KMO Deletion Preserves Non-Associative Learning and SVZ Neurogenesis in Aging Mice

Abstract

Activation of the kynurenine pathway (KP) by age-related chronic inflammation has been linked with cognitive decline and altered neurogenesis. When upregulated, kynurenine 3-monooxygenase (KMO), a rate-limiting enzyme, drives the KP toward oxidative metabolism and the production of neurotoxic metabolites. Here, we show that genetic deletion of KMO preserves olfactory habituation and dishabituation, a form of non-associative learning, and alters subventricular zone (SVZ) proliferative and neurogenic activity in aging mice. Olfactory habituation and dishabituation were assessed in young (4–5 months), middle-aged (9–12 months), and old (22–30 months) wild-type (WT) and KMO^−/− male and female mice. WT mice displayed progressive habituation decline, with deficits to non-social odors emerging in middle age and extending to social odors in old age. In contrast, KMO^−/− mice maintained habituation and dishabituation to both odor types across age groups, despite differences in exploration strategies. At the cellular level, SVZ cell proliferation declined with age in WT mice. In contrast, KMO^−/− mice exhibited greater proliferative activity and increased neuroblast-associated labeling compared to WT controls. Our findings suggest a previously unrecognized role for KMO in non-associative learning and in shaping SVZ cellular indices during aging. These results support further investigation into KMO-dependent KP metabolism as a modulator of behavioral and neurogenic processes across the lifespan.

Graphical abstract

1. Introduction

The kynurenine pathway (KP) is the primary route of tryptophan (TRP) metabolism and plays a central role in age-related physiological changes, cognitive decline, and neurodegenerative diseases. Under normal physiological conditions, the KP is a primary source of nicotinamide adenine dinucleotide (NAD⁺), an essential coenzyme required for cellular energy production. The KP generates important neuroactive metabolites, including kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK) and quinolinic acid (QA), which regulate processes such as inflammation, immune function, gene expression, and cellular stress responses in the brain ^1,2. During aging, increased proinflammatory signaling is associated with upregulation of key KP enzymes, including indoleamine 2,3-dioxygenase (IDO) and kynurenine 3-monooxygenase (KMO). As a result, KP metabolic balance shifts toward oxidative metabolism, which increases oxidative stress, promotes lipid peroxidation, and contributes to neurotoxicity ^3–5. At the synaptic level, this oxidative burden disrupts NMDA receptor signaling, calcium-dependent pathways, and activity-dependent gene expression required for learning^6,7. Determining if these changes contribute to age-related declines in non-associative learning and memory is essential for identifying mechanisms of age-related cognitive decline (Figure 1A).

Figure 1.

(A) The kynurenine pathway - Pro-inflammatory cytokines upregulate KMO, driving the KP towards oxidative, neurotoxic metabolites. Inhibiting KMO shifts metabolism toward neuroprotection. (B) Habituation and dishabituation - Habituation reduces behavioral responses to repeated stimuli, while dishabituation restores the response upon introduction of a novel stimulus.

In the brain, KP metabolic enzymes are expressed in a cell type-dependent manner. Microglia and peripherally derived macrophages mainly express KMO, the rate-limiting enzyme that directs KYN metabolism toward 3-HK and QA and ultimately NAD⁺. When dysregulated, the KP in microglia have been shown to promote oxidative stress and lipid peroxidation through free radical generation and contribute to excitotoxicity via direct and indirect activation of NMDA receptors. In contrast, astrocytes primarily express kynurenine aminotransferases (KATs 1–4), which convert KYN into KYNA, an NMDA receptor antagonist with neuroprotective properties^8–10. However, age-related chronic inflammation is associated with increased KMO expression, favoring production of neurotoxic metabolites such as 3-HK and QA. These metabolites amplify oxidative stress, cytokine signaling, and excitotoxic pathways, reinforcing a feedback loop in which KP activation and neuroinflammation reciprocally sustain one another^1,6. This metabolic shift toward oxidative stress and neurotoxicity has been implicated in neurological disorders and neurodegenerative diseases, and KMO inhibition has been proposed as a strategy to reduce neuroinflammation and oxidative damage ^3,11,12. Regulation of the KP is a complex balance between its two main metabolic branches, one producing neuroprotective KYNA and the other generating neurotoxic QA (Figure 1A). Although KYNA is generally considered to be neuroprotective, excessive levels in young rodent models have been linked to cognitive deficits, depression-like and schizophrenia-like behaviors, and disruptions in learning, memory, and sleep architecture highlighting the importance for precise regulation of KP activity ^13–15. These findings suggest that the functional impact of KP metabolites depends on biological context, including age-related shifts in inflammatory tone and redox balance.

Beyond its effects on cognition, KP metabolites may play a key role in regulating neural stem cells (NSCs) and neurogenesis ¹⁶. In vitro studies with human embryonic stem cells and induced pluripotent stem cells suggest that KYN and KYNA activation of the aryl hydrocarbon receptor (AhR) may play a critical role in maintaining self-renewal signaling in these cells ¹⁷. However, in vivo studies indicate that KYN signaling through the AhR becomes upregulated in the ventricular-subventricular zone (SVZ) and dentate gyrus (DG) of the hippocampus, following ischemic stroke in young mice, and is associated with increased gliosis and reduced neurogenesis ¹⁸. These studies reveal distinct and seemingly opposing roles for KYN/KYNA activation of AhR depending on the cellular context and physiological state. Nonetheless, adult neurogenesis declines progressively with age, particularly in the SVZ, and this decline has been associated with reduced plasticity and impairments in olfactory learning and memory ^19,20. Neural stem cells in the SVZ generate neuroblasts that migrate to the olfactory bulb, where they integrate into local circuits and support the plasticity required for associative and non-associative learning^{6 21}. Age-related declines in this process likely contribute to impairments in learning. Even so, activation of the KP by proinflammatory markers is known to impair NSC function and contributing to neurogenic decline ^22,23. Given the sensitivity of NSCs to inflammatory and oxidative signals, sustained KP activation may represent one mechanism contributing to age-related neurogenic decline. Despite evidence linking KP dysregulation to cognitive and neurogenic decline, most work has focused on young models or acute injury paradigms, leaving it unclear how KP activation, and specifically KMO activity, may impact SVZ neurogenesis and non-associative learning during aging. Here we investigated whether lifelong KMO deletion influences SVZ neurogenesis and olfactory habituation across the lifespan.

Habituation, a form of non-associative learning, enables organisms to ignore repetitive, non-salient stimuli ²⁴. Non-associative learning differs from associative learning, such as classical conditioning, which forms associations between stimuli ²⁵. In contrast, habituation is the reduction in behavioral responses to a single repeated stimulus, functioning as an attentional filter that supports higher-order cognition by shifting neural resources to more salient stimuli, thereby regulating adaptive behaviors. Unlike sensory adaptation or motor fatigue, habituation is further characterized by dishabituation, where the habituated response is recovered after a novel stimulus is introduced ²⁶ (Figure 1B). Moreover, habituation deficits occur in neurological disorders such as autism spectrum disorder and posttraumatic stress disorder, and also decline with age in neurodegenerative diseases like Huntington’s and Parkinson’s disease ^27,28. Although the mechanisms underlying habituation deficits remain unclear, we hypothesize that age-related KP dysregulation may contribute by altering the neuromodulatory balance required for this process. Neurotoxic metabolites produced by KMO upregulation may disrupt glutamatergic and GABAergic signaling, both essential for habituation, and KP-mediated oxidative stress may impair neural circuits and plasticity involved in olfactory habituation ^2,3,21,29 . Investigating these potential links may clarify how KP activation contributes to habituation decline during aging. To our knowledge, no studies have examined this possible connection, making it an unexplored aspect of KP-related cognitive decline.

Here, we report that genetic deletion of KMO is associated with stable olfactory habituation and dishabituation across aging. Whereas WT mice exhibit progressive impairments in this form of learning, KMO^−/− mice show little evidence of age-related decline. This behavioral preservation parallels cellular findings in the SVZ, where aging influences proliferative and DCX-positive measures, and KMO^−/− mice exhibit higher overall levels of these indices compared to WT controls. Together, these results suggest a previously unrecognized role for KMO in non-associative learning and in shaping SVZ cellular measures during aging and suggest that modulation of KMO activity may influence neural function later in life. Our findings position KMO inhibition as a potential strategy for mitigating age-related cognitive decline.

2. Methods

2.1. Animals

All procedures followed the Guide for the Care and Use of Laboratory Animals (NRC, 8th edition) and were approved by the Institutional Animal Care and Use Committee at UT Health San Antonio. Male and female C57BL/6J wild-type (WT) mice (Jackson Laboratory, stock #000664) and KMO^−/− mice, generated as described in ³⁰, were used. Mice were tested at three ages: young (4–5 months), middle-aged (9–12 months), or old (22–30 months). Both male and female mice were included across all genotype and age groups, with comparable representation. Mice were group-housed under a reverse 12-hour light/dark cycle with ad libitum food and water. Health was monitored daily by veterinary and research staff.

2.2. Behavior Testing

All behavioral testing was conducted in age-matched young, aged, and old cohorts as noted. The same testing procedures were repeated in independent cohorts, and additional animals were included to increase sample size within each age group. The buried food-seeking test was adapted from ³¹. Mice were fasted for 12 hours with water ad libitum. A fresh 3g chow pellet was buried 2 cm beneath sanitized bedding in the same corner of a plexiglass test box (29 × 18 × 12 cm) for all trials. Mice acclimated in their home cages for 1 hour in the test room before testing. Then they were placed in the text box in the opposite corner and given 10 minutes to locate the pellet. Trials ended when the mouse touched the pellet with its forepaws or mouth; those failing to find it within 600 seconds received the maximum latency score. A new test box, bedding, and food pellet were used for each mouse.

An automated olfactory habituation and dishabituation system, modified from ³², was used to standardize odor delivery, minimize animal stress, and ensure unbiased scoring. Mice were tested in clear plexiglass boxes (38 × 16 × 10 cm) with ventilated lids featuring a 6 mm hole at the front for odor delivery. Three boxes were fixed side by side using a PVC frame, separated by opaque barriers (40 × 12 cm) to prevent visual contact. Logitech C920 HD cameras, mounted 50 cm above each box on the same PVC frame to ensure consistent tracking. Mice acclimated to the test room for an hour and then to the test boxes for 30 minutes before testing. Odorants were applied to sterile, cotton-tipped swabs and introduced through the 6 mm hole via a modified 1000 mL pipette tip. New swabs were used for every trial. The presentation sequence included distilled water, used as an initial stimulus to acclimate mice to the odor presentation procedure, followed by almond and banana extracts and two distinct social odors from soiled cages of sex-matched mice. Water trials were not included in experimental analyses. Each odor was presented three times for 2 minutes, with 1-minute intervals. Investigation time, defined as head oriented towards the odor source within 2 cm, was recorded automatically using ANY-maze software (version 7.3, Stoelting) for simultaneous, high-throughput tracking.

2.3. Immunohistochemistry

Mice received intraperitoneal EdU (50 mg/kg) 2 hours before euthanasia. Transcardial perfusion was performed with cold heparinized saline followed by isotonic 4% paraformaldehyde (PFA) solution. Brains were post-fixed in 4% PFA overnight at 4°C, then transferred to 30% sucrose until saturation (2–3 days), embedded in OCT, frozen, and cryosectioned sagittally at 50 μm. EdU incorporation was detected using the Click-iT EdU Imaging Kit (Invitrogen). Sections were blocked/permeabilized in 10% NDS in 0.2% Triton X100 in 1X PBS for 1 hour, then incubated overnight at 4°C with anti-Doublecortin (DCX, 1:400, Cell Signaling Technology) in blocking solution. After three PBST washes, sections were incubated for 1 hour at room temperature in Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500, Jackson Immuno-Research), protected from light. After three additional PBST washes, DAPI (1:1000) counterstaining was performed for 5 minutes, followed by 3 PBST washes. Sections were mounted with ProLong Gold Antifade (Invitrogen), cured overnight in the dark, and stored at −20°C. Images were acquired using a Zeiss Axio Observer.D1 microscope with a 10X objective. The SVZ was identified in sagittal sections using DAPI and the entire lateral wall of the SVZ was imaged. EdU⁺ cells were quantified manually, whereas DCX signal was quantified from fluorescent images using Fiji (ImageJ) by applying a consistent threshold to isolate DCX-positive signal, subtracting background, and expressing labeling as percentage of DCX-positive pixel area within the SVZ. This approach was used due to the dense and overlapping nature of DCX-positive neuroblast processes, which precludes reliable individual cell counting and yields an approximate measure of DCX labeling. All quantifications were performed double-blinded and averaged across 8–12 sections per animal.

2.4. Statistical analysis

Statistical analyses were performed using GraphPad Prism 10.4. Data normality was assessed with the Shapiro-Wilk test. Buried food-seeking test data (parametric) were analyzed using two-way ANOVA with Tukey’s post hoc test, with outliers removed via Grubbs’ test. Olfactory Habituation and Dishabituation test data (non-parametric) were analyzed using the Kruskal-Wallis test with Dunn’s post hoc test, and outliers were identified using the interquartile range (IQR) method. Outlier detection methods were selected based on distributional assumptions: Grubbs’ test was applied to normally distributed parametric data, whereas IQR-based detection was used for nonparametric datasets. Exclusion criteria were defined a priori and applied uniformly across genotypes and age groups. Trials were excluded if investigation time was zero in Trial 1 or across all three trials, indicating non-participation. The habituation index for each odor set was calculated as: Habituation Index = ((Trial 1 − Trial 3) / Trial 1) × 100. The relationship between habituation index and age was assessed via Spearman’s rank correlation with linear regression. EdU⁺ cell counts and DCX-positive pixel area data met assumptions of normality as assessed by the Shapiro–Wilk test and were analyzed using two-way ANOVA with Tukey’s post hoc test. Sex was included as a biological variable in all behavioral analyses; no statistically significant differences between males and females were detected within any genotype or age group in the buried food-seeking test or olfactory habituation and dishabituation task.

3. Results

3.1. Young WT Mice Locate Buried Food Faster Than KMO^−/− Mice, with No Differences in Older Groups.

The buried food-seeking test (BFST) was used to evaluate olfactory function, foraging, and locomotor abilities among young, middle-aged, and old WT and KMO^−/− mice. A significant interaction between genotype and age was observed (Figure 2, two-way ANOVA, F(2, 165) = 5.834, p = 0.0036), primarily driven by a notable difference between young WT and KMO^−/− mice. Young WT mice located the buried food pellet significantly faster than young KMO^−/− mice (p = 0.0020). However, there were no significant main effects of genotype (F(1, 165) = 1.502, p = 0.2222) or age (F(2, 165) = 1.168, p = 0.3135) as no differences were detected between the genotypes in the middle-aged (p > 0.999) or old groups (p = 0.438). While young WT mice outperformed young KMO^−/− mice in the BFST, both genotypes were able to locate the buried food pellet across all age groups. Analysis of individual data points revealed substantial inter individual variability within groups, likely reflecting natural variability in exploratory and foraging strategies, rather than discrete behavioral subgroups.

Figure 2. Young WT mice locate buried food faster than KMO^−/− mice, with no differences in older groups.

Young WT mice located the buried food pellet significantly faster than young KMO^−/− mice. However, no significant differences in latency were observed between WT and KMO^−/− mice in the middle-aged or old groups, indicating that both genotypes retained the ability to locate the food pellet across all age groups. Sample sizes: young n ≥ 34, middle-aged n ≥ 23, old n ≥ 24 (males and females). Data represent mean ± SEM.

3.2. Young WT and KMO^−/− Mice Habituate and Dishabituate to Non-social and Social Odors but Display Differences in Investigation Strategies.

The olfactory habituation and dishabituation test (OHDT) is a widely used assay to evaluate olfactory function as well as non-associative learning and memory in rodents³². The OHDT assesses the ability of mice to habituate to repeated presentations of the same non-social and social odors and to discriminate and dishabituate to the presentation of a novel odor. In the context of aging, the OHDT is particularly valuable for detecting potential declines in non-associative learning and memory by comparing olfactory behavior across different age groups.

Young WT mice repeatedly exposed to the two non-social odors, Almond (AL) and Banana (BA), showed a significant effect across the six odor presentations (Figure 3A, Kruskal-Wallis, H = 17.42, p = 0.0038). A significant decrease in investigation time between AL1 and AL3 (p = 0.0243) that defines habituation, whereas significantly increased investigation time between AL3 and BA1 (p = 0.0006) reflects dishabituation to the novel non-social odor. Next, a significant decrease between BA1 and BA3 (p = 0.0440) indicates habituation to the new odor. For social odors, Social Odor 1 (SO1) and Social Odor 2 (SO2), WT mice displayed significant differences in investigation times across the six presentations (Figure 3A, Kruskal-Wallis, H = 26.75, p < 0.0001). Significant habituation was observed between SO1–1 and SO1–3 (p = 0.0026), dishabituation between SO1–3 and SO2–1 (p = 0.0019), and habituation between SO2–1 and SO2–3 (p = 0.0005).

Figure 3. Young WT and KMO^−/− mice habituate and dishabituate to non-social and social odors, but display differences in investigation strategies.

(A-B) Both young WT and KMO^−/− mice show robust habituation (decreased investigation time) and dishabituation (increased investigation time) to non-social and social odors. (C) Young KMO^−/− mice spent less time investigating non-social odors compared to WT mice, but investigation times for social odors were similar between both groups (D) Young KMO^−/− mice enter the odor zone less frequently than young WT mice for non-social odors but not social odors. (E) Latency to investigate odors is longer in young KMO^−/− mice for both non-social and social odors compared to young WT mice. (F) Young WT mice show a lower habituation index for non-social odors, indicating weaker habituation, whereas social odor habituation does not differ between genotypes. Sample sizes: young n ≥ 34, (males and females). Data represent median ± 95% CI.

In young KMO^−/− mice, habituation plots show that time investigating non-social odors indicated significant differences across all trials (Figure 3B, Kruskal-Wallis, H = 56.90, p < 0.0001). Young KMO^−/− mice displayed significant habituation between AL1 and AL3 (p < 0.0001) and dishabituation between AL3 and BA1 (p = 0.0001), reflecting clear discrimination and increased interest in the novel non-social odor. Habituation was observed again between BA1 and BA3 (p < 0.0001), indicating a decline in investigation time with repeated exposure. Significant differences were also observed across the six odor social odor presentations (Figure 3B, Kruskal-Wallis, H = 29.69, p < 0.0001). Significant habituation was observed between SO1–1 and SO1–3 (p = 0.0004) and dishabituation between SO1–3 and SO2–1 (p = 0.0173), followed by habituation to the novel social odor between SO2–1 and SO2–3 (p = 0.0033). Both young WT and KMO^−/− mice displayed robust habituation and dishabituation to non-social and social odors, though KMO^−/− mice consistently spent less time, in absolute terms, investigating odors than WT controls.

To explore the lower investigation time observed in KMO^−/− mice, we analyzed additional metrics, starting with mean total time of investigation. WT mice spent significantly more time investigating non-social odors compared to KMO^−/− mice (Figure 3C, Kruskal-Wallis, H = 47.51, p < 0.0001; p = 0.0008). No significant differences were observed in total investigation times for social odors (p = 0.2018), suggesting that functional deletion of KMO influences investigation time for non-social but not social odors in young mice.

The number of entries into the target zone (area where odors are presented) showed similar results (Figure 3D, Kruskal-Wallis, H = 49.80, p < 0.0001). WT mice made significantly more entries for non-social odors compared to KMO^−/− mice (p < 0.0001), while no significant differences were observed for social odors (p = 0.2930). As with the mean total time investigating, the number of entries suggest that genotype influences these metrics for non-social odors but not social odors. KMO^−/− mice investigated non-social odors less than WT mice, suggesting that WT mice may require more time to habituate. Latency to investigate both odor types differed significantly between genotypes (Figure 3E, Kruskal-Wallis, H = 31.96, p < 0.0001). KMO^−/− mice displayed significantly longer latencies to investigate both non-social odors (p < 0.0001) and social odors (p = 0.0068) compared to WT mice, possibly suggesting a reduced exploratory drive or delayed arousal response in KMO^−/− mice. Although KMO^−/− mice showed reduced overall odor investigation and longer latencies to investigate, habituation indices were preserved across odors and ages, indicating intact stimulus discrimination despite altered exploratory strategy.

The habituation index (HI), which quantifies the magnitude of the habituation response, was used to normalize differences in baseline investigation times. A higher HI value indicates stronger habituation, while lower values reflect weaker habituation. The HI in young mice revealed significant effect on habituation (Figure 3F, Kruskal-Wallis, H = 19.58, p = 0.0002). KMO^−/− mice exhibited a significantly higher HI for non-social odors compared to WT mice (p = 0.0001), indicating more robust habituation. However, no significant differences were observed for social odors (p = 0.6618), suggesting that genotype primarily affects habituation for non-social but not for social odors. Young WT and KMO^−/− mice displayed similar patterns of habituation and dishabituation, however, WT mice required longer investigation times to habituate to social odors compared to KMO^−/− mice.

3.3. Middle-aged KMO^−/− Mice Habituate to Non-social Odors, but Middle-aged WT Mice Do Not.

Investigation times for middle-aged WT mice exposed to non-social odors revealed a significant effect related to odor presentations (Figure 4A, Kruskal-Wallis, H = 17.14, p = 0.0042). In middle-aged WT mice, no significant habituation was observed between AL1 and AL3 (p = 0.8562). However, investigation time increased significantly between AL3 and BA1 (p = 0.0037), indicating discrimination of the novel odors but not dishabituation as no habituation occurred between AL1 and AL3. No changes in investigation time were detected between BA1 vs. BA3, (p > 0.9999), again suggesting a lack of habituation in middle-aged WT mice to non-social odors.

Figure 4. Middle-aged KMO^−/− mice habituate to non-social odors, but middle-aged WT mice do not.

(A) Middle-aged WT mice fail to habituate to non-social odors but show intact habituation and dishabituation to social odors, whereas (B) middle-aged KMO^−/− mice exhibit intact habituation and dishabituation to both odor types similar to young KMO^−/− mice. (C) Middle-aged KMO^−/− mice spend less time investigating both non-social and social odors compared to middle-aged WT controls. (D) Middle-aged KMO^−/− mice enter the target zone fewer times than middle-aged WT mice. (E) Middle-aged KMO^−/− mice take longer to initiate odor investigation middle-aged WT mice. (F) Middle-aged WT mice show a lower habituation index than middle-aged KMO^−/− mice. Sample sizes: middle-aged n ≥ 23 (males and females). Data represent median ± 95% CI.

Middle-aged WT mice showed a statistically significant effect when exposed to social odors (Figure 4A, Kruskal-Wallis, H = 39.58, p < 0.0001). Related to differences between trials, middle-aged WT mice displayed clear habituation between SO1–1 and SO1–3 (p = 0.0004), dishabituation between SO1–3 and SO2–1, (p = 0.0299), and habituation between SO2–1 and SO2–3 (p = 0.0002. Middle-aged WT failed to habituate to non-social odors while young WT mice habituated to non-social odors. However, their ability to habituate social odors remained comparable to young WT mice. This pattern indicates potential age-related declines in non-associative learning and memory in middle-aged WT mice.

Habituation and dishabituation analysis of middle-aged KMO^−/− mice exposed to non-social odors revealed a significant difference across all non-social odor trials (Figure 4B, Kruskal-Wallis, H = 24.91, p = 0.0001). Middle-aged KMO^−/− mice displayed significantly reduced investigation time between AL1 and AL3 (p = 0.0063), indicating robust habituation, while increased investigation time between AL3 and BA1 suggests clear dishabituation (p = 0.0045). Additionally, significant habituation was detected between BA1 and BA3 (p = 0.0356). Like their younger counterparts middle-aged KMO^−/− successfully habituated and dishabituated to social odors.

Investigation times in middle-aged KMO^−/− mice differed significantly across all social odor trials (Figure 4B; Kruskal–Wallis, H = 59.13, p < 0.0001). KMO^−/− mice habituated between SO1–1 and SO1–3 (p < 0.0001), dishabituated between SO1–3 and SO2–1 (p = 0.0002) and subsequently habituated between SO2–1 and SO2–3 (p = 0.0002), once again indicating that middle-aged KMO^−/− mice, like young KMO^−/− mice, effectively habituated and dishabituated to social odors. Both middle-aged WT and KMO^−/− mice showed habituation and dishabituation to social odors; however, only KMO^−/− mice retained these responses to non-social odors, indicating an age-related decline in WT mice.

Mean total time investigating non-social and social odors was statistically different between middle-aged WT and KMO^−/− mice (Figure 4C, Kruskal-Wallis, H = 59.80, p < 0.0001). Middle-aged WT mice spent more time investigating non-social odors compared to middle-aged KMO^−/− mice (p = 0.0057) yet failed to habituate to non-social odors despite increased investigation time. While young WT and KMO^−/− mice did not differ in their investigation times for social odors, a significant difference emerged in middle age (p < 0.0001), suggesting an age-related shift in habituation in WT mice that was not observed in KMO^−/− mice.

The frequency of entries into the target zone in middle-aged WT and KMO^−/− mice revealed a significant overall difference between groups for both non-social and social odors (Figure 4D, Kruskal-Wallis, H = 58.62, p < 0.0001). Middle-aged WT mice made significantly more entries to the target zone than middle-aged KMO^−/− mice for non-social odors (p = 0.0010) and social odors (p < 0.0001). In the young groups, differences in number of entries were observed only for non-social odors. However, middle-aged WT mice required more visits to the target zone to achieve habituation compared to KMO^−/− mice.

Like the young groups, the latency to investigate the target zone for both non-social and social odors remained statistically different between middle-aged WT and KMO^−/− mice (Figure 4E, Kruskal-Wallis, H = 49.05, p < 0.0001). Middle-aged KMO^−/− mice took significantly longer to initiate investigation of non-social odors (p = 0.0021) and social odors (p < 0.0001) compared to middle-aged WT mice controls, indicating a persistent genotype-related delay.

The HI for middle-aged WT and KMO^−/− mice revealed significant overall differences in both non-social and social odors (Figure 4F, Kruskal-Wallis, H = 46.39, p < 0.0001). Whereas, in the young groups, differences in HI were detected in non-social odors but not social odors. Middle-aged KMO^−/− mice exhibited significantly higher HI for both non-social (p = 0.0001) and social odors (p = 0.0318) compared to middle-aged WT mice.

Results from the middle-aged groups suggest that KMO^−/− mice retain habituation and dishabituation to both non-social and social odors with age, whereas WT mice maintain these responses only for social odors. Additionally, both young and middle-aged KMO^−/− mice required less investigation time and fewer visits to the target zone to reach habituation compared to their WT counterparts. To further investigate these age-related differences, we next examined habituation and dishabituation patterns in old WT and KMO^−/− mice.

3.4. Age-related habituation deficits in old WT mice are absent in Old KMO^−/− mice

Old WT mice showed a significant overall effect in odor investigation (Figure 5A, Kruskal-Wallis, H = 11.94, p = 0.0356), but no significant habituation between AL1 and AL3 was detected (AL1 and AL3, p = 0.5485). Despite their ability to discriminate between the AL3 and BA1 (p = 0.0122), the lack of habituation in the first odor (AL) suggests no dishabituation. No significant reduction in investigation was detected between exposures of BA1 and BA3 (, p = 0.6622). For social odors, old WT mice exhibited a significant overall effect (Figure 5A, Kruskal-Wallis, H = 29.43, p < 0.0001). They successfully habituated between SO1–1 and SO1–3 (p = 0.0081) but failed to exhibit dishabituation when SO2 was introduced (p > 0.9999). Additionally, a non-significant trend for habituation was observed between SO2–1 and SO2–3 (p = 0.0784). The absence of habituation in old WT mice reflects an age-dependent disruption of non-associative learning mechanisms.

Figure 5. Age-related habituation deficits in old WT mice are absent in Old KMO^−/− mice.

(A) Old WT mice fail to habituate and dishabituate to non-social and social odors. (B) Old KMO^−/− mice habituation and dishabituate like their younger KMO^−/− counterparts. (C) Old WT mice spend more time investigating odors compared to old KMO^−/− mice. (D) Equally, old WT mice visit the target zone more often than old KMO^−/− mice. (E) Old KMO^−/− mice display longer latencies to investigate the target zone compared to old WT mice. (F) Old WT mice show a significantly lower habituation index than old KMO^−/− mice. Sample sizes: middle-aged n ≥ 24 (males and females). Data represent median ± 95% CI.

Unlike old WT mice, old KMO^−/− mice demonstrated robust habituation and dishabituation to non-social odors (Figure 5B, Kruskal-Wallis, H = 40.66, p < 0.0001). Significant habituation occurred between AL1 and AL3 (p = 0.0002), followed by successful dishabituation between AL3 and BA1 (p = 0.0078), and subsequent habituation between BA1 and BA3 (p = 0.0004). Similarly, old KMO^−/− mice exhibited strong habituation responses to social odors (Figure 5B, Kruskal-Wallis, H = 42.57, p < 0.0001). Old KMO^−/− mice showed clear habituation between SO1–1 and SO1–3 (p < 0.0001) and dishabituation between SO1–3 and SO2–1 (p = 0.0020), followed by successful habituation to the second social odor SO2–1 and SO2–3 (p = 0.0011). Unlike old WT mice, old KMO^−/− mice maintain intact habituation and dishabituation responses to both non-social and social odors.

Mean total investigation time continued to differ significantly between groups (Figure 5C, Kruskal-Wallis, H = 43.28, p < 0.0001), with old WT mice spending significantly more time investigating both non-social (p = 0.0164) and social odors (p < 0.0001) compared to old KMO^−/− mice. Similarly, entry frequency into the target zone was considerably higher in old WT mice (Figure 5D, Kruskal-Wallis, H = 49.13, p < 0.0001), with post hoc tests confirming greater entries for both non-social (p = 0.0001) and social odors (p < 0.0001). As with young and middle-aged KMO^−/− mice, exploratory activity remains low in old mutants compared to old WT mice. The latency to initiate odor investigation remained significantly different between old WT and KMO^−/− mice (Figure 5E, Kruskal-Wallis, H = 40.23, p < 0.0001), with old KMO^−/− mice displaying significantly longer latencies for both non-social (p = 0.0006) and social odors (p < 0.0001) compared to old WT mice.

The HI revealed a persistent and significant overall difference between the genotypes (Figure 5F, Kruskal-Wallis, H = 42.45, p < 0.0001). Old WT mice had statistically lower HI than old KMO^−/− mice for both non-social (p = 0.0020) and social odors (p < 0.0001), indicating weaker habituation and dishabituation responses in WT mice, but not old KMO^−/− mice. In old WT mice, habituation impairments that initially affected only non-social odors in middle age extended to include social odors, whereas old KMO^−/− mice maintained robust habituation and dishabituation to both odor types. Additionally, the reduced exploratory drive observed in young and middle-aged KMO^−/− mice persists in old KMO^−/− mice.

3.5. Habituation declines with age in WT mice but remains stable in KMO^−/− mice

To assess the relationship between the HI and age, we performed Spearman correlation and linear regression analyses separately for WT and KMO^−/− mice. In WT mice, a significant negative correlation was detected between HI and age (Figure 6A, Spearman r = −0.1660, p = 0.0028), indicating a decline in habituation with age. Linear regression analysis showed a significant negative slope (p = 0.0293), consistent with a gradual reduction in HI over time. However, the modest correlation coefficient suggests that while age contributes to this decline, other factors may also influence the habituation decline in WT mice.

Figure 6. Habituation declines with age in WT mice but remains stable in KMO^−/− mice.

(A) In WT mice, habituation index negatively correlates with age, indicating a decline in habituation over time. (B) In contrast, KMO^−/− mice show no significant correlation between habituation index and age, suggesting that their habituation ability remains stable with aging.

Conversely, no significant correlation was observed between age and HI in KMO^−/− mice (Figure 6B, Spearman r = −0.01507, p = 0.8123), suggesting that their habituation ability remains stable throughout aging. Linear regression analysis showed a non-significant slope (p = 0.8601) indicating no decline in habituation with age in KMO^−/− mice. The low R² value (0.000125) further highlights the lack of a meaningful relationship between these variables in KMO^−/− mice. Correlation analysis suggests that while habituation deteriorates with age in WT mice, it is preserved in KMO^−/− mice.

3.6. SVZ proliferation and neuroblast marker expression are influenced by age and genotype

Representative fluorescent microscopy images of the SVZ from young, middle-aged, and old WT and KMO^−/− mice are shown in Figure 7A–F. In WT mice, EdU⁺ cells decrease visibly with advancing age, whereas DCX labeling increases. A similar pattern is observed in KMO^−/− mice, with EdU⁺ cells declining across age while DCX-positive area appears greater in older animals, and overall labeling remains higher than in age-matched WT mice. Quantification of SVZ proliferation revealed a significant main effect of age (Figure 7G, two-way ANOVA, F(2,41) = 13.89, P < 0.0001) and genotype (F(1,41) = 34.13, P < 0.0001), with no significant age × genotype interaction (P = 0.1276). Post hoc analysis of the age effect demonstrated that EdU⁺ cell counts were significantly reduced in middle-aged compared to young mice (P < 0.0001) and in old compared to young mice (P = 0.0003), whereas middle-aged and old groups did not differ (P = 0.4154). Across age groups, KMO^−/− mice exhibited higher EdU⁺ cell counts than WT mice. DCX-positive pixel area in the SVZ was also significantly influenced by age (F(2,39) = 31.87, P < 0.0001) and genotype (Figure 7H, two-way ANOVA, F(1,39) = 26.27, P < 0.0001), with no significant age × genotype interaction (P = 0.8146). Post hoc comparisons revealed that DCX labeling was significantly higher in old mice compared to both young (P < 0.0001) and middle-aged mice (P < 0.0001), while young and middle-aged groups did not differ (P = 0.5638). Across ages, KMO^−/− mice exhibited greater DCX-positive area than WT mice.

Figure 7. Age and genotype effects on SVZ cell proliferation and DCX-positive area in WT and KMO^−/− mice.

(A–F) Representative immunohistochemical images of the subventricular zone (SVZ) in young, middle-aged, and old cohorts. Proliferating cells were labeled with EdU (green); immature neurons were labeled with DCX (red). Inset in (B) shows higher magnification of DCX-positive neuroblasts. (G) Quantification of mean EdU⁺ cells per SVZ demonstrated significant main effects of genotype and age, with no significant interaction. Tukey's multiple comparisons test revealed a significant decline in proliferation from young to middle-aged and young to old cohorts. (H) Quantification of DCX+ pixel count percentages indicated significant main effects of genotype and age, with no significant interaction. Post hoc analysis showed a significant increase in DCX-positive area in the old cohort compared to both young and middle-aged mice. Data are represented as mean ± SEM. *** p < 0.001 and **** p < 0.0001 for age main effects. Scale bar = 100μm. LV = lateral ventricle, SVZ = subventricular zone, CP = caudate putamen, CC = corpus collosum, DG = dentate gyrus.

4. Discussion

This study is the first to demonstrate that deletion of KMO may preserve olfactory habituation and dishabituation, key forms of non-associative learning. Whereas associative learning paradigms, including classical and operant conditioning, involve forming links between stimuli and outcomes, non-associative learning like habituation is a fundamental process that reduces behavioral responses to repeated sensory input ²⁵. Though often considered a rudimentary form of learning, habituation functions as an attentional filter critical for cognitive and behavioral plasticity ^24,26. Deficits in habituation have been reported across a range of neuropsychiatric and neurodegenerative disorders, including autism spectrum disorder, depression, post-traumatic stress disorder, schizophrenia, Huntington’s disease and Parkinson’s disease ^27,28. Habituation reduces behavioral responses to repetitive, insignificant stimuli, allowing biologically costly neural resources to be allocated more efficiently to salient inputs. This adaptive filtering relies on stable synaptic modulation within sensory circuits, processes that are particularly sensitive to redox imbalance. In this way, habituation serves as a foundational cognitive mechanism that supports higher-order processing ²⁶. While age-related shifts in the KP toward oxidative, neurotoxic metabolism have been widely linked to impairments in associative learning and memory ^3,4,9,33 our findings implicate these KP shifts in deficits of non-associative learning and memory as well.

Here we report that in WT mice, habituation impairments emerged progressively with age. Young mice habituated and dishabituated to both social and non-social odors, but middle-aged mice showed habituation deficits specific to non-social odors, and old mice failed to habituate to non-social odors altogether. However, WT mice retained the ability, albeit with increasing variability in older mice, to habituate and dishabituate to social odors in all age groups, which may reflect the biological salience of social odors. Nonetheless, in WT mice a clear pattern emerged where habituation to non-social odors progressively disappeared as these mice progressed to old age.

Unlike WT mice, KMO^−/− mice of all ages maintained intact habituation and dishabituation to both odor types. Although their exploration strategy differed, characterized by fewer total visits to odors, reduced overall investigation time, and longer latencies to investigate odors, these mice consistently demonstrated preserved habituation and dishabituation into old age. The altered exploration behavior may reflect an underlying anxiety-like phenotype previously reported in KMO^−/− mice ^8,15,34. Despite these differences in exploratory strategies, WT mice showed an age-related decline in the habituation index, whereas KMO^−/− mice maintained consistently higher indices across age, indicating preserved non-associative learning into old age. Sex was included as a biological variable in the olfactory habituation and dishabituation analyses, and no significant differences were detected between males and females across genotype or age groups. Together, these behavioral findings suggest a putative novel role for KMO in non-associative learning and memory in aging mice.

Preserved non-associative learning in aging KMO^−/− mice was observed in the context of chronic, age-related inflammation, a condition known to increase expression of key KP enzymes such as IDO and KMO and to favor a shift toward oxidative, neurotoxic metabolism. This shift moves TRP and KYN metabolism away from the production of neuroprotective KYNA and toward neurotoxic metabolites like 3-HK and QA ^2,35. The resulting increase in oxidative stress and excitotoxicity has been linked to cognitive function decline and reduced neurogenesis during aging ^3,11,16,33. At the cellular level, oxidative stress can impair synaptic plasticity by disrupting NMDA receptor signaling, calcium-dependent pathways, and activity-dependent transcription required for adaptive learning^6,7,36. Consistent with this framework, we and others have previously reported that deletion of KMO increases KYNA, reduces 3-HK and QA thereby decreasing oxidative stress ^37–39. While metabolites were not quantified in the present study, our findings are consistent with prior studies implicating KMO-dependent KP metabolism as an effector of age-dependent functional decline ^12,33 . Specifically, our behavioral findings suggest that genetic deletion of KMO may preserve non-associative learning, possibly allowing aged and old KMO^−/− mice to habituate and dishabituate to both odor types and to maintain higher habituation indices across their lifespan, unlike WT controls.

To identify a potential cellular substrate for the preserved behavioral function observed in aging KMO^−/− mice, we turned to the subventricular zone (SVZ), a neurogenic niche that remains active in adulthood. The rodent SVZ generates new interneurons that integrate into the olfactory bulb through the rostral migratory stream, supporting olfactory processing throughout life ^40–42. However, the ability of the SVZ to produce new neurons declines with age ^20,22,43. Given this link between SVZ neurogenesis and olfactory function, we examined whether KMO deletion is associated with maintenance of neurogenic capacity across age, in parallel with the behavioral phenotype observed in aging KMO^−/− mice.

In WT controls, SVZ cell proliferation declined with age, as reflected by reduced EdU⁺ cell counts in middle-aged and old mice relative to young animals. A similar age-related reduction in EdU labeling was observed in KMO^−/− mice. However, across age groups, KMO^−/− mice exhibited higher overall EdU⁺ cell counts than WT controls, indicating a genotype effect on proliferative activity independent of age. DCX-positive area was also influenced by both age and genotype. DCX labeling was greater in old mice compared to young and middle-aged animals, and KMO^−/− mice showed higher overall DCX-positive area than WT mice. The absence of an interaction between age and genotype indicates that aging-related changes in proliferation and DCX labeling occur in both genotypes, while KMO deletion is associated with higher baseline levels of these measures across the lifespan. These cellular differences may contribute to the behavioral phenotypes observed, although the present data do not establish a causal relationship between SVZ measures and olfactory habituation.

Our published work has shown that KMO deletion shifts KP metabolism toward a neuroprotective profile, with KMO^−/− mice exhibiting significantly reduced levels of neurotoxic metabolites such as 3-HK and QA, and elevated levels of the neuroprotective metabolite KYN and KYNA in brain and plasma. These effects have been demonstrated both in vivo following systemic immune activation in adult mice ³⁰ and in vitro using primary microglia cultures from KMO^−/− mice ³⁷. These KP shifts have been directly linked to reduced microglial activation, oxidative stress, and decreased expression of pro-inflammatory cytokines, including IL-1β, TNFα, and IL-6, following immune challenge in vivo and in microglial cultures^30,44. Because inflammatory signaling and KP activation are bidirectionally coupled, modulation of KMO activity has the potential to influence this feedback loop. Our findings are consistent with studies showing that KMO deletion or inhibition reduces levels of neurotoxic metabolites such as 3-HK and QA, increases KYNA, attenuating neuroinflammatory responses. For example, Erhardt et al.³⁴ demonstrated that KMO^−/− mice have markedly reduced 3-HK and elevated KYNA in both cerebrum and cerebellum, while Tufvesson-Alm et al.¹⁵ reported increased KYNA under basal conditions accompanied by an absence of microglial activation. Imbeault et al.⁴⁵ similarly observed shifts in KP metabolism toward a neuroprotective profile, and Pocivavsek et al.² showed that KMO deletion decreases 3-HK and QUIN, increases KYNA, attenuates cytokine expression, and protects against inflammation-induced behavioral deficits. Thus, while KP metabolites were not directly assayed in this study, the findings summarized above demonstrate that KMO deletion shifts KP metabolism away from neurotoxic intermediates and toward increased KYN and KYNA. Such metabolic differences may influence the SVZ microenvironment, including inflammatory tone and oxidative stress, factors known to regulate neural progenitor activity. Future studies directly integrating regional KP metabolite measurements with SVZ cellular analyses and behavioral outcomes will be necessary to clarify mechanistic links.

Our findings suggesting that KMO deletion may benefit aging-related cognitive function contrast with previous reports of cognitive impairments in young KMO^−/− mice, pointing to a possible temporal dimension in the effects of KP modulation. While young adult KMO^−/− mice have been shown to display cognitive deficits associated with elevated KYNA ^15,45,46, our results indicate that in aging, KMO^−/− mice retain olfactory habituation and dishabituation, a form of learning, unlike WT controls. This contrast suggests that the impact of KP metabolites may depend on biological context and developmental stage, with KYNA elevations that are disruptive early in life potentially exerting protective effects later in aging.

To better contextualize these findings, we considered how elevated KYN and KYNA levels in KMO^−/− mice may relate to the maintained olfactory habituation and dishabituation observed across age. These mice exhibit chronically elevated levels of both metabolites throughout development, which may influence the behavioral outcomes observed here. KYNA accumulation in aging could buffer the brain against inflammation and excitotoxicity. As an endogenous antagonist of NMDA and α7 nicotinic acetylcholine receptors, KYNA may reduce neural excitability and dampen neuroinflammatory signaling ^1,2,11. Both KYN and KYNA are ligands for the aryl hydrocarbon receptor (AhR), a pathway involved in immunoregulation and neural stem cell maintenance ^17,18,47. Through AhR activation, these metabolites may alter the expression of genes involved in oxidative stress, synaptic maintenance, and neurogenesis in a manner that depends on both timing and context^18,47,48. Although high KYN levels have been linked to psychiatric disorders and schizophrenia-like phenotypes in rodents, KYN may exert context-dependent benefits in aging. These findings suggest that elevated KYN and KYNA in the aging rodent brain may promote neuroprotection through immunoregulatory and neurogenic mechanisms. Despite previous associations between KYNA and cognitive disruption early in life, our results indicate that inhibition of KMO activity may help alters both behavioral performance and SVZ cellular indices across age. However, because this study used a conventional global KMO knockout model, temporal or cell-type-specific effects could not be resolved. Future work using conditional or inducible KMO knockouts may help clarify the mechanisms involved.

In summary, our findings indicate that age and KMO deletion independently influence non-associative learning and SVZ cellular measures. These results support a role for KMO-dependent KP metabolism in shaping both behavioral function and neurogenic markers during aging. The higher overall SVZ proliferation and neurogenesis observed in KMO^−/− mice, together with preserved olfactory habituation, suggest that KMO activity may contribute to age-related changes in neural function. These findings highlight a potential cellular context in which KMO-dependent KP metabolism intersects with behavioral aging and support further investigation into the therapeutic modulation of KMO for maintaining neural function later in life.

Highlights:

• KMO^−/− mice exhibit stable olfactory habituation and dishabituation across age, but WT mice do not.

• Aged KMO^−/− mice show attenuated declines in SVZ proliferation and neuroblast marker expression compared to WT.

• Findings suggest a novel role for KMO of age-related changes in non-associative learning and SVZ plasticity.