Sex Differences in the Modulation of Anxiety- and Depression-like Behaviors by Matrix Metalloproteinase-9 Expression Levels in Mice

Sex Differences in the Modulation of Anxiety- and Depression-like Behaviors by Matrix Metalloproteinase-9 Expression Levels in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sex Differences in the Modulation of Anxiety- and Depression-like Behaviors by Matrix Metalloproteinase-9 Expression Levels in Mice Júlia Senserrich, Elena Castro, Eva Florensa-Zanuy, Álvaro Díaz, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5820273/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 May, 2025 Read the published version in Biology of Sex Differences → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Major depressive disorder is one of the main causes of disability worldwide, but its etiopathology remains largely unknown, although several hypotheses have been proposed. Recent studies suggest a potential role for matrix metalloproteinase 9 (MMP-9) in depression, as it is overexpressed in the plasma of depressed patients and normalizes following chronic antidepressant treatment. This study aimed to characterize anxiety and depression-like behaviors in transgenic MMP-9 mice, as well as the expression of different neuroplasticity markers associated withdepression, in both sexes. Methods: In this study, we characterized the behavioral phenotypes of both MMP-9 knockout and MMP-9-overexpressing male and female mice. Here, we used a battery of tests to assess anxiety (open field, light‒dark box, elevated plus maze, and novelty‒suppressedfeeding tests), depressive-like (tail suspension and social interaction tests), and cognitive (T-maze) behaviors. Results: MMP-9 knockout female mice displayed increasedinnate anxiety (open field test), decreasedbehavioral despair (tail suspension test), and increased sociability (social interaction test). This increased sociability was also observed in male MMP-9 knockout mice. Compared with control mice, female MMP-9 knockout mice presented increased levels of different neuroplasticity markers in the hippocampus. With respect to MMP-9-overexpressing mice, females presented decreasedinnate anxiety (elevated plus maze and light‒dark box). Male MMP-9-overexpressing mice presented greaterconflict-based anxiety (novelty-suppressed feeding test) and lower working memory (T-maze) than control mice did. These male mice presented a reduction in mTOR pathway activation and increased PSD95 hippocampal levels. Conclusions: MMP-9 levels may have a sex-dependent impact on the anxious/depressive-like phenotype, as well as on neuroplasticity markers in the hippocampus. These findings reinforce the sex differences in the etiopathology of depression. Depression Anxiety Matrix metalloproteinase-9 Transgenic mice Sex Neuroplasticity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 HIGHLIGHTS Matrix metalloproteinase 9 (MMP-9) overexpression male mice exhibit conflictive anxiety and low spatial memory. MMP-9 overexpression female mice display low innate anxiety. MMP-9 knockout female mice display high innate anxiety and low depressive-like behavior. MMP-9 deletion in female mice show higher neuroplasticity markers expression. PLAIN ENGLISH SUMMARY Major depressive disorder is one of the most common neuropsychiatric diseases, which affects a two-fold number of women than men. The cause of this disorder is not known, however, impairments in neurotransmitters and neuroplasticity are some of the suggested causes. Specifically, some studies indicate that the expression of the matrix metalloproteinase 9 (MMP-9), one protein implicated in neuroplasticity, is increased in patients with depression, and these levels are reduced after antidepressant treatment. However, to date it has not been studied the differential implication of MMP-9 in the sex-differences observed in this pathology. In our study, we evaluated the impact of the elimination or the overexpression of MMP-9 in anxious and depressive-like behavior in male and female mice. We also evaluated how neuroplasticity was affected in these animals. We observed in female mice an opposite effect to the expected phenotype regarding anxiety, with higher anxiety in mice lacking MMP-9 and lower anxiety in mice that overexpress this protein. Moreover, the lower behavioral despair expected in mice that lack MMP-9 was only observed in females, which was parallel to the higher expression of some neuroplasticity markers. In addition, the cognitive impairment expected in mice that overexpress MMP-9 was only present in male mice. In conclusion, we demonstrate a sex-differential effect of MMP-9 expression levels on anxious/depressive-like behavior, and the impact on neuroplasticity. BACKGROUND Major depressive disorder (MDD) is a chronic psychiatric condition characterized by low mood, anhedonia, psychomotor alterations, feelings of guilt, and suicidal ideation. Patients can also present cognitive impairment and reduced libido [ 1 ]. According to the World Health Organization (WHO), MDD affects 3.8% of the population, which includes 5% adults, and has a higher prevalence in women than in men [ 2 ]. Moreover, the WHO reported that the COVID-19 pandemic induced a 25% increase in depression and anxiety cases [ 3 ]. The etiopathogenesis of MDD is not fully known, although multiple risk factors are involved, including biological, genetic, and psychosocial factors. Since the formulation of the classical “monoaminergic” hypothesis [ 4 ], which proposes the existence of a dysfunctional brain monoaminergic system associated with depression, different hypotheses, including the neuroplastic, glutamatergic , and neuroinflammatory hypotheses, have been proposed to explain the neurobiology of this disease. Understanding the mechanisms underlying the pathophysiology of MDD is highly important for the development of novel therapeutic approaches. Matrix metalloproteinases (MMPs) are endopeptidases responsible for the cleavage of the extracellular matrix and basement membrane components, cell surface receptors, cell adhesion molecules, growth factors, cytokines, and other proteases. Interestingly, they act as regulators of not only extracellular but also intracellular signaling networks [ 5 , 6 ]. One of the most studied MMPs is MMP-9, a gelatinase expressed in both the peripheral and central nervous systems. It is released from glutamatergic excitatory synapses upon the activation of NMDA receptors [ 7 ]. MMP-9 plays a pivotal role in the regulation of synaptic plasticity, learning and memory, allowing the growth and maturation of dendritic spines and the accumulation and immobilization of AMPA receptors, which makes excitatory synapses more efficient in modulating the AMPA/NMDAR ratio [ 8 , 9 ]. Moreover, MMP-9 can catalyze the activation of brain-derived neurotrophic factor (BDNF) and proinflammatory cytokines, which participate in the processes of neurogenesis and inflammation, respectively [ 7 ]. All these biological functions explain the involvement of MMP-9 in the etiopathogenesis of certain neurodegenerative disorders, such as epilepsy [ 10 ] and multiple sclerosis [ 11 ]. MDD patients present increased blood levels of the matrix metalloproteinases MMP-2, MMP-7, and MMP-9 [ 12 , 13 , 14 ]. Serum MMP-9 levels are positively correlated with the severity of symptoms [ 15 , 16 , 17 , 18 ]. In addition, electroconvulsive therapy reduces the serum levels of MMP-9 in depressed patients, resulting in a therapeutic response [ 19 ]. Increased MMP-9 activity has also been shown in postmortem hippocampal samples from MDD patients [ 20 ], whereas other authors reported no differences in MMP-9 protein levels in the prefrontal cortex of untreated MDD patients [ 21 ]. Moreover, different MMP-9 gene polymorphisms, which are associated with high MMP-9 levels, have been associated with depressive symptoms in bipolar disorder patients (rs17576) [ 22 ] and anxiety disorders (rs3918242) [ 23 ]. These findings highlight the importance of MMP-9 as a potential diagnostic marker of depression [ 24 ], which is correlated with treatment response and disease progression. Regarding the role of brain MMP-9 in animal models of depression, we recently reported increased expression and activity of MMP-9 in the hippocampus and cortex of a chronic corticosterone mouse model of depression [ 25 ]. Similar results have been shown in other models of depression, such as the chronic stress model [ 26 ], the chronic unpredictable stress model (CUS) in mice [ 20 ], and a neuroinflammation model [ 27 ]. In the last few years, the use of female animal models has increased, supporting the urge to address preclinical research to define optimal female models of neuropsychiatric diseases and their use for a deeper knowledge of sex bias. Moreover, a better understanding of the role of MMP-9 in the development of a depressive/anxious-like phenotype would help to elucidate the role of this protein in the neurobiology of depression. Therefore, in this study, we characterized the anxious and depressive-like behavioral and molecular phenotypes of male and female mice of two transgenic models: MMP-9 knockout (MMP-9 KO) mice, which have a nonfunctional MMP-9 protein, and mice that overexpress the human MMP-9 (MMP-9 OE) in the central nervous system. METHODS 1. ANIMALS The MMP-9 knockout (Jackson Laboratory, Maine, USA) and MMP-9 overexpression (kindly donated by Dr. A.K. Tzinia) male and female mice, 2–3 months old, were group-housed (4–5 mice per cage) with a 12 h light‒dark cycle and food and water ad libitum . All procedures were carried out with the previous approval of the Animal Care Committee of the University of Cantabria and according to Spanish legislation (RD 53/2013) and the European Communities Council Directive on “Protection of Animals Used in Experimental and Other Scientific Purposes” (2010/63/UE). 1.1. MMP-9 knockout mice B6. FVB(Cg)-Mmp9tm1Tvu/J (MMP-9 KO) mice (#007084, The Jackson Laboratory, Maine, USA) were generated by inserting a neomycin resistance gene driven by the mouse phosphoglycerate kinase promoter. This cassette replaces most of exon 2 and all of intron 2, disrupting MMP-9 functionality. 1.2. MMP-9-overexpressing mice TgMMP9 (MMP-9 OE) mice were generated with the PDGF-B/MMP9 transgene of human origin, a gene encoding human pro-MMP-9 under the control of the human neuron-specific PDGF-B promoter [ 28 ]. 2. BEHAVIORAL TESTS All the behavioral tests were performed during the light phase. Mice were placed in the experimental room one hour before the evaluation to allow them to acclimatize. Two different batches of animals were used to avoid an overload of stress due to the battery of behavioral tests assessed. Three hours after the last experiment, the mice were sacrificed via cervical dislocation. The brains were rapidly removed, and the hippocampus was dissected on an ice-cold platform and kept at -80°C until use for western blot experiments. 2.1. Open field test (OFT) The OFT was performed as previously described by Breviario et al. [ 25 ]. Mice were placed in a corner of the arena (40 × 40 × 30 cm) with an illuminated (50 lx) center (20 × 20 cm). The central time and total distance traveled were video tracked (Any-Maze™ tracking software version 4.99; Stoelting Co., Wood Dale, USA) for 5 min. 2.2. Elevated plus maze (EPM) Mice were placed in the center (5 × 5 cm) of the apparatus (50 cm from the floor), which consists of two open arms (25 × 5 × 0.5 cm) and two perpendicular closed arms (25 × 5 × 16 cm). Mice were allowed to explore freely for 5 min. The time spent in each arm was video tracked (Any-Maze™). 2.3. Light-Dark Box (LDB) The apparatus consists of a square box (40 × 40 × 20 cm) divided into two halves, one illuminated (50 lx) area and one dark area, connected by an opening. Mice were placed in a corner of the illuminated compartment, and the animal was allowed to freely explore the apparatus [ 29 ]. The time spent in each area was video tracked (Any-Maze™) for 5 min. 2.4. Novelty-suppressed feeding (NSF) test NSF was performed as previously described by Vidal et al. [ 29 ]. Mice were food deprived for 24 hours. A food pellet was placed in the illuminated center (50 lx) of an arena (40 × 40 × 30 cm) covered with a woodchip. The mouse was placed in a corner, and the latency to eat the pellet was recorded via video-tracking software (Any-Maze™) for a maximum duration of 10 min. After the test, the mice were placed back in their home cages, and the amount of food consumed for 5 min was measured. The animals that showed no food intake in their home cages were excluded from the data analysis. 2.5. Tail suspension test (TST) Mice were suspended by the tail for 6 min. Video-tracking software (Any-Maze™) was used to record the test results, and the time spent immobile was determined manually for the last 4 min by an observer blinded to the experimental groups. 2.6. Social interaction test (SIT) Mice were placed in an open field arena (40 × 40 × 30 cm) with an empty wire cylinder (7 cm diameter) placed in a corner. The experimental mice were allowed to explore the arena freely for 2.5 min. Immediately after, an unfamiliar mouse of a different strain (CD-1) was placed inside the wire cylinder, and the experimental mice were allowed to explore for 2.5 min. The time in the interaction zone (20 cm diameter) was assessed via video-tracking software (Any-Maze™). 2.7. T-maze The T-maze was adapted from a previously described protocol [ 30 ]. The apparatus consists of three arms, two opposite (30 × 5 × 15 cm) and one perpendicular (35 × 5 × 15 cm), creating a “T” shape. First, a training trial was assessed in which the left arm of the T-maze was blocked. The mice were placed at the end of the perpendicular arm and allowed to explore for 8 min. After the training trial, mice were removed from the apparatus and left to rest for an intertrial interval of 1 h. Then, for the experimental trial, the left arm was unlocked, recovering the “T” shape. A 3 min test was performed by placing the mice at the end of the perpendicular arm and allowing them to freely explore the apparatus. Mice were video tracked (Any-Maze™), and the time spent in the novel arm during the first minute of the test was measured. 3. WESTERN BLOT 3.1. Synaptoneurosomal protein extraction Protein extraction was performed according to the protocol previously described by Li et al. [ 31 ]. Briefly, the hippocampi were homogenized (1:15 w/v) in homogenization buffer (0.32 M sucrose, 20 mM HEPES pH 7.4, 1 mM EDTA, 1:100 protease inhibitor cocktail, 5 mM NaF and 1 mM Na 3 VO 4 ). The samples were subsequently centrifuged at 800xg for 10 min at 4°C. The pellet was removed, and the supernatant was centrifuged at 15 300xg for 10 min at 4°C. The pellet was resuspended in 150 µl of protein lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 1 mM Na 3 VO 4 , 5 mM NaF and 1:100 protease inhibitor cocktail) and sonicated for 1 min. The protein concentration was quantified via the Lowry method. The samples were prepared with loading buffer containing β-mercaptoethanol, boiled at 100°C for 5 min, cooled on ice for 3 min, and centrifuged at 1000xg for 5 min at 4°C. The supernatant was stored at -20°C until use. 3.2. Western blot Fifty µg of protein per sample was loaded in duplicate on 8.5% or 15% SDS‒PAGE gels. The electrophoresis mixture was run at 100 V for 15 min and then at 160 V for 50 min and then transferred to nitrocellulose membranes (GE Healthcare Europe GmbH, Munich, Germany). The membranes were blocked with 5% (w/v) powder skim milk in TBS-T (50 mM Tris‐HCl pH 7.6, 150 mM NaCl, 0.05% Tween‐20) for 1 h. For phosphorylated proteins, the blocking solution used was 3% powder skim milk in TBS‐T supplemented with phosphatase inhibitors (1 mM Na 3 VO 4 and 1 mM NaF). The following primary antibodies were incubated with the corresponding blocking solution at 4°C overnight: BDNF (1:500, ab108319, Abcam, Cambridge, MA, USA), mTOR (1:1000, #4517, Cell Signaling, Massachusetts, USA), phospho-Ser2448-mTOR (1:1000, #2971, Cell Signaling, Massachusetts, USA), PSD95 (1:200, sc-8575, Santa Cruz Biotechnology, Texas, USA), synapsin I (1:200, sc-390867, Santa Cruz Biotechnology, Texas, USA), and β-tubulin III (1:20000, T2200/T8660, Sigma‒Aldrich, Missouri, USA). The membranes were washed with TBS-T and then incubated with the corresponding secondary antibody conjugated to an NIR fluorophore (1:15 000, LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. After washing, the specific signal was visualized via an Odyssey® CLx Imaging System (LI-COR Bioscience, Lincoln, USA) and quantified via Image Studio™ Software (LI‐COR Biosciences, Lincoln, USA). The densitometric values were normalized to those of the housekeeping protein β‐tubulin III. STATISTICAL ANALYSIS The values are expressed as the means ± standard errors of the means (S.E.M.). For the molecular studies, the means of the sample replicates were normalized to those of the WT mice (100%). The data were analyzed via two-way ANOVA followed by an uncorrected Fisher's LSD post hoc test. The statistical analyses and identification of outliers were performed via GraphPad Prism 10 (GraphPad Software, Inc., California, USA). Statistical significance was set at p < 0.05. RESULTS 1. Innate anxiety in MMP-9 KO and OE mice In the open field test (OFT), MMP-9 KO female mice spent less time in the center than WT female mice ( p < 0.01), with no differences in MMP-9 KO male mice (Figure 1A). Compared with male WT mice, female WT mice spent more time in the center ( p < 0.001). Two‐way ANOVA revealed a significant effect of sex [F(1,33) = 33.4, p < 0.001] and the interaction sex × genotype [F(1,33) = 10.4, p < 0.01]. In the OFT, MMP-9 OE male and female mice did not significantly differ in the time spent in the center compared with their respective WT mice. However, the MMP-9 OE female mice spent more time in the center of the arena than the MMP-9 OE male mice ( p < 0.05, Figure 1D). Two‐way ANOVA revealed a trend toward the interaction sex × genotype [F(1,33) = 3.95, p = 0.06]. In the elevated plus maze (EPM) and light‒dark box (LDB) tests, no significant differences were found in the time spent in the open arms in the EPM (Figure 1B) or the time spent in the light zone of the LDB (Figure 1C) in male and female MMP-9 KO mice, compared to their WT counterparts. WT female mice spent less time in the light zone than WT male mice ( p < 0.05, Fig. 1C). In the LDB test, two‐way ANOVA revealed a significant effect of sex [F(1,33) = 7.2, p < 0.05]. In both the EPM and LDB tests, MMP-9 OE female mice spent more time in the open arms ( p < 0.01, Figure 1E) and in the light zone ( p < 0.05, Figure 1F). There were no differences in MMP-9 OE male mice in either test. Compared with MMP-9 OE male mice, MMP-9 OE female mice spent more time in the open arms ( p < 0.05, Fig. 1E). In the EPM test, two‐way ANOVA revealed a significant effect of genotype [F(1,33) = 4.9, p < 0.05] and the interaction sex × genotype [F(1,33) = 7.2, p < 0.05]. Figure 1. Innate anxiety in MMP-9 KO (A-C) and MMP-9 OE (D-F) male and female mice. Time spent in the center (A and D) in the OFT, time spent in the open arms in the EPM (B and E), and time spent in the lit compartment in the LDB (C and F). The data are expressed as the means ± SEMs. Two-way ANOVA followed by an uncorrected Fisher's LSD post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001. n = 7–10 animals per group. OFT: open field test; EPM: elevated plus maze; LDB: light‒dark box test. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice. 2. Conflict-based anxiety in MMP-9 KO and OE mice In the novelty-suppressed feeding (NSF) test, no significant differences were observed in the latency to feed in the male and female MMP-9 KO mice (Figure 2A) and OE female mice (Figure 2B). However, male MMP-9 OE mice presented a greater latency to feed ( p < 0.01, Figure 2B). Two‐way ANOVA revealed a significant effect of genotype [F(1,33) = 9.4, p < 0.01]. Additionally, the amount of food consumed after the test did not significantly differ (see Figure S1). Figure 2. Conflict-based anxiety in MMP-9 KO (A) and MMP-9 OE (B) male and female mice assessed by the latency to feeding in the novelty-suppressed feeding test. The data are expressed as the means ± SEMs. Two-way ANOVA followed by an uncorrected Fisher's LSD post hoc test. ** p < 0.01. n = 7–10 animals per group. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice. 3. Depressive-like behavior in MMP-9 KO and OE mice In the tail suspension test (TST), MMP-9 KO female mice displayed a lower immobility time than WT female and MMP-9 KO male mice ( p < 0.01, Figure 3A). No differences were observed in the MMP-9 KO males (Figure 3A). Two‐way ANOVA revealed a significant effect of sex [F(1,32) = 6.0, p < 0.05] and the interaction sex × genotype [F(1,32) = 5.6, p < 0.05]. Both male and female MMP-9 OE mice did not significantly differ (Figure 3D). In the social interaction test (SIT), male and female MMP-9 KO mice were more sociable than their corresponding WT mice, as evidenced by the greater amount of time spent in the interaction zone ( p < 0.05, Figure 3B). Two‐way ANOVA revealed a significant effect of genotype on MMP-9 KO mice [F(1,32) = 8.7, p < 0.01]. Compared with their WT counterparts, male and female MMP-9 OE mice did not significantly differ (Figure 3D). Figure 3. Depressive-like behavior in MMP-9 KO (A and B) and MMP-9 OE (C and D) male and female mice. Immobility time in the TST (A and C) and social interaction time (B and D) in the SIT. The data are expressed as the means ± SEMs. Two-way ANOVA followed by an uncorrected Fisher's LSD post hoc test. * p < 0.05, ** p < 0.01. n = 7–10 animals per group. TST: tail suspension test; SIT: social interaction test; WT: wild-type; KO: MMP-9 knockout; OE: MMP-9-overexpressing. 4. Cognitive behavior in MMP-9 KO and OE mice No significant differences in the time spent in the new arm in the T-maze test were detected between male and female MMP-9 KO mice and their WT counterparts (Figure 4A). Compared with their wild-type counterparts, male MMP-9 OE transgenic mice spent less time in the novel arm ( p < 0.05), whereas no significant differences were detected in female mice (Figure 4B). Two‐way ANOVA revealed a significant effect of genotype [F(1,31) = 4.5, p < 0.05]. Figure 4. Spatial working memory in MMP-9 KO (A) and MMP-9 OE (B) male and female mice assessed by the time spent in the novel arm in the T-maze. The results are expressed as the means ± S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher's LSD post hoc test. * p < 0.05. n= 8 ‐ 10 animals per group. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice. 5. Neuroplasticity markers in the hippocampus of MMP-9 KO and OE mice In the hippocampus, MMP-9 KO male and female mice did not show differences in BDNF expression levels compared with their WT counterparts (Figure 5A), although a trend toward higher BDNF expression was observed in female MMP-9 KO mice than in their WT counterparts ( p = 0.05). Compared with their WT counterparts, the female MMP-9 KO group also presented increased mTOR ( p < 0.05, Figure 5B), p-mTOR ( p < 0.05, Figure 5C), PSD95 ( p < 0.001, Figure 5D) and synapsin l ( p < 0.01, Figure 5E) expression levels. Compared with the male MMP-9 KO group, the female MMP-9 KO group also presented higher BDNF ( p < 0.01, Figure 5A), mTOR ( p < 0.05, Figure 5B), p-mTOR ( p < 0.01, Figure 5C), and PSD95 ( p < 0.001, Figure 5D) levels. Two‐way ANOVA revealed a significant effect of sex [F(1,27) = 6.5, p < 0.05] and the interaction sex × genotype [F(1,27) = 6.5, p < 0.05] on BDNF values. Two‐way ANOVA revealed a significant effect of sex [F(1,28) = 6.1, p < 0.05] and the interaction of genotype × sex [F(1,28) = 6.1, p < 0.05] on phospho-mTOR values. Two‐way ANOVA revealed significant effects of sex [F(1,28) = 10.0, p < 0.01], genotype [F(1,28) = 4.2, p < 0.05] and the interaction of genotype × sex [F(1,28) = 10.0, p < 0.01] on PSD95 values. Two‐way ANOVA revealed a significant effect of genotype [F(1,28) = 11.1, p < 0.01] on synapsin I values. Figure 5. Neuroplasticity markers expression in the hippocampus of male and female MMP-9 KO mice. BDNF (A), mTOR (B), phospho-mTOR (C), PSD95 (D), and synapsin I (E) expression in MMP-9 KO mice and their corresponding wild-type counterparts. Representative western blot bands are shown. The results are expressed as percentages versus the WT group and as the means ± S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher's LSD post hoc test. * p < 0.05, ** p < 0.01 *** p < 0.001. n = 7–8 animals per group. BDNF: brain-derived neurotrophic factor; mTOR: mammalian target of rapamycin; PSD95: postsynaptic density 95 protein; WT: wild-type; KO: MMP-9 knockout. The hippocampal levels of BDNF (Figure 6A), mTOR (Figure 6B), and synapsin I (Figure 6E) were not significantly different in male MMP-9 OE mice. A lower level of phosphorylated mTOR protein was observed in male MMP-9 OE mice than in their WT counterparts ( p < 0.05, Figure 6C). Two‐way ANOVA revealed a significant effect of genotype [F(1,21) = 4.4, p < 0.05]. PSD95 expression was greater in MMP-9 OE males than in their WT counterparts ( p < 0.05) and in the MMP-9 OE female group ( p < 0,01, Figure 6D). Two‐way ANOVA revealed a significant effect of sex [F(1,22) = 4.9, p < 0.05] and the interaction of genotype × sex [F(1,22) = 4.9, p < 0.05] on PSD95 expression levels. MMP-9 OE female mice did not present differences in the levels of the neuroplasticity markers studied (Figure 6A‒E). Figure 6. Neuroplasticity markers expression in the hippocampus of male and female MMP-9 OE mice. BDNF (A), mTOR (B), p-mTOR (C), PSD95 (D), and synapsin I (E) expression in MMP-9 OE mice and their corresponding wild-type counterparts. Representative western blot bands are shown. The results are expressed as percentages versus the WT group and as the means ± S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher's LSD post hoc test. * p < 0.05, ** p < 0.01. n = 6–7 animals per group. BDNF: brain-derived neurotrophic factor; mTOR: mammalian target of rapamycin; PSD95: postsynaptic density 95 protein; WT: wild-type mice; OE: MMP-9-overexpressing mice. DISCUSSION The present study focused on the behavioral and molecular parameters of male and female transgenic MMP-9 KO and OE mice. We did not observe any statistically significant differences in innate anxiety in male MMP-9 KO mice, which is consistent with previous studies that reported no alterations in anxiety in MMP-9 KO mice [32,33,34]. Interestingly, female MMP-9 KO mice presented increased innate anxiety, but only in the open field test. This finding might be partially attributed to the lower baseline anxiety levels observed in female wild-type mice than in males, as reported in previous studies in naïve rats [35,36,37]. These findings suggest a sex-dependent effect on certain manifestations of innate anxiety in MMP-9 KO mice, which has not been previously described, as other studies present pooled data from both male and female mice [34]. In contrast to the more anxious phenotype observed only in the open field test in female MMP-9 KO mice, female MMP-9 OE mice consistently displayed lower innate anxiety across different behavioral tests, including the elevated plus maze and light‒dark box tests. This finding contrasts with previous results in which no differences in innate anxiety were observed in pooled male and female MMP-9 OE mice [28]. Some studies have suggested a link between MMP-9 levels and the basal anxious phenotype in female mice of various strains. For example, female C57BL/6J mice, which exhibit higher basal MMP-9 brain levels, show lower anxiety in the elevated plus-maze test than other strains with lower MMP-9 brain levels and greater anxiety [38]. The lower level of innate anxiety observed in our female MMP-9 OE mice aligns with these findings [38]. Furthermore, increased resilience to anxiety has been reported in female mice in various depression models, such as chronic corticosterone exposure [39,40] and acute lipopolysaccharide administration [41]. Taken together, these results suggest that MMP-9 plays a sex-dependent role in anxiety. In male mice, the overexpression of MMP-9 led to increased conflict-based anxiety, which is consistent with the increased anxiety levels observed in the novelty-suppressed feeding test in animal models of depression, such as the chronic corticosterone [25] and obesity [42] models, both of which are characterized by elevated MMP-9 brain levels. However, female MMP-9 OE mice appeared to be protected from developing this conflict-based phenotype. These results support the involvement of MMP-9 in anxiety-related disorders, such as post-traumatic stress disorder [43]. With respect to depressive-like behaviors, female MMP-9 KO mice exhibited decreased behavioral despair, which is consistent with the antidepressant effects observed in various animal models following MMP-9 inhibition [44,45,46]. Interestingly, this reduced behavioral despair was not observed in male MMP-9 KO mice. Additionally, our MMP-9 OE mice did not show alterations in behavioral despair, which contrasts with the depressive-related behavior typically associated with increased MMP-9 expression and activity in this paradigm [20,25]. The lack of observed differences in our transgenic mice may be due to compensatory changes, which are common in constitutive transgenic mice [7]. Depressive disorders are often associated with impaired social functioning [47]. In our study, the overexpression of MMP-9 did not negatively affect social interaction. However, both male and female MMP-9 KO mice presented increased sociability, which could suggest a resilient or less vulnerable phenotype with respect to depressive-like manifestations. Our findings contrast with studies reporting no differences in social behavior in MMP-9 KO mice [33]. These discrepancies may arise from differences in the social interaction protocols used. In line with our findings, several studies in animal models with high MMP-9 levels suggest that the normalization or reduction of MMP-9 levels can improve sociability [48,49]. Moreover, transient overexpression of nectin-3, a proteolytic target of MMP-9, is able to reverse stress-induced social deficits [26]. Furthermore, it is important to note that depressive symptomatology is often accompanied by cognitive deficits, both in humans [50,51] and in animal models of depression [21]. In this study, male MMP-9 OE mice displayed working memory deficits, which is consistent with a negative correlation between hippocampal MMP-9 levels and working memory in male rats [52], although the impact of sex has not been extensively evaluated. Depressive disorders are associated with changes in neuroplasticity markers in brain regions such as the hippocampus and prefrontal cortex, which are correlated with symptom severity. Treatment with antidepressant drugs can reverse these changes in neuroplasticity. In this study, we examined neuroplasticity markers associated with depression and the mechanism of action of antidepressant drugs in the hippocampus. Our findings revealed that female MMP-9 KO mice presented increased activation of the mTOR pathway, increased levels of PSD95 and synapsin l, and a trend toward increased BDNF levels, whereas male MMP-9 mice did not show differences. These findings indicate a sex-dependent effect of MMP-9 on depression-related neuroplasticity markers. Several murine models of depression show reduced hippocampal levels of mTOR, PSD95, and synapsin l, which are restored by antidepressant drug treatments, such as serotonin selective reuptake inhibitors (SSRIs) [53,54,55], ketamine [56,57], and others [58,59]. In fact, antidepressant drugs such as fluoxetine and paroxetine normalize mTOR and PSD95 levels in the hippocampus in chronic unpredictable mild stress [53] and chronic social defeat stress models [54]. Thus, the increased levels of mTOR, its phosphorylated form and the pre- and postsynaptic proteins synapsin I and PSD95 observed in our MMP-9 KO female mice support the decreased behavioral despair observed in the tail suspension test. The high BDNF levels in these mice are also consistent with antidepressant-like behavior [60,61], in good agreement with the lower behavioral despair elicited by these animals. However, some studies have reported that acute intrahippocampal BDNF administration can induce an anxiogenic effect [61,62], which might explain the increased innate anxiety observed in our female MMP-9 KO mice. In MMP-9 OE male mice, the decreased mTOR pathway activation in the hippocampus could be linked to the increased conflict-based anxiety observed, which is consistent with findings from other studies [63,64,65,66]. In association with lower hippocampal mTOR pathway activation, we did not observe depressive-like behavior, contrary to previous reports [53,55], or lower levels of PSD95, which have been associated with depressive-like behavior in other animal models [67]. These discrepancies may be due to compensatory changes in our constitutive transgenic mice. Additionally, other signaling pathways, such as the PLC, PI3K, and MAPK/ERK pathways, may also play a role in the regulation of PSD95 expression [68]. CONCLUSIONS Our study highlights the sex-dependent role of MMP-9 in anxiety and depression. However, the regulation of MMP-9 levels does not appear to be the sole factor driving the modulation of anxiety and depression-like manifestations. These findings emphasize the importance of including female subjects in preclinical and clinical studies of depression and anxiety, as the incidence of these disorders is generally higher in women than in men. Abbreviations BDNF brain-derived neurotrophic factor EPM elevated plus maze KO knockout transgenic mouse LDB light‒dark box test MDD major depressive disorder MMP matrix metalloproteinase mTOR mammalian target of rapamycin NSF novelty-suppressed feeding test OE overexpression transgenic mouse OFT open-field test PSD95 postsynaptic density protein 95 SIT social interaction test TST tail suspension test WT wild-type mouse Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE All procedures were carried out with the previous approval of the Animal Care Committee of the University of Cantabria and according to Spanish legislation (RD 53/2013) and the European Communities Council Directive on “Protection of Animals Used in Experimental and Other Scientific Purposes” (2010/63/UE). Authorized project no. PI-03-19. CONSENT FOR PUBLICATION Not applicable. AVAILABILITY OF DATA AND MATERIALS The data that support the findings of this study are available upon request from the corresponding author. COMPETING INTERESTS The authors declare that they have no competing interests. FUNDING This research was funded by the Ministerio de Ciencia, Innovación y Universidades (RTI2018-097534-B-I00), Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), the Instituto de Salud Carlos III (FIS Grant PI19-00170), which were co-funded by the European Regional Development Fund (‘A way to build Europe’), and the Spanish Network for Stress Research RED2022-134191-T financed by MCIN/AEI/10.13039/501100011033. 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Front Synaptic Neurosci. 2014. 10.3389/fnsyn.2014.00006 . Additional Declarations No competing interests reported. Supplementary Files SenserrichetalSupplementaryMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 22 May, 2025 Read the published version in Biology of Sex Differences → Version 1 posted Editorial decision: Revision requested 18 Feb, 2025 Reviews received at journal 18 Feb, 2025 Reviews received at journal 14 Feb, 2025 Reviewers agreed at journal 29 Jan, 2025 Reviewers agreed at journal 27 Jan, 2025 Reviewers invited by journal 24 Jan, 2025 Editor assigned by journal 14 Jan, 2025 Submission checks completed at journal 14 Jan, 2025 First submitted to journal 13 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5820273","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":401966803,"identity":"3d11e23a-4284-4611-87ff-19d2dba68f68","order_by":0,"name":"Júlia Senserrich","email":"","orcid":"","institution":"Departamento de Señalización Molecular y Celular, Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC","correspondingAuthor":false,"prefix":"","firstName":"Júlia","middleName":"","lastName":"Senserrich","suffix":""},{"id":401966804,"identity":"ec31d2b8-162e-4bd0-8724-358af78c1376","order_by":1,"name":"Elena Castro","email":"","orcid":"","institution":"Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Castro","suffix":""},{"id":401966805,"identity":"821346f1-3146-4475-9cd9-da95a7c62e9e","order_by":2,"name":"Eva Florensa-Zanuy","email":"","orcid":"","institution":"Departamento de Señalización Molecular y Celular, Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"","lastName":"Florensa-Zanuy","suffix":""},{"id":401966806,"identity":"e00a3d5d-e461-439f-a884-0ae8bcea37f0","order_by":3,"name":"Álvaro Díaz","email":"","orcid":"","institution":"Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria","correspondingAuthor":false,"prefix":"","firstName":"Álvaro","middleName":"","lastName":"Díaz","suffix":""},{"id":401966807,"identity":"0f648a33-92d7-47bc-beaf-7ca084569dd8","order_by":4,"name":"Ángel Pazos","email":"","orcid":"","institution":"Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria","correspondingAuthor":false,"prefix":"","firstName":"Ángel","middleName":"","lastName":"Pazos","suffix":""},{"id":401966808,"identity":"50247f7d-98ac-4d08-b8d7-8b21100d8c54","order_by":5,"name":"Albert Adell","email":"","orcid":"","institution":"Departamento de Señalización Molecular y Celular, Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC","correspondingAuthor":false,"prefix":"","firstName":"Albert","middleName":"","lastName":"Adell","suffix":""},{"id":401966809,"identity":"33f433b3-9998-4579-b59c-e358f6106de1","order_by":6,"name":"Athina Tzinia","email":"","orcid":"","institution":"Institute of Biosciences and Applications, National Center for Scientific Research \"Demokritos\", Agia Paraskevi","correspondingAuthor":false,"prefix":"","firstName":"Athina","middleName":"","lastName":"Tzinia","suffix":""},{"id":401966810,"identity":"860864b6-e11d-4683-bf26-10470c4eab59","order_by":7,"name":"Fuencisla Pilar-Cuéllar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDACZuYGICknx8DA2ECsFrBKY2MStEBUGicSrZ7BnJ2x8XNljkH6htvNjR9+7mCQkyek2bKZsVny7DaD3A13DjZL9p5hMDY4QECLwWHGBsnGbX9yN9xIbGPgbWNI3EDIYUAtzT8btxmkGwC1MP5tY6ifT8hhQC1tQFsMEkBamIG2JDAQchjQL22WQC2GM28kNkvLtkkYbiCkxZz/8OGbQC3yfDfSH35822YjTzDEDND4EgTUY9EyCkbBKBgFowATAADTYkDb4wAG7wAAAABJRU5ErkJggg==","orcid":"","institution":"Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria","correspondingAuthor":true,"prefix":"","firstName":"Fuencisla","middleName":"","lastName":"Pilar-Cuéllar","suffix":""}],"badges":[],"createdAt":"2025-01-13 13:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5820273/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5820273/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13293-025-00716-5","type":"published","date":"2025-05-22T15:57:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74074636,"identity":"723e825d-e00f-41d2-b1f1-419c11d09e44","added_by":"auto","created_at":"2025-01-17 13:27:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":506313,"visible":true,"origin":"","legend":"\u003cp\u003eInnate anxiety in MMP-9 KO (A-C) and MMP-9 OE (D-F) male and female mice. Time spent in the center (A and D) in the OFT, time spent in the open arms in the EPM (B and E), and time spent in the lit compartment in the LDB (C and F). The data are expressed as the means ± SEMs. Two-way ANOVA followed by an uncorrected Fisher's LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. n = 7–10 animals per group. OFT: open field test; EPM: elevated plus maze; LDB: light‒dark box test. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5820273/v1/aa61c5d7f1604842e10a425d.png"},{"id":74074632,"identity":"2055bb6d-0b20-476d-b5c7-c9b25e8d09af","added_by":"auto","created_at":"2025-01-17 13:27:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128770,"visible":true,"origin":"","legend":"\u003cp\u003eConflict-based anxiety in MMP-9 KO (A) and MMP-9 OE (B) male and female mice assessed by the latency to feeding in the novelty-suppressed feeding test. The data are expressed as the means ± SEMs. Two-way ANOVA followed by an uncorrected Fisher's LSD \u003cem\u003epost hoc\u003c/em\u003e test. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01. n = 7–10 animals per group. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5820273/v1/4f074c35bed3056515a914d8.png"},{"id":74076628,"identity":"8860fbc2-14a9-4b68-baff-4b80f22ef216","added_by":"auto","created_at":"2025-01-17 13:43:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221251,"visible":true,"origin":"","legend":"\u003cp\u003eDepressive-like behavior in MMP-9 KO (A and B) and MMP-9 OE (C and D) male and female mice. Immobility time in the TST (A and C) and social interaction time (B and D) in the SIT. The data are expressed as the means ± SEMs. Two-way ANOVA followed by an uncorrectedFisher's LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. n = 7–10animals per group. TST: tail suspension test; SIT: social interaction test; WT: wild-type; KO: MMP-9 knockout; OE: MMP-9-overexpressing.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5820273/v1/c70d5c224a7f3090786cbc42.png"},{"id":74074635,"identity":"8cd6a38f-860b-4421-a40f-39bde1dd51fa","added_by":"auto","created_at":"2025-01-17 13:27:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":126080,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial working memory in MMP-9 KO (A) and MMP-9 OE (B) male and female mice assessed by the time spent in the novel arm in the T-maze. The results are expressed as the means ± S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher's LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. n= 8 ‐ 10 animals per group. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5820273/v1/ee5f9e94678fef56ea336ea8.png"},{"id":74074640,"identity":"6f321e05-af6f-4662-bf5a-405fbb651aea","added_by":"auto","created_at":"2025-01-17 13:27:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":593540,"visible":true,"origin":"","legend":"\u003cp\u003eNeuroplasticity markers expression in the hippocampus of male and female MMP-9 KO mice. BDNF (A), mTOR (B), phospho-mTOR (C), PSD95 (D), and synapsin I (E) expression in MMP-9 KO mice and their corresponding wild-typecounterparts. Representative western blot bands are shown. The results are expressed as percentages \u003cem\u003eversus\u003c/em\u003e the WT group and as the means ± S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher's LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. n = 7–8animals per group. BDNF: brain-derived neurotrophic factor; mTOR: mammalian target of rapamycin; PSD95: postsynapticdensity 95 protein; WT: wild-type; KO: MMP-9 knockout.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5820273/v1/607d01fc6ce58f58a7c89c05.png"},{"id":74074642,"identity":"926a8827-1e02-4e39-87c8-0bab77eed70a","added_by":"auto","created_at":"2025-01-17 13:27:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":504335,"visible":true,"origin":"","legend":"\u003cp\u003eNeuroplasticity markers expression in the hippocampus of male and female MMP-9 OE mice. BDNF (A), mTOR (B), p-mTOR (C), PSD95 (D), and synapsin I (E) expression in MMP-9 OE mice and their corresponding wild-type counterparts. Representative western blot bands are shown. The results are expressed as percentages \u003cem\u003eversus\u003c/em\u003ethe WT group and as the means ± S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher's LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. n = 6–7 animals per group. BDNF: brain-derived neurotrophic factor; mTOR: mammalian target of rapamycin; PSD95: postsynaptic density 95 protein; WT: wild-type mice; OE: MMP-9-overexpressing mice.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5820273/v1/6349fbaa227ceb946bd6ef4f.png"},{"id":83460201,"identity":"11547385-15a3-428d-a535-8bc2f9423a8e","added_by":"auto","created_at":"2025-05-26 16:12:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4165034,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5820273/v1/51f43a1f-caec-470b-889b-ec9695ea46b3.pdf"},{"id":74074633,"identity":"b0a37152-abdc-42d4-874e-3dc87d1366fe","added_by":"auto","created_at":"2025-01-17 13:27:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":97999,"visible":true,"origin":"","legend":"","description":"","filename":"SenserrichetalSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5820273/v1/90a4e25e7a7a72db7d0cab7a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sex Differences in the Modulation of Anxiety- and Depression-like Behaviors by Matrix Metalloproteinase-9 Expression Levels in Mice","fulltext":[{"header":"HIGHLIGHTS","content":"\u003cul\u003e\n \u003cli\u003eMatrix metalloproteinase 9 (MMP-9) overexpression male mice exhibit conflictive anxiety and low spatial memory.\u003c/li\u003e\n \u003cli\u003eMMP-9 overexpression female mice display low innate anxiety.\u003c/li\u003e\n \u003cli\u003eMMP-9 knockout female mice display high innate anxiety and low depressive-like behavior.\u003c/li\u003e\n \u003cli\u003eMMP-9 deletion in female mice show higher neuroplasticity markers expression.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"PLAIN ENGLISH SUMMARY","content":"\u003cp\u003eMajor depressive disorder is one of the most common neuropsychiatric diseases, which affects a two-fold number of women than men. The cause of this disorder is not known, however, impairments in neurotransmitters and neuroplasticity are some of the suggested causes. Specifically, some studies indicate that the expression of the matrix metalloproteinase 9 (MMP-9), one protein implicated in neuroplasticity, is increased in patients with depression, and these levels are reduced after antidepressant treatment. However, to date it has not been studied the differential implication of MMP-9 in the sex-differences observed in this pathology.\u003c/p\u003e\n\u003cp\u003eIn our study, we evaluated the impact of the elimination or the overexpression of MMP-9 in anxious and depressive-like behavior in male and female mice. We also evaluated how neuroplasticity was affected in these animals. We observed in female mice an opposite effect to the expected phenotype regarding anxiety, with higher anxiety in mice lacking MMP-9 and lower anxiety in mice that overexpress this protein. Moreover, the lower behavioral despair expected in mice that lack MMP-9 was only observed in females, which was parallel to the higher expression of some neuroplasticity markers. In addition, the cognitive impairment expected in mice that overexpress MMP-9 was only present in male mice.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we demonstrate a sex-differential effect of MMP-9 expression levels on anxious/depressive-like behavior, and the impact on neuroplasticity.\u003c/p\u003e"},{"header":"BACKGROUND","content":"\u003cp\u003eMajor depressive disorder (MDD) is a chronic psychiatric condition characterized by low mood, anhedonia, psychomotor alterations, feelings of guilt, and suicidal ideation. Patients can also present cognitive impairment and reduced libido [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to the World Health Organization (WHO), MDD affects 3.8% of the population, which includes 5% adults, and has a higher prevalence in women than in men [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, the WHO reported that the COVID-19 pandemic induced a 25% increase in depression and anxiety cases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe etiopathogenesis of MDD is not fully known, although multiple risk factors are involved, including biological, genetic, and psychosocial factors. Since the formulation of the classical “monoaminergic” hypothesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which proposes the existence of a dysfunctional brain monoaminergic system associated with depression, different hypotheses, including the \u003cem\u003eneuroplastic, glutamatergic\u003c/em\u003e, and \u003cem\u003eneuroinflammatory\u003c/em\u003e hypotheses, have been proposed to explain the neurobiology of this disease. Understanding the mechanisms underlying the pathophysiology of MDD is highly important for the development of novel therapeutic approaches.\u003c/p\u003e \u003cp\u003eMatrix metalloproteinases (MMPs) are endopeptidases responsible for the cleavage of the extracellular matrix and basement membrane components, cell surface receptors, cell adhesion molecules, growth factors, cytokines, and other proteases. Interestingly, they act as regulators of not only extracellular but also intracellular signaling networks [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. One of the most studied MMPs is MMP-9, a gelatinase expressed in both the peripheral and central nervous systems. It is released from glutamatergic excitatory synapses upon the activation of NMDA receptors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. MMP-9 plays a pivotal role in the regulation of synaptic plasticity, learning and memory, allowing the growth and maturation of dendritic spines and the accumulation and immobilization of AMPA receptors, which makes excitatory synapses more efficient in modulating the AMPA/NMDAR ratio [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, MMP-9 can catalyze the activation of brain-derived neurotrophic factor (BDNF) and proinflammatory cytokines, which participate in the processes of neurogenesis and inflammation, respectively [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. All these biological functions explain the involvement of MMP-9 in the etiopathogenesis of certain neurodegenerative disorders, such as epilepsy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and multiple sclerosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMDD patients present increased blood levels of the matrix metalloproteinases MMP-2, MMP-7, and MMP-9 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Serum MMP-9 levels are positively correlated with the severity of symptoms [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, electroconvulsive therapy reduces the serum levels of MMP-9 in depressed patients, resulting in a therapeutic response [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Increased MMP-9 activity has also been shown in \u003cem\u003epostmortem\u003c/em\u003e hippocampal samples from MDD patients [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], whereas other authors reported no differences in MMP-9 protein levels in the prefrontal cortex of untreated MDD patients [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, different MMP-9 gene polymorphisms, which are associated with high MMP-9 levels, have been associated with depressive symptoms in bipolar disorder patients (rs17576) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and anxiety disorders (rs3918242) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings highlight the importance of MMP-9 as a potential diagnostic marker of depression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which is correlated with treatment response and disease progression.\u003c/p\u003e \u003cp\u003eRegarding the role of brain MMP-9 in animal models of depression, we recently reported increased expression and activity of MMP-9 in the hippocampus and cortex of a chronic corticosterone mouse model of depression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similar results have been shown in other models of depression, such as the chronic stress model [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the chronic unpredictable stress model (CUS) in mice [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and a neuroinflammation model [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the last few years, the use of female animal models has increased, supporting the urge to address preclinical research to define optimal female models of neuropsychiatric diseases and their use for a deeper knowledge of sex bias. Moreover, a better understanding of the role of MMP-9 in the development of a depressive/anxious-like phenotype would help to elucidate the role of this protein in the neurobiology of depression. Therefore, in this study, we characterized the anxious and depressive-like behavioral and molecular phenotypes of male and female mice of two transgenic models: MMP-9 knockout (MMP-9 KO) mice, which have a nonfunctional MMP-9 protein, and mice that overexpress the human MMP-9 (MMP-9 OE) in the central nervous system.\u003c/p\u003e "},{"header":"METHODS","content":"\u003ch3\u003e1. ANIMALS\u003c/h3\u003e\u003cp\u003eThe MMP-9 knockout (Jackson Laboratory, Maine, USA) and MMP-9 overexpression (kindly donated by Dr. A.K. Tzinia) male and female mice, 2–3 months old, were group-housed (4–5 mice per cage) with a 12 h light‒dark cycle and food and water \u003cem\u003ead libitum\u003c/em\u003e. All procedures were carried out with the previous approval of the Animal Care Committee of the University of Cantabria and according to Spanish legislation (RD 53/2013) and the European Communities Council Directive on “Protection of Animals Used in Experimental and Other Scientific Purposes” (2010/63/UE).\u003c/p\u003e\u003cp\u003e1.1. MMP-9 knockout mice\u003c/p\u003e\u003cp\u003eB6. FVB(Cg)-Mmp9tm1Tvu/J (MMP-9 KO) mice (#007084, The Jackson Laboratory, Maine, USA) were generated by inserting a neomycin resistance gene driven by the mouse phosphoglycerate kinase promoter. This cassette replaces most of exon 2 and all of intron 2, disrupting MMP-9 functionality.\u003c/p\u003e\u003cp\u003e1.2. MMP-9-overexpressing mice\u003c/p\u003e\u003cp\u003eTgMMP9 (MMP-9 OE) mice were generated with the PDGF-B/MMP9 transgene of human origin, a gene encoding human pro-MMP-9 under the control of the human neuron-specific PDGF-B promoter [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2. BEHAVIORAL TESTS\u003c/h2\u003e\u003cp\u003eAll the behavioral tests were performed during the light phase. Mice were placed in the experimental room one hour before the evaluation to allow them to acclimatize. Two different batches of animals were used to avoid an overload of stress due to the battery of behavioral tests assessed. Three hours after the last experiment, the mice were sacrificed via cervical dislocation. The brains were rapidly removed, and the hippocampus was dissected on an ice-cold platform and kept at -80°C until use for western blot experiments.\u003c/p\u003e\u003cp\u003e2.1. Open field test (OFT)\u003c/p\u003e\u003cp\u003eThe OFT was performed as previously described by Breviario et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Mice were placed in a corner of the arena (40 × 40 × 30 cm) with an illuminated (50 lx) center (20 × 20 cm). The central time and total distance traveled were video tracked (Any-Maze™ tracking software version 4.99; Stoelting Co., Wood Dale, USA) for 5 min.\u003c/p\u003e\u003cp\u003e2.2. Elevated plus maze (EPM)\u003c/p\u003e\u003cp\u003eMice were placed in the center (5 × 5 cm) of the apparatus (50 cm from the floor), which consists of two open arms (25 × 5 × 0.5 cm) and two perpendicular closed arms (25 × 5 × 16 cm). Mice were allowed to explore freely for 5 min. The time spent in each arm was video tracked (Any-Maze™).\u003c/p\u003e\u003cp\u003e2.3. Light-Dark Box (LDB)\u003c/p\u003e\u003cp\u003eThe apparatus consists of a square box (40 × 40 × 20 cm) divided into two halves, one illuminated (50 lx) area and one dark area, connected by an opening. Mice were placed in a corner of the illuminated compartment, and the animal was allowed to freely explore the apparatus [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The time spent in each area was video tracked (Any-Maze™) for 5 min.\u003c/p\u003e\u003cp\u003e2.4. Novelty-suppressed feeding (NSF) test\u003c/p\u003e\u003cp\u003eNSF was performed as previously described by Vidal et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Mice were food deprived for 24 hours. A food pellet was placed in the illuminated center (50 lx) of an arena (40 × 40 × 30 cm) covered with a woodchip. The mouse was placed in a corner, and the latency to eat the pellet was recorded via video-tracking software (Any-Maze™) for a maximum duration of 10 min. After the test, the mice were placed back in their home cages, and the amount of food consumed for 5 min was measured. The animals that showed no food intake in their home cages were excluded from the data analysis.\u003c/p\u003e\u003cp\u003e2.5. Tail suspension test (TST)\u003c/p\u003e\u003cp\u003eMice were suspended by the tail for 6 min. Video-tracking software (Any-Maze™) was used to record the test results, and the time spent immobile was determined manually for the last 4 min by an observer blinded to the experimental groups.\u003c/p\u003e\u003cp\u003e2.6. Social interaction test (SIT)\u003c/p\u003e\u003cp\u003eMice were placed in an open field arena (40 × 40 × 30 cm) with an empty wire cylinder (7 cm diameter) placed in a corner. The experimental mice were allowed to explore the arena freely for 2.5 min. Immediately after, an unfamiliar mouse of a different strain (CD-1) was placed inside the wire cylinder, and the experimental mice were allowed to explore for 2.5 min. The time in the interaction zone (20 cm diameter) was assessed via video-tracking software (Any-Maze™).\u003c/p\u003e\u003cp\u003e2.7. T-maze\u003c/p\u003e\u003cp\u003eThe T-maze was adapted from a previously described protocol [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The apparatus consists of three arms, two opposite (30 × 5 × 15 cm) and one perpendicular (35 × 5 × 15 cm), creating a “T” shape. First, a training trial was assessed in which the left arm of the T-maze was blocked. The mice were placed at the end of the perpendicular arm and allowed to explore for 8 min. After the training trial, mice were removed from the apparatus and left to rest for an intertrial interval of 1 h. Then, for the experimental trial, the left arm was unlocked, recovering the “T” shape. A 3 min test was performed by placing the mice at the end of the perpendicular arm and allowing them to freely explore the apparatus. Mice were video tracked (Any-Maze™), and the time spent in the novel arm during the first minute of the test was measured.\u003c/p\u003e\u003ch3\u003e3. WESTERN BLOT\u003c/h3\u003e\u003cp\u003e3.1. Synaptoneurosomal protein extraction\u003c/p\u003e\u003cp\u003eProtein extraction was performed according to the protocol previously described by Li et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Briefly, the hippocampi were homogenized (1:15 w/v) in homogenization buffer (0.32 M sucrose, 20 mM HEPES pH 7.4, 1 mM EDTA, 1:100 protease inhibitor cocktail, 5 mM NaF and 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e). The samples were subsequently centrifuged at 800xg for 10 min at 4°C. The pellet was removed, and the supernatant was centrifuged at 15 300xg for 10 min at 4°C. The pellet was resuspended in 150 µl of protein lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 5 mM NaF and 1:100 protease inhibitor cocktail) and sonicated for 1 min. The protein concentration was quantified via the Lowry method. The samples were prepared with loading buffer containing β-mercaptoethanol, boiled at 100°C for 5 min, cooled on ice for 3 min, and centrifuged at 1000xg for 5 min at 4°C. The supernatant was stored at -20°C until use.\u003c/p\u003e\u003cp\u003e3.2. Western blot\u003c/p\u003e\u003cp\u003eFifty µg of protein per sample was loaded in duplicate on 8.5% or 15% SDS‒PAGE gels. The electrophoresis mixture was run at 100 V for 15 min and then at 160 V for 50 min and then transferred to nitrocellulose membranes (GE Healthcare Europe GmbH, Munich, Germany). The membranes were blocked with 5% (w/v) powder skim milk in TBS-T (50 mM Tris‐HCl pH 7.6, 150 mM NaCl, 0.05% Tween‐20) for 1 h. For phosphorylated proteins, the blocking solution used was 3% powder skim milk in TBS‐T supplemented with phosphatase inhibitors (1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e and 1 mM NaF). The following primary antibodies were incubated with the corresponding blocking solution at 4°C overnight: BDNF (1:500, ab108319, Abcam, Cambridge, MA, USA), mTOR (1:1000, #4517, Cell Signaling, Massachusetts, USA), phospho-Ser2448-mTOR (1:1000, #2971, Cell Signaling, Massachusetts, USA), PSD95 (1:200, sc-8575, Santa Cruz Biotechnology, Texas, USA), synapsin I (1:200, sc-390867, Santa Cruz Biotechnology, Texas, USA), and β-tubulin III (1:20000, T2200/T8660, Sigma‒Aldrich, Missouri, USA). The membranes were washed with TBS-T and then incubated with the corresponding secondary antibody conjugated to an NIR fluorophore (1:15 000, LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. After washing, the specific signal was visualized via an Odyssey® CLx Imaging System (LI-COR Bioscience, Lincoln, USA) and quantified via Image Studio™ Software (LI‐COR Biosciences, Lincoln, USA). The densitometric values were normalized to those of the housekeeping protein β‐tubulin III.\u003c/p\u003e\u003ch2\u003eSTATISTICAL ANALYSIS\u003c/h2\u003e\u003cp\u003eThe values are expressed as the means ± standard errors of the means (S.E.M.). For the molecular studies, the means of the sample replicates were normalized \u003cem\u003eto those of\u003c/em\u003e the WT mice (100%). The data were analyzed via two-way ANOVA followed by an uncorrected Fisher's LSD \u003cem\u003epost hoc\u003c/em\u003e test. The statistical analyses and identification of outliers were performed via GraphPad Prism 10 (GraphPad Software, Inc., California, USA). Statistical significance was set at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e1. Innate anxiety in MMP-9 KO and OE mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the open field test (OFT), MMP-9 KO female mice spent less time in the center than WT female mice (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01), with no differences in MMP-9 KO male mice (Figure 1A). Compared with male WT mice, female WT mice spent more time in the center (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Two‐way ANOVA revealed a significant effect of sex [F(1,33) = 33.4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001] and the interaction sex \u0026times; genotype [F(1,33) = 10.4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01].\u003c/p\u003e\n\u003cp\u003eIn the OFT, MMP-9 OE male and female mice did not significantly differ in the time spent in the center compared with their respective WT mice. However, the MMP-9 OE female mice spent more time in the center of the arena than the MMP-9 OE male mice (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, Figure 1D). Two‐way ANOVA revealed a trend toward the interaction sex \u0026times; genotype [F(1,33) = 3.95, \u003cem\u003ep\u003c/em\u003e = 0.06].\u003c/p\u003e\n\u003cp\u003eIn the elevated plus maze (EPM) and light‒dark box (LDB) tests, no significant differences were found in the time spent in the open arms in the EPM (Figure 1B) or the time spent in the light zone of the LDB (Figure 1C) in male and female MMP-9 KO mice, compared to their WT counterparts. WT female mice spent less time in the light zone than WT male mice (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, Fig. 1C). In the LDB test, two‐way ANOVA revealed a significant effect of sex [F(1,33) = 7.2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05].\u003c/p\u003e\n\u003cp\u003eIn both the EPM and LDB tests, MMP-9 OE female mice\u0026nbsp;spent more time in the open arms (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01, Figure 1E) and in the light zone (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05,\u0026nbsp;Figure 1F). There were no differences in\u0026nbsp;MMP-9 OE male mice\u0026nbsp;in either test. Compared with MMP-9 OE male mice,\u0026nbsp;MMP-9 OE female mice spent more time in the open\u0026nbsp;arms\u0026nbsp;(\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, Fig. 1E). In the EPM test, two‐way ANOVA revealed a significant effect of genotype [F(1,33) = 4.9, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] and the interaction sex \u0026times; genotype [F(1,33) = 7.2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05].\u003c/p\u003e\n\u003cp\u003eFigure 1. Innate anxiety in MMP-9 KO (A-C) and MMP-9 OE (D-F) male and female mice. Time spent in the center (A and D) in the OFT, time spent in the open arms in the EPM (B and E), and time spent in the lit compartment in the LDB (C and F). The data are expressed as the means \u0026plusmn; SEMs. Two-way ANOVA followed by an uncorrected Fisher\u0026apos;s LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. n = 7\u0026ndash;10 animals per group. OFT: open field test; EPM: elevated plus maze; LDB: light‒dark box test. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2. Conflict-based anxiety in MMP-9 KO and OE mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the novelty-suppressed feeding (NSF) test, no significant differences were observed in the latency to feed in the male and female MMP-9 KO mice (Figure 2A) and OE female mice (Figure 2B). However, male MMP-9 OE mice presented a greater latency to feed (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01, Figure 2B). Two‐way ANOVA revealed a significant effect of genotype [F(1,33) = 9.4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01]. Additionally, the amount of food consumed after the test did not significantly differ (see Figure S1).\u003c/p\u003e\n\u003cp\u003eFigure 2. Conflict-based anxiety in MMP-9 KO (A) and MMP-9 OE (B) male and female mice assessed by the latency to feeding in the novelty-suppressed feeding test. The data are expressed as the means \u0026plusmn; SEMs. Two-way ANOVA followed by an uncorrected Fisher\u0026apos;s LSD \u003cem\u003epost hoc\u003c/em\u003e test. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. n = 7\u0026ndash;10 animals per group. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3. Depressive-like behavior in MMP-9 KO and OE mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the tail suspension test (TST), MMP-9 KO female mice displayed a lower immobility time than WT female and MMP-9 KO male mice (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Figure 3A). No differences were observed in the MMP-9 KO males (Figure 3A). Two‐way ANOVA revealed a significant effect of sex [F(1,32) = 6.0, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] and the interaction sex \u0026times; genotype [F(1,32) = 5.6, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05]. Both male and female MMP-9 OE mice did not significantly differ (Figure 3D).\u003c/p\u003e\n\u003cp\u003eIn the social interaction test (SIT), male and female MMP-9 KO mice were more sociable than their corresponding WT mice, as evidenced by the greater amount of time spent in the interaction zone (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 3B). Two‐way ANOVA revealed a significant effect of genotype on MMP-9 KO mice [F(1,32) = 8.7, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01]. Compared with their WT counterparts, male and female MMP-9 OE mice did not significantly differ (Figure 3D).\u003c/p\u003e\n\u003cp\u003eFigure 3. Depressive-like behavior in MMP-9 KO (A and B) and MMP-9 OE (C and D) male and female mice. Immobility time in the TST (A and C) and social interaction time (B and D) in the SIT. The data are expressed as the means \u0026plusmn; SEMs. Two-way ANOVA followed by an uncorrected Fisher\u0026apos;s LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. n = 7\u0026ndash;10 animals per group. TST: tail suspension test; SIT: social interaction test; WT: wild-type; KO: MMP-9 knockout; OE: MMP-9-overexpressing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4. Cognitive behavior in MMP-9 KO and OE mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo significant differences in the time spent in the new arm in the T-maze test\u0026nbsp;were detected between male and female MMP-9 KO mice and their WT counterparts (Figure 4A).\u003c/p\u003e\n\u003cp\u003eCompared with their wild-type counterparts, male MMP-9 OE transgenic mice spent less time in the novel arm (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), whereas no significant differences were detected in female mice (Figure 4B). Two‐way ANOVA revealed a significant effect of genotype [F(1,31) = 4.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05].\u003c/p\u003e\n\u003cp\u003eFigure 4. Spatial working memory in MMP-9 KO (A) and MMP-9 OE (B) male and female mice assessed by the time spent in the novel arm in the T-maze. The results are expressed as the means \u0026plusmn; S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher\u0026apos;s LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. n= 8 ‐ 10 animals per group. WT: wild-type mice; KO: MMP-9 knockout mice; OE: MMP-9-overexpressing mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e5. Neuroplasticity markers in the hippocampus of MMP-9 KO and OE mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the hippocampus, MMP-9 KO male and female mice did not show differences in BDNF expression levels compared with their WT counterparts (Figure 5A), although a trend toward higher BDNF expression was observed in female MMP-9 KO mice than in their WT counterparts (\u003cem\u003ep\u003c/em\u003e = 0.05). Compared with their WT counterparts, the female MMP-9 KO group also presented increased mTOR (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 5B), p-mTOR (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 5C), PSD95 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Figure 5D) and synapsin l (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Figure 5E) expression levels. Compared with the male MMP-9 KO group, the female MMP-9 KO group also presented higher BDNF (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Figure 5A), mTOR (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 5B), p-mTOR (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Figure 5C), and PSD95 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Figure 5D) levels. Two‐way ANOVA revealed a significant effect of sex [F(1,27) = 6.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] and the interaction sex \u0026times; genotype [F(1,27) = 6.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] on BDNF values. Two‐way ANOVA revealed a significant effect of sex [F(1,28) = 6.1, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] and the interaction of genotype \u0026times; sex [F(1,28) = 6.1, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] on phospho-mTOR values. Two‐way ANOVA revealed significant effects of sex [F(1,28) = 10.0, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01], genotype [F(1,28) = 4.2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] and the interaction of genotype \u0026times; sex [F(1,28) = 10.0, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01] on PSD95 values. Two‐way ANOVA revealed a significant effect of genotype [F(1,28) = 11.1, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01] on synapsin I values.\u003c/p\u003e\n\u003cp\u003eFigure 5. Neuroplasticity markers expression in the hippocampus of male and female MMP-9 KO mice. BDNF (A), mTOR (B), phospho-mTOR (C), PSD95 (D), and synapsin I (E) expression in MMP-9 KO mice and their corresponding wild-type counterparts. Representative western blot bands are shown. The results are expressed as percentages \u003cem\u003eversus\u003c/em\u003e the WT group and as the means \u0026plusmn; S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher\u0026apos;s LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. n = 7\u0026ndash;8 animals per group. BDNF: brain-derived neurotrophic factor; mTOR: mammalian target of rapamycin; PSD95: postsynaptic density 95 protein; WT: wild-type; KO: MMP-9 knockout.\u003c/p\u003e\n\u003cp\u003eThe hippocampal levels of BDNF (Figure 6A), mTOR (Figure 6B), and synapsin I (Figure 6E) were not significantly different in male MMP-9 OE mice. A lower level of phosphorylated mTOR protein was observed in male MMP-9 OE mice than in their WT counterparts (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 6C). Two‐way ANOVA revealed a significant effect of genotype [F(1,21) = 4.4, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05]. PSD95 expression was greater in MMP-9 OE males than in their WT counterparts (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and in the MMP-9 OE female group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0,01, Figure 6D). Two‐way ANOVA revealed a significant effect of sex [F(1,22) = 4.9, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] and the interaction of genotype \u0026times; sex [F(1,22) = 4.9, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05] on PSD95 expression levels. MMP-9 OE female mice did not present differences in the levels of the neuroplasticity markers studied (Figure 6A‒E).\u003c/p\u003e\n\u003cp\u003eFigure 6. Neuroplasticity markers expression in the hippocampus of male and female MMP-9 OE mice. BDNF (A), mTOR (B), p-mTOR (C), PSD95 (D), and synapsin I (E) expression in MMP-9 OE mice and their corresponding wild-type counterparts. Representative western blot bands are shown. The results are expressed as percentages \u003cem\u003eversus\u003c/em\u003e the WT group and as the means \u0026plusmn; S.E.M.s. Two-way ANOVA followed by an uncorrected Fisher\u0026apos;s LSD \u003cem\u003epost hoc\u003c/em\u003e test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. n = 6\u0026ndash;7 animals per group. BDNF: brain-derived neurotrophic factor; mTOR: mammalian target of rapamycin; PSD95: postsynaptic density 95 protein; WT: wild-type mice; OE: MMP-9-overexpressing mice.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study focused on the behavioral and molecular parameters of male and female transgenic MMP-9 KO and OE mice. We did not observe any statistically significant differences in innate anxiety in male MMP-9 KO mice, which is consistent with previous studies that reported no alterations in anxiety in MMP-9 KO mice [32,33,34]. Interestingly, female MMP-9 KO mice presented increased innate anxiety, but only in the open field test. This finding might be partially attributed to the lower baseline anxiety levels observed in female wild-type mice than in males, as reported in previous studies in na\u0026iuml;ve rats [35,36,37]. These findings suggest a sex-dependent effect on certain manifestations of innate anxiety in MMP-9 KO mice, which has not been previously described, as other studies present pooled data from both male and female mice [34].\u003c/p\u003e\n\u003cp\u003eIn contrast to the more anxious phenotype observed only in the open field test in female MMP-9 KO mice, female MMP-9 OE mice consistently displayed lower innate anxiety across different behavioral tests, including the elevated plus maze and light‒dark box tests. This finding contrasts with previous results in which no differences in innate anxiety were observed in pooled male and female MMP-9 OE mice [28]. Some studies have suggested a link between MMP-9 levels and the basal anxious phenotype in female mice of various strains. For example, female C57BL/6J mice, which exhibit higher basal MMP-9 brain levels, show lower anxiety in the elevated plus-maze test than other strains with lower MMP-9 brain levels and greater anxiety [38]. The lower level of innate anxiety observed in our female MMP-9 OE mice aligns with these findings [38]. Furthermore, increased resilience to anxiety has been reported in female mice in various depression models, such as chronic corticosterone exposure [39,40] and acute lipopolysaccharide administration [41]. Taken together, these results suggest that MMP-9 plays a sex-dependent role in anxiety.\u003c/p\u003e\n\u003cp\u003eIn male mice, the overexpression of MMP-9 led to increased conflict-based anxiety, which is consistent with the increased anxiety levels observed in the novelty-suppressed feeding test in animal models of depression, such as the chronic corticosterone [25] and obesity [42] models, both of which are characterized by elevated MMP-9 brain levels. However, female MMP-9 OE mice appeared to be protected from developing this conflict-based phenotype. These results support the involvement of MMP-9 in anxiety-related disorders, such as post-traumatic stress disorder [43].\u003c/p\u003e\n\u003cp\u003eWith respect to depressive-like behaviors, female MMP-9 KO mice exhibited decreased behavioral despair, which is consistent with the antidepressant effects observed in various animal models following MMP-9 inhibition [44,45,46]. Interestingly, this reduced behavioral despair was not observed in male MMP-9 KO mice. Additionally, our MMP-9 OE mice did not show alterations in behavioral despair, which contrasts with the depressive-related behavior typically associated with increased MMP-9 expression and activity in this paradigm [20,25]. The lack of observed differences in our transgenic mice may be due to compensatory changes, which are common in constitutive transgenic mice [7].\u003c/p\u003e\n\u003cp\u003eDepressive disorders are often associated with impaired social functioning [47]. In our study, the overexpression of MMP-9 did not negatively affect social interaction. However, both male and female MMP-9 KO mice presented increased sociability, which could suggest a resilient or less vulnerable phenotype with respect to depressive-like manifestations. Our findings contrast with studies reporting no differences in social behavior in MMP-9 KO mice [33]. These discrepancies may arise from differences in the social interaction protocols used. In line with our findings, several studies in animal models with high MMP-9 levels suggest that the normalization or reduction of MMP-9 levels can improve sociability [48,49]. Moreover, transient overexpression of nectin-3, a proteolytic target of MMP-9, is able to reverse stress-induced social deficits [26].\u003c/p\u003e\n\u003cp\u003eFurthermore, it is important to note that depressive symptomatology is often accompanied by cognitive deficits, both in humans [50,51] and in animal models of depression [21]. In this study, male MMP-9 OE mice displayed working memory deficits, which is consistent with a negative correlation between hippocampal MMP-9 levels and working memory in male rats [52], although the impact of sex has not been extensively evaluated.\u003c/p\u003e\n\u003cp\u003eDepressive disorders are associated with changes in neuroplasticity markers in brain regions such as the hippocampus and prefrontal cortex, which are correlated with symptom severity. Treatment with antidepressant drugs can reverse these changes in neuroplasticity. In this study, we examined neuroplasticity markers associated with depression and the mechanism of action of antidepressant drugs in the hippocampus. Our findings revealed that female MMP-9 KO mice presented increased activation of the mTOR pathway, increased levels of PSD95 and synapsin l, and a trend toward increased BDNF levels, whereas male MMP-9 mice did not show differences. These findings indicate a sex-dependent effect of MMP-9 on depression-related neuroplasticity markers. Several murine models of depression show reduced hippocampal levels of mTOR, PSD95, and synapsin l, which are restored by antidepressant drug treatments, such as serotonin selective reuptake inhibitors (SSRIs) [53,54,55], ketamine [56,57], and others [58,59]. In fact, antidepressant drugs such as fluoxetine and paroxetine normalize mTOR and PSD95 levels in the hippocampus in chronic unpredictable mild stress [53] and chronic social defeat stress models [54]. Thus, the increased levels of mTOR, its phosphorylated form and the pre- and postsynaptic proteins synapsin I and PSD95 observed in our MMP-9 KO female mice support the decreased behavioral despair observed in the tail suspension test. The high BDNF levels in these mice are also consistent with antidepressant-like behavior [60,61], in good agreement with the lower behavioral despair elicited by these animals. However, some studies have reported that acute intrahippocampal BDNF administration can induce an anxiogenic effect [61,62], which might explain the increased innate anxiety observed in our female MMP-9 KO mice.\u003c/p\u003e\n\u003cp\u003eIn MMP-9 OE male mice, the decreased mTOR pathway activation in the hippocampus could be linked to the increased conflict-based anxiety observed, which is consistent with findings from other studies [63,64,65,66]. In association with lower hippocampal mTOR pathway activation, we did not observe depressive-like behavior, contrary to previous reports [53,55], or lower levels of PSD95, which have been associated with depressive-like behavior in other animal models [67]. These discrepancies may be due to compensatory changes in our constitutive transgenic mice. Additionally, other signaling pathways, such as the PLC, PI3K, and MAPK/ERK pathways, may also play a role in the regulation of PSD95 expression [68].\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eOur study highlights the sex-dependent role of MMP-9 in anxiety and depression. However, the regulation of MMP-9 levels does not appear to be the sole factor driving the modulation of anxiety and depression-like manifestations. These findings emphasize the importance of including female subjects in preclinical and clinical studies of depression and anxiety, as the incidence of these disorders is generally higher in women than in men.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBDNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebrain-derived neurotrophic factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEPM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelevated plus maze\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eknockout transgenic mouse\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elight‒dark box test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emajor depressive disorder\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ematrix metalloproteinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emTOR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emammalian target of rapamycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enovelty-suppressed feeding test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoverexpression transgenic mouse\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOFT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eopen-field test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePSD95\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epostsynaptic density protein 95\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSIT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esocial interaction test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etail suspension test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewild-type mouse\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/p\u003e\n\u003cp\u003eAll procedures were carried out with the previous approval of the Animal Care Committee of the University of Cantabria and according to Spanish legislation (RD 53/2013) and the European Communities Council Directive on \u0026ldquo;Protection of Animals Used in Experimental and Other Scientific Purposes\u0026rdquo; (2010/63/UE). Authorized project no. PI-03-19.\u003c/p\u003e\n\u003cp\u003eCONSENT FOR PUBLICATION\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003eCOMPETING INTERESTS\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFUNDING\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades (RTI2018-097534-B-I00), Centro de Investigaci\u0026oacute;n Biom\u0026eacute;dica en Red de Salud Mental (CIBERSAM), the Instituto de Salud Carlos III (FIS Grant PI19-00170), which were co-funded by the European Regional Development Fund (\u0026lsquo;A way to build Europe\u0026rsquo;), and the Spanish Network for Stress Research RED2022-134191-T financed by MCIN/AEI/10.13039/501100011033.\u003c/p\u003e\n\u003cp\u003eAUTHORS\u0026rsquo; CONTRITUTIONS\u003c/p\u003e\n\u003cp\u003eJ.S.: performed the experiments, analyzed and interpreted the data, and wrote the original draft; E.C.: designed the study, performed the experiments, analyzed and interpreted the data, and wrote the original draft; E.F.-Z.: performed the experiments, methodology and data analysis; A.D.: conceptualization and writing review; A.P.: funding acquisition and writing review; A.A.: conceptualization and writing review; A.T.: writing review; and F.P.-C. : conceptualization, funding acquisition, project administration, writing original draft.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmerican Psychiatric Association. 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Front Synaptic Neurosci. 2014. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fnsyn.2014.00006\u003c/span\u003e\u003cspan address=\"10.3389/fnsyn.2014.00006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biology-of-sex-differences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bosd","sideBox":"Learn more about [Biology of Sex Differences](http://bsd.biomedcentral.com)","snPcode":"13293","submissionUrl":"https://submission.nature.com/new-submission/13293/3","title":"Biology of Sex Differences","twitterHandle":"@BiologySexDiff","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Depression, Anxiety, Matrix metalloproteinase-9; Transgenic mice, Sex, Neuroplasticity","lastPublishedDoi":"10.21203/rs.3.rs-5820273/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5820273/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Major depressive disorder is one of the main causes of disability worldwide, but its etiopathology remains largely unknown, although several hypotheses have been proposed. Recent studies suggest a potential role for matrix metalloproteinase 9 (MMP-9) in depression, as it is overexpressed in the plasma of depressed patients and normalizes following chronic antidepressant treatment. This study aimed to characterize anxiety and depression-like behaviors in transgenic MMP-9 mice, as well as the expression of different neuroplasticity markers associated withdepression, in both sexes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e In this study, we characterized the behavioral phenotypes of both MMP-9 knockout and MMP-9-overexpressing male and female mice. Here, we used a battery of tests to assess anxiety (open field, light‒dark box, elevated plus maze, and novelty‒suppressedfeeding tests), depressive-like (tail suspension and social interaction tests), and cognitive (T-maze) behaviors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eMMP-9 knockout female mice displayed increasedinnate anxiety (open field test), decreasedbehavioral despair (tail suspension test), and increased sociability (social interaction test). This increased sociability was also observed in male MMP-9 knockout mice. Compared with control mice, female MMP-9 knockout mice presented increased levels of different neuroplasticity markers in the hippocampus.\u003c/p\u003e\n\u003cp\u003eWith respect to MMP-9-overexpressing mice, females presented decreasedinnate anxiety (elevated plus maze and light‒dark box). Male MMP-9-overexpressing mice presented greaterconflict-based anxiety (novelty-suppressed feeding test) and lower working memory (T-maze) than control mice did. These male mice presented a reduction in mTOR pathway activation and increased PSD95 hippocampal levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e MMP-9 levels may have a sex-dependent impact on the anxious/depressive-like phenotype, as well as on neuroplasticity markers in the hippocampus. These findings reinforce the sex differences in the etiopathology of depression.\u003c/p\u003e","manuscriptTitle":"Sex Differences in the Modulation of Anxiety- and Depression-like Behaviors by Matrix Metalloproteinase-9 Expression Levels in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-17 13:27:53","doi":"10.21203/rs.3.rs-5820273/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-18T16:30:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-18T12:36:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-14T11:47:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266106180035671236028563087746874774043","date":"2025-01-29T17:14:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84374539869962973242658482711109669545","date":"2025-01-27T16:33:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-24T23:38:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-14T10:49:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-14T10:49:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biology of Sex Differences","date":"2025-01-13T13:09:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biology-of-sex-differences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bosd","sideBox":"Learn more about [Biology of Sex Differences](http://bsd.biomedcentral.com)","snPcode":"13293","submissionUrl":"https://submission.nature.com/new-submission/13293/3","title":"Biology of Sex Differences","twitterHandle":"@BiologySexDiff","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f25acf32-71e1-42fe-9ead-8a8cf2f6d52f","owner":[],"postedDate":"January 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-26T16:06:20+00:00","versionOfRecord":{"articleIdentity":"rs-5820273","link":"https://doi.org/10.1186/s13293-025-00716-5","journal":{"identity":"biology-of-sex-differences","isVorOnly":false,"title":"Biology of Sex Differences"},"publishedOn":"2025-05-22 15:57:54","publishedOnDateReadable":"May 22nd, 2025"},"versionCreatedAt":"2025-01-17 13:27:53","video":"","vorDoi":"10.1186/s13293-025-00716-5","vorDoiUrl":"https://doi.org/10.1186/s13293-025-00716-5","workflowStages":[]},"version":"v1","identity":"rs-5820273","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5820273","identity":"rs-5820273","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}