Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov 3;4:e2666.
doi: 10.7717/peerj.2666. eCollection 2016.

Hypergravity Hinders Axonal Development of Motor Neurons in Caenorhabditis elegans

Affiliations
Free PMC article

Hypergravity Hinders Axonal Development of Motor Neurons in Caenorhabditis elegans

Saraswathi Subbammal Kalichamy et al. PeerJ. .
Free PMC article

Abstract

As space flight becomes more accessible in the future, humans will be exposed to gravity conditions other than our 1G environment on Earth. Our bodies and physiology, however, are adapted for life at 1G gravity. Altering gravity can have profound effects on the body, particularly the development of muscles, but the reasons and biology behind gravity's effect are not fully known. We asked whether increasing gravity had effects on the development of motor neurons that innervate and control muscle, a relatively unexplored area of gravity biology. Using the nematode model organism Caenorhabditis elegans, we examined changes in response to hypergravity in the development of the 19 GABAergic DD/VD motor neurons that innervate body muscle. We found that a high gravity force above 10G significantly increases the number of animals with defects in the development of axonal projections from the DD/VD neurons. We showed that a critical period of hypergravity exposure during the embryonic/early larval stage was sufficient to induce defects. While characterizing the nature of the axonal defects, we found that in normal 1G gravity conditions, DD/VD axonal defects occasionally occurred, with the majority of defects occurring on the dorsal side of the animal and in the mid-body region, and a significantly higher rate of error in the 13 VD axons than the 6 DD axons. Hypergravity exposure increased the rate of DD/VD axonal defects, but did not change the distribution or the characteristics of the defects. Our study demonstrates that altering gravity can impact motor neuron development.

Keywords: Axon; C. elegans; Gravity; Motor neurons; Space flight.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. In 100G, hypergravity induces axonal defects in DD/VD motor neurons.
(A) Harvested embryos were exposed to hypergravity by centrifugation and then analyzed by microscopy as adults for neuronal defects. (B) (p)unc-25::GFP control animal at 1G gravity shows normal axonal commissures. Bar = 100 µm (C) Magnified view of boxed area in (B). (D) (p)unc-25::GFP animal exposed to 100G hypergravity shows multiple axon commissural defects (white triangles) Bar = 100 µm. (E) Axonal defects in 100G exposed animals. Bar = 50 µm. (F) 3D extended depth of field (EDF) image of (p)unc-25::GFP exposed to 100G hypergravity. Ventral in front, dorsal in back. Bar = 100 µm. (G) Magnified image of white box in (F) showing normal axonal commissures. Note the circumferential axons traveling along the body wall from the ventral to dorsal side during development. (H) Magnified image of white box in (F) showing defective axon that turned and extended and formed branches before it approached the dorsal side of the animal. (I) Percent animals that display at least one axon defect for 1G and 100G. Error bars represent SE. T-test was performed and ** indicates statistical significance p < 0.005. (J) Percent axons that are defective in 1G and 100G. T-test was performed and *** indicates statistical significance p < 0.0005. Error bars represent SE.
Figure 2
Figure 2. Hypergravity force and exposure time affect DD/VD neuron axon development.
(A) Animals were exposed to 1–500G hypergravity for 60 h after embryo harvesting. Gravity force of over 10G increased axonal defects. T-test was performed and ** indicates statistical significance p < 0.0005. Error bars represent SE. (B) Animals were exposed to 100G hypergravity for various times after embryo harvesting, or to 3 h or 60 h during adulthood (far right bars). Grey bars = 1G control, black bars = 100G. Single-factor ANOVA and post-hoc Bonferroni correction was performed, ** indicates statistical significance p < 0.005. (C) Data in (B) represented by exposure time over the major developmental events of C. elegans (top of graph). Green represents axonal defects induced, red represents axonal defects not induced, yellow represents axonal defects slightly induced. Bars represent SE.
Figure 3
Figure 3. Spatial distribution of axonal defects in 1G and 100G hypergravity exposed animals.
(A) Location of the DD and VD axon commissures along the anterior-posterior axis of the worm. VD2 and DD1 are overlapped commissures and are indistinguishable. (B) Axon defects for each VD/DD commissure. Top table shows the contribution of axon defects for each commissure to the total number of defects for 1G and 100G. The percents for each commissure add up to 100% for 1G and 100G, respectively. Heat maps in purple/lavender shades show the percents indicated in the legend on the right. N = number of worms. Bottom table shows the percent defects for each individual commissure for 1G and 100G. Heat maps in orange/yellow shades show the percents indicated in the legend on the right. (C) Localization of defects in the dorsal-ventral direction for 1G animals. Defects were categorized into the four regions listed and percent of defects in each region is shown. (D) Localization of defects in the dorsal-ventral direction for 100G hypergravity exposed animals.
Figure 4
Figure 4. Qualitative characterization of axonal defects in 1G and 100G hypergravity exposed animals.
(A) Axonal defects were categorized into the four groups shown here. (B) Quantification of axonal defects by category for 1G and 100G exposed animals. T-test was performed and none of the values were shown to be significantly different. Bars represent SE.

Similar articles

See all similar articles

References

    1. Blanchette CR, Perrat PN, Thackeray A, Bénard CY. Glypican is a modulator of netrin-mediated axon guidance. PLOS Biology. 2015;13(7):e2666 doi: 10.1371/journal.pbio.1002183. - DOI - PMC - PubMed
    1. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71–94. - PMC - PubMed
    1. Cáceres IDC, Valmas N, Hilliard MA, Lu H. Laterally orienting C. elegans using geometry at microscale for high-throughput visual screens in neurodegeneration and neuronal development studies. PLoS ONE. 2012;7(4):e2666 doi: 10.1371/journal.pone.0035037. - DOI - PMC - PubMed
    1. Colavita A, Krishna S, Zheng H, Padgett RW, Culotti JG. Pioneer axon guidance by UNC-129, a C. elegans TGF-β. Science. 1998;281(5377):706–709. doi: 10.1126/science.281.5377.706. - DOI - PubMed
    1. DeFelipe J, Arellano JI, Merchán-Pérez A, González-Albo MC, Walton K, Llinás R. Spaceflight induces changes in the synaptic circuitry of the postnatal developing neocortex. Cerebral Cortex. 2002;12(8):883–891. doi: 10.1093/cercor/12.8.883. - DOI - PubMed

Grant support

This work was supported by a New Investigator Grant (2014R1A1A1005553) from the National Research Foundation of Korea (NRF) to J.I.L. Strains were provided by Jeong-Hoon Cho at Chosun University, and the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Feedback