Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

Domain: neuroscience
Published: 2026-01-16
Editorial outcome: major_revision

Abstract
Chronic stress promotes blood-brain barrier (BBB) integrity loss leading to passage of inflammatory mediators in mood-regulating brain areas and establishment of depressive behaviors. Conversely, neurovascular adaptations favoring stress resilience and preventive strategies to promote them are undetermined. We report that environmental enrichment dampens stress-induced loss of endothelial tight junction Claudin-5 (Cldn5) along with anxiety- and depression-like behaviors in male mice via an increase in fibroblast growth factor 2 (Fgf2). Coping with voluntary physical exercise also protects the BBB from stress deleterious effects by increasing Fgf2. Fgf2 is mostly expressed by glial cells, and viral-mediated astrocyte-specific Fgf2 upregulation prevents stress-induced social avoidance while downregulation increases stress susceptibility and blunts physical exercise benefits. Treatment of mouse and human endothelial cells with Fgf2 prior an immune challenge reduces BBB dysfunction, Cldn5 loss, and altered signaling supporting its protective role. Circulating FGF2 level is linked with depression severity and symptomatology in men and women reinforcing involvement of this growth factor in mood disorders.

Introduction
Major depressive disorder (MDD) is a psychiatric condition affecting >300 million people worldwide, representing a growing burden on global health systems1. Women are twice as likely to be diagnosed, and MDD presents sex differences in symptomatology, treatment responses and brain transcriptional profiles2–5. Common antidepressants are ineffective for 30–50% of depressed individuals suggesting that underlying causal mechanisms remain unaddressed6,7. First evidence of blood-brain barrier (BBB) leakiness in human depression by detection of brain-related proteins in the blood was reported 60 years ago8. Indeed, cerebrospinal fluid serum ratio of various peripheral markers are altered in individuals with MDD suggesting compromised BBB function6,9,10. The BBB is a dynamic frontier mediating communication between the periphery and the brain, composed of an intricate cellular network, including astrocytes, pericytes, and endothelial cells connected by specialized tight junctions7,11. This distinctive composition enables metabolic supply while also ensuring a selective permeability which protects the brain from bloodstream harmful toxins and inflammatory factors12,13. The BBB is necessary to maintain neural activity, and disruption can lead to neuroinflammation, neuronal death, and severe cognitive deficits7. For decades, mechanisms leading to neurovascular pathology in MDD remained elusive.

In the last years, several groups reported that in mice, chronic stress exposure, MDD main environmental risk factor, is associated with a sustained elevation of circulating inflammatory mediators such as interleukin-1β, interleukin-6 (IL-6) and tumor necrosis factor α (TNFα)6,14. This increase results in activation of brain endothelial cells via elevated cytokine expression, leukocyte adhesion, and degradation of tight junction protein Claudin-5 (Cldn5) leading to increased barrier permeability as assessed by passage of tracers15–17. BBB alterations and tight junction loss occur in a sex- and brain region-specific manner with the BBB being more vulnerable in the female prefrontal cortex (PFC) vs nucleus accumbens (NAc) for males15,18. The NAc is a forebrain nucleus playing key roles in reward and mood regulation while the PFC is involved in social behaviors, executive function and decision making19,20. Importantly, region-specific reduced expression of endothelial cell tight junction CLDN5 was confirmed in postmortem human brain samples from MDD men21 and women18 supporting a direct link between neurovascular health, stress vulnerability and human depression7,15,16,18,22. In other mental conditions like bipolar disorder and schizophrenia, the degree of BBB damage appears to correlate with age of onset as well as disease severity23,24, suggesting that the neurovasculature could represent an innovative target for effective intervention against mood disorder development and progression.

Environmental conditions that promote stress resilience and neurovascular health offer a promising approach to identify therapeutic sites. Indeed, stimulating environments are well known to positively alter the adult brain, including vascularization and BBB function25–28. Further, circumstantial factors such as socioeconomic status and physical activity are negatively correlated with depression risk in humans29,30, while in mice, access to nesting material, shelter, and toys (enriched environment, EE) or a running wheel (physical exercise, PE) attenuates depression-like behaviors following stress exposure31,32. The involvement of the BBB in these outcomes has not yet been assessed; however, we recently observed neurovascular changes after learning and memory tasks depending on environmental conditions33. The present study expands this idea by combining behavioral experiments performed in male and female mice with in vivo functional manipulations and in vitro cell signaling work to identify functional and transcriptional adaptations of the BBB involved in the pro-resilient effects of EE and PE during chronic stress. We report that environmental intervention can rescue stress-induced deficits in social behavior and expression of tight junction protein Cldn5 in both sexes. We identify fibroblast growth factor 2 (Fgf2) as a sex-specific protective factor upregulated in response to stress in the NAc of males with access to either EE or voluntary PE. Fgf2 is mostly expressed by astrocytes in both the mouse and human brain34,35 and it is involved in neuronal and glial cell signaling, proliferation, and wound healing36,37. This growth factor can induce angiogenesis by stimulating endothelial cell proliferation in pathological context38,39 and thus we hypothesized that it could be actively involved in pro-resilient neurovascular adaptations. Indeed, viral-mediated increase of astrocytic Fgf2 expression in the male NAc prevents stress-induced social avoidance, while downregulation in the same brain area promotes stress susceptibility. Fgf2 can dampen inflammatory activation of brain endothelial cells, as well as subsequent loss of barrier integrity by increasing Cldn5 expression, suggesting a potential protective mechanism. Finally, we associate a change in circulating FGF2 with depressive symptoms in human cohorts and highlight sex differences as well as the impact of university education, an important indicator of socioeconomic status.

Results
Social stress has been extensively studied in humans since higher prevalence of mood disorders and suicide attempts have been reported in victims of bullying40. The chronic social defeat stress (CSDS) paradigm is a commonly used mouse model of depression in which C57Bl/6 mice are exposed daily (5 min/day) for 10 days to a physical bout with a larger aggressive CD-1 mouse41. The day after the last stress bout, a social interaction (SI) test is performed, and behavioral phenotype is defined as the time spent in the interaction zone test when the aggressor is present divided by absent (Fig. 1A)41. In standard conditions with no supplements such as toys, shelter, or nesting material, about two-thirds of stressed mice display social avoidance (SI ratio <1) and are classified as stress-susceptible (SS), while the other third, showing no behavioral deficits, are considered resilient (RES) with a SI ratio >141. To evaluate if EE has an impact on stress-induced BBB alterations, male C57Bl/6 were given access to nesting material, a shelter, and plastic chew toy on their side of the CSDS cage throughout the stress paradigm (Fig. 1A). EE increased the proportion of mice classified as resilient to more than half (Fig. 1B, stress x environment effect: **p=0.0029, 57.9% with a SI ratio >1 vs 41% for standard housing conditions). As expected, almost all unstressed control (CTRL) mice were resilient (Fig. 1B). Importantly, stressed EE mice spent similar time in the corners during the SI test compared to unstressed controls, while mice subjected to CSDS in a standard environment developed social avoidance (Fig. 1C, stress×environment effect: ****p<0.0001). No difference in exploration time was noted as measured by the distance traveled in the arena when the aggressor was either present or absent excluding locomotor issues (Supp. Fig. 1A).

To probe a role for the BBB in these positive effects, we next investigated stress-induced changes for transcription of BBB-related genes involved in cell proliferation, vascular remodeling, tight junction formation, or markers of astrocyte, pericyte, and neuroinflammation, in the NAc and PFC, two brain regions involved in the onset of depressive-like behaviors in mice and MDD in humans (Fig. 1D and Supp. Fig. 1B for behavioral data). Genes were selected based on previous work from our group and others showing changes in expression following stress exposure15,18,42,43. Standard CSDS reduces the expression of Cldn5, a key tight junction protein, specifically in the male NAc, and increases expression of inflammatory cytokine Il-615. In contrast, we found in the NAc of stressed EE mice a general increase in gene expression associated with vascular remodeling and tight junction formation relative to unstressed EE control, including for Cldn5 (Fig. 1D, left, *p=0.0253), as well as growth factors, particularly Fgf2 (*p=0.038) which is linked to BBB integrity44,45 and antidepressant behavioral effects43,46–49. No change in Cldn5 was observed in the male PFC in line with intact BBB integrity15, but expression of several growth factors, including Fgf2 was upregulated (Fig. 1D, right, *p=0.0196), suggesting adaptive mechanisms. The FGF family consists of ~20 ligands with a subset expressed in the brain. Most are involved in cortical patterning during development. Fgf2 is notable for its high expression in non-neuronal cells in mice and humans34 (Supp. Fig. 1C), angiogenic properties36,44, and implication in MDD43,48–52. We assessed the expression of Fgf1, 2, 4, 8, 9, 10, and 21, the most abundant of the FGF family, in the PFC and NAc (Supp. Fig. 1D). Fgf4, 8 and 21 were undetectable in both brain areas while Fgf9 and 10 expression levels could not be detected in cultured BBB-related astrocytes or endothelial cells (Supp. Fig. 1E) as expected from a cell-specific brain RNA-seq atlas34. Stress exposure had no impact on the expression of Fgf1 in the NAc (Supp. Fig. 1F). Comparison of NAc gene expression from standard CSDS and EE cohorts indicates a beneficial effect of EE through prevention of both Cldn5 loss (Fig. 1E, ****p<0.0001) and increased inflammatory cytokine IL-6 (*p=0.0318) following CSDS. Previous reports from standard CSDS show that SS and RES mice display substantially different transcriptional patterns21. Principal component analysis (PCA), used to identify strong patterns from the gene transcripts in our EE cohort, revealed that SS and RES mice strongly overlap (Fig. 1F). Indeed, when comparing gene expression patterns from our EE mice with previously published standard environment CSDS15, we found evidence for close clustering of all behavioral phenotypes (CTRL, SS, and RES) based on BBB-related gene expression, in EE but not standard housed CSDS cohorts (Fig. 1F and Supp. Fig. 2D), indicating EE dampens stress effect on BBB-related transcription.

To confirm gene expression changes at the protein level, immunofluorescent staining was performed to label for Cldn5, Fgf2, and CD31, a marker of blood vessels, in brain slices from the NAc and PFC of unstressed CTRL, SS and RES mice (Fig. 1G and Supp. Fig. 2A for behavioral data). In standard CSDS, Cldn5 coverage of blood vessels is reduced by ~50% in the NAc of SS male mice15. With EE, this loss is dampened and not significant anymore (p=0.1322), supporting compensatory changes (Fig. 1G, Supp. Fig. 2B for CD31). In this line, while chronic stress has been reported to reduce Fgf2 expression in a standard environment in the PFC50, stressed mice in our EE cohort had increased Fgf2 in the NAc (*p=0.0317), and this tends to correlate with the degree of Cldn5 loss (r=−0.4882, p=0.0648) (Fig. 1G). No change was observed in the PFC in EE conditions (Supp. Fig. 2C). Altogether, our findings suggest that access to an enriched environment has a protective effect on stress-induced BBB alterations, possibly via an elevation in Fgf2.

While CSDS is a standard protocol to induce social stress in male mice, it is not as relevant for female mice who do not commonly experience aggression in the wild53. Artificial methods for inducing social defeat in females exist, including application of male urine to promote aggressive bouts54, but it introduces sensory information which may interfere with enrichment effects. In our hands, still only ~30% of female mice become susceptible to 10-day CSDS with this protocol, hampering the possibility to test preventive or protective approaches18. Thus, to evaluate whether EE could promote resilience in females, we took advantage of the subchronic variable stress (SCVS) paradigm, an established protocol producing anxiety and anhedonia after 6 days in females18,55 along with BBB changes in the PFC18. Female mice assigned to control or SCVS groups had access to EE as described above and the SCVS group was exposed to a series of three repeated stressors, namely foot shock, tail suspension, and tube restraint (Fig. 2A). Following SCVS, a cohort was subjected to a battery of behavioral tests to assess the impact of EE on stress responses (Supp. Fig. 3A). As expected, exposure to SCVS in standard conditions induced anhedonia, a core feature of MDD (Supp. Fig. 3B, **p=0.0042). Compared to 6-d SCVS with standard housing18, access to EE prevented stress-induced reduction in time spent in the elevated plus maze (EPM) open arms (Fig. 2B, ***p=0.0002), increased social interactions (*p=0.0297), and sucrose preference (****p<0.0001) (Supp. Fig. 3B-F). Exposure to 6-d SCVS is generally not sufficient to induce anxiety- or depression-like behaviors in males56. Nevertheless, to be consistent a cohort of male mice was subjected to the same stress paradigm without or with access to EE (Supp. Fig. 4A). No difference in behaviors was observed except for a significant effect of the environment for the SI ratio, regardless of stress exposure, due to increased time spent in the interaction zone when the CD-1 was absent (Supp. Fig. 4B-E). For tissue collection, a 2nd cohort of female EE+SCVS was tested in the EPM only to confirm normal behaviors despite stress exposure, then brain tissue was collected 24 h later (Fig. 2A and Supp. Fig. 5A, B for behavioral data) so 48 h after the last stressor, like for males (Fig. 1A). No difference in estrous cycle stage was observed between CTRL and SCVS groups at tissue collection (Supp. Fig. 5C).

Neurovascular disruption in the PFC underlies the development of anxiety- and depression-like behaviors in female mice and a loss of CLDN5 was noted in this brain area in postmortem samples from women with MDD18. Gene expression analysis showed that EE stabilizes BBB transcriptomic patterns in the PFC (Fig. 2C), leading to maintenance of Cldn5 expression vs SCVS in standard housing (Fig. 2D, **p=0.0024) and normal behaviors despite stress exposure. We also observed that expression of Fgf1 and Fgf2 is similar in the NAc and PFC of female and male mice at baseline (Supp. Fig. 5D). Next, BBB-related gene expression patterns in the female PFC were compared after SCVS in standard vs EE conditions with PCA analysis. We revealed that with standard housing, CTRL and SCVS mice form distinct clusters, while with EE they are more closely grouped (Fig. 2E). Because EE reduces transcriptional differences between control and stressed mice in both males and females, gene expression patterns were compared in the NAc and PFC across sexes. Even if the behavioral outcomes are similar, BBB-related transcriptomic profiles of males and females differ, suggesting that environmental influence on the neurovasculature could be sex-specific (Fig. 2F, Supp. Fig. 5E, F). Given the implication of Fgf2 as a protective factor in males, we assessed its protein level along with Cldn5 and Cd31 in females. Immunofluorescent staining confirmed an absence of stress-induced changes in Cldn5 but also Fgf2 in both PFC (Fig. 2G, Supp. Fig. 5G) and NAc (Supp. Fig. 5H) in the female EE cohort. These results suggest that females benefit from an EE however, elevation of Fgf2 might be a male specific protective mechanism for pro-resilient effects on the BBB.

Fgf2 is sensitive to environmental conditions, and modifiable lifestyle factors such as physical exercise (PE) have been linked to elevated expression of this growth factor in the brain57. Voluntary PE has been proposed as a critical variable for disease prevention and stress resilience associated with environmental enrichment experiments58. PE has benefits for neurovascular health, however, it is unknown if it could protect from chronic social stress-induced BBB alterations. After running wheel habituation, male mice were then randomly assigned to either control or stress groups which both had free voluntary access to running wheels in their home cage throughout the CSDS protocol (Fig. 3A). Access to voluntary physical exercise during stress exposure increased the proportion of RES mice after CSDS to 53.6% (Fig. 3B), and like EE (Fig. 1C), strongly reduced social avoidance as measured by the time spent in the corners during the social interaction test (Fig. 3C, ****p<0.0001 and Supp. Fig. 6A-C for additional behavioral data). As expected, mice ran mostly during the dark cycle with distance increasing throughout the 10-d CSDS paradigm for the stressed mice to reach a total distance significant effect when compared to unstressed controls (Fig. 3D-E, *p=0.0241). Intriguingly, RES mice ran more than other groups during the light cycle (Fig. 3F, **p=0.0061), including right after the defeat bout (Fig. 3G, ****p<0.0001 vs CTRL and *p=0.0136 vs SS), suggesting that PE may represent an active coping strategy when facing social stress.

Brains were collected 48 h after the last stressor (Fig. 3A) and BBB-related genes in the NAc and PFC analyzed and compared between groups. Few changes were noted between CSDS and control mice with access to PE, with the exception of a strong increase in Cldn5 transcript levels in both the NAc (**p=0.005) and PFC (**p=0.0012) of stressed mice and this effect was driven by RES animals (Fig. 3H, *p=0.0206 for NAc and *p=0.0233 for PFC). Conversely to our EE cohort where stressed mice displayed gene expression patterns distinct from controls, PCA analysis of the PE CSDS cohort revealed that CTRL, SS, and RES mice all form similar clusters, though stressed mice exhibit more variance (Fig. 3I, Supp. Fig. 6E). At the protein level, CSDS did not affect Cldn5 levels in the NAc of our PE cohort, whereas all stressed PE mice exhibited an increase in Fgf2 staining compared to controls (Fig. 3J, Supp. Fig. 6D-G). Altogether, our results indicate a protective effect of voluntary PE on stress-induced BBB changes in the male NAc, dampening social avoidance and favoring resilience.

Since Fgf2 is mostly expressed by astrocytes in the mouse and human brain34,35, and its level is increased in this cell type particularly along blood vessels in the context of physical exercise (Fig. 4A, B), we designed an adeno-associated virus (AAV) vector driving astrocytic Fgf2 expression via the gfaABC1D promoter, and then tested its impact on social behaviors after CSDS exposure. First, we validated increased Fgf2 protein level in the NAc of AAV5-gfaABC1D-mFgf2-P2A-GFP-injected mice when compared to animals injected with the control virus (AAV5-gfaABC1D-GFP) (Fig. 4C, D, left, **p=0.0067). Intriguingly, the elevation in Fgf2 expression was mostly observed along blood vessels stained with the endothelial marker CD31 (Fig. 4D, right, *p=0.0163). Next, a cohort of male mice was bilaterally injected with either the control AAV5-gfaABC1D-GFP or AAV5-gfaABC1D-mFgf2-P2A-GFP viruses and then subjected to 10-day CSDS 4 weeks later to allow maximal Fgf2 expression (Fig. 4E). Astrocytic increase in Fgf2 did not alter the SI ratio, phenotype distribution (Fig. 4F, G) or locomotion (Fig. 4H), however, it reduced social avoidance as defined by the time spent in the corners of the arena (Fig. 4I). Stressed mice spent more time in the corners, an effect driven by the AAV5-gfaABC1D-GFP-injected animals (Fig. 4I, left, *p=0.0407) with no difference observed for AAV5-gfaABC1D-mFgf2-P2A-GFP mice (p=0.5267). In fact, only SS mice injected with the control virus displayed social avoidance when compared to AAV5-gfaABC1D-GFP unstressed control and RES groups (Fig. 4I, right, **p=0.0089 for SS vs CTRL; *p=0.0453 for RES vs SS) or AAV5-gfaABC1D-mFgf2-P2A-GFP animals categorized as SS due to a SI ratio <1 (*p=0.0258). Tissue collection after behavioral assessment confirmed higher Fgf2 level in the NAc of mice injected with the AAV5-gfaABC1D-mFgf2-P2A-GFP virus (Fig. 4J). These results suggest that increasing Fgf2 in astrocytes can prevent CSDS-induced social avoidance, mimicking the beneficial effect of access to an enriched environment (Fig. 1C) or voluntary physical exercise (Fig. 3C) while facing chronic social stress exposure.

Next, to confirm that elevation of astrocytic Fgf2 is involved in stress resilience, we designed an AAV vector with a short hairpin RNA (shRNA) blocking Fgf2 expression under the gfaABC1D astrocyte-specific promoter (Fig. 5A). We first validated decreased Fgf2 protein level in the NAc of AAV5-gfaABC1D-shRNA-mFgf2-GFP-injected mice when compared to animals injected with the control virus (AAV5-gfaABC1D-shRNA-scramble-GFP) in a cohort that did not go through behavioral testing (Fig. 5B, *p=0.0163). A cohort of male mice was bilaterally injected with either the control AAV5-gfaABC1D-shRNA-scramble-GFP or AAV5-gfaABC1D-shRNA-mFgf2-GFP viruses and then subjected to 10-day CSDS 4 weeks later to allow maximal viral expression (Fig. 5C). Reducing astrocytic Fgf2 in the NAc altered the SI ratio after CSDS exposure, with a higher proportion of SS mice (Fig. 5D, E, *p=0.0499). This was not due to a change in locomotion (Fig. 5F). Another cohort of mice was bilaterally injected with either the control AAV5-gfaABC1D-shRNA-scramble-GFP or AAV5-gfaABC1D-shRNA-mFgf2-GFP viruses and subjected to 10-day CSDS 4 weeks later but with access to running wheels (Fig. 5G). Stressed mice from both groups displayed social avoidance, as measured by lower SI ratio (***p=0.0005) and higher number of entries in the corners when the AGG was present (*p=0.0223), supporting our hypothesis that an elevation in NAc astrocytic Fgf2 may contribute to the benefits of PE (Fig. 5H, I). Behavioral alterations were not due to impaired locomotion (Fig. 5J). In fact, stressed mice injected with the control virus ran increasing distance across days after the first defeat bout (Fig. 5K) as observed in non-injected animals (Fig. 3D). This was not the case for AAV5-gfaABC1D-shRNA-mFgf2-GFP-injected mice (Fig. 5L), suggesting that motivation could be affected, with the NAc playing a crucial role in reward processing19. In line with these behavioral findings, we observed an elevation of Fgf2 expression in the stressed PE cohort injected with the control AAV5-gfaABC1D-shRNA-scramble-GFP virus (**p=0.0024) (Fig. 5M). In contrast, AAV-driven reduced Fgf2 expression was similar in the NAc of AAV5-gfaABC1D-shRNA-mFgf2-GFP-injected mice without (**p=0.0025) or with access to running wheels (***p<0.0001) (Fig. 5M), indicating that Fgf2 may be involved in the rewarding effects of PE.

BBB disruption is associated with stress-induced behavioral deficits in mice15,16,18,22 and psychiatric disorders, including MDD in humans15,18,21,23,24,59. Fgf2 can mediate the formation of tight junctions in endothelial cells44,45, nevertheless, it is undetermined if Fgf2 could protect against stress-related neurovascular damage. Our findings indicate a role for Fgf2 in protective effects of EE on the NAc BBB, specifically in male mice, with upregulation of this growth factor occurring in parallel with increased Cldn5 expression at both gene and protein level in stressed mice with access to EE (Fig. 1). Therefore, we investigated whether Fgf2 can preserve brain endothelial cell properties in vitro using treatment with the proinflammatory cytokine TNF-α as a biological stressor. Indeed, TNF-α is elevated in the blood of mice after CSDS and individuals with MDD60–62, while downstream signaling through TNF receptors is linked to stress-induced BBB breakdown in mice21,22. To ensure translational relevance we exposed both human (HBEC-5i) and mouse (bEnd.3) brain endothelial cell lines to either acute (<24 h) or chronic (up to 7 days) periods of inflammatory challenge with 10 ng/mL of TNF-α (Fig. 6A). Doses of TNF-α and time points for cell collection were determined according to previous publications describing impact of this cytokine treatment on endothelial cells63–65. Cells were pretreated for 1 h with FGF2 (10 ng/mL38) to mimic habituation with EE/PE, before co-stimulation with TNF-α and/or FGF2. Following TNF-α treatment, expression of genes associated with tight junctions, FGF2 signaling, and proinflammatory activation of endothelial cells was evaluated at several time points (1, 3, 6, or 24 h). Acute TNF-α stimulation induced strong endothelial cell activation characterized by downregulation of tight junction proteins Cldn5/CLDN5 and Ocln/OCLN, as well as expression of inflammatory factors such as IL-6 (HBEC-5i) and Vcam-1 (bEnd.3) (Fig. 6B, C). In both cell types, FGF2 attenuated TNF-α-induced loss of CLDN5, with protective effects especially prominent after 6 h of treatment (Fig. 6D, E, *p=0.0119 for mouse and ***p=0.0004 for human endothelial cells).

Since FGF2 could reverse the effects of acute TNF-α treatment on CLDN5 expression, we next tested if it could prevent loss in barrier integrity following long-term TNF-α exposure. Chronic stimulation with TNF-α over 7 days altered endothelial monolayer integrity as measured with trans-endothelial electrical resistance (TEER) when compared to control wells, by day 1 in bEnd.3 and day 3 in HBEC-5i, and this effect was prevented by FGF2 co-treatment (Fig. 6F, G, **p=0.0014 for mouse and ****p<0.0001 for human endothelial cells). FGF2 is a potent mitogen thus, an MTT assay was conducted to rule out potential changes in cell number which could influence TEER. Once confluent, FGF2 did not influence cell number vs controls (Supp. Fig. 7). On day 7 of the TEER protocol, cells were fixed and stained for Cldn5 to visualize tight junctions. bEnd.3 cells treated with TNF-α showed disruption of tight junction structure, including presence of spikes, discontinuities, and membrane ruffling which are signs of endothelial dysfunction66; however, this morphology was not observed for cells co-stimulated with Fgf2 (Fig. 6H).

To further gain mechanistic insights, the molecular mechanisms underlying the rescue of TNF-α-induced Cldn5 loss by Fgf2 were investigated. An integral part of TNF-α response in endothelial cells is activation of the Akt/ERK pathways, which slow and eventually terminate the inflammatory signaling cascade65. Fgf2 is a potent activator of these pathways67, and we evaluated whether the protective effects of Fgf2 on endothelial cells could be a result of rapid resolution of inflammatory signaling. Akt phosphorylates GSK3β serine residues, inhibiting this redox-sensitive enzyme and crucial mediator of TNF-α signaling known to disrupt tight junction integrity when activated in endothelial cells63,64,68. Therefore, we performed western blotting to assess the effects of TNF-α and Fgf2 on phosphorylation of GSK3β in the human HBEC-5i cell line (Supp. Fig. 8). We found that 1 h pretreatment with FGF2 induced GSK3β serine-9 phosphorylation (Fig. 7A, *p=0.0332), but it did not prevent the rapid (5 min) dephosphorylation response upon TNF-α stimulation (Fig. 7A). β-catenin is a downstream target of GSK3β, which normally interacts with cadherins to promote tight junction integrity, but in the context of stress and inflammation, it can be internalized to promote deleterious signaling21,69,70. In concordance with GSK3β inhibition, 1 h of FGF2 treatment tended to reduce serine/threonine β-catenin phosphorylation (Fig. 7B, p=0.0747). However, while TNF-α treatment initially reduced β-catenin phosphorylation (**p=0.0031), when co-administered with Fgf2 it led to a strong increase in β-catenin phosphorylation peaking 1 h following TNF-α introduction (***p=0.0004) (Fig. 7B). These results suggest that FGF2 can regulate β-catenin dynamics during inflammatory activation of brain endothelial cells which could mitigate stress-induced BBB alterations (Fig. 7C).

With endothelial β-catenin signaling essential for tight junction regulation and BBB integrity, we assessed if changes in inflammation-induced β-catenin phosphorylation mediated by FGF2 treatment could affect β-catenin cellular distribution and tight junction morphology. β-catenin was stained by immunofluorescence in human HBEC-5i cells in control conditions or following 30 min of TNF-α treatment with or without FGF2. TNF-α-treated cells displayed tight junction spikes and discontinuities indicative of ultrastructural disruption as well as possible β-catenin internalization, whereas this morphology was not observed in control or TNF-α/FGF2 co-treated cells (Fig. 7D, E). Furthermore, TNF-α induced distension of β-catenin-labeled tight junction strands compared to vehicle-treated wells, as the width of β-catenin labeled tight junctions, was increased in TNF-α-treated wells (Fig. 7D, E). This diffusion was normalized when cells were co-administered FGF2, suggesting a role for this growth factor in stabilizing β-catenin at sites of cell adhesion and preserving normal tight junction morphology. Since both Fgf2 and β-catenin have been implicated in vascular remodeling and wound repair71,72, a scratch wound test was performed to evaluate endothelial healing responses after TNF-α treatment (Fig. 7F). As expected, TNF-α substantially reduced wound healing (Fig. 7F). Interestingly, while FGF2 alone did not affect wound repair compared to control, it did improve TNF-α-related reduction in healing rate to result in a higher percentage of total healed area after 12 h (Fig. 7F). This finding suggests that this growth factor not only attenuates TNF-α-induced inflammatory signaling in endothelial cells, but it can restore functional and healing properties of the BBB which may underlie beneficial impact of EE in preventing stress-induced neurovascular alterations and promoting resilience.

To this day, mood disorders, including MDD, are still diagnosed with questionnaires only. Identification of biomarkers with potential to inform clinicians for diagnosis and treatment choice is greatly needed and blood immune and vascular markers have received increasing attention in recent years6,18,73–75. To validate the importance of environmental conditions in stress-induced behavioral responses and underlying biology, we first conducted a hierarchical clustering analysis of all our mouse cohorts for BBB gene expression in stress-sensitive brain areas. It revealed that the environment is a key factor influencing BBB gene expression in both male (Fig. 8A) and female (Fig. 8B) mice. Indeed, groups tend to cluster by environmental conditions as opposed to stress exposure or phenotype. With this in mind, we explored if circulating FGF2 could be associated with human MDD when considering socioeconomic factors. Clinical evidence and post-mortem studies related to FGF2 in the context of MDD are inconsistent51,52,76–80 and this could be due to sampling heterogeneity. Blood serum FGF2 level was measured by ELISA with a high sensitivity kit in samples from men and women with a diagnosis of MDD and compared to matched controls. MDD diagnosis and severity of symptoms had a significant impact on FGF2 level (Fig. 8C, D, *p=0.026), which may explain previously reported discrepancies. Socioeconomic position, as measured by education, income, and occupational prestige, is a strong determinant of MDD onset81. Accordingly, in our cohort, having a university diploma and being employed was associated with lower depression rates (Fig. 8E, F) so we tested the impact on FGF2 blood level. In individuals with no university diploma, an increase in circulating FGF2 was measured for MDD samples (Fig. 8E, **p=0.006). An elevation in circulating FGF2 could be linked to MDD-associated neurovascular alterations and an attempt to repair the leaky BBB82. Importantly, a change in circulating FGF2 was associated with MDD in all men as assessed by the overall Patient Health Questionnaire (PHQ-9) score, a widely used screen test for depression (Fig. 8G, right). Blood level of FGF2 was significantly correlated with multiple symptoms of this psychiatric condition in both women and men (Fig. 8G). Sex-specific symptomatology was noted with men with MDD reporting depressed mood, insomnia, and devaluation while women mentioned anhedonia, fatigue, a change in appetite, and concentration capacities, all correlated with changes in blood FGF2 (Fig. 8G). To sum up, circulating FGF2 may represent a promising blood biomarker of mood disorders, however, it is highly influenced by MDD severity, symptomatology, sex, environmental and socioeconomic factors.

Methods
Animals
Male and female C57BL/6 mice aged 8 weeks of age at arrival (Charles River Laboratories, Québec, Canada) were used for all experiments. Retired male CD-1 breeders were used as resident aggressors (AGG) for social defeat and social interaction tests. All mice were group housed in 27×21×14 cm polypropylene cages upon their arrival and left undisturbed for at least three days prior to experimentation. Mice were maintained on a 12-h light–dark cycle (light on from 0700 to 1900 h) with constant temperature, humidity (22°C, 63%) and free access to water and food (Teklad Irradiated Laboratory Animal Diet, Madison, USA). All experimental procedures were approved by the Animal Care and Use Committee of Université Laval (2022-1061-1) and met the guidelines set out by the Canadian Council on Animal Care.

Housing conditions
Control and stressed EE mice were housed in a standard cage supplemented with a plastic house, nesting material, and a small plastic toy. When animals were moved between cages during CSDS, they maintained their original enrichment materials. Control and stress animals in the PE cohorts were housed in standard cages and habituated with battery-powered, wireless running wheels (Med Associates) for 5 days prior to the beginning of CSDS. This habituation period is based on previous reports as well as our observations (not shown) that running activity per day reaches a plateau after five days. Each animal was assigned a wheel which it was kept with throughout cage changes during the 10d CSDS protocol. Data was collected and exported at 1-min intervals using Wheel Manager software (Med Associates).

Chronic social defeat stress (CSDS)
Male C57/Bl6 mice underwent CSDS as previously described41. AGG mice underwent 3 days of screening for aggression profile and were conditioned in social defeat cages separated halfway by a clear, perforated divider for 24 h prior to experiments. Experimental mice were subject to physical interaction with a novel CD-1 for 5 min a day over 10 consecutive days and subsequently housed in defeat cages opposite the CD-1 with the divider preventing physical altercation but allowing sensory contact. Interactions were stopped before the 5-min period elapsed if attacks were repeated and severe, or if wounding occurred. Unstressed controls were co-housed in social defeat cages on each side of a divider and were moved every other day. After the last bout of interaction, the experimental mice were single-housed in standard cages for 24 h before undergoing a social interaction (SI) test, and tissue was collected 24 h after that (Fig. 1A).

Chronic variable stress (CVS)
Female C57/Bl6 mice were housed in groups of four in standard cages for 6d CVS protocol18. Briefly, stressed mice were subject to three different alternating stressors, one per day, in the following order: 100 random mild foot shocks (0.45 mA) for 1 h, tail suspension for 1 h, and tube restraint within home cage for 1 h. Unstressed controls were handled every day. After the last stressor, mice were single-housed for behavioral testing and tissue was collected 24 h after the last test (Fig. 2A).

Social interaction (SI) test
SI tests to assess social preference were performed under red light conditions15,41. Mice were placed in an open field arena with a small wire cage at one end for 150 s. Mice were then removed and the arena was cleaned, a CD-1 (AGG) was placed in the wire cage, and experimental mice were again allowed 150 s to freely explore the arena. Behavior in presence and absence of social target was tracked with AnyMaze software. Interaction zone (IZ) is defined as the area around the mesh cage. SI ratio was calculated by dividing the time in interaction zone in presence vs. absence of AGG. Mice with SI <1 were classified as stress-susceptible (SS), while SI = 1 or >1 were resilient (RES).

Elevated plus maze (EPM)
The EPM apparatus is a cross-shaped plexiglass arena with 4 arms (12 cm width × 50 cm length) 1 m above ground level, where two arms had tall black walls (closed arms) and two were unprotected (open arms). Under red light, mice were placed in the middle of the maze and allowed to explore for 300 s. Behavior was automatically tracked (AnyMaze 6.1, Stoelting Co.). Time in closed arms is taken as a measure of anxiety-like behavior.

Sucrose preference test
Water bottles in standard cages were replaced with two 50 mL conical tubes containing water for a 48-h habituation. Next, water from one of the tubes was replaced with 1% sucrose and mice were allowed to drink ad libitum. Tubes were switched after 24 h to account for place bias, and weights were recorded at 0 h, 24 h, and 48 h. Sucrose preference after 24 h was calculated by dividing weight of sucrose consumed by the total weight of liquid.

Forced swim test (FST)
Forced swim is used to evaluate learned helplessness as a measure of depression. Mice were placed in a 4 L glass beaker filled with 3 L lukewarm water under bright light for 360 s. Video of each session was manually evaluated for time spent immobile, defined as no movement or small hind-leg gestures needed to stay afloat, by blinded observers.

Viral experiments
At 5 weeks of age, mice were injected either AAV5-gfaABC1D-GFP, AAV5-gfaABC1D-mFgf2-P2A-GFP, AAV5-gfaABC1D-shRNAmir(-scramble)-GFP, or AAV5-gfaABC1D-shRNAmir(mFgf2)-GFP (Sigma Gene) viruses via stereotaxic injection. All surgeries were performed under aseptic conditions using anesthetic as previously described15,18. Mice were anesthetised with isoflurane (3-4% in O2 for induction and 1–1.5% in O2 for maintenance at 1 L/min flow rate) and positioned in a small stereotaxic instrument (Harvard Apparatus). Skull was exposed, and 0.5 ul (0.2 ul mere viral solution + 0.3 ul saline) was bilaterally infused in the NAc (bregma coordinates: anteroposterior +1.6 mm; mediolateral ±1.5 mm; dorsoventral −4.4 mm), at a rate of 0.1 ul/min. For FGF2 staining to confirm FGF2 over-expression, mice were allowed to recover for 4 weeks, then brains were collected and flash frozen with isopentane on dry ice and store in −80°C until use. For viral injection + CSDS experiment, mice were allowed to recover for ~4 weeks, then went under CSDS as described above. Viral groups were divided between unstressed controls group and stressed group (n=6 and 12, respectively). SI was performed after 10 days CSDS, then the following day 24 h later, mice were sacrificed by rapid decapitation, and their brains were collected via NAc bilateral punches of 2 mm from 1 mm slices.

Cell culture
The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150°C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell). Culture surfaces were precoated with 0.1% gelatin and cells were passaged when confluent (3–5 days) by washing with PBS, detaching with TrpLE Dissociation Reagent (Gibco), and seeding on gelatin coated flasks at desired concentration. Cells were used for experiments between passages 3 and 7, seeded at a density of 5×10^4 cells/cm2. For immunofluorescence, cells were grown on gelatin-coated 12 mm glass coverslips (Marienfeld), which were previously hydrophilized by 10 min treatment with 0.1 M hydrochloric acid and sterilized for 10 min with 100% ethanol.

TNF-α and Fgf2 treatment
Human recombinant TNF-α and FGF2 (Gibco) were dissolved in sterile water per manufacturer’s instructions and stored at −20°C. Experiments were performed using HBEC-5i and bEnd.3 after 4–5 days in vitro. Cells were pretreated for 1 h with 10 ng/mL human recombinant FGF-2 (Gibco) or vehicle (sterile water). At the start of treatment, existing medium was completely aspirated and replaced with HBEC-5i cell culture medium containing either sterile water (vehicle), 10 ng/mL FGF2+vehicle, vehicle+10 ng/mL TNF-α, or 10 ng/mL FGF2+10 ng/mL TNF-α. Acute inflammation studies looked at the response to a single stimulus up to 24 h, while chronic inflammation was assessed by replacing the medium each day with fresh medium containing TNF-α and/or FGF2 over a period of up to 7 days.

Gene expression analysis
Mice were anesthetized by decapitation and brains were rapidly removed. 2.0 mm punches were taken from NAc and PFC in both hemispheres and frozen at -80°C until use. HBEC-5i and bEnd.3 in a 6-well plate were pretreated for 1 h with Fgf2 or sterile water (vehicle) and then stimulated with TNF-α or TNF-α+Fgf2 for 0 h (control), 1 h, 3 h, 6 h, or 24 h (3 wells/condition/timepoint). RNA was extracted from brain punches as well as HBEC-5i and bEnd.3 cells in 6-well plates using TRIzol (Invitrogen) homogenization and phase separation with chloroform. The clear RNA phase was processed further with the Pure Link RNA MINI Kit (Life Technologies) and assessed for purity and concentration with NanoDrop (Thermo Fisher Scientific). Complementary DNA (cDNA) was obtained with a reverse transcriptase reaction using Maxima-H-minus cDNA synthesis kit (Fisher Scientific). For qPRC reactions, each well of a 384-well plate contained 3 μL of sample cDNA, 1 μL qPCR primer (see Supp. Table 1 for primer list), 5 μL Powerup SYBR green (Fisher Scientific), and 1 μL distilled H2O. In a thermocycler, samples were heated to 95°C followed by 40 cycles of 95°C for 15 s, 60°C for 33 s and 72°C for 33 s. Ct values were converted to normalized expression using the 2−ΔΔCt method124 with Gapdh as the reference gene.

Immunofluorescent staining
Whole brains of mice after rapid decapitation were flash frozen with isopentane on dry ice and stored at −80°C until use. Frozen brains were embedded in OCT Compound (Thermo Fisher Scientific) and slices from PFC and NAc were collected using a cryostat (Leica) at 20 μm thickness or 30 μm for AAV5-Fgf2-related experiments. Brain slices and cells cultured on glass coverslips were post-fixed for 10 min in ice-cold methanol. After 3×5 min wash with PBS brain slices were incubated for 2 h in blocking solution (1% bovine serum albumin, 4% normal donkey serum, and 0.03% Triton X-100 in PBS) before overnight incubation with primary antibodies in blocking solution (see Supp. Table 2 for antibodies and dilutions). Samples were then washed 3×5 min with PBS and incubated with fluorophore-conjugated secondary antibodies in PBS, then washed again and stained with DAPI to visualize nuclei. Coverslips were mounted on slides using Prolong Diamond Antifade Mountant (Invitrogen). Z-stack images of the NAc and PFC were taken at 20× (Z=10 μm) and 40× (Z=3 μm or 4.8 μm for AAV5-Fgf2-related experiments) on an epifluorescence microscope (Carl Zeiss).

Fluorescent image analysis
Analysis of brain tissue images was automatically performed in batches using Fiji ImageJ software125. For each channel, maximum intensity projection was performed and resulting images were processed with rolling ball background subtraction followed by noise removal of bright artefacts less than 2 μm. In CD31 channel only, continuity of blood vessels was ensured by 2D Gaussian blur, σ=2 μm. Processed images were binarized using automatic thresholding algorithms to measure positive staining area. β-catenin distribution at tight junctions in HBEC-5i cells was assessed by a blinded experimenter who sampled one tight junction from each quadrant of each image for a total of 48 TJs per condition. β-catenin distribution was assessed by measuring fluorescence intensity across a 10 μm line drawn perpendicular to the junction plane.

Cell viability assay
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) (Millipore Sigma) is converted to water-insoluble product formazan by reduction at mitochondrial complex II of the electron transport chain. This reaction can therefore act as a proxy for mitochondrial respiration and cell viability126. Briefly, cells in a 24-well plate were treated with 500 μM MTT and incubated (37°C and 5% CO2) for 2 h. Media was removed, and formazan crystals were dissolved in 500 μL dimethyl sulfoxide for absorbance readings at 570 nm using a spectrophotometer. Viability is calculated as a percent of the control reading.

Trans-endothelial electrical resistance (TEER)
For TEER studies, HBEC-5i were seeded on transwell polycarbonate culture inserts (Millicell) with 12 mm diameter and 3 μm pore size. TEER measurements were taken using the Millicell® ERS-2 Electrical Resistance System. Gelatin-coated insert with no cells was used as a blank. Electrodes were habituated in complete growth media at room temp for 10 min before reading resistance across cell monolayers. TEER was calculated as resistance of sample minus resistance of blank, multiplied by membrane surface area (0.6 cm2). TEER measurements were normalized to baseline for each well and are presented as percentage of control; raw TEER values are available in Supp. Fig. 7.

Protein extraction and western blot
HBEC-5i and bEnd.3 in a 6-well plate were pretreated for 1 h with Fgf2 or sterile water (vehicle) and then stimulated with TNF-α or TNF-α+Fgf2 for 0 h (control), 5 min, 1 h, or 6 h (3 wells/condition/timepoint). Protein was extracted at desired time points by washing with ice-cold PBS and then lysing cells with 200 μL cell lysis buffer (Cell Signaling Cat. No. 9803) supplemented with protease inhibitor cocktail (Cell Signaling Cat. No. 5871). Samples were sonicated in ice-cold water, centrifuged for 10 min (17,949×g, 4°C), and supernatant transferred to a new tube. Protein level was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Cat. No. 23250) per manufacturers instructions. Samples were diluted 1:10 and absorbance values were read on a spectrophotometer at 562 nm. Protein levels were calculated from standard curve. Samples were prepared for gel electrophoresis by adding a volume containing 20 μg protein to 3 μL 1M DTT and 7.5 μL 4X Laemmli sample buffer (BioRad Cat. No. 1610747) and topping up to 30 μL with deionized water. Samples and protein ladder (10–250 kDa, Cell Signaling Cat. No. 74124) were pipetted into wells of an 18-well Criterion™ TGX Stain-Free™ pre-cast gel (4–15%, BioRad Cat. No. 5678084) and separated by SDS-PAGE. Protein was transferred to a polyvinylidene difluoride membrane with 60 min of 90V current in transfer buffer (25 mM Tris base, 192 mM glycine, 20% methanol in deionized water). Membranes were blocked in Tris-buffered Saline (TBS) supplemented with 0.1% Tween (TBST) and 0.5% BSA and incubated overnight with primary antibodies in blocking solution at 4°C (see Supp. Table 2 for antibodies and dilutions). Membranes were washed 3×10 min in TBST and incubated 1 h with horseradish-peroxidase (HRP)-conjugated secondary antibodies in TBST. Membranes were washed again and signals on blots were revealed by enhanced chemiluminescence (ECL) reagents (BioRad Cat. No. 1705060) in a ChemiDoc imaging system (BioRad). Band intensity was estimated in Fiji ImageJ by removing background separately for each lane and measuring volume of the peak signal127,128. Phosphorylated GSK3β and β-Catenin levels were normalized to total GSK3β and β-Catenin. Total protein was measured with Stain-Free™ imaging technology and used as a loading control for Cldn5 levels.

Scratch wound assay
HBEC-5i in 24-well plates were pre-treated with Fgf2 for 1 h and subsequently stimulated with Fgf2, TNF-α, Fgf2+TNF-α, or sterile H2O as vehicle (CTRL) for 24 h prior to wound induction. Scratch wound was induced using a sterile 200 μL pipette tip aligned with a custom 3D-printed plastic template to ensure all scratch sizes were equal, following the recommendations of previous publications129. Wounded cells were washed twice with DMEM/F12 and subsequently incubated with Fgf2, TNF-α, Fgf2+TNF-α, or sterile H2O as vehicle. Images were taken at 5× on the brightfield setting of an epifluorescence microscope at 2 h intervals beginning immediately after scratch and continuing until 12 h. Wound size was manually evaluated using the Wound Healing Size Tool plugin for Fiji129.

Human serum sample collection
All human blood samples were provided by Signature Bank from the Centre de recherche de l’Institut universitaire en santé mentale de Montréal (CR-IUSMM) under approval of the institution’s Ethics Committee. Samples from volunteers with major depressive disorder were collected at the emergency room of the Institut universitaire en santé mentale de Montréal of CIUSSS de l’Est-de-Montreal, and samples from healthy volunteers at the CR-IUSMM. All donors provided informed consent and signed a 7-page document detailing the goals of the Signature Bank, participants involvement (questionnaires and tissue sampling), advantages vs risks, compensation, confidentiality measures, rights as participant and contact information. Subjects with known history of drug abuse were excluded. Demographic characteristics associated with each sample are listed in Supp. Table 3 (sex was self-reported). Depressive behaviors were assessed by the Patient Health Questionnaire (PHQ-9), which scores each of the nine Diagnostic and Statistical Manual of Mental Disorders (DSM) IV criteria130. All experiments were performed under the approval of Université Laval and CERVO Brain Research Center Ethics Committee Neuroscience et santé mentale (Project #2019-1540).

Enzyme-linked immunosorbent assay (ELISA)
Serum levels of FGF2 were assessed with the Human FGF basic/FGF2/bFGF Quantikine® high sensitivity ELISA kit from R&D systems (Cat. No. HSFB00D, sensitivity 0.07 pg/mL, assay range 0.3-20 pg/mL), following manufacturers instructions. Serum samples were diluted 1:2 and absorbance read at 490 nm nm with wavelength correction at 690 nm on the microplate/spectrophotometer Agilent Biotek Epoch 2. Concentrations were calculated from a 4PL standard curve using the R package drc.

Statistical analysis
Statistical comparisons were performed using GraphPad Prism 9 software. Each dataset was tested for normality (Shapiro-Wilk test, alpha=0.05) and outliers (Grubbs’ test, alpha=0.05). Animals identified as outliers in two or more distinct behavioral measures were removed from further analysis. Two-group comparisons were performed using two-tailed unpaired Welch’s t-test (normal distribution) or Mann-Whitney U test (non-gaussian distribution). Multiple group comparisons were assessed with one- and two-way analysis of variance (ANOVA) or multiple permutations (non-equal variances) followed by Bonferroni post-hoc testing (normal distribution) or Kruskal-Wallis test with Dunn’s post-hoc test (non-gaussian distribution). Principal component analysis (PCA) was performed using the R software, package FactoMineR, and missing values were imputed with missMDA.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an interesting study that provide new mechanistic insights into the FGF2-mediated protective effect of BBB by preventing Cldn5 loss and mitigating TNFa-mediated neuroinflammation and vascular inflammation. Exogenous delivery of AAV-FGF-2 improves depression-like behavior, zindlujcing social avoidance. Finally, they correlated the circulating FGF-2 levels with MDD in humans and reported a significant difference between men and women and the degree of MDD severity. In general, these findings are interesting and clinically relevant. I have major concerns regarding the FGF-2 biology in relation to MDD.\n1. It seems to me that three growth factors, including VEGFA, PDGF, and FGF2, are somehow predetermined signaling molecules owing to their previously known involvements in BBB. This is a biased selection of growth factors. The authors should have deployed an unbiased approach to identify growth factors or cytokines that play key roles in their animal models, but not too much affected by what have been known.\n2. The FGF family consists of more 20 members and some of the these members have distinct expression patterns in the brain. I am uncertain if the FGF-2 is the only FGF expressed in PFC and NAc. Given the key sex difference in their stress models, it is hard to believe that FGF-2 would execute the protective function in both cases. Other FGF members would likely to play a crucial role. It would have been really valuable if the authors looked into the differential expression profiling of growth factors in PFC and NAc areas as the initial step for the project.\n3. FGF-2 is not a classical secretory protein. It lacks a signal peptide and remains as an intracellularly cytoplasmic protein. While the authors detected transcription expression of Fgf2 mRNA in their models, it does not mean export of FGF-2 to the extracellular space.\n4. In the AVV system for the enforced expression of FGF-2, the authors should have introduced a signal peptide to ensure secretion of FGF-2 and have replaced the cysteine residues with other animo acids. Otherwise, the structure of FGF-2 would be changed by forming disulfide bonds.\n5. Detection of FGF-2 in the plasma/serum usually represents noisy backgrounds. In this case, subtle differences between 5-7.5 pg/ml should not be considered as real differences. Owing to its extremely high affinity to heparin, the circulating FGF-2 would not be detectable because the FGF-2 molecules will be trapped by interaction with heparin sulfate proteoglycans in the extracellular matrix and on the cell surface. In fact, injection of FGF2 into the blood stream results in a rapid turnover.\nIn summary, while approaching the stress disorders using experimental models is extremely important and these authors have tremendous expertise in this field, identification of signaling molecules and mechanistic insights into their functions remain very vague. Much more rigors are needed to justify their conclusions.", "reviewer_2": "The manuscript, “Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2” by S.E.J. Paton, J.L. Solano, A. Cadoret, A. Collington, L. Bandeira Binder, E. Richer, F. Coulombe-Rozon, L. Dion-Albert, K.A. Dudek, Signature Consortium, M. Lebel & C. Ménard is a potentially exciting examination of the sex-specific protective effects of Fgf2 in attenuating stress-induced loss of the endothelial tight junction protein, Claudin-5, decreasing anxiety- and depressive-like behaviours and protection against an inflammatory challenge. The authors suggest that the protective effects of an enriched environment and voluntary physical exercise on psychological stress are, in part, mediated through an increase in the astrocyte-derived growth factor, Fgf2, which has been previously reported to have anxiolytic and antidepressant effects. Furthermore, the authors demonstrate that this increase in Fgf2 is related to BBB changes, which is known to be impaired in individuals with MDD.\nChanges in the BBB in MDD are clearly understudied and are likely important to both the etiology and treatment of MDD. In theory, this manuscript could provide a novel step forward in understanding precisely how environmental factors may protect against many stress-induced effects, particularly through changes in BBB function. However, some conclusions made in the manuscript are an overreach for the data presented. In addition, there are important technical limitations that need to be remediated.\nMajor issues:\n-The authors correctly note that sex differences in MDD are marked and it is appreciated that they investigate these given the lack of studies in females in preclinical work. However, in this case, they chose to employ two different stress models, with males exposed to CSDS and females to SCVS, and then go on to make conclusions about sex differences in response to EE. CSDS and SCVS models are not interchangeable as they model different types of stress-induced depressive phenotypes. EE may have different effects in each of these models, regardless of sex and therefore conclusions cannot be made as to sex differences. Importantly, the authors again correctly note that CSDS is not easily transferable to females, however SCVS can be employed in males and therefore it is not clear why they do not include males in their SCVS study to make direct conclusions about sex differences in EE effectiveness.\n-In several statements (including the title), the authors conclude that “Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2”, however they do not test whether blocking FGF2 blocks the effects of EE, PE and the BBB alterations. They show that FGF2 coincidently goes up with PE and EE and they show that increasing FGF2 in the male NAc “mimics” the effects of PE and EE on social avoidance, however they do not show that FGF2 is causally involved in the effect of PE and EE.\n-Why does FGF2 prevent social avoidance in CSDS but not SI? EE alone changes SI and social avoidance, so is FGF2 really mimicking EE? Importantly, the levels of FGF2 reported with their AAV are quite high and therefore it is striking that the behavioural effects are limited. Are changes in social avoidance behaviour alone meaningful? Is this potentially an off-target effect? For example, does increased FGF2 change activity levels in general? FGF2 KOs show decreased centre time in the open field, so is this simply a reduction in anxiety/increased exploration?\n-Figure captions and methods for calculating scores need to be expanded. For example, the authors report that they use control data from past studies to compare with EE and PE. Were any controls included in the current work? How do they ensure that behavioural baselines in controls are consistent across studies?\n-It appears as though the authors used the DFB50 kit from R&D to measure FGF2 levels and not the high sensitivity (HS) kit. This kit is normed for clinical oncology samples with high levels of FGF2 and is sensitive from 10-640 pg/ml and is not sensitive for low levels of FGF2 typically seen in non-oncology samples. The HS kit is typically used for samples with low (or non-pathological) FGF2 levels as its sensitivity is between 0.3-20 pg/ml. Levels of FGF2 in Figure 7 are clearly below the sensitive range for the kit employed and therefore results may be confounded by a floor effect and conclusions cannot be drawn. Samples should be re-processed with the correct assay in order to have enough sensitivity to detect effects. Importantly, this may also help to resolve the inconsistencies in previous findings that the authors note.\n-The data with respect to diploma and second language seems slightly out of left field. While social determinants of mental health are indeed critical, it is not clear what these two variables mean in this context?\n-It might be interesting to correlate FGF2 levels to symptom clusters rather than these social determinants given previous work with FGF2 relating to specific phenotypes in humans.\nMinor issues:\n-How were the doses and timing of FGF2 and TNF-a chosen?\n-Figure 6B- it appears as though p-B-catenin is increased at 0 hours with TNFa and not with TNFa + FGF2? Also, what does CTRL refer to?\n-The authors refer to the PFC generally but may want to specify as it looks as though they are investigating the mPFC?\n-Page 2 citation 34 does not refer to the human brain.\n-Page 3 Figure 1G- this correlation is not significant and therefore conclusions should not be drawn\n-Figure 2- how do they explain the lack of significant differences on the FST (ie. supplemental figure 2B)?\n-SuppF3B- the authors state that no differences in the estrous cycle stage were noted, however, it looks as though almost double the number of control animals were in Metestrus. There are a number of studies showing changes in social investigation and distance travelled across the cycle, which could skew these results. Again, while it is appreciated that these factors are even considered in the current work, more probing as to the effects of cycle stage on behaviour may help to clarify effects (or non-effects) in females.\n-Page 7 lines 241-6 are confusing and don’t seem to match the graphs?\n-Are the mouse and human cell lines from males, females or mixed?\n-The authors may want to add a statement to the discussion about the EE employed. Arguably the EE employed may be considered basic housing and that the standard environment is actually an impoverished environment.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We are pleased to submit our revised manuscript titled ″Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2″ for publication as an Article in Nature Communications. We addressed all comments as described in this point-by-point response and were pleased that the 3 reviewers considered our work \"interesting, clinically relevant, exciting and potentially a step forward in understanding precisely how environmental factors may protect against stress-induced effects\". The text and Figures were revised according to their recommendations (changes in blue), and we did perform additional experiments to reinforce our conclusions leading to the addition of 3 new Main and Supplementary Figures and revision of 3 more Figures. The Discussion was also revised to expand on limitations and refine data interpretation. The manuscript is now much stronger, and we are grateful for the careful reviews and thoughtful suggestions, which have greatly improved our study.\nReviewer 1 (Remarks to the Author):\nThis is an interesting study that provide new mechanistic insights into the FGF2-mediated protective effect of BBB by preventing Cldn5 loss and mitigating TNFa-mediated neuroinflammation and vascular inflammation. Exogenous delivery of AAV-FGF-2 improves depression-like behavior, inducing social avoidance. Finally, they correlated the circulating FGF-2 levels with MDD in humans and reported a significant difference between men and women and the degree of MDD severity. In general, these findings are interesting and clinically relevant. I have major concerns regarding the FGF-2 biology in relation to MDD.\nWe do thank the reviewer for considering our work interesting and clinically relevant.\n1.1 It seems to me that three growth factors, including VEGFA, PDGF, and FGF2, are somehow predetermined signaling molecules owing to their previously known involvements in BBB. This is a biased selection of growth factors. The authors should have deployed an unbiased approach to identify growth factors or cytokines that play key roles in their animal models, but not too much affected by what have been known.\nResponse: Genes of interest in the PCR screen were determined by previous work from our group and others showing changes in expression following stress (Salmaso et al. Biological Psychiatry, 2016; Menard et al., Nature Neuroscience, 2017; Dion-Albert et al., Nature Communications, 2022; Matsuno et al., Molecular Psychiatry, 2022; Dion-Albert et al., Research Square, 2024). The growth factors chosen (Fgf2, Vegfa, Pdgf) are of interest since they are highly expressed by non-neuronal cells in the CNS and have been implicated in vascular pathologies and depression (Salmaso et al. Biological Psychiatry, 2016; Matsuno et al., Molecular Psychiatry, 2022; Dion-Albert et al., Research Square, 2024). A sentence with relevant references was added in the results section (page 3, lines 112-113) to clarify why these genes/growth factors were selected.\n1.2 The FGF family consists of more 20 members and some of the these members have distinct expression patterns in the brain. I am uncertain if the FGF-2 is the only FGF expressed in PFC and NAc. Given the key sex difference in their stress models, it is hard to believe that FGF-2 would execute the protective function in both cases. Other FGF members would likely to play a crucial role. It would have been really valuable if the authors looked into the differential expression profiling of growth factors in PFC and NAc areas as the initial step for the project.\nFurthermore, we assessed the expression of Fgf1, 2, 4, 8, 9, 10, and 21, the most abundant of the FGF family, in the prefrontal cortex (PFC) and nucleus accumbens (NAc) of naïve male and female mice (Supp.Fig.1D, Supp.Fig.5D). Fgf4, 8, 10 and 21 were indetectable in both brain areas while Fgf9 and 10 were indetectable in cultured astrocytes and endothelial cells, our cell types of interest (Supp.Fig.1D-E) as expected from a brain RNA-seq atlas (Zhang et al., J Neurosci, 2014). Importantly, stress exposure had no impact on the expression of Fgf1 in the male NAc (Supp.Fig.1F). These new results were added in the revised manuscript (page 3, lines 125-129; page 6, line 206-208).\n1.3 FGF-2 is not a classical secretory protein. It lacks a signal peptide and remains as an intracellularly cytoplasmic protein. While the authors detected transcription expression of Fgf2 mRNA in their models, it does not mean export of FGF-2 to the extracellular space.\nResponse: This is a valid point. Fgf2 is secreted via an unconventional secretory pathway and due to the lack of a signal peptide, it is transported into the extracellular space by an ER/Golgi-independent mechanism (Steringer et al., eLife, 2017). Immunofluorescence staining allowed us to quantify Fgf2 protein level in the mouse NAc and PFC, but it is true that we cannot claim that Fgf2 is secreted in the extracellular space. The Discussion was revised to briefly describe Fgf2 unique secretory mechanism, acknowledge this limitation of our study, and suggest that future studies could take advantage of super-resolution microscopy which was used to map the FGF receptor network (Schroder et al., Methods, 2021) (page 22, line 638-644).\n1.4 In the AVV system for the enforced expression of FGF-2, the authors should have introduced a signal peptide to ensure secretion of FGF-2 and have replaced the cysteine residues with other animo acids. Otherwise, the structure of FGF-2 would be changed by forming disulfide bonds.\nResponse: This is an interesting suggestion. Our rationale was to mimic the endogenous increase of Fgf2 gene expression in the NAc triggered by physical exercise (PE) or access to an enriched environment (EE). Adding a signal peptide to promote Fgf2 secretion would certainly ensure it gets released but it may interfere with normal secretion rhythm (Murgo et al., Cell Commun Signal, 2024). Moreover, it was shown that consistent Fgf2 overexpression increases seizure susceptibility in transgenic mice (Zucchini et al., J Neurosci, 2008) which would create a confounding factor for behavioral studies. This is now mentioned in the revised Discussion (page 22, line 646-651).\nTo confirm that the elevation of Fgf2 is involved in stress resilience, we designed a new viral vector allowing astrocyte-specific downregulation of this growth factor expression. Cohorts of male mice were injected in the NAc and impact on social interactions was evaluated without or with access to physical exercise (Fig.5). We observed increased stress susceptibility and social avoidance in AAV5-gfaABC1D-shRNA-mFgf2-Gfp-injected mice, even when they add access to running wheels, supporting our hypothesis that expression of this growth factor is involved in stress responses and benefits of PE. The text was revised to include these new findings (page 12-14, line 339-388).\n2\n1.5 Detection of FGF-2 in the plasma/serum usually represents noisy backgrounds. In this case, subtle differences between 5-7.5 pg/ml should not be considered as real differences. Owing to its extremely high affinity to heparin, the circulating FGF-2 would not be detectable because the FGF-2 molecules will be trapped by interaction with heparin sulfate proteoglycans in the extracellular matrix and on the cell surface. In fact, injection of FGF2 into the blood stream results in a rapid turnover.\nResponse: The reviewer is correct that quantification of FGF-2 in the plasma/serum is usually noisy. As recommended by Reviewer 2, we re-analyzed all our human serum samples with a more sensitive ELISA kit (Human FGF basic/FGF2/bFGF Quantikine high sensitive) with a detection range of 0.3-20 pg/mL and sensitivity of 0.07 pg/mL. Fig.8C-G panels were revised according to the new results which did not change our overall conclusions (page 18, line 513-529). We also added a sentence in the Discussion about the challenges related to FGF2 detection in plasma/serum and recommend highly sensitive immunoassays (page 24, line 754-756).\nIn summary, while approaching the stress disorders using experimental models is extremely important and these authors have tremendous expertise in this field, identification of signaling molecules and mechanistic insights into their functions remain very vague. Much more rigors are needed to justify their conclusions.\nWe are confident that the new experiments performed, including evaluation of other Fgf ligands’ expression, design of a new viral vector downregulating astrocytic Fgf2 and assessment of impact on social behaviors in the context of stress exposure and physical exercise, and confirmation of human FGF2 serum level changes with a highly sensitive kit, strengthen our conclusions. We thank the reviewer for recognizing relevance of our experimental approaches and expertise in the stress and BBB field and for highlighting limitations to our study that are now discussed.\nReviewer 2 (Remarks to the Author):\nThe manuscript, “Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2” by S.E.J. Paton, J.L. Solano, A. Cadoret, A. Collington, L. Bandeira Binder, E. Richer, F. Coulombe-Rozon, L. Dion-Albert, K.A. Dudek, Signature Consortium, M. Lebel & C. Ménard is a potentially exciting examination of the sex-specific protective effects of Fgf2 in attenuating stress-induced loss of the endothelial tight junction protein, Claudin-5, decreasing anxiety- and depressive-like behaviours and protection against an inflammatory challenge. The authors suggest that the protective effects of an enriched environment and voluntary physical exercise on psychological stress are, in part, mediated through an increase in the astrocyte-derived growth factor, Fgf2, which has been previously reported to have anxiolytic and antidepressant effects. Furthermore, the authors demonstrate that this increase in Fgf2 is related to BBB changes, which is known to be impaired in individuals with MDD.\nChanges in the BBB in MDD are clearly understudied and are likely important to both the etiology and treatment of MDD. In theory, this manuscript could provide a novel step forward in understanding precisely how environmental factors may protect against many stress-induced effects, particularly through changes in BBB function. However, some conclusions made in the manuscript are an overreach for the data presented. In addition, there are important technical limitations that need to be remediated.\nWe do thank the Reviewer for their positive comments and useful suggestions which we believe reinforce our conclusions.\n2.1 The authors correctly note that sex differences in MDD are marked and it is appreciated that they investigate these given the lack of studies in females in preclinical work. However, in this case, they chose to employ two different stress models, with males exposed to CSDS and females to SCVS, and then go on to make conclusions about sex differences in response to EE. CSDS and SCVS models are not interchangeable as they model different types of stress-induced depressive phenotypes. EE may have different effects in each of these models, regardless of sex and therefore conclusions cannot be made as to sex differences. Importantly, the authors again correctly note that CSDS is not easily transferable to females, however SCVS can be employed in males and therefore it is not clear why they do not include males in their SCVS study to make direct conclusions about sex differences in EE effectiveness.\nResponse: This is a valid point raised by the Reviewer. To address it, a cohort of male mice was subjected to the 6-day subchronic variable stress (SCVS) paradigm with or without an enriched environment (n=6-7/group) then a battery of behavioral tests was performed like for females (Supp.Fig.4A). No difference in behaviors was observed due to stress (Supp.Fig.4B-F) in line with previous publications showing that exposure to 6-d SCVS is not sufficient to induce anxiety- and depression-like behaviors in males (Hodes et al., J Neurosci, 2015). A significant effect of the environment was noted for the social interaction (SI) ratio, independent of stress, due to lower time spent in the interaction zone when the CD-1 aggressor was absent for mice with access to an EE (Supp.Fig.4C). The text was revised to add these new findings (page 5-6, line 192-197; page 21, line 612-614). We also now mention in the Discussion that it would be relevant to perform a stress paradigm known to consistently induce anxiety- and depression-like behaviors in animals of both sexes, like 21-day CVS (Labonte et al., Nature Medicine, 2017), in future studies to enable direct comparisons of protective mechanisms (page 21, line 628-631).\n2.2 In several statements (including the title), the authors conclude that “Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2”, however they do not test whether blocking FGF2 blocks the effects of EE, PE and the BBB alterations. They show that FGF2 coincidently goes up with PE and EE and they show that increasing FGF2 in the male NAc “mimics” the effects of PE and EE on social avoidance, however they do not show that FGF2 is causally involved in the effect of PE and EE.\nResponse: As suggested, we tested whether blocking Fgf2 expression alters social behaviors and impacts the beneficial effects of PE by designing and optimizing a new AAV downregulating Fgf2 expression specifically in astrocytes (AAV5-gfaABC1D-shRNA-mFgf2-GFP). For the current revised manuscript, we chose to focus on PE and males only, in line with the results of Fig.3-4. We do plan to explore the impact of manipulating Fgf2 in the context of EE in a future study, in the NAc but also the PFC, which is more vulnerable to stress exposure in females (Dion-Albert et al., Nature Communications, 2022). As shown on the new Fig.5 (page 12-14, line 339-388), after validation of viral efficiency (Fig.5A-B), male mice were injected bilaterally in the NAc with either the new virus or a control AAV5-gfaABC1D-shRNA-scramble-GFP viral vector then subjected to 10-day CSDS (Fig.5C). We observed increased stress susceptibility in mice injected with the virus downregulating Fgf2 in astrocytes which was not due to impaired locomotion (Fig.5D-F). Another cohort of mice was injected with the same viruses and exposed to 10-d CSDS but this time, with access to running wheels (Fig.5G). In contrast to Fig.3C and Fig.4I, downregulation of astrocytic Fgf2 in the NAc induced social avoidance as measured by decreased SI ratio but also increased number of entries in the corners when the aggressor was present (Fig.5H-J), supporting our hypothesis that an elevation of this growth factor may contribute to the benefits of PE. In fact, we noticed that stressed mice injected with the control virus ran increasing distance across days after the first defeat bout (Fig.5K). This was not the case for AAV5-gfaABC1D-shRNA-mFgf2-GFP-injected mice (Fig.5L), suggesting that motivation could be affected, with the NAc playing a crucial role in reward processing. This is something we will explore in the future by performing behavioral tests related to cognition like the progressive ratio task, reward-based paradigms, etc. Finally, we validate AAV-driven reduced Fgf2 expression in both cohorts of mice without and with access to PE (Fig.5M).\n2.3 Why does FGF2 prevent social avoidance in CSDS but not SI? EE alone changes SI and social avoidance, so is FGF2 really mimicking EE? Importantly, the levels of FGF2 reported with their AAV are quite high and therefore it is striking that the behavioural effects are limited. Are changes in social avoidance behaviour alone meaningful? Is this potentially an off-target effect? For example, does increased FGF2 change activity levels in general? FGF2 KOs show decreased centre time in the open field, so is this simply a reduction in anxiety/increased exploration?\nResponse: Previous studies exploring the role of Fgf2 in behavioral responses focused largely on anxiolytic effects of Fgf2 injection since it can act on glucocorticoid receptors to modify the hypothalamic-pituitary-adrenal axis activity (Perez et al., J Neurosci, 2009; Salmaso et al., Biol Psychiatry, 2016). Indeed, Simard et al. (2018) reported that FGF2 KO mice spend less time in the center of the open field test and that knocking down FGF2 has an impact on exploratory behaviour (open field, EPM) with no effect on affective behaviours as measured with sucrose consumption or the forced swim test. As recommended, we evaluated if manipulating Fgf2 could impact activity level at baseline and in the context of social stress exposure. Upregulation of Fgf2 expression in the NAc astrocytes did no alter locomotion/exploration during the SI test when the aggressor was absent with similar distance travelled for unstressed and stressed mice injected with either the control or AAV5-gfaABC1D-mFgf2-P2A-GFP virus. This important control was added on revised Fig.3H. Similarly, viral-mediated Fgf2 downregulation in the NAc astrocytes had no effect on locomotion/exploratory behaviors (Fig.5F, J). However, it could impact motivation and reward, as mentioned above, and this something we will investigate in the future with the viral tools developed here.\nThe Discussion was also revised to acknowledge limitations of our study regarding the unconventional secretion mechanism of Fgf2 (Steringer et al., eLife, 2017), and possible compensatory changes from other growth factors as reported before in transgenic mice (Miller et al., Mol Cell Biol, 2000; Yoshimura et al., PNAS, 2001; Beenken & Mohammadi, Nature Review Drug Discov, 2009) (page 22, line 640-654).\n2.3 Figure captions and methods for calculating scores need to be expanded. For example, the authors report that they use control data from past studies to compare with EE and PE. Were any controls included in the current work? How do they ensure that behavioural baselines in controls are consistent across studies?\nResponse: Legends of Fig.1-3 were expanded to provide additional information about social interaction scoring and categorization of stress-susceptible (SS) vs resilient (RES) subgroups (page 5, line 147-150; page 10, line 277-280). The total number of words allowed for each Figure Legend is limited to 350 with Fig.1 and 3 currently at 351 words. We want to clarify that control unstressed groups of mice, labeled CTRL, are included in all stress paradigms and behavioral studies throughout the manuscript. The reviewer may refer to the cohorts of mice exposed to CSDS or 6-d SCVS with standard cages – no EE or PE – from our previously published studies (Menard et al., Nature Neuroscience, 2017; Dion-Albert et al., Nature Communications, 2022). These cohorts were produced using the exact same experimental protocols, defined in detail in Golden et al., Nature Protocols, 2011 for CSDS and Hodes et al., J Neurosci, 2015 for 6-d SCVS, which give reproducible results across labs worldwide. Furthermore, additional cohorts of mice with standard cages are included in the current work for both EE (Supp.Fig.4), PE (Fig.5) and viral-mediated manipulations (Fig.4-5).\n2.4 It appears as though the authors used the DFB50 kit from R&D to measure FGF2 levels and not the high sensitivity (HS) kit. This kit is normed for clinical oncology samples with high levels of FGF2 and is sensitive from 10-640 pg/ml and is not sensitive for low levels of FGF2 typically seen in non-oncology samples. The HS kit is typically used for samples with low (or non-pathological) FGF2 levels as its sensitivity is between 0.3-20 pg/ml. Levels of FGF2 in Figure 7 are clearly below the sensitive range for the kit employed and therefore results may be confounded by a floor effect and conclusions cannot be drawn. Samples should be re-processed with the correct assay in order to have enough sensitivity to detect effects. Importantly, this may also help to resolve the inconsistencies in previous findings that the authors note.\nResponse: We do thank the reviewer for this important suggestion. As recommended, all human blood serum samples were re-analyzed with the more sensitive ELISA kit (Human FGF basic/FGF2/bFGF Quantikine high sensitive) with a detection range of 0.3-20 pg/mL and sensitivity of 0.07 pg/mL. Fig.8C-G panels were revised according to the new results which did not change our overall conclusions (page 18, line 513-529). We also added a sentence in the Discussion about the challenges related to FGF2 detection in plasma/serum and recommend highly sensitive immunoassays (page 24, line 754-756).\n2.5 The data with respect to diploma and second language seems slightly out of left field. While social determinants of mental health are indeed critical, it is not clear what these two variables mean in this context?\nResponse: We did aim to validate the importance of environmental conditions, as identified by hierarchical clustering in mice (Fig.8A-B), with socioeconomic factors available in our human cohort. Education and employment are modifiable lifestyle factors, not necessarily related to genetics, that we found could modify prevalence of MDD (Fig.8F). In line with this, socioeconomic position, as measured by education, income, and occupational prestige, is a strong determinant of MDD onset (Hoveling et al. J Affect Disord, 2022). This rationale was added to the revised manuscript (page 18, line 516-519). In Supp.Table 3, we also show data for the number of languages spoken, which did not relate to Fgf2 levels or MDD severity. Unfortunately, our cohort did not include information about housing situation or activity level.\n2.6 It might be interesting to correlate FGF2 levels to symptom clusters rather than these social determinants given previous work with FGF2 relating to specific phenotypes in humans.\nResponse: This is an excellent suggestion. A new column (All) was added on the Spearman correlation heat map for both men and women MDD groups and matched healthy controls (Fig.8G).\nMinor:\n1. How were the doses and timing of FGF2 and TNF-a chosen?\nResponse: Doses of TNF- and timepoints for cell collection were determined according to previous publications describing impact of this cytokine treatment on endothelial cells (Cross et al., Nature; 1995; Eto et al., Circulation, 2005; Zhou et al., Am J Physiol Renal Physiol, 2008). Cells were pretreated for 1h with Fgf2 (10 ng/mL as described in Seghezzi et al., J Cell Biol, 1998) to mimic habituation with EE/PE, before co-stimulation with TNF- and/or Fgf2. These details are now clarified in the Results section (page 14, line 404-407).\n6\n2. Figure 6B- it appears as though p-B-catenin is increased at 0 hours with TNFa and not with TNFa + FGF2? Also, what does CTRL refer to?\nResponse: In now Fig.7A-B, CTRL refers to 0h with no FGF2 pretreatment. The Figure panels and Legend were modified to add this precision (page 17, line 483-487). Control lanes are darker in Fig.7B for the TNF- treated vs. TNF-a + FGF2 treated gels, however, brightness is arbitrary for both figures and analysis is relative to CTRL (not between gels).\n3. The authors refer to the PFC generally but may want to specify as it looks as though they are investigating the mPFC?\nResponse: This is a good point. We used PFC to be consistent with our previous publications (Dion-Albert et al., Nature Communications, 2022; Dudek et al., Nature Neuroscience, 2025).\n4. Page 2 citation 34 does not refer to the human brain.\nResponse: This is right, Ref. 34 (Zhang et al., J Neurosci, 2024) refers to mouse RNA seq only. Thus, the 2016 human RNA seq reference was added (Zhang et al., Neuron, 2016) (page 2, line 72-75).\n5. Page 3 Figure 1G- this correlation is not significant and therefore conclusions should not be drawn\nResponse: We do not mention a significant correlation but only a trend (now page 5, line 168-171).\n6. Figure 2- how do they explain the lack of significant differences on the FST (ie. supplemental figure 2B)?\nResponse: Female mice were subjected to a battery of behavioral tests as described in our previous publication (Dion-Albert et al., Nature Communications, 2022). Forced swim test (FST) was the last test performed and 6-day SCVS is considered a mild stressor, so it is possible that the stress effect had worn off at that time. Moreover, the validity of FST is increasingly contested as a screen for depressive-like behaviors and it could be more associated with stress coping strategies (Commons et al., ACS Chem Neurosci., 2017).\n7. SuppF3B- the authors state that no differences in the estrous cycle stage were noted, however, it looks as though almost double the number of control animals were in Metestrus. There are a number of studies showing changes in social investigation and distance travelled across the cycle, which could skew these results. Again, while it is appreciated that these factors are even considered in the current work, more probing as to the effects of cycle stage on behaviour may help to clarify effects (or non-effects) in females.\nResponse: We do agree with the reviewer that it will be important to investigate further how hormonal regulation, notably the estrus cycle and testosterone level, can affect behavioral responses with sex hormones known to be potent modulators of BBB integrity. This is something we have been advocating for in the brain barriers field as highlighted in these publications: Dion-Albert et al., Front Neuroendocrinol, 2022; Collignon et al., Fluids Barriers CNS, 2024. The Discussion was revised accordingly (page 21, line 626-628).\n7\n8. Page 7 lines 241-6 are confusing and don’t seem to match the graphs?\nResponse: The text was revised to better describe the findings. The sentence “This implies that while EE alters more broadly the transcriptional stress response at the neurovasculature, PE more precisely targets Cldn5 expression” was removed while “At protein level, CSDS did not affect Cldn5 levels in the NAc of our PE cohort, whereas all stressed PE mice exhibited an increase in Fgf2 staining when compared to controls”, was revised (now page 8, line 266-268).\n9. Are the mouse and human cell lines from males, females or mixed?\nResponse: The HBEC-5i cell line is from a male donor (source: https://www.cellosaurus.org/CVCL_4D10) (page 36, line 1283-1285). This information is unfortunately not available for the bEnd.3 cell line.\n10. The authors may want to add a statement to the discussion about the EE employed. Arguably the EE employed may be considered basic housing and that the standard environment is actually an impoverished environment.\nResponse: The Discussion was revised as recommended (page 21, line 593-600).\nReviewer 3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\n8"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have largely satisfied my previous concerns. I congratulate on the authors for contribution of this excellent work to NC.", "reviewer_2": "The revised manuscript, “Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2” by S.E.J. Paton, J.L. Solano, A. Cadoret, A. Collington, L. Bandeira Binder, B. Daigle, L.M. Bevilacqua, E. Richer, F. Coulombe-Rozon, L. Dion-Albert, K.A. Dudek, Signature Consortium, M. Lebel & C. Ménard examines the sex-specific protective effects of Fgf2 in attenuating stress-induced loss of the endothelial tight junction protein, Claudin-5, decreasing anxiety- and depressive-like behaviours and protection against an inflammatory challenge. In this revision, the authors have done an excellent job of addressing each of the reviewer comments and have added critical experiments that show causal evidence for the involvement of FGF2 in mediating the effects of stress resilience by directly testing knockdown of astrocytic FGF2 in the nucleus accumbens (NA). In addition, they have added appropriate controls as requested, they consider other FGF ligands, show FGF ligand expression levels and develop their rationale to focus on FGF2. They have also confirmed their FGF2 ELISA results using an appropriate high-sensitivity assay. As such, I believe that their revisions greatly strengthen the paper and their conclusions. Two minor points to edit: the AAV knockdown of FGF in Figure 5 is not huge in magnitude, and it looks like there is quite a bit of variability, where a few mice have levels comparable to controls, suggesting inadequate knockdown in these mice. As such, the authors should consider removing these mice from the behavioural studies. In addition, a correlation between FGF2 levels and behavioural outputs could be added. Finally, the NA is a small region and some images that show the extent of regional knockdown (not just cellular specificity) should be added.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Referees\nWe are pleased to submit our revised manuscript titled ″Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2″ for publication as an Article in Nature Communications. We addressed all remaining reviewers’ comments, as described in this point-by-point response.\nReviewer 1 (Remarks to the Author):\nThe authors have largely satisfied my previous concerns. I congratulate on the authors for contribution of this excellent work to NC.\nReviewer 2 (Remarks to the Author):\nThe revised manuscript, “Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2” by S.E.J. Paton, J.L. Solano, A. Cadoret, A. Collington, L. Bandeira Binder, B. Daigle, L.M. Bevilacqua, E. Richer, F. Coulombe-Rozon, L. Dion-Albert, K.A. Dudek, Signature Consortium, M. Lebel & C. Ménard examines the sex-specific protective effects of Fgf2 in attenuating stress-induced loss of the endothelial tight junction protein, Claudin-5, decreasing anxiety- and depressive-like behaviours and protection against an inflammatory challenge. In this revision, the authors have done an excellent job of addressing each of the reviewer comments and have added critical experiments that show causal evidence for the involvement of FGF2 in mediating the effects of stress resilience by directly testing knockdown of astrocytic FGF2 in the nucleus accumbens (NA). In addition, they have added appropriate controls as requested, they consider other FGF ligands, show FGF ligand expression levels and develop their rationale to focus on FGF2. They have also confirmed their FGF2 ELISA results using an appropriate high-sensitivity assay. As such, I believe that their revisions greatly strengthen the paper and their conclusions.\nTwo minor points to edit: the AAV knockdown of FGF in Figure 5 is not huge in magnitude, and it looks like there is quite a bit of variability, where a few mice have levels comparable to controls, suggesting inadequate knockdown in these mice. As such, the authors should consider removing these mice from the behavioural studies. In addition, a correlation between FGF2 levels and behavioural outputs could be added. Finally, the NA is a small region and some images that show the extent of regional knockdown (not just cellular specificity) should be added.\nThe reviewer is possibly referring to Fig.5B in which Fgf2 knockdown was confirmed in astrocytes in a cohort of mice that did not go through behavioral testing. The text was revised to clarify (page 6, line 262-265). For behavioral studies, viral-mediated downregulation of astrocytic Fgf2 was confirmed by qPCR (Fig.5M). As expected, most mice are stress-susceptible in the AAV5-gfaABC1D-shRNA-mFgf2 group (17/24) without or with access to running wheels (Fig.5D and 5H) and accordingly, Fgf2 is down-regulated by 20-50% in 22/24 animals. With so few resilient animals and a bias toward stress susceptibility driven by the viral manipulation, we do not believe that correlative studies are appropriate. However, we should mention that we do observe a positive correlation between Fgf2 levels and social interaction ratio for resilient mice (*p=0.0222) and a trend for unstressed controls (*p=0.0547) when the viruses are combined.\nFinally, the NA is a small region and some images that show the extent of regional knockdown (not just cellular specificity) should be added.\nAs recommended, Fig.5B was revised to add lower magnification images of NAc viral infection.\nReviewer 3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}]

Decision letter