Dissection of C. elegans behavioral genetics in 3-D environments

The nematode Caenorhabditis elegans is a widely used model for genetic dissection of animal behaviors. Despite extensive technical advances in imaging methods, it remains challenging to visualize and quantify C. elegans behaviors in three-dimensional (3-D) natural environments. Here we developed an innovative 3-D imaging method that enables quantification of C. elegans behavior in 3-D environments. Furthermore, for the first time, we characterized 3-D-specific behavioral phenotypes of mutant worms that have defects in head movement or mechanosensation. This approach allowed us to reveal previously unknown functions of genes in behavioral regulation. We expect that our 3-D imaging method will facilitate new investigations into genetic basis of animal behaviors in natural 3-D environments

Synchrotron x-ray imaging of pulmonary alveoli in respiration in live intact mice

Despite nearly a half century of studies, it has not been fully understood how pulmonary alveoli, the elementary gas exchange units in mammalian lungs, inflate and deflate during respiration. Understanding alveolar dynamics is crucial for treating patients with pulmonary diseases. In-vivo, real-time visualization of the alveoli during respiration has been hampered by active lung movement. Previous studies have been therefore limited to alveoli at lung apices or subpleural alveoli under open thorax conditions. Here we report direct and real-time visualization of alveoli of live intact mice during respiration using tracking X-ray microscopy. Our studies, for the first time, determine the alveolar size of normal mice in respiration without positive end expiratory pressure as 58 ± 14 (mean ± s.d.) μm on average, accurately measured in the lung bases as well as the apices. Individual alveoli of normal lungs clearly show heterogeneous inflation from zero to ~25% (6.7 ± 4.7% (mean ± s.d.)) in size. The degree of inflation is higher in the lung bases (8.7 ± 4.3% (mean ± s.d.)) than in the apices (5.7 ± 3.2% (mean ± s.d.)). The fraction of the total tidal volume allocated for alveolar inflation is 34 ± 3.8% (mean ± s.e.m). This study contributes to the better understanding of alveolar dynamics and helps to develop potential treatment options for pulmonary diseases

3-D Worm Tracker for Freely Moving C. elegans

The manner in which the nervous system regulates animal behaviors in natural environments is a fundamental issue in biology. To address this question, C. elegans has been widely used as a model animal for the analysis of various animal behaviors. Previous behavioral assays have been limited to two-dimensional (2-D) environments, confining the worm motion to a planar substrate that does not reflect three-dimensional (3-D) natural environments such as rotting fruits or soil. Here, we develop a 3-D worm tracker (3DWT) for freely moving C. elegans in 3-D environments, based on a stereoscopic configuration. The 3DWT provides us with a quantitative trajectory, including the position and movement direction of the worm in 3-D. The 3DWT is also capable of recording and visualizing postures of the moving worm in 3-D, which are more complex than those in 2-D. Our 3DWT affords new opportunities for understanding the nervous system function that regulates animal behaviors in natural 3-D environments.X11119sciescopu

3-D Worm Tracker for Freely Moving <em>C. elegans</em>

<div><p>The manner in which the nervous system regulates animal behaviors in natural environments is a fundamental issue in biology. To address this question, <i>C. elegans</i> has been widely used as a model animal for the analysis of various animal behaviors. Previous behavioral assays have been limited to two-dimensional (2-D) environments, confining the worm motion to a planar substrate that does not reflect three-dimensional (3-D) natural environments such as rotting fruits or soil. Here, we develop a 3-D worm tracker (3DWT) for freely moving <i>C. elegans</i> in 3-D environments, based on a stereoscopic configuration. The 3DWT provides us with a quantitative trajectory, including the position and movement direction of the worm in 3-D. The 3DWT is also capable of recording and visualizing postures of the moving worm in 3-D, which are more complex than those in 2-D. Our 3DWT affords new opportunities for understanding the nervous system function that regulates animal behaviors in natural 3-D environments.</p> </div

The principle of the 3-D worm tracker (3DWT).

<p>(A) Experimental scheme of the stereoscopic recording. Two imaging assemblies at right angles with the same focal point are used to synchronously record images of <i>C. elegans</i> from two perpendicular directions. (B) Stereomatching of skeletons. The two X-Z and Y-Z skeletons, extracted from the two projection images of the worm, are combined using stereomatching to reconstruct the 3-D skeleton. (C) Volume rendering of the 3-D skeleton. The 3-D volume of the worm is rendered by assigning particles that are centered on the 3D skeleton points.</p

Volume-rendered images in three views.

<p>(A) 3-D posture of a worm showing bends in various directions (gray arrows) with only slight changes in the moving direction (green arrow) during crawling. (B) 3-D posture of another worm showing a significant change in the moving direction (green arrow). The asterisk in each image indicates the head of the worm.</p

Bending vector analysis of worm motion.

<p>(A) Schematic of the bending vector in a 3-D skeleton. The arrows indicate the bending vector at a skeletal point, P_i. (B) Variable bending vectors of a second skeletal point from the head of a representative worm during crawling. (C) The X-Y projections of bending vectors along the whole body for 5.5 s of crawling. The colors of arrows represent the directions of the bending vectors by linearly converting the components of normalized bending vectors (−1.0∼1.0) to 8-bit colors (0∼255): the X-component is displayed in red (R), the Y-component in green (G), and the Z-component in blue (B).</p

3-D trajectory and velocity of a worm.

<p>(A) 3-D trajectory of the worm. Red, blue, and green arrows are movement directions of a worm for 12 s, 14 s, and 12 s, respectively. (B) The x-, y-, and z-components of the worm's velocity.</p