Dependence of locomotion paralysis on light intensity.

<p>Animals expressing Arch::GFP under muscle-specific (<i>myo-3p</i>) and pan-neuronal (<i>aex-3p</i> and <i>F25B3.3p</i>) promoters were illuminated with green light at varying intensities. Animals with <i>nc3034Ex[F25B3.3p::Arch::gfp]</i> (squares) and <i>nc3026Ex[aex-3p::Arch::gfp]</i> (diamonds) exhibited higher responsiveness at lower light intensities than <i>nc3031Ex[myo-3p::Arch::gfp]</i> (triangles) animals did. (mean±SEM; n = 3. Five animals were examined for each trial.).</p

Expression of Arch::GFP in <i>C. elegans</i> driven by various promoters.

<p>(A) A fluorescent micrograph of an <i>nc3031Ex[myo-3p::Arch::gfp]</i> animal. Arch::GFP is expressed in longitudinal bands composed of body wall muscles (arrow). (B) An enlarged view of body wall muscle cells. GFP signal is observed along the outline of muscle cells (arrow). Vesicular structures visualized with GFP are localized close to the cell membrane (arrow head). Weak GFP signal is detected in the cytoplasm (asterisk). (C) Expression of Arch::GFP in an <i>nc3034Ex[F25B3.3p::Arch::gfp]</i> animal. Arch::GFP is expressed in head neurons (arrow head), tail neurons (open arrow head) and the ventral nerve cord (arrow). (D) The head of an animal carrying <i>F25B3.3p::Arch::gfp</i>. Arch::GFP is expressed in the axon (arrow) and the cell body (arrow head) of head neurons. (E) Expression of Arch::GFP in an <i>nc3026Ex[aex-3p::Arch::gfp]</i> animal. Arch::GFP is expressed in head neurons (arrow), tail neurons (arrow head), and the ventral nerve cord (open arrow head). (F) The head of an animal carrying <i>aex-3p::Arch::gfp</i>. Arch::GFP is expressed in the axon of head neurons in a punctured pattern (arrow). GFP is seen on the cell membrane of a cell body (arrow head). (G) Expression of Arch::GFP in an <i>nc3003Ex[hsp-16.2p::Arch::gfp]</i> animal. Arch::GFP is expressed everywhere in the body. (H) The head of an animal carrying <i>hsp-16.2p:: Arch::gfp</i>. Arch::GFP is expressed in neurons (arrow). (I) Body wall muscle of an <i>hsp-16.2p::Arch::gfp</i> animal. GFP is clearly localized at the cell membrane (arrow). Scale bar: A, C, E, G = 100 µm; B, D, F, H I = 10 µm. Anterior is toward the right except for (G).</p

Comparison of light-elicited locomotory paralysis mediated by Arch and Arch::GFP.

<p>(A) Dependence of locomotion paralysis of <i>Ex[hsp-16.2p::Arch]</i>animals (black squares) and <i>Ex[hsp-16.2p::Arch::gfp]</i> animals (open circles) to light intensity was measured 12h after heat shock. (B) Time course of light-elicited locomotory paralysis after heat-shock induction of Arch and Arch::GFP. Heat-shocked <i>Ex[hsp-16.2p::Arch]</i> (black squares) and <i>Ex hsp-16.2p::Arch::gfp]</i> (open circles) animals were examined at each time point as shown in Fig. 4. A(i). Three independently isolated lines were used for each transgene: <i>ncEx3002, ncEx3003</i> and <i>ncEx3004</i> for <i>Ex[hsp-16.2p::Arch::gfp]</i>, and <i>ncEx3039, ncEx3040</i> and <i>ncEx3041</i> for <i>Ex[hsp16-2p::Arch]</i>. Shown are the mean±SEM of 9 trials consisting of 3 trials for each line. Five animals were examined for each trial.</p

Locomotion assay using heat shock-mediated induction of Arch.

<p>(A) Scheme showing the schedule of transferring worms to plates with or without all-trans-retinal (ATR) in heat shock induction experiments. i: Animals were cultivated in the presence of ATR throughout the experiments. (Figs. 3B, 4B) ii: Animals were transferred to ATR-supplemented plates 24 h after heat shock (Fig. 4B). iii: Animals were cultivated in the presence of ATR and transferred to ATR-free plates 24 h after heat shock. (B) Time course of light-elicited locomotory paralysis after heat-shock induction of Arch::GFP. Heat-shocked <i>nc3003Ex[hsp-16.2p::Arch::gfp]</i> animals cultivated in the presence of ATR throughout the experiment (circles) (Fig. 4. A(i)) or transferred from ATR-free to ATR-supplemented plates 24 h after heat shock (triangles) (Fig. 4. A(ii)) were examined at each time point. When ATR was present throughout the experiment (circles), paralysis of worms was first noticed 6 h after heat shock. The paralysis rate reached a plateau 12 h after heat shock, and remained constant for 48 h. (mean±SEM; n = 3, Five animals were examined for each trial.) Worms cultivated in the absence of ATR throughout the experiment did not respond to illumination at any time point. When animals were grown and heat shocked in the absence of ATR and then transferred to ATR-supplemented plates 24 h later (triangles), half of them were paralyzed by illumination 1.5 h after transfer, and the paralysis rate reached a plateau within 3 h. (mean±SEM; n = 4, Five animals were examined for each trial.).</p

Defects in the locomotory behavior caused by silencing subsets of motor neurons.

<p>(A) A crawling track of an <i>ncEx2351[unc-47p::Arch::gfp]</i> animal. When the freely moving animal was illuminated with green light for 1 minute, it performed loopy movement (dotted square). After turning off the green light, it resumed normal sinusoidal movement. Scale bar = 500 µm (B-D) Locomotory behavior of worms expressing Arch in motor neurons subsets. Arch::GFP was expressed in D-type (VD, DD), A-type (VA, DA), B-type (VB, DB) and VA +VB motor neurons in <i>ncEx2351[unc-47p::Arch::gfp], ncEx2371[acr-5p::Arch::gfp], ncEx3068[unc-4p::Arch::gfp]</i> and <i>ncEx2365[del-1p::Arch::gfp]</i> animals, respectively. Locomotion of <i>unc-47(e307)</i> animals was also scored. For all transgenic strains, animals behaviors under green light illumination (ON = open box) and those without illumination (OFF = filled box) differed significantly (p<0.001, Fisher's exact test for the locomotion assay under the freely moving condition and Student's <i>t</i> test for the touch response assay). Exceptions are forward movement of <i>ncEx3068[unc-4p::Arch::gfp]</i> animals, which did not change significantly under illumination, and forward movement of <i>ncEx2365[del-1p::Arch::gfp]</i> animals elicited by gentle posterior touch (p = 0.012). Error bars indicate ±SEM. (B) Forward (F) and backward movement (B) was scored in worms moving freely. Percentage of animals exhibiting the “Class 3 (severe)”, “Class2 (mild)” and “Class1 (no)” phenotype in locomotory behaviors when they were illuminated with green light is shown. Defects were classified as “Class 3″ when no corresponding movement or response was observed. Other abnormalities, such as retardation or decrease in the extent of movement, are classified as “Class2”. (C) Forward movement (F) to gentle posterior touch and backward (B) movement to gentle anterior touch were scored. Responses out of five touches are shown. (D) Forward (F) and backward movement (B) to harsh touch was scored, and was shown additively in each bar. Responses out of five touches are shown.</p