All twenty mice flown on the first foray of the NASA Rodent Habitat on ISS maintained excellent health during the mission. Qualitative observations indicated that spaceflight mice readily adapted to the RH, propelling their bodies freely and actively throughout the habitat, utilizing the entire volume of space available to them. Over time, the spaceflight mice began to move more quickly throughout the habitat, translating with ease through open spaces, but also anchoring their bodies using tails and/or paws. Anchoring allowed mice to feed, self-groom, huddle, and engage in social interactions. Mice remained active and mobile throughout the experiment, exploring their environment and occupying all areas of the habitat. The unique circling behavior emerged during the second mission quarter and progressed from a relatively solitary behavior to a highly coordinated group activity.

Here we report that spaceflight mice preferred the spacious Filter area over the smaller Lixit area of the habitat. This was evidenced by increased numbers of flight versus control animals in the Filter view beginning during the second mission week. With its larger expanse, the cage volume captured by the Filter camera provides greater opportunities for mobility relative to the Lixit area, and is therefore uniquely permissive for circling behavior. Further, circling emerged at the same time that mice increased their presence in the Filter area of the cage, raising the possibility that spaceflight mice developed a preference in flight for the larger cage area where circling could be performed. It is noteworthy that circling has been observed in at least one past shuttle mission11 utilizing the Animal Enclosure Module (AEM), a precursor to the RH with considerable design overlap. This behavior has also been reported in mice flown on ISS in the RH on missions following RR110.

Studies of mice flown in habitats other than the AEM or RH have not reported either circling or increased levels of physical activity or ambulation during spaceflight relative to 1 g controls7,8,9. This could be related to the design of the RH, that in contrast to some other habitats, is configured with multiple surfaces that mice can grab and utilize that may facilitate ambulation throughout the habitat. However there could be other explanations for the circling behavior. In the present study, circling by older Experimental mice was less than 1 percent of the numbers of circling laps observed in younger Validation mice. Age-related decline in physical activity in laboratory rodents, even across the range of young to middle-age, is well-established12 and may have been a determinant of circling behavior in 16- but not 32-week-old mice reported here. This interpretation is supported by reports on subsequent RR flights of circling behavior in younger mice acquired from both Jackson Laboratories and Taconic Biosciences suggesting that age plays a role in the emergence of circling rather than strain differences. While additional experiments are clearly needed to ascertain the precise factors underlying mouse circling on orbit, here we consider several possible explanations.

Circling could represent the emergence of stereotyped motor behavior or abnormal repetitive behaviors (ARBs). Repetition comprises an important feature of normal behavioral functioning across animal phyla13, however stereotypic behaviors are repetitive, unvarying, and apparently functionless behavior patterns3,14. Stereotypies are generally thought to reflect impaired welfare as they tend to spontaneously appear in barren or restricted housing conditions3,14,15,16. Common stereotypies3,14,16 that have been observed include pacing in birds, prosimians and large carnivores, crib- and bar-biting in horses, pigs and mice, vertical jumping in mice, rocking in primates, and self-injurious behaviors in parrots and primates.

Although less common in mice, “somersaulting”, “route-tracing”, and other forms of repetitive, unvarying and functionless locomotor have been observed3. Indeed, circling exhibited by Validation spaceflight mice shares some common characteristics with stereotypic behavior, viz., highly repetitive, somewhat invariant, with no obvious goal. In addition to the rapid, smooth circling trajectories, mice occasionally exhibited rapid back-flipping, not unlike somersaulting reported in the mouse husbandry literature. Free-fall is stressful and can activate immune responses17, and stereotypies can be triggered or intensified by emotional stress18. Notably, ground control mice housed in the RH did not exhibit somersaulting or any other identifiable motor behavior. This is similar to looping or somersaulting reported in tadpoles, fish and birds exposed to microgravity during parabolic or spaceflights19. These observations indicate that mouse circling in flight was not due to a housing effect alone but raises the distinct possibility of an interactive influence of housing and weightlessness.

Environment enrichment is a biologically relevant resource or structuring of the cage that facilitates the occurrence of highly motivated natural behavior20. As such, enrichment can successfully reduce or prevent the occurrence of maladaptive behaviors such as ARBs3, and improves translation animal studies to humans4. For mice, bedding is considered a highly effective form of environment enrichment that enables species-typical foraging and nest-building behaviors, and promotes warmth15. An animal is likely to be under a state of stress when its ability to perform natural, species-typical behaviors, such as foraging or nest-building, are prevented3,20,21. In this study, the RH was not configured with environmental enrichment typical in terrestrial laboratories. However identically-housed ground controls did not exhibit analogous behavior, therefore a lack of enrichment and/or foraging opportunities alone does not support the interpretation of circling as a motor stereotypy. Microgravity was clearly a necessary condition for the emergence of circling. Notably, mice prefer a three-dimensional, complex cage structure that facilitates climbing and locomotion20. The ability of mice to utilize the full volume of the RH under microgravity conditions could, in and of itself, serve as an effective enrichment.

The mices’ circling behavior has some similarities to their use of running wheels in terrestrial studies. Wheel running is considered to be a paramount form of enrichment for rodents, one that occurs at far greater rates in barren cages as compared to enriched ones22,23 or under stressful conditions23,24,25. Mice given the opportunity to use a running wheel in the wild will do so with bout lengths comparable to captive mice26 suggesting that running wheel activity in the laboratory setting is an elective behavior. It has been argued, however, that voluntary wheel running in the wild is not sufficient to preclude the possibility that wheel running in the laboratory itself represents a motor stereotypy21 with the potential to become compulsive and self-reinforcing27,28.

Terrestrial laboratory animals housed in colony environments often experience environmental stress29. It’s likely that hypergravity during launch, and microgravity (free-fall), weightlessness, increased airflow, low ambient temperature, increased CO2 and other environmental changes in space are stressful. Circling behavior in this study could have emerged as a stress response, however it would need to be argued spaceflight was uniquely stressful for the Validation mice that were younger in age and acquired from the Jackson Labs as compared to older Experimental mice acquired from Taconic Biosciences. Importantly, none of the Validation mice showed overt physiological signs of chronic stress or compromised health or welfare raising doubt that spaceflight mice circled due to stress.

For example, amounts of time spent feeding, and post-flight body weights were comparable in flight and ground control mice in both age groups. Antagonistic behaviors were observed at low levels in all study groups. Post-flight examination of the carcasses preserved on-orbit confirmed excellent coat condition with no evidence of ‘barbering’, a mouse obsessive/compulsive behavior involving abnormal whisker- and/or fur-plucking that is homologous to trichotillomania, human compulsive hair pulling30.

There is increasing recognition that physical exercise exerts positive, rewarding effects on brain and behavioral health, and can help combat anxiety, depression and cognitive impairment31,32. Voluntary running in young adult mice reduces depressive and anxiety-like behavior33,34,35. Stress-protective effects of exercise arising from changes to neural systems include enhanced galanin-mediated suppression of brain norepinephrine36,37 and cortical regulation of polyunsaturated fatty acids (PFAs)38,39 that exert positive effects on mood40,41.

Voluntary wheel running has been shown to be rewarding and to activate brain reward pathways with effects on the brain mimicking those induced by natural rewards and drugs of abuse42,43. The dopamine system (viz., D2 and D3 receptor activation) involved in movement, and to a lesser extent, in enhanced corticosterone synthesis, appear to contribute to exercise motivation44. Physical exercise, including locomotor activity in mice and rats, and flying in birds, exerts significant influence(s) on growth factors and neuropeptides leading to structural changes in the brain45,46,47 including enhanced hippocampal neurogenesis and angiogenesis, and substantial increases in gray and white matter volume in multiple cortical area and hippocampus45,46,47. Circling behavior may relate to one or more potentially rewarding or anxiety-reducing changes in rodent brains. Future research comparing brain morphological, physiological and biochemical changes in mice that circle in space is warranted.

Microgravity exposure eliminates vestibular sensory input to the otolith organs (i.e. the utricle and saccule) that detect and respond to head static position and linear acceleration48. The otoliths also contribute to the perception of verticality and spatial navigation in the neocortex and limbic system, including the hippocampus47. Changes in vestibular reflexive function and perception commonly occur during spaceflight Ground-based studies of rodents in which Bilateral Vestibular Labrynthectomy (BVL) is performed (sur- gical, chemical, or genetic lesions induced) report post-operative emergence of persistent motor abnormalities including hyperactivity, circling, and moderate ataxia49,50,51. The activity of forebrain dopamine systems was thought to play a central role in motor abnormalities following BVL, however the findings have been inconsistent51,52 and vestibular loss-related motor disorders cannot be solely explained by dopaminergic (DA) alterations53. Interestingly, mice flown for 30-days on the Bion-M1 mission showed decreased expression of crucial genes involved in DA synthesis and degradation, as well as the D1 receptor54. Other studies have focused on the orexin/hypocretin system implicated in central motor control55, a role that is supported by findings that altering OXA expression may contribute to hyper-locomotion following an acute vestibular lesion51. Anxiety-like responses have also been reported following BVL49,52, raising the possibility that circling served as an anxiolytic to offset effects of otolith unloading.

Fish exposed to microgravity exhibit somersaulting and looping (swimming in tight circles) and spin- ning56,57,58. In a simple, but clever, experiment, Anken and colleagues analyzed Video images of fish looping behavior during a parabolic flight in relation to post-landing otolith weights56. The asymmetry in otolith weight was highly correlated with the direction and extent of the behavior looping. Thus microgravity reveals the natural asymmetry in neural structures that have been neutralized in gravity through adaptation.

An intriguing possibility is that, in the absence of gravitational input to the otoliths, circling behavior by the Validation spaceflight mice generated biologically-relevant amounts of vestibular sensory input. It has been hypothesized that stimulation of the vestibular system during self-motion could play a regulatory role in brain changes associated with physical activity45. The canals are functional in microgravity and presumed to be actively monitoring circling movements. It is reasonable to postulate that self-motion behaviors evoke vestibular stimulation in space.

The novel idea that natural, everyday activities generate measurable vestibular stimulation was recently examined in mice59 by recording mouse head movements using a lightweight module combining three linear accelerometers to measure linear accelerations, and three gyroscopes to measure angular accelerations during species-typical behaviors (e.g., walking, running, foraging, grooming, eating, climbing, etc). The intensity of the stimuli ranged from approximately 500 to 1300 deg s-1 angular velocity and 1–4.5 G linear acceleration providing evidence that the animals’ behavioral repertoire generates natural vestibular signals reaching the vestibular end organs. Thus it is feasible to suggest that self-motion could provide biologically-relevant amounts of stimulation to the vestibular end-organs during spaceflight. In the present study, we calculated from the RR1 in-flight Video images from L + 12 through L + 35 the average circling speed for the Validation mice. Assuming an ellipsoidal path around the habitat measuring an average of 27.2 inches in circumference, we estimated an average acceleration at a range of 1.16 m/s2 to 1.82 m/s2 (0.12 g-0.186 g). Vestibular self-stimulation poses a viable explanation for the circling behavior reported in this study. Spaceflight studies comparing mouse circling in different cage configurations (e.g., round vs oval) are needed to explain why mice are circling on orbit.

The behavioral analysis reported here provides a deeper understanding of how mammals acclimate to extended spaceflight, and sets the stage for identifying the physiological, cellular and molecular events driving mouse circling behavior in space. Stereotyped motor behavior, rewarding effects of physical exer- cise, and vestibular sensation produced via self-motion are controlled by distinct sensory-motor and brain areas for which specific, testable hypotheses can be generated. For example, signaling in stress pathways (hypothalamic-pituitary-adrenal axis; HPAA) of circling mice would suggest classification of circling behavior as a motor stereotypy whereas reward centers (primarily the cortico-basal ganglia-thalamo-cortical loop) would be involved if circling exerts a positive reinforcing effect. Vestibular self-stimulation would involve activity within the otolith organs and central nuclei of the balance system and may overlap with activation of stress and/or reward pathways. Behavioral research is vital for ensuring fidelity in translating rodent studies to human health concerns in space. Affording mice the opportunity to grab and run in the RH resembles physical activities that the crew participate in routinely. Our approach is yielding an interesting analogue for better understanding human responses to spaceflight, and providing the opportunity to begin to address how physical movement influences responses to microgravity.