Targeting Serotonin Pathways for Astronaut Safety and Performance
INTRODUCTION: Exposure to microgravity has physiological consequences that can impair astronaut safety and performance. Many can be directly linked to fluctuations in plasma serotonin levels on Earth, like bone loss, nausea, and fatigue. Yet the metabolic activity of serotonin in space is not well known. This study measured plasma serotonin levels and bone density in the mouse hindlimb unloading (HU) model, an established Earth analog of microgravity-induced bone loss.
METHODS: The HU model has been used for decades to simulate axial unloading and fluidic shifts experienced in microgravity. Over a 30-d period, mice were suspended by their tails, with blood plasma collected at days 1, 15, and 30. Plasma was assessed for the presence of serotonin protein using an enzyme-linked immunosorbent assay and quantified. At day 30, microcomputed tomography of femur structural changes in HU mice was correlated with plasma serotonin increases.
RESULTS: Serotonin in plasma from HU mice showed increases in plasma serotonin at every timepoint compared to normally loaded mice. Between days 15–30, there was a 1.87-fold increase in serotonin levels found for normal mice while a significantly larger increase of 2.5-fold was found in the HU mice.
DISCUSSION: The HU mouse model showed plasma serotonin is elevated in HU mice, which corresponds to cortical and trabecular bone loss. These data suggest that elevated plasma serotonin may have a role in microgravity-induced bone loss. Specific serotonin receptor antagonists may be a safer countermeasure than currently used bisphosphonates to protect against astronaut bone loss.
Casey TJ, Kubik AJ, Allen NG, Zilberman AH, Whitman BM, Hunt JC, Juran CM, French J, Blaber EA. Targeting serotonin pathways for astronaut safety and performance. Aerosp Med Hum Perform. 2025; 96(11):969–975.
Spaceflight has heralded a new frontier for human exploration and scientific discovery, but has revealed significant challenges and inadequacies in the human capacity for life beyond Earth. For decades, clinical researchers and astronauts have documented the deleterious effects of long-term exposure to microgravity on various aspects of human physiology, notably the neurological and musculoskeletal systems. Many of these pathophysiological challenges are known to involve the neurohormone serotonin on Earth.1,2 Surprisingly, little is known about the metabolic activity of serotonin in microgravity.
The amount of bone loss experienced by astronauts is about 1% of total bone mass per month,3 which is roughly 10 times the level observed in postmenopausal women from decreased estrogen levels.4 Historically, NASA has used various methods to combat bone loss, including intense, frequent exercise while in space, which has been inadequate for mitigation of bone loss.5 Mineral/hormonal supplementation with vitamin D and estrogen, respectively, have also been tried, as have bisphosphonates, but these have had limited success.6 Exogenous estrogen has been shown to promote a state of hypercoagulability that can lead to deep venous thromboses.7 This is particularly problematic in space, where it has been found that microgravity independently leads to a state of hypercoagulability.8 It is thought to be due to cephalad fluid shifts, causing altered blood flow dynamics and promoting stasis.9 Additionally, bisphosphonates can cause nausea/vomiting, hypocalcemia, and mandibular osteonecrosis.10
The mechanism by which osteoporosis occurs on Earth is thought to be due in large part to the elevated plasma levels of serotonin or 5-hydroxytryptamine (5-HT). Specifically, gut-derived serotonin, as compared to brain-derived, is linked to bone metabolic imbalances. Primarily produced in the enterochromaffin cells of the intestines, 5-HT aids in digestion and bone regulation.11,12 Gut derived 5-HT is then released into the plasma by food moving in the lumen of the gut during peristalsis, likely by the mechanosensitive Piezo 2 receptors, where it is reabsorbed by platelets in the intestinal veins.13 Platelets then move 5-HT into bone, where it activates the 5-HT1b receptors on progenitor osteoblasts to inhibit osteoblast formation,14 thereby serving as an important means to regulate bone formation. Osteoblasts serve to build bone from calcium ions in the blood, while osteoclasts break bone down and release calcium back into the bloodstream, which can induce hypercalcemia, kidney stones, and nausea.15 Given that bone demineralization is a potential issue for astronaut safety and performance, particularly on long duration missions, the lack of information about 5-HT in microgravity is a critical knowledge gap that needs to be addressed.
Because hypercalcemia and inefficient calcium levels may lead to severe health concerns, the level of calcium in the blood is tightly regulated by several hormones. Parathyroid hormone is produced by the parathyroid glands in the neck and serves to raise calcium levels. Conversely, the hormone calcitonin, made by parafollicular C cells, depresses calcium levels. Physiologically, calcium plays a vital role in musculoskeletal function by controlling the regulatory proteins actin and myosin in sarcomeres. At the presynaptic junction in nerves, calcium ions permit neurotransmitter release, allowing the action potential to fire. High levels of extracellular calcium decrease neuromuscular membrane excitability and can lead to cardiac arrhythmias and nephrolithiasis.16 Hypercalcemia may also contribute to the downregulation of somatosensory function following initial and prolonged exposure to microgravity. Clinically, hypercalcemia presents as nausea, emesis, lethargy, fatigue, neuromuscular incoordination, nephrolithiasis, abdominal pain, and psychiatric symptoms.17 Many of these symptoms have been experienced by astronauts during or immediately upon return from spaceflight.18
Researchers have used the hindlimb unloading (HU) model in rodents to simulate microgravity on Earth. Originally developed in the 1980s, HU involves suspending the hind limbs in the air, mirroring the axial unloading and cephalad fluid shifts astronauts experience in space.19 We were unable to find any prior literature directly studying the effects of 5-HT on microgravity-induced challenges such as bone loss, nor could we find any information on 5-HT levels in the HU model. In this paper, we focused on evaluating the possibility that simulated microgravity exposure could be linked to an elevation in plasma 5-HT concomitant with an increase in bone demineralization.
METHODS
Animals
All animal experiments were conducted with prior approval from the Rensselaer Polytechnic University Institutional Animal Care and Use Committee (IACUC # BLA-001-22). Using a custom HU cage modified for social housing, 16-wk-old B6/129SF2/J female mice (N = 10 per condition) underwent HU and were maintained that way for 30 d. Corresponding normally loaded (NL) control mice were maintained in identical housing conditions to the HU mice, except for the tail suspension apparatus and associated hardware. All animals were acclimated to the test cages for 3 d prior to the attachment of orthopedic traction tape. Animals were fed standard chow (Prolab® IsoPro® RMH 3000, LabDiet, St. Louis, MO, United States) and continuously provided water with twice daily health checks to ensure consumption and drinking. If an animal was deemed unfit to continue the study, it was removed. A 12-h light/dark cycle and temperature between 23–25°C were maintained throughout the experiment. After 30 d, the animals were euthanized by carbon dioxide inhalation and secondary cervical dislocation. Immediately following euthanasia, blood and other tissues were collected and stabilized accordingly.
Procedure
Peripheral blood was collected in EDTA coated tubes. The tubes were centrifuged at 2000 × g for 10 min. Plasma samples were collected and assessed for serotonin concentration using three biological replicates and three technical replicates at a 1:4 dilution by the Novus Serotonin colorimetric ELISA accompanied by concentration standard curve.
Microcomputed tomography (µCT) was used to image and analyze cortical bone morphometric parameters of the femoral shaft and trabecular parameters of the femoral head and distal femur. The right hindlimbs were dissected at the femoral head and fixed in 4% paraformaldehyde for 48 h. Following fixation, hindlimbs were washed twice with 1X phosphate buffered saline without calcium and magnesium additives (PBS-/-) and stored in a final volume of PBS with calcium and magnesium additives (PBS+/+) at 4°C until scanning.
Fully intact hindlimbs were wrapped in PBS-soaked gauze to prevent drying and mounted horizontally inside 0.6 mL microcentrifuge tubes for high resolution µCT scanning (Bruker SkyScan 1276, Kontich, Belgium) at the University of Massachusetts Amherst Animal Imaging core facility. The µCT was set to operate at a source voltage of 65 kV and a tube current of 166 µA using a 0.25 mm aluminum filter. All bones were scanned at a pixel resolution of 4.05 µm using an exposure time of 710 ms/frame with three averaging frames. Images were captured using a rotation step of 0.5° through a rotational angle of 180°.
Raw cross-sectional images were processed through the NRecon reconstruction software (SkyScan, version 1.7.4.2; Bruker, Kontich, Belgium) to generate a stack of 2D cross-sectional slices using a modified Feldkamp algorithm with geometrical correction. Reconstruction was carried out with the following parameters: automatic misalignment compensation, a Gaussian smoothing kernel of 2, a beam hardening correction of 36%, a ring artifact correction of 6, and a dynamic contrast range of 0–0.10. The reconstructed images were visualized in 3D using the volume rendering software CTVox (Bruker, version 3.3.1) and each specimen was manually rotated in DataViewer (Bruker, version 1.5.6.2) to align the bone along the appropriate analysis axis. The CTAnalyser software, CTAn (Bruker, version 1.20.3.0), was then used to define the appropriate volume of interest (VOI). For analysis of the cortical femur, VOIs were defined as a 140-slice region (1 mm) around the midpoint of the femur. Distal femur VOIs were defined as a 140-slice trabecular region (1 mm) at the distal metaphysis, beginning 5 slices above the distal epiphyseal growth plate (distal physis) and extending proximally. Lastly, femoral head VOIs were defined as a 25-slice cancellous region (0.1 mm) around the midpoint between the start of the femoral head and the proximal epiphyseal growth plate. Standardized task lists were performed using BatchMan and cortical and trabecular parameters were computed. Cortical parameters measured were total cross-sectional area (T.Ar), cortical bone area (Ct.Ar), bone area fraction (B.Ar/T.Ar), cortical thickness (Ct.Th), endosteal perimeter (Es.Pm), and periosteal perimeter (P.Pm). Cancellous parameters reported include bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), structure model index (SMI), connectivity density (Conn.Dn), and trabecular pattern factor (Tb.Pf).
Statistical Analysis
All statistical analyses were performed using GraphPad Prism (version 9.5, GraphPad Software, Boston, MA). Two types of data were analyzed: serotonin concentration and microCT-derived bone metrics. For microCT-derived bone metrics, measurements were obtained from nine biological replicates per group, and for serotonin concentration, measurements for eight biological replicates were assessed. Data were assessed for normality and analyzed using unpaired, two-tailed t-tests to compare normally loaded (control) and hindlimb unloaded (experimental) groups. Results are reported as mean ± SD, and significance was defined as P < 0.05.
RESULTS
Serotonin in plasma from NL and HU mice showed increases at every time point as shown in Fig. 1. Increased concentration in the NL mice is likely associated with changes in muscle activity due to the altered cage environment. Vivarium comparisons to controls from several previous studies have concluded the HU cage can induce biologically significant changes in the cardiac and musculoskeletal systems without suspension. Fig. 1 shows that on day 1 of HU, serotonin levels were already 14.2% higher in plasma concentration than NL controls and elevated at both day 14 and 30 (36.4% and 47.6%, respectively). Comparative analysis of serotonin between days 1 and 14 illustrates that serotonin concentration in HU mice increases at a faster rate (1.66×) than the NL animals (1.25×).
Citation: Aerospace Medicine and Human Performance 96, 11; 10.3357/AMHP.6712.2025
Between day 15 and 30 the NL animals continue to increase plasma serotonin concentration by 1.87× while the HU increased 2.5×. The average serotonin plasma concentration quadratic trajectory expression change, fit from day 1–30, show the HU mice have a trend toward continued increase while the NL trend shows a lesser growth curve and earlier plateau (Fig. 2).
Citation: Aerospace Medicine and Human Performance 96, 11; 10.3357/AMHP.6712.2025
Trabecular and cortical bone architectures were evaluated by µCT to investigate the potential correlation between elevated 5-HT levels and bone loss during unloading. In the trabecular bone of the distal femur, bone volume fraction (BV/TV%), trabecular number, and trabecular thickness all demonstrated a significant decrease in the HU animals [t(48) = 2.3, P = 0.026]. As shown in Fig. 3, BV/TV% was reduced to 1.119 ± 0.3398 [t(18) = 3.292, P = 0.004] and the trabecular number dropped to 0.3912 ± 0.129 [t(18) = 3.235, P = 0.0046]. Trabecular thickness decreased to 0.001538 ± 0.0006612 mm [t(18) = 2.326, P = 0.0319]. Interestingly, results also demonstrate an HU-associated decrease in Conn.Dn of 166 ± 68.35 [t(18) = 2.428, P = 0.0259], while Tb.Pf and SMI showed no significant changes between either experimental group. These data demonstrate that cancellous bone is the principally load-sensitive architecture of the femur and may be the structure most affected by plasma serotonin due to dysregulation of osteogenic metabolism. Within the cortical shaft of the femur, no significant sources of variation were detected within B.Ar/T.Ar, P.Pm, or Es.Pm between HU and NL animals. Ct.Th of the femur shaft showed a trend toward cortical thinning, although this did not reach statistical significance (Fig. 4).
Citation: Aerospace Medicine and Human Performance 96, 11; 10.3357/AMHP.6712.2025 Citation: Aerospace Medicine and Human Performance 96, 11; 10.3357/AMHP.6712.2025
DISCUSSION
There is compelling data that gut serotonin has a powerful effect on bone metabolism. The source of serotonin to the bone is also known to be a regulatory factor, with brain-derived serotonin promoting osteoblast function and increased bone deposition,20 while gut-derived serotonin reduces pre-osteoblast proliferation and encourages osteoclast specialization. Pre-osteoblast proliferation involves gut-derived serotonin receptors, including Hrt1b (5-hydroxytryptamine receptor in mice and humans), which can inhibit proliferation when bound. Elevated gut-derived serotonin has also been shown to prevent association of Forkhead box protein O1 with cAMP response element binding protein, resulting in suppressed osteoblast proliferation. Serotonin can additionally increase osteoclast differentiation by mechanisms which include amplifying receptor activator of nuclear factor kappa B ligand via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation of NF-kB pathways.
Serotonin regulated bone metabolism would, therefore, favor osteoblast inhibition and encourage osteoclast resorption of bone matrix, resulting in the release of calcium into the blood stream. In fact, this process of calcium allocation is part of fetal development. For example, during pregnancy maternal bone calcium is known to spike to support fetal bone growth, and maternal recovery occurs after breast feeding is concluded.
Surprisingly, little is known about plasma serotonin activity in microgravity. Using an analog of microgravity-induced bone loss, the mouse HU model, we were able to show that increases in plasma serotonin correlates to femur architecture changes. These architectural changes demonstrate a weakening trabecular morphology in HU, but not NL mice despite rising serotonin levels in NL animals. The HU cage may cause a stress response as the floor of the cage is a grate structure, which poses challenges to normal ambulation in the mice. Additional research comparing serotonin levels under conditions of HU and NL to a baseline would be beneficial to explore if unloading is the primary contributor to elevation of serotonin in space, as well as to define how severely unloading compounds serotonin increases due to other stresses. A longer 60-d duration of unloading would also resolve if the plasma serotonin concentration of the HU animals continues to outpace the NL condition. Other stress-inducers in space, including radiation exposure and circadian dysregulation, have established patterns of serotonin disruption that lead to muscle loss, depression, nausea, and vomiting. Combined with the physical and mental stresses of spaceflight, this may additively increase serotonin in plasma and result in significant bone mineral density loss.
An additional next step for investigating if serotonin and bone mineral density loss are causally linked would be to determine if selective 5-HT blockers are able to slow or diminish the bone loss observed in the HU model. A successful outcome would support use in human microgravity trials to determine the effectiveness of a targeted pharmaceutical intervention of the 5-HT related bone loss mechanisms.
New discoveries about gut-derived 5-HT are frequently made as more research is directed to understanding its role in calcium regulation. Much is yet to be learned and 5-HT blockers may have additional protective benefits to biological systems known as key risk factors for extended space travel. It is thus plausible that hypercalcemia in microgravity is the result of the effects of elevated 5-HT release on 5-HT3 and 5-HT1b receptors. We can postulate a mechanism for this. The immediate effects of entry into microgravity environments lead to central nervous system misinterpretation of sensory information, leading to space motion sickness that can potentially jeopardize astronaut performance.21 Most astronauts overcome the initial nausea in a few days, but occasional bouts do return. If the emetic response could be blocked, it may be possible to reduce other issues relating to hypercalcemia, such as neurological and musculoskeletal dysfunction.22 It is well known that the 5-HT3 blockers are effective mitigations for radiation-induced nausea and vomiting on Earth. It is also relevant to point out that bone loss is associated with 5-HT elevating compounds like fluoxetine and other SSRI drugs.23 The 5-HT3 antagonists like ondansetron carry fewer and less severe side effects than bisphosphonates as a potential regulator of bone loss, or the other antiemetic agents promethazine and scopolamine that are currently in use by NASA. These effects point to the contention that 5-HT blockers may be useful in targeting specific 5-HT pathways to help mitigate serotonin mediated microgravity challenges to astronauts. The actions of serotonin are widespread and touch on many physiological functions, in addition to bone loss, that are important in spaceflight.
Another key player in calcium homeostasis is melatonin, an active metabolite of 5-HT that is also produced in the pineal gland and retina. Exogenous melatonin can cause fatigue and act as a chronobiotic to reset circadian rhythms.24 Elevated levels of 5-HT from continued, periodic nausea could lead to elevated melatonin in cells that can produce melatonin from 5-HT. Moreover, it is possible that the persistent fatigue experienced by astronauts in space is a consequence of elevated melatonin from elevated 5-HT. Fatigue from motion sickness is called sopite syndrome and can also be due to elevated melatonin, which would require elevated serotonin. Calcium extraction by melatonin is evidenced by the calcification of the pineal gland seen in patients over the age of 40, conveniently serving as a biomarker for radiology. It follows that melatonin would be able to extract calcium from other tissue like bone and prior studies have shown elevated melatonin levels can cause bone loss.25 Melatonin and 5-HT’s roles extend beyond calcium regulation, encompassing glucose metabolism, circadian rhythms, and immune function, underscoring the importance of melatonin in maintaining overall physiological balance.
In this study, we used an accepted animal model of microgravity-induced bone loss to demonstrate the correlation between hyperserotonemia and corresponding loss of trabecular and cortical bone architecture representative of many bone loss conditions, including osteopenia or early osteoporosis. These results argue that targeting specific serotonin systems with pharmaceuticals could help mitigate bone loss and other pathophysiological challenges, including nausea, fatigue, and muscle dysfunction induced by microgravity, that threaten astronaut safety and effectiveness, particularly for long duration missions.
Serotonin in plasma from normally loaded (NL) and hindlimb unloaded (HU) mice are shown. Bars plot the mean concentration in ng · ml−1 and SD error bars. Individual data points are coplotted to demonstrate data spread. Unpaired Student t-tests with significance of P < 0.05 were used.
Serotonin levels plotted as a trend showing serotonin levels in the NL animals. Parabolic growth cure metrics are reported in the insert table (R2 = 0.9077 and 0.7052, respectively).
The bone volume/trabecular volume, trabecular thickness, and trabecular number are shown in the HU animals compared to NL. Data shown are median levels ± the max/min levels and individual measurements (dots) to illustrate the data spread.
Cortical bone levels in HU compared to NL are shown. Data shown are median levels ± the max/min levels and individual measurements (dots) to illustrate the data spread.
Contributor Notes
These authors contributed equally.
