Astronauts on a Mars mission will be exposed to potentially harmful levels of charged-particle radiation. Thirty years of basic research with ground-based charged-particle radiation has provided overwhelming evidence that the rodent Central Nervous System (CNS) and behavior are negatively affected by charged-particles, suggesting this is a concern for astronaut health. However, the current literature has several caveats, including using young rather than fully-adult rodents, lack of studies focused on the female rodent CNS, unrealistic single-particle radiation simulations, lack of feasible therapeutic countermeasures, and behavioral tests with low translational potential and high handling- or experimenter-induced variability. Additionally, no published CNS study has yet assessed the effects of charged-particle radiation in combination with spaceflight-relevant stressors.

We will address these limitations by exposing male and female mice of astronaut age to a novel, complex, but realistic Galactic Cosmic Ray simulation (GCR) then examining network-dependent behavioral performance on a highly-translational, appetitive touch-screen platform, including a behavioral test battery that can be viewed as analogous to the tests currently used on astronauts aboard the International Space Station. A separate group of mice will be examined for the influence of GCR on their ability to cope with social and physical stress in a validated mouse model of depression. Finally, we will test a promising anti-inflammatory drug and its ability to prevent GCR-induced deficits in cognition and social defeat stress.

This proposal will address a wide range of NASA-defined knowledge gaps in its Human Research Roadmap. Specifically, we strive to provide a model basis for identifying individuals who are resilient to the extreme spaceflight conditions, validate a measure for monitoring behavioral health, determine radiation dose thresholds for behavioral measures in a domain basis and in combination with social defeat stress, all of which will help inform NASA’s risk models for a crewed mission to Mars.

Aim 1. Define GCR effects on translationally-relevant cognitive tests in mice.

Aim 2. Define the outcomes of GCR on adaptive, social-stress-mediated behavior.

2. Key Findings

To accomplish aim 1, we have exposed 6-month-old male and female mice to an acute, whole-body 750mGy dose of the NSRL’s 33-GCR. To assess CDDO-EA as a GCR countermeasure, we gave a subset in mice receiving either Sham or GCR ad libitum access to a CDDO-EA-containing diet (400mg/kg); whereas, non-countermeasure mice received a vehicle (Veh) diet of the same excipient preparation omitting CDDO-EA.

Four months after radiation, male mice were tested for locomotor activity with activity chambers, anxiety with open field (OF) and elevated-plus mazes (EPM), sociability and social memory with the 3 chamber social interaction (3-CSI) test and object memory, with the novel object recognition (NOR) test. We observed no effects of diet on weights among the four groups of male mice for the duration of the experiment. We similarly did not observe significant differences in mouse survival between groups. Following suit, behavioral analyses revealed no major differences in gross locomotion, anxiety, or object memory following radiation. However, in 3-CSI CDDO-EA/33-GCR, mice failed to spend more time exploring a stranger mouse vs. nothing, suggesting sociability deficits, and Veh/33-GCR and CDDO-EA/Sham mice failed to discriminate between a stranger vs. familiar mouse, suggesting social memory deficits. CDDO-EA did not attenuate the 33-GCR-induced social memory deficits. Our findings suggest that radiation poses a risk to socio-cognitive behavior, and that CDDO-EA was not an effective GCR countermeasure in male mice. Future elucidation of the mechanisms underlying 33-GCR-induced social memory deficits will improve risk analysis for astronauts which may, in turn, improve countermeasures.

Female mice similarly maintained similar weights among groups for the duration of the experiment. They were next trained for a touchscreen-based appetitive behavioral platform (ABET II, Lafayette Neuroscience) for one month. Mice were next tested for visuo-spatial pattern separation, by the Location Discrimination task. For both the “easy” and “hard” separation settings, Veh/33-GCR completed fewer daily trials throughout the three month-long testing period. Mice were next tested for a rule-based acquisition test, and subsequently for extinction learning. Veh/33-GCR took nearly three times longer to reach acquisition criteria than the other groups. However, we observed no differences among groups in extinction learning. Touchscreen testing was followed by arena testing for anxiety (EPM, OF), sociability and social memory (3-CSI), object memory (NOR), and compulsive-like behavior (marble-burying and nestlet-digging). We observed no differences in social, cognitive, or compulsive behavior; however, Veh/33-GCR and CDDO-EA/33-GCR mice explored the center of the OF for shorter durations than non-33-GCR mice. Overall, 33-GCR exposure resulted in pattern separation and rule-based acquisition deficits in mice, which was prevented by CDDO-EA. 33-GCR also caused lowered open field exploration, which was not prevented by CDDO-EA.

To fulfill aim 2, we exposed six-month-old male and female mice to 3 x 6.7 cGy of 56Fe (600MeV/n) every other day for five days. Six months after exposure, mice were tested for social hierarchy and dominance behavior via the tube dominance test across 16 days. Next, mice were tested for anxious behavior (OF), sociability and social memory (3-CSI), and despair response with the Forced Swim Test. We observed sex-specific responses to social hierarchy and dominance behavior. Radiation lowered rank attainment, and lowered rank stability in males, but improved these measures in females. Rank and other behavioral and histological analyses are ongoing.

3. Impact of Key Findings on Hypotheses

Guided by a literature of simple GCR models, it was hypothesized that a complex ground-based GCR simulation would result in numerous behavioral deficits in male and female mice of astronaut-equivalent age. Overall, we did not observe the commonly reported behavioral deficits described in the literature, such as anxious behavior and object memory. However, male mice nevertheless displayed social memory deficits, and female mice displayed lowered pattern separation, rule-based acquisition, and increased anxious behavior, suggesting our hypothesis that radiation would result in behavioral deficits is accepted.

Contrary to our hypotheses, CDDO-EA was an ineffective countermeasure for radiation-induced social memory deficits in male mice, and when given in combination with 33-GCR, appeared to induce sociability deficits. However, in female mice, CDDO-EA prevented radiation-induced deficits to cognitive behaviors (pattern separation and rule-based acquisition). CDDO-EA did not, however, prevent 33-GCR-induced OF anxiety in females, suggesting partial success as a radiation countermeasure in female, but not male mice.

4. Proposed Research Plan for the Coming Year

Early analyses of tube test data indicate sex-specific responses to hierarchy and stress-mediated social conflict resolution. To understand what might give rise to these differences, we will extrapolate individual mouse behaviors across the testing period using deep learning-based automated ethograms to quantify within-tube behaviors such as conflict lengths, methods of resolution (passive vs. conflict-based), number of pushes, retreats, etc. To investigate the brain regions involved in tube-based conflicts we will process collected brain tissues from behaviorally naive, but acutely tube-tested mice. Tissues will be stained for immediate early genes for insight into the neuronal ensembles involved in this task, and quantified to understand if there are radiation differences in the regions involved in tube testing between sham and irradiated mice, and whether these vary by sex, or social rank.

Astronauts on a Mars mission will be exposed to potentially harmful levels of charged-particle radiation. Thirty years of basic research with ground-based charged-particle radiation has provided overwhelming evidence that the rodent Central Nervous System (CNS) and behavior are negatively affected by charged-particles, suggesting this is a concern for astronaut health. However, the current literature has several caveats, including using young rather than fully-adult rodents, lack of studies focused on the female rodent CNS, unrealistic single-particle radiation simulations, lack of feasible therapeutic countermeasures, and behavioral tests with low translational potential and high handling- or experimenter-induced variability. Additionally, no published CNS study has yet assessed the effects of charged-particle radiation in combination with spaceflight-relevant stressors.

We will address these limitations by exposing male and female mice of astronaut age to a novel, complex, but realistic Galactic Cosmic Ray simulation (GCR) and then examining network-dependent behavioral performance on a highly-translational, appetitive touch-screen platform, including a behavioral test battery that can be viewed as analogous to the tests currently used on astronauts aboard the International Space Station. A separate group of mice will be examined for the influence of GCR on their ability to cope with social and physical stress in a validated mouse model of depression. Finally, we will test a promising anti-inflammatory drug, its ability to prevent GCR-induced deficits in cognition and social defeat stress.

This proposal will address a wide range of NASA-defined knowledge gaps in its Human Research Roadmap. Specifically, we strive to provide a model basis for identifying individuals who are resilient to the extreme spaceflight conditions, validate a measure for monitoring behavioral health, determine radiation dose thresholds for behavioral measures in a domain basis and in combination with social defeat stress, all of which will help inform NASA’s risk models for a crewed mission to Mars.

Astronauts on a Mars mission will be exposed to potentially harmful levels of charged-particle radiation. Thirty years of basic research with ground-based charged-particle radiation has provided overwhelming evidence that the rodent Central Nervous System (CNS) and behavior are negatively affected by charged-particles, suggesting this is a concern for astronaut health. However, the current literature has several caveats, including using young rather than fully-adult rodents, lack of studies focused on the female rodent CNS, unrealistic single-particle radiation simulations, lack of feasible therapeutic countermeasures, and behavioral tests with low translational potential and high handling- or experimenter-induced variability. Additionally, no published CNS study has yet assessed the effects of charged-particle radiation in combination with spaceflight-relevant stressors. We will address these limitations by exposing male and female mice of astronaut age to a novel, complex, but realistic Galactic Cosmic Ray simulation (GCR) and then examining network-dependent behavioral performance on a highly-translational, appetitive touch-screen platform, including a behavioral test battery that can be viewed as analogous to the tests currently used on astronauts aboard the International Space Station (ISS). A separate group of mice will be examined for the influence of GCR on their ability to cope with social and physical stress in a validated mouse model of depression. Finally, we will test a promising anti-inflammatory drug for its ability to prevent GCR-induced deficits in cognition and social defeat stress. This proposal will address a wide range of NASA-defined knowledge gaps in its Human Research Roadmap. Specifically, we strive to provide a model basis for identifying individuals who are resilient to the extreme spaceflight conditions, validate a measure for monitoring behavioral health, determine radiation dose thresholds for behavioral measures in a domain basis and in combination with social defeat stress, all of which will help inform NASA's risk models for a crewed mission to Mars. We are working to achieve these goals by fulfilling gaps in knowledge addressed in our specific aims:

Aim 1. Define GCR effects on translationally-relevant cognitive tests in mice.

Aim 2. Define the outcomes of GCR on adaptive, social-stress-mediated behavior.

2. Key Findings

We have exposed 6-month-old male and female mice to an acute 750 mGy dose of the NSRL's 33-beam GCR simulation (NSRL 19A, collaboration with Jerry Shay, University of Texas Southwestern). 4 months following exposure, we tested males for open field activity, anxious behavior via the elevated-plus maze, object memory via the Novel Object Recognition test, and sociability and social recognition by the 3-Chamber Social Interaction test. Female mice were trained and tested on an appetitive touchscreen platform for an object-based location discrimination task, a simple, rule-based operant acquisition test, and an extinction learning test. Following touchscreen-based testing, females were subsequently tested on the same behavioral tasks as the male mice, with additional marble burying, and nestlet digging tests. We are currently processing and validating these behavioral data targeted at fulfilling Aim 1.

3. Proposed Research Plan for the Coming Year

Our Aim 2 approach was initially to define the outcomes of GCR on adaptive, social-stress-mediated behavior using the social defeat-stress model. Due to the COVID-19 pandemic lab shut-down and limitations on animal case staff, we were unable to maintain our colony of mice needed to perform social defeat stress (e.g., screened CD-1 retired breeders which we used as aggressors). While the labs re-opened in June to 25% capacity as long as personnel were masked and at least 6-feet apart, we still were unable to perform social defeat stress since a) it would take ~2month to re-order and screen aggressors, at which point the experimental mice may be too old to use for social defeat; and b) we are unable to perform the screening and the subsequent social defeat stress with the personnel restrictions in place. Therefore, we did research to assess an alternative behavioral model in which to define the outcomes of GCR on adaptive, social-stress-mediated behavior. After intense consideration, we pivoted to the tube dominance test for social hierarchy, a test that also involves social stress as an experimental condition. We are currently conducting behavioral experiments including the tube dominance test in male and female mice that during NSRL 19C received 3 x 67 mGy fractions of 56Fe (600 MeV/n) over the course of 5 days. The resulting data will help address how prior 56Fe exposure (a significant dose-contributing component of GCR) at 6 months of age influences social-stress-mediated behavior 9 months later. We are also preparing a future experiment assessing the effects of a chronic proton exposure equivalent to the NSRL GCR simulation on hippocampal neurogenesis.

Astronauts on a Mars mission will be exposed to potentially harmful levels of charged-particle radiation. Thirty years of basic research with ground-based charged-particle radiation has provided overwhelming evidence that the rodent Central Nervous System (CNS) and behavior are negatively affected by charged-particles, suggesting this is a concern for astronaut health. However, the current literature has several caveats, including using young rather than fully-adult rodents, lack of studies focused on the female rodent CNS, unrealistic single-particle radiation simulations, lack of feasible therapeutic countermeasures, and behavioral tests with low translational potential and high handling- or experimenter-induced variability. Additionally, no published CNS study has yet assessed the effects of charged-particle radiation in combination with spaceflight-relevant stressors.

We will address these limitations by exposing male and female mice of astronaut age to a novel, complex, but realistic Galactic Cosmic Ray simulation (GCR) then examining network-dependent behavioral performance on a highly-translational, appetitive touch-screen platform, including a behavioral test battery that can be viewed as analogous to the tests currently used on astronauts aboard the International Space Station. A separate group of mice will be examined for the influence of GCR on their ability to cope with social and physical stress in a validated mouse model of depression. Finally, we will test a promising anti-inflammatory drug, its ability to prevent GCR-induced deficits in cognition and social defeat stress.

This proposal will address a wide range of NASA-defined knowledge gaps in its Human Research Roadmap. Specifically, we strive to provide a model basis for identifying individuals who are resilient to the extreme spaceflight conditions, validate a measure for monitoring behavioral health, determine radiation dose thresholds for behavioral measures in a domain basis and in combination with social defeat stress, all of which will help inform NASA’s risk models for a crewed mission to Mars.