Galactic cosmic radiation produces sex-specific, circuit-selective cognitive vulnerability: countermeasure trade-offs revealed by multi-domain assessment

Galactic cosmic radiation produces sex-specific, circuit-selective cognitive vulnerability: countermeasure trade-offs revealed by multi-domain assessment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Galactic cosmic radiation produces sex-specific, circuit-selective cognitive vulnerability: countermeasure trade-offs revealed by multi-domain assessment Sheridan A. O’Connor, Pragatee Narain, Amishi Mahajan, Grace L. Bancroft, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9476558/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Astronauts on deep space missions face chronic exposure to galactic cosmic radiation (GCR). However, it remains unknown whether mission-relevant multi-ion GCR produces global or circuit-selective cognitive vulnerabilities and whether candidate countermeasures protect uniformly or show domain-dependent trade-offs. Here we used a 33-ion GCR simulation with concurrent countermeasure treatment to address both questions in male and female mice. C57BL/6J mice received 33-GCR (0.75 Gy) or sham radiation with the Nrf2-activating compound CDDO-EA or vehicle, followed by multi-domain behavioral assessment across the hippocampal-nucleus accumbens-prefrontal circuit. Under very high memory load, male Veh/33-GCR mice showed enhanced pattern separation compared to Veh/Sham males, an effect normalized by CDDO-EA. Female mice showed no radiation-induced changes in pattern separation but weighed more than Veh/Sham females and had reduced locomotor activity. Reward-based learning differed by sex: males showed no changes, while female Veh/33-GCR mice displayed enhanced reward anticipation, with both treatments contributing to elevated goal-tracking. For behavioral flexibility, CDDO-EA impaired reversal learning in males regardless of radiation, while 33-GCR impaired reversal learning in females regardless of CDDO-EA. Principal component analysis revealed CDDO-EA under 33-GCR specifically disrupted the balance between stimulus-driven and executive control processes and altered goal-directed behavior, while hippocampal-dependent discrimination maintained its functional relationships with other cognitive domains — confirming circuit-selective rather than global vulnerability. In a preliminary fiber photometry cohort, irradiated males showed enhanced dentate gyrus encoding activity under high memory load. At the cellular level, combined CDDO-EA/33-GCR selectively reduced dentate gyrus progenitors in females. Together, these findings reveal distinct, circuit-selective vulnerability patterns in males and females that would have been invisible to single-sex, single-endpoint designs. CDDO-EA proved a double-edged sword: protecting one cognitive domain while impairing another, a trade-off invisible to single-endpoint assessment and directly relevant to astronaut risk assessment. Biological sciences/Neuroscience Biological sciences/Physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Astronauts on deep space missions beyond Earth's magnetosphere face chronic galactic cosmic radiation (GCR) exposure, a complex mixture of high-energy particles with documented risks to cognitive performance and mission success 1 – 3 . GCR consists predominantly of protons (85%), helium nuclei (14%), and HZE ions (≤ 1%); particles such as ¹⁶O, ¹²C, ⁵⁶Fe, and ²⁸Si are notable for their high linear energy transfer, which causes DNA damage, oxidative stress, and inflammation 1 , 4 – 6 . The CNS is highly vulnerable to these effects, with space radiation inducing persistent structural and functional alterations across multiple brain regions 7 – 11 . Understanding how GCR affects the brain requires assessing multiple cognitive domains simultaneously 12 , yet most ground-based studies have used single-ion exposures, limited behavioral endpoints, and male-only designs, a limitation shared broadly across neuroscience 5 , 13 , 14 .These approaches neither replicate deep space conditions nor capture how radiation affects integrated cognitive function across sexes. Cognitive functions central to astronaut performance — spatial navigation, reward processing, and behavioral flexibility — rely on coordinated activity across the hippocampus, nucleus accumbens (NAc), and prefrontal cortex (PFC) 15 – 18 . These regions function as an integrated circuit; disruption at any node can cascade system-wide 19 – 25 .The hippocampus provides spatial and contextual information, with the dentate gyrus (DG) specialized for pattern separation, the ability to form distinct representations of similar experiences 26 , 27 . Through projections to the NAc and PFC, the hippocampus integrates contextual information with reward value and executive control 28 , 29 . The NAc serves as a hub where hippocampal information converges with reward signals to guide motivated behavior 30 – 32 ; Pavlovian autoshaping paradigms provide a well-validated measure of this function 33 , 34 . The PFC provides top-down executive control, receiving hippocampal input for context-guided decision-making while modulating NAc activity to regulate behavioral flexibility 28 , 35 – 39 . Within this circuit, the DG is a particularly vulnerable node. Adult-born granule neurons contribute to pattern separation and memory formation 40 – 42 , but their high metabolic activity and low antioxidant capacity render them susceptible to oxidative damage 43 , 44 . DG dysfunction can disrupt downstream signaling to the NAc and PFC, with consequences for circuit-wide function 28 . All three regions show vulnerability to radiation-induced changes, including altered dopaminergic signaling, reduced dendritic complexity, and disrupted synaptic plasticity 7 , 45 – 50 . Prior work demonstrates that GCR impairs hippocampal-dependent learning and neurogenesis and induces neuroinflammation 51 – 57 . However, the relative vulnerability of the hippocampus, NAc, and PFC to multi-ion GCR tested within a single study remains unknown. Critical knowledge gaps persist. Prior GCR studies examining hippocampal-dependent tasks have shown inconsistent effects — deficits and enhancements alike — depending on dose and paradigm 3 , 48 , 58 . Recent multi-ion studies demonstrate impairments in hippocampal long-term memory 51 , 55 and PFC-based attention 59 , while single-ion work has shown effects on NAc and reward circuitry 49 , 60 – 66 . No study has assessed how multi-ion GCR affects the integrated hippocampal-NAc-PFC circuit across multiple cognitive domains, or whether effects are global versus domain-selective. Sex differences in space radiation effects remain poorly characterized 3 , 58 , 67 , 68 , and most studies have tested only males, despite evidence that males and females may show different vulnerability patterns 69 . Given that disruption at any circuit node can cascade system-wide 19 – 25 , understanding multi-domain cognitive outcomes in both sexes is essential for astronaut risk assessment 1 , 70 . No adequate shielding currently exists for deep space radiation, making pharmacological countermeasures essential 68 , 71 , 72 . Because GCR-induced oxidative damage and inflammation can compromise multiple nodes within the hippocampal-NAc-PFC circuit 59 , effective countermeasures must provide broad neuroprotection. CDDO-EA (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid ethyl amide) is a candidate countermeasure: a synthetic triterpenoid that activates the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway 73 , 74 . Nrf2 is a master regulator of cellular antioxidant responses, inducing cytoprotective gene expression 75 , 76 . CDDO-EA and related triterpenoids have demonstrated neuroprotective effects in injury and neurological disorder models, with Nrf2 activators mitigating radiation-induced damage across multiple organ systems 76 – 82 . The structurally related Nrf2-activating compound omaveloxolone (Skyclarys) recently received FDA approval for treating Friedreich's ataxia 83 , supporting the translational potential of this compound class. Our laboratory demonstrated that CDDO-EA provides cognitive protection in female mice exposed to 33-GCR, including enhanced pattern separation and improved reversal learning 84 . However, that study used a single behavioral endpoint in males 85 , leaving open whether domain-specific effects were missed. This raises the question: does CDDO-EA protect uniformly, or does protection in one domain come at the cost of impairment in another? Here we investigated how 33-ion GCR exposure affects cognition across multiple domains in male and female mice, and whether CDDO-EA provides broad neuroprotection or domain-selective effects. Based on prior work 3 , 58 , 67 , we hypothesized that 33-GCR would produce domain-specific rather than global cognitive effects that differ between sexes, with hippocampal-dependent functions showing particular vulnerability in males 86 , 87 , and metabolic or locomotor effects predominating in females 3 , 58 , 67 . We further hypothesized that CDDO-EA would show domain-dependent rather than uniform protection, with potential trade-offs across circuit nodes 84 , 85 . To test these hypotheses, we assessed four cognitive domains (hippocampal-dependent pattern separation, reward-based learning, behavioral flexibility, and anxiety-related behavior), alongside body weight, locomotor activity, hippocampal neurogenesis markers, and neural activity over 7.25 months post-irradiation (post-IRR). Neural activity and cellular markers were assessed at later timepoints to determine whether radiation-induced alterations persist beyond the behavioral testing window. Our findings reveal circuit-selective rather than global effects, with distinct vulnerability patterns in males and females and countermeasure trade-offs invisible to single-endpoint assessment — findings with direct implications for astronaut risk assessment on missions beyond Earth orbit. Results Male and female C57BL/6J mice received 33-ion GCR simulation (0.75 Gy) or sham irradiation with concurrent CDDO-EA or vehicle treatment, followed by multi-domain behavioral testing over 7.25 months post-IRR (Fig. 1 ). 33-GCR enhances pattern separation in males under very high memory load; CDDO-EA normalizes this effect Hippocampal-dependent cognition was assessed using the spontaneous location recognition (SLR) task with varying memory loads (Fig. 2 A). Task difficulty increased as objects were placed closer together: dissimilar (d-SLR, low load), similar (s-SLR, high load), and extra-similar (xs-SLR, very high load). Males began testing at 3.25 months post-IRR. Under low and high memory loads (d-SLR, s-SLR), all groups showed similar object exploration during the Sample phase and comparable discrimination and locomotion during the Test phase (Fig. 2 B-H, Supplementary Table 1; all post-hoc p > 0.05). Group differences emerged under very high memory load (xs-SLR). During the Sample phase, males in all groups explored objects similarly (Fig. 2 J; 3 -way RM ANOVA, IRR p 0.05). During the Test phase, Veh/33-GCR males showed a positive d2 ratio (0.20 ± 0.09) while Veh/Sham males showed a negative d2 ratio (− 0.11 ± 0.10), indicating successful discrimination of the novel location in irradiated but not control males (Fig. 2 K; 2 -way ANOVA, Drug p < 0.05, IRR×Drug p < 0.05; post-hoc Veh/Sham vs Veh/33-GCR, p < 0.05). CDDO-EA prevented this radiation-induced enhancement: CDDO-EA/33-GCR males showed a negative d2 ratio (− 0.17 ± 0.08), similar to Veh/Sham controls and less than Veh/33-GCR males (post-hoc p 0.05). In sum, 33-GCR enhanced pattern separation in males specifically under very high cognitive demand, an effect normalized by CDDO-EA. Female mice show no radiation-induced changes in pattern separation despite reduced locomotion during testing In contrast to males, female mice showed no group differences in pattern separation at any memory load. Females began testing at 3.75 months post-IRR. Across all three memory loads, female groups showed similar object exploration during the Sample phase and comparable discrimination during the Test phase (Fig. 3 B-C, F-G, J-K, Supplementary Table 1 ; all post-hoc p > 0.05). However, locomotion during SLR testing differed among female groups. Under d-SLR, Veh/33-GCR females traveled 19.6% shorter distances during the Test phase than Veh/Sham females (Fig. 3 D; 2 -way ANOVA, IRR p < 0.01, Drug p < 0.05; post-hoc p < 0.05). Under s-SLR, CDDO-EA/Sham females traveled 20.6% shorter distances than Veh/Sham females (Fig. 3 H; Drug p < 0.01; post-hoc p < 0.05). Under xs-SLR, IRR affected locomotion overall but no post-hoc differences reached significance (Fig. 3 L; IRR p 0.05). Thus, neither 33-GCR nor CDDO-EA affected pattern separation in females, despite both treatments independently reducing locomotion during testing. 33-GCR produces physiological effects in females that dissociate from cognitive outcomes Since females showed reduced locomotion during SLR testing without cognitive impairment (Fig. 3 ), we assessed body weight in both sexes throughout the 7.25-month experiment and measured home cage locomotor activity at 2.25 months post-IRR to determine whether 33-GCR and CDDO-EA produced systemic physiological effects. All male mice gained weight progressively throughout the experiment, with no weight differences among the four groups (Fig. 4 A, Supplementary Table 1 ; male: 3-way mixed effect RM ANOVA, Time F(6,258) = 353.618, p 0.05). Female mice also gained weight but, unlike males, showed group differences in weight trajectory (Fig. 4 B, Supplementary Table 1 ; female: 3-way RM ANOVA, Time F(6,258) = 250.06, p < 0.001, IRR F(1,43) = 6.28, p < 0.05; Drug × Time F(6,258) = 3.25, p < 0.01, IRR × Time F(6,258) = 12.13, p < 0.001). At 1 month post-IRR, CDDO-EA/Sham females transiently weighed 8.6% more than Veh/Sham females (post-hoc p < 0.01). From 2 months post-IRR through study completion, Veh/33-GCR females weighed 9.3–18.3% more than Veh/Sham females (post-hoc p < 0.05 at months 2–4; p < 0.01 at months 6–7). This weight difference was present before the onset of appetitive touchscreen testing (months 2–3, 9.3–11.2% increase) and became more pronounced during the food-restricted touchscreen period (months 4–7, 13.4–18.3% increase), suggesting that caloric restriction may have amplified rather than driven the effect. Home cage locomotor activity, measured over 18 hours including 12 hours of the dark cycle, was consistent with the locomotion differences observed during SLR testing. In males, Veh/Sham and Veh/33-GCR mice made a similar number of total beam breaks, indicating no effect of radiation alone on home cage activity. However, CDDO-EA/Sham males made 21.9% fewer total beam breaks compared to Veh/Sham males (Fig. 4 C, Supplementary table 1 and Supplementary Fig. 1 ; Kruskal-Wallis p < 0.05; post-hoc Veh/Sham vs. CDDO-EA/Sham, p < 0.05). This reduction was driven by 26.2% less ambulatory movement in CDDO-EA/Sham males compared to Veh/Sham males (Fig. 4 D; Kruskal-Wallis p < 0.05; post-hoc p 0.05). Veh/33-GCR males also made fewer total and ambulatory beam breaks compared to CDDO-EA/Sham males (Fig. 4 C-D; post-hoc p < 0.05), consistent with CDDO-EA reducing activity regardless of radiation status. In females, both 33-GCR and CDDO-EA independently reduced home cage locomotor activity. Veh/33-GCR females made 26.0% fewer total beam breaks than Veh/Sham females (Fig. 4 F, Supplementary Table 1 ; Kruskal-Wallis p < 0.05; post-hoc p < 0.05), with 31.1% fewer ambulatory beam breaks (Fig. 4 G; post-hoc p < 0.05) and 15.9% fewer fine movement beam breaks (Fig. 4 H; post-hoc p < 0.01). Similarly, CDDO-EA/Sham females made 24.7% fewer total beam breaks than Veh/Sham females (Fig. 4 F; post-hoc p < 0.05), with 31.5% fewer ambulatory beam breaks (Fig. 4 G; post-hoc p < 0.05) and a trend toward 12.9% fewer fine movement beam breaks (Fig. 4 H; post-hoc p = 0.06). These data show that 33-GCR exposure produced sustained weight gain and reduced home cage activity in female mice, while CDDO-EA reduced home cage activity in both sexes. These physiological effects dissociated from hippocampal-dependent cognition: females showed no radiation-induced pattern separation deficits despite pronounced weight and activity changes, while males showed radiation-enhanced pattern separation without corresponding weight or home cage activity differences. 33-GCR and CDDO-EA independently enhance goal tracking in females; males show no treatment effects on reward-based learning We next examined whether 33-GCR affected reward-based learning, given its distinct effects on hippocampal cognition in males versus physiological outcomes in females. Mice were tested on touchscreen-based Pavlovian autoshaping over 11 days (Fig. 5 A). This paradigm measures goal tracking (approaches to the reward magazine during CS+ presentation) and sign tracking (preferential approach to CS+ over CS−). In males, all groups increased reward magazine approaches over acquisition days (Fig. 5 B, Supplementary Table 1; 3 -way RM ANOVA, Time p < 0.0001). Within-group increases from early to late acquisition were confirmed in Veh/33-GCR and CDDO-EA/Sham groups (Days 4–5 vs. Days 9 or 11; post-hoc: Veh/33-GCR Day 4 vs Day 11 and Day 5 vs. Day 11, p < 0.05; CDDO-EA/Sham Day 4 vs Day 9 and Day 4 vs Day 11, p < 0.05), with similar numerical trends in Veh/Sham and CDDO-EA/33-GCR groups that did not reach significance. All male groups also progressively increased their preferential approach to CS+ over CS− (Fig. 5 C; Time p < 0.0001) and showed similar accuracy in approaching CS+ (Fig. 5 D; Time p < 0.001). Thus, neither 33-GCR nor CDDO-EA affected Pavlovian learning in males. In contrast, female mice showed treatment-dependent differences in goal tracking (Fig. 5 E, Supplementary Table 1; 3 -way RM ANOVA, Time p < 0.0001, Drug p < 0.01, Time×IRR p < 0.05). Veh/33-GCR females made more reward magazine approaches than Veh/Sham females on Day 9 (167% more; post-hoc p < 0.05), with similar trends on Day 5 (78% more; p = 0.056) and Day 7 (200% more; p = 0.062). CDDO-EA also independently increased goal tracking: CDDO-EA/Sham females made 109% (Day 5) and 191% (Day 6) more approaches than Veh/Sham females (post-hoc p < 0.05). CDDO-EA/33-GCR females also made 181% (Day 6) and 267% (Day 10) more approaches than Veh/33-GCR females (post-hoc p < 0.05). These combined effects suggest additive influences on reward anticipation, with each treatment independently contributing to enhanced goal-tracking. Despite these goal tracking differences, all female groups showed similar sign tracking (Fig. 5 F; Time p < 0.0001; no group differences) and CS+ accuracy (Fig. 5 G; Time p < 0.01; no group differences). Thus, both 33-GCR and CDDO-EA enhanced goal tracking in females, with effects accumulating across treatments, while sign tracking remained unaffected in both sexes. This pattern of results — altered hippocampal function in males but altered reward processing in females — indicates that 33-GCR does not produce uniform effects across interconnected circuit nodes. CDDO-EA impairs reversal learning in males; 33-GCR impairs reversal learning in females Following acquisition, mice underwent reversal learning in which CS + and CS− locations were switched (Fig. 6 A). Males were tested for 5 days; females for 6 days. In males, all groups showed similar numbers of approaches to the new CS+ location across reversal days (Fig. 6 B, Supplementary Table 1; 3 -way RM ANOVA, Time p 0.05). However, CDDO-EA impaired reversal accuracy regardless of irradiation status (Fig. 6 C; Time p < 0.01, Drug p < 0.05). On Day 1, CDDO-EA/33-GCR males showed 45% lower accuracy than Veh/33-GCR males (post-hoc p < 0.01). On Days 2 and 3, CDDO-EA/Sham males showed 36–37% lower accuracy than Veh/Sham males (post-hoc p < 0.05). Thus, CDDO-EA impaired behavioral flexibility in males independent of radiation exposure. In females, 33-GCR impaired reversal learning regardless of CDDO-EA treatment. On Day 6, both irradiated groups made fewer approaches to the new CS+ than their respective sham controls: Veh/33-GCR females made 26% fewer approaches than Veh/Sham females, and CDDO-EA/33-GCR females made 16% fewer approaches than CDDO-EA/Sham females (Fig. 6 D, Supplementary Table 1; 3 -way RM ANOVA, Time p < 0.0001, Time×IRR p < 0.05; post-hoc p < 0.05 for both comparisons). Similarly, 33-GCR reduced reversal accuracy: CDDO-EA/33-GCR females showed 33% lower accuracy than CDDO-EA/Sham females on Day 4 (post-hoc p < 0.05), and Veh/33-GCR females showed 27% lower accuracy than Veh/Sham females on Day 6 (Fig. 6 E; Time p < 0.0001, Time×IRR p < 0.05; post-hoc p < 0.05). The source of behavioral flexibility impairment thus differed between sexes: CDDO-EA impaired reversal learning in males regardless of radiation status, while 33-GCR impaired reversal learning in females regardless of CDDO-EA treatment. This is a direct countermeasure trade-off: CDDO-EA normalized radiation-enhanced pattern separation in males (Fig. 2 ) while independently impairing reversal learning in the same animals. This effect would be missed entirely by any study assessing only a single cognitive endpoint. Neither 33-GCR nor CDDO-EA affects anxiety-like behavior; combined treatment reduces locomotion in females To determine whether the locomotor reductions observed across testing contexts (Figs. 3 , 4 ) reflected anxiety, mice were tested on the elevated plus maze (EPM) at 6.5 months (males) and 6.75 months (females) post-IRR. Males showed no group differences in time spent in open arms, open arm entries, open-to-closed arm ratio, or total distance traveled (Fig. 7 A-D, Supplementary Table 1; 2 -way ANOVA, all p > 0.05 or post-hoc p > 0.05). In females, Drug×IRR interactions emerged for open arm time and entries (Fig. 7 E-F; Drug×IRR p < 0.05 and p 0.05), indicating no true anxiety-like phenotype. Rather, these interactions reflected locomotor differences: CDDO-EA/33-GCR females traveled 22% shorter distances than CDDO-EA/Sham females (Fig. 7 H; IRR p < 0.01, Drug×IRR p < 0.05; post-hoc p < 0.01), consistent with the home cage activity and SLR locomotion patterns described above. These data indicate that neither 33-GCR nor CDDO-EA induced anxiety-like behavior in either sex. The locomotor reductions observed throughout the study therefore reflect a separable physiological effect rather than anxiety-driven changes in exploration. Principal component analysis reveals circuit-specific treatment effects across cognitive domains To examine how 33-GCR and CDDO-EA affected relationships among interconnected cognitive domains, we performed principal component analysis (PCA) on six behavioral measures: pattern separation at three difficulty levels (d-SLR, s-SLR, xs-SLR), goal tracking, sign tracking, and reversal learning (Fig. 8 ). The first three principal components explained 57.6% of total variance (PC1: 20.8%, PC2: 19.7%, PC3: 17.2%; Fig. 8 A). PC1 captured an axis contrasting stimulus-driven responding with executive control. Sign tracking loaded positively (+ 0.582) while reversal learning (− 0.485) and discrimination tasks (d-SLR: −0.463; s-SLR: −0.410) loaded negatively (Fig. 8 B). Vector angles confirmed competitive interactions (> 135°) between sign tracking and reversal/discrimination tasks (Fig. 8 C, inset, orange arcs). CDDO-EA/33-GCR animals showed higher PC1 scores than Veh/33-GCR animals (Fig. 8 D; 2 -way ANOVA, Treatment p < 0.05; post-hoc p < 0.05), indicating a shift toward stimulus-driven responding in the combined treatment group. PC2 captured an axis contrasting hippocampal-dependent discrimination with reversal learning. The discrimination tasks loaded positively (xs-SLR: +0.835; s-SLR: +0.604) while reversal learning loaded negatively (− 0.459). Vector angles between discrimination tasks were 0.05), indicating that hippocampal discrimination capacity was preserved despite radiation and drug treatment. PC3 captured goal-directed behavior, with goal tracking loading strongly positive (+ 0.702) and sign tracking (− 0.385) and reversal (− 0.328) loading negatively. Vector angles showed goal tracking oriented ~ 90° from sign tracking (Fig. 8 C, inset, purple arcs), indicating functional independence. PC3 scores differed across treatment groups (Fig. 8 F; 2 -way ANOVA, Treatment p < 0.05), consistent with the selective goal-tracking changes observed in females. These data demonstrate that CDDO-EA treatment under 33-GCR exposure selectively altered specific circuit relationships, particularly the balance between stimulus-driven and goal-directed behavior (PC1), while still preserving hippocampal discrimination capacity (PC2). Instead, the behavioral effects observed in males reflect altered circuit communication involving NAc and PFC nodes rather than hippocampal damage per se . This selective vulnerability with preserved function is consistent with the domain-specific patterns observed in individual behavioral measures (Figs. 2 – 6 ). 33-GCR produces persistent alterations in dentate gyrus activity during memory encoding To assess whether the domain-specific behavioral effects reflected stable changes in hippocampal circuit function, we performed fiber photometry in a subset of male Veh/Sham and Veh/33-GCR mice at 7.25 months post-IRR (Fig. 9 A-B). Ca²⁺ transients were recorded from DG glutamatergic neurons during SLR testing. Behaviorally, in this small surgical cohort (n = 3–4/group), both groups showed positive d2 ratios under d-SLR. Under xs-SLR, the directional pattern mirrored the main cohort: Veh/Sham mice showed a negative d2 ratio (− 0.15 ± 0.17) while Veh/33-GCR mice showed a positive d2 ratio (+ 0.21 ± 0.06; Fig. 9 C). This did not reach statistical significance, consistent with the reduced power of this subset (all p > 0.05). During the Sample phase (encoding), Veh/33-GCR mice showed enhanced DG activity compared to Veh/Sham mice. Under d-SLR, Ca²⁺ transient rates were similar between groups (p > 0.05; Fig. 9 E), but Veh/33-GCR mice showed 127% higher signal amplitudes at Object zone 3 than Veh/Sham mice (Kruskal-Wallis p < 0.0001; post-hoc p < 0.0001 at all zones; Fig. 9 F). Under xs-SLR, Veh/33-GCR mice showed 29% lower transient rates at Object 3 than Veh/Sham mice (Kruskal-Wallis p < 0.001; post-hoc p < 0.05; Fig. 9 G) but 210–516% higher amplitudes across all object zones (Object p < 0.01, IRR p < 0.0001; post-hoc p < 0.0001 at all zones; Fig. 9 H). During the Test phase (retrieval), Veh/33-GCR mice showed 40% lower transient rates at the Novel location than Veh/Sham mice under d-SLR (Kruskal-Wallis p < 0.01; post-hoc p < 0.01; Fig. 9 I) and 103% lower amplitudes at the Familiar location (p < 0.0001; post-hoc p 0.05; Fig. 9 K). Signal amplitudes showed a significant group effect (Kruskal-Wallis p < 0.001; Fig. 9 L): Veh/33-GCR mice showed higher amplitude at the Novel versus Familiar location (p < 0.001), whereas Veh/Sham mice showed no location-specific difference. Given the small cohort size (n = 3–4/group), these findings should be interpreted with caution and treated as preliminary; the patterns reported here are intended to motivate future work with adequately powered samples rather than to support mechanistic conclusions. These findings are consistent with 33-GCR producing persistent alterations in DG circuit activity detectable 7 months post-IRR, well beyond the behavioral testing window. The enhanced encoding amplitudes and location-selective retrieval patterns suggest that hippocampal circuit dynamics may be persistently shifted rather than transiently perturbed. Combined CDDO-EA and 33-GCR reduces dentate gyrus progenitor cells in females despite intact pattern separation To assess whether treatments affected cellular indices relevant to hippocampal function, we quantified doublecortin-immunoreactive (DCX+) cells in the dentate gyrus at 7.25 months post-IRR (Fig. 10 A). In males, all groups showed similar numbers of DCX+ immature neurons (Fig. 10 B, Supplementary Table 1; 2 -way ANOVA, all p > 0.05), with comparable distribution across the rostro-caudal axis (Fig. 10 C; 3 -way RM ANOVA, Bregma p 0.05), with no differences along the rostro-caudal axis (Fig. 10 G; Bregma p 0.05). In females, DCX+ immature neuron numbers were similar across all groups (Fig. 10 D; 2 -way ANOVA, all p > 0.05), with comparable rostro-caudal distribution (Fig. 10 E; 3 -way RM ANOVA, Bregma p < 0.0001). However, 33-GCR reduced DCX+ progenitor cells in CDDO-EA-treated females. CDDO-EA/33-GCR females had 21% fewer total DCX+ progenitor cells than CDDO-EA/Sham females (Fig. 10 H; 2 -way ANOVA, IRR p < 0.05; post-hoc p < 0.05). This reduction was particularly evident in the dorsal DG: at Bregma − 1.90, CDDO-EA/33-GCR females had significantly fewer progenitors than CDDO-EA/Sham females (Fig. 10 I; 3 -way RM ANOVA, Bregma p < 0.0001, IRR p < 0.05; post-hoc p 0.05), indicating that this progenitor reduction required the combination of CDDO-EA and 33-GCR. This suggests that Nrf2 activation may alter the cellular context in which radiation affects progenitor populations. These data demonstrate that combined CDDO-EA and 33-GCR exposure reduced DCX+ progenitor cells in females, particularly in the dorsal hippocampus, despite intact pattern separation performance in these animals (Fig. 3 ). Pattern separation was assessed at 3.75 months post-IRR while DCX+ cells were quantified at 7.25 months post-IRR; these measurements are therefore temporally decoupled and cannot be interpreted as a direct cellular-behavioral dissociation. Discussion Prior GCR studies have assessed single cognitive endpoints in single sexes, leaving open whether radiation effects are global or circuit-selective and whether countermeasures protect uniformly or trade off across domains. The present data resolve both questions, and the answers are more complex than either framing anticipated. Here we show that 33-GCR produced domain-specific rather than global cognitive effects, with distinct vulnerability patterns in males and females. In addition, CDDO-EA acted as a double-edged sword: normalizing some radiation effects while impairing others, a trade-off invisible to single-endpoint assessment. In males, 33-GCR enhanced pattern separation specifically under very high memory load, consistent with prior single-ion studies showing improved pattern separation following ⁵⁶Fe or ²⁸Si exposure in males 86 , 87 . The convergence across radiation types and testing paradigms suggests this reflects a genuine radiation effect rather than a paradigm artifact. CDDO-EA normalized this enhancement, to our knowledge the first demonstration that this compound can reverse radiation-induced enhancements rather than solely prevent impairments. Enhanced pattern separation does not necessarily indicate improved cognitive function: in clinical populations, overly distinct memory representations can interfere with generalization and flexible cognition 88 . In females, pattern separation did not differ among groups at any memory load, contrasting with prior work reporting impairments at earlier timepoints using a different paradigm 51 . That task required spatial memory retention over ≥ 24 hours, whereas the SLR task tests discrimination within ~ 35 minutes, a fundamental difference in memory demand. Furthermore, females in the prior work showed hippocampal-dependent deficits during training before pattern separation testing 51 , raising the possibility that pre-existing impairments confounded performance. Consistent with the present findings, a prior 33-GCR study in females using touchscreen-based tasks also found no radiation effect on pattern separation 84 . Fiber photometry revealed enhanced DG signal amplitudes during encoding in irradiated males, particularly under very high memory load, with location-selective retrieval patterns at 7 months post-IRR. To our knowledge, this is the first use of in vivo Ca²⁺ imaging to assess hippocampal activity during cognitive testing in the context of space radiation. The stage-specific nature of these alterations, enhanced encoding but shifted retrieval, points to changes in circuit dynamics rather than uniform suppression or excitation⁹ 89,90 . Increased encoding amplitude may strengthen memory formation and contribute to more distinct representations 26 , 27 , 91 , 92 , though causal relationships cannot be established from these data. These alterations were detectable 7 months post-IRR, suggesting stable rather than transient changes in circuit function. The small cohort size (n = 3–4/group) limits interpretation; these findings are best treated as preliminary. DCX+ progenitor cells were reduced in CDDO-EA/33-GCR females at 7.25 months post-IRR, but not in Veh/33-GCR females, indicating this reduction required combined treatment. This contrasts with our prior work showing that 33-GCR alone reduces DCX+ immature neurons but not progenitors at 14.25 months post-IRR 84 , suggesting CDDO-EA may alter the temporal trajectory of radiation effects on neurogenesis-related populations: combined treatment affects earlier-stage progenitor cells at intermediate timepoints, whereas radiation alone affects later-stage immature neurons at extended timepoints. Since pattern separation was assessed at 3.75 months post-IRR and DCX+ cells at 7.25 months post-IRR, these measurements are temporally decoupled and cannot be interpreted as a direct cellular-behavioral dissociation; rather, they reveal a late-emerging cellular consequence of combined treatment not captured by behavioral assessment alone. Female mice often show resilience to space radiation-induced cognitive deficits relative to males 3 , 58 , 67 , 68 , 93 – 97 , and the present findings are consistent with this pattern. In males, 33-GCR altered hippocampal-dependent cognition without affecting body weight or home cage activity, suggesting effects concentrated in cognitive circuits rather than distributed across metabolic and motor systems 86 , 98 , 99 . The mechanisms underlying female resilience are not established; proposed factors include differences in neuroimmune responses, antioxidant capacity, and gonadal hormone signaling 3 , 100 – 102 , though which, if any, contribute here cannot be determined from the present data. Preserved cognition in females may nonetheless come at a metabolic cost: persistent weight gain in Veh/33-GCR females suggests radiation-induced disruption of energy homeostasis 48 , 103 , 104 , and reduced locomotor activity may reflect metabolic changes or altered motivation rather than cognitive impairment 84 . These distinct patterns between males and females underscore why single-sex studies, still common in radiation neuroscience 3 , 58 , 67 , 93 and in neuroscience more broadly 14 , can produce incomplete or misleading conclusions about radiation effects on cognition. Locomotor reductions were observed across multiple contexts in both sexes but followed sex-specific patterns: in males, CDDO-EA alone reduced home cage activity while radiation had no additional effect, whereas in females both 33-GCR and CDDO-EA reduced activity across home cage, SLR, and EPM contexts. These reductions did not confound cognitive outcomes: pattern separation was enhanced in irradiated males despite no locomotor changes, and intact in females despite pronounced locomotor reductions. The EPM confirmed these reductions do not reflect anxiety, supporting the interpretation that locomotor and cognitive effects represent separable consequences of treatment. The autoshaping paradigm dissociates sign-tracking (dorsolateral striatum/amygdala-dependent), goal-tracking (NAc/PFC-dependent), and reversal learning (OFC-dependent) 36 , 105 – 110 . The selective enhancement of goal-tracking in females, with no autoshaping effects in males, points to regional rather than uniform circuit vulnerability. Goal-tracking changes without corresponding hippocampal alterations may reflect the NAc's integration of multiple input streams beyond the hippocampus, including direct cortical and amygdala inputs 111 – 113 . CDDO-EA enhanced goal-tracking in females but had no reward-related effects in males, indicating sex-specific modulation of NAc-PFC circuitry by Nrf2 activation 114 . This sex specificity extended to the combined treatment: CDDO-EA and 33-GCR produced additive goal-tracking enhancement in females, with no parallel effect in males. This raises the possibility that GCR and Nrf2 activation converge on shared circuitry in a sex-dependent manner, consistent with evidence that opponent striatal monoamine signaling modulates reinforcement behavior 113 . The specificity of these effects to goal-tracking rather than sign-tracking suggests CDDO-EA preferentially modulates NAc-PFC circuits 36 , 105 – 110 , possibly through sex-dependent differences in dopaminergic signaling or Nrf2 expression 76 . In males, CDDO-EA impaired reversal accuracy without affecting initial acquisition, pointing to selective disruption of behavioral updating, the ability to revise a learned response when contingencies change, rather than reward learning itself. In females the picture was different; the drug was not the culprit, radiation was, and the impairment likely reflects the learning context established during acquisition. Enhanced goal-tracking in irradiated females may have created strong reward-location associations that made updating cue-reward contingencies particularly difficult, given that sign-tracking and goal-tracking behaviors resist contingency changes 115 – 118 . Reversal impairment looked the same on the surface in both sexes but arose from distinct circuit-level processes: behavioral updating in males, acquisition strategy in females. The autoshaping reversal impairment in females contrasts with our previous finding that CDDO-EA/33-GCR females showed enhanced cognitive flexibility in touchscreen-based spatial discrimination reversals 84 . This dissociation is reconcilable: the touchscreen task requires spatial discrimination without competing goal-tracking strategies, so reversal does not require overcoming a strongly established reward-location habit. Irradiated females thus retain capacity for flexible behavioral updating; the impairment is context-dependent, not a signature of broad OFC dysfunction. PCA quantified what the individual behavioral results suggested qualitatively. The PC2 null result is particularly informative: pattern separation tasks and reversal learning maintained their relationship across all treatment groups, indicating the hippocampus itself was not globally disrupted. The behavioral effects seen in males therefore reflect altered signaling at NAc and PFC nodes rather than hippocampal damage per se. PC1 and PC3, by contrast, shifted with treatment, capturing the rebalancing of stimulus-driven versus goal-directed behavior. Some circuit relationships were disrupted; others held. That selective pattern, rather than uniform impairment, is the defining feature of circuit-selective vulnerability. Domain-specific drug-induced cognitive effects are well recognized in clinical pharmacology 119 ; preclinical countermeasure evaluation has rarely followed suit, largely because single cognitive endpoints remain the norm 120 – 122 . Region-specific neural effects of space radiation are equally well established 12 , 123 , but the corollary, that countermeasures may show domain-dependent trade-offs, has not been systematically tested. The practical consequences are stark: a study assessing only pattern separation would conclude CDDO-EA is protective; a study assessing only reversal learning would conclude it is harmful. Neither conclusion captures the reality that CDDO-EA's effects are circuit-dependent, sex-dependent, and only fully visible through multi-domain assessment. Three limitations warrant mention. Fiber photometry and DCX+ quantification were conducted at 7–7.25 months post-IRR while behavioral testing occurred at 3.25–4.5 months; concurrent measurements at matched timepoints are needed to establish whether cellular changes relate directly to behavioral performance. Fiber photometry was conducted only in males; extending these recordings to females would clarify whether distinct circuit activity patterns underlie preserved pattern separation in that sex. The separate statistical analyses of males and females, while appropriate given divergent response profiles, preclude direct statistical comparison of sex differences, a limitation shared broadly in the field 14 . Male and female mice showed distinct, circuit-selective vulnerability patterns that would have been invisible to single-task or single-sex designs. The core finding is not simply that radiation affects cognition, but that its effects are circuit-specific and sex-dependent. A candidate countermeasure can simultaneously protect one function while impairing another, a trade-off invisible to single-endpoint assessment. As space agencies plan missions beyond Earth orbit, these findings have immediate relevance for astronaut risk assessment. Any stressor or neuroprotective intervention evaluated with limited behavioral endpoints risks missing exactly these trade-offs; the present framework provides a template for doing better. Materials and methods Animals Male (M, n = 124) and female (F, n = 48) C57BL/6J mice ( Mus musculus C57BL/6J, 4.5-5 months old; RRID:IMSR_JAX:000664; Jackson Laboratory, Bar Harbor, ME) were shipped to Brookhaven Laboratory Animal Facility (BLAF) at Brookhaven National Laboratory (BNL, Upton, NY). Males arrived in variable group sizes (2 cages of 5 mice, 23 cages of 4 mice, 1 cage of 3 mice, 8 cages of 2 mice, and 3 single-housed mice), while females arrived in uniform groups (12 cages of 4 mice). Upon arrival, mice were maintained with their original cagemates throughout the study. After three days of acclimation, mice received ear punch followed by first weight measurement. Two days later, mice were transported to the NASA Space Radiation Laboratory (NSRL) within BLAF for treatment with either 33-GCR IRR or Sham IRR and returned to BLAF the following day. At both facilities, mice were housed 4/cage in HEPA-filtered, closed airflow vivarium systems under a 12:12 h light/dark cycle (06:00 light on) at 22°C, 30–70% humidity with standard rodent chow (5015; Lab Diet, cat# 0001328) and water ad libitum. Two days post-IRR, mice were transported by ground to Children's Hospital of Philadelphia (CHOP) and held in quarantine for 6 weeks with ad libitum access to medicated chow (13 PPM ivermectin and 150 PPM fenbendazole; Test Diet, custom cat# 1813527[5SKU]). Behavioral testing began at 9 weeks post-IRR when mice were released from quarantine and returned to standard chow (5015). At CHOP, mice were housed in HEPA-filtered, closed airflow vivarium systems (Enviro-Gard™ A; Lab Products Inc.) under a 12:12 h light/dark cycle (06:15 light on) at 20–23°C, 30–40% humidity. Each cage received a nestlet square at cage changes; no other enrichment was provided. All procedures were approved by IACUCs at BNL and CHOP in accordance with AAALAC and NIH guidelines (CHOP: AAALAC #000427, PHS D16-00280 [OLAW A3442-01]; BNL: AAALAC #000048, PHS D16-00067 [OLAW A3106-01]). Our reporting adheres to ARRIVE 2.0 guidelines 124 . Drug administration Cages were sequentially preassigned to treatment groups in rotating order (Veh/Sham, Veh/33-GCR, CDDO-EA/Sham, CDDO-EA/33-GCR), balanced for mean cage body weight and cage size (number of mice per cage). Drug treatment groups consisted of mice receiving either CDDO-EA (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid ethylamide, 4 mg/kg IP; MedChemExpress, cat# HY-12213; n = 83, M:59, F:24) or vehicle control (matching volume IP; n = 89, M:65, F:24). The vehicle solution was prepared according to the manufacturer's instructions and consisted of 5% DMSO (Sigma-Aldrich, cat# D2650) and 20% Sulfobutylether-β-Cyclodextrin (SBE; MedChemExpress, cat# HY-17031) in 0.9% saline solution (Grainger, cat# 3PWK4). Both CDDO-EA and vehicle were administered intraperitoneally once daily between 8:00–10:00 AM for three consecutive days. IRR was performed on Day 2 of the treatment regimen (one day after the first injection, concurrent with the second injection, and one day before the final injection). Irradiation (IRR) On the second day of drug treatment (Day 0 of IRR), mice underwent IRR during the BNL 22A campaign as previously described 84 . Mice were placed with cagemates in well-ventilated polycarbonate containers (10 × 10 × 4.5 cm), with 2 mice per container when possible or individually housed if no cagemate was available. Irradiated mice were exposed to 75 cGy of NASA's whole-body 33-beam GCR simulation delivered over a 60 × 60 cm field for approximately 1.25 hours beginning at 11:45 AM. Beam uniformity and dosimetry were monitored by NSRL staff. Sham-irradiated mice were placed in containers with cagemates (or individually) for the same duration but were not exposed to the beam. This resulted in four experimental groups: Veh/Sham (M:32, F:12), Veh/33-GCR (M:33, F:12), CDDO-EA/Sham (M:30, F:12), and CDDO-EA/33-GCR (M:29, F:12). Surgery C57BL/6J male mice (3–4 mice/group) underwent viral infusion and fiber implantation at 6 months post-IRR. Before surgery, each mouse was weighed and the head was shaved using an electric clipper (Wahl, cat# 41590-0438). Anesthesia was induced in an induction chamber with 4–5% isoflurane (Piramal Pharma Limited, cat# 66794-013-25) in 100% oxygen and maintained at 1–3% isoflurane during surgery. Ophthalmic lubricant (Dechra, cat# B00HGMZ7RQ) was applied to both eyes, and buprenorphine ER (1 mg/kg, s.c.) was administered before incision. Following aseptic preparation of the surgical site with betadine (Avrio Health, cat# 67618-155-16) and 70% isopropyl alcohol, a midline incision was made. The skull was scored with a scalpel blade and treated with 30% hydrogen peroxide solution (Sigma Aldrich, cat# MKCJ1024). A burr hole was drilled above the target site using a surgical electric drill with a ⅛ inch engraving bit (Dremel, cat# 7350). AAV9-CaMKII-GCaMP6f (Penn Vector Core, 100834) was unilaterally infused into the hilus of the dorsal dentate gyrus (DG) hilus (A/P -2.0 mm, M/L -1.4 mm, D/V -2.2 mm from bregma) using a 33-gauge Hamilton syringe (Hamilton, cat# 2141205) at 0.1 µl/min 125 . An optic fiber (Thorlabs, #CFML22L05, Ø1.25 mm SS ferrule, Ø200 µm core, 0.22 NA, L = 2 mm) was implanted in the molecular layer of the DG middle/outer molecular layer (A/P -2.0 mm, M/L -1.4 mm, D/V -1.8 mm from bregma). The fiber was secured to the skull with light-cured resin (Ivoclar Vivadent AG, cat# 595979US) followed by light-cured adhesive (Pearson, cat# 595979). Following surgery, the incision was sutured, triple-antibiotic ointment was applied topically, and meloxicam (5 mg/kg, s.c.; Norbrook, cat# 5552904010) was administered. Mice were monitored daily for 48 hours post-operatively. Body weight monitoring and health observations Mouse body weight was measured monthly from arrival at BNL (4.5 months of age) through tissue collection (12.25 months of age). Health status and cage conditions were monitored during weighing sessions and biweekly cage changes by the Children's Hospital of Philadelphia (CHOP) Department of Veterinary Resources, with documentation of fighting, illness, or loss of cagemates. When aggressive behavior was identified, aggressor mice were isolated into separate cages to prevent injury. Seven male cages required splitting due to aggression (3 Veh/Sham, 3 CDDO-EA/Sham, 1 Veh/33-GCR). All mice from these disrupted cages were excluded from behavioral testing to maintain consistent social housing conditions across experimental groups. Behavioral testing Overview of behavioral testing Behavioral testing groups were selected in rotating order across treatment groups (Veh/Sham, Veh/33-GCR, CDDO-EA/Sham, CDDO-EA/33-GCR) and balanced for mean cage body weight. Most cages contained 4 mice per cage when behavioral testing began, except one female cage with 3 mice. Two deaths occurred: female #28 (CDDO-EA/Sham) died at 1 month post-IRR before behavioral testing began, and male #9 (CDDO-EA/Sham) died at 2.5 months post-IRR during locomotor testing. Both deaths reduced their respective cages to 3 mice. Despite these losses, both affected cages were retained for behavioral testing due to limited female availability and to avoid excluding the male cagemates who had already begun testing. Both deceased mice were excluded from analyses. This resulted in 47 mice per sex (total n = 94) for behavioral testing. Following release from quarantine at 2 months post-IRR, behavioral testing began at 2.25 months post-IRR with locomotor activity recording (Fig. 1 A). Gentle handling (2 min/day) began at 2.5 months post-IRR and continued for 5 days. Male mice underwent arena habituation for 5 days beginning at 3 months post-IRR to acclimate to the Spontaneous Location Recognition (SLR) arena, followed by SLR testing at 3.25 months post-IRR. Males then completed the Autoshaping task (Pavlovian learning) at 3.5 months post-IRR and anxiety behavioral testing at 6.5 months post-IRR. Female mice underwent arena habituation for one week beginning at 3.5 months post-IRR, followed by SLR testing at 3.75 months post-IRR. Females then completed the Autoshaping task at 4.5 months post-IRR and anxiety behavioral testing at 6.75 months post-IRR. Home cage activity monitoring Each mouse was individually placed in a clean mouse conventional cage containing fresh bedding to record 18 hours of locomotion activity from 4pm to 10am. This cage was positioned between 4×8 photocells, with identical lighting parameters to home housing room dim/red lighting during the light cycle and red lighting during the dark cycle. Their movement across the XY plane was monitored by a computer-controlled photobeam activity system (San Diego Instruments), which recorded photocell beam breaks in 15-minute intervals over a period of 18 hours 126 . One male mouse from (mouse ID #10, group: CDDO/Sham) was excluded due to equipment failure. Spontaneous location recognition (SLR) The spontaneous location recognition (SLR) task was performed following established protocols 89 . Arena Setup and Objects. The testing arena consisted of a circular open field with 20 marked segments radiating from the center, etched by laser cutting at 18° intervals on the base and covered with corncob bedding during testing. Objects consisted of 50 ml conical centrifuge tubes containing three blue latex gloves, secured to the arena base with screws and nuts. Each arena was surrounded by three-sided black cardboard barriers displaying three distinct visual cues, with two additional cues mounted on the wall. These spatial cues remained consistent throughout testing to provide reliable landmark references. Habituation . Prior to SLR testing, mice underwent gentle handling (2 min/day) for 5 days to reduce stress, followed by arena habituation beginning at 3.0 months post-IRR for males and 3.5 months post-IRR for females. Subjects were placed in the arena with spatial cues for 10 minutes daily over five consecutive days. Testing Procedures . On test days, mice were transported to the testing room and acclimated in their home cages for 30 minutes. Testing time was kept consistent across subjects. Between subjects, one scoop of bedding was removed and replaced with clean bedding, and arena floors and walls were wiped with 10% ethanol solution to eliminate olfactory cues. The experimenter exited the room during testing sessions. Experimental Design and Memory Loads. The SLR experiment evaluated three memory loads based on spacing between objects 2 and 3: Dissimilar (d-; low memory load, 108° apart), Similar (s-; high memory load, 72° apart), and Extra similar (xs-; very high memory load, 36° apart). During the sample phase (10 min), three identical objects were placed at predetermined distances corresponding to one of the three memory load conditions, and mice freely explored the arena. Following a 35-minute retention interval in home cages, mice were returned for the test phase (5 min) with two objects: one at a familiar location (matching object 1 from the sample phase) and one at a novel location (positioned midway between the original locations of objects 2 and 3) 89 . Data Analysis . Time spent in object zones was calculated based on nose position, and movement distance was calculated based on center body position using EthoVision XT 12 (Noldus Information Technology). The discrimination index (d2 ratio) was calculated as d2 ratio = Time spent in (novel location - familiar location) / Time spent in both locations Autoshaping Autoshaping was performed between 8:00 AM and 2:00 PM daily (Monday-Friday) at CHOP using the Bussey-Saksida operant touchscreen platform (Lafayette Life Sciences, cat# 80614A) equipped with the ABET Core Intelli-Interface (Lafayette Life Sciences, cat# 81430) and ABET II Software (Lafayette Life Sciences, cat# 89509) 127 , 128 . The operant chamber contained a light, auditory cue speaker, food dispenser, and two white vertical rectangular response windows (6.5 × 14 cm) positioned left and right of the reward dispenser. Two infrared photobeams detected approaches to the touchscreen and entries/exits to the food magazine. Behavioral tasks and data collection were controlled by ABET II Autoshaping Software (cat# 89544). The reward stimulus was Strawberry Ensure® Nutrition Shake (Abbott Laboratories) delivered without food deprivation. Habituation. Mice were handled for one minute daily for three days prior to habituation. During Hab 1 (one day), mice were placed in the chamber for 10 minutes with all lights off while strawberry milkshake was delivered into the food tray for 2800 ms (70 µl). The number of broken photobeams was recorded to assess locomotor activity. During Hab 2, mice remained in the chamber for 30 minutes while milkshake (280 ms, 7 µl) was delivered after variable intervals (0–30 seconds), accompanied by tray light illumination and a tone. Once reward was delivered, the program waited for the mouse to enter the food tray before restarting the variable interval. Upon tray entry, the tray light was turned off and the procedure repeated. The criterion for Hab 2 was 30/40 trials within the 30-minute session, which most mice achieved within 2 days. Acquisition Training. Following habituation, mice underwent 11 days of autoshaping acquisition to associate one side of the screen as a positive conditioned stimulus (CS+) and the other side as a negative conditioned stimulus (CS-), counterbalanced between animals. Trials were conducted in pairs presenting both CS + and CS- stimuli in random order, ensuring no more than two consecutive presentations of the same stimulus type and preventing the same side from being illuminated first in trial pairs more than 3 times consecutively. After a variable interval (10–40 seconds), the chosen stimulus was presented for 10 seconds when the animal was breaking the rear beam. For CS- trials, another variable interval (10–40 seconds) followed before the other stimulus was presented. For CS+ trials, reward was delivered, tray entry was awaited, then a 10–40 second variable interval preceded the other stimulus presentation. Sessions lasted 30 minutes. The criterion was completing at least 25 trials within the 30-minute session for two out of three consecutive days. Reversal Learning. After 11 days of acquisition training, mice underwent reversal learning for 5 (males) or 6 (females) days using identical procedures except CS + and CS- assignments were switched. The difference in reversal duration between sexes was due to equipment availability. Data (the number of reward chamber approaches, approach difference between CS + and CS- approach, accuracy to CS+ approaches) were collected by ABET II Software (cat# 89509). Elevated plus maze Anxiety behavior in the EPM (Harvard Apparatus, cat# 760075) was assessed 6 mon post-IRR. The EPM apparatus (99cm elevation; 2 open and 2 closed arms each measuring L 67cm x W 6cm, closed arm walls H 17cm) was constructed as described previously 84 , 86 . Mice were placed in the center of the apparatus pseudorandomly facing one of two open arms, and allowed 5 minutes of free exploration under white lighting conditions (200 lux). Ethovision ver 12 software (Noldus Information Technology) was used to record the time spent in the open arms, closed arms, and center zone, as well as the frequency of entries into each area. From these measurements, an exploration index was calculated: The ratio of the time spent in open arms vs closed arms: Ratio of time open: closed = Time in open arms / Time in closed arms Fiber photometry recording A separate in vivo imaging cohort (n = 7 males, 3 Veh/Sham and 4 Veh/33-GCR mice) received AAV9-CaMKIIa-GCaMP6f viral infusion in the DG hilus and optic fiber implantation in the molecular layer of the DG at 6 months post-IRR to monitor DG granule cell activity as described in Surgery section. This group performed the SLR with fiber photometry (FP) recording at 7.25 months post-IRR. Recording System and Setup. Three weeks post-surgery, fiber photometry recordings were conducted using the Neurophotometrics FP3002 system (Neurophotometrics LTD, CA) with customized Bonsai software during the SLR paradigm as previously described 129 – 131 . A patch cord (Doric Lenses, D204-80052, BBP(2)_200/220/900 − 0.37_2m_SMA-2xMF1.25) was connected unilaterally to the implanted optic fiber. Calcium-dependent fluorescence changes were recorded at 470 nm excitation, while calcium-independent fluorescence was captured at 415 nm excitation to control for motion artifacts and photobleaching. Data acquisition occurred continuously during both the Sample phase (10 min) and Test phase (5 min) of the SLR paradigm, with simultaneous behavioral video recording. Behavioral Video Analysis . Behavioral videos were analyzed using Social LEAP Estimates Animal Poses (SLEAP v1.4.1) for pose estimation 132 , followed by Simple Behavioral Analysis (SimBA version 3.2.8 with Python 3.10) for behavioral quantification 133 . For SLEAP model training, 461 frames from 19 videos were annotated, achieving mean Average Precision of 0.854 and mean Average Recall of 0.881. All experimental videos were analyzed using this validated model. SLEAP output files (.csv format) were imported into SimBA for exploratory behavior quantification. Regions of interest were defined as: (1) a 5-cm radius circle centered at the base of each object, and (2) a 29-cm radius circle encompassing the entire arena. Behavioral metrics extracted included time spent in each zone, zone entry counts (calculated relative to nose position), total movement distance, and average velocity (calculated relative to center body position). Frame-by-frame Boolean values indicating zone occupancy were extracted for temporal alignment with photometry data using custom MATLAB code (MathWorks). Fluorescence changes (ΔF/F) were calculated as previously described 134 : ΔF/F = (Raw fluorescence - Fitted fluorescence) / Baseline fluorescence (F₀) The ΔF/F signals were normalized using Z-score transformation: Z-score ΔF/F=[(ΔF/F) - mean(ΔF/F)]/ standard deviation(ΔF/F). Calcium transient peaks were identified using a noise threshold defined by the Median Absolute Deviation (MAD) method. Peaks were defined as local maxima with: (1) amplitude greater than 0.1×MAD above baseline, and (2) minimum separation of 0 data points between events. Peak frequency and amplitude were quantified from these identified events. For event-locked analysis, ΔF/F traces were aligned to specific behavioral events, including entry into the Familiar Object zone and Novel Object zone during the Test phase of the SLR paradigm. Tissue collection and processing Brain tissue was collected at 7.25 months post-IRR for the behavioral cohort and at 8.75 months post-IRR for the in vivo imaging cohort. Following decapitation, brains were immersed in 4% paraformaldehyde (PFA; Sigma-Aldrich, cat# P6148) in PBS for 3 days 135 , 136 , then cryoprotected by immersion in 30% sucrose (Fisher Scientific, cat# S5-3) containing 0.01% sodium azide (NaN₃; Sigma-Aldrich, cat# S8032) at 4°C for 24 hours until complete equilibration. Brains were coronally sectioned at 40 µm thickness using a freezing microtome (Leica SM2010R). The left hemisphere was marked with a 26-gauge needle on the dorsal cortical area for consistent orientation. Serial sections were collected systematically throughout the hippocampus for stereological assessment as previously reported 87 , 135 , 137 . Sectioned tissue was stored in 1× PBS containing 0.01% NaN₃ at 4°C until further processing. Immunohistochemistry (IHC) IHC was performed on slide-mounted coronal brain sections as previously described 84 , 136 . Sections underwent antigen retrieval by placement in near-boiling citric acid (pH 6.0; Fisher Chemical, cat# A940-500). Endogenous peroxidase activity was quenched by incubation in 0.3% hydrogen peroxide (Sigma, cat# H-1009) in PBS. Non-specific binding was blocked with 3% normal donkey serum (Jackson ImmunoResearch, cat# 017-000-121) in 0.3% Triton X-100 in PBS. Sections were incubated overnight at room temperature with primary antibody against doublecortin (goat anti-DCX; Santa Cruz Biotechnology, cat# SC-8066; 1:500) or GFP (chicken anti-GFP; Aves Labs, cat# GFP-1020; 1:3000) diluted in 3% normal donkey serum with 0.3% Tween-20 in PBS. Following PBS washes, sections were incubated for 1 hour at room temperature with biotinylated secondary antibodies at 1:200 (donkey anti-goat IgG for DCX, Jackson ImmunoResearch, cat# 705-065-003; donkey anti-chicken IgG for GFP, Jackson ImmunoResearch, cat# 703-065-155; 1:200). After additional PBS washes, signal amplification was achieved using avidin-biotin complex (ABC; Vector Laboratories, cat# PK-6100) for HRP conjugation. The HRP signal was visualized using 3,3'-diaminobenzidine (DAB; Thermo Fisher Scientific, cat# 1856090) for DCX and Cy3-conjugated Tyramide Signal Amplification substrate (PerkinElmer, cat# FP1050) for GFP. Nuclei were counterstained with Fast Red (Vector Laboratories, cat# H-3403) for DCX IHC and DAPI (Roche, cat# 236276) for GFP IHC. Sections were dehydrated through a graded ethanol series, cleared in Citrasolv, and coverslipped using DPX mounting medium (Electron Microscopy Services, cat# 13512) with 24×60 mm coverglasses (VWR, cat# 48393). Stereological cell counts DCX-immunoreactive (DCX+) cells were quantified by an observer blinded to experimental conditions using an Olympus BX-51 brightfield microscope at 400× magnification. DCX+ cells in the subgranular zone (SGZ) of the dentate gyrus granule cell layer (GCL) were counted in the right hemisphere of each section, with left hemisphere counted only if right hemisphere damage occurred. Stereological principles were applied as previously described [44,111]. Quantification was conducted along the entire anterior-posterior hippocampal axis (-0.82 to -4.33 mm from bregma). Two cell classifications were recorded: (1) DCX+ immature neurons, defined as brown-stained soma in the SGZ with neurite and dendrite outgrowth containing at least one dendritic branching node, and (2) DCX+ progenitor cells, defined as brown-stained soma in the SGZ lacking neurite extension. Total cell populations were calculated using the following stereological formula 138 . The raw cell counts for each bregma level are presented separately. Total population of cells = Total cells counted x 1/ssf x 1/asf x 1/hsf where ssf is the section sampling fraction (1/9 for one hemisphere analysis), asf is the area sampling fraction (1, as all cells were counted in sampled sections), and hsf is the height sampling fraction (1, given minimal edge artifact effects in counting soma < 10 µm with ssf 1/18), as described previously 136 – 138 . Since only one hemisphere was counted, the total population for both hemispheres was calculated as: Total population of cells in both hemispheres = (Total cells counted × 9) × 2 Principal component analysis Principal component analysis (PCA) was performed on six behavioral measures: spatial discrimination (low [dSLR] and high [sSLR]: mean d2 ratio), pattern separation (very high [xsSLR]: mean d2 ratio), goal tracking (mean reward chamber approaches, days 4–7), sign tracking (mean CS + vs. CS- approach difference, days 4–7), and reversal learning (mean CS+ approaches after reversal, days 1–4). For Pavlovian conditioning measures (goal and sign tracking), performance was averaged across days 4–7 of acquisition when animals reached maximal performance. For reversal learning, performance was averaged across days 1–4 post-reversal to capture initial acquisition of the new contingency when cognitive flexibility demands were highest. All measures were z-score normalized across all subjects (grand mean = 0, SD = 1) prior to analysis. PCA was conducted using scikit-learn (version 1.3.0) in Python 3.10 139 . Component loadings (eigenvectors × √eigenvalues) represent variable-component correlations; loadings >|0.40| were considered substantial. The first three principal components explained 57.6% of total variance (PC1: 20.8%, PC2: 19.7%, PC3: 17.2%). To quantify functional relationships between behavioral domains, vector angles from the three-dimensional loading space (PC1, PC2, PC3) were calculated using the arccosine of normalized dot products. Angles 135° indicated competitive interactions. Computer scripts Custom analysis scripts were developed in MATLAB and Python 3.10 (Google Colaboratory). A MATLAB script integrated Bonsai Ca²⁺ signal output with SLEAP pose estimation data to calculate zone-specific transient rates and amplitudes during fiber photometry recordings. Python scripts processed 15-min interval photobeam locomotion data and performed principal component analysis (scikit-learn 1.3.0) on multi-domain behavioral measures, including vector angle calculations and visualization. Blinding, subject number, and data removal All behavioral testing, tissue collection, and data analysis were conducted by investigators blinded to treatment conditions. Two mice were found dead during the study: one female (CDDO-EA/Sham) at 1 month post-IRR prior to data collection, and one male (CDDO-EA/Sham) at 2.5 months post-IRR after home-cage locomotion recording but before SLR testing. These subjects were excluded from all analyses. No additional mice were removed due to husbandry issues or veterinary recommendations. Task-specific subject numbers with exclusion criteria are detailed below and summarized in Supplementary Table 2. Treatment groups are ordered as Veh/Sham, Veh/33-GCR, CDDO-EA/Sham, and CDDO-EA/33-GCR throughout. Home-cage locomotion recording: One male CDDO-EA/Sham subject was not recorded due to technology failure. Final male subject numbers: n = 12, 12, 10, and 12. Final female subject numbers: n = 12, 12, 11, and 12. SLR: Subjects were excluded if they failed to meet predetermined criteria during the sample phase (> 2 seconds/object, > 10 seconds total exploration, equal percent exploration time per object) or test phase (> 1 second/object, > 5 seconds total exploration). Final male subject numbers: n = 10, 10, 9, and 10. Final female subject numbers: n = 12, 12, 9, and 11. Autoshaping: Subjects were excluded if they failed to reach acquisition criteria (≥ 25 trials in 2 out of 3 consecutive days during 11 days of testing). Final male subject numbers: n = 10, 11, 10, and 10. Final female subject numbers: n = 12, 12, 10, and 11. Statistical analysis Data normality was assessed using D'Agostino-Pearson and Shapiro-Wilk tests with QQ plot visualization. Behavioral measures meeting normality assumptions were analyzed using ANOVA with drug (Veh, CDDO-EA) and irradiation (Sham, 33-GCR) as between-subjects factors. Two-way ANOVA was used for SLR discrimination indices (d2 ratio), SLR test locomotion, EPM measures (time in open arms, entries, exploration index, total distance), and DCX+ total cell counts. Three-way repeated measures ANOVA was used for SLR sample phase object exploration (drug × irradiation × object for objects 1, 2, 3), autoshaping measures (drug × irradiation × day for reward approaches, delta approaches, and percent accuracy during acquisition and reversal), and DCX+ cell distribution across bregma positions (drug × irradiation × bregma). Three-way repeated measures ANOVA was also used for body weight (drug × irradiation × time). Mixed-effects models were used when data were missing. Home cage locomotion (total, ambulatory, and fine beam breaks during both phase and interval testing) failed normality and was analyzed using Kruskal-Wallis tests with Dunn's post-hoc correction. Fiber photometry data were analyzed using two-way ANOVA (load × irradiation for d2 ratio) or Kruskal-Wallis tests (for signal rate and amplitude measures). Significant ANOVA effects were followed by Tukey's or Bonferroni post-hoc tests corrected for multiple comparisons. Statistical significance was set at α = 0.05. Data are presented as mean ± SEM. Analyses were performed using R Studio, and Python 3.10 (scikit-learn 1.3.0, NumPy 1.24.3, SciPy 1.11.1, statsmodels 0.14.0). Graphs and figures Graphs were generated using GraphPad Prism 10 (GraphPad Software, San Diego, CA) and Python 3.10 (for PCA analysis). Brightfield photomicrographs were acquired using an Olympus digital camera with cellSens Standard software. Figures were assembled using Adobe Illustrator. Declarations Data availability All data, including individual animal-level data, necessary to replicate the findings of this study are publicly available. Code availability Behavioral data (spontaneous location recognition, autoshaping, reversal learning, elevated plus maze), home cage locomotor activity data, body weight data, fiber photometry recordings, and stereological cell counts have been deposited in Zenodo: https://doi.org/10.5281/zenodo.19157801. Analysis scripts for fiber photometry, locomotor activity, Statistical output and principal component analysis are available at https://github.com/EischLab/NSRL22A. Author Contributions S.A.O. contributed to Formal analysis, Investigation, Data Curation, Writing – Original Draft, and Visualization. P.N. contributed to Software, Formal analysis, Data Curation, Writing – Original Draft, and Visualization. A.M. contributed to Formal analysis and Investigation. G.L.B. contributed to Formal analysis, Investigation and Data Curation. H.A.H. contributed to Formal analysis, Investigation and Data Curation. E.W-F. contributed to Investigation. S.V. contributed to Software. H.T. contributed to Software. F.C.K. contributed to Conceptualization and Investigation and Writing – Review & Editing. A.J.E. contributed to Conceptualization, Methodology, Investigation, Resources, Writing – Original Draft, Writing – Review & Editing, Supervision, Project administration, and Funding acquisition. S.Y. contributed to Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization, Supervision, Project administration, and Funding acquisition. All authors read and approved the final manuscript. Acknowledgments We thank the team members at BNL/NSRL for their assistance with the 22A irradiation campaign, particularly Adam Rusek and Peter Guida. We are grateful to an anonymous donor for support of the Eisch Lab and this project. This work was supported by the following funding sources: S.Y. was supported by a 2019 NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation, a 2020 Penn Undergraduate Research Foundation grant, NASA HERO grant 80NSSC21K0814, a 2022 Foerderer Fund for Excellence Award, two CHOP Junior Faculty Awards (2021, PI: Bhoj; 2023, PI: Van Batavia), and NIH awards MH076690 (PI: Tamminga), MH107945 (PI: Eisch), and MH129970 (PI: Eisch). F.K. was supported by the Translational Research Institute for Space Health (TRISH) through NASA cooperative agreement NNX16AO69A, a Penn Provost/CHOP Postdoctoral Fellowship for Academic Diversity, and a Perelman School of Medicine Department of Radiation Oncology Pilot Grant (PIs: Fan and Eisch). A.J.E. was supported by NIH awards MH129970, NS007413, DA007290, DA023555, DA016765, and MH107945; NASA awards NNX07AP84G, NNX12AB55G, and NNX15AE09G; and NIH NS126279 (PI: Ahrens-Nicklas). S.Y. and A.J.E. were also supported by NIH DK135871 (PI: Zderic), NIH NS088555 (PI: Stowe), and NIH MH117628 (PI: Lambert). H.H. was supported by the 2021 Penn Undergraduate Research Mentoring Program (PURM), the 2022 Summer Undergraduate Internship Program (SUIP), the 2023 Penn College Alumni Society Board of Managers, a Penn President's Undergraduate Research Grant, and an augmentation award to NASA HERO grant 80NSSC21K0814 (PI: Yun). 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Supplementary Files OConnoretal.Suppinfo.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 08 May, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers invited by journal 29 Apr, 2026 Editor assigned by journal 22 Apr, 2026 Submission checks completed at journal 22 Apr, 2026 First submitted to journal 20 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9476558","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":635540928,"identity":"34960439-fce0-4efa-890e-02aefb942625","order_by":0,"name":"Sheridan A. 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Bancroft","email":"","orcid":"","institution":"The Children's Hospital of Philadelphia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Grace","middleName":"L.","lastName":"Bancroft","suffix":""},{"id":635540937,"identity":"84ec81fd-a8b3-42fa-91a1-3b01df7dda43","order_by":4,"name":"Harley A. Haas","email":"","orcid":"","institution":"The Children's Hospital of Philadelphia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Harley","middleName":"A.","lastName":"Haas","suffix":""},{"id":635540938,"identity":"3aeb0ad8-9788-40d0-bc5f-b94c22d2f456","order_by":5,"name":"Elise Wallen-Friedman","email":"","orcid":"","institution":"The Children's Hospital of Philadelphia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Elise","middleName":"","lastName":"Wallen-Friedman","suffix":""},{"id":635540941,"identity":"e9617012-0f49-470b-845b-253763c3affd","order_by":6,"name":"Shubha Vasisht","email":"","orcid":"","institution":"The Children's Hospital of Philadelphia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Shubha","middleName":"","lastName":"Vasisht","suffix":""},{"id":635540942,"identity":"7afb458d-a0b1-4f7e-9e0f-e7d7f47cd05c","order_by":7,"name":"Hajime Takano","email":"","orcid":"","institution":"The Children's Hospital of Philadelphia (CHOP) Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Hajime","middleName":"","lastName":"Takano","suffix":""},{"id":635540943,"identity":"e2f5e579-d99e-40cc-9434-efafe8de1411","order_by":8,"name":"Frederico C. Kiffer","email":"","orcid":"","institution":"The Children's Hospital of Philadelphia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Frederico","middleName":"C.","lastName":"Kiffer","suffix":""},{"id":635540944,"identity":"6860d224-ef98-4f11-9615-0ebad2dc09c7","order_by":9,"name":"Amelia J. Eisch","email":"","orcid":"","institution":"The Children's Hospital of Philadelphia Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Amelia","middleName":"J.","lastName":"Eisch","suffix":""},{"id":635540945,"identity":"5625ca9a-2a0c-481a-903d-97d8dd1fe339","order_by":10,"name":"Sanghee Yun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAn0lEQVRIiWNgGAWjYLCCDwwHwLQE0ToYZ5CshZmHJC0GN3IPf7apuSNnzsB88DYPcVry0qRzjj0ztmxgS7YmUkuOGXNuw+HEDQd4zKSJ1WL82RKshf8b0VoMpBkhtrARp0XyzBszyZ5jh40tm9mMLecQo4XveI7xhx81h+XM2Zsf3nhDjBaFAzAXMhOjHATkG2BaiNUxCkbBKBgFIw8AAMAEMnWcgI20AAAAAElFTkSuQmCC","orcid":"","institution":"The Children's Hospital of Philadelphia Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Sanghee","middleName":"","lastName":"Yun","suffix":""}],"badges":[],"createdAt":"2026-04-20 21:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9476558/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9476558/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108806252,"identity":"6ec3a9c6-5d4f-499f-9c7e-8b31c70bec39","added_by":"auto","created_at":"2026-05-08 15:28:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental timeline showing treatment and behavioral testing schedule over 7.25 months. \u003c/strong\u003eMale and female C57BL/6J mice (n=47/sex) received 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-ethylamide (CDDO-EA; 4 mg/kg, i.p.) or vehicle (Veh) for 3 days surrounding administration of whole-body 33-ion galactic cosmic radiation (33-GCR, 0.75 Gy) or sham irradiation at 4.5–5 months of age. Behavioral assessments included home cage activity, spontaneous location recognition (SLR), touchscreen-based Pavlovian learning, and elevated plus maze testing over 7.25 months post-irradiation. Abbreviations: 33-GCR, 33-ion galactic cosmic radiation; CDDO-EA, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-ethylamide; EPM, elevated plus maze; IRR, irradiation; SLR, spontaneous location recognition; Veh, vehicle.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/3b6b6903a7d6e09a471c888c.png"},{"id":108807031,"identity":"58b8d7c0-5c7d-43d1-962a-c8ff7bcb08e3","added_by":"auto","created_at":"2026-05-08 15:29:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e33-GCR enhances male pattern separation under very high memory load; CDDO-EA normalizes this. (A) \u003c/strong\u003eSchematic of the d-SLR (dissimilar: low memory load) task. Mice explored three identical objects (objects 2 and 3, 108° apart) during the Sample phase (10 min), then after a 35-min delay explored two objects—one at a familiar location [F] and one at a novel location [N]—during the Test phase (5 min). \u003cstrong\u003e(B) \u003c/strong\u003ed-SLR Sample: percent time exploring each object location. \u003cstrong\u003e(C)\u003c/strong\u003e d-SLR Test: discrimination index (d2 ratio). \u003cstrong\u003e(D)\u003c/strong\u003ed-SLR Test: total distance traveled (cm). \u003cstrong\u003e(E)\u003c/strong\u003e Schematic of the s-SLR (similar: high memory load) task (objects 2 and 3, 72° apart). \u003cstrong\u003e(F)\u003c/strong\u003e s-SLR Sample: percent time exploring each location. \u003cstrong\u003e(G)\u003c/strong\u003e s-SLR Test: d2 ratio. (H) s-SLR Test: total distance traveled. \u003cstrong\u003e(I) \u003c/strong\u003eSchematic of the xs-SLR (extra-similar: very high memory load) task (objects 2 and 3, 36° apart). \u003cstrong\u003e(J)\u003c/strong\u003exs-SLR Sample: percent time exploring each location. \u003cstrong\u003e(K)\u003c/strong\u003e xs-SLR Test: d2 ratio. \u003cstrong\u003e(L) \u003c/strong\u003exs-SLR Test: total distance traveled. Data are presented as mean ± SEM (n=9–10/group). Statistical analysis: 3-way RM ANOVA (IRR × Drug × Object) in \u003cstrong\u003eB,\u003c/strong\u003e \u003cstrong\u003eF\u003c/strong\u003e, J; 2-way ANOVA (IRR × Drug) in\u003cstrong\u003e C–D\u003c/strong\u003e, \u003cstrong\u003eG–H\u003c/strong\u003e, \u003cstrong\u003eK–L\u003c/strong\u003e. *p\u0026lt;0.05, **p\u0026lt;0.01. Post-hoc: Veh/Sham vs Veh/33-GCR, ᵃ p\u0026lt;0.05; Veh/33-GCR vs CDDO-EA/33-GCR, ᵉ p\u0026lt;0.05. Complete subject numbers and detailed statistical analyses are provided in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/931f89333b4d232cc00a2c9b.png"},{"id":108806629,"identity":"4e13043c-e100-4a8b-ad2d-e8befd310609","added_by":"auto","created_at":"2026-05-08 15:29:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFemale mice show no radiation-induced pattern separation changes despite reduced locomotion.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic of the d-SLR task. \u003cstrong\u003e(B)\u003c/strong\u003e d-SLR Sample: percent time exploring each object location. \u003cstrong\u003e(C) \u003c/strong\u003ed-SLR Test: discrimination index (d2 ratio). \u003cstrong\u003e(D) \u003c/strong\u003ed-SLR Test: total distance traveled (cm). \u003cstrong\u003e(E) \u003c/strong\u003eSchematic of the s-SLR task. \u003cstrong\u003e(F) \u003c/strong\u003es-SLR Sample: percent time exploring each location.\u003cstrong\u003e (G) \u003c/strong\u003es-SLR Test: d2 ratio. \u003cstrong\u003e(H)\u003c/strong\u003e s-SLR Test: total distance traveled.\u003cstrong\u003e (I) \u003c/strong\u003eSchematic of the xs-SLR task. \u003cstrong\u003e(J)\u003c/strong\u003exs-SLR Sample: percent time exploring each location. \u003cstrong\u003e(K) \u003c/strong\u003exs-SLR Test: d2 ratio.\u003cstrong\u003e (L)\u003c/strong\u003e xs-SLR Test: total distance traveled. Data are presented as mean ± SEM (n=9–12/group). Statistical analysis: 3-way RM ANOVA (IRR × Drug × Object) in \u003cstrong\u003eB, F, J\u003c/strong\u003e; 2-way ANOVA (IRR × Drug) in \u003cstrong\u003eC–D, G–H, K–L\u003c/strong\u003e. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. Post-hoc: Veh/Sham vs Veh/33-GCR, ᵃ p\u0026lt;0.05; Veh/Sham vs CDDO-EA/Sham, ᵇ p\u0026lt;0.05. Complete subject numbers and detailed statistical analyses are provided in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/f60ca4a3ebb646f9de04f62d.png"},{"id":108807034,"identity":"2b9d578f-c8ff-4e9e-b69d-9d090c1f7f20","added_by":"auto","created_at":"2026-05-08 15:29:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":244374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e33-GCR produces physiological effects in females that dissociate from cognitive outcomes.\u003cbr\u003e\n \u0026nbsp;(A–B) \u003c/strong\u003eBody weights recorded monthly throughout the experiment in male \u003cstrong\u003e(A)\u003c/strong\u003e and female \u003cstrong\u003e(B) \u003c/strong\u003emice (n=11–12/group). \u003cstrong\u003e(C–E)\u003c/strong\u003e Male home cage locomotor activity measured over 18 hours (4pm–10am) via 4×8 photobeam breaks in the xy-plane at 2.25 months post-IRR: total \u003cstrong\u003e(C)\u003c/strong\u003e, ambulatory\u003cstrong\u003e (D)\u003c/strong\u003e, and fine\u003cstrong\u003e (E) \u003c/strong\u003ebeam breaks. \u003cstrong\u003e(F–H) \u003c/strong\u003eFemale home cage locomotor activity: total \u003cstrong\u003e(F)\u003c/strong\u003e, ambulatory\u003cstrong\u003e (G)\u003c/strong\u003e, and fine \u003cstrong\u003e(H)\u003c/strong\u003e beam breaks (n=10–12/group). Body weight data are presented as mean ± SEM; locomotor data are presented as median ± IQR. Statistical analysis: mixed-effects 3-way RM ANOVA in \u003cstrong\u003eA\u003c/strong\u003e; 3-way RM ANOVA in \u003cstrong\u003eB\u003c/strong\u003e; Kruskal-Wallis test with Dunn's post-hoc comparisons in \u003cstrong\u003eC–H\u003c/strong\u003e. Main effect and/or interaction denoted by *p\u0026lt;0.05. Post-hoc comparisons for body weight: Veh/Sham vs Veh/33-GCR, ᵃ p\u0026lt;0.05, ᵃ' p\u0026lt;0.01; Veh/Sham vs CDDO-EA/Sham, ᵇ' p\u0026lt;0.01 \u003cstrong\u003e(B)\u003c/strong\u003e. Post-hoc comparisons for locomotor activity: Veh/Sham vs Veh/33-GCR, ᵃ p\u0026lt;0.05, ᵃ' p\u0026lt;0.01; Veh/Sham vs CDDO-EA/Sham, ᵇ p\u0026lt;0.05; Veh/33-GCR vs CDDO-EA/Sham, ᵈ p\u0026lt;0.05; # 0.05\u0026lt;p\u0026lt;0.08. Complete subject numbers and detailed statistical analyses are provided in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/a596e60b3e70d6bd77056f34.png"},{"id":108807033,"identity":"7187ec48-1575-4ec4-b6ee-c658ab1f074e","added_by":"auto","created_at":"2026-05-08 15:29:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":412879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e33-GCR and CDDO-EA independently enhance female goal-tracking; males are unaffected.\u003c/strong\u003e \u003cbr\u003e\n \u003cstrong\u003e(A)\u003c/strong\u003e Schematic of the autoshaping task. During acquisition, a conditioned stimulus (CS+, illuminated panel) is paired with reward delivery (strawberry milkshake). Mice learn the CS-reward association over 11 days. \u003cstrong\u003e(B, E) \u003c/strong\u003eGoal-tracking: approaches to the reward magazine during CS+ presentation in males \u003cstrong\u003e(B)\u003c/strong\u003eand females \u003cstrong\u003e(E)\u003c/strong\u003e. \u003cstrong\u003e(C, F) \u003c/strong\u003eSign-tracking: difference in approaches to CS+ vs CS− in males \u003cstrong\u003e(C)\u003c/strong\u003e and females\u003cstrong\u003e (F)\u003c/strong\u003e. \u003cstrong\u003e(D, G)\u003c/strong\u003e Accuracy: percent CS+ approaches out of total CS+ trials in males\u003cstrong\u003e (D) \u003c/strong\u003eand females \u003cstrong\u003e(G)\u003c/strong\u003e. Data are presented as mean ± SEM (n=10–12/group). Statistical analysis: 3-way RM ANOVA (IRR × Drug × Time). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. Post-hoc: timepoints in Veh/GCR, ² p\u0026lt;0.05; timepoints in CDDO-EA/Sham, ³ p\u0026lt;0.05; Veh/Sham vs Veh/33-GCR, ᵃ p\u0026lt;0.05; Veh/Sham vs CDDO-EA/Sham, ᵇ p\u0026lt;0.05; Veh/33-GCR vs CDDO-EA/33-GCR, ᵉ p\u0026lt;0.05; CDDO-EA/Sham vs CDDO-EA/33-GCR, ᶠ p\u0026lt;0.05; # 0.05\u0026lt;p\u0026lt;0.08. Complete subject numbers and detailed statistical analyses are provided in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/f0cbbe5365b95fce3da0891f.png"},{"id":108701976,"identity":"676bdfd0-f123-444f-8e2f-9511dd588007","added_by":"auto","created_at":"2026-05-07 12:52:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":261479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCDDO-EA impairs male reversal learning; 33-GCR impairs female reversal learning. (A) \u003c/strong\u003eSchematic of the reversal learning task. Following 11 days of acquisition, the CS+ and CS− locations were switched. Males underwent 5 days of reversal; females underwent 6 days.\u003cstrong\u003e (B–C) \u003c/strong\u003eMale reversal learning: approaches to the new CS+\u003cstrong\u003e (B) \u003c/strong\u003eand accuracy\u003cstrong\u003e (C). (D–E) \u003c/strong\u003eFemale reversal learning: approaches to the new CS+ \u003cstrong\u003e(D) \u003c/strong\u003eand accuracy\u003cstrong\u003e (E). \u003c/strong\u003eData are presented as mean ± SEM (n=10–12/group). Statistical analysis: 3-way RM ANOVA (IRR × Drug × Time). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. Post-hoc: Veh/Sham vs Veh/33-GCR, ᵃ p\u0026lt;0.05; Veh/Sham vs CDDO-EA/Sham, ᵇ p\u0026lt;0.05; Veh/33-GCR vs CDDO-EA/33-GCR, ᵉ' p\u0026lt;0.01; CDDO-EA/Sham vs CDDO-EA/33-GCR, ᶠ p\u0026lt;0.05. Complete subject numbers and detailed statistical analyses are provided in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/7e339747a139a12124847e63.png"},{"id":108701973,"identity":"c0edab5f-892c-4802-8754-146d51f2cf89","added_by":"auto","created_at":"2026-05-07 12:52:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":196237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeither 33-GCR nor CDDO-EA affects anxiety-like behavior; combined treatment reduces locomotion in females.\u003c/strong\u003e \u003cbr\u003e\n \u003cstrong\u003e(A–H)\u003c/strong\u003e Mice were recorded during 5 minutes of free exploration under 200 lux in an elevated plus maze (EPM) with male mice at 6.5 months post-IRR \u003cstrong\u003e(A–D)\u003c/strong\u003eand female mice at 6.75 months post-IRR \u003cstrong\u003e(E–H)\u003c/strong\u003e. Anxiety-like behaviors were measured by time spent\u003cstrong\u003e (A, E) \u003c/strong\u003eand number of entries \u003cstrong\u003e(B, F)\u003c/strong\u003ein open arms, and the ratio of open to closed arm time \u003cstrong\u003e(C, G)\u003c/strong\u003e. Locomotor activity was measured by total movement distance (cm) in the entire arena \u003cstrong\u003e(D, H)\u003c/strong\u003e. Data are presented as mean ± SEM. Statistical analysis: 2-way ANOVA (IRR × Drug) in \u003cstrong\u003eA–H\u003c/strong\u003e. Main effect and/or interaction denoted by *p\u0026lt;0.05, **p\u0026lt;0.01; post-hoc: Veh/Sham vs CDDO-EA/Sham, ᵇ' p\u0026lt;0.01; Veh/Sham vs CDDO-EA/33-GCR, ᶜ p\u0026lt;0.05; CDDO-EA/Sham vs CDDO-EA/33-GCR, ᶠ p\u0026lt;0.05, ᶠ' p\u0026lt;0.01. Complete subject numbers and detailed statistical analyses are provided in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/b178b2a8805a45dfe47f14a6.png"},{"id":108805732,"identity":"6da2a07d-26e2-4b70-b68d-dfe158e4fd72","added_by":"auto","created_at":"2026-05-08 15:26:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":330059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrincipal component analysis reveals circuit-specific treatment effects across cognitive domains.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A) \u003c/strong\u003eScree plot showing variance explained by each PC. Blue bars: individual variance; orange circles/line: cumulative variance; dashed line: 80% threshold\u003cstrong\u003e. (B)\u003c/strong\u003e Component loading heatmap for the first four PCs across six behavioral variables: d2 ratio (d-SLR, s-SLR, xs-SLR), sign-tracking (CS+ vs CS− approach difference, Days 4–7), goal-tracking (reward chamber approaches, Days 4–7), and reversal learning (CS+ approaches, Days 1–4). Red: positive loading; blue: negative loading. \u003cstrong\u003e(C)\u003c/strong\u003e Three-dimensional biplot showing individual animals across PC1 × PC2 × PC3 (57.6% total variance). Black arrows: loading vectors. Points color-coded by group (gray: Veh/Sham; orange: Veh/33-GCR; light blue: CDDO-EA/Sham; purple: CDDO-EA/33-GCR) and shape (circles: males; squares: females). Inset: vector angle relationships indicating competitive (\u0026gt;135°, orange), independent (~90°, pink), and synergistic (\u0026lt;60°, blue) domain interactions. \u003cstrong\u003e(D–F)\u003c/strong\u003e Treatment effects on PC scores shown separately for males (blue) and females (pink). Statistical analysis: 2-way ANOVA (Treatment × Sex). *p\u0026lt;0.05. Post-hoc: Veh/33-GCR vs CDDO-EA/33-GCR, * p\u0026lt;0.05. Complete subject numbers and detailed statistical analyses are provided in Supplementary table 1.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/064cb9836e1cd375ba09a614.png"},{"id":108806046,"identity":"9fe2f102-343a-44d8-97ca-c4fdc602b78f","added_by":"auto","created_at":"2026-05-08 15:27:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":263791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e33-GCR produces persistent alterations in dentate gyrus activity during memory encoding.\u003c/strong\u003e \u003cbr\u003e\n \u003cstrong\u003e(A) \u003c/strong\u003eExperimental timeline. A subset of male Veh/Sham and Veh/33-GCR mice (n=3–4/group) underwent AAV-CaMKIIα-GCaMP6f viral infusion in the DG hilus and optic fiber implantation in the molecular layer at 6 months post-IRR. Mice performed SLR with simultaneous fiber photometry recording at 7.25 months post-IRR. \u003cstrong\u003e(B)\u003c/strong\u003e GFP expression in the DG granule cell layer; dotted line indicates fiber track. Scale bar: 100 μm. \u003cstrong\u003e(C)\u003c/strong\u003e Discrimination index (d2 ratio) in d-SLR and xs-SLR. \u003cstrong\u003e(D)\u003c/strong\u003e Representative Ca²⁺ signal (ΔF/F) during the Test phase. Purple: novel location; beige: familiar location. Scale bar: 5% ΔF/F, 2.5 s. \u003cstrong\u003e(E–H)\u003c/strong\u003e Sample phase Ca²⁺ signals: transient rate\u003cstrong\u003e(E)\u003c/strong\u003e and amplitude \u003cstrong\u003e(F)\u003c/strong\u003e in d-SLR; transient rate (G) and amplitude \u003cstrong\u003e(H)\u003c/strong\u003ein xs-SLR. \u003cstrong\u003e(I–L)\u003c/strong\u003e Test phase Ca²⁺ signals: transient rate\u003cstrong\u003e(I) \u003c/strong\u003eand amplitude\u003cstrong\u003e (J)\u003c/strong\u003e in d-SLR; transient rate\u003cstrong\u003e (K) \u003c/strong\u003eand amplitude\u003cstrong\u003e (L) \u003c/strong\u003ein xs-SLR. Data are presented as mean ± SEM \u003cstrong\u003e(C, H)\u003c/strong\u003eor median ± IQR \u003cstrong\u003e(E–G, I–L)\u003c/strong\u003e. Each symbol indicates an animal \u003cstrong\u003e(C) \u003c/strong\u003eor detected event \u003cstrong\u003e(E–L)\u003c/strong\u003e. Statistical analysis: 2-way RM ANOVA \u003cstrong\u003e(C, H)\u003c/strong\u003e; Kruskal-Wallis with Dunn's post-hoc \u003cstrong\u003e(E–G, I–L)\u003c/strong\u003e. **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. Post-hoc: Veh/Sham vs Veh/33-GCR, ᵃ p\u0026lt;0.05, ᵃ' p\u0026lt;0.01, ᵃ'' p\u0026lt;0.001, ᵃ''' p\u0026lt;0.0001. Complete subject numbers and detailed statistical analyses are provided in\u003cstrong\u003e \u003c/strong\u003eSupplementary Table 1.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/8725a3beb0cca5a508fe94fa.png"},{"id":108805789,"identity":"fee10a28-6a96-4a09-b642-7befc78ccf39","added_by":"auto","created_at":"2026-05-08 15:26:54","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":445399,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined CDDO-EA and 33-GCR reduces DCX+ progenitor cells in females despite intact pattern separation.\u003c/strong\u003e \u003cbr\u003e\n \u003cstrong\u003e(A)\u003c/strong\u003e Representative photomicrographs of DCX+ cells in the DG at 100× (i) and higher magnification (ii). Immature neurons were identified by oval cell bodies with extensive dendritic processes containing at least one dendritic node (arrowheads). Progenitor cells were identified by irregular cell bodies with short processes lacking dendritic nodes (arrows). \u003cstrong\u003e(B–I)\u003c/strong\u003eQuantification of DCX+ cells using unbiased stereology in males \u003cstrong\u003e(B, C, F, G) \u003c/strong\u003eand females \u003cstrong\u003e(D, E, H, I)\u003c/strong\u003e. Total DCX+ immature neurons\u003cstrong\u003e (B, D) \u003c/strong\u003eand distribution across the rostro-caudal axis \u003cstrong\u003e(C, E)\u003c/strong\u003e. Total DCX+ progenitor cells\u003cstrong\u003e (F, H)\u003c/strong\u003e and distribution across the rostro-caudal axis \u003cstrong\u003e(G, I)\u003c/strong\u003e. Data are presented as mean ± SEM (n=8–12/group). Statistical analysis: 2-way ANOVA (IRR × Drug) in \u003cstrong\u003eB, D, F, H\u003c/strong\u003e; 3-way RM ANOVA (Bregma × IRR × Drug) in \u003cstrong\u003eC, E, G, I\u003c/strong\u003e. *p\u0026lt;0.05, ****p\u0026lt;0.0001. Post-hoc: CDDO-EA/Sham vs CDDO-EA/33-GCR, ᶠ p\u0026lt;0.05. Scale bars: 200 μm (i), 100 μm (ii). Complete subject numbers and detailed statistical analyses are provided in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/b28e29c6cfc666b76c2a0c65.png"},{"id":108810172,"identity":"83745f79-3036-42aa-b063-273382ffe399","added_by":"auto","created_at":"2026-05-08 15:57:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2900063,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/20e175d6-b751-4fc0-944b-3d5e46522724.pdf"},{"id":108701969,"identity":"4e2cbc7a-9105-4a46-89c4-d5502fd86d95","added_by":"auto","created_at":"2026-05-07 12:52:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1302769,"visible":true,"origin":"","legend":"","description":"","filename":"OConnoretal.Suppinfo.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9476558/v1/4de57365bce4e8d942d66db3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Galactic cosmic radiation produces sex-specific, circuit-selective cognitive vulnerability: countermeasure trade-offs revealed by multi-domain assessment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAstronauts on deep space missions beyond Earth's magnetosphere face chronic galactic cosmic radiation (GCR) exposure, a complex mixture of high-energy particles with documented risks to cognitive performance and mission success\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. GCR consists predominantly of protons (85%), helium nuclei (14%), and HZE ions (\u0026le;\u0026thinsp;1%); particles such as \u0026sup1;⁶O, \u0026sup1;\u0026sup2;C, ⁵⁶Fe, and \u0026sup2;⁸Si are notable for their high linear energy transfer, which causes DNA damage, oxidative stress, and inflammation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The CNS is highly vulnerable to these effects, with space radiation inducing persistent structural and functional alterations across multiple brain regions\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Understanding how GCR affects the brain requires assessing multiple cognitive domains simultaneously\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, yet most ground-based studies have used single-ion exposures, limited behavioral endpoints, and male-only designs, a limitation shared broadly across neuroscience\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.These approaches neither replicate deep space conditions nor capture how radiation affects integrated cognitive function across sexes.\u003c/p\u003e \u003cp\u003eCognitive functions central to astronaut performance \u0026mdash; spatial navigation, reward processing, and behavioral flexibility \u0026mdash; rely on coordinated activity across the hippocampus, nucleus accumbens (NAc), and prefrontal cortex (PFC)\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These regions function as an integrated circuit; disruption at any node can cascade system-wide\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.The hippocampus provides spatial and contextual information, with the dentate gyrus (DG) specialized for pattern separation, the ability to form distinct representations of similar experiences\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Through projections to the NAc and PFC, the hippocampus integrates contextual information with reward value and executive control\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The NAc serves as a hub where hippocampal information converges with reward signals to guide motivated behavior\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e; Pavlovian autoshaping paradigms provide a well-validated measure of this function\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The PFC provides top-down executive control, receiving hippocampal input for context-guided decision-making while modulating NAc activity to regulate behavioral flexibility\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan additionalcitationids=\"CR36 CR37 CR38\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWithin this circuit, the DG is a particularly vulnerable node. Adult-born granule neurons contribute to pattern separation and memory formation\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, but their high metabolic activity and low antioxidant capacity render them susceptible to oxidative damage\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. DG dysfunction can disrupt downstream signaling to the NAc and PFC, with consequences for circuit-wide function\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. All three regions show vulnerability to radiation-induced changes, including altered dopaminergic signaling, reduced dendritic complexity, and disrupted synaptic plasticity\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR46 CR47 CR48 CR49\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Prior work demonstrates that GCR impairs hippocampal-dependent learning and neurogenesis and induces neuroinflammation\u003csup\u003e\u003cspan additionalcitationids=\"CR52 CR53 CR54 CR55 CR56\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, the relative vulnerability of the hippocampus, NAc, and PFC to multi-ion GCR tested within a single study remains unknown.\u003c/p\u003e \u003cp\u003eCritical knowledge gaps persist. Prior GCR studies examining hippocampal-dependent tasks have shown inconsistent effects \u0026mdash; deficits and enhancements alike \u0026mdash; depending on dose and paradigm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Recent multi-ion studies demonstrate impairments in hippocampal long-term memory\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e and PFC-based attention\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, while single-ion work has shown effects on NAc and reward circuitry\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan additionalcitationids=\"CR61 CR62 CR63 CR64 CR65\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. No study has assessed how multi-ion GCR affects the integrated hippocampal-NAc-PFC circuit across multiple cognitive domains, or whether effects are global versus domain-selective. Sex differences in space radiation effects remain poorly characterized\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, and most studies have tested only males, despite evidence that males and females may show different vulnerability patterns\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Given that disruption at any circuit node can cascade system-wide\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, understanding multi-domain cognitive outcomes in both sexes is essential for astronaut risk assessment\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNo adequate shielding currently exists for deep space radiation, making pharmacological countermeasures essential \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Because GCR-induced oxidative damage and inflammation can compromise multiple nodes within the hippocampal-NAc-PFC circuit\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, effective countermeasures must provide broad neuroprotection. CDDO-EA (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid ethyl amide) is a candidate countermeasure: a synthetic triterpenoid that activates the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Nrf2 is a master regulator of cellular antioxidant responses, inducing cytoprotective gene expression\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. CDDO-EA and related triterpenoids have demonstrated neuroprotective effects in injury and neurological disorder models, with Nrf2 activators mitigating radiation-induced damage across multiple organ systems\u003csup\u003e\u003cspan additionalcitationids=\"CR77 CR78 CR79 CR80 CR81\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. The structurally related Nrf2-activating compound omaveloxolone (Skyclarys) recently received FDA approval for treating Friedreich's ataxia\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e, supporting the translational potential of this compound class. Our laboratory demonstrated that CDDO-EA provides cognitive protection in female mice exposed to 33-GCR, including enhanced pattern separation and improved reversal learning\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. However, that study used a single behavioral endpoint in males\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e, leaving open whether domain-specific effects were missed. This raises the question: does CDDO-EA protect uniformly, or does protection in one domain come at the cost of impairment in another?\u003c/p\u003e \u003cp\u003eHere we investigated how 33-ion GCR exposure affects cognition across multiple domains in male and female mice, and whether CDDO-EA provides broad neuroprotection or domain-selective effects. Based on prior work\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, we hypothesized that 33-GCR would produce domain-specific rather than global cognitive effects that differ between sexes, with hippocampal-dependent functions showing particular vulnerability in males\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e, and metabolic or locomotor effects predominating in females\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. We further hypothesized that CDDO-EA would show domain-dependent rather than uniform protection, with potential trade-offs across circuit nodes\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e,\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. To test these hypotheses, we assessed four cognitive domains (hippocampal-dependent pattern separation, reward-based learning, behavioral flexibility, and anxiety-related behavior), alongside body weight, locomotor activity, hippocampal neurogenesis markers, and neural activity over 7.25 months post-irradiation (post-IRR). Neural activity and cellular markers were assessed at later timepoints to determine whether radiation-induced alterations persist beyond the behavioral testing window. Our findings reveal circuit-selective rather than global effects, with distinct vulnerability patterns in males and females and countermeasure trade-offs invisible to single-endpoint assessment \u0026mdash; findings with direct implications for astronaut risk assessment on missions beyond Earth orbit.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eMale and female C57BL/6J mice received 33-ion GCR simulation (0.75 Gy) or sham irradiation with concurrent CDDO-EA or vehicle treatment, followed by multi-domain behavioral testing over 7.25 months post-IRR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e33-GCR enhances pattern separation in males under very high memory load; CDDO-EA normalizes this effect\u003c/p\u003e \u003cp\u003eHippocampal-dependent cognition was assessed using the spontaneous location recognition (SLR) task with varying memory loads (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Task difficulty increased as objects were placed closer together: dissimilar (d-SLR, low load), similar (s-SLR, high load), and extra-similar (xs-SLR, very high load). Males began testing at 3.25 months post-IRR.\u003c/p\u003e \u003cp\u003eUnder low and high memory loads (d-SLR, s-SLR), all groups showed similar object exploration during the Sample phase and comparable discrimination and locomotion during the Test phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-H, Supplementary Table\u0026nbsp;1; all post-hoc p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eGroup differences emerged under very high memory load (xs-SLR). During the Sample phase, males in all groups explored objects similarly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-way RM ANOVA, IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; all post-hoc p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). During the Test phase, Veh/33-GCR males showed a positive d2 ratio (0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09) while Veh/Sham males showed a negative d2 ratio (\u0026minus;\u0026thinsp;0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10), indicating successful discrimination of the novel location in irradiated but not control males (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-way ANOVA, Drug p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, IRR\u0026times;Drug p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc Veh/Sham vs Veh/33-GCR, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CDDO-EA prevented this radiation-induced enhancement: CDDO-EA/33-GCR males showed a negative d2 ratio (\u0026minus;\u0026thinsp;0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08), similar to Veh/Sham controls and less than Veh/33-GCR males (post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Locomotion did not differ among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL; all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eIn sum, 33-GCR enhanced pattern separation in males specifically under very high cognitive demand, an effect normalized by CDDO-EA.\u003c/p\u003e \u003cp\u003eFemale mice show no radiation-induced changes in pattern separation despite reduced locomotion during testing\u003c/p\u003e \u003cp\u003eIn contrast to males, female mice showed no group differences in pattern separation at any memory load. Females began testing at 3.75 months post-IRR.\u003c/p\u003e \u003cp\u003eAcross all three memory loads, female groups showed similar object exploration during the Sample phase and comparable discrimination during the Test phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C, F-G, J-K, \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e; all post-hoc p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eHowever, locomotion during SLR testing differed among female groups. Under d-SLR, Veh/33-GCR females traveled 19.6% shorter distances during the Test phase than Veh/Sham females (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-way ANOVA, IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Drug p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Under s-SLR, CDDO-EA/Sham females traveled 20.6% shorter distances than Veh/Sham females (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH; Drug p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Under xs-SLR, IRR affected locomotion overall but no post-hoc differences reached significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL; IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; all post-hoc p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThus, neither 33-GCR nor CDDO-EA affected pattern separation in females, despite both treatments independently reducing locomotion during testing.\u003c/p\u003e \u003cp\u003e33-GCR produces physiological effects in females that dissociate from cognitive outcomes\u003c/p\u003e \u003cp\u003eSince females showed reduced locomotion during SLR testing without cognitive impairment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we assessed body weight in both sexes throughout the 7.25-month experiment and measured home cage locomotor activity at 2.25 months post-IRR to determine whether 33-GCR and CDDO-EA produced systemic physiological effects.\u003c/p\u003e \u003cp\u003eAll male mice gained weight progressively throughout the experiment, with no weight differences among the four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e; male: 3-way mixed effect RM ANOVA, Time F(6,258)\u0026thinsp;=\u0026thinsp;353.618, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; all other main effects and interactions p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Female mice also gained weight but, unlike males, showed group differences in weight trajectory (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e; female: 3-way RM ANOVA, Time F(6,258)\u0026thinsp;=\u0026thinsp;250.06, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, IRR F(1,43)\u0026thinsp;=\u0026thinsp;6.28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Drug \u0026times; Time F(6,258)\u0026thinsp;=\u0026thinsp;3.25, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, IRR \u0026times; Time F(6,258)\u0026thinsp;=\u0026thinsp;12.13, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At 1 month post-IRR, CDDO-EA/Sham females transiently weighed 8.6% more than Veh/Sham females (post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). From 2 months post-IRR through study completion, Veh/33-GCR females weighed 9.3\u0026ndash;18.3% more than Veh/Sham females (post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 at months 2\u0026ndash;4; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 at months 6\u0026ndash;7). This weight difference was present before the onset of appetitive touchscreen testing (months 2\u0026ndash;3, 9.3\u0026ndash;11.2% increase) and became more pronounced during the food-restricted touchscreen period (months 4\u0026ndash;7, 13.4\u0026ndash;18.3% increase), suggesting that caloric restriction may have amplified rather than driven the effect.\u003c/p\u003e \u003cp\u003eHome cage locomotor activity, measured over 18 hours including 12 hours of the dark cycle, was consistent with the locomotion differences observed during SLR testing. In males, Veh/Sham and Veh/33-GCR mice made a similar number of total beam breaks, indicating no effect of radiation alone on home cage activity. However, CDDO-EA/Sham males made 21.9% fewer total beam breaks compared to Veh/Sham males (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cb\u003eSupplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;1\u003c/b\u003e; Kruskal-Wallis p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc Veh/Sham vs. CDDO-EA/Sham, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This reduction was driven by 26.2% less ambulatory movement in CDDO-EA/Sham males compared to Veh/Sham males (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD; Kruskal-Wallis p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while fine movement did not differ among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE; Kruskal-Wallis p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Veh/33-GCR males also made fewer total and ambulatory beam breaks compared to CDDO-EA/Sham males (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with CDDO-EA reducing activity regardless of radiation status.\u003c/p\u003e \u003cp\u003eIn females, both 33-GCR and CDDO-EA independently reduced home cage locomotor activity. Veh/33-GCR females made 26.0% fewer total beam breaks than Veh/Sham females (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e; Kruskal-Wallis p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with 31.1% fewer ambulatory beam breaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 15.9% fewer fine movement beam breaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, CDDO-EA/Sham females made 24.7% fewer total beam breaks than Veh/Sham females (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with 31.5% fewer ambulatory beam breaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a trend toward 12.9% fewer fine movement beam breaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH; post-hoc p\u0026thinsp;=\u0026thinsp;0.06).\u003c/p\u003e \u003cp\u003eThese data show that 33-GCR exposure produced sustained weight gain and reduced home cage activity in female mice, while CDDO-EA reduced home cage activity in both sexes. These physiological effects dissociated from hippocampal-dependent cognition: females showed no radiation-induced pattern separation deficits despite pronounced weight and activity changes, while males showed radiation-enhanced pattern separation without corresponding weight or home cage activity differences.\u003c/p\u003e \u003cp\u003e33-GCR and CDDO-EA independently enhance goal tracking in females; males show no treatment effects on reward-based learning\u003c/p\u003e \u003cp\u003eWe next examined whether 33-GCR affected reward-based learning, given its distinct effects on hippocampal cognition in males versus physiological outcomes in females. Mice were tested on touchscreen-based Pavlovian autoshaping over 11 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This paradigm measures goal tracking (approaches to the reward magazine during CS+ presentation) and sign tracking (preferential approach to CS+ over CS\u0026minus;).\u003c/p\u003e \u003cp\u003eIn males, all groups increased reward magazine approaches over acquisition days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003eSupplementary Table\u0026nbsp;1; 3\u003c/b\u003e-way RM ANOVA, Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Within-group increases from early to late acquisition were confirmed in Veh/33-GCR and CDDO-EA/Sham groups (Days 4\u0026ndash;5 vs. Days 9 or 11; post-hoc: Veh/33-GCR Day 4 vs Day 11 and Day 5 vs. Day 11, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; CDDO-EA/Sham Day 4 vs Day 9 and Day 4 vs Day 11, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with similar numerical trends in Veh/Sham and CDDO-EA/33-GCR groups that did not reach significance. All male groups also progressively increased their preferential approach to CS+ over CS\u0026minus; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and showed similar accuracy in approaching CS+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD; Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Thus, neither 33-GCR nor CDDO-EA affected Pavlovian learning in males.\u003c/p\u003e \u003cp\u003eIn contrast, female mice showed treatment-dependent differences in goal tracking (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cb\u003eSupplementary Table\u0026nbsp;1; 3\u003c/b\u003e-way RM ANOVA, Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Drug p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Time\u0026times;IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Veh/33-GCR females made more reward magazine approaches than Veh/Sham females on Day 9 (167% more; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with similar trends on Day 5 (78% more; p\u0026thinsp;=\u0026thinsp;0.056) and Day 7 (200% more; p\u0026thinsp;=\u0026thinsp;0.062). CDDO-EA also independently increased goal tracking: CDDO-EA/Sham females made 109% (Day 5) and 191% (Day 6) more approaches than Veh/Sham females (post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CDDO-EA/33-GCR females also made 181% (Day 6) and 267% (Day 10) more approaches than Veh/33-GCR females (post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These combined effects suggest additive influences on reward anticipation, with each treatment independently contributing to enhanced goal-tracking. Despite these goal tracking differences, all female groups showed similar sign tracking (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF; Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; no group differences) and CS+ accuracy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG; Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; no group differences).\u003c/p\u003e \u003cp\u003eThus, both 33-GCR and CDDO-EA enhanced goal tracking in females, with effects accumulating across treatments, while sign tracking remained unaffected in both sexes. This pattern of results \u0026mdash; altered hippocampal function in males but altered reward processing in females \u0026mdash; indicates that 33-GCR does not produce uniform effects across interconnected circuit nodes.\u003c/p\u003e \u003cp\u003eCDDO-EA impairs reversal learning in males; 33-GCR impairs reversal learning in females\u003c/p\u003e \u003cp\u003eFollowing acquisition, mice underwent reversal learning in which CS\u0026thinsp;+\u0026thinsp;and CS\u0026minus; locations were switched (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Males were tested for 5 days; females for 6 days.\u003c/p\u003e \u003cp\u003eIn males, all groups showed similar numbers of approaches to the new CS+ location across reversal days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cb\u003eSupplementary Table\u0026nbsp;1; 3\u003c/b\u003e-way RM ANOVA, Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Drug, IRR, and interactions all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, CDDO-EA impaired reversal accuracy regardless of irradiation status (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC; Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Drug p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On Day 1, CDDO-EA/33-GCR males showed 45% lower accuracy than Veh/33-GCR males (post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). On Days 2 and 3, CDDO-EA/Sham males showed 36\u0026ndash;37% lower accuracy than Veh/Sham males (post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Thus, CDDO-EA impaired behavioral flexibility in males independent of radiation exposure.\u003c/p\u003e \u003cp\u003eIn females, 33-GCR impaired reversal learning regardless of CDDO-EA treatment. On Day 6, both irradiated groups made fewer approaches to the new CS+ than their respective sham controls: Veh/33-GCR females made 26% fewer approaches than Veh/Sham females, and CDDO-EA/33-GCR females made 16% fewer approaches than CDDO-EA/Sham females (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cb\u003eSupplementary Table\u0026nbsp;1; 3\u003c/b\u003e-way RM ANOVA, Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Time\u0026times;IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both comparisons). Similarly, 33-GCR reduced reversal accuracy: CDDO-EA/33-GCR females showed 33% lower accuracy than CDDO-EA/Sham females on Day 4 (post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Veh/33-GCR females showed 27% lower accuracy than Veh/Sham females on Day 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE; Time p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Time\u0026times;IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe source of behavioral flexibility impairment thus differed between sexes: CDDO-EA impaired reversal learning in males regardless of radiation status, while 33-GCR impaired reversal learning in females regardless of CDDO-EA treatment. This is a direct countermeasure trade-off: CDDO-EA normalized radiation-enhanced pattern separation in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) while independently impairing reversal learning in the same animals. This effect would be missed entirely by any study assessing only a single cognitive endpoint.\u003c/p\u003e \u003cp\u003eNeither 33-GCR nor CDDO-EA affects anxiety-like behavior; combined treatment reduces locomotion in females\u003c/p\u003e \u003cp\u003eTo determine whether the locomotor reductions observed across testing contexts (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) reflected anxiety, mice were tested on the elevated plus maze (EPM) at 6.5 months (males) and 6.75 months (females) post-IRR. Males showed no group differences in time spent in open arms, open arm entries, open-to-closed arm ratio, or total distance traveled (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D, \u003cb\u003eSupplementary Table\u0026nbsp;1; 2\u003c/b\u003e-way ANOVA, all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 or post-hoc p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In females, Drug\u0026times;IRR interactions emerged for open arm time and entries (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-F; Drug\u0026times;IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively). However, the open-to-closed arm ratio, a locomotion-independent measure of anxiety, did not differ among female groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG; all post-hoc p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating no true anxiety-like phenotype. Rather, these interactions reflected locomotor differences: CDDO-EA/33-GCR females traveled 22% shorter distances than CDDO-EA/Sham females (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH; IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Drug\u0026times;IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), consistent with the home cage activity and SLR locomotion patterns described above. These data indicate that neither 33-GCR nor CDDO-EA induced anxiety-like behavior in either sex. The locomotor reductions observed throughout the study therefore reflect a separable physiological effect rather than anxiety-driven changes in exploration.\u003c/p\u003e \u003cp\u003ePrincipal component analysis reveals circuit-specific treatment effects across cognitive domains\u003c/p\u003e \u003cp\u003eTo examine how 33-GCR and CDDO-EA affected relationships among interconnected cognitive domains, we performed principal component analysis (PCA) on six behavioral measures: pattern separation at three difficulty levels (d-SLR, s-SLR, xs-SLR), goal tracking, sign tracking, and reversal learning (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The first three principal components explained 57.6% of total variance (PC1: 20.8%, PC2: 19.7%, PC3: 17.2%; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003ePC1 captured an axis contrasting stimulus-driven responding with executive control. Sign tracking loaded positively (+\u0026thinsp;0.582) while reversal learning (\u0026minus;\u0026thinsp;0.485) and discrimination tasks (d-SLR: \u0026minus;0.463; s-SLR: \u0026minus;0.410) loaded negatively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Vector angles confirmed competitive interactions (\u0026gt;\u0026thinsp;135\u0026deg;) between sign tracking and reversal/discrimination tasks (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, inset, orange arcs). CDDO-EA/33-GCR animals showed higher PC1 scores than Veh/33-GCR animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-way ANOVA, Treatment p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating a shift toward stimulus-driven responding in the combined treatment group.\u003c/p\u003e \u003cp\u003ePC2 captured an axis contrasting hippocampal-dependent discrimination with reversal learning. The discrimination tasks loaded positively (xs-SLR: +0.835; s-SLR: +0.604) while reversal learning loaded negatively (\u0026minus;\u0026thinsp;0.459). Vector angles between discrimination tasks were \u0026lt;\u0026thinsp;60\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, inset, blue arcs), indicating coordinated, rather than competing, operations. PC2 scores did not differ across treatment groups or sex (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-way ANOVA, all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that hippocampal discrimination capacity was preserved despite radiation and drug treatment.\u003c/p\u003e \u003cp\u003ePC3 captured goal-directed behavior, with goal tracking loading strongly positive (+\u0026thinsp;0.702) and sign tracking (\u0026minus;\u0026thinsp;0.385) and reversal (\u0026minus;\u0026thinsp;0.328) loading negatively. Vector angles showed goal tracking oriented\u0026thinsp;~\u0026thinsp;90\u0026deg; from sign tracking (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, inset, purple arcs), indicating functional independence. PC3 scores differed across treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-way ANOVA, Treatment p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with the selective goal-tracking changes observed in females.\u003c/p\u003e \u003cp\u003eThese data demonstrate that CDDO-EA treatment under 33-GCR exposure selectively altered specific circuit relationships, particularly the balance between stimulus-driven and goal-directed behavior (PC1), while still preserving hippocampal discrimination capacity (PC2). Instead, the behavioral effects observed in males reflect altered circuit communication involving NAc and PFC nodes rather than hippocampal damage \u003cem\u003eper se\u003c/em\u003e. This selective vulnerability with preserved function is consistent with the domain-specific patterns observed in individual behavioral measures (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e33-GCR produces persistent alterations in dentate gyrus activity during memory encoding\u003c/p\u003e \u003cp\u003eTo assess whether the domain-specific behavioral effects reflected stable changes in hippocampal circuit function, we performed fiber photometry in a subset of male Veh/Sham and Veh/33-GCR mice at 7.25 months post-IRR (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-B). Ca\u0026sup2;⁺ transients were recorded from DG glutamatergic neurons during SLR testing. Behaviorally, in this small surgical cohort (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4/group), both groups showed positive d2 ratios under d-SLR. Under xs-SLR, the directional pattern mirrored the main cohort: Veh/Sham mice showed a negative d2 ratio (\u0026minus;\u0026thinsp;0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17) while Veh/33-GCR mice showed a positive d2 ratio (+\u0026thinsp;0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). This did not reach statistical significance, consistent with the reduced power of this subset (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eDuring the Sample phase (encoding), Veh/33-GCR mice showed enhanced DG activity compared to Veh/Sham mice. Under d-SLR, Ca\u0026sup2;⁺ transient rates were similar between groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE), but Veh/33-GCR mice showed 127% higher signal amplitudes at Object zone 3 than Veh/Sham mice (Kruskal-Wallis p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 at all zones; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). Under xs-SLR, Veh/33-GCR mice showed 29% lower transient rates at Object 3 than Veh/Sham mice (Kruskal-Wallis p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG) but 210\u0026ndash;516% higher amplitudes across all object zones (Object p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 at all zones; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eDuring the Test phase (retrieval), Veh/33-GCR mice showed 40% lower transient rates at the Novel location than Veh/Sham mice under d-SLR (Kruskal-Wallis p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI) and 103% lower amplitudes at the Familiar location (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eJ).\"Under xs-SLR, transient rates did not differ across groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eK). Signal amplitudes showed a significant group effect (Kruskal-Wallis p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eL): Veh/33-GCR mice showed higher amplitude at the Novel versus Familiar location (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas Veh/Sham mice showed no location-specific difference.\u003c/p\u003e \u003cp\u003eGiven the small cohort size (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4/group), these findings should be interpreted with caution and treated as preliminary; the patterns reported here are intended to motivate future work with adequately powered samples rather than to support mechanistic conclusions. These findings are consistent with 33-GCR producing persistent alterations in DG circuit activity detectable 7 months post-IRR, well beyond the behavioral testing window. The enhanced encoding amplitudes and location-selective retrieval patterns suggest that hippocampal circuit dynamics may be persistently shifted rather than transiently perturbed.\u003c/p\u003e \u003cp\u003eCombined CDDO-EA and 33-GCR reduces dentate gyrus progenitor cells in females despite intact pattern separation\u003c/p\u003e \u003cp\u003eTo assess whether treatments affected cellular indices relevant to hippocampal function, we quantified doublecortin-immunoreactive (DCX+) cells in the dentate gyrus at 7.25 months post-IRR (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn males, all groups showed similar numbers of DCX+ immature neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB, \u003cb\u003eSupplementary Table\u0026nbsp;1; 2\u003c/b\u003e-way ANOVA, all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), with comparable distribution across the rostro-caudal axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-way RM ANOVA, Bregma p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; no group differences). DCX+ progenitor cell numbers were also similar across groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eF; all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), with no differences along the rostro-caudal axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eG; Bregma p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; all post-hoc p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eIn females, DCX+ immature neuron numbers were similar across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-way ANOVA, all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), with comparable rostro-caudal distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eE; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-way RM ANOVA, Bregma p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). However, 33-GCR reduced DCX+ progenitor cells in CDDO-EA-treated females. CDDO-EA/33-GCR females had 21% fewer total DCX+ progenitor cells than CDDO-EA/Sham females (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eH; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-way ANOVA, IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This reduction was particularly evident in the dorsal DG: at Bregma\u0026thinsp;\u0026minus;\u0026thinsp;1.90, CDDO-EA/33-GCR females had significantly fewer progenitors than CDDO-EA/Sham females (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eI; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-way RM ANOVA, Bregma p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, IRR p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; post-hoc p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Veh/33-GCR females did not differ from Veh/Sham females (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that this progenitor reduction required the combination of CDDO-EA and 33-GCR. This suggests that Nrf2 activation may alter the cellular context in which radiation affects progenitor populations.\u003c/p\u003e \u003cp\u003eThese data demonstrate that combined CDDO-EA and 33-GCR exposure reduced DCX+ progenitor cells in females, particularly in the dorsal hippocampus, despite intact pattern separation performance in these animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Pattern separation was assessed at 3.75 months post-IRR while DCX+ cells were quantified at 7.25 months post-IRR; these measurements are therefore temporally decoupled and cannot be interpreted as a direct cellular-behavioral dissociation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrior GCR studies have assessed single cognitive endpoints in single sexes, leaving open whether radiation effects are global or circuit-selective and whether countermeasures protect uniformly or trade off across domains. The present data resolve both questions, and the answers are more complex than either framing anticipated. Here we show that 33-GCR produced domain-specific rather than global cognitive effects, with distinct vulnerability patterns in males and females. In addition, CDDO-EA acted as a double-edged sword: normalizing some radiation effects while impairing others, a trade-off invisible to single-endpoint assessment.\u003c/p\u003e \u003cp\u003eIn males, 33-GCR enhanced pattern separation specifically under very high memory load, consistent with prior single-ion studies showing improved pattern separation following ⁵⁶Fe or \u0026sup2;⁸Si exposure in males\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. The convergence across radiation types and testing paradigms suggests this reflects a genuine radiation effect rather than a paradigm artifact. CDDO-EA normalized this enhancement, to our knowledge the first demonstration that this compound can reverse radiation-induced enhancements rather than solely prevent impairments. Enhanced pattern separation does not necessarily indicate improved cognitive function: in clinical populations, overly distinct memory representations can interfere with generalization and flexible cognition\u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn females, pattern separation did not differ among groups at any memory load, contrasting with prior work reporting impairments at earlier timepoints using a different paradigm\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. That task required spatial memory retention over \u0026ge;\u0026thinsp;24 hours, whereas the SLR task tests discrimination within ~\u0026thinsp;35 minutes, a fundamental difference in memory demand. Furthermore, females in the prior work showed hippocampal-dependent deficits during training before pattern separation testing\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, raising the possibility that pre-existing impairments confounded performance. Consistent with the present findings, a prior 33-GCR study in females using touchscreen-based tasks also found no radiation effect on pattern separation\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFiber photometry revealed enhanced DG signal amplitudes during encoding in irradiated males, particularly under very high memory load, with location-selective retrieval patterns at 7 months post-IRR. To our knowledge, this is the first use of in vivo Ca\u0026sup2;⁺ imaging to assess hippocampal activity during cognitive testing in the context of space radiation. The stage-specific nature of these alterations, enhanced encoding but shifted retrieval, points to changes in circuit dynamics rather than uniform suppression or excitation⁹\u003csup\u003e89,90\u003c/sup\u003e. Increased encoding amplitude may strengthen memory formation and contribute to more distinct representations\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e,\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e, though causal relationships cannot be established from these data. These alterations were detectable 7 months post-IRR, suggesting stable rather than transient changes in circuit function. The small cohort size (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4/group) limits interpretation; these findings are best treated as preliminary.\u003c/p\u003e \u003cp\u003eDCX+ progenitor cells were reduced in CDDO-EA/33-GCR females at 7.25 months post-IRR, but not in Veh/33-GCR females, indicating this reduction required combined treatment. This contrasts with our prior work showing that 33-GCR alone reduces DCX+ immature neurons but not progenitors at 14.25 months post-IRR\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e, suggesting CDDO-EA may alter the temporal trajectory of radiation effects on neurogenesis-related populations: combined treatment affects earlier-stage progenitor cells at intermediate timepoints, whereas radiation alone affects later-stage immature neurons at extended timepoints. Since pattern separation was assessed at 3.75 months post-IRR and DCX+ cells at 7.25 months post-IRR, these measurements are temporally decoupled and cannot be interpreted as a direct cellular-behavioral dissociation; rather, they reveal a late-emerging cellular consequence of combined treatment not captured by behavioral assessment alone.\u003c/p\u003e \u003cp\u003eFemale mice often show resilience to space radiation-induced cognitive deficits relative to males\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan additionalcitationids=\"CR94 CR95 CR96\" citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e, and the present findings are consistent with this pattern. In males, 33-GCR altered hippocampal-dependent cognition without affecting body weight or home cage activity, suggesting effects concentrated in cognitive circuits rather than distributed across metabolic and motor systems\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e,\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e,\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. The mechanisms underlying female resilience are not established; proposed factors include differences in neuroimmune responses, antioxidant capacity, and gonadal hormone signaling\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan additionalcitationids=\"CR101\" citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u003c/sup\u003e, though which, if any, contribute here cannot be determined from the present data. Preserved cognition in females may nonetheless come at a metabolic cost: persistent weight gain in Veh/33-GCR females suggests radiation-induced disruption of energy homeostasis\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e,\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/sup\u003e, and reduced locomotor activity may reflect metabolic changes or altered motivation rather than cognitive impairment\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. These distinct patterns between males and females underscore why single-sex studies, still common in radiation neuroscience\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e and in neuroscience more broadly\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, can produce incomplete or misleading conclusions about radiation effects on cognition.\u003c/p\u003e \u003cp\u003eLocomotor reductions were observed across multiple contexts in both sexes but followed sex-specific patterns: in males, CDDO-EA alone reduced home cage activity while radiation had no additional effect, whereas in females both 33-GCR and CDDO-EA reduced activity across home cage, SLR, and EPM contexts. These reductions did not confound cognitive outcomes: pattern separation was enhanced in irradiated males despite no locomotor changes, and intact in females despite pronounced locomotor reductions. The EPM confirmed these reductions do not reflect anxiety, supporting the interpretation that locomotor and cognitive effects represent separable consequences of treatment.\u003c/p\u003e \u003cp\u003eThe autoshaping paradigm dissociates sign-tracking (dorsolateral striatum/amygdala-dependent), goal-tracking (NAc/PFC-dependent), and reversal learning (OFC-dependent)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan additionalcitationids=\"CR106 CR107 CR108 CR109\" citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u003c/sup\u003e. The selective enhancement of goal-tracking in females, with no autoshaping effects in males, points to regional rather than uniform circuit vulnerability. Goal-tracking changes without corresponding hippocampal alterations may reflect the NAc's integration of multiple input streams beyond the hippocampus, including direct cortical and amygdala inputs\u003csup\u003e\u003cspan additionalcitationids=\"CR112\" citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCDDO-EA enhanced goal-tracking in females but had no reward-related effects in males, indicating sex-specific modulation of NAc-PFC circuitry by Nrf2 activation\u003csup\u003e\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e\u003c/sup\u003e. This sex specificity extended to the combined treatment: CDDO-EA and 33-GCR produced additive goal-tracking enhancement in females, with no parallel effect in males. This raises the possibility that GCR and Nrf2 activation converge on shared circuitry in a sex-dependent manner, consistent with evidence that opponent striatal monoamine signaling modulates reinforcement behavior\u003csup\u003e\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e\u003c/sup\u003e. The specificity of these effects to goal-tracking rather than sign-tracking suggests CDDO-EA preferentially modulates NAc-PFC circuits\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan additionalcitationids=\"CR106 CR107 CR108 CR109\" citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u003c/sup\u003e, possibly through sex-dependent differences in dopaminergic signaling or Nrf2 expression\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn males, CDDO-EA impaired reversal accuracy without affecting initial acquisition, pointing to selective disruption of behavioral updating, the ability to revise a learned response when contingencies change, rather than reward learning itself. In females the picture was different; the drug was not the culprit, radiation was, and the impairment likely reflects the learning context established during acquisition. Enhanced goal-tracking in irradiated females may have created strong reward-location associations that made updating cue-reward contingencies particularly difficult, given that sign-tracking and goal-tracking behaviors resist contingency changes\u003csup\u003e\u003cspan additionalcitationids=\"CR116 CR117\" citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e\u003c/sup\u003e. Reversal impairment looked the same on the surface in both sexes but arose from distinct circuit-level processes: behavioral updating in males, acquisition strategy in females.\u003c/p\u003e \u003cp\u003eThe autoshaping reversal impairment in females contrasts with our previous finding that CDDO-EA/33-GCR females showed enhanced cognitive flexibility in touchscreen-based spatial discrimination reversals\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. This dissociation is reconcilable: the touchscreen task requires spatial discrimination without competing goal-tracking strategies, so reversal does not require overcoming a strongly established reward-location habit. Irradiated females thus retain capacity for flexible behavioral updating; the impairment is context-dependent, not a signature of broad OFC dysfunction.\u003c/p\u003e \u003cp\u003ePCA quantified what the individual behavioral results suggested qualitatively. The PC2 null result is particularly informative: pattern separation tasks and reversal learning maintained their relationship across all treatment groups, indicating the hippocampus itself was not globally disrupted. The behavioral effects seen in males therefore reflect altered signaling at NAc and PFC nodes rather than hippocampal damage per se. PC1 and PC3, by contrast, shifted with treatment, capturing the rebalancing of stimulus-driven versus goal-directed behavior. Some circuit relationships were disrupted; others held. That selective pattern, rather than uniform impairment, is the defining feature of circuit-selective vulnerability.\u003c/p\u003e \u003cp\u003eDomain-specific drug-induced cognitive effects are well recognized in clinical pharmacology\u003csup\u003e\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e\u003c/sup\u003e; preclinical countermeasure evaluation has rarely followed suit, largely because single cognitive endpoints remain the norm\u003csup\u003e\u003cspan additionalcitationids=\"CR121\" citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e\u003c/sup\u003e. Region-specific neural effects of space radiation are equally well established\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e\u003c/sup\u003e, but the corollary, that countermeasures may show domain-dependent trade-offs, has not been systematically tested. The practical consequences are stark: a study assessing only pattern separation would conclude CDDO-EA is protective; a study assessing only reversal learning would conclude it is harmful. Neither conclusion captures the reality that CDDO-EA's effects are circuit-dependent, sex-dependent, and only fully visible through multi-domain assessment.\u003c/p\u003e \u003cp\u003eThree limitations warrant mention. Fiber photometry and DCX+ quantification were conducted at 7\u0026ndash;7.25 months post-IRR while behavioral testing occurred at 3.25\u0026ndash;4.5 months; concurrent measurements at matched timepoints are needed to establish whether cellular changes relate directly to behavioral performance. Fiber photometry was conducted only in males; extending these recordings to females would clarify whether distinct circuit activity patterns underlie preserved pattern separation in that sex. The separate statistical analyses of males and females, while appropriate given divergent response profiles, preclude direct statistical comparison of sex differences, a limitation shared broadly in the field\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMale and female mice showed distinct, circuit-selective vulnerability patterns that would have been invisible to single-task or single-sex designs. The core finding is not simply that radiation affects cognition, but that its effects are circuit-specific and sex-dependent. A candidate countermeasure can simultaneously protect one function while impairing another, a trade-off invisible to single-endpoint assessment. As space agencies plan missions beyond Earth orbit, these findings have immediate relevance for astronaut risk assessment. Any stressor or neuroprotective intervention evaluated with limited behavioral endpoints risks missing exactly these trade-offs; the present framework provides a template for doing better.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eAnimals\u003c/p\u003e \u003cp\u003eMale (M, n\u0026thinsp;=\u0026thinsp;124) and female (F, n\u0026thinsp;=\u0026thinsp;48) C57BL/6J mice (\u003cem\u003eMus musculus\u003c/em\u003e C57BL/6J, 4.5-5 months old; RRID:IMSR_JAX:000664; Jackson Laboratory, Bar Harbor, ME) were shipped to Brookhaven Laboratory Animal Facility (BLAF) at Brookhaven National Laboratory (BNL, Upton, NY). Males arrived in variable group sizes (2 cages of 5 mice, 23 cages of 4 mice, 1 cage of 3 mice, 8 cages of 2 mice, and 3 single-housed mice), while females arrived in uniform groups (12 cages of 4 mice). Upon arrival, mice were maintained with their original cagemates throughout the study. After three days of acclimation, mice received ear punch followed by first weight measurement. Two days later, mice were transported to the NASA Space Radiation Laboratory (NSRL) within BLAF for treatment with either 33-GCR IRR or Sham IRR and returned to BLAF the following day. At both facilities, mice were housed 4/cage in HEPA-filtered, closed airflow vivarium systems under a 12:12 h light/dark cycle (06:00 light on) at 22\u0026deg;C, 30\u0026ndash;70% humidity with standard rodent chow (5015; Lab Diet, cat# 0001328) and water ad libitum. Two days post-IRR, mice were transported by ground to Children's Hospital of Philadelphia (CHOP) and held in quarantine for 6 weeks with ad libitum access to medicated chow (13 PPM ivermectin and 150 PPM fenbendazole; Test Diet, custom cat# 1813527[5SKU]). Behavioral testing began at 9 weeks post-IRR when mice were released from quarantine and returned to standard chow (5015). At CHOP, mice were housed in HEPA-filtered, closed airflow vivarium systems (Enviro-Gard\u0026trade; A; Lab Products Inc.) under a 12:12 h light/dark cycle (06:15 light on) at 20\u0026ndash;23\u0026deg;C, 30\u0026ndash;40% humidity. Each cage received a nestlet square at cage changes; no other enrichment was provided. All procedures were approved by IACUCs at BNL and CHOP in accordance with AAALAC and NIH guidelines (CHOP: AAALAC #000427, PHS D16-00280 [OLAW A3442-01]; BNL: AAALAC #000048, PHS D16-00067 [OLAW A3106-01]). Our reporting adheres to ARRIVE 2.0 guidelines \u003csup\u003e\u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDrug administration\u003c/p\u003e \u003cp\u003eCages were sequentially preassigned to treatment groups in rotating order (Veh/Sham, Veh/33-GCR, CDDO-EA/Sham, CDDO-EA/33-GCR), balanced for mean cage body weight and cage size (number of mice per cage). Drug treatment groups consisted of mice receiving either CDDO-EA (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid ethylamide, 4 mg/kg IP; MedChemExpress, cat# HY-12213; n\u0026thinsp;=\u0026thinsp;83, M:59, F:24) or vehicle control (matching volume IP; n\u0026thinsp;=\u0026thinsp;89, M:65, F:24).\u003c/p\u003e \u003cp\u003e The vehicle solution was prepared according to the manufacturer's instructions and consisted of 5% DMSO (Sigma-Aldrich, cat# D2650) and 20% Sulfobutylether-β-Cyclodextrin (SBE; MedChemExpress, cat# HY-17031) in 0.9% saline solution (Grainger, cat# 3PWK4). Both CDDO-EA and vehicle were administered intraperitoneally once daily between 8:00\u0026ndash;10:00 AM for three consecutive days. IRR was performed on Day 2 of the treatment regimen (one day after the first injection, concurrent with the second injection, and one day before the final injection).\u003c/p\u003e \u003cp\u003eIrradiation (IRR)\u003c/p\u003e \u003cp\u003eOn the second day of drug treatment (Day 0 of IRR), mice underwent IRR during the BNL 22A campaign as previously described \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Mice were placed with cagemates in well-ventilated polycarbonate containers (10 \u0026times; 10 \u0026times; 4.5 cm), with 2 mice per container when possible or individually housed if no cagemate was available. Irradiated mice were exposed to 75 cGy of NASA's whole-body 33-beam GCR simulation delivered over a 60 \u0026times; 60 cm field for approximately 1.25 hours beginning at 11:45 AM. Beam uniformity and dosimetry were monitored by NSRL staff. Sham-irradiated mice were placed in containers with cagemates (or individually) for the same duration but were not exposed to the beam. This resulted in four experimental groups: Veh/Sham (M:32, F:12), Veh/33-GCR (M:33, F:12), CDDO-EA/Sham (M:30, F:12), and CDDO-EA/33-GCR (M:29, F:12).\u003c/p\u003e \u003cp\u003eSurgery\u003c/p\u003e \u003cp\u003eC57BL/6J male mice (3\u0026ndash;4 mice/group) underwent viral infusion and fiber implantation at 6 months post-IRR. Before surgery, each mouse was weighed and the head was shaved using an electric clipper (Wahl, cat# 41590-0438). Anesthesia was induced in an induction chamber with 4\u0026ndash;5% isoflurane (Piramal Pharma Limited, cat# 66794-013-25) in 100% oxygen and maintained at 1\u0026ndash;3% isoflurane during surgery. Ophthalmic lubricant (Dechra, cat# B00HGMZ7RQ) was applied to both eyes, and buprenorphine ER (1 mg/kg, s.c.) was administered before incision. Following aseptic preparation of the surgical site with betadine (Avrio Health, cat# 67618-155-16) and 70% isopropyl alcohol, a midline incision was made. The skull was scored with a scalpel blade and treated with 30% hydrogen peroxide solution (Sigma Aldrich, cat# MKCJ1024). A burr hole was drilled above the target site using a surgical electric drill with a ⅛ inch engraving bit (Dremel, cat# 7350).\u003c/p\u003e \u003cp\u003eAAV9-CaMKII-GCaMP6f (Penn Vector Core, 100834) was unilaterally infused into the hilus of the dorsal dentate gyrus (DG) hilus (A/P -2.0 mm, M/L -1.4 mm, D/V -2.2 mm from bregma) using a 33-gauge Hamilton syringe (Hamilton, cat# 2141205) at 0.1 \u0026micro;l/min \u003csup\u003e\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e\u003c/sup\u003e. An optic fiber (Thorlabs, #CFML22L05, \u0026Oslash;1.25 mm SS ferrule, \u0026Oslash;200 \u0026micro;m core, 0.22 NA, L\u0026thinsp;=\u0026thinsp;2 mm) was implanted in the molecular layer of the DG middle/outer molecular layer (A/P -2.0 mm, M/L -1.4 mm, D/V -1.8 mm from bregma). The fiber was secured to the skull with light-cured resin (Ivoclar Vivadent AG, cat# 595979US) followed by light-cured adhesive (Pearson, cat# 595979). Following surgery, the incision was sutured, triple-antibiotic ointment was applied topically, and meloxicam (5 mg/kg, s.c.; Norbrook, cat# 5552904010) was administered. Mice were monitored daily for 48 hours post-operatively.\u003c/p\u003e \u003cp\u003eBody weight monitoring and health observations\u003c/p\u003e \u003cp\u003eMouse body weight was measured monthly from arrival at BNL (4.5 months of age) through tissue collection (12.25 months of age). Health status and cage conditions were monitored during weighing sessions and biweekly cage changes by the Children's Hospital of Philadelphia (CHOP) Department of Veterinary Resources, with documentation of fighting, illness, or loss of cagemates. When aggressive behavior was identified, aggressor mice were isolated into separate cages to prevent injury. Seven male cages required splitting due to aggression (3 Veh/Sham, 3 CDDO-EA/Sham, 1 Veh/33-GCR). All mice from these disrupted cages were excluded from behavioral testing to maintain consistent social housing conditions across experimental groups.\u003c/p\u003e \u003cp\u003eBehavioral testing\u003c/p\u003e\n\u003ch3\u003eOverview of behavioral testing\u003c/h3\u003e\n\u003cp\u003eBehavioral testing groups were selected in rotating order across treatment groups (Veh/Sham, Veh/33-GCR, CDDO-EA/Sham, CDDO-EA/33-GCR) and balanced for mean cage body weight. Most cages contained 4 mice per cage when behavioral testing began, except one female cage with 3 mice. Two deaths occurred: female #28 (CDDO-EA/Sham) died at 1 month post-IRR before behavioral testing began, and male #9 (CDDO-EA/Sham) died at 2.5 months post-IRR during locomotor testing. Both deaths reduced their respective cages to 3 mice. Despite these losses, both affected cages were retained for behavioral testing due to limited female availability and to avoid excluding the male cagemates who had already begun testing. Both deceased mice were excluded from analyses. This resulted in 47 mice per sex (total n\u0026thinsp;=\u0026thinsp;94) for behavioral testing. Following release from quarantine at 2 months post-IRR, behavioral testing began at 2.25 months post-IRR with locomotor activity recording (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Gentle handling (2 min/day) began at 2.5 months post-IRR and continued for 5 days. Male mice underwent arena habituation for 5 days beginning at 3 months post-IRR to acclimate to the Spontaneous Location Recognition (SLR) arena, followed by SLR testing at 3.25 months post-IRR. Males then completed the Autoshaping task (Pavlovian learning) at 3.5 months post-IRR and anxiety behavioral testing at 6.5 months post-IRR. Female mice underwent arena habituation for one week beginning at 3.5 months post-IRR, followed by SLR testing at 3.75 months post-IRR. Females then completed the Autoshaping task at 4.5 months post-IRR and anxiety behavioral testing at 6.75 months post-IRR.\u003c/p\u003e\n\u003ch3\u003eHome cage activity monitoring\u003c/h3\u003e\n\u003cp\u003eEach mouse was individually placed in a clean mouse conventional cage containing fresh bedding to record 18 hours of locomotion activity from 4pm to 10am. This cage was positioned between 4\u0026times;8 photocells, with identical lighting parameters to home housing room dim/red lighting during the light cycle and red lighting during the dark cycle. Their movement across the XY plane was monitored by a computer-controlled photobeam activity system (San Diego Instruments), which recorded photocell beam breaks in 15-minute intervals over a period of 18 hours \u003csup\u003e\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e\u003c/sup\u003e. One male mouse from (mouse ID #10, group: CDDO/Sham) was excluded due to equipment failure.\u003c/p\u003e\n\u003ch3\u003eSpontaneous location recognition (SLR)\u003c/h3\u003e\n\u003cp\u003eThe spontaneous location recognition (SLR) task was performed following established protocols \u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eArena Setup and Objects.\u003c/span\u003e The testing arena consisted of a circular open field with 20 marked segments radiating from the center, etched by laser cutting at 18\u0026deg; intervals on the base and covered with corncob bedding during testing. Objects consisted of 50 ml conical centrifuge tubes containing three blue latex gloves, secured to the arena base with screws and nuts. Each arena was surrounded by three-sided black cardboard barriers displaying three distinct visual cues, with two additional cues mounted on the wall. These spatial cues remained consistent throughout testing to provide reliable landmark references. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eHabituation\u003c/span\u003e. Prior to SLR testing, mice underwent gentle handling (2 min/day) for 5 days to reduce stress, followed by arena habituation beginning at 3.0 months post-IRR for males and 3.5 months post-IRR for females. Subjects were placed in the arena with spatial cues for 10 minutes daily over five consecutive days. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eTesting Procedures\u003c/span\u003e. On test days, mice were transported to the testing room and acclimated in their home cages for 30 minutes. Testing time was kept consistent across subjects. Between subjects, one scoop of bedding was removed and replaced with clean bedding, and arena floors and walls were wiped with 10% ethanol solution to eliminate olfactory cues. The experimenter exited the room during testing sessions. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eExperimental Design and Memory Loads.\u003c/span\u003e The SLR experiment evaluated three memory loads based on spacing between objects 2 and 3: Dissimilar (d-; low memory load, 108\u0026deg; apart), Similar (s-; high memory load, 72\u0026deg; apart), and Extra similar (xs-; very high memory load, 36\u0026deg; apart). During the sample phase (10 min), three identical objects were placed at predetermined distances corresponding to one of the three memory load conditions, and mice freely explored the arena. Following a 35-minute retention interval in home cages, mice were returned for the test phase (5 min) with two objects: one at a familiar location (matching object 1 from the sample phase) and one at a novel location (positioned midway between the original locations of objects 2 and 3) \u003csup\u003e89\u003c/sup\u003e. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eData Analysis\u003c/span\u003e. Time spent in object zones was calculated based on nose position, and movement distance was calculated based on center body position using EthoVision XT 12 (Noldus Information Technology). The discrimination index (d2 ratio) was calculated as\u003c/p\u003e \u003cp\u003ed2 ratio\u0026thinsp;=\u0026thinsp;Time spent in (novel location - familiar location) / Time spent in both locations\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAutoshaping\u003c/h2\u003e \u003cp\u003eAutoshaping was performed between 8:00 AM and 2:00 PM daily (Monday-Friday) at CHOP using the Bussey-Saksida operant touchscreen platform (Lafayette Life Sciences, cat# 80614A) equipped with the ABET Core Intelli-Interface (Lafayette Life Sciences, cat# 81430) and ABET II Software (Lafayette Life Sciences, cat# 89509)\u003csup\u003e\u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e,\u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e\u003c/sup\u003e. The operant chamber contained a light, auditory cue speaker, food dispenser, and two white vertical rectangular response windows (6.5 \u0026times; 14 cm) positioned left and right of the reward dispenser. Two infrared photobeams detected approaches to the touchscreen and entries/exits to the food magazine. Behavioral tasks and data collection were controlled by ABET II Autoshaping Software (cat# 89544). The reward stimulus was Strawberry Ensure\u0026reg; Nutrition Shake (Abbott Laboratories) delivered without food deprivation. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eHabituation.\u003c/span\u003e Mice were handled for one minute daily for three days prior to habituation. During Hab 1 (one day), mice were placed in the chamber for 10 minutes with all lights off while strawberry milkshake was delivered into the food tray for 2800 ms (70 \u0026micro;l). The number of broken photobeams was recorded to assess locomotor activity. During Hab 2, mice remained in the chamber for 30 minutes while milkshake (280 ms, 7 \u0026micro;l) was delivered after variable intervals (0\u0026ndash;30 seconds), accompanied by tray light illumination and a tone. Once reward was delivered, the program waited for the mouse to enter the food tray before restarting the variable interval. Upon tray entry, the tray light was turned off and the procedure repeated. The criterion for Hab 2 was 30/40 trials within the 30-minute session, which most mice achieved within 2 days. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eAcquisition Training.\u003c/span\u003e Following habituation, mice underwent 11 days of autoshaping acquisition to associate one side of the screen as a positive conditioned stimulus (CS+) and the other side as a negative conditioned stimulus (CS-), counterbalanced between animals. Trials were conducted in pairs presenting both CS\u0026thinsp;+\u0026thinsp;and CS- stimuli in random order, ensuring no more than two consecutive presentations of the same stimulus type and preventing the same side from being illuminated first in trial pairs more than 3 times consecutively. After a variable interval (10\u0026ndash;40 seconds), the chosen stimulus was presented for 10 seconds when the animal was breaking the rear beam. For CS- trials, another variable interval (10\u0026ndash;40 seconds) followed before the other stimulus was presented. For CS+ trials, reward was delivered, tray entry was awaited, then a 10\u0026ndash;40 second variable interval preceded the other stimulus presentation. Sessions lasted 30 minutes. The criterion was completing at least 25 trials within the 30-minute session for two out of three consecutive days. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eReversal Learning.\u003c/span\u003e After 11 days of acquisition training, mice underwent reversal learning for 5 (males) or 6 (females) days using identical procedures except CS\u0026thinsp;+\u0026thinsp;and CS- assignments were switched. The difference in reversal duration between sexes was due to equipment availability. Data (the number of reward chamber approaches, approach difference between CS\u0026thinsp;+\u0026thinsp;and CS- approach, accuracy to CS+ approaches) were collected by ABET II Software (cat# 89509).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eElevated plus maze\u003c/h3\u003e\n\u003cp\u003eAnxiety behavior in the EPM (Harvard Apparatus, cat# 760075) was assessed 6 mon post-IRR. The EPM apparatus (99cm elevation; 2 open and 2 closed arms each measuring L 67cm x W 6cm, closed arm walls H 17cm) was constructed as described previously \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e,\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. Mice were placed in the center of the apparatus pseudorandomly facing one of two open arms, and allowed 5 minutes of free exploration under white lighting conditions (200 lux). Ethovision ver 12 software (Noldus Information Technology) was used to record the time spent in the open arms, closed arms, and center zone, as well as the frequency of entries into each area. From these measurements, an exploration index was calculated:\u003c/p\u003e \u003cp\u003eThe ratio of the time spent in open arms vs closed arms:\u003c/p\u003e \u003cp\u003eRatio of time open: closed\u0026thinsp;=\u0026thinsp;Time in open arms / Time in closed arms\u003c/p\u003e\n\u003ch3\u003eFiber photometry recording\u003c/h3\u003e\n\u003cp\u003eA separate \u003cem\u003ein vivo\u003c/em\u003e imaging cohort (n\u0026thinsp;=\u0026thinsp;7 males, 3 Veh/Sham and 4 Veh/33-GCR mice) received AAV9-CaMKIIa-GCaMP6f viral infusion in the DG hilus and optic fiber implantation in the molecular layer of the DG at 6 months post-IRR to monitor DG granule cell activity as described in Surgery section. This group performed the SLR with fiber photometry (FP) recording at 7.25 months post-IRR. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eRecording System and Setup.\u003c/span\u003e Three weeks post-surgery, fiber photometry recordings were conducted using the Neurophotometrics FP3002 system (Neurophotometrics LTD, CA) with customized Bonsai software during the SLR paradigm as previously described \u003csup\u003e\u003cspan additionalcitationids=\"CR130\" citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR131\" class=\"CitationRef\"\u003e131\u003c/span\u003e\u003c/sup\u003e. A patch cord (Doric Lenses, D204-80052, BBP(2)_200/220/900\u0026thinsp;\u0026minus;\u0026thinsp;0.37_2m_SMA-2xMF1.25) was connected unilaterally to the implanted optic fiber. Calcium-dependent fluorescence changes were recorded at 470 nm excitation, while calcium-independent fluorescence was captured at 415 nm excitation to control for motion artifacts and photobleaching. Data acquisition occurred continuously during both the Sample phase (10 min) and Test phase (5 min) of the SLR paradigm, with simultaneous behavioral video recording. \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eBehavioral Video Analysis\u003c/span\u003e. Behavioral videos were analyzed using Social LEAP Estimates Animal Poses (SLEAP v1.4.1) for pose estimation \u003csup\u003e\u003cspan citationid=\"CR132\" class=\"CitationRef\"\u003e132\u003c/span\u003e\u003c/sup\u003e, followed by Simple Behavioral Analysis (SimBA version 3.2.8 with Python 3.10) for behavioral quantification \u003csup\u003e\u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e133\u003c/span\u003e\u003c/sup\u003e. For SLEAP model training, 461 frames from 19 videos were annotated, achieving mean Average Precision of 0.854 and mean Average Recall of 0.881. All experimental videos were analyzed using this validated model. SLEAP output files (.csv format) were imported into SimBA for exploratory behavior quantification. Regions of interest were defined as: (1) a 5-cm radius circle centered at the base of each object, and (2) a 29-cm radius circle encompassing the entire arena. Behavioral metrics extracted included time spent in each zone, zone entry counts (calculated relative to nose position), total movement distance, and average velocity (calculated relative to center body position). Frame-by-frame Boolean values indicating zone occupancy were extracted for temporal alignment with photometry data using custom MATLAB code (MathWorks).\u003c/p\u003e \u003cp\u003eFluorescence changes (ΔF/F) were calculated as previously described \u003csup\u003e\u003cspan citationid=\"CR134\" class=\"CitationRef\"\u003e134\u003c/span\u003e\u003c/sup\u003e:\u003c/p\u003e \u003cp\u003eΔF/F = (Raw fluorescence - Fitted fluorescence) / Baseline fluorescence (F₀)\u003c/p\u003e \u003cp\u003eThe ΔF/F signals were normalized using Z-score transformation:\u003c/p\u003e \u003cp\u003eZ-score ΔF/F=[(ΔF/F) - mean(ΔF/F)]/ standard deviation(ΔF/F).\u003c/p\u003e \u003cp\u003eCalcium transient peaks were identified using a noise threshold defined by the Median Absolute Deviation (MAD) method. Peaks were defined as local maxima with: (1) amplitude greater than 0.1\u0026times;MAD above baseline, and (2) minimum separation of 0 data points between events. Peak frequency and amplitude were quantified from these identified events. For event-locked analysis, ΔF/F traces were aligned to specific behavioral events, including entry into the Familiar Object zone and Novel Object zone during the Test phase of the SLR paradigm.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection and processing\u003c/h2\u003e \u003cp\u003eBrain tissue was collected at 7.25 months post-IRR for the behavioral cohort and at 8.75 months post-IRR for the in vivo imaging cohort. Following decapitation, brains were immersed in 4% paraformaldehyde (PFA; Sigma-Aldrich, cat# P6148) in PBS for 3 days \u003csup\u003e\u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e135\u003c/span\u003e,\u003cspan citationid=\"CR136\" class=\"CitationRef\"\u003e136\u003c/span\u003e\u003c/sup\u003e, then cryoprotected by immersion in 30% sucrose (Fisher Scientific, cat# S5-3) containing 0.01% sodium azide (NaN₃; Sigma-Aldrich, cat# S8032) at 4\u0026deg;C for 24 hours until complete equilibration. Brains were coronally sectioned at 40 \u0026micro;m thickness using a freezing microtome (Leica SM2010R). The left hemisphere was marked with a 26-gauge needle on the dorsal cortical area for consistent orientation. Serial sections were collected systematically throughout the hippocampus for stereological assessment as previously reported \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e,\u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e135\u003c/span\u003e,\u003cspan citationid=\"CR137\" class=\"CitationRef\"\u003e137\u003c/span\u003e\u003c/sup\u003e. Sectioned tissue was stored in 1\u0026times; PBS containing 0.01% NaN₃ at 4\u0026deg;C until further processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eIHC was performed on slide-mounted coronal brain sections as previously described \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e,\u003cspan citationid=\"CR136\" class=\"CitationRef\"\u003e136\u003c/span\u003e\u003c/sup\u003e. Sections underwent antigen retrieval by placement in near-boiling citric acid (pH 6.0; Fisher Chemical, cat# A940-500). Endogenous peroxidase activity was quenched by incubation in 0.3% hydrogen peroxide (Sigma, cat# H-1009) in PBS. Non-specific binding was blocked with 3% normal donkey serum (Jackson ImmunoResearch, cat# 017-000-121) in 0.3% Triton X-100 in PBS. Sections were incubated overnight at room temperature with primary antibody against doublecortin (goat anti-DCX; Santa Cruz Biotechnology, cat# SC-8066; 1:500) or GFP (chicken anti-GFP; Aves Labs, cat# GFP-1020; 1:3000) diluted in 3% normal donkey serum with 0.3% Tween-20 in PBS. Following PBS washes, sections were incubated for 1 hour at room temperature with biotinylated secondary antibodies at 1:200 (donkey anti-goat IgG for DCX, Jackson ImmunoResearch, cat# 705-065-003; donkey anti-chicken IgG for GFP, Jackson ImmunoResearch, cat# 703-065-155; 1:200). After additional PBS washes, signal amplification was achieved using avidin-biotin complex (ABC; Vector Laboratories, cat# PK-6100) for HRP conjugation. The HRP signal was visualized using 3,3'-diaminobenzidine (DAB; Thermo Fisher Scientific, cat# 1856090) for DCX and Cy3-conjugated Tyramide Signal Amplification substrate (PerkinElmer, cat# FP1050) for GFP. Nuclei were counterstained with Fast Red (Vector Laboratories, cat# H-3403) for DCX IHC and DAPI (Roche, cat# 236276) for GFP IHC. Sections were dehydrated through a graded ethanol series, cleared in Citrasolv, and coverslipped using DPX mounting medium (Electron Microscopy Services, cat# 13512) with 24\u0026times;60 mm coverglasses (VWR, cat# 48393).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStereological cell counts\u003c/h2\u003e \u003cp\u003eDCX-immunoreactive (DCX+) cells were quantified by an observer blinded to experimental conditions using an Olympus BX-51 brightfield microscope at 400\u0026times; magnification. DCX+ cells in the subgranular zone (SGZ) of the dentate gyrus granule cell layer (GCL) were counted in the right hemisphere of each section, with left hemisphere counted only if right hemisphere damage occurred. Stereological principles were applied as previously described [44,111]. Quantification was conducted along the entire anterior-posterior hippocampal axis (-0.82 to -4.33 mm from bregma).\u003c/p\u003e \u003cp\u003eTwo cell classifications were recorded: (1) DCX+ immature neurons, defined as brown-stained soma in the SGZ with neurite and dendrite outgrowth containing at least one dendritic branching node, and (2) DCX+ progenitor cells, defined as brown-stained soma in the SGZ lacking neurite extension.\u003c/p\u003e \u003cp\u003eTotal cell populations were calculated using the following stereological formula \u003csup\u003e\u003cspan citationid=\"CR138\" class=\"CitationRef\"\u003e138\u003c/span\u003e\u003c/sup\u003e. The raw cell counts for each bregma level are presented separately.\u003c/p\u003e \u003cp\u003eTotal population of cells\u0026thinsp;=\u0026thinsp;Total cells counted x 1/ssf x 1/asf x 1/hsf\u003c/p\u003e \u003cp\u003ewhere ssf is the section sampling fraction (1/9 for one hemisphere analysis), asf is the area sampling fraction (1, as all cells were counted in sampled sections), and hsf is the height sampling fraction (1, given minimal edge artifact effects in counting soma\u0026thinsp;\u0026lt;\u0026thinsp;10 \u0026micro;m with ssf 1/18), as described previously \u003csup\u003e\u003cspan additionalcitationids=\"CR137\" citationid=\"CR136\" class=\"CitationRef\"\u003e136\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR138\" class=\"CitationRef\"\u003e138\u003c/span\u003e\u003c/sup\u003e. Since only one hemisphere was counted, the total population for both hemispheres was calculated as:\u003c/p\u003e \u003cp\u003eTotal population of cells in both hemispheres = (Total cells counted \u0026times; 9) \u0026times; 2\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis\u003c/h2\u003e \u003cp\u003ePrincipal component analysis (PCA) was performed on six behavioral measures: spatial discrimination (low [dSLR] and high [sSLR]: mean d2 ratio), pattern separation (very high [xsSLR]: mean d2 ratio), goal tracking (mean reward chamber approaches, days 4\u0026ndash;7), sign tracking (mean CS\u0026thinsp;+\u0026thinsp;vs. CS- approach difference, days 4\u0026ndash;7), and reversal learning (mean CS+ approaches after reversal, days 1\u0026ndash;4). For Pavlovian conditioning measures (goal and sign tracking), performance was averaged across days 4\u0026ndash;7 of acquisition when animals reached maximal performance. For reversal learning, performance was averaged across days 1\u0026ndash;4 post-reversal to capture initial acquisition of the new contingency when cognitive flexibility demands were highest. All measures were z-score normalized across all subjects (grand mean\u0026thinsp;=\u0026thinsp;0, SD\u0026thinsp;=\u0026thinsp;1) prior to analysis. PCA was conducted using scikit-learn (version 1.3.0) in Python 3.10 \u003csup\u003e139\u003c/sup\u003e. Component loadings (eigenvectors \u0026times; \u0026radic;eigenvalues) represent variable-component correlations; loadings \u0026gt;|0.40| were considered substantial. The first three principal components explained 57.6% of total variance (PC1: 20.8%, PC2: 19.7%, PC3: 17.2%). To quantify functional relationships between behavioral domains, vector angles from the three-dimensional loading space (PC1, PC2, PC3) were calculated using the arccosine of normalized dot products. Angles\u0026thinsp;\u0026lt;\u0026thinsp;60\u0026deg; indicated synergistic operations, angles\u0026thinsp;~\u0026thinsp;90\u0026deg; indicated functional independence, and angles\u0026thinsp;\u0026gt;\u0026thinsp;135\u0026deg; indicated competitive interactions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eComputer scripts\u003c/h2\u003e \u003cp\u003eCustom analysis scripts were developed in MATLAB and Python 3.10 (Google Colaboratory). A MATLAB script integrated Bonsai Ca\u0026sup2;⁺ signal output with SLEAP pose estimation data to calculate zone-specific transient rates and amplitudes during fiber photometry recordings. Python scripts processed 15-min interval photobeam locomotion data and performed principal component analysis (scikit-learn 1.3.0) on multi-domain behavioral measures, including vector angle calculations and visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBlinding, subject number, and data removal\u003c/h2\u003e \u003cp\u003eAll behavioral testing, tissue collection, and data analysis were conducted by investigators blinded to treatment conditions. Two mice were found dead during the study: one female (CDDO-EA/Sham) at 1 month post-IRR prior to data collection, and one male (CDDO-EA/Sham) at 2.5 months post-IRR after home-cage locomotion recording but before SLR testing. These subjects were excluded from all analyses. No additional mice were removed due to husbandry issues or veterinary recommendations. Task-specific subject numbers with exclusion criteria are detailed below and summarized in Supplementary Table\u0026nbsp;2. Treatment groups are ordered as Veh/Sham, Veh/33-GCR, CDDO-EA/Sham, and CDDO-EA/33-GCR throughout. Home-cage locomotion recording: One male CDDO-EA/Sham subject was not recorded due to technology failure. Final male subject numbers: n\u0026thinsp;=\u0026thinsp;12, 12, 10, and 12. Final female subject numbers: n\u0026thinsp;=\u0026thinsp;12, 12, 11, and 12. SLR: Subjects were excluded if they failed to meet predetermined criteria during the sample phase (\u0026gt;\u0026thinsp;2 seconds/object, \u0026gt;\u0026thinsp;10 seconds total exploration, equal percent exploration time per object) or test phase (\u0026gt;\u0026thinsp;1 second/object, \u0026gt;\u0026thinsp;5 seconds total exploration). Final male subject numbers: n\u0026thinsp;=\u0026thinsp;10, 10, 9, and 10. Final female subject numbers: n\u0026thinsp;=\u0026thinsp;12, 12, 9, and 11. Autoshaping: Subjects were excluded if they failed to reach acquisition criteria (\u0026ge;\u0026thinsp;25 trials in 2 out of 3 consecutive days during 11 days of testing). Final male subject numbers: n\u0026thinsp;=\u0026thinsp;10, 11, 10, and 10. Final female subject numbers: n\u0026thinsp;=\u0026thinsp;12, 12, 10, and 11.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData normality was assessed using D'Agostino-Pearson and Shapiro-Wilk tests with QQ plot visualization. Behavioral measures meeting normality assumptions were analyzed using ANOVA with drug (Veh, CDDO-EA) and irradiation (Sham, 33-GCR) as between-subjects factors. Two-way ANOVA was used for SLR discrimination indices (d2 ratio), SLR test locomotion, EPM measures (time in open arms, entries, exploration index, total distance), and DCX+ total cell counts. Three-way repeated measures ANOVA was used for SLR sample phase object exploration (drug \u0026times; irradiation \u0026times; object for objects 1, 2, 3), autoshaping measures (drug \u0026times; irradiation \u0026times; day for reward approaches, delta approaches, and percent accuracy during acquisition and reversal), and DCX+ cell distribution across bregma positions (drug \u0026times; irradiation \u0026times; bregma). Three-way repeated measures ANOVA was also used for body weight (drug \u0026times; irradiation \u0026times; time). Mixed-effects models were used when data were missing. Home cage locomotion (total, ambulatory, and fine beam breaks during both phase and interval testing) failed normality and was analyzed using Kruskal-Wallis tests with Dunn's post-hoc correction. Fiber photometry data were analyzed using two-way ANOVA (load \u0026times; irradiation for d2 ratio) or Kruskal-Wallis tests (for signal rate and amplitude measures). Significant ANOVA effects were followed by Tukey's or Bonferroni post-hoc tests corrected for multiple comparisons. Statistical significance was set at α\u0026thinsp;=\u0026thinsp;0.05. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Analyses were performed using R Studio, and Python 3.10 (scikit-learn 1.3.0, NumPy 1.24.3, SciPy 1.11.1, statsmodels 0.14.0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGraphs and figures\u003c/h2\u003e \u003cp\u003eGraphs were generated using GraphPad Prism 10 (GraphPad Software, San Diego, CA) and Python 3.10 (for PCA analysis). Brightfield photomicrographs were acquired using an Olympus digital camera with cellSens Standard software. Figures were assembled using Adobe Illustrator.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data, including individual animal-level data, necessary to replicate the findings of this study are publicly available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Behavioral data (spontaneous location recognition, autoshaping, reversal learning, elevated plus maze), home cage locomotor activity data, body weight data, fiber photometry recordings, and stereological cell counts have been deposited in Zenodo: https://doi.org/10.5281/zenodo.19157801. Analysis scripts for fiber photometry, locomotor activity, Statistical output and principal component analysis are available at\u003ca href=\"https://github.com/EischLab/NSRL22A\"\u003e\u0026nbsp;\u003c/a\u003ehttps://github.com/EischLab/NSRL22A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;S.A.O. contributed to Formal analysis, Investigation, Data Curation, Writing \u0026ndash; Original Draft, and Visualization. P.N. contributed to Software, \u0026nbsp;Formal analysis, Data Curation, Writing \u0026ndash; Original Draft, and Visualization. A.M. contributed to Formal analysis and Investigation. G.L.B. contributed to Formal analysis, Investigation and Data Curation. H.A.H. contributed to Formal analysis, Investigation and Data Curation. E.W-F. contributed to Investigation. S.V. contributed to Software. H.T. contributed to Software. F.C.K. contributed to Conceptualization and Investigation and Writing \u0026ndash; Review \u0026amp; Editing. A.J.E. contributed to Conceptualization, Methodology, Investigation, Resources, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing, Supervision, Project administration, and Funding acquisition. S.Y. contributed to Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data Curation, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing, Visualization, Supervision, Project administration, and Funding acquisition. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the team members at BNL/NSRL for their assistance with the 22A irradiation campaign, particularly Adam Rusek and Peter Guida. We are grateful to an anonymous donor for support of the Eisch Lab and this project. This work was supported by the following funding sources: S.Y. was supported by a 2019 NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation, a 2020 Penn Undergraduate Research Foundation grant, NASA HERO grant 80NSSC21K0814, a 2022 Foerderer Fund for Excellence Award, two CHOP Junior Faculty Awards (2021, PI: Bhoj; 2023, PI: Van Batavia), and NIH awards MH076690 (PI: Tamminga), MH107945 (PI: Eisch), and MH129970 (PI: Eisch). F.K. was supported by the Translational Research Institute for Space Health (TRISH) through NASA cooperative agreement NNX16AO69A, a Penn Provost/CHOP Postdoctoral Fellowship for Academic Diversity, and a Perelman School of Medicine Department of Radiation Oncology Pilot Grant (PIs: Fan and Eisch). A.J.E. was supported by NIH awards MH129970, NS007413, DA007290, DA023555, DA016765, and MH107945; NASA awards NNX07AP84G, NNX12AB55G, and NNX15AE09G; and NIH NS126279 (PI: Ahrens-Nicklas). S.Y. and A.J.E. were also supported by NIH DK135871 (PI: Zderic), NIH NS088555 (PI: Stowe), and NIH MH117628 (PI: Lambert). H.H. was supported by the 2021 Penn Undergraduate Research Mentoring Program (PURM), the 2022 Summer Undergraduate Internship Program (SUIP), the 2023 Penn College Alumni Society Board of Managers, a Penn President\u0026apos;s Undergraduate Research Grant, and an augmentation award to NASA HERO grant 80NSSC21K0814 (PI: Yun). \u0026nbsp;A.M. was supported by the Penn Career Services Summer Funding award during Summer 2022 and the Vagelos Molecular Life Sciences Program (2021-2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNelson, G. A. Space radiation and human exposures, A primer. \u003cem\u003eRadiat. Res.\u003c/em\u003e 185, 349\u0026ndash;358 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCherry, J. 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Med.\u003c/em\u003e 30, 1667\u0026ndash;1679 (2024).\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9476558/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9476558/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAstronauts on deep space missions face chronic exposure to galactic cosmic radiation (GCR). However, it remains unknown whether mission-relevant multi-ion GCR produces global or circuit-selective cognitive vulnerabilities and whether candidate countermeasures protect uniformly or show domain-dependent trade-offs. Here we used a 33-ion GCR simulation with concurrent countermeasure treatment to address both questions in male and female mice. C57BL/6J mice received 33-GCR (0.75 Gy) or sham radiation with the Nrf2-activating compound CDDO-EA or vehicle, followed by multi-domain behavioral assessment across the hippocampal-nucleus accumbens-prefrontal circuit. Under very high memory load, male Veh/33-GCR mice showed enhanced pattern separation compared to Veh/Sham males, an effect normalized by CDDO-EA. Female mice showed no radiation-induced changes in pattern separation but weighed more than Veh/Sham females and had reduced locomotor activity. Reward-based learning differed by sex: males showed no changes, while female Veh/33-GCR mice displayed enhanced reward anticipation, with both treatments contributing to elevated goal-tracking. For behavioral flexibility, CDDO-EA impaired reversal learning in males regardless of radiation, while 33-GCR impaired reversal learning in females regardless of CDDO-EA. Principal component analysis revealed CDDO-EA under 33-GCR specifically disrupted the balance between stimulus-driven and executive control processes and altered goal-directed behavior, while hippocampal-dependent discrimination maintained its functional relationships with other cognitive domains \u0026mdash; confirming circuit-selective rather than global vulnerability. In a preliminary fiber photometry cohort, irradiated males showed enhanced dentate gyrus encoding activity under high memory load. At the cellular level, combined CDDO-EA/33-GCR selectively reduced dentate gyrus progenitors in females. Together, these findings reveal distinct, circuit-selective vulnerability patterns in males and females that would have been invisible to single-sex, single-endpoint designs. CDDO-EA proved a double-edged sword: protecting one cognitive domain while impairing another, a trade-off invisible to single-endpoint assessment and directly relevant to astronaut risk assessment.\u003c/p\u003e","manuscriptTitle":"Galactic cosmic radiation produces sex-specific, circuit-selective cognitive vulnerability: countermeasure trade-offs revealed by multi-domain assessment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 12:52:44","doi":"10.21203/rs.3.rs-9476558/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-08T14:50:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217391915346291628593887094392996128203","date":"2026-04-29T19:04:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-29T19:01:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-22T16:49:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-22T16:49:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Microgravity","date":"2026-04-20T21:28:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d8d976f-15f1-4d26-8909-719e32a62e86","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-08T14:50:23+00:00","index":16,"fulltext":""},{"type":"reviewerAgreed","content":"217391915346291628593887094392996128203","date":"2026-04-29T19:04:55+00:00","index":11,"fulltext":""},{"type":"reviewersInvited","content":"5","date":"2026-04-29T19:01:59+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67641255,"name":"Biological sciences/Neuroscience"},{"id":67641256,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-05-07T12:52:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 12:52:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9476558","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9476558","identity":"rs-9476558","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}