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.

Dendritic planarity of Purkinje cells is independent of Reelin signaling.

The dendritic planarity of Purkinje cells is critical for cerebellar circuit formation. In the absence of Crk and CrkL, the Reelin pathway does not function resulting in partial Purkinje cell migration and defective dendritogenesis. However, the relationships among Purkinje cell migration, dendritic development and Reelin signaling have not been clearly delineated. Here, we use synchrotron X-ray microscopy to obtain 3-D images of Golgi-stained Purkinje cell dendrites. Purkinje cells that failed to migrate completely exhibited conical dendrites with abnormal 3-D arborization and reduced dendritic complexity. Furthermore, their spines were fewer in number with a distorted morphology. In contrast, Purkinje cells that migrated successfully displayed planar dendritic and spine morphologies similar to normal cells, despite reduced dendritic complexity. These results indicate that, during cerebellar formation, Purkinje cells migrate into an environment that supports development of dendritic planarity and spine formation. While Reelin signaling is important for the migration process, it does not make a direct major contribution to dendrite formation.

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.

Tracking X-ray microscopy for alveolar dynamics in live intact mice.

Here we report a tracking X-ray microscopy (TrXM) as a novel methodology by using upper right lung apices alveoli in live intact mice. By enabling tracking of individual alveolar movements during respiration, TrXM identifies alveolar dynamics: individual alveoli in the upper lung apices show a small size increment as 4.9 ± 0.4% (mean ± s.e.m.) during respiration while their shapes look almost invariant. TrXM analysis in alveolar dynamics would be significant for better understanding of alveolar-based diseases, for instance, ventilator induced lung injury (VILI) in acute respiratory distress syndrome (ARDS).

Altered branching patterns of Purkinje cells in mouse model for cortical development disorder.

Disrupted cortical cytoarchitecture in cerebellum is a typical pathology in reeler. Particularly interesting are structural problems at the cellular level: dendritic morphology has important functional implication in signal processing. Here we describe a combinatorial imaging method of synchrotron X-ray microtomography with Golgi staining, which can deliver 3-dimensional(3-D) micro-architectures of Purkinje cell(PC) dendrites, and give access to quantitative information in 3-D geometry. In reeler, we visualized in 3-D geometry the shape alterations of planar PC dendrites (i.e., abnormal 3-D arborization). Despite these alterations, the 3-D quantitative analysis of the branching patterns showed no significant changes of the 77 ± 8° branch angle, whereas the branch segment length strongly increased with large fluctuations, comparing to control. The 3-D fractal dimension of the PCs decreased from 1.723 to 1.254, indicating a significant reduction of dendritic complexity. This study provides insights into etiologies and further potential treatment options for lissencephaly and various neurodevelopmental disorders.