Name: assaying &beta;-amyloid toxicity using a transgenic c. elegans model
Uploaded: 2010-10-09T17:00:00
Duration: 13 min 59 s
Description: Watch this Scientific Journal Video about Assaying Î²-amyloid Toxicity using a Transgenic C. elegans Model at JoVE.com

We would like to thank current and former members of the Link lab who helped develop the protocols described here, particularly Gin Fonte, who developed the original procedure. This work has been supported by awards from the NIH (AG12423) and the Alzheimer's Association (Zenith Award) to C.D.L.

1) Preparing Nematode Growth Media plates for paralysis assay.
Paralysis assays are performed on the standard Nematode Growth Media (NGM) plates routinely used for C. elegans propagation and genetics (Stiernagle, 2006).
<ol>
 <li>Autoclave the NGM solution containing NaCl, Agar and Peptone in an Erlenmeyer flask and add the appropriate amounts of sterile CaCl2, Uracil, Cholesterol, 1M MgSO4 and 1M KPO4. Aliquot 10 mL of liquid NGM into each 60 mm x 15 mm Petri Dish. Allow NGM to solidify overnight at room temperature.</li>
 <li>If testing for the effects of compounds/extracts, aliquot the extract onto each plate and use a spreader to evenly distribute the extract over the surface of the plate. Allow extract to dry overnight at room temperature. Volumes over 1 mL will require more time to dry. </li>
 <li> Spot each plate with 250 &mu;L of E. coli strain OP5O grown in LB Media overnight for an optical density of 0.4-0.6. Allow bacteria to dry overnight at room temperature.</li>
</ol>
Relevant variables
The age of plates has a significant affect on the paralysis assay, as older plates tend to dry out. Use plates poured within one week of paralysis assay. For ideal results, pour plates 3-4 days before initiation of synchronous worm populations [see (2) below]. For any experiment, only use plates poured from the same batch.
Altering the size of the lawn (i.e. spotted vs. spread) and/or the type of bacteria will also alter the behavior of the worms. Paralysis assays can be performed using alternative E. coli strains as a food source (e.g., the HT115 strain used in feeding RNAi experiments), but this can change the kinetics of paralysis, thus requiring appropriate internal controls.
2) Preparation of Age-synchronous Worm Populations
Strain CL4176 (smg-1(cc546ts) I; dvIs27 [myo-3/ A&beta; minigene + rol-6(su1006) marker gene] X is most commonly used for assaying A&beta;-induced paralysis. This strain and other transgenic models are available to academic researchers for a nominal fee from the Caenorhabditis Genetics Center (http://www.cbs.umn.edu/CGC/).
<ol>
 <li>To maximize the age synchrony of the test population it is preferable to start with a synchronized parental generation. A week before the initiating the paralysis assay of the test population, use a platinum worm picker to transfer 20-30 gravid adults onto several 10 cm NGM plates spread with OP50 for a 2 hour egg lay at 16&deg;C (the permissive temperature for CL4176). Remove the gravid adults and allow the progeny to grow for 7 days, by which time they will be "second day" gravid adults. </li>
 <li>(Day 1) The second day gravid adults are used to prepare the age-synchronous test populations. For each experimental condition the object is to generate triplicate plates containing 50-75 age-synchronous worms. Using a platinum worm picker, transfer 10-12 of the day 2 gravid adults onto 60 mm (10 mL volume) NGM plates spotted with OP50. Allow the worms to lay eggs at 16&deg;C for 2 hours, then pick off the adults and allow eggs to hatch and grow at 16&deg;C. At this point it is best to have someone who will not be scoring the plates to code them, so the scorer will be blind to experimental conditions.</li>
</ol>
Relevant variables
The stage of embryonic development when eggs are laid (~ the 26-cell stage for wild type worms under optimal conditions) is a function of adult age and nutrition. If the gravid adults used for preparation of the test population are older or starved, later stage eggs will be laid, reducing the age synchrony of the population and the reproducibility of paralysis kinetics. It is essential than mono-axenic (i.e., only OP50 bacteria) cultures are used (microbial contaminants can severely affect this assay), and reproducibility is highest if the triplicate plates have similar numbers of worms.
3) Transgene Induction and Paralysis Assay
<ol>
 <li>(Day 3) Upshift plates to 25&deg;C 48 hours after the end of the synchronous egg lay; worms should be at the third larval stage (L3). Plates should be arranged without stacking in a 25&deg;C incubator to allow all plates to reach 25&deg;C at the same time.
</li>
 <li>(Day 4) Begin scoring the paralysis of worms (strain CL4176) at 18-20 hrs after the initiation of upshift. At this point, all worms in the population should have reached the fourth larval (L4) stage. Continue scoring in two-hour increments until all worms on each of the plates are paralyzed. For unknown reasons, paralysis is not even across the body length: the head region is the last part of the worm to cease moving. Thus, worms that have recently initiated paralysis cannot translate across the plate, but can move their heads, thereby clearing bacteria around their anterior and leaving a "halo" of cleared bacteria. These worms will invariably become completely paralyzed, and therefore worms with halos are categorized as paralyzed. Some worms will not have halos but will also not show spontaneous movement; these are tested by prodding with the worm picker. If a prodded worm cannot undergo a full body wave propagation upon prodding, it is also scored as paralyzed. For efficient scoring, it is easiest to move the paralyzed worms to an unspotted sector of the plate so that they will not be unintentionally rescored the next scoring period. (This also allows mis-categorized worms to demonstrate movement, allowing a scoring correction).</li>
 <li> To generate a paralysis curve, at each time point the fraction of worms on each plate that have not been paralyzed is converted to a percentage, and the average "unparalyzed" percentage is plotted against time from upshift initiation. (The rationale for plotting in this manner is that the resulting curve is formally analogous to a survival curve, and thus can be analyzed using standard non-parametric survival statistics.)</li>
</ol>
Relevant variables
The upshift of transgenic myo-3/A&beta; worms must occur before they reach the mid L4 stage for paralysis to be initiated, as the myo-3 promoter is developmentally regulated, and has significantly reduced expression in late L4 or adult worms. Similarly, expression of this transgene is blocked if worms become starved, so it is essential that the worms are well-fed throughout the experiment. We have never observed a paralyzed worm to recover under any conditions, and after 12-16 hr of upshift, all CL4176 worms will paralyze, even if they are downshifted to 16&deg;C. In strain CL4176, sufficient upregulation of transgene expression to cause paralysis can occur at lower temperatures (i.e., 20-25&deg;C) although the timing of paralysis will be altered. The paralysis rate depends on the specific transgenic strain used (we have constructed myo-3/A&beta; strains with both faster and slower rates than CL4176), so with alternative strains the scoring window may need to be adjusted.
4) Representative Results
Shown in Figure 1 is a plot of a paralysis curve for CL4176, both untreated and exposed to 6.27 mM caffeine (final concentration in NGM plate). This treatment results in a highly statistically significant delay in paralysis of approximately 4 hrs, which we interpret as indicative of suppression of A&beta; toxicity in this model. Shown in Figure 2 is an independent experiment assaying the effects of cafestol, another compound found in coffee. This plot is typical for treatments that do not impact A&beta; toxicity. Note that in both experiments about half the untreated CL4176 populations are paralyzed at 24 hr, and paralysis is complete by 28 hr. These are typical timeframes for paralysis of control CL4176 populations. Significant deviations from this timing are indicative of altered environmental conditions or spontaneous deletion of transgene copies. (CL4176 contains a chromosomally-integrated transgenic array with multiple copies of the myo-3/A&beta; transgene. Although this transgenic array is normally quite stable, any propagation of the strain at temperatures &gt;16&deg;C will select for rare worms that have undergone a spontaneous deletion of transgenes, resulting in variants that will have reduced A&beta; expression and slower paralysis results.)
<img src="/files/ftp_upload/2252/2252fig1.jpg" alt="Figure 1" /> Figure 1. Paralysis curve for strain CL4176 worms untreated or exposed to 6.27 mM caffeine (Sigma). The indicated times are from initiation of temperature upshift. Error bars = S.E.M.
<img src="/files/ftp_upload/2252/2252fig2.jpg" alt="Figure 2" /> Figure 2. Paralysis curve for CL4176 worms exposed to 0.48 mM cafestol (Sigma). Error bars = S.E.M.

<ol>
	<li>Arya, U., Dwivedi, H., Subramaniam, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reserpine+ameliorates+Abeta+toxicity+in+the+Alzheimer's+disease+model+in+Caenorhabditis+elegans.">Reserpine ameliorates Abeta toxicity in the Alzheimer's disease model in Caenorhabditis elegans.</a> Exp Gerontol. 44, 462-466 (2009).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313, 1604-1610 (2006).</li><li>Dosanjh, L. E., Brown, M. K., Rao, G., Link, C. D., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+of+a+transgenic+C.+elegans+expressing+neuronal+amyloid-beta.">Behavioral phenotyping of a transgenic C. elegans expressing neuronal amyloid-beta.</a> J Alzheimers Dis. , (2009).</li><li>Drake, J., Link, C. D., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+precedes+fibrillar+deposition+of+Alzheimer's+disease+amyloid+beta-peptide+(1-42)+in+a+transgenic+Caenorhabditis+elegans+model.">Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model.</a> Neurobiol Aging. 24, 415-4120 (2003).</li><li>Fay, D. S., Fluet, A., Johnson, C. J., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+aggregation+of+beta-amyloid+peptide+variants.">In vivo aggregation of beta-amyloid peptide variants.</a> J Neurochem. 71, 1616-1625 (1998).</li><li>Florez-McClure, M. L., Hohsfield, L. A., Fonte, G., Bealor, M. T., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+insulin-receptor+signaling+promotes+the+autophagic+degradation+of+beta-amyloid+peptide+in+C.+elegans.">Decreased insulin-receptor signaling promotes the autophagic degradation of beta-amyloid peptide in C. elegans.</a> Autophagy. 3, 569-580 (2007).</li><li>Fonte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+in+vivo+beta-amyloid+peptide+toxicity+by+overexpression+of+the+HSP-16.2+small+chaperone+protein.">Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-16.2 small chaperone protein.</a> J Biol Chem. 283, 784-7891 (2008).</li><li>Hassan, W. M., Merin, D. A., Fonte, V., Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AIP-1+ameliorates+beta-amyloid+peptide+toxicity+in+a+Caenorhabditis+elegans+Alzheimer's+disease+model.">AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model.</a> Hum Mol Genet. 18, 2739-2747 (2009).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+beta-amyloid+peptide+in+transgenic+Caenorhabditis+elegans.">Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 92, 9368-9372 (1995).</li><li>Link, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+in+a+transgenic+Caenorhabditis+elegans+Alzheimer's+disease+model.">Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's disease model.</a> Neurobiol Aging. 24, 397-413 (2003).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta-induced+pathological+behaviors+are+suppressed+by+Ginkgo+biloba+extract+EGb+761+and+ginkgolides+in+transgenic+Caenorhabditis+elegans.">Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans.</a> J Neurosci. 26, 13102-13113 (2006).</li></ol>

No conflicts of interest declared.

The protocol we have described can be effectively used to assay the effects of compounds on A&beta; toxicity. Published examples include the effects of Ginkgo biloba extract constituents (Wu et al. 2006) and reserpine (Arya et al. 2009). Compounds that have been protective against A&beta; toxicity in other systems have also been shown to be protective in this model (e.g., memantine, our unpublished results). An important rationale for establishing this model is that the well-developed genetic and molecular tools available in C. elegans can be used to investigate mechanisms of A&beta; toxicity. Thus, the effects specific mutations on A&beta; toxicity can be assayed (e.g., the effect of reducing insulin-like signaling by introducing a daf-2 loss-of-function mutation, Florez-McClure et al. 2007), and this model can also be used to examine the effects of over-expression transgenes (Fonte et al. 2008). We have recently made extensive use of this model to examine the effects on toxicity of single amino acid substitutions in A&beta; (unpublished data).
The transgenic model described here assays the effect of acute A&beta; expression on muscle cell function. At the time of paralysis muscle cells and their sarcomere are intact; the paralysis phenotype is not the result of muscle cell death. However, after paralysis ( ~48 hr after initial upshift) there is a breakdown of muscle integrity and eventual death of the worms. We have not investigated this downstream effect of A&beta; expression or used it as a toxicity marker.
The inducible A&beta; expression model described here is not appropriate for investigating treatments that modulate &beta;-amyloid formation per se. Although transgenic worms with constitutive A&beta; expression form readily detectable amyloid deposits (Fay et al. 1998; Link et al. 2001), transgenic worms with inducible A&beta; expression rarely have amyloid deposits and the paralysis phenotype appears independent of amyloid deposition (Drake et al. 2003). Our results are consistent with the view that acute toxicity of induced A&beta; expression results from the accumulation of soluble oligomeric A&beta;, not amyloid deposition.

<table border="1">
<tbody>
 <tr>
 <td>Reagents</td>
 <td>Quantity/2L</td>
 <td>Company/Catalogue #</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>6g</td>
 <td>Sigma-Aldrich / S9888</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>40g</td>
 <td>Sigma-Aldrich / A7002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Peptone</td>
 <td>5g</td>
 <td>Sigma-Aldrich / P0556</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ddH2O</td>
 <td>2L</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1M CaCl2</td>
 <td>2mL</td>
 <td>&nbsp;</td>
 <td>147.02mg/mL in ddH2O</td>
 </tr>
 <tr>
 <td>Uracil</td>
 <td>2mL</td>
 <td>&nbsp;</td>
 <td>2mg/mL in ddH2O</td>
 </tr>
 <tr>
 <td>Cholesterol</td>
 <td>2mL</td>
 <td>&nbsp;</td>
 <td>5mg/mL in ethanol (do not autoclave)</td>
 </tr>
 <tr>
 <td>1M MgSO4</td>
 <td>2mL</td>
 <td>&nbsp;</td>
 <td>246.5mg/mL in ddH2O</td>
 </tr>
 <tr>
 <td>1M KPO4</td>
 <td>50mL</td>
 <td>&nbsp;</td>
 <td>98mg KH2PO4.mL, 48mg K2HPO4/mL in ddH2O, pH</td>
 </tr>
 <tr>
 <td>Erlenmeyer Flask</td>
 <td>&nbsp;</td>
 <td>VWR</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

assaying &beta;-amyloid toxicity using a transgenic c. elegans model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Watch this Scientific Journal Video about Assaying Î²-amyloid Toxicity using a Transgenic C. elegans Model at JoVE.com

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

assaying-amyloid-toxicity-using-a-transgenic-c-elegans-model

Research

JoVE Journal

Neuroscience

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...