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. 2013 Mar 21:7:20.
doi: 10.3389/fnbeh.2013.00020. eCollection 2013.

Evaluation of Head Movement Periodicity and Irregularity during Locomotion of Caenorhabditis elegans

Ryuzo Shingai  1 , Morimichi Furudate, Katsunori Hoshi, Yuishi Iwasaki
Affiliations

Evaluation of Head Movement Periodicity and Irregularity during Locomotion of Caenorhabditis elegans

Ryuzo Shingai et al. Front Behav Neurosci. .

Abstract

Caenorhabditis elegans is suitable for studying the nervous system, which controls behavior. C. elegans shows sinusoidal locomotion on an agar plate. The head moves not only sinusoidally but also more complexly, which reflects regulation of the head muscles by the nervous system. The head movement becomes more irregular with senescence. To date, the head movement complexity has not been quantitatively analyzed. We propose two simple methods for evaluation of the head movement regularity on an agar plate using image analysis. The methods calculate metrics that are a measure of how the head end movement is correlated with body movement. In the first method, the length along the trace of the head end on the agar plate between adjacent intersecting points of the head trace and the quasi-midline of the head trace, which was made by sliding an averaging window of 1/2 the body wavelength, was obtained. Histograms of the lengths showed periodic movement of the head and deviation from it. In the second method, the intersections between the trace of the head end and the trace of the 5 (near the pharynx) or 50% (the mid-body) point from the head end in the centerline length of the worm image were marked. The length of the head trace between adjacent intersections was measured, and a histogram of the lengths was produced. The histogram for the 5% point showed deviation of the head end movement from the movement near the pharynx. The histogram for the 50% point showed deviation of the head movement from the sinusoidal movement of the body center. Application of these methods to wild type and several mutant strains enabled evaluation of their head movement periodicity and irregularity, and revealed a difference in the age-dependence of head movement irregularity between the strains. A set of five parameters obtained from the histograms reliably identifies differences in head movement between strains.

Keywords: behavior; image analysis; mutant; nematode; senescence.

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Figures

Figure 1
Figure 1
Trace of head and Type-1 segment-length. (A) Superimposed images (gray) of the wild type worm at the A1 stage with the trace of the head movement (red) and the quasi-midline (yellow). A Type-1 segment-length is indicated by SL-1. (B) Superimposed worm images (gray) of an unc-25(e156) mutant worm at the A1 and A10 stages with the head trace (red) and the quasi-midline (yellow). (A,B) The worms moved from the left to the right.
Figure 2
Figure 2
Histograms of Type-1 segment-length. (A) (Left) Histogram of Type-1 segment-length without normalization by the length of each worm. (Right) nSL-1 histogram after normalization of the length of the worm to 1 mm. The worms are wild type at the L4–A10 stages. Numbers of worms are listed in Table 2. (B) Comparison of histogram made from the 30-min data set with that from the 10-min data set, which was taken from the initial part of the 30-min data set of wild type, unc-25, and unc-49 at the A1 stage. There was no statistical difference between the histograms of the 10 and 30-min data sets.
Figure 3
Figure 3
nSL-1 histograms of mutant strains at various ages. (A,B) unc-25(e156). (C) unc-49(e382). (D) glr-1(n2461). (E) nmr-1(ak4). (F) tph-1(mg280). (G) cat-1(e1111). (H) cat-2(e1112). (A–C) Ages between L4 and A10. (D–H) Ages between A1 and A10. (B) Histogram of the middle point of the body of unc-25(e156). All the other histograms are of the head end. Numbers of worms are listed in Table 2.
Figure 4
Figure 4
nSL-1 histograms of forward movement of the wild type and mutant strains at the A3 stage. Normalized histograms of Type-1 segment-length (nSL-1 histograms) at A3. (A) GABAergic mutants unc-25(e156) and unc-49(e382). (B) Glutamatergic mutants glr-1(n2461) and nmr-1(ak4). (C) Monoaminergic mutants cat-1(e1111), cat-2(e1112), and tph-1(mg280). (A–C) For comparison, the histogram of the wild type at the A3 stage is shown by a dotted line. (D) Peak values of nSL-1 histograms. (E) Fractions of the small segment-length (<0.18 mm) of nSL-1 histograms. (D,E) Statistically indifferent (p ≥ 0.01) values are connected by lines. Error bar indicates SE (A–E). Numbers of worms are listed in Table 2.
Figure 5
Figure 5
Type-2 segment-length. (A) An image of a wild type worm at the A1 stage. The blue line is the centerline of the image. The three yellow points indicate the head end, the 5 and 50% points on the centerline from the head end. (B–D) Superimposed images of a worm (gray), the head trace (red), and the trace of the 5 or 50% point (blue). The yellow points indicate the intersections of the two traces. The length of the head trace between adjacent intersections is the Type-2 segment-length; an example is indicated by SL-2 in (C). (B) Intersections and the trace of the 5% point of the wild type worm at the A1 stage. (C) Intersections and the trace of the 50% point of the wild type worm at the A1 stage. (D) Trace of the 50% point of the tph-1 mutant worm at A1.
Figure 6
Figure 6
nSL-2 histograms (5%) of the wild type and mutant strains at various ages. Normalized histograms of Type-2 segment-length for the 5% point [nSL-2 histograms (5%)]. (A) Wild type (N2). (B) unc-25(e156). (C) unc-49(e382). (D) glr-1(n2461). (E) nmr-1(ak4). (F) tph-1(mg280). (G) cat-1(e1111). (H) cat-2(e1112). (A–C) Ages L4–A10. (D–H) Ages A1–A10. Numbers of worms are listed in Table 2.
Figure 7
Figure 7
nSL-2 histograms (50%) of the wild type and mutant strains at various ages. Normalized histograms of Type-2 segment-length for the 50% point [nSL-2 histograms (50%)]. (A) Wild type (N2). (B) unc-25(e156). (C) unc-49(e382). (D) glr-1(n2461). (E) nmr-1(ak4). (F) tph-1(mg280). (G) cat-1(e1111). (H) cat-2(e1112). (A–C) Ages L4–A10. (D–H) Ages A1–A10. Numbers of worms are listed in Table 2.
Figure 8
Figure 8
Age changes in peak values and small segment-length fractions of nSL-1 histograms. (A–C) Peak values of the nSL-1 histograms. (D–F) Small segment-length fractions (<0.18 mm) in the nSL-1 histograms. (A,D) Wild type (N2), unc-25(e156), and unc-49(e382). (B,E) glr-1(n2461) and nmr-1(ak4). (C,F) tph-1(mg280), cat-1(e1111), and cat-2(e1112). *Indicates a statistically significant difference (p < 0.01) between the wild type and unc-49 in (A,D); between glr-1 and nmr-1 in (B,E); and between tph-1 and cat-1 in (C,F). #Indicates a statistically significant difference (p < 0.01) between unc-25 and unc-49 in (A,D); and between cat-1 and cat-2 in (C,F). Numbers of worms are listed in Table 2. Error bar indicates SE.
Figure 9
Figure 9
Age changes in peak values of nSL-2 histograms. (A–C) Peak values (closed symbols) in the nSL-2 histograms (5%) around 0.06–0.10 mm. (D–F) Peak values (closed symbols) in the nSL-2 histograms (50%) around 0.3–0.4 mm. When there was no peak around 0.06–0.10 mm in the nSL-2 (5%) histogram or 0.3–0.4 mm in the nSL-2 (50%) histogram, the histogram value at the same segment-length as that of the peak in the closest age histogram having a peak was plotted (open symbols). * and # have the same meaning as in Figure 8. Numbers of worms are listed in Table 2. Error bar indicates SE.
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