyzed meningeal cytokine penetrance into the amygdala by administering biotinylated IL-lb via i.c.m. injection (Fig. 4k). We detected significantly more IL-lb+puncta in the BLA of 18-day stressed mice relative to controls (Fig. 4k), consistent with reports of blood-brain barrier leakiness following chronic stress6 15. In line w ith our genomic and functional studies predicting the regulation of EGFR+astrocytes by IL-lb, we also detected increased IL-1R expression in BLA astrocytes in 18-day stressed mice relative to controls. These data suggest inflammatory monocytes mobilized in response to chronic stress may modulate fear behavior and amygdala transcriptional responses, in line with previous studies6,44.Attorney Docket No. 29618-0538WO1 / BWH 2023-538

[0255] To generate hypotheses surrounding the regulation of meningeally-recruited monocytes, we performed bulk RNA-seq, which predicted lower activity of serotonin signaling in meningeal monocytes after 18 days of restraint stress relative to controls (Fig. 41) Serotonin signaling impacts multiple tissues, and is not only modulated by the neurotransmitter serotonin, but also by psychedelic drugs such as psilocybin45and MDMA46. Moreover, psilocybin or MDMA administration modulates affective behaviors governed by limbic areas in animals as well as in MDD and PTSD patients47 51. As independent validation, we detected the expression of several receptors that regulate the effects of psychedelics in immune cell subsets from the mouse ImmGen atlas. To functionally test whether myeloid cell subsets respond to psilocybin or MDMA, we treated primary CD1 lb+splenic cells with 200 ng / mL corticosterone and 10 pM psilocybin or 10 pM MDMA racemate, which downregulated the chemokine receptors Cxcr3 and Cxcr4, and was reversed by treatment with a 5-HT2R antagonist. Treatment with 10 pM psilocybin or 10 pM MDMA racemate also decreased II lb expression in LPS-treated primary CDllb+splenic cell cultures, suggesting that psilocybin and MDMA can modulate some immune cell responses.

[0256] To test whether psilocybin or MDMA affected monocyte recruitment or responses in vivo, we treated mice that underw ent 18 days of restraint stress with vehicle, 1 mg / kg psilocybin, or 10 mg / kg MDMA racemate, since these doses roughly correspond to those used in humans48,49, and have been used extensively in mice47,50,51. We detected a decrease in meningeal monocyte recruitment, meningeal cytokine production, and fear behavior in mice treated with psilocybin or MDMA relative to controls (Figs. 4m, n).

[0257] We found that in addition to inflammatory monocytes, psilocybin and MDMA treatment regulated the abundance of multiple immune cell classes in the meninges following 18 days of restraint stress (Fig. 7a), with more modest effects observed in the spleen and dCLN (Figs. 7b, c). Hence, these data suggest psilocybin and MDMA may influence meningeal immune cell abundance through non-cell autonomous mechanisms. In fact, psychedelics are known to induce vasoconstriction52while chronic stress induces vascular dysfunction6,38,53. We therefore examined whether psilocybin or MDMA might exert effects on the vasculature to indirectly influence immune cell abundance. Indeed, we detected expression of type-2 serotonin receptorsAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0258] and Ntrk2 in non-hematopoietic meningeal cells. Next, we measured vascularity using a fluorescent dye that binds to the vasculature and detected a significant increase in fluorescent signal following 18 days of restraint stress, which was reduced upon administration of psilocybin or MDMA racemate. To determine whether vasoconstriction influenced inflammatory monocyte abundance, we treated 18-day stressed mice with psilocybin or MDMA racemate together with the systemic vasodilator nifedipine and observed increased numbers of meningeal inflammatory monocytes in the presence of nifedipine relative to psilocybin or MDMA alone. By contrast, we did not observe sustained decreases in peripheral corticosterone levels following psychedelic treatment nor did we observe that psychedelic-induced immune cell redistribution was exclusively controlled by the sympathetic nervous system. Beyond the vasculature, we also detected transcription of type-2 serotonin receptors and Ntrk2 in primal)' astrocyte cultures (Fig. 7d). Functionally, psilocybin or MDMA racemate treatment also reduced astrocyte Ptprs expression in vitro (Fig. 7e).

[0259] Altogether, these data point to potential direct and indirect mechanisms by which psychedelics could influence physiological responses to chronic stress and neuroimmune interactions.

[0260] Example 5. Validation in humans and MDD patients

[0261] To validate our findings in human cells, we treated primary human astrocytes with TNFa, IL-6, or IL-1 b in the presence of cortisol and found the combination of IL-lb and cortisol increased PTPRS expression, similar to our data in mice (Fig. 5a).

[0262] We also detected decreased EGFR expression in primary' astrocytes treated with IL-lb and cortisol (Fig. 5b). Next, we validated in the ImmGen human immune cell atlas expression of HTR2A. HTR2B, HTR2C, and NTRK2 in human immune cell subsets in blood, including monocytes. We then cultured primary human monocytes in the presence or absence of 100 ng / mL LPS with or without 10 pM MDMA racemate or 10 pM psilocybin and quantified by bulk RNA-seq that treatment with either drug reduced CXCR4 expression and other transcriptional modules (Figs. 5c, akin to our findings in mice (Figs.4m, n).

[0263] Finally, to validate our observations in human tissues, we analyzed by single nucleus RNA-seq (snRNA-seq) the amygdalae of 6 patients with MDD and 6 healthy controls, matched by sex, race, and age. We recovered 13,408 cells that we assigned to 6 different cell types including astrocytes, microglia, excitatory’ neurons, inhibitoryAttorney Docket No. 29618-0538WO1 / BWH 2023-538

[0264] neurons, oligodendrocyte precursor cells, and oligodendrocytes. Next, we subclustered astrocytes to identify subsets associated with MDD, and found a population expanded in MDD marked by downregulated EGFR signaling. We also examined the pathways altered in cluster 1 and cluster 6 astrocytes, which are down-and upregulated in MDD, respectively. In these subsets, we identified altered metabolic, neurotransmitter, and extracellular matrix signaling, which have previously been implicated in MDD54. We orthogonally validated downregulated EGFR expression in MDD by qPCR of bulk patient tissue and by immunostaining of amygdala astrocytes in MDD patients. Moreover, subclustering of excitatory neurons revealed a group expanded in MDD expressing NR2F2 and SLITRK2 and predicted to be controlled by elevated NR2F2-driven transcriptional programs (Figs. 5e).

[0265] Altogether, these data suggest the neuroimmune mechanisms we initially defined in mice may be relevant in humans, and potentially in MDD.

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[0415] OTHER EMBODIMENTS

[0416] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Atorney Docket No. 29618-0538WO1 / BWH 2023-538WHAT IS CLAIMED IS:

1. A method for identifying a potential candidate neuroimmune target transcript for modifying neuroimmunity in a mammal, the method comprising:subjecting anon-human animal model to a behavioral stress condition; obtaining first samples comprising cells from the CNS, and second samples immune cells from the animal;determining RNA transcriptomic profiles from the first and second samples, and comparing the RNA transcriptomic profiles from the first and second samples to RNA transcriptomic profiles from first and second samples determined from a control animal that was not subjected to the behavioral stress condition, to identify transcripts altered in the animal model as compared to the control animal; selecting transcripts that are altered in the first samples and second samples, and identifying one or a subset of transcripts that are altered in the first samples that are related to transcripts that are altered in the second samples,thereby identifying a potential candidate target transcript for modifying neuroimmunity in a mammal.

2. The method of claim 1, further comprising using cell sorting or computational sorting to identify cell subtypes within the first and second samples, and identifying transcripts altered in one or more of the subtypes.

3. The method of claim 2, wherein computational sorting is performed by clustering based on gene expression.

4. The method of any of claims 1 to 3, wherein the cells from the CNS comprise astrocytes, microglia, excitatory neurons, inhibitory neurons, oligodendrocyte precursor cells, and oligodendrocytes.

5. The method of any of claims 1 to 4, wherein the immune cells comprise astrocytes from the CNS.

6. The method of claim 1, wherein identifying one or a subset of transcripts that are altered in the first samples that are related to transcripts that are altered in the second samples comprises using a database to identify transcripts in the firstAtorney Docket No. 29618-0538WO1 / BWH 2023-538samples that affect expression of transcripts that are altered in the second samples, or vice versa.

7. The method of claim 1, further comprising:providing an in vitro model of the CNS cells or immune cells;perturbing the potential candidate target transcript; evaluating an effect of the perturbation on related transcripts, andselecting a potential candidate target transcript that alters the related transcripts as a candidate target.

8. The method of claim 7, wherein perturbing the candidate target transcript comprises using a genetic or pharmacological tool to reduce or increase expression of the candidate target transcript.

9. The method of claim 8, wherein the genetic tool is selected from CRISPR knockout, or administration of an antisense oligonucleotide (ASO), Cre recombinase based deletion, small interfering RNA (siRNA), small hairpin RNA (shRNA).

10. The method of claim 8, wherein the pharmacological tool is selected from contacting the cells with a small molecule or antibody that binds to the protein product of the candidate transcript.

11. The method of claim 7, further comprising:subjecting a second non-human animal model to a behavioral stress condition in the presence of a perturbation of the candidate transcript, wherein the second non- human animal model is the same as the non-human animal model used in claim 1; evaluating a parameter of the behavior of the animal in the presence of the perturbation as compared to the parameter of the behavior in the absence of the perturbation; andidentifying a transcript that alters the parameter when perturbed as a potential therapeutic candidate.

12. The method of claim 11, further comprising:obtaining first samples comprising cells from the CNS, and second samples immune cells from the animal;Atorney Docket No. 29618-0538WO1 / BWH 2023-538determining RNA transcriptomic profiles from the first and second samples, and comparing the RNA transcriptomic profiles from the first and second samples in the presence of the perturbation of the candidate transcript to RNA transcriptomic profiles from first and second samples determined from a control animal in the absence of a perturbation of the candidate transcript, to determine an effect of the perturbation of the candidate transcript;selecting transcripts that are altered in the first samples and second samples, and identifying one or a subset of transcripts that are altered in the first samples that are related to transcripts that are altered in the second samples, and determining whether the perturbation of the candidate transcript alters related transcripts in the second samples;thereby identifying a potential therapeutic candidate transcript for modifying neuroimmunity in a mammal.

13. The method of claims 11 or 12, further comprising:obtaining a first human sample comprising human CNS cells and a second human sample comprising human immune cells;perturbing the potential therapeutic candidate transcript in the first and second human samples;determining whether the perturbation of the potential therapeutic candidate transcript alters related transcripts in the second human samples and selecting a potential therapeutic candidate transcript when the perturbation alters the related transcripts as a therapeutic candidate.

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