Negr1 Deficiency Alters Glutamate Signalling and Kynurenine Pathway in a Mouse Model of Psychiatric Disorders

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In this study, we expanded on these findings by investigating the behavioural response to NMDA receptor antagonism, expression of NMDA receptor subunits, and kynurenine pathway metabolites in a Negr1 -deficient mouse model. Male and female wild-type and Negr1 -deficient mice received daily injections of MK-801, a non-competitive NMDA receptor antagonist, until behavioural tolerance developed in the open field test (after 9 days in males and 5 days in females). In drug-naive animals, acute MK-801 administration (0.2 mg/kg) elicited a stronger motor response in Negr1 -deficient males compared to wild-type controls. However, with repeated dosing, Negr1 -deficient males exhibited a blunted behavioural response and attenuated progression of rapid behavioural tolerance during every-second-day MK-801 administration, suggesting altered receptor sensitivity. Gene expression analysis revealed sex- and brain region-specific changes in NMDA receptor subunit expression. Additionally, kynurenine pathway metabolites showed genotype- and sex-dependent alterations. These findings suggest that Negr1 modulates NMDA receptor function and tryptophan metabolism in a sex-dependent manner, highlighting the importance of considering both genetic background and sex in models of glutamatergic dysfunction relevant to neuropsychiatric disorders. Health sciences/Diseases Biological sciences/Neuroscience Negr1 NMDA MK-801 kynurenine pathway behavioural tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Psychiatric disorders such as anxiety, major depression, bipolar disorder, and schizophrenia affect around 800 million people worldwide, often impairing quality of life [1][2]. Genetic factors, including polymorphisms in specific genes, contribute to susceptibility. One such gene is neuronal growth regulator ( NEGR1) . This gene encodes for a cell adhesion molecule involved in neural development, synapse formation, and plasticity [3][4][5][6]. Genome-wide association studies (GWAS) have identified NEGR1 as a risk gene for several psychiatric and neurodevelopmental disorders [7][8][9][10][11][12]. However, the mechanisms through which NEGR1 influences behaviour and neurotransmitter systems remain poorly understood. Previous findings have shown that MK-801 (dizocilpine) binding density at NMDA receptors is higher in hippocampal sections of Negr1 -deficient mice compared to wild-type (WT) controls, suggesting increased N-methyl-D-aspartate (NMDA) receptor availability in the Negr1 -deficient brain [4]. Given the central role of NMDA receptors in synaptic plasticity, learning, and memory [13][14][15], the glutamatergic system emerges as a potential pathway linking Negr1 to psychiatric phenotypes. The NMDA receptor is composed of multiple subunits (e.g., GluN1, GluN2A, GluN2B), and changes in their expression have been associated with cognitive and emotional dysregulation [16][17]. Notably, GluN1 and GluN2A subunits serve as binding sites for D-serine, a molecule that can act as either a co-agonist or antagonist depending on the site [18]. D-serine levels are regulated by serine racemase (Srr), an enzyme critical for NMDA receptor function, and disruptions in Srr activity have been implicated in schizophrenia spectrum disorders [19][20][21]. Dysregulation of NMDA receptor subunits and Srr activity may therefore provide a mechanistic link between Negr1 deficiency and the behavioural abnormalities observed in psychiatric conditions. Our previous work demonstrated that Negr1 -deficient mice exhibit heightened behavioural sensitivity to amphetamine, including exaggerated motor and stereotypic responses, along with altered expression of dopaminergic markers [8]. These findings suggest that Negr1 influences dopaminergic reactivity and behavioural sensitisation. Building on prior in vitro findings of increased MK-801 binding to NMDA receptors in Negr1 -deficient brain tissue, the present study investigates how MK-801, a non-competitive NMDA receptor antagonist known to mimic glutamatergic dysfunction and interfere with sensitisation processes [22][23][24], affects behaviour and molecular markers in Negr1 -deficient mice. In addition to direct glutamatergic modulation, we considered the role of the kynurenine pathway (KP), which metabolises tryptophan into neuroactive compounds such as kynurenic acid (KYNA) and quinolinic acid (QUIN). KYNA acts as an NMDA receptor antagonist at GluN1 subunits, while QUIN acts as an agonist at GluN2A and GluN2B subunits [17][25][26][27][28][29]. Imbalances in these metabolites have been associated with psychiatric and neurodegenerative disorders [17][30][31], suggesting that KP dysregulation may influence NMDA receptor function and excitatory signalling. Despite growing evidence implicating the KP in neuropsychiatric conditions, the relationships between Negr1, NMDA receptor signalling, KP metabolites, and glutamate levels remain poorly defined. Thus, the present study aims to elucidate how Negr1 deficiency affects behaviour and its underlying molecular mechanisms, focusing specifically on glutamatergic signalling and kynurenine pathway metabolism. Using a Negr1- deficient mouse model, we examined the expression of key NMDA receptor subunits (GluN1, GluN2A, GluN2B) and serine racemase (Srr) in the hippocampus and frontal cortex—regions crucial for learning, memory, and higher cognitive functions that depend on NMDA receptor-mediated plasticity [13][32][33]. Additionally, we measured kynurenine pathway metabolites and glutamate levels, both known modulators of NMDA receptor activity [34][35] and implicated in neuropsychiatric disease [35][36][37]. To evaluate behavioural and molecular sensitivity to glutamatergic disruption, we assessed responses to repeated MK-801 administration. Finally, we investigated sex differences in these outcomes to determine whether Negr1-related effects differ between male and female mice. By linking behavioural phenotypes with glutamatergic and metabolic alterations, this study provides new insights into the neurobiological mechanisms underlying psychiatric disorders associated with NEGR1 (Fig. 2 ). Results Effect of Repeated Treatment with MK-801 (0.2 mg/kg) on Locomotor Activity in Male Wild-Type and Negr1 -Deficient Mice Based on the dose–response experiments performed in male mice, the optimal dose for behavioural activation was determined to be 0.2 mg/kg (Supplementary Fig. S1 A-C). Acute administration of MK-801 at this dose produced a significantly stronger motor activity response in Negr1 -deficient ( Negr1 −/− ) mice compared to wild-type controls (total distance covered - p < 0.0001; distance covered in corners - p < 0.05). Interestingly, this enhanced response was not observed during the first day of testing in the repeated administration experiment (Fig. 1 A-C). Notably, the same cohort of mice used for the dose-response curve – following a one-week washout period – was also used for the repeated administration protocol. As a result, these mice were not completely drug-naive at the start of the repeated treatment. We hypothesised that the heightened acute response to MK-801 is specific to drug-naive Negr1 -deficient mice. To test this, the acute administration experiment was repeated in an independent cohort of drug-naive male mice. Consistent with our hypothesis, Negr1 -deficient mice in this new cohort again showed a stronger motor activity response, as measured by the total distance covered (p < 0.05) (Supplementary Fig. S1 D). Interestingly, during repeated MK-801 administration in males, Negr1 −/− mice exhibited a blunted behavioural response over time, suggesting altered sensitivity or tolerance development. Namely, repeated administration of MK-801 elicited distinct locomotor activity patterns in male mice across treatment days, highlighting both genotype-dependent and temporal effects (Fig. 1 – 2 , Supplementary Fig. S2-S3). Day 1 MK-801 significantly increased total distance (p < 0.0001 for both WT and Negr1 −/− mice), distance in corners (p < 0.001 for WT; p < 0.05 for Negr1 −/− mice), and rotations (p < 0.0001 for both). Day 2 Response diminished; distance still increased (p < 0.05 for both), but only Negr1 −/− mice showed increased rotations (p < 0.05) and WT showed increased corner activity (p < 0.01). Day 3 A strong stimulatory effect re-emerged. MK-801 increased total distance (p < 0.00001 WT; p < 0.001 Negr1 −/− ), rotations (p < 0.0001 WT; p < 0.001 Negr1 −/− ), and corner distance (p < 0.0001 WT; p < 0.05 Negr1 −/− ). Day 4 Effects waned. Small but significant increases in distance (p < 0.05 WT; p < 0.01 Negr1 −/− ) and corner activity (p < 0.01 for both). Rotations increased only in Negr1 −/− mice (p < 0.01). Day 5 : WT mice showed peak activity — distance (p < 1×10⁻⁷), rotations (p < 1×10⁻⁶), and corner distance (p < 0.0001). Negr1 −/− mice showed no such increase, which resulted in significant genotype differences in all parameters (distance: p < 0.01; corners: p < 0.05; rotations: p < 0.05). Day 6 MK-801 effects declined markedly in WT and even more in Negr1 −/− mice, nearing Day 2 and 4 levels. Days 7–9 Gradual attenuation continued. By Day 9, activity in both genotypes had dropped significantly from peak levels (Days 3 and 5). Effect of Repeated Treatment with MK-801 (0.2 mg/kg) on Locomotor Activity in Female Wild-Type and Negr1 -Deficient Mice Female mice displayed a distinct locomotor response profile compared to males, characterised by rapid attenuation of MK-801’s effects (Fig. 1 – 2 , Supplementary Fig. S2-S3), but no genotype effect was present. Day 1 MK-801 significantly increased distance (p < 0.001 for both genotypes), rotations (p < 0.01), and corner distance (p < 0.001). Day 2 : Reduced response. Significant increases only in Negr1 −/− mice (distance: p < 0.01; rotations: p < 0.05; corners: p < 0.05). Day 3 Partial response. Distance (p < 0.05 for both), corners (p < 0.01 for both); only Negr1 −/− showed increased rotations (p < 0.05). Days 4 and 5 MK-801 effects are nearly absent. All parameters declined to saline-control levels, indicating tolerance development and leading to discontinuation of treatment in females. Sex Differences in Response to MK-801 Administering MK-801 resulted in both rapid and general behavioural tolerance, which was measured daily as motor activity in the open field. The drug’s effect diminished every second day in both sexes. General tolerance became evident on day 9 in males and on day 5 in females, leading to sex-specific treatment durations. Notably, the genotype effect was present only in male but not in female mice. The effects of MK-801 on locomotor activity revealed significant gender-dependent differences, particularly with repeated administrations. These differences became most pronounced by Day 5, prompting a comparative analysis of Days 1 and 5. Distance covered: On Day 5, wild-type (WT) males showed a significant increase (p < 0.05) while Negr1 -deficient males did not. In females, MK-801's effect decreased significantly by Day 5 in both WT (p < 0.001) and Negr1 −/− mice (p < 0.0001). Female mice also showed significantly lower responses compared to their male counterparts (p < 0.0001 for WT; p < 0.01 for Negr1 −/− ). Rotations: WT males showed increased rotations on Day 5 (p < 0.05); no change occurred in Negr1 −/− males. In females, MK-801 reduced rotations by Day 5 in WT (p < 0.01) and Negr1 −/− (p < 0.001) mice. Female mice also exhibited significantly fewer rotations than males on Day 5 (p < 1*10 − 5 for WT; p < 0.01 for Negr1 −/− ). Distance covered in corners: WT males had increased corner activity on Day 5 (p < 0.05); Negr1 −/− males, again, showed no change. In females, MK-801 reduced corner distance in Negr1 −/− mice (p < 0.0001) and to a lesser extent in WT (p < 0.05). WT females had a significantly lower response than males (p < 0.01); no sex difference was found in Negr1 −/− mice. Changes in NMDA-Related Gene Expression Due to Repeated MK-801 Treatment Frontal cortex In male mice, NMDA-related gene expression tended to be lower than in female littermates. However, MK-801 treatment did not cause significant alterations in gene expression in male mice (Fig. 3 A-C). In the frontal cortex of female mice, the expression of the Grin2a gene was unaffected by MK-801 administration (Fig. 3 B). For Grin1 , a significant treatment effect was observed (F₁,₃₃ = 13.12, p < 0.001). Post hoc analysis (Tukey HSD test) revealed a significant reduction in Grin1 expression in Negr1 -deficient mice (p < 0.01), but not in wild-type animals (Fig. 3 A). For Grin2b , significant effects of genotype (F₁,₃₁ = 6.06, p < 0.05) and treatment (F₁,₃₁ = 31.16, p < 0.00001) were identified (Fig. 3 C). Post hoc analysis showed a significant reduction in Grin2b expression in wild-type (p < 0.01) and Negr1 -deficient mice (p < 0.001). For Srr , MK-801 treatment had a significant effect (F₁,₃₃ = 6.89, p < 0.05), but post hoc analysis did not reveal specific group differences (Supplementary Fig. S5). Hippocampus In female mice, MK-801 treatment did not result in significant changes in NMDA-related gene expression in the hippocampus (Fig. 3 D-F). In male mice, a significant change was observed for Grin2a expression, with a treatment effect (F₁,₃₃ = 6.08, p < 0.05) and genotype × treatment interaction (F₁,₃₃ = 7.12, p < 0.01). Post hoc analysis showed a significant increase in Grin2a expression in Negr1 -deficient mice who were given physiological solution compared to the mice who received MK-801 (p < 0.001) and wild-type mice (p < 0.05) (Fig. 3 E). Regarding Grin2b expression, a significant change was seen with a genotype × treatment interaction (F₁,₃₃ = 4.56, p < 0.05). The levels of Grin2b expression were increased in Negr1 -deficient mice (p < 0.05) who were given physiological solution compared to the mice who received MK-801 (Fig. 3 F). Correlational Analysis of Kynurenine Pathway Metabolites, Tryptophan and Glutamate Across Brain Regions and Blood Plasma In the correlation analysis, we compared WT and Negr1 -deficient mice, both male and female. Analysis included seven metabolites, which we determined most relevant to this paper’s topic: tryptophan, kynurenine, kynurenic acid, quinolinic acid, picolinic acid, xanthurenic acid and glutamate. The data was gathered from blood plasma and four brain regions: frontal cortex, hippocampus, hypothalamus and ventral striatum (Supplementary Fig. S6 and Fig. S7, Supplementary tables S2-S5). Similarities between groups Analysis revealed that xanthurenic acid in hippocampus significantly correlated with quinolinic acid in hippocampus in both female Negr1 -/- and female wild-type groups, demonstrating a consistent positive association across these conditions. Additionally, xanthurenic acid in blood plasma displayed multiple significant correlations with various metabolites in male wild-type and male Negr1 -/- groups. These associations encompassed both positive and negative directions. Another notable relationship was observed between glutamate and xanthurenic acid in the hippocampus in both male and female Negr1 -/- groups, suggesting a mutation-specific link between glutamate and xanthurenic acid metabolism. Moreover, glutamate exhibited significant correlations with xanthurenic acid in plasma in male Negr1 -/- mice and female wild-type controls. Interestingly, the directionality of these correlations diverged: a positive correlation in male Negr1 -/- mice contrasted with a negative one in female wild-type mice. Differences between groups Distinct patterns emerged when comparing male and female groups. Male mice exhibited a higher number of significant correlations involving xanthurenic acid in plasma, while female mice showed a greater emphasis on xanthurenic acid correlations in the hippocampus. Notably, female Negr1 -/- mice demonstrated particularly strong associations, such as between quinolinic acid and xanthurenic acid in the frontal cortex (r = 0.86, p < 0.001). When comparing Negr1 -/- and wild-type mice groups, several differentiating features became apparent. Male WT mice showed classic tryptophan and kynurenine pathway correlations, whereas Negr1 -/- groups displayed stronger associations involving glutamate and quinolinic acid. Furthermore, female Negr1 -/- mice exhibited more robust and pronounced correlation values than their wild-type counterparts. Changes in the Kynurenine Pathway and Glutamate Levels of Negr1 -Deficient Mice As a result of the correlation analysis, we focused on kynurenic acid, quinolinic acid, xanthurenic acid and glutamate in the frontal cortex, hippocampus and blood plasma (Figs. 4 and 5 ). We also looked at the levels of these metabolites in hypothalamus and ventral striatum (Supplementary Fig. S8 - Fig. S17). There was no statistically significant difference in the kynurenic acid and quinolinic acid levels between WT and Negr1 -deficient male mice in the frontal cortex, although there seemed to be a trend for an increase among mutant mice compared to the WT controls (Fig. 4 A and B). The xanthurenic acid (p < 0.01) and glutamate levels (p < 0.05), however, were significantly increased in the Negr1 -deficient male mice (Fig. 4 C and D). The level of kynurenic acid remained unchanged for the female mice in the frontal cortex (Fig. 4 A), but contrary to the male mice, the levels of quinolinic acid (p < 0.01), xanthurenic acid (p < 0.01) and glutamate (p < 0.05) were considerably reduced (Fig. 4 B-D). The levels of measured metabolites in the hippocampus of Negr1 -deficient male and female mice were not significantly altered compared to the wild-type mice (Fig. 4 E-H). The blood plasma analysis included two cohorts to estimate the dynamics of the biochemical shifts during ageing: cohort 2 consisted of 5-month-old mice, and cohort 3 of 7-month-old mice (Fig. 5 ). Our results indicate that older mice were more strongly influenced by genotype. Specifically, male Negr1 −/− mice showed a significant decrease in quinolinic acid levels (p < 0.001) (Fig. 5 B), and female Negr1 −/− mice exhibited a significant increase in kynurenic acid levels (p < 0.05) (Fig. 5 E) compared to age-matched wild-type controls (5-month-olds). The reduction in quinolinic acid levels in male mice remained significant when the two age groups were combined (p < 0.01), whereas the increase in kynurenic acid in females did not (Supplementary Fig. S14). Xanthurenic acid and glutamate levels remained relatively unchanged across sex, age, and genotype groups. Discussion This study is the first to demonstrate a link between Negr1 , NMDA receptor function, and kynurenine pathway metabolites, resulting in significant behavioural alterations. While NEGR1 has been associated with various psychiatric disorders [38], we extended this research using MK-801, a non-competitive NMDA receptor antagonist, to model glutamatergic imbalances observed in neuropsychiatric and neurodegenerative conditions [22][23][24]. Behavioural analyses revealed significant differences between saline- and MK-801-treated mice. Acute MK-801 administration elicited a heightened motor response in drug-naive Negr1 -deficient males compared to wild-type controls. However, with repeated exposure, Negr1 -deficient males displayed a blunted response, indicating altered NMDA receptor sensitivity or tolerance development. The most pronounced changes were observed in total distance covered, distance covered in corners, and rotational behaviour. MK-801–induced hyperlocomotion is attributed to its action on GABAergic interneurons; NMDA receptor blockade reduces inhibitory tone and indirectly enhances excitatory output [39]. The exaggerated initial response in Negr1 -deficient mice may reflect a baseline reduction in GABAergic tone [5][40], amplifying the disinhibitory effects of MK-801, consistent with prior findings of disrupted excitatory and inhibitory balance in models of psychiatric disease [41]. An unexpected zig-zag pattern in behavioural responsiveness emerged, marked by reduced activity every other day, suggesting the rapid development of behavioural tolerance to daily MK-801 administration. The underlying mechanism remains unclear but may involve residual drug accumulation due to MK-801’s long half-life [24] or transient NMDA receptor desensitisation [42]. Repeated exposure could trigger rapid yet reversible neuroadaptive processes, such as receptor upregulation or alterations in downstream signalling [14]. After a brief recovery period, receptor sensitivity may reset, restoring responsiveness. Although this pattern was evident in both genotypes, Negr1 -deficient mice showed a stronger progression of tolerance, indicating altered NMDA receptor sensitivity. In addition, Negr1 -deficient male mice exhibited a stronger acute response to MK-801 but developed tolerance more rapidly with repeated dosing. Behavioural suppression — seen as reduced locomotion and stereotypy — diminished more quickly in Negr1 −/− mice compared to wild-type controls, particularly across treatment intervals (delta days 1–2, 3–4, and 5–6; Fig. 2 and supplementary Fig. S3). These findings suggest that Negr1 deficiency alters NMDA receptor function or regulation, potentially due to increased receptor availability [4]. Elevated baseline NMDA receptor density may heighten initial MK-801 sensitivity while accelerating desensitization or downstream adaptations during repeated exposure. Overall, the data indicate dysregulated NMDA receptor dynamics in Negr1 -deficient mice, influencing both acute responsiveness and the trajectory of tolerance development. At the molecular level, our data indicate a complex, sex- and region-specific modulation of NMDA receptor subunit expression. Previous studies have shown that receptors with a higher GluN2B-to-GluN2A ( Grin2b -to- Grin2a ) ratio are more susceptible to quinolinic acid–induced neurotoxicity due to their predominant expression in immature neurons and extrasynaptic sites, where they can promote excitotoxicity [43][44][45]. In the present study, a similar pattern appeared in the frontal cortex of adult female mice but was not observed in the hippocampus or in male mice. However, some studies have reported contrasting findings — highlighting a critical role for GluN2B in intracellular signalling and excitotoxicity and suggesting that both GluN2A and GluN2B subunits contribute equally to extrasynaptic signalling [46][47]. Furthermore, we found that female Negr1 -deficient mice treated with MK-801 exhibited reduced expression of GluN1 ( Grin1 ) in the frontal cortex. Together, these findings suggest a sex- and brain region-specific interaction between Negr1 deficiency and NMDA receptor regulation In male mice, expression levels of NMDA receptor subunit genes in the frontal cortex did not differ significantly between genotypes. In contrast, in the hippocampus, Grin2a and Grin2b were significantly upregulated in Negr1 -deficient mice treated with physiological solution compared to wild-type controls. This finding aligns with earlier evidence of increased NMDA receptor binding density in the hippocampus of Negr1 -deficient animals [4], suggesting elevated baseline receptor availability in this brain region under non-challenged conditions. Interestingly, MK-801 administration normalised the expression of these subunits to levels comparable with wild-type controls. This pattern may reflect a compensatory mechanism, wherein Negr1 -deficient mice upregulate NMDA receptor subunits to counterbalance impaired receptor function or altered inhibitory signalling. Alternatively, increased expression could serve to maintain excitatory-inhibitory homeostasis in the context of disrupted GABAergic tone. MK-801 treatment may override this compensatory adaptation by saturating receptor activity and externally shifting the excitatory-inhibitory balance. However, previous studies have shown that overexpression of Grin2a and Grin2b can exacerbate neuronal vulnerability [47], and GluN2A overexpression has been associated with impaired synaptic structure and function [48]. In contrast, GluN2B overexpression has been linked to improved learning and memory [49][50][51]. These contrasting outcomes highlight the complexity of NMDA receptor regulation and emphasise the need for further research to determine whether such subunit overexpression is neuroprotective or detrimental in the context of Negr1 deficiency. Although gene expression was assessed after behavioural adaptation to MK-801, this reflects a typical compromise in longitudinal study designs. Future studies could build on these findings by targeting more specific time points — such as day 5 in males and day 3 in females — when behavioural phenotypes diverge most clearly. These adjustments would help to refine the temporal resolution of gene expression dynamics and strengthen causal interpretations. One of the most notable findings of this study was the emergence of clear sex differences, underscoring the importance of including both male and female animals in neurobiological research [52][53]. Previous studies have reported sex-specific differences in NMDA receptor function and responses to NMDA receptor antagonists [54][55][56]. Our results extend these observations by showing that sex differences in Negr1 -deficient mice are evident not only in behaviour but also in kynurenine pathway metabolites and glutamate levels. Over the course of five days, wild-type males displayed a progressive increase in locomotor activity following repeated MK-801 administration, indicative of sensitisation. In contrast, Negr1 -deficient males showed minimal behavioural change, suggesting altered receptor responsiveness or adaptation. Female mice, regardless of genotype, exhibited more rapid tolerance and sensitisation to MK-801, reflected by a decline in locomotor activity over time. These findings highlight a dynamic interplay between sex, genotype, and NMDA receptor function and point to sex-specific mechanisms of behavioural plasticity in response to glutamatergic disruption. Although we anticipated that kynurenic acid (KYNA) and quinolinic acid (QUIN) levels would directly influence NMDA receptor function in Negr1 -deficient mice, our findings suggest a more nuanced relationship. While levels of kynurenine pathway metabolites were altered in the Negr1 -deficient group, these changes did not appear to drive NMDA receptor-related behavioural outcomes directly. This may indicate that NMDA receptor function was maintained through compensatory mechanisms involving other co-agonists or modulatory systems. In addition, correlation analyses revealed that kynurenine pathway metabolite profiles were region-specific. The frontal cortex was the most affected by Negr1 deficiency, whereas other brain regions exhibited few significant changes (Supplementary Fig. S6–S7; Supplementary Table S2–S5; Supplementary Fig. S8–S17). These findings emphasise the importance of spatial context when studying neuroimmune-metabolic interactions and suggest that the impact of Negr1 on kynurenine metabolism may be anatomically selective. Furthermore, the effects of Negr1 deficiency became more pronounced with age, with older mice showing stronger genotype-related shifts in kynurenine pathway metabolites. This suggests that ageing may exacerbate or unmask metabolic consequences of Negr1 deficiency. Conclusion This study demonstrates that Negr1 deficiency leads to pronounced, sex-specific alterations in glutamatergic signalling, behavioural responses to NMDA receptor antagonism, and kynurenine pathway metabolism. These effects were both brain region– and sex–dependent, underscoring the importance of considering biological sex and genetic background when modelling neuropsychiatric disorders. Our findings suggest that Negr1 influences NMDA receptor availability and dynamics, contributing to altered sensitivity and tolerance to glutamatergic disruption. Moreover, the observed region-specific changes in kynurenine metabolites highlight a possible link between neuroimmune metabolism and glutamatergic function in the Negr1 -deficient brain. Taken together, these results provide novel insights into the neurobiological mechanisms associated with Negr1 and support its relevance as a molecular node connecting genetic risk, glutamate dysregulation, and sex-dependent vulnerability in psychiatric disorders. Targeting Negr1 -related pathways may open new avenues for understanding and eventually mitigating glutamate-related dysfunction in mental illness. Methods Animals Adult male and female wild-type (WT) mice and their homozygous Negr1 -deficient littermates ( Negr1 −/− ), previously generated and described by Lee et al. (2012), were used in this study. All mice were on an F2 hybrid background: ((129S5/SvEvBrd × C57BL/6N) × (129S5/SvEvBrd × C57BL/6N)). Animals were group-housed (10 per cage) in standard laboratory cages (42.5 × 26.6 × 15.5 cm) under controlled environmental conditions (22 ± 1°C; 12:12 h light/dark cycle, with lights off at 19:00). Each cage contained a 2 cm layer of aspen bedding and 0.5 L of aspen nesting material (Tapvei, Paekna, Estonia), which were changed weekly. Food pellets (R70, Lactamin AB, Kimstad, Sweden) and water were provided ad libitum . Breeding and maintenance were carried out at the animal facility of the Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia. All behavioural testing was conducted between 8:00 a.m. and 5:00 p.m. Prior to testing, mice were kept in group housing conditions to minimise stress. Three separate mouse cohorts were used in this study (Fig. 6 ): Cohort 1 Included 2-month-old male and female mice, with equal representation of WT and Negr1 −/− genotypes. The age of the mice was chosen to match the age of mice used in Singh et al. (2018) where the differential receptor sensitivity to MK-801 was shown in vitro in Negr1 −/− hippocampal slices. Half of the mice in each genotype group received the NMDA receptor antagonist MK-801, while the remaining animals received physiological solution (saline). This cohort was used to investigate the role of NMDA receptor function in a schizophrenia spectrum disorder model. Cohort 2 Comprised 5-month-old male and female WT and Negr1 −/− mice. Brain tissues and blood plasma were collected for analysis of tryptophan pathway metabolites and glutamate. Cohort 3 Included 7-month-old male and female mice of both genotypes. These mice were handled identically to Cohort 2, though at a different time point. Blood plasma was collected for additional tryptophan pathway metabolites and glutamate analysis. Older mice were used to estimate the dynamics of the biochemical shifts during ageing. All animal procedures were carried out in accordance with the European Communities Directive (2010/63/EU) and approved by the Laboratory Animal Centre at the Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia. The study was conducted under a permit from the Estonian National Board of Animal Experiments (Permit No. 150, 27 September 2019). We confirm this study is reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines as outlined at https://arriveguidelines.org . MK-801 treatment In the dose response experiment, mice received MK-801 (dizocilpine) in three different dosages: 0.1 mg/kg, 0.2 mg/kg and 0.4 mg/kg (Supplementary Fig. S1 ). A concentration of 0.2 mg/kg was chosen for the chronic MK-801 experiment. All participating mice received an intraperitoneal injection. Control mice received a corresponding injection of physiological solution (saline). Open field test Locomotor activity of individual mice was measured with the illumination level of 450 lx for 30 min in soundproof photoelectric motility boxes (44.8 × 44.8 × 45 cm) connected to a computer (TSE, Technical & Scientific Equipment GmbH, Berlin, Germany). The floor of the testing apparatus was cleaned with 70% ethanol and dried thoroughly after each mouse. The system automatically registered the movement of the animal and the time it took to do all the following activities: the distance covered in total, and in corners of the box, the number of rearings, rotations (clockwise + counterclockwise) and corner visits. RT-qPCR Analysis in Mouse Brain Areas Gene expression was determined by two-step RT-qPCR. Total RNA was extracted from each tissue sample by using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. First-strand cDNA was synthesized by using FIREScript® RT cDNA synthesis MIX with Oligo (dT) and Random primers (Solis BioDyne, Tartu, Estonia) according to the manufacturer’s protocol. In qPCR, four NMDA receptor subunit-related genes were studied: glutamate ionotropic receptor NMDA type subunit 1 (GluN1, gene Grin1 ), glutamate ionotropic receptor NMDA type subunit 2a (GluN2A, gene Grin2a ), glutamate ionotropic receptor NMDA type subunit 2b (GluN2B, gene Grin2b ) and serine racemase (Srr). HPRT (hypoxanthine guanine phosphoribosyltransferase) was used as a housekeeper gene. The same primers have been previously described in Varul et al., 2021. Primer sequences can be found in Supplementary Table S6. For qPCR, all reactions were performed in a final volume of 10 µL, using 5 ng of cDNA and HOT FIREPol® EvaGreen® qPCR Supermix (Solis BioDyne). Every reaction was made in four parallel replicates to minimise possible errors. ABI Prism 7900HT Sequence Detection System with ABI Prism 7900 SDS 2.4.2 software (Applied Biosystems) was used for qPCR detection. Data in the Figures is presented on a linear scale, calculated as 2 −ΔCT , where ΔCT is the difference in cycle threshold (CT) between the target genes and the housekeeper gene. Measurement of biomarkers From all the second and third cohorts’ mice’s blood plasma, the levels of 8 different tryptophan pathway metabolites and glutamate were measured using high-performance liquid chromatography-mass spectrometry (Waters Xevo TQ-XS with Acquity H-class UPLC). From the second cohort, the same metabolite levels were also measured in the frontal cortex, hippocampus, hypothalamus and ventral striatum. For quantification 10 µl of plasma or tissue homogenate was mixed with internal standards (D 4 -nicotinic acid, 13 C 10 -kynurenine, D 4 -dopamine) and derivatized with phenylisothiocyanate for 1 h at room temperature. After drying under a stream of nitrogen the samples were extracted with methanol and diluted with water to 50%. Standard curves from known concentrations of commercial compounds were created. In addition to separate measurements, the blood plasma data was also pooled together from the second and third cohort to see more significant differences between the Negr1- deficient mice and the wild-type control mice. Statistical analysis Data are presented as mean values ± standard error of the mean (SEM). Before the analyses, an outlier test was performed on all the data. Log-transformation was used to normalise the data before analysis. Normality of data distribution was assessed using the Shapiro–Wilk test. Brain metabolite levels were analysed using Student’s t -test or the Mann–Whitney U test for non-parametric data. Blood plasma metabolites and qPCR data were evaluated using two-way ANOVA followed by Tukey’s post hoc test. (In the supplementary, one-way ANOVA was used for blood plasma to allow pooling the data.) Statistical analyses for behavioural experiments and metabolite measurements, as well as correlation plot generation, were conducted using R (version 4.3.1). Analysis of qPCR data and generation of all other graphs (excluding correlation plots) were performed using GraphPad Prism (version 10.2.1). Z-scores were calculated when necessary to standardise and compare data across groups. Statistical significance was defined as p < 0.05. Illustrative figures were created using BioRender.com. Declarations Conflict of interests The authors declare no conflict of interest. Funding This research was funded by the investigation grant PRG2544 from the Estonian Research Council (E.V.). Author Contribution Conceptualisation: M-A.P., E.V.; Methodology: C.K., K.M., K.K., M.K., M.J., N.M., G.I., E.L., M-A.P; Analysis: C.K, K.M., K.K., M-A.P.; Writing—original draft preparation: C.K., M-A.P., E.V.; Writing—review and editing: C.K., K.M., K.K., M.K., M.J., N.M., G.I., E.L., M-A.P, E.V.; Prepared figures: C.K, M-A.P. Funding acquisition: M-A.P., E.V. All authors critically revised the manuscript for intellectual content and approved the final version for publication. Data Availability The data that support the findings of this study are available upon reasonable request to the corresponding author. References Rakofsky, J. & Rapaport, M. Mood Disorders. Continuum (Minneap Minn.) 24, 804–827 (2018). World Health Organization. Mental disorders [Fact sheet]. https://www.who.int/news-room/fact-sheets/detail/mental-disorders (2022). Carboni, L. et al. Depression‑Associated Gene Negr1‑Fgfr2 Pathway Is Altered by Antidepressant Treatment. Cells 9, 1818; https://doi.org/10.3390/cells9081818 (2020). Singh, K. et al. Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease‑Associated Negr1 Gene. Front. Mol. Neurosci. 11, 30; https://doi.org/10.3389/fnmol.2018.00030 (2018). Singh, K. et al. Neural cell adhesion molecule Negr1 deficiency in mouse results in structural brain endophenotypes and behavioral deviations related to psychiatric disorders. Sci. Rep. 9, 5457; https://doi.org/10.1038/s41598-019-41849-7 (2019). Vanaveski, T. et al. Promoter‑Specific Expression and Genomic Structure of IgLON Family Genes in Mouse. Front. Neurosci. 11, 38; https://doi.org/10.3389/fnins.2017.00038 (2017). Howard, D. M. et al. Genome‑wide meta‑analysis of depression identifies 102 independent variants and highlights the importance of the prefrontal brain regions. Nat. Neurosci. 22, 343–352 (2019). Kaare, M. et al. Depression‑Associated Negr1 Gene‑Deficiency Induces Alterations in the Monoaminergic Neurotransmission Enhancing Time‑Dependent Sensitization to Amphetamine in Male Mice. Brain Sci. 12, 1696; https://doi.org/10.3390/brainsci12121696 (2022). Karis, K. et al. Altered Expression Profile of IgLON Family of Neural Cell Adhesion Molecules in the Dorsolateral Prefrontal Cortex of Schizophrenic Patients. Front. Mol. Neurosci. 11, 8; https://doi.org/10.3389/fnmol.2018.00008 (2018). Levey, D. F. et al. Bi‑ancestral depression GWAS in the Million Veteran Program and meta‑analysis in > 1.2 million individuals highlight new therapeutic directions. Nat. Neurosci. 24, 954–963 (2021). Noh, K. et al. Negr1 controls adult hippocampal neurogenesis and affective behaviors. Mol. Psychiatry 24, 1189–1205 (2019). Trubetskoy, V. et al. Mapping genomic loci implicates genes and synaptic biology in schizophrenia. Nature 604, 502–508 (2022). Baez, M. V., Cercato, M. C. & Jerusalinsky, D. A. NMDA Receptor Subunits Change after Synaptic Plasticity Induction and Learning and Memory Acquisition. Neural Plast. 2018, 5093048; https://doi.org/10.1155/2018/5093048 (2018). Hunt, D. L. & Castillo, P. E. Synaptic plasticity of NMDA receptors: mechanisms and functional implications. Curr. Opin. Neurobiol. 22, 496–508 (2012). Kemp, J. A. & McKernan, R. M. NMDA receptor pathways as drug targets. Nat. Neurosci. 5, 1039–1042 (2002). Yamamoto, H. et al. Specific Roles of NMDA Receptor Subunits in Mental Disorders. Curr. Mol. Med. 15, 193–205 (2015). Myint, A. M. & Halaris, A. Imbalances in Kynurenines as Potential Biomarkers in the Diagnosis and Treatment of Psychiatric Disorders. Front. Psychiatry 13, 913303; https://doi.org/10.3389/fpsyt.2022.913303 (2022). Yovanno, R. A. et al. Excitatory and inhibitory D‑serine binding to the NMDA receptor. eLife 11, e77645; https://doi.org/10.7554/eLife.77645 (2022). Coyle, J. T. & Balu, D. T. The Role of Serine Racemase in the Pathophysiology of Brain Disorders. Adv. Pharmacol. 82, 35–56 (2018). Labrie, V. et al. Serine racemase is associated with schizophrenia susceptibility in humans and in a mouse model. Hum. Mol. Genet. 18, 3227–3243 (2009). Wolosker, H. et al. Serine racemase: a glial enzyme synthesizing D‑serine to regulate glutamate‑N‑methyl‑D‑aspartate neurotransmission. Proc. Natl Acad. Sci. USA 96, 13409–13414 (1999). Janus, A. et al. MK‑801 and cognitive functions: Investigating the behavioral effects of a non‑competitive NMDA receptor antagonist. Psychopharmacology 240, 2435–2457 (2023). Latysheva, N. V. & Rayevsky, K. S. Chronic neonatal N‑methyl‑D‑aspartate receptor blockade induces learning deficits and transient hypoactivity in young rats. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 787–794 (2003). Wong, E. H. et al. The anticonvulsant MK‑801 is a potent N‑methyl‑D‑aspartate antagonist. Proc. Natl Acad. Sci. USA 83, 7104–7108 (1986). Birch, P. J. et al. Kynurenic acid antagonises responses to NMDA via an action at the strychnine‑insensitive glycine receptor. Eur. J. Pharmacol. 154, 85–87 (1988). Danysz, W. & Parsons, C. G. Glycine and N‑methyl‑D‑aspartate receptors: physiological significance and possible therapeutic applications. Pharmacol. Rev. 50, 597–664 (1998). de Carvalho, L. P. et al. The endogenous agonist quinolinic acid and the non‑endogenous homoquinolinic acid discriminate between NMDAR2 receptor subunits. Neurochem. Int. 28, 445–452 (1996). Lugo‑Huitrón, R. et al. Quinolinic acid: an endogenous neurotoxin with multiple targets. Oxid. Med. Cell Longev. 2013, 104024; https://doi.org/10.1155/2013/104024 (2013). Savitz, J. The kynurenine pathway: a finger in every pie. Mol. Psychiatry 25, 131–147 (2020). Dauvermann, M. R. et al. Glutamatergic regulation of cognition and functional brain connectivity: insights from pharmacological, genetic and translational schizophrenia research. Br. J. Pharmacol. 174, 3136–3160 (2017). Kuuskmäe, C. et al. Kynurenine pathway dynamics in patients with schizophrenia spectrum disorders across the disease trajectory. Psychiatry Res. 328, 115423; https://doi.org/10.1016/j.psychres.2023.115423 (2023). Goto, Y. et al. Functional and dysfunctional synaptic plasticity in prefrontal cortex: roles in psychiatric disorders. Biol. Psychiatry 67, 199–207 (2010). Morris, R. G. et al. Hippocampal synaptic plasticity and NMDA receptors: a role in information storage? Philos. Trans. R. Soc. Lond., B, Biol. Sci. 329, 187–204 (1990). Perkins, M. N. & Stone, T. W. An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid. Brain Res. 247, 184–187 (1982). Deutschenbaur, L. et al. Role of calcium, glutamate and NMDA in major depression and therapeutic application. Prog. Neuropsychopharmacol. Biol. Psychiatry 64, 325–333 (2016). Braidy, N. et al. Mechanism for quinolinic acid cytotoxicity in human astrocytes and neurons. Neurotoxicol. Res. 16, 77–86 (2009). Ostapiuk, A. & Urbanska, E. M. Kynurenic acid in neurodegenerative disorders‑unique neuroprotection or double‑edged sword? CNS Neurosci. Ther. 28, 19–35 (2022). Cross‑Disorder Group of the Psychiatric Genomics Consortium. Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders. Cell 179, 1469–1482.e11; https://doi.org/10.1016/j.cell.2019.11.020 (2019). Cohen, S. M. et al. The impact of NMDA receptor hypofunction on GABAergic neurons in the pathophysiology of schizophrenia. Schizophr. Res. 167, 98–107 (2015). Su, F. et al. Neuronal growth regulator 1 (NEGR1) promotes the synaptic targeting of glutamic acid decarboxylase 65 (GAD65). J. Neurochem. 169, e16279; https://doi.org/10.1111/jnc.16279 (2025). Gandal, M. J. et al. GABAB‑mediated rescue of altered excitatory‑inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR‑hypofunction. Transl. Psychiatry 2, e142; https://doi.org/10.1038/tp.2012.69 (2012). Rosenmund, C. et al. Synaptic NMDA receptor channels have a low open probability. J. Neurosci. 15, 2788–2795 (1995). Hardingham, G. E. et al. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut‑off and cell death pathways. Nat. Neurosci. 5, 405–414 (2002). Liu, Y. et al. NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J. Neurosci. 27, 2846–2857 (2007). Tovar, K. R. & Westbrook, G. L. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J. Neurosci. 19, 4180–4188 (1999). Stanika, R. I. et al. Coupling diverse routes of calcium entry to mitochondrial dysfunction and glutamate excitotoxicity. Proc. Natl Acad. Sci. USA 106, 9854–9859 (2009). Zhou, X. et al. Involvement of the GluN2A and GluN2B subunits in synaptic and extrasynaptic N‑methyl‑D‑aspartate receptor function and neuronal excitotoxicity. J. Biol. Chem. 288, 24151–24159 (2013). Wang, G. et al. GluN2A: A Promising Target for Developing Novel Antidepressants. Int. J. Neuropsychopharmacol. 27, pyae037; https://doi.org/10.1093/ijnp/pyae037 (2024). Brim, B. L. et al. Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor. Behav. Brain Res. 238, 211–226 (2013). Cui, Y. et al. Forebrain NR2B overexpression facilitating the prefrontal cortex long‑term potentiation and enhancing working memory function in mice. PLoS One 6, e20312; https://doi.org/10.1371/journal.pone.0020312 (2011). Sun, Y. Y. et al. Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats. Sci. Rep. 6, 30743; https://doi.org/10.1038/srep30743(2016). Philips, M. A. et al. Myg1‑deficient mice display alterations in stress‑induced responses and reduction of sex‑dependent behavioural differences. Behav. Brain Res. 207, 182–195 (2010). Kaare, M. et al. High‑Fat Diet Induces Pre‑Diabetes and Distinct Sex‑Specific Metabolic Alterations in Negr1‑Deficient Mice. Biomedicines 9, 1148; https://doi.org/10.3390/biomedicines9091148 (2021). Holter, K. M. et al. Use of Quantitative Electroencephalography to Inform Age‑ and Sex‑Related Differences in NMDA Receptor Function Following MK‑801 Administration. Pharmaceuticals 17, 237; https://doi.org/10.3390/ph17020237 (2024). Kniffin, A. R. & Briand, L. A. Sex differences in glutamate transmission and plasticity in reward related regions. Front. Behav. Neurosci. 18, 1455478; https://doi.org/10.3389/fnbeh.2024.1455478 (2024). Neuhäusel, T. S. & Gerevich, Z. Sex‑specific effects of subchronic NMDA receptor antagonist MK‑801 treatment on hippocampal gamma oscillations. Front. Neurosci. 18, 1425323; https://doi.org/10.3389/fnins.2024.1425323 (2024). Lee, A. W. et al. Functional inactivation of the genome‑wide association study obesity gene neuronal growth regulator 1 in mice causes a body mass phenotype. PLoS One 7, e41537; https://doi.org/10.1371/journal.pone.0041537(2012). Varul, J. et al. Dopamine System, NMDA Receptor and EGF Family Expressions in Brain Structures of Bl6 and 129Sv Strains Displaying Different Behavioral Adaptation. Brain Sci. 11, 725; https://doi.org/10.3390/brainsci11060725(2021). Additional Declarations No competing interests reported. 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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-7023014","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":480355274,"identity":"b82d8679-c94f-4153-a5eb-51544f00f948","order_by":0,"name":"Carolin Kuuskmäe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYLCCBwYMDBJAmrEBSPATpSUBWYtkAzMxWhiQtBgcIKCFv/3swwcJBXcYJGfkGH+cmWMnZ3wj/wDDhwrcWiTOpBsbJBg8Y5CWyDGT3Lgt2djsRjID44wzuLUYMKSxSSQYHGaQA2phfLiNOXEbUAszbxseLfzP4FqMPz7cVl+/eQZQy99/eLRIQG0BOswA6LDDCQYSQC3ggMDllxvPmIF+Ocwj2fOsTHLmtuOGM848NjjYcwy3Fv7+NMYHH/4clpM4nrz5Y++2ann+9sSHD37U4NYCAzwovAOENYyCUTAKRsEowAcAHPdN4higsQAAAAAASUVORK5CYII=","orcid":"","institution":"University of Tartu","correspondingAuthor":true,"prefix":"","firstName":"Carolin","middleName":"","lastName":"Kuuskmäe","suffix":""},{"id":480355275,"identity":"bb047114-d411-4f35-b465-cf5089385cf8","order_by":1,"name":"Kaie Mikheim","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Kaie","middleName":"","lastName":"Mikheim","suffix":""},{"id":480355276,"identity":"e11a9598-3e70-4720-98c4-023d5f7ec3eb","order_by":2,"name":"Narges Mohammadrahimi","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Narges","middleName":"","lastName":"Mohammadrahimi","suffix":""},{"id":480355277,"identity":"4a7cefff-53bf-4306-a13e-ebe60ca88858","order_by":3,"name":"Kalle Kilk","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Kalle","middleName":"","lastName":"Kilk","suffix":""},{"id":480355278,"identity":"ea2d7482-ea0a-4b56-b0a2-ab392ad3a663","order_by":4,"name":"Maria Kaare","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Kaare","suffix":""},{"id":480355279,"identity":"82342fd0-9bfb-4eec-9ff9-a8b2d52dc3b9","order_by":5,"name":"Mohan Jayaram","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Mohan","middleName":"","lastName":"Jayaram","suffix":""},{"id":480355280,"identity":"330f2a5d-72b3-44d0-86bc-5b695b1968c4","order_by":6,"name":"German Ilnitski","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"German","middleName":"","lastName":"Ilnitski","suffix":""},{"id":480355281,"identity":"b81d6a4f-691f-46f4-b80c-965ab516c844","order_by":7,"name":"Este Leidmaa","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Este","middleName":"","lastName":"Leidmaa","suffix":""},{"id":480355282,"identity":"0efc80d8-a88f-4d34-a341-6807d1a75a45","order_by":8,"name":"Mari-Anne Philips","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Mari-Anne","middleName":"","lastName":"Philips","suffix":""},{"id":480355283,"identity":"ca6e874f-3baf-4caa-b261-b84c697f5446","order_by":9,"name":"Eero Vasar","email":"","orcid":"","institution":"University of Tartu","correspondingAuthor":false,"prefix":"","firstName":"Eero","middleName":"","lastName":"Vasar","suffix":""}],"badges":[],"createdAt":"2025-07-01 18:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7023014/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7023014/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-35968-7","type":"published","date":"2026-01-16T16:30:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86023620,"identity":"b3209511-627a-4fc5-8ce6-4416cadb5071","added_by":"auto","created_at":"2025-07-04 12:37:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":449426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMK-801 effect on wild-type (WT) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNegr1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-deficient mice’s behaviour in the open field test. \u003c/strong\u003eFigure shows the total distance covered (A, D), distance covered in corners (B, E) and total rotations made (C, F) by both male and female mice until behavioural tolerance developed (after 9 days in males and 5 days in females). Each dot represents the day’s average (males n = 10, females n = 8-16), whiskers show SEM. Main effects, calculated using three-way ANOVA (Tukey HSD test), are depicted as symbols above graphs: * - treatment, # - genotype, \u0026amp; - day, $ - day and treatment interaction, € - genotype and treatment interaction. One symbol - p \u0026lt; 0.05, two symbols - p \u0026lt; 0.01, three symbols - p \u0026lt; 0.001, four symbols p \u0026lt; 0.0001. Exact values can be found in the Supplementary Table S1.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7023014/v1/77dc068a591a08fa27e48799.png"},{"id":86023616,"identity":"f4e8fad5-936b-4053-b01c-7b5eb3383dae","added_by":"auto","created_at":"2025-07-04 12:37:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":903811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNMDA receptor structure and blunted progression of rapid behavioural tolerance during every-second-day MK-801 administration in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNegr1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-deficient mice. (A)\u003c/strong\u003e Schematic representation of the NMDA receptor subunit composition relevant to this study. The receptor consists of GluN1 (encoded by \u003cem\u003eGrin1\u003c/em\u003e), GluN2A (encoded by \u003cem\u003eGrin2a\u003c/em\u003e), and GluN2B (encoded by \u003cem\u003eGrin2b\u003c/em\u003e) subunits (other subunits, such as GluN3A, are known but were not investigated here). The GluN1 subunit binds glycine, D-serine, and kynurenic acid, while GluN2A and GluN2B bind glutamate, NMDA, and quinolinic acid. At high concentrations, D-serine may also bind to GluN2A. MK-801 is a reversible, non-competitive antagonist that blocks the NMDA receptor by binding within its open ion channel. Figure created using BioRender.com.\u003cstrong\u003e (B–H)\u003c/strong\u003e MK-801-induced locomotor activity and stereotypic behaviour showed a consistent reduction every second day during chronic administration. Delta values for days 1–2 (B–C), 3–4 (D–E), and 5–6 (F–H, available for males only) represent the change in activity observed on each alternate day. \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice exhibited smaller reductions in behaviour compared to wild-type (WT) controls, indicating a blunted progression of rapid behavioural tolerance. These genotype-dependent fluctuations suggest altered NMDA receptor sensitivity in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7023014/v1/8edd7d875795198caa52b71d.png"},{"id":86024574,"identity":"ce696590-cfd2-4e37-aedc-8c057d3feaab","added_by":"auto","created_at":"2025-07-04 12:45:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":149103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in NMDA-related gene expression in the frontal cortex and hippocampus of mice. \u003c/strong\u003eThe gene expression of glutamate receptor subunit GluN1 encoded by \u003cem\u003eGrin1 \u003c/em\u003e(A, D), subunit GluN2A encoded by \u003cem\u003eGrin2a\u003c/em\u003e (B, E), and GluN2B encoded by \u003cem\u003eGrin2b\u003c/em\u003e (C, F) are depicted for both male and female mice. There are four groups in each graph: WT mice injected with physiological solution (saline), WT mice injected with MK-801, \u003cem\u003eNegr1\u003c/em\u003e-deficient mice injected with physiological solution and \u003cem\u003eNegr1\u003c/em\u003e-deficient mice injected with MK-801. In the frontal cortex (A-C), statistically significant changes were observed only among female mice with \u003cem\u003eGrin1\u003c/em\u003e and \u003cem\u003eGrin2b \u003c/em\u003egenes showing sex, genotype and treatment effects. In the hippocampus (D-F), statistically significant changes were observed only among male mice with \u003cem\u003eGrin2a\u003c/em\u003e and \u003cem\u003eGrin2b \u003c/em\u003egenes showing sex, genotype, and treatment effects. We also measured the expression of these genes in the ventral striatum (Supplementary Fig. S4) and looked at the \u003cem\u003eSrr\u003c/em\u003e expression (Supplementary Fig. S5), but found no significant differences between groups. In each group n = 8-10. Data represent mean ± SEM, ordinary two-way ANOVA (Tukey HSD test). WT – wild-type. * - p \u0026lt; 0.05, ** - p \u0026lt; 0.01, *** - p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7023014/v1/eb7289e222f22f2c1c50b363.png"},{"id":86023619,"identity":"49983df0-2c34-46bc-94d4-4345c52b107a","added_by":"auto","created_at":"2025-07-04 12:37:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":128965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in the frontal cortex and hippocampus metabolite levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNegr1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-deficient mice (Cohort 2). \u003c/strong\u003eThe figure depicts z-scores of the kynurenic acid (A, E), quinolinic acid (B, F), xanthurenic acid (C, G) and glutamate (D, H) levels in wild-type and \u003cem\u003eNegr1\u003c/em\u003e-deficient male and female mice. Results show significant differences in the xanthurenic acid and glutamate levels between \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and WT male and female mice in the frontal cortex, with the levels being elevated in \u003cem\u003eNegr1-/- \u003c/em\u003emales and diminished in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003efemales. There was also a statistically significant decline in the quinolinic acid levels of \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003efemale mice. No statistically significant differences were observed in the measured metabolite levels between WT and \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice in the hippocampus. Data represents mean ± SEM, unpaired t-test results, n = 12 - 14. WT – wild type. * - p \u0026lt; 0.05, ** - p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7023014/v1/5bf52851db19d92e0a087ad1.png"},{"id":86024575,"identity":"eaccdc77-9979-4941-9736-78001d234d42","added_by":"auto","created_at":"2025-07-04 12:45:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":513591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in the blood plasma metabolite levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNegr1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-deficient mice (Cohorts 2 and 3). \u003c/strong\u003eThe figure depicts z-scores of the kynurenic acid (A, E), quinolinic acid (B, F), xanthurenic acid (C, G) and glutamate (D, H) levels in wild-type and \u003cem\u003eNegr1\u003c/em\u003e-deficient male and female mice. Shown are data from cohort 2 (5-month-olds) and cohort 3 (7-month-olds). There was a significant decrease in the quinolinic acid level among the older \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emale\u003cem\u003e \u003c/em\u003emice and an increase in kynurenic acid level of older \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003efemale\u003cem\u003e \u003c/em\u003emice. Xanthurenic acid and glutamate levels remained approximately the same for both genders. Data represents mean ± SEM, 2-way ANOVA results (Tukey HSD test), n = (6)8 - 14. WT – wild type. * - p \u0026lt; 0.05, ** - p \u0026lt; 0.01, *** - p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7023014/v1/0c672572095828b5b906aeb6.png"},{"id":86023621,"identity":"3dd33a16-f75e-4386-9864-81efa0107d95","added_by":"auto","created_at":"2025-07-04 12:37:29","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":435849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneral description of the study.\u003c/strong\u003eCreated in BioRender. https://BioRender.com/z11r035.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7023014/v1/08e03d953fc546d9f6c01a23.jpeg"},{"id":100614790,"identity":"0382e4be-4b50-4c0f-841f-0aa15f58364d","added_by":"auto","created_at":"2026-01-19 17:25:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3256240,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7023014/v1/7b59f874-0efd-4dcf-b7f6-fc2d0468c5b0.pdf"},{"id":86023622,"identity":"7bba97b9-5a9b-446b-86d1-90d5d995c21d","added_by":"auto","created_at":"2025-07-04 12:37:29","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5165907,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7023014/v1/89bf49621e0aa0611edddcb5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Negr1 Deficiency Alters Glutamate Signalling and Kynurenine Pathway in a Mouse Model of Psychiatric Disorders","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePsychiatric disorders such as anxiety, major depression, bipolar disorder, and schizophrenia affect around 800\u0026nbsp;million people worldwide, often impairing quality of life [1][2]. Genetic factors, including polymorphisms in specific genes, contribute to susceptibility. One such gene is neuronal growth regulator (\u003cem\u003eNEGR1)\u003c/em\u003e. This gene encodes for a cell adhesion molecule involved in neural development, synapse formation, and plasticity [3][4][5][6]. Genome-wide association studies (GWAS) have identified \u003cem\u003eNEGR1\u003c/em\u003e as a risk gene for several psychiatric and neurodevelopmental disorders [7][8][9][10][11][12]. However, the mechanisms through which NEGR1 influences behaviour and neurotransmitter systems remain poorly understood.\u003c/p\u003e \u003cp\u003ePrevious findings have shown that MK-801 (dizocilpine) binding density at NMDA receptors is higher in hippocampal sections of \u003cem\u003eNegr1\u003c/em\u003e-deficient mice compared to wild-type (WT) controls, suggesting increased N-methyl-D-aspartate (NMDA) receptor availability in the \u003cem\u003eNegr1\u003c/em\u003e-deficient brain [4]. Given the central role of NMDA receptors in synaptic plasticity, learning, and memory [13][14][15], the glutamatergic system emerges as a potential pathway linking \u003cem\u003eNegr1\u003c/em\u003e to psychiatric phenotypes. The NMDA receptor is composed of multiple subunits (e.g., GluN1, GluN2A, GluN2B), and changes in their expression have been associated with cognitive and emotional dysregulation [16][17]. Notably, GluN1 and GluN2A subunits serve as binding sites for D-serine, a molecule that can act as either a co-agonist or antagonist depending on the site [18].\u003c/p\u003e \u003cp\u003eD-serine levels are regulated by serine racemase (Srr), an enzyme critical for NMDA receptor function, and disruptions in Srr activity have been implicated in schizophrenia spectrum disorders [19][20][21]. Dysregulation of NMDA receptor subunits and Srr activity may therefore provide a mechanistic link between \u003cem\u003eNegr1\u003c/em\u003e deficiency and the behavioural abnormalities observed in psychiatric conditions.\u003c/p\u003e \u003cp\u003eOur previous work demonstrated that \u003cem\u003eNegr1\u003c/em\u003e-deficient mice exhibit heightened behavioural sensitivity to amphetamine, including exaggerated motor and stereotypic responses, along with altered expression of dopaminergic markers [8]. These findings suggest that Negr1 influences dopaminergic reactivity and behavioural sensitisation. Building on prior \u003cem\u003ein vitro\u003c/em\u003e findings of increased MK-801 binding to NMDA receptors in \u003cem\u003eNegr1\u003c/em\u003e-deficient brain tissue, the present study investigates how MK-801, a non-competitive NMDA receptor antagonist known to mimic glutamatergic dysfunction and interfere with sensitisation processes [22][23][24], affects behaviour and molecular markers in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice.\u003c/p\u003e \u003cp\u003eIn addition to direct glutamatergic modulation, we considered the role of the kynurenine pathway (KP), which metabolises tryptophan into neuroactive compounds such as kynurenic acid (KYNA) and quinolinic acid (QUIN). KYNA acts as an NMDA receptor antagonist at GluN1 subunits, while QUIN acts as an agonist at GluN2A and GluN2B subunits [17][25][26][27][28][29]. Imbalances in these metabolites have been associated with psychiatric and neurodegenerative disorders [17][30][31], suggesting that KP dysregulation may influence NMDA receptor function and excitatory signalling.\u003c/p\u003e \u003cp\u003eDespite growing evidence implicating the KP in neuropsychiatric conditions, the relationships between Negr1, NMDA receptor signalling, KP metabolites, and glutamate levels remain poorly defined.\u003c/p\u003e \u003cp\u003eThus, the present study aims to elucidate how \u003cem\u003eNegr1\u003c/em\u003e deficiency affects behaviour and its underlying molecular mechanisms, focusing specifically on glutamatergic signalling and kynurenine pathway metabolism. Using a \u003cem\u003eNegr1-\u003c/em\u003edeficient mouse model, we examined the expression of key NMDA receptor subunits (GluN1, GluN2A, GluN2B) and serine racemase (Srr) in the hippocampus and frontal cortex\u0026mdash;regions crucial for learning, memory, and higher cognitive functions that depend on NMDA receptor-mediated plasticity [13][32][33]. Additionally, we measured kynurenine pathway metabolites and glutamate levels, both known modulators of NMDA receptor activity [34][35] and implicated in neuropsychiatric disease [35][36][37]. To evaluate behavioural and molecular sensitivity to glutamatergic disruption, we assessed responses to repeated MK-801 administration. Finally, we investigated sex differences in these outcomes to determine whether Negr1-related effects differ between male and female mice. By linking behavioural phenotypes with glutamatergic and metabolic alterations, this study provides new insights into the neurobiological mechanisms underlying psychiatric disorders associated with NEGR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of Repeated Treatment with MK-801 (0.2 mg/kg) on Locomotor Activity in Male Wild-Type and\u003c/b\u003e \u003cb\u003eNegr1\u003c/b\u003e\u003cb\u003e-Deficient Mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the dose\u0026ndash;response experiments performed in male mice, the optimal dose for behavioural activation was determined to be 0.2 mg/kg (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C). Acute administration of MK-801 at this dose produced a significantly stronger motor activity response in \u003cem\u003eNegr1\u003c/em\u003e-deficient (\u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice compared to wild-type controls (total distance covered - p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; distance covered in corners - p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interestingly, this enhanced response was not observed during the first day of testing in the repeated administration experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). Notably, the same cohort of mice used for the dose-response curve \u0026ndash; following a one-week washout period \u0026ndash; was also used for the repeated administration protocol. As a result, these mice were not completely drug-naive at the start of the repeated treatment.\u003c/p\u003e \u003cp\u003eWe hypothesised that the heightened acute response to MK-801 is specific to drug-naive \u003cem\u003eNegr1\u003c/em\u003e-deficient mice. To test this, the acute administration experiment was repeated in an independent cohort of drug-naive male mice. Consistent with our hypothesis, \u003cem\u003eNegr1\u003c/em\u003e-deficient mice in this new cohort again showed a stronger motor activity response, as measured by the total distance covered (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eInterestingly, during repeated MK-801 administration in males, \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited a blunted behavioural response over time, suggesting altered sensitivity or tolerance development. Namely, repeated administration of MK-801 elicited distinct locomotor activity patterns in male mice across treatment days, highlighting both genotype-dependent and temporal effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Fig. S2-S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDay 1\u003c/strong\u003e \u003cp\u003eMK-801 significantly increased total distance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both WT and \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice), distance in corners (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for WT; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice), and rotations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDay 2\u003c/strong\u003e \u003cp\u003eResponse diminished; distance still increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both), but only \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed increased rotations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and WT showed increased corner activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDay 3\u003c/strong\u003e \u003cp\u003eA strong stimulatory effect re-emerged. MK-801 increased total distance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001 WT; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e), rotations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 WT; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e), and corner distance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 WT; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDay 4\u003c/strong\u003e \u003cp\u003eEffects waned. Small but significant increases in distance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 WT; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) and corner activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for both). Rotations increased only in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDay 5\u003c/b\u003e: WT mice showed peak activity \u0026mdash; distance (p\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026times;10⁻⁷), rotations (p\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026times;10⁻⁶), and corner distance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed no such increase, which resulted in significant genotype differences in all parameters (distance: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; corners: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; rotations: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDay 6\u003c/strong\u003e \u003cp\u003eMK-801 effects declined markedly in WT and even more in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, nearing Day 2 and 4 levels.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDays 7\u0026ndash;9\u003c/strong\u003e \u003cp\u003eGradual attenuation continued. By Day 9, activity in both genotypes had dropped significantly from peak levels (Days 3 and 5).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Repeated Treatment with MK-801 (0.2 mg/kg) on Locomotor Activity in Female Wild-Type and\u003c/b\u003e \u003cb\u003eNegr1\u003c/b\u003e\u003cb\u003e-Deficient Mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFemale mice displayed a distinct locomotor response profile compared to males, characterised by rapid attenuation of MK-801\u0026rsquo;s effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Fig. S2-S3), but no genotype effect was present.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDay 1\u003c/strong\u003e \u003cp\u003eMK-801 significantly increased distance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both genotypes), rotations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and corner distance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDay 2\u003c/b\u003e: Reduced response. Significant increases only in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (distance: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; rotations: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; corners: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDay 3\u003c/strong\u003e \u003cp\u003ePartial response. Distance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both), corners (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for both); only \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e showed increased rotations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDays 4 and 5\u003c/strong\u003e \u003cp\u003eMK-801 effects are nearly absent. All parameters declined to saline-control levels, indicating tolerance development and leading to discontinuation of treatment in females.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSex Differences in Response to MK-801\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAdministering MK-801 resulted in both rapid and general behavioural tolerance, which was measured daily as motor activity in the open field. The drug\u0026rsquo;s effect diminished every second day in both sexes. General tolerance became evident on day 9 in males and on day 5 in females, leading to sex-specific treatment durations. Notably, the genotype effect was present only in male but not in female mice.\u003c/p\u003e \u003cp\u003eThe effects of MK-801 on locomotor activity revealed significant gender-dependent differences, particularly with repeated administrations. These differences became most pronounced by Day 5, prompting a comparative analysis of Days 1 and 5.\u003c/p\u003e \u003cp\u003eDistance covered: On Day 5, wild-type (WT) males showed a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) while \u003cem\u003eNegr1\u003c/em\u003e-deficient males did not. In females, MK-801's effect decreased significantly by Day 5 in both WT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Female mice also showed significantly lower responses compared to their male counterparts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for WT; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eRotations: WT males showed increased rotations on Day 5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); no change occurred in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e males. In females, MK-801 reduced rotations by Day 5 in WT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) mice. Female mice also exhibited significantly fewer rotations than males on Day 5 (p\u0026thinsp;\u0026lt;\u0026thinsp;1*10\u0026thinsp;\u0026minus;\u0026thinsp;5 for WT; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eDistance covered in corners: WT males had increased corner activity on Day 5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e males, again, showed no change. In females, MK-801 reduced corner distance in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and to a lesser extent in WT (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). WT females had a significantly lower response than males (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); no sex difference was found in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in NMDA-Related Gene Expression Due to Repeated MK-801 Treatment\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFrontal cortex\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn male mice, NMDA-related gene expression tended to be lower than in female littermates. However, MK-801 treatment did not cause significant alterations in gene expression in male mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003eIn the frontal cortex of female mice, the expression of the \u003cem\u003eGrin2a\u003c/em\u003e gene was unaffected by MK-801 administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). For \u003cem\u003eGrin1\u003c/em\u003e, a significant treatment effect was observed (F₁,₃₃ = 13.12, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Post hoc analysis (Tukey HSD test) revealed a significant reduction in \u003cem\u003eGrin1\u003c/em\u003e expression in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but not in wild-type animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eGrin2b\u003c/em\u003e, significant effects of genotype (F₁,₃₁ = 6.06, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and treatment (F₁,₃₁ = 31.16, p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001) were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Post hoc analysis showed a significant reduction in \u003cem\u003eGrin2b\u003c/em\u003e expression in wild-type (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and \u003cem\u003eNegr1\u003c/em\u003e-deficient mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eSrr\u003c/em\u003e, MK-801 treatment had a significant effect (F₁,₃₃ = 6.89, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but post hoc analysis did not reveal specific group differences (Supplementary Fig. S5).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHippocampus\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn female mice, MK-801 treatment did not result in significant changes in NMDA-related gene expression in the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003eIn male mice, a significant change was observed for \u003cem\u003eGrin2a\u003c/em\u003e expression, with a treatment effect (F₁,₃₃ = 6.08, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and genotype \u0026times; treatment interaction (F₁,₃₃ = 7.12, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Post hoc analysis showed a significant increase in \u003cem\u003eGrin2a\u003c/em\u003e expression in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice who were given physiological solution compared to the mice who received MK-801 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and wild-type mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Regarding \u003cem\u003eGrin2b\u003c/em\u003e expression, a significant change was seen with a genotype \u0026times; treatment interaction (F₁,₃₃ = 4.56, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The levels of \u003cem\u003eGrin2b\u003c/em\u003e expression were increased in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) who were given physiological solution compared to the mice who received MK-801 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrelational Analysis of Kynurenine Pathway Metabolites, Tryptophan and Glutamate Across Brain Regions and Blood Plasma\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the correlation analysis, we compared WT and \u003cem\u003eNegr1\u003c/em\u003e-deficient mice, both male and female. Analysis included seven metabolites, which we determined most relevant to this paper\u0026rsquo;s topic: tryptophan, kynurenine, kynurenic acid, quinolinic acid, picolinic acid, xanthurenic acid and glutamate. The data was gathered from blood plasma and four brain regions: frontal cortex, hippocampus, hypothalamus and ventral striatum (Supplementary Fig. S6 and Fig. S7, Supplementary tables S2-S5).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSimilarities between groups\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAnalysis revealed that xanthurenic acid in hippocampus significantly correlated with quinolinic acid in hippocampus in both female \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and female wild-type groups, demonstrating a consistent positive association across these conditions. Additionally, xanthurenic acid in blood plasma displayed multiple significant correlations with various metabolites in male wild-type and male \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e groups. These associations encompassed both positive and negative directions.\u003c/p\u003e \u003cp\u003eAnother notable relationship was observed between glutamate and xanthurenic acid in the hippocampus in both male and female \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e groups, suggesting a mutation-specific link between glutamate and xanthurenic acid metabolism. Moreover, glutamate exhibited significant correlations with xanthurenic acid in plasma in male \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice and female wild-type controls. Interestingly, the directionality of these correlations diverged: a positive correlation in male \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice contrasted with a negative one in female wild-type mice.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferences between groups\u003c/em\u003e \u003c/p\u003e \u003cp\u003eDistinct patterns emerged when comparing male and female groups. Male mice exhibited a higher number of significant correlations involving xanthurenic acid in plasma, while female mice showed a greater emphasis on xanthurenic acid correlations in the hippocampus. Notably, female \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice demonstrated particularly strong associations, such as between quinolinic acid and xanthurenic acid in the frontal cortex (r\u0026thinsp;=\u0026thinsp;0.86, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eWhen comparing \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and wild-type mice groups, several differentiating features became apparent. Male WT mice showed classic tryptophan and kynurenine pathway correlations, whereas \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e groups displayed stronger associations involving glutamate and quinolinic acid. Furthermore, female \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice exhibited more robust and pronounced correlation values than their wild-type counterparts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in the Kynurenine Pathway and Glutamate Levels of\u003c/b\u003e \u003cb\u003eNegr1\u003c/b\u003e\u003cb\u003e-Deficient Mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs a result of the correlation analysis, we focused on kynurenic acid, quinolinic acid, xanthurenic acid and glutamate in the frontal cortex, hippocampus and blood plasma (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We also looked at the levels of these metabolites in hypothalamus and ventral striatum (Supplementary Fig. S8 - Fig. S17).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere was no statistically significant difference in the kynurenic acid and quinolinic acid levels between WT and \u003cem\u003eNegr1\u003c/em\u003e-deficient male mice in the frontal cortex, although there seemed to be a trend for an increase among mutant mice compared to the WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). The xanthurenic acid (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and glutamate levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), however, were significantly increased in the \u003cem\u003eNegr1\u003c/em\u003e-deficient male mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003eThe level of kynurenic acid remained unchanged for the female mice in the frontal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), but contrary to the male mice, the levels of quinolinic acid (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), xanthurenic acid (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and glutamate (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were considerably reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eThe levels of measured metabolites in the hippocampus of \u003cem\u003eNegr1\u003c/em\u003e-deficient male and female mice were not significantly altered compared to the wild-type mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe blood plasma analysis included two cohorts to estimate the dynamics of the biochemical shifts during ageing: cohort 2 consisted of 5-month-old mice, and cohort 3 of 7-month-old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Our results indicate that older mice were more strongly influenced by genotype. Specifically, male \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed a significant decrease in quinolinic acid levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and female \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice exhibited a significant increase in kynurenic acid levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) compared to age-matched wild-type controls (5-month-olds). The reduction in quinolinic acid levels in male mice remained significant when the two age groups were combined (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas the increase in kynurenic acid in females did not (Supplementary Fig. S14). Xanthurenic acid and glutamate levels remained relatively unchanged across sex, age, and genotype groups.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study is the first to demonstrate a link between \u003cem\u003eNegr1\u003c/em\u003e, NMDA receptor function, and kynurenine pathway metabolites, resulting in significant behavioural alterations. While \u003cem\u003eNEGR1\u003c/em\u003e has been associated with various psychiatric disorders [38], we extended this research using MK-801, a non-competitive NMDA receptor antagonist, to model glutamatergic imbalances observed in neuropsychiatric and neurodegenerative conditions [22][23][24].\u003c/p\u003e \u003cp\u003eBehavioural analyses revealed significant differences between saline- and MK-801-treated mice. Acute MK-801 administration elicited a heightened motor response in drug-naive \u003cem\u003eNegr1\u003c/em\u003e-deficient males compared to wild-type controls. However, with repeated exposure, \u003cem\u003eNegr1\u003c/em\u003e-deficient males displayed a blunted response, indicating altered NMDA receptor sensitivity or tolerance development. The most pronounced changes were observed in total distance covered, distance covered in corners, and rotational behaviour. MK-801\u0026ndash;induced hyperlocomotion is attributed to its action on GABAergic interneurons; NMDA receptor blockade reduces inhibitory tone and indirectly enhances excitatory output [39]. The exaggerated initial response in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice may reflect a baseline reduction in GABAergic tone [5][40], amplifying the disinhibitory effects of MK-801, consistent with prior findings of disrupted excitatory and inhibitory balance in models of psychiatric disease [41].\u003c/p\u003e \u003cp\u003eAn unexpected zig-zag pattern in behavioural responsiveness emerged, marked by reduced activity every other day, suggesting the rapid development of behavioural tolerance to daily MK-801 administration. The underlying mechanism remains unclear but may involve residual drug accumulation due to MK-801\u0026rsquo;s long half-life [24] or transient NMDA receptor desensitisation [42]. Repeated exposure could trigger rapid yet reversible neuroadaptive processes, such as receptor upregulation or alterations in downstream signalling [14]. After a brief recovery period, receptor sensitivity may reset, restoring responsiveness. Although this pattern was evident in both genotypes, \u003cem\u003eNegr1\u003c/em\u003e-deficient mice showed a stronger progression of tolerance, indicating altered NMDA receptor sensitivity.\u003c/p\u003e \u003cp\u003eIn addition, \u003cem\u003eNegr1\u003c/em\u003e-deficient male mice exhibited a stronger acute response to MK-801 but developed tolerance more rapidly with repeated dosing. Behavioural suppression \u0026mdash; seen as reduced locomotion and stereotypy \u0026mdash; diminished more quickly in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice compared to wild-type controls, particularly across treatment intervals (delta days 1\u0026ndash;2, 3\u0026ndash;4, and 5\u0026ndash;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and supplementary Fig. S3).\u003c/p\u003e \u003cp\u003eThese findings suggest that \u003cem\u003eNegr1\u003c/em\u003e deficiency alters NMDA receptor function or regulation, potentially due to increased receptor availability [4]. Elevated baseline NMDA receptor density may heighten initial MK-801 sensitivity while accelerating desensitization or downstream adaptations during repeated exposure. Overall, the data indicate dysregulated NMDA receptor dynamics in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice, influencing both acute responsiveness and the trajectory of tolerance development.\u003c/p\u003e \u003cp\u003eAt the molecular level, our data indicate a complex, sex- and region-specific modulation of NMDA receptor subunit expression. Previous studies have shown that receptors with a higher GluN2B-to-GluN2A (\u003cem\u003eGrin2b\u003c/em\u003e-to-\u003cem\u003eGrin2a\u003c/em\u003e) ratio are more susceptible to quinolinic acid\u0026ndash;induced neurotoxicity due to their predominant expression in immature neurons and extrasynaptic sites, where they can promote excitotoxicity [43][44][45]. In the present study, a similar pattern appeared in the frontal cortex of adult female mice but was not observed in the hippocampus or in male mice. However, some studies have reported contrasting findings \u0026mdash; highlighting a critical role for GluN2B in intracellular signalling and excitotoxicity and suggesting that both GluN2A and GluN2B subunits contribute equally to extrasynaptic signalling [46][47]. Furthermore, we found that female \u003cem\u003eNegr1\u003c/em\u003e-deficient mice treated with MK-801 exhibited reduced expression of GluN1 (\u003cem\u003eGrin1\u003c/em\u003e) in the frontal cortex. Together, these findings suggest a sex- and brain region-specific interaction between \u003cem\u003eNegr1\u003c/em\u003e deficiency and NMDA receptor regulation\u003c/p\u003e \u003cp\u003eIn male mice, expression levels of NMDA receptor subunit genes in the frontal cortex did not differ significantly between genotypes. In contrast, in the hippocampus, \u003cem\u003eGrin2a\u003c/em\u003e and \u003cem\u003eGrin2b\u003c/em\u003e were significantly upregulated in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice treated with physiological solution compared to wild-type controls. This finding aligns with earlier evidence of increased NMDA receptor binding density in the hippocampus of \u003cem\u003eNegr1\u003c/em\u003e-deficient animals [4], suggesting elevated baseline receptor availability in this brain region under non-challenged conditions. Interestingly, MK-801 administration normalised the expression of these subunits to levels comparable with wild-type controls. This pattern may reflect a compensatory mechanism, wherein \u003cem\u003eNegr1\u003c/em\u003e-deficient mice upregulate NMDA receptor subunits to counterbalance impaired receptor function or altered inhibitory signalling. Alternatively, increased expression could serve to maintain excitatory-inhibitory homeostasis in the context of disrupted GABAergic tone. MK-801 treatment may override this compensatory adaptation by saturating receptor activity and externally shifting the excitatory-inhibitory balance. However, previous studies have shown that overexpression of \u003cem\u003eGrin2a\u003c/em\u003e and \u003cem\u003eGrin2b\u003c/em\u003e can exacerbate neuronal vulnerability [47], and GluN2A overexpression has been associated with impaired synaptic structure and function [48]. In contrast, GluN2B overexpression has been linked to improved learning and memory [49][50][51]. These contrasting outcomes highlight the complexity of NMDA receptor regulation and emphasise the need for further research to determine whether such subunit overexpression is neuroprotective or detrimental in the context of \u003cem\u003eNegr1\u003c/em\u003e deficiency. Although gene expression was assessed after behavioural adaptation to MK-801, this reflects a typical compromise in longitudinal study designs. Future studies could build on these findings by targeting more specific time points \u0026mdash; such as day 5 in males and day 3 in females \u0026mdash; when behavioural phenotypes diverge most clearly. These adjustments would help to refine the temporal resolution of gene expression dynamics and strengthen causal interpretations.\u003c/p\u003e \u003cp\u003eOne of the most notable findings of this study was the emergence of clear sex differences, underscoring the importance of including both male and female animals in neurobiological research [52][53]. Previous studies have reported sex-specific differences in NMDA receptor function and responses to NMDA receptor antagonists [54][55][56]. Our results extend these observations by showing that sex differences in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice are evident not only in behaviour but also in kynurenine pathway metabolites and glutamate levels. Over the course of five days, wild-type males displayed a progressive increase in locomotor activity following repeated MK-801 administration, indicative of sensitisation. In contrast, \u003cem\u003eNegr1\u003c/em\u003e-deficient males showed minimal behavioural change, suggesting altered receptor responsiveness or adaptation. Female mice, regardless of genotype, exhibited more rapid tolerance and sensitisation to MK-801, reflected by a decline in locomotor activity over time. These findings highlight a dynamic interplay between sex, genotype, and NMDA receptor function and point to sex-specific mechanisms of behavioural plasticity in response to glutamatergic disruption.\u003c/p\u003e \u003cp\u003eAlthough we anticipated that kynurenic acid (KYNA) and quinolinic acid (QUIN) levels would directly influence NMDA receptor function in \u003cem\u003eNegr1\u003c/em\u003e-deficient mice, our findings suggest a more nuanced relationship. While levels of kynurenine pathway metabolites were altered in the \u003cem\u003eNegr1\u003c/em\u003e-deficient group, these changes did not appear to drive NMDA receptor-related behavioural outcomes directly. This may indicate that NMDA receptor function was maintained through compensatory mechanisms involving other co-agonists or modulatory systems. In addition, correlation analyses revealed that kynurenine pathway metabolite profiles were region-specific. The frontal cortex was the most affected by \u003cem\u003eNegr1\u003c/em\u003e deficiency, whereas other brain regions exhibited few significant changes (Supplementary Fig. S6\u0026ndash;S7; Supplementary Table S2\u0026ndash;S5; Supplementary Fig. S8\u0026ndash;S17). These findings emphasise the importance of spatial context when studying neuroimmune-metabolic interactions and suggest that the impact of \u003cem\u003eNegr1\u003c/em\u003e on kynurenine metabolism may be anatomically selective. Furthermore, the effects of \u003cem\u003eNegr1\u003c/em\u003e deficiency became more pronounced with age, with older mice showing stronger genotype-related shifts in kynurenine pathway metabolites. This suggests that ageing may exacerbate or unmask metabolic consequences of \u003cem\u003eNegr1\u003c/em\u003e deficiency.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that \u003cem\u003eNegr1\u003c/em\u003e deficiency leads to pronounced, sex-specific alterations in glutamatergic signalling, behavioural responses to NMDA receptor antagonism, and kynurenine pathway metabolism. These effects were both brain region– and sex–dependent, underscoring the importance of considering biological sex and genetic background when modelling neuropsychiatric disorders. Our findings suggest that \u003cem\u003eNegr1\u003c/em\u003e influences NMDA receptor availability and dynamics, contributing to altered sensitivity and tolerance to glutamatergic disruption. Moreover, the observed region-specific changes in kynurenine metabolites highlight a possible link between neuroimmune metabolism and glutamatergic function in the \u003cem\u003eNegr1\u003c/em\u003e-deficient brain. Taken together, these results provide novel insights into the neurobiological mechanisms associated with \u003cem\u003eNegr1\u003c/em\u003e and support its relevance as a molecular node connecting genetic risk, glutamate dysregulation, and sex-dependent vulnerability in psychiatric disorders. Targeting \u003cem\u003eNegr1\u003c/em\u003e-related pathways may open new avenues for understanding and eventually mitigating glutamate-related dysfunction in mental illness.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e \u003cem\u003eAnimals\u003c/em\u003e \u003c/p\u003e\u003cp\u003eAdult male and female wild-type (WT) mice and their homozygous \u003cem\u003eNegr1\u003c/em\u003e-deficient littermates (\u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e), previously generated and described by Lee et al. (2012), were used in this study. All mice were on an F2 hybrid background: ((129S5/SvEvBrd × C57BL/6N) × (129S5/SvEvBrd × C57BL/6N)). Animals were group-housed (10 per cage) in standard laboratory cages (42.5 × 26.6 × 15.5 cm) under controlled environmental conditions (22 ± 1°C; 12:12 h light/dark cycle, with lights off at 19:00). Each cage contained a 2 cm layer of aspen bedding and 0.5 L of aspen nesting material (Tapvei, Paekna, Estonia), which were changed weekly. Food pellets (R70, Lactamin AB, Kimstad, Sweden) and water were provided \u003cem\u003ead libitum\u003c/em\u003e. Breeding and maintenance were carried out at the animal facility of the Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia.\u003c/p\u003e\u003cp\u003eAll behavioural testing was conducted between 8:00 a.m. and 5:00 p.m. Prior to testing, mice were kept in group housing conditions to minimise stress.\u003c/p\u003e\u003cp\u003eThree separate mouse cohorts were used in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e):\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eCohort 1\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eIncluded 2-month-old male and female mice, with equal representation of WT and \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e genotypes. The age of the mice was chosen to match the age of mice used in Singh et al. (2018) where the differential receptor sensitivity to MK-801 was shown in vitro in \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e hippocampal slices. Half of the mice in each genotype group received the NMDA receptor antagonist MK-801, while the remaining animals received physiological solution (saline). This cohort was used to investigate the role of NMDA receptor function in a schizophrenia spectrum disorder model.\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eCohort 2\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eComprised 5-month-old male and female WT and \u003cem\u003eNegr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e mice. Brain tissues and blood plasma were collected for analysis of tryptophan pathway metabolites and glutamate.\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eCohort 3\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eIncluded 7-month-old male and female mice of both genotypes. These mice were handled identically to Cohort 2, though at a different time point. Blood plasma was collected for additional tryptophan pathway metabolites and glutamate analysis. Older mice were used to estimate the dynamics of the biochemical shifts during ageing.\u003c/p\u003e\u003cp\u003e All animal procedures were carried out in accordance with the European Communities Directive (2010/63/EU) and approved by the Laboratory Animal Centre at the Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia. The study was conducted under a permit from the Estonian National Board of Animal Experiments (Permit No. 150, 27 September 2019). We confirm this study is reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines as outlined at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e \u003cem\u003eMK-801 treatment\u003c/em\u003e \u003c/p\u003e\u003cp\u003eIn the dose response experiment, mice received MK-801 (dizocilpine) in three different dosages: 0.1 mg/kg, 0.2 mg/kg and 0.4 mg/kg (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A concentration of 0.2 mg/kg was chosen for the chronic MK-801 experiment. All participating mice received an intraperitoneal injection. Control mice received a corresponding injection of physiological solution (saline).\u003c/p\u003e\u003cp\u003e \u003cem\u003eOpen field test\u003c/em\u003e \u003c/p\u003e\u003cp\u003eLocomotor activity of individual mice was measured with the illumination level of 450 lx for 30 min in soundproof photoelectric motility boxes (44.8 × 44.8 × 45 cm) connected to a computer (TSE, Technical \u0026amp; Scientific Equipment GmbH, Berlin, Germany). The floor of the testing apparatus was cleaned with 70% ethanol and dried thoroughly after each mouse. The system automatically registered the movement of the animal and the time it took to do all the following activities: the distance covered in total, and in corners of the box, the number of rearings, rotations (clockwise + counterclockwise) and corner visits.\u003c/p\u003e\u003cp\u003e \u003cem\u003eRT-qPCR Analysis in Mouse Brain Areas\u003c/em\u003e \u003c/p\u003e\u003cp\u003eGene expression was determined by two-step RT-qPCR. Total RNA was extracted from each tissue sample by using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. First-strand cDNA was synthesized by using FIREScript® RT cDNA synthesis MIX with Oligo (dT) and Random primers (Solis BioDyne, Tartu, Estonia) according to the manufacturer’s protocol.\u003c/p\u003e\u003cp\u003eIn qPCR, four NMDA receptor subunit-related genes were studied: glutamate ionotropic receptor NMDA type subunit 1 (GluN1, gene \u003cem\u003eGrin1\u003c/em\u003e), glutamate ionotropic receptor NMDA type subunit 2a (GluN2A, gene \u003cem\u003eGrin2a\u003c/em\u003e), glutamate ionotropic receptor NMDA type subunit 2b (GluN2B, gene \u003cem\u003eGrin2b\u003c/em\u003e) and serine racemase (Srr). \u003cem\u003eHPRT\u003c/em\u003e (hypoxanthine guanine phosphoribosyltransferase) was used as a housekeeper gene. The same primers have been previously described in Varul et al., 2021. Primer sequences can be found in Supplementary Table S6. For qPCR, all reactions were performed in a final volume of 10 µL, using 5 ng of cDNA and HOT FIREPol® EvaGreen® qPCR Supermix (Solis BioDyne). Every reaction was made in four parallel replicates to minimise possible errors. ABI Prism 7900HT Sequence Detection System with ABI Prism 7900 SDS 2.4.2 software (Applied Biosystems) was used for qPCR detection. Data in the Figures is presented on a linear scale, calculated as 2\u003csup\u003e−ΔCT\u003c/sup\u003e, where ΔCT is the difference in cycle threshold (CT) between the target genes and the housekeeper gene.\u003c/p\u003e\u003cp\u003e \u003cem\u003eMeasurement of biomarkers\u003c/em\u003e \u003c/p\u003e\u003cp\u003eFrom all the second and third cohorts’ mice’s blood plasma, the levels of 8 different tryptophan pathway metabolites and glutamate were measured using high-performance liquid chromatography-mass spectrometry (Waters Xevo TQ-XS with Acquity H-class UPLC). From the second cohort, the same metabolite levels were also measured in the frontal cortex, hippocampus, hypothalamus and ventral striatum.\u003c/p\u003e\u003cp\u003eFor quantification 10 µl of plasma or tissue homogenate was mixed with internal standards (D\u003csub\u003e4\u003c/sub\u003e-nicotinic acid, \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e10\u003c/sub\u003e-kynurenine, D\u003csub\u003e4\u003c/sub\u003e-dopamine) and derivatized with phenylisothiocyanate for 1 h at room temperature. After drying under a stream of nitrogen the samples were extracted with methanol and diluted with water to 50%. Standard curves from known concentrations of commercial compounds were created. In addition to separate measurements, the blood plasma data was also pooled together from the second and third cohort to see more significant differences between the \u003cem\u003eNegr1-\u003c/em\u003edeficient mice and the wild-type control mice.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean values ± standard error of the mean (SEM). Before the analyses, an outlier test was performed on all the data. Log-transformation was used to normalise the data before analysis. Normality of data distribution was assessed using the Shapiro–Wilk test. Brain metabolite levels were analysed using Student’s \u003cem\u003et\u003c/em\u003e-test or the Mann–Whitney \u003cem\u003eU\u003c/em\u003e test for non-parametric data. Blood plasma metabolites and qPCR data were evaluated using two-way ANOVA followed by Tukey’s post hoc test. (In the supplementary, one-way ANOVA was used for blood plasma to allow pooling the data.)\u003c/p\u003e\u003cp\u003eStatistical analyses for behavioural experiments and metabolite measurements, as well as correlation plot generation, were conducted using R (version 4.3.1). Analysis of qPCR data and generation of all other graphs (excluding correlation plots) were performed using GraphPad Prism (version 10.2.1). Z-scores were calculated when necessary to standardise and compare data across groups. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. Illustrative figures were created using BioRender.com.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the investigation grant PRG2544 from the Estonian Research Council (E.V.).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualisation: M-A.P., E.V.; Methodology: C.K., K.M., K.K., M.K., M.J., N.M., G.I., E.L., M-A.P; Analysis: C.K, K.M., K.K., M-A.P.; Writing\u0026mdash;original draft preparation: C.K., M-A.P., E.V.; Writing\u0026mdash;review and editing: C.K., K.M., K.K., M.K., M.J., N.M., G.I., E.L., M-A.P, E.V.; Prepared figures: C.K, M-A.P. Funding acquisition: M-A.P., E.V. All authors critically revised the manuscript for intellectual content and approved the final version for publication.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available upon reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRakofsky, J. \u0026amp; Rapaport, M. Mood Disorders. \u003cem\u003eContinuum (Minneap Minn.)\u003c/em\u003e 24, 804\u0026ndash;827 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization. Mental disorders [Fact sheet]. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://www.who.int/news-room/fact-sheets/detail/mental-disorders\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarboni, L. et al. Depression‑Associated Gene Negr1‑Fgfr2 Pathway Is Altered by Antidepressant Treatment. \u003cem\u003eCells\u003c/em\u003e 9, 1818; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3390/cells9081818\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh, K. et al. Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease‑Associated Negr1 Gene. \u003cem\u003eFront. Mol. Neurosci.\u003c/em\u003e 11, 30; https://doi.org/10.3389/fnmol.2018.00030 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh, K. et al. Neural cell adhesion molecule Negr1 deficiency in mouse results in structural brain endophenotypes and behavioral deviations related to psychiatric disorders. \u003cem\u003eSci. Rep.\u003c/em\u003e 9, 5457; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1038/s41598-019-41849-7\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVanaveski, T. et al. Promoter‑Specific Expression and Genomic Structure of IgLON Family Genes in Mouse. \u003cem\u003eFront. Neurosci.\u003c/em\u003e 11, 38; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3389/fnins.2017.00038\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoward, D. M. et al. Genome‑wide meta‑analysis of depression identifies 102 independent variants and highlights the importance of the prefrontal brain regions. \u003cem\u003eNat. Neurosci.\u003c/em\u003e 22, 343\u0026ndash;352 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaare, M. et al. Depression‑Associated Negr1 Gene‑Deficiency Induces Alterations in the Monoaminergic Neurotransmission Enhancing Time‑Dependent Sensitization to Amphetamine in Male Mice. \u003cem\u003eBrain Sci.\u003c/em\u003e 12, 1696; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3390/brainsci12121696\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaris, K. et al. Altered Expression Profile of IgLON Family of Neural Cell Adhesion Molecules in the Dorsolateral Prefrontal Cortex of Schizophrenic Patients. \u003cem\u003eFront. Mol. Neurosci.\u003c/em\u003e 11, 8; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3389/fnmol.2018.00008\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevey, D. F. et al. Bi‑ancestral depression GWAS in the Million Veteran Program and meta‑analysis in \u0026gt;\u0026thinsp;1.2\u0026nbsp;million individuals highlight new therapeutic directions. \u003cem\u003eNat. Neurosci.\u003c/em\u003e 24, 954\u0026ndash;963 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoh, K. et al. Negr1 controls adult hippocampal neurogenesis and affective behaviors. \u003cem\u003eMol. Psychiatry\u003c/em\u003e 24, 1189\u0026ndash;1205 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrubetskoy, V. et al. Mapping genomic loci implicates genes and synaptic biology in schizophrenia. \u003cem\u003eNature\u003c/em\u003e 604, 502\u0026ndash;508 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaez, M. V., Cercato, M. C. \u0026amp; Jerusalinsky, D. A. NMDA Receptor Subunits Change after Synaptic Plasticity Induction and Learning and Memory Acquisition. \u003cem\u003eNeural Plast.\u003c/em\u003e 2018, 5093048; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1155/2018/5093048\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunt, D. L. \u0026amp; Castillo, P. E. Synaptic plasticity of NMDA receptors: mechanisms and functional implications. \u003cem\u003eCurr. Opin. Neurobiol.\u003c/em\u003e 22, 496\u0026ndash;508 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKemp, J. A. \u0026amp; McKernan, R. M. NMDA receptor pathways as drug targets. \u003cem\u003eNat. Neurosci.\u003c/em\u003e 5, 1039\u0026ndash;1042 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamamoto, H. et al. Specific Roles of NMDA Receptor Subunits in Mental Disorders. \u003cem\u003eCurr. Mol. Med.\u003c/em\u003e 15, 193\u0026ndash;205 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMyint, A. M. \u0026amp; Halaris, A. Imbalances in Kynurenines as Potential Biomarkers in the Diagnosis and Treatment of Psychiatric Disorders. \u003cem\u003eFront. Psychiatry\u003c/em\u003e 13, 913303; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3389/fpsyt.2022.913303\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYovanno, R. A. et al. Excitatory and inhibitory D‑serine binding to the NMDA receptor. \u003cem\u003eeLife\u003c/em\u003e 11, e77645; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.7554/eLife.77645\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoyle, J. T. \u0026amp; Balu, D. T. The Role of Serine Racemase in the Pathophysiology of Brain Disorders. \u003cem\u003eAdv. Pharmacol.\u003c/em\u003e82, 35\u0026ndash;56 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabrie, V. et al. Serine racemase is associated with schizophrenia susceptibility in humans and in a mouse model. \u003cem\u003eHum. Mol. Genet.\u003c/em\u003e 18, 3227\u0026ndash;3243 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolosker, H. et al. Serine racemase: a glial enzyme synthesizing D‑serine to regulate glutamate‑N‑methyl‑D‑aspartate neurotransmission. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e 96, 13409\u0026ndash;13414 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanus, A. et al. MK‑801 and cognitive functions: Investigating the behavioral effects of a non‑competitive NMDA receptor antagonist. \u003cem\u003ePsychopharmacology\u003c/em\u003e 240, 2435\u0026ndash;2457 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLatysheva, N. V. \u0026amp; Rayevsky, K. S. Chronic neonatal N‑methyl‑D‑aspartate receptor blockade induces learning deficits and transient hypoactivity in young rats. \u003cem\u003eProg. Neuropsychopharmacol. Biol. Psychiatry\u003c/em\u003e 27, 787\u0026ndash;794 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong, E. H. et al. The anticonvulsant MK‑801 is a potent N‑methyl‑D‑aspartate antagonist. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e 83, 7104\u0026ndash;7108 (1986).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirch, P. J. et al. Kynurenic acid antagonises responses to NMDA via an action at the strychnine‑insensitive glycine receptor. \u003cem\u003eEur. J. Pharmacol.\u003c/em\u003e 154, 85\u0026ndash;87 (1988).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanysz, W. \u0026amp; Parsons, C. G. Glycine and N‑methyl‑D‑aspartate receptors: physiological significance and possible therapeutic applications. \u003cem\u003ePharmacol. Rev.\u003c/em\u003e 50, 597\u0026ndash;664 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Carvalho, L. P. et al. The endogenous agonist quinolinic acid and the non‑endogenous homoquinolinic acid discriminate between NMDAR2 receptor subunits. \u003cem\u003eNeurochem. Int.\u003c/em\u003e 28, 445\u0026ndash;452 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLugo‑Huitr\u0026oacute;n, R. et al. Quinolinic acid: an endogenous neurotoxin with multiple targets. \u003cem\u003eOxid. Med. Cell Longev.\u003c/em\u003e2013, 104024; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1155/2013/104024\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavitz, J. The kynurenine pathway: a finger in every pie. \u003cem\u003eMol. Psychiatry\u003c/em\u003e 25, 131\u0026ndash;147 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDauvermann, M. R. et al. Glutamatergic regulation of cognition and functional brain connectivity: insights from pharmacological, genetic and translational schizophrenia research. \u003cem\u003eBr. J. Pharmacol.\u003c/em\u003e 174, 3136\u0026ndash;3160 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuuskm\u0026auml;e, C. et al. Kynurenine pathway dynamics in patients with schizophrenia spectrum disorders across the disease trajectory. \u003cem\u003ePsychiatry Res.\u003c/em\u003e 328, 115423; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1016/j.psychres.2023.115423\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoto, Y. et al. Functional and dysfunctional synaptic plasticity in prefrontal cortex: roles in psychiatric disorders. \u003cem\u003eBiol. Psychiatry\u003c/em\u003e 67, 199\u0026ndash;207 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris, R. G. et al. Hippocampal synaptic plasticity and NMDA receptors: a role in information storage? \u003cem\u003ePhilos. Trans. R. Soc. Lond., B, Biol. Sci.\u003c/em\u003e 329, 187\u0026ndash;204 (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerkins, M. N. \u0026amp; Stone, T. W. An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid. \u003cem\u003eBrain Res.\u003c/em\u003e 247, 184\u0026ndash;187 (1982).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeutschenbaur, L. et al. Role of calcium, glutamate and NMDA in major depression and therapeutic application. \u003cem\u003eProg. Neuropsychopharmacol. Biol. Psychiatry\u003c/em\u003e 64, 325\u0026ndash;333 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBraidy, N. et al. Mechanism for quinolinic acid cytotoxicity in human astrocytes and neurons. \u003cem\u003eNeurotoxicol. Res.\u003c/em\u003e 16, 77\u0026ndash;86 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOstapiuk, A. \u0026amp; Urbanska, E. M. Kynurenic acid in neurodegenerative disorders‑unique neuroprotection or double‑edged sword? \u003cem\u003eCNS Neurosci. Ther.\u003c/em\u003e 28, 19\u0026ndash;35 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCross‑Disorder Group of the Psychiatric Genomics Consortium. Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders. \u003cem\u003eCell\u003c/em\u003e 179, 1469\u0026ndash;1482.e11; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1016/j.cell.2019.11.020\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen, S. M. et al. The impact of NMDA receptor hypofunction on GABAergic neurons in the pathophysiology of schizophrenia. \u003cem\u003eSchizophr. Res.\u003c/em\u003e 167, 98\u0026ndash;107 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu, F. et al. Neuronal growth regulator 1 (NEGR1) promotes the synaptic targeting of glutamic acid decarboxylase 65 (GAD65). \u003cem\u003eJ. Neurochem.\u003c/em\u003e 169, e16279; https://doi.org/10.1111/jnc.16279 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGandal, M. J. et al. GABAB‑mediated rescue of altered excitatory‑inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR‑hypofunction. \u003cem\u003eTransl. Psychiatry\u003c/em\u003e 2, e142; https://doi.org/10.1038/tp.2012.69 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenmund, C. et al. Synaptic NMDA receptor channels have a low open probability. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 15, 2788\u0026ndash;2795 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHardingham, G. E. et al. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut‑off and cell death pathways. \u003cem\u003eNat. Neurosci.\u003c/em\u003e 5, 405\u0026ndash;414 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y. et al. NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 27, 2846\u0026ndash;2857 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTovar, K. R. \u0026amp; Westbrook, G. L. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 19, 4180\u0026ndash;4188 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanika, R. I. et al. Coupling diverse routes of calcium entry to mitochondrial dysfunction and glutamate excitotoxicity. \u003cem\u003eProc. Natl Acad. Sci. USA\u003c/em\u003e 106, 9854\u0026ndash;9859 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, X. et al. Involvement of the GluN2A and GluN2B subunits in synaptic and extrasynaptic N‑methyl‑D‑aspartate receptor function and neuronal excitotoxicity. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e 288, 24151\u0026ndash;24159 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, G. et al. GluN2A: A Promising Target for Developing Novel Antidepressants. \u003cem\u003eInt. J. Neuropsychopharmacol.\u003c/em\u003e27, pyae037; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1093/ijnp/pyae037\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrim, B. L. et al. Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor. \u003cem\u003eBehav. Brain Res.\u003c/em\u003e 238, 211\u0026ndash;226 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui, Y. et al. Forebrain NR2B overexpression facilitating the prefrontal cortex long‑term potentiation and enhancing working memory function in mice. \u003cem\u003ePLoS One\u003c/em\u003e 6, e20312; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1371/journal.pone.0020312\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, Y. Y. et al. Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats. \u003cem\u003eSci. Rep.\u003c/em\u003e 6, 30743; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1038/srep30743(2016).\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhilips, M. A. et al. Myg1‑deficient mice display alterations in stress‑induced responses and reduction of sex‑dependent behavioural differences. \u003cem\u003eBehav. Brain Res.\u003c/em\u003e 207, 182\u0026ndash;195 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaare, M. et al. High‑Fat Diet Induces Pre‑Diabetes and Distinct Sex‑Specific Metabolic Alterations in Negr1‑Deficient Mice. \u003cem\u003eBiomedicines\u003c/em\u003e 9, 1148; https://doi.org/10.3390/biomedicines9091148 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolter, K. M. et al. Use of Quantitative Electroencephalography to Inform Age‑ and Sex‑Related Differences in NMDA Receptor Function Following MK‑801 Administration. \u003cem\u003ePharmaceuticals\u003c/em\u003e 17, 237; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3390/ph17020237\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKniffin, A. R. \u0026amp; Briand, L. A. Sex differences in glutamate transmission and plasticity in reward related regions. \u003cem\u003eFront. Behav. Neurosci.\u003c/em\u003e 18, 1455478; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3389/fnbeh.2024.1455478\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeuh\u0026auml;usel, T. S. \u0026amp; Gerevich, Z. Sex‑specific effects of subchronic NMDA receptor antagonist MK‑801 treatment on hippocampal gamma oscillations. \u003cem\u003eFront. Neurosci.\u003c/em\u003e 18, 1425323; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3389/fnins.2024.1425323\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, A. W. et al. Functional inactivation of the genome‑wide association study obesity gene neuronal growth regulator 1 in mice causes a body mass phenotype. \u003cem\u003ePLoS One\u003c/em\u003e 7, e41537; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.1371/journal.pone.0041537(2012).\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarul, J. et al. Dopamine System, NMDA Receptor and EGF Family Expressions in Brain Structures of Bl6 and 129Sv Strains Displaying Different Behavioral Adaptation. \u003cem\u003eBrain Sci.\u003c/em\u003e 11, 725; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehttps://doi.org/10.3390/brainsci11060725(2021).\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Negr1, NMDA, MK-801, kynurenine pathway, behavioural tolerance","lastPublishedDoi":"10.21203/rs.3.rs-7023014/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7023014/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe NEGR1 gene has been implicated in several psychiatric disorders, and increased NMDA receptor binding density has been demonstrated \u003cem\u003ein vitro\u003c/em\u003e in hippocampal slices from \u003cem\u003eNegr1\u003c/em\u003e-deficient mice. In this study, we expanded on these findings by investigating the behavioural response to NMDA receptor antagonism, expression of NMDA receptor subunits, and kynurenine pathway metabolites in a \u003cem\u003eNegr1\u003c/em\u003e-deficient mouse model.\u003c/p\u003e \u003cp\u003eMale and female wild-type and \u003cem\u003eNegr1\u003c/em\u003e-deficient mice received daily injections of MK-801, a non-competitive NMDA receptor antagonist, until behavioural tolerance developed in the open field test (after 9 days in males and 5 days in females). In drug-naive animals, acute MK-801 administration (0.2 mg/kg) elicited a stronger motor response in \u003cem\u003eNegr1\u003c/em\u003e-deficient males compared to wild-type controls. However, with repeated dosing, \u003cem\u003eNegr1\u003c/em\u003e-deficient males exhibited a blunted behavioural response and attenuated progression of rapid behavioural tolerance during every-second-day MK-801 administration, suggesting altered receptor sensitivity.\u003c/p\u003e \u003cp\u003eGene expression analysis revealed sex- and brain region-specific changes in NMDA receptor subunit expression. Additionally, kynurenine pathway metabolites showed genotype- and sex-dependent alterations. These findings suggest that Negr1 modulates NMDA receptor function and tryptophan metabolism in a sex-dependent manner, highlighting the importance of considering both genetic background and sex in models of glutamatergic dysfunction relevant to neuropsychiatric disorders.\u003c/p\u003e","manuscriptTitle":"Negr1 Deficiency Alters Glutamate Signalling and Kynurenine Pathway in a Mouse Model of Psychiatric Disorders","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-04 12:37:24","doi":"10.21203/rs.3.rs-7023014/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-14T12:35:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T15:41:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247420514050414950036953956354475018049","date":"2025-09-28T19:26:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T00:05:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69243209922434134826894923890175981973","date":"2025-07-07T21:15:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21726986495497947453787820315306439195","date":"2025-07-03T19:02:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-02T16:16:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-02T16:09:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-02T14:52:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-02T05:45:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-01T18:38:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"435eb0b0-48e1-4ee5-9724-32b6f360d7a6","owner":[],"postedDate":"July 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51014056,"name":"Health sciences/Diseases"},{"id":51014057,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-01-19T16:49:38+00:00","versionOfRecord":{"articleIdentity":"rs-7023014","link":"https://doi.org/10.1038/s41598-026-35968-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-16 16:30:34","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-07-04 12:37:24","video":"","vorDoi":"10.1038/s41598-026-35968-7","vorDoiUrl":"https://doi.org/10.1038/s41598-026-35968-7","workflowStages":[]},"version":"v1","identity":"rs-7023014","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7023014","identity":"rs-7023014","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}