Loss of physical contact in space alters the dopamine system in C. elegans

Abstract

Progressive neuromuscular decline in microgravity is a prominent health concern preventing interplanetary human habitation. We establish functional dopamine-mediated impairments as a consistent feature across multiple spaceflight exposures and during simulated microgravity in C. elegans. Animals grown continuously in these conditions display reduced movement and body length. Loss of mechanical contact stimuli in microgravity elicits decreased endogenous dopamine and comt-4 (catechol-O-methyl transferase) expression levels. The application of exogenous dopamine reverses the movement and body length defects caused by simulated microgravity. In addition, increased physical contact made comt-4 and dopamine levels rise. It also increased muscular cytoplasmic Ca²⁺ firing. In dop-3 (D2-like receptor) mutants, neither decrease in movement nor in body length were observed during simulated microgravity growth. These results strongly suggest that targeting the dopamine system through manipulation of the external environment (contact stimuli) prevents muscular changes and is a realistic and viable treatment strategy to promote safe human deep-space travel.

Keywords: Aerospace Engineering; Space medicine.

Graphical abstract

Figure 1

Decreased comt-4 expression and endogenous dopamine levels of C. elegans grown under space microgravity and artificial microgravity with 3D clinorotation (A) Changes in gene expression (fold change values with DNA microarray analyses) of eft-1 and comt-4 across all 15 independent specimens grown under microgravity and artificial 1G conditions or ground control (detailed in Figure S1B). (B) Endogenous DA levels in wild-type N2 adult hermaphrodites grown on the ground (1G) and space microgravity (μG). (C) Expression levels of comt-4 were analyzed by real-time RT-PCR with eft-2 internal standard in N2 adults grown on the ground (1G) and simulated μG with 3D clinorotation (3D). (D) Endogenous DA levels in wild-type N2 adults grown on the ground (1G) and simulated μG (3D). Data are shown as box and whiskers to indicate median and SD. Statistical analysis was performed in each condition using Student’s t test. ∗p< 0.05, ∗∗p< 0.01, and ∗∗∗p< 0.001.

Figure 2

Supply of exogenous dopamine restored moving activity and physique loss under artificial microgravity (A–D) Maximal bending angle (n = 15 each), (B) moving frequency as thrashing rate (n = 30 per condition), (C) body length (n = 30 per condition), and (D) expression levels of comt-4 were analyzed in wild-type N2 day 1 adult hermaphrodites grown on the ground (1G) and simulated μG (3D) with or without a final 50μM DA treatment (3,4-dihydroxyphenethylamine hydrochloride). DA was added at the start of culture at the L1 larval stage (+ DA) or in adult animals 24 h before observation (+DA (24 h BO) for only comt-4 analysis). Data are shown as box and whiskers to indicate median and standard deviations. Statistical analysis was performed in each condition using one-way ANOVA followed by Tukey post hoc test. Different letters indicate statistically significant differences at p< 0.05.

Figure 3

D2-like receptor dop-3 mutation restored moving activity and physique loss under artificial microgravity (A–C) Maximal bending angle (n = 15 per condition), (B) moving frequency as thrashing rate (n = 30 per condition), and (C) body length (n = 30 per condition) were measured in day 1 adults of N2 and dop-3 (vs106) deletion mutant cultured parallelly under normal gravity (1G) and simulated μG (3D) for 4 days. Data are shown as box and whiskers to indicate median and standard deviations. Statistical analysis was performed in each condition using one-way ANOVA followed by Tukey post hoc test. Different letters indicate statistically significant differences at p< 0.05.

Figure 4

Simulated microgravity did not change DA neuron morphology (A) Day 4 adults (vtIs1) under 1G or 3D clinorotation. Scale bars: 20 μm. (B) DA neurodegeneration, as an increase in the number of blebs along dendrites in animals under 1G or 3D clinorotation. Data are shown as box and whiskers to indicate median and standard deviation. Statistical analysis was performed in each condition using Student’s t test. ns: not significant.

Figure 5

Increased contact stimuli with the supply of microbeads restored locomotion activity and loss of physique under artificial microgravity (A) D1 adults (vtIs1) grown in culture bags with or without microspheres under 3D clinorotation (Video S4). (B–F) Expression levels of comt-4 by real-time RT-PCR with eft-2 internal standard, (C) endogenous DA levels, (D) maximal bending angle (n = 15 per condition), (E) moving frequency as thrashing rate (n = 30 per condition), and (F) body length (n = 30 per condition) was measured in day 1 adults (vtIs1) parallelly cultured for 4 days under normal gravity (1G) and simulated μG (3D) in the absence or presence of microspheres (‘+ beads’). Data are shown as box and whiskers to indicate median and standard deviations. Statistical analysis was performed in each condition using one-way ANOVA followed by Tukey post hoc test. Different letters indicate statistically significant differences at p< 0.05.

Figure 6

Contact stimulation increases muscle cytoplasmic Ca²⁺ firing (A) Changes in muscular cytoplasmic Ca²⁺ levels of day 1 adults (goeIs3: Pmyo-3::GCaMP) grown at 1G or μG (3D) for 4 days with or without microsphere beads (‘+ beads’) were captured under the same fluorescent intensities from video images (Video S5). GFP fluorescent signals were converted using image J royal color. Scale bars: 100 μm. (B) Maximal signal intensity of muscle cytoplasmic Ca^2 + levels of contract site under swimming behavior and contact beads (n = 16 animals per condition) were measured using image J software. Data are shown as box and whiskers to indicate median and standard deviation. Statistical analysis was performed in each condition using one-way ANOVA followed by Tukey post hoc test. Different letters indicate statistically significant differences at p< 0.05.