Long-Term Imaging of <i>Caenorhabditis elegans</i> Using Nanoparticle-Mediated Immobilization

<div><p>One advantage of the nematode <i>Caenorhabditis elegans</i> as a model organism is its suitability for <i>in vivo</i> optical microscopy. Imaging <i>C</i>. <i>elegans</i> often requires animals to be immobilized to avoid movement-related artifacts. Immobilization has been performed by application of anesthetics or by introducing physical constraints using glue or specialized microfluidic devices. Here we present a method for immobilizing <i>C. elegans</i> using polystyrene nanoparticles and agarose pads. Our technique is technically simple, does not expose the worm to toxic substances, and allows recovery of animals. We evaluate the method and show that the polystyrene beads increase friction between the worm and agarose pad. We use our method to quantify calcium transients and long-term regrowth in single neurons following axotomy by a femtosecond laser.</p></div

Nanoparticles reduce locomotory rate in a surface-dependent manner.

<p>Locomotory frequency of freely swimming worms and worms in contact with 0.5% agarose pads, in the presence (NP) and absence (NGM) of nanoparticles. n = 30 for each group. **p<0.001.</p

Quantifying movement during immobilization.

<p>(a) Fluorescence of AFD soma in a nanoparticle-immobilized <i>Pgcy-8</i>::YC3.60 worm. Centroid of cell body indicated by crosshairs. Scale bar: 20 µm. (b) NP: Mean absolute displacement of AFD neuron cell body during 10 s intervals in animals immobilized with 0.1 µm diameter nanoparticles, as a function of agarose concentration in pad. NGM: no beads. (c) Mean absolute displacement of buccal cavity during 10 s intervals for adult worms on 10% agarose pads and varied bead diameters; L1, L3, and adult (Ad) worms immobilized with 0.1 µm beads on 10% agarose pads.</p

<i>In vivo</i> laser axotomy and time-lapse imaging.

<p>a) Images from a continuously immobilized <i>C. elegans</i>, showing an ALM neuron severed 20 µm from the cell soma (top panel) immediately following laser axotomy and (bottom panel) after 10 hr. Arrow indicates lesion point. b) Average regenerative outgrowth of ALM neurons severed at the indicated distances. Outgrowth was measured from images taken at 10 hr post surgery and categorized as initiating from the lesion point or from the cell soma (* indicates p<0.05 by Student’s t-test). c) Average FRET signals measuring cellular calcium response to laser axotomy. At each time point, two channel fluorescent FRET images of the target neuron where averaged across the cell soma and along the axon within 5 µm of the lesion point to measure intracellular calcium levels in those regions. Arrows indicate time of laser damage (t = 0 s). Shaded regions indicate standard error on the mean at each time point. n indicates number of worms assayed.</p

CED-3 caspase contributes to early dynamics of axonal regeneration.

<p>(a) Time-lapse regenerative outgrowth measurements during the 0–5 h time period following laser surgery for WT (grey) and <i>ced-3(n2433)</i> (red). Data points indicate outgrowth of individual neurons, and lines indicate average outgrowth (shaded region areas ± s.e.m.). The insert shows total outgrowth rates over the 0–5 h time period (calculated using a regression fit of the displayed outgrowth data, restricted to pass through the origin). (b) Mean time of initial outgrowth after laser surgery for WT (grey) and <i>ced-3(n2433)</i> (red) mutant worms as determined from time lapse measurements. (c) Mean number of individual exploratory processes generated during the 0–45 min and 0–5 h time periods following laser surgery. (d) Representative images showing numerous exploratory outgrowths, sprouting of small often short-lived processes, in the WT background, compared to (e) relatively few such protrusions in the <i>ced-3(n2433)</i> mutant background. Green arrows mark new exploratory processes, green arrowheads mark stunted or stalled processes, purple arrow marks an exploratory process from the disconnected distal axon segment, and time is indicated in minutes post-laser surgery. For bar graphs, data are expressed as mean ± s.e.m. *<i>p</i><0.05, **<i>p</i><0.005 versus wild type by Student's <i>t</i> test.</p

CED-3 caspase contributes to reconnection to the dissociated distal fragment.

<p>(a) Photo-bleaching test for successful reconnection (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001331#s4" target="_blank">Materials and Methods</a> for details). (i, ii) Compressed z-stack images 12 h post-laser surgery of neurons displaying apparent reconnection. Red arrow indicates the original cut point. (iii, iv) Magnified image of distal segment (single z-frame). (v, vi) A second laser cut (yellow arrow) is followed by selective photo-bleaching between the two cut points. (vii) Recovery of GFP fluorescence in the original distal segment within 15 min indicates the existence of a fusion between the regenerating proximal axon segment and the distal segment, and (viii) a lack of fluorescence recovery indicates no such reconnection (with a cutoff of <7.7% fluorescence score, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001331#s4" target="_blank">Materials and Methods</a>). White brackets indicate the portion of process analyzed for fluorescence recovery; numbers indicate percent recovery of fluorescence. (b) Percent of re-growing axons that track to the place of the dissociated distal fragment at 12 h (i.e., appear to be in physical contact). Note that although <i>ced-3</i> mutant axons tend to track less often to the dissociated distal fragment, the differences are not statistically significant (<i>p</i> = 0.217). (c) Percent of neurons, of those that track to the dissociated distal fragment, that are also scored to have reconnected at 12 h. Specific reconnection events (in addition to poor tracking) appear delayed in <i>ced-3</i> mutant axons. (d) Percentage of total neurons at 12 h post-surgery, for which the regenerating proximal axon successfully reconnected with the disconnected distal axon segment. (e) Percentage of total neurons at 72 h post-surgery, for which the regenerating proximal axon successfully reconnected with the disconnected distal axon segment. Note that reconnected axons do not show filopodial extensions, suggesting this trait might be suppressed in reconnected neurons as well as in intact neurons. All comparisons are by Fisher's exact test, with *<i>p</i><0.05.</p

CED-3 caspase activity is needed for efficient axonal regeneration.

<p>(a) Representative images of a <i>p_mec-4</i>GFP-labeled ALM neuron were taken before (i), immediately after (ii, red arrow indicates cut point at 20 µm from the cell body), and 24 h after laser axotomy in WT (iii) and in the <i>ced-3(n2433)</i> active site mutant (iv). Images are projected z-stacks. The green traces indicate the observed regenerative outgrowth for WT (v) and <i>ced-3(n2433)</i> (vi); scale bar: 10 µm. Regenerative outgrowth was measured 24 h after surgery in (b) ALM neurons (four independent <i>ced-3</i> alleles including deletion allele <i>n2452</i> and active site mutant allele <i>n2433</i>) and (c) D-motor neurons for young adult animals. (d) Regenerative outgrowth was measured 3 d after surgery in ALM neurons in WT and <i>ced-3(n2433)</i> (no statistical difference between the two by <i>t</i> test). (e) Comparison of WT (grey) and <i>ced-3(n2433)</i> (red) 24 h regenerative outgrowth in ALM neurons for different age animals (L3 and L4 larvae, young adults, and 2-d-old adult). (f) Cell autonomy test for <i>ced-3</i> rescue of regeneration outgrowth phenotype. The length of ALM regenerative outgrowth was measured 24 h after surgery in young adult animals for the control transgenic strains, bearing the <i>unc-119(+)</i> marker of transformation, <i>Is[unc-119(+)]</i> and <i>ced-3(n2433); Is[unc-119(+)]</i> as well as transgenic strains expressing <i>ced-3</i> in the touch neurons <i>Is[p_mec-4ced-3]</i> and <i>ced-3(n2433); Is[p_mec-4ced-3]</i>. See notes on strain construction and <i>ced-3</i> transgene expression toxicity in touch neurons in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001331#pbio.1001331.s002" target="_blank">Figure S2</a>. The <i>unc-119</i> integrated copy (<i>Is[unc-119(+)]</i>) did not affect the <i>ced-3(n2433)</i> defect in regeneration, and expression of <i>p_mec-4ced-3</i> (<i>Is[p_mec-4ced-3]</i>) in the mechanosensory neurons rescues the <i>ced-3(n2433)</i> defect, despite some neurotoxicity. <i>p_mec-4ced-3</i> expression in wild type does not induce excessive regeneration (panel f, third bar), and thus does not appear sufficient to promote regeneration, although we cannot rule that toxicity of elevated caspase activation could mask a potential beneficial outcome. All bar graphs depict mean ± s.e.m. The Student's <i>t</i> test, with a Dunn-Sidak adjustment for multiple comparisons, was used to determine the statistical significance of differences versus WT in each panel, except in (f) where brackets indicate direct Student's <i>t</i> test between two specific values; *<i>p</i><0.05, **<i>p</i><0.005 in all cases. Number of animals assayed is indicated in (or above) each bar for this and all other figures.</p

The <i>ced-4</i> core apoptotic gene, but not known <i>C. elegans</i> upstream regulators of developmental, germline, or radiation-induced apoptosis, are needed for efficient axonal regeneration.

<p>(a) We measured mean regenerative outgrowth in ALM neurons 24 h after laser surgery for WT and mutant strains affecting <i>ced-3</i> and <i>ced-4</i> (two independent alleles) core apoptotic genes, compound mutants, and in <i>ced-4; Is[p_mec-4ced-3]</i>. Because some studies <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001331#pbio.1001331-Hammarlund1" target="_blank">[9]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001331#pbio.1001331-Yan1" target="_blank">[10]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001331#pbio.1001331-Nix1" target="_blank">[18]</a> documented mutant neurons that show virtually no post-axotomy regeneration and we find that the <i>kgb-1 ced-3</i> double mutant exhibits lower regeneration than the <i>ced-3</i> single mutant (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001331#pbio.1001331.s006" target="_blank">Figure S6</a>), the partial phenotype can become more severe in compound mutants. (b) Regenerative outgrowth was measured 3 d after surgery in ALM neurons in WT and <i>ced-4(n1162)</i> (no statistical difference by <i>t</i> test). (c) Regenerative outgrowth was measured 24 h after surgery in ALM neurons for mutants in upstream apoptosis regulators as well as compound mutants (which show no statistical difference by one-way ANOVA test). Bar graphs depict mean ± s.e.m. For (a), the Student's <i>t</i> test, with a Dunn-Sidak adjustment for multiple comparisons, was used to determine the statistical significance of differences versus WT, with brackets indicating direct Student's <i>t</i> test between two specific values. *<i>p</i><0.05, **<i>p</i><0.005.</p

List of strains used in this study.

<p>Mutations are loss-of-function unless otherwise indicated. We confirmed lesions in all <i>ced-3</i>, <i>ced-4</i>, and <i>dlk-1</i> constructs by DNA sequence analysis.</p

CED-3 caspase acts in a calreticulin-, calcium-dependent pathway for efficient axonal regeneration.

<p>(a) Intracellular calcium dynamics in the ALM neurons during laser axotomy. Two different variants of the FRET-based calcium-sensitive fluorophore cameleon were used: Left panel, YC2.12; right panel, YC3.60. Differences in the wild type response (amplitude and shape) are due in part to the lower calcium affinity and larger dynamic range of the YC3.60 fluorophore compared to that of YC2.12 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001331#pbio.1001331-Nagai1" target="_blank">[58]</a>. All laser axotomies were performed 20 µm from the cell soma at time = 0 s (red arrow). Traces represent average response at the cell soma (≥9 trials per trace), and shaded regions indicate s.e.m. (b) Mean regenerative outgrowth in ALM neurons measured 24 h after laser surgery for the indicated mutant strains and compound mutant strains defective in the ER calcium-binding chaperone calreticulin, including in the context of <i>ced-3</i> expression in touch neurons (indicated as <i>crt-1; Is[p_mec-4ced-3]</i>). (c) Regenerative outgrowth was measured 3 d after surgery in ALM neurons in <i>crt-1(bz29)</i> (no statistical difference was found by Student's <i>t</i> test). <i>crt-1(bz29)</i> and <i>ced-3(n2433); crt-1(bz29)</i> double mutant were compared with WT and <i>ced-3</i>. (d) Mean time of initial outgrowth after laser surgery, and (e) mean number of individual exploratory processes generated during the 0–45 min and 0–5 h time periods following laser surgery. Bar graphs depict mean ± s.e.m. For (b), (c), (d), and (e) the Student's <i>t</i> test, with a Dunn-Sidak adjustment for multiple comparison, was used to determine the statistical significance of differences versus WT in each panel, with brackets indicating direct Student's <i>t</i> test between two specific values; *<i>p</i><0.05, **<i>p</i><0.005.</p