Cosmic radiation causes long-term cognitive dysfunction

To assess the functional consequences of cosmic radiation exposure on the brain, we behaviorally tested mice 12 weeks after exposure to 48Ti or 16O (Fig. 1). While impairments on the novel object recognition (NOR) task can be caused by multiple underlying causes, it can indicate changes in the functional connectivity of the hippocampus and the medial prefrontal cortex (mPFC) and requires the animal to distinguish a novel object from a familiar object. Analysis of this preference for novelty in NOR testing showed a significant group effect as indicated by a reduced DI [F (4, 51) = 2.897, P = 0.03]. Multiple comparison testing showed that exposure to 30 cGy 48Ti significantly reduced recognition memory (P = 0.03), while 5 cGy 48Ti or 16O and 30 cGy 16O had no effect on memory retention (Fig. 1a). Following the NOR task, mice were habituated and tested on the object in place (OiP) task which is also dependent on intact hippocampal and prefrontal and perirhinal cortical functions. In this case, functionally intact mice exhibit a preference towards objects that have been moved to a novel location. The results of this test show statistically significant group difference, again indicated by a markedly reduced preference to explore novelty [F (4, 42) = 3.427, P = 0.02] (Fig. 1b). Individual analysis showed that 48Ti 30 cGy irradiation impaired OiP memory (P < 0.004), while 5 cGy 48Ti or 16O and 30 cGy 16O again had no effect on spatial memory function. Lastly, the mice were tested on the temporal order (TO) task where animals were familiarized with two sets of objects, 4 hours apart. Mice with intact hippocampal function show a preference for the first object explored rather than the more recent object. The results of this test demonstrate that cosmic radiation exposure impaired recency memory as reflected by a reduced preference for less recently presented object (Fig. 1c). Analysis of recency discrimination in the TO task revealed an overall group effect [F (4, 43) = 7.753; P < 0.0001]. Further multiple comparison analyses showed that all HZE particle exposures impaired recency memory, 5 and 30 cGy 16O (P < 0.0001 and P = 0.01, respectively) and 5 and 30 cGy 48Ti (P = 0.005 and P = 0.05, respectively). Total exploration times of mice for each of these tasks is provided in Supplementary Table 1 (S1A–C). The inability of these irradiated animals to react to novelty after exposure to space relevant doses of cosmic radiation demonstrates the persistence of cognitive decrements in learning and memory. These data extend our past findings obtained at 6 weeks post exposure16, and demonstrate that charged particle irradiation results in robust and persistent deficits in recognition and temporal order memory 12 weeks later.

Figure 1 Cognitive deficits evaluated 12 weeks after cosmic radiation exposure. (a) Analysis of preference for novelty on a Novel Object Recognition (NOR) task shows that 30 cGy 48Ti particle irradiation significantly reduced recognition memory. (b) Performance on an Object in Place (OiP) task shows decrements in spatial memory retention for mice exposed to 30 cGy 48Ti particles as manifested in a reduced preference to explore an object found in a novel location. (c) All 48Ti and 16O irradiations significantly impaired recency memory as evident by a reduced preference for the less recently explored object in the Temporal Order task (TO). *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison post hoc analysis. (d) Attentional set shifting performance of adult male Wistar rats at 12 weeks post irradiation. Number of attempts required to reach criterion in the Simple Discrimination (SD); Compound Discrimination (CD) and Compound Discrimination Reversal (CDR) paradigms. Graphs show means ± SEM for control rats or rats exposed to 5 cGy 1 GeV/n 48Ti ions. *P = 0.048 (Mann-Whitney, compared to respective control values). Full size image

To determine further the extent and nature of cosmic radiation-induced behavioral deficits, a different rodent model (male Wistar rats) was used to assess executive function through their capability to perform attentional set shifting17. For these studies, exposure to GCR relevant low doses (5 cGy) of 48Ti caused significant decrements (P = 0.048, Mann-Whitney) in compound discrimination (CD) when assessed at the same 12 week post-irradiation time point (Fig. 1d). Deficits in CD, indicated by the increased number of attempts required to successfully reach criterion (6 consecutive accurate choices) indicate the relative inability of those animals to identify and focus on task relevant cues. Other tasks involving simple discrimination (SD) and compound discrimination reversal (CDR) were not impaired at this post-irradiation time. These results extend our cognitive findings considerably and now demonstrate that charged particle exposure compromises executive function and temporal order memory that can be linked to impaired perirhinal cortex function over extended post-irradiation times.

Given the wide-ranging cognitive deficits found 12 weeks after cosmic radiation exposure, we assessed the persistence of these decrements 24 weeks after irradiation (Fig. 2). Analysis at 24 weeks revealed overall significant group differences for the preference of novelty for the NOR task [F (4, 39) = 3.601, P = 0.02]. 48Ti particle irradiation was again most damaging, significantly reducing the DI of the 5 and 30 cGy irradiated mice, as compared to controls (P = 0.03), while neither dose of 16O had an effect on recognition memory (Fig. 2a). Performance on the OiP task showed significant decrements in spatial memory retention, again indicated by a markedly reduced preference to explore novelty [F (4, 38) = 5.018, P = 0.002]. Multiple comparison analysis showed that the preference for novelty in OiP was significantly reduced following 30 cGy 16O (P = 0.02) 5 and 30 cGy 48Ti (P = 0.001 and P = 0.02, respectively) irradiation when compared to controls (Fig. 2b). At 24 weeks, HZE particle exposure was again shown to impair TO memory as demonstrated by reduced recency discriminations [F (4, 44) = 5.012, P = 0.002] following irradiation. Multiple comparison testing showed that exposure to cosmic radiation significantly reduced preference for the less recently presented, sample phase 1 object, 5 and 30 cGy 16O (P = 0.04 and P = 0.007, respectively) and 5 and 30 cGy 48Ti (P = 0.003 and P = 0.05, respectively; Fig. 2c). Total exploration times of mice for each of these tasks is again provided in Supplementary Table 2 (S2A–C). These data suggest that the effects of irradiation on cognition persist over extended times with no apparent reduction in severity. These longer-term changes in performance were again found to manifest dose independence, suggesting an absence of, or a lower dose threshold (≤5 cGy) for charged particle-induced cognitive impairment. These data demonstrate that space-relevant fluences of charged particles can elicit surprisingly long-term cognitive decrements in learning and memory that persist for at least 6 months.

Figure 2 Cognition remains significantly impaired 24 weeks following exposure to cosmic radiation. (a) Analysis of the preference for novelty in the Novel Object Recognition (NOR) task demonstrates that charged particle irradiation continues to impair object recognition memory 6 months following exposure to low doses of 48Ti particles, while animals exposed to 16O remain unaffected. (b) Performance on the Object in Place (OiP) task, however, shows significant decrements in spatial memory retention following exposure to 30 cGy 16O, and 5 and 30 cGy 48Ti particles when compared to controls. (c) Analysis of preference for the Temporal Order (TO) task shows all 48Ti and 16O irradiations significantly impaired recency memory as shown by a reduced preference for the less recently explored object. (d) Irradiation using 30 cGy of 48Ti particles did not impair the acquisition of conditioned fear as demonstrated by similar freezing times observed by tone-shock trial 5 for both control and exposed mice. All mice showed a gradual decrease in freezing behavior over the extinction training on day 3, however the time spent freezing was significantly greater for the irradiated mice as compared to controls. Control mice successful abolish fear memory as demonstrated by reduced freezing behavior in the memory retrieval test when compared to irradiated mice. (e) Irradiated mice showed robust freezing between the first and last extinction training session as compared to controls, demonstrating that irradiated mice have a compromised ability to relearn. (f) Elevated Plus Maze (EPM) testing reveals that charged particle irradiation enhances anxiety-like behavior as demonstrated by reduced numbers of entries and time spent to open arms when compared controls. (g) Irradiated mice exhibiting severe extinction impairment also had increased anxiety as compared to control mice that were able extinguished fear memories. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Bonferroni’s multiple comparison post hoc analysis; ###P = 0.001 compared to 1st extinction training, paired t test. Full size image

To ascertain whether cosmic radiation exposure was associated with additional behavioral consequences, a separate cohort of animals was tested for fear extinction and anxiety 24 weeks following exposure to 48Ti particles (30 cGy, Fig. 2d,e). Fear extinction refers to an active process that involves the dissociation of a learned response to a prior adverse event. Irradiation had no effect on associative learning as indicated by robust freezing following exposure [Fig. 2d; Two-way RM ANOVA, F (1, 16) = 0.9210, P = 0.4]. Unirradiated mice exhibited a gradual decrease in freezing behavior over the extinction training [Fig. 2d, Two-way RM ANOVA, F (1, 18) = 14.61, P = 0.001]. Furthermore, time spent in freezing was statistically indistinguishable between control and irradiated groups on day 3 (sessions 11–15) [Multiple t test, DF = 12, P = 0.001]. Control mice showed abolished fear memory as demonstrated by reduced freezing behavior in the memory retrieval test when compared to irradiated animals [Fig. 2d, Unpaired t test, DF = 12, P = 0.02]. Unirradiated mice exhibited significant extinction or re-learning as evidenced by reduced freezing between first to last (15th) extinction training session [Fig. 2e Paired t test, t = 5.035, DF = 6, P = 0.002], while irradiated mice continued to show robust freezing [paired t test, DF = 6, P = 0.1] during both 1st and 15th trials. Baseline freezing values for this task are provided in Supplementary Table 3 (S3).

The ability to dissociate certain events from adverse outcomes (inhibitory learning) over time helps maintain cognitive health by minimizing stress18. When irradiation compromises the process of extinction, increased anxiety may result. To quantify anxiety, animals exposed to 48Ti particles (30 cGy) were subjected to an elevated plus maze (EPM) that provides animals the choice of remaining in either “open” or more protected, “closed”, arms of the maze. HZE particle irradiation enhances anxiety-like behavior as evident by reduced numbers of entries into the open arms [Fig. 2f, Unpaired t test, DF = 20, P = 0.01] and less time spent in the open arms [Unpaired t test, DF = 16, P = 0.001] for the irradiated mice as compared to the control group. Correlations between memory extinction and EPM data show the relationship between heightened anxiety and reduced rates of extinction (Fig. 2g). The ratio of the number of entries and time spent in closed arms versus open arms are provided in Supplementary Table 4 (S4). These data demonstrate that increased anxiety may also contribute to the inability to properly engage “unlearning” processes, representing yet another cognitive risk factor associated with cosmic radiation exposure.

Cosmic radiation exposure reduces the dendritic complexity of mPFC neurons

Cognitive changes may be predictive of structural alterations, and based on the behavioral paradigms used we postulated that neurons within the mPFC would exhibit indications of radiation-induced damage. Therefore, following cognitive testing, the morphometric assessment of neurons in the prelimbic layer of the mPFC was conducted. This analysis was facilitated by the enhanced green fluorescent protein (EGFP) expressed in neurons of the Thy1-EGFP transgenic strain of mice, allowing for high resolution imaging of select neurons throughout the brain (14, 15). Digital reconstructions of confocal Z-stacks exhibited marked reduction in the dendritic arborization (Fig. 3, green) of mPFC neurons throughout the prelimbic cortical layers (I–VI) 15 weeks after exposure to charged particles. Quantitative analysis of dendritic branching patterns and length showed significant reductions in the number of dendritic branches, branch points, and overall dendritic length for every dosing paradigm used (Fig. 3). As was found for the cognitive endpoints, none of these long-term structural changes were dose-responsive, suggesting either lower dose thresholds or the absence thereof. Again, these data validate our past findings observed at 6 weeks post exposure7, and further demonstrate the marked and persistent deterioration of neuronal structure following cosmic radiation exposure.

Figure 3 Reduced dendritic complexity of neurons in the prelimbic layer of the mPFC 15 weeks following exposure to cosmic radiation. Digitally reconstructed EGFP-positive neurons from control and irradiated mice showing dendrites (green) and spines (red). Quantification of dendritic parameters (bar charts) shows that dendritic branching and length are significantly reduced 15 weeks after exposure to 5 or 30 cGy of 16O or 48Ti particles. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison post hoc analysis. Full size image

Cosmic radiation exposure reduces spine density along mPFC neurons

To determine the effects of cosmic radiation exposure on dendritic spines, higher-resolution analysis of reconstructed dendritic segments was performed. Comparison of control animals to animals exposed to charged particle radiation showed a marked reduction in dendritic spines 15 weeks later (Fig. 4a, red). When normalized to dendritic length, (i.e. 10 μm), each charged particle type and dose was found to elicit reduced yields of total dendritic spines and spine density that were dose-independent (Fig. 4a). Consistent with our past results7, our present findings highlight yet another structural parameter of neurons that remain compromised at protracted post irradiation times.

Figure 4 Reduced dendritic spine density in the mPFC 15 weeks following exposure to cosmic radiation. (a) Representative digital images of 3D reconstructed dendritic segments (green) containing spines (red) in unirradiated (top left panel) and irradiated (bottom panels) brains. Multiple comparisons show that total spine numbers (left bar chart) and spine density (right bar chart) are significantly reduced after exposure to 5 or 30 cGy of 16O or 48Ti particles. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; ANOVA. (b) Memory deficits correlate with reduced spine density in irradiated mice. Dendritic spine density (per 1.2 mm2) is plotted against the corresponding performance of each animal on the OiP task. Radiation-induced reductions in spine number correlate with reduced DI for novelty after exposure to 16O (5 cGy, P = 0.01; left panel) and 48Ti (5 and 30 cGy, P = 0.01; right panel). Full size image

Functional and structural correlations after cosmic radiation exposure

To validate the functional relevance of morphometric analyses, the individual behavioral performance of each mouse (i.e. DI value) was plotted against its respective spine density (1.2 mm2) for all irradiation paradigms. Correlating dendritic spine density against the corresponding performance of each animal subjected to the OiP task revealed consistent and significant trends (Fig. 4b). For 16O exposures, Spearman correlations are as follows: r = 0.29 for 0 Gy controls (P = 0.48), r = 1.0 for 5 cGy (*P = 0.01) and r = 0.90 for 30 cGy (P = 0.08). For 48Ti exposures, Spearman correlations are as follows: r = 0.29 for 0 Gy controls (P = 0.48), r = 1.0 for 5 cGy (**P = 0.01) and r = 1.0 for 30 cGy (**P = 0.01). With the exception of animals subjected to 30 cGy 16O particles, reduced spine density was correlated significantly with lower DI values for all irradiated cohorts when compared to controls. These data clearly demonstrate the importance of correlating persistent radiation-induced changes in neuronal morphometry to behavioral performance, where certain structural changes in neurons correspond to select deficits in cognition.

Cosmic radiation exposure reduces specific spine types

To analyze further potential differences in the susceptibility of morphologically distinct spines to cosmic radiation exposure, distinct subclasses of spines were categorized and quantified 15 weeks following irradiation (Fig. 5). Reconstructed dendritic segments were scrutinized for changes in specific subclasses of spines after irradiation, as shown in representative images. Dendritic spines were classified as filopodia, long, mushroom, or stubby based on rigorous morphometric criteria as described previously19. The data clearly illustrate that cosmic radiation exposure caused dose-independent reductions in multiple immature spine types along mPFC neurons. Significantly lower numbers of filopodia (30–37%), thin (32–35%) and mushroom (21–34%) spines types were found after 16O or 48Ti particle exposures, while mature stubby spines were more radioresistant. These data clearly show that spines of defined morphology exhibit differential susceptibility to cosmic irradiation.

Figure 5 Differential radiosensitivity of dendritic spines. Representative dendritic segments (green) showing immature filopodia (white), long (pink) and mushroon (red) spine types along with more mature stubby (blue) spines. Exposure to 16O (upper panel) or 48Ti (lower panel) particles leads to significant reductions in the number of immature spines with no effect on mature spines. Quantification of each morphological type of dendritic spine are expressed as the total number of spines for each class. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus control; ANOVA. Full size image

Increased PSD-95 synaptic puncta after irradiation of mPFC neurons

To complement structural analyses, the levels of postsynaptic density protein 95 (PSD-95) puncta were quantified from deconvoluted confocal images of immunohistochemically stained tissue sections (Fig. 6). High-resolution imaging of brain tissue (layer II of the mPFC) revealed consistent and significant increases in the yield of PSD-95 puncta after all charged particle irradiations (Fig. 6a). Exposure to either dose of 16O or 48Ti particles increased PSD-95 levels by ~1.2–1.4 fold along neurons in the mPFC in a dose-independent manner (Fig. 6b). These data indicate that, in addition to structural changes, charged particle exposure elicits persistent and significant alterations in the amount and distribution of specific synaptic proteins that remain 15 weeks following acute exposures. Data are also consistent with past data sets obtained at 6 weeks7, and indicate that similar irradiation paradigms elicit long lasting changes in critical synaptic proteins.

Figure 6 Cosmic radiation exposure induces persistent increases in PSD-95 puncta in the mPFC. (a) Representative fluorescence micrographs showing PSD-95 puncta (red). (b) Quantitative analyses show that exposure to 5 or 30 cGy of 16O or 48Ti particles leads to increased numbers of PSD-95 puncta in mPFC neurons as compared to control (15 weeks, left panel; 27 weeks, right panel). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; ANOVA. (c) Overexpression of PSD-95 correlates with cognitive decrements in irradiated mice. PSD-95 puncta (per 400 μm2) are plotted against the corresponding performance of each animal on the OiP task. Increased levels of PSD-95 puncta are associated with decreased behavioral performance following exposure to 16O (30 cGy, P = 0.01) and 48Ti (5 and 30 cGy, P = 0.01). Full size image

Correlating synaptic and functional changes after cosmic radiation exposure

To determine how the observed changes in synaptic PSD-95 puncta corresponded with changes in cognition, similar correlation analyses were performed as those described for spine density (Fig. 6c). For these comparisons, elevated PSD-95 levels significantly correlated with reduced DI for all conditions except those for the animals exposed to 5 cGy 16O particles. For 16O exposures, Spearman correlations are as follows: r = −0.27 for 0 Gy controls (P = 0.48), r = −0.90 for 5 cGy (P = 0.08) and r = −1.0 for 30 cGy (**P = 0.01). For 48Ti exposures, Spearman correlations are as follows: r = −0.15 for 0 Gy controls (P = 0.69), r = −1.0 for 5 cGy (**P = 0.01) and r = −1.0 for 30 cGy (**P = 0.01). Thus, with the exception of animals subjected to 5 cGy 16O particles, individual performance on the OiP task was significantly lower and inversely correlated with elevated PSD-95 puncta, an effect that was most pronounced for animals irradiated with 30 cGy of 48Ti particles. These data again indicate additional consistent and persistent charged particle-induced effects in the brain. While the functional significance of elevated PSD-95 puncta is less certain, changes in synaptic protein levels have proven to be reliable markers of cosmic and terrestrial radiation exposure of the brain.

Cosmic radiation-induced neuroinflammation

While morphometric alterations to neurons are likely to play a pivotal role in neurocognitive outcomes, other factors may directly or indirectly impair neurotransmission following cosmic radiation exposure. To ascertain whether charged particle irradiation caused persistent alterations in the levels of activated microglia, ED1 immunopositive cells were quantified 15 and 27 weeks later. For all irradiation conditions and time points, the number of ED1 positive cells increased significantly (Fig. 7). At 15 weeks post-irradiation ED1 levels were increased by ~1.2–1.6 fold and further elevated to ~2-fold at 27 weeks. The persistence of microglial activation is noteworthy and suggests that low dose charged particle exposure elicits an increase in the number of inflammatory cells that prune neuronal processes, thereby disrupting neurotransmission and cognition.